eIF4B stimulates translation of long mRNAs with structured 5′ UTRs and low closed-loop potential but weak dependence on eIF4G

PubMed Central

Sen, Neelam Dabas; Zhou, Fujun; Harris, Michael S.; Ingolia, Nicholas T.

2016-01-01

DEAD-box RNA helicases eukaryotic translation initiation factor 4A (eIF4A) and Ded1 promote translation by resolving mRNA secondary structures that impede preinitiation complex (PIC) attachment to mRNA or scanning. Eukaryotic translation initiation factor 4B (eIF4B) is a cofactor for eIF4A but also might function independently of eIF4A. Ribosome profiling of mutants lacking eIF4B or with impaired eIF4A or Ded1 activity revealed that eliminating eIF4B reduces the relative translational efficiencies of many more genes than does inactivation of eIF4A, despite comparable reductions in bulk translation, and few genes display unusually strong requirements for both factors. However, either eliminating eIF4B or inactivating eIF4A preferentially impacts mRNAs with longer, more structured 5′ untranslated regions (UTRs). These findings reveal an eIF4A-independent role for eIF4B in addition to its function as eIF4A cofactor in promoting PIC attachment or scanning on structured mRNAs. eIF4B, eIF4A, and Ded1 mutations also preferentially impair translation of longer mRNAs in a fashion mitigated by the ability to form closed-loop messenger ribonucleoprotein particles (mRNPs) via eIF4F–poly(A)-binding protein 1 (Pab1) association, suggesting cooperation between closed-loop assembly and eIF4B/helicase functions. Remarkably, depleting eukaryotic translation initiation factor 4G (eIF4G), the scaffold subunit of eukaryotic translation initiation factor 4F (eIF4F), preferentially impacts short mRNAs with strong closed-loop potential and unstructured 5′ UTRs, exactly the opposite features associated with hyperdependence on the eIF4B/helicases. We propose that short, highly efficient mRNAs preferentially depend on the stimulatory effects of eIF4G-dependent closed-loop assembly. PMID:27601676

Clock-controlled output gene Dbp is a regulator of Arnt/Hif-1β gene expression in pancreatic islet β-cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakabayashi, Hiroko; Ohta, Yasuharu, E-mail: yohta@yamaguchi-u.ac.jp; Yamamoto, Masayoshi

2013-05-03

Highlights: •Arnt mRNA expressed in a circadian manner in mouse pancreatic islets. •Expressions of Dbp and Arnt damped in the islets of a diabetic model mouse. •DBP and E4BP4 regulate Arnt promoter activity by direct binding. •Arnt may have a role in connecting circadian rhythm and metabolism. -- Abstract: Aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia inducible factor-1β (HIF-1β) has emerged as a potential determinant of pancreatic β-cell dysfunction and type 2 diabetes in humans. An 82% reduction in Arnt expression was observed in islets from type 2 diabetic donors as compared to non-diabetic donors. However, few regulators of Arnt expressionmore » have been identified. Meanwhile, disruption of the clock components CLOCK and BMAL1 is known to result in hypoinsulinemia and diabetes, but the molecular details remain unclear. In this study, we identified a novel molecular connection between Arnt and two clock-controlled output genes, albumin D-element binding protein (Dbp) and E4 binding protein 4 (E4bp4). By conducting gene expression studies using the islets of Wfs1{sup −/−} A{sup y}/a mice that develop severe diabetes due to β-cell apoptosis, we demonstrated clock-related gene expressions to be altered in the diabetic mice. Dbp mRNA decreased by 50%, E4bp4 mRNA increased by 50%, and Arnt mRNA decreased by 30% at Zeitgever Time (ZT) 12. Mouse pancreatic islets exhibited oscillations of clock gene expressions. E4BP4, a D-box negative regulator, oscillated anti-phase to DBP, a D-box positive regulator. We also found low-amplitude circadian expression of Arnt mRNA, which peaked at ZT4. Over-expression of DBP raised both mRNA and protein levels of ARNT in HEK293 and MIN6 cell lines. Arnt promoter-driven luciferase reporter assay in MIN6 cells revealed that DBP increased Arnt promoter activity by 2.5-fold and that E4BP4 competitively inhibited its activation. In addition, on ChIP assay, DBP and E4BP4 directly bound to D-box elements within the Arnt promoter in MIN6 cells. These results suggest that in mouse pancreatic islets mRNA expression of Arnt fluctuates significantly in a circadian manner and that the down-regulation of Dbp and up-regulation E4bp4 contribute to direct suppression of Arnt expression in diabetes.« less

Inhibition of Group I Metabotropic Glutamate Receptors Reverses Autistic-Like Phenotypes Caused by Deficiency of the Translation Repressor eIF4E Binding Protein 2

PubMed Central

Aguilar-Valles, Argel; Matta-Camacho, Edna; Khoutorsky, Arkady; Gkogkas, Christos; Nader, Karim

2015-01-01

Exacerbated mRNA translation during brain development has been linked to autism spectrum disorders (ASDs). Deletion of the eukaryotic initiation factor 4E (eIF4E)-binding protein 2 gene (Eif4ebp2), encoding the suppressor of mRNA translation initiation 4E-BP2, leads to an imbalance in excitatory-to-inhibitory neurotransmission and ASD-like behaviors. Inhibition of group I metabotropic glutamate receptors (mGluRs) mGluR1 and mGluR5 reverses the autistic phenotypes in several ASD mouse models. Importantly, these receptors control synaptic physiology via activation of mRNA translation. We investigated the potential reversal of autistic-like phenotypes in Eif4ebp2−/− mice by using antagonists of mGluR1 (JNJ16259685) or mGluR5 (fenobam). Augmented hippocampal mGluR-induced long-term depression (LTD; or chemically induced mGluR-LTD) in Eif4ebp2−/− mice was rescued by mGluR1 or mGluR5 antagonists. While rescue by mGluR5 inhibition occurs through the blockade of a protein synthesis-dependent component of LTD, normalization by mGluR1 antagonists requires the activation of protein synthesis. Synaptically induced LTD was deficient in Eif4ebp2−/− mice, and this deficit was not rescued by group I mGluR antagonists. Furthermore, a single dose of mGluR1 (0.3 mg/kg) or mGluR5 (3 mg/kg) antagonists in vivo reversed the deficits in social interaction and repetitive behaviors (marble burying) in Eif4ebp2−/− mice. Our results demonstrate that Eif4ebp2−/− mice serve as a relevant model to test potential therapies for ASD symptoms. In addition, we provide substantive evidence that the inhibition of mGluR1/mGluR5 is an effective treatment for physiological and behavioral alterations caused by exacerbated mRNA translation initiation. SIGNIFICANCE STATEMENT Exacerbated mRNA translation during brain development is associated with several autism spectrum disorders (ASDs). We recently demonstrated that the deletion of a negative regulator of mRNA translation initiation, the eukaryotic initiation factor 4E-binding protein 2, leads to ASD-like behaviors and increased excitatory synaptic activity. Here we demonstrated that autistic behavioral and electrophysiological phenotypes can be treated in adult mice with antagonists of group I metabotropic glutamate receptors (mGluRs), which have been previously used in other ASD models (i.e., fragile X syndrome). These findings support the use of group I mGluR antagonists as a potential therapy that extends to autism models involving exacerbated mRNA translation initiation. PMID:26245973

Inhibition of Group I Metabotropic Glutamate Receptors Reverses Autistic-Like Phenotypes Caused by Deficiency of the Translation Repressor eIF4E Binding Protein 2.

PubMed

Aguilar-Valles, Argel; Matta-Camacho, Edna; Khoutorsky, Arkady; Gkogkas, Christos; Nader, Karim; Lacaille, Jean-Claude; Sonenberg, Nahum

2015-08-05

Exacerbated mRNA translation during brain development has been linked to autism spectrum disorders (ASDs). Deletion of the eukaryotic initiation factor 4E (eIF4E)-binding protein 2 gene (Eif4ebp2), encoding the suppressor of mRNA translation initiation 4E-BP2, leads to an imbalance in excitatory-to-inhibitory neurotransmission and ASD-like behaviors. Inhibition of group I metabotropic glutamate receptors (mGluRs) mGluR1 and mGluR5 reverses the autistic phenotypes in several ASD mouse models. Importantly, these receptors control synaptic physiology via activation of mRNA translation. We investigated the potential reversal of autistic-like phenotypes in Eif4ebp2(-/-) mice by using antagonists of mGluR1 (JNJ16259685) or mGluR5 (fenobam). Augmented hippocampal mGluR-induced long-term depression (LTD; or chemically induced mGluR-LTD) in Eif4ebp2(-/-) mice was rescued by mGluR1 or mGluR5 antagonists. While rescue by mGluR5 inhibition occurs through the blockade of a protein synthesis-dependent component of LTD, normalization by mGluR1 antagonists requires the activation of protein synthesis. Synaptically induced LTD was deficient in Eif4ebp2(-/-) mice, and this deficit was not rescued by group I mGluR antagonists. Furthermore, a single dose of mGluR1 (0.3 mg/kg) or mGluR5 (3 mg/kg) antagonists in vivo reversed the deficits in social interaction and repetitive behaviors (marble burying) in Eif4ebp2(-/-) mice. Our results demonstrate that Eif4ebp2(-/-) mice serve as a relevant model to test potential therapies for ASD symptoms. In addition, we provide substantive evidence that the inhibition of mGluR1/mGluR5 is an effective treatment for physiological and behavioral alterations caused by exacerbated mRNA translation initiation. Exacerbated mRNA translation during brain development is associated with several autism spectrum disorders (ASDs). We recently demonstrated that the deletion of a negative regulator of mRNA translation initiation, the eukaryotic initiation factor 4E-binding protein 2, leads to ASD-like behaviors and increased excitatory synaptic activity. Here we demonstrated that autistic behavioral and electrophysiological phenotypes can be treated in adult mice with antagonists of group I metabotropic glutamate receptors (mGluRs), which have been previously used in other ASD models (i.e., fragile X syndrome). These findings support the use of group I mGluR antagonists as a potential therapy that extends to autism models involving exacerbated mRNA translation initiation. Copyright © 2015 the authors 0270-6474/15/3511126-08$15.00/0.

Pharmacogenetic inhibition of eIF4E-dependent Mmp9 mRNA translation reverses fragile X syndrome-like phenotypes.

PubMed

Gkogkas, Christos G; Khoutorsky, Arkady; Cao, Ruifeng; Jafarnejad, Seyed Mehdi; Prager-Khoutorsky, Masha; Giannakas, Nikolaos; Kaminari, Archontia; Fragkouli, Apostolia; Nader, Karim; Price, Theodore J; Konicek, Bruce W; Graff, Jeremy R; Tzinia, Athina K; Lacaille, Jean-Claude; Sonenberg, Nahum

2014-12-11

Fragile X syndrome (FXS) is the leading genetic cause of autism. Mutations in Fmr1 (fragile X mental retardation 1 gene) engender exaggerated translation resulting in dendritic spine dysmorphogenesis, synaptic plasticity alterations, and behavioral deficits in mice, which are reminiscent of FXS phenotypes. Using postmortem brains from FXS patients and Fmr1 knockout mice (Fmr1(-/y)), we show that phosphorylation of the mRNA 5' cap binding protein, eukaryotic initiation factor 4E (eIF4E), is elevated concomitant with increased expression of matrix metalloproteinase 9 (MMP-9) protein. Genetic or pharmacological reduction of eIF4E phosphorylation rescued core behavioral deficits, synaptic plasticity alterations, and dendritic spine morphology defects via reducing exaggerated translation of Mmp9 mRNA in Fmr1(-/y) mice, whereas MMP-9 overexpression produced several FXS-like phenotypes. These results uncover a mechanism of regulation of synaptic function by translational control of Mmp-9 in FXS, which opens the possibility of new treatment avenues for the diverse neurological and psychiatric aspects of FXS. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Dietary vitamin C deficiency depressed the gill physical barriers and immune barriers referring to Nrf2, apoptosis, MLCK, NF-κB and TOR signaling in grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare.

PubMed

Xu, Hui-Jun; Jiang, Wei-Dan; Feng, Lin; Liu, Yang; Wu, Pei; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

2016-11-01

This study explored the effects of vitamin C on the physical barriers and immune barriers, and relative mRNA levels of signaling molecules in the gill of grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare. The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) (1) increased reactive oxygen species, malondialdehyde and protein carbonyl (PC) contents (P < 0.05), decreased the copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities and mRNA levels (P < 0.05), and glutathione and vitamin C contents (P < 0.05), down-regulated NF-E2-related factor 2 mRNA level (P < 0.05), and up-regulated Kelch-like ECH-associating protein (Keap) 1a (rather than Keap1b) mRNA level (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency induced oxidative injury in fish gill; (2) up-regulated caspase-3, -7, -8, -9, Fas ligand, B-cell lymphoma protein 2 associated X protein, apoptotic protease activating factor-1 mRNA levels (P < 0.05), and down-regulated inhibitor of apoptosis protein and B-cell lymphoma-2 (rather than myeloid cell leukemia-1) mRNA level (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency aggravated cell apoptosis in fish gill; (3) up-regulated pore-forming TJs Claudin-12, 15a, -15b, and related signaling molecules myosin light chain kinase, p38 mitogen-activated protein kinase (rather than c-Jun N-terminal kinases) mRNA levels (P < 0.05), and down-regulated barrier-forming TJs Occludin, zonula occludens (ZO) 1, ZO-2, Claudin-c, -3c, -7a, -7b mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency disrupted tight junctional complexes in fish gill; (4) decreased lysozyme and acid phosphatase (ACP) activities, and complement 3 (C3), C4 and IgM contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, Hepcidin, β-defensin mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency decrease fish gill immune function; (5) down-regulated the mRNA levels of anti-inflammatory cytokines-related factors interleukin 10 (IL-10), IL-11, transforming growth factor (TGF) β1, TGF-β2, inhibitor of κBa and eIF4E-binding protein 1 (4E-BP1) (rather than 4E-BP2) (P < 0.05), and up-regulated pro-inflammatory cytokines-related factors interferon γ2, IL-1β, IL-6, IL-8, IL-12 P35, IL-12 P40, nuclear factor κB (NF-κB) p65 (rather than NF-κB p52), IκB kinases (IKK) (only IKKα and IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency aggravated fish gill inflammation. In conclusion, vitamin C deficiency disrupted physical barriers and immune barriers, and regulated relative mRNA levels of signaling molecules in fish gill. The vitamin C requirement for against gill rot morbidity of grass carp (264-1031 g) was estimated to be 156.0 mg/kg diet. In addition, based on the gill biochemical indices (antioxidant indices MDA, PC and vitamin C contents, and immune indices LA and ACP activity) the vitamin C requirements for grass carp (264-1031 g) were estimated to be 116.8, 156.6, 110.8, 57.8 and 134.9 mg/kg diet, respectively. Copyright © 2016 Elsevier Ltd. All rights reserved.

Multi-functional regulation of 4E-BP gene expression by the Ccr4-Not complex.

PubMed

Okada, Hirokazu; Schittenhelm, Ralf B; Straessle, Anna; Hafen, Ernst

2015-01-01

The mechanistic target of rapamycin (mTOR) signaling pathway is highly conserved from yeast to humans. It senses various environmental cues to regulate cellular growth and homeostasis. Deregulation of the pathway has been implicated in many pathological conditions including cancer. Phosphorylation cascades through the pathway have been extensively studied but not much is known about the regulation of gene expression of the pathway components. Here, we report that the mRNA level of eukaryotic translation initiation factor (eIF) subunit 4E-binding protein (4E-BP) gene, one of the key mTOR signaling components, is regulated by the highly conserved Ccr4-Not complex. RNAi knockdown of Not1, a putative scaffold protein of this protein complex, increases the mRNA level of 4E-BP in Drosophila Kc cells. Examination of the gene expression mechanism using reporter swap constructs reveals that Not1 depletion increases reporter mRNAs with the 3'UTR of 4E-BP gene, but decreases the ones with the 4E-BP promoter region, suggesting that Ccr4-Not complex regulates both degradation and transcription of 4E-BP mRNA. These results indicate that the Ccr4-Not complex controls expression of a single gene at multiple levels and adjusts the magnitude of the total effect. Thus, our study reveals a novel regulatory mechanism of a key component of the mTOR signaling pathway at the level of gene expression.

Probing the closed-loop model of mRNA translation in living cells

PubMed Central

Archer, Stuart K; Shirokikh, Nikolay E; Hallwirth, Claus V; Beilharz, Traude H; Preiss, Thomas

2015-01-01

The mRNA closed-loop, formed through interactions between the cap structure, poly(A) tail, eIF4E, eIF4G and PAB, features centrally in models of eukaryotic translation initiation, although direct support for its existence in vivo is not well established. Here, we investigated the closed-loop using a combination of mRNP isolation from rapidly cross-linked cells and high-throughput qPCR. Using the interaction between these factors and the opposing ends of mRNAs as a proxy for the closed-loop, we provide evidence that it is prevalent for eIF4E/4G-bound but unexpectedly sparse for PAB1-bound mRNAs, suggesting it primarily occurs during a distinct phase of polysome assembly. We observed mRNA-specific variation in the extent of closed-loop formation, consistent with a role for polysome topology in the control of gene expression. PMID:25826658

Molar loss and powder diet leads to memory deficit and modifies the mRNA expression of brain-derived neurotrophic factor in the hippocampus of adult mice.

PubMed

Takeda, Yosuke; Oue, Hiroshi; Okada, Shinsuke; Kawano, Akira; Koretake, Katsunori; Michikawa, Makoto; Akagawa, Yasumasa; Tsuga, Kazuhiro

2016-12-05

It is known that tooth loss is known to be a risk factor for Alzheimer's disease and soft diet feeding induces memory impairment. Recent studies have shown that brain-derived neurotrophic factor (BDNF) is associated with tooth loss or soft diet in young animal model, and that BDNF expression is decreased in patients with Alzheimer's disease. However, single or combined effect of tooth loss and/or soft diet on brain function has not fully understood. Here we examined the effect of molar loss and powder diet on memory ability and the expression of BDNF mRNA in the hippocampus of adult C57BL/6J mice. Twenty eight-weeks-old C57BL/6J mice were divided into intact molar group and extracted molar group. They were randomly divided into the I/S group (Intact upper molar teeth/Solid diet feeding), the E/S group (Extracted upper molar teeth/Solid diet feeding), the I/P group (Intact upper molar teeth/Powder diet feeding), and the E/P group (Extracted upper molar teeth/Powder diet feeding). The observation periods were 4 and 16-week. To analyze the memory ability, the step-through passive avoidance test was conducted. BDNF-related mRNA in the hippocampus was analyzed by real-time polymerase chain reaction (RT-PCR). At 4 weeks later, we performed memory test and isolated brains to analyze. There were no differences in memory function and BDNF mRNA level between these four groups. However, at 16 weeks later, E/S and E/P group showed memory impairment, and decreased level of BDNF mRNA. Whereas, the powder diet had no effect on memory function and BDNF mRNA level even at 16 weeks later. These results suggest that the effect of molar loss and powder diet on memory function and BDNF mRNA levels were different, molar loss may have a greater long-term effect on memory ability than powder diet does.

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs.

PubMed

Rong, Liwei; Livingstone, Mark; Sukarieh, Rami; Petroulakis, Emmanuel; Gingras, Anne-Claude; Crosby, Katherine; Smith, Bradley; Polakiewicz, Roberto D; Pelletier, Jerry; Ferraiuolo, Maria A; Sonenberg, Nahum

2008-07-01

Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs

PubMed Central

Rong, Liwei; Livingstone, Mark; Sukarieh, Rami; Petroulakis, Emmanuel; Gingras, Anne-Claude; Crosby, Katherine; Smith, Bradley; Polakiewicz, Roberto D.; Pelletier, Jerry; Ferraiuolo, Maria A.; Sonenberg, Nahum

2008-01-01

Eukaryotic initiation factor (eIF) 4E, the mRNA 5′-cap-binding protein, mediates the association of eIF4F with the mRNA 5′-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (∼30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras–expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization. PMID:18515545

TGF-β1 expression in wound healing is acutely affected by experimental malnutrition and early enteral feeding.

PubMed

Alves, Claudia Cristina; Torrinhas, Raquel Susana; Giorgi, Ricardo; Brentani, Maria Mitzi; Logullo, Angela Flavia; Waitzberg, Dan Linetzky

2014-10-01

Malnutrition is associated with the delay or failure of healing. We assessed the effect of experimental malnutrition and early enteral feeding with standard diet or diet supplemented with arginine and antioxidants on the levels of mRNA encoding growth factors in acute, open wound healing. Standardised cutaneous dorsal wounds and gastrostomies for enteral feeding were created in malnourished (M, n = 27) and eutrophic control (E, n = 30) Lewis male adult rats. Both M and E rats received isocaloric and isonitrogenous regimens with oral chow and saline (C), standard (S) or supplemented (A) enteral diets. On post-trauma day 7, mRNA levels of growth factor genes were analysed in wound granulation tissue by reverse transcription polymerase chain reaction (RT-PCR). M(C) rats had significantly lower transforming growth factor β(TGF-β1 ) mRNA levels than E(C) rats (2·58 ± 0·83 versus 3·53 ± 0·57, P < 0·01) and in comparison with M(S) and M(A) rats (4·66 ± 2·49 and 4·61 ± 2·11, respectively; P < 0·05). VEGF and KGF-7 mRNA levels were lower in M(A) rats than in E(A) rats (0·74 ± 0·16 versus 1·25 ± 0·66; and 1·07 ± 0·45 versus 1·79 ± 0·89, respectively; P≤ 0·04), but did not differ from levels in E(C) and M(C) animals. In experimental open acute wound healing, previous malnutrition decreased local mRNA levels of TGF-β1 genes, which was minimised by early enteral feeding with standard or supplemented diets. © 2012 The Authors. International Wound Journal © 2012 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

Plant growth and fertility requires functional interactions between specific PABP and eIF4G gene family members

PubMed Central

2018-01-01

The initiation of protein synthesis requires the involvement of the eukaryotic translation initiation factor (eIF) 4G to promote assembly of the factors needed to recruit a 40S ribosomal subunit to an mRNA. Although many eukaryotes express two eIF4G isoforms that are highly similar, those in plants, referred to as eIF4G and eIFiso4G, are highly divergent in size, sequence, and domain organization. Species of the Brassicaceae and the Cleomaceae also express a divergent eIFiso4G isoform, referred to as eIFiso4G2, not found elsewhere in the plant kingdom. Despite their divergence, eIF4G and eIFiso4G interact with eIF4A, eIF4B, and eIF4E isoforms needed for binding an mRNA. eIF4G and eIFiso4G also interact with the poly(A)-binding protein (PABP) which promotes ribosome recruitment to an mRNA. Increasing the complexity of such an interaction, however, Arabidopsis also expresses three PABP isoforms (PAB2, PAB4, and PAB8) in vegetative and reproductive tissues. In this study, the functional interactions among the eIF4G and the widely-expressed PABP isoforms were examined. Loss of PAB2 or PAB8 in combination with loss of eIF4G or eIFiso4G had little to no effect on growth or fertility whereas pab2 pab8 eif4g or pab2 pab8 eifiso4g1/2 mutants exhibited smaller stature and reduced fertility. Although the pab4 eifiso4g1 mutant grows normally and is fertile, pab4 eif4g or pab4 eifiso4g2 mutants could not be isolated. Even pab4/PAB4 eif4g/eIF4G heterozygous plants exhibited growth defects and low fertility. Mutant co-inheritance analysis in reciprocal crosses with wild-type plants revealed that most ovaries and pollen from pab4/PAB4 eif4g/eIF4G plants were PAB4 eif4g. Similarly, co-inheritance studies with pab4/PAB4 eifiso4g2/eIFiso4G2 plants suggested most ovaries were PAB4 eifiso4g2. These results suggest that a functional interaction between PAB4 and eIF4G and between PAB4 and eIFiso4G2 is required for growth and normal fertility. PMID:29381712

Eukaryotic Initiation Factor eIFiso4G1 and eIFiso4G2 Are Isoforms Exhibiting Distinct Functional Differences in Supporting Translation in Arabidopsis*

PubMed Central

Gallie, Daniel R.

2016-01-01

The eukaryotic translation initiation factor (eIF) 4G is required during protein synthesis to promote the assembly of several factors involved in the recruitment of a 40S ribosomal subunit to an mRNA. Although many eukaryotes express two eIF4G isoforms that are highly similar, the eIF4G isoforms in plants, referred to as eIF4G and eIFiso4G, are highly divergent in size, sequence, and domain organization but both can interact with eIF4A, eIF4B, eIF4E isoforms, and the poly(A)-binding protein. Nevertheless, eIF4G and eIFiso4G from wheat exhibit preferences in the mRNAs they translate optimally. For example, mRNA containing the 5′-leader (called Ω) of tobacco mosaic virus preferentially uses eIF4G in wheat germ lysate. In this study, the eIF4G isoform specificity of Ω was used to examine functional differences of the eIF4G isoforms in Arabidopsis. As in wheat, Ω-mediated translation was reduced in an eif4g null mutant. Loss of the eIFiso4G1 isoform, which is similar in sequence to wheat eIFiso4G, did not substantially affect Ω-mediated translation. However, loss of the eIFiso4G2 isoform substantially reduced Ω-mediated translation. eIFiso4G2 is substantially divergent from eIFiso4G1 and is present only in the Brassicaceae, suggesting a recent evolution. eIFiso4G2 isoforms exhibit sequence-specific differences in regions representing partner protein and RNA binding sites. Loss of any eIF4G isoform also resulted in a substantial reduction in reporter transcript level. These results suggest that eIFiso4G2 appeared late in plant evolution and exhibits more functional similarity with eIF4G than with eIFiso4G1 during Ω-mediated translation. PMID:26578519

Cot/tpl2-MKK1/2-Erk1/2 controls mTORC1-mediated mRNA translation in Toll-like receptor–activated macrophages

PubMed Central

López-Pelaéz, Marta; Fumagalli, Stefano; Sanz, Carlos; Herrero, Clara; Guerra, Susana; Fernandez, Margarita; Alemany, Susana

2012-01-01

Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor–activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow–derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP–eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene–encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene–encoding mRNA translation in Toll-like receptor–activated macrophages. PMID:22675026

Cot/tpl2-MKK1/2-Erk1/2 controls mTORC1-mediated mRNA translation in Toll-like receptor-activated macrophages.

PubMed

López-Pelaéz, Marta; Fumagalli, Stefano; Sanz, Carlos; Herrero, Clara; Guerra, Susana; Fernandez, Margarita; Alemany, Susana

2012-08-01

Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor-activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow-derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP-eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene-encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene-encoding mRNA translation in Toll-like receptor-activated macrophages.

Canonical Initiation Factor Requirements of the Myc Family of Internal Ribosome Entry Segments▿ †

PubMed Central

Spriggs, Keith A.; Cobbold, Laura C.; Jopling, Catherine L.; Cooper, Rebecca E.; Wilson, Lindsay A.; Stoneley, Mark; Coldwell, Mark J.; Poncet, Didier; Shen, Ya-Ching; Morley, Simon J.; Bushell, Martin; Willis, Anne E.

2009-01-01

Initiation of protein synthesis in eukaryotes requires recruitment of the ribosome to the mRNA and its translocation to the start codon. There are at least two distinct mechanisms by which this process can be achieved; the ribosome can be recruited either to the cap structure at the 5′ end of the message or to an internal ribosome entry segment (IRES), a complex RNA structural element located in the 5′ untranslated region (5′-UTR) of the mRNA. However, it is not well understood how cellular IRESs function to recruit the ribosome or how the 40S ribosomal subunits translocate from the initial recruitment site on the mRNA to the AUG initiation codon. We have investigated the canonical factors that are required by the IRESs found in the 5′-UTRs of c-, L-, and N-myc, using specific inhibitors and a tissue culture-based assay system, and have shown that they differ considerably in their requirements. The L-myc IRES requires the eIF4F complex and the association of PABP and eIF3 with eIF4G for activity. The minimum requirements of the N- and c-myc IRESs are the C-terminal domain of eIF4G to which eIF4A is bound and eIF3, although interestingly this protein does not appear to be recruited to the IRES RNA via eIF4G. Finally, our data show that all three IRESs require a ternary complex, although in contrast to c- and L-myc IRESs, the N-myc IRES has a lesser requirement for a ternary complex. PMID:19124605

Yeast eIF4A enhances recruitment of mRNAs regardless of their structural complexity

PubMed Central

Yourik, Paul; Aitken, Colin Echeverría; Zhou, Fujun; Gupta, Neha

2017-01-01

eIF4A is a DEAD-box RNA-dependent ATPase thought to unwind RNA secondary structure in the 5'-untranslated regions (UTRs) of mRNAs to promote their recruitment to the eukaryotic translation pre-initiation complex (PIC). We show that eIF4A's ATPase activity is markedly stimulated in the presence of the PIC, independently of eIF4E•eIF4G, but dependent on subunits i and g of the heteromeric eIF3 complex. Surprisingly, eIF4A accelerated the rate of recruitment of all mRNAs tested, regardless of their degree of structural complexity. Structures in the 5'-UTR and 3' of the start codon synergistically inhibit mRNA recruitment in a manner relieved by eIF4A, indicating that the factor does not act solely to melt hairpins in 5'-UTRs. Our findings that eIF4A functionally interacts with the PIC and plays important roles beyond unwinding 5'-UTR structure is consistent with a recent proposal that eIF4A modulates the conformation of the 40S ribosomal subunit to promote mRNA recruitment. PMID:29192585

Modulation of tumor eIF4E by antisense inhibition: A phase I/II translational clinical trial of ISIS 183750-an antisense oligonucleotide against eIF4E-in combination with irinotecan in solid tumors and irinotecan-refractory colorectal cancer.

PubMed

Duffy, A G; Makarova-Rusher, O V; Ulahannan, S V; Rahma, O E; Fioravanti, S; Walker, M; Abdullah, S; Raffeld, M; Anderson, V; Abi-Jaoudeh, N; Levy, E; Wood, B J; Lee, S; Tomita, Y; Trepel, J B; Steinberg, S M; Revenko, A S; MacLeod, A R; Peer, C J; Figg, W D; Greten, T F

2016-10-01

The eukaryotic translation initiation factor 4E (eIF4E) is a potent oncogene that is found to be dysregulated in 30% of human cancer, including colorectal carcinogenesis (CRC). ISIS 183750 is a second-generation antisense oligonucleotide (ASO) designed to inhibit the production of the eIF4E protein. In preclinical studies we found that EIF4e ASOs reduced expression of EIF4e mRNA and inhibited proliferation of colorectal carcinoma cells. An additive antiproliferative effect was observed in combination with irinotecan. We then performed a clinical trial evaluating this combination in patients with refractory cancer. No dose-limiting toxicities were seen but based on pharmacokinetic data and tolerability the dose of irinotecan was reduced to 160 mg/m(2) biweekly. Efficacy was evaluated in 15 patients with irinotecan-refractory colorectal cancer. The median time of disease control was 22.1 weeks. After ISIS 183750 treatment, peripheral blood levels of eIF4E mRNA were decreased in 13 of 19 patients. Matched pre- and posttreatment tumor biopsies showed decreased eIF4E mRNA levels in five of nine patients. In tumor tissue, the intracellular and stromal presence of ISIS 183750 was detected by IHC in all biopsied patients. Although there were no objective responses stable disease was seen in seven of 15 (47%) patients who were progressing before study entry, six of whom were stable at the time of the week 16 CT scan. We were also able to confirm through mandatory pre- and posttherapy tumor biopsies penetration of the ASO into the site of metastasis. © 2016 UICC.

La-related Protein 1 (LARP1) Represses Terminal Oligopyrimidine (TOP) mRNA Translation Downstream of mTOR Complex 1 (mTORC1).

PubMed

Fonseca, Bruno D; Zakaria, Chadi; Jia, Jian-Jun; Graber, Tyson E; Svitkin, Yuri; Tahmasebi, Soroush; Healy, Danielle; Hoang, Huy-Dung; Jensen, Jacob M; Diao, Ilo T; Lussier, Alexandre; Dajadian, Christopher; Padmanabhan, Niranjan; Wang, Walter; Matta-Camacho, Edna; Hearnden, Jaclyn; Smith, Ewan M; Tsukumo, Yoshinori; Yanagiya, Akiko; Morita, Masahiro; Petroulakis, Emmanuel; González, Jose L; Hernández, Greco; Alain, Tommy; Damgaard, Christian K

2015-06-26

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5'TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

La-related Protein 1 (LARP1) Represses Terminal Oligopyrimidine (TOP) mRNA Translation Downstream of mTOR Complex 1 (mTORC1)*

PubMed Central

Fonseca, Bruno D.; Zakaria, Chadi; Jia, Jian-Jun; Graber, Tyson E.; Svitkin, Yuri; Tahmasebi, Soroush; Healy, Danielle; Hoang, Huy-Dung; Jensen, Jacob M.; Diao, Ilo T.; Lussier, Alexandre; Dajadian, Christopher; Padmanabhan, Niranjan; Wang, Walter; Matta-Camacho, Edna; Hearnden, Jaclyn; Smith, Ewan M.; Tsukumo, Yoshinori; Yanagiya, Akiko; Morita, Masahiro; Petroulakis, Emmanuel; González, Jose L.; Hernández, Greco; Alain, Tommy; Damgaard, Christian K.

2015-01-01

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5′TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1. PMID:25940091

Translation initiation on mRNAs bound by nuclear cap-binding protein complex CBP80/20 requires interaction between CBP80/20-dependent translation initiation factor and eukaryotic translation initiation factor 3g.

PubMed

Choe, Junho; Oh, Nara; Park, Sungjin; Lee, Ye Kyung; Song, Ok-Kyu; Locker, Nicolas; Chi, Sung-Gil; Kim, Yoon Ki

2012-05-25

In the cytoplasm of mammalian cells, either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF) 4E can direct the initiation of translation. Although the recruitment of ribosomes to mRNAs during eIF4E-dependent translation (ET) is well characterized, the molecular mechanism for CBP80/20-dependent translation (CT) remains obscure. Here, we show that CBP80/20-dependent translation initiation factor (CTIF), which has been shown to be preferentially involved in CT but not ET, specifically interacts with eIF3g, a component of the eIF3 complex involved in ribosome recruitment. By interacting with eIF3g, CTIF serves as an adaptor protein to bridge the CBP80/20 and the eIF3 complex, leading to efficient ribosome recruitment during CT. Accordingly, down-regulation of CTIF using a small interfering RNA causes a redistribution of CBP80 from polysome fractions to subpolysome fractions, without significant consequence to eIF4E distribution. In addition, down-regulation of eIF3g inhibits the efficiency of nonsense-mediated mRNA decay, which is tightly coupled to CT but not to ET. Moreover, the artificial tethering of CTIF to an intercistronic region of dicistronic mRNA results in translation of the downstream cistron in an eIF3-dependent manner. These findings support the idea that CT mechanistically differs from ET.

Identification and Characterization of Functionally Critical, Conserved Motifs in the Internal Repeats and N-terminal Domain of Yeast Translation Initiation Factor 4B (yeIF4B)*

PubMed Central

Zhou, Fujun; Walker, Sarah E.; Mitchell, Sarah F.; Lorsch, Jon R.; Hinnebusch, Alan G.

2014-01-01

eIF4B has been implicated in attachment of the 43 S preinitiation complex (PIC) to mRNAs and scanning to the start codon. We recently determined that the internal seven repeats (of ∼26 amino acids each) of Saccharomyces cerevisiae eIF4B (yeIF4B) compose the region most critically required to enhance mRNA recruitment by 43 S PICs in vitro and stimulate general translation initiation in yeast. Moreover, although the N-terminal domain (NTD) of yeIF4B contributes to these activities, the RNA recognition motif is dispensable. We have now determined that only two of the seven internal repeats are sufficient for wild-type (WT) yeIF4B function in vivo when all other domains are intact. However, three or more repeats are needed in the absence of the NTD or when the functions of eIF4F components are compromised. We corroborated these observations in the reconstituted system by demonstrating that yeIF4B variants with only one or two repeats display substantial activity in promoting mRNA recruitment by the PIC, whereas additional repeats are required at lower levels of eIF4A or when the NTD is missing. These findings indicate functional overlap among the 7-repeats and NTD domains of yeIF4B and eIF4A in mRNA recruitment. Interestingly, only three highly conserved positions in the 26-amino acid repeat are essential for function in vitro and in vivo. Finally, we identified conserved motifs in the NTD and demonstrate functional overlap of two such motifs. These results provide a comprehensive description of the critical sequence elements in yeIF4B that support eIF4F function in mRNA recruitment by the PIC. PMID:24285537

Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

PubMed Central

Aitken, Colin Echeverría; Beznosková, Petra; Vlčkova, Vladislava; Chiu, Wen-Ling; Zhou, Fujun; Valášek, Leoš Shivaya; Hinnebusch, Alan G; Lorsch, Jon R

2016-01-01

Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA. DOI: http://dx.doi.org/10.7554/eLife.20934.001 PMID:27782884

Signalling to eIF4E in cancer

PubMed Central

Siddiqui, Nadeem; Sonenberg, Nahum

2015-01-01

Translational control plays a critical role in the regulation of gene expression in eukaryotes and affects many essential cellular processes, including proliferation, apoptosis and differentiation. Under most circumstances, translational control occurs at the initiation step at which the ribosome is recruited to the mRNA. The eukaryotic translation initiation factor 4E (eIF4E), as part of the eIF4F complex, interacts first with the mRNA and facilitates the recruitment of the 40S ribosomal subunit. The activity of eIF4E is regulated at many levels, most profoundly by two major signalling pathways: PI3K (phosphoinositide 3-kinase)/Akt (also known and Protein Kinase B, PKB)/mTOR (mechanistic/mammalian target of rapamycin) and Ras (rat sarcoma)/MAPK (mitogen-activated protein kinase)/Mnk (MAPK-interacting kinases). mTOR directly phosphorylates the 4E-BPs (eIF4E-binding proteins), which are inhibitors of eIF4E, to relieve translational suppression, whereas Mnk phosphorylates eIF4E to stimulate translation. Hyperactivation of these pathways occurs in the majority of cancers, which results in increased eIF4E activity. Thus, translational control via eIF4E acts as a convergence point for hyperactive signalling pathways to promote tumorigenesis. Consequently, recent works have aimed to target these pathways and ultimately the translational machinery for cancer therapy. PMID:26517881

The expression analysis of Sfrs10 and Celf4 during mouse retinal development

PubMed Central

Karunakaran, Devi Krishna Priya; Congdon, Sean; Guerrette, Thomas; Banday, Abdul Rouf; Lemoine, Christopher; Chhaya, Nisarg; Kanadia, Rahul

2013-01-01

Processing of mRNAs including, alternative splicing (AS), mRNA transport and translation regulation are crucial to eukaryotic gene expression. For example, >90% of the gene in the human genome are known to undergo alternative splicing thereby expanding the proteome production capacity of a limited number of genes. Similarly, mRNA export and translation regulation plays a vital role in regulating protein production. Thus, it is important to understand how these RNA binding proteins including alternative splicing factors (ASFs) and mRNA transport and translation factors regulate these processes. Here we report the expression of an ASF, Serine-arginine rich splicing factor 10 (Sfrs10) and a mRNA translation regulation factor, CUGBP, elav like family member 4 (Celf4) in the developing mouse retina. Sfrs10 was expressed throughout postnatal (P) retinal development and was observed progressively in newly differentiating neurons. Immunofluorescence (IF) showed Sfrs10 in retinal ganglion cells (RGCs) at P0, followed by amacrine and bipolar cells, and at P8 it was enriched in red/green cone photoreceptor cells. By P22, Sfrs10 was observed in rod photoreceptors in a peri-nuclear pattern. Like Sfrs10, Celf4 was also observed in the developing retina, but with two distinct retinal isoforms. In situ hybridization (ISH) showed progressive expression of Celf4 in differentiating neurons, which was confirmed by IF that showed a dynamic shift in Celf4 localization. Early in development Celf4 expression was restricted to the nuclei of newly differentiating RGCs and later (E16 onwards) it was observed in the initial segments of RGC axons. Later, during postnatal development, Celf4 was observed in amacrine and bipolar cells, but here it was predominantly cytoplasmic and enriched in the two synaptic layers. Specifically, at P14, Celf4 was observed in the synaptic boutons of rod bipolar cells marked by Pkc-α. Thus, Celf4 might be regulating AS early in development besides its known role of regulating mRNA localization/translation. In all, our data suggests an important role for AS and mRNA localization/translation in retinal neuron differentiation. PMID:23932931

Transcriptome-wide studies uncover the diversity of modes of mRNA recruitment to eukaryotic ribosomes.

PubMed

Shatsky, Ivan N; Dmitriev, Sergey E; Andreev, Dmitri E; Terenin, Ilya M

2014-01-01

The conventional paradigm of translation initiation in eukaryotes states that the cap-binding protein complex eIF4F (consisting of eIF4E, eIF4G and eIF4A) plays a central role in the recruitment of capped mRNAs to ribosomes. However, a growing body of evidence indicates that this paradigm should be revised. This review summarizes the data which have been mostly accumulated in a post-genomic era owing to revolutionary techniques of transcriptome-wide analysis. Unexpectedly, these techniques have uncovered remarkable diversity in the recruitment of cellular mRNAs to eukaryotic ribosomes. These data enable a preliminary classification of mRNAs into several groups based on their requirement for particular components of eIF4F. They challenge the widely accepted concept which relates eIF4E-dependence to the extent of secondary structure in the 5' untranslated regions of mRNAs. Moreover, some mRNA species presumably recruit ribosomes to their 5' ends without the involvement of either the 5' m(7)G-cap or eIF4F but instead utilize eIF4G or eIF4G-like auxiliary factors. The long-standing concept of internal ribosome entry site (IRES)-elements in cellular mRNAs is also discussed.

Long-term treatment of bile duct-ligated rats with rapamycin (sirolimus) significantly attenuates liver fibrosis: analysis of the underlying mechanisms.

PubMed

Biecker, Erwin; De Gottardi, Andrea; Neef, Markus; Unternährer, Matthias; Schneider, Vreni; Ledermann, Monika; Sägesser, Hans; Shaw, Sidney; Reichen, Jürg

2005-06-01

Rapamycin is an immunosuppressant with antiproliferative properties. We investigated whether rapamycin treatment of bile duct-ligated (BDL) rats is capable of inhibiting liver fibrosis and thereby affecting hemodynamics. Following BDL, rats were treated for 28 days with rapamycin (BDL SIR). BDL animals without drug treatment (BDL CTR) and sham-operated animals served as controls. After 28 days, hemodynamics were measured, and livers were harvested for histology/immunohistochemistry. Liver mRNA levels of transforming growth factor (TGF)-beta1, connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF)-beta, cyclin-dependent kinase inhibitor p27(kip) (p27), and cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (p21) were quantified by real-time polymerase chain reaction. Liver protein levels of p27, p21, p70 S6 kinase (p70(s6k)), phosphorylated p70(s6k) (p-p70(s6k)), eukaryotic initiation factor 4E-binding protein (4E-BP1), p-4E-BP1 (Thr37/46), and p-4E-BP1 (Ser65/Thr70) were determined by Western blotting. Portal vein pressure was lower in BDL SIR than in BDL CTR animals. Volume fractions of connective tissue, bile duct epithelial, and desmin- and actin-positive cells were lower in BDL SIR than in BDL CTR rats. On the mRNA level, TGF-beta1, CTGF, and PDGF were decreased by rapamycin. p27 and p21 mRNA did not differ. On the protein level, rapamycin increased p27 and decreased p21 levels. Levels of nonphosphorylated p70(s6k) and 4E-BP1 did not vary between groups, but levels of p-p70(s6k) were decreased by rapamycin. Rapamycin had no effect on p-4E-BP1 (Thr37/46) and p-4E-BP1 (Ser65/Thr70) levels. In BDL rats, rapamycin inhibits liver fibrosis and ameliorates portal hypertension. This is paralleled by decreased levels of TGF-beta1, CTGF, and PDGF. Rapamycin influences the cell cycle by up-regulation of p27, down-regulation of p21, and inhibition of p70(s6k) phosphorylation.

Translation Initiation Factor AteIF(iso)4E Is Involved in Selective mRNA Translation in Arabidopsis Thaliana Seedlings

PubMed Central

Martínez-Silva, Ana Valeria; Aguirre-Martínez, César; Flores-Tinoco, Carlos E.; Alejandri-Ramírez, Naholi D.; Dinkova, Tzvetanka D.

2012-01-01

One of the most regulated steps of translation initiation is the recruitment of mRNA by the translation machinery. In eukaryotes, this step is mediated by the 5′end cap-binding factor eIF4E bound to the bridge protein eIF4G and forming the eIF4F complex. In plants, different isoforms of eIF4E and eIF4G form the antigenically distinct eIF4F and eIF(iso)4F complexes proposed to mediate selective translation. Using a microarray analysis of polyribosome- and non-polyribosome-purified mRNAs from 15 day-old Arabidopsis thaliana wild type [WT] and eIF(iso)4E knockout mutant [(iso)4E-1] seedlings we found 79 transcripts shifted from polyribosomes toward non-polyribosomes, and 47 mRNAs with the opposite behavior in the knockout mutant. The translationally decreased mRNAs were overrepresented in root-preferentially expressed genes and proteins from the endomembrane system, including several transporters such as the phosphate transporter PHOSPHATE1 (PHO1), Sucrose transporter 3 (SUC3), ABC transporter-like with ATPase activity (MRP11) and five electron transporters, as well as signal transduction-, protein modification- and transcription-related proteins. Under normal growth conditions, eIF(iso)4E expression under the constitutive promoter 35 S enhanced the polyribosomal recruitment of PHO1 supporting its translational preference for eIF(iso)4E. Furthermore, under phosphate deficiency, the PHO1 protein increased in the eIF(iso)4E overexpressing plants and decreased in the knockout mutant as compared to wild type. In addition, the knockout mutant had larger root, whereas the 35 S directed expression of eIF(iso)4E caused shorter root under normal growth conditions, but not under phosphate deficiency. These results indicate that selective translation mediated by eIF(iso)4E is relevant for Arabidopsis root development under normal growth conditions. PMID:22363683

5'-Phosphorothiolate Dinucleotide Cap Analogues: Reagents for Messenger RNA Modification and Potent Small-Molecular Inhibitors of Decapping Enzymes.

PubMed

Wojtczak, Blazej A; Sikorski, Pawel J; Fac-Dabrowska, Kaja; Nowicka, Anna; Warminski, Marcin; Kubacka, Dorota; Nowak, Elzbieta; Nowotny, Marcin; Kowalska, Joanna; Jemielity, Jacek

2018-05-09

The 5' cap consists of 7-methylguanosine (m 7 G) linked by a 5'-5'-triphosphate bridge to messenger RNA (mRNA) and acts as the master regulator of mRNA turnover and translation initiation in eukaryotes. Cap analogues that influence mRNA translation and turnover (either as small molecules or as part of an RNA transcript) are valuable tools for studying gene expression, which is often also of therapeutic relevance. Here, we synthesized a series of 15 dinucleotide cap (m 7 GpppG) analogues containing a 5'-phosphorothiolate (5'-PSL) moiety (i.e., an O-to-S substitution within the 5'-phosphoester) and studied their biological properties in the context of three major cap-binding proteins: translation initiation factor 4E (eIF4E) and two decapping enzymes, DcpS and Dcp2. While the 5'-PSL moiety was neutral or slightly stabilizing for cap interactions with eIF4E, it significantly influenced susceptibility to decapping. Replacing the γ-phosphoester with the 5'-PSL moiety (γ-PSL) prevented β-γ-pyrophosphate bond cleavage by DcpS and conferred strong inhibitory properties. Combining the γ-PSL moiety with α-PSL and β-phosphorothioate (PS) moiety afforded first cap-derived hDcpS inhibitor with low nanomolar potency. Susceptibility to Dcp2 and translational properties were studied after incorporation of the new analogues into mRNA transcripts by RNA polymerase. Transcripts containing the γ-PSL moiety were resistant to cleavage by Dcp2. Surprisingly, superior translational properties were observed for mRNAs containing the α-PSL moiety, which were Dcp2-susceptible. The overall protein expression measured in HeLa cells for this mRNA was comparable to mRNA capped with the translation augmenting β-PS analogue reported previously. Overall, our study highlights 5'-PSL as a synthetically accessible cap modification, which, depending on the substitution site, can either reduce susceptibility to decapping or confer superior translational properties on the mRNA. The 5'-PSL-analogues may find application as reagents for the preparation of efficiently expressed mRNA or for investigation of the role of decapping enzymes in mRNA processing or neuromuscular disorders associated with decapping.

Synthesis of Toll-like receptor 4 in Kupffer cells and its role in alcohol-induced liver disease.

PubMed

Zuo, Guoqing; Gong, Jianping; Liu, Chang'an; Wu, Chuanxin; Li, Shengwei; Dai, Lili

2003-02-01

To observe the synthesis of Toll-like receptor (TLR) 4 protein and its mRNA expression in Kupffer cells (KCs) and evaluate the role of TLR 4 in liver injury to rats through alcohol-induced liver disease. Twenty-eight Wistar rats were divided into two groups: ethanol-fed (group E) and control (group C). Group E rats were given ethanol at a dose of 5 - 12 g x kg(-1) x d(-1), while group C received dextrose. Animals from both groups were killed at 4 and 8 weeks. The KCs were isolated and synthesis of TLR 4 protein was determined by laser scanning confocal microscopy. TLR 4 mRNA expression in KCs was determined by reverse transcription polymerase chain reaction (RT-PCR) analysis. The levels of endotoxin, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in plasma were determined. Changes in liver pathology were observed. Laser scanning confocal microscopy showed that the intensity of fluorescence of TLR 4 protein in group E was stronger than group C. Ethanol administration led to a significant increase in TLR 4 mRNA expression in group E compared with group C (P < 0.05). The concentrations of plasma endotoxin, TNF-alpha and IL-6 were higher in group E than in group C (P < 0.05). Liver sections from rats in group E demonstrated marked pathological changes. Ethanol administration can lead to the synthesis of TLR 4 protein and its gene expression in KCs, indicating that TLR 4 may play a major role in the development of alcohol-induced liver injury.

mRNA-Selective Translation Induced by FSH in Primary Sertoli Cells

PubMed Central

Musnier, Astrid; León, Kelly; Morales, Julia; Reiter, Eric; Boulo, Thomas; Costache, Vlad; Vourc'h, Patrick; Heitzler, Domitille; Oulhen, Nathalie; Poupon, Anne; Boulben, Sandrine; Cormier, Patrick

2012-01-01

FSH is a key hormonal regulator of Sertoli cell secretory activity, required to optimize sperm production. To fulfil its biological function, FSH binds a G protein-coupled receptor, the FSH-R. The FSH-R-transduced signaling network ultimately leads to the transcription or down-regulation of numerous genes. In addition, recent evidence has suggested that FSH might also regulate protein translation. However, this point has never been demonstrated conclusively yet. Here we have addressed this issue in primary rat Sertoli cells endogenously expressing physiological levels of FSH-R. We observed that, within 90 min of stimulation, FSH not only enhanced overall protein synthesis in a mammalian target of rapamycin-dependent manner but also increased the recruitment of mRNA to polysomes. m7GTP pull-down experiments revealed the functional recruitment of mammalian target of rapamycin and p70 S6 kinase to the 5′cap, further supported by the enhanced phosphorylation of one of p70 S6 kinase targets, the eukaryotic initiation factor 4B. Importantly, the scaffolding eukaryotic initiation factor 4G was also recruited, whereas eukaryotic initiation factor 4E-binding protein, the eukaryotic initiation factor 4E generic inhibitor, appeared to play a minor role in translational regulations induced by FSH, in contrast to what is generally observed in response to anabolic factors. This particular regulation of the translational machinery by FSH stimulation might support mRNA-selective translation, as shown here by quantitative RT-PCR amplification of the c-fos and vascular endothelial growth factor mRNA but not of all FSH target mRNA, in polysomal fractions. These findings add a new level of complexity to FSH biological roles in its natural target cells, which has been underappreciated so far. PMID:22383463

Eukaryotic Initiation Factor (eIF) 4F Binding to Barley Yellow Dwarf Virus (BYDV) 3′-Untranslated Region Correlates with Translation Efficiency*

PubMed Central

Banerjee, Bidisha; Goss, Dixie J.

2014-01-01

Eukaryotic initiation factor (eIF) 4F binding to mRNA is the first committed step in cap-dependent protein synthesis. Barley yellow dwarf virus (BYDV) employs a cap-independent mechanism of translation initiation that is mediated by a structural BYDV translation element (BTE) located in the 3′-UTR of its mRNA. eIF4F bound the BTE and a translationally inactive mutant with high affinity, thus questioning the role of eIF4F in translation of BYDV. To examine the effects of eIF4F in BYDV translation initiation, BTE mutants with widely different in vitro translation efficiencies ranging from 5 to 164% compared with WT were studied. Using fluorescence anisotropy to obtain quantitative data, we show 1) the equilibrium binding affinity (complex stability) correlated well with translation efficiency, whereas the “on” rate of binding did not; 2) other unidentified proteins or small molecules in wheat germ extract prevented eIF4F binding to mutant BTE but not WT BTE; 3) BTE mutant-eIF4F interactions were found to be both enthalpically and entropically favorable with an enthalpic contribution of 52–90% to ΔG° at 25 °C, suggesting that hydrogen bonding contributes to stability; and 4) in contrast to cap-dependent and tobacco etch virus internal ribosome entry site interaction with eIF4F, poly(A)-binding protein did not increase eIF4F binding. Further, the eIF4F bound to the 3′ BTE with higher affinity than for either m7G cap or tobacco etch virus internal ribosome entry site, suggesting that the 3′ BTE may play a role in sequestering host cell initiation factors and possibly regulating the switch from replication to translation. PMID:24379412

Region-specific expression and hormonal regulation of the first exon variants of rat prolactin receptor mRNA in rat brain and anterior pituitary gland.

PubMed

Nogami, H; Hoshino, R; Ogasawara, K; Miyamoto, S; Hisano, S

2007-08-01

Recent studies have revealed the occurrence of five first exon variants of the rat prolactin receptor mRNA, suggesting that multiple promoters direct prolactin receptor transcription in response to different regulatory factors. In the present study, regional expression of these first exon variants, as well as two prolactin receptor subtypes generated by alternative splicing, was examined in the brains and anterior pituitary glands of female rats. Expression of the long-form was detected in the choroid plexus, hypothalamus, hippocampus, cerebral cortex and anterior pituitary gland, whereas the short form was detected only in the choroid plexus. E1-3 mRNA, a first exon variant, was detected in the choroid plexus, hypothalamus, and anterior pituitary gland, whereas E1-4 was detected only in the choroid plexus. Other variants were not detectable by the polymerase chain reaction protocol employed in this study. Ovariectomy increased the short form in the choroid plexus and the E1-3 expression in the choroid plexus and pituitary gland, but changes in the long-form and E1-4 expression were minimal. Replacement of oestrogens and prolactin suggest that oestrogens down-regulate E1-3 expression in the choroid plexus and pituitary gland, and that the negative effect of oestrogen is mediated by prolactin in the pituitary gland. The present results revealed the region-specific promoter usage in prolactin receptor mRNA transcription, as well as the involvement of oestrogens in the regulation of E1-3 mRNA expression in the brain and pituitary gland.

[Effect of lipopolysaccharides from Porphyromonas endodontalis on the expression of macrophage colony stimulating factor in mouse osteoblasts].

PubMed

Yu, Yaqiong; Qiu, Lihong; Guo, Jiajie; Qu, Liu; Xu, Liya; Zhong, Ming

2014-09-01

To investigate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on the expression of macrophage colony stimulating factor (M-CSF) mRNA and protein in MC3T3-E1 cells and the role of nucler factor-κB (NF-κB) in the process. MC3T3-E1 cells were treated with different concentrations of Pe-LPS (0-50 mg/L) and 10 mg/L Pe-LPS for different hours (0-24 h). The expression of M-CSF mRNA and protein was detected by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunoadsordent assay (ELISA). The cells untreated by Pe-LPS served as control. The expression of M- CSF mRNA and protein was also detected in 10 mg/L Pe- LPS treated MC3T3-E1 cells after pretreated with BAY 11-7082 for 1 h, a special NF-κB inhibitor. The groups were divided as follows, control group, BAY group (10 µmol/L BAY 11-7082 treated alone MC3T3-E1 cells), Pe-LPS group (10 mg/L Pe-LPS stimulated MC3T3-E1 cells for 6 h), BAY combine with Pe-LPS group (10 µmol/L BAY 11-7082 pretreated cells for 1 h and 10 mg/L of Pe-LPS stimulated MC3T3-E1 cells for 6 h). The level of M- CSF mRNA and protein increased significantly after treatment with different concentrations of Pe-LPS (0-50 mg/L), which indicated that Pe-LPS induced osteoblasts to express M-CSF mRNA and protein in dose dependent manners. The expression of M-CSF protein increased from (35 ± 2) ng/L (control group) to (170 ± 8) ng/L (50 mg/L group). Maximal induction of M-CSF mRNA expression was found in the MC3T3- E1 cells treated with 10 mg/L Pe-LPS for 6 h. After 6 h, the expression of M-CSF mRNA decreased gradually. The expression of M-CSF protein also increased with the treatment of 10 mg/L Pe-LPS for 10 h [(122 ± 4) ng/L]. After 10 h, the expression of M-CSF protein decreased gradually. The mRNA and proteins of M-CSF decreased significantly after pretreatment with 10 µmol/L BAY 11-7082 for 1 h. There was no significant difference between BAY group and the control. Pe-LPS may induce the expression of M-CSF mRNA and protein in MC3T3-E1 cells through the signaling of NF-κB.

Stage of perinatal development regulates skeletal muscle mitochondrial biogenesis and myogenic regulatory factor genes with little impact of growth restriction or cross-fostering.

PubMed

Laker, R C; Wadley, G D; McConell, G K; Wlodek, M E

2012-02-01

Foetal growth restriction impairs skeletal muscle development and adult muscle mitochondrial biogenesis. We hypothesized that key genes involved in muscle development and mitochondrial biogenesis would be altered following uteroplacental insufficiency in rat pups, and improving postnatal nutrition by cross-fostering would ameliorate these deficits. Bilateral uterine vessel ligation (Restricted) or sham (Control) surgery was performed on day 18 of gestation. Males and females were investigated at day 20 of gestation (E20), 1 (PN1), 7 (PN7) and 35 (PN35) days postnatally. A separate cohort of Control and Restricted pups were cross-fostered onto a different Control or Restricted mother and examined at PN7. In both sexes, peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α), cytochrome c oxidase subunits 3 and 4 (COX III and IV) and myogenic regulatory factor 4 expression increased from late gestation to postnatal life, whereas mitochondrial transcription factor A, myogenic differentiation 1 (MyoD), myogenin and insulin-like growth factor I (IGF-I) decreased. Foetal growth restriction increased MyoD mRNA in females at PN7, whereas in males IGF-I mRNA was higher at E20 and PN1. Cross-fostering Restricted pups onto a Control mother significantly increased COX III mRNA in males and COX IV mRNA in both sexes above controls with little effect on other genes. Developmental age appears to be a major factor regulating skeletal muscle mitochondrial and developmental genes, with growth restriction and cross-fostering having only subtle effects. It therefore appears that reductions in adult mitochondrial biogenesis markers likely develop after weaning.

Characterization of a novel RNA-binding region of eIF4GI critical for ribosomal scanning

PubMed Central

Prévôt, Déborah; Décimo, Didier; Herbreteau, Cécile H.; Roux, Florence; Garin, Jérôme; Darlix, Jean-Luc; Ohlmann, Théophile

2003-01-01

The eukaryotic translation initiation factor eIF4GI binds several proteins and acts as a scaffold to promote preinitiation complex formation on the mRNA molecule (48S). Following mRNA attachment this complex scans along the messenger in a 5′ to 3′ direction until it locates and recognizes the initiation start codon. By using a combination of retroviral and picornaviral proteases (HIV-2 and L respectively) in the reticulocyte lysate system, we have characterized a 40 amino acid (aa) region of eIF4GI (aa 642–681) that exhibits general RNA-binding properties. Removal of this domain by proteolytic processing followed by translational assays showed virtually no inhibition of internal ribosome entry on the encephalomyocarditis virus, but resulted in drastic impairment of ribosome scanning as demonstrated by studying poliovirus and foot-and-mouth disease virus translation. Based on these findings, we propose that this 40 aa motif of eIF4GI is critical for ribosome scanning. PMID:12682023

An Anacardiaceae preparation reduces the expression of inflammation-related genes in murine macrophages.

PubMed

Leiro, J; García, D; Arranz, J A; Delgado, R; Sanmartín, M L; Orallo, F

2004-08-01

This study investigated the effects of an aqueous extract of the stem bark of Mangifera indica L. (Anacardiaceae; Vimang), which contains a defined mixture of components including polyphenols (principally mangiferin, MA), triterpenes, phytosteroids, fatty acids and microelements, on expression of inflammation mediators in inflammatory murine macrophages after stimulation in vitro with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). In vitro treatment with Vimang at 4 microg/ml reduced levels of NOS-2 mRNA and NOS-2, while treatment at 40 microg/ml also reduced levels of COX-2 mRNA, COX-2, and prostaglandin E2 (PGE2). Results suggested that MA is involved in these effects. In vitro treatment with Vimang at 40 microg/ml also inhibited mRNA levels of the proinflammatory cytokines interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha) and colony-stimulating factor (GM-CSF), but did not affect mRNA levels of IL-6 or tumor growth factor-beta (TGF-beta). Extracellular release of TNF-alpha by inflammatory macrophages was inhibited by in vitro treatment with Vimang at the same concentrations that showed inhibition of TNF-alpha mRNA levels. The inhibition of TNF-alpha production appears to be at least partially attributable to MA. Vimang at 4 microg/ml decreased mRNA levels of nuclear factor-kappaB (NF-kappaB) but did not affect expression of the NF-kappaB inhibitor (IkappaB). These data indicate that the potent anti-inflammatory effects of Vimang are due to selective modulation of the expression of inflammation-related genes, leading to attenuation of macrophage activation.

The light-induced transcriptome of the zebrafish pineal gland reveals complex regulation of the circadian clockwork by light

PubMed Central

Ben-Moshe, Zohar; Alon, Shahar; Mracek, Philipp; Faigenbloom, Lior; Tovin, Adi; Vatine, Gad D.; Eisenberg, Eli; Foulkes, Nicholas S.; Gothilf, Yoav

2014-01-01

Light constitutes a primary signal whereby endogenous circadian clocks are synchronized (‘entrained’) with the day/night cycle. The molecular mechanisms underlying this vital process are known to require gene activation, yet are incompletely understood. Here, the light-induced transcriptome in the zebrafish central clock organ, the pineal gland, was characterized by messenger RNA (mRNA) sequencing (mRNA-seq) and microarray analyses, resulting in the identification of multiple light-induced mRNAs. Interestingly, a considerable portion of the molecular clock (14 genes) is light-induced in the pineal gland. Four of these genes, encoding the transcription factors dec1, reverbb1, e4bp4-5 and e4bp4-6, differentially affected clock- and light-regulated promoter activation, suggesting that light-input is conveyed to the core clock machinery via diverse mechanisms. Moreover, we show that dec1, as well as the core clock gene per2, is essential for light-entrainment of rhythmic locomotor activity in zebrafish larvae. Additionally, we used microRNA (miRNA) sequencing (miR-seq) and identified pineal-enhanced and light-induced miRNAs. One such miRNA, miR-183, is shown to downregulate e4bp4-6 mRNA through a 3′UTR target site, and importantly, to regulate the rhythmic mRNA levels of aanat2, the key enzyme in melatonin synthesis. Together, this genome-wide approach and functional characterization of light-induced factors indicate a multi-level regulation of the circadian clockwork by light. PMID:24423866

Cyclin B Translation Depends on mTOR Activity after Fertilization in Sea Urchin Embryos

PubMed Central

Boulben, Sandrine; Glippa, Virginie; Morales, Julia; Cormier, Patrick

2016-01-01

The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism. PMID:26962866

Relative mRNA expression of prostate-derived E-twenty-six factor and E-twenty-six variant 4 transcription factors, and of uridine phosphorylase-1 and thymidine phosphorylase enzymes, in benign and malignant prostatic tissue

PubMed Central

CAVAZZOLA, LUCIANE ROSTIROLA; CARVALHAL, GUSTAVO FRANCO; DEVES, CANDIDA; RENCK, DAIANA; ALMEIDA, RICARDO; SANTOS, DIóGENES SANTIAGO

2015-01-01

Prostate cancer is the most frequent urological tumor, and the second most common cancer diagnosed in men. Incidence and mortality are variable and appear to depend on behavioral factors and genetic predisposition. The prostate-derived E-twenty-six factor (PDEF) and E-twenty-six variant 4 (ETV4) transcription factors, and the thymidine phosphorylase (TP) and uridine phosphorylase-1 (UP-1) enzymes, are reported to be components of the pathways leading to tumorigenesis and/or metastasis in a number of tumors. The present study aimed to analyze the mRNA expression levels of these proteins in prostatic cancerous and benign tissue, and their association with clinical and pathological variables. Using quantitative reverse transcription polymerase chain reaction, the mRNA expression levels of PDEF, ETV4, TP and UP-1 were studied in 52 tissue samples (31 of benign prostatic hyperplasia and 21 of prostate adenocarcinomas) obtained from patients treated by transurethral resection of the prostate or by radical prostatectomy. Relative expression was assessed using the ∆-CT method. Data was analyzed using Spearman's tests for correlation. P<0.05 was considered to indicate a statistically significant difference. The results revealed that PDEF, ETV4, UP-1 and TP were expressed in 85.7, 90.5, 95.2 and 100% of the prostate cancer samples, and in 90.3, 96.8, 90.3 and 96.8% of the benign samples, respectively. PDEF and ETV4 exhibited a significantly higher relative expression level in the tumor samples compared with their benign counterparts. The relative expression of TP and UP-1 did not differ significantly between benign and cancerous prostate tissues. The relative expression of TP was moderately and significantly correlated with the expression of ETV4 in the benign tissues. The relative expression of UP-1 was significantly lower in T3 compared with T1 and T2 cancers. These findings indicate that PDEF, ETV4, TP and UP-1 are typically expressed in benign and malignant prostatic tissues. Further studies are necessary to define the role of these proteins as therapeutic targets in prostate cancer. PMID:26137165

Two highly similar DEAD box proteins, OsRH2 and OsRH34, homologous to eukaryotic initiation factor 4AIII, play roles of the exon junction complex in regulating growth and development in rice.

PubMed

Huang, Chun-Kai; Sie, Yi-Syuan; Chen, Yu-Fu; Huang, Tian-Sheng; Lu, Chung-An

2016-04-12

The exon junction complex (EJC), which contains four core components, eukaryotic initiation factor 4AIII (eIF4AIII), MAGO/NASHI (MAGO), Y14/Tsunagi/RNA-binding protein 8A, and Barentsz/Metastatic lymph node 51, is formed in both nucleus and cytoplasm, and plays important roles in gene expression. Genes encoding core EJC components have been found in plants, including rice. Currently, the functional characterizations of MAGO and Y14 homologs have been demonstrated in rice. However, it is still unknown whether eIF4AIII is essential for the functional EJC in rice. This study investigated two DEAD box RNA helicases, OsRH2 and OsRH34, which are homologous to eIF4AIII, in rice. Amino acid sequence analysis indicated that OsRH2 and OsRH34 had 99 % identity and 100 % similarity, and their gene expression patterns were similar in various rice tissues, but the level of OsRH2 mRNA was about 58-fold higher than that of OsRH34 mRNA in seedlings. From bimolecular fluorescence complementation results, OsRH2 and OsRH34 interacted physically with OsMAGO1 and OsY14b, respectively, which indicated that both of OsRH2 and OsRH34 were core components of the EJC in rice. To study the biological roles of OsRH2 and OsRH34 in rice, transgenic rice plants were generated by RNA interference. The phenotypes of three independent OsRH2 and OsRH34 double-knockdown transgenic lines included dwarfism, a short internode distance, reproductive delay, defective embryonic development, and a low seed setting rate. These phenotypes resembled those of mutants with gibberellin-related developmental defects. In addition, the OsRH2 and OsRH34 double-knockdown transgenic lines exhibited the accumulation of unspliced rice UNDEVELOPED TAPETUM 1 mRNA. Rice contains two eIF4AIII paralogous genes, OsRH2 and OsRH34. The abundance of OsRH2 mRNA was about 58-fold higher than that of OsRH34 mRNA in seedlings, suggesting that the OsRH2 is major eIF4AIII in rice. Both OsRH2 and OsRH34 are core components of the EJC, and participate in regulating of plant height, pollen, and seed development in rice.

Diagnostic performance of HPV E6/E7 mRNA assay for detection of cervical high-grade intraepithelial neoplasia and cancer among women with ASCUS Papanicolaou smears.

PubMed

Ren, Chenchen; Zhu, Yuanhang; Yang, Li; Zhang, Xiaoan; Liu, Ling; Ren, Chunying

2018-02-01

The aim of this study was to investigate the clinical performance of high risk (HR) HPV E6/E7 mRNA assay in detecting cervical high-grade intraepithelial neoplasia and cancer among women with atypical squamous cells of undetermined significance (ASCUS) Papanicolaou (Pap) smears. A total of 160 patients with ASCUS who underwent HR-HPV DNA assay, HR-HPV E6/E7 mRNA assay and colposcopy biopsy at Third Affiliated Hospital of Zhengzhou University, China, from December 2015 to March 2017, were enrolled. Logistic regression analysis was used to evaluate the relationship between pathological results with clinical biologic factors. Univariate analysis showed that the qualitative results of HR-HPV DNA, qualitative results of HR-HPV E6/E7 mRNA and expression levels of HR-HPV E6/E7 mRNA were risk factors of high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer (all P < 0.05). Multivariable analysis found that only the expression levels of HR-HPV E6/E7 mRNA was associated with high-grade CIN and cervical cancer (OR = 8.971, 95% CI = 2.572-31.289, P = 0.001). An optimal cut-off value of ≥ 558.26 copies/ml was determined using receiver operating characteristic curve, and specificity of cut-off value were higher than E6/E7 mRNA qualitative assay and DNA qualitative assay. HPV E6/E7 mRNA quantitative assay may be a valuable tool in triage of ASCUS pap smears. A high specificity of E6/E7 mRNA quantitative assay as a triage test in women with ASCUS can be translated into a low referral for colposcopy.

How does a scanning ribosomal particle move along the 5'-untranslated region of eukaryotic mRNA? Brownian Ratchet model.

PubMed

Spirin, Alexander S

2009-11-17

A model of the ATP-dependent unidirectional movement of the 43S ribosomal initiation complex (=40S ribosomal subunit + eIF1 + eIF1A + eIF2.GTP.Met-tRNA(i) + eIF3) during scanning of the 5'-untranslated region of eukaryotic mRNA is proposed. The model is based on the principles of molecular Brownian ratchet machines and explains several enigmatic data concerning the scanning complex. In this model, the one-dimensional diffusion of the ribosomal initiation complex along the mRNA chain is rectified into the net-unidirectional 5'-to-3' movement by the Feynman ratchet-and-pawl mechanism. The proposed mechanism is organized by the heterotrimeric protein eIF4F (=eIF4A + eIF4E + eIF4G), attached to the scanning ribosomal particle via eIF3, and the RNA-binding protein eIF4B that is postulated to play the role of the pawl. The energy for the useful work of the ratchet-and-pawl mechanism is supplied from ATP hydrolysis induced by the eIF4A subunit: ATP binding and its hydrolysis alternately change the affinities of eIF4A for eIF4B and for mRNA, resulting in the restriction of backward diffusional sliding of the 43S ribosomal complex along the mRNA chain, while stochastic movements ahead are allowed.

Natural antioxidants exhibit chemopreventive characteristics through the regulation of CNC b-Zip transcription factors in estrogen-induced breast carcinogenesis.

PubMed

Chatterjee, Anwesha; Ronghe, Amruta; Singh, Bhupendra; Bhat, Nimee K; Chen, Jie; Bhat, Hari K

2014-12-01

The objective of the present study was to characterize the role of resveratrol (Res) and vitamin C (VC) in prevention of estrogen-induced breast cancer through regulation of cap "n"collar (CNC) b-zip transcription factors. Human breast epithelial cell line MCF-10A was treated with 17β-estradiol (E2) and VC or Res with or without E2. mRNA and protein expression levels of CNC b-zip transcription factors nuclear factor erythroid 2-related factor 1 (Nrf1), nuclear factor erythroid 2 related factor 2 (Nrf2), nuclear factor erythroid 2 related factor 3 (Nrf3), and Nrf2-regulated antioxidant enzymes superoxide dismutase 3 (SOD3) and quinone oxidoreductase 1 (NQO1) were quantified. The treatment with E2 suppressed, whereas VC and Res prevented E2-mediated decrease in the expression levels of SOD3, NQO1, Nrf2 mRNA, and protein in MCF-10A cells. The treatment with E2, Res, or VC significantly increased mRNA and protein expression levels of Nrf1. 17β-Estradiol treatment significantly increased but VC or Res decreased Nrf3 mRNA and protein expression levels. Our studies demonstrate that estrogen-induced breast cancer might be prevented through upregulation of antioxidant enzymes via Nrf-dependent pathways. © 2014 Wiley Periodicals, Inc.

Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms.

PubMed

Kahvejian, Avak; Svitkin, Yuri V; Sukarieh, Rami; M'Boutchou, Marie-Noël; Sonenberg, Nahum

2005-01-01

Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5'-end of the mRNA to promote the recruitment of the ribosome. Although the 3' poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (approximately 65% vs. approximately 35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5'-end of mRNA.

Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms

PubMed Central

Kahvejian, Avak; Svitkin, Yuri V.; Sukarieh, Rami; M'Boutchou, Marie-Noël; Sonenberg, Nahum

2005-01-01

Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5′-end of the mRNA to promote the recruitment of the ribosome. Although the 3′ poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (∼65% vs. ∼35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5′-end of mRNA. PMID:15630022

The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ziehr, Ben; Lenarcic, Erik; Cecil, Chad

Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compoundmore » that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. - Highlights: • The host eIF4AIII RNA helicase is required for efficient HCMV replication. • Depleting eIF4AIII inhibited the nuclear export of HCMV mRNAs. • HCMV mRNAs did not require eIF4AIII to associate with polyribosomes. • The eIF4A family helicases may be new targets for host-directed antiviral drugs.« less

Biological insights into the expression of translation initiation factors from recombinant CHOK1SV cell lines and their relationship to enhanced productivity.

PubMed

Mead, Emma J; Masterton, Rosalyn J; Feary, Marc; Obrezanova, Olga; Zhang, Lin; Young, Robert; Smales, C Mark

2015-12-15

Translation initiation is on the critical pathway for the production of monoclonal antibodies (mAbs) by mammalian cells. Formation of a closed loop structure comprised of mRNA, a number of eukaryotic initiation factors (eIFs) and ribosomal proteins has been proposed to aid re-initiation of translation and therefore increase global translational efficiency. We have determined mRNA and protein levels of the key components of the closed loop, eIFs (eIF3a, eIF3b, eIF3c, eIF3h, eIF3i and eIF4G1), poly(A)-binding protein (PABP) 1 and PABP-interacting protein 1 (PAIP1), across a panel of 30 recombinant mAb-producing GS-CHOK1SV cell lines with a broad range of growth characteristics and production levels of a model recombinant mAb. We have used a multi-level statistical approach to investigate the relationship between key performance indicators (cell growth and recombinant antibody productivity) and the intracellular amounts of target translation initiation factor proteins and the mRNAs encoding them. We show that high-producing cell lines maintain amounts of the translation initiation factors involved in the formation of the closed loop mRNA, maintaining these proteins at appropriate levels to deliver enhanced recombinant protein production. We then utilize knowledge of the amounts of these factors to build predictive models for and use cluster analysis to identify, high-producing cell lines. The present study therefore defines the translation initiation factor amounts that are associated with highly productive recombinant GS-CHOK1SV cell lines that may be targets for screening highly productive cell lines or to engineer new host cell lines with the potential for enhanced recombinant antibody productivity. © 2015 Authors; published by Portland Press Limited.

Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction.

PubMed

Ray, Swagat; Anderson, Emma C

2016-03-03

The RNA binding protein Unr, which contains five cold shock domains, has several specific roles in post-transcriptional control of gene expression. It can act as an activator or inhibitor of translation initiation, promote mRNA turnover, or stabilise mRNA. Its role depends on the mRNA and other proteins to which it binds, which includes cytoplasmic poly(A) binding protein 1 (PABP1). Since PABP1 binds to all polyadenylated mRNAs, and is involved in translation initiation by interaction with eukaryotic translation initiation factor 4G (eIF4G), we investigated whether Unr has a general role in translational control. We found that Unr strongly stimulates translation in vitro, and mutation of cold shock domains 2 or 4 inhibited its translation activity. The ability of Unr and its mutants to stimulate translation correlated with its ability to bind RNA, and to interact with PABP1. We found that Unr stimulated the binding of PABP1 to mRNA, and that Unr was required for the stable interaction of PABP1 and eIF4G in cells. siRNA-mediated knockdown of Unr reduced the overall level of cellular translation in cells, as well as that of cap-dependent and IRES-dependent reporters. These data describe a novel role for Unr in regulating cellular gene expression.

Minimum-noise production of translation factor eIF4G maps to a mechanistically determined optimal rate control window for protein synthesis

PubMed Central

Meng, Xiang; Firczuk, Helena; Pietroni, Paola; Westbrook, Richard; Dacheux, Estelle; Mendes, Pedro; McCarthy, John E.G.

2017-01-01

Gene expression noise influences organism evolution and fitness. The mechanisms determining the relationship between stochasticity and the functional role of translation machinery components are critical to viability. eIF4G is an essential translation factor that exerts strong control over protein synthesis. We observe an asymmetric, approximately bell-shaped, relationship between the average intracellular abundance of eIF4G and rates of cell population growth and global mRNA translation, with peak rates occurring at normal physiological abundance. This relationship fits a computational model in which eIF4G is at the core of a multi-component–complex assembly pathway. This model also correctly predicts a plateau-like response of translation to super-physiological increases in abundance of the other cap-complex factors, eIF4E and eIF4A. Engineered changes in eIF4G abundance amplify noise, demonstrating that minimum stochasticity coincides with physiological abundance of this factor. Noise is not increased when eIF4E is overproduced. Plasmid-mediated synthesis of eIF4G imposes increased global gene expression stochasticity and reduced viability because the intrinsic noise for this factor influences total cellular gene noise. The naturally evolved eIF4G gene expression noise minimum maps within the optimal activity zone dictated by eIF4G's mechanistic role. Rate control and noise are therefore interdependent and have co-evolved to share an optimal physiological abundance point. PMID:27928055

Induced expression of mRNA for IL-5, IL-6, TNF-alpha, MIP-2 and IFN-gamma in immunologically activated rat peritoneal mast cells: inhibition by dexamethasone and cyclosporin A.

PubMed

Williams, C M; Coleman, J W

1995-10-01

We examined the capacity of purified rat peritoneal connective tissue-type mast cells (PMC) to express mRNA for several cytokines. Stimulation of PMC with anti-IgE for 4 hr induced the expression of mRNA encoding interleukin-5 (IL-5), IL-6, tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Unstimulated PMC expressed detectable mRNA for TNF-alpha but not for the other four cytokines. Incubation of PMC with cyclosporin A (CsA) or dexamethasone (DEX), each at 10(-6) M for 24 hr, significantly inhibited the induced expression of mRNA for each of the five cytokines, and also inhibited release of biologically active TNF-alpha. Throughout these experiments mRNA levels of the housekeeping gene G3PDH were not altered by stimulation with anti-IgE or incubation with CsA or DEX. We conclude that immunological activation of rat PMC induces gene expression of several cytokines and that expression of these genes can be inhibited by immunosuppressive drugs.

Induced expression of mRNA for IL-5, IL-6, TNF-alpha, MIP-2 and IFN-gamma in immunologically activated rat peritoneal mast cells: inhibition by dexamethasone and cyclosporin A.

PubMed Central

Williams, C M; Coleman, J W

1995-01-01

We examined the capacity of purified rat peritoneal connective tissue-type mast cells (PMC) to express mRNA for several cytokines. Stimulation of PMC with anti-IgE for 4 hr induced the expression of mRNA encoding interleukin-5 (IL-5), IL-6, tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Unstimulated PMC expressed detectable mRNA for TNF-alpha but not for the other four cytokines. Incubation of PMC with cyclosporin A (CsA) or dexamethasone (DEX), each at 10(-6) M for 24 hr, significantly inhibited the induced expression of mRNA for each of the five cytokines, and also inhibited release of biologically active TNF-alpha. Throughout these experiments mRNA levels of the housekeeping gene G3PDH were not altered by stimulation with anti-IgE or incubation with CsA or DEX. We conclude that immunological activation of rat PMC induces gene expression of several cytokines and that expression of these genes can be inhibited by immunosuppressive drugs. Images Figure 1 Figure 2 Figure 3 PMID:7490125

HPV E6/E7 mRNA versus HPV DNA biomarker in cervical cancer screening of a group of Macedonian women.

PubMed

Duvlis, Sotirija; Popovska-Jankovic, Katerina; Arsova, Zorica Sarafinovska; Memeti, Shaban; Popeska, Zaneta; Plaseska-Karanfilska, Dijana

2015-09-01

High risk types of human papillomaviruses E6/E7 oncogenes and their association with tumor suppressor genes products are the key factors of cervical carcinogenesis. This study proposed them as specific markers for cervical dysplasia screening. The aim of the study is to compare the clinical and prognostic significance of HPV E6/E7 mRNA as an early biomarker versus HPV DNA detection and cytology in triage of woman for cervical cancer. The study group consists of 413 women: 258 NILM, 26 ASC-US, 81 LSIL, 41 HSIL, and 7 unsatisfactory cytology. HPV4AACE screening, real-time multiplex PCR and MY09/11 consensus PCR primers methods were used for the HPV DNA detection. The real-time multiplex nucleic acid sequence-based assay (NucliSENS EasyQ HPV assay) was used for HPV E6/E7 mRNA detection of the five most common high risk HPV types in cervical cancer (16, 18, 31, 33, and 45). The results show that HPV E6/E7 mRNA testing had a higher specificity 50% (95% CI 32-67) and positive predictive value (PPV) 62% (95% CI 46-76) for CIN2+ compared to HPV DNA testing that had specificity of 18% (95% CI 7-37) and PPV 52% (95% CI 39-76) respectively. The higher specificity and PPV of HPV E6/E7 mRNA testing are valuable in predicting insignificant HPV DNA infection among cases with borderline cytological finding. It can help in avoiding aggressive procedures (biopsies and over-referral of transient HPV infections) as well as lowering patient's anxiety and follow up period. © 2015 Wiley Periodicals, Inc.

Changes in the mRNA expression of structural proteins, hormone synthesis and secretion from bovine placentome sections after DDT and DDE treatment.

PubMed

Wojciechowska, A; Mlynarczuk, J; Kotwica, J

2017-01-15

Disorders in the barrier function and secretory activity of the placenta can be caused by xenobiotics (XB) present in the environment and their accumulation in tissues of living organisms. Thus, the aim of this study was to investigate the effect of 1,1,1-trichloro-2,2,-bis-4-chlorophenyl-ethane (DDT) and its metabolite 1,1-dichloro-2,2-bis-4-chlorophenyl-ethene (DDE) (for 24 or 48h) at doses of 1, 10 or 100ng/ml on the function of cow placentome sections in the second trimester of pregnancy. DDT and DDE affected neither (P>0.05) the viability nor hypoxia inducible factor 1 (HIF1α) mRNA expression of the sections. XB decreased (P<0.05) connexin (Cx) 26, 32, 43 and placenta-specific 1 (PLAC-1) mRNA expression but did not affect (P>0.05) keratin 8 (KRT8) mRNA expression. DDT and DDE also reduced (P<0.05) prostaglandin F2α (PGF2α) synthase (PGFS) mRNA expression, while DDT increased (P<0.05) prostaglandin E2 (PGE2) synthase (PGES) mRNA expression. Neither cyclooxygenase 2 (COX-2) mRNA expression nor PGF2α and PGE2 secretion were affected. Both DDT and DDE increased (P<0.05) neurophysin I/oxytocin (NP1/OT) mRNA expression and oxytocin (OT), oestradiol (E2) and progesterone (P4) secretion while DDT stimulated only 3β-hydroxysteroid dehydrogenase (3βHSD) and cholesterol side-chain cleavage enzyme (CYP11A1) mRNA expression (P<0.05). In summary, DDT and DDE impaired the barrier function and secretory activity of the placenta. Thus, these compounds can disrupt trophoblast invasion, myometrium contractility and gas/nutrient exchange throughout pregnancy in cows. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The MNK–eIF4E Signaling Axis Contributes to Injury-Induced Nociceptive Plasticity and the Development of Chronic Pain

PubMed Central

Asiedu, Marina N.; Megat, Salim; Burton, Michael D.; Burgos-Vega, Carolina C.; Melemedjian, Ohannes K.; Boitano, Scott; Vagner, Josef; Pancrazio, Joseph J.; Mogil, Jeffrey S.; Dussor, Gregory

2017-01-01

Injury-induced sensitization of nociceptors contributes to pain states and the development of chronic pain. Inhibiting activity-dependent mRNA translation through mechanistic target of rapamycin and mitogen-activated protein kinase (MAPK) pathways blocks the development of nociceptor sensitization. These pathways convergently signal to the eukaryotic translation initiation factor (eIF) 4F complex to regulate the sensitization of nociceptors, but the details of this process are ill defined. Here we investigated the hypothesis that phosphorylation of the 5′ cap-binding protein eIF4E by its specific kinase MAPK interacting kinases (MNKs) 1/2 is a key factor in nociceptor sensitization and the development of chronic pain. Phosphorylation of ser209 on eIF4E regulates the translation of a subset of mRNAs. We show that pronociceptive and inflammatory factors, such as nerve growth factor (NGF), interleukin-6 (IL-6), and carrageenan, produce decreased mechanical and thermal hypersensitivity, decreased affective pain behaviors, and strongly reduced hyperalgesic priming in mice lacking eIF4E phosphorylation (eIF4ES209A). Tests were done in both sexes, and no sex differences were found. Moreover, in patch-clamp electrophysiology and Ca2+ imaging experiments on dorsal root ganglion neurons, NGF- and IL-6-induced increases in excitability were attenuated in neurons from eIF4ES209A mice. These effects were recapitulated in Mnk1/2−/− mice and with the MNK1/2 inhibitor cercosporamide. We also find that cold hypersensitivity induced by peripheral nerve injury is reduced in eIF4ES209A and Mnk1/2−/− mice and following cercosporamide treatment. Our findings demonstrate that the MNK1/2–eIF4E signaling axis is an important contributing factor to mechanisms of nociceptor plasticity and the development of chronic pain. SIGNIFICANCE STATEMENT Chronic pain is a debilitating disease affecting approximately one in three Americans. Chronic pain is thought to be driven by changes in the excitability of peripheral nociceptive neurons, but the precise mechanisms controlling these changes are not elucidated. Emerging evidence demonstrates that mRNA translation regulation pathways are key factors in changes in nociceptor excitability. Our work demonstrates that a single phosphorylation site on the 5′ cap-binding protein eIF4E is a critical mechanism for changes in nociceptor excitability that drive the development of chronic pain. We reveal a new mechanistic target for the development of a chronic pain state and propose that targeting the upstream kinase, MAPK interacting kinase 1/2, could be used as a therapeutic approach for chronic pain. PMID:28674170

Endoplasmic reticulum stress-mediated neuronal apoptosis by acrylamide exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Komoike, Yuta, E-mail: komoike@research.twmu.ac.jp

Acrylamide (AA) is a well-known neurotoxic compound in humans and experimental animals. However, intracellular stress signaling pathways responsible for the neurotoxicity of AA are still not clear. In this study, we explored the involvement of the endoplasmic reticulum (ER) stress response in AA-induced neuronal damage in vitro and in vivo. Exposure of SH-SY5Y human neuroblastoma cells to AA increased the levels of phosphorylated form of eukaryotic translation initiation factor 2α (eIF2α) and its downstream effector, activating transcription factor 4 (ATF4), indicating the induction of the unfolded protein response (UPR) by AA exposure. Furthermore, AA exposure increased the mRNA level ofmore » c/EBP homologous protein (CHOP), the ER stress-dependent apoptotic factor, and caused the accumulation of reactive oxygen species (ROS) in SH-SY5Y cells. Treatments of SH-SY5Y cells with the chemical chaperone, 4-phenylbutyric acid and the ROS scavenger, N-acetyl-cysteine reduced the AA-induced expression of ATF4 protein and CHOP mRNA, and resulted in the suppression of apoptosis. In addition, AA-induced eIF2α phosphorylation was also suppressed by NAC treatment. In consistent with in vitro study, exposure of zebrafish larvae at 6-day post fertilization to AA induced the expression of chop mRNA and apoptotic cell death in the brain, and also caused the disruption of brain structure. These findings suggest that AA exposure induces apoptotic neuronal cell death through the ER stress and subsequent eIF2α–ATF4–CHOP signaling cascade. The accumulation of ROS by AA exposure appears to be responsible for this ER stress-mediated apoptotic pathway. - Highlights: • Exposure of SH-SY5Y cells to AA activates the eIF2α–ATF4 pathway of the UPR. • Exposure of SH-SY5Y cells to AA induces the CHOP expression and apoptosis. • Exposure of zebrafish to AA induces the chop expression and apoptosis in the brain. • AA possibly induces apoptotic neuronal cell death through the ER stress response. • AA-induced ROS production is involved in this ER stress response.« less

The acute angiogenic signalling response to low-load resistance exercise with blood flow restriction.

PubMed

Ferguson, Richard A; Hunt, Julie E A; Lewis, Mark P; Martin, Neil R W; Player, Darren J; Stangier, Carolin; Taylor, Conor W; Turner, Mark C

2018-04-01

This study investigated protein kinase activation and gene expression of angiogenic factors in response to low-load resistance exercise with or without blood flow restriction (BFR). In a repeated measures cross-over design, six males performed four sets of bilateral knee extension exercise at 20% 1RM (reps per set = 30:15:15:continued to fatigue) with BFR (110 mmHg) and without (CON). Muscle biopsies were obtained from the vastus lateralis before, 2 and 4 h post-exercise. mRNA expression was determined using real-time RT-PCR. Protein phosphorylation/expression was determined using Western blot. p38MAPK phosphorylation was greater (p = 0.05) at 2 h following BFR (1.3 ± 0.8) compared to CON (0.4 ± 0.3). AMPK phosphorylation remained unchanged. PGC-1α mRNA expression increased at 2 h (5.9 ± 1.3 vs. 2.1 ± 0.8; p = 0.03) and 4 h (3.2 ± 0.8 vs. 1.5 ± 0.4; p = 0.03) following BFR exercise with no change in CON. PGC-1α protein expression did not change following either exercise. BFR exercise enhanced mRNA expression of vascular endothelial growth factor (VEGF) at 2 h (5.2 ± 2.8 vs 1.7 ± 1.1; p = .02) and 4 h (6.8 ± 4.9 vs. 2.5 ± 2.7; p = .01) compared to CON. mRNA expression of VEGF-R2 and hypoxia-inducible factor 1α increased following BFR exercise but only eNOS were enhanced relative to CON. Matrix metalloproteinase-9 mRNA expression was not altered in response to either exercise. Acute low-load resistance exercise with BFR provides a targeted angiogenic response potentially mediated through enhanced ischaemic and shear stress stimuli.

Ethanol-induced changes in Poly (ADP ribose) Polymerase and neuronal developmental gene expression

PubMed Central

Gavin, David P.; Kusumo, Handojo; Sharma, Rajiv P.; Guizzetti, Marina

2016-01-01

Prenatal alcohol exposure has profound effects on neuronal growth and development. Poly-ADP Ribose Polymerase (PARP) enzymes are perhaps unique in the field of epigenetics in that they directly participate in histone modifications, transcription factor modifications, DNA methylation/demethylation and are highly inducible by ethanol. It was our hypothesis that ethanol would induce PARP enzymatic activity leading to alterations in neurodevelopmental gene expression. Mouse E18 cortical neurons were treated with ethanol, PARP inhibitors, and nuclear hormone receptor transcription factor PPARγ agonists and antagonists. Subsequently, we measured PARP activity and changes in Bdnf, OKSM (Oct4, Klf4, Sox2, c-Myc), DNA methylating/demethylating factors, and Pparγ mRNA expression, promoter 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC), and PPARγ promoter binding. We found that ethanol reduced Bdnf4, 9a, and Klf4 mRNA expression, and increased c-Myc expression. These changes were reversed with a PARP inhibitor. In agreement with its role in DNA demethylation PARP inhibition increased 5MC levels at the c-Myc promoter. In addition, we found that elevated PARP enzymatic activity reduced PPARγ promoter binding, and this corresponded to decreased Bdnf and Klf4 mRNA expression. Our results suggest that PARP participates in DNA demethylation and reduces PPARγ promoter binding. The current study underscores the importance of PARP in ethanol-induced changes to neurodevelopmental gene expression. PMID:27497606

Ethanol-induced changes in poly (ADP ribose) polymerase and neuronal developmental gene expression.

PubMed

Gavin, David P; Kusumo, Handojo; Sharma, Rajiv P; Guizzetti, Marina

2016-11-01

Prenatal alcohol exposure has profound effects on neuronal growth and development. Poly-ADP Ribose Polymerase (PARP) enzymes are perhaps unique in the field of epigenetics in that they directly participate in histone modifications, transcription factor modifications, DNA methylation/demethylation and are highly inducible by ethanol. It was our hypothesis that ethanol would induce PARP enzymatic activity leading to alterations in neurodevelopmental gene expression. Mouse E18 cortical neurons were treated with ethanol, PARP inhibitors, and nuclear hormone receptor transcription factor PPARγ agonists and antagonists. Subsequently, we measured PARP activity and changes in Bdnf, OKSM (Oct4, Klf4, Sox2, c-Myc), DNA methylating/demethylating factors, and Pparγ mRNA expression, promoter 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC), and PPARγ promoter binding. We found that ethanol reduced Bdnf4, 9a, and Klf4 mRNA expression, and increased c-Myc expression. These changes were reversed with a PARP inhibitor. In agreement with its role in DNA demethylation PARP inhibition increased 5MC levels at the c-Myc promoter. In addition, we found that inhibition of PARP enzymatic activity increased PPARγ promoter binding, and this corresponded to increased Bdnf and Klf4 mRNA expression. Our results suggest that PARP participates in DNA demethylation and reduces PPARγ promoter binding. The current study underscores the importance of PARP in ethanol-induced changes to neurodevelopmental gene expression. Published by Elsevier Ltd.

Identification of a Kinase in Wheat Germ that Phosphorylates the Large Subunit of Initiation Factor 4F 1

PubMed Central

Humphreys, Jean; Browning, Karen S.; Ravel, Joanne M.

1988-01-01

A kinase has been isolated from wheat (Triticum aestivum) germ that phosphorylates the 220 kilodaltons (kD) subunit of wheat germ initiation factor (eIF) 4F, the 80 kD subunit of eIF-4B (an isozyme form of eIF-4F) and eIF-4G (the functional equivalent to mammalian eIF-4B). The kinase elutes from Sephacryl S-200 slightly in front of ovalbumin. The kinase phosphorylates casein and histone IIA to a small extent, but does not phosphorylate phosvitin. Of the wheat germ initiation factors, elongation factors, and small and large ribosomal subunits, only eIF-4F, eIF-4B, and eIF-4G are phosphorylated to a significant extent. The kinase phosphorylates eIF-4F to the extent of two phosphates per mole of the 220 kD subunit and phosphorylates eIF-4B to the extent of one phosphate per mole of the 80 kD subunit. The 26 kD subunit of eIF-4F and the 28 kD subunit of eIF-4B are not phosphorylated by the kinase. The kinase phosphorylates the 59 kD component of eIF-4G to the extent of 0.25 phosphate per mole of eIF-4G. Phosphorylation of eIF-4F and eIF-4B does not affect their ability to support the binding of mRNA to small ribosomal subunits in vitro. Images Fig. 2 Fig. 3 PMID:16666331

Distinct effects of 4-nonylphenol and estrogen-17β on expression of estrogen receptor α gene in smolting sockeye salmon

USGS Publications Warehouse

Luo, Qiong; Ban, Massatoshi; Ando, Hironori; Kitahashi, Takashi; Bhandari, Ramji K.; McCormick, Stephen D.; Urano, Akihisa

2005-01-01

Xenoestrogens such as 4-nonylphenol (4-NP) have been shown to affect the parr–smolt transformation, but their mechanisms of action are not known. We therefore examined effects of 4-NP and estradiol-17β (E2) on expression of estrogen receptor (ER) α gene in the liver, gill, pituitary and brain of sockeye salmon to elucidate molecular mechanisms of 4-NP and E2 and developmental differences in response during smolting. Fish were treated twice within a week with 4-NP (15 and 150 mg/kg BW), E2 (2 mg/kg BW) or only vehicle at three stages of smolting, pre-smolting in March, early smolting in April and late smolting in May. The absolute amounts of ERα mRNA were determined by real-time PCR. The basal amounts of ERα mRNA peaked in April in the liver, gill and pituitary. In March, E2 extensively increased the amounts in the liver, while 4-NP had no effects at this stage. In contrast, 4-NP (but not E2) decreased liver ERα mRNA in April. 4-NP also decreased the amount of ERα mRNA in the gill in April. In the pituitary, 4-NP increased ERα mRNA in March but decreased it in May. There were no significant effects in the brain. Changes in basal ERα mRNA observed in this study indicate that estrogen responsiveness of tissues may change during salmon smolting. Furthermore, 4-NP and E2 have different effects on expression of ERα gene in the liver and gill during smolting, and the response is dependent on smolt stage.

Analysis of eIF4E and 4EBP1 mRNAs in head and neck cancer.

PubMed

Sunavala-Dossabhoy, Gulshan; Palaniyandi, Senthilnathan; Clark, Cheryl; Nathan, Cherie-Ann O; Abreo, Fleurette W; Caldito, Gloria

2011-10-01

The eukaryotic translation initiation factor 4E (eIF4E) in conjunction with its binding protein, 4EBP1, regulates the translation of cap-dependent mRNAs. An aberrant increase in eIF4E shifts the balance in favor of translation of transcripts that promote cell proliferation and malignancy. eIF4E protein is commonly elevated in head and neck squamous cell carcinomas (HNSCC), and its overexpression is associated with increased recurrence. An underlying mechanism for eIF4E overexpression is gene amplification, and we wanted to determine whether eIF4E mRNA could serve as a prognostic maker of HNSCC. Tumor specimens from 26 HNSCC patients and oral tissues from 17 control subjects were examined for eIF4E and 4EBP1 by semiquantitative RT-PCR and correlated with clinical and pathologic findings. Unlike eIF4E mRNA alone, expression of eIF4E relative to 4EBP1 was a more precise predictor of HNSCC and its progression (P < .01, Wilcoxon rank sum test). Eight of 26 patients (31%) had elevated eIF4E:4EBP1 (4E:4EBP1; >25), and 7 of these (87.5%) had recurrence. Alternately, from 18 patients with low 4E:4EBP1 (<25; 69%), only 5 patients had recurrence (30.1%). To determine the probability of no recurrence, Kaplan-Meier analysis showed significantly poor disease-free survival in patients with elevated 4E:4EBP1 than those with low ratios (P < .01, log rank test). Elevated 4E:4EBP1 significantly correlated with increased disease recurrence. Because 4EBP1 modulates eIF4E activity, our results highlight the importance of incorporating a joint analysis of eIF4E and 4EBP1 mRNAs in HNSCC patient care decisions. Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.

Analysis of the Glucocorticoid Receptor in Spontaneous and Drug-induced Steroid Resistant Mutants Isolated from the Human Leukemic Cell Line CEM-C7

DTIC Science & Technology

1991-01-04

be due to defects in GR function. However, the loss of GR function in x" S49 cells is paralleled by the loss of GR mRNA which is not seen in x’ OEM ...in the protein transcription factor IIIA from Xepopus oocytes. EMBO J. 4: 1609-1614. Milgrom. E.; Atger, M.; Baulieu, E.-E. 1973 Acidophilic

Therapeutic suppression of translation initiation factor eIF4E expression reduces tumor growth without toxicity

PubMed Central

Graff, Jeremy R.; Konicek, Bruce W.; Vincent, Thomas M.; Lynch, Rebecca L.; Monteith, David; Weir, Spring N.; Schwier, Phil; Capen, Andrew; Goode, Robin L.; Dowless, Michele S.; Chen, Yuefeng; Zhang, Hong; Sissons, Sean; Cox, Karen; McNulty, Ann M.; Parsons, Stephen H.; Wang, Tao; Sams, Lillian; Geeganage, Sandaruwan; Douglass, Larry E.; Neubauer, Blake Lee; Dean, Nicholas M.; Blanchard, Kerry; Shou, Jianyong; Stancato, Louis F.; Carter, Julia H.; Marcusson, Eric G.

2007-01-01

Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers. PMID:17786246

The Effect of Repeated Electroacupuncture Analgesia on Neurotrophic and Cytokine Factors in Neuropathic Pain Rats

PubMed Central

Wang, Junying; Duanmu, Chenlin; Feng, Xiumei; Yan, Yaxia

2016-01-01

Chronic pain is a common disability influencing quality of life. Results of previous studies showed that acupuncture has a cumulative analgesic effect, but the relationship with spinal cytokines neurotrophic factors released by astrocytes remains unknown. The present study was designed to observe the effect of electroacupuncture (EA) treatment on spinal cytokines neurotrophic factors in chronic neuropathic pain rats. The chronic neuropathic pain was established by chronic constrictive injury (CCI). EA treatment was applied at Zusanli (ST36) and Yanglingquan (GB34) (both bilateral) once a day, for 30 min. IL-1β mRNA, TNF-α mRNA, and IL-1 mRNA were detected by quantitative real-time PCR, and the proteins of BDNF, NGF, and NT3/4 were detected by Western blot. The expression levels of cytokines such as IL-1β mRNA, TNF-α mRNA, IL-6 mRNA, and neurotrophic factors such as BDNF, NGF, and NT3/4 in the spinal cord were increased significantly after CCI. The astrocytes released more IL-1β and BDNF after CCI. Repeated EA treatment could suppress the elevated expression of IL-1β mRNA, TNFα mRNA, and BDNF, NGF, and NT3/4 but had no effect on IL-6 mRNA. It is suggested that cytokines and neurotrophic factors which may be closely associated with astrocytes participated in the process of EA relieving chronic pain. PMID:27800006

Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations

PubMed Central

Laudenbach, Beatrice Theres; Martínez-Montero, Saúl; Cencic, Regina; Habjan, Matthias; Pichlmair, Andreas; Damha, Masad J.; Pelletier, Jerry; Nagar, Bhushan

2017-01-01

IFIT1 (IFN-induced protein with tetratricopeptide repeats-1) is an effector of the host innate immune antiviral response that prevents propagation of virus infection by selectively inhibiting translation of viral mRNA. It relies on its ability to compete with the translation initiation factor eIF4F to specifically recognize foreign capped mRNAs, while remaining inactive against host mRNAs marked by ribose 2′-O methylation at the first cap-proximal nucleotide (N1). We report here several crystal structures of RNA-bound human IFIT1, including a 1.6-Å complex with capped RNA. IFIT1 forms a water-filled, positively charged RNA-binding tunnel with a separate hydrophobic extension that unexpectedly engages the cap in multiple conformations (syn and anti) giving rise to a relatively plastic and nonspecific mode of binding, in stark contrast to eIF4E. Cap-proximal nucleotides encircled by the tunnel provide affinity to compete with eIF4F while allowing IFIT1 to select against N1 methylated mRNA. Gel-shift binding assays confirm that N1 methylation interferes with IFIT1 binding, but in an RNA-dependent manner, whereas translation assays reveal that N1 methylation alone is not sufficient to prevent mRNA recognition at high IFIT1 concentrations. Structural and functional analysis show that 2′-O methylation at N2, another abundant mRNA modification, is also detrimental for RNA binding, thus revealing a potentially synergistic role for it in self- versus nonself-mRNA discernment. Finally, structure-guided mutational analysis confirms the importance of RNA binding for IFIT1 restriction of a human coronavirus mutant lacking viral N1 methylation. Our structural and biochemical analysis sheds new light on the molecular basis for IFIT1 translational inhibition of capped viral RNA. PMID:28251928

MiR-30e suppresses proliferation of hepatoma cells via targeting prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feng, Guoxing; Shi, Hui; Li, Jiong

Aberrant microRNA expression has been shown to be characteristic of many cancers. It has been reported that the expression levels of miR-30e are decreased in liver cancer tissues. However, the role of miR-30e in hepatocellular carcinoma remains poorly understood. In the present study, we investigated the significance of miR-30e in hepatocarcinogenesis. Bioinformatics analysis reveals a putative target site of miR-30e in the 3′-untranslated region (3′UTR) of prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA. Moreover, luciferase reporter gene assays verified that miR-30e directly targeted 3′UTR of P4HA1 mRNA. Then, we demonstrated that miR-30e was able to reduce the expression of P4HA1 atmore » the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis. Enforced expression of miR-30e suppressed proliferation of HepG2 cells by 5-ethynyl-2-deoxyuridine (EdU) assay and reduced colony formation of these cells by colony formation analysis. Conversely, anti-miR-30e enhanced the proliferation of hepatoma cells in vitro. Interestingly, the ectopic expression of P4HA1 could efficiently rescue the inhibition of cell proliferation mediated by miR-30e in HepG2 cells. Meanwhile, silencing of P4HA1 abolished the anti-miR-30e-induced proliferation of cells. Clinically, quantitative real-time PCR showed that miR-30e was down-regulated in liver tumor tissues relative to their peritumor tissues. The expression levels of miR-30e were negatively correlated to those of P4HA1 mRNA in clinical liver tumor tissues. Thus, we conclude that miR-30e suppresses proliferation of hepatoma cells through targeting P4HA1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis. - Highlights: • P4HA1 is a novel target gene of miR-30e. • P4HA1 is increased in clinical HCC tissues. • MiR-30e is negatively correlated with P4HA1 in clinical HCC tissues. • MiR-30e suppresses the proliferation of HCC cells through targeting P4HA1 mRNA.« less

The Antagonistic Effect of Selenium on Cadmium-Induced Damage and mRNA Levels of Selenoprotein Genes and Inflammatory Factors in Chicken Kidney Tissue.

PubMed

Wang, Xinyue; Bao, Rongkun; Fu, Jing

2018-02-01

Selenium (Se) is a necessary trace mineral in the diet of humans and animals. Cadmium (Cd) is a toxic heavy metal that can damage animal organs, especially the kidneys. Antagonistic interactions between Se and Cd have been reported in previous studies. However, little is known about the effects of Se against Cd toxicity and on the mRNA levels of 25 selenoprotein genes and inflammatory factors in chicken kidneys. In the current study, we fed chickens with a Se-treated, Cd-treated, or Se/Cd treated diet for 90 days. We then analyzed the mRNA expression of inflammatory factors (including prostaglandin E synthase (PTGES), nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2)) and 25 selenoprotein genes (Gpx1, Gpx2, Gpx3, Gpx4, Txnrd1, Txnrd2, Txnrd3, Dio1, Dio2, Dio3, SPS2, Sepp1, SelPb, Sep15, Selh, Seli, Selm, Selo, Sels, Sepx1, Selu, Selk, Selw, Seln, Selt). The results demonstrated that Cd exposure increased the Cd content in the chicken kidneys, renal tubular epithelial cells underwent denaturation and necrosis, and the tubules became narrow or disappeared. However, Se supplementation reduced the Cd content in chicken kidneys and induced normal development of renal tubular epithelial cells. In addition, we also observed that Se alleviated the Cd-induced increase in the mRNA levels of inflammatory factors and ameliorated the Cd-induced downtrend in the mRNA levels of 25 selenoprotein genes in chicken kidneys.

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae.

PubMed

Huch, Susanne; Müller, Maren; Muppavarapu, Mridula; Gommlich, Jessie; Balagopal, Vidya; Nissan, Tracy

2016-10-15

The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization. © 2016. Published by The Company of Biologists Ltd.

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae

PubMed Central

Huch, Susanne; Müller, Maren; Muppavarapu, Mridula; Gommlich, Jessie; Balagopal, Vidya; Nissan, Tracy

2016-01-01

ABSTRACT The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization. PMID:27543059

Transcription factor Brn-3α mRNA in cancers, relationship with AR, ER receptors and AKT/m-TOR pathway components

NASA Astrophysics Data System (ADS)

Spirina, L. V.; Gorbunov, A. K.; Chigevskaya, S. Y.; Usynin, Y. A.; Kondakova, I. V.; Slonimskaya, E. M.; Usynin, E. A.; Choinzonov, E. L.; Zaitseva, O. S.

2017-09-01

Transcription factors POU4F1 (neurogenic factor Brn-3α) play a pivotal role in cancers development. The aim of the study was to reveal the Brn-3α expression, AR, ER expression in cancers development, association with AKT/mTOR pathway activation. 30 patients with locally advanced prostate cancer, 20 patients with papillary thyroid cancer, T2-3N0-1M0 stages and 40 patients with renal cell cancer T2-3N0M0-1 were involved into the study. The expressions of Brn-3α, AR, ERα, components of AKT/m-TOR signaling pathway genes were performed by real-time PCR. The dependence of Brn-3α expression on mRNA levels of steroid hormone receptors and components of AKT/m-TOR signaling pathway in studied cancers were shown. High levels of mRNA of nuclear factor, steroid hormone receptors were found followed by the activation of this signaling pathway in prostate cancer tissue. The reduction of transcription factor Brn-3α was accompanied with tumor invasive growth with increasing rates of AR, ER and 4E-BP1 mRNA. Thyroid cancer development happened in a case of a Brn-3α and steroid hormone receptors decrease. The activation of AKT/m-TOR signaling pathway was established in the metastatic renal cancers, accompanied with the increase of ER mRNA. But there was no correlation between the steroid receptor and Brn-3α. One-direction changes of Brn-3α were observed in the development of prostate and thyroid cancer due to its effect on the steroid hormone receptors and the activation of AKT/m-TOR signaling pathway components. The influence of this factor on the development of the kidney cancer was mediated through m-TOR activity modifications, the key enzyme of oncogenesis.

Expression of TIGIT and FCRL3 is Altered in T Cells from Patients with Distinct Patterns of Chronic Autoimmune Thyroiditis.

PubMed

Štefanić, Mario; Tokić, Stana; Suver-Stević, Mirjana; Glavaš-Obrovac, Ljubica

2018-06-11

Co-inhibitory receptors (IR), such as TIGIT and FCRL3, provide a checkpoint against highly destructive immune responses. Co-expression of TIGIT and FCRL3, in particular, has been linked to the HELIOS + subset of regulatory CD4 + FOXP3 + T-cells. Of these, CD4 + FOXP3-exon(E)2 + cells have higher expression of IR and exhibit strongest suppressive properties. Nevertheless, how the expression of TIGIT, FCRL3, HELIOS, and FOXP3E2 is regulated in chronic autoimmune thyroiditis (AT), is not known. Thirty patients with AT [encompassing spontaneously euthyroid (euAT), hypothyroid-untreated and L-thyroxine-treated cases)] and 10 healthy controls (HC) were recruited. FCRL3, TIGIT, HELIOS and FOXP3E2 mRNA expression levels in peripheral blood (PB) T cells were measured via quantitative real-time PCR and compared to clinicopathological factors. The TIGIT and FCRL3 expression levels from T cells of AT cases were inversely related to the thyroid volume, and were significantly increased in hypothyroid patients (on+off L-thyroxine), but not euAT cases. The FCRL3 expression in PB T cells positively correlated with thyroid-peroxidase autoantibody levels; by contrast, T cells from aged AT patients and combined samples (AT+HC) accumulated more TIGIT mRNA. The patients with higher TIGIT mRNA levels had a greater prevalence of hypothyroidism, showing higher peak thyrotropin levels at diagnosis or at follow-up. Multiple IR, namely FCRL3 and TIGIT, but not the transcription factors HELIOS and FOXP3E2, showed increased mRNA levels in PB T cells from end-stage, long-standing and/or more aggressive AT, in proportion to disease severity. A relation with major clinical subphenotypes was observed, thereby identifying IR as potentially important players in AT. © Georg Thieme Verlag KG Stuttgart · New York.

Effects of single amino acid deficiency on mRNA translation are markedly different for methionine versus leucine.

PubMed

Mazor, Kevin M; Dong, Leiming; Mao, Yuanhui; Swanda, Robert V; Qian, Shu-Bing; Stipanuk, Martha H

2018-05-24

Although amino acids are known regulators of translation, the unique contributions of specific amino acids are not well understood. We compared effects of culturing HEK293T cells in medium lacking either leucine, methionine, histidine, or arginine on eIF2 and 4EBP1 phosphorylation and measures of mRNA translation. Methionine starvation caused the most drastic decrease in translation as assessed by polysome formation, ribosome profiling, and a measure of protein synthesis (puromycin-labeled polypeptides) but had no significant effect on eIF2 phosphorylation, 4EBP1 hyperphosphorylation or 4EBP1 binding to eIF4E. Leucine starvation suppressed polysome formation and was the only tested condition that caused a significant decrease in 4EBP1 phosphorylation or increase in 4EBP1 binding to eIF4E, but effects of leucine starvation were not replicated by overexpressing nonphosphorylatable 4EBP1. This suggests the binding of 4EBP1 to eIF4E may not by itself explain the suppression of mRNA translation under conditions of leucine starvation. Ribosome profiling suggested that leucine deprivation may primarily inhibit ribosome loading, whereas methionine deprivation may primarily impair start site recognition. These data underscore our lack of a full understanding of how mRNA translation is regulated and point to a unique regulatory role of methionine status on translation initiation that is not dependent upon eIF2 phosphorylation.

A new MIF4G domain-containing protein, CTIF, directs nuclear cap-binding protein CBP80/20-dependent translation

PubMed Central

Kim, Kyoung Mi; Cho, Hana; Choi, Kobong; Kim, Jaedong; Kim, Bong-Woo; Ko, Young-Gyu; Jang, Sung Key; Kim, Yoon Ki

2009-01-01

During or right after mRNA export via the nuclear pore complex (NPC) in mammalian cells, mRNAs undergo translation mediated by nuclear cap-binding proteins 80 and 20 (CBP80/20). After CBP80/20-dependent translation, CBP80/20 is replaced by cytoplasmic cap-binding protein eIF4E, which directs steady-state translation. Nonsense-mediated mRNA decay (NMD), one of the best-characterized mRNA surveillance mechanisms, has been shown to occur on CBP80/20-bound mRNAs. However, despite the tight link between CBP80/20-dependent translation and NMD, the underlying molecular mechanism and cellular factors that mediate CBP80/20-dependent translation remain obscure. Here, we identify a new MIF4G domain-containing protein, CTIF (CBP80/20-dependent translation initiation factor). CTIF interacts directly with CBP80 and is part of the CBP80/20-dependent translation initiation complex. Depletion of endogenous CTIF from an in vitro translation system selectively blocks the translation of CBP80-bound mRNAs, while addition of purified CTIF restores it. Accordingly, down-regulation of endogenous CTIF abrogates NMD. Confocal microscopy shows that CTIF is localized to the perinuclear region. Our observations demonstrate the existence of CBP80/20-dependent translation and support the idea that CBP80/20-dependent translation is mechanistically different from steady-state translation through identification of a specific cellular protein, CTIF. PMID:19648179

Quantitative studies of mRNA recruitment to the eukaryotic ribosome.

PubMed

Fraser, Christopher S

2015-07-01

The process of peptide bond synthesis by ribosomes is conserved between species, but the initiation step differs greatly between the three kingdoms of life. This is illustrated by the evolution of roughly an order of magnitude more initiation factor mass found in humans compared with bacteria. Eukaryotic initiation of translation is comprised of a number of sub-steps: (i) recruitment of an mRNA and initiator methionyl-tRNA to the 40S ribosomal subunit; (ii) migration of the 40S subunit along the 5' UTR to locate the initiation codon; and (iii) recruitment of the 60S subunit to form the 80S initiation complex. Although the mechanism and regulation of initiation has been studied for decades, many aspects of the pathway remain unclear. In this review, I will focus discussion on what is known about the mechanism of mRNA selection and its recruitment to the 40S subunit. I will summarize how the 43S preinitiation complex (PIC) is formed and stabilized by interactions between its components. I will discuss what is known about the mechanism of mRNA selection by the eukaryotic initiation factor 4F (eIF4F) complex and how the selected mRNA is recruited to the 43S PIC. The regulation of this process by secondary structure located in the 5' UTR of an mRNA will also be discussed. Finally, I present a possible kinetic model with which to explain the process of mRNA selection and recruitment to the eukaryotic ribosome. Copyright © 2015 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

eIF4E and eIF4GI have distinct and differential imprints on multiple myeloma's proteome and signaling

PubMed Central

Attar-Schneider, Oshrat; Pasmanik-Chor, Metsada; Tartakover-Matalon, Shelly

2015-01-01

Accumulating data indicate translation plays a role in cancer biology, particularly its rate limiting stage of initiation. Despite this evolving recognition, the function and importance of specific translation initiation factors is unresolved. The eukaryotic translation initiation complex eIF4F consists of eIF4E and eIF4G at a 1:1 ratio. Although it is expected that they display interdependent functions, several publications suggest independent mechanisms. This study is the first to directly assess the relative contribution of eIF4F components to the expressed cellular proteome, transcription factors, microRNAs, and phenotype in a malignancy known for extensive protein synthesis-multiple myeloma (MM). Previously, we have shown that eIF4E/eIF4GI attenuation (siRNA/Avastin) deleteriously affected MM cells' fate and reduced levels of eIF4E/eIF4GI established targets. Here, we demonstrated that eIF4E/eIF4GI indeed have individual influences on cell proteome. We used an objective, high throughput assay of mRNA microarrays to examine the significance of eIF4E/eIF4GI silencing to several cellular facets such as transcription factors, microRNAs and phenotype. We showed different imprints for eIF4E and eIF4GI in all assayed aspects. These results promote our understanding of the relative contribution and importance of eIF4E and eIF4GI to the malignant phenotype and shed light on their function in eIF4F translation initiation complex. PMID:25717031

Eukaryotic Translation Initiation Factor 4E Availability Controls the Switch between Cap-Dependent and Internal Ribosomal Entry Site-Mediated Translation†

PubMed Central

Svitkin, Yuri V.; Herdy, Barbara; Costa-Mattioli, Mauro; Gingras, Anne-Claude; Raught, Brian; Sonenberg, Nahum

2005-01-01

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5′ end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection. PMID:16287867

Eukaryotic translation initiation factor 4E availability controls the switch between cap-dependent and internal ribosomal entry site-mediated translation.

PubMed

Svitkin, Yuri V; Herdy, Barbara; Costa-Mattioli, Mauro; Gingras, Anne-Claude; Raught, Brian; Sonenberg, Nahum

2005-12-01

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5' end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.

Sox2 is translationally activated by eukaryotic initiation factor 4E in human glioma-initiating cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ge, Yuqing; Zhou, Fengbiao; Chen, Hong

2010-07-09

Sox2, a master transcription factor, contributes to the generation of induced pluripotent stem cells and plays significant roles in sustaining the self-renewal of neural stem cells and glioma-initiating cells. Understanding the functional differences of Sox2 between glioma-initiating cells and normal neural stem cells would contribute to therapeutic approach for treatment of brain tumors. Here, we first demonstrated that Sox2 could contribute to the self-renewal and proliferation of glioma-initiating cells. The following experiments showed that Sox2 was activated at translational level in a subset of human glioma-initiating cells compared with the normal neural stem cells. Further investigation revealed there was amore » positive correlation between Sox2 and eukaryotic initiation factor 4E (eIF4E) in glioma tissues. Down-regulation of eIF4E decreased Sox2 protein level without altering its mRNA level in glioma-initiating cells, indicating that Sox2 was activated by eIF4E at translational level. Furthermore, eIF4E was presumed to regulate the expression of Sox2 by its 5' untranslated region (5' UTR) sequence. Our results suggest that the eIF4E-Sox2 axis is a novel mechanism of unregulated self-renewal of glioma-initiating cells, providing a potential therapeutic target for glioma.« less

2,4,6-Trichlorophenol cytotoxicity involves oxidative stress, endoplasmic reticulum stress, and apoptosis.

PubMed

Zhang, Xiaoning; Zhang, Xiaona; Niu, Zhidan; Qi, Yongmei; Huang, Dejun; Zhang, Yingmei

2014-01-01

This study aims to evaluate the cytotoxicity and potential mechanisms of 2,4,6-trichlorophenol (2,4,6-TCP) in mouse embryonic fibroblasts. Our results show that 2,4,6-TCP causes morphological changes and reduces cell viability. The overproduction of reactive oxygen species, the upregulation of nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase 1 (HMOX1) messenger RNA (mRNA) expressions, and the nuclear translocation of Nrf2 protein demonstrate that 2,4,6-TCP induces oxidative stress, and the Nrf2/HMOX1 pathway might be involved in 2,4,6-TCP-induced antioxidative response. Simultaneously, our data also demonstrate that 2,4,6-TCP upregulates the expressions of binding immunoglobulin protein, inositol-requiring enzyme/endonuclease 1α, and C/EBP homologous protein; stimulates α subunit of eukaryotic translation initiation factor 2 phosphorylation; and induces the splicing of Xbp1 mRNA, suggesting that endoplasmic reticulum (ER) stress is triggered. Moreover, 2,4,6-TCP alters the mitochondrial membrane potential and increases the apoptosis rate, the caspase 3 activity, and the Bax/Bcl-2 ratio, demonstrating that the mitochondrial pathway is involved in the 2,4,6-TCP-induced apoptosis. Thus, these results show that 2,4,6-TCP induces oxidative stress, ER stress, and apoptosis, which together contribute to its cytotoxicity in vitro. © The Author(s) 2014.

Cortisol response to waterborne 4-nonylphenol exposure leads to increased brain POMC and HSP70 mRNA expressions and reduced total antioxidant capacity in juvenile sole (Solea solea).

PubMed

Palermo, Francesco Alessandro; Cocci, Paolo; Nabissi, Massimo; Polzonetti-Magni, Alberta; Mosconi, Gilberto

2012-11-01

4-Nonylphenol (4-NP) is a breakdown product of alkylphenolpolyethoxylates and can be found in almost all environmental water matrices. 4-NP can act as environmental stressor on fish, typically causing modulation of hypothalamic-pituitary-interrenal axis (HPI). To examine the effects of the xenoestrogen 4-NP or 17β-estradiol (E2) on induction of stress response mechanisms by evaluating the levels of proopiomelanocortin (POMC) mRNA, heat shock protein 70 (HSP70) mRNA and plasma cortisol, we exposed juvenile sole (Solea solea), under static condition for 7 day, to either 10(-6) or 10(-8) M 4-NP, or 10(-8) M E2. In addition, plasma cortisol titers were correlated to the total antioxidant capacity (TAC), one of the oxidative stress parameters. 4-NP treatments resulted in high levels of POMC mRNA, HSP70 mRNA and plasma cortisol. On the contrary, E2 basically down-regulated POMC expression. Moreover, elevated cortisol levels in fish exposed to the highest dose of 4-NP were accompanied by low TAC. These results suggest that 4-NP modulates the sole HPI axis inducing a cortisol-mediated stress response. Specifically, we suggest that 4-NP affects brain POMC mRNA levels via non-estrogen receptor (ER)-mediated mechanism further supporting the ability of 4-NP to target multiple receptor systems. Copyright © 2012 Elsevier Inc. All rights reserved.

Effect of interferon-gamma on complement gene expression in different cell types.

PubMed

Lappin, D F; Guc, D; Hill, A; McShane, T; Whaley, K

1992-01-15

We have studied the expression of the complement components C2, C3, factor B, C1 inhibitor (C1-inh), C4-binding protein (C4-bp) and factor H in human peripheral blood monocytes, skin fibroblasts, umbilical vein endothelial cells (HUVEC) and the human hepatoma cell line G2 (Hep G2) in the absence and the presence of interferon-gamma (IFN-gamma). E.l.i.s.a. performed on culture fluids, run-on transcription assays, Northern blot and double-dilution dot-blot techniques confirmed that monocytes expressed all six components, whereas fibroblasts, HUVEC and HepG2 each expressed five of the six components. Fibroblasts and HUVEC did not synthesize C4-bp, and Hep G2 did not produce factor H. In addition to these differences, the synthesis rates of C3, C1-inh and factor H were not the same in all cell types. However, the synthesis rates of C2 and factor B were similar in all four cell types. The half-lives of the mRNAs were shorter in monocytes than in other cell types. Monocyte factor H mRNA had a half-life of 12 min in monocytes, compared with over 3 h in fibroblasts and HUVEC. The instability of factor H mRNA in monocytes may contribute to their low factor H secretion rate. IFN-gamma produced dose-dependent stimulation of C2, factor B, C1-inh, C4-bp and factor H synthesis by all cell types expressing these proteins, but decreased C3 synthesis in all four cell types. Cell-specific differences in the response to IFN-gamma were observed. The increased rates of transcription of the C1-inh and factor H genes in HUVEC were greater than in other cell types, while the increased rate of transcription of the C2, factor B and C1-inh genes in Hep G2 cells was less than in other cell types. IFN-gamma did not affect the stability of C3, factor H or C4 bp mRNAs, but increased the stability of factor B and C1-inh mRNAs and decreased the stability of C2 mRNA. Although these changes occurred in all four cell types studied, the half-life of C1-inh mRNA in monocytes was increased almost 4-fold, whereas the increases in the other cell types were less than 30%. These data show that the constitutive synthesis rates of complement components may vary in the different cell types. They also show that the degree of change in synthesis rates in response to IFN-gamma in each of the cell types often varies due to differences in transcriptional response, sometimes in association with changes in mRNA stability.

Dietary vitamin C deficiency depresses the growth, head kidney and spleen immunity and structural integrity by regulating NF-κB, TOR, Nrf2, apoptosis and MLCK signaling in young grass carp (Ctenopharyngodon idella).

PubMed

Xu, Hui-Jun; Jiang, Wei-Dan; Feng, Lin; Liu, Yang; Wu, Pei; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

2016-05-01

This study investigated the effects of dietary vitamin C on the growth, and head kidney, spleen and skin immunity, structural integrity and related signaling molecules mRNA expression levels of young grass carp (Ctenopharyngodon idella). A total of 540 grass carp (264.37 ± 0.66 g) were fed six diets with graded levels of vitamin C (2.9, 44.2, 89.1, 133.8, 179.4 and 224.5 mg/kg diet) for 10 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila and the survival rate recorded for 14 days. The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) decreased lysozyme (LA) and acid phosphatase (ACP) activities, and complement 3 and complement 4 (C4) contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides [liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, hepcidin, β-defensin] and anti-inflammatory cytokines-related factors, interleukin (IL) 4/13A, IL-4/13B (only in head kidney), IL-10, IL-11, transforming growth factor (TGF) β1, TGF-β2, inhibitor of κBα and eIF4E-binding protein 1 (P < 0.05), and up-regulated pro-inflammatory cytokines-related factors, tumor necrosis factor α, interferon γ2, IL-1β, IL-6, IL-8, IL-12 P35 (only in spleen), IL-12 P40, IL-15, IL-17D, nuclear factor κB p65, IκB kinases (IKKα, IKKβ, IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the head kidney and spleen under injection fish of A. hydrophila, suggesting that vitamin C deficiency could decrease fish head kidney and spleen immunity and cause inflammation. Meanwhile, compared with optimal vitamin C supplementation, vitamin C deficiency decreased the activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase, glutathione S-transferases and glutathione reductase (P < 0.05), and down-regulated zonula occludens (ZO) 1, ZO-2, Claudin-b, -c, -3c, -7a, -7b, B-cell lymphoma-2, inhibitor of apoptosis protein, NF-E2-related factor 2 mRNA levels (P < 0.05), increased reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl contents (P < 0.05), and up-regulated Claudin-12, 15a, -15b, Fas ligand, mitogen-activated protein kinase kinase 6, p38 mitogen-activated protein kinase, B-cell lymphoma protein 2 associated X protein, apoptotic protease activating factor-1, caspase-3, -7, -8, -9, Kelch-like ECH-associating protein (Keap) 1a and Keap 1b mRNA levels (P < 0.05) in the head kidney and spleen under injection fish of A. hydrophila, suggesting that vitamin C deficiency could decrease fish head kidney and spleen structural integrity through depression of antioxidative ability, induction of apoptosis and disruption of tight junctional complexes. In addition, except the activities of ACP and MnSOD, and mRNA expression levels of TGF-β1, Occludin and MnSOD, the effect of vitamin C on fish head kidney, spleen and skin immunity and structural integrity other indicators model are similar under infection of A. hydrophila. Finally, the vitamin C requirement for the growth performance (PWG) of young grass carp was estimated to be 92.8 mg/kg diet. Meanwhile, the vitamin C requirement for against skin lesion morbidity of young grass carp was estimated to be 122.9 mg/kg diet. In addition, based on the biochemical indices [immune indices (LA activity in the head kidney and C4 content in the spleen) and antioxidant indices (MDA content in the head kidney and ROS content in the spleen)] the vitamin C requirements for young grass carp were estimated to be 131.2, 137.5, 135.8 and 129.8 mg/kg diet, respectively. Copyright © 2016. Published by Elsevier Ltd.

Amicoumacin A inhibits translation by stabilizing mRNA interaction with the ribosome

PubMed Central

Polikanov, Yury S.; Osterman, Ilya A.; Szal, Teresa; Tashlitsky, Vadim N.; Serebryakova, Marina V.; Kusochek, Pavel; Bulkley, David; Malanicheva, Irina A.; Efimenko, Tatyana A.; Efremenkova, Olga V.; Konevega, Andrey L.; Shaw, Karen J.; Bogdanov, Alexey A.; Rodnina, Marina V.; Dontsova, Olga A.; Mankin, Alexander S.; Steitz, Thomas A.; Sergiev, Petr V.

2014-01-01

SUMMARY We demonstrate that the antibiotic amicoumacin A (AMI) whose cellular target was unknown, is a potent inhibitor of protein synthesis. Resistance mutations in helix 24 of the 16S rRNA mapped the AMI binding site to the small ribosomal subunit. The crystal structure of bacterial ribosome in complex with AMI solved at 2.4 Å resolution revealed that the antibiotic makes contacts with universally conserved nucleotides of 16S rRNA in the E site and the mRNA backbone. Simultaneous interactions of AMI with 16S rRNA and mRNA and the in vivo experimental evidence suggest that it may inhibit the progression of the ribosome along mRNA. Consistent with this proposal, binding of AMI interferes with translocation in vitro. The inhibitory action of AMI can be partly compensated by mutations in the translation elongation factor G. PMID:25306919

Embryotrophic factor-3 from human oviductal cells affects the messenger RNA expression of mouse blastocyst.

PubMed

Lee, Y L; Lee, K F; Xu, J S; Kwok, K L; Luk, J M; Lee, W M; Yeung, W S B

2003-02-01

Our previous results showed that embryotrophic factor-3 (ETF-3) from human oviductal cells increased the size and hatching rate of mouse blastocysts in vitro. The present study investigated the production of ETF-3 by an immortalized human oviductal cell line (OE-E6/E7) and the effects of ETF-3 on the mRNA expression of mouse embryos. The ETF-3 was purified from primary oviductal cell conditioned media using sequential liquid chromatographic systems, and antiserum against ETF-3 was raised. The ETF-3-supplemented Chatot-Ziomek-Bavister medium was used to culture Day 1 MF1 x BALB/c mouse embryos for 4 days. The ETF-3 treatment significantly enhanced the mouse embryo blastulation and hatching rate. The antiserum, at concentrations of 0.03-3%, abolished the embryotrophic effect of ETF-3. Positive ETF-3 immunoreactivity was detected in the primary oviductal cells, OE-E6/E7, and blastocysts derived from ETF-3 treatment. Vero cells (African Green Monkey kidney cell line), fibroblasts, and embryos cultured in control medium did not possess ETF-3 immunoreactivity. The mRNA expression patterns of the treated embryos were studied at the blastocyst stage by mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR). The DDRT-PCR showed that some of the mRNAs were differentially expressed after ETF-3 treatment. Twelve of the differentially expressed mRNAs that had high homology with cDNA sequences in the GenBank were selected for further characterization. The differential expression of seven of these mRNAs (ezrin, heat shock 70-kDa protein, cytochrome c oxidase subunit VIIa-L precursor, proteinase-activated receptor 2, eukaryotic translation initiation factor 2beta, cullin 1, and proliferating cell nuclear antigen) was confirmed by semiquantitative RT-PCR. In conclusion, immortalized oviductal cells produce ETF-3, which influences mRNA expression of mouse blastocyst.

Postnatal handling does not normalize hypothalamic corticotropin-releasing factor mRNA levels in animals prenatally exposed to ethanol.

PubMed

Gabriel, Kara I; Glavas, Maria M; Ellis, Linda; Weinberg, Joanne

2005-06-09

Postnatal handling has been shown to attenuate some of the deficits in developmental outcome observed following prenatal ethanol exposure (E) although it appears to be ineffective at ameliorating the hypothalamic-pituitary-adrenal (HPA) hyperresponsiveness to stressors that has been observed in adult E animals. However, the effects of postnatal handling on central regulation of HPA activity in E animals, particularly with regard to alterations in steady-state hypothalamic corticotropin-releasing factor (CRF) activity, have not been examined. In the present study, offspring from E, pair-fed (PF), and ad-libitum-fed control (C) groups were exposed to daily handling during the first 2 weeks of life (H) or were left entirely undisturbed until weaning (NH). Basal CRF and arginine vasopressin (AVP) mRNA in the parvocellular portion of the paraventricular nucleus (pPVN) of the hypothalamus were assessed at 90-110 days of age. Prenatal ethanol exposure resulted in elevated basal pPVN CRF mRNA levels compared to those in ad-libitum-fed controls. Handling altered CRF mRNA levels in a sex-specific and prenatal treatment-specific manner. Females showed no significant effects of handling. In contrast, handling decreased CRF mRNA levels in PF and C but not E males compared to their NH counterparts. There were no effects of prenatal ethanol or postnatal handling on AVP mRNA levels. These findings indicate that prenatal ethanol exposure results in elevated basal CRF mRNA levels in adulthood and that handling appears to be ineffective in normalizing those elevations, supporting the suggestion that altered basal HPA regulation in E animals may, at least in part, underlie their HPA hyperresponsiveness to stressors.

The effects of estradiol and selective estrogen receptor modulators on gene expression and messenger RNA stability in immortalized sheep endometrial stromal cells and human endometrial adenocarcinoma cells.

PubMed

Farnell, Yuhua Z; Ing, Nancy H

2003-03-01

The purpose of this study was to identify an endometrial cell line that maintained the E2 up-regulation of estrogen receptor (ER) mRNA by enhanced message stability and to assess its dependence on ER protein. Estradiol (E2) effects on gene expression were measured in three cell lines: one immortalized from sheep endometrial stroma (ST) and two from human endometrial adenocarcinomas (Ishikawa and ECC-1). E2 up-regulated ER mRNA levels in ST and Ishikawa cells, but down-regulated ER mRNA levels in ECC-1 cells. E2 up-regulated progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and transforming growth factor-alpha (TGF-alpha) in both Ishikawa and ECC-1 cells. The selective estrogen receptor modulator ICI 182,780 antagonized the E2-induced up-regulation of ER and/or PR mRNA levels in all three cells, while another, GW 5638, antagonized the up-regulation of PR mRNA in Ishikawa and ECC-1 cells. In mechanistic studies, E2 had no effect on ER mRNA stability in ST cells and it destabilized ER mRNA in ECC-1 cells. Thus, Ishikawa cells appear to be the most physiologically relevant cell line in which to study the up-regulation of ER mRNA levels by enhanced mRNA stability. Its antagonism by ICI 182,780 reveals that ER protein is involved in this E2 response.

FOXP3, CBLB and ITCH gene expression and cytotoxic T lymphocyte antigen 4 expression on CD4+CD25high T cells in multiple sclerosis

PubMed Central

Sellebjerg, F; Krakauer, M; Khademi, M; Olsson, T; Sørensen, P S

2012-01-01

Expression of the forkhead box protein 3 (FoxP3) transcription factor is regulated by the E3 ubiquitin ligases Itch and Cbl-b and induces regulatory activity CD4+CD25high T cells. Treatment with interferon (IFN)-β enhances regulatory T cell activity in multiple sclerosis (MS). We studied the phenotype of CD4+CD25high T cells in MS by flow cytometry and its relationship with expression of the FOXP3, ITCH and CBLB genes. We found that untreated MS patients had lower cell surface expression of cytotoxic T lymphocyte antigen 4 (CTLA-4) on CD4+CD25high T cells and higher intracellular CTLA-4 expression than healthy controls. Cell surface expression of CTLA-4 on CD4+CD25high T cells correlated with expression of FOXP3 mRNA in untreated patients and increased significantly with time from most recent injection in patients treated with IFN-β. FOXP3 mRNA expression correlated with CBLB and ITCH and T helper type 2 cytokine mRNA expression in MS patients. These data link expression of FOXP3, CBLB and ITCH mRNA and CTLA-4 expression on the surface of CD4+CD25high T cell in MS. We hypothesize that this may reflect alterations in the inhibitory effect of CTLA-4 or in regulatory T cell function. PMID:23039885

Cardiomyogenic Differentiation in Cardiac Myxoma Expressing Lineage-Specific Transcription Factors

PubMed Central

Kodama, Hiroaki; Hirotani, Takashi; Suzuki, Yusuke; Ogawa, Satoshi; Yamazaki, Kazuto

2002-01-01

We investigated five cases of cardiac myxoma and one case of cardiac undifferentiated sarcoma by light and electron microscopy, in situ hybridization, immunohistochemical staining, and reverse transcriptase-polymerase chain reaction for cardiomyocyte-specific transcription factors, Nkx2.5/Csx, GATA-4, MEF2, and eHAND. Conventional light microscopy revealed that cardiac myxoma and sarcoma cells presented variable cellular arrangements and different histological characteristics. Ultrastructurally, some of the myxoma cells exhibited endothelium-like or immature mesenchymal cell differentiation. Immunohistochemistry for Nkx2.5/Csx, GATA-4, and eHAND was slightly to intensely positive in all myxoma cases. MEF2 immunoreactivity was observed in all cases including the case of sarcoma, thus suggesting myogenic differentiation of myxoma or sarcoma cells. In situ hybridization for Nkx2.5/Csx also revealed that all myxoma cells, but not sarcoma cells, expressed mRNA of the cardiac homeobox gene, Nkx2.5/Csx. Furthermore, nested reverse transcriptase-polymerase chain reaction from formalin-fixed, paraffin-embedded tissue was performed and demonstrated that the Nkx2.5/Csx and eHAND gene product to be detected in all cases, and in three of six cases, respectively. In conclusion, cardiac myxoma cells were found to express various amounts of cardiomyocyte-specific transcription factor gene products at the mRNA and protein levels, thus suggesting cardiomyogenic differentiation. These results support the concept that cardiac myxoma might arise from mesenchymal cardiomyocyte progenitor cells. PMID:12163362

La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs.

PubMed

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-04-07

The 5'terminal oligopyrimidine (5'TOP) motif is a cis -regulatory RNA element located immediately downstream of the 7-methylguanosine [m 7 G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5'TOP motif, a cap analog (m 7 GTP), and a capped cytidine (m 7 GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5'TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5'TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.

mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells.

PubMed

Jossé, Lyne; Xie, Jianling; Proud, Christopher G; Smales, C Mark

2016-12-15

Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1. © 2016 The Author(s).

A novel mechanism for Bcr-Abl action: Bcr-Abl-mediated induction of the eIF4F translation initiation complex and mRNA translation.

PubMed

Prabhu, S; Saadat, D; Zhang, M; Halbur, L; Fruehauf, J P; Ong, S T

2007-02-22

The oncogenic kinase Bcr-Abl is thought to cause chronic myelogenous leukemia (CML) by altering the transcription of specific genes with growth- and survival-promoting functions. Recently, Bcr-Abl has also been shown to activate an important regulator of protein synthesis, the mammalian target of rapamycin (mTOR), which suggests that dysregulated translation may also contribute to CML pathogenesis. In this study, we found that both Bcr-Abl and the rapamycin-sensitive mTORC1 complex contribute to the phosphorylation (inactivation) of 4E-BP1, an inhibitor of the eIF4E translation initiation factor. Experiments with rapamycin and the Bcr-Abl inhibitor, imatinib mesylate, in Bcr-Abl-expressing cell lines and primary CML cells indicated that Bcr-Abl and mTORC1 induced formation of the translation initiation complex, eIF4F. This was characterized by reduced 4E-BP1 binding and increased eIF4G binding to eIF4E, two events that lead to the assembly of eIF4F. One target transcript is cyclin D3, which is regulated in Bcr-Abl-expressing cells by both Bcr-Abl and mTORC1 in a translational manner. In addition, the combination of imatinib and rapamycin was found to act synergistically against committed CML progenitors from chronic and blast phase patients. These experiments establish a novel mechanism of action for Bcr-Abl, and they provide insights into the modes of action of imatinib mesylate and rapamycin in treatment of CML. They also suggest that aberrant cap-dependent mRNA translation may be a therapeutic target in Bcr-Abl-driven malignancies.

Pulsed low-level infrared laser alters mRNA levels from muscle repair genes dependent on power output in Wistar rats

NASA Astrophysics Data System (ADS)

Trajano, L. A. S. N.; Trajano, E. T. L.; Thomé, A. M. C.; Sergio, L. P. S.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

2017-10-01

Satellite cells are present in skeletal muscle functioning in the repair and regeneration of muscle injury. Activation of these cells depends on the expression of myogenic factor 5 (Myf5), myogenic determination factor 1(MyoD), myogenic regulatory factor 4 (MRF4), myogenin (MyoG), paired box transcription factors 3 (Pax3), and 7 (Pax7). Low-level laser irradiation accelerates the repair of muscle injuries. However, data from the expression of myogenic factors have been controversial. Furthermore, the effects of different laser beam powers on the repair of muscle injuries have been not evaluated. The aim of this study was to evaluate the effects of low-level infrared laser at different powers and in pulsed emission mode on the expression of myogenic regulatory factors and on Pax3 and Pax7 in injured skeletal muscle from Wistar rats. Animals that underwent cryoinjury were divided into three groups: injury, injury laser 25 Mw, and injury laser 75 mW. Low-level infrared laser irradiation (904 nm, 3 J cm-2, 5 kHz) was carried out at 25 and 75 mW. After euthanasia, skeletal muscle samples were withdrawn and the total RNA was extracted for the evaluation of mRNA levels from the MyoD, MyoG, MRF4, Myf5, Pax3, and Pax7 gene. Pax 7 mRNA levels did not alter, but Pax3 mRNA levels increased in the injured and laser-irradiated group at 25 mW. MyoD, MyoG, and MYf5 mRNA levels increased in the injured and laser-irradiated animals at both powers, and MRF4 mRNA levels decreased in the injured and laser-irradiated group at 75 mW. In conclusion, exposure to pulsed low-level infrared laser, by power-dependent effect, could accelerate the muscle repair process altering mRNA levels from paired box transcription factors and myogenic regulatory factors.

Nitric oxide-sensitive guanylyl cyclase is differentially regulated by nuclear and non-nuclear estrogen pathways in anterior pituitary gland.

PubMed

Cabilla, Jimena P; Nudler, Silvana I; Ronchetti, Sonia A; Quinteros, Fernanda A; Lasaga, Mercedes; Duvilanski, Beatriz H

2011-01-01

17β-estradiol (E2) regulates hormonal release as well as proliferation and cell death in the pituitary. The main nitric oxide receptor, nitric oxide sensitive- or soluble guanylyl cyclase (sGC), is a heterodimer composed of two subunits, α and β, that catalyses cGMP formation. α1β1 is the most abundant and widely expressed heterodimer, showing the greater activity. Previously we have shown that E2 decreased sGC activity but exerts opposite effects on sGC subunits increasing α1 and decreasing β1 mRNA and protein levels. In the present work we investigate the mechanisms by which E2 differentially regulates sGC subunits' expression on rat anterior pituitary gland. Experiments were performed on primary cultures of anterior pituitary cells from adult female Wistar rats at random stages of estrous cycle. After 6 h of E2 treatment, α1 mRNA and protein expression is increased while β1 levels are down-regulated. E2 effects on sGC expression are partially dependent on de novo transcription while de novo translation is fully required. E2 treatment decreased HuR mRNA stabilization factor and increased AUF1 p37 mRNA destabilization factor. E2-elicited β1 mRNA decrease correlates with a mRNA destabilization environment in the anterior pituitary gland. On the other hand, after 6 h of treatment, E2-BSA (1 nM) and E2-dendrimer conjugate (EDC, 1 nM) were unable to modify α1 or β1 mRNA levels, showing that nuclear receptor is involved in E2 actions. However, at earlier times (3 h), 1 nM EDC causes a transient decrease of α1 in a PI3k-dependent fashion. Our results show for the first time that E2 is able to exert opposite actions in the anterior pituitary gland, depending on the activation of classical or non-classical pathways. Thus, E2 can also modify sGC expression through membrane-initiated signals bringing to light a new point of regulation in NO/sGC pathway. © 2011 Cabilla et al.

[Influence of macrophages on the expression of vascular endothelial growth factor receptor mRNA, homeobox B2 mRNA, and integrin alpha nu beta3 in vascular endothelial strain].

PubMed

Liu, Liang; Liu, Chang; Zhang, Xiao-qi; Ming, Jia; Liu, Xu-sheng; Xu, Hui; Cheng, Tian-min

2005-06-01

To investigate the influence of macrophages on the expression of the vascular endothelial growth factor (VEGF) receptor (KDR) mRNA, homeobox B2 (HOXB2) mRNA, and integrin alpha nu beta3 in vitro in vascular endothelial strain. Human umbilical vein cells (ECV304) were cultured in vitro and divided into 4 groups, i.e. (1) ECV304 group, (2) ECV304 + conA group [with conA (25 microg/ml in culture) added to ECV304], (3) ECV304 + U937 group (with 1 x 10(5)/ml of U937 cells added to 1 x 10(5)/ml ECV 304), (4) ECV304 + U937 + conA group [with 1 x 10(5)/ml of U937 cells and conA (25 microg/ml in culture)] groups. Forty-eight hours after culturing, the expression of integrin receptor alpha nu beta3 and the changes in the expression of KDR mRNA and HOXB2 mRNA in each group were determined by immunofluorescent technique and RT-PCR, respectively. The expression of integrin receptor alpha nu beta3, KDR mRNA, and HOXB2 mRNA in ECV304 group were 6.7 +/- 1.5, 0.633 +/- 0.012, and 0.674 +/- 0.004, respectively, while those in ECV304 + U937 + conA group (10.2 +/- 1.7, 0.879 +/- 0.003, 0.947 +/- 0.003) were obviously more upregulated when compared with those in ECV304 group (P < 0.01). No difference in the above indices was found between ECV304 and ECV304 + conA, ECV304 + U937 groups (P > 0.05). Macrophages activated by ConA can accelerate the proliferation, migration and adhesion to the basement membrane matrix of vascular endothelial cells through the influence on the expression of KDR mRNA, HOXB2 mRNA and integrin alpha nu beta3, and through this pathway the angiogenesis is modulated.

La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs

PubMed Central

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-01-01

The 5’terminal oligopyrimidine (5’TOP) motif is a cis-regulatory RNA element located immediately downstream of the 7-methylguanosine [m7G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5’TOP motif, a cap analog (m7GTP), and a capped cytidine (m7GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5’TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5’TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis. DOI: http://dx.doi.org/10.7554/eLife.24146.001 PMID:28379136

CUP promotes deadenylation and inhibits decapping of mRNA targets

PubMed Central

Igreja, Catia; Izaurralde, Elisa

2011-01-01

CUP is an eIF4E-binding protein (4E-BP) that represses the expression of specific maternal mRNAs prior to their posterior localization. Here, we show that CUP employs multiple mechanisms to repress the expression of target mRNAs. In addition to inducing translational repression, CUP maintains mRNA targets in a repressed state by promoting their deadenylation and protects deadenylated mRNAs from further degradation. Translational repression and deadenylation are independent of eIF4E binding and require both the middle and C-terminal regions of CUP, which collectively we termed the effector domain. This domain associates with the deadenylase complex CAF1–CCR4–NOT and decapping activators. Accordingly, in isolation, the effector domain is a potent trigger of mRNA degradation and promotes deadenylation, decapping and decay. However, in the context of the full-length CUP protein, the decapping and decay mediated by the effector domain are inhibited, and target mRNAs are maintained in a deadenylated, repressed form. Remarkably, an N-terminal regulatory domain containing a noncanonical eIF4E-binding motif is required to protect CUP-associated mRNAs from decapping and further degradation, suggesting that this domain counteracts the activity of the effector domain. Our findings indicate that the mode of action of CUP is more complex than previously thought and provide mechanistic insight into the regulation of mRNA expression by 4E-BPs. PMID:21937713

Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

PubMed Central

Smits, A.; Funa, K.; Vassbotn, F. S.; Beausang-Linder, M.; af Ekenstam, F.; Heldin, C. H.; Westermark, B.; Nistér, M.

1992-01-01

Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protein was found in the malignant tumors, and also in some benign lesions, such as dermatofibroma. In all these cases, benign as well as malignant, the PDGF B-chain mRNA, and less clearly, the PDGF A-chain mRNA, were coexpressed with the beta-receptor. In contrast, high expression of PDGF alpha-receptor mRNA was only found in fully malignant lesions, i.e., malignant fibrous histiocytoma. These data indicate that an autocrine growth stimulation via the PDGF beta-receptor could occur in an early phase of tumorigenesis, and may be a necessary but insufficient event for the progression into fully malignant human connective tissue lesions. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:1372158

Downregulated Translation Initiation Signaling Predisposes Low-Birth-Weight Neonatal Pigs to Slower Rates of Muscle Protein Synthesis

PubMed Central

Chen, Ying; McCauley, Sydney R.; Johnson, Sally E.; Rhoads, Robert P.; El-Kadi, Samer W.

2017-01-01

Low-birth-weight (LBWT) neonates experience restricted muscle growth in their perinatal life. Our aim was to investigate the mechanisms that contribute to slower skeletal muscle growth of LBWT neonatal pigs. Twenty-four 1-day old male LBWT (816 ± 55 g) and normal-birth-weight (NBWT; 1,642 ± 55 g) littermates (n = 12) were euthanized to collect blood and longissimus dorsi (LD) muscle subsamples. Plasma glucose, insulin, and insulin-like growth factor-I (IGF-I) were lower in LBWT compared with NBWT pigs. Muscle IGF-I mRNA expression were lower in LBWT than NBWT pigs. However, IGF-I receptor mRNA and protein abundance was greater in LD of LBWT pigs. Abundance of myostatin and its receptors, and abundance and phosphorylation of smad3 were lower in LBWT LD by comparison with NBWT LD. Abundance of eukaryotic initiation factor (eIF) 4E binding protein 1 and mitogen-activated protein kinase-interacting kinases was lower in muscle of LBWT pigs compared with NBWT siblings, while eIF4E abundance and phosphorylation did not differ between the two groups. Furthermore, phosphorylation of ribosomal protein S6 kinase 1 (S6K1) was less in LBWT muscle, possibly due to lower eIF3e abundance. In addition, abundance and phosphorylation of eIF4G was reduced in LBWT pigs by comparison with NBWT littermates, suggesting translation initiation complex formation is compromised in muscle of LBWT pigs. In conclusion, diminished S6K1 activation and translation initiation signaling are likely the major contributors to impaired muscle growth in LBWT neonatal pigs. The upregulated IGF-I R expression and downregulated myostatin signaling seem to be compensatory responses for the reduction in protein synthesis signaling. PMID:28744224

Eukaryotic Translation Initiation Factor eIFiso4G Is Required to Regulate Violaxanthin De-epoxidase Expression in Arabidopsis*

PubMed Central

Chen, Zhong; Jolley, Blair; Caldwell, Christian; Gallie, Daniel R.

2014-01-01

The eukaryotic translation initiation factor (eIF) 4G is a scaffold protein that organizes the assembly of those initiation factors needed to recruit the 40 S ribosomal subunit to an mRNA. Plants, like many eukaryotes, express two eIF4G isoforms. eIFiso4G, one of the isoforms specific to plants, is unique among eukaryotic eIF4G proteins in that it is highly divergent and unusually small in size, raising the possibility of functional specialization. In this study, the role of eIFiso4G in plant growth was investigated using null mutants for the eIF4G isoforms in Arabidopsis. eIFiso4G loss of function mutants exhibited smaller cell, leaf, plant size, and biomass accumulation that correlated with its reduced photosynthetic activity, phenotypes not observed with the eIF4G loss of function mutant. Although no change in photorespiration or dark respiration was observed in the eIFiso4G loss of function mutant, a reduction in chlorophyll levels and an increase in the level of nonphotochemical quenching were observed. An increase in xanthophyll cycle activity and the generation of reactive oxygen species contributed to the qE and qI components of nonphotochemical quenching, respectively. An increase in the transcript and protein levels of violaxanthin de-epoxidase in the eIFiso4G loss of function mutant and an increase in its xanthophyll de-epoxidation state correlated with the higher qE associated with loss of eIFiso4G expression. These observations indicate that eIFiso4G expression is required to regulate violaxanthin de-epoxidase expression and to support photosynthetic activity. PMID:24706761

Importin 8 mediates m7G cap-sensitive nuclear import of the eukaryotic translation initiation factor eIF4E

PubMed Central

Volpon, Laurent; Culjkovic-Kraljacic, Biljana; Osborne, Michael J.; Ramteke, Anup; Sun, Qingxiang; Niesman, Ashley; Chook, Yuh Min; Borden, Katherine L. B.

2016-01-01

Regulation of nuclear-cytoplasmic trafficking of oncoproteins is critical for growth homeostasis. Dysregulated trafficking contributes to malignancy, whereas understanding the process can reveal unique therapeutic opportunities. Here, we focus on eukaryotic translation initiation factor 4E (eIF4E), a prooncogenic protein highly elevated in many cancers, including acute myeloid leukemia (AML). Typically, eIF4E is localized to both the nucleus and cytoplasm, where it acts in export and translation of specific methyl 7-guanosine (m7G)–capped mRNAs, respectively. Nuclear accumulation of eIF4E in patients who have AML is correlated with increased eIF4E-dependent export of transcripts encoding oncoproteins. The subcellular localization of eIF4E closely correlates with patients’ responses. During clinical responses to the m7G-cap competitor ribavirin, eIF4E is mainly cytoplasmic. At relapse, eIF4E reaccumulates in the nucleus, leading to elevated eIF4E-dependent mRNA export. We have identified importin 8 as a factor that directly imports eIF4E into the nucleus. We found that importin 8 is highly elevated in untreated patients with AML, leading to eIF4E nuclear accumulation. Importin 8 only imports cap-free eIF4E. Cap-dependent changes to the structure of eIF4E underpin this selectivity. Indeed, m7G cap analogs or ribavirin prevents nuclear entry of eIF4E, which mirrors the trafficking phenotypes observed in patients with AML. Our studies also suggest that nuclear entry is important for the prooncogenic activity of eIF4E, at least in this context. These findings position nuclear trafficking of eIF4E as a critical step in its regulation and position the importin 8–eIF4E complex as a novel therapeutic target. PMID:27114554

The unique Leishmania EIF4E4 N-terminus is a target for multiple phosphorylation events and participates in critical interactions required for translation initiation.

PubMed

de Melo Neto, Osvaldo P; da Costa Lima, Tamara D C; Xavier, Camila C; Nascimento, Larissa M; Romão, Tatiany P; Assis, Ludmila A; Pereira, Mariana M C; Reis, Christian R S; Papadopoulou, Barbara

2015-01-01

The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4's partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa.

The unique Leishmania EIF4E4 N-terminus is a target for multiple phosphorylation events and participates in critical interactions required for translation initiation

PubMed Central

de Melo Neto, Osvaldo P; da Costa Lima, Tamara D C; Xavier, Camila C; Nascimento, Larissa M; Romão, Tatiany P; Assis, Ludmila A; Pereira, Mariana M C; Reis, Christian R S; Papadopoulou, Barbara

2015-01-01

The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4's partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa. PMID:26338184

Quantivirus® HPV E6/E7 RNA 3.0 assay (bDNA) is as sensitive, but less specific than Hybrid Capture 2 test.

PubMed

Shen, Yong; Gong, Jiaomei; He, Yanxia; Cheng, Guomei; Okunieff, Paul; Li, Xiaofu

2013-02-01

Human papillomavirus (HPV) infection is the primary cause of cervical cancer. The Quantivirus(®) HPV E6/E7 RNA 3.0 assay (DiaCarta, CA, USA) detects E6/E7 mRNA of 13 high risk subtypes and 6 low risk subtypes. Cervical specimens collected in PreservCyt were processed for HPV detection. Cervical biopsies were taken only from those women with abnormal colposcopy. 200 out of 272 (73.5%) cases were mRNA positive. The percentage of HPV E6/E7 mRNA positive samples increases with the severity of the cytological diagnosis, but not in histological diagnosis. In 146 patients with both tests, the E6/E7 mRNA assay had significant higher positivity rate than the Hybrid Capture 2 assay (75.3% versus 62.3%). The HPV mRNA assay and the HC2 assay had the same sensitivity of high grade cervical intraepithelial neoplasia (CIN 2+), 82.4% (14/17) (95% confidence interval [CI], 64.3, 100). However, the specificity of CIN 2+ for the HPV mRNA assay was significantly lower than HC2 assay. Receiver operating characteristic curve analysis was used to compare the diagnostic performance of the E6/E7 mRNA and HC2. E6/E7 mRNA achieved 58.8% sensitivity with 74.1% specificity, HC2, achieved 47.1% sensitivity with 70.7% specificity. The overall performance of HPV E6/E7 mRNA assay for detecting CIN 2+ was lower than HC2. This study does not support the use of this assay in screening for cervical cancer prevention alone. Copyright © 2012 Elsevier B.V. All rights reserved.

Identification of the Drosophila eIF4A gene as a target of the DREF transcription factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ida, Hiroyuki; Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585; Yoshida, Hideki

2007-12-10

The DNA replication-related element-binding factor (DREF) regulates cell proliferation-related gene expression in Drosophila. We have carried out a genetic screening, taking advantage of the rough eye phenotype of transgenic flies that express full-length DREF in the eye imaginal discs and identified the eukaryotic initiation factor 4A (eIF4A) gene as a dominant suppressor of the DREF-induced rough eye phenotype. The eIF4A gene was here found to carry three DRE sequences, DRE1 (- 40 to - 47), DRE2 (- 48 to - 55), and DRE3 (- 267 to - 274) in its promoter region, these all being important for the eIF4A genemore » promoter activity in cultured Drosophila Kc cells and in living flies. Knockdown of DREF in Drosophila S2 cells decreased the eIF4A mRNA level and the eIF4A gene promoter activity. Furthermore, specific binding of DREF to genomic regions containing DRE sequences was demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. Band mobility shift assays using Kc cell nuclear extracts revealed that DREF could bind to DRE1 and DRE3 sequences in the eIF4A gene promoter in vitro, but not to the DRE2 sequence. The results suggest that the eIF4A gene is under the control of the DREF pathway and DREF is therefore involved in the regulation of protein synthesis.« less

Endocrine disruptors and human reproductive failure: the in vitro effect of phthalates on human luteal cells.

PubMed

Romani, Federica; Tropea, Anna; Scarinci, Elisa; Federico, Alex; Dello Russo, Cinzia; Lisi, Lucia; Catino, Stefania; Lanzone, Antonio; Apa, Rosanna

2014-09-01

To evaluate the influence of phthalates on human luteal cell function. Laboratory study. University hospital. Twenty-three normally menstruating patients in the midluteal phase. Human luteal cells isolated from corpora lutea for primary cultures. Progesterone (P4) and prostaglandin release assayed by enzyme immunoassay, vascular endothelial growth factor (VEGF) secretion by enzyme-linked immunosorbent assay (ELISA), and VEGF mRNA expression by real-time polymerase chain reaction. We investigated the effect of di(2-ethylhexyl)phthalate (DEHP), di-n-butyl phthalate (DBP), and butyl benzyl phthalate (BBP) on basal and hCG-induced progesterone (P4) release, as well as DEHP effect on the balance between prostaglandin (PG) E2, vascular endothelial growth factor (VEGF)-luteotrophic factors, and the luteolitic PGF2α in isolated human steroidogenc cells. Phthalates influence on VEGF expression has been also evaluated. DEHP, DBP, and BBP were able to reduce both basal and hCG-stimulated P4 as well as PGE2 release. PGF2α release was reduced after DEHP incubation. VEGF protein release was decreased by the incubation with the tested phthalates. VEGF mRNA expression was not affected by DEHP, DBP, and BBP. As expected, both hCG and cobalt chloride were able to induce P4 release and VEGF release and mRNA expression in human luteal cells respectively. The results show the ability of phthalates to affect luteal steroidogenesis as well as the balance between luteotrophic and luteolytic factors suggesting an interference of phthalates in human luteal function. These data may contribute to clarify the classically known impaired reproductive health observed after phthalates exposure. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Up-regulated expression of cartilage intermediate-layer protein and ANK in articular hyaline cartilage from patients with calcium pyrophosphate dihydrate crystal deposition disease.

PubMed

Hirose, Jun; Ryan, Lawrence M; Masuda, Ikuko

2002-12-01

Excess accumulation of extracellular inorganic pyrophosphate (ePPi) in aged human cartilage is crucial in calcium pyrophosphate dihydrate (CPPD) crystal formation in cartilage matrix. Two sources of ePPi are ePPi-generating ectoenzymes (NTPPPH) and extracellular transport of intracellular PPi by ANK. This study was undertaken to evaluate the role of NTPPPH and ANK in ePPi elaboration, by investigating expression of NTPPPH enzymes (cartilage intermediate-layer protein [CILP] and plasma cell membrane glycoprotein 1 [PC-1]) and ANK in human chondrocytes from osteoarthritic (OA) articular cartilage containing CPPD crystals and without crystals. Chondrocytes were harvested from knee cartilage at the time of arthroplasty (OA with CPPD crystals [CPPD], n = 8; OA without crystals [OA], n = 10). Normal adult human chondrocytes (n = 1) were used as a control. Chondrocytes were cultured with transforming growth factor beta1 (TGFbeta1), which stimulates ePPi elaboration, and/or insulin-like growth factor 1 (IGF-1), which inhibits ePPi elaboration. NTPPPH and ePPi were measured in the media at 48 hours. Media CILP, PC-1, and ANK were determined by dot-immunoblot analysis. Chondrocyte messenger RNA (mRNA) was extracted for reverse transcriptase-polymerase chain reaction to study expression of mRNA for CILP, PC-1, and ANK. NTPPPH and ANK mRNA and protein were also studied in fresh frozen cartilage. Basal ePPi elaboration and NTPPPH activity in conditioned media from CPPD chondrocytes were elevated compared with normal chondrocytes, and tended to be higher compared with OA chondrocytes. Basal expression of mRNA for CILP (chondrocytes) and ANK (cartilage) was higher in both CPPD chondrocytes and CPPD cartilage extract than in OA or normal samples. PC-1 mRNA was less abundant in CPPD chondrocytes and cartilage extract than in OA chondrocytes and extract, although the difference was not significant. CILP, PC-1, and ANK protein levels were similar in CPPD, OA, and normal chondrocytes or cartilage extracts. Both CILP and ANK mRNA expression and ePPi elaboration were stimulated by TGFbeta1 and inhibited by IGF-1 in chondrocytes from all sources. CILP and ANK mRNA expression correlates with chondrocyte ePPi accumulation around CPPD and OA chondrocytes, and all respond similarly to growth factor stimulation. These findings suggest that up-regulated CILP and ANK expression contributes to higher ePPi accumulation from CPPD crystal-forming cartilage.

Cell-cell contact regulates gene expression in CDK4-transformed mouse podocytes.

PubMed

Sakairi, Toru; Abe, Yoshifusa; Jat, Parmijit S; Kopp, Jeffrey B

2010-10-01

We transformed mouse podocytes by ectopic expression of cyclin-dependent kinase 4 (CDK4). Compared with podocytes transformed with a thermo-sensitive SV40 large T antigen mutant tsA58U19 (tsT podocytes), podocytes transformed with CDK4 (CDK4 podocytes) exhibited significantly higher expression of nephrin mRNA. Synaptopodin mRNA expression was significantly lower in CDK4 podocytes and in tsT podocytes under growth-permissive conditions (33°C) compared with tsT podocytes under growth-restricted conditions (37°C), which suggests a role for cell cycle arrest in synaptopodin mRNA expression. Confluent CDK4 podocytes showed significantly higher mRNA expression levels for nephrin, synaptopodin, Wilms tumor 1, podocalyxin, and P-cadherin compared with subconfluent cultures. We carried out experiments to clarify roles of various factors in the confluent podocyte cultures; our findings indicate that cell-cell contact promotes expression of five podocyte marker genes studied, that cellular quiescence increases synaptopodin and podocalyxin mRNA expression, and that soluble factors play a role in nephrin mRNA expression. Our findings suggest that CDK4 podocytes are useful tools to study podocyte biology. Furthermore, the role of cell-cell contact in podocyte gene expression may have relevance for podocyte function in vivo.

Progesterone and 17β-estradiol regulate expression of nesfatin-1/NUCB2 in mouse pituitary gland.

PubMed

Chung, Yiwa; Kim, Jinhee; Im, Eunji; Kim, Heejeong; Yang, Hyunwon

2015-01-01

Nesfatin-1 was first shown to be involved in the control of appetite and energy metabolism in the hypothalamus. Many recent reports have shown nesfatin-1 expression in various tissues including the pituitary gland, but its expression and regulation mechanisms in the pituitary gland are unclear. Therefore, first, we investigated the mRNA and protein expression of nesfatin-1 in the pituitary using qRT-PCR and Western blotting, respectively. Expression of NUCB2 mRNA and nesfatin-1 protein was higher in the pituitary gland than in other organs, and nesfatin-1 protein was localized in many cells in the anterior pituitary gland. Next, we investigated whether NUCB2 mRNA expression in the pituitary gland was regulated by sex steroid hormones secreted by the ovary. Mice were ovariectomized and injected with progesterone (P4) and 17β-estradiol (E2). The expression of NUCB2 in the pituitary gland was dramatically decreased after ovariectomy and increased with injection of P4 and E2, respectively. The in vitro experiment to elucidate the direct effect of P4 and E2 on NUCB2 mRNA expression showed NUCB2 mRNA expression was significantly increased with E2 and decreased with P4 alone and P4 plus E2 in cultured pituitary tissue. The present study demonstrated that nesfatin-1/NUCB2 was highly expressed in the mouse pituitary and was regulated by P4 and E2. These data suggest that reproductive-endocrine regulation through hypothalamus-pituitary-ovary axis may contribute to nesfatin-1/NUCB2 expression in the pituitary gland. Copyright © 2014 Elsevier Inc. All rights reserved.

Shikonin, a constituent of Lithospermum erythrorhizon exhibits anti-allergic effects by suppressing orphan nuclear receptor Nr4a family gene expression as a new prototype of calcineurin inhibitors in mast cells.

PubMed

Wang, Xiaoyu; Hayashi, Shusaku; Umezaki, Masahito; Yamamoto, Takeshi; Kageyama-Yahara, Natsuko; Kondo, Takashi; Kadowaki, Makoto

2014-12-05

Over the last few decades, food allergy (FA) has become a common disease in infants in advanced countries. However, anti-allergic medicines available in the market have no effect on FA, and consequently effective drug therapies for FA are not yet available. We have already demonstrated that mucosal mast cells play an essential role in the development of FA in a murine model. Thus, we screened many constituents from medicinal herbs for the ability to inhibit rat basophilic leukemia-2H3 mast-like cell degranulation, and found that shikonin, a naphthoquinone dye from Lithospermum erythrorhizon, exhibited the most potent inhibitory effect among them. Furthermore, shikonin extremely inhibited the IgE/antigen-induced and calcium ionophore-induced upregulation of tumor necrosis factor (TNF)-α mRNA expression in mucosal-type bone marrow-derived mast cells (mBMMCs). Global gene expression analysis confirmed by real-time PCR revealed that shikonin drastically inhibited the IgE/antigen-induced and calcium ionophore-induced upregulation of mRNA expression of the nuclear orphan receptor 4a family (Nr4a1, Nr4a2 and Nr4a3) in mBMMCs, and knockdown of Nr4a1 or Nr4a2 suppressed the IgE/antigen-induced upregulation of TNF-α mRNA expression. Computational docking simulation of a small molecule for a target protein is a useful technique to elucidate the molecular mechanisms underlying the effects of drugs. Therefore, the simulation revealed that the predicted binding sites of shikonin to immunophilins (cyclophilin A and FK506 binding protein (FKBP) 12) were almost the same as the binding sites of immunosuppressants (cyclosporin A and FK506) to immunophilins. Indeed, shikonin inhibited the calcineurin activity to a similar extent as cyclosporin A that markedly suppressed the IgE/antigen-enhanced mRNA expression of TNF-α and the Nr4a family in mBMMCs. These findings suggest that shikonin suppresses mucosal mast cell activation by reducing Nr4a family gene expression through the inhibition of calcineurin activity. Therefore, shikonin has therapeutic potential for the treatment of allergic diseases as a new calcineurin inhibitor. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

The clinical application of HPV E6/E7 mRNA testing in triaging women with atypical squamous cells of undetermined significance or low-grade squamous intra-epithelial lesion Pap smear: A meta-analysis.

PubMed

Yang, Li; Zhu, Yuanhang; Bai, Yang; Zhang, Xiaoan; Ren, Chenchen

2017-01-01

The aim is to evaluate the clinical application value and correlation with cervical lesions' progression of human papillomavirus (HPV) E6/E7 mRNA test in women with atypical squamous cells of undetermined significance (ASCUS/borderline) or low-grade squamous intraepithelial lesions (LSILs/mild dyskaryosis) cytological abnormalities. A meta-analysis was conduct by searching China National Knowledge Infrastructure (1979-2016), Wanfang Date (1998-2016), VIP (1989-2016), PubMed (1950-2016), Web of Science (1950-2016) and Elsevier Science Direct (1998-2016), for studies on effect of HPV E6/E7 mRNA detection in women with ASCUS/LSIL/dyskaryosis. Study selection and appraisal were conducted independently by three authors, according to inclusive and exclusive criteria. Then, a meta-analysis was performed using the RevMan4.2 software. The subgroups analysis was conducted according to women's initial HPV DNA test results. Six articles with a total of 1024 subjects were included in the study. It was concluded that a positive HPV E6/E7 mRNA tested result have a higher risk of progressing to CIN2+ in future 2 years than a negative result. The pooled relative risk (RR) is 3.08, (95% confidence interval [CI] = 1.57-6.07, P < 0.05). The same situation was also observed in the subgroup of HPV DNA tested positive group and HPV DNA tested unlimited group. The pooled RR value of the two subgroups was, respectively, 1.98, (95% CI = 1.19-1.19, P < 0.05) and 7.58, (95% CI = 3.64-3.64, P < 0.05). A positive HPV E6/E7 mRNA testing result suggested the women with ASCUS, or LSIL Pap smear was in a truly dangerous position, which is an adverse prognostic factor. It suggested that cervical lesions stay in a progressing status and these women should be referred for colposcopy and strengthen follow-up promptly. Whereas, women with a negative HPV E6/E7 mRNA testing result can increase follow-up interval, by comprehensively considering their situation, thus, avoiding unnecessary colposcopy and reducing the rate of colposcopy and biopsy.

Cap analogs modified with 1,2-dithiodiphosphate moiety protect mRNA from decapping and enhance its translational potential

PubMed Central

Strenkowska, Malwina; Grzela, Renata; Majewski, Maciej; Wnek, Katarzyna; Kowalska, Joanna; Lukaszewicz, Maciej; Zuberek, Joanna; Darzynkiewicz, Edward; Kuhn, Andreas N.; Sahin, Ugur; Jemielity, Jacek

2016-01-01

Along with a growing interest in mRNA-based gene therapies, efforts are increasingly focused on reaching the full translational potential of mRNA, as a major obstacle for in vivo applications is sufficient expression of exogenously delivered mRNA. One method to overcome this limitation is chemically modifying the 7-methylguanosine cap at the 5′ end of mRNA (m7Gppp-RNA). We report a novel class of cap analogs designed as reagents for mRNA modification. The analogs carry a 1,2-dithiodiphosphate moiety at various positions along a tri- or tetraphosphate bridge, and thus are termed 2S analogs. These 2S analogs have high affinities for translation initiation factor 4E, and some exhibit remarkable resistance against the SpDcp1/2 decapping complex when introduced into RNA. mRNAs capped with 2S analogs combining these two features exhibit high translation efficiency in cultured human immature dendritic cells. These properties demonstrate that 2S analogs are potentially beneficial for mRNA-based therapies such as anti-cancer immunization. PMID:27903882

Time course expression of Foxo transcription factors in skeletal muscle following corticosteroid administration

PubMed Central

Cho, John E.; Fournier, Mario; Da, Xiaoyu

2010-01-01

Increased expression of forkhead box O (Foxo) transcription factors were reported in cultured myotubes and mouse limb muscle with corticosteroid (CS) treatment. We previously reported that administration of CS to rats resulted in muscle fiber atrophy only by day 7. The aim of this study, therefore, was to evaluate the time-course changes in the expression of Foxo transcription factors and muscle-specific ubiquitin E3 ligases in rat limb muscle following CS administration. Triamcinolone (TRI; 1 mg · kg−1 · day−1 im) was administered for 1, 3, or 7 days. Control (CTL) rats were given saline. Muscle mRNA was analyzed by real-time RT-PCR. Compared with CTL, body weights of TRI-treated animals decreased by 3, 12, and 21% at days 1, 3, and 7, respectively. Muscle IGF-1 mRNA levels decreased by 33, 65, and 58% at days 1, 3, and 7 in TRI-treated rats compared with CTL. Levels of phosphorylated Akt were 28, 50, and 36% lower in TRI animals at these time points. Foxo1 mRNA increased progressively by 1.2-, 1.4-, and 2.5-fold at days 1, 3, and 7 in TRI animals. Similar changes were noted in the expression of Foxo3a mRNA (1.3-, 1.4-, and 2.6-fold increments). By contrast, Foxo4 mRNA was not significantly changed in TRI animals. With TRI, muscle atrophy F box/Atrogin-1 increased by 1.8-, 4.1-, and 7.5-fold at days 1, 3, and 7 compared with CTL rats. By contrast, muscle RING finger 1 increased only from day 7 (2.7-fold). Gradual reduction in IGF-I expression with TRI over the time series paralleled that of Akt. These findings are consistent with a progressive stimulus to muscle protein degradation and the need to process/remove disassembled muscle proteins via the ubiquitin-proteasome system. Elucidating the dynamic catabolic responses to CS challenge is important in understanding the mechanisms underlying muscle atrophy and therapeutic measures to offset this. PMID:19850732

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation.

PubMed

Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed; Pelletier, Jerry

2006-10-01

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2alpha phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2alpha phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2alpha to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2alpha phosphorylation-dependent and -independent pathways that target translation initiation.

Inhibition of Ribosome Recruitment Induces Stress Granule Formation Independently of Eukaryotic Initiation Factor 2α Phosphorylation

PubMed Central

Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J.; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed

2006-01-01

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2α phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2α phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2α to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2α phosphorylation-dependent and -independent pathways that target translation initiation. PMID:16870703

Enhance tumor radiosensitivity by intracellular delivery of eukaryotic translation initiation factor 4E binding proteins.

PubMed

Tian, Shuang; Li, Xiu-Li; Shi, Mei; Yao, Yuan-Qing; Li, Li-Wen; Xin, Xiao-Yan

2011-02-01

PTEN (phosphatase and tensin homologue deleted on chromosome ten)/PI3K (phosphatidylinositol 3-kinase)/Akt/mTOR (mammalian target of rapamycin) signaling pathway, which is commonly dysregulated in a broad array of human malignancies, controls the assembly of eukaryotic translation initiation factor 4F (eIF4F) complex through regulation of eIF4E binding proteins (4E-BPs) phosphorylation. And accumulated data over the past two decades implicated eIF4F complex as one of the promising targets for anticancer therapy. It has been confirmed that the translation initiation of mRNA coding for hypoxia-inducible factor-1α (HIF-1α) and survivin, which had been considered as the two major determinants of tumor radiosensitivity, are both controlled by eIF4F complex. Also, eIF4F complex controls the expression of VEGF and bFGF, the two well-known pro-angiogenic factors involved in developing radioresistance. Therefore eIF4F complex plays a pivotal role in regulation of radiosensitivity. In this article, we postulate that cell-permeable, phosphorylation-defective 4E-BP fusion proteins, which could be prepared by substituting the mTOR recognition motif located in N-terminal of 4E-BPs with protein transduction domain from HIV-1 TAT, HSV-1 VP22 or PTD4, could not only inhibit tumor growth but also enhance tumor response to radiation therapy through disruption of eIF4F complex assembly. In our opinion, the recombinant fusion proteins are superior to mTOR inhibitors for they do not cause immunosuppression, do not lead to Akt activation, and could be easily prepared by prokaryotic expression. If the hypothesis was proved to be practical, the cell-permeable, phosphorylation-defective 4E-BP fusion proteins would be widely used in clinical settings to improve tumor response to radiotherapy in the near future. Copyright © 2010 Elsevier Ltd. All rights reserved.

[Effects of lipopolysaccharides from various Porphyromonas on the expression of CD14 and TLRs in mouse osteoblast].

PubMed

Jia, Ge; Xue, Ming; Li, Ren; Lv, You; Qiu, Li-hong

2011-12-01

To observe the effect of lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) and Porphyromonas gingivals(P.g) on the expression of CD14 and TLRs in osteoblast. MC3T3-E1 cells were stimulated with 10μg/mL P.e-LPS and P.g-LPS. The change of CD14,TLR2 and TLR4 mRNA was observed at different time point (0,1,3,6,12,24h) using RT-PCR,and the expression of CD14,TLR2 and TLR4 protein was measured by flow cytometry at 24-hour. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS11.0 software package. MC3T3-E1 cells were stimulated with 10μg/mL P.e-LPS for 1h,the expression of CD14 and TLR4 mRNA increased significantly. There was no increase of TLR2 mRNA with stimulation of P.e-LPS. The CD14,TLR2 and TLR4 mRNA expression increased significantly after stimulation with 10μg/mL P.g-LPS. Flow cytometry showed that CD14 and TLR4 protein increased significantly after stimulation with 10μg/mL P.e-LPS. CD14,TLR2 and TLR4 protein increased significantly after treatment with 10μg/mL P.g-LPS. CD14,TLR4 receptors are involved in P.e-LPS effect and CD14,TLR2 and TLR4 receptors are involved in P.g-LPS effect in mouse osteoblast.

miR-328 Functions as an RNA Decoy to Modulate hnRNP E2 Regulation of mRNA Translation in Leukemic Blasts

PubMed Central

Eiring, Anna M.; Harb, Jason G.; Neviani, Paolo; Garton, Christopher; Oaks, Joshua J.; Spizzo, Riccardo; Liu, Shujun; Schwind, Sebastian; Santhanam, Ramasamy; Hickey, Christopher J.; Becker, Heiko; Chandler, Jason C.; Andino, Raul; Cortes, Jorge; Hokland, Peter; Huettner, Claudia S.; Bhatia, Ravi; Roy, Denis C.; Liebhaber, Stephen A.; Caligiuri, Michael A.; Marcucci, Guido; Garzon, Ramiro; Croce, Carlo M.; Calin, George A.; Perrotti, Danilo

2010-01-01

SUMMARY MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNA’s seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins. PMID:20211135

Lipoxin A4-Induced Heme Oxygenase-1 Protects Cardiomyocytes against Hypoxia/Reoxygenation Injury via p38 MAPK Activation and Nrf2/ARE Complex

PubMed Central

Chen, Xiao-Qing; Wu, Sheng-Hua; Zhou, Yu; Tang, Yan-Rong

2013-01-01

Objective To investigate whether lipoxin A4 (LXA4) increases expression of heme oxygenase-1(HO-1) in cardiomyocytes, whether LXA4-induced HO-1 protects cardiomyocytes against hypoxia/reoxygenation (H/R) injury, and what are the mechanisms involved in the LXA4-induced HO-1 induction. Methods Rat cardiomyocytes were exposed to H/R injury with or without preincubation with LXA4 or HO-1 inhibitor ZnPP-IX or various signal molecule inhibitors. Expressions of HO-1 protein and mRNA were analyzed by using Western blot and RT-PCR respectively. Activity of nuclear factor E2-related factor 2 (Nrf2) binding to the HO-1 E1 enhancer was assessed by chromatin immunoprecipitation. Nrf2 binding to the HO-1 antioxidant responsive element (ARE) were measured by using electrophoretic mobility shift assay. Results Pretreatment of the cells undergoing H/R lesion with LXA4 significantly reduced the lactate dehydrogenase and creatine kinase productions, increased the cell viability, and increased the expressions of HO-1 protein and mRNA and HO-1 promoter activity. HO-1 inhibition abolished the protective role of LXA4 on the cells undergoing H/R lesion. LXA4 increased p38 mitogen-activated protein kinase (p38 MAPK) activation, nuclear translocation of Nrf2, Nrf2 binding to the HO-1 ARE and E1 enhancer in cardiomyocytes with or without H/R exposure. Conclusion The protection role of LXA4 against H/R injury of cardiomyocytes is related to upregulation of HO-1, via activation of p38 MAPK pathway and nuclear translocation of Nrf2 and Nrf2 binding to the HO-1 ARE and E1 enhancer, but not via activation of phosphatidyinositol-3-kinase or extracellular signal-regulated kinase pathway. PMID:23826208

Factors regulating collagen synthesis and degradation during second-intention healing of wounds in the thoracic region and the distal aspect of the forelimb of horses.

PubMed

Schwartz, Anne J; Wilson, David A; Keegan, Kevin G; Ganjam, Venkataseshu K; Sun, Yao; Weber, Karl T; Zhang, Jiakun

2002-11-01

To determine significant molecular and cellular factors responsible for differences in second-intention healing in thoracic and metacarpal wounds of horses. 6 adult mixed-breed horses. A full-thickness skin wound on the metacarpus and another such wound on the pectoral region were created, photographed, and measured, and tissue was harvested from these sites weekly for 4 weeks. Gene expression of type-I collagen, transforming growth factor (TGF)-beta1, matrix metalloproteinase (MMP)-1, and tissue inhibitor of metalloproteinase (TIMP)-1 were determined by quantitative in situ hybridization. Myofibroblasts were detected by immunohistochemical labeling with alpha-smooth muscle actin (alpha-SMA). Collagen accumulation was detected by use of picrosirius red staining. Tissue morphology was examined by use of H&E staining. Unlike thoracic wounds, forelimb wounds enlarged during the first 2 weeks. Myofibroblasts, detected by week 1, remained abundant with superior organization in thoracic wounds. Type-I collagen mRNA accumulated progressively in both wounds. More type-I collagen and TGF-beta1 mRNA were seen in forelimb wounds. Volume of MMP-1 mRNA decreased from day 0 in both wounds. By week 3, TIMP-1 mRNA concentration was greater in thoracic wounds. Greater collagen synthesis in metacarpal than thoracic wounds was documented by increased concentrations of myofibroblasts, type-I collagen mRNA,TGF-beta1 mRNA, and decreased collagen degradation (ie, MMP-1). Imbalanced collagen synthesis and degradation likely correlate with development of exuberant granulation tissue, delaying healing in wounds of the distal portions of the limbs. Factors that inhibit collagen synthesis or stimulate collagenase may provide treatment options for horses with exuberant granulation tissue.

Dietary low or excess levels of lipids reduced growth performance, and impaired immune function and structure of head kidney, spleen and skin in young grass carp (Ctenopharyngodon idella) under the infection of Aeromonas hydrophila.

PubMed

Ni, Pei-Jun; Jiang, Wei-Dan; Wu, Pei; Liu, Yang; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Feng, Lin

2016-08-01

Our study explored the effect of dietary lipids on growth and immunity and structure (head kidney, spleen and skin) of young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp with an average initial weight of 261.41 ± 0.53 g were fed diets containing six graded levels of lipids at 5.9-80.1 g/kg diet for 8 weeks. After that, a challenge trial was conducted by injection of Aeromonas hydrophila over 2 weeks. The results indicated that compared with optimal lipids supplementation, low and excess levels of lipids down-regulated the mRNA levels of antimicrobial peptides, anti-inflammatory cytokines, inhibitor of κBα (IκBα) and ribosomal p70S6 kinase (S6K1), and up-regulated pro-inflammatory cytokines, nuclear factor κB p65 (NF-κB p65), NF-κB c-Rel (not p52), IκB kinase α (IKKα), IKKβ, IKKγ, and eIF4E-binding protein (4EBP) mRNA levels in the head kidney and spleen of young grass carp (P < 0.05). Low or excess levels of lipids also increased reactive oxygen species (ROS) production and malondialdehyde (MDA) and protein carbonyl (PC) contents, reduced the activities of antioxidant enzymes (P < 0.05), down-regulate the relative mRNA levels of antioxidant enzymes and NF-E2-related factor 2 (Nrf2), and up-regulated the expression levels of Kelch-like ECH-associating protein 1a (Keap1a) and Keap1b in the head kidney and spleen. In addition, low or excess levels of lipids down-regulated the mRNA levels of B-cell lymphoma-2 (Bcl-2) and inhibitor of apoptosis protein (IAP) in the head kidney and spleen, whereas up-regulated the mRNA levels of apoptotic protease activating factor-1 (Apaf-1), caspase 3, 7, 8 and 9 mRNA levels in the head kidney and spleen and Fas ligand (FasL) mRNA levels in the spleen of young grass carp, suggesting that low or excess levels of lipids could decrease the head kidney and spleen immune function, induce oxidative damage and apoptosis and impair antioxidant system of young grass carp. At last, low or excess levels of lipids also impaired the immune function and structure in the skin of young grass carp. Based on the quadratic regression analysis for PWG, skin haemorrhage and lesions morbidity and IgM content, the dietary lipids requirements for young grass carp were estimated to be 43.7, 60.2, 55.0 and 52.1 g/kg diet, respectively. Copyright © 2016. Published by Elsevier Ltd.

[Mechanism of TNF-α in bone defect of chronic apical periodontitis].

PubMed

Yu, Ya-Qiong; Qu, Liu; Qiu, Li-Hong; Guo, Jia-Jie; Ma, Nan; Zhu, Li

2016-08-01

To investigate the effect of lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) on the expression of tumor necrosis factor-α(TNF-α) mRNA in MC3T3-E1 cells and the role of NF-κB signaling on the expression of macrophage colony stimulating factor (M-CSF) induced by TNF-α in MC3T3-El cells． METHODS: MC3T3-E1 cells were treated with different concentrations of P.e-LPS(0-50 mg/L) and 10 mg/L P.e-LPS for different time (0-24 h). The expression of TNF-α mRNA was detected by reverse transcription polymerase chain reaction(RT-PCR). MC3T3-E1 cells were treated with different concentrations of TNF-α(0-10 ng/L) for 6 h. The expression of M-CSF mRNA and protein was detected by RT-PCR and enzyme-linked immunoadsordent assay(ELISA).The expression of M-CSF protein was also detected in 10 ng/L TNF-α treated MC3T3-E1 cells after pretreated with BAY 11-7082 for 1 h, a special NF-κB inhibitor . Statistical analysis was performed using Multi-way ANOVA and Dunnett t test with SPSS 13.0 software package. The level of TNF-α mRNA increased significantly after treatment with different concentrations of P.e-LPS(0-50 mg/L)，which indicated that P.e-LPS induced osteoblasts to express TNF-α mRNA in dose dependent manners. Maximal induction of TNF-α mRNA expression was seen in the MC3T3-E1 cells treated with 10 mg/L P.e-LPS for 6 h. After 6 h, the expression of TNF-α mRNA decreased gradually .The expression of M-CSF mRNA and protein was increased in a does- dependent manner by different concentrations of TNF-α treatment（0-10 ng/L）. The expression of M-CSF protein increased from (37±2) ng/L(control group) to (301±8) ng/L(10 ng/L group).The protein of M-CSF decreased significantly after pretreatment with 10 μmol/L BAY 11-7082 for 1 h, and the expression of M-CSF proteins was reduced from (253±14) ng/L to (154±2) ng/L .BAY group had no significant difference from the control group. The expression of TNF-α mRNA was increased by P. endodontalis LPS treatment in osteoblast. TNF-α may induce the expression of M-CSF in MC3T3-E1 cells through the signaling of NF-κB. It suggests that TNF-α affect osteoblasts through autocrine way for bone destruction in chronic apical periodontitis induced by P.e-LPS.

Transcriptional Repression of ATF4 Gene by CCAAT/Enhancer-binding Protein β (C/EBPβ) Differentially Regulates Integrated Stress Response*

PubMed Central

Dey, Souvik; Savant, Sudha; Teske, Brian F.; Hatzoglou, Maria; Calkhoven, Cornelis F.; Wek, Ronald C.

2012-01-01

Different environmental stresses induce the phosphorylation of eIF2 (eIF2∼P), repressing global protein synthesis coincident with preferential translation of ATF4. ATF4 is a transcriptional activator of genes involved in metabolism and nutrient uptake, antioxidation, and regulation of apoptosis. Because ATF4 is a common downstream target that integrates signaling from different eIF2 kinases and their respective stress signals, the eIF2∼P/ATF4 pathway is collectively referred to as the integrated stress response. Although eIF2∼P elicits translational control in response to many different stresses, there are selected stresses, such as exposure to UV irradiation, that do not increase ATF4 expression despite robust eIF2∼P. The rationale for this discordant induction of ATF4 expression and eIF2∼P in response to UV irradiation is that transcription of ATF4 is repressed, and therefore ATF4 mRNA is not available for preferential translation. In this study, we show that C/EBPβ is a transcriptional repressor of ATF4 during UV stress. C/EBPβ binds to critical elements in the ATF4 promoter, resulting in its transcriptional repression. Expression of C/EBPβ increases in response to UV stress, and the liver-enriched inhibitory protein (LIP) isoform of C/EBPβ, but not the liver-enriched activating protein (LAP) version, represses ATF4 transcription. Loss of the liver-enriched inhibitory protein isoform results in increased ATF4 mRNA levels in response to UV irradiation and subsequent recovery of ATF4 translation, leading to enhanced expression of its target genes. Together these results illustrate how eIF2∼P and translational control combined with transcription factors regulated by alternative signaling pathways can direct programs of gene expression that are specifically tailored to each environmental stress. PMID:22556424

Chemokine-like factor 1 (CLFK1) is over-expressed in patients with atopic dermatitis.

PubMed

Yang, Gao-Yun; Chen, Xue; Sun, Ya-Chun; Ma, Chen-Li; Qian, Ge

2013-01-01

Human chemokine-like factor 1 (CKLF1), a recently discovered chemokine, has a broad spectrum of biological functions in immune-mediated diseases. It is highly expressed on Th2 lymphocytes and is a functional ligand for human CCR4. CKLF1 has a major role in the recruitment and activation of leucocytes, which plays an important role in the pathogenesis of allergic diseases. The present study was designed to determine the expression of CKLF1 in skin and serum in patients with atopic dermatitis (AD). The CKLF1 protein expression in skin lesion was analyzed by immunohistochemistry and ELISA. The mRNA expression of CKLF1 in skin lesion was detected by Real-time PCR. The serum levels of CKLF1, IgE, eotaxin, IL-4, IL-5, and IL-13 were measured by ELISA. Histopathological changes in the skin of AD patients showed local inflammation with epidermal thickening and significant inflammatory cellular infiltration. Immunohistochemistry results demonstrated that CKLF1-staining positive cells were located in the epidermal and dermis, and that the CKLF1 expression in AD patients was significantly higher than that in normal control. The CKLF1 mRNA expression in AD patients was significantly higher than that in healthy controls. Serum CKLF1 and IgE levels were significantly increased in AD patients, as were the serum levels of IL-4, IL-5, IL-13 and eotaxin. Both CKLF1 protien and mRNA levels are overexpressed in the skin lesion of AD patients, along with an increase in serum CKLF1 level, indicating that CKLF1 may play an important role in the development of atopic dermatitis.

Duplex unwinding and ATPase activities of the DEAD-box helicase eIF4A are coupled by eIF4G and eIF4B

PubMed Central

Özeş, Ali R.; Feoktistova, Kateryna; Avanzino, Brian C.; Fraser, Christopher S.

2011-01-01

Eukaryotic initiation factor 4A (eIF4A) is a DEAD-box helicase that stimulates translation initiation by unwinding mRNA secondary structure. The accessory proteins, eIF4G, eIF4B, and eIF4H enhance the duplex unwinding activity of eIF4A, but the extent to which they modulate eIF4A activity is poorly understood. Here, we use real time fluorescence assays to determine the kinetic parameters of duplex unwinding and ATP hydrolysis by these initiation factors. To ensure efficient duplex unwinding, eIF4B and eIF4G cooperatively activate the duplex unwinding activity of eIF4A. Our data reveal that eIF4H is much less efficient at stimulating eIF4A unwinding activity than eIF4B, implying that eIF4H is not able to completely substitute for eIF4B in duplex unwinding. By monitoring unwinding and ATPase assays using identical conditions, we demonstrate that eIF4B couples the ATP hydrolysis cycle of eIF4A with strand separation, thereby minimizing non-productive unwinding events. Using duplex substrates with altered GC contents, but with similar predicted thermal stabilities, we further show that the rate of formation of productive unwinding complexes is strongly influenced by the local stability per base pair in addition to the stability of the entire duplex. This finding explains how a change in the GC content of a hairpin while maintaining overall predicted thermal stability is able to influence translation initiation. PMID:21840318

[Changes of FoxP3, CD4(+)CD25(+) regulatory T cells, TLR2 and TLR9 in children with infectious mononucleosis].

PubMed

Wang, Qiang; Wang, Zuo-Feng; Cao, Mei; Wang, Zhi-Ying

2013-04-01

The aim of this study was to investigate the effects of TLR2, TLR9, CD4(+)CD25(+) regulatory T cells (Treg) and transcription factor FoxP3 in the pathogenesis of children with infectious mononucleosis (IM). Thirty-five acute IM patients admitted in our hospital from April 2010 to January 2011 were enrolled in this study. Thirty-five healthy subjects were taken as control. The thirty-five patients before treatment were considered as patients in acute stage, after treatment and without clinical symptom they were thought as patients in recovery stage. The expression levels of TLR2, TLR9 and FoxP3 mRNA were detected by real time PCR using SYBR Green I. The expression of T lymphocyte subset CD4(+)CD25(+) in peripheral blood mononuclear cells was detected by flow cytometry. The results showed that the relative levels of TLR2 mRNA (4.03 ± 0.56), TLR9 mRNA (8.88 ± 1.56) in peripheral blood mononuclear cells of IM patients in acute stage were significantly higher than those of the controls [TLR2 mRNA (2.22 ± 0.57), TLR9 mRNA (3.63 ± 1.30)] and IM patients in recovery stage [TLR2 mRNA (2.76 ± 0.83), TLR9 mRNA (5.34 ± 1.60)] (P < 0.01). The result of CD4(+)CD25(+) (2.38 ± 1.32%) and relative level of FoxP3 mRNA(2.82 ± 0.90) in peripheral blood mononuclear cells of IM patients in acute stage were lower than those of the control [CD4(+)CD25(+) (7.85 ± 1.97%), FoxP3 mRNA (4.65 ± 1.23) ] (P < 0.01). There was no significant difference in CD4(+)CD25(+) (6.81 ± 1.84%), FoxP3 mRNA(4.11 ± 1.37) levels between IM patients in recovery stage and the controls (P > 0.05). It is concluded that the expression of CD4(+)CD25(+)regulatory T cells is reduced, and its special transcription factor FoxP3 mRNA is down-regulated, but expression levels of TLR2 mRNA, TLR9 mRNA are up-regulated in IM patients of acute stage.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murooka, Thomas T.; Rahbar, Ramtin; Department of Immunology, University of Toronto, Ont.

The proliferative capacity of cancer cells is regulated by factors intrinsic to cancer cells and by secreted factors in the microenvironment. Here, we investigated the proto-oncogenic potential of the chemokine receptor, CCR5, in MCF-7 breast cancer cell lines. At physiological levels, CCL5, a ligand for CCR5, enhanced MCF-7.CCR5 proliferation. Treatment with the mTOR inhibitor, rapamycin, inhibited this CCL5-inducible proliferation. Because mTOR directly modulates mRNA translation, we investigated whether CCL5 activation of CCR5 leads to increased translation. CCL5 induced the formation of the eIF4F translation initiation complex through an mTOR-dependent process. Indeed, CCL5 initiated mRNA translation, shown by an increase inmore » high-molecular-weight polysomes. Specifically, we show that CCL5 mediated a rapid up-regulation of protein expression for cyclin D1, c-Myc and Dad-1, without affecting their mRNA levels. Taken together, we describe a mechanism by which CCL5 influences translation of rapamycin-sensitive mRNAs, thereby providing CCR5-positive breast cancer cells with a proliferative advantage.« less

Brd4 modulates the innate immune response through Mnk2-eIF4E pathway-dependent translational control of IκBα.

PubMed

Bao, Yan; Wu, Xuewei; Chen, Jinjing; Hu, Xiangming; Zeng, Fuxing; Cheng, Jianjun; Jin, Hong; Lin, Xin; Chen, Lin-Feng

2017-05-16

Bromodomain-containing factor Brd4 has emerged as an important transcriptional regulator of NF-κB-dependent inflammatory gene expression. However, the in vivo physiological function of Brd4 in the inflammatory response remains poorly defined. We now demonstrate that mice deficient for Brd4 in myeloid-lineage cells are resistant to LPS-induced sepsis but are more susceptible to bacterial infection. Gene-expression microarray analysis of bone marrow-derived macrophages (BMDMs) reveals that deletion of Brd4 decreases the expression of a significant amount of LPS-induced inflammatory genes while reversing the expression of a small subset of LPS-suppressed genes, including MAP kinase-interacting serine/threonine-protein kinase 2 ( Mknk2 ). Brd4 -deficient BMDMs display enhanced Mnk2 expression and the corresponding eukaryotic translation initiation factor 4E (eIF4E) activation after LPS stimulation, leading to an increased translation of IκBα mRNA in polysomes. The enhanced newly synthesized IκBα reduced the binding of NF-κB to the promoters of inflammatory genes, resulting in reduced inflammatory gene expression and cytokine production. By modulating the translation of IκBα via the Mnk2-eIF4E pathway, Brd4 provides an additional layer of control for NF-κB-dependent inflammatory gene expression and inflammatory response.

Krüppel homolog 1 and E93 mediate Juvenile hormone regulation of metamorphosis in the common bed bug, Cimex lectularius.

PubMed

Gujar, Hemant; Palli, Subba Reddy

2016-05-17

The common bed bug is an obligate hematophagous parasite of humans. We studied the regulation of molting and metamorphosis in bed bugs with a goal to identify key players involved. qRT-PCR studies on the expression of genes known to be involved in molting and metamorphosis showed high levels of Krüppel homolog 1 [Kr-h1, a transcription factor that plays key roles in juvenile hormone (JH) action] mRNA in the penultimate nymphal stage (N4). However, low levels of Kr-h1 mRNA were detected in the fifth and last nymphal stage (N5). Knockdown of Kr-h1 in N4 resulted in a precocious development of adult structures. Kr-h1 maintains the immature stage by suppressing E93 (early ecdysone response gene) in N4. E93 expression increases during the N5 in the absence of Kr-h1 and promotes the development of adult structures. Knockdown of E93 in N5 results in the formation of supernumerary nymphs. The role of JH in the suppression of adult structures through interaction with Kr-h1 and E93 was also studied by the topical application of JH analog, methoprene, to N5. Methoprene induced Kr-h1 and suppressed E93 and induced formation of the supernumerary nymph. These data show interactions between Kr-h1, E93 and JH in the regulation of metamorphosis in the bed bugs.

Krüppel homolog 1 and E93 mediate Juvenile hormone regulation of metamorphosis in the common bed bug, Cimex lectularius

PubMed Central

Gujar, Hemant; Palli, Subba Reddy

2016-01-01

The common bed bug is an obligate hematophagous parasite of humans. We studied the regulation of molting and metamorphosis in bed bugs with a goal to identify key players involved. qRT-PCR studies on the expression of genes known to be involved in molting and metamorphosis showed high levels of Krüppel homolog 1 [Kr-h1, a transcription factor that plays key roles in juvenile hormone (JH) action] mRNA in the penultimate nymphal stage (N4). However, low levels of Kr-h1 mRNA were detected in the fifth and last nymphal stage (N5). Knockdown of Kr-h1 in N4 resulted in a precocious development of adult structures. Kr-h1 maintains the immature stage by suppressing E93 (early ecdysone response gene) in N4. E93 expression increases during the N5 in the absence of Kr-h1 and promotes the development of adult structures. Knockdown of E93 in N5 results in the formation of supernumerary nymphs. The role of JH in the suppression of adult structures through interaction with Kr-h1 and E93 was also studied by the topical application of JH analog, methoprene, to N5. Methoprene induced Kr-h1 and suppressed E93 and induced formation of the supernumerary nymph. These data show interactions between Kr-h1, E93 and JH in the regulation of metamorphosis in the bed bugs. PMID:27185064

Inflammatory mediator mRNA expression by adenovirus E1A-transfected bronchial epithelial cells.

PubMed

Higashimoto, Yuji; Elliott, W Mark; Behzad, Ali R; Sedgwick, Edward G; Takei, Tatsuo; Hogg, James C; Hayashi, Shizu

2002-07-15

Lung tissue from patients with emphysema and airway obstruction carries excess adenoviral E1A DNA that is expressed as protein in airway surface epithelium and is associated with an increased inflammatory response. To examine mechanisms by which latent adenoviral infection might amplify the inflammatory process, we transfected primary human bronchial epithelial (HBE) cells from three separate patients undergoing lung resection so that they stably expressed adenovirus E1A. Lipopolysaccharide stimulation of the E1A-transfected HBE cells increased intercellular adhesion molecule-1 and interleukin-8 mRNA and protein expression compared with control cells from the same patient. It also induced greater intercellular adhesion molecule-1 promoter activity and greater nuclear factor-kappa B binding activity of nuclear extracts in E1A transfectants than controls. E1A-positive transfectants constitutively expressed transforming growth factor-beta 1 mRNA and protein, whereas this expression was either very low or not detected in control cells. We conclude that adenoviral E1A transfection transforms primary HBE cells and upregulates their production of mediators that are clinically relevant to the pathogenesis of chronic obstructive pulmonary disease.

Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

PubMed

Xie, Fang-Fei; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Hong; Wu, Jian; Guo, Yu-Fan; Zeng, Ke-Qin; Wang, Ming-Jun; Zhu, Xiao-Wei; Xia, Wei; Wang, Lan; He, Pei; Bing, Peng-Fei; Lu, Xin; Zhang, Yong-Hong; Lei, Shu-Feng

2018-01-01

DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1 ) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P < 0.05 and more than 5.96% genes presented very strong correlation (R T4  > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

The RNA recognition motif of eukaryotic translation initiation factor 3g (eIF3g) is required for resumption of scanning of posttermination ribosomes for reinitiation on GCN4 and together with eIF3i stimulates linear scanning.

PubMed

Cuchalová, Lucie; Kouba, Tomás; Herrmannová, Anna; Dányi, István; Chiu, Wen-Ling; Valásek, Leos

2010-10-01

Recent reports have begun unraveling the details of various roles of individual eukaryotic translation initiation factor 3 (eIF3) subunits in translation initiation. Here we describe functional characterization of two essential Saccharomyces cerevisiae eIF3 subunits, g/Tif35 and i/Tif34, previously suggested to be dispensable for formation of the 48S preinitiation complexes (PICs) in vitro. A triple-Ala substitution of conserved residues in the RRM of g/Tif35 (g/tif35-KLF) or a single-point mutation in the WD40 repeat 6 of i/Tif34 (i/tif34-Q258R) produces severe growth defects and decreases the rate of translation initiation in vivo without affecting the integrity of eIF3 and formation of the 43S PICs in vivo. Both mutations also diminish induction of GCN4 expression, which occurs upon starvation via reinitiation. Whereas g/tif35-KLF impedes resumption of scanning for downstream reinitiation by 40S ribosomes terminating at upstream open reading frame 1 (uORF1) in the GCN4 mRNA leader, i/tif34-Q258R prevents full GCN4 derepression by impairing the rate of scanning of posttermination 40S ribosomes moving downstream from uORF1. In addition, g/tif35-KLF reduces processivity of scanning through stable secondary structures, and g/Tif35 specifically interacts with Rps3 and Rps20 located near the ribosomal mRNA entry channel. Together these results implicate g/Tif35 and i/Tif34 in stimulation of linear scanning and, specifically in the case of g/Tif35, also in proper regulation of the GCN4 reinitiation mechanism.

Role of p70S6K1-mediated phosphorylation of eIF4B and PDCD4 proteins in the regulation of protein synthesis.

PubMed

Dennis, Michael D; Jefferson, Leonard S; Kimball, Scot R

2012-12-14

Modulation of mRNA binding to the 40 S ribosomal subunit during translation initiation controls not only global rates of protein synthesis but also regulates the pattern of protein expression by allowing for selective inclusion, or exclusion, of mRNAs encoding particular proteins from polysomes. The mRNA binding step is modulated by signaling through a protein kinase known as the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 directly phosphorylates the translational repressors eIF4E binding proteins (4E-BP) 1 and 2, releasing them from the mRNA cap binding protein eIF4E, thereby promoting assembly of the eIF4E·eIF4G complex. mTORC1 also phosphorylates the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1), which subsequently phosphorylates eIF4B, and programmed cell death 4 (PDCD4), which sequesters eIF4A from the eIF4E·eIF4G complex, resulting in repressed translation of mRNAs with highly structured 5'-untranslated regions. In the present study, we compared the role of the 4E-BPs in the regulation of global rates of protein synthesis to that of eIF4B and PDCD4. We found that maintenance of eIF4E interaction with eIF4G was not by itself sufficient to sustain global rates of protein synthesis in the absence of mTORC1 signaling to p70S6K1; phosphorylation of both eIF4B and PDCD4 was additionally required. We also found that the interaction of eIF4E with eIF4G was maintained in the liver of fasted rats as well as in serum-deprived mouse embryo fibroblasts lacking both 4E-BP1 and 4E-BP2, suggesting that the interaction of eIF4G with eIF4E is controlled primarily through the 4E-BPs.

Association of Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) and Thyroglobulin (TG) Genetic Variants with Autoimmune Hypothyroidism

PubMed Central

Patel, Hinal; Mansuri, Mohmmad Shoab; Singh, Mala; Begum, Rasheedunnisa; Shastri, Minal; Misra, Ambikanandan

2016-01-01

Autoimmune hypothyroidism is known to be caused by immune responses related to the thyroid gland and its immunological feature includes presence of autoimmune antibodies. Therefore the aim was to analyze presence of anti-TPO antibodies in hypothyroidism patients in Gujarat. Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) is one of the susceptibility genes for various autoimmune diseases. Hence, exon1 +49A/G and 3’UTR CT60A/G single nucleotide polymorphisms (SNPs) in CTLA4 and its mRNA expression levels were investigated in autoimmune hypothyroidism patients. Thyroglobulin (TG) is known to be associated with autoimmune thyroid disorders and thus exon 33 (E33) SNP in TG was investigated. We analyzed the presence of anti-TPO antibodies in the plasma samples of 84 hypothyroidism patients and 62 controls by ELISA. PCR-RFLP technique was used for genotyping of polymorphisms. sCTLA4 and flCTLA4 mRNA expression levels were assessed by real time PCR. 59.52% of hypothyroid patients had anti-TPO antibodies in their circulation. The genotype and allele frequencies differed significantly for +49A/G (p = 0.0004 for +49AG, p = 0.0019 for +49GG & p = 0.0004 for allele), CT60 (p = 0.0110 for CT60AG, p = 0.0005 for CT60GG & p<0.0001 for allele) and TG E33 (p = 0.0003 for E33TC p<0.0001 for E33CC& p<0.0001 for allele) SNPs between patients and controls. Patients had significantly decreased mRNA levels of both sCTLA4 (p = 0.0017) and flCTLA4 (p<0.0001) compared to controls. +49A/G and CT60 polymorphisms of CTLA4 were in moderate linkage disequilibrium. Logistic regression analysis indicated significant association of CT49A/G, CT60A/G and TG exon 33 polymorphisms with susceptibility to autoimmune hypothyroidism when adjusted for age and gender. Our results suggest +49A/G and CT60 polymorphism of CTLA4 and E33 polymorphism of TG may be genetic risk factors for autoimmune hypothyroidism susceptibility and down regulation of both forms of CTLA4 advocates the crucial role of CTLA4 in pathogenesis of autoimmune hypothyroidism. PMID:26963610

mRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma.

PubMed

Bache, Matthias; Rot, Swetlana; Keßler, Jacqueline; Güttler, Antje; Wichmann, Henri; Greither, Thomas; Wach, Sven; Taubert, Helge; Söling, Ariane; Bilkenroth, Udo; Kappler, Matthias; Vordermark, Dirk

2015-06-01

The roles of hypoxia-induced and stem cell-associated genes in the development of malignancy and tumour progression are well known. However, there are a limited number of studies analysing the impact of mRNA expression levels of hypoxia-induced and stem cell-associated genes in the tissues of brain tumours and glioblastoma patients. In this study, tumour tissues from patients with glioblastoma multiforme and tumour adjacent tissues were analysed. We investigated mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1) and osteopontin (OPN), and stem cell-associated genes survivin, epidermal growth factor receptor (EGFR), human telomerase reverse transcriptase (hTERT), Nanog and octamer binding transcription factor 4 (OCT4) using quantitative real-time polymerase chain reaction (qRT-PCR). Our data revealed higher mRNA expression levels of hypoxia-induced and stem cell-associated genes in tumour tissue than levels in the tumour adjacent tissues in patients with glioblastoma multiforme. A strong positive correlation between the mRNA expression levels of HIF-2α, CA9, VEGF, GLUT-1 and OPN suggests a specific hypoxia-associated profile of mRNA expression in glioblastoma multiforme. Additionally, the results indicate the role of stem-cell-related genes in tumour hypoxia. Kaplan-Maier analysis revealed that high mRNA expression levels of hypoxia-induced markers showed a trend towards shorter overall survival in glioblastoma patients (P=0.061). Our data suggest that mRNA expression levels of hypoxia-induced genes are important tumour markers in patients with glioblastoma multiforme.

Antiallergic effect of fisetin on IgE-mediated mast cell activation in vitro and on passive cutaneous anaphylaxis (PCA).

PubMed

Jo, Woo-Ri; Park, Hye-Jin

2017-10-01

Fisetin (3,7,3',4'-tetrahydroxyflavone), a naturally occurring bioactive flavonoid, has been shown to inhibit inflammation. However, little is known about the effect of fisetin on immunoglobulin E (IgE)-mediated allergic responses. In this study, the effect of fisetin on rat basophilic leukemia (RBL-2H3) cell-mediated allergic reactions was investigated. Fisetin inhibited β-hexosaminidase release and decreased the level of interleukin-4 and tumor necrosis factor-α mRNA in IgE/antigen (IgE/Ag)-stimulated RBL-2H3 cells. To elucidate the antiallergic mechanism, we examined the levels of signaling molecules responsible for degranulation and release of inflammatory cytokines. Fisetin decreased the levels of activated spleen tyrosine kinase, Gab2 proteins, linker of activated T cells, extracellular signal-related kinase 1/2 in the IgE/Ag-stimulated RBL2H3 cells, and NFκB and STAT3 proteins activated in the ear tissue of mice with passive cutaneous anaphylaxis (PCA). In addition, fisetin significantly lowered of FcɛRI α-subunit mRNA expression. Consistent with the cellular data, fisetin markedly suppressed RBL-2H3 cell-dependent PCA in IgE/Ag-sensitized mice. These results suggest that fisetin may have potential as a therapeutic agent for the treatment of allergic diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Conceptual Modeling in Systems Biology Fosters Empirical Findings: The mRNA Lifecycle

PubMed Central

Dori, Dov; Choder, Mordechai

2007-01-01

One of the main obstacles to understanding complex biological systems is the extent and rapid evolution of information, way beyond the capacity individuals to manage and comprehend. Current modeling approaches and tools lack adequate capacity to model concurrently structure and behavior of biological systems. Here we propose Object-Process Methodology (OPM), a holistic conceptual modeling paradigm, as a means to model both diagrammatically and textually biological systems formally and intuitively at any desired number of levels of detail. OPM combines objects, e.g., proteins, and processes, e.g., transcription, in a way that is simple and easily comprehensible to researchers and scholars. As a case in point, we modeled the yeast mRNA lifecycle. The mRNA lifecycle involves mRNA synthesis in the nucleus, mRNA transport to the cytoplasm, and its subsequent translation and degradation therein. Recent studies have identified specific cytoplasmic foci, termed processing bodies that contain large complexes of mRNAs and decay factors. Our OPM model of this cellular subsystem, presented here, led to the discovery of a new constituent of these complexes, the translation termination factor eRF3. Association of eRF3 with processing bodies is observed after a long-term starvation period. We suggest that OPM can eventually serve as a comprehensive evolvable model of the entire living cell system. The model would serve as a research and communication platform, highlighting unknown and uncertain aspects that can be addressed empirically and updated consequently while maintaining consistency. PMID:17849002

Effect of eccentric exercise on the healing process of injured patellar tendon in rats.

PubMed

Nakamura, Kenichi; Kitaoka, Katsuhiko; Tomita, Katsuro

2008-07-01

Earlier studies have reported positive results from eccentric training in patients with tendon disorders. The reasons for the beneficial clinical effects of eccentric training are not known. Vascularization followed by regression of the vasculature enhances the healing response of injured tendons. Eccentric exercise induces a more beneficial healing response than concentric exercise. Sixty rats with patellar tendon injuries were divided into three groups: nonexercise controls (group N; n = 20); concentric exercise group (group C; n = 20); eccentric exercise group (group E; n = 20). Each rat was taught to run uphill or downhill for 14 days. Patellar tendons were removed 1, 4, 7, 10, and 14 days following injury. Vascular endothelial growth factor (VEGF), angiopoietin-1, and angiopoietin-2 were measured by reverse transcription polymerase chain reaction. In group C, VEGF mRNA was increased 1 and 4 days following injury but was decreased on days 7, 10, and 14. In group E, VEGF mRNA was elevated only on day 1. In group N, VEGF mRNA remained at a low level throughout all 14 days. The angiopoietin-2/angiopoietin-1 ratio was higher for group C than for group E. In the presence of VEGF, angiopoietin-1 promotes vessel stability, whereas angiopoietin-2 has the opposite effect. Eccentric exercise contributes to stabilized angiogenesis during the early phase of tendon injury. Conversely, concentric exercise, which induces destabilized angiogenesis, leads to a delayed healing response. Initiation of eccentric exercise immediately after tendon injury may help improve healing by reducing vascularity.

Cis-Regulatory Variants Affect CHRNA5 mRNA Expression in Populations of African and European Ancestry

PubMed Central

Wang, Jen-Chyong; Spiegel, Noah; Bertelsen, Sarah; Le, Nhung; McKenna, Nicholas; Budde, John P.; Harari, Oscar; Kapoor, Manav; Brooks, Andrew; Hancock, Dana; Tischfield, Jay; Foroud, Tatiana; Bierut, Laura J.; Steinbach, Joe Henry; Edenberg, Howard J.; Traynor, Bryan J.; Goate, Alison M.

2013-01-01

Variants within the gene cluster encoding α3, α5, and β4 nicotinic receptor subunits are major risk factors for substance dependence. The strongest impact on risk is associated with variation in the CHRNA5 gene, where at least two mechanisms are at work: amino acid variation and altered mRNA expression levels. The risk allele of the non-synonymous variant (rs16969968; D398N) primarily occurs on the haplotype containing the low mRNA expression allele. In populations of European ancestry, there are approximately 50 highly correlated variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster and the adjacent PSMA4 gene region that are associated with CHRNA5 mRNA levels. It is not clear which of these variants contribute to the changes in CHRNA5 transcript level. Because populations of African ancestry have reduced linkage disequilibrium among variants spanning this gene cluster, eQTL mapping in subjects of African ancestry could potentially aid in defining the functional variants that affect CHRNA5 mRNA levels. We performed quantitative allele specific gene expression using frontal cortices derived from 49 subjects of African ancestry and 111 subjects of European ancestry. This method measures allele-specific transcript levels in the same individual, which eliminates other biological variation that occurs when comparing expression levels between different samples. This analysis confirmed that substance dependence associated variants have a direct cis-regulatory effect on CHRNA5 transcript levels in human frontal cortices of African and European ancestry and identified 10 highly correlated variants, located in a 9 kb region, that are potential functional variants modifying CHRNA5 mRNA expression levels. PMID:24303001

Brain region-specific effects of immobilization stress on cholinesterases in mice.

PubMed

Valuskova, Paulina; Farar, Vladimir; Janisova, Katerina; Ondicova, Katarina; Mravec, Boris; Kvetnansky, Richard; Myslivecek, Jaromir

2017-01-01

Brain acetylcholinesterase (AChE) variant AChE R expression increases with acute stress, and this persists for an extended period, although the timing, strain and laterality differences, have not been explored previously. Acute stress transiently increases acetylcholine release, which in turn may increase activity of cholinesterases. Also the AChE gene contains a glucocorticoid response element (GRE), and stress-inducible AChE transcription and activity changes are linked to increased glucocorticoid levels. Corticotropin-releasing hormone knockout (CRH-KO) mice have basal glucocorticoid levels similar to wild type (WT) mice, but much lower levels during stress. Hence we hypothesized that CRH is important for the cholinesterase stress responses, including butyrylcholinesterase (BChE). We used immobilization stress, acute (30 or 120 min) and repeated (120 min daily × 7) in 48 male mice (24 WT and 24 CRH-KO) and determined AChE R , AChE and BChE mRNA expression and AChE and BChE activities in left and right brain areas (as cholinergic signaling shows laterality). Immobilization decreased BChE mRNA expression (right amygdala, to 0.5, 0.3 and 0.4, × control respectively) and AChE R mRNA expression (to 0.5, 0.4 and 0.4, × control respectively). AChE mRNA expression increased (1.3, 1.4 and 1.8-fold, respectively) in the left striatum (Str). The AChE activity increased in left Str (after 30 min, 1.2-fold), decreased in right parietal cortex with repeated stress (to 0.5 × control). BChE activity decreased after 30 min in the right CA3 region (to 0.4 × control) but increased (3.8-fold) after 120 min in the left CA3 region. The pattern of changes in CRH-KO differed from that in WT mice.

Molecular Diagnosis for Nodal Metastasis in Endoscopically Managed Cervical Cancer: The Accuracy of the APTIMA Test to Detect High-risk Human Papillomavirus Messenger RNA in Sentinel Lymph Nodes.

PubMed

Köhler, Christhardt; Le, Xin; Dogan, Nasuh Utku; Pfiffer, Tatiana; Schneider, Achim; Marnitz, Simone; Bertolini, Julia; Favero, Giovanni

2016-01-01

To evaluate the feasibility and accuracy of a commercially available test to detect E6/E7 mRNA of 14 subtypes of high-risk HPVs (APTIMA; Hologic, Bedford, MA) in the sentinel lymph nodes of CC patients laparoscopically operated. Prospective pilot study. The study was conducted in the Department of Advanced Operative and Oncologic Gynecology, Asklepios Hospital, Hamburg, Germany. 54 women with HPV-positive CC submitted to laparoscopic sentinel node biopsy alone or sentinel node biopsy followed by systematic pelvic and/or para-aortic endoscopic lymphadenectomy. All removed sentinel lymph nodes (SLNs) underwent sample collection by cytobrush for the APTIMA assay before frozen section. Results obtained with the HPV mRNA test were compared with the definitive histopathological analysis of the SLNs and additional lymph nodes removed. A total of 125 SLNs (119 pelvic and 6 paraaortic) were excised with a mean number of 2.3 SLNs per patient. Final histopathologic analysis confirmed nodal metastases in 10 SLNs from 10 different patients (18%). All the histologically confirmed metastatic lymph nodes were also HPV E6/E7 mRNA positive, resulting in a sensitivity of 100%. Four histologically free sentinel nodes were positive for HPV E6/E7 mRNA, resulting in a specificity of 96.4%. The HPV E6/E7 mRNA assay in the SLNs of patients with CC is feasible and highly accurate. The detection of HPV mRNA in 4 women with negative SLNs might denote a shift from microscopic identification of metastasis to the molecular level. The prognostic value of this findings awaits further verification. Copyright © 2016. Published by Elsevier Inc.

Progesterone increases ex vivo testosterone production and decreases the expression of progestin receptors and steroidogenic enzymes in the fathead minnow (Pimephales promelas) ovary.

PubMed

Chishti, Yasmin Z; Feswick, April; Martyniuk, Christopher J

2014-04-01

Progesterone (P4) is a metabolic precursor for a number of steroids, including estrogens and androgens. P4 also has diverse roles within the vertebrate ovary that include oocyte growth and development. The objectives of this study were to measure the effects of P4 on testosterone (T) and 17β-estradiol (E2) production in the fathead minnow (FHM) ovary and on the mRNA abundance of transcripts involved in steroidogenesis and steroid receptor signaling. Ovary explants were treated with P4 (10(-6)M) for 6 and 12h. P4 administration significantly increased T production ∼3-fold at both 6 and 12h, whereas E2 production was not affected, consistent with the hypothesis that excess P4 is not converted to terminal estrogens in the mature ovary. Nuclear progesterone receptor mRNA was decreased at 6h and membrane progesterone receptor gamma-2 mRNA was significantly down-regulated at both 6 and 12h; however there was no change in membrane progesterone receptor alpha or beta mRNA levels. Androgen receptor (ar) and estrogen receptor 2a (esr2a) mRNA were significantly reduced at 6h with P4 treatment, but there was no change in esr2b mRNA at either time point. Transcripts for enzymes in the steroid pathway (star, hsd11b2) were significantly lower at 6h compared to controls, whereas cyp17a and cyp19a mRNA abundance did not change with treatments at either time point. These data suggest that P4 incubation can lead to increased T production in the FHM ovary without a concomitant change in E2, and that the membrane bound progestin receptors are differentially regulated by P4 in the teleost ovary. As environmental progestins have received increased attention due to their suspected role as endocrine disruptors, mechanistic data on the role of exogenous P4 treatments in the male and female gonad is warranted. Copyright © 2014 Elsevier Inc. All rights reserved.

The decreased growth performance and impaired immune function and structural integrity by dietary iron deficiency or excess are associated with TOR, NF-κB, p38MAPK, Nrf2 and MLCK signaling in head kidney, spleen and skin of grass carp (Ctenopharyngodon idella).

PubMed

Guo, Yan-Lin; Jiang, Wei-Dan; Wu, Pei; Liu, Yang; Zhou, Xiao-Qiu; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Feng, Lin

2017-06-01

This study was conducted to investigate the effects of dietary iron on the growth, and immune function and structural integrity in head kidney, spleen and skin as well as the underlying signaling of young grass carp (Ctenopharyngodon idella). Total 630 grass carp (242.32 ± 0.58 g) were fed diets containing graded levels of iron at 12.15 (basal diet), 35.38, 63.47, 86.43, 111.09, 136.37 mg/kg (diets 2-6 were added with ferrous fumarate) and 73.50 mg/kg (diet 7 was added with ferrous sulfate) diet for 60 days. Then, a challenge test was conducted by infection of Aeromonas hydrophila for 14 days. The results firstly showed that compared with optimal iron level, iron deficiency decreased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents and down-regulated the mRNA levels of antibacterial peptides, anti-inflammatory cytokines, inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1), whereas up-regulated the mRNA levels of pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) p65, IκB kinases β (IKKβ) and eIF4E-binding protein (4E-BP) in head kidney and spleen of young grass carp (P < 0.05), indicating that iron deficiency impaired immune function in head kidney and spleen of fish. Secondly, iron deficiency down-regulated the mRNA levels of B-cell lymphoma-2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), and inhibitor of apoptosis protein (IAP), and decreased activities and mRNA levels of antioxidant enzymes, down-regulated the mRNA levels of NF-E2-related factor 2 (Nrf2) and tight junction complexes, and up-regulated mRNA levels of cysteinyl aspartic acid-protease (caspase) -2, -3, -7, -8, -9, apoptotic protease activating factor-1 (Apaf-1), Bcl-2 associated X protein (Bax), Fas ligand (FasL), p38 mitogen-activated protein kinase (p38MAPK), Kelch-like ECH-associating protein (Keap) 1a, Keap1b, claudin-12 and myosin light chain kinase (MLCK), and increased malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) contents in head kidney and spleen of young grass carp (P < 0.05), indicating that iron deficiency impaired structural integrity in head kidney and spleen of fish. Thirdly, iron deficiency increased skin hemorrhage and lesion morbidity, and impaired immune function and structural integrity in skin of fish. Fourthly, iron excess decreased growth and impaired the immune function and structural integrity in head kidney, spleen and skin of fish. Besides, in young grass carp, based on PWG and ability against skin hemorrhage and lesion, the efficacy of ferrous fumarate relative to ferrous sulfate was 140.32% and 126.48%, respectively, and the iron requirements based on PWG, ability against skin hemorrhage and lesion, ACP activities and MDA contents in head kidney and spleen were estimated to be 75.65, 87.03, 79.74, 78.93, 83.17 and 82.14 mg/kg diet (based on ferrous fumarate), respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hippuristanol - A potent steroid inhibitor of eukaryotic initiation factor 4A

PubMed Central

Cencic, Regina; Pelletier, Jerry

2016-01-01

ABSTRACT Protein synthesis and its regulatory signaling pathways play essential roles in the initiation and maintenance of the cancer phenotype. Insight obtained over the last 3 decades on the mechanisms regulating translation in normal and transformed cells have revealed that perturbed control in cancer cells may offer an Achilles' heel for the development of novel anti-neoplastic agents. Several small molecule inhibitors have been identified and characterized that target translation initiation – more specifically, the rate-limiting step where ribosomes are recruited to mRNA templates. Among these, hippuristanol, a polyhydroxysteroid from the gorgonian Isis hippuris has been found to inhibit translation initiation by blocking the activity of eukaryotic initiation factor (eIF) 4A, an essential RNA helicase involved in this process. Herein, we highlight the biological properties of this compound, its potential development as an anti-cancer agent, and its use to validate eIF4A as an anti-neoplastic target. PMID:27335721

[Effect of CD-14 and toll like receptors on the expression of interleukin-6 induced by lipopolysaccharides of Porphyromonas endodontalis].

PubMed

Jia, Ge; Qiu, Li-Hong; Li, Ren; Lü, You; Yu, Ya-Qiong; Zhong, Ming

2011-09-01

To evaluate the effect of cluster of differentiation 14 (CD-14) and Toll like receptors (TLR) on the expression of interleukin-6 (IL-6) mRNA induced by Porphyromonas endodontalis (Pe) lipopolysaccharides (LPS). MC3T3-E1 cells were treated with 10 mg/L Pe-LPS for different hours, and the cells uninvolved by anything as the blank group. The expression of IL-6 was detected by reverse transcription polymerse chain reaction (RT-PCR) and enzyme-liked immunosorbent assay (ELISA). The expression of CD-14, TLR-2 and TLR-4 mRNA was observed at different time point (0 - 24 h) by RT-PCR. The protein of CD-14, TLR-2 and TLR-4 was analyzed with a flow cytometer. MC3T3-E1 cells were pretreated with anti-CD-14, anti-TLR-2 and anti-TLR-4 antibody for 1 h, and then cells were stimulated with 10 mg/L Pe-LPS for 6 h. The expression of IL-6 mRNA was examined by RT-PCR. Statistical analysis was performed using one-way ANOVA Dunnett-t test with SPSS 11.0 software package. The IL-6 mRNA and proteins increased significantly after treatment with Pe-LPS. When MC3T3-E1 cells treated by Pe-LPS for 6 h, the expression of proteins soared from (11.696 ± 0.672) ng/L to (36.534 ± 0.574) ng/L (P < 0.01); In the control group, the CD-14 and TLR-4 mRNA are ambly-expression, and the ratios of CD-14 and TLR-4 positive cells were (39.038 ± 3.131)% and (11.438 ± 0.385)% respectively in MC3T3-E1. After treatment by Pe-LPS, the expression of CD-14 and TLR-4 mRNA increased significantly, and the ratios of CD-14 and TLR-4 positive cells markedly increased to (62.407 ± 1.800)% and (21.367 ± 2.271)%. TLR-2 expression did not change apparently after Pe-LPS treatment. The expression of IL-6 mRNA was partly inhibited by anti-CD-14 or anti-TLR-4 antibody, but not by TLR-2. Pe-LPS can induce the expression of IL-6 in osteoblast MC3T3-E1 through CD-14 and TLR-4, but not TLR-2.

Luteal function during the estrous cycle in arginine-treated ewes fed different planes of nutrition.

PubMed

Bass, Casie S; Redmer, Dale A; Kaminski, Samantha L; Grazul-Bilska, Anna T

2017-03-01

Functions of corpus luteum (CL) are influenced by numerous factors including hormones, growth and angiogenic factors, nutritional plane and dietary supplements such as arginine (Arg), a semi-essential amino acid and precursor for proteins, polyamines and nitric oxide (NO). The aim of this study was to determine if Arg supplementation to ewes fed different planes of nutrition influences: (1) progesterone (P4) concentrations in serum and luteal tissue, (2) luteal vascularity, cell proliferation, endothelial NO synthase (eNOS) and receptor (R) soluble guanylate cyclase β protein and mRNA expression and (3) luteal mRNA expression for selected angiogenic factors during the estrous cycle. Ewes (n = 111) were categorized by weight and randomly assigned to one of three nutritional planes: maintenance control (C), overfed (2× C) and underfed (0.6× C) beginning 60 days prior to onset of estrus. After estrus synchronization, ewes from each nutritional plane were assigned randomly to one of two treatments: Arg or saline. Serum and CL were collected at the early, mid and late luteal phases. The results demonstrated that: (1) nutritional plane affected ovulation rates, luteal vascularity, cell proliferation and NOS3, GUCY1B3, vascular endothelial growth factor (VEGF) and VEGFR2 mRNA expression, (2) Arg affected luteal vascularity, cell proliferation and NOS3, GUCY1B3, VEGF and VEGFR2 mRNA expression and (3) luteal vascularity, cell proliferation and the VEGF and NO systems depend on the stage of the estrous cycle. These data indicate that plane of nutrition and/or Arg supplementation can alter vascularization and expression of selected angiogenic factors in luteal tissue during the estrous cycle in sheep. © 2017 Society for Reproduction and Fertility.

Gper and ESRs are expressed in rat round spermatids and mediate oestrogen-dependent rapid pathways modulating expression of cyclin B1 and Bax.

PubMed

Chimento, A; Sirianni, R; Zolea, F; Bois, C; Delalande, C; Andò, S; Maggiolini, M; Aquila, S; Carreau, S; Pezzi, V

2011-10-01

Spermatogenesis is a precisely controlled and timed process, comprising mitotic divisions of spermatogonia, meiotic divisions of spermatocytes, maturation and differentiation of haploid spermatids giving rise to spermatozoa. It is well known that the maintenance of spermatogenesis is controlled by gonadotrophins and testosterone, the effects of which are modulated by a complex network of locally produced factors, including oestrogens. However, it remains uncertain whether oestrogens are able to activate rapid signalling pathways directly in male germ cells. Classically, oestrogens act by binding to oestrogen receptors (ESRs) 1 and 2. Recently, it has been demonstrated that rapid oestrogen action can also be mediated by the G-protein-coupled oestrogen receptor 1 (Gper). The aim of the present study was to investigate ESRs and Gper expression in primary cultures of adult rat round spermatids (RS) and define if oestradiol (E2) is able to activate, through these receptors, pathways involved in the regulation of genes controlling rat RS apoptosis and/or maturation. In this study, we demonstrated that rat RS express ESR1, ESR2 and Gper. Short-time treatment of RS with E2, the selective Gper agonist G1 and the selective ESR1 and ERβ agonists, 4,4',4"-(4-propyl-[1H]pyrazole-1,3,5-triyl) trisphenol (PPT) and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), respectively, determined activation of Extra-cellular signal-regulated kinase (ERK1/2) through the involvement of epidermal growth factor receptor transactivation. In addition, we investigated the effects of ESRs and Gper pathway activation on factors involved in RS maturation. Expression of cyclin B1 mRNA was downregulated by E2, G1 and PPT, but not by DPN. A concomitant and inverse regulation of the pro-apoptotic factor Bax mRNA expression was observed in the same conditions, with DPN being the only one determining an increase in this factor expression. Collectively, these data demonstrate that E2 activates, through ESRs and Gper, pathways involved in the regulation of genes controlling rat RS apoptosis and differentiation such as cyclin B1 and Bax. © 2010 The Authors. International Journal of Andrology © 2011 European Academy of Andrology.

The eIF4F and eIFiso4F Complexes of Plants: An Evolutionary Perspective

PubMed Central

Patrick, Ryan M.; Browning, Karen S.

2012-01-01

Translation initiation in eukaryotes requires a number of initiation factors to recruit the assembled ribosome to mRNA. The eIF4F complex plays a key role in initiation and is a common target point for regulation of protein synthesis. Most work on the translation machinery of plants to date has focused on flowering plants, which have both the eIF4F complex (eIF4E and eIF4G) as well as the plant-specific eIFiso4F complex (eIFiso4E and eIFiso4G). The increasing availability of plant genome sequence data has made it possible to trace the evolutionary history of these two complexes in plants, leading to several interesting discoveries. eIFiso4G is conserved throughout plants, while eIFiso4E only appears with the evolution of flowering plants. The eIF4G N-terminus, which has been difficult to annotate, appears to be well conserved throughout the plant lineage and contains two motifs of unknown function. Comparison of eIFiso4G and eIF4G sequence data suggests conserved features unique to eIFiso4G and eIF4G proteins. These findings have answered some questions about the evolutionary history of the two eIF4F complexes of plants, while raising new ones. PMID:22611336

Effects of Intermittent Alcohol Exposure on Emotion and Cognition: A Potential Role for the Endogenous Cannabinoid System and Neuroinflammation

PubMed Central

Sanchez-Marin, Laura; Pavon, Francisco J.; Decara, Juan; Suarez, Juan; Gavito, Ana; Castilla-Ortega, Estela; Rodriguez de Fonseca, Fernando; Serrano, Antonia

2017-01-01

Intermittent alcohol exposure is a common pattern of adolescent alcohol use that can lead to binge drinking episodes. Alcohol use is known to modulate the endocannabinoid system (ECS), which is involved in neuronal communication, neuroplasticity, neuroinflammation and behavior. Adolescent male Wistar rats were exposed to 4-week intermittent alcohol intoxication (3 g/kg injections for 4 days/week) or saline (N = 12 per group). After alcohol deprivation, adult rats were assessed for emotionality and cognition and the gene expression of the ECS and other factors related to behavior and neuroinflammation was examined in the brain. Alcohol-exposed rats exhibited anxiogenic-like responses and impaired recognition memory but no motor alterations. There were brain region-dependent changes in the mRNA levels of the ECS and molecular signals compared with control rats. Thus, overall, alcohol-exposed rats expressed higher mRNA levels of endocannabinoid synthetic enzymes (N-acyl-phosphatidylethanolamine phospholipase D and diacylglycerol lipases) in the medial-prefrontal cortex (mPFC) but lower mRNA levels in the amygdala. Furthermore, we observed lower mRNA levels of receptors CB1 CB2 and peroxisome proliferator-activated receptor-α in the striatum. Regarding neuropeptide signaling, alcohol-exposed rats displayed lower mRNA levels of the neuropeptide Y signaling, particularly NPY receptor-2, in the amygdala and hippocampus and higher mRNA levels of corticotropin-releasing factor in the hippocampus. Additionally, we observed changes of several neuroinflammation-related factors. Whereas, the mRNA levels of toll-like receptor-4, tumor necrosis factor-α, cyclooxygenase-2 and glial fibrillary acidic protein were significantly increased in the mPFC, the mRNA levels of cyclooxygenase-2 and glial fibrillary acidic protein were decreased in the striatum and hippocampus. However, nuclear factor-κβ mRNA levels were lower in the mPFC and striatum and allograft inflammatory factor-1 levels were differentially expressed in the amygdala and hippocampus. In conclusion, rats exposed to adolescent intermittent alcohol displayed anxiety-like behavior and cognitive deficits in adulthood and these alterations were accompanied by brain region-dependent changes in the gene expression of the ECS and other signals associated with neuroinflammation and behavior. An intermittent adolescent alcohol exposure has behavioral and molecular consequences in the adult brain, which might be linked to higher vulnerability to addictive behaviors and psychopathologies. PMID:28223925

Control of transcription of the Bacillus subtilis spoIIIG gene, which codes for the forespore-specific transcription factor sigma G.

PubMed

Sun, D X; Cabrera-Martinez, R M; Setlow, P

1991-05-01

The Bacillus subtilis spoIIIG gene codes for a sigma factor termed sigma G which directs transcription of genes expressed only in the forespore compartment of the sporulating cell. Use of spoIIIG-lacZ transcriptional fusions showed that spoIIIG is cotranscribed with the spoIIG operon beginning at t0.5-1 of sporulation. However, this large mRNA produced little if any sigma G, and transferring the spoIIIG gene without the spoIIG promoter into the amyE locus resulted in a Spo+ phenotype. Significant translation of spoIIIG began at t2.5-3 with use of an mRNA whose 5' end is just upstream of the spoIIIG coding sequence. Synthesis of this spoIIIG-specific mRNA was not abolished by a deletion in spoIIIG itself. Similar results were obtained when a spoIIIG-lacZ translational fusion lacking the spoIIG promoter was integrated at the amyE locus. These data suggest that synthesis of sigma G is dependent neither on transcription from the spoIIG promoter nor on sigma G itself but can be due to another transcription factor. This transcription factor may be sigma F, the product of the spoIIAC locus, since a spoIIAC mutation blocked spoIIIG expression, and sequences upstream of the 5' end of the spoIIIG-specific mRNA agree well with the recognition sequence for sigma F. RNA polymerase containing sigma F (E sigma F) initiated transcription in vitro on a spoIIIG template at the 5' end found in vivo, as did E sigma G. However, E sigma F showed a greater than 20-fold preference for spoIIIG over a known sigma G-dependent gene compared with the activity of E sigma G.

Identification of the Transformational Properties and Transcriptional Targets of the Oncogenic SRY Transcription Factor SOX4

DTIC Science & Technology

2009-01-01

has also been implicated in tumorigenesis of multiple tumor types and has been shown by our lab to be upregulated in prostate cancer. However, the...mobility group (HMG) DNA-binding domain (DBD) related to the TCF/LEF family of transcription factors. Our lab has previously shown SOX4 mRNA and...protein to be overexpressed in prostate cancer, and this expression is correlated with increasing Gleason score. Other labs have shown SOX4 mRNA to be

Preprotachykinin A mRNA expression in the rat brain during development.

PubMed

Brené, S; Lindefors, N; Friedman, W J; Persson, H

1990-12-15

Expression of preprotachykinin A (PPT-A) mRNA was analyzed by northern blots using mRNA prepared from rat brain at 12 different developmental stages ranging from embryonic day 15 (E15) to adult. A single PPT-A mRNA of 1.3 kb was detected throughout development. PPT-A mRNA was detected as early as E15 and an approximately 3-fold increase occurred at birth. This amount remained until 3 weeks of age when the level increased, reaching a peak at 5 weeks of age. Adult amounts were approximately 3-fold higher than the levels at birth. The distribution of PPT-A mRNA-expressing cells in rat brain was studied by in situ hybridization on sections from embryonic day 20, postnatal days 4 and 7 as well as adult. Cells expressing PPT-A mRNA were detected in the forebrain at all 4 ages analyzed. However, the hybridization pattern and the labeling intensity varied in different brain regions during development. In cingulate cortex, intense labeling was seen in numerous cells at embryonic day 20 and postnatal days 4 and 7, whereas in the adult cingulate cortex only a few scattered labeled cells were observed. In frontoparietal cortex labeled cells were found from postnatal day 4 to adult, with the highest density of labeled cells at P7. Developmental differences in both the distribution of PPT-A mRNA-expressing cells and the level of PPT-A mRNA expression were also found in caudate-putamen, lateral hypothalamus and amygdala. Thus, our results show several changes in PPT-A mRNA expression during ontogeny, indicating a region and time-specific regulation of PPT-A mRNA expression during brain maturation.

4E-BP is a target of the GCN2–ATF4 pathway during Drosophila development and aging

PubMed Central

Park, Jung-Eun; Zeng, Xiaomei

2017-01-01

Reduced amino acid availability attenuates mRNA translation in cells and helps to extend lifespan in model organisms. The amino acid deprivation–activated kinase GCN2 mediates this response in part by phosphorylating eIF2α. In addition, the cap-dependent translational inhibitor 4E-BP is transcriptionally induced to extend lifespan in Drosophila melanogaster, but through an unclear mechanism. Here, we show that GCN2 and its downstream transcription factor, ATF4, mediate 4E-BP induction, and GCN2 is required for lifespan extension in response to dietary restriction of amino acids. The 4E-BP intron contains ATF4-binding sites that not only respond to stress but also show inherent ATF4 activity during normal development. Analysis of the newly synthesized proteome through metabolic labeling combined with click chemistry shows that certain stress-responsive proteins are resistant to inhibition by 4E-BP, and gcn2 mutant flies have reduced levels of stress-responsive protein synthesis. These results indicate that GCN2 and ATF4 are important regulators of 4E-BP transcription during normal development and aging. PMID:27979906

Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Zidong Donna; Klaassen, Curtis D., E-mail: cklaasse@kumc.edu

2014-01-01

Calorie restriction (CR) is one of the most effective anti-aging interventions in mammals. A modern theory suggests that aging results from a decline in detoxification capabilities and thus accumulation of damaged macromolecules. The present study aimed to determine how short-term CR alters mRNA profiles of genes that encode metabolism and detoxification machinery in the liver. Male C57BL/6 mice were fed CR (0, 15, 30, or 40%) diets for one month, followed by mRNA quantification of 98 xenobiotic processing genes (XPGs) in the liver, including 7 uptake transporters, 39 phase-I enzymes, 37 phase-II enzymes, 10 efflux transporters, and 5 transcription factors.more » In general, 15% CR did not alter mRNAs of most XPGs, whereas 30 and 40% CR altered over half of the XPGs (32 increased and 29 decreased). CR up-regulated some phase-I enzymes (fold increase), such as Cyp4a14 (12), Por (2.3), Nqo1 (1.4), Fmo2 (5.4), and Fmo3 (346), and numerous number of phase-II enzymes, such as Sult1a1 (1.2), Sult1d1 (2.0), Sult1e1 (33), Sult3a1 (2.2), Gsta4 (1.3), Gstm2 (1.3), Gstm3 (1.7), and Mgst3 (2.2). CR feminized the mRNA profiles of 32 XPGs in livers of male mice. For instance, CR decreased the male-predominantly expressed Oatp1a1 (97%) and increased the female-predominantly expressed Oatp1a4 (11). In conclusion, short-term CR alters the mRNA levels of over half of the 98 XPGs quantified in livers of male mice, and over half of these alterations appear to be due to feminization of the liver. - Highlights: • Utilized a graded CR model in male mice • The mRNA profiles of xenobiotic processing genes (XPGs) in liver were investigated. • CR up-regulates many phase-II enzymes. • CR tends to feminize the mRNA profiles of XPGs.« less

C/EBPβ-LAP*/LAP Expression Is Mediated by RSK/eIF4B-Dependent Signalling and Boosted by Increased Protein Stability in Models of Monocytic Differentiation

PubMed Central

Christmann, Martin; Friesenhagen, Judith; Westphal, Andreas; Pietsch, Daniel; Brand, Korbinian

2015-01-01

The transcription factor C/EBPβ plays a key role in monocytic differentiation and inflammation. Its small isoform LIP is associated with proliferation at early premonocytic developmental stages and regulated via mTOR-dependent signalling. During later stages of (pre)monocytic differentiation there is a considerable increase in the large C/EBPβ isoforms LAP*/LAP which inhibit proliferation thus supporting terminal differentiation. Here, we showed in different models of monocytic differentiation that this dramatic increase in the LAP*/LAP protein and LAP/LIP ratio was accompanied by an only modest/retarded mRNA increase suggesting an important role for (post)translational mechanisms. We found that LAP*/LAP formation was induced via MEK/RSK-dependent cascades, whereas mTOR/S6K1 were not involved. Remarkably, LAP*/LAP expression was dependent on phosphorylated eIF4B, an acceleratory protein of RNA helicase eIF4A. PKR inhibition reduced the expression of eIF4B and C/EBPβ in an eIF2α-independent manner. Furthermore, under our conditions a marked stabilisation of LAP*/LAP protein occurred, accompanied by reduced chymotrypsin-like proteasome/calpain activities and increased calpastatin levels. Our study elucidates new signalling pathways inducing LAP*/LAP expression and indicates new alternative PKR functions in monocytes. The switch from mTOR- to RSK-mediated signalling to orchestrate eIF4B-dependent LAP*/LAP translation, accompanied by increased protein stability but only small mRNA changes, may be a prototypical example for the regulation of protein expression during selected processes of differentiation/proliferation. PMID:26646662

Azuki bean (Vigna angularis) extract inhibits the development of experimentally induced atopic dermatitis-like skin lesions in NC/Nga mice.

PubMed

Collantes, Therese Marie; Rho, Mun-Chual; Kwon, Hyoung-Jun; Jung, Bock-Gie; Alfajaro, Mia Madel; Kim, Deok-Song; Kim, Hyun-Jeong; Hosmillo, Myra; Park, Jun-Gyu; Son, Kyu-Yeol; Park, Sang-Ik; Kang, Mun-Il; Park, Su-Jin; Lee, Seung Woong; Lee, Woo-Song; Cho, Kyoung-Oh

2012-06-01

The present study investigated the effects of azuki bean (Vigna angularis) extract (VAE) on the progress of atopic dermatitis (AD)-like skin lesions in NC/Nga mice induced by 1-chloro-2,4-dinitrobenzene. The efficacy of VAE in NC/Nga mice was determined by measuring gross and histological skin lesions, serum IgE levels, eosinophil ratio in peripheral leucocytes, and mRNA expression levels of interleukin (IL)-4, tumour necrosis factor (TNF)-α and interferon (IFN)-γ in splenocytes. Continuous ingestion of VAE inhibited the development of the AD-like skin lesions in a dose-dependent manner. In the VAE-treated mice, the numbers of mast cells in the skin, eosinophil ratio in peripheral leucocytes, relative mRNA expression of inflammatory cytokines in the spleen, and serum IgE levels were significantly reduced. Results suggest that VAE can inhibit the development of AD-like skin lesions in NC/Nga mice by regulating immune mediators and cells, and may be an effective alternative therapy for AD. Copyright © 2011 Elsevier Ltd. All rights reserved.

Multiple PAR and E4BP4 bZIP transcription factors in zebrafish: diverse spatial and temporal expression patterns.

PubMed

Ben-Moshe, Zohar; Vatine, Gad; Alon, Shahar; Tovin, Adi; Mracek, Philipp; Foulkes, Nicholas S; Gothilf, Yoav

2010-09-01

Circadian rhythms of physiology and behavior are generated by an autonomous circadian oscillator that is synchronized daily with the environment, mainly by light input. The PAR subfamily of transcriptional activators and the related E4BP4 repressor belonging to the basic leucine zipper (bZIP) family are clock-controlled genes that are suggested to mediate downstream circadian clock processes and to feedback onto the core oscillator. Here, the authors report the characterization of these genes in the zebrafish, an increasingly important model in the field of chronobiology. Five novel PAR and six novel e4bp4 zebrafish homolog genes were identified using bioinformatic tools and their coding sequences were cloned. Based on their evolutionary relationships, these genes were annotated as ztef2, zhlf1 and zhlf2, zdbp1 and zdbp2, and ze4bp4-1 to -6. The spatial and temporal mRNA expression pattern of each of these factors was characterized in zebrafish embryos in the context of a functional circadian clock and regulation by light. Nine of the factors exhibited augmented and rhythmic expression in the pineal gland, a central clock organ in zebrafish. Moreover, these genes were found to be regulated, to variable extents, by the circadian clock and/or by light. Differential expression patterns of multiple paralogs in zebrafish suggest multiple roles for these factors within the vertebrate circadian clock. This study, in the genetically accessible zebrafish model, lays the foundation for further research regarding the involvement and specific roles of PAR and E4BP4 transcription factors in the vertebrate circadian clock mechanism.

Activation of the growth hormone/insulin-like growth factor axis by treatment with 17 alpha-methyltestosterone and seawater rearing in the tilapia, Oreochromis mossambicus.

PubMed

Riley, Larry G; Richman, Nurney H; Hirano, Tetsuya; Gordon Grau, E

2002-07-01

Effects of 17 alpha-methyltestosterone (MT) treatment and environmental salinity on the growth hormone (GH)/insulin-like growth factor (IGF) axis were examined in the euryhaline tilapia, Oreochromis mossambicus. Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to 1 of 4 groups: FW, MT treatment in FW, SW, and MT treatment in seawater (SW). After 147 days, FW controls had the lowest levels of GH mRNA followed by FW fish treated with MT and SW control fish. Seawater fish fed with a diet containing MT, which grew the fastest, had significantly higher levels of GH mRNA than all the other groups. A significant correlation was observed between GH mRNA and the size of the individual fish. By contrast, plasma GH levels did not vary significantly among the groups. Pituitary GH mRNA levels, plasma IGF-I levels, and fish size varied in a correlated pattern, i.e., SW+MT>FW+MT=SW control>FW control. The tilapia pituitary produces two prolactins (PRLs), PRL(177) and PRL(188). Prolactin(177), but not PRL(188), exhibits growth-promoting actions in FW tilapia. Pituitary mRNA levels of both PRLs were significantly higher in fish reared in FW than those reared in SW. Treatment with MT significantly increased mRNA levels of both PRLs in FW, but had no effect on SW fish. No correlation was seen between plasma PRL levels and growth or between PRL mRNA levels and growth. These results indicate that SW rearing and MT treatment stimulate the GH/IGF-I axis, and suggest that pituitary GH mRNA at this stage of development is a better indicator of growth than plasma levels of GH and IGF-I.

Use of Cytology, E6/E7 mRNA, and p16INK4a-Ki-67 to Define the Management of Human Papillomavirus (HPV)-Positive Women in Cervical Cancer Screening.

PubMed

Gustinucci, Daniela; Giorgi Rossi, Paolo; Cesarini, Elena; Broccolini, Massimo; Bulletti, Simonetta; Carlani, Angela; D'angelo, Valentina; D'amico, Maria Rosaria; Di Dato, Eugenio; Galeazzi, Paola; Malaspina, Morena; Martinelli, Nadia; Spita, Nicoletta; Tintori, Beatrice; Giaimo, Maria Donata; Passamonti, Basilio

2016-01-01

We measured the accuracy of p16(INK4a)-Ki67 (CINtec PLUS, Roche, Mannheim, Germany), and E6/E7mRNA (types 16/18/31/33/45 NucliSENS easyQ, bioMérieux, Boxtel, The Netherlands) as triage test, alone and combined with cytology. Six thousand two hundred and seventy two women were recruited in a population-based screening using HPV DNA as primary test; 396 were positive and were tested for cytology and biomarkers. All tests were performed on the same sample. Cytology-positive women were referred to colposcopy; cytology-negative women were referred to one-year HPV re-testing. The endpoint was CIN2+ at baseline or follow up. Sensitivity was 77.6% (95% confidence interval (CI) 65.3-86.7) and 53.2% (95%CI: 40.3-65.4) for cytology at atypical squamous cells of undetermined significance (ASC-US) and high-grade threshold, and 87.6% (95%CI:75.7-93.6), and 80.8% (95%CI: 67.6-89.8) for p16INK4a-Ki67, and E6/E7mRNA, respectively. Colposcopy referral was 36% (95%CI: 31.2-40.9) and 11.2% (95%CI: 7.8-14.1) for cytology at ASC-US and high-grade threshold, respectively, and 36.0% (95%CI: 29.9-29.6), and 47.5% (95%CI: 32.5-42.4) for p16(INK4a)-Ki67, and E6/E7mRNA, respectively. Strategies referring high-grade cytology or biomarker positive women to colposcopy reached sensitivity close to 100%, with modest increase in colposcopy referral. The high sensitivity of combined strategies probably allows longer intervals in HPV-positive, triage-negative women. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Effects of Acupuncture on 1-chloro-2,4-dinitrochlorobenzene-induced Allergic Contact Dermatitis in Mice.

PubMed

Chen, Cuicui; Liu, Xiaozhu; Li, Yinfeng; Liang, Huankun; Li, Kangyan; Li, Jiali; Cheng, Chengwu; Liu, Xianpan; Zhong, Shuhai; Li, Laiqing; Wang, Yan

2017-08-01

Allergic contact dermatitis (ACD) is a chronic inflammatory skin disease. Topical corticosteroids are the first-line therapy for ACD despite their significant adverse effects. Acupuncture has been widely used in the treatment of various skin diseases, but its underlying mechanism remains unrevealed. In this study, we investigated the characteristics of acupuncture treatment based on effectiveness and mechanism. BALB/c mice received 1-chloro-2,4-dinitrobenzene (DNCB) application to build AD-like model. Results showed that acupuncture was an effective treatment method in inhibiting inflammatory conditions, serum IgE levels, and expression of proinflammatory cytokine Th2 (IL-4, IL-6), and Th2 (IL-1β, TNF-α) mRNA compared with DNCB treatment. Acupuncture treatment also inhibited nuclear factor-κB p65, phosphorylation of IκBα, and phosphorylation of occludin proteins expression. Furthermore, it could improve the expression of epidermal growth factor in both mRNA and protein levels. These results suggest that acupuncture, as an alternative therapy treatment for its no significant side effects, was effective in alleviating ACD by reducing proinflammatory cytokines and changing proteins' expression. Copyright © 2017. Published by Elsevier B.V.

Vibrational force alters mRNA expression in osteoblasts

NASA Technical Reports Server (NTRS)

Tjandrawinata, R. R.; Vincent, V. L.; Hughes-Fulford, M.

1997-01-01

Serum-deprived mouse osteoblastic (MC3T3E1) cells were subjected to a vibrational force modeled by NASA to simulate a space shuttle launch (7.83 G rms). The mRNA levels for eight genes were investigated to determine the effect of vibrational force on mRNA expression. The mRNA levels of two growth-related protooncogenes, c-fos and c-myc, were up-regulated significantly within 30 min after vibration, whereas those of osteocalcin as well as transforming growth factor-beta1 were decreased significantly within 3 h after vibration. No changes were detected in the levels of beta-actin, histone H4, or cytoplasmic phospholipase A2 after vibration. No basal levels of cyclooxygenase-2 expression were detected. In addition, the extracellular concentrations of prostaglandin E2 (PGE2), a potent autocrine/paracrine growth factor in bone, were not significantly altered after vibration most likely due to the serum deprivation state of the osteoblasts. In comparison with the gravitational launch profile, vibrational-induced changes in gene expression were greater both in magnitude and number of genes activated. Taken together, these data suggest that the changes in mRNA expression are due to a direct mechanical effect of the vibrational force on the osteoblast cells and not to changes in the local PGE2 concentrations. The finding that launch forces induce gene expression is of utmost importance since many of the biological experiments do not dampen vibrational loads on experimental samples. This lack of dampening of vibrational forces may partially explain why 1-G onboard controls sometimes do not reflect 1-G ground controls. These data may also suggest that scientists use extra ground controls that are exposed to launch forces, have these forces dampened on launched samples, or use facilities such as Biorack that provide an onboard 1-G centrufuge in order to control for space shuttle launch forces.

Transcriptome and Proteome Analyses of TNFAIP8 Knockdown Cancer Cells Reveal New Insights into Molecular Determinants of Cell Survival and Tumor Progression.

PubMed

Day, Timothy F; Mewani, Rajshree R; Starr, Joshua; Li, Xin; Chakravarty, Debyani; Ressom, Habtom; Zou, Xiaojun; Eidelman, Ofer; Pollard, Harvey B; Srivastava, Meera; Kasid, Usha N

2017-01-01

Tumor necrosis factor-α-inducible protein 8 (TNFAIP8) is the first discovered oncogenic and an anti-apoptotic member of a conserved TNFAIP8 or TIPE family of proteins. TNFAIP8 mRNA is induced by NF-kB, and overexpression of TNFAIP8 has been correlated with poor prognosis in many cancers. Downregulation of TNFAIP8 expression has been associated with decreased pulmonary colonization of human tumor cells, and enhanced sensitivities of tumor xenografts to radiation and docetaxel. Here we have investigated the effects of depletion of TNFAIP8 on the mRNA, microRNA and protein expression profiles in prostate and breast cancers and melanoma. Depending on the tumor cell type, knockdown of TNFAIP8 was found to be associated with increased mRNA expression of several antiproliferative and apoptotic genes (e.g., IL-24, FAT3, LPHN2, EPHA3) and fatty acid oxidation gene ACADL, and decreased mRNA levels of oncogenes (e.g., NFAT5, MALAT1, MET, FOXA1, KRAS, S100P, OSTF1) and glutamate transporter gene SLC1A1. TNFAIP8 knockdown cells also exhibited decreased expression of multiple onco-proteins (e.g., PIK3CA, SRC, EGFR, IL5, ABL1, GAP43), and increased expression of the orphan nuclear receptor NR4A1 and alpha 1 adaptin subunit of the adaptor-related protein complex 2 AP2 critical to clathrin-mediated endocytosis. TNFAIP8-centric molecules were found to be predominately implicated in the hypoxia-inducible factor-1α (HIF-1α) signaling pathway, and cancer and development signaling networks. Thus TNFAIP8 seems to regulate the cell survival and cancer progression processes in a multifaceted manner. Future validation of the molecules identified in this study is likely to lead to new subset of molecules and functional determinants of cancer cell survival and progression.

Critical Role of PPAR-α in Perfluorooctanoic Acid– and Perfluorodecanoic Acid–Induced Downregulation of Oatp Uptake Transporters in Mouse Livers

PubMed Central

Cheng, Xingguo; Klaassen, Curtis D.

2008-01-01

Perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) have been detected globally in wildlife and humans. Data from a gene array indicate that PFOA decreases organic anion transporting polypeptides (Oatps) in liver. Na+-taurocholate cotransporting polypeptide (Ntcp) and Oatp1a1, 1a4, and 1b2 are major transporters responsible for uptake of bile acids (BAs) and other organic compounds into liver. The purpose of the present study was to determine the effects of two perfluorinated fatty acids, PFOA and PFDA, on mRNA and protein expression of hepatic uptake transporters Oatps and Ntcp, and to determine the underlying regulatory mechanisms by using peroxisome proliferator-activated receptor alpha (PPAR-α), constitutive androstane receptor, pregnane-X receptor, NF-E2–related factor 2, and farnesoid X receptor-null mouse models. After 2 days following a single i.p. administration, PFOA did not alter serum BA concentrations, but PFDA increased serum BA concentrations 300%. Furthermore, PFOA decreased mRNA and protein expression of Oatp1a1, 1a4, and 1b2, but not Ntcp in mouse liver. In contrast, PFDA decreased mRNA and protein expression of all four transporters, and decreased the mRNA expression in a dose-dependent manner, with the decrease of Oatp1a4 occurring at lower doses than the other three transporters. Multiple mechanisms are likely involved in the down-regulation of mouse Oatps and Ntcp by PFDA. By using the various transcription factor-null mice, PPAR-α was shown to play a central role in the down-regulation of Oatp1a1, 1a4, 1b2, and Ntcp by PFDA. The current studies provide important insight into understanding the mechanisms by which PFDA regulate the expression of hepatic uptake transporters. In conclusion, PFOA and PFDA decrease mouse liver uptake transporters primarily via activation of PPAR-α. PMID:18703564

Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes

PubMed Central

Kowalska, Joanna; Wypijewska del Nogal, Anna; Darzynkiewicz, Zbigniew M.; Buck, Janina; Nicola, Corina; Kuhn, Andreas N.; Lukaszewicz, Maciej; Zuberek, Joanna; Strenkowska, Malwina; Ziemniak, Marcin; Maciejczyk, Maciej; Bojarska, Elzbieta; Rhoads, Robert E.; Darzynkiewicz, Edward; Sahin, Ugur; Jemielity, Jacek

2014-01-01

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5′,5′-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m27,3′-OGpppG. Higher expression of cancer antigens would make mRNAs containing m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2 favorable for anticancer immunization. PMID:25150148

Role of Akt/PKB and PFKFB isoenzymes in the control of glycolysis, cell proliferation and protein synthesis in mitogen-stimulated thymocytes.

PubMed

Houddane, Amina; Bultot, Laurent; Novellasdemunt, Laura; Johanns, Manuel; Gueuning, Marie-Agnès; Vertommen, Didier; Coulie, Pierre G; Bartrons, Ramon; Hue, Louis; Rider, Mark H

2017-06-01

Proliferating cells depend on glycolysis mainly to supply precursors for macromolecular synthesis. Fructose 2,6-bisphosphate (Fru-2,6-P 2 ) is the most potent positive allosteric effector of 6-phosphofructo-1-kinase (PFK-1), and hence of glycolysis. Mitogen stimulation of rat thymocytes with concanavalin A (ConA) led to time-dependent increases in lactate accumulation (6-fold), Fru-2,6-P 2 content (4-fold), 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase isoenzyme 3 and 4 (PFKFB3 and PFKFB4) protein levels (~2-fold and ~15-fold, respectively) and rates of cell proliferation (~40-fold) and protein synthesis (10-fold) after 68h of incubation compared with resting cells. After 54h of ConA stimulation, PFKFB3 mRNA levels were 45-fold higher than those of PFKFB4 mRNA. Although PFKFB3 could be phosphorylated at Ser461 by protein kinase B (PKB) in vitro leading to PFK-2 activation, PFKFB3 Ser461 phosphorylation was barely detectable in resting cells and only increased slightly in ConA-stimulated cells. On the other hand, PFKFB3 and PFKFB4 mRNA levels were decreased (90% and 70%, respectively) by exposure of ConA-stimulated cells to low doses of PKB inhibitor (MK-2206), suggesting control of expression of the two PFKFB isoenzymes by PKB. Incubation of thymocytes with ConA resulted in increased expression and phosphorylation of the translation factors eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) and ribosomal protein S6 (rpS6). Treatment of ConA-stimulated thymocytes with PFK-2 inhibitor (3PO) or MK-2206 led to significant decreases in Fru-2,6-P 2 content, medium lactate accumulation and rates of cell proliferation and protein synthesis. These data were confirmed by using siRNA knockdown of PFKFB3, PFKFB4 and PKB α/β in the more easily transfectable Jurkat E6-1 cell line. The findings suggest that increased PFKFB3 and PFKFB4 expression, but not increased PFKFB3 Ser461 phosphorylation, plays a role in increasing glycolysis in mitogen-stimulated thymocytes and implicate PKB in the upregulation of PFKFB3 and PFKFB4. The results also support a role for Fru-2,6-P 2 in coupling glycolysis to cell proliferation and protein synthesis in this model. Copyright © 2017 Elsevier Inc. All rights reserved.

An mRNA decapping mutant deficient in P body assembly limits mRNA stabilization in response to osmotic stress.

PubMed

Huch, Susanne; Nissan, Tracy

2017-03-14

Yeast is exposed to changing environmental conditions and must adapt its genetic program to provide a homeostatic intracellular environment. An important stress for yeast in the wild is high osmolarity. A key response to this stress is increased mRNA stability primarily by the inhibition of deadenylation. We previously demonstrated that mutations in decapping activators (edc3∆ lsm4∆C), which result in defects in P body assembly, can destabilize mRNA under unstressed conditions. We wished to examine whether mRNA would be destabilized in the edc3∆ lsm4∆C mutant as compared to the wild-type in response to osmotic stress, when P bodies are intense and numerous. Our results show that the edc3∆ lsm4∆C mutant limits the mRNA stability in response to osmotic stress, while the magnitude of stabilization was similar as compared to the wild-type. The reduced mRNA stability in the edc3∆ lsm4∆C mutant was correlated with a shorter PGK1 poly(A) tail. Similarly, the MFA2 mRNA was more rapidly deadenylated as well as significantly stabilized in the ccr4∆ deadenylation mutant in the edc3∆ lsm4∆C background. These results suggest a role for these decapping factors in stabilizing mRNA and may implicate P bodies as sites of reduced mRNA degradation.

The Krüppel-like factor 2 and Krüppel-like factor 4 genes interact to maintain endothelial integrity in mouse embryonic vasculogenesis

PubMed Central

2013-01-01

Background Krüppel-like Factor 2 (KLF2) plays an important role in vessel maturation during embryonic development. In adult mice, KLF2 regulates expression of the tight junction protein occludin, which may allow KLF2 to maintain vascular integrity. Adult tamoxifen-inducible Krüppel-like Factor 4 (KLF4) knockout mice have thickened arterial intima following vascular injury. The role of KLF4, and the possible overlapping functions of KLF2 and KLF4, in the developing vasculature are not well-studied. Results Endothelial breaks are observed in a major vessel, the primary head vein (PHV), in KLF2-/-KLF4-/- embryos at E9.5. KLF2-/-KLF4-/- embryos die by E10.5, which is earlier than either single knockout. Gross hemorrhaging of multiple vessels may be the cause of death. E9.5 KLF2-/-KLF4+/- embryos do not exhibit gross hemorrhaging, but cross-sections display disruptions of the endothelial cell layer of the PHV, and these embryos generally also die by E10.5. Electron micrographs confirm that there are gaps in the PHV endothelial layer in E9.5 KLF2-/-KLF4-/- embryos, and show that the endothelial cells are abnormally bulbous compared to KLF2-/- and wild-type (WT). The amount of endothelial Nitric Oxide Synthase (eNOS) mRNA, which encodes an endothelial regulator, is reduced by 10-fold in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos. VEGFR2, an eNOS inducer, and occludin, a tight junction protein, gene expression are also reduced in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos. Conclusions This study begins to define the roles of KLF2 and KLF4 in the embryonic development of blood vessels. It indicates that the two genes interact to maintain an intact endothelial layer. KLF2 and KLF4 positively regulate the eNOS, VEGFR2 and occludin genes. Down-regulation of these genes in KLF2-/-KLF4-/- embryos may result in the observed loss of vascular integrity. PMID:24261709

Estrogen promotes the brain metastatic colonization of triple negative breast cancer cells via an astrocyte-mediated paracrine mechanism.

PubMed

Sartorius, C A; Hanna, C T; Gril, B; Cruz, H; Serkova, N J; Huber, K M; Kabos, P; Schedin, T B; Borges, V F; Steeg, P S; Cittelly, D M

2016-06-02

Brain metastases (BM) are a devastating consequence of breast cancer. BM occur more frequently in patients with estrogen receptor-negative (ER-) breast cancer subtypes; HER2 overexpressing (HER2+) tumors and triple-negative (TN) (ER-, progesterone receptor-negative (PR-) and normal HER2) tumors. Young age is an independent risk factor for the development of BM, thus we speculated that higher circulating estrogens in young, pre-menopausal women could exert paracrine effects through the highly estrogen-responsive brain microenvironment. Using a TN experimental metastases model, we demonstrate that ovariectomy decreased the frequency of magnetic resonance imaging-detectable lesions by 56% as compared with estrogen supplementation, and that the combination of ovariectomy and letrozole further reduced the frequency of large lesions to 14.4% of the estrogen control. Human BM expressed 4.2-48.4% ER+ stromal area, particularly ER+ astrocytes. In vitro, E2-treated astrocytes increased proliferation, migration and invasion of 231BR-EGFP cells in an ER-dependent manner. E2 upregulated epidermal growth factor receptor (EGFR) ligands Egf, Ereg and Tgfa mRNA and protein levels in astrocytes, and activated EGFR in brain metastatic cells. Co-culture of 231BR-EGFP cells with E2-treated astrocytes led to the upregulation of the metastatic mediator S100 Calcium-binding protein A4 (S100A4) (1.78-fold, P<0.05). Exogenous EGF increased S100A4 mRNA levels in 231BR-EGFP cells (1.40±0.02-fold, P<0.01 compared with vehicle control) and an EGFR/HER2 inhibitor blocked this effect, suggesting that S100A4 is a downstream effector of EGFR activation. Short hairpin RNA-mediated S100A4 silencing in 231BR-EGFP cells decreased their migration and invasion in response to E2-CM, abolished their increased proliferation in co-cultures with E2-treated astrocytes and decreased brain metastatic colonization. Thus, S100A4 is one effector of the paracrine action of E2 in brain metastatic cells. These studies provide a novel mechanism by which estrogens, acting through ER+ astrocytes in the brain microenvironment, can promote BM of TN breast cancers, and suggests existing endocrine agents may provide some clinical benefit towards reducing and managing BM.

Factors of transforming growth factor beta signalling are co-regulated in human hepatocellular carcinoma.

PubMed

Longerich, Thomas; Breuhahn, Kai; Odenthal, Margarete; Petmecky, Katharina; Schirmacher, Peter

2004-12-01

Transforming growth factor beta (TGFbeta) is a central mitoinhibitory factor for epithelial cells, and alterations of TGFbeta signalling have been demonstrated in many different human cancers. We have analysed human hepatocellular carcinomas (HCCs) for potential pro-tumourigenic alterations in regard to expression of Smad4 and mutations and expression changes of the pro-oncogenic transcriptional co-repressors Ski and SnoN, as well as mRNA levels of matrix metalloproteinase-2 (MMP2), which is transcriptionally regulated by TGFbeta. Smad4 mRNA was detected in all HCCs; while, using immunohistology, loss of Smad4 expression was found in 10% of HCCs. Neither mutations in the transformation-relevant sequences nor significant pro-tumourigenic expression changes of the Ski and SnoN genes were detected. In HCC cell lines, expression of both genes was regulated, potentially involving phosphorylation. Ski showed a distinct nuclear speckled pattern, indicating recruitment to active transcription complexes. MMP2 mRNA levels were increased in 19% of HCCs, whereas MMP2 mRNA was not detectable in HCC cell lines, suggesting that MMP2 was derived only from tumour stroma cells. Transcript levels of Smad4, Ski, SnoN and MMP2 correlated well. These data argue against a significant role of Ski and SnoN in human hepatocarcinogenesis and suggest that, in the majority of HCCs, the analysed factors are co-regulated by an upstream mechanism, potentially by TGFbeta itself.

Phylogenetic analysis of eIF4E-family members

PubMed Central

Joshi, Bhavesh; Lee, Kibwe; Maeder, Dennis L; Jagus, Rosemary

2005-01-01

Background Translation initiation in eukaryotes involves the recruitment of mRNA to the ribosome which is controlled by the translation factor eIF4E. eIF4E binds to the 5'-m7Gppp cap-structure of mRNA. Three dimensional structures of eIF4Es bound to cap-analogues resemble 'cupped-hands' in which the cap-structure is sandwiched between two conserved Trp residues (Trp-56 and Trp-102 of H. sapiens eIF4E). A third conserved Trp residue (Trp-166 of H. sapiens eIF4E) recognizes the 7-methyl moiety of the cap-structure. Assessment of GenBank NR and dbEST databases reveals that many organisms encode a number of proteins with homology to eIF4E. Little is understood about the relationships of these structurally related proteins to each other. Results By combining sequence data deposited in the Genbank databases, we have identified sequences encoding 411 eIF4E-family members from 230 species. These sequences have been deposited into an internet-accessible database designed for sequence comparisons of eIF4E-family members. Most members can be grouped into one of three classes. Class I members carry Trp residues equivalent to Trp-43 and Trp-56 of H. sapiens eIF4E and appear to be present in all eukaryotes. Class II members, possess Trp→Tyr/Phe/Leu and Trp→Tyr/Phe substitutions relative to Trp-43 and Trp-56 of H. sapiens eIF4E, and can be identified in Metazoa, Viridiplantae, and Fungi. Class III members possess a Trp residue equivalent to Trp-43 of H. sapiens eIF4E but carry a Trp→Cys/Tyr substitution relative to Trp-56 of H. sapiens eIF4E, and can be identified in Coelomata and Cnidaria. Some eIF4E-family members from Protista show extension or compaction relative to prototypical eIF4E-family members. Conclusion The expansion of sequenced cDNAs and genomic DNAs from all eukaryotic kingdoms has revealed a variety of proteins related in structure to eIF4E. Evolutionarily it seems that a single early eIF4E gene has undergone multiple gene duplications generating multiple structural classes, such that it is no longer possible to predict function from the primary amino acid sequence of an eIF4E-family member. The variety of eIF4E-family members provides a source of alternatives on the eIF4E structural theme that will benefit structure/function analyses and therapeutic drug design. PMID:16191198

Phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) in mammary tissue of Holstein cows during the periparturient period is associated with mRNA abundance of antioxidant gene networks.

PubMed

Han, L Q; Zhou, Z; Ma, Y; Batistel, F; Osorio, J S; Loor, J J

2018-04-18

Changes in the production of reactive oxygen species in the mammary gland of dairy cows during the periparturient period could lead to oxidative stress and potentially impair mammary function. Phosphorylation of the transcription factor nuclear factor erythroid 2-like 2 (NFE2L2), also known as nuclear factor-E2-related factor 2, controls mRNA abundance of genes encoding antioxidant proteins and enzymes. The hypothesis was that NFE2L2 phosphorylation status and target gene mRNA abundance in the mammary gland of dairy cows is altered around parturition. Total NFE2L2 protein, phosphorylated protein (p-NFE2L2), and ratio of p-NFE2L2 to NFE2L2 along with mRNA abundance of 24 genes related to the NFE2L2 signaling pathway, apoptosis, and cell proliferation were measured in mammary tissue samples from Holstein cows at -30, 1, 15, and 30 d relative to parturition. Although total NFE2L2 protein abundance did not differ, p-NFE2L2 and p-NFE2L2-to-NFE2L2 ratio were greater after parturition. The upregulation of DNA damage inducible transcript 3 (DDIT3) postpartum indicated a localized oxidative stress state. Among genes evaluated, thioredoxin (TXN), glutathione peroxidase 1 (GPX1), and glutathione S-transferase mu 1 (GSTM1) had the highest (37.1, 15.1, and 4.8% of total mRNA measured, respectively) abundance. The mRNA abundance of various target genes with detoxifying enzymatic functions and free radical scavenging activities [glutamate-cysteine ligase catalytic subunit (GCLC); glutathione reductase (GSR); ferrochelatase (FECH); TXN; thioredoxin reductase 1 (TXNRD1); and NAD(P)H quinone dehydrogenase 1 (NQO1)] were consistently upregulated (linear effect of time) as parturition approached and lactation began. Among the transcription regulators, NFE2L2 had the highest mRNA abundance (7.3% of total mRNA measured). Abundance of NFE2L2 and other transcription factors [nuclear factor kappa B subunit 1 (NFKB1), retinoid X receptor α (RXRA), and mitogen-activated protein kinase 14 (MAPK14)] were upregulated (linear effect of time) from -30 d to 30 d relative to parturition. Overall, NFE2L2 phosphorylation and downstream signaling leading to postpartal upregulation of genes associated with oxidative stress and inflammation in the mammary gland seem to be key components of normal cellular function to maintain proper redox homeostasis. However, if the longitudinal increases in mRNA and protein abundance of these antioxidant mechanisms are a reflection of cellular oxidative stress, then the likelihood of protein and DNA damage would be greater and might be one factor compromising cell viability and potentially lactation persistency. The actual cues coordinating these molecular responses remain to be determined. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The expression of Apoc3 mRNA is regulated by HNF4α and COUP-TFII, but not acute retinoid treatments, in primary rat hepatocytes and hepatoma cells.

PubMed

Howell, Meredith; Li, Rui; Zhang, Rui; Li, Yang; Chen, Wei; Chen, Guoxun

2014-02-01

Vitamin A status regulates obesity development, hyperlipidemia, and hepatic lipogenic gene expression in Zucker fatty (ZF) rats. The development of hyperlipidemia in acne patients treated with retinoic acid (RA) has been attributed to the induction of apolipoprotein C-III expression. To understand the role of retinoids in the development of hyperlipidemia in ZF rats, the expression levels of several selected RA-responsive genes in the liver and isolated hepatocytes from Zucker lean (ZL) and ZF rats were compared using real-time PCR. The Rarb and Srebp-1c mRNA levels are higher in the liver and isolated hepatocytes from ZF than ZL rats. The Apoc3 mRNA level is only higher in the isolated hepatocytes from ZF than ZL rats. To determine whether dynamic RA production acutely regulates Apoc3 expression, its mRNA levels in response to retinoid treatments or adenovirus-mediated overexpression of hepatocyte nuclear factor 4 alpha (HNF4α) and chicken ovalbumin upstream-transcription factor II (COUP-TFII) were analyzed. Retinoid treatments for 2-6 h did not induce the expression of Apoc3 mRNA. The overexpression of HNF4α or COUP-TFII induced or inhibited Apoc3 expression, respectively. We conclude that short-term retinoid treatments could not induce Apoc3 mRNA expression, which is regulated by HNF4α and COUP-TFII in hepatocytes.

Coupled Evolution of Transcription and mRNA Degradation

PubMed Central

Dori-Bachash, Mally; Shema, Efrat; Tirosh, Itay

2011-01-01

mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor binding motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast. PMID:21811398

Stress Granule-Inducing Eukaryotic Translation Initiation Factor 4A Inhibitors Block Influenza A Virus Replication

PubMed Central

Slaine, Patrick D.; Kleer, Mariel; Smith, Nathan K.; Khaperskyy, Denys A.

2017-01-01

Eukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5′ untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) protein synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection resulted in SG formation, arrest of viral protein synthesis and failure to replicate the viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiviral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of viral protein synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates that the core host protein synthesis machinery can be targeted to block viral replication. PMID:29258238

Detection of active human papilloma virus-16 in head and neck cancers of Asian North Indian patients.

PubMed

Sannigrahi, M K; Singh, V; Sharma, R; Panda, N K; Radotra, B D; Khullar, M

2016-01-01

Head and neck cancers (HNC) are one of the most common cancers in India. Human papillomavirus (HPV) has been identified as an emerging risk factor for HNC. The present study was carried out to determine the active form of HPV-16 using a combination of PCR, viral load determination, HPV-16 E7 mRNA expression, p16, p53, and pRB immuno-histochemistry (IHC). A total of 226 HNC patients were enrolled in the present study. Sixty-seven (29.7%) of HNC cases were found to be HPV DNA positive. Thirty-two (14%) cases were HPV-16 DNA positive and 20 (9%) cases expressed HPV-16 E7 mRNA. HPV-16 mRNA/p16 positive cases had significantly increased viral load and integrated HPV-16 DNA. In summary, of total HNC patients, 6% cases were positive for both HPV-16 DNA and p16, and 5% were positive for both E7 mRNA and p16 IHC. We observed similar HPV-16 DNA/E7mRNA prevalence in oropharynx and oral cavity sites, however, oropharynx SCC had significantly higher viral load. Our results show low prevalence of active HPV-16 in North Indian HNC patients. HPV-16 E7 mRNA expression correlated with p16 nuclear positivity and increased viral load. Therefore, E7 mRNA expression may be used as a good surrogate indicator for active form of HPV-16 infection. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Oxytocin and tumor necrosis factor alpha stimulate expression of prostaglandin E2 synthase and secretion of prostaglandin E2 by luminal epithelial cells of the porcine endometrium during early pregnancy.

PubMed

Waclawik, Agnieszka; Blitek, Agnieszka; Ziecik, Adam J

2010-10-01

Oxytocin (OXT) and tumor necrosis factor α (TNF) have been implicated in the control of luteolysis by stimulating endometrial secretion of luteolytic prostaglandin F(2α) (PGF(2α)). Nevertheless, OXT concentration in porcine uterine lumen increases markedly on days 11-12 of pregnancy, and TNF is expressed in endometrium during pregnancy. The objective of the study was to determine the effect of OXT and TNF on expression of the enzymes involved in PG synthesis: PG-endoperoxide synthase 2 (PTGS2), PGE(2) synthase (mPGES-1) and PGF synthase, and PGE(2) receptor (PTGER2), as well as on PG secretion by endometrial luminal epithelial cells (LECs) on days 11-12 of the estrous cycle and pregnancy. LECs isolated from gilts on days 11-12 of the estrous cycle (n=8) and pregnancy (n=7) were treated with OXT (100  nmol/l) and TNF (0.6  nmol/l) for 24  h. OXT increased PTGS2 mRNA and mPGES-1 protein contents, as well as PGE(2) secretion but only on days 11-12 of pregnancy. TNF stimulated PTGS2 and mPGES-1 mRNA, as well as mPGES-1 protein expression and PGE(2) release on days 11-12 of pregnancy and the estrous cycle. In addition, expressions of PTGER2 and PTGER4 were determined in corpus luteum (CL). Abundance of PTGER2 mRNA and PTGER4 protein in CL was upregulated on day 14 of pregnancy versus day 14 of the estrous cycle. This study indicates that TNF and OXT regulate PGE(2) synthesis in LECs during early pregnancy. PGE(2) secreted by LECs, after reaching ovaries, could have a luteoprotective effect through luteal PTGER2 and PTGER4, or may directly promote uterine function and conceptus development.

Overcoming food allergy through acquired tolerance conferred by transfer of Tregs in a murine model.

PubMed

Yamashita, H; Takahashi, K; Tanaka, H; Nagai, H; Inagaki, N

2012-02-01

The number of food allergy patients is increasing. Some children outgrow their food allergies through tolerance, whereas others remain susceptible throughout their lives. We aimed to contribute to food allergy therapeutics by understanding induction of oral tolerance in a murine food allergy model. We modified an existing murine food allergy model by using ovalbumin (OVA) to induce oral tolerance, either by pretreating mice with OVA or by transferring mesenteric lymph node (MLN) cells or T cells derived from mice treated with OVA. Pretreatment with OVA prevented food allergy, with complete suppression of OVA-specific immunoglobulin (Ig)E and IgA antibody production and interleukin (IL)-4, IL-10, and IL-9 mRNA expression. The proportion of regulatory T cells (Tregs) in MLN cells and expression of transforming growth factor-β mRNA increased. In the transfer model, anaphylaxis secondary to OVA intake was suppressed by transfer of whole MLN cells and Tregs from OVA-treated mice. However, OVA-specific IgE and IgA expressions were partially attenuated by transfer of antigen-specific and nonspecific Tregs, but not by whole MLN cells from OVA-treated mice. In the Treg transfer model, IL-4 and IL-10 mRNA expression decreased, but IL-9 mRNA expression increased. We concluded that oral tolerance for food antigens is induced in two ways: (i) by initial exposure to antigen, or inherent tolerance, and (ii) by transfer of Tregs, or acquired tolerance. Because food allergies occur when inherent tolerance is absent, understanding of acquired tolerance is important for the development of therapies for food allergy. © 2011 John Wiley & Sons A/S.

Rescue of impaired sociability and anxiety-like behavior in adult cacna1c-deficient mice by pharmacologically targeting eIF2α

PubMed Central

Kabir, ZD; Che, A; Fischer, DK; Rice, RC; Rizzo, BK; Byrne, M; Glass, MJ; De Marco Garcia, NV; Rajadhyaksha, AM

2018-01-01

CACNA1C, encoding the Cav1.2 subunit of L-type Ca2+ channels, has emerged as one of the most prominent and highly replicable susceptibility genes for several neuropsychiatric disorders. Cav1.2 channels play a crucial role in calcium-mediated processes involved in brain development and neuronal function. Within the CACNA1C gene, disease-associated single-nucleotide polymorphisms have been associated with impaired social and cognitive processing and altered prefrontal cortical (PFC) structure and activity. These findings suggest that aberrant Cav1.2 signaling may contribute to neuropsychiatric-related disease symptoms via impaired PFC function. Here, we show that mice harboring loss of cacna1c in excitatory glutamatergic neurons of the forebrain (fbKO) that we have previously reported to exhibit anxiety-like behavior, displayed a social behavioral deficit and impaired learning and memory. Furthermore, focal knockdown of cacna1c in the adult PFC recapitulated the social deficit and elevated anxiety-like behavior, but not the deficits in learning and memory. Electrophysiological and molecular studies in the PFC of cacna1c fbKO mice revealed higher E/I ratio in layer 5 pyramidal neurons and lower general protein synthesis. This was concurrent with reduced activity of mTORC1 and its downstream mRNA translation initiation factors eIF4B and 4EBP1, as well as elevated phosphorylation of eIF2α, an inhibitor of mRNA translation. Remarkably, systemic treatment with ISRIB, a small molecule inhibitor that suppresses the effects of phosphorylated eIF2α on mRNA translation, was sufficient to reverse the social deficit and elevated anxiety-like behavior in adult cacna1c fbKO mice. ISRIB additionally normalized the lower protein synthesis and higher E/I ratio in the PFC. Thus this study identifies a novel Cav1.2 mechanism in neuropsychiatric-related endophenotypes and a potential future therapeutic target to explore. PMID:28584287

Identification of factors required for m6 A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI.

PubMed

Růžička, Kamil; Zhang, Mi; Campilho, Ana; Bodi, Zsuzsanna; Kashif, Muhammad; Saleh, Mária; Eeckhout, Dominique; El-Showk, Sedeer; Li, Hongying; Zhong, Silin; De Jaeger, Geert; Mongan, Nigel P; Hejátko, Jan; Helariutta, Ykä; Fray, Rupert G

2017-07-01

N6-adenosine methylation (m 6 A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m 6 A writer proteins in Arabidopsis thaliana. The components required for m 6 A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m 6 A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m 6 A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m 6 A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Differential regulation of membrane-associated mucins in the human ocular surface epithelium.

PubMed

Hori, Yuichi; Spurr-Michaud, Sandra; Russo, Cindy Leigh; Argüeso, Pablo; Gipson, Ilene K

2004-01-01

Membrane-associated mucins present in the apical cells of the ocular surface epithelium (MUC1, -4, and -16) are believed to contribute to the maintenance of a hydrated and wet-surfaced epithelial phenotype. Serum and retinoic acid (RA) have been used to treat drying ocular surface diseases. The goal of this study was to determine whether serum or RA regulates the production of membrane-associated mucins in human conjunctival epithelial cells. A telomerase-immortalized human conjunctival epithelial cell line (HCjE) was used. Cells were cultured in serum-free medium to confluence and then cultured with either 10% calf serum or with 100 nM RA for 0 to 72 hours. Conventional RT-PCR was used to determine the expression of retinoic acid receptors (RARs) and quantitative real-time PCR was used to investigate the mRNA expression of MUC1, -4, and -16. Protein levels were assayed by immunoblot analysis, using the antibodies HMFG-2, 1G8, or OC125, which are specific to MUC1, -4 and -16, respectively. To determine whether RA-associated MUC4 mRNA induction is a direct or indirect effect, HCjE cells were treated with RA and the protein synthesis inhibitor cycloheximide (1.0 microg/mL) for 12 hours. MUC1 and -16, but not -4, mRNAs were detectable in HCjE cells grown in serum-free medium. Real-time PCR revealed that MUC4 mRNA was significantly induced by serum 3 hours after its addition, and that MUC1 and MUC16 mRNA levels were significantly upregulated at 72 hours. Western blot analysis demonstrated that the MUC1, -4, and -16 proteins increased over time after addition of serum. Conventional RT-PCR analysis demonstrated that RAR-alpha and -gamma mRNA were expressed in native human conjunctival tissue as well as in the HCjE cells. Treatment with RA upregulated the expression of both MUC4 and -16 mRNA and protein, but MUC1 was unaffected. Because the protein synthesis inhibitor cycloheximide did not prevent the RA-associated induction of MUC4 mRNA, the action of RA on the MUC4 promoter may be direct. The membrane-associated mucins of the ocular surface epithelia, MUC1, -4, and -16, are differentially regulated by serum and RA in the telomerase-immortalized human conjunctival epithelial cell line. Serum derived from vessels in the conjunctiva may play an important role in mucin regulation in the ocular surface epithelia. These data also support the clinical efficacy of autologous serum and RA application in patients with ocular surface diseases. Furthermore, the data suggest that MUC4 and -16 are particularly important hydrophilic molecules involved in maintenance of a healthy ocular surface.

Comparison of Individual and Combined Effects of Four Endocrine Disruptors on Estrogen Receptor Beta Transcription in Cerebellar Cell Culture: The Modulatory Role of Estradiol and Triiodo-Thyronine

PubMed Central

Jocsak, Gergely; Kiss, David Sandor; Toth, Istvan; Goszleth, Greta; Bartha, Tibor; Frenyo, Laszlo V.; Horvath, Tamas L.; Zsarnovszky, Attila

2016-01-01

Background: Humans and animals are continuously exposed to a number of environmental substances that act as endocrine disruptors (EDs). While a growing body of evidence is available to prove their adverse health effects, very little is known about the consequences of simultaneous exposure to a combination of such chemicals; Methods: Here, we used an in vitro model to demonstrate how exposure to bisphenol A, zearalenone, arsenic, and 4-methylbenzylidene camphor, alone or in combination, affect estrogen receptor β (ERβ) mRNA expression in primary cerebellar cell cultures. Additionally, we also show the modulatory role of intrinsic biological factors, such as estradiol (E2), triiodo-thyronine (T3), and glial cells, as potential effect modulators; Results: Results show a wide diversity in ED effects on ERβ mRNA expression, and that the magnitude of these ED effects highly depends on the presence or absence of E2, T3, and glial cells; Conclusion: The observed potency of the EDs to influence ERβ mRNA expression, and the modulatory role of E2, T3, and the glia suggests that environmental ED effects may be masked as long as the hormonal milieu is physiological, but may tend to turn additive or superadditive in case of hormone deficiency. PMID:27338438

Ras-dva Is a Novel Pit-1- and Glucocorticoid-Regulated Gene in the Embryonic Anterior Pituitary Gland

PubMed Central

Ellestad, Laura E.

2013-01-01

Glucocorticoids play a role in functional differentiation of pituitary somatotrophs and lactotrophs during embryogenesis. Ras-dva was identified as a gene regulated by anterior neural fold protein-1/homeobox expressed in embryonic stem cells-1, a transcription factor known to be critical in pituitary development, and has an expression profile in the chicken embryonic pituitary gland that is consistent with in vivo regulation by glucocorticoids. The objective of this study was to characterize expression and regulation of ras-dva mRNA in the developing chicken anterior pituitary. Pituitary ras-dva mRNA levels increased during embryogenesis to a maximum on embryonic day (e) 18 and then decreased and remained low or undetectable after hatch. Ras-dva expression was highly enriched in the pituitary gland on e18 relative to other tissues examined. Glucocorticoid treatment of pituitary cells from mid- and late-stage embryos rapidly increased ras-dva mRNA, suggesting it may be a direct transcriptional target of glucocorticoids. A reporter construct driven by 4 kb of the chicken ras-dva 5′-flanking region, containing six putative pituitary-specific transcription factor-1 (Pit-1) binding sites and two potential glucocorticoid receptor (GR) binding sites, was highly activated in embryonic pituitary cells and up-regulated by corticosterone. Mutagenesis of the most proximal Pit-1 site decreased promoter activity in chicken e11 pituitary cells, indicating regulation of ras-dva by Pit-1. However, mutating putative GR binding sites did not substantially reduce induction of ras-dva promoter activity by corticosterone, suggesting additional DNA elements within the 5′-flanking region are responsible for glucocorticoid regulation. We have identified ras-dva as a glucocorticoid-regulated gene that is likely expressed in cells of the Pit-1 lineage within the developing anterior pituitary gland. PMID:23161868

Ras-dva is a novel Pit-1- and glucocorticoid-regulated gene in the embryonic anterior pituitary gland.

PubMed

Ellestad, Laura E; Porter, Tom E

2013-01-01

Glucocorticoids play a role in functional differentiation of pituitary somatotrophs and lactotrophs during embryogenesis. Ras-dva was identified as a gene regulated by anterior neural fold protein-1/homeobox expressed in embryonic stem cells-1, a transcription factor known to be critical in pituitary development, and has an expression profile in the chicken embryonic pituitary gland that is consistent with in vivo regulation by glucocorticoids. The objective of this study was to characterize expression and regulation of ras-dva mRNA in the developing chicken anterior pituitary. Pituitary ras-dva mRNA levels increased during embryogenesis to a maximum on embryonic day (e) 18 and then decreased and remained low or undetectable after hatch. Ras-dva expression was highly enriched in the pituitary gland on e18 relative to other tissues examined. Glucocorticoid treatment of pituitary cells from mid- and late-stage embryos rapidly increased ras-dva mRNA, suggesting it may be a direct transcriptional target of glucocorticoids. A reporter construct driven by 4 kb of the chicken ras-dva 5'-flanking region, containing six putative pituitary-specific transcription factor-1 (Pit-1) binding sites and two potential glucocorticoid receptor (GR) binding sites, was highly activated in embryonic pituitary cells and up-regulated by corticosterone. Mutagenesis of the most proximal Pit-1 site decreased promoter activity in chicken e11 pituitary cells, indicating regulation of ras-dva by Pit-1. However, mutating putative GR binding sites did not substantially reduce induction of ras-dva promoter activity by corticosterone, suggesting additional DNA elements within the 5'-flanking region are responsible for glucocorticoid regulation. We have identified ras-dva as a glucocorticoid-regulated gene that is likely expressed in cells of the Pit-1 lineage within the developing anterior pituitary gland.

TSHB mRNA is linked to cholesterol metabolism in adipose tissue.

PubMed

Moreno-Navarrete, José María; Moreno, María; Ortega, Francisco; Xifra, Gemma; Hong, Shangyu; Asara, John M; Serrano, José C E; Jové, Mariona; Pissios, Pavlos; Blüher, Matthias; Ricart, Wifredo; Portero-Otin, Manuel; Fernández-Real, José Manuel

2017-10-01

Subclinical hypothyroidism is known to be associated with increased serum cholesterol. Since thyroid-stimulating hormone (TSH) exerts an inductor effect on cholesterol biosynthesis, we aimed to investigate the relationship between TSH mRNA and cholesterol metabolism in human adipose tissue (AT). Cross-sectionally, AT TSH-β ( TSHB ) mRNA was evaluated in 4 independent cohorts in association with serum total and LDL cholesterol, and AT lipidomics. Longitudinally, the effects of statins and of diet and exercise on AT TSHB mRNA were also examined. The bidirectional relationship between cholesterol and TSHB were studied in isolated human adipocytes. TSHB mRNA was consistently detected in AT from euthyroid subjects, and positively associated with serum total- and LDL-cholesterol, and with AT-specific cholesterol metabolism-associated lipids [arachidonoyl cholesteryl ester, C8-dihydroceramide, N -stearoyl-d-sphingosine, and GlcCer(18:0, 24:1)]. Reduction of cholesterol with statins and with diet and exercise interventions led to decreased TSHB mRNA in human AT, whereas excess cholesterol up-regulated TSHB mRNA in human adipocytes. In addition, recombinant human TSH α/β administration resulted in increased HMGCR mRNA levels in human adipocytes. In mice, subcutaneous AT Tshb expression levels correlated directly with circulating cholesterol levels. In summary, current results provide novel evidence of TSHB as a paracrine factor that is modulated in parallel with cholesterol metabolism in human AT.-Moreno-Navarrete, J. M., Moreno, M., Ortega, F., Xifra, G., Hong, S., Asara, J. M., Serrano, J. C. E., Jové, M., Pissios, P., Blüher, M., Ricart, W., Portero-Otin, M., Fernández-Real, J. M. TSHB mRNA is linked to cholesterol metabolism in adipose tissue. © FASEB.

Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice.

PubMed

Fu, Zidong Donna; Klaassen, Curtis D

2014-01-01

Calorie restriction (CR) is one of the most effective anti-aging interventions in mammals. A modern theory suggests that aging results from a decline in detoxification capabilities and thus accumulation of damaged macromolecules. The present study aimed to determine how short-term CR alters mRNA profiles of genes that encode metabolism and detoxification machinery in the liver. Male C57BL/6 mice were fed CR (0, 15, 30, or 40%) diets for one month, followed by mRNA quantification of 98 xenobiotic processing genes (XPGs) in the liver, including 7 uptake transporters, 39 phase-I enzymes, 37 phase-II enzymes, 10 efflux transporters, and 5 transcription factors. In general, 15% CR did not alter mRNAs of most XPGs, whereas 30 and 40% CR altered over half of the XPGs (32 increased and 29 decreased). CR up-regulated some phase-I enzymes (fold increase), such as Cyp4a14 (12), Por (2.3), Nqo1 (1.4), Fmo2 (5.4), and Fmo3 (346), and numerous number of phase-II enzymes, such as Sult1a1 (1.2), Sult1d1 (2.0), Sult1e1 (33), Sult3a1 (2.2), Gsta4 (1.3), Gstm2 (1.3), Gstm3 (1.7), and Mgst3 (2.2). CR feminized the mRNA profiles of 32 XPGs in livers of male mice. For instance, CR decreased the male-predominantly expressed Oatp1a1 (97%) and increased the female-predominantly expressed Oatp1a4 (11). In conclusion, short-term CR alters the mRNA levels of over half of the 98 XPGs quantified in livers of male mice, and over half of these alterations appear to be due to feminization of the liver. Copyright © 2013 Elsevier Inc. All rights reserved.

Transcriptional and translational control of ornithine decarboxylase during Ras transformation.

PubMed Central

Shantz, Lisa M

2004-01-01

ODC (ornithine decarboxylase) activity is induced following ras activation. However, the Ras effector pathways responsible are unknown. These experiments used NIH-3T3 cells expressing partial-loss-of-function Ras mutants to activate selectively pathways downstream of Ras and examined the contribution of each pathway to ODC induction. Overexpression of Ras12V, a constitutively active mutant, resulted in ODC activities up to 20-fold higher than controls. Stable transfections of Ras partial-loss-of-function mutants and constitutively active forms of MEK (MAPK kinase) and Akt indicated that activation of more than one Ras effector pathway is necessary for the complete induction of ODC activity. The increase in ODC activity in Ras12V-transformed cells is not owing to a substantial change in ODC protein half-life, which increased by <2-fold. Northern-blot analysis and reporter assays suggested that the mechanism of ODC induction involves both a modest increase in the transcription of ODC mRNA and a much more considerable increase in the translation of mRNA into protein. ODC transcription was controlled through a pathway dependent on Raf/MEK/ERK (where ERK stands for extracellular-signal-regulated kinase) activation, whereas activation of the phosphoinositide 3-kinase and the Raf/MEK/ERK pathways were necessary for translational regulation of ODC. The increase in ODC synthesis was accompanied by changes in phosphorylation of eukaryotic initiation factor 4E and its binding protein 4E-BP1. Results show that the phosphoinositide 3-kinase pathway regulates phosphorylation of both proteins, whereas the Raf/MEK/ERK pathway affects only the eukaryotic initiation factor 4E phosphorylation. PMID:14519103

Covalent Chemical 5'-Functionalization of RNA with Diazo Reagents.

PubMed

Gampe, Christian M; Hollis-Symynkywicz, Micah; Zécri, Frédéric

2016-08-22

Functionalization of RNA at the 5'-terminus is important for analytical and therapeutic purposes. Currently, these RNAs are synthesized de novo starting with a chemically functionalized 5'-nucleotide, which is incorporated into RNA using chemical synthesis or biochemical techniques. Methods for direct chemical modification of native RNA would provide an attractive alternative but are currently underexplored. Herein, we report that diazo compounds can be used to selectively alkylate the 5'-phosphate of ribo(oligo)nucleotides to give RNA labelled through a native phosphate ester bond. We applied this method to functionalize oligonucleotides with biotin and an orthosteric inhibitor of the eukaryotic initiation factor 4E (eIF4E), an enzyme involved in mRNA recognition. The modified RNA binds to eIF4E, demonstrating the utility of this labelling technique to modulate biological activity of RNA. This method complements existing techniques and may be used to chemically introduce a broad range of functional handles at the 5'-end of RNA. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Distinct mRNAs for Translation*

PubMed Central

Timpano, Sara; Uniacke, James

2016-01-01

Translation initiation is a focal point of translational control and requires the binding of eIF4E to the 5′ cap of mRNA. Under conditions of extreme oxygen depletion (hypoxia), human cells repress eIF4E and switch to an alternative cap-dependent translation mediated by a homolog of eIF4E, eIF4E2. This homolog forms a complex with the oxygen-regulated hypoxia-inducible factor 2α and can escape translation repression. This complex mediates cap-dependent translation under cell culture conditions of 1% oxygen (to mimic tumor microenvironments), whereas eIF4E mediates cap-dependent translation at 21% oxygen (ambient air). However, emerging evidence suggests that culturing cells in ambient air, or “normoxia,” is far from physiological or “normal.” In fact, oxygen in human tissues ranges from 1–11% or “physioxia.” Here we show that two distinct modes of cap-dependent translation initiation are active during physioxia and act on separate pools of mRNAs. The oxygen-dependent activities of eIF4E and eIF4E2 are elucidated by observing their polysome association and the status of mammalian target of rapamycin complex 1 (eIF4E-dependent) or hypoxia-inducible factor 2α expression (eIF4E2-dependent). We have identified oxygen conditions where eIF4E is the dominant cap-binding protein (21% normoxia or standard cell culture conditions), where eIF4E2 is the dominant cap-binding protein (1% hypoxia or ischemic diseases and cancerous tumors), and where both cap-binding proteins act simultaneously to initiate the translation of distinct mRNAs (1–11% physioxia or during development and stem cell differentiation). These data suggest that the physioxic proteome is generated by initiating translation of mRNAs via two distinct but complementary cap-binding proteins. PMID:27002144

Depletion of mRNA export regulator DBP5/DDX19, GLE1 or IPPK that is a key enzyme for the production of IP6, resulting in differentially altered cytoplasmic mRNA expression and specific cell defect

PubMed Central

Okamura, Masumi; Yamanaka, Yasutaka; Shigemoto, Maki; Kitadani, Yuya; Kobayashi, Yuhko; Kambe, Taiho; Nagao, Masaya; Kobayashi, Issei; Okumura, Katsuzumi

2018-01-01

DBP5, also known as DDX19, GLE1 and inositol hexakisphosphate (IP6) function in messenger RNA (mRNA) export at the cytoplasmic surface of the nuclear pore complex in eukaryotic cells. DBP5 is a DEAD-box RNA helicase, and its activity is stimulated by interactions with GLE1 and IP6. In addition, these three factors also have unique role(s). To investigate how these factors influenced the cytoplasmic mRNA expression and cell phenotype change, we performed RNA microarray analysis to detect the effect and function of DBP5, GLE1 and IP6 on the cytoplasmic mRNA expression. The expression of some cytoplasmic mRNA subsets (e.g. cell cycle, DNA replication) was commonly suppressed by the knock-down of DBP5, GLE1 and IPPK (IP6 synthetic enzyme). The GLE1 knock-down selectively reduced the cytoplasmic mRNA expression required for mitotic progression, results in an abnormal spindle phenotype and caused the delay of mitotic process. Meanwhile, G1/S cell cycle arrest was observed in DBP5 and IPPK knock-down cells. Several factors that function in immune response were also down-regulated in DBP5 or IPPK knock-down cells. Thereby, IFNβ-1 mRNA transcription evoked by poly(I:C) treatment was suppressed. These results imply that DBP5, GLE1 and IP6 have a conserved and individual function in the cytoplasmic mRNA expression. Variations in phenotype are due to the difference in each function of DBP5, GLE1 and IPPK in intracellular mRNA metabolism. PMID:29746542

Smad4-Mediated Signaling Inhibits Intestinal Neoplasia by Inhibiting Expression of β-Catenin

PubMed Central

Freeman, Tanner J.; Smith, J. Joshua; Chen, Xi; Washington, M. Kay; Roland, Joseph T.; Means, Anna L.; Eschrich, Steven A.; Yeatman, Timothy J.; Deane, Natasha G.; Beauchamp, R. Daniel

2012-01-01

Background & Aims Mutational inactivation of APC is an early event in colorectal cancer (CRC) progression that affects the stability and increases the activity of β-catenin, a mediator of Wnt signaling. CRC progression also involves inactivation of signaling via transforming growth factor (TGF)β and bone morphenogenic protein (BMP), which are tumor suppressors. However, the interactions between these pathways are not clear. We investigated the effects of loss of the transcription factor Smad4 loss on levels of β-catenin mRNA and Wnt signaling. Methods We used microarray analysis to associate levels of Smad4 and β-catenin mRNA in colorectal tumor samples from 250 patients. We performed oligonucleotide-mediated knockdown of Smad4 in human embryonic kidney (HEK293T) and in HCT116 colon cancer cells and transgenically expressed Smad4 in SW480 colon cancer cells. We analyzed adenomas from (APCΔ1638/+) and (APCΔ1638/+)x(K19CreERT2Smad4lox/lox) mice using laser-capture microdissection. Results In human CRC samples, reduced levels of Smad4 correlated with increased levels of β-catenin mRNA. In Smad4-depleted cell lines, levels of β-catenin mRNA and Wnt signaling increased. Inhibition of BMP or depletion of Smad4 in HEK293T cells increased binding of RNA polymerase II to the β-catenin gene. Expression of Smad4 in SW480 cells reduced Wnt signaling and levels of β-catenin mRNA. In mice with heterozygous disruption of Apc(APCΔ1638/+), Smad4-deficient intestinal adenomas had increased levels of β-catenin mRNA and expression of Wnt target genes, compared with adenomas from APCΔ1638/+mice that expressed Smad4. Conclusions Transcription of β-catenin is inhibited by BMP signaling to Smad4. These findings provide important information about the interaction among TGF-β, BMP, and Wnt signaling pathways in CRC progression. PMID:22115830

Metabolomic profiles delineate the potential role of glycine in gold nanorod-induced disruption of mitochondria and blood-testis barrier factors in TM-4 cells

NASA Astrophysics Data System (ADS)

Xu, Bo; Chen, Minjian; Ji, Xiaoli; Mao, Zhilei; Zhang, Xuemei; Wang, Xinru; Xia, Yankai

2014-06-01

Gold nanorods (GNRs) are commonly used nanomaterials with potential harmful effects on male reproduction. However, the mechanism by which GNRs affect male reproduction remains largely undetermined. In this study, the metabolic changes in spermatocyte-derived cells GC-2 and Sertoli cell line TM-4 were analyzed after GNR treatment for 24 h. Metabolomic analysis revealed that glycine was highly decreased in TM-4 cells after GNR-10 nM treatment while there was no significant change in GC-2 cells. RT-PCR showed that the mRNA levels of glycine synthases in the mitochondrial pathway decreased after GNR treatment, while there was no significant difference in mRNA levels of glycine synthases in the cytoplasmic pathway. High content screening (HCS) showed that GNRs decreased membrane permeability and mitochondrial membrane potential of TM-4 cells, which was also confirmed by JC-1 staining. In addition, RT-PCR and Western blot indicated that the mRNA and protein levels of blood-testis barrier (BTB) factors (ZO-1, occludin, claudin-5, and connexin-43) in TM-4 cells were also disrupted by GNRs. After glycine was added into the medium, the GNR-induced harmful effects on mitochondria and BTB factors were recovered in TM-4 cells. Our results showed that even low doses of GNRs could induce significant toxic effects on mitochondria and BTB factors in TM-4 cells. Furthermore, we revealed that glycine was a potentially important metabolic intermediary for the changes of membrane permeability, mitochondrial membrane potential and BTB factors after GNR treatment in TM-4 cells.Gold nanorods (GNRs) are commonly used nanomaterials with potential harmful effects on male reproduction. However, the mechanism by which GNRs affect male reproduction remains largely undetermined. In this study, the metabolic changes in spermatocyte-derived cells GC-2 and Sertoli cell line TM-4 were analyzed after GNR treatment for 24 h. Metabolomic analysis revealed that glycine was highly decreased in TM-4 cells after GNR-10 nM treatment while there was no significant change in GC-2 cells. RT-PCR showed that the mRNA levels of glycine synthases in the mitochondrial pathway decreased after GNR treatment, while there was no significant difference in mRNA levels of glycine synthases in the cytoplasmic pathway. High content screening (HCS) showed that GNRs decreased membrane permeability and mitochondrial membrane potential of TM-4 cells, which was also confirmed by JC-1 staining. In addition, RT-PCR and Western blot indicated that the mRNA and protein levels of blood-testis barrier (BTB) factors (ZO-1, occludin, claudin-5, and connexin-43) in TM-4 cells were also disrupted by GNRs. After glycine was added into the medium, the GNR-induced harmful effects on mitochondria and BTB factors were recovered in TM-4 cells. Our results showed that even low doses of GNRs could induce significant toxic effects on mitochondria and BTB factors in TM-4 cells. Furthermore, we revealed that glycine was a potentially important metabolic intermediary for the changes of membrane permeability, mitochondrial membrane potential and BTB factors after GNR treatment in TM-4 cells. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr01035c

Decreased FOXP3 mRNA expression in children with atopic asthma and IgE-mediated food allergy.

PubMed

Krogulska, Aneta; Polakowska, Ewa; Wąsowska-Królikowska, Krystyna; Małachowska, Beata; Młynarski, Wojciech; Borowiec, Maciej

2015-11-01

The role of T regulatory lymphocytes has been investigated in various allergic diseases. However, the precise relation between the phenotype and severity of allergic diseases and the changes in FOXP3 mRNA expression are not fully understood. To compare the expression of FOXP3 mRNA in children with asthma with and without concomitant food allergy (FA) with healthy children and children with only FA. The study included 82 children: 15 with atopic asthma and IgE-dependent FA, 27 with atopic asthma without FA, 20 with IgE-dependent FA without asthma, and 20 healthy children without atopy. Reverse transcription was performed using a commercially available High Capacity cDNA Archive Kit (Applied Biosystems, Carlsbad, California). Analysis was carried out with a 7900HT real-time polymerase chain reaction system (Applied Biosystems). The average level of the FOXP3 gene expression in children with allergy was significantly lower compared with healthy children (2.2 ± 1.3 vs 4.2 ± 4.2; P = .014). The lowest mean level of FOXP3 mRNA expression (1.9 ± 1.6) was recorded in children with asthma and FA, and the highest level (4.2 ± 4.2) was recorded in healthy children without atopy (P = .036). A milder course of asthma or the degree of allergic reaction after a food challenge was associated with higher FOXP3 mRNA expression. Significantly lower levels of FOXP3 gene expression, observed more commonly in children with asthma and IgE-dependent FA than in healthy controls, were associated with a more severe clinical course. Therefore, FOXP3 expression could serve as an indicator of severe asthma with concomitant atopic conditions such as IgE-dependent FA. Copyright © 2015 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Patterns of mRNA and protein expression during minus-lens compensation and recovery in tree shrew sclera.

PubMed

Gao, Hong; Frost, Michael R; Siegwart, John T; Norton, Thomas T

2011-04-12

To increase our understanding of the mechanisms that remodel the sclera during the development of lens-induced myopia, when the sclera responds to putative "go" signals of retinal origin, and during recovery from lens-induced myopia, when the sclera responds to retinally-derived "stop" signals. Seven groups of tree shrews were used to examine mRNA levels during minus lens compensation and recovery. Starting 24 days after eye opening (days of visual experience [VE]) lens compensation animals wore a monocular -5D lens for 1, 4, or 11 days. Recovery animals wore the -5D lens for 11 days, which was then removed for 1 or 4 days. Normal animals were examined at 24 and 38 days of VE. All groups contained 8 animals. Scleral mRNA levels were examined in the treated and contralateral control eyes with quantitative real-time polymerase chain reaction (qPCR) for 27 genes divided into four categories: 1) signaling molecules, 2) matricellular proteins, 3) metalloproteinases (MPs) and tissue inhibitors of metalloproteinases (TIMPs), and 4) cell adhesion and other proteins. Four groups (n=5 per group) were used to examine protein levels. One group wore a -5D lens for 4 days. A second group recovered for 4 days after 11 days of -5D lens treatment. Two groups were used to examine age-matched normal protein levels at 28 and 39 days of VE. The levels of six scleral proteins that showed differential mRNA expression were examined with quantitative western blots. Nineteen of the genes showed differential (treated eye versus control eye) expression of mRNA levels in at least one group of animals. Which genes showed differential expression differed after 1 and 4 days of compensation and after 1 or 4 days of recovery. The mRNA level for one gene, a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), was upregulated in the treated eyes after 1 day of compensation. After 4 days, transforming growth factor beta receptor 3 (TGFBR3), transforming growth factor-beta-induced protein ig-h3 (TGFBI), and matrix metalloproteinase 14 (MMP14) mRNA levels were upregulated. Downregulated were mRNA levels for transforming growth factor beta-1 (TGFB1), transforming growth factor beta-2 (TGFB2), thrombospondin 1 (THBS1), tenascin (TNC), osteonectin (SPARC), osteopontin (SPP1), tissue inhibitor of metalloproteinases 3 (TIMP3), and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5). After 11 days of lens wear, there was no differential expression. During recovery, after 1 day, treated-eye mRNA downregulation was found for TGFB2, TGFBR1, TGFBR2, TGFBR3, SPARC, ADAMTS1, ADAMTS5, syndecan 4 (SDC4), and collagen type VI, alpha 1 (COL6A1). After 4 days, TGFB1, TGFB2, TGFB3, THBS2, and TIMP3 mRNA levels were upregulated in the recovering eye. Significant downregulation, relative to normal eyes, was found in both the control and treated eyes for most genes after 1 day of compensation; a similar decrease was found, compared to lens-compensated eyes, after one day of recovery. Protein levels for THBS1 showed positive correlation with the differential mRNA levels and TGFBR3 showed a negative correlation. No differential protein expression was found for TGFB2, TGFBI, MMP14, and TIMP3. The different patterns of differential mRNA expression during minus lens compensation (hyperopia) and recovery (myopia) show that scleral fibroblasts distinguish between "go" and "stop" conditions. There is evidence of binocular global downregulation of genes at the start of both lens wear and recovery. As additional information accumulates about changes in gene expression that occur during compensation and recovery the "signature" of differential changes may help us to understand in more detail how the sclera responds in "go" and "stop" conditions.

Expression and regulation of anti-mullerian hormone in an oviparous species, the hen.

PubMed

Johnson, P A; Kent, T R; Urick, M E; Giles, J R

2008-01-01

Anti-mullerian hormone (AMH) has a critical role in regression of the mullerian duct system during development in male mammalian and avian species and in regression of the right oviduct in female avian species. AMH in adult female birds has not been investigated. Chicken-specific cDNA primers were used to isolate Amh by RT-PCR. This probe was used in Northern blot analysis to identify a 2.8-kb band with expression in total ovarian RNA and in granulosa cell RNA. Quantitative real-time PCR was used to assess Amh expression in follicles of different maturity (1, 3, 5, and 6-12 mm and the largest F1 follicle; n = 4-6 of each size). There was an increased amount of Amh mRNA in the granulosa layer of the smaller follicles and a lower amount in the granulosa layer of the larger follicles (P < 0.01). There was no difference in granulosa Amh expression between the germinal disc and non-germinal disc region of 6- to 12-mm follicles, although expression differed with follicle size (P < 0.01). To examine hormone regulation of Amh, granulosa cells (from 6- to 8-mm follicles) were cultured with various concentrations of estradiol (E(2)) and progesterone (P(4)), and Amh mRNA was assessed. Neither E(2) nor P(4) influenced Amh mRNA accumulation. Granulosa cells were also cultured in the presence of oocyte-conditioned medium (OCM), which decreased Amh mRNA expression in a dose-related manner (P < 0.05); FSH receptor expression was not affected. Heat treatment of OCM abolished the effect, but growth differentiation factor 9 antiserum did not block the suppression. Immunohistochemistry confirmed that the granulosa layer was the predominant source of AMH in the small follicles of the hen and indicated that AMH was present early in follicle development, with expression in very small follicles (approximately 150 mum).

Presence of High-Risk HPV mRNA in Relation to Future High-Grade Lesions among High-Risk HPV DNA Positive Women with Minor Cytological Abnormalities

PubMed Central

Johansson, Hanna; Bjelkenkrantz, Kaj; Darlin, Lotten; Dilllner, Joakim; Forslund, Ola

2015-01-01

Objective Continuous expression of E6- and E7-oncogenes of high-risk human papillomavirus (HPV) types is necessary for the development and maintenance of the dysplastic phenotype. The aim of the study was to determine the sensitivity and specificity of the APTIMA HPV mRNA assay (Hologic) in predicting future development of high-grade cervical intraepithelial neoplasia (CIN) among high-risk HPV-DNA-positive women with atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous epithelial lesion (LSIL) cytology. Methods Archived SurePath cervical samples of women ≥ 35 years of age with high-risk HPV DNA-positive ASCUS (n = 211) or LSIL, (n = 131) were tested for the presence of high-risk HPV E6/E7 mRNA using the APTIMA HPV assay, and the women were monitored for development of histopathologically verified CIN2+. Results Twenty-nine percent (61/211) of the women in the ASCUS group, and 34.3% (45/131) in the LSIL group developed CIN2+ within 4.5 years of follow-up. The prevalence of HPV mRNA was 90.0% (95% CI 85.9-94.0) among women with ASCUS and 95.4% (95% CI 91.8-99.0) among women with LSIL. The presence of HPV E6/E7 mRNA was associated with future development of CIN2+ among women with ASCUS and LSIL (p=0.02). The mRNA assay demonstrated high sensitivity in predicting future CIN2+ and CIN3 for index ASCUS (96.7%; 95% CI 87.6-99.4 and 100%; 95% CI 82.2-100, respectively) and LSIL (97.8%, 95% CI 86.8-99.9 and 100%, 95% CI 79.9-100, respectively). The corresponding specificity was low, 12.7% (95% CI 7.9-19.3) and 5.8% (95% CI 2.2-13.6), for future CIN2+, respectively. The negative predictive value of the HPV mRNA assay for detecting future CIN3 was 100%, since no mRNA-negative woman developed CIN3 (0/27) as compared to 13.6% (43/315) of the mRNA-positive women (p = 0.03). Conclusion The APTIMA mRNA assay demonstrated high sensitivity but low specificity in predicting future CIN2+ among women with minor cytological abnormalities. The assay had high negative predictive value for future CIN3, indicating that HPV-mRNA-negative women are at low risk of progression to high grade CIN. PMID:25893988

Transcription elongation factors are involved in programming hormone production in pituitary neuroendocrine GH4C1 cells.

PubMed

Fujita, Toshitsugu; Piuz, Isabelle; Schlegel, Werner

2010-05-05

Transcription elongation of many eukaryotic genes is regulated. Two negative transcription elongation factors, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) are known to stall collaboratively RNA polymerase II promoter proximally. We discovered that DSIF and NELF are linked to hormone expression in rat pituitary GH4C1 cells. When NELF-E, a subunit of NELF or Spt5, a subunit of DSIF was stably knocked-down, prolactin (PRL) expression was increased both at the mRNA and protein levels. In contrast, stable knock-down of only Spt5 abolished growth hormone (GH) expression. Transient NELF-E knock-down increased coincidentally PRL expression and enhanced transcription of a PRL-promoter reporter gene. However, no direct interaction of NELF with the PRL gene could be demonstrated by chromatin immuno-precipitation. Thus, NELF suppressed PRL promoter activity indirectly. In conclusion, transcription regulation by NELF and DSIF is continuously involved in the control of hormone production and may contribute to neuroendocrine cell differentiation. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Rapid changes in ovarian mRNA induced by brief photostimulation in Siberian hamsters (Phodopus sungorus)

PubMed Central

Shahed, Asha; McMichael, Carling F.; Young, Kelly A.

2017-01-01

This study sought to characterize the rapid intraovarian mRNA response of key folliculogenic factors that may contribute to the restoration of folliculogenesis during 2-10 days of photostimulation in Siberian hamsters. Adult hamsters were exposed to short photoperiod (8L:16D) for 14 weeks (SD). A subset were then transferred to long photoperiod (16L:8D) for 2(PT day-2), 4(PT day-4), or 10 days (PT day-10). Quantitative real-time PCR was used to measure intraovarian mRNA expression of: gonadotropin releasing hormone (GnRH), follicle stimulating hormone β-subunit (FSHβ-subunit), luteinizing hormone β-subunit (LHβ-subunit), FSH and LH receptors, estrogen receptorsα and β (Esr1 and Esr2), matrix metalloproteinase (MMP)-2 and -9, anti-Müllerian hormone (AMH), inhibin-α subunit, fibroblast growth factor-2 (FGF-2) and proliferating cell nuclear antigen (PCNA). Compared to SD, plasma FSH concentrations increased on PT day-4 and the number of antral follicles and corpora lutea increased on PT day-10. FSHR and inhibin-α mRNA expression also increased on PT day-4, whereas LHR and proliferation marker PCNA both increased on PT day-10 as compared to SD. Esr1 mRNA increased on PT day-2 and remained significantly increased as compared to SD, whereas Esr1 mRNA increased only on PT day-2, similar to FGF-2 and MMP-2 results. No differences were observed in mRNA expression in ovarian GnRH, FSHβ- and LHβ-subunits, AMH, and MMP-9 mRNA with 2-10 days of photostimulation. Rapid increases in intraovarian FSHR and inhibin-α mRNA and antral follicle/corpora lutea numbers suggest that the ovary is primed to react quickly to the FSH released in response to brief periods of photostimulation. PMID:26174001

Promotion of mouse fibroblast collagen gene expression by mast cells stimulated via the Fc epsilon RI. Role for mast cell-derived transforming growth factor beta and tumor necrosis factor alpha

PubMed Central

1994-01-01

Chronic allergic diseases and other disorders associated with mast cell activation can also be associated with tissue fibrosis, but a direct link between mast cell mediator release and fibroblast collagen gene expression has not been established. Using in situ hybridization, we show that the elicitation of an IgE-dependent passive cutaneous anaphylaxis (PCA) reaction in mice results in a transient, but marked augmentation of steady state levels of type alpha-1 (I) collagen mRNA in the dermis. While peak levels of collagen mRNA expression in the skin are observed 16-24 h after mast cell activation, substantial numbers of dermal cells are strongly positive for collagen mRNA at 1 and 2 h after antigen challenge, before circulating inflammatory cells are recruited into the tissues. Furthermore, experiments in mast cell- reconstituted or genetically mast cell-deficient WBB6F1-W/Wv mice demonstrate that the increased expression of collagen mRNA at sites of PCA reactions is entirely mast cell dependent. In vitro studies show that the supernatants of mouse serosal mast cells activated via the Fc epsilon RI markedly increase type alpha-1 (I) collagen mRNA levels in mouse embryonic skin fibroblasts, and also upregulate collagen secretion by these cells. The ability of mast cell supernatants to induce increased steady state levels of collagen mRNA in mouse skin fibroblasts is markedly diminished by absorption with antibodies specific for either of two mast cell-derived cytokines, transforming growth factor beta (TGF-beta 1) or tumor necrosis factor alpha (TNF- alpha), and is eliminated entirely by absorption with antibodies against both cytokines. Taken together, these findings demonstrate that IgE-dependent mouse mast cell activation can induce a transient and marked increase in steady state levels of type alpha-1 (I) collagen mRNA in dermal fibroblasts and that mast cell-derived TGF-beta 1 and TNF-alpha importantly contribute to this effect. PMID:7964480

Host control of plasmid replication: requirement for the sigma factor sigma 32 in transcription of mini-F replication initiator gene.

PubMed Central

Wada, C; Imai, M; Yura, T

1987-01-01

Replication of F factor or mini-F plasmid is strongly inhibited in the rpoH (htpR) mutants of Escherichia coli deficient in the sigma factor (sigma 32) known to be required for heat shock gene expression. Transcription of the mini-F repE gene encoding a replication initiator protein (E protein) was examined by operon fusion and by direct determination of repE mRNA. The synthesis rate and the level of repE mRNA were found to increase transiently upon temperature upshift (30 degrees C to 42 degrees C) in wild-type cells but to decrease rapidly in the rpoH mutants. Thus sigma 32 appeared to be directly involved in transcription of repE whose product, E protein, in turn activates DNA replication from the mini-F ori2 region. This scheme of host-controlled plasmid replication is further supported by the analysis of transcription in vitro: RNA synthesis can be initiated from the repE promoter by a minor form of RNA polymerase containing sigma 32 but not by the major polymerase containing the normal sigma factor sigma 70. The sigma 32-mediated transcription from the repE promoter is strongly inhibited by the E protein. We conclude that transcription of the mini-F repE gene is mediated by the host transcription factor sigma 32 and is negatively controlled by its own product. Images PMID:2447584

Snail, Slug, and Smad-interacting protein 1 as novel parameters of disease aggressiveness in metastatic ovarian and breast carcinoma.

PubMed

Elloul, Sivan; Elstrand, Mari Bukholt; Nesland, Jahn M; Tropé, Claes G; Kvalheim, Gunnar; Goldberg, Iris; Reich, Reuven; Davidson, Ben

2005-04-15

It was demonstrated previously that the Snail family of transcription factors and Smad-interacting protein 1 (Sip1) regulate E-cadherin and matrix metalloproteinase 2 (MMP-2) expression, cellular morphology, and invasion in carcinoma. For the current study, the authors analyzed the relation between the expression of Snail, Slug, and Sip1; the expression of MMP-2 and E-cadherin; and clinical parameters in patients with metastatic ovarian and breast carcinoma. One hundred one fresh-frozen, malignant effusions from patients who were diagnosed with gynecologic carcinomas (78 ovarian carcinomas and 23 breast carcinomas) were studied for mRNA expression of Snail, Slug, Sip1, MMP-2, and E-cadherin using reverse transcriptase-polymerase chain reaction analysis. Snail mRNA and E-cadherin protein expression levels also were studied in ovarian carcinoma effusions using in situ hybridization and immunocytochemistry. The results were analyzed for possible correlation with clinicopathologic parameters in both tumor types. E-cadherin mRNA expression was lower in breast carcinoma (P = 0.001), whereas Snail expression was higher (P = 0.003). The Snail/E-cadherin ratio (P < 0.001) and the Sip1/E-cadherin ratio (P = 0.002) were higher in breast carcinomas. Sip1 mRNA expression (P < 0.001) and Slug mRNA expression (P < 0.001) were correlated with the expression of MMP-2 in ovarian carcinomas. The Sip1/E-cadherin ratio was higher in primary ovarian carcinomas at the time of diagnosis compared with postchemotherapy ovarian carcinoma effusions (P = 0.003), higher in Stage IV tumors compared with Stage III tumors (P = 0.049), and higher in pleural effusions compared with peritoneal effusions (P = 0.044). In a univariate survival analysis of patients with ovarian carcinoma, a high Sip1/E-cadherin ratio predicted poor overall survival (P = 0.018). High E-cadherin mRNA expression predicted better disease-free survival (P = 0.023), with a similar trend for a low Slug/E-cadherin ratio (P = 0.07). High Snail mRNA expression predicted shorter effusion-free survival (P = 0.008), disease-free survival (P = 0.03), and overall survival (P = 0.008) in patients with breast carcinoma. Transcription factors that regulate E-cadherin were expressed differentially in metastatic ovarian and breast carcinoma. Snail may predict a poor outcome in patients who have breast carcinoma metastatic to effusions. E-cadherin expression generally was conserved in effusions from patients with ovarian carcinoma, but the subset of patients with postulated Sip1-induced repression of this adhesion molecule had a significantly worse outcome. This finding was in agreement with the stronger suppression of E-cadherin by Snail and Sip1 in breast carcinoma effusions, a clinical condition associated with extremely poor survival. (c) 2005 American Cancer Society.

Tachykinin receptor and neutral endopeptidase gene expression in the rat uterus: characterization and regulation in response to ovarian steroid treatment.

PubMed

Pinto, F M; Armesto, C P; Magraner, J; Trujillo, M; Martín, J D; Candenas, M L

1999-06-01

Tachykinin neuropeptides, such as substance P, are localized to a population of sensory fibers that innervate the mammalian female reproductive tract. In the present study, we have characterized tachykinin NK1 receptor (NK1R), NK2 receptor (NK2R), and NK3 receptor (NK3R) gene expression by semiquantitative RT-PCR in uteri from ovariectomized rats and studied their regulation in response to 17beta-estradiol (E2), progesterone (P4), or a combination of both. In addition, we analyzed the expression and regulation of the neutral endopeptidase 24.11 (NEP), the most important enzyme involved in tachykinin degradation in the rat uterus. In uteri from control (olive oil-treated) rats, RT-PCR assays revealed single bands corresponding to the expected product sizes encoding complementary DNA for NK1R (232 bp), NK2R (491 bp), NK3R (325 bp), and NEP (221 bp). The identity of the amplified fragments was confirmed by DNA sequence analysis. Compared with control rats, NK1R messenger RNA (mRNA) was increased by 2-fold in uteri from rats treated with E2, was decreased by 3.3-fold in rats treated with P4, and was decreased by 1.8-fold in rats treated with both E2 and P4. Uterine NK2R mRNA levels were not altered by any steroid treatment. E2 treatment decreased by 15-fold NK3R mRNA. P4 was without effect if administered alone and did not influence the E2-induced decrease in NK3R mRNA. NEP mRNA levels were about 4-fold lower in E2-treated than in P4-treated rats. Functional studies were carried out in uteri from E2- or P4-treated ovariectomized rats to characterize the contractile response evoked by the selective tachykinin receptor agonists [Sar9Met(O2)11]substance P (NK1R selective), [Nle10]NKA-(4-10) (NK2R selective), and [MePhe7]NKB (NK3R selective) in the presence of the NEP inhibitor phosphoramidon (1 microM). A marked correlation was observed between the magnitude of the contractile response to each agonist and the level of expression determined by RT-PCR for each tachykinin receptor. The present findings show that tachykinin NK1R, NK2R, NK3R, and NEP are expressed in the rat uterus and that ovarian steroids differentially regulate their expression.

IL-4 mRNA Is Downregulated in the Liver of Pancreatic Cancer Patients Suffering from Cachexia.

PubMed

Prokopchuk, Olga; Steinacker, Jürgen M; Nitsche, Ulrich; Otto, Stephanie; Bachmann, Jeannine; Schubert, Elaine C; Friess, Helmut; Martignoni, Marc E

2017-01-01

Interleukin-4 (IL-4) together with interleukin-13 (IL-13) play an important role in inflammation and wound repair, and are known to be upregulated in human skeletal muscle after strenuous physical exercise. Additionally, these cytokines may act as autocrine growth factors in pancreatic cancer cells. We hypothesize that IL-4, IL-13, and their corresponding receptors are involved in mechanism of cancer cachexia. Tissue samples from human skeletal muscle, white fat, liver, healthy pancreas, and pancreatic ductal adenocarcinoma were analyzed by quantitative real-time polymerase chain reaction for mRNA expression levels of IL-4, IL-13, IL-4 receptor α, and IL-13 receptor α1. We demonstrate for the first time that liver IL-4 mRNA is downregulated in vivo in patients with pancreatic cancer and cachexia. Additionally, IL-4 mRNA in the liver inversely correlated with musculus psoas thickness. We speculate that suppression of IL-4 is involved in cancer cachexia, although the exact mechanisms have to be further elucidated.

GIGYF1/2 proteins use auxiliary sequences to selectively bind to 4EHP and repress target mRNA expression

PubMed Central

Peter, Daniel; Weber, Ramona; Sandmeir, Felix; Wohlbold, Lara; Helms, Sigrun; Bawankar, Praveen; Valkov, Eugene; Igreja, Cátia; Izaurralde, Elisa

2017-01-01

The eIF4E homologous protein (4EHP) is thought to repress translation by competing with eIF4E for binding to the 5′ cap structure of specific mRNAs to which it is recruited through interactions with various proteins, including the GRB10-interacting GYF (glycine–tyrosine–phenylalanine domain) proteins 1 and 2 (GIGYF1/2). Despite its similarity to eIF4E, 4EHP does not interact with eIF4G and therefore fails to initiate translation. In contrast to eIF4G, GIGYF1/2 bind selectively to 4EHP but not eIF4E. Here, we present crystal structures of the 4EHP-binding regions of GIGYF1 and GIGYF2 in complex with 4EHP, which reveal the molecular basis for the selectivity of the GIGYF1/2 proteins for 4EHP. Complementation assays in a GIGYF1/2-null cell line using structure-based mutants indicate that 4EHP requires interactions with GIGYF1/2 to down-regulate target mRNA expression. Our studies provide structural insights into the assembly of 4EHP–GIGYF1/2 repressor complexes and reveal that rather than merely facilitating 4EHP recruitment to transcripts, GIGYF1/2 proteins are required for repressive activity. PMID:28698298

Impaired Bone Resorption by Lipopolysaccharide In Vivo in Mice Deficient in the Prostaglandin E Receptor EP4 Subtype

PubMed Central

Sakuma, Yoko; Tanaka, Kiyoshi; Suda, Michio; Komatsu, Yasato; Yasoda, Akihiro; Miura, Masako; Ozasa, Ami; Narumiya, Shuh; Sugimoto, Yukihiko; Ichikawa, Atsushi; Ushikubi, Fumitaka; Nakao, Kazuwa

2000-01-01

In a previous study we showed that the involvement of EP4 subtype of the prostaglandin E (PGE) receptor is crucial for lipopolysaccharide (LPS)-induced osteoclast formation in vitro. The present study was undertaken to test whether EP4 is actually associated with LPS-induced bone resorption in vivo. In wild-type (WT) mice, osteoclast formation in vertebrae and tibiae increased 5 days after systemic LPS injection, and urinary excretion of deoxypyridinoline, a sensitive marker for bone resorption, statistically increased 10 days after injection. In EP4 knockout (KO) mice, however, LPS injection caused no significant changes in these parameters throughout the experiment. LPS exposure for 4 h strongly induced osteoclast differentiation factor (ODF) mRNA expression in primary osteoblastic cells (POB) both from WT and EP4 KO mice, and this expression was not inhibited by indomethacin, suggesting prostaglandin (PG) independence. LPS exposure for 24 h further induced ODF expression in WT POB, but not in EP4 KO POB. Indomethacin partially inhibited ODF expression in WT POB, but not in EP4 KO POB. These data suggest that ODF is induced both PG dependently and PG independently. LPS exposure for 24 h induced slightly greater osteoclastgenesis inhibitory factor (OCIF) mRNA expression in EP4 KO than in WT POB. These findings suggest that the reduced ODF expression and apparently increased OCIF expression also are responsible for the markedly reduced LPS-induced osteoclast formation in EP4 KO mice. Our results show that the EP4 subtype of the PGE receptor is involved in LPS-induced bone resorption in vivo also. Since LPS is considered to be largely involved in bacterially induced bone loss, such as in periodontitis and osteomyelitis, our study is expected to help broaden our understanding of the pathophysiology of these conditions. PMID:11083800

Neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-HSD type 4

PubMed Central

2010-01-01

Background Steroids affect many tissues, including the brain. In the zebra finch, the estrogenic steroid estradiol (E2) is especially effective at promoting growth of the neural circuit specialized for song. In this species, only the males sing and they have a much larger and more interconnected song circuit than females. Thus, it was surprising that the gene for 17β-hydroxysteroid dehydrogenase type 4 (HSD17B4), an enzyme that converts E2 to a less potent estrogen, had been mapped to the Z sex chromosome. As a consequence, it was likely that HSD17B4 was differentially expressed in males (ZZ) and females (ZW) because dosage compensation of Z chromosome genes is incomplete in birds. If a higher abundance of HSD17B4 mRNA in males than females was translated into functional enzyme in the brain, then contrary to expectation, males could produce less E2 in their brains than females. Results Here, we used molecular and biochemical techniques to confirm the HSD17B4 Z chromosome location in the zebra finch and to determine that HSD17B4 mRNA and activity were detectable in the early developing and adult brain. As expected, HSD17B4 mRNA expression levels were higher in males compared to females. This provides further evidence of the incomplete Z chromosome inactivation mechanisms in birds. We detected HSD17B4 mRNA in regions that suggested a role for this enzyme in the early organization and adult function of song nuclei. We did not, however, detect significant sex differences in HSD17B4 activity levels in the adult brain. Conclusions Our results demonstrate that the HSD17B4 gene is expressed and active in the zebra finch brain as an E2 metabolizing enzyme, but that dosage compensation of this Z-linked gene may occur via post-transcriptional mechanisms. PMID:20359329

Studies on Pentoxifylline and Tocopherol Combination for Radiation-Induced Heart Disease in Rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Hui; Department of Radiotherapy, Cancer Center, Sun Yat-sen University, Guangzhou, Guangdong; Xiong Mai

Purpose: To investigate whether the application of pentoxifylline (PTX) and tocopherol l (Vit. E) could modify the development of radiation-induced heart disease and downregulate the expression of transforming growth factor (TGF)-{beta}1mRNA in rats. Methods and Materials: A total of 120 Sprague-Dawley rats were separated into four groups: control group, irradiated group, experimental group 1, and experiment group 2. Supplementation was started 3 days before irradiation; in experimental group 1, injection of PTX (15 mg/kg/d) and Vit. E (5.5 mg/kg/d) continued till the 12th week postirradiation, whereas in experimental group 2 it was continued until the 24th week postirradiation. All ratsmore » were administrated a single dose of 20 Gy irradiation to the heart except the control group. Histopathologic evaluation was performed at various time points (Days 1, 2, 4, 8, and 12 and 24th week) up to 24 weeks after irradiation. Changes of levels of TGF-{beta}1 mRNA expression were also investigated at the same time points using competitive polymerase chain reaction. Results: Compared with the irradiated group, levels of TGF-{beta}1 mRNA of the rat hearts were relatively low in the two experimental groups on the 12th week postirradiation. In experimental group 1, there was a rebound expression of TGF-{beta}1 mRNA on the 24th week postirradiation, whereas that of the experimental group 2 remained low (p < 0.05). The proportions of collagen fibers of the two experimental groups were lower than that of irradiated group (p < 0.05). A rebound could be observed in the experimental group 1. Conclusion: PTX and Vit. E downregulated the expression of TGF-{beta}1 mRNA. The irradiated rat hearts showed a marked pathologic response to the drugs. The withdrawal of drugs in the 12th week postirradiation could cause rebound effects of the development of fibrosis.« less

Activating transcription factor 4 regulates stearate-induced vascular calcification.

PubMed

Masuda, Masashi; Ting, Tabitha C; Levi, Moshe; Saunders, Sommer J; Miyazaki-Anzai, Shinobu; Miyazaki, Makoto

2012-08-01

Previously, we reported that stearate, a saturated fatty acid, promotes osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC). In this study, we examined the molecular mechanisms by which stearate promotes vascular calcification. ATF4 is a pivotal transcription factor in osteoblastogenesis and endoplasmic reticulum (ER) stress. Increased stearate by either supplementation of exogenous stearic acid or inhibition of stearoyl-CoA desaturase (SCD) by CAY10566 induced ATF4 mRNA, phosphorylated ATF4 protein, and total ATF4 protein. Induction occurred through activation of the PERK-eIF2α pathway, along with increased osteoblastic differentiation and mineralization of VSMCs. Either stearate or the SCD inhibitor but not oleate or other fatty acid treatments also increased ER stress as determined by the expression of p-eIF2α, CHOP, and the spliced form of XBP-1, which were directly correlated with ER stearate levels. ATF4 knockdown by lentiviral ATF4 shRNA blocked osteoblastic differentiation and mineralization induced by stearate and SCD inhibition. Conversely, treatment of VSMCs with an adenovirus containing ATF4 induced vascular calcification. Our results demonstrated that activation of ATF4 mediates vascular calcification induced by stearate.

Activating transcription factor 4 regulates stearate-induced vascular calcification

PubMed Central

Masuda, Masashi; Ting, Tabitha C.; Levi, Moshe; Saunders, Sommer J.; Miyazaki-Anzai, Shinobu; Miyazaki, Makoto

2012-01-01

Previously, we reported that stearate, a saturated fatty acid, promotes osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC). In this study, we examined the molecular mechanisms by which stearate promotes vascular calcification. ATF4 is a pivotal transcription factor in osteoblastogenesis and endoplasmic reticulum (ER) stress. Increased stearate by either supplementation of exogenous stearic acid or inhibition of stearoyl-CoA desaturase (SCD) by CAY10566 induced ATF4 mRNA, phosphorylated ATF4 protein, and total ATF4 protein. Induction occurred through activation of the PERK-eIF2α pathway, along with increased osteoblastic differentiation and mineralization of VSMCs. Either stearate or the SCD inhibitor but not oleate or other fatty acid treatments also increased ER stress as determined by the expression of p-eIF2α, CHOP, and the spliced form of XBP-1, which were directly correlated with ER stearate levels. ATF4 knockdown by lentiviral ATF4 shRNA blocked osteoblastic differentiation and mineralization induced by stearate and SCD inhibition. Conversely, treatment of VSMCs with an adenovirus containing ATF4 induced vascular calcification. Our results demonstrated that activation of ATF4 mediates vascular calcification induced by stearate. PMID:22628618

Targeted gene expression profiling in European sea bass (Dicentrarchus labrax, L.) follicles from primary growth to late vitellogenesis.

PubMed

García-López, Angel; Sánchez-Amaya, María Isabel; Prat, Francisco

2011-11-01

A real-time PCR-based gene expression survey was performed on isolated European sea bass follicles from primary growth to late vitellogenesis. Expression levels of 18 transcripts with demonstrated relevance during oogenesis, encoding gonadotropin, thyrotropin, estrogen, androgen, and vitellogenin receptors, steroidogenesis-related as well as growth and transcription factors were measured. Primary oocytes showed high mRNA levels of insulin-like growth factors 1 and 2, bone morphogenetic protein 4, estrogen receptor 2b, androgen receptor b, and SRY-box containing gene 17 together with low transcript amounts of gonadotropin receptors. Follicles at the lipid vesicles stage (i.e., the beginning of the secondary growth phase) showed elevated mRNA amounts of follicle stimulating hormone receptor (fshr) and anti-Mullerian hormone. Early-to-mid vitellogenic follicles showed high mRNA levels of fshr and cytochrome P450, family 19, subfamily A, polypeptide 1a while mid-to-late vitellogenic follicles expressed increasing transcript amounts of luteinizing hormone/choriogonadotropin receptor, steroidogenic acute regulatory protein, and estrogen receptors 1 and 2a. The molecular data presented here may serve as a solid base for future studies focused on unraveling the specific mechanisms orchestrating follicular development in teleost fish. Copyright © 2011 Elsevier Inc. All rights reserved.

Clomiphene citrate versus letrozole: molecular analysis of the endometrium in women with polycystic ovary syndrome.

PubMed

Wallace, Kedra L; Johnson, Venessia; Sopelak, Victoria; Hines, Randall

2011-10-01

To compare the effect of clomiphene citrate (CC) and letrozole on endometrial receptivity in women with polycystic ovary syndrome (PCOS). A randomized controlled trial. University teaching hospital. Ten anovulatory women with PCOS and 5 fertile ovulatory women. Patients received 2.5 mg of letrozole on cycle days 3-7 (5 patients, 1 cycle) or 50 mg of CC on cycle days 5-9 (5 patients, 1 cycle). Serum estrogen (E) and progesterone (P) endometrial protein and messenger RNA (mRNA) expression of leukemia inhibitory factor (LIF), dickkhopf homolog 1 (DKK-1), fibroblast growth factor 22 (FGF-22), and endometrial mRNA expression of LIF/GP130 receptor (LIFR). No statistically significant differences were observed between groups compared with fertile ovulatory women when serum E and P were examined, or between body mass index (BMI), and cycle day at time of biopsy. Letrozole increased mRNA expression of LIF, DKK1, LIFR, and FGF-22, whereas CC only increased endometrial mRNA expression of LIF. Letrozole mRNA expression directly translated into increased protein expression of like genes in the endometrium. The CC protein expression of DKK-1 was significantly decreased compared with controls. Letrozole positively influences a number of markers of endometrial receptivity compared with CC. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

EBP1 is a novel E2F target gene regulated by transforming growth factor-β.

PubMed

Judah, David; Chang, Wing Y; Dagnino, Lina

2010-11-10

Regulation of gene expression requires transcription factor binding to specific DNA elements, and a large body of work has focused on the identification of such sequences. However, it is becoming increasingly clear that eukaryotic transcription factors can exhibit widespread, nonfunctional binding to genomic DNA sites. Conversely, some of these proteins, such as E2F, can also modulate gene expression by binding to non-consensus elements. E2F comprises a family of transcription factors that play key roles in a wide variety of cellular functions, including survival, differentiation, activation during tissue regeneration, metabolism, and proliferation. E2F factors bind to the Erb3-binding protein 1 (EBP1) promoter in live cells. We now show that E2F binding to the EBP1 promoter occurs through two tandem DNA elements that do not conform to typical consensus E2F motifs. Exogenously expressed E2F1 activates EBP1 reporters lacking one, but not both sites, suggesting a degree of redundancy under certain conditions. E2F1 increases the levels of endogenous EBP1 mRNA in breast carcinoma and other transformed cell lines. In contrast, in non-transformed primary epidermal keratinocytes, E2F, together with the retinoblastoma family of proteins, appears to be involved in decreasing EBP1 mRNA abundance in response to growth inhibition by transforming growth factor-β1. Thus, E2F is likely a central coordinator of multiple responses that culminate in regulation of EBP1 gene expression, and which may vary depending on cell type and context.

Comparative Sigma Factor-mRNA Levels in Mycobacterium marinum under Stress Conditions and during Host Infection

PubMed Central

Pettersson, B. M. Fredrik; Das, Sarbashis; Behra, Phani Rama Krishna; Jordan, Heather R.; Ramesh, Malavika; Mallick, Amrita; Root, Kate M.; Cheramie, Martin N.; de la Cruz Melara, Irma; Small, Pamela L. C.; Dasgupta, Santanu; Ennis, Don G.; Kirsebom, Leif A.

2015-01-01

We have used RNASeq and qRT-PCR to study mRNA levels for all σ-factors in different Mycobacterium marinum strains under various growth and stress conditions. We also studied their levels in M. marinum from infected fish and mosquito larvae. The annotated σ-factors were expressed and transcripts varied in relation to growth and stress conditions. Some were highly abundant such as sigA, sigB, sigC, sigD, sigE and sigH while others were not. The σ-factor mRNA profiles were similar after heat stress, during infection of fish and mosquito larvae. The similarity also applies to some of the known heat shock genes such as the α-crystallin gene. Therefore, it seems probable that the physiological state of M. marinum is similar when exposed to these different conditions. Moreover, the mosquito larvae data suggest that this is the state that the fish encounter when infected, at least with respect to σ-factor mRNA levels. Comparative genomic analysis of σ-factor gene localizations in three M. marinum strains and Mycobacterium tuberculosis H37Rv revealed chromosomal rearrangements that changed the localization of especially sigA, sigB, sigD, sigE, sigF and sigJ after the divergence of these two species. This may explain the variation in species-specific expression upon exposure to different growth conditions. PMID:26445268

Cooperative modulation by eIF4G of eIF4E-binding to the mRNA 5' cap in yeast involves a site partially shared by p20.

PubMed Central

Ptushkina, M; von der Haar, T; Vasilescu, S; Frank, R; Birkenhäger, R; McCarthy, J E

1998-01-01

Interaction between the mRNA 5'-cap-binding protein eIF4E and the multiadaptor protein eIF4G has been demonstrated in all eukaryotic translation assemblies examined so far. This study uses immunological, genetic and biochemical methods to map the surface amino acids on eIF4E that contribute to eIF4G binding. Cap-analogue chromatography and surface plasmon resonance (SPR) analyses demonstrate that one class of mutations in these surface regions disrupts eIF4E-eIF4G association, and thereby polysome formation and growth. The residues at these positions in wild-type eIF4E mediate positive cooperativity between the binding of eIF4G to eIF4E and the latter's cap-affinity. Moreover, two of the mutations confer temperature sensitivity in eIF4G binding to eIF4E which correlates with the formation of large numbers of inactive ribosome 80S couples in vivo and the loss of cellular protein synthesis activity. The yeast 4E-binding protein p20 is estimated by SPR to have a ten times lower binding affinity than eIF4G for eIF4E. Investigation of a second class of eIF4E mutations reveals that p20 shares only part of eIF4G's binding site on the cap-binding protein. The results presented provide a basis for understanding how cycling of eIF4E and eIF4G occurs in yeast translation and explains how p20 can act as a fine, but not as a coarse, regulator of protein synthesis. PMID:9707439

Downregulation in GATA4 and Downstream Structural and Contractile Genes in the db/db Mouse Heart

PubMed Central

Broderick, Tom L.; Jankowski, Marek; Wang, Donghao; Danalache, Bogdan A.; Parrott, Cassandra R.; Gutkowska, Jolanta

2012-01-01

Reduced expression of GATA4, a transcriptional factor for structural and cardioprotective genes, has been proposed as a factor contributing to the development of cardiomyopathy. We investigated whether the reduction of cardiac GATA4 expression reported in diabetes alters the expression of downstream genes, namely, atrial natriuretic peptide (ANP), B-type natriuretic, peptide (BNP), and α- and β-myosin heavy chain (MHC). db/db mice, a model of type 2 diabetes, with lean littermates serving as controls, were studied. db/db mice exhibited obesity, hyperglycemia, and reduced protein expression of cardiac GLUT4 and IRAP (insulin-regulated aminopeptidase), the structural protein cosecreted with GLUT4. Hearts from db/db mice had reduced protein expression of GATA4 (~35%) with accompanying reductions in mRNA expression of ANP (~40%), BNP (~85%), and α-MHC mRNA (~50%) whereas expression of β-MHC mRNA was increased by ~60%. Low GATA4 was not explained by an increased ligase or atrogin1 expression. CHIP protein content was modestly downregulated (27%) in db/db mice whereas mRNA and protein expression of the CHIP cochaperone HSP70 was significantly decreased in db/db hearts. Our results indicate that low GATA4 in db/db mouse heart is accompanied by reduced expression of GATA4-regulated cardioprotective and structural genes, which may explain the development of cardiomyopathy in diabetes. PMID:22474596

[Modified Cheng's Juanbi Decoction downregulates expression of prostaglandin E receptor 4 in synovial tissue in rats with adjuvant arthritis].

PubMed

Xu, Xia; Cheng, Hui; Cao, Jian; DU, Huan; Meng, Qingwei; Guo, Mengyuan

2017-06-01

Objective To investigate the effect of modified Cheng's Juanbi Decoction on the expression of prostaglandin E receptor 4 (PTGER4), the T cell receptor in the synovial tissues, in rats with adjuvant arthritis (AA). Methods A rat model of AA was established by subcutaneous injection of Freund's complete adjuvant at the vola pedis combined with ice-water bath and blowing. The degree of joint swelling and arthritis index were determined in each group. The quantitative real-time PCR was performed to assess the effect of modified Cheng's Juanbi Decoction on the mRNA expression of PTGER4in the synovial tissue. Results Cheng's Juanbi Decoction significantly alleviated the damage in the joints and synovial tissues in the AA rats. High-dose (the content of crude drug: 4 g/mL) Cheng's Juanbi Decoction significantly reduced the mRNA expression of PTGER4 in the synovial tissues. Conclusion Cheng's Juanbi Decoction can reduce the level of PTGER4 mRNA in the synovial tissue in AA rats.

Expression of Leaf Nitrate Reductase Genes from Tomato and Tobacco in Relation to Light-Dark Regimes and Nitrate Supply

PubMed Central

Galangau, Fabienne; Daniel-Vedele, Françoise; Moureaux, Thérèse; Dorbe, Marie-France; Leydecker, Marie-Thérèse; Caboche, Michel

1988-01-01

The influence of light-dark cycles and nitrate supply on nitrate reductase (NR) mRNA levels was studied in two plant species, tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) using specific NR DNA probes. In the same series of experiments, changes in the levels of NR protein (NRP) by enzyme-linked immunosorbent assay and changes in the level of NADH-nitrate reductase activity (NRA) were also followed. During a light-dark cycle, it was found that in both tomato and tobacco, NR mRNA accumulation increased rapidly during the dark period and reached a maximum at the beginning of the day, while NRP reached a peak 2 and 4 hours after mRNA peaked, for tomato and tobacco, respectively. At the end of the day, the amount of mRNA was decreased by a factor of at least 100 compared to sunrise in both species. These results demonstrate that light is involved, although probably not directly, in the regulation of the NR gene expression at the mRNA level. The peak of NRA in tobacco coincided with the peak in NR mRNA accumulation (i.e. sunrise), whereas in tomato the peak of NRA was approximately 5 to 6 hours after sunrise. There is no obvious correlation between NRP and NRA levels during the day. In nitrogen starvation experiments, a rapid decrease of NRP and NRA was detected, while NR mRNA levels were not significantly altered. Upon nitrate replenishment, nitrogen-starved plants accumulated NR mRNA rapidly. These results suggest that the availability of nitrogen affects the expression of NR activity at the transcriptional as well as at the post-transcriptional levels. Images Fig. 3 Fig. 5 Fig. 6 PMID:16666313

The human insulin mRNA is partly translated via a cap- and eIF4A-independent mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fred, Rikard G., E-mail: Rikard.Fred@mcb.uu.se; Sandberg, Monica; Pelletier, Jerry

Highlights: {yields} The polypyrimidine tract binding protein binds to the 5'-UTR of the insulin mRNA. {yields} Insulin mRNA can be translated via a cap-independent mechanism. {yields} The fraction cap-independent insulin synthesis increases during conditions of stress. {yields} The {beta}-cell is able to uphold basal insulin biosynthesis under conditions of stress. -- Abstract: The aim of this study was to investigate whether cap-independent insulin mRNA translation occurs in human pancreatic islets at basal conditions, during stimulation at a high glucose concentration and at conditions of nitrosative stress. We also aimed at correlating cap-independent insulin mRNA translation with binding of the IRESmore » trans-acting factor polypyrimidine tract binding protein (PTB) to the 5'-UTR of insulin mRNA. For this purpose, human islets were incubated for 2 h in the presence of low (1.67 mM) or high glucose (16.7 mM). Nitrosative stress was induced by addition of 1 mM DETA/NO and cap-dependent mRNA translation was inhibited with hippuristanol. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. PTB affinity to insulin mRNA 5'-UTR was assessed by a magnetic micro bead pull-down procedure. We observed that in the presence of 1.67 mM glucose, approximately 70% of the insulin mRNA translation was inhibited by hippuristanol. Corresponding value from islets incubated at 16.7 mM glucose was 93%. DETA/NO treatment significantly decreased the translation of insulin by 85% in high glucose incubated islets, and by 50% at a low glucose concentration. The lowered insulin biosynthesis rates of DETA/NO-exposed islets were further suppressed by hippuristanol with 55% at 16.7 mM glucose but not at 1.67 mM glucose. Thus, hippuristanol-induced inhibition of insulin biosynthesis was less pronounced in DETA/NO-treated islets as compared to control islets. We observed also that PTB bound specifically to the insulin mRNA 5'-UTR in vitro, and that this binding corresponded well with rates of cap-independent insulin biosynthesis at the different conditions. In conclusion, our studies show that insulin biosynthesis is mainly cap-dependent at a high glucose concentration, but that the cap-independent biosynthesis of insulin can constitute as much as 40-100% of all insulin biosynthesis during conditions of nitrosative stress. These data suggest that the pancreatic {beta}-cell is able to uphold basal insulin synthesis at conditions of starvation and stress via a cap- and eIF4A-independent mechanism, possibly mediated by the binding of PTB to the 5'-UTR of the human insulin mRNA.« less

Orf virus interleukin-10 and vascular endothelial growth factor-E modulate gene expression in cultured equine dermal fibroblasts.

PubMed

Wise, Lyn M; Bodaan, Christa J; Mercer, Andrew A; Riley, Christopher B; Theoret, Christine L

2016-10-01

Wounds in horses often exhibit sustained inflammation and inefficient vascularization, leading to excessive fibrosis and clinical complications such as "proud flesh". Orf virus-derived proteins, vascular endothelial growth factor (VEGF)-E and interleukin (ovIL)-10, enhance angiogenesis and control inflammation and fibrosis in skin wounds of laboratory animals. The study aimed to determine if equine dermal cells respond to VEGF-E and ovIL-10. Equine dermal cells are expected to express VEGF and IL-10 receptors, so viral protein treatment is likely to alter cellular gene expression and behaviour in a manner conducive to healing. Skin samples were harvested from the lateral thoracic wall of two healthy thoroughbred horses. Equine dermal cells were isolated using a skin explant method and their phenotype assessed by immunofluorescence. Cells were treated with recombinant proteins, with or without inflammatory stimuli. Gene expression was examined using standard and quantitative reverse transcriptase PCR. Cell behaviour was evaluated in a scratch assay. Cultured cells were half vimentin(+ve) fibroblasts and half alpha smooth muscle actin(+ve) and vimentin(+ve) myofibroblasts. VEGF-E increased basal expression of IL-10 mRNA, whereas VEGF-A and collagenase-1 mRNA expression was increased by ovIL-10. In cells exposed to inflammatory stimulus, both treatments dampened tumour necrosis factor mRNA expression, and ovIL-10 exacerbated expression of monocyte chemoattractant protein. Neither viral protein influenced cell migration greatly. This study shows that VEGF-E and ovIL-10 are active on equine dermal cells and exert anti-inflammatory and anti-fibrotic effects that may enhance skin wound healing in horses. © 2016 ESVD and ACVD.

The alveolate translation initiation factor 4E family reveals a custom toolkit for translational control in core dinoflagellates.

PubMed

Jones, Grant D; Williams, Ernest P; Place, Allen R; Jagus, Rosemary; Bachvaroff, Tsvetan R

2015-02-10

Dinoflagellates are eukaryotes with unusual cell biology and appear to rely on translational rather than transcriptional control of gene expression. The eukaryotic translation initiation factor 4E (eIF4E) plays an important role in regulating gene expression because eIF4E binding to the mRNA cap is a control point for translation. eIF4E is part of an extended, eukaryote-specific family with different members having specific functions, based on studies of model organisms. Dinoflagellate eIF4E diversity could provide a mechanism for dinoflagellates to regulate gene expression in a post-transcriptional manner. Accordingly, eIF4E family members from eleven core dinoflagellate transcriptomes were surveyed to determine the diversity and phylogeny of the eIF4E family in dinoflagellates and related lineages including apicomplexans, ciliates and heterokonts. The survey uncovered eight to fifteen (on average eleven) different eIF4E family members in each core dinoflagellate species. The eIF4E family members from heterokonts and dinoflagellates segregated into three clades, suggesting at least three eIF4E cognates were present in their common ancestor. However, these three clades are distinct from the three previously described eIF4E classes, reflecting diverse approaches to a central eukaryotic function. Heterokonts contain four clades, ciliates two and apicomplexans only a single recognizable eIF4E clade. In the core dinoflagellates, the three clades were further divided into nine sub-clades based on the phylogenetic analysis and species representation. Six of the sub-clades included at least one member from all eleven core dinoflagellate species, suggesting duplication in their shared ancestor. Conservation within sub-clades varied, suggesting different selection pressures. Phylogenetic analysis of eIF4E in core dinoflagellates revealed complex layering of duplication and conservation when compared to other eukaryotes. Our results suggest that the diverse eIF4E family in core dinoflagellates may provide a toolkit to enable selective translation as a strategy for controlling gene expression in these enigmatic eukaryotes.

Differential mRNA expression of neuroinflammatory modulators in the spinal cord and thalamus of type 2 diabetic monkeys.

PubMed

Ding, Huiping; Kiguchi, Norikazu; Kishioka, Shiroh; Ma, Tao; Peters, Christopher M; Ko, Mei-Chuan

2018-05-11

Given that diabetes-associated complications are closely associated with neuroinflammation, it is imperative to study potential changes in neuroinflammatory modulators in the central nervous system of diabetic primates. The mRNA levels of pro- and anti-inflammatory cytokines, toll-like receptors (TLRs), growth factors, and cannabinoid receptors were compared in the spinal dorsal horn (SDH) and thalamus of naturally occurring type 2 diabetic monkeys and an age-matched control group using reverse transcription and quantitative real-time polymerase chain reaction. In the SDH of diabetic monkeys, mRNA levels of proinflammatory cytokines (i.e. interleukin [IL]-1β and tumor necrosis factor [TNF] α), TLR1, and TLR2 were increased, whereas mRNA levels of IL-10, an anti-inflammatory cytokine, were decreased. No changes were observed in the mRNA levels of growth factors and cannabinoid receptors. In line with the mRNA data, TNFα immunoreactivity was significantly increased in diabetic monkeys. Moreover, mRNA expression levels of IL-1β, TNFα, TLR1, and TLR2 in the SDH were positively correlated with plasma glucose concentrations in all monkeys. Several ligands and receptors involved in neuroinflammation are simultaneously dysregulated in the spinal cord of diabetic monkeys. This primate disease model will facilitate the design of novel treatment approaches to ameliorate neuroinflammation-driven adverse effects in diabetic patients. © 2018 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

Butyric acid regulates progesterone and estradiol secretion via cAMP signaling pathway in porcine granulosa cells.

PubMed

Lu, Naisheng; Li, Mengjiao; Lei, Hulong; Jiang, Xueyuan; Tu, Weilong; Lu, Yang; Xia, Dong

2017-09-01

Butyric acid (BA), one of the short chain fatty acids (SCFAs), has positive actions on the metabolism, inflammation, etc. However, whether it influences the reproductive physiology and if so the detail mechanism involved has not yet been determined. In this study, the porcine granulosa cells (PGCs) were treated with gradient concentrations of BA. After 24h culture, 0.05mM BA significantly stimulated the progesterone (P 4 ) secretion (P<0.05), 5mM and 10mM BA significantly inhibited the P 4 secretion (P<0.05). Simultaneously, BA up-regulated the estradiol (E 2 ) secretion in a dose dependent manner, 5mM and 10mM BA significantly promoted the E 2 level (P<0.05). In addition, 10mM BA significantly promoted the G-protein-coupled receptor 41/43 mRNA (P<0.05). Interestingly, 5mM BA treatment significantly down-regulated cyclic adenosine monophosphate (cAMP) content (P<0.05), steroidogenic acute regulatory (StAR), steroidogenic factor 1 (SF1), P450scc in the mRNA and/or protein level (P<0.05), and these actions were reversed by cAMP activator forskolin (FK). Moreover, the co-treatment of 5mM BA and bupivacaine (BPC, the cAMP inhibitor) significantly accumulated the inhibition action of BPC on cAMP, the secretion of P 4 , and the abundance of StAR mRNA (P<0.05), inhibited the up-regulation of 5mM BA on the E 2 secretion (P<0.05). Further, the Global Proteome and KEGG pathway analysis found that 5mM BA significantly up-regulated the I3LM80 proteins (P<0.05), which is involved in the steroid biosynthesis signaling pathway. 5mM BA significantly decreased the F2Z5G3 protein level (P<0.05), and the cAMP signaling pathway. In conclusion, present findings for the first time demonstrated that BA could regulate the P 4 and E 2 hormone synthesis in PGCs via the cAMP signaling pathway. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Translation initiation mediated by nuclear cap-binding protein complex.

PubMed

Ryu, Incheol; Kim, Yoon Ki

2017-04-01

In mammals, cap-dependent translation of mRNAs is initiated by two distinct mechanisms: cap-binding complex (CBC; a heterodimer of CBP80 and 20)-dependent translation (CT) and eIF4E-dependent translation (ET). Both translation initiation mechanisms share common features in driving cap- dependent translation; nevertheless, they can be distinguished from each other based on their molecular features and biological roles. CT is largely associated with mRNA surveillance such as nonsense-mediated mRNA decay (NMD), whereas ET is predominantly involved in the bulk of protein synthesis. However, several recent studies have demonstrated that CT and ET have similar roles in protein synthesis and mRNA surveillance. In a subset of mRNAs, CT preferentially drives the cap-dependent translation, as ET does, and ET is responsible for mRNA surveillance, as CT does. In this review, we summarize and compare the molecular features of CT and ET with a focus on the emerging roles of CT in translation. [BMB Reports 2017; 50(4): 186-193].

Structural insights into the role of diphthamide on elongation factor 2 in messenger RNA reading frame maintenance.

PubMed

Pellegrino, Simone; Demeshkina, Natalia; Mancera-Martinez, Eder; Melnikov, Sergey; Simonetti, Angelita; Myasnikov, Alexander; Yusupov, Marat; Yusupova, Gulnara; Hashem, Yaser

2018-06-07

One of the most critical steps of protein biosynthesis is the coupled movement of messenger RNA (mRNA), that encodes genetic information, with transfer RNAs (tRNAs) on the ribosome. In eukaryotes this process is catalyzed by a conserved G-protein, the elongation factor 2 (eEF2), which carries a unique post-translational modification, called diphthamide, found in all eukaryotic species. Here we present near-atomic resolution cryo-EM structures of yeast 80S ribosome complexes containing mRNA, tRNA and eEF2 trapped in different GTP-hydrolysis states which provide further structural insights on the role of diphthamide in the mechanism of translation fidelity in eukaryotes. Copyright © 2018. Published by Elsevier Ltd.

Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis.

PubMed

Wilson, Fiona A; Suryawan, Agus; Orellana, Renán A; Nguyen, Hanh V; Jeyapalan, Asumthia S; Gazzaneo, Maria C; Davis, Teresa A

2008-10-01

Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 microg x kg(-1) x day(-1)) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) (P<0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level (P<0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1.eIF4E complex association, and increased active eIF4E.eIF4G complex formation (P<0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex.

Intronic SNP in ESR1 encoding human estrogen receptor alpha is associated with brain ESR1 mRNA isoform expression and behavioral traits.

PubMed

Pinsonneault, Julia K; Frater, John T; Kompa, Benjamin; Mascarenhas, Roshan; Wang, Danxin; Sadee, Wolfgang

2017-01-01

Genetic variants of ESR1 have been implicated in multiple diseases, including behavioral disorders, but causative variants remain uncertain. We have searched for regulatory variants affecting ESR1 expression in human brain, measuring allelic ESR1 mRNA expression in human brain tissues with marker SNPs in exon4 representing ESR1-008 (or ESRα-36), and in the 3'UTR of ESR1-203, two main ESR1 isoforms in brain. In prefrontal cortex from subjects with bipolar disorder, schizophrenia, and controls (n = 35 each; Stanley Foundation brain bank), allelic ESR1 mRNA ratios deviated from unity up to tenfold at the exon4 marker SNP, with large allelic ratios observed primarily in bipolar and schizophrenic subjects. SNP scanning and targeted sequencing identified rs2144025, associated with large allelic mRNA ratios (p = 1.6E10-6). Moreover, rs2144025 was significantly associated with ESR1 mRNA levels in the Brain eQTL Almanac and in brain regions in the Genotype-Tissue Expression project. In four GWAS cohorts, rs2104425 was significantly associated with behavioral traits, including: hypomanic episodes in female bipolar disorder subjects (GAIN bipolar disorder study; p = 0.0004), comorbid psychological symptoms in both males and females with attention deficit hyperactivity disorder (GAIN ADHD, p = 0.00002), psychological diagnoses in female children (eMERGE study of childhood health, subject age ≥9, p = 0.0009), and traits in schizophrenia (e.g., grandiose delusions, GAIN schizophrenia, p = 0.0004). The first common ESR1 variant (MAF 12-33% across races) linked to regulatory functions, rs2144025 appears conditionally to affect ESR1 mRNA expression in the brain and modulate traits in behavioral disorders.

Intronic SNP in ESR1 encoding human estrogen receptor alpha is associated with brain ESR1 mRNA isoform expression and behavioral traits

PubMed Central

Kompa, Benjamin; Mascarenhas, Roshan; Wang, Danxin; Sadee, Wolfgang

2017-01-01

Genetic variants of ESR1 have been implicated in multiple diseases, including behavioral disorders, but causative variants remain uncertain. We have searched for regulatory variants affecting ESR1 expression in human brain, measuring allelic ESR1 mRNA expression in human brain tissues with marker SNPs in exon4 representing ESR1-008 (or ESRα-36), and in the 3’UTR of ESR1-203, two main ESR1 isoforms in brain. In prefrontal cortex from subjects with bipolar disorder, schizophrenia, and controls (n = 35 each; Stanley Foundation brain bank), allelic ESR1 mRNA ratios deviated from unity up to tenfold at the exon4 marker SNP, with large allelic ratios observed primarily in bipolar and schizophrenic subjects. SNP scanning and targeted sequencing identified rs2144025, associated with large allelic mRNA ratios (p = 1.6E10-6). Moreover, rs2144025 was significantly associated with ESR1 mRNA levels in the Brain eQTL Almanac and in brain regions in the Genotype-Tissue Expression project. In four GWAS cohorts, rs2104425 was significantly associated with behavioral traits, including: hypomanic episodes in female bipolar disorder subjects (GAIN bipolar disorder study; p = 0.0004), comorbid psychological symptoms in both males and females with attention deficit hyperactivity disorder (GAIN ADHD, p = 0.00002), psychological diagnoses in female children (eMERGE study of childhood health, subject age ≥9, p = 0.0009), and traits in schizophrenia (e.g., grandiose delusions, GAIN schizophrenia, p = 0.0004). The first common ESR1 variant (MAF 12–33% across races) linked to regulatory functions, rs2144025 appears conditionally to affect ESR1 mRNA expression in the brain and modulate traits in behavioral disorders. PMID:28617822

Uroepithelial cells are part of a mucosal cytokine network.

PubMed Central

Hedges, S; Agace, W; Svensson, M; Sjögren, A C; Ceska, M; Svanborg, C

1994-01-01

This study compared the cytokine production of uroepithelial cell lines in response to gram-negative bacteria and inflammatory cytokines. Human kidney (A498) and bladder (J82) epithelial cell lines were stimulated with either Escherichia coli Hu734, interleukin 1 alpha (IL-1 alpha), or tumor necrosis factor alpha (TNF-alpha). Supernatant samples were removed, and the RNA was extracted from cells at 0, 2, 6, and 24 h. The secreted cytokine levels were determined by bioassay or immunoassay; mRNA was examined by reverse transcription-PCR. The two cell lines secreted IL-6 and IL-8 constitutively. IL-6 and IL-8 mRNA were constitutively produced in both cell lines; IL-1 beta mRNA was detected in J82 cells. IL-1 alpha induced significantly higher levels of IL-6 secretion than did E. coli Hu734 or TNF-alpha. IL-1 alpha and TNF-alpha induced significantly higher levels of IL-8 secretion than did E. coli Hu734. Secreted IL-1 beta was not detected; IL-1 alpha and TNF-alpha were not detected above the levels used for stimulation. IL-1 alpha, IL-1 beta, IL-6, and IL-8 mRNAs were detected in both cell lines after exposure to the stimulants. TNF-alpha mRNA was occasionally detected in the J82 cell line after TNF-alpha stimulation. Cytokine (IL-6 and IL-8) and control (glyceraldehyde 3-phosphate dehydrogenase [G3PDH] and beta-actin) mRNA concentrations were quantitated with internal PCR standards. Cytokine mRNA levels relative to beta-actin mRNA levels were the highest in E. coli-stimulated cells. In comparison, the cytokine mRNA levels relative to G3PDH mRNA levels were the highest in IL-1 alpha-stimulated cells. beta-Actin mRNA levels decreased after bacterial stimulation but not after cytokine stimulation, while G3PDH mRNA levels increased in response to all of the stimulants tested. These results suggested that E. coli Hu734 lowered the beta-actin mRNA levels in uroepithelial cells, thus distorting the IL-6 and IL-8 mRNA levels relative to this control. In summary, E. coli IL-1 alpha and TNF-alpha were found to activate the de novo synthesis and secretion of IL-6 and IL-8 in uroepithelial cells. These results emphasize the role of epithelial cells in cytokine-mediated responses during the early stages of infection. Images PMID:8188354

Multiparametric in situ mRNA hybridization analysis to predict disease recurrence in patients with colon carcinoma.

PubMed Central

Kitadai, Y.; Ellis, L. M.; Tucker, S. L.; Greene, G. F.; Bucana, C. D.; Cleary, K. R.; Takahashi, Y.; Tahara, E.; Fidler, I. J.

1996-01-01

We examined the expression level of several genes that regulate different steps of metastasis in formalin-fixed, paraffin-embedded archival specimens of primary human colon carcinomas from patients with at least 5 years of follow-up. The expression of epidermal growth factor receptor, basic fibroblast growth factor, type IV collagenase, E-cadherin, and multidrug resistance (mdr-1) was examined by a colorimetric in situ mRNA hybridization technique concentrating on reactivity at the periphery of the neoplasms. The in situ hybridization technique revealed inter- and intratumor heterogeneity for expression of the metastasis-related genes. The expression of basic fibroblast growth factor, collagenase type IV, epidermal growth factor receptor, and mdr-1 mRNA was higher in Dukes's stage D than in Dukes' stage B tumors. Among the 22 Dukes' stage B neoplasms, 5 specimens exhibited a high expression level of epidermal growth factor receptor, basic fibroblast growth factor, and collagenase type IV. Clinical outcome data (5-year follow-up) revealed that all 5 patients with Dukes' stage B tumors developed distant metastasis (recurrent disease), whereas the other 17 patients with Dukes' stage B tumors expressing low levels of the metastasis-related genes were disease-free. Multivariate analysis identified high levels of expression of collagenase type IV and low levels of expression of E-cadherin as independent factors significantly associated with metastasis or recurrent disease. More specifically, metastatic or recurrent disease was associated with a high ratio (> 1.35) of expression of collagenase type IV to E-cadherin (specificity of 95%). Collectively, the data show that multiparametric in situ hybridization analysis for several metastasis-related genes may predict the metastatic potential, and hence the clinical outcome, of individual lymph-node-negative human colon cancers. Images Figure 1 Figure 2 PMID:8909244

The effect of thyroid hormone and a long-acting somatostatin analogue on TtT-97 murine thyrotropic tumors.

PubMed

Woodmansee, W W; Gordon, D F; Dowding, J M; Stolz, B; Lloyd, R V; James, R A; Wood, W M; Ridgway, E C

2000-07-01

Thyroid hormone inhibits thyrotropin (TSH) production and thyrotrope growth. Somatostatin has been implicated as a synergistic factor in the inhibition of thyrotrope function. We have previously shown that pharmacological doses of thyroid hormone (levothyroxine [LT4]) inhibit growth of murine TtT-97 thyrotropic tumors in association with upregulation of somatostatin receptor type 5 (sst5) mRNA and somatostatin receptor binding. In the current study, we examined the effect of physiological thyroid hormone replacement alone or in combination with the long-acting somatostatin analogue, Sandostatin LAR, on thyrotropic tumor growth, thyrotropin growth factor-beta (TSH-beta), and sst5 mRNA expression, as well as somatostatin receptor binding sites. Physiological LT4 replacement therapy resulted in tumor shrinkage in association with increased sst5 mRNA levels, reduced TSH-beta mRNA levels and enhanced somatostatin receptor binding. Sandostatin LAR alone had no effect on any parameter measured. However, Sandostatin LAR combined with LT4 synergistically inhibited TSH-beta mRNA production and reduced final tumor weights to a greater degree. In this paradigm, Sandostatin LAR required a euthyroid status to alter thyrotrope parameters. These data suggest an important interaction between the somatostatinergic system and thyroid hormone in the regulation of thyrotrope cell structure and function.

Altered PIWI-LIKE 1 and PIWI-LIKE 2 mRNA expression in ejaculated spermatozoa of men with impaired sperm characteristics.

PubMed

Giebler, Maria; Greither, Thomas; Müller, Lisa; Mösinger, Carina; Behre, Hermann M

2018-01-01

In about half the cases of involuntary childlessness, a male infertility factor is involved. The PIWI-LIKE genes, a subclade of the Argonaute protein family, are involved in RNA silencing and transposon control in the germline. Knockout of murine Piwi-like 1 and 2 homologs results in complete infertility in males. The aim of this study was to analyze whether the mRNA expression of human PIWI-LIKE 1-4 genes is altered in ejaculated spermatozoa of men with impaired sperm characteristics. Ninety male participants were included in the study, among which 47 were with normozoospermia, 36 with impaired semen characteristics according to the World Health Organization (WHO) manual, 5 th edition, and 7 with azoospermia serving as negative control for the PIWI-LIKE 1-4 mRNA expression in somatic cells in the ejaculate. PIWI-LIKE 1-4 mRNA expression in the ejaculated spermatozoa of the participants was measured by quantitative real-time PCR. In nonazoospermic men, PIWI-LIKE 1-4 mRNA was measurable in ejaculated spermatozoa in different proportions. PIWI-LIKE 1 (100.0%) and PIWI-LIKE 2 (49.4%) were more frequently expressed than PIWI-LIKE 3 (9.6%) and PIWI-LIKE 4 (15.7%). Furthermore, a decreased PIWI-LIKE 2 mRNA expression showed a significant correlation with a decreased sperm count (P = 0.022) and an increased PIWI-LIKE 1 mRNA expression with a decreased progressive motility (P = 0.048). PIWI-LIKE 1 and PIWI-LIKE 2 mRNA expression exhibited a significant association with impaired sperm characteristics and may be a useful candidate for the evaluation of the impact of PIWI-LIKE 1-4 mRNA expression on male infertility.

Targeting the eIF4F translation initiation complex: a critical nexus for cancer development.

PubMed

Pelletier, Jerry; Graff, Jeremy; Ruggero, Davide; Sonenberg, Nahum

2015-01-15

Elevated protein synthesis is an important feature of many cancer cells and often arises as a consequence of increased signaling flux channeled to eukaryotic initiation factor 4F (eIF4F), the key regulator of the mRNA-ribosome recruitment phase of translation initiation. In many cellular and preclinical models of cancer, eIF4F deregulation results in changes in translational efficiency of specific mRNA classes. Importantly, many of these mRNAs code for proteins that potently regulate critical cellular processes, such as cell growth and proliferation, enhanced cell survival and cell migration that ultimately impinge on several hallmarks of cancer, including increased angiogenesis, deregulated growth control, enhanced cellular survival, epithelial-to-mesenchymal transition, invasion, and metastasis. By being positioned as the molecular nexus downstream of key oncogenic signaling pathways (e.g., Ras, PI3K/AKT/TOR, and MYC), eIF4F serves as a direct link between important steps in cancer development and translation initiation. Identification of mRNAs particularly responsive to elevated eIF4F activity that typifies tumorigenesis underscores the critical role of eIF4F in cancer and raises the exciting possibility of developing new-in-class small molecules targeting translation initiation as antineoplastic agents. ©2014 American Association for Cancer Research.

Mutational Analysis of Plant Cap-Binding Protein eIF4E Reveals Key Amino Acids Involved in Biochemical Functions and Potyvirus Infection▿

PubMed Central

German-Retana, Sylvie; Walter, Jocelyne; Doublet, Bénédicte; Roudet-Tavert, Geneviève; Nicaise, Valérie; Lecampion, Cécile; Houvenaghel, Marie-Christine; Robaglia, Christophe; Michon, Thierry; Le Gall, Olivier

2008-01-01

The eukaryotic translation initiation factor 4E (eIF4E) (the cap-binding protein) is involved in natural resistance against several potyviruses in plants. In lettuce, the recessive resistance genes mo11 and mo12 against Lettuce mosaic virus (LMV) are alleles coding for forms of eIF4E unable, or less effective, to support virus accumulation. A recombinant LMV expressing the eIF4E of a susceptible lettuce variety from its genome was able to produce symptoms in mo11 or mo12 varieties. In order to identify the eIF4E amino acid residues necessary for viral infection, we constructed recombinant LMV expressing eIF4E with point mutations affecting various amino acids and compared the abilities of these eIF4E mutants to complement LMV infection in resistant plants. Three types of mutations were produced in order to affect different biochemical functions of eIF4E: cap binding, eIF4G binding, and putative interaction with other virus or host proteins. Several mutations severely reduced the ability of eIF4E to complement LMV accumulation in a resistant host and impeded essential eIF4E functions in yeast. However, the ability of eIF4E to bind a cap analogue or to fully interact with eIF4G appeared unlinked to LMV infection. In addition to providing a functional mutational map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle might be distinct from its physiological function in cellular mRNA translation. PMID:18480444

LARP4 Is Regulated by Tumor Necrosis Factor Alpha in a Tristetraprolin-Dependent Manner

PubMed Central

Mattijssen, Sandy

2015-01-01

LARP4 is a protein with unknown function that independently binds to poly(A) RNA, RACK1, and the poly(A)-binding protein (PABPC1). Here, we report on its regulation. We found a conserved AU-rich element (ARE) in the human LARP4 mRNA 3′ untranslated region (UTR). This ARE, but not its antisense version or a point-mutated version, significantly decreased the stability of β-globin reporter mRNA. We found that overexpression of tristetraprolin (TTP), but not its RNA binding mutant or the other ARE-binding proteins tested, decreased cellular LARP4 levels. RNA coimmunoprecipitation showed that TTP specifically associated with LARP4 mRNA in vivo. Consistent with this, mouse LARP4 accumulated to higher levels in TTP gene knockout (KO) cells than in control cells. Stimulation of WT cells with tumor necrosis factor alpha (TNF-α), which rapidly induces TTP, robustly decreased LARP4 with a coincident time course but had no such effect on LARP4B or La protein or on LARP4 in the TTP KO cells. The TNF-α-induced TTP pulse was followed by a transient decrease in LARP4 mRNA that was quickly followed by a subsequent transient decrease in LARP4 protein. Involvement of LARP4 as a target of TNF-α–TTP regulation provides a clue as to how its functional activity may be used in a physiologic pathway. PMID:26644407

Expression and Functional Activities of Selected Sulfotransferase Isoforms in BeWo Cells and Primary Cytotrophoblast Cells

PubMed Central

Mitra, Pallabi; Audus, Kenneth L.

2009-01-01

Several cytosolic sulfotransferase enzyme isoforms are functional in placenta but there is limited information available on the utility of cultured trophoblast cells for studying sulfation. The trophoblast cell layer constitutes the rate-determining barrier for trans-placental transfer. The objective of this work was to examine the mRNA expression and enzyme activities of four sulfotransferase isoforms reported to be functional in human placenta (SULT1A1, SULT1A3, SULT1E1, and SULT2A1) in primary cytotrophoblast cells and the trophoblast-like BeWo cell line. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine mRNA expression. Enzyme activities were assessed using the following substrates: 4-nitrophenol for SULT1A1, dopamine for SULT1A3, 17β-estradiol for SULT1E1, and dehydroepiandrosterone for SULT2A1. For 4-nitrophenol and dopamine sulfation, apparent Km values, response to inhibitors (2,6-dichloro-4-nitrophenol and sodium chloride), and thermal stability profiles indicated that 4-nitrophenol and dopamine sulfation in BeWo cells were being mediated by SULT1A1 and SULT1A3, respectively. SULT1A1 and SULT1A3 were also functional in the cytotrophoblast cells. Both at the protein and at the mRNA levels, SULT1A1 was more abundant in BeWo cells in comparison to the primary cytotrophoblast cells. SULT1E1 and SULT2A1 mRNA were not detected in the cytotrophoblasts. SULT1E1 mRNA was weakly expressed in BeWo but there was negligible functional activity. Although SULT2A1 mRNA was abundantly expressed in BeWo, Western blot and enzyme activities revealed that the protein is not expressed in BeWo cells. The results suggest that the BeWo cells and the cytotrophoblast cells can be used to examine the roles of SULT1A1 and SULT1A3 in placental metabolism. PMID:19646966

Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation.

PubMed

Suryawan, Agus; Jeyapalan, Asumthia S; Orellana, Renan A; Wilson, Fiona A; Nguyen, Hanh V; Davis, Teresa A

2008-10-01

Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E.eIF4G complex and increased eIF4E.4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein beta-subunit-like protein (GbetaL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors.

Chemical chaperon 4-phenylbutyrate protects against the endoplasmic reticulum stress-mediated renal fibrosis in vivo and in vitro.

PubMed

Liu, Shing-Hwa; Yang, Ching-Chin; Chan, Ding-Cheng; Wu, Cheng-Tien; Chen, Li-Ping; Huang, Jenq-Wen; Hung, Kuan-Yu; Chiang, Chih-Kang

2016-04-19

Renal tubulointerstitial fibrosis is the common and final pathologic change of kidney in end-stage renal disease. Interesting, endoplasmic reticulum (ER) stress is known to contribute to the pathophysiological mechanisms during the development of renal fibrosis. Here, we investigated the effects of chemical chaperon sodium 4-phenylbutyrate (4-PBA) on renal fibrosis in vivo and in vitro. In a rat unilateral ureteral obstruction (UUO) model, 4-PBA mimicked endogenous ER chaperon in the kidneys and significantly reduced glucose regulated protein 78 (GRP78), CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), activating transcription factor 4 (ATF4), and phosphorylated JNK protein expressions as well as restored spliced X-box-binding protein 1 (XBP1) expressions in the kidneys of UUO rats. 4-PBA also attenuated the increases of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) protein expressions, tubulointerstitial fibrosis, and apoptosis in the kidneys of UUO rats. Moreover, transforming growth factor (TGF)-β markedly increased ER stress-associated molecules, profibrotic factors, and apoptotic markers in the renal tubular cells (NRK-52E), all of which could be significantly counteracted by 4-PBA treatment. 4-PBA also diminished TGF-β-increased CTGF promoter activity and CTGF mRNA expression in NRK-52E cells. Taken together, our results indicated that 4-PBA acts as an ER chaperone to ameliorate ER stress-induced renal tubular cell apoptosis and renal fibrosis.

The effects of estrogen receptors α- and β-specific agonists and antagonists on cell proliferation and energy metabolism in human bone cell line.

PubMed

Somjen, D; Katzburg, S; Sharon, O; Grafi-Cohen, M; Knoll, E; Stern, N

2011-02-01

In cultured human osteoblasts estradiol-17β (E2) modulated DNA synthesis, the specific activity of creatine kinase BB (CK), 12 and 15 lipoxygenase (LO) mRNA expression and formation of 12- and 15-hydroxyeicosatetraenoic acid (HETE). We now investigate the response of human bone cell line (SaOS2) to phytoestrogens and estrogen receptors (ER)-specific agonists and antagonists. Treatment of SaSO2 with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ-specific agonist), 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα-specific agonist), biochainin A (BA), daidzein (D), genistein (G) and raloxifene (Ral) showed increased DNA synthesis and CK. Ral inhibited completely all stimulations except DPN and to some extent D. The ERα-specific antagonist methyl-piperidino-pyrazole (MPP) and the ERβ-specific antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a]pyrimidin-3-yl] phenol (PTHPP) inhibited DNA synthesis, CK and reactive oxygen species (ROS) formation induced by estrogens according to their receptors affinity. The LO inhibitor baicaleine inhibited only E2, DPN and G's effects. E2 and Ral unlike all other compounds had no effect on ERα mRNA expression, while ERβ mRNA expression was stimulated by all compounds. All compounds modulated the expression of 12LO and 15LO mRNA, except E2, PPT and Ral for 12LO, and 12- and 15-HETE productions and stimulated ROS formation which was inhibited by NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and N-acetyl cysteine and the estrogen inhibitor ICI. DPI did not affect hormonal-induced DNA and CK. In conclusion, we provide evidence for the separation of mediation via ERα and ERβ pathways in the effects of estrogenic compounds on osteoblasts, but the role of LO/HETE/ROS is unclear. Copyright © 2010 Wiley-Liss, Inc.

Circadian clock-dependent and -independent posttranscriptional regulation underlies temporal mRNA accumulation in mouse liver

PubMed Central

Wang, Jingkui; Yeung, Jake; Gobet, Cédric; Sobel, Jonathan; Lück, Sarah; Molina, Nacho; Naef, Felix

2018-01-01

The mammalian circadian clock coordinates physiology with environmental cycles through the regulation of daily oscillations of gene expression. Thousands of transcripts exhibit rhythmic accumulations across mouse tissues, as determined by the balance of their synthesis and degradation. While diurnally rhythmic transcription regulation is well studied and often thought to be the main factor generating rhythmic mRNA accumulation, the extent of rhythmic posttranscriptional regulation is debated, and the kinetic parameters (e.g., half-lives), as well as the underlying regulators (e.g., mRNA-binding proteins) are relatively unexplored. Here, we developed a quantitative model for cyclic accumulations of pre-mRNA and mRNA from total RNA-seq data, and applied it to mouse liver. This allowed us to identify that about 20% of mRNA rhythms were driven by rhythmic mRNA degradation, and another 15% of mRNAs regulated by both rhythmic transcription and mRNA degradation. The method could also estimate mRNA half-lives and processing times in intact mouse liver. We then showed that, depending on mRNA half-life, rhythmic mRNA degradation can either amplify or tune phases of mRNA rhythms. By comparing mRNA rhythms in wild-type and Bmal1−/− animals, we found that the rhythmic degradation of many transcripts did not depend on a functional BMAL1. Interestingly clock-dependent and -independent degradation rhythms peaked at distinct times of day. We further predicted mRNA-binding proteins (mRBPs) that were implicated in the posttranscriptional regulation of mRNAs, either through stabilizing or destabilizing activities. Together, our results demonstrate how posttranscriptional regulation temporally shapes rhythmic mRNA accumulation in mouse liver. PMID:29432155

Vascular Endothelial Growth Factor (VEGF) mRNA Isoforms are Altered in Bovine Granulosa Cells (GC) by Circulating Progestin Concentrations (P4) and May Indicate Follicle Status and Oocyte Competence

USDA-ARS?s Scientific Manuscript database

Previously, Melengestrol Acetate (MGA) fed for 14 d (0.5mg/cow/d; < 1 ng/ml P4) resulted in persistent follicles with increased size, decreased number of GC/follicular fluid (FF) volume, and less fertile oocytes. An experiment was conducted to determine effects of circulating P4 on amount of mRNA fo...

Nuclear factor-E2-related factor 2 is a major determinant of bile acid homeostasis in the liver and intestine

PubMed Central

Weerachayaphorn, Jittima; Mennone, Albert; Soroka, Carol J.; Harry, Kathy; Hagey, Lee R.; Kensler, Thomas W.

2012-01-01

The transcription factor nuclear factor-E2-related factor 2 (Nrf2) is a key regulator for induction of hepatic detoxification and antioxidant mechanisms, as well as for certain hepatobiliary transporters. To examine the role of Nrf2 in bile acid homeostasis and cholestasis, we assessed the determinants of bile secretion and bile acid synthesis and transport before and after bile duct ligation (BDL) in Nrf2−/− mice. Our findings indicate reduced rates of biliary bile acid and GSH excretion, higher levels of intrahepatic bile acids, and decreased expression of regulators of bile acid synthesis, Cyp7a1 and Cyp8b1, in Nrf2−/− compared with wild-type control mice. The mRNA expression of the bile acid transporters bile salt export pump (Bsep) and organic solute transporter (Ostα) were increased in the face of impaired expression of the multidrug resistance-associated proteins Mrp3 and Mrp4. Deletion of Nrf2 also decreased ileal apical sodium-dependent bile acid transporter (Asbt) expression, leading to reduced bile acid reabsorption and increased loss of bile acid in feces. Finally, when cholestasis is induced by BDL, liver injury was not different from that in wild-type BDL mice. These Nrf2−/− mice also had increased pregnane X receptor (Pxr) and Cyp3a11 mRNA expression in association with enhanced hepatic bile acid hydroxylation. In conclusion, this study finds that Nrf2 plays a major role in the regulation of bile acid homeostasis in the liver and intestine. Deletion of Nrf2 results in a cholestatic phenotype but does not augment liver injury following BDL. PMID:22345550

β-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies.

PubMed

Kraan, Claudine M; Cornish, Kim M; Bui, Quang M; Li, Xin; Slater, Howard R; Godler, David E

2018-01-01

Relationships between Fragile X Mental Retardation 1 (FMR1) mRNA levels in blood and intragenic FMR1 CGG triplet expansions support the pathogenic role of RNA gain of function toxicity in premutation (PM: 55-199 CGGs) related disorders. Real-time PCR (RT-PCR) studies reporting these findings normalised FMR1 mRNA level to a single internal control gene called β-glucuronidase (GUS). This study evaluated FMR1 mRNA-CGG correlations in 33 PM and 33 age- and IQ-matched control females using three normalisation strategies in peripheral blood mononuclear cells (PBMCs): (i) GUS as a single internal control; (ii) the mean of GUS, Eukaryotic Translation Initiation Factor 4A2 (EIF4A2) and succinate dehydrogenase complex flavoprotein subunit A (SDHA); and (iii) the mean of EIF4A2 and SDHA (with no contribution from GUS). GUS mRNA levels normalised to the mean of EIF4A2 and SDHA mRNA levels and EIF4A2/SDHA ratio were also evaluated. FMR1mRNA level normalised to the mean of EIF4A2 and SDHA mRNA levels, with no contribution from GUS, showed the most significant correlation with CGG size and the greatest difference between PM and control groups (p = 10-11). Only 15% of FMR1 mRNA PM results exceeded the maximum control value when normalised to GUS, compared with over 42% when normalised to the mean of EIF4A2 and SDHA mRNA levels. Neither GUS mRNA level normalised to the mean RNA levels of EIF4A2 and SDHA, nor to the EIF4A2/SDHA ratio were correlated with CGG size. However, greater variability in GUS mRNA levels were observed for both PM and control females across the full range of CGG repeat as compared to the EIF4A2/SDHA ratio. In conclusion, normalisation with multiple control genes, excluding GUS, can improve assessment of the biological significance of FMR1 mRNA-CGG size relationships.

Transforming growth factor-beta 1 (TGF-beta1) promotes IL-2 mRNA expression through the up-regulation of NF-kappaB, AP-1 and NF-AT in EL4 cells.

PubMed

Han, S H; Yea, S S; Jeon, Y J; Yang, K H; Kaminski, N E

1998-12-01

Transforming growth factor beta1 (TGF-beta1) has been previously shown to modulate interleukin 2 (IL-2) secretion by activated T-cells. In the present studies, we determined that TGF-beta1 induced IL-2 mRNA expression in the murine T-cell line EL4, in the absence of other stimuli. IL-2 mRNA expression was significantly induced by TGF-beta1 (0.1-1 ng/ml) over a relatively narrow concentration range, which led to the induction of IL-2 secretion. Under identical condition, we examined the effect of TGF-beta1 on the activity of nuclear factor AT (NF-AT), nuclear factor kappaB (NF-kappaB), activator protein-1 (AP-1) and octamer, all of which contribute to the regulation of IL-2 gene expression. Electrophoretic mobility shift assays showed that TGF-beta1 markedly increased NF-AT, NF-kappaB and AP-1 binding to their respective cognate DNA binding sites, whereas octamer binding remained constant, as compared with untreated cells. Employing a reporter gene expression system with p(NF-kappaB)3-CAT, p(NF-AT)3-CAT and p(AP-1)3-CAT, TGF-beta1 treatment of transfected EL4 cells induced a dose-related increase in chloramphenicol acetyltransferase activity that correlated well with the DNA binding profile found in the electrophoretic mobility shift assay studies. These results show that TGF-beta1, in the absence of any additional stimuli, up-regulates the activity of key transcription factors involved in IL-2 gene expression, including NF-AT, NF-kappaB and AP-1, to help promote IL-2 mRNA expression by EL4 cells.

Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus

NASA Technical Reports Server (NTRS)

Krumenacker, J. S.; Hyder, S. M.; Murad, F.

2001-01-01

Previous reports that investigated the regulation of the NO/soluble guanylyl cyclase (sGC)/cGMP pathway by estrogenic compounds have focused primarily on the levels of NO, NO-producing enzymes, and cGMP in various tissues. In this study, we demonstrate that 17beta-estradiol (E2) regulates the alpha(1) and beta(1) subunits of the NO receptor, sGC, at the mRNA and protein levels in rat uterus. Using real-time quantitative PCR, we found that within 1 h of in vivo E2 administration to rats, sGC mRNA levels begin to diminish. After 3 h, there is a maximal diminution of sGC mRNA expression (sGC alpha(1) 10% and sGC beta(1) 33% of untreated). This effect was blocked by the estrogen receptor antagonist, ICI 182,780, indicating that estrogen receptor is required. The effect of E2 also was observed in vitro with incubations of uterine tissue, indicating that the response does not depend on the secondary release of other hormones or factors from other tissues. Puromycin did not block the effect, suggesting the effects occur because of preexisting factors in uterine tissues and do not require new protein synthesis. Using immunoblot analysis, we found that sGC protein levels also were reduced by E2 over a similar time course as the sGC mRNA. We conclude that sGC plays a vital role in the NO/sGC/cGMP regulatory pathway during conditions of elevated estrogen levels in the rat uterus as a result of the reduction of sGC expression.

Expression of early growth response factor-1 in rats with cerulein-induced acute pancreatitis and its significance

PubMed Central

Gong, Lan-Bo; He, Li; Liu, Yang; Chen, Xue-Qing; Jiang, Bo

2005-01-01

AIM: To observe the expressions of early growth response factor-1 (Egr-1) and tissue factor (TF) in rats with cerulein-induced acute pancreatitis and to explore its significance. METHODS: A large dose of cerulein was used to create the experimental acute pancreatitis model in rats. The changes of Egr-1 mRNA and protein in rats were observed during 30 min to 4 h after the treatment and immunohistochemical method was used to observe the localized expression of Egr-1 in tissues. In addition to the mRNA expression of Egr-1 target gene, TF was also observed. A blank control group, and a bombesin-administered group were used for comparison. RESULTS: After the stimulation of a large dose of cerulein, the rats showed typical inflammatory changes of acute pancreatitis. Thirty minutes after the stimulation, the mRNA expression of Egr-1 in the pancreatic tissue reached its peak and then declined, while the expression of Egr-1 protein reached its peak 2 h after the stimulation. Histologically, 2 h after the stimulation, almost all pancreatic acinar cells had the expression of Egr-1 protein, which was focused in the nuclei. The mRNA expression of TF occurred 1 h after the stimulation and gradually increased within 4 h. However, a large dose of bombesin only stimulated the pancreatic tissue to produce a little mRNA expression of Egr-1 and no mRNA expression of Egr-1 protein and TF. CONCLUSION: Egr-1 as a pro-inflammatory transcription factor may play an important role in the pathogenesis of acute pancreatitis by modulating the expression of TF. PMID:16124058

Triage of women with low-grade cervical lesions--HPV mRNA testing versus repeat cytology.

PubMed

Sørbye, Sveinung Wergeland; Arbyn, Marc; Fismen, Silje; Gutteberg, Tore Jarl; Mortensen, Elin Synnøve

2011-01-01

In Norway, women with low-grade squamous intraepithelial lesions (LSIL) are followed up after six months in order to decide whether they should undergo further follow-up or be referred back to the screening interval of three years. A high specificity and positive predictive value (PPV) of the triage test is important to avoid unnecessary diagnostic and therapeutic procedures. At the University Hospital of North Norway, repeat cytology and the HPV mRNA test PreTect HPV-Proofer, detecting E6/E7 mRNA from HPV types 16, 18, 31, 33 and 45, are used in triage of women with ASC-US and LSIL. In this study, women with LSIL cytology in the period 2005-2008 were included (n = 522). Two triage methods were evaluated in two separate groups: repeat cytology only (n = 225) and HPV mRNA testing in addition to repeat cytology (n = 297). Histologically confirmed cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) was used as the study endpoint. Of 522 women with LSIL, 207 had biopsies and 125 of them had CIN2+. The sensitivity and specificity of repeat cytology (ASC-US or worse) were 85.7% (95% confidence interval (CI): 72.1, 92.2) and 54.4 % (95% CI: 46.9, 61.9), respectively. The sensitivity and specificity of the HPV mRNA test were 94.2% (95% CI: 88.7, 99.7) and 86.0% (95% CI: 81.5, 90.5), respectively. The PPV of repeat cytology was 38.4% (95% CI: 29.9, 46.9) compared to 67.0% (95% CI: 57.7, 76.4) of the HPV mRNA test. HPV mRNA testing was more sensitive and specific than repeat cytology in triage of women with LSIL cytology. In addition, the HPV mRNA test showed higher PPV. These data indicate that the HPV mRNA test is a better triage test for women with LSIL than repeat cytology.

Sestrins are differentially expressed with age in the skeletal muscle of men: A cross-sectional analysis.

PubMed

Zeng, Nina; D'Souza, Randall F; Mitchell, Cameron J; Cameron-Smith, David

2018-05-08

A gradual loss of skeletal muscle mass is a common feature of aging, leading to impaired insulin sensitivity and mobility. Sestrin1, 2, 3 are multifunctional proteins that regulate the mammalian target of rapamycin complex (mTORC1), autophagy and redox homeostasis. It is unclear how aging affects Sestrins and their downstream targets in human, therefore this study examined the basal expression of Sestrins in three age groups, young, middle-aged and older men and explored the mTORC1 pathway, autophagy markers and antioxidant regulation. Older men had less Sestrin1 and 3 protein and a different pattern of Sestrin2 electrophoretic mobility. The mRNA expression of SESN1 was highly upregulated in older men, but the discrepancy was not by microRNA expression. Although protein expressions of Sestrins were downregulated with aging, phosphorylation of AMP-dependent protein kinase (AMPKα Thr172 ) and read-outs of mTORC1 activation, ribosomal protein S6 kinase 1 (p70S6K1 Thr421/Ser424 ) and 4E-binding protein 1 (4E-BP1) mobility shift were unaltered. However, total p70S6K1 and 4E-BP1 were reduced in middle-age and older men. The mRNA expressions of autophagic markers including microtubule-associated protein 1 light chain 3 (LC3) and BCL2 interacting protein 3 (BNIP3) were upregulated in middle-age and older men. Although nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was upregulated in older men, the protein and mRNA expressions of its downstream antioxidants were either increased, decreased or unaltered. No clear relationship was observed between Sestrins and their downstream targets, yet it can be concluded that Sestrins proteins are clearly downregulated with aging. Copyright © 2017. Published by Elsevier Inc.

Inhibitory effect of 10-hydroxydecanoic acid on lipopolysaccharide-induced nitric oxide production via translational downregulation of interferon regulatory factor-1 in RAW264 murine macrophages.

PubMed

Takahashi, Keita; Sugiyama, Tsuyoshi; Tokoro, Shunji; Neri, Paol; Mori, Hiroshi

2013-08-01

Toll-like receptors (TLRs) play a critical role in innate immunity by recognizing pathogen-associated molecular patterns. Various environmental materials including lipids may affect TLR signaling and modulate innate immune responses. We previously reported that 10-hydroxy-trans-2-decenoic acid (10H2DA) inhibits lipopolysaccharide (LPS)-induced interleukin (IL)-6 and nitric oxide (NO) production via inhibiting NF-κB activation. In this study, we investigated the effect of 10-hydroxydecanoic acid (10HDA), a saturated fatty acid of 10H2DA, on LPS-induced cytokines/chemokines and NO production. 10HDA inhibited LPS-induced NO production, but not tumor necrosis factor-α or IL-6 production. LPS-induced activation of interferon (IFN)-stimulated response element, but not NF-κB, was inhibited by 10HDA. Phosphorylation of STAT1 and STAT2 was not affected, but IFN-regulatory factor (IRF)-1 production was significantly reduced by 10HDA. The LPS-induced increase of IRF-1 mRNA, however, was not affected by 10HDA. We found that IRF-1 mRNA level in the polysomal fraction was significantly decreased by 10HDA. Further, LPS-induced phosphorylation of Akt and 4E-BP1, which control mRNA translation, was markedly decreased. These results suggest that 10HDA inhibited LPS-induced NO production through inhibiting IRF-1 translation. These findings elucidate a novel mechanism for anti-inflammatory activity of medium-chain fatty acid 10HDA.

Comparing triage algorithms using HPV DNA genotyping, HPV E7 mRNA detection and cytology in high-risk HPV DNA-positive women.

PubMed

Luttmer, Roosmarijn; Berkhof, Johannes; Dijkstra, Maaike G; van Kemenade, Folkert J; Snijders, Peter J F; Heideman, Daniëlle A M; Meijer, Chris J L M

2015-06-01

High-risk human papillomavirus (hrHPV) DNA positive women require triage testing to identify those with high-grade cervical intraepithelial neoplasia or cancer (≥CIN2). Comparing three triage algorithms (1) E7 mRNA testing following HPV16/18/31/33/45/52/58 genotyping (E7 mRNA test), (2) HPV16/18 DNA genotyping and (3) cytology, for ≥CIN2 detection in hrHPV DNA-positive women. hrHPV DNA-positive women aged 18-63 years visiting gynecology outpatient clinics were included in a prospective observational cohort study. From these women a cervical scrape and colposcopy-directed biopsies were obtained. Cervical scrapes were evaluated by cytology, HPV DNA genotyping by bead-based multiplex genotyping of GP5+6+-PCR-products, and presence of HPV16/18/31/33/45/52/58 E7 mRNA using nucleic acid sequence-based amplification (NASBA) in DNA positive women for respective HPV types. Sensitivities and specificities for ≥CIN2 were compared between E7 mRNA test and HPV16/18 DNA genotyping in the total group (n=348), and E7 mRNA test and cytology in a subgroup of women referred for non-cervix-related gynecological complaints (n=133). Sensitivity for ≥CIN2 of the E7 mRNA test was slightly higher than that of HPV16/18 DNA genotyping (66.9% versus 60.9%; ratio 1.10, 95% CI: 1.0002-1.21), at similar specificity (54.8% versus 52.3%; ratio 1.05, 95% CI: 0.93-1.18). Neither sensitivity nor specificity of the E7 mRNA test differed significantly from that of cytology (sensitivity: 68.8% versus 75.0%; ratio 0.92, 95% CI: 0.72-1.17; specificity: 59.4% versus 65.3%; ratio 0.91, 95% CI: 0.75-1.10). For detection of ≥CIN2 in hrHPV DNA-positive women, an algorithm including E7 mRNA testing following HPV16/18/31/33/45/52/58 DNA genotyping performs similar to HPV16/18 DNA genotyping or cytology. Copyright © 2015 Elsevier B.V. All rights reserved.

Peripheral mRNA expression of pluripotency markers in bipolar disorder and the effect of long-term lithium treatment.

PubMed

Ferensztajn-Rochowiak, Ewa; Tarnowski, Maciej; Samochowiec, Jerzy; Michalak, Michal; Ratajczak, Mariusz Z; Rybakowski, Janusz K

2016-10-01

The aim was to evaluate the peripheral mRNA expression of pluripotency master transcriptional factors such as octamer-binding transcription factor 4 (Oct4), sex-determining region Y-box 2 (Sox2) and homeobox protein Nanog, in patients with bipolar disorder (BD), and the effect of long-term lithium treatment. Fifteen BD patients (aged 53±7years) not treated with lithium, with duration of illness>10years, 15 BD patients (aged 55±6years) treated with lithium for 8-40 years (mean 16years) and 15 control subjects (aged 50±5years) were included. Assessment of the mRNA levels of pluripotency markers (Oct-4, Sox 2 and Nanog) was performed, using the Real-time quantitative reverse transcription PCR (RQ-PCR) procedure, and the number of CD34+ very small embryonic-like stem cells (VSELs) was measured by flow cytometric analysis. In those BD patients not treated with lithium the expression of all three pluripotency genes was significantly higher than that in the control subjects. Oct-4, Sox2 and Nanog also positively correlated with the number of CD34+ VSELs/[ul] in this group. In the lithium-treated patients the mRNA levels of Nanog were significantly higher than in the control individuals and correlated with the number and % of CD34+ VSELs. The overexpression of the pluripotency master transcriptional factors in patients with a long duration of BD not treated with lithium, may contribute to the pathogenesis of the illness and make them potential biological markers of BD. Long-term lithium treatment may attenuate these excessive regenerative processes, especially in relation to the transcription factors Oct-4 and Sox2. Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.

Regulation of Kruppel-like factor 4, 9, and 13 genes and the steroidogenic genes LDLR, StAR, and CYP11A in ovarian granulosa cells.

PubMed

Natesampillai, Sekar; Kerkvliet, Jason; Leung, Peter C K; Veldhuis, Johannes D

2008-02-01

Kruppel-like factors (KLFs) are important Sp1-like eukaryotic transcriptional proteins. The LDLR, StAR, and CYP11A genes exhibit GC-rich Sp1-like sites, which have the potential to bind KLFs in multiprotein complexes. We now report that KLF4, KLF9, and KLF13 transcripts are expressed in and regulate ovarian cells. KLF4 and 13, but not KLF9, mRNA expression was induced and then repressed over time (P < 0.001). Combined LH and IGF-I stimulation increased KLF4 mRNA at 2 h (P < 0.01), whereas LH decreased KLF13 mRNA at 6 h (P < 0.05), and IGF-I reduced KLF13 at 24 h (P < 0.01) compared with untreated control. KLF9 was not regulated by either hormone. Transient transfection of KLF4, KLF9, and KLF13 suppressed LDLR/luc, StAR/luc, and CYP11A/luc by 80-90% (P < 0.001). Histone-deacetylase (HDAC) inhibitors stimulated LDLR/luc five- to sixfold and StAR/luc and CYP11A/luc activity twofold (P < 0.001) and partially reversed suppression by all three KLFs (P < 0.001). Deletion of the zinc finger domain of KLF13 abrogated repression of LDLR/luc. Lentiviral overexpression of the KLF13 gene suppressed LDLR mRNA (P < 0.001) and CYP11A mRNA (P = 0.003) but increased StAR mRNA (P = 0.007). Collectively, these data suggest that KLFs may recruit inhibitory complexes containing HDAC corepressors, thereby repressing LDLR and CYP11A transcription. Conversely, KLF13 may recruit unknown coactivators or stabilize StAR mRNA, thereby explaining enhancement of in situ StAR gene expression. These data introduce new potent gonadal transregulators of genes encoding proteins that mediate sterol uptake and steroid biosynthesis.

Effects of prenatal stress and monoaminergic perturbations on the expression of serotonin 5-HT₄ and adrenergic β₂ receptors in the embryonic mouse telencephalon.

PubMed

Chen, Angela; Kelley, Lauren D S; Janušonis, Skirmantas

2012-06-12

The serotonin 5-HT(4) receptor (5-HT(4)R) is coded by a complex gene that produces four mRNA splice variants in mice (5-HT(4(a))R, 5-HT(4(b))R, 5-HT(4(e))R, 5-HT(4(f))R). This receptor has highly dynamic expression in brain development and its splice variants differ in their developmental trajectories. Since 5-HT(4)Rs are important in forebrain function (including forebrain control of serotonergic activity in the brainstem), we investigated the susceptibility of 5-HT(4)R expression in the mouse embryonic telencephalon to prenatal maternal stress and altered serotonin (5-hydroxytryptamine, 5-HT) levels. Because the gene coding the adrenergic β(2) receptor (β(2)AR) is embedded in the 5-HT(4)R gene, we also investigated whether 5-HT(4)R mRNA levels were modulated by selective β(2)AR agents. Timed-pregnant C57BL/6 mice were treated beginning at embryonic day (E) 14 and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to assess the mRNA levels of all 5-HT(4)R splice variants and β(2)AR in the embryonic telencephalon at E17. Maternal prenatal stress and 5-HT depletion with pCPA, a tryptophan hydroxylase inhibitor, reduced the levels of the 5-HT(4(b))R splice variant. Terbutaline (a selective β(2)AR agonist) and ICI 118,551 (a selective β(2)AR antagonist) had no effect on β(2)AR and 5-HT(4)R mRNA levels. These results show that prenatal stress and reduced 5-HT levels can alter 5-HT(4)R expression in the developing forebrain and that some 5-HT(4)R splice variants may be more susceptible than others. Copyright © 2012 Elsevier B.V. All rights reserved.

[Studies on the correlation between the expression of Toll-like receptor 4 and the synovitis of the temporomandibular joint in rats].

PubMed

Kong, Jingjing; Wu, Qingting; Wang, Xiaohui; Yang, Yingying; Lin, Xuefen; Ji, Ping

2014-08-01

To investigate the expression of the Toll-like receptor-4 (TLR-4) in temporo-mandibular joint synovitis in rats, and to discuss the correlation between the expression of TLR-4 and the synovitis. Sixty male wistar rats were randomly divided into five groups, 12 each. Group A was the control group in which the rats were given normal diet.In Group B, the rats' bilateral masseter muscles were cut off (masseter resection group). In Group C, An cast metal crown were bonded on the mandibular right first molar of each rat (occlusal interference group). In Group D, occlusal pad were bonded on maxillary molars of each rat (occlusal dimension increase group). In Group E, rats' bilateral masseter muscles were re-sected and occlusal pads were bonded on their maxillary molars (masseter resection and occlusal dimension increase group). Pathological changes of synovium were observed using hematoxylin and eosin (HE) stains and pathology scores were evaluated. The expression of TLR- 4 were determined by immunohistochemical stains, and the expression of TLR-4 mRNA were determined by real-time PCR. The correlation between the expression of TLR-4, TLR-4 mRNA and the pathological score were analyzed using Spearman analysis. The pathological scores of Group A-E were 0.5 ± 0.5, 2.5 ± 1.0, 2.7 ± 1.0, 3.0 ± 0.9, 5.3 ± 1.2 respectively. The expression of TLR-4 were (3.2 ± 1.5)%, (16.± 2.6)%, (15.8 ± 2.1)%, (17.5 ± 2.4)%, (38.2 ± 4.4) %. The expression of TLR-4 mRNA were 1.07 ± 0.09, 2.12 ± 0.33, 2.07 ± 0.29, 2.17 ± 0.34, 4.53 ± 0.46. Compared with group A, groups B- E showed significant higher pathology score (P < 0.05) and increased expression of both TLR-4 (P < 0.05) and TLR-4 mRNA (P < 0.05). An significant positive correlation was found between the expression of TLR- 4 and the pathology score (r = 0.785, P < 0.05), and between the expression of TLR- 4 mRNA and the pathology score (r = 0.720, P < 0.05). TLR-4 may be closely associated with the development of the synovitis of TMJ of rats.

Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis

PubMed Central

Wilson, Fiona A.; Suryawan, Agus; Orellana, Renán A.; Nguyen, Hanh V.; Jeyapalan, Asumthia S.; Gazzaneo, Maria C.; Davis, Teresa A.

2008-01-01

Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 μg·kg−1·day−1) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) (P < 0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level (P < 0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1·eIF4E complex association, and increased active eIF4E·eIF4G complex formation (P < 0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex. PMID:18682537

Green tea polyphenols improve cardiac muscle mRNA and protein levels of signal pathways related to insulin and lipid metabolism and inflammation in insulin-resistant rats.

PubMed

Qin, Bolin; Polansky, Marilyn M; Harry, Dawson; Anderson, Richard A

2010-05-01

Epidemiological studies indicate that the consumption of green tea polyphenols (GTP) may reduce the risk of coronary artery disease. To explore the underlying mechanisms of action at the molecular level, we examined the effects of GTP on the cardiac mRNA and protein levels of genes involved in insulin and lipid metabolism and inflammation. In rats fed a high-fructose diet, supplementation with GTP (200 mg/kg BW daily dissolved in distilled water) for 6 wk, reduced systemic blood glucose, plasma insulin, retinol-binding protein 4, soluble CD36, cholesterol, triglycerides, free fatty acids and LDL-C levels, as well as the pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and IL-6. GTP did not affect food intake, bodyweight and heart weight. In the myocardium, GTP also increased the insulin receptor (Ir), insulin receptor substrate 1 and 2 (Irs1 and Irs2), phosphoinositide-3-kinase (Pi3k), v-akt murine thymoma viral oncogene homolog 1 (Akt1), glucose transporter 1 and 4 (Glut1 and Glut4) and glycogen synthase 1 (Gys1) expression but inhibited phosphatase and tensin homolog deleted on chromosome ten (Pten) expression and decreased glycogen synthase kinase 3beta (Gsk3beta) mRNA expression. The sterol regulatory element-binding protein-1c (Srebp1c) mRNA, microsomal triglyceride transfer protein (Mttp) mRNA and protein, Cd36 mRNA and cluster of differentiation 36 protein levels were decreased and peroxisome proliferator-activated receptor (Ppar)gamma mRNA levels were increased. GTP also decreased the inflammatory factors: Tnf, Il1b and Il6 mRNA levels, and enhanced the anti-inflammatory protein, zinc-finger protein, protein and mRNA expression. In summary, consumption of GTP ameliorated the detrimental effects of high-fructose diet on insulin signaling, lipid metabolism and inflammation in the cardiac muscle of rats.

Influence of elastin-derived peptides, glucose, LDL and oxLDL on nitric oxide synthase expression in human umbilical artery endothelial cells.

PubMed

Garczorz, Wojciech; Francuz, Tomasz; Gmiński, Jan; Likus, Wirginia; Siemianowicz, Krzysztof; Jurczak, Teresa; Strzałka-Mrozik, Barbara

2011-01-01

Endothelial dysfunction plays an important role in the development of atherosclerosis. Elastin-derived peptides (EDP), hyperglycemia, hypercholesterolemia and oxidized LDL have a proven proatherosclerotic potential. Nitric oxide generated by endothelial nitric oxide synthase (eNOS; EC 1.14.13.39) is an important vasorelaxant. Here we studied the influence of those proatherosclerotic factors on eNOS gene and protein expression in artery-derived endothelial cells. Human umbilical artery endothelial cells (HUAEC) were incubated with or without: glucose (270 mg/dl), LDL (200 mg/dl), oxidized LDL (oxLDL 25 mg/dl) or κ-elastin (0.5 and 2.5 µg/ml). Gene expression was assessed by real time RT-PCR, whilst the eNOS protein by ELISA. In cells incubated with 2.5 µg/ml of κ-elastin, a 31 % increase of eNOS mRNA expression was observed, but the protein level remained unchanged. OxLDL, LDL and glucose decreased the eNOS protein level by 74 %, 37 % and 29 %, respectively. OxLDL decreased eNOS mRNA by 42 %. LDL non-significantly decreased eNOS mRNA expression. An elevated glucose level did not affect the eNOS mRNA expression. Hyperglycemia and an elevated level of LDL, particularly oxLDL, decreased the level of eNOS protein in endothelial cells. As κ-elastin did not decrease the expression of eNOS gene in HUAEC, the proatherogenic properties of elastin-derived peptides are unlikely to be due to their influence on eNOS.

Ribonuclease inhibitor 1 regulates erythropoiesis by controlling GATA1 translation.

PubMed

Chennupati, Vijaykumar; Veiga, Diogo Ft; Maslowski, Kendle M; Andina, Nicola; Tardivel, Aubry; Yu, Eric Chi-Wang; Stilinovic, Martina; Simillion, Cedric; Duchosal, Michel A; Quadroni, Manfredo; Roberts, Irene; Sankaran, Vijay G; MacDonald, H Robson; Fasel, Nicolas; Angelillo-Scherrer, Anne; Schneider, Pascal; Hoang, Trang; Allam, Ramanjaneyulu

2018-04-02

Ribosomal proteins (RP) regulate specific gene expression by selectively translating subsets of mRNAs. Indeed, in Diamond-Blackfan anemia and 5q- syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anemia. Little is known about the molecular mechanisms of selective mRNA translation and involvement of ribosomal-associated factors in this process. Ribonuclease inhibitor 1 (RNH1) is a ubiquitously expressed protein that binds to and inhibits pancreatic-type ribonucleases. Here, we report that RNH1 binds to ribosomes and regulates erythropoiesis by controlling translation of the erythroid transcription factor GATA1. Rnh1-deficient mice die between embryonic days E8.5 and E10 due to impaired production of mature erythroid cells from progenitor cells. In Rnh1-deficient embryos, mRNA levels of Gata1 are normal, but GATA1 protein levels are decreased. At the molecular level, we found that RNH1 binds to the 40S subunit of ribosomes and facilitates polysome formation on Gata1 mRNA to confer transcript-specific translation. Further, RNH1 knockdown in human CD34+ progenitor cells decreased erythroid differentiation without affecting myelopoiesis. Our results reveal an unsuspected role for RNH1 in the control of GATA1 mRNA translation and erythropoiesis.

mRNA Cap Methyltransferase, RNMT-RAM, Promotes RNA Pol II-Dependent Transcription.

PubMed

Varshney, Dhaval; Lombardi, Olivia; Schweikert, Gabriele; Dunn, Sianadh; Suska, Olga; Cowling, Victoria H

2018-05-01

mRNA cap addition occurs early during RNA Pol II-dependent transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II transcription independent of mRNA capping and translation. In cells, sublethal suppression of RNMT-RAM reduces RNA Pol II occupancy, net mRNA synthesis, and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independent of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription-associated factors including the RNA Pol II subunits SPT4, SPT6, and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2BK120 ubiquitination, H3K4 and H3K36 methylation, RNA Pol II CTD S5 and S2 phosphorylation, and PAFc recruitment. These findings suggest that multiple interactions among RNMT-RAM, RNA Pol II factors, and RNA along the transcription unit stimulate transcription. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Uromodulin mRNA from Urinary Extracellular Vesicles Correlate to Kidney Function Decline in Type 2 Diabetes Mellitus.

PubMed

Yamamoto, Cindy M; Murakami, Taku; Oakes, Melanie L; Mitsuhashi, Masato; Kelly, Colleen; Henry, Robert R; Sharma, Kumar

2018-05-18

Extracellular vesicles (EVs) enclose mRNA derived from their cell of origin and are considered a source of potential biomarkers. We examined urinary EV mRNA from individuals with diabetic kidney disease (DKD), chronic kidney disease, type 2 diabetes (T2DM), and obese and healthy controls to determine if such biomarkers had the potential to classify kidney disease and predict patients at higher risk of renal function decline. A total of 242 participants enrolled in this study. Urinary EV mRNA from all subjects were isolated by a filter-based platform, and the expression of 8 target genes were determined by quantitative polymerase chain reaction (qPCR). Changes in estimated glomerular filtration rate (eGFR) in 161 T2DM patients were evaluated for 2 consecutive years and compared with EV RNA profiles at baseline. We observe that mild and severe DKD groups show a significant 3.2- and -4.4-fold increase in UMOD compared to healthy controls and expression increases linearly from healthy, diabetic, and DKD subjects. UMOD expression is significantly correlated to albumin creatinine ratio (ACR), eGFR, and HbA1c. Using linear discriminant analyses with mRNA from severe DKD and T2DM as training data, a multi-gene signature classified DKD and -non-DKD with a sensitivity of 93% and specificity of 73% with area under the receiver operating characteristic (ROC) curve (AUC) = 0.90. Although 6% of T2DM were determined to have a > 80% posterior probability of developing DKD based on this mRNA profile, eGFR changes observed within the 2-year follow-up did not reveal a decline in kidney function. Urinary EV UMOD mRNA levels are progressively elevated from T2DM to DKD groups and correlate with widely used eGFR and ACR diagnostic criteria. An EV mRNA signature could identify DKD with greater than 90% sensitivity and 70% specificity. © 2018 S. Karger AG, Basel.

A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs

PubMed Central

Gromadzka, Agnieszka M.; Steckelberg, Anna-Lena; Singh, Kusum K.; Hofmann, Kay; Gehring, Niels H.

2016-01-01

The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs. PMID:26773052

Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation

PubMed Central

Suryawan, Agus; Jeyapalan, Asumthia S.; Orellana, Renan A.; Wilson, Fiona A.; Nguyen, Hanh V.; Davis, Teresa A.

2008-01-01

Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E·eIF4G complex and increased eIF4E·4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein β-subunit-like protein (GβL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors. PMID:18682538

Antihypertensive methyldopa, labetalol, hydralazine, and clonidine reversed tumour necrosis factor-α inhibited endothelial nitric oxide synthase expression in endothelial-trophoblast cellular networks.

PubMed

Xu, Bei; Bobek, Gabriele; Makris, Angela; Hennessy, Annemarie

2017-03-01

Medications used to control hypertension in pregnancy also improve trophoblast and endothelial cellular interaction in vitro. Tumour necrosis factor-α (TNF-α) inhibits trophoblast and endothelial cellular interactions and simultaneously decreases endothelial nitric oxide synthase (eNOS) expression. This study investigated whether antihypertensive medications improved these cellular interactions by modulating eNOS and inducible nitric oxide synthase (iNOS) expression. Human uterine myometrial microvascular endothelial cells (UtMVECs) were pre-incubated with (or without) low dose TNF-α (0.5 ng/mL) or TNF-α plus soluble fms-like tyrosine kinase-1 (sFlt-1) (100 ng/mL). The endothelial cells were cultured on Matrigel. After endothelial cellular networks appeared, trophoblast derived HTR-8/SVneo cells were co-cultured in the presence of clinically relevant doses of methyldopa, labetalol, hydralazine or clonidine for 24 hours. Cells were retrieved from the Matrigel to extract mRNA and eNOS and iNOS expression were examined by quantitative PCR. Methyldopa, labetalol, hydralazine and clonidine reversed the inhibitory effect of TNF-α on eNOS mRNA expression. After pre-incubating endothelial cells with TNF-α and sFlt-1, all the medications except methyldopa lost their effect on eNOS mRNA expression. In the absence of TNF-α, antihypertensive medications did not change eNOS expression. The mRNA expression of iNOS was not affected by TNF-α or any medications. This study shows that selected antihypertensive medications used in the treatment of hypertension in pregnancy increase eNOS expression in vitro when induced by the inflammatory TNF-α. The anti-angiogenic molecule sFlt-1 may antagonise the potential benefit of these medications by interfering with the NOS pathway. © 2016 John Wiley & Sons Australia, Ltd.

Quantifying Extrinsic Noise in Gene Expression Using the Maximum Entropy Framework

PubMed Central

Dixit, Purushottam D.

2013-01-01

We present a maximum entropy framework to separate intrinsic and extrinsic contributions to noisy gene expression solely from the profile of expression. We express the experimentally accessible probability distribution of the copy number of the gene product (mRNA or protein) by accounting for possible variations in extrinsic factors. The distribution of extrinsic factors is estimated using the maximum entropy principle. Our results show that extrinsic factors qualitatively and quantitatively affect the probability distribution of the gene product. We work out, in detail, the transcription of mRNA from a constitutively expressed promoter in Escherichia coli. We suggest that the variation in extrinsic factors may account for the observed wider-than-Poisson distribution of mRNA copy numbers. We successfully test our framework on a numerical simulation of a simple gene expression scheme that accounts for the variation in extrinsic factors. We also make falsifiable predictions, some of which are tested on previous experiments in E. coli whereas others need verification. Application of the presented framework to more complex situations is also discussed. PMID:23790383

Quantifying extrinsic noise in gene expression using the maximum entropy framework.

PubMed

Dixit, Purushottam D

2013-06-18

We present a maximum entropy framework to separate intrinsic and extrinsic contributions to noisy gene expression solely from the profile of expression. We express the experimentally accessible probability distribution of the copy number of the gene product (mRNA or protein) by accounting for possible variations in extrinsic factors. The distribution of extrinsic factors is estimated using the maximum entropy principle. Our results show that extrinsic factors qualitatively and quantitatively affect the probability distribution of the gene product. We work out, in detail, the transcription of mRNA from a constitutively expressed promoter in Escherichia coli. We suggest that the variation in extrinsic factors may account for the observed wider-than-Poisson distribution of mRNA copy numbers. We successfully test our framework on a numerical simulation of a simple gene expression scheme that accounts for the variation in extrinsic factors. We also make falsifiable predictions, some of which are tested on previous experiments in E. coli whereas others need verification. Application of the presented framework to more complex situations is also discussed. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Asporin and transforming growth factor-beta gene expression in osteoblasts from subchondral bone and osteophytes in osteoarthritis.

PubMed

Sakao, Kei; Takahashi, Kenji A; Arai, Yuji; Saito, Masazumi; Honjyo, Kuniaki; Hiraoka, Nobuyuki; Kishida, Tsunao; Mazda, Osam; Imanishi, Jiro; Kubo, Toshikazu

2009-11-01

To clarify the significance of subchondral bone and osteophytes in the pathology of osteoarthritis (OA), we investigated the expression of asporin (ASPN), transforming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF-beta3, and runt-related transcription factor-2 (Runx2) genes involved in bone metabolism. Osteoblasts were isolated from 19 patients diagnosed with knee OA and from 4 patients diagnosed with femoral neck fracture. Osteoblast expression of mRNA encoding ASPN, TGF-beta1, TGF-beta2, TGF-beta3, and Runx2 was analyzed using real-time RT-PCR. Expression of ASPN, TGF-beta1, and TGF-beta3 mRNA in the subchondral bone and osteophytes of OA patients increased compared with that of non-OA patients. The ratio of ASPN to TGF-beta1 mRNA in patients with severe cartilage damage was higher than that in patients with mild cartilage damage. The increased ratio of ASPN mRNA to TGF-beta1 mRNA in patients with severe relative to mild cartilage damage indicates that increased ASPN mRNA expression was significantly associated with the severity of cartilage degeneration. This finding suggests that ASPN may regulate TGF-beta1-mediated factors in the development of OA, which may provide clues as to the underlying pathology of OA.

Antisense myb inhibition of purified erythroid progenitors in development and differentiation is linked to cycling activity and expression of DNA polymerase alpha

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valtieri, M.; Venturelli, D.; Care, A.

These studies aimed to determine the expression and functional role of c-myb in erythroid progenitors with different cycling activities. In the first series of experiments the erythroid burst-forming unit (BFU-E) and colony-forming unit (CFU-E) populations from adult peripheral blood (PB), bone marrow (BM), and embryonic-fetal liver (FL) were treated with either c-myb antisense oligomers or 3H-thymidine (3H-TdR). A direct correlation was always observed between the inhibitory effect of anti-myb oligomers and the level of cycling activity. Thus, the inhibitory effect of antisense c-myb on the number of BFU-E colonies was 28.3% +/- 15.8% in PB, 53.4% +/- 9.3% in BM,more » and 68.2% +/- 24.5% in FL. Both adult and embryonic CFU-E were markedly inhibited. Using purified PB progenitors, we observed a similar pattern, although with slightly lower inhibitory effects. In the 3H-TdR suicide assay the killing index of BFU-E was 8.9% +/- 4.2% in PB, 29.4% +/- 6.5% in BM, and 40.1% +/- 9.6% in FL. The values for adult and embryonic CFU-E were 55.7% +/- 7.9% and 60.98% +/- 6.6%, respectively. We then investigated the kinetics of c-myb mRNA level during the erythroid differentiation of purified adult PB and FL BFU-E, as evaluated in liquid-phase culture by reverse transcription-polymerase chain reaction. Adult erythroid precursors showed a gradual increase of c-myb mRNA from day 4 through day 8 of culture and a sharp decrease at later times, whereas the expression of c-myb mRNA and protein in differentiation embryonic precursors peaked 2 days earlier. In both cases, c-myb mRNA level peaked at the CFU-E stage of differentiation. Finally, highly purified adult PB BFU-E were stimulated into cycling by a 3-day treatment with interleukin-3 in liquid phase: both the sensitivity to c-myb antisense oligomers and the 3H-TdR suicide index showed a gradual, strictly parallel increase.« less

Time- and dose-dependent differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase enzymatic activity and mRNA level by vitamin E in rat blood cells.

PubMed

Hajiani, Maliheh; Razi, Farideh; Golestani, Aboualfazl; Frouzandeh, Mehdi; Owji, Ali Akbar; Khaghani, Shahnaz; Ghannadian, Naghmeh; Shariftabrizi, Ahmad; Pasalar, Parvin

2012-01-01

Vitamin E is the most important lipid-soluble antioxidant. Recently, it has been proposed as a gene regulator, and its gene modulation effects have been observed at different levels of gene expression and cell signaling. This study was performed to investigate the effects of vitamin E on the activity and expression of the most important endogenous antioxidant enzyme, superoxide dismutase (SOD), in rat plasma. Twenty-eight male Sprauge-Dawley rats were divided into four groups: control group and three dosing groups. The control group received the vehicle (liquid paraffin), and the dosing groups received twice-weekly intraperitoneal injections of 10, 30, and 100 mg/kg of vitamin E ((±)-α-Tocopherol) for 6 weeks. Quantitative real-time reverse transcription-polymerase chain reaction and enzyme assays were used to assess the levels of Cu/Zn-SOD and Mn-SOD mRNA and enzyme activity levels in blood cells at 0, 2, 4, and 6 weeks following vitamin E administration. Catalase enzyme activity and total antioxidant capacity were also assessed in plasma at the same time intervals. Mn-SOD activity was significantly increased in the 100 and 30 mg/kg dosing groups after 4 and 6 weeks, with corresponding significant increase in their mRNA levels. Cu/Zn-SOD activity was not significantly changed in response to vitamin E administration at any time points, whereas Cu/Zn-SOD mRNA levels were significantly increased after longer time points with high doses (30 and 100 mg/kg) of vitamin E. Catalase enzyme activity was transiently but significantly increased after 4 weeks of vitamin E treatment in 30 and 100 mg/kg dosing groups. Total antioxidant status was significantly increased after 4 and 6 weeks in the 100 mg/kg dosing group. Only the chronic administration of higher doses of alpha-tocopherol is associated with the increased activity and expression of Mn-SOD in rats. Cu/Zn-SOD activity and expression does not dramatically change in response to vitamin E.

Adverse early life experience and social stress during adulthood interact to increase serotonin transporter mRNA expression

PubMed Central

Gardner, Katherine L.; Hale, Matthew W.; Lightman, Stafford L.; Plotsky, Paul M.; Lowry, Christopher A.

2009-01-01

Anxiety disorders, depression and animal models of vulnerability to a depression-like syndrome have been associated with dysregulation of serotonergic systems in the brain. To evaluate the effects of early life experience, adverse experiences during adulthood, and potential interactions between these factors on serotonin transporter (slc6a4) mRNA expression, we investigated in rats the effects of maternal separation (180 min/day from days 2–14 of life; MS180), neonatal handing (15 min/day from days 2–14 of life; MS15), or normal animal facility rearing control conditions (AFR) with or without subsequent exposure to adult social defeat on slc6a4 mRNA expression in the dorsal raphe nucleus (DR) and caudal linear nucleus. At the level of specific subdivisions of the DR, there were no differences in slc6a4 mRNA expression between MS15 and AFR rats. Among rats exposed to a novel cage control condition, increased slc6a4 mRNA expression was observed in the dorsal part of the DR in MS180 rats, relative to AFR control rats. In contrast, MS180 rats exposed to social defeat as adults had increased slc6a4 mRNA expression throughout the DR compared to both MS15 and AFR controls. Social defeat increased slc6a4 mRNA expression, but only in MS180 rats and only in the “lateral wings” of the DR. Overall these data demonstrate that early life experience and stressful experience during adulthood interact to determine slc6a4 mRNA expression. These data support the hypothesis that early life experience and major stressful life events contribute to dysregulation of serotonergic systems in stress-related neuropsychiatric disorders. PMID:19781533

Methylphenidate and Atomoxetine-Responsive Prefrontal Cortical Genetic Overlaps in "Impulsive" SHR/NCrl and Wistar Rats.

PubMed

Dela Peña, Ike; Dela Peña, Irene Joy; de la Peña, June Bryan; Kim, Hee Jin; Shin, Chan Young; Han, Doug Hyun; Kim, Bung-Nyun; Ryu, Jong Hoon; Cheong, Jae Hoon

2017-09-01

Impulsivity, the predisposition to act prematurely without foresight, is associated with a number of neuropsychiatric disorders, including attention-deficit/hyperactivity disorder (ADHD). Identifying genetic underpinnings of impulsive behavior may help decipher the complex etiology and neurobiological factors of disorders marked by impulsivity. To identify potential genetic factors of impulsivity, we examined common differentially expressed genes (DEGs) in the prefrontal cortex (PFC) of adolescent SHR/NCrl and Wistar rats, which showed marked decrease in preference for the large but delayed reward, compared with WKY/NCrl rats, in the delay discounting task. Of these DEGs, we examined drug-responsive transcripts whose mRNA levels were altered following treatment (in SHR/NCrl and Wistar rats) with drugs that alleviate impulsivity, namely, the ADHD medications methylphenidate and atomoxetine. Prefrontal cortical genetic overlaps between SHR/NCrl and Wistar rats in comparison with WKY/NCrl included genes associated with transcription (e.g., Btg2, Fos, Nr4a2), synaptic plasticity (e.g., Arc, Homer2), and neuron apoptosis (Grik2, Nmnat1). Treatment with methylphenidate and/or atomoxetine increased choice of the large, delayed reward in SHR/NCrl and Wistar rats and changed, in varying degrees, mRNA levels of Nr4a2, Btg2, and Homer2, genes with previously described roles in neuropsychiatric disorders characterized by impulsivity. While further studies are required, we dissected potential genetic factors that may influence impulsivity by identifying genetic overlaps in the PFC of "impulsive" SHR/NCrl and Wistar rats. Notably, these are also drug-responsive transcripts which may be studied further as biomarkers to predict response to ADHD drugs, and as potential targets for the development of treatments to improve impulsivity.

Estrogenic environmental contaminants alter the mRNA abundance profiles of genes involved in gonadal differentiation of the American bullfrog

PubMed Central

Wolff, Stephanie E.; Veldhoen, Nik; Helbing, Caren C.; Ramirez, Claire A.; Malpas, Janae M.; Propper, Catherine R.

2015-01-01

Wildlife and human populations are exposed to anthropogenic mixtures of chemicals in the environment that may adversely influence normal reproductive function and development. We determined the effects of exposure to estrogenic chemicals and wastewater effluent (WWE) on developing gonads of the American bullfrog, Rana (Lithobates) catesbeiana, a species whose widespread distribution make it an ideal model for environmental monitoring for endocrine effects of chemical contaminants. Premetamorphic bullfrog tadpoles were exposed to treatment vehicle, 17β-estradiol (E2; 10−9 M) or 4-tert-octylphenol (OP; 10−9 M, 10−8 M, and 10−7 M). Additionally, gonadal differentiation was evaluated in bullfrog tadpoles from a WWE-containing site versus those from a reference location receiving no WWE. In both studies, phenotypic sex, steroidogenic factor-1 (nr5a1), and aromatase (cyp19a1) mRNA levels using quantitative real-time PCR were determined. Exposure to E2 or OP did not alter sex ratios. In controls, both nr5a1 and cyp19a1 transcript levels exhibited sexual dimorphism, with males demonstrating higher levels of nr5a1 and females greater abundance of cyp19a1. However, E2 exposure increased cyp19a1 mRNA abundance in testes and decreased levels in ovaries, eliminating the sexual dimorphism observed in controls. E2-exposed males exhibited increased nr5a1 transcript levels in the testes compared to controls, while females demonstrated no E2 effect. OP treatment had no effect on female cyp19a1 mRNA abundance, but exposure to 10−7 M OP increased testicular transcript levels. Treatment with 10−9 and 10−8 M OP, but not 10−7 M, resulted in decreased abundance of nr5a1 transcript in both ovaries and testes. Animals from the field had sexually dimorphic gonadal levels of cyp19a1, but both sexes from the WWE site exhibited elevated cyp19a1 transcript abundance compared to the reference location. Individual chemical compounds and anthropogenic wastewater effluent dispersed within the environment influence the levels of gonadal mRNA encoding key proteins involved in gonadal differentiation. PMID:25863316

Reduction in the mRNA expression of sVEGFR1 and sVEGFR2 is associated with the selection of dominant follicle in cows.

PubMed

Ortega Serrano, P V; Guzmán, A; Hernández-Coronado, C G; Castillo-Juárez, H; Rosales-Torres, A M

2016-12-01

The vascular endothelial growth factor (VEGF) is essential for follicular development by promoting follicular angiogenesis, as well as for the proliferation and survival of granulosa cells. The biological effects of VEGF are regulated by two membrane receptors, VEGFR1 and VEGFR2, and two soluble receptors, sVEGFR1 and sVEGFR2, which play an antagonistic role. Thus, the objective of this study was to identify the mRNA expression pattern of total VEGF, VEGFR1, VEGFR2, sVEGFR1 and sVEGFR2 in bovine preselected follicles (PRF) and post-selected follicles (POF). The mRNA expression of these five genes in both granulosa cells (GC) and theca cells (TC) was compared between follicles classified as PRF and POF based on their diameter and on their ratio of estradiol/progesterone (E2/P4). Results showed a lower expression of mRNA of sVEGFR1 and sVEGFR2 in POF than in PRF (p < .05). Regarding the mRNA expression of total VEGF, VEGFR1 and VEGFR2, there was no difference between POF and PRF follicles (p > .05). Our results showed that the mRNA expression of VEGFR2 and sVEGFR1 was more abundant than the expression of VEGFR1 and sVEGFR2, while GC was the main source of mRNA for total VEGF. On the other hand, TC was the follicular compartment where the receptors were most expressed. Our results suggest that non-dominant follicles maintain a greater concentration of the mRNA expression of both membrane and soluble VEGF receptors. On the other hand, follicular dominance is related to a reduction in the mRNA expression of sVEGFR1 and sVEGFR2, which may favour VEGF binding with VEGFR2 and, hence, improve the follicular health and development. © 2016 Blackwell Verlag GmbH.

The suppressive effect of immune stress on LH secretion is absent in the early neonatal period in rats.

PubMed

Munkhzaya, Munkhsaikhan; Matsuzaki, Toshiya; Iwasa, Takeshi; Tungalagsuvd, Altankhuu; Kawami, Takako; Kato, Takeshi; Kuwahara, Akira; Irahara, Minoru

2015-11-01

Some physiological functions display weak responses to stress in the early neonatal period; i.e., they exhibit stress hyporesponse periods. In this study, we evaluated whether gonadotropin regulatory factors exhibit stress hyporesponsive periods in male and female rats. Rats were intraperitoneally injected with lipopolysaccharide (100μg/kg) (LPS group) or saline (control group) on postnatal day (PND) 5, 10, 15, or 25. Then, their serum luteinizing hormone (LH) concentrations and hypothalamic mRNA levels of gonadotropin regulatory factors; i.e., kisspeptin (Kiss1), the kisspeptin receptor (Kiss1r), and gonadotropin-releasing hormone (GnRH), were measured at 2h after the injection. The hypothalamic mRNA levels of pro-inflammatory cytokines were also measured because they suppress gonadotropin secretion. The serum LH concentration of the LPS group was lower than that of the control group at PND25 in both sexes, but no such difference was seen at PND5, 10, or 15 in either sex. In both sexes, the hypothalamic tumor necrosis factor (TNF)α and interleukin (IL)-6 mRNA expression levels of the LPS group were higher than those of the control group at PND25, but not at PND5 or 10. The hypothalamic IL-1β mRNA expression level of the LPS group was higher than that of the control group at all time points. The hypothalamic Kiss1, Kiss1r, and GnRH mRNA expression levels of the LPS and control groups did not differ at any time point in either sex. These findings suggest that gonadotropin regulatory factors exhibit stress hyporesponse periods. The hypothalamic-pituitary-gonadal axis (HPG) might become responsive to immune stress between PND15 and 25, which could be related to enhanced hypothalamic cytokine expression. The avoidance of infectious stress during the early neonatal period might be important for normal development of the HPG axis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Triage of HR-HPV positive women with minor cytological abnormalities: a comparison of mRNA testing, HPV DNA testing, and repeat cytology using a 4-year follow-up of a population-based study.

PubMed

Persson, Maria; Elfström, K Miriam; Brismar Wendel, Sophia; Weiderpass, Elisabete; Andersson, Sonia

2014-01-01

Expression of the viral E6/E7 oncogenes of high-risk human papillomaviruses (HR-HPV) is necessary for malignant conversion and maintenance in cervical tissue. In order to determine whether HR-HPV E6/E7 mRNA testing more effectively predicts precancerous lesions and invasive cervical cancer than HR-HPV DNA testing, we aimed to compare triage using HR-HPV E6/E7 mRNA testing by APTIMA HPV Assay (APTIMA) to HPV16 DNA testing, HPV16/18 DNA testing, and repeat cytology. Liquid-based (PreservCyt) cell samples were obtained from HR-HPV-positive women diagnosed with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL) within the framework of the population-based cervical cancer screening program in Stockholm, Sweden. Samples were tested for HR-HPV E6/E7 mRNA by APTIMA (Gene-Probe Inc., San Diego, CA, USA). Women were followed up for 4 years after the index cytology via medical and laboratory records, and the Stockholm Oncology Center. Nine of 25 (36%) women in the ASCUS group, and 64 of 180 (36%) women in the LSIL group developed cervical intraepithelial neoplasia (CIN) grade 2 or worse during 4 years of follow-up. 162 (74%) women were APTIMA-positive, and APTIMA had the highest sensitivity to predict CIN2 or worse and CIN3 or worse in the ASCUS (77.8% and 100%) and LSIL (78.1 and 75.8%) groups, although specificity was insufficient (<50%). HPV16 DNA testing and repeat cytology were more specific than APTIMA. The results of this population-based study with comprehensive follow-up support the use of APTIMA as a triage test for women with ASCUS. More focused investigation is required for women with LSIL.

Triage of HR-HPV Positive Women with Minor Cytological Abnormalities: A Comparison of mRNA Testing, HPV DNA Testing, and Repeat Cytology Using a 4-Year Follow-Up of a Population-Based Study

PubMed Central

Persson, Maria; Elfström, K. Miriam; Brismar Wendel, Sophia; Weiderpass, Elisabete; Andersson, Sonia

2014-01-01

Objective Expression of the viral E6/E7 oncogenes of high-risk human papillomaviruses (HR-HPV) is necessary for malignant conversion and maintenance in cervical tissue. In order to determine whether HR-HPV E6/E7 mRNA testing more effectively predicts precancerous lesions and invasive cervical cancer than HR-HPV DNA testing, we aimed to compare triage using HR-HPV E6/E7 mRNA testing by APTIMA HPV Assay (APTIMA) to HPV16 DNA testing, HPV16/18 DNA testing, and repeat cytology. Methods Liquid-based (PreservCyt) cell samples were obtained from HR-HPV-positive women diagnosed with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL) within the framework of the population-based cervical cancer screening program in Stockholm, Sweden. Samples were tested for HR-HPV E6/E7 mRNA by APTIMA (Gene-Probe Inc., San Diego, CA, USA). Women were followed up for 4 years after the index cytology via medical and laboratory records, and the Stockholm Oncology Center. Results Nine of 25 (36%) women in the ASCUS group, and 64 of 180 (36%) women in the LSIL group developed cervical intraepithelial neoplasia (CIN) grade 2 or worse during 4 years of follow-up. 162 (74%) women were APTIMA-positive, and APTIMA had the highest sensitivity to predict CIN2 or worse and CIN3 or worse in the ASCUS (77.8% and 100%) and LSIL (78.1 and 75.8%) groups, although specificity was insufficient (<50%). HPV16 DNA testing and repeat cytology were more specific than APTIMA. Conclusion The results of this population-based study with comprehensive follow-up support the use of APTIMA as a triage test for women with ASCUS. More focused investigation is required for women with LSIL. PMID:24587193

Cell-autonomous CCL5 transcription by memory CD8 T cells is regulated by IL-4.

PubMed

Marçais, Antoine; Coupet, Charles-Antoine; Walzer, Thierry; Tomkowiak, Martine; Ghittoni, Raffaella; Marvel, Jacqueline

2006-10-01

Immunological memory is associated with the display of improved effector functions. The maintenance by CD8 memory cells of high levels of untranslated CCL5 mRNA allows these cells to immediately secrete this chemokine upon Ag stimulation. Untranslated mRNA storage is a newly described process supporting the immediate display of an effector function by memory lymphocytes. We have tested the capacity of different cytokines to regulate the memorization of CCL5 by memory CD8 T cells. We found that IL-4 treatment of murine CD8 T cells impairs immediate CCL5 secretion capacity by inhibiting CCL5 mRNA transcription through a STAT6-dependent pathway. The inhibition by IL-4 is reversible, as memory CD8 T cells reconstitute their CCL5 mRNA stores and reacquire their immediate CCL5 secretion capacity when IL-4 is withdrawn. This recovery is cell autonomous because it proceeds in culture medium in the absence of exogenous growth factors, suggesting that CCL5 expression by memory CD8 T cells is a default process. Overall, these results indicate that the expression of CCL5 is an intrinsic property acquired by memory CD8 T cells that is regulated by environmental factors.

Human adenovirus Ad36 and its E4orf1 gene enhance cellular glucose uptake even in the presence of inflammatory cytokines.

PubMed

Na, Ha-Na; Dubuisson, Olga; Hegde, Vijay; Nam, Jae-Hwan; Dhurandhar, Nikhil V

2016-05-01

Aging and obesity are associated with elevated pro-inflammatory cytokines such as monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor (TNF)α, which are linked to insulin resistance. Anti-inflammatory agents have marginal effect in improving insulin resistance. Hence, agents are needed to improve glycemic control despite the inflammation. Ad36, a human adenovirus, increases TNFα and MCP1 mRNA in adipose tissue, yet improves glycemic control in mice. Ad36 via its E4orf1 gene, up-regulates AKT/glucose transporter (Glut)-4 signaling to enhance cellular glucose uptake. Directly test a role of Ad36, or E4orf1 in enhancing cellular glucose uptake in presence of inflammatory cytokines. Experiment 1: 3T3-L1 preadipocytes were treated with 0, 10 or 100 ng/mL lipopolysaccharides (LPS), and infected with 0 or 5 plaque forming units (PFU) of Ad36/cell. 3T3-L1 cells that stably and inducibly express E4orf1 or a null vector (pTRE-E4orf1 or pTRE-null cells), were similarly treated with LPS and then with doxycycline, to induce E4orf1. Experiment 2: 3T3L1 preadipocytes were treated with 25 nM MCP1 or 20 nM TNFα for 16 h, followed by infection with 0 or 5 PFU of Ad36/cell. Experiment 3: pTRE-E4orf1 or -null cells were similarly treated with MCP1 or TNFα followed by doxycycline to induce E4orf1. Cellular glucose uptake and cellular signaling were determined 72 h post-Ad36 infection or E4orf1-induction, in continued presence of MCP1 or TNFα. In 3T3-L1 preadipocytes, Ad36, but not E4orf1, increased MCP1 and TNFα mRNA, in presence of LPS stimulation. Ad36 or E4orf1 up-regulated AKT-phosphorylation and Glut4 and increased glucose uptake (P < 0.05) in the presence of MCP1 or TNFα. Unlike Ad36, E4orf1 does not appear to stimulate inflammatory response. Ad36 and E4orf1 both enhance cellular glucose uptake even in presence of inflammation. Further research is needed to harness this novel and beneficial property of E4orf1 to improve hyperglycemia despite chronic inflammation that is commonly present in aging and obesity. Copyright © 2014 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Signal transduction involved in lipoxin A4-induced protection of tubular epithelial cells against hypoxia/reoxygenation injury

PubMed Central

Wu, Sheng-Hua; Wang, Ming-Jie; Lü, Jing; Chen, Xiao-Qing

2017-01-01

Previous studies have reported that lipoxin A4 (LXA4) may exert a renoprotective effect on ischemia/reperfusion injury in various animal models. The underlying mechanism of LXA4-induced renoprotection during ischemia/reperfusion injury remains to be elucidated. The present study investigated LXA4-induced protection on renal tubular cells subjected to hypoxia/reoxygenation (H/R) injury, and determined the effects of peroxisome proliferator-activated receptor-γ (PPARγ) and heme oxygenase-1 (HO-1) on LXA4 treatment. HK-2 human tubular epithelial cells exposed to H/R injury were pretreated with LXA4, signal molecule inhibitors or the HO-1 inhibitor zinc protoporphyrin-IX, or were transfected with PPARγ small interfering RNA (siRNA) or nuclear factor E2-related factor 2 (Nrf2) siRNA. The protein and mRNA expression levels of PPARγ and HO-1 were analyzed using western blotting and reverse transcription-quantitative polymerase chain reaction. Binding activity of Nrf2 to the HO-1 E1 enhancer was determined using chromatin immunoprecipitation. Nrf2 binding to the HO-1 antioxidant responsive element (ARE) was assessed using electrophoretic mobility shift assay. Preincubation of cells with LXA4 exposed to H/R injury led to a decreased production of inducible nitrogen oxide synthase, malondialdehyde, γ-glutamyl transpeptidase, leucine aminopeptidase and N-acetyl-β-glucosaminidase. In addition, LXA4 pretreatment increased cell viability, protein and mRNA expression levels of PPARγ and HO-1 and PPARγ and HO-1 promoter activity. SB20358 is a p38 mitogen-activated protein kinase (p38 MAPK) pathway inhibitor, which reduced LXA4-induced PPARγ expression levels. LXA4 treatment upregulated p38 MAPK activation, Nrf2 nuclear translocation and increased binding activity of Nrf2 to HO-1 ARE and E1 enhancer in cells exposed to H/R injury. Transfection of the cells with PPARγ siRNA reduced the LXA4-induced Nrf2 translocation. Transfection of the cells with PPARγ siRNA or Nrf2 siRNA also reduced the LXA4-induced increase in HO-1 expression. In conclusion, LXA4-induced protection of renal tubular cells against H/R injury was associated with the induction of PPARγ and HO-1, via activation of the p38 MAPK pathway, as well as Nrf2 nuclear translocation and binding to HO-1 ARE and E1 enhancer. Therefore, LXA4-induced renoprotection is associated with activation of the p38 MAPK/PPARγ/Nrf2-ARE/HO-1 pathway. PMID:28259922

PGE2 released by primary sensory neurons modulates Toll-like receptor 4 activities through an EP4 receptor-dependent process.

PubMed

Tse, Kai-Hei; Chow, Kevin B S; Wise, Helen

2016-04-15

Exogenous prostaglandin E2 (PGE2) displays mixed regulatory properties with regard to inflammatory gene expression in dorsal root ganglion (DRG) cells. We show here that endogenously-produced nanomolar concentrations of PGE2, such as that generated in response to Toll-like receptor 4 (TLR4) stimulation, inhibits both cyclooxygenase-2 (COX-2) and tumour necrosis factor alpha (TNFα) mRNA expression in DRG cells in an EP4 receptor-dependent manner. DRG neurons appear to be the major source of PGE2 in the DRG and likely serve as both an autocrine and paracrine system for limiting over-activation of both DRG neurons and glial cells in response to TLR4 stimulation. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular analysis of globin gene expression in different thalassaemia disorders: individual variation of β(E) pre-mRNA splicing determine disease severity.

PubMed

Tubsuwan, Alisa; Munkongdee, Thongperm; Jearawiriyapaisarn, Natee; Boonchoy, Chanikarn; Winichagoon, Pranee; Fucharoen, Suthat; Svasti, Saovaros

2011-09-01

Thalassaemia is characterized by the reduced or absent production of globins in the haemoglobin molecule leading to imbalanced α-globin/non α-globin chains. HbE, the result of a G to A mutation in codon 26 of the HBB (β-globin) gene, activates a cryptic 5' splice site in codon 25 leading to a reduction of correctly spliced β(E) -globin (HBB:c.79G>A) mRNA and consequently β(+) -thalassaemia. A wide range of clinical severities in bothα- and β-thalassaemia syndromes, from nearly asymptomatic to transfusion-dependent, has been observed. The correlation between clinical heterogeneity in various genotypes of thalassaemia and the levels of globin gene expression and β(E) -globin pre-mRNA splicing were examined using multiplex quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) and allele-specific RT-qPCR. The α-globin/non α-globin mRNA ratio was demonstrated to be a good indicator for disease severity among different thalassaemia disorders. However, the α-globin/non α-globin mRNA ratio ranged widely in β-thalassaemia/HbE patients, with no significant difference between mild and severe phenotypes. Interestingly, the correctly to aberrantly spliced β(E) -globin mRNA ratio in 30% of mild β-thalassaemia/HbE patients was higher than that of the severe patients. The splicing process of β(E) -globin pre-mRNA differs among β-thalassaemia/HbE patients and serves as one of the modifying factors for disease severity. © 2011 Blackwell Publishing Ltd.

Over-expression of the miRNA cluster at chromosome 14q32 in the alcoholic brain correlates with suppression of predicted target mRNA required for oligodendrocyte proliferation.

PubMed

Manzardo, A M; Gunewardena, S; Butler, M G

2013-09-10

We examined miRNA expression from RNA isolated from the frontal cortex (Broadman area 9) of 9 alcoholics (6 males, 3 females, mean age 48 years) and 9 matched controls using both the Affymetrix GeneChip miRNA 2.0 and Human Exon 1.0 ST Arrays to further characterize genetic influences in alcoholism and the effects of alcohol consumption on predicted target mRNA expression. A total of 12 human miRNAs were significantly up-regulated in alcohol dependent subjects (fold change≥1.5, false discovery rate (FDR)≤0.3; p<0.05) compared with controls including a cluster of 4 miRNAs (e.g., miR-377, miR-379) from the maternally expressed 14q32 chromosome region. The status of the up-regulated miRNAs was supported using the high-throughput method of exon microarrays showing decreased predicted mRNA gene target expression as anticipated from the same RNA aliquot. Predicted mRNA targets were involved in cellular adhesion (e.g., THBS2), tissue differentiation (e.g., CHN2), neuronal migration (e.g., NDE1), myelination (e.g., UGT8, CNP) and oligodendrocyte proliferation (e.g., ENPP2, SEMA4D1). Our data support an association of alcoholism with up-regulation of a cluster of miRNAs located in the genomic imprinted domain on chromosome 14q32 with their predicted gene targets involved with oligodendrocyte growth, differentiation and signaling. Copyright © 2013 Elsevier B.V. All rights reserved.

Different dietary energy intake affects skeletal muscle development through an Akt-dependent pathway in Dorper × Small Thin-Tailed crossbred ewe lambs.

PubMed

Zhao, J X; Liu, X D; Li, K; Liu, W Z; Ren, Y S; Zhang, J X

2016-10-01

The objective of this experiment was to investigate the mechanisms through which different levels of dietary energy affect postnatal skeletal muscle development in ewe lambs. Twelve Dorper × Small Thin-Tailed crossbred ewe lambs (100 d of age; 20 ± 0.5 kg BW) were selected randomly and divided into 2 groups in a completely randomized design. Animals were offered identical diets at 100% or 65% of ad libitum intake. Lambs were euthanized when BW in the ad libitum group reached 35 kg and the semitendinosus muscle was sampled. Final BW and skeletal muscle weight were decreased (P < 0.01) by feed restriction. Both muscle fiber size distribution and myofibril cross-sectional area were altered by feed restriction. Insulin-like growth factor 1 (IGF-1) messenger RNA (mRNA) content was decreased (P < 0.05) when lambs were underfed, whereas no difference for IGF-2 mRNA expression was observed (P > 0.05). Feed restriction altered phosphor-Akt protein abundance (P < 0.01). Moreover, the mammalian target of rapamycin (mTOR) pathway was inhibited by feed restriction, which was associated with decreased phosphor-mTOR, phosphorylated eukaryotic initiation factor 4E binding protein 1 (phosphor-4EBP1), and phosphorylated ribosomal protein S6 kinase (phosphor-S6K). Both mRNA expression of myostatin and its protein content were elevated in feed-restricted ewe lambs (P < 0.05). In addition, mRNA expression of both muscle RING finger 1 and muscle atrophy F-box was increased when ewe lambs were underfed. In summary, feed restriction in young growing ewe lambs attenuates skeletal muscle hypertrophy by inhibiting protein synthesis and increasing protein degradation, which may act through the Akt-dependent pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

High-level mRNA quantification of proliferation marker pKi-67 is correlated with favorable prognosis in colorectal carcinoma.

PubMed

Ihmann, Thomas; Liu, Jian; Schwabe, Wolfgang; Häusler, Peter; Behnke, Detlev; Bruch, Hans-Peter; Broll, Rainer; Windhövel, Ute; Duchrow, Michael

2004-12-01

The present study retrospectively examines the expression of pKi-67 mRNA and protein in colorectal carcinoma and their correlation to the outcome of patients. Immunohistochemistry and quantitative RT-PCR were used to analyze the expression of pKi-67 in 43 archival specimens of patients with curatively resected primary colorectal carcinoma, who were not treated with neo-adjuvant therapy. We determined a median pKi-67 (MIB-1) labeling index of 31.3% (range 10.3-66.4%), and a mean mRNA level of 0.1769 (DeltaC(T): range 0.01-0.69); indices and levels did not correlate. High pKi-67 mRNA DeltaC(T) values were associated with a significantly favorable prognosis, while pKi-67 labeling indices were not correlated to prognostic outcome. A multivariate analysis of clinical and biological factors indicated that tumor stage (UICC) and pKi-67 mRNA expression level were independent prognostic factors. Quantitatively determined pKi-67 mRNA can be a good and new prognostic indicator for primary resected colorectal carcinoma.

Role of insulin-like growth factor-1 (IGF-1) in regulating cell cycle progression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Qi-lin; Yang, Tian-lun; Yin, Ji-ye

2009-11-06

Aims: Insulin-like growth factor-1 (IGF-1) is a polypeptide protein hormone, similar in molecular structure to insulin, which plays an important role in cell migration, cell cycle progression, cell survival and proliferation. In this study, we investigated the possible mechanisms of IGF-1 mediated cell cycle redistribution and apoptosis of vascular endothelial cells. Method: Human umbilical vein endothelial cells (HUVECs) were pretreated with 0.1, 0.5, or 2.5 {mu}g/mL of IGF-1 for 30 min before the addition of Ang II. Cell cycle redistribution and apoptosis were examined by flow cytometry. Expression of Ang II type 1 (AT{sub 1}) mRNA and cyclin E proteinmore » were determined by RT-PCR and Western blot, respectively. Results: Ang II (1 {mu}mol/L) induced HUVECs arrested at G{sub 0}/G{sub 1}, enhanced the expression level of AT{sub 1} mRNA in a time-dependent manner, reduced the enzymatic activity of nitric oxide synthase (NOS) and nitric oxide (NO) content as well as the expression level of cyclin E protein. However, IGF-1 enhanced NOS activity, NO content, and the expression level of cyclin E protein, and reduced the expression level of AT{sub 1} mRNA. L-NAME significantly counteracted these effects of IGF-1. Conclusions: Our data suggests that IGF-1 can reverse vascular endothelial cells arrested at G{sub 0}/G{sub 1} and apoptosis induced by Ang II, which might be mediated via a NOS-NO signaling pathway and is likely associated with the expression levels of AT1 mRNA and cyclin E proteins.« less

The Human T-Lymphotropic Virus Type 1 Tax Protein Inhibits Nonsense-Mediated mRNA Decay by Interacting with INT6/EIF3E and UPF1

PubMed Central

Mocquet, Vincent; Neusiedler, Julia; Rende, Francesca; Cluet, David; Robin, Jean-Philippe; Terme, Jean-Michel; Duc Dodon, Madeleine; Wittmann, Jürgen; Morris, Christelle; Le Hir, Hervé; Ciminale, Vincenzo

2012-01-01

In this report, we analyzed whether the degradation of mRNAs by the nonsense-mediated mRNA decay (NMD) pathway was affected in human T-lymphotropic virus type 1 (HTLV-1)-infected cells. This pathway was indeed strongly inhibited in C91PL, HUT102, and MT2 cells, and such an effect was also observed by the sole expression of the Tax protein in Jurkat and HeLa cells. In line with this activity, Tax binds INT6/EIF3E (here called INT6), which is a subunit of the translation initiation factor eukaryotic initiation factor 3 (eIF3) required for efficient NMD, as well as the NMD core factor upstream frameshift protein 1 (UPF1). It was also observed that Tax expression alters the morphology of processing bodies (P-bodies), the cytoplasmic structures which concentrate RNA degradation factors. The presence of UPF1 in these subcellular compartments was increased by Tax, whereas that of INT6 was decreased. In line with these effects, the level of the phosphorylated form of UPF1 was increased in the presence of Tax. Analysis of several mutants of the viral protein showed that the interaction with INT6 is necessary for NMD inhibition. The alteration of mRNA stability was observed to affect viral transcripts, such as that coding for the HTLV-1 basic leucine zipper factor (HBZ), and also several cellular mRNAs sensitive to the NMD pathway. Our data indicate that the effect of Tax on viral and cellular gene expression is not restricted to transcriptional control but can also involve posttranscriptional regulation. PMID:22553336

The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

PubMed Central

Halter, David; Collart, Martine A.; Panasenko, Olesya O.

2014-01-01

Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome. PMID:24465968

Ethanol- and acetaldehyde-induced cholinergic imbalance in the hippocampus of Aldh2-knockout mice does not affect nerve growth factor or brain-derived neurotrophic factor.

PubMed

Jamal, Mostofa; Ameno, Kiyoshi; Ruby, Mostofa; Miki, Takanori; Tanaka, Naoko; Nakamura, Yu; Kinoshita, Hiroshi

2013-11-20

Neurotrophins, including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), play an important role in the maintenance of cholinergic-neuron function. The objective of this study was to investigate whether ethanol (EtOH)- and acetaldehyde (AcH)- induced cholinergic effects would cause neurotrophic alterations in the hippocampus of mice. We used Aldh2 knockout (Aldh2-KO) mice, a model of aldehyde dehydrogenase 2 (ALDH2)-deficiency in humans, to examine the effects of acute administration of EtOH and the role of AcH. Hippocampal slices were collected and the mRNA and protein levels of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), NGF and BDNF were analyzed 30 min after the i.p. administration of EtOH (0.5, 1.0, or 2.0 g/kg). We show that treatment with 2.0 g/kg of EtOH decreased ChAT mRNA and protein levels in Aldh2-KO mice but not in wild-type (WT) mice, which suggests a role for AcH in the mechanism of action of EtOH. The administration of 2.0 g/kg of EtOH increased AChE mRNA in both strains of mice. EtOH failed to change the levels of NGF or BDNF at any dose. Aldh2-KO mice exhibited a distinctly lower expression of ChAT and a higher expression of NGF both at mRNA and protein levels in the hippocampus compared with WT mice. Our observations suggest that administration of EtOH and elevated AcH can alter cholinergic markers in the hippocampus of mice, and this effect did not change the levels of NGF or BDNF. © 2013 Elsevier B.V. All rights reserved.

Estrogen Responsiveness of the TFIID Subunit TAF4B in the Normal Mouse Ovary and in Ovarian Tumors1

PubMed Central

Wardell, Jennifer R.; Hodgkinson, Kendra M.; Binder, April K.; Seymour, Kimberly A.; Korach, Kenneth S.; Vanderhyden, Barbara C.; Freiman, Richard N.

2013-01-01

ABSTRACT Estrogen signaling in the ovary is a fundamental component of normal ovarian function, and evidence also indicates that excessive estrogen is a risk factor for ovarian cancer. We have previously demonstrated that the gonadally enriched TFIID subunit TAF4B, a paralog of the general transcription factor TAF4A, is required for fertility in mice and for the proliferation of ovarian granulosa cells following hormonal stimulation. However, the relationship between TAF4B and estrogen signaling in the normal ovary or during ovarian tumor initiation and progression has yet to be defined. Herein, we show that Taf4b mRNA and TAF4B protein, but not Taf4a mRNA or TAF4A protein, are increased in whole ovaries and granulosa cells of the ovary after exposure to 17beta-estradiol or the synthetic estrogen diethylstilbestrol and that this response occurs within hours after stimulation. Furthermore, this increase occurs via nuclear estrogen receptors both in vivo and in a mouse granulosa cancer cell line, NT-1. We observe a significant increase in Taf4b mRNA in estrogen-supplemented mouse ovarian tumors, which correlates with diminished survival of these mice. These data highlight the novel response of the general transcription factor TAF4B to estrogen in the normal ovary and during ovarian tumor progression in the mouse, suggesting its potential role in regulating actions downstream of estrogen stimulation. PMID:24068106

Estrogen responsiveness of the TFIID subunit TAF4B in the normal mouse ovary and in ovarian tumors.

PubMed

Wardell, Jennifer R; Hodgkinson, Kendra M; Binder, April K; Seymour, Kimberly A; Korach, Kenneth S; Vanderhyden, Barbara C; Freiman, Richard N

2013-11-01

Estrogen signaling in the ovary is a fundamental component of normal ovarian function, and evidence also indicates that excessive estrogen is a risk factor for ovarian cancer. We have previously demonstrated that the gonadally enriched TFIID subunit TAF4B, a paralog of the general transcription factor TAF4A, is required for fertility in mice and for the proliferation of ovarian granulosa cells following hormonal stimulation. However, the relationship between TAF4B and estrogen signaling in the normal ovary or during ovarian tumor initiation and progression has yet to be defined. Herein, we show that Taf4b mRNA and TAF4B protein, but not Taf4a mRNA or TAF4A protein, are increased in whole ovaries and granulosa cells of the ovary after exposure to 17beta-estradiol or the synthetic estrogen diethylstilbestrol and that this response occurs within hours after stimulation. Furthermore, this increase occurs via nuclear estrogen receptors both in vivo and in a mouse granulosa cancer cell line, NT-1. We observe a significant increase in Taf4b mRNA in estrogen-supplemented mouse ovarian tumors, which correlates with diminished survival of these mice. These data highlight the novel response of the general transcription factor TAF4B to estrogen in the normal ovary and during ovarian tumor progression in the mouse, suggesting its potential role in regulating actions downstream of estrogen stimulation.

Apple FLOWERING LOCUS T proteins interact with transcription factors implicated in cell growth and organ development.

PubMed

Mimida, Naozumi; Kidou, Shin-Ichiro; Iwanami, Hiroshi; Moriya, Shigeki; Abe, Kazuyuki; Voogd, Charlotte; Varkonyi-Gasic, Erika; Kotoda, Nobuhiro

2011-05-01

Understanding the flowering process in apple (Malus × domestica Borkh.) is essential for developing methods to shorten the breeding period and regulate fruit yield. It is known that FLOWERING LOCUS T (FT) acts as a transmissible floral inducer in the Arabidopsis flowering network system. To clarify the molecular network of two apple FT orthologues, MdFT1 and MdFT2, we performed a yeast two-hybrid screen to identify proteins that interact with MdFT1. We identified several transcription factors, including two members of the TCP (TEOSINTE BRANCHED1, CYCLOIDEA and PROLIFERATING CELL FACTORs) family, designated MdTCP2 and MdTCP4, and an Arabidopsis thaliana VOZ1 (Vascular plant One Zinc finger protein1)-like protein, designated MdVOZ1. MdTCP2 and MdVOZ1 also interacted with MdFT2 in yeast. The expression domain of MdTCP2 and MdVOZ1 partially overlapped with that of MdFT1 and MdFT2, most strikingly in apple fruit tissue, further suggesting a potential interaction in vivo. Constitutive expression of MdTCP2, MdTCP4 and MdVOZ1 in Arabidopsis affected plant size, leaf morphology and the formation of leaf primordia on the adaxial side of cotyledons. On the other hand, chimeric MdTCP2, MdTCP4 and MdVOZ1 repressors that included the ethylene-responsive transcription factors (ERF)-associated amphiphilic repression (EAR) domain motif influenced reproduction and inflorescence architecture in transgenic Arabidopsis. These results suggest that MdFT1 and/or MdFT2 might be involved in the regulation of cellular proliferation and the formation of new tissues and that they might affect leaf and fruit development by interacting with TCP- and VOZ-family proteins. DDBJ accession nos. AB531019 (MdTCP2a mRNA), AB531020 (MdTCP2b mRNA), AB531021 (MdTCP4a mRNA), AB531022 (MdTCP4b mRNA) and AB531023 (MdVOZ1a mRNA). © The Author 2011. Published by Oxford University Press. All rights reserved.

The Expression Alteration of BC1 RNA and its Interaction with Eukaryotic Translation Initiation Factor eIF4A Post-Status Epilepticus.

PubMed

Zeng, Xiangchang; Zong, Wenjing; Gao, Qing; Chen, Siyu; Chen, Lulu; Zeng, Guirong; Huang, Weihua; Li, Zhenyu; Zeng, Chang; Xie, Yuanyuan; Li, Xiaohui; Xiao, Bo; Dongsheng-Ouyang; Hu, Kai

2018-05-17

Abnormal dendritic sprouting and synaptic remodelling are important pathological features of temporal lobe epilepsy. BC1 RNA is a translation repressor involved in the regulation of the dendritic protein synthesis and mRNA transport, which is essential for dendritic development and plasticity. The expression alteration of BC1 RNA in the pilocarpine induced epilepsy model remains unknown. It is unclear if the interactions between BC1 RNA and eukaryotic initiation factor 4A (eIF4A) exists in this model. The purpose of this study was to investigate the expression changes of BC1 RNA and its interactions with eIF4A post-status epilepticus (SE). Chloride lithium and pilocarpine were used to induce the SE rat model. Either a whole brain or hippocampus tissues were collected at different time points after SE. The expression patterns of BC1 was detected by qPCR and in situ hybridization. The levels of eIF4AI/II protein expression were analyzed via western blotting and immunohistochemistry. The BC1 RNA-eIF4AI/II interaction was determined by electrophoretic mobility shift assay (EMSA). We found that the BC1 RNA levels decreased in hippocampus 3d, 1w and 2w post-SE before the levels recovered. The eIF4AI/II began to rise 3d post-SE and reached the maximum level 1w post-SE. After 1w post-SE the levels decreased in the hippocampal CA1, CA3 and DG subregions. EMSA analysis showed that BC1 RNA specifically interacted with the eIF4AI/II. The BC1 RNA-eIF4AI/II complex reduced to the lowest level 1w post-SE. Our results suggested that BC1 has a negative regulatory correlation with eIF4AI/II, where BC1 RNA could be involved in epileptogenesis by regulating dendritic protein synthesis.

Histone mRNA degradation in vivo: the first detectable step occurs at or near the 3' terminus.

PubMed Central

Ross, J; Peltz, S W; Kobs, G; Brewer, G

1986-01-01

The first detectable step in the degradation of human H4 histone mRNA occurs at the 3' terminus in a cell-free mRNA decay system (J. Ross and G. Kobs, J. Mol. Biol. 188:579-593, 1986). Most or all of the remainder of the mRNA is then degraded in a 3'-to-5' direction. The experiments described here were designed to determine whether a similar degradation pathway is followed in whole cells. Two sets of short-lived histone mRNA decay products were detected in logarithmically growing erythroleukemia (K562) cells. These products, designated the -5 and -12 RNAs, were generated by the loss of approximately 4 to 6 and 11 to 13 nucleotides, respectively, from the 3' terminus of histone mRNA. The same decay products were observed after a brief incubation in vitro. They were in low abundance or absent from cells that were not degrading histone mRNA. In contrast, they were readily detectable in cells that degraded the mRNA at an accelerated rate, i.e., in cells cultured with a DNA synthesis inhibitor, either cytosine arabinoside or hydroxyurea. During the initial stages of the decay process, as the 3' terminus of the mRNA was being degraded, the 5'-terminal region remained intact. These results indicate that the first detectable step in human H4 histone mRNA decay occurs at the 3' terminus and that degradation proceeds 3' to 5', both in cells and in cell-free reactions. Images PMID:3467177

Histone mRNA degradation in vivo: the first detectable step occurs at or near the 3' terminus.

PubMed

Ross, J; Peltz, S W; Kobs, G; Brewer, G

1986-12-01

The first detectable step in the degradation of human H4 histone mRNA occurs at the 3' terminus in a cell-free mRNA decay system (J. Ross and G. Kobs, J. Mol. Biol. 188:579-593, 1986). Most or all of the remainder of the mRNA is then degraded in a 3'-to-5' direction. The experiments described here were designed to determine whether a similar degradation pathway is followed in whole cells. Two sets of short-lived histone mRNA decay products were detected in logarithmically growing erythroleukemia (K562) cells. These products, designated the -5 and -12 RNAs, were generated by the loss of approximately 4 to 6 and 11 to 13 nucleotides, respectively, from the 3' terminus of histone mRNA. The same decay products were observed after a brief incubation in vitro. They were in low abundance or absent from cells that were not degrading histone mRNA. In contrast, they were readily detectable in cells that degraded the mRNA at an accelerated rate, i.e., in cells cultured with a DNA synthesis inhibitor, either cytosine arabinoside or hydroxyurea. During the initial stages of the decay process, as the 3' terminus of the mRNA was being degraded, the 5'-terminal region remained intact. These results indicate that the first detectable step in human H4 histone mRNA decay occurs at the 3' terminus and that degradation proceeds 3' to 5', both in cells and in cell-free reactions.

Locally applied leptin induces regional aortic wall degeneration preceding aneurysm formation in apolipoprotein E-deficient mice.

PubMed

Tao, Ming; Yu, Peng; Nguyen, Binh T; Mizrahi, Boaz; Savion, Naphtali; Kolodgie, Frank D; Virmani, Renu; Hao, Shuai; Ozaki, C Keith; Schneiderman, Jacob

2013-02-01

Leptin promotes atherosclerosis and vessel wall remodeling. As abdominal aortic aneurysm (AAA) formation involves tissue remodeling, we hypothesized that local leptin synthesis initiates and promotes this process. Human surgical AAA walls were analyzed for antigen and mRNA levels of leptin and leptin receptor, as well as mRNA for matrix metalloproteinases (MMP)-9 and MMP-12. Leptin and leptin receptor antigen were evident in all AAAs, and leptin, MMP-9, and MMP-12 mRNA was increased relative to age-matched nondilated controls. To simulate in vivo local leptin synthesis, ApoE(-/-) mice were subjected to a paravisceral periaortic application of low-dose leptin. Leptin-treated aortas exhibited decreased transforming growth factor-β and increased MMP-9 mRNA levels 5 days after surgery, and leptin receptor mRNA was upregulated by day 28. Serial ultrasonography demonstrated accelerated regional aortic diameter growth after 28 days, correlating with local medial degeneration, increased MMP-9, MMP-12, and periadventitial macrophage clustering. Furthermore, the combination of local periaortic leptin and systemic angiotensin II administration augmented medial MMP-9 synthesis and aortic aneurysm size. Leptin is locally synthesized in human AAA wall. Paravisceral aortic leptin in ApoE(-/-) mice induces local medial degeneration and augments angiotensin II-induced AAA, thus suggesting novel mechanistic links between leptin and AAA formation.

Regression of vessels in the tunica vasculosa lentis is initiated by coordinated endothelial apoptosis: a role for vascular endothelial growth factor as a survival factor for endothelium.

PubMed

Mitchell, C A; Risau, W; Drexler, H C

1998-11-01

The development of the embryonic lens is dependent on the formation and regression of the tunica vasculosa lentis (TVL), which is a transiently occurring capillary plexus that surrounds the posterior part of the lens. In this study, by using the terminal deoxy-nucleotidyl transferase mediated nick end-labelling technique (TUNEL), electron microscopy, radioactive end-labelling of DNA extracted from TVL, and the Comet assay, we show that widespread apoptosis of the endothelial cells that constitute the TVL is occurring already at embryonic day 17.5 (E17.5) of mouse development, much earlier than was reported previously (Jack [1972a] Am. J. Ophthalmol. 74:261-272; Lang [1997] Cell Death Diff. 4:12-20). In addition to apoptotic cell death, regression of this structure is associated with loss of capillary integrity, leakage of erythrocytes into the vitreal compartment, and phagocytosis of the apoptotic endothelium by tissue macrophages (hyalocytes). In situ hybridization experiments with probes for the flk-1 receptor and its high-affinity ligand, vascular endothelial growth factor (VEGF; Terman et al. [1992] Biochem. Biophys. Res. Commun. 187:1579-1586; Millauer et al. [1993] Cell 72:835-846), revealed strong endothelial cell expression for flk-1 in the eyes of E13.5-E17.5 embryos. VEGF mRNA was detected in lens epithelial cells located at the posterior pole of the developing lens in E13.5 embryos, in close proximity to the TVL capillaries. At later times (E14.5-E17.5), when the lens epithelial cells have differentiated into primary lens fiber cells, and a thick lenticular capsule is formed, the expression of VEGF mRNA becomes restricted to the anterior and equatorial portions of the lens. The physical separation of the VEGF-producing cells from the flk-1-expressing endothelium (due to the differentiation of the lens epithelial cells into lens fiber cells and the formation of the lenticular capsule) may deprive the endothelium of an essential survival factor and, thus, may constitute the primary mechanism that is responsible for the induction of endothelial cell apoptosis in this model.

An algorithm for the classification of mRNA patterns in eosinophilic esophagitis: Integration of machine learning.

PubMed

Sallis, Benjamin F; Erkert, Lena; Moñino-Romero, Sherezade; Acar, Utkucan; Wu, Rina; Konnikova, Liza; Lexmond, Willem S; Hamilton, Matthew J; Dunn, W Augustine; Szepfalusi, Zsolt; Vanderhoof, Jon A; Snapper, Scott B; Turner, Jerrold R; Goldsmith, Jeffrey D; Spencer, Lisa A; Nurko, Samuel; Fiebiger, Edda

2018-04-01

Diagnostic evaluation of eosinophilic esophagitis (EoE) remains difficult, particularly the assessment of the patient's allergic status. This study sought to establish an automated medical algorithm to assist in the evaluation of EoE. Machine learning techniques were used to establish a diagnostic probability score for EoE, p(EoE), based on esophageal mRNA transcript patterns from biopsies of patients with EoE, gastroesophageal reflux disease and controls. Dimensionality reduction in the training set established weighted factors, which were confirmed by immunohistochemistry. Following weighted factor analysis, p(EoE) was determined by random forest classification. Accuracy was tested in an external test set, and predictive power was assessed with equivocal patients. Esophageal IgE production was quantified with epsilon germ line (IGHE) transcripts and correlated with serum IgE and the T h 2-type mRNA profile to establish an IGHE score for tissue allergy. In the primary analysis, a 3-class statistical model generated a p(EoE) score based on common characteristics of the inflammatory EoE profile. A p(EoE) ≥ 25 successfully identified EoE with high accuracy (sensitivity: 90.9%, specificity: 93.2%, area under the curve: 0.985) and improved diagnosis of equivocal cases by 84.6%. The p(EoE) changed in response to therapy. A secondary analysis loop in EoE patients defined an IGHE score of ≥37.5 for a patient subpopulation with increased esophageal allergic inflammation. The development of intelligent data analysis from a machine learning perspective provides exciting opportunities to improve diagnostic precision and improve patient care in EoE. The p(EoE) and the IGHE score are steps toward the development of decision trees to define EoE subpopulations and, consequently, will facilitate individualized therapy. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Promoter-dependent and -independent activation of insulin-like growth factor binding protein-5 gene expression by prostaglandin E2 in primary rat osteoblasts

NASA Technical Reports Server (NTRS)

McCarthy, T. L.; Casinghino, S.; Mittanck, D. W.; Ji, C. H.; Centrella, M.; Rotwein, P.

1996-01-01

Insulin-like growth factor (IGF) action is mediated by high affinity cell surface IGF receptors and modulated by a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of six IGFBPs characterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E2 (PGE2), a local factor that mediates certain effects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain. In this study, we show that transcriptional and post-transcriptional events initiated by PGE2 collaborate to enhance IGFBP-5 gene expression in primary fetal rat osteoblast cultures. PGE2 treatment stimulated up to a 7-fold rise in steady-state levels of IGFBP-5 mRNA throughout 32 h of incubation. Analysis of nascent IGFBP-5 mRNA suggested that PGE2 had only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter-reporter genes confirmed that PGE2 enhanced promoter activity by approximately 2-fold. Similar stimulatory effects were seen with forskolin. A DNA fragment with only 51 base pairs of the 5'-flanking sequence retained hormonal responsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts with the mRNA transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that PGE2 enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t1/2 from 9 to 18 h. The effects of PGE2 on steady-state IGFBP-5 transcripts were abrogated by preincubating cells with cycloheximide, indicating that the effects of PGE2 on both gene transcription and mRNA stability required ongoing protein synthesis. Therefore, both promoter-dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE2 in osteoblasts.

Role of thymosin beta 4 in hair growth.

PubMed

Gao, Xiao-Yu; Hou, Fang; Zhang, Zhi-Peng; Nuo, Ming-Tu; Liang, Hao; Cang, Ming; Wang, Zhi-Gang; Wang, Xin; Xu, Teng; Yan, Le-Yan; Guo, Xu-Dong; Liu, Dong-Jun

2016-08-01

Although thymosin beta 4 (Tβ4) is known to play a role in hair growth, its mechanism of action is unclear. We examined the levels of key genes in a Tβ4 epidermal-specific over-expressing mouse model and Tβ4 global knockout mouse model to explore how Tβ4 affects hair growth. By depilation and histological examination of the skin, we confirmed the effect of Tβ4 on hair growth, the number of hair shafts and hair follicle (HF) structure. The mRNA and protein expression of several genes involved in hair growth were detected by real-time PCR and western blotting, respectively. Changes in the expression of β-catenin and Lef-1, the two key molecules in the Wnt signaling pathway, were similar to the changes observed in Tβ4 expression. We also found that compared to the control mice, the mRNA and protein expression of MMP-2 and VEGF were increased in the Tβ4 over-expressing mice, while the level of E-cadherin (E-cad) remained the same. Further, in the Tβ4 global knockout mice, the mRNA and protein levels of MMP-2 and VEGF decreased dramatically and the level of E-cad was stable. Based on the above results, we believe that Tβ4 may regulate the levels of VEGF and MMP-2 via the Wnt/β-catenin/Lef-1 signaling pathway to influence the growth of blood vessels around HFs and to activate cell migration. Tβ4 may have potential for the treatment of hair growth problems in adults, and its effects should be further confirmed in future studies.

Toll-Like Receptor 4 in Paraventricular Nucleus Mediates Visceral Hypersensitivity Induced by Maternal Separation

PubMed Central

Tang, Hui-Li; Zhang, Gongliang; Ji, Ning-Ning; Du, Lei; Chen, Bin-Bin; Hua, Rong; Zhang, Yong-Mei

2017-01-01

Neonatal maternal separation (MS) is a major early life stress that increases the risk of emotional disorders, visceral pain perception and other brain dysfunction. Elevation of toll-like receptor 4 (TLR4) signaling in the paraventricular nucleus (PVN) precipitates early life colorectal distension (CRD)-induced visceral hypersensitivity and pain in adulthood. The present study aimed to investigate the role of TLR4 signaling in the pathogenesis of postnatal MS-induced visceral hypersensitivity and pain during adulthood. The TLR4 gene was selectively knocked out in C57BL/10ScSn mice (Tlr4-/-). MS was developed by housing the offspring alone for 6 h daily from postnatal day 2 to day 15. Visceral hypersensitivity and pain were assessed in adulthood. Tlr4+/+, but not Tlr4-/-, mice that had experienced neonatal MS showed chronic visceral hypersensitivity and pain. TLR4 immunoreactivity was observed predominately in microglia in the PVN, and MS was associated with an increase in the expression of protein and/or mRNA levels of TLR4, corticotropin-releasing factor (CRF), CRF receptor 1 (CRFR1), tumor necrosis factor-α, and interleukin-1β in Tlr4+/+ mice. These alterations were not observed in Tlr4-/- mice. Local administration of lipopolysaccharide, a TLR4 agonist, into the lateral cerebral ventricle elicited visceral hypersensitivity and TLR4 mRNA expression in the PVN, which could be prevented by NBI-35965, an antagonist to CRFR1. The present results indicate that neonatal MS induces a sensitization and upregulation of microglial TLR4 signaling activity, which facilitates the neighboring CRF neuronal activity and, eventually, precipitates visceral hypersensitivity in adulthood. Highlights (1)Neonatal MS does not induce chronic visceral hypersensitivity and pain in Tlr4-/- mice.(2)Neonatal MS increases the expression of TLR4 mRNA, CRF protein and mRNA, CRFR1 protein, TNF-α protein, and IL-1β protein in Tlr4+/+ mice.(3)TLR4 agonist LPS (i.c.v.) elicits visceral hypersensitivity and TLR4 mRNA expression in the PVN. PMID:28611665

Conformational Differences between Open and Closed States of the Eukaryotic Translation Initiation Complex

PubMed Central

Llácer, Jose L.; Hussain, Tanweer; Marler, Laura; Aitken, Colin Echeverría; Thakur, Anil; Lorsch, Jon R.; Hinnebusch, Alan G.; Ramakrishnan, V.

2015-01-01

Summary Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5′ end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition. PMID:26212456

[Effects of lipopolysaccharides extracted from Porphyromonas endodontalis on the expression of IL-1beta mRNA and IL-6 mRNA in osteoblasts].

PubMed

Yang, Di; Li, Ren; Qiu, Li-Hong; Li, Chen

2009-04-01

To quantify the IL-1 beta mRNA and IL-6 mRNA expression induced by lipopolysaccharides (LPS)extracted from Porphyromonas endodontalis(P.e) in osteoblasts, and to relate P.e-LPS to bone absorption pathogenesis in lesions of chronical apical periodontitis. MG63 was treated with different concentrations of P.e-LPS(0-50 microg/mL) for different hours(0-24h). The expression of IL-1 beta mRNA and IL-6 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR).Statistical analysis was performed using one- way ANOVA and Dunnett t test with SPSS11.0 software package. The level of IL-1 beta mRNA and IL-6 mRNA increased significantly after treatment with P.e-LPS at more than 5 microg/mL (P<0.01)and for more than 1 hour (P<0.01), which indicated that P.e-LPS induced osteoblasts to express IL-1 beta mRNA and IL-6 mRNA in dose and time dependent manners. P.e-LPS may promote bone resorption in lesions of chronical apical periodontitis by inducing IL-1 beta mRNA and IL-6 mRNA expression in osteoblasts.

Gene expression of glucose transporter (GLUT) 1, 3 and 4 in bovine follicle and corpus luteum.

PubMed

Nishimoto, H; Matsutani, R; Yamamoto, S; Takahashi, T; Hayashi, K-G; Miyamoto, A; Hamano, S; Tetsuka, M

2006-01-01

Glucose is the main energy substrate in the bovine ovary, and a sufficient supply of it is necessary to sustain the ovarian activity. Glucose cannot permeate the plasma membrane, and its uptake is mediated by a number of glucose transporters (GLUT). In the present study, we investigated the gene expression of GLUT1, 3 and 4 in the bovine follicle and corpus luteum (CL). Ovaries were obtained from Holstein x Japanese Black F1 heifers. Granulosa cells and theca interna layers were harvested from follicles classified into five categories by their physiologic status: follicular size (>or= 8.5 mm: dominant; < 8.5 mm: subordinate), ratio of estradiol (E(2)) to progesterone in follicular fluid (>or= 1: E(2) active;<1: E(2) inactive), and stage of estrous cycle (luteal phase, follicular phase). CL were also classified by the stage of estrous cycle. Expression levels of GLUT1, 3 and 4 mRNA were quantified by a real-time PCR. The mRNA for GLUT1 and 3 were detected in the bovine follicle and CL at comparable levels to those in classic GLUT-expressing organs such as brain and heart. Much lower but appreciable levels of GLUT4 were also detected in these tissues. The gene expression of these GLUT showed tissue- and stage-specific patterns. Despite considerable differences in physiologic conditions, similar levels of GLUT1, 3 and 4 mRNA were expressed in subordinate follicles as well as dominant E(2)-active follicles in both luteal and follicular phases, whereas a notable increase in the gene expression of these GLUT was observed in dominant E(2)-inactive follicles undergoing the atretic process. In these follicles, highly significant negative correlations were observed between the concentrations of glucose in follicular fluid and the levels of GLUT1 and 3 mRNA in granulosa cells, implying that the local glucose environment affects glucose uptake of follicles. These results indicate that GLUT1 and 3 act as major transporters of glucose while GLUT4 may play a supporting role in the bovine follicle and CL.

What determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame?

PubMed Central

Pöyry, Tuija A.A.; Kaminski, Ann; Jackson, Richard J.

2004-01-01

If the 5′-proximal AUG triplet in a mammalian mRNA is followed by a short open reading frame (sORF), a significant fraction of ribosomes resume scanning after termination of sORF translation, and reinitiate at a downstream AUG. To examine the underlying mechanism, we examined reinitiation in vitro using a series of mRNAs that differed only in the 5′-untranslated region (UTR). Efficient reinitiation was found to occur only if the eIF4F complex, or at a minimum the central one-third fragment of eIF4G, participated in the primary initiation event at the sORF initiation codon. It did not occur, however, when sORF translation was driven by the classical swine fever virus or cricket paralysis virus internal ribosome entry sites (IRESs), which do not use eIF4A, 4B, 4E, or 4G. A critical test was provided by an mRNA with an unstructured 5′-UTR, which is translated by scanning but does not absolutely need eIF4G and eIF4A: There was efficient reinitiation in a standard reticulocyte lysate, when initiation would be largely driven by eIF4F, but no reinitiation in an eIF4G-depleted lysate. These results suggest that resumption of scanning may depend on the interaction between eIF4F (or the eIF4G central domain) and the ribosome being maintained while the ribosome translates the sORF. PMID:14701882

Triage of Women with Low-Grade Cervical Lesions - HPV mRNA Testing versus Repeat Cytology

PubMed Central

Sørbye, Sveinung Wergeland; Arbyn, Marc; Fismen, Silje; Gutteberg, Tore Jarl; Mortensen, Elin Synnøve

2011-01-01

Background In Norway, women with low-grade squamous intraepithelial lesions (LSIL) are followed up after six months in order to decide whether they should undergo further follow-up or be referred back to the screening interval of three years. A high specificity and positive predictive value (PPV) of the triage test is important to avoid unnecessary diagnostic and therapeutic procedures. Materials and Methods At the University Hospital of North Norway, repeat cytology and the HPV mRNA test PreTect HPV-Proofer, detecting E6/E7 mRNA from HPV types 16, 18, 31, 33 and 45, are used in triage of women with ASC-US and LSIL. In this study, women with LSIL cytology in the period 2005–2008 were included (n = 522). Two triage methods were evaluated in two separate groups: repeat cytology only (n = 225) and HPV mRNA testing in addition to repeat cytology (n = 297). Histologically confirmed cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) was used as the study endpoint. Results Of 522 women with LSIL, 207 had biopsies and 125 of them had CIN2+. The sensitivity and specificity of repeat cytology (ASC-US or worse) were 85.7% (95% confidence interval (CI): 72.1, 92.2) and 54.4 % (95% CI: 46.9, 61.9), respectively. The sensitivity and specificity of the HPV mRNA test were 94.2% (95% CI: 88.7, 99.7) and 86.0% (95% CI: 81.5, 90.5), respectively. The PPV of repeat cytology was 38.4% (95% CI: 29.9, 46.9) compared to 67.0% (95% CI: 57.7, 76.4) of the HPV mRNA test. Conclusion HPV mRNA testing was more sensitive and specific than repeat cytology in triage of women with LSIL cytology. In addition, the HPV mRNA test showed higher PPV. These data indicate that the HPV mRNA test is a better triage test for women with LSIL than repeat cytology. PMID:21918682

Detection of PIK3CA gene mutations with HRM analysis and association with IGFBP-5 expression levels in breast cancer.

PubMed

Dirican, Ebubekir; Kaya, Zehra; Gullu, Gokce; Peker, Irem; Ozmen, Tolga; Gulluoglu, Bahadir M; Kaya, Handan; Ozer, Ayse; Akkiprik, Mustafa

2014-01-01

Breast cancer is the second most common cancer and second leading cause of cancer deaths in women. Phosphatidylinositol-3-kinase (PI3K)/AKT pathway mutations are associated with cancer and phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene mutations have been observed in 25-45% of breast cancer samples. Insulin growth factor binding protein-5 (IGFBP-5) can show different effects on apoptosis, cell motility and survival in breast cancer. We here aimed to determine the association between PIK3CA gene mutations and IGFBP-5 expressions for the first time in breast cancer patients. Frozen tumor samples from 101 Turkish breast cancer patients were analyzed with high resolution melting (HRM) for PIK3CA mutations (exon 9 and exon 20) and 37 HRM positive tumor samples were analyzed by DNA sequencing, mutations being found in 31. PIK3CA exon 9 mutations (Q546R, E542Q, E545K, E542K and E545D) were found in 10 tumor samples, exon 20 mutations (H1047L, H1047R, T1025T and G1049R) in 21, where only 1 tumor sample had two exon 20 mutations (T1025T and H1047R). Moreover, we detected one sample with both exon 9 (E542Q) and exon 20 (H1047R) mutations. 35% of the tumor samples with high IGFBP-5 mRNA expression and 29.4% of the tumor samples with low IGFBP-5 mRNA expression had PIK3CA mutations (p=0.9924). This is the first study of PIK3CA mutation screening results in Turkish breast cancer population using HRM analysis. This approach appears to be a very effective and reliable screening method for the PIK3CA exon 9 and 20 mutation detection. Further analysis with a greater number of samples is needed to clarify association between PIK3CA gene mutations and IGFBP-5 mRNA expression, and also clinical outcome in breast cancer patients.

Hypotonic stress induces RANKL via transient receptor potential melastatin 3 (TRPM3) and vaniloid 4 (TRPV4) in human PDL cells.

PubMed

Son, G Y; Yang, Y M; Park, W S; Chang, I; Shin, D M

2015-03-01

Bone remodeling occurs in response to various types of mechanical stress. The periodontal ligament (PDL) plays an important role in mechanical stress-mediated alveolar bone remodeling. However, the underlying mechanism at the cellular level has not been extensively studied. In this study, we investigated the effect of shear stress on the expression of bone remodeling factors, including receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) and osteoprotegerin (OPG), as well as its upstream signaling pathway in primary human PDL cells. We applied hypotonic stress to reproduce shear stress to PDL cells. Hypotonic stress induced the messenger RNA (mRNA) and protein expression of RANKL but not OPG. It also increased intracellular Ca(2+) concentration ([Ca(2+)]i). Extracellular Ca(2+) depletion and nonspecific plasma membrane Ca(2+) channel blockers completely inhibited the increase in both [Ca(2+)]i and RANKL mRNA expression. We identified the expression and activation of transient receptor potential melastatin 3 (TRPM3) and vaniloid 4 (TRPV4) channels in PDL cells. Pregnenolone sulfate (PS) and 4α-phorbol 12, 13-didecanoate (4α-PDD), which are agonists of TRPM3 and TRPV4, augmented Ca(2+) influx and RANKL mRNA expression. Both pharmacological (2-aminoethoxydiphenyl borate [2-APB], ruthenium red [RR], ononetin [Ono], and HC 067047 [HC]) and genetic (small interfering RNA [siRNA]) inhibitors of TRPM3 and TRPV4 reduced the hypotonic stress-mediated increase in [Ca(2+)]i and RANKL mRNA expression. Our study shows that hypotonic stress induced RANKL mRNA expression via TRPM3- and TRPV4-mediated extracellular Ca(2+) influx and RANKL expression. This signaling pathway in PDL cells may play a critical role in mechanical stress-mediated alveolar bone remodeling. © International & American Associations for Dental Research 2015.

Effects of different dietary intake on mRNA levels of MSTN, IGF-I, and IGF-II in the skeletal muscle of Dorper and Hu sheep hybrid F1 rams.

PubMed

Xing, H J; Wang, Z Y; Zhong, B S; Ying, S J; Nie, H T; Zhou, Z R; Fan, Y X; Wang, F

2014-07-24

MSTN, IGF-І(insulin-like growth factor-І) and IGF-II (insulin-like growth factor-II) regulate skeletal muscle growth. This study investigated the effects of different dietary intake levels on skeletal muscles. Sheep was randomly assigned to 3 feeding groups: 1) the maintenance diet (M), 2) 1.4 x the maintenance diet (1.4M), and 3) 2.15 x the maintenance diet (2.15M). Before slaughtering the animals, blood samples were collected to measure plasma urea, growth hormone, and insulin concentrations. After slaughtering, the longissimus dorsi, semitendinosus, semimembranosus, gastrocnemius, soleus, and chest muscle were removed to record various parameters, including the mRNA expression levels of MSTN and IGFs, in addition to skeletal muscle fiber diameter and cross-sectional area. The result showed that as dietary intake improved, the mRNA expression levels of MSTN and IGF-II decreased, whereas IGF-Іexpression increased. The mRNA expression levels of MSTN and IGFs were significantly different in the same skeletal muscle under different dietary intake. The skeletal muscle fiber diameter and cross-sectional area increased with greater dietary intake, as observed for the mRNA expression of IGF-І; however, it contrasted to that observed for the mRNA expression of MSTN and IGF-II. In conclusion, dietary intake levels have a certain influence on MSTN and IGFs mRNA expression levels, in addition to skeletal muscle fiber diameter and cross-sectional area. This study contributes valuable information for enhancing the molecular-based breeding of sheep.

Maternal Factors Are Associated with the Expression of Placental Genes Involved in Amino Acid Metabolism and Transport

PubMed Central

Day, Pricilla E.; Ntani, Georgia; Crozier, Sarah R.; Mahon, Pam A.; Inskip, Hazel M.; Cooper, Cyrus; Harvey, Nicholas C.; Godfrey, Keith M.; Hanson, Mark A.; Lewis, Rohan M.; Cleal, Jane K.

2015-01-01

Introduction Maternal environment and lifestyle factors may modify placental function to match the mother’s capacity to support the demands of fetal growth. Much remains to be understood about maternal influences on placental metabolic and amino acid transporter gene expression. We investigated the influences of maternal lifestyle and body composition (e.g. fat and muscle content) on a selection of metabolic and amino acid transporter genes and their associations with fetal growth. Methods RNA was extracted from 102 term Southampton Women’s Survey placental samples. Expression of nine metabolic, seven exchange, eight accumulative and three facilitated transporter genes was analyzed using quantitative real-time PCR. Results Increased placental LAT2 (p = 0.01), y + LAT2 (p = 0.03), aspartate aminotransferase 2 (p = 0.02) and decreased aspartate aminotransferase 1 (p = 0.04) mRNA expression associated with pre-pregnancy maternal smoking. Placental mRNA expression of TAT1 (p = 0.01), ASCT1 (p = 0.03), mitochondrial branched chain aminotransferase (p = 0.02) and glutamine synthetase (p = 0.05) was positively associated with maternal strenuous exercise. Increased glutamine synthetase mRNA expression (r = 0.20, p = 0.05) associated with higher maternal diet quality (prudent dietary pattern) pre-pregnancy. Lower LAT4 (r = -0.25, p = 0.05) and aspartate aminotransferase 2 mRNA expression (r = -0.28, p = 0.01) associated with higher early pregnancy diet quality. Lower placental ASCT1 mRNA expression associated with measures of increased maternal fat mass, including pre-pregnancy BMI (r = -0.26, p = 0.01). Lower placental mRNA expression of alanine aminotransferase 2 associated with greater neonatal adiposity, for example neonatal subscapular skinfold thickness (r = -0.33, p = 0.001). Conclusion A number of maternal influences have been linked with outcomes in childhood, independently of neonatal size; our finding of associations between placental expression of transporter and metabolic genes and maternal smoking, physical activity and diet raises the possibility that their effects are mediated in part through alterations in placental function. The observed changes in placental gene expression in relation to modifiable maternal factors are important as they could form part of interventions aimed at maintaining a healthy lifestyle for the mother and for optimal fetal development. PMID:26657885

Genetic evaluation of childhood acute lymphoblastic leukemia in Iraq using FTA cards.

PubMed

Al-Kzayer, Lika'a Fasih Y; Sakashita, Kazuo; Matsuda, Kazuyuki; Al-Hadad, Salma Abbas; Al-Jadiry, Mazin Faisal; Abed, Wisam Majeed; Abdulkadhim, Jaafar M H; Al-Shujairi, Tariq Abadi; Hasan, Janan Ghalib; Al-Abdullah, Hussam M Salih; Al-Ani, Mouroge H; Saber, Paiman Ali I; Inoshita, Toshi; Kamata, Minoru; Koike, Kenichi

2012-09-01

Genetic examination of childhood leukemia has not been available in Iraq. We here report the frequency of TEL-AML1, E2A-PBX1, MLL-AF4, and BCR-ABL chimeric transcripts in 264 Iraqi children newly diagnosed with acute lymphoblastic leukemia (ALL), using FTA cards impregnated with bone marrow aspirate or whole blood. The diagnosis of ALL was made according to standard French-American-British morphologic criteria. Based on the results of storage temperature and duration, most of the FTA samples were preserved at 4°C for up to 6 weeks in five Iraqi hospitals and then transferred to Japan for molecular analysis. Nested reverse transcription-polymerase chain reaction was adopted for the analysis. TEL-AML1 chimeric transcript product was found in 32 (12.1%) of 264 ALL patients. Eleven (4.2%) patients, 4 (1.5%) patients, and 11 (4.2%) patients had E2A-PBX1 mRNA, MLL-AF4 mRNA, and BCR-ABL mRNA, respectively. One patient had both TEL-AML1 and E2A-PBX1 fusion genes. The incidence of TEL-AML1 in Iraqi ALL children appears to be similar to or slightly higher than those of Jordan (12%) and Kuwait (7%). The prevalence and clinical findings of ALL patients with either E2A-PBX1 or BCR-ABL were comparable to the data reported elsewhere. International collaboration via FTA cards may be helpful to improve diagnosis and management of patients with hematological malignancies in low-income and underdeveloped countries. Copyright © 2012 Wiley Periodicals, Inc.

Stimulation by thyroid-stimulating hormone and Grave's immunoglobulin G of vascular endothelial growth factor mRNA expression in human thyroid follicles in vitro and flt mRNA expression in the rat thyroid in vivo.

PubMed

Sato, K; Yamazaki, K; Shizume, K; Kanaji, Y; Obara, T; Ohsumi, K; Demura, H; Yamaguchi, S; Shibuya, M

1995-09-01

To elucidate the pathogenesis of thyroid gland hypervascularity in patients with Graves' disease, we studied the expression of mRNAs for vascular endothelial growth factor (VEGF) and its receptor, Flt family, using human thyroid follicles in vitro and thiouracil-fed rats in vivo. Human thyroid follicles, cultured in the absence of endothelial cells, secreted de novo-synthesized thyroid hormone in response to thyroid-stimulating hormone (TSH) and Graves' IgG. The thyroid follicles produced VEGF mRNA but not flt-1 mRNA. The expression of VEGF mRNA was enhanced by insulin, tumor-promoting phorbol ester, calcium ionophore, dibutyryl cAMP, TSH, and Graves' IgG. When rats were fed thiouracil for 4 wk, their serum levels of TSH were increased at day 3. VEGF mRNA was also increased on day 3, accompanied by an increase in flt family (flt-1 and KDR/ flk-1) mRNA expression. These in vitro and in vivo findings suggest that VEGF is produced by thyroid follicles in response to stimulators of TSH receptors, via the protein kinase A and C pathways. VEGF, a secretable angiogenesis factor, subsequently stimulates Flt receptors on endothelial cells in a paracrine manner, leading to their proliferation and producing hypervascularity of the thyroid gland, as seen in patients with Graves' disease.

Modulatory effects of steroid hormones, oxytocin, arachidonic acid, forskolin and cyclic AMP on the expression of aquaporin 1 and aquaporin 5 in the porcine uterus during placentation.

PubMed

Skowronska, A; Mlotkowska, P; Okrasa, S; Nielsen, S; Skowronski, M T

2016-04-01

Aquaporins (AQPs) are proteins forming trans-membrane channels responsible for water transport. AQP1 and AQP5 are present in structures of the female reproductive system. In the uterus, these AQPs are involved in water movement between the intraluminal, interstitial and capillary compartments and their uterine expression is essential throughout the pregnancy, including its early stages. Thus, the study aimed to assess the influence of P4 (progesterone), E2 (estradiol), OT (oxytocin), AA (arachidonic acid), cAMP and FSK (forskolin) on the AQP1 and AQP5 mRNA and protein expression in the uterine tissue of gilts on Days 30 - 32 of gestation (the placentation period), following short (3 h) and long (24 h) incubations. Steroid hormones influenced the expression of AQP1 and AQP5; E2 up-regulated, but P4 down-regulated mRNAs of these AQPs, whereas the protein level of studied AQPs was increased by both steroids. OT treatment decreased AQP1 (after 24 h), but increased AQP5 (after 3 h) mRNA expression. Treatment with AA significantly reduced the AQP1 expression at the mRNA level, but stimulated at the protein level. The expression of AQP5 mRNA and protein was stimulated by AA. FSK markedly decreased AQP1 mRNA, but increased of AQP5 after 3-h incubation. In turn, cAMP stimulated and inhibited transcription of AQP5 after 3- and 24-h incubations, respectively. Immunohistochemical analysis confirmed the uterine localization of AQP1 in the apical and basal membranes of endothelial cells and AQP5 in the apical membranes of epithelial cells under control condition. Treatments with P4, E2, AA, cAMP or FSK have caused additional appearance of AQP5 labeling in the basolateral membranes of epithelial cells. These results suggest a participation of steroid hormones (P4 and E2), AA derivatives and cAMP in controlling the expression of AQP1 and AQP5 as well as the distribution of AQP5 in the uterine tissue of pregnant gilts during placentation (Days 30 - 32 of gestation).

Translational control of aberrant stress responses as a hallmark of cancer.

PubMed

El-Naggar, Amal M; Sorensen, Poul H

2018-04-01

Altered mRNA translational control is emerging as a critical factor in cancer development and progression. Targeting specific elements of the translational machinery, such as mTORC1 or eIF4E, is emerging as a new strategy for innovative cancer therapy. While translation of most mRNAs takes place through cap-dependent mechanisms, a sub-population of cellular mRNA species, particularly stress-inducible mRNAs with highly structured 5'-UTR regions, are primarily translated through cap-independent mechanisms. Intriguingly, many of these mRNAs encode proteins that are involved in tumour cell adaptation to microenvironmental stress, and thus linked to aggressive behaviour including tumour invasion and metastasis. This necessitates a rigorous search for links between microenvironmental stress and aggressive tumour phenotypes. Under stress, cells block global protein synthesis to preserve energy while maintaining selective synthesis of proteins that support cell survival. One highly conserved mechanism to regulate protein synthesis under cell stress is to sequester mRNAs into cytosolic aggregates called stress granules (SGs), where their translation is silenced. SGs confer survival advantages and chemotherapeutic resistance to tumour cells under stress. Recently, it has been shown that genetically blocking SG formation dramatically reduces tumour invasive and metastatic capacity in vivo. Therefore, targeting SG formation might represent a potential treatment strategy to block cancer metastasis. Here, we present the critical link between selective mRNA translation, stress adaptation, SGs, and tumour progression. Further, we also explain how deciphering mechanisms of selective mRNA translation occurs under cell stress holds great promise for the identification of new targets in the treatment of cancer. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Delta ribozyme has the ability to cleave in transan mRNA.

PubMed Central

Roy, G; Ananvoranich, S; Perreault, J P

1999-01-01

We report here the first demonstration of the cleavage of an mRNA in trans by delta ribozyme derived from the antigenomic version of the human hepatitis delta virus (HDV). We characterized potential delta ribozyme cleavage sites within HDV mRNA sequence (i.e. C/UGN6), using oligonucleotide binding shift assays and ribonuclease H hydrolysis. Ribozymes were synthesized based on the structural data and then tested for their ability to cleave the mRNA. Of the nine ribozymes examined, three specifically cleaved a derivative HDV mRNA. All three active ribozymes gave consistent indications that they cleaved single-stranded regions. Kinetic characterization of the ability of ribozymes to cleave both the full-length mRNA and either wild-type or mutant small model substrate suggests: (i) delta ribozyme has turnovers, that is to say, several mRNA molecules can be successively cleaved by one ribozyme molecule; and (ii) the substrate specificity of delta ribozyme cleavage is not restricted to C/UGN6. Specifically, substrates with a higher guanosine residue content upstream of the cleavage site (i.e. positions -4 to -2) were always cleaved more efficiently than wild-type substrate. This work shows that delta ribozyme constitutes a potential catalytic RNA for further gene-inactivation therapy. PMID:9927724

Expression of ZNF804A in human brain and alterations in schizophrenia, bipolar disorder, and major depressive disorder: a novel transcript fetally regulated by the psychosis risk variant rs1344706.

PubMed

Tao, Ran; Cousijn, Helena; Jaffe, Andrew E; Burnet, Philip W J; Edwards, Freya; Eastwood, Sharon L; Shin, Joo Heon; Lane, Tracy A; Walker, Mary A; Maher, Brady J; Weinberger, Daniel R; Harrison, Paul J; Hyde, Thomas M; Kleinman, Joel E

2014-10-01

The single-nucleotide polymorphism rs1344706 in the zinc finger protein 804A gene (ZNF804A) shows genome-wide association with schizophrenia and bipolar disorder. Little is known regarding the expression of ZNF804A and the functionality of rs1344706. To characterize ZNF804A expression in human brain and to investigate how it changes across the life span and how it is affected by rs1344706, schizophrenia, bipolar disorder, and major depressive disorder. Molecular and immunochemical methods were used to study ZNF804A messenger RNA (mRNA) and ZNF804A protein, respectively. ZNF804A transcripts were investigated using next-generation sequencing and polymerase chain reaction-based methods, and ZNF804A protein was investigated using Western blots and immunohistochemistry. Samples of dorsolateral prefrontal cortex and inferior parietal lobe tissue were interrogated from 697 participants between 14 weeks' gestational age and age 85 years, including patients with schizophrenia, bipolar disorder, or major depressive disorder. Quantitative measurements of ZNF804A mRNA and immunoreactivity, and the effect of diagnosis and rs1344706 genotype. ZNF804A was expressed across the life span, with highest expression prenatally. An abundant and developmentally regulated truncated ZNF804A transcript was identified, missing exons 1 and 2 (ZNF804AE3E4) and predicted to encode a protein lacking the zinc finger domain. rs1344706 influenced expression of ZNF804AE3E4 mRNA in fetal brain (P = .02). In contrast, full-length ZNF804A showed no association with genotype (P > .05). ZNF804AE3E4 mRNA expression was decreased in patients with schizophrenia (P = .006) and increased in those with major depressive disorder (P < .001), and there was a genotype-by-diagnosis interaction in bipolar disorder (P = .002). ZNF804A immunoreactivity was detected in fetal and adult human cerebral cortex. It was localized primarily to pyramidal neurons, with cytoplasmic as well as dendritic and nuclear staining. No differences in ZNF804A-immunoreactive neurons were seen in schizophrenia or related to rs1344706 (P > .05). rs1344706 influences the expression of ZNF804AE3E4, a novel splice variant. The effect is limited to fetal brain and to this isoform. It may be part of the mechanism by which allelic variation in ZNF804A affects risk of psychosis. ZNF804A is translated in human brain, where its functions may extend beyond its predicted role as a transcription factor.

The mAb against adipocyte fatty acid-binding protein 2E4 attenuates the inflammation in the mouse model of high-fat diet-induced obesity via toll-like receptor 4 pathway.

PubMed

Miao, Xiaoliang; Wang, Ying; Wang, Wang; Lv, Xiaobo; Wang, Min; Yin, Hongping

2015-03-05

Adipocyte fatty acid-binding protein (A-FABP) plays an important role in fatty acid-mediated processes and related metabolic and inflammatory responses. In this study, we prepared a novel monoclonal antibody against A-FABP, designated 2E4. Our data showed that 2E4 specifically binded to the recombinant A-FABP and native A-FABP of mice adipose tissue. Furthermore, we investigated the effect of 2E4 on metabolic and inflammatory responses in C57BL/6J obese mice fed on a high fat diet. 2E4 administration improved glucose response in high-fat-diet induced obese mice. The 2E4 treated groups exhibited lower free fatty acids, cholesterol, and triglycerides in a concentration-dependent manner. These changes were accompanied by down-regulated expression of pro-inflammatory cytokines in adipose tissue, including tumor necrosis factor α, monocyte chemotactic protein-1, and interleukin-6. Meanwhile, our data demonstrated that 2E4 significantly decreased the mRNA and protein levels of A-FABP in adipose tissue of mice. Further experiments showed that 2E4 notably suppressed the phosphorylation of IκBα and jun-N-terminal kinase through toll-like receptor 4 signaling pathway. Taken together, 2E4 is an effective monoclonal antibody against A-FABP, which attenuated the inflammatory responses induced in the high-fat-diet mice. These findings may provide scientific insight into the treatment of chronic low-grade inflammation in obesity. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Insulin alleviates degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system in septic rats.

PubMed

Chen, Qiyi; Li, Ning; Zhu, Weiming; Li, Weiqin; Tang, Shaoqiu; Yu, Wenkui; Gao, Tao; Zhang, Juanjuan; Li, Jieshou

2011-06-03

Hypercatabolism is common under septic conditions. Skeletal muscle is the main target organ for hypercatabolism, and this phenomenon is a vital factor in the deterioration of recovery in septic patients. In skeletal muscle, activation of the ubiquitin-proteasome system plays an important role in hypercatabolism under septic status. Insulin is a vital anticatabolic hormone and previous evidence suggests that insulin administration inhibits various steps in the ubiquitin-proteasome system. However, whether insulin can alleviate the degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system under septic condition is unclear. This paper confirmed that mRNA and protein levels of the ubiquitin-proteasome system were upregulated and molecular markers of skeletal muscle proteolysis (tyrosine and 3-methylhistidine) simultaneously increased in the skeletal muscle of septic rats. Septic rats were infused with insulin at a constant rate of 2.4 mU.kg-1.min-1 for 8 hours. Concentrations of mRNA and proteins of the ubiquitin-proteasome system and molecular markers of skeletal muscle proteolysis were mildly affected. When the insulin infusion dose increased to 4.8 mU.kg-1.min-1, mRNA for ubiquitin, E2-14 KDa, and the C2 subunit were all sharply downregulated. At the same time, the levels of ubiquitinated proteins, E2-14KDa, and the C2 subunit protein were significantly reduced. Tyrosine and 3-methylhistidine decreased significantly. We concluded that the ubiquitin-proteasome system is important skeletal muscle hypercatabolism in septic rats. Infusion of insulin can reverse the detrimental metabolism of skeletal muscle by inhibiting the ubiquitin-proteasome system, and the effect is proportional to the insulin infusion dose.

[Effects of different mechanical stretch conditions on differentiation of rat tendon stem cells].

PubMed

Li, Pao; Gao, Shang; Zhou, Mei; Tang, Hong; Mu, Miduo; Zhang, Jiqiang; Tang, Kanglai

2017-04-01

To investigate the effects of different mechanical stretch conditions on the differentiation of rat tendon stem cells (TSCs), to find the best uniaxial cyclic stretching for TSCs tenogenic differentiation, osteogenic differentiation, and adipogenic differentiation. TSCs were isolated from the Achilles tendons of 8-week-old male Sprague Dawley rats by enzymatic digestion method and cultured. The TSCs at passage 3 were randomly divided into 5 groups: group A (stretch strength of 4% and frequency of 1 Hz), group B (stretch strength of 4% and frequency of 2 Hz), group C (stretch strength of 8% and frequency of 1 Hz), group D (stretch strength of 8% and frequency of 2 Hz), and group E (static culture). At 12, 24, and 48 hours after mechanical stretch, the mRNA expressions of the tenogenic differentiation related genes [Scleraxis (SCX) and Tenascin C (TNC)], the osteogenic differentiation related genes [runt related transcription factor 2 (RUNX2) and distal-less homeobox 5 (DLX5)], and the adipogenic differentiation related genes [CCAAT-enhancer-binding protein-α (CEBPα) and lipoprteinlipase (LPL)] were detected by real-time fluorescent quantitative PCR and the protein expressions of TNC, CEBPα, and RUNX2 were detected by Western blot. The mRNA expressions of SCX and TNC in group B were significantly higher than those in groups A, C, D, and E at 24 hours after mechanical stretch ( P <0.05). The mRNA expressions of CEBPα and LPL in group D were significantly higher than those in groups A, B, C, and E at 48 hours after mechanical stretch ( P <0.05). The mRNA expressions of RUNX2 and DLX5 in group C were significantly higher than those in groups A, B, D, and E at 24 hours after mechanical stretch ( P <0.05). Western blot detection showed that higher protein expression of TNC in group B than group E at each time point after mechanical stretch ( P <0.05), and the protein expression of CEBPα was significantly inhibited when compared with group E at 24 hours after mechanical stretch ( P <0.05). At 24 hours after mechanical stretch, the protein expression of RUNX2 in group C was significantly higher than that in group E ( P <0.05); and the protein expression of TNC was significantly lower than that in group E at 24 and 48 hours after mechanical stretch ( P <0.05). At 48 hours after mechanical stretch, the protein expression of CEBPα was significantly increased and the protein expression of TNC was significantly decreased in group D when compared with group E ( P <0.05), but no significant difference was found in the protein expression of RUNX2 between groups D and E ( P >0.05). The mechanical strain could promote differentiation of TSCs, and different parameter of stretch will lead to different differentiation. The best stretch condition for tenogenic differentiation is 4% strength and 2 Hz frequency for 24 hours; the best stretch condition for osteogenic differentiation is 8% strength and 1 Hz frequency for 24 hours; and the best stretch condition for adipogenic differentiation is 8% strength and 2 Hz frequency for 48 hours.

Urinary podocyte-associated mRNA levels correlate with proximal tubule dysfunction in early diabetic nephropathy of type 2 diabetes mellitus.

PubMed

Petrica, Ligia; Ursoniu, Sorin; Gadalean, Florica; Vlad, Adrian; Gluhovschi, Gheorghe; Dumitrascu, Victor; Vlad, Daliborca; Gluhovschi, Cristina; Velciov, Silvia; Bob, Flaviu; Matusz, Petru; Milas, Oana; Secara, Alina; Simulescu, Anca; Popescu, Roxana

2017-01-01

The study assessed mRNA expression of podocyte-associated molecules in urinary sediments of patients with type 2 diabetes mellitus (DM) in relation to urinary podocytes, biomarkers of podocyte injury and of proximal tubule (PT) dysfunction. A total of 76 patients with type 2 DM and 20 healthy subjects were enrolled in a cross-sectional study, and assessed concerning urinary podocytes, urinary mRNA of podocyte-associated genes, urinary biomarkers of podocyte damage and of PT dysfunction. We found significant differences between urinary mRNA of podocyte-associated molecules in relation with albuminuria stage. In multivariable regression analysis, urinary mRNA of nephrin, podocin, alpha-actinin-4, CD2-associated protein, glomerular epithelial protein 1 (GLEPP1), ADAM 10, and NFκB correlated directly with urinary podocytes, albuminuria, urinary alpha 1 -microglobulin, urinary kidney-injury molecule-1, nephrinuria, urinary vascular endothelial growth factor, urinary advanced glycation end-products (AGE), and indirectly with eGFR (p < 0.0001, R 2  = 0.808; p < 0.0001, R 2  = 0.825; p < 0.0001, R 2  = 0.805; p < 0.0001, R 2  = 0.663; p < 0.0001, R 2  = 0.726; p < 0.0001, R 2  = 0.720; p < 0.0001, R 2  = 0.724). In patients with type 2 DM there is an association between urinary mRNA of podocyte-associated molecules, biomarkers of podocyte damage, and of PT dysfunction. GLEPP1, ADAM10, and NFκB may be considered additional candidate molecules indicative of early diabetic nephropathy. AGE could be involved in this association.

The increase in the expression and hypomethylation of MUC4 gene with the progression of pancreatic ductal adenocarcinoma.

PubMed

Zhu, Yi; Zhang, Jing-jing; Zhu, Rong; Zhu, Yan; Liang, Wen-biao; Gao, Wen-tao; Yu, Jun-bo; Xu, Ze-kuan; Miao, Yi

2011-12-01

The MUC4 gene could have a key role in the progression of pancreatic cancer, but the quantitative measurement of its expression in clinical tissue samples remains a challenge. The correlations between MUC4 promoter methylation status in vivo and either pancreatic cancer progression or MUC4 mRNA expression need to be demonstrated. We used the techniques of quantitative real-time PCR and DNA methylation-specific PCR combined microdissection to precisely detect MUC4 expression and promoter methylation status in 116 microdissected foci from 57 patients with pancreatic ductal adenocarcinoma. Both mRNA expression and hypomethylation frequency increased from normal to precancerous lesions to pancreatic cancer. Multivariate Cox regression analysis showed that high-level MUC4 expression (P = 0.008) and tumor-node-metastasis staging (P = 0.038) were significant independent risk factors for predicting the prognosis of 57 patients. The MUC4 mRNA expression was not significantly correlated with promoter methylation status in 30 foci of pancreatic ductal adenocarcinoma. These results suggest that high mRNA expression and hypomethylation of the MUC4 gene could be involved in carcinogenesis and in the malignant development of pancreatic ductal adenocarcinoma. The MUC4 mRNA expression may become a new prognostic marker for pancreatic cancer. Microdissection-based quantitative real-time PCR and methylation-specific PCR contribute to the quantitative detection of MUC4 expression in clinical samples and reflect the epigenetic regulatory mechanisms of MUC4 in vivo.

Induction of cancer chemopreventive enzymes by coffee is mediated by transcription factor Nrf2. Evidence that the coffee-specific diterpenes cafestol and kahweol confer protection against acrolein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Higgins, Larry G.; Cavin, Christophe; Itoh, Ken

2008-02-01

Mice fed diets containing 3% or 6% coffee for 5 days had increased levels of mRNA for NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase class Alpha 1 (GSTA1) of between 4- and 20-fold in the liver and small intestine. Mice fed 6% coffee also had increased amounts of mRNA for UDP-glucuronosyl transferase 1A6 (UGT1A6) and the glutamate cysteine ligase catalytic (GCLC) subunit of between 3- and 10-fold in the small intestine. Up-regulation of these mRNAs was significantly greater in mice possessing Nrf2 (NF-E2 p45 subunit-related factor 2) than those lacking the transcription factor. Basal levels of mRNAs for NQO1, GSTA1,more » UGT1A6 and GCLC were lower in tissues from nrf2{sup -/-} mice than from nrf2{sup +/+} mice, but modest induction occurred in the mutant animals. Treatment of mouse embryonic fibroblasts (MEFs) from nrf2{sup +/+} mice with either coffee or the coffee-specific diterpenes cafestol and kahweol (C + K) increased NQO1 mRNA up to 9-fold. MEFs from nrf2{sup -/-} mice expressed less NQO1 mRNA than did wild-type MEFs, but NQO1 was induced modestly by coffee or C + K in the mutant fibroblasts. Transfection of MEFs with nqo1-luciferase reporter constructs showed that induction by C + K was mediated primarily by Nrf2 and required the presence of an antioxidant response element in the 5'-upstream region of the gene. Luciferase reporter activity did not increase following treatment of MEFs with 100 {mu}mol/l furan, suggesting that this ring structure within C + K is insufficient for gene induction. Priming of nrf2{sup +/+} MEFs, but not nrf2{sup -/-} MEFs, with C + K conferred 2-fold resistance towards acrolein.« less

Sensitivity, Specificity, and Clinical Value of Human Papillomavirus (HPV) E6/E7 mRNA Assay as a Triage Test for Cervical Cytology and HPV DNA Test ▿

PubMed Central

Benevolo, Maria; Vocaturo, Amina; Caraceni, Donatella; French, Deborah; Rosini, Sandra; Zappacosta, Roberta; Terrenato, Irene; Ciccocioppo, Lucia; Frega, Antonio; Rossi, Paolo Giorgi

2011-01-01

There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of HPV DNA positive-mRNA negative cases. PMID:21525231

Soyabean glycinin depresses intestinal growth and function in juvenile Jian carp (Cyprinus carpio var Jian): protective effects of glutamine.

PubMed

Jiang, Wei-Dan; Hu, Kai; Zhang, Jin-Xiu; Liu, Yang; Jiang, Jun; Wu, Pei; Zhao, Juan; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Feng, Lin

2015-11-28

This study investigated the effects of glycinin on the growth, intestinal oxidative status, tight junction components, cytokines and apoptosis signalling factors of fish. The results showed that an 80 g/kg diet of glycinin exposure for 42 d caused poor growth performance and depressed intestinal growth and function of juvenile Jian carp (Cyprinus carpio var. Jian). Meanwhile, dietary glycinin exposure induced increases in lipid peroxidation and protein oxidation; it caused reductions in superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activities; and it increased MnSOD, CuZnSOD, GPx1b and GPx4a mRNA levels, suggesting an adaptive mechanism against stress in the intestines of fish. However, dietary glycinin exposure decreased both the activity and mRNA levels of nine isoforms of glutathione-S-transferase (GST) (α, μ, π, ρ, θ, κ, mGST1, mGST2 and mGST3), indicating toxicity to this enzyme activity and corresponding isoform gene expressions. In addition, glycinin exposure caused partial disruption of intestinal cell-cell tight junction components, disturbances of cytokines and induced apoptosis signalling in the distal intestines>mid intestines>proximal intestines of fish. Glycinin exposure also disturbed the mRNA levels of intestinal-related signalling factors Nrf2, Keap1a, Keap1b, eleven isoforms of protein kinase C and target of rapamycin/4E-BP. Interestingly, glutamine was observed to partially block those negative influences. In conclusion, this study indicates that dietary glycinin exposure causes intestinal oxidative damage and disruption of intestinal physical barriers and functions and reduces fish growth, but glutamine can reverse those negative effects in fish. This study provides some information on the mechanism of glycinin-induced negative effects.

Determinants of Viral Oncogene E6-E7 mRNA Overexpression in a Population-Based Large Sample of Women Infected by High-Risk Human Papillomavirus Types

PubMed Central

Bisanzi, Simonetta; Allia, Elena; Mongia, Alessandra; Carozzi, Francesca; Gillio-Tos, Anna; De Marco, Laura; Ronco, Guglielmo; Gustinucci, Daniela; Del Mistro, Annarosa; Frayle, Helena; Iossa, Anna; Fantacci, Giulia; Pompeo, Giampaolo; Cesarini, Elena; Bulletti, Simonetta; Passamonti, Basilio; Rizzi, Martina; Penon, Maria Gabriella; Barca, Alessandra; Benevolo, Maria

2017-01-01

ABSTRACT Cervical cancer screening by human papillomavirus (HPV) DNA testing with cytology triage is more effective than cytology testing. Compared to cytology, the HPV DNA test's higher sensitivity, which allows better protection with longer intervals, makes it necessary to triage the women with a positive result to compensate its lower specificity. We are conducting a large randomized clinical trial (New Technologies for Cervical Cancer 2 [NTCC2]) within organized population-based screening programs in Italy using HPV DNA as the primary screening test to evaluate, by the Aptima HPV assay (Hologic), the use of HPV E6-E7 mRNA in a triage test in comparison to cytology. By the end of June 2016, data were available for 35,877 of 38,535 enrolled women, 2,651 (7.4%) of whom were HPV DNA positive. Among the samples obtained, 2,453 samples were tested also by Aptima, and 1,649 (67.2%) gave a positive result. The proportion of mRNA positivity was slightly higher among samples tested for HPV DNA by the Cobas 4800 HPV assay (Roche) than by the Hybrid Capture 2 (HC2) assay (Qiagen). In our setting, the observed E6-E7 mRNA positivity rate, if used as a triage test, would bring a rate of immediate referral to colposcopy of about 4 to 5%. This value is higher than that observed with cytology triage for both immediate and delayed referrals to colposcopy. By showing only a very high sensitivity and thus allowing a longer interval for HPV DNA-positive/HPV mRNA-negative women, a triage by this test might be more efficient than by cytology. PMID:28100595

Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: a new gene-silencing approach.

PubMed

Marsollier, Anne-Charlotte; Ciszewski, Lukasz; Mariot, Virginie; Popplewell, Linda; Voit, Thomas; Dickson, George; Dumonceaux, Julie

2016-04-15

Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

PubMed

Smits, A; Funa, K; Vassbotn, F S; Beausang-Linder, M; af Ekenstam, F; Heldin, C H; Westermark, B; Nistér, M

1992-03-01

Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protein was found in the malignant tumors, and also in some benign lesions, such as dermatofibroma. In all these cases, benign as well as malignant, the PDGF B-chain mRNA, and less clearly, the PDGF A-chain mRNA, were coexpressed with the beta-receptor. In contrast, high expression of PDGF alpha-receptor mRNA was only found in fully malignant lesions, i.e., malignant fibrous histiocytoma. These data indicate that an autocrine growth stimulation via the PDGF beta-receptor could occur in an early phase of tumorigenesis, and may be a necessary but insufficient event for the progression into fully malignant human connective tissue lesions.

Mucosal immunization in macaques upregulates the innate APOBEC 3G anti-viral factor in CD4(+) memory T cells.

PubMed

Wang, Yufei; Bergmeier, Lesley A; Stebbings, Richard; Seidl, Thomas; Whittall, Trevor; Singh, Mahavir; Berry, Neil; Almond, Neil; Lehner, Thomas

2009-02-05

APOBEC3G is an innate intracellular anti-viral factor which deaminates retroviral cytidine to uridine. In vivo studies of APOBEC3G (A3G) were carried out in rhesus macaques, following mucosal immunization with SIV antigens and CCR5 peptides, linked to the 70kDa heat shock protein. A progressive increase in A3G mRNA was elicited in PBMC after each immunization (p<0.0002 to p< or =0.02), which was maintained for at least 17 weeks. Analysis of memory T cells showed a significant increase in A3G mRNA and protein in CD4(+)CCR5(+) memory T cells in circulating (p=0.0001), splenic (p=0.0001), iliac lymph nodes (p=0.002) and rectal (p=0.01) cells of the immunized compared with unimmunized macaques. Mucosal challenge with SIVmac 251 showed a significant increase in A3G mRNA in the CD4(+)CCR5(+) circulating cells (p<0.01) and the draining iliac lymph node cells (p<0.05) in the immunized uninfected macaques, consistent with a protective effect exerted by A3G. The results suggest that mucosal immunization in a non-human primate can induce features of a memory response to an innate anti-viral factor in CCR5(+)CD4(+) memory and CD4(+)CD95(+)CCR7(-) effector memory T cells.

Estrogen promotes the brain metastatic colonization of triple negative breast cancer cells via an astrocyte-mediated paracrine mechanism

PubMed Central

Sartorius, Carol A.; Hanna, Colton T.; Gril, Brunilde; Cruz, Hazel; Serkova, Natalie J.; Huber, Kendra M.; Kabos, Peter; Schedin, Troy B.; Borges, Virginia F.; Steeg, Patricia S.; Cittelly, Diana M.

2015-01-01

Brain metastases (BM) are a devastating consequence of breast cancer. BM occur more frequently in patients with estrogen receptor-negative (ER−) breast cancer subtypes; HER2 overexpressing (HER2+) tumors and triple-negative (TN) (ER−, progesterone receptor-negative (PR−) and normal HER2) tumors. Young age is an independent risk factor for development of BM, thus we speculated that higher circulating estrogens in young, pre-menopausal women could exert paracrine effects through the highly estrogen-responsive brain microenvironment. Using a TN experimental metastases model, we demonstrate that ovariectomy decreased the frequency of MRI detectable lesions by 56% as compared to estrogen supplementation, and that the combination of ovariectomy and letrozole further reduced the frequency of large lesions to 14.4% of the estrogen control. Human BM expressed 4.2-48.4% ER+ stromal area, particularly ER+ astrocytes. In vitro, E2-treated astrocytes increased proliferation, migration and invasion of 231BR-EGFP cells in an ER-dependent manner. E2 upregulated EGFR ligands Egf, Ereg, and Tgfa mRNA and protein levels in astrocytes, and activated EGFR in brain metastatic cells. Co-culture of 231BR-EGFP cells with E2-treated astrocytes led to upregulation of the metastatic mediator S100 Calcium-binding protein A4 (S100A4) (1.78-fold, P<0.05). Exogenous EGF increased S100A4 mRNA levels in 231BR-EGFP cells (1.40±0.02 fold, P<0.01 compared to vehicle-control) and an EGFR/HER2 inhibitor blocked this effect, suggesting that S100A4 is a downstream effector of EGFR activation. ShRNA-mediated S100A4 silencing in 231BR-EGFP cells decreased their migration and invasion in response to E2-CM, abolished their increased proliferation in co-cultures with E2-treated astrocytes, and decreased brain metastatic colonization. Thus, S100A4 is one effector of the paracrine action of E2 in brain metastatic cells. These studies provide a novel mechanism by which estrogens, acting through ER+ astrocytes in the brain microenvironment, can promote BM of TN breast cancers, and suggests existing endocrine agents may provide some clinical benefit towards reducing and managing BM. PMID:26411365

Apocynin suppresses the progression of atherosclerosis in apoE-deficient mice by inactivation of macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kinoshita, Hiroyuki; Matsumura, Takeshi, E-mail: takeshim@gpo.kumamoto-u.ac.jp; Ishii, Norio

Highlights: ► We examined the anti-athrogenic effect of apocynin in atherosclerotic model mice. ► Apocynin prevented atherosclerotic lesion formation. ► Apocynin suppressed ROS production in aorta and in macrophages. ► Apocynin suppressed cytokine expression and cell proliferation in macrophages. ► Apocynin may be beneficial compound for the prevention of atherosclerosis. -- Abstract: Production of reactive oxygen species (ROS) and other proinflammatory substances by macrophages plays an important role in atherogenesis. Apocynin (4-hydroxy-3-methoxy-acetophenone), which is well known as a NADPH oxidase inhibitor, has anti-inflammatory effects including suppression of the generation of ROS. However, the suppressive effects of apocynin on the progressionmore » of atherosclerosis are not clearly understood. Thus, we investigated anti-atherosclerotic effects of apocynin using apolipoprotein E-deficient (apoE{sup –/–}) mice in vivo and in mouse peritoneal macrophages in vitro. In atherosclerosis-prone apoE{sup –/–} mice, apocynin suppressed the progression of atherosclerosis, decreased 4-hydroxynonenal-positive area in atherosclerotic lesions, and mRNA expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) in aorta. In mouse peritoneal macrophages, apocynin suppressed the Ox-LDL-induced ROS generation, mRNA expression of MCP-1, IL-6 and granulocyte/macrophage colony-stimulating factor, and cell proliferation. Moreover, immunohistochemical studies revealed that apocynin decreased the number of proliferating cell nuclear antigen-positive macrophages in atherosclerotic lesions of apoE{sup –/–} mice. These results suggested that apocynin suppressed the formation of atherosclerotic lesions, at least in part, by inactivation of macrophages. Therefore, apocynin may be a potential therapeutic material to prevent the progression of atherosclerosis.« less

A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs.

PubMed

Gromadzka, Agnieszka M; Steckelberg, Anna-Lena; Singh, Kusum K; Hofmann, Kay; Gehring, Niels H

2016-03-18

The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Progesterone dose-dependently modulates hepatocyte growth factor production in 3T3-L1 mouse preadipocytes.

PubMed

Ito, Tomoki; Yamaji, Daisuke; Kamikawa, Akihiro; Abd Eldaim, Mabrouk Attia; Okamatsu-Ogura, Yuko; Terao, Akira; Saito, Masayuki; Kimura, Kazuhiro

2017-08-30

It is well documented that estrogen is predominant inducer of hepatocyte growth factor (HGF) in a variety of cell types. However, the effect of progesterone (P) remains to be elusive. Thus, in the present study, we examined the effect of P and combined effect of P and 17β-estradiol (E2) on HGF expression and production in 3T3-L1 fibroblastic preadipocytes and mature adipocytes, as a model of stromal cells. Northern blot analysis showed that hgf mRNA expressed in preadipocytes was notably higher than that of mature adipocytes, and increased by treatment of preadipocytes with E2 or 10 nM P, but not with 1,000 nM P. The E2-induced hgf mRNA expression was enhanced by 10 nM P, but suppressed by 1,000 nM P. Western blot analysis revealed that biological active forms of HGF protein was found in the preadipocyte culture medium, while the lesser amount of HGF precursor protein was detected in the mature adipocyte culture medium. The amounts of HGF were changed dependently on the hgf mRNA expression levels. These results indicate that HGF production is intricately regulated by E2 and P at the transcriptional levels in 3T3-L1 cells, and may explain the changes in the HGF production during the mammary gland development, especially decrease in HGF expression during pregnancy when P concentration is high.

Expression and localization of insulin-like growth factor system in corpus luteum during different stages of estrous cycle in water buffaloes (Bubalus bubalis) and the effect of insulin-like growth factor I on production of vascular endothelial growth factor and progesterone in luteal cells cultured in vitro.

PubMed

Uniyal, S; Panda, R P; Chouhan, V S; Yadav, V P; Hyder, I; Dangi, S S; Gupta, M; Khan, F A; Sharma, G T; Bag, S; Sarkar, M

2015-01-01

This study investigated the expression and localization of insulin-like growth factor (IGF) system at different stages of buffalo CL and the role of IGF-I in stimulating vascular endothelial growth factor (VEGF) and progesterone (P4) production in cultured luteal cells. The mRNA expression of IGF system, VEGF, steroidogenic acute regulatory protein, P450scc, and hydroxysteroid dehydrogenase (HSD) was investigated by quantitative real-time polymerase chain reaction (PCR). Protein expression of IGF was demonstrated by Western blot and localization by immunohistochemistry. Progesterone and VEGF production was assayed using RIA and ELISA. A relatively high mRNA expression of IGF-I and IGF-II in early, mid- and late luteal phases with immunoreactivity mostly restricted to cytoplasm of large luteal cells indicates their autocrine role, whereas very weak immunoreactivity in endothelial cells during the mid-luteal phase indicates their paracrine role. Insulin-like growth factor receptors, IGF-IR and IGF-IIR, were restricted to large luteal cells with high mRNA and protein expressions in the mid-luteal phase. The significantly higher expression of insulin-like growth factor binding protein (IGFBP)-1, -3, -5, and -6 in the early or mid-luteal phase suggested their stimulatory role, whereas that of IGFBP-2 and -4 in mid-, late, and regressive luteal stages implied their inhibitory role. The mRNA expressions of key steroidogenic factors and VEGF were significantly higher (P < 0.05) when the culture medium was supplemented with 100 ng/mL of IGF-I for 72 hours. Moreover, IGF-I at a dose of 100 ng/mL increased P4 and VEGF production (P < 0.05). It can be concluded that IGF family members via their autocrine and paracrine effect play significant roles in promoting angiogenesis through the production of VEGF in luteal cells and steroid synthesis through the production of key steroidogenic factors. Copyright © 2015 Elsevier Inc. All rights reserved.

Non-Eosinophilic Nasal Polyps Shows Increased Epithelial Proliferation and Localized Disease Pattern in the Early Stage.

PubMed

Kim, Dong-Kyu; Jin, Hong Ryul; Eun, Kyoung Mi; Mutusamy, Somasundran; Cho, Seong H; Oh, Sohee; Kim, Dae Woo

2015-01-01

Non-eosinophilic nasal polyps (NPs) show less inflammatory changes and are less commonly associated with lower airway inflammatory disorders such as asthma, compared with eosinophilic NPs. However, the development of non-eosinophilic NPs which is a predominant subtype in Asian population still remains unclear. A total of 81 patients (45 with non-eosinophilic NPs and 36 with eosinophilic NPs) were enrolled. Clinical information and computed tomography (CT), endoscopic, and histological findings were investigated. Tissue samples were analyzed for total IgE levels and for mRNA expression levels of interleukin (IL)-4, IL-5, IL-13, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-17A, IL-22, IL-23p19, transforming growth factor (TGF)-β1, TGF-β2, TGF-β3, and periostin. Immunostaining assessment of Ki-67 as a proliferation marker was performed. We found that epithelial in-growing patterns such as pseudocysts were more frequently observed in histological and endoscopic evaluations of non-eosinophilic NPs, which was linked to increase epithelial staining of Ki-67, a proliferating marker. Eosinophilic NPs were characterized by high infiltration of inflammatory cells, compared with non-eosinophilic NPs. To investigate the developmental course of each subtype, CT was analyzed according to CT scores and subtypes. Non-eosinophilic NPs showed more localized pattern and maxillary sinus involvement, but lesser olfactory involvement in early stage whereas eosinophilic NPs were characterized by diffuse ethmoidal and olfactory involvement. In addition, high ethmoidal/maxillary (E/M) CT scores, indicating ethmoidal dominant involvement, were one of surrogate markers for eosinophilic NP. E/M CT scores was positively correlated with levels of TH2 inflammatory markers, including IL-4, IL-5, periostin mRNA expression and total IgE levels in NPs, whereas levels of the TH1 cytokine, IFN- γ were inversely correlated. Moreover, if the combinatorial algorithm meet the three of the four markers, including IL-5 (<2.379), periostin (<3.889), IFN-γ (>0.316), and E/M ratio (<2.167), non-eosinophilic CRSwNP are diagnosed with a sensitivity of 84.4% and a specificity of 84.8%. Histologic, immunologic and clinical data suggest that non-eosinophilic NPs showed enhanced epithelial alteration and more localized maxillary involvement. Combination of cutoff value on IL-5, periostin, IFN-γ, and E/M scores may be one of surrogate markers for non-eosinophil NP subtype.

Differential regulation and impact of fucosyltransferase VII and core 2 β1,6-N-acetyl-glycosaminyltransferase for generation of E-selectin and P-selectin ligands in murine CD4+ T cells

PubMed Central

Schroeter, Micha F; Ratsch, Boris A; Lehmann, Jeanette; Baumgrass, Ria; Hamann, Alf; Syrbe, Uta

2012-01-01

Ligands for E-selectin and P-selectin (E-lig and P-lig) are induced on CD4+ T cells upon differentiation into effector T cells. Glycosyltransferases, especially α 1,3-fucosyltransferase VII (FucT-VII) and core 2 β1,6-N-acetyl-glycosaminyltransferase I (C2GlcNAcT-I), are critical for their synthesis. We here analysed the signals that control the expression of E-lig, P-lig and mRNA coding for FucT-VII and C2GlcNAcT-I. In line with previous reports, we found that P-lig expression correlates with the regulation of C2GlcNAcT-I, whereas E-lig expression can occur at low levels of C2GlcNAcT-I mRNA but requires high FucT-VII mRNA expression. Interestingly, the two enzymes are regulated by different signals. Activation-induced C2GlcNAcT-I up-regulation under permissive (T helper type 1) conditions was strongly reduced by cyclosporin A (CsA), suggesting the involvement of T-cell receptor-dependent, calcineurin/NFAT-dependent signals in combination with interleukin-12 (IL-12) -mediated signals in the regulation of C2GlcNAcT-I. In contrast, expression of FucT-VII mRNA was not significantly inhibited by CsA. Interleukin-4 inhibited the expression of FucT-VII but IL-2 and IL-7 were found to support induction of FucT-VII and E-lig. E-selectin, P-selectin and their ligands initially appeared to have rather overlapping functions. These findings however, unravel striking differences in the regulation of E-lig and P-lig expression, dictated by the dominance of FucT-VII and C2GlcNAcT-I, respectively, and their dependency on signals from either promiscuous or homeostatic cytokines (FucT-VII) or a strong T-cell receptor signal in combination with inflammatory cytokines in case of C2GlcNAcT-I. PMID:23039181

Time-dependent progression from the acute to chronic phases in atopic dermatitis induced by epicutaneous allergen stimulation in NC/Nga mice.

PubMed

Kim, Ji-Yun; Jeong, Mi Sook; Park, Mi Kyung; Lee, Mi-Kyung; Seo, Seong Jun

2014-01-01

Atopic dermatitis (AD) is a complicated skin condition influenced by genetic background and environmental factors. In this study, we applied Dermatophagoides farinae body extract (DfE) to the barrier-disrupted skin of NC/Nga mice twice a week for 8 weeks to identify the clinical and immunological factors in AD progression. Repeated application of the DfE to the skin of NC/Nga mice showed the similar consequences for the natural course of progression in human AD, histologically and immunologically. We confirmed that the AD-like skin lesions in NC/Nga mice did not last for the whole period of our experiment in spite of repeated topical applications of DfE twice a week. Topical DfE stimulation increased the skin mRNA expressions of Th1-, Th2- and Th17-related cytokines in the acute phase. The expression patterns of IL-4 and IL-13 in splenic T cells and skin lesions were consistent with the time course alterations of clinical features of AD-like skin symptoms. We also showed that there was a remission phase either just before or right after the chronic phase in this experimental model. Interestingly, splenic T-cell-derived IL-5 expression began to increase in the chronic phase, while skin-derived IL-5 mRNA expression increased in the acute phase. In conclusion, our results suggest that we should pay attention to the characteristics of each stage of AD progression and choose a suitable corresponding stage of animal model not only to elucidate the pathogenesis of AD but also to develop and evaluate therapeutic drugs for AD. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Evidences for involvement of growth hormone and insulin-like growth factor in ovarian development of starry flounder (Platichthys stellatus).

PubMed

Xu, Yongjiang; Wang, Bin; Liu, Xuezhou; Shi, Bao; Zang, Kun

2017-04-01

Although gonadotrophins are major regulators of ovarian function in teleosts and other vertebrates, accumulating evidence indicates that the growth hormone (GH)-insulin-like growth factor (IGF) axis also plays an important role in fish reproduction. As a first step to understand the physiological role of the GH-IGF system in the ovarian development of starry flounder (Platichthys stellatus), the expression profiles of GH and IGF messenger RNAs (mRNAs) and plasma GH, IGF-I, estradiol-17β (E2), and testosterone (T) levels during the ovarian development were investigated. The developmental stages of ovaries were divided into five stages (II, III, IV, V, and VI) by histological analysis. The hepatosomatic index (HSI) and gonadosomatic index (GSI) values increased and peaked at stage IV and stage V, respectively, and then declined at stage VI. Pituitary GH mRNA levels decreased sharply at stage III and raised to top level at stage VI. The hepatic IGF-I mRNA levels ascended to maximum value at stage V and then declined significantly at stage VI. However, the hepatic IGF-II mRNA levels remained stable and increased significantly at stage VI. In contrast, the ovarian IGF-I mRNA levels increased gradually and peaked at stage VI. The ovarian IGF-II mRNA levels were initially stable and increased significantly at stage V until the top level at stage VI. Consistent with the pituitary GH mRNA levels, plasma GH levels reduced sharply at stage III and remained depressed until stage V and then raised remarkably at stage VI. Plasma IGF-I level peaked at stage V and then declined to initial level. Plasma E2 level peaked at stage IV and then dramatically descended to the basal level. Plasma T level peaked at stage V and then declined significantly back to the basal level. Based on statistical analysis, significant positive correlations between hepatic IGF-I mRNA and GSI, ovarian IGF-II mRNA and hepatic IGF-II mRNA, ovarian IGF-I mRNA and ovarian IGF-II mRNA, and plasma IGF-I and plasma T were observed, respectively. These results suggest that the GH-IGF system may be involved in the ovarian development of starry flounder; GH and IGFs appear to play distinct roles in the regulation of the ovarian development in paracrine/autocrine manners. These findings extend our knowledge of the roles of the GH-IGF axis on reproduction regulation in fish.

RNA Modulates the Interaction between Influenza A Virus NS1 and Human PABP1.

PubMed

Arias-Mireles, Bryan H; de Rozieres, Cyrus M; Ly, Kevin; Joseph, Simpson

2018-05-25

Nonstructural protein 1 (NS1) is a multifunctional protein involved in preventing host-interferon response in influenza A virus (IAV). Previous studies have indicated that NS1 also stimulates the translation of viral mRNA by binding to conserved sequences in the viral 5'-UTR. Additionally, NS1 binds to poly(A) binding protein 1 (PABP1) and eukaryotic initiation factor 4G (eIF4G). The interaction of NS1 with the viral 5'-UTR, PABP1, and eIF4G has been suggested to specifically enhance the translation of viral mRNAs. In contrast, we report that NS1 does not directly bind to sequences in the viral 5'-UTR, indicating that NS1 is not responsible for providing the specificity to stimulate viral mRNA translation. We also monitored the interaction of NS1 with PABP1 using a new, quantitative FRET assay. Our data show that NS1 binds to PABP1 with high affinity; however, the binding of double-stranded RNA (dsRNA) to NS1 weakens the binding of NS1 to PABP1. Correspondingly, the binding of PABP1 to NS1 weakens the binding of NS1 to double-stranded RNA (dsRNA). In contrast, the affinity of PABP1 for binding to poly(A) RNA is not significantly changed by NS1. We propose that the modulation of NS1·PABP1 interaction by dsRNA may be important for the viral cycle.

Apolipoprotein E3 (ApoE3) but Not ApoE4 Protects against Synaptic Loss through Increased Expression of Protein Kinase Cϵ

PubMed Central

Sen, Abhik; Alkon, Daniel L.; Nelson, Thomas J.

2012-01-01

Synaptic loss is the earliest pathological change in Alzheimer disease (AD) and is the pathological change most directly correlated with the degree of dementia. ApoE4 is the major genetic risk factor for the age-dependent form of AD, which accounts for 95% of cases. Here we show that in synaptic networks formed from primary hippocampal neurons in culture, apoE3, but not apoE4, prevents the loss of synaptic networks produced by amyloid β oligomers (amylospheroids). Specific activators of PKCϵ, such as 8-(2-(2-pentyl-cyclopropylmethyl)-cyclopropyl)-octanoic acid methyl ester and bryostatin 1, protected against synaptic loss by amylospheroids, whereas PKCϵ inhibitors blocked this synaptic protection and also blocked the protection by apoE3. Blocking LRP1, an apoE receptor on the neuronal membrane, also blocked the protection by apoE. ApoE3, but not apoE4, induced the synthesis of PKCϵ mRNA and expression of the PKCϵ protein. Amyloid β specifically blocked the expression of PKCϵ but had no effect on other isoforms. These results suggest that protection against synaptic loss by apoE is mediated by a novel intracellular PKCϵ pathway. This apoE pathway may account for much of the protective effect of apoE and reduced risk for the age-dependent form of AD. This finding supports the potential efficacy of newly developed therapeutics for AD. PMID:22427674

Pomegranate protects liver against cecal ligation and puncture-induced oxidative stress and inflammation in rats through TLR4/NF-κB pathway inhibition.

PubMed

Makled, Mirhan N; El-Awady, Mohammed S; Abdelaziz, Rania R; Atwan, Nadia; Guns, Emma T; Gameil, Nariman M; Shehab El-Din, Ahmed B; Ammar, Elsayed M

2016-04-01

Acute liver injury secondary to sepsis is a major challenge in intensive care unit. This study was designed to investigate potential protective effects of pomegranate against sepsis-induced acute liver injury in rats and possible underlying mechanisms. Pomegranate was orally given (800mg/kg/day) for two weeks before sepsis induction by cecal ligation and puncture (CLP). Pomegranate improved survival and attenuated liver inflammatory response, likely related to downregulation of mRNA expression of toll like recptor-4, reduced nuclear translocation and DNA binding activity of proinflammatory transcription factor NF-κB subunit p65, decreased mRNA and protein expression of tumor necrosis factor-alpha and reduction in myeloperoxidase activity and mRNA expression. Pomegranate also decreased CLP-induced oxidative stress as reflected by decreased malondialdehyde content, and increased reduced glutathione level and superoxide dismutase activity. These results confirm the antiinflammatory and antioxidant effects of pomegranate in CLP-induced acute liver injury mediated through inhibiting TLR4/NF-κB pathway, lipid peroxidation and neutrophil infiltration. Copyright © 2016 Elsevier B.V. All rights reserved.

The E4 protein; structure, function and patterns of expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doorbar, John, E-mail: jdoorba@nimr.mrc.ac.uk

2013-10-15

The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genomemore » amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1{sup ∧}E4, these kinases regulate one of the E1{sup ∧}E4 proteins main functions, the association with the cellular keratin network, and eventually also its cleavage by the protease calpain which allows assembly into amyloid-like fibres and reorganisation of the keratin network. Although the E4 proteins of different HPV types appear divergent at the level of their primary amino acid sequence, they share a recognisable modular organisation and pattern of expression, which may underlie conserved functions and regulation. Assembly into higher-order multimers and suppression of cell proliferation are common to all E4 proteins examined. Although not yet formally demonstrated, a role in virus release and transmission remains a likely function for E4. - Highlights: • E4 gene products have a modular structure, and are expressed from the E1{sup ∧}E4 spliced mRNA. • E4 proteins are modified during epithelial differentiation by phosphorylation and proteolysis. • The E4 proteins contribute to genome amplification-efficiency and virus synthesis. • E4 proteins are abundantly expressed and may facilitate efficient virus release and transmission. • High-risk E4 proteins are deposited as amyloid fibres and can be used as infection biomarkers.« less

Anti-Interleukin-9 Antibody Increases the Effect of Allergen-Specific Immunotherapy in Murine Allergic Rhinitis.

PubMed

Shin, Ji Hyeon; Kim, Do Hyun; Kim, Boo Young; Kim, Sung Won; Hwang, Se Hwan; Lee, Joohyung; Kim, Soo Whan

2017-05-01

Interleukin (IL)-9 induces allergic responses; however, the roles of anti-IL-9 antibody in the induction of tolerance remain unclear. This study investigated the effects of anti-IL-9 antibody on oral tolerance (OT) in a mouse model of allergic rhinitis (AR). BALB/c mice were divided into 4 groups: the control, AR, OT, and OT with anti-IL-9 antibody (OT+IL9AB) groups. Ovalbumin (OVA) was used for sensitization and challenge. Mice in the OT and OT+IL9AB groups were fed OVA for immunotherapy. During immunotherapy, OT+IL9AB mice were injected with anti-IL-9 antibody. Allergic symptoms, tissue eosinophil counts, and serum OVA-specific immunoglobulin E (IgE) were measured. The mRNA expressions of cytokines and transcription factors of T cells of nasal mucosa were determined by real-time polymerase chain reaction (PCR). The protein levels of GATA3, ROR-γt, and Foxp3 in nasal mucosa were determined by Western blot. CD4⁺CD25⁺Foxp3⁺ T cells in the spleen were analyzed by flow cytometry. Administration of anti-IL-9 antibody decreased allergic symptoms, OVA-specific IgE levels, and eosinophil counts. In addition, it inhibited T-helper (Th) 2 responses, but had no effect on Th1 responses. Protein levels of ROR-γt and mRNA levels of PU.1 and ROR-γt were reduced by anti-IL-9 antibody. Anti-IL-9 antibody increased Foxp3 and IL-10 mRNA expression, Foxp3 protein, and induction of CD4⁺CD25⁺Foxp3⁺ T cells. Anti-IL-9 antibody decreased allergic inflammation through suppression of Th2 and Th17 cells. Anti-IL-9 antibody enhanced the tolerogenic effects of regulatory T cells. These results suggest that anti-IL-9 antibody might represent a potential therapeutic agent for allergen immunotherapy in patients with uncontrolled allergic airway disease. Copyright © 2017 The Korean Academy of Asthma, Allergy and Clinical Immunology · The Korean Academy of Pediatric Allergy and Respiratory Disease

Garlic Extract Diallyl Sulfide (DAS) Activates Nuclear Receptor CAR to Induce the Sult1e1 Gene in Mouse Liver

PubMed Central

Sueyoshi, Tatsuya; Green, William D.; Vinal, Kellie; Woodrum, Tyler S.; Moore, Rick; Negishi, Masahiko

2011-01-01

Constituent chemicals in garlic extract are known to induce phase I and phase II enzymes in rodent livers. Here we have utilized Car +/+ and Car −/− mice to demonstrate that the nuclear xenobiotic receptor CAR regulated the induction of the estrogen sulfotransferase Sult1e1 gene by diallyl sulfide (DAS) treatment in mouse liver. DAS treatment caused CAR accumulation in the nucleus, resulting in a remarkable increase of SULT1E1 mRNA (3,200 fold) and protein in the livers of Car +/+ females but not of Car −/− female mice. DAS also induced other CAR-regulated genes such as Cyp2b10, Cyp3a11 and Gadd45β. Compared with the rapid increase of these mRNA levels, which began as early as 6 hourrs after DAS treatment, the levels of SULT1E1 mRNA began increasing after 24 hours. This slow response to DAS suggested that CAR required an additional factor to activate the Sult1e1 gene or that this activation was indirect. Despite the remarkable induction of SULT1E1, there was no decrease in the serum levels of endogenous E2 or increase of estrone sulfate while the clearance of exogenously administrated E2 was accelerated in DAS treated mice. PMID:21698271

Garlic extract diallyl sulfide (DAS) activates nuclear receptor CAR to induce the Sult1e1 gene in mouse liver.

PubMed

Sueyoshi, Tatsuya; Green, William D; Vinal, Kellie; Woodrum, Tyler S; Moore, Rick; Negishi, Masahiko

2011-01-01

Constituent chemicals in garlic extract are known to induce phase I and phase II enzymes in rodent livers. Here we have utilized Car(+/+) and Car(-/-) mice to demonstrate that the nuclear xenobiotic receptor CAR regulated the induction of the estrogen sulfotransferase Sult1e1 gene by diallyl sulfide (DAS) treatment in mouse liver. DAS treatment caused CAR accumulation in the nucleus, resulting in a remarkable increase of SULT1E1 mRNA (3,200 fold) and protein in the livers of Car(+/+) females but not of Car(-/-) female mice. DAS also induced other CAR-regulated genes such as Cyp2b10, Cyp3a11 and Gadd45β. Compared with the rapid increase of these mRNA levels, which began as early as 6 hours after DAS treatment, the levels of SULT1E1 mRNA began increasing after 24 hours. This slow response to DAS suggested that CAR required an additional factor to activate the Sult1e1 gene or that this activation was indirect. Despite the remarkable induction of SULT1E1, there was no decrease in the serum levels of endogenous E2 or increase of estrone sulfate while the clearance of exogenously administrated E2 was accelerated in DAS treated mice.

Reduction of carbon tetrachloride-induced rat liver injury by IRFI 042, a novel dual vitamin E-like antioxidant.

PubMed

Campo, G M; Squadrito, F; Ceccarelli, S; Calò, M; Avenoso, A; Campo, S; Squadrito, G; Altavilla, D

2001-04-01

Carbon tetrachloride (CCl4 )-induced hepatotoxicity is likely the result of a CCl4 -induced free radical production which causes membrane lipid peroxidation and activation of transcription factors regulating both the TNF-alpha gene and the early-immediate genes involved in tissue regeneration. IRFI 042 is a novel vitamin E-like compound having a masked sulphydryl group in the aliphatic side chain. We studied the effect of IRFI 042 on CCl4 -induced liver injury. Liver damage was induced in male rats by an intraperitoneal injection of CCl4 (1 ml/kg in vegetal oil). Serum alanine aminotransferase (ALT) activity, liver malondialdehyde (MAL), hydroxyl radical formation (OH*), calculated indirectly by a trapping agent, hepatic reduced glutathione (GSH) concentration, plasma TNF-alpha, liver histology and hepatic mRNA levels for TNF-alpha were evaluated 48 h after CCl4 administration. Hepatic vitamin E (VE) levels were evaluated, in a separate group of animals, 2 h after CCl4 injection. A control group with vitamin E (100 mg/kg) was also treated in order to evaluate the differences versus the analogue treated groups. Intraperitoneal injection of carbon tetrachloride produced a marked increase in serum ALT activity (CCl4 = 404.61 +/- 10.33 U/L; Controls= 28.54 +/- 4.25 U/L), liver MAL (CCl4 = 0.67 +/- 0.16 nmol/mg protein; Controls= 0.13 +/- 0.06 nmol/mg protein), OH(7) levels assayed as 2,3-DHBA (CCl4 = 8.73 +/- 1.46 microM; Controls= 0.45 +/- 0.15 microM) and 2,5-DHBA (CCl4 = 24.61 +/- 3.32 microM; Controls= 2.75 +/- 0.93 microM), induced a severe depletion of GSH (CCl4 = 3.26 +/- 1.85 micromol/g protein; Controls= 17.82 +/- 3.13 micromol/g protein) and a marked decrease in VE levels (CCl4 = 5.67 +/- 1.22 nmol/g tissue; Controls= 13.47 +/- 3.21 nmol/g tissue), caused liver necrosis, increased plasma TNF-alpha levels (CCl4 = 57.36 +/- 13.24 IU/ml; Controls= 7.26 +/- 2.31 IU/ml) and enhanced hepatic mRNA for TNF-alpha (CCl4 = 19.22 +/- 4.38 a.u.; Controls= 0.76 +/- 0.36 a.u.). IRFI 042 (100 mg/kg, 30 min after CCl4 injection) blunted liver MAL (0.32 +/- 0.17 nmol/mg protein), decreased the serum levels of ALT (128.71 +/- 13.23 U/L), and restored the hepatic concentrations of VE (9.52 +/- 3.21 nmol/g tissue), inhibited OH* production (2,3-DHBA= 3.54 +/- 1.31 microM; 2,5-DHBA= 7.37 +/- 2.46 microM), restored the endogenous antioxidant GSH (12.77 +/- 3.73 mmol/g protein) and improved histology. Furthermore IRFI 042 treatment suppressed plasma TNF-alpha concentrations (31.47 +/- 18.25 IU/ml) and hepatic TNF-alpha mRNA levels (11.65 +/- 3.21 a.u.). The acute treatment with vitamin E failed to exert any protective effect against CCl4 -induced hepatotoxicity. These investigations suggest that IRFI 042 treatment may be of benefit during free radical-mediated liver injury.

Expression and localization of members of the thrombospondin family during final follicle maturation and corpus luteum formation and function in the bovine ovary

PubMed Central

BERISHA, Bajram; SCHAMS, Dieter; RODLER, Daniela; SINOWATZ, Fred; PFAFFL, Michael W.

2016-01-01

The aim of this study was to characterize the expression patterns and localization of the thrombospondin family members (THBS1, THBS2) and their receptors (CD36 and CD47) in bovine ovaries. First, the antral follicles were classified into 5 groups based on the follicle size and estradiol-17beta (E2) concentration in the follicular fluid (< 0.5, 0.5–5, 5–40, 40–180 and >180 E2 ng/ml). Second, the corpus luteum (CL) was assigned to the following stages: days 1–2, 3–4, 5–7, 8–12, 13–16 and >18 of the estrous cycle and of pregnancy (month 1–2, 3–4, 6–7 and > 8). Third, the corpora lutea were collected by transvaginal ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after inducing luteolysis by injecting a prostaglandin F2alpha analog. The mRNA expression of examined factors was measured by RT-qPCR, steroid hormone concentration by EIA, and localization by immunohistochemistry. The mRNA expression of THBS1, THBS2, CD36, and CD47 in the granulosa cells and theca interna was high in the small follicles and reduced in the preovulatory follicles. The mRNA expression of THBS1, THBS2, and CD47 in the CL during the estrous cycle was high, but decreased significantly during pregnancy. After induced luteolysis, thrombospondins increased significantly to reach the maximum level at 12 h for THBS1, 24 h for THBS2, and 48 h for CD36. The temporal expression and localization pattern of the thrombospondins and their specific receptors in the antral follicles and corpora lutea during the different physiological phases of the estrous cycle and induced luteolysis appear to be compatible with their inhibitory role in the control of ovarian angiogenesis. PMID:27396384

Vitamin D receptor expression and potential role of vitamin D on cell proliferation and steroidogenesis in goat ovarian granulosa cells.

PubMed

Yao, Xiaolei; Zhang, Guomin; Guo, Yixuan; Ei-Samahy, Mohamed; Wang, Shuting; Wan, Yongjie; Han, Le; Liu, Zifei; Wang, Feng; Zhang, Yanli

2017-10-15

This study aimed to investigate the expression of the vitamin D receptor (VDR) in goat follicles and to determine the effects of Vit D 3 supplementation on goat granulosa cells (GCs) function linked to follicular development. The results demonstrated that VDR was prominently localized in GCs, with expression increasing with follicle diameter. Addition of Vit D 3 (1α,25-(OH) 2 VD 3 ; 10 nM) to GCs caused an increase in VDR and in steroidogenic acute regulator (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA expression. Additionally, Vit D 3 increased the cyclic adenosine monophosphate (cAMP), estradiol (E 2 ), and progesterone (P 4 ) levels, while it decreased anti-müllerian hormone receptor (AMHR) and follicle-stimulating hormone receptor (FSHR) mRNA expression (P < 0.05). Addition of FSH remarkably increased E 2, P 4 , and cAMP levels (P < 0.05), and Vit D 3 further enhanced the E 2 and cAMP levels in the presence of FSH (P < 0.05). Vit D 3 significantly induced the mRNA expression of CDK4 and CyclinD1, and downregulated P21 gene expression (P < 0.05). In addition, Vit D 3 significantly decreased reactive oxygen species (ROS) production and increased the mRNA and protein expression of superoxide dismutase 2 (SOD2) and catalase (CAT) (P < 0.05). In conclusion, VDR is expressed in GCs of the goat ovaries and Vit D 3 might play an important role in GCs proliferation by regulating cellular oxidative stress and cell cycle-related genes. Meanwhile, Vit D 3 enhances the E 2 and P 4 output of GCs by regulating the expression of 3β-HSD and StAR and the level of cAMP, which regulate steroidogenesis, supporting a potential role for Vit D 3 in follicular development. Copyright © 2017 Elsevier Inc. All rights reserved.

(+/-)-3-[4-(2-dimethylamino-1-methylethoxy)-phenyl]-1H-pyrazolo[3,4- B]pyridine-1-acetic acid (Y-25510) stimulates production of IL-1 beta and IL-6 at the level of messenger RNA expression in cultured human monocytes.

PubMed

Kusuhara, H; Komatsu, H; Hisadome, M; Ikeda, Y

1996-12-01

(+/-)-3-[4-(2-Dimethylamino-1-methylethoxy)phenyl]-1H-pyrazolo[3, 4-b]pyridine-1-acetic acid (Y-25510) stimulated the mRNA expression for interleukin-1 beta (IL-1 beta), and enhanced the expression induced by lipopolysaccharide (LPS) in cultured human peripheral blood mononuclear cells (PBMC) and THP-1 cells, a cell-line derived from human monocytic leukemia. Y-25510 also stimulated the mRNA expression for IL-6 in both types of the cells, however, the stimulation required the presence of LPS. In THP-1 cells, the stimulation of IL-1 beta mRNA expression by Y-25510 was suppressed by cycloheximide, an inhibitor of protein synthesis. This phenomenon indicates that the stimulation requires de norv protein synthesis. In contrast, the stimulation of mRNA expression for IL-6 by Y-25510 was not suppressed by cycloheximide but suppressed by N alpha-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of nuclear transcription factor-kappa B (NF-kappa B) activation, in the presence of LPS, suggesting that the stimulation requires NF-kappa activation. These results demonstrate that Y-25510 stimulates the mRNA expression for IL-1 beta and IL-6 by different mechanisms. Dexamethasone suppressed the LPS-induced expression of mRNA for IL-1 beta and IL-6 in THP-1 cells, whereas the drug never suppressed the mRNA expression for these cytokines in the presence of Y-25510. The result indicates that Y-25510 stimulates the mRNA expression for IL-1 beta and IL-6 by different mechanisms from those of LPS.

TRIM25 Is Required for the Antiviral Activity of Zinc Finger Antiviral Protein

PubMed Central

Zheng, Xiaojiao; Wang, Xinlu; Tu, Fan; Wang, Qin; Fan, Zusen

2017-01-01

ABSTRACT Zinc finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses by binding to viral mRNAs and repressing the translation and/or promoting the degradation of target mRNA. In addition, ZAP regulates the expression of certain cellular genes. Here, we report that tripartite motif-containing protein 25 (TRIM25), a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 abolished ZAP's antiviral activity. The E3 ligase activity of TRIM25 is required for this regulation. TRIM25 mediated ZAP ubiquitination, but the ubiquitination of ZAP itself did not seem to be required for its antiviral activity. Downregulation of endogenous ubiquitin or overexpression of the deubiquitinase OTUB1 impaired ZAP's activity. We provide evidence indicating that TRIM25 modulates the target RNA binding activity of ZAP. These results uncover a mechanism by which the antiviral activity of ZAP is regulated. IMPORTANCE ZAP is a host antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1, Sindbis virus, and Ebola virus. ZAP binds directly to target mRNA, and it represses the translation and promotes the degradation of target mRNA. While the mechanisms by which ZAP posttranscriptionally inhibits target RNA expression have been extensively studied, how its antiviral activity is regulated is not very clear. Here, we report that TRIM25, a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 remarkably abolished ZAP's activity. TRIM25 is required for ZAP optimal binding to target mRNA. These results help us to better understand how the antiviral activity of ZAP is regulated. PMID:28202764

TRIM25 Is Required for the Antiviral Activity of Zinc Finger Antiviral Protein.

PubMed

Zheng, Xiaojiao; Wang, Xinlu; Tu, Fan; Wang, Qin; Fan, Zusen; Gao, Guangxia

2017-05-01

Zinc finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses by binding to viral mRNAs and repressing the translation and/or promoting the degradation of target mRNA. In addition, ZAP regulates the expression of certain cellular genes. Here, we report that tripartite motif-containing protein 25 (TRIM25), a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 abolished ZAP's antiviral activity. The E3 ligase activity of TRIM25 is required for this regulation. TRIM25 mediated ZAP ubiquitination, but the ubiquitination of ZAP itself did not seem to be required for its antiviral activity. Downregulation of endogenous ubiquitin or overexpression of the deubiquitinase OTUB1 impaired ZAP's activity. We provide evidence indicating that TRIM25 modulates the target RNA binding activity of ZAP. These results uncover a mechanism by which the antiviral activity of ZAP is regulated. IMPORTANCE ZAP is a host antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1, Sindbis virus, and Ebola virus. ZAP binds directly to target mRNA, and it represses the translation and promotes the degradation of target mRNA. While the mechanisms by which ZAP posttranscriptionally inhibits target RNA expression have been extensively studied, how its antiviral activity is regulated is not very clear. Here, we report that TRIM25, a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 remarkably abolished ZAP's activity. TRIM25 is required for ZAP optimal binding to target mRNA. These results help us to better understand how the antiviral activity of ZAP is regulated. Copyright © 2017 American Society for Microbiology.

Effects of tiletamine on the adenosine monophosphate-activated protein kinase signaling pathway in the rat central nervous system.

PubMed

Su, Li-Xue; Shi, Xing-Xing; Yang, Peng; Chen, Hao; Li, Xin; Fan, Hong-Gang; Wang, Hong-Bin

2017-10-01

The dissociative anesthetic tiletamine, which acts on the central nervous system (CNS), is widely used in veterinary medicine and animal experiments. Recent studies indicate that adenosine 5'-monophosphate activated protein kinase (AMPK) plays a key role in the analgesic action of tiletamine. In the present study, the effects of tiletamine on the AMPK signaling pathway in rats were investigated. Sprague-Dawley rats were injected intraperitoneally with tiletamine and executed at 10, 20, 40 and 60min post injection. The cerebral cortex, hippocampus, thalamus, cerebellum and brainstem were immediately taken out to evaluate the mRNA and protein phosphorylation levels of liver kinase B1 (LKB1), AMPKα and eIF4E-binding protein 1 (4EBP1) using quantitative real-time polymerase chain reaction and western blot analysis. Tiletamine increased AMPK mRNA expression in the rat brain (P<0.01). Increased mRNA expression of AMPK was accompanied by an increase in phosphorylation of LKB1, resulting in significant decreases in the phosphorylation levels of 4EBP1 in the corresponding brain regions (P<0.01). In summary, the findings indicate that tiletamine regulates the mRNA expression and protein phosphorylation levels of LKB1, AMPK and 4EBP1 in the CNS, suggesting that the analgesic effect of the anesthetic is mediated, at least in part, by the AMPK signaling pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

Protection by Nigella sativa against carbon tetrachloride-induced downregulation of hepatic cytochrome P450 isozymes in rats.

PubMed

Ibrahim, Zein S; Ishizuka, Mayumi; Soliman, Mohamed; ElBohi, Khlood; Sobhy, Wageh; Muzandu, Kaampwe; Elkattawy, Azza M; Sakamoto, Kentaro Q; Fujita, Shoichi

2008-11-01

Nigella sativa (family Ranunculaceae) is an annual plant that has been traditionally used on the Indian subcontinent and in Middle Eastern countries. In this study, we investigated the effect of N. sativa oil on the drug-metabolizing cytochrome P450 (CYP) enzymes and whether it has a protective effect against the acute hepatotoxicity of CCl4. Intraperitoneal injection of rats with CCl4 drastically decreased CYP2E1, CYP2B, CYP3A2, CYP2C11, and CYP1A2 mRNA and protein expressions. Oral administration of 1 ml/kg N. sativa oil every day for one week prior to CCl4 injection alleviated CCl4-induced suppression of CYP2B, CYP3A2, CYP2C11, and CYP1A2. Moreover, CCl4 increased iNOS and TNFalpha mRNA, while N. sativa oil administration for one week prior to CCl4 injection downregulated the CCl4-induced iNOS mRNA and up-regulated IL-10 mRNA. These results indicate that N. sativa oil administration has a protective effect against the CCl4-mediated suppression of hepatic CYPs and that this protective effect is partly due to the downregulation of NO production and up-regulation of the anti-inflammatory IL-10.

Precision cancer immunotherapy: optimizing dendritic cell-based strategies to induce tumor antigen-specific T-cell responses against individual patient tumors.

PubMed

Osada, Takuya; Nagaoka, Koji; Takahara, Masashi; Yang, Xiao Yi; Liu, Cong-Xiao; Guo, Hongtao; Roy Choudhury, Kingshuk; Hobeika, Amy; Hartman, Zachary; Morse, Michael A; Lyerly, H Kim

2015-05-01

Most dendritic cell (DC)-based vaccines have loaded the DC with defined antigens, but loading with autologos tumor-derived antigens would generate DCs that activate personalized tumor-specific T-cell responses. We hypothesized that DC matured with an optimized combination of reagents and loaded with tumor-derived antigens using a clinically feasible electroporation strategy would induce potent antitumor immunity. We first studied the effects on DC maturation and antigen presentation of the addition of picibanil (OK432) to a combination of zoledronic acid, tumor necrosis factor-α, and prostaglandin E2. Using DC matured with the optimized combination, we tested 2 clinically feasible sources of autologous antigen for electroloading, total tumor mRNA or total tumor lysate, to determine which stimulated more potent antigen-specific T cells in vitro and activated more potent antitumor immunity in vivo. The combination of tumor necrosis factor-α/prostaglandin E2/zoledronic acid/OK432 generated DC with high expression of maturation markers and antigen-specific T-cell stimulatory function in vitro. Mature DC electroloaded with tumor-derived mRNA [mRNA electroporated dendritic cell (EPDC)] induced greater expansion of antigen-specific T cells in vitro than DC electroloaded with tumor lysate (lysate EPDC). In a therapeutic model of MC38-carcinoembryonic antigen colon cancer-bearing mice, vaccination with mRNA EPDC induced the most efficient anti-carcinoembryonic antigen cellular immune response, which significantly suppressed tumor growth. In conclusion, mature DC electroloaded with tumor-derived mRNA are a potent cancer vaccine, especially useful when specific tumor antigens for vaccination have not been identified, allowing autologous tumor, and if unavailable, allogeneic cell lines to be used as an unbiased source of antigen. Our data support clinical testing of this strategy.

Forskolin Inhibits Lipopolysaccharide-Induced Modulation of MCP-1 and GPR120 in 3T3-L1 Adipocytes through an Inhibition of NFκB

PubMed Central

Chiadak, Jeanne Durendale; Arsenijevic, Tatjana; Verstrepen, Kevin; Gregoire, Françoise; Bolaky, Nargis; Delforge, Valérie; Flamand, Véronique

2016-01-01

In an obese state, Toll-like receptor-4 (TLR-4) upregulates proinflammatory adipokines secretion including monocyte chemotactic protein-1 (MCP-1) in adipose tissue. In contrast, G-protein coupled receptor 120 (GPR120) mediates antiobesity effects. The aim of this study was to determine the signaling pathway by which Forskolin (FK), a cyclic adenosine monophosphate- (cAMP-) promoting agent causing positive changes in body composition in overweight and obese adult men, affects MCP-1 and GPR120 expression during an inflammatory response induced by lipopolysaccharide (LPS) in adipocytes, such as in an obese state. 3T3-L1 cells differentiated into adipocytes (DC) were stimulated with LPS in the absence or presence of FK and inhibitors of TLR-4 and inhibitor of kappa B (IκBα). In DC, LPS increased MCP-1, TLR-4, and nuclear factor-κB1 (NFκB1) mRNA levels, whereas it decreased GPR120 mRNA levels. In DC, FK inhibited the LPS-induced increase in MCP-1, TLR-4, and NFκB1 mRNA levels and the LPS-induced decrease in GPR120 mRNA. BAY11-7082 and CLI-095 abolished these LPS-induced effects. In conclusion, FK inhibits LPS-induced increase in MCP-1 mRNA levels and decrease in GPR120 mRNA levels in adipocytes and may be a potential treatment for inflammation in obesity. Furthermore, TLR-4-induced activation of NFκB may be involved in the LPS-induced regulation of these genes. PMID:27881903

Forskolin Inhibits Lipopolysaccharide-Induced Modulation of MCP-1 and GPR120 in 3T3-L1 Adipocytes through an Inhibition of NFκB.

PubMed

Chiadak, Jeanne Durendale; Arsenijevic, Tatjana; Verstrepen, Kevin; Gregoire, Françoise; Bolaky, Nargis; Delforge, Valérie; Flamand, Véronique; Perret, Jason; Delporte, Christine

2016-01-01

In an obese state, Toll-like receptor-4 (TLR-4) upregulates proinflammatory adipokines secretion including monocyte chemotactic protein-1 (MCP-1) in adipose tissue. In contrast, G-protein coupled receptor 120 (GPR120) mediates antiobesity effects. The aim of this study was to determine the signaling pathway by which Forskolin (FK), a cyclic adenosine monophosphate- (cAMP-) promoting agent causing positive changes in body composition in overweight and obese adult men, affects MCP-1 and GPR120 expression during an inflammatory response induced by lipopolysaccharide (LPS) in adipocytes, such as in an obese state. 3T3-L1 cells differentiated into adipocytes (DC) were stimulated with LPS in the absence or presence of FK and inhibitors of TLR-4 and inhibitor of kappa B (I κ B α ). In DC, LPS increased MCP-1, TLR-4, and nuclear factor- κ B1 (NF κ B1) mRNA levels, whereas it decreased GPR120 mRNA levels. In DC, FK inhibited the LPS-induced increase in MCP-1, TLR-4, and NF κ B1 mRNA levels and the LPS-induced decrease in GPR120 mRNA. BAY11-7082 and CLI-095 abolished these LPS-induced effects. In conclusion, FK inhibits LPS-induced increase in MCP-1 mRNA levels and decrease in GPR120 mRNA levels in adipocytes and may be a potential treatment for inflammation in obesity. Furthermore, TLR-4-induced activation of NF κ B may be involved in the LPS-induced regulation of these genes.

Dissecting the expression of EEF1A1/2 genes in human prostate cancer cells: the potential of EEF1A2 as a hallmark for prostate transformation and progression.

PubMed

Scaggiante, B; Dapas, B; Bonin, S; Grassi, M; Zennaro, C; Farra, R; Cristiano, L; Siracusano, S; Zanconati, F; Giansante, C; Grassi, G

2012-01-03

In prostate adenocarcinoma, the dissection of the expression behaviour of the eukaryotic elongation factors (eEF1A1/2) has not yet fully elucidated. The EEF1A1/A2 expressions were investigated by real-time PCR, western blotting (cytoplasmic and cytoskeletal/nuclear-enriched fractions) and immunofluorescence in the androgen-responsive LNCaP and the non-responsive DU-145 and PC-3 cells, displaying a low, moderate and high aggressive phenotype, respectively. Targeted experiments were also conducted in the androgen-responsive 22Rv1, a cell line marking the progression towards androgen-refractory tumour. The non-tumourigenic prostate PZHPV-7 cell line was the control. Compared with PZHPV-7, cancer cells showed no major variations in EEF1A1 mRNA; eEF1A1 protein increased only in cytoskeletal/nuclear fraction. On the contrary, a significant rise of EEF1A2 mRNA and protein were found, with the highest levels detected in LNCaP. Eukaryotic elongation factor 1A2 immunostaining confirmed the western blotting results. Pilot evaluation in archive prostate tissues showed the presence of EEF1A2 mRNA in near all neoplastic and perineoplastic but not in normal samples or in benign adenoma; in contrast, EEF1A1 mRNA was everywhere detectable. Eukaryotic elongation factor 1A2 switch-on, observed in cultured tumour prostate cells and in human prostate tumour samples, may represent a feature of prostate cancer; in contrast, a minor involvement is assigned to EEF1A1. These observations suggest to consider EEF1A2 as a marker for prostate cell transformation and/or possibly as a hallmark of cancer progression.

[Promoting effect of cyclin D1 overexpression on proliferation and epithelial mesenchymal transition of cervical squamous cell carcinoma SiHa cells].

PubMed

Wang, P; Liu, S; Cheng, B; Wu, X Z; Ding, S S; Xu, L; Liu, Y; Duan, L; Sun, S Z

2017-03-08

Objective: To study effects of cyclin D1 overexpression on the proliferation and differentiation of cervical squamous cell carcinoma SiHa cells and to investigate related signaling molecules. Methods: Primers were designed to amplify the full length of cyclin D1 gene and cyclin D1 gene was amplified by PCR for constructing pcDNA3.1 plasmid vector. The construct was then transfected into SiHa cells, and the cells with stable overexpression of cyclin D1 were established, cyclin D1 gene and protein expression were detected by RT-PCR and Western blot, respectively. Cell growth curve was documented by MTT assay. CK7, E-cadherin, vimentin, Snail gene and protein expression in transfected cells were detected by RT-PCR and Western blot. RT-PCR was used to detect the mRNA expression of proliferation and differentiation-related genes like CDK4, CDK2, p21, p27, cyclin E, Rb, E2F, E6/E7 and Ki-67. After synchronization of cells, RT-PCR was used to detect of cyclin D1 and p21 mRNA expression at different time points of the cell cycle. Results: The G-3 cells with cyclin D1 overexpression were successfully established. The growth curve and Ki-67 mRNA expression accelerated in G-3 cells.Vimentin and Snail expression significantly increased at both gene and protein levels, while E-cadherin, CK7 gene and protein expression significantly decreased, indicating epithelial mesenchymal transitionoccurred in G-3 cells.Meanwhile, mRNA expression of cyclin D1, CDK4, CDK2, p21, p27, cyclin E, E2F and Rb increased, while E6/E7 and p16 showed no significant change. The expression trends of p21 and cyclin D1 were almost identical with fluctuation at different time points in the cell cycle. Conclusions: Overexpression of cyclin D1 induced by gene transfection promotes proliferation and epithelial mesenchymal transition in SiHa cells.The process is accompanied by up-regulation of CDK4, CDK2, p21, p27 and cyclin E genes.p21 expression increases synchronously with cyclin D1, suggesting a regulatory role in epithelial mesenchymal transition by affecting expression of vimentin in G-3 cells.

Effects of huperzine A on secretion of nerve growth factor in cultured rat cortical astrocytes and neurite outgrowth in rat PC12 cells.

PubMed

Tang, Li-li; Wang, Rui; Tang, Xi-can

2005-06-01

To study the effects of huperzine A (HupA) on neuritogenic activity and the expression of nerve growth factor (NGF). After being treated with 10 micromol/L HupA, neurite outgrowth of PC12 cells was observed and counted under phase-contrast microscopy. Mitogenic activity was assayed by [3H]thymidine incorporation. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH) release. AChE activity, mRNA and protein expression were measured by the Ellman method, RT-PCR, and Western blot, respectively. NGF mRNA and protein levels were determined by RT-PCR and ELISA assays. Treatment of PC12 cells with 10 micromol/L HupA for 48 h markedly increased the number of neurite-bearing cells, but caused no significant alteration in cell viability or other signs of cytotoxicity. In addition to inhibiting AChE activity, 10 micromol/L HupA also increased the mRNA and protein levels of this enzyme. In addition, following 2 h exposure of the astrocytes to 10 micromol/L HupA, there was a significant up-regulation of mRNA for NGF and P75 low-affinity NGF receptor. The protein level of NGF was also increased after 24 h treatment with HupA. Our findings demonstrate for the first time that HupA has a direct or indirect neurotrophic activity, which might be beneficial in treatment of neurodegenerative disorders such as Alzheimer disease.

Integrative comparison of mRNA expression patterns in breast cancers from Caucasian and Asian Americans with implications for precision medicine

PubMed Central

Wang, Jianan; He, Max M; Li, Liren; Zhang, Jinfeng

2016-01-01

Asian Americans (AS) have significantly lower incidence and mortality rates of breast cancer (BRCA) than Caucasian Americans (CA). While this racial disparity has been documented the underlying pathogenetic factors explaining it are obscure. We addressed this issue by an integrative genomics approach to compare mRNA expression between AS and CA cases of BRCA. RNA-seq data from the Cancer Genome Atlas showed that mRNA expression revealed significant differences at gene and pathway levels. Increased susceptibility and severity in CA patients were likely the result of synergistic environmental and genetic risk factors, with arachidonic acid metabolism and PPAR signaling pathways implicated in linking environmental and genetic factors. An analysis that also added eQTL data from the Genotype-Tissue Expression Project and single nucleotide polymorphism (SNP) data from the 1000 Genomes Project identified several SNPs associated with differentially expressed genes. Overall, the associations we identified may enable a more focused study of genotypic differences that may help explain the disparity in BRCA incidence and mortality rates in CA and AS populations and inform precision medicine. PMID:28069798

[Regulatory role of hypoxia inducible factor-1 alpha in the changes of contraction of vascular smooth muscle cell induced by hypoxia].

PubMed

Zhang, Yuan; Liu, Liang-ming; Ming, Jia; Yang, Guang-ming; Chen, Wei

2007-11-01

To observe the regulatory role and mechanism of hypoxia inducible factor-1 alpha (HIF-1 alpha) in the contractile changes of vascular smooth muscle cell (VSMC) induced by hypoxia. Cells were divided into three groups: normal, hypoxia and oligomycin treated groups. VSMC and vascular endothelial cell (VEC) were co-cultured in Transwell models with the hypoxic time of 0, 0.5, 1, 2, 3, 4 and 6 hours respectively. The contractile response of VSMC to norepinephrine were determined by measuring the fluorescent infiltration rate in the lower chamber. The mRNA expression of HIF-1 alpha, endothelial-nitric oxide synthase (eNOS), inducible-nitric oxide synthase(iNOS), heme oxygenase-1 (HO-1) and cyclooxygenase-2 (COX-2) were determined by reverse transcription-polymerase chain reaction (RT-PCR). VSMC contraction was increased at the early stage of hypoxia with the 1.53-fold increase at 0.5 hour as compared to the normal group (P<0 .01), and decreased gradually at the prolonged period of hypoxia with the drop of 30% at 6 hours as compared to the normal group (P<0.05). Oligomycin treatment significantly inhibited the increase of VSMC contraction at early stage, while improved it at late hypoxic period with the 6 hours increase of 12.8% (P<0.05). HIF-1 alpha, iNOS, COX-2 and HO-1 mRNA exhibited a time-dependent increase following hypoxia, and peaked at 6, 2, 3 and 4 hours respectively, they were increased 1.62, 3.23, 2.26 and 2.86-folds as compared with normal group (all P<0.01). iNOS, COX-2 and HO-1 mRNA expression were fluctuated in the normal range following oligomycin administration (all P>0.05). Hypoxia can elicit a biphasic changes of VSMC contraction, and HIF-1 alpha seems to play an important role in the regulation of VSMC contraction induced by hypoxia by regulating eNOS, iNOS, COX-2 and HO-1 expression.

Ovarian steroids, oxytocin, and tumor necrosis factor modulate equine oviduct function.

PubMed

Pinto-Bravo, P; Galvão, A; Rebordão, M R; Amaral, A; Ramilo, D; Silva, E; Szóstek-Mioduchowska, A; Alexandre-Pires, G; Roberto da Costa, R; Skarzynski, D J; Ferreira-Dias, G

2017-10-01

The oviduct plays important roles in the early reproductive process. The aim of this study was to evaluate gene transcription and protein expression of progesterone receptor (PGR), estrogen receptors 1 (ESR1) and 2 (ESR2); oxytocin receptor (OXTR); prostaglandin F2α synthase (AKR1C3), and prostaglandin E2 synthase (Ptges) in mare oviduct in different estrous cycle stages. Estradiol (E 2 ), progesterone (P 4 ), oxytocin (OXT), and tumor necrosis factor α (TNF) effect on in vitro PGE 2 and prostaglandin F2α (PGF 2α ) secretion by equine oviduct explants or by oviductal epithelial cells (OECs) were also assessed. During the breeding season, oviduct tissue was obtained post mortem from cyclic mares. Protein of ESR1, ESR2, PGR, AKR1C3, and Ptges was present in OECs, whereas OXTR was shown in oviduct stroma. In follicular phase, protein expression of ESR1, ESR2, PGR, and OXTR increased in oviduct explants (P < 0.05), whereas no estrous cycle effect was noted for AKR1C3 or Ptges. In follicular phase, mRNA transcription was upregulated for Pgr but downregulated for Oxtr, Ptges, and Akr1c3 (P < 0.05). Nevertheless, Esr1 and Esr2 mRNA levels did not change with the estrous cycle. In the ampulla, Esr1, Esr2, and Oxtr mRNA transcription increased, but not for Pgr or Ptges. In contrast, Akr1c3 mRNA level was upregulated in the infundibulum (P < 0.05). In follicular phase, E 2 , P 4 , and OXT downregulated PGE 2 production by OEC (P < 0.05), but no difference was observed in mid-luteal phase. Explants production of PGE 2 rose when treated with OXT in follicular phase; with TNF or OXT in early luteal phase; or with TNF, OXT, or P 4 in mid-luteal phase. PGF 2α production by OEC was downregulated by all treatments in follicular phase but upregulated in mid-luteal phase (P < 0.05). Oviduct explants PGF 2α production was stimulated by TNF or OXT in all estrous cycle phases. In conclusion, this work has shown that ESR1, ESR2, OXTR, Ptges, and AKRLC3 gene transcription and/or translation is estrous cycle dependent and varies with oviduct portion (infundibulum vs ampulla) and cell type. Ovarian steroid hormones, OXT and TNF stimulation of PGF 2α and/or PGE 2 production is also estrous cycle dependent and varies in the different portions of mare oviduct. Differential transcription level and protein localization in various portions of the oviduct throughout the estrous cycle, as well as PG production, suggest coordinated physiologic actions and mechanisms of steroid hormones, OXT, and TNF in the equine oviduct. Copyright © 2017 Elsevier Inc. All rights reserved.

Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lazarus, Kyren A.; Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122; Zhao, Zhe

2013-08-30

Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels highermore » in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.« less

Black tea high-molecular-weight polyphenol stimulates exercise training-induced improvement of endurance capacity in mouse via the link between AMPK and GLUT4.

PubMed

Eguchi, Tomoaki; Kumagai, Chiaki; Fujihara, Takashi; Takemasa, Thoru; Ozawa, Tetsuo; Numata, Osamu

2013-01-01

Aerobic exercise can promote "fast-to-slow transition" in skeletal muscles, i.e. an increase in oxidative fibers, mitochondria, and myoglobin and improvement in glucose and lipid metabolism. Here, we found that mice administered Mitochondria Activation Factor (MAF) combined with exercise training could run longer distances and for a longer time compared with the exercise only group; MAF is a high-molecular-weight polyphenol purified from black tea. Furthermore, MAF intake combined with exercise training increased phosphorylation of AMPK and mRNA level of glucose transporter 4 (GLUT4). Thus, our data demonstrate for the first time that MAF activates exercise training-induced intracellular signaling pathways that involve AMPK, and improves endurance capacity.

Mechanisms of impaired nephrogenesis with fetal growth restriction: altered renal transcription and growth factor expression

PubMed Central

Abdel-Hakeem, Ahmed K; Henry, Tasmia Q; Magee, Thomas R; Desai, Mina; Ross, Michael; Mansano, Roy; Torday, John; Nast, Cynthia C.

2010-01-01

Objective Maternal food restriction during pregnancy results in growth restricted newborns and reduced glomerular number, contributing to programmed offspring hypertension. We investigated whether reduced nephrogenesis may be programmed by dysregulation of factors controlling ureteric bud branching and mesenchyme to epithelial transformation. Study Design 10 to 20 days gestation, Sprague Dawley pregnant rats (n=6/group) received ad libitum food; FR rats were 50% food restricted. At embryonic day 20, mRNA and protein expression of WT1, Pax2, FGF2, GDNF, cRET, WNT4, WNT11, BMP4, BMP7, and FGF7 were determined by real-time PCR and Western blotting. Results Maternal FR resulted in up-regulated mRNA expression for WT1, FGF2, and BMP7 whereas Pax2, GDNF, FGF7, BMP4, WNT4, and WNT11 mRNAs were down-regulated. Protein expression was concordant for WT1, GDNF, Pax2, FGF7, BMP4 and WNT4. Conclusion Maternal FR altered gene expression of fetal renal transcription and growth factors, and likely contributes to development of offspring hypertension. PMID:18639218

TET1-mediated DNA hypomethylation regulates the expression of MUC4 in lung cancer

PubMed Central

Yokoyama, Seiya; Higashi, Michiyo; Tsutsumida, Hideaki; Wakimoto, Jouji; Hamada, Tomofumi; Wiest, Edwin; Matsuo, Kei; Kitazono, Ikumi; Goto, Yuko; Guo, Xin; Hamada, Taiji; Yamada, Sohsuke; Hiraki, Tsubasa; Yonezawa, Suguru; Batra, Surinder K.; Hollingsworth, Michael A.; Tanimoto, Akihide

2017-01-01

Lung cancer remains a disease of high mortality, despite advanced diagnostic techniques. Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in lung neoplasms. Our immunohistochemistry (IHC) studies have shown that high MUC4 expression correlates with a poor outcome. We have also shown that the expression of several mucin genes in cancer cell lines is regulated by DNA methylation. We evaluated the expression level of MUC4, mRNA and several DNA hypomethylation factors in lung tissue samples from 33 patients with various lung lesions. The results indicated that the DNA methylation status of MUC4 matched the expression level of mRNA. In addition, the TET1 (Ten-Eleven Translocation) mRNA showed a significant correlation with the status of DNA methylation of MUC4. Furthermore, the treatment of a lung cancer cell line with TET1 siRNA caused a reduction in MUC4 mRNA expression. Thus, we suggest that TET1 mediated DNA hypomethylation plays a key role in the expression of MUC4. This is the first report that TET1 mediated DNA hypomethylation regulates the expression of MUC4 in lung cancer. The analysis of these epigenetic changes may be useful for diagnosing carcinogenic risk. PMID:28680536

TET1-mediated DNA hypomethylation regulates the expression of MUC4 in lung cancer.

PubMed

Yokoyama, Seiya; Higashi, Michiyo; Tsutsumida, Hideaki; Wakimoto, Jouji; Hamada, Tomofumi; Wiest, Edwin; Matsuo, Kei; Kitazono, Ikumi; Goto, Yuko; Guo, Xin; Hamada, Taiji; Yamada, Sohsuke; Hiraki, Tsubasa; Yonezawa, Suguru; Batra, Surinder K; Hollingsworth, Michael A; Tanimoto, Akihide

2017-03-01

Lung cancer remains a disease of high mortality, despite advanced diagnostic techniques. Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in lung neoplasms. Our immunohistochemistry (IHC) studies have shown that high MUC4 expression correlates with a poor outcome. We have also shown that the expression of several mucin genes in cancer cell lines is regulated by DNA methylation. We evaluated the expression level of MUC4, mRNA and several DNA hypomethylation factors in lung tissue samples from 33 patients with various lung lesions. The results indicated that the DNA methylation status of MUC4 matched the expression level of mRNA. In addition, the TET1 (Ten-Eleven Translocation) mRNA showed a significant correlation with the status of DNA methylation of MUC4 . Furthermore, the treatment of a lung cancer cell line with TET1 siRNA caused a reduction in MUC4 mRNA expression. Thus, we suggest that TET1 mediated DNA hypomethylation plays a key role in the expression of MUC4. This is the first report that TET1 mediated DNA hypomethylation regulates the expression of MUC4 in lung cancer. The analysis of these epigenetic changes may be useful for diagnosing carcinogenic risk.

Role of S100A1 in hypoxia-induced inflammatory response in cardiomyocytes via TLR4/ROS/NF-κB pathway.

PubMed

Yu, Jiangkun; Lu, Yanyu; Li, Yapeng; Xiao, Lili; Xing, Yu; Li, Yanshen; Wu, Leiming

2015-09-01

S100A1 plays a crucial role in hypoxia-induced inflammatory response in cardiomyocytes. However, the role of S100A1 in hypoxia-induced inflammatory response in cardiomyocytes is still unknown. enzyme-linked immunosorbent assay (ELISA) was performed for the determination of inflammatory cytokines. Immunocytochemistry and immunofluorescence, Western blot analysis and Real-time polymerase chain reaction (RT-PCR) were conducted to assess protein or mRNA expressions. Fluorogenic probe dihydroethidium (DHE) was used to evaluate the generation of reactive oxygen species (ROS) while Hoechst 33342 staining for apoptosis. Small interfering RNA (siRNA) for S100A1 was used to evaluate the role of S100A1. The levels of ROS and inflammatory cytokine including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-8 in H9c2 cells were increased remarkably by hypoxia. However, IL-37 protein or mRNA levels were decreased significantly. Both Toll-like receptor 4 (TLR4) inhibitor Ethyl (6R)-6-[N-(2-Chloro-4fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242) treatment or siRNA S100A1 downregulated TLR4 expression and inflammatory cytokine level and mRNA in H9c2 cells, as well as weakening ROS and phospho-p65 Nuclear factor (NF)-κB levels. Further, S100A1 treatment significantly reduced TNF-α protein or mRNA level whereas enhanced IL-37 protein or mRNA level, and could attenuate ROS and phospho-p65 NF-κB levels. Our results demonstrate that S100A1 can regulate the inflammatory response and oxidative stress in H9C2 cells via TLR4/ROS/NF-κB pathway. These findings provide an interesting strategy for protecting cardiomyocytes from hypoxia-induced inflammatory response. © 2015 Royal Pharmaceutical Society.

Alteration in cardiac uncoupling proteins and eNOS gene expression following high-intensity interval training in favor of increasing mechanical efficiency.

PubMed

Fallahi, Ali Asghar; Shekarfroush, Shahnaz; Rahimi, Mostafa; Jalali, Amirhossain; Khoshbaten, Ali

2016-03-01

High-intensity interval training (HIIT) increases energy expenditure and mechanical energy efficiency. Although both uncoupling proteins (UCPs) and endothelial nitric oxide synthase (eNOS) affect the mechanical efficiency and antioxidant capacity, their effects are inverse. The aim of this study was to determine whether the alterations of cardiac UCP2, UCP3, and eNOS mRNA expression following HIIT are in favor of increased mechanical efficiency or decreased oxidative stress. Wistar rats were divided into five groups: control group (n=12), HIIT for an acute bout (AT1), short term HIIT for 3 and 5 sessions (ST3 and ST5), long-term training for 8 weeks (LT) (6 in each group). The rats of the training groups were made to run on a treadmill for 60 min in three stages: 6 min running for warm-up, 7 intervals of 7 min running on treadmill with a slope of 5° to 20° (4 min with an intensity of 80-110% VO2max and 3 min at 50-60% VO2max), and 5-min running for cool-down. The control group did not participate in any exercise program. Rats were sacrificed and the hearts were extracted to analyze the levels of UCP2, UCP3 and eNOS mRNA by RT-PCR. UCP3 expression was increased significantly following an acute training bout. Repeated HIIT for 8 weeks resulted in a significant decrease in UCPs mRNA and a significant increase in eNOS expression in cardiac muscle. This study indicates that Long term HIIT through decreasing UCPs mRNA and increasing eNOS mRNA expression may enhance energy efficiency and physical performance.

Alteration in cardiac uncoupling proteins and eNOS gene expression following high-intensity interval training in favor of increasing mechanical efficiency

PubMed Central

Fallahi, Ali Asghar; Shekarfroush, Shahnaz; Rahimi, Mostafa; Jalali, Amirhossain; Khoshbaten, Ali

2016-01-01

Objective(s): High-intensity interval training (HIIT) increases energy expenditure and mechanical energy efficiency. Although both uncoupling proteins (UCPs) and endothelial nitric oxide synthase (eNOS) affect the mechanical efficiency and antioxidant capacity, their effects are inverse. The aim of this study was to determine whether the alterations of cardiac UCP2, UCP3, and eNOS mRNA expression following HIIT are in favor of increased mechanical efficiency or decreased oxidative stress. Materials and Methods: Wistar rats were divided into five groups: control group (n=12), HIIT for an acute bout (AT1), short term HIIT for 3 and 5 sessions (ST3 and ST5), long-term training for 8 weeks (LT) (6 in each group). The rats of the training groups were made to run on a treadmill for 60 min in three stages: 6 min running for warm-up, 7 intervals of 7 min running on treadmill with a slope of 5° to 20° (4 min with an intensity of 80-110% VO2max and 3 min at 50-60% VO2max), and 5-min running for cool-down. The control group did not participate in any exercise program. Rats were sacrificed and the hearts were extracted to analyze the levels of UCP2, UCP3 and eNOS mRNA by RT-PCR. Results: UCP3 expression was increased significantly following an acute training bout. Repeated HIIT for 8 weeks resulted in a significant decrease in UCPs mRNA and a significant increase in eNOS expression in cardiac muscle. Conclusion: This study indicates that Long term HIIT through decreasing UCPs mRNA and increasing eNOS mRNA expression may enhance energy efficiency and physical performance. PMID:27114795

Metabolism of sex steroids is influenced by acquired adiposity-A study of young adult male monozygotic twin pairs.

PubMed

Vihma, Veera; Naukkarinen, Jussi; Turpeinen, Ursula; Hämäläinen, Esa; Kaprio, Jaakko; Rissanen, Aila; Heinonen, Sini; Hakkarainen, Antti; Lundbom, Jesper; Lundbom, Nina; Mikkola, Tomi S; Tikkanen, Matti J; Pietiläinen, Kirsi H

2017-09-01

Obesity and ageing are associated with lower serum testosterone levels in men. How fat distribution or adipose tissue metabolism, independent of genetic factors and age, are related to sex steroid metabolism is less clear. We studied the associations between adiposity and serum sex hormone concentrations, and mRNA expression of genes regulating sex hormone metabolism in adipose tissue in young adult male monozygotic (MZ) twin pairs. The subjects [n=18 pairs; mean age, 32 years; individual body mass indexes (BMIs) 22-36kg/m 2 ] included 9 male MZ twin pairs discordant for BMI [intra-pair difference (Δ) in BMI ≥3kg/m 2 ]. Sex steroid concentrations were determined by liquid chromatography-tandem mass spectrometry, body composition by dual-energy X-ray absorptiometry and magnetic resonance imaging, and mRNA expressions from subcutaneous adipose tissue by Affymetrix. In BMI-discordant pairs (mean ΔBMI=5.9kg/m 2 ), serum dihydrotestosterone (DHT) was lower [mean 1.9 (SD 0.7) vs. 2.4 (1.0) nmol/l, P=0.040] and mRNA expressions of DHT-inactivating AKR1C2 (P=0.021) and cortisol-producing HSD11B1 (P=0.008) higher in the heavier compared to the leaner co-twins. Serum free 17β-estradiol (E2) was higher [2.3 (0.5) vs. 1.9 (0.5) pmol/l, P=0.028], and in all twin pairs, serum E2 and estrone concentrations were higher in the heavier than in the leaner co-twins [107 (28) vs. 90 (22) pmol/l, P=0.006; and 123 (43) vs. 105 (27) pmol/l, P=0.025]. Within all twin pairs, i.e. independent of genetic effects and age, 1) the amount of subcutaneous fat inversely correlated with serum total and free testosterone, DHT, and sex hormone-binding globulin (SHBG) concentrations (P<0.01 for all), 2) intra-abdominal fat with total testosterone and SHBG (P<0.05), and 3) liver fat with SHBG (P=0.006). Also, 4) general and intra-abdominal adiposity correlated positively with mRNA expressions of AKR1C2, HSD11B1, and aromatase in adipose tissue (P<0.05). In conclusion, acquired adiposity was associated with decreased serum DHT and increased estrogen concentrations, independent of genetic factors and age. The reduction of DHT could be linked to its increased degradation (by AKR1C2 and HSD11B1) and increased estrogen levels to increased adiposity-related expression of aromatase in adipose tissue. Copyright © 2017 Elsevier Ltd. All rights reserved.

Actinobacillus actinomycetemcomitans Y4 capsular polysaccharide induces IL-1β mRNA expression through the JNK pathway in differentiated THP-1 cells

PubMed Central

Iwata, T; Mitani, A; Ishihara, Y; Tanaka, S; Yamamoto, G; Kikuchi, T; Naganawa, T; Matsumura, Y; Suga, T; Koide, M; Sobue, T; Suzuki, T; Noguchi, T

2005-01-01

Capsular polysaccharide from Actinobacillus actinomycetemcomitans Y4 (Y4 CP) induces bone resorption in a mouse organ culture system and osteoclast formation in mouse bone marrow cultures, as reported in previous studies. We also found that Y4 CP inhibits the release of interleukin (IL)-6 and IL-8 from human gingival fibroblast (HGF). Thus Y4 CP induces various responses in localized tissue and leads to the secretion of several cytokines. However, the effects of Y4 CP on human monocytes/macrophages are still unclear. In this study, THP-1 cells, which are a human monocytic cell line, were stimulated with Y4 CP, and we measured gene expression in inflammatory cytokine and signal transduction pathways. IL-1β and tumour necrosis factor (TNF)-α mRNA were induced from Y4 CP-treated THP-1 cells. IL-1β mRNA expression was increased according to the dose of Y4 CP, and in a time-dependent manner. IL-1β mRNA expression induced by Y4 CP (100 µg/ml) was approximately 7- to 10-fold greater than that in the control by real-time PCR analysis. Furthermore, neither PD98059, a specific inhibitor of extracellular signal-regulated kinase nor SB203580, a specific inhibitor of p38 kinase prevented the IL-1β expression induced by Y4 CP. However, JNK Inhibitor II, a specific inhibitor of c-Jun N-terminal kinase (JNK) prevented the IL-1β mRNA expression induced by Y4 CP in a concentration-dependent manner. These results indicate that Y4 CP-mediated JNK pathways play an important role in the regulation of IL-1β mRNA. Therefore, Y4 CP-transduced signals for IL-1β induction in the antibacterial action of macrophages may provide a therapeutic strategy for periodontitis. PMID:15996190

[Experimental study on the Der f 1 mRNA molecules derived from dermatophagoides farinae for specific immunotherapy on murine model of asthma].

PubMed

Jiang, Yu-xin; Yin, Kang; Jin, Wen-jie; Wu, Lu-yi; Li, Chao-pin

2014-08-01

To investigate the effect of Der f 1 mRNA molecules for specific immunotherapy on murine model of asthma. Fifty BALB/c mice were randomly divided into 5 groups: PBS group, Der f 1 sensitization group, Der f 1 specific immunotherapy (SIT) group, beta-actin mRNA SIT group, and Derf 1 mRNA SIT group. On days 0, 7 and 14, mice in PBS group received PBS injection; mice in the other groups were intraperitoneally injected with 10 microg Derf 1. At day 21, the mice in the 4 experimental groups were challenged with a 30-min inhaled dose of Der f 1 (100 microg/ml) for 7 successive days. Two weeks after the final sensitization, the mice in the above five groups were im- munized by intradermal injection with PBS, 1 microg Der f 1, 10 microg Der f 1, 2 microg beta-actin mRNA, and 2 microg Der f 1 mRNA, respectively for 3 times at one-week intervals. Two weeks after the last intradermal injection, all mice were sacrificed and bronchoalveolar lavage fluid (BALF) was collected. ELISA was performed to detect the levels of IFN-gamma and IL-13 in BALF, the number of eosinophils in the BALF was recorded. Splenocytes were prepared, and cultured with Der f 1 al- lergen (10 Jg/ml) for 72 h. Splenocytes of PBS group was cultured without Derf 1 allergen. The levels of IFN-gamma and IL-13 in splenocyte culture supernatant were measured by ELISA, as well as serum antibody levels of total IgE, allergen- specific IgE (sIgE), sIgG1, and sIgG2a. Lung sections were stained in hematoxylin and eosin, and observed under the microsope. Except for PBS group, mice in the other 4 group showed symptoms of acute asthma attack. Com- pared with Derf 1 sensitization group [(897.56 +/- 105.73) pg/ml] and beta-actin mRNA SIT group [(219.47 +/- 64.72) pg/ml], the level of IFN-gamma in BALF from Der f 1 mRNA SIT group [(897.56 +/- 105.73) pg/ml] and Derfl SIT group [(864.48 +/- 70.62)pg/ml] significantly increased (P<0.01). However, the level of IL-13 in BALF from Derf 1 mRNA SIT group [(241.64 +/- 31.41) pg/ml] and Derf 1 SIT group [(321.94 +/- 41.07)pg/ml] was significantly lower than that of Der f 1 sensitization group [(520.62 +/- 43.77) pg/ml] and beta-actin mRNA SIT group [(507.22 +/- 42.26) pg/ml](P<0.01). The number of eosinophils in Der f 1 mRNA SIT group [(1.33 +/- 0.44) x 10(5)/ml] and Der f 1 SIT group [(1.48 +/- 0.39) x 10(5)/ml] was also lower than that of Der f 1 sensitization group [(3.54 +/- 0.52)x10(5)/ml] and beta-actin mRNA SIT group [(2.98-0.53) x 10(5)/ml] (P<0.01). The levels of IFN-GAMMA and IL-13 in splenocyte culture supernatant showed that IFN-gamma level in Der f 1 mRNA SIT group [(420.91+69.92) pg/ml] and Der f 1 SIT group [(334.92 +/- 43.72) pg/ml] was significantly higher than that of Der f 1 sensitization group[(123.75 +/- 5.48) pg/ml] and beta-actin mRNA SIT group[(128.84 +/- 59.00) pg/ml] (P<0.01). However, IL-13 level of Der f 1 mRNA SIT group [(268.51 +/- 40.42) pg/ml] and Der f 1 SIT group [(285.26 +/- 62.21) pg/ml] was significantly lower than that of Derf 1 sensitization group [(613.89 +/- 51.54) pg/ml] and beta-actin mRNA SIT group [(524.05 +/- 39.12) pg/ml] (P<0.01). Compared with Der f 1 sensitization group [total IgE: (94.34 +/- 11.66) ng/ml, sIgE: (65.67 +/- 9.47) ng/ml, sIgG1: (75.18 +/- 9.52) ng/ml, sIgG2a: (2.81 +/- 1.17) ng/ml] and beta-actin mRNA SIT group[total IgE: (86.48 +/- 10.26) ng/ml, sIgE: (62.36 +/- 8.35) ng/ml, sIgG1: (69.51 +/- 8.98) ng/ml, IgG2a: (1.06 +/- 0.11) ng/ml], the serum antibody levels of total IgE [(33.72 +/- 9.78) ng/ml], sIgE [(22.76 +/- 8.09) ng/ml], sIgG1 [(17.87 +/- 7.59) ng/ml] of Der f 1 mRNA SIT group decreased significantly (P<0.01), whereas the level of IgG% [(7.74 +/- 0.88) ng/ml] increased (P<0.01). Compared with Der f 1 sensitization group, the asthmatic symptoms were relieved after immunization with Der f 1 mRNA for specific immunotherapy, including intact structure of respiratory and alveolar epithelial cells, decreased inflammatory cell infiltration, and similar to those in Der f 1 SIT group. However, the breakage and detachment of bronchial epithelial cells occurred in beta-actin mRNA SIT group. Derf 1 mRNA vaccine can correct Th1 and Th2 imbalance.

Expression of Cytochrome P450s in the Liver of Rats Administered with Socheongryong-tang, a Traditional Herbal Formula

PubMed Central

Jin, Seong Eun; Ha, Hyekyung; Seo, Chang-Seob; Shin, Hyeun-Kyoo; Jeong, Soo-Jin

2016-01-01

Objective: The purpose of this study was to investigate the potential influences of Socheongryong-tang (SCRT) on the messenger ribonucleic acid (mRNA) and protein expression of cytochrome P450 (CYP450) in vivo. Materials and Methods: SCRT was orally administered to either male or female Sprague-Dawley rats once daily at doses of 0, 1000, 2000, or 5000 mg/kg/day for 13 weeks. The mRNA expression of CYP450s (CYP1A1, 1A2, 2B1/2, 2C11, 2E1, 3A1, 3A2, and 4A1) in liver tissues was measured by reverse transcription polymerase chain reaction. And then, the protein expression of CYP1A1 and CYP2B1/2 in liver tissues was analyzed by the Western blot. Results: We found no significant influence in the mRNA expression of hepatic CYP1A2, 2C11, 2E1, 3A1, 3A2, and 4A1 after repeated administration of SCRT for 13 weeks. By contrast, the mRNA and protein expression of hepatic CYP1A1 was increased by repeated SCRT treatment in male rats, but not in female rats. The mRNA and protein expression of hepatic CYP2B1/2 in both genders was increased by administration of SCRT. Conclusion: A caution is needed when SCRT is co-administered with substrates of CYP2B1/2 for clinical usage. In case of male, an attention is also required when SCRT and drugs metabolized by CYP1A1 are taken together. Our findings provide information regarding the safety and effectiveness of SCRT when combined with conventional drugs. SUMMARY Oral administration of Socheongryong-tang for 13 weeks did not affect the mRNA expression of hepatic CYP1A2, 2C11, 2E1, 3A1, 3A2, and 4A1In male rats, oral administration of Socheongryong-tang for 13 weeks induced the mRNA and protein expression of hepatic CYP1A1 and CYP2B1/2In female rats, oral administration of Socheongryong-tang for 13 weeks induced the mRNA and protein expression of hepatic CYP2B1/2. Abbreviations used: SCRT: Socheongryong-tang, CYP450: Cytochrome P450, HPLC: High performance liquid chromatography, RT-PCR: Reverse transcription polymerase chain reaction. PMID:27601852

Differentiation of embryonic stem cells into hepatocytes that coexpress coagulation factors VIII and IX.

PubMed

Cao, Jun; Shang, Chang-zhen; Lü, Li-hong; Qiu, De-chuan; Ren, Meng; Chen, Ya-jin; Min, Jun

2010-11-01

To establish an efficient culture system to support embryonic stem (ES) cell differentiation into hepatocytes that coexpress F-VIII and F-IX. Mouse E14 ES cells were cultured in differentiation medium containing sodium butyrate (SB), basic fibroblast growth factor (bFGF), and/or bone morphogenetic protein 4 (BMP4) to induce the differentiation of endoderm cells and hepatic progenitor cells. Hepatocyte growth factor, oncostatin M, and dexamethasone were then used to induce the maturation of ES cell-derived hepatocytes. The mRNA expression levels of endoderm-specific genes and hepatocyte-specific genes, including the levels of F-VIII and F-IX, were detected by RT-PCR and real-time PCR during various stages of differentiation. Protein expression was examined by immunofluorescence and Western blot. At the final stage of differentiation, flow cytometry was performed to determine the percentage of cells coexpressing F-VIII and F-IX, and ELISA was used to detect the levels of F-VIII and F-IX protein secreted into the culture medium. The expression of endoderm-specific and hepatocyte-specific markers was upregulated to highest level in response to the combination of SB, bFGF, and BMP4. Treatment with the three inducers during hepatic progenitor differentiation significantly enhanced the mRNA and protein levels of F-VIII and F-IX in ES cell-derived hepatocytes. More importantly, F-VIII and F-IX were coexpressed with high efficiency at the final stage of differentiation, and they were also secreted into the culture medium. We have established a novel in vitro differentiation protocol for ES-derived hepatocytes that coexpress F-VIII and F-IX that may provide a foundation for stem cell replacement therapy for hemophilia.

Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Monaco, L.; Murtagh, J.J.; Newman, K.B.

1990-03-01

ADP-ribosylation factors (ARFs) are {approx}20-kDa proteins that act as GTP-dependent allosteric activators of cholera toxin. With deoxyinosine-containing degenerate oligonucleotide primers corresponding to conserved GTP-binding domains in ARFs, the polymerase chain reaction (PCR) was used to amplify simultaneously from human DNA portions of three ARF genes that include codons for 102 amino acids, with intervening sequences. Amplification products that differed in size because of differences in intron sizes were separated by agarose gel electrophoresis. One amplified DNA contained no introns and had a sequence different from those of known AFRs. Based on this sequence, selective oligonucleotide probes were prepared and usedmore » to isolate clone {Psi}ARF 4, a putative ARF pseudogene, from a human genomic library in {lambda} phage EMBL3. Reverse transcription-PCR was then used to clone from human poly(A){sup +} RNA the cDNA corresponding to the expressed homolog of {Psi}ARF 4, referred to as human ARF 4. It appears that {Psi}ARF 4 arose during human evolution by integration of processed ARF 4 mRNA into the genome. Human ARF 4 differs from previously identified mammalian ARFs 1, 2, and 3. Hybridization of ARF 4-specific oligonucleotide probes with human, bovine, and rat RNA revealed a single 1.8-kilobase mRNA, which was clearly distinguished from the 1.9-kilobase mRNA for ARF 1 in these tissues. The PCR provides a powerful tool for investigating diversity in this and other multigene families, especially with primers targeted at domains believed to have functional significance.« less

Biphasic Modulation of NOS Expression, Protein and Nitrite Products by Hydroxocobalamin Underlies Its Protective Effect in Endotoxemic Shock: Downstream Regulation of COX-2, IL-1β, TNF-α, IL-6, and HMGB1 Expression

PubMed Central

Sampaio, André L. F.; Dalli, Jesmond; Brancaleone, Vincenzo; D'Acquisto, Fulvio; Perretti, Mauro; Wheatley, Carmen

2013-01-01

Background. NOS/•NO inhibitors are potential therapeutics for sepsis, yet they increase clinical mortality. However, there has been no in vivo investigation of the (in vitro) •NO scavenger, cobalamin's (Cbl) endogenous effects on NOS/•NO/inflammatory mediators during the immune response to sepsis. Methods. We used quantitative polymerase chain reaction (qPCR), ELISA, Western blot, and NOS Griess assays, in a C57BL/6 mouse, acute endotoxaemia model. Results. During the immune response, pro-inflammatory phase, parenteral hydroxocobalamin (HOCbl) treatment partially inhibits hepatic, but not lung, iNOS mRNA and promotes lung eNOS mRNA, but attenuates the LPS hepatic rise in eNOS mRNA, whilst paradoxically promoting high iNOS/eNOS protein translation, but relatively moderate •NO production. HOCbl/NOS/•NO regulation is reciprocally associated with lower 4 h expression of TNF-α, IL-1β, COX-2, and lower circulating TNF-α, but not IL-6. In resolution, 24 h after LPS, HOCbl completely abrogates a major late mediator of sepsis mortality, high mobility group box 1 (HMGB1) mRNA, inhibits iNOS mRNA, and attenuates LPS-induced hepatic inhibition of eNOS mRNA, whilst showing increased, but still moderate, NOS activity, relative to LPS only. experiments (LPS+D-Galactosamine) HOCbl afforded significant, dose-dependent protection in mice Conclusions. HOCbl produces a complex, time- and organ-dependent, selective regulation of NOS/•NO during endotoxaemia, corollary regulation of downstream inflammatory mediators, and increased survival. This merits clinical evaluation. PMID:23781123

Gene expression signature of benign prostatic hyperplasia revealed by cDNA microarray analysis.

PubMed

Luo, Jun; Dunn, Thomas; Ewing, Charles; Sauvageot, Jurga; Chen, Yidong; Trent, Jeffrey; Isaacs, William

2002-05-15

Despite the high prevalence of benign prostatic hyperplasia (BPH) in the aging male, little is known regarding the etiology of this disease. A better understanding of the molecular etiology of BPH would be facilitated by a comprehensive analysis of gene expression patterns that are characteristic of benign growth in the prostate gland. Since genes differentially expressed between BPH and normal prostate tissues are likely to reflect underlying pathogenic mechanisms involved in the development of BPH, we performed comparative gene expression analysis using cDNA microarray technology to identify candidate genes associated with BPH. Total RNA was extracted from a set of 9 BPH specimens from men with extensive hyperplasia and a set of 12 histologically normal prostate tissues excised from radical prostatectomy specimens. Each of these 21 RNA samples was labeled with Cy3 in a reverse transcription reaction and cohybridized with a Cy5 labeled common reference sample to a cDNA microarray containing 6,500 human genes. Normalized fluorescent intensity ratios from each hybridization experiment were extracted to represent the relative mRNA abundance for each gene in each sample. Weighted gene and random permutation analyses were performed to generate a subset of genes with statistically significant differences in expression between BPH and normal prostate tissues. Semi-quantitative PCR analysis was performed to validate differential expression. A subset of 76 genes involved in a wide range of cellular functions was identified to be differentially expressed between BPH and normal prostate tissues. Semi-quantitative PCR was performed on 10 genes and 8 were validated. Genes consistently upregulated in BPH when compared to normal prostate tissues included: a restricted set of growth factors and their binding proteins (e.g. IGF-1 and -2, TGF-beta3, BMP5, latent TGF-beta binding protein 1 and -2); hydrolases, proteases, and protease inhibitors (e.g. neuropathy target esterase, MMP2, alpha-2-macroglobulin); stress response enzymes (e.g. COX2, GSTM5); and extracellular matrix molecules (e.g. laminin alpha 4 and beta 1, chondroitin sulfate proteoglycan 2, lumican). Genes consistently expressing less mRNA in BPH than in normal prostate tissues were less commonly observed and included the transcription factor KLF4, thrombospondin 4, nitric oxide synthase 2A, transglutaminase 3, and gastrin releasing peptide. We identified a diverse set of genes that are potentially related to benign prostatic hyperplasia, including genes both previously implicated in BPH pathogenesis as well as others not previously linked to this disease. Further targeted validation and investigations of these genes at the DNA, mRNA, and protein levels are warranted to determine the clinical relevance and possible therapeutic utility of these genes. Copyright 2002 Wiley-Liss, Inc.

Hepatic effects of a methionine-choline-deficient diet in hepatocyte RXRalpha-null mice.

PubMed

Gyamfi, Maxwell Afari; Tanaka, Yuji; He, Lin; Klaassen, Curtis D; Wan, Yu-Jui Yvonne

2009-01-15

Retinoid X receptor-alpha (RXRalpha) is an obligate partner for several nuclear hormone receptors that regulate important physiological processes in the liver. In this study the impact of hepatocyte RXRalpha deficiency on methionine and choline deficient (MCD) diet-induced steatosis, oxidative stress, inflammation, and hepatic transporters gene expression were examined. The mRNA of sterol regulatory element-binding protein (SREBP)-regulated genes, important for lipid synthesis, were not altered in wild type (WT) mice, but were increased 2.0- to 5.4-fold in hepatocyte RXRalpha-null (H-RXRalpha-null) mice fed a MCD diet for 14 days. Furthermore, hepatic mRNAs and proteins essential for fatty acid beta-oxidation were not altered in WT mice, but were decreased in the MCD diet-fed H-RXRalpha-null mice, resulting in increased hepatic free fatty acid levels. Cyp2e1 enzyme activity and lipid peroxide levels were induced only in MCD-fed WT mice. In contrast, hepatic mRNA levels of pro-inflammatory factors were increased only in H-RXRalpha-null mice fed the MCD diet. Hepatic uptake transporters Oatp1a1 and Oatp1b2 mRNA levels were decreased in WT mice fed the MCD diet, whereas the efflux transporter Mrp4 was increased. However, in the H-RXRalpha-null mice, the MCD diet only moderately decreased Oatp1a1 and induced both Oatp1a4 and Mrp4 gene expression. Whereas the MCD diet increased serum bile acid levels and alkaline phosphatase activity in both WT and H-RXRalpha-null mice, serum ALT levels were induced (2.9-fold) only in the H-RXRalpha-null mice. In conclusion, these data suggest a critical role for RXRalpha in hepatic fatty acid homeostasis and protection against MCD-induced hepatocyte injury.

Hypoperfusion induces overexpression of beta-amyloid precursor protein mRNA in a focal ischemic rodent model.

PubMed

Shi, J; Yang, S H; Stubley, L; Day, A L; Simpkins, J W

2000-01-17

Silent stroke is one of the risk factors of dementia. In the present study, we used a novel focal ischemic animal model to investigate the effects of comparatively small changes of cerebral blood flow (CBF) on the expression of beta-amyloid precursor protein (APP) mRNA. Focal ischemia was achieved by introducing a 4-0 monofilament to the bifurcation of anterior and middle cerebral arteries. Brain samples were harvested from ischemic core and penumbra of cortices at 1, 4 and 7 days following ischemia. The expression of APP mRNA was assessed by RT-PCR. The CBF was decreased to 50% for 1 day after stroke and recovered to 90% at the fourth day after stroke. The changes of CBF were accompanied by an increase in the expression of APP mRNA. APP mRNA increased to 208% and 152% in the penumbra and core ischemic regions, respectively, on the fourth day after MCAO and remained high through the seventh day of ischemia. This study suggests brain hypoperfusion enhances APP mRNA expression and may contribute to the progression of cognitive impairment after silent stroke.

Cinnamon extract regulates plasma levels of adipose-derived factors and expression of multiple genes related to carbohydrate metabolism and lipogenesis in adipose tissue of fructose-fed rats.

PubMed

Qin, B; Polansky, M M; Anderson, R A

2010-03-01

We reported earlier that dietary cinnamon extract (CE) improves systemic insulin sensitivity and dyslipidemia by enhancing insulin signaling. In the present study, we have examined the effects of CE on several biomarkers including plasma levels of adipose-derived adipokines, and the potential molecular mechanisms of CE in epididymal adipose tissue (EAT). In Wistar rats fed a high-fructose diet (HFD) to induce insulin resistance, supplementation with a CE (Cinnulin PF, 50 mg/kg daily) for 8 weeks reduced blood glucose, plasma insulin, triglycerides, total cholesterol, chylomicron-apoB48, VLDL-apoB100, and soluble CD36. CE also inhibited plasma retinol binding protein 4 (RBP4) and fatty acid binding protein 4 (FABP4) levels. CE-induced increases in plasma adiponectin were not significant. CE did not affect food intake, bodyweight, and EAT weight. In EAT, there were increases in the insulin receptor ( IR) and IR substrate 2 ( IRS2) mRNA, but CE-induced increases in mRNA expression of IRS1, phosphoinositide-3-kinase, AKT1, glucose transporters 1 and 4 , and glycogen synthase 1 expression and decreased trends in mRNA expression of glycogen synthase kinase 3beta were not statistically significant. CE also enhanced the mRNA levels of ADIPOQ, and inhibited sterol regulatory element binding protein-1c mRNA levels. mRNA and protein levels of fatty acid synthase and FABP4 were inhibited by CE and RBP4, and CD36 protein levels were also decreased by CE. These results suggest that CE effectively ameliorates circulating levels of adipokines partially mediated via regulation of the expression of multiple genes involved in insulin sensitivity and lipogenesis in the EAT.

[miR-503-5p inhibits the proliferation of T24 and EJ bladder cancer cells by interfering with the Rb/E2F signaling pathway].

PubMed

Li, Xiaohui; Han, Xingtao; Yang, Jinhui; Sun, Jiantao; Wei, Pengtao

2017-10-01

Objective To observe the effect of microRNA-503-5p (miR-503-5p) on the growth of T24 and EJ bladder cancer cells, and explore the possible molecular mechanism. Methods The miR-504-5p mimics or miR-NC was transfected into T24 and EJ cells. The target gene of miR-503-5p was predicted by bioinformatics. The expressions of E2F transcription factor 3 (E2F3) mRNA and Rb/E2F signaling pathway mRNA were detected by the real-time quantitative PCR (qPCR). The expressions of Rb/E2F signal pathway proteins E2F3, cyclin E, CDK2, Rb and p-Rb were detected by Western blotting. The cell cycle of bladder cancer cell lines was determined by flow cytometry. MTT assay and plate cloning assay were performed to observe the proliferation ability of bladder cancer cells. Results After miR-503-5p mimics transfection, the expression of miR-503-5p in bladder cancer cells significantly increased. The increased expression of miR-503-5p significantly reduced the expressions of E2F3 mRNA and Rb/E2F signaling pathway mRNA in bladder cancer cells. What's more, the expressions of Rb/E2F signal pathway proteins were down-regulated. The bladder cancer cells were arrested in G0/G1 phase, and their growth was significantly inhibited by miR-503-5p. Conclusion The miR-503-5p over-expression can inhibit the growth of bladder cancer cell lines T24 and EJ by down-regulating the expression of the Rb/E2F signaling pathway.

Changes in mRNA levels for brain-derived neurotrophic factor after wheel running in rats selectively bred for high- and low-aerobic capacity

PubMed Central

Groves-Chapman, Jessica L.; Murray, Patrick S.; Stevens, Kristin L.; Monroe, Derek; Koch, Lauren G.; Britton, Steven L.; Holmes, Philip V.

2012-01-01

We evaluated levels of exercise-induced brain-derived neurotrophic factor (BDNF) messenger RNA (mRNA) within the hippocampal formation in rats selectively bred for 1) high intrinsic (i.e., untrained) aerobic capacity (High Capacity Runners, HCR), 2) low intrinsic aerobic capacity (Low Capacity Runners, LCR), and 3) unselected Sprague-Dawley (SD) rats with or without free access to running wheels for three weeks. The specific aim of the study was to determine whether a dose-response relationship exists between cumulative running distance and levels of BDNF mRNA. No additional treatments or behavioral manipulations were used. HCR, LCR, and SD rats were grouped by strain and randomly assigned to sedentary or activity (voluntary access to activity wheel) conditions. Animals were killed after 21 days of exposure to the assigned conditions. Daily running distances (mean ± standard deviation meters/d) during week three were: HCR (4726 ± 3220), SD (2293 ± 3461), LCR (672 ± 323). Regardless of strain, levels of BDNF mRNA in CA1 were elevated in wheel runners compared to sedentary rats and this difference persisted after adjustment for age (p=0.040). BDNF mRNA was not affected by intrinsic aerobic capacity and was not related to total running distance. The results support that BDNF mRNA expression is increased by unlimited access to activity wheel running for 3 weeks but is not dependent upon accumulated running distance. PMID:22024546

Lipoxin A4 inhibits UV radiation-induced skin inflammation and oxidative stress in mice.

PubMed

Martinez, R M; Fattori, V; Saito, P; Melo, C B P; Borghi, S M; Pinto, I C; Bussmann, A J C; Baracat, M M; Georgetti, S R; Verri, W A; Casagrande, R

2018-04-27

Lipoxin A4 (LXA 4 ) is a metabolic product of arachidonic acid. Despite potent anti-inflammatory and pro-resolution activities, it remains to be determined if LXA 4 has effect on ultraviolet (UV) radiation-induced skin inflammation. To investigate the effects of systemic administration with LXA 4 on UV radiation-induced inflammation and oxidative damage in the skin of mice. Varied parameters of inflammation and oxidative stress in the skin of mice were evaluated after UV radiation (4.14 J/cm 2 ). Pretreatment with LXA 4 significantly inhibited UV radiation-induced skin edema and myeloperoxidase activity. LXA 4 efficacy was enhanced by increasing the time of pre-treatment to up to 72 h. LXA 4 reduced UV radiation-induced skin edema, neutrophil recruitment (myeloperoxidase activity and LysM-eGFP + cells), MMP-9 activity, deposition of collagen fibers, epidermal thickness, sunburn cell counts, and production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-33). Depending on the time point, LXA 4 increased the levels of anti-inflammatory cytokines (TGF-β and IL-10). LXA 4 significantly attenuated UV radiation-induced oxidative damage returning the oxidative status to baseline levels in parameters such as ferric reducing ability, scavenging of free radicals, GSH levels, catalase activity and superoxide anion production. LXA 4 also reduced UV radiation-induced gp91 phox [nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) subunit] mRNA expression and enhanced nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target enzyme nicotinamide adenine dinucleotide (phosphate) quinone oxidoreductase (Nqo1) mRNA expression. LXA 4 inhibited UV radiation-induced skin inflammation by diminishing pro-inflammatory cytokine production and oxidative stress as well as inducing anti-inflammatory cytokines and Nrf2. Copyright © 2018. Published by Elsevier B.V.

Downregulation of Programmed Cell Death 4 by Inflammatory Conditions Contributes to the Generation of the Tumor Promoting Microenvironment

PubMed Central

Yasuda, Michiko; Schmid, Tobias; Rübsamen, Daniela; Colburn, Nancy H.; Irie, Kazuhiro; Murakami, Akira

2012-01-01

Ample evidence has shown key roles of inflammation in tumor promotion and carcinogenesis, and tumor-associated macrophages are known to promote tumor growth and dissemination. Programmed cell death 4 (Pdcd4) is a novel tumor suppressor, and although various studies have revealed that the functions and expression mechanisms of Pdcd4 in tumor promotion, those in regard to inflammation remain unclear. In the present study, we examined whether inflammatory stimuli regulate Pdcd4 expression. 12-O-tetradecanoylphorbol 13-acetate (TPA) suppressed expression of pdcd4 mRNA in human monocytic cell lines (U937, THP-1). Similarly, the bacterial endotoxin lipopolysaccharide (LPS) downregulated pdcd4 level in mouse RAW264.7 and peritoneal macrophages. Furthermore, conditioned medium from LPS-stimulated RAW264.7 macrophages suppressed pdcd4 mRNA in RAW264.7 macrophages, and findings obtained with recombinant tumor necrosis factor-α (TNF-α) and TNF-α-specific siRNA suggested that TNF-α partly mediates LPS-triggered Pdcd4 downregulation via an autocrine mechanism. Specific inhibitors of phosphoinositide-3-kinase (PI3K) and c-jun N-terminus kinase (JNK) restored LPS-abolished pdcd4 mRNA. Consistently, in MCF7 mammary carcinoma cells, conditioned medium from TPA-differentiated/activated U937 cells suppressed pdcd4 mRNA. Additionally, knockdown of pdcd4 in RAW264.7 macrophages using siRNA significantly enhanced LPS-induced TNF-α protein production, and interferon-γ, CC chemokine ligand (Ccl) 1, Ccl20, and interleukin-10 mRNA expression. These results suggest that Pdcd4 suppresses the induction of these inflammatory mediators. Taken together, loss of Pdcd4 in macrophages may be a critical step in establishing the inflammatory environment while that in tumor cells contributes to tumor progression. PMID:20607724

Replenishment of RANTES mRNA expression in activated eosinophils fromatopic asthmatics

PubMed Central

Velazquez, J R; Lacy, P; Moqbel, R

2000-01-01

Eosinophils have been shown to express the gene encoding regulated upon activation, normal T‐cell expressed and secreted (RANTES), a potent eosinophilotactic chemokine. RANTES protein expression in eosinophils has previously been shown to be up‐regulated by a number of agonists, including complement‐dependent factors (C3b/iC3b) and interferon‐γ (IFN‐γ). We hypothesized that gene expression of RANTES is regulated in these cells by eosinophil‐specific agonists. We analysed RANTES mRNA expression by reverse‐transcription polymerase chain reaction (RT‐PCR) in human peripheral blood eosinophils obtained from mild atopic asthmatics following stimulation over time. In resting eosinophils, a low level of RANTES mRNA was found to be constitutively expressed in all the atopic donors tested in this study (n = 6). Following stimulation with C3b/iC3b (serum‐coated surfaces), eosinophils released measurable levels of RANTES, while sustained transcript expression was detected for up to 24 hr of stimulation. In contrast, IFN‐γ (5 ng/ml) transiently and significantly (P < 0·05, n = 3) depleted relative amounts of RANTES PCR product (compared with β2‐microglobulin) after 1–4 hr of stimulation. RANTES transcript was again detectable after 24 hr of IFN‐γ incubation, suggesting that the pool of RANTES mRNA had been replenished. Other eosinophil‐active cytokines, interleukin‐3 (IL‐3), IL‐4, IL‐5 and granulocyte–macrophage colony‐stimulating factor, did not appear to modulate RANTES mRNA expression after 1 hr of incubation. The effect of IFN‐γ on RANTES mRNA was reversed by cycloheximide, suggesting that IFN‐γ may act by increasing the rate of translation of RANTES mRNA. These findings indicate that IFN‐γ may induce a rapid and transient effect on the translation and replenishment of RANTES mRNA in eosinophils. This novel observation supports the notion that eosinophils have the potential to replenish their stored and released bioactive proteins. PMID:10792507

Molecular Dynamics Simulation of the Allosteric Regulation of eIF4A Protein from the Open to Closed State, Induced by ATP and RNA Substrates

PubMed Central

Meng, Hongqing; Li, Chaoqun; Wang, Yan; Chen, Guangju

2014-01-01

Background Eukaryotic initiation factor 4A (eIF4A) plays a key role in the process of protein translation initiation by facilitating the melting of the 5′ proximal secondary structure of eukaryotic mRNA for ribosomal subunit attachment. It was experimentally postulated that the closed conformation of the eIF4A protein bound by the ATP and RNA substrates is coupled to RNA duplex unwinding to promote protein translation initiation, rather than an open conformation in the absence of ATP and RNA substrates. However, the allosteric process of eIF4A from the open to closed state induced by the ATP and RNA substrates are not yet fully understood. Methodology In the present work, we constructed a series of diplex and ternary models of the eIF4A protein bound by the ATP and RNA substrates to carry out molecular dynamics simulations, free energy calculations and conformation analysis and explore the allosteric properties of eIF4A. Results The results showed that the eIF4A protein completes the conformational transition from the open to closed state via two allosteric processes of ATP binding followed by RNA and vice versa. Based on cooperative allosteric network analysis, the ATP binding to the eIF4A protein mainly caused the relative rotation of two domains, while the RNA binding caused the proximity of two domains via the migration of RNA bases in the presence of ATP. The cooperative binding of ATP and RNA for the eIF4A protein plays a key role in the allosteric transition. PMID:24465900

Variable liver fat concentration as a proxy for body fat mobilization postpartum has minor effects on insulin-induced changes in hepatic gene expression related to energy metabolism in dairy cows.

PubMed

Weber, C; Schäff, C T; Kautzsch, U; Börner, S; Erdmann, S; Bruckmaier, R M; Röntgen, M; Kuhla, B; Hammon, H M

2017-02-01

The liver plays a central role in adaptation for energy requirements around calving, and changes in the effects of insulin on hepatic energy metabolism contribute to metabolic adaptation in dairy cows. Hepatic insulin effects may depend on body fat mobilization. The objective of this study was to investigate the effects of insulin on the hepatic gene expression of enzymes involved in energy metabolism and factors related to nutrition partitioning in cows with high and low total liver fat concentration (LFC) after calving. Holstein cows were retrospectively grouped according to their LFC after calving as a proxy for body fat mobilization. Cows were classified as low (LLFC; LFC <24% fat/dry matter; n = 9) and high (HLFC; LFC >24.4% fat/dry matter; n = 10) fat-mobilizing after calving. Euglycemic-hyperinsulinemic clamps [6 mU/(kg × min) of insulin for 6 h] were performed in wk 5 antepartum (ap) and wk 3 postpartum (pp). Before and at the end of the euglycemic-hyperinsulinemic clamps, liver biopsies were taken to measure the mRNA abundance of enzymes involved in carbohydrate and lipid metabolism, expression related to the somatotropic axis, and adrenergic and glucocorticoid receptors. The mRNA abundance of pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase (PEPCK; PCK1), acyl-CoA-dehydrogenase very long chain (ACADVL), and hydroxyl-methyl-glutaryl-CoA-synthase 1 increased, but the mRNA abundance of solute carrier family 2 (SLC2A2 and SLC2A4), growth hormone receptor 1A (GHR1A), insulin-like growth factor 1 (IGF1), sterol regulatory element binding factor 1, adrenoceptor α 1A, and glucocorticoid receptor decreased from ap to pp. Insulin treatment was associated with decreased PCK1, mitochondrial PEPCK, glucose-6-phosphatase, propionyl-CoA-carboxylase α, carnitine-palmitoyl-transferase 1A, ACADVL, and insulin receptor mRNA, but increased IGF1 and SLC2A4 mRNA ap and pp and GHR1A mRNA pp. The mRNA abundance of SLC2A4 was greater, and the mRNA abundance of GHR1A and IGF1 tended to be lower in LLFC than in HLFC. Administration of insulin, albeit at a supraphysiological dose, was associated with inhibition of gene expression related to glucose production and β-oxidation, but we observed variable effects in the degree of insulin depression of individual genes. Insulin status is important for regulation of nutrient partitioning, but different LFC pp had very little influence on changes in hepatic gene expression following administration of insulin. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Induction of the plasticity-associated immediate early gene Arc by stress and hallucinogens: role of brain-derived neurotrophic factor.

PubMed

Benekareddy, Madhurima; Nair, Amrita R; Dias, Brian G; Suri, Deepika; Autry, Anita E; Monteggia, Lisa M; Vaidya, Vidita A

2013-03-01

Exposure to stress and hallucinogens in adulthood evokes persistent alterations in neurocircuitry and emotional behaviour. The structural and functional changes induced by stress and hallucinogen exposure are thought to involve transcriptional alterations in specific effector immediate early genes. The immediate early gene, activity regulated cytoskeletal-associated protein (Arc), is important for both activity and experience dependent plasticity. We sought to examine whether trophic factor signalling through brain-derived neurotrophic factor (BDNF) contributes to the neocortical regulation of Arc mRNA in response to distinct stimuli such as immobilization stress and the hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI). Acute exposure to either immobilization stress or DOI induced Arc mRNA levels within the neocortex. BDNF infusion into the neocortex led to a robust up-regulation of local Arc transcript expression. Further, baseline Arc mRNA expression in the neocortex was significantly decreased in inducible BDNF knockout mice with an adult-onset, forebrain specific BDNF loss. The induction of Arc mRNA levels in response to both acute immobilization stress or a single administration of DOI was significantly attenuated in the inducible BDNF knockout mice. Taken together, our results implicate trophic factor signalling through BDNF in the regulation of cortical Arc mRNA expression, both under baseline conditions and following stress and hallucinogen exposure. These findings suggest the possibility that the regulation of Arc expression via BDNF provides a molecular substrate for the structural and synaptic plasticity observed following stimuli such as stress and hallucinogens.

Expression of ATF4 and VEGF in chorionic villus tissue in early spontaneous abortion.

PubMed

Chai, Luwei; Ling, Kang; He, Xiaoxi; Yang, Rong

2013-10-01

To explore the relationship between early spontaneous abortion (SA) and the expression of activating transcription factor 4 (ATF4) and vascular endothelial growth factor (VEGF). The expression of ATF4 and VEGF protein and mRNA in villi from first trimester spontaneous abortion (SA, n=30) and normal pregnancy (NP, n=30) were detected by immunohistochemistry and fluorescent quantitative polymerase chain reaction (FQ-PCR). Both protein and mRNA expressions of ATF4 and VEGF in the SA group were significantly lower than in the NP group (P<0.01). Their proteins are expressed mainly in syncytiotrophoblast, cytotrophoblast and villous stromal cells. Correlation analysis showed that the expression of ATF4 was positively correlated with that of VEGF in the SA group (r=0.717, P<0.01). Lower expression of ATF4 and VEGF genes in chorionic villus tissue may participate in the pathogenesis of spontaneous abortion. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Repeated Exposure to D-Amphetamine Decreases Global Protein Synthesis and Regulates the Translation of a Subset of mRNAs in the Striatum

PubMed Central

Biever, Anne; Boubaker-Vitre, Jihane; Cutando, Laura; Gracia-Rubio, Irene; Costa-Mattioli, Mauro; Puighermanal, Emma; Valjent, Emmanuel

2017-01-01

Repeated psychostimulant exposure induces persistent gene expression modifications that contribute to enduring changes in striatal GABAergic spiny projecting neurons (SPNs). However, it remains unclear whether changes in the control of mRNA translation are required for the establishment of these durable modifications. Here we report that repeated exposure to D-amphetamine decreases global striatal mRNA translation. This effect is paralleled by an enhanced phosphorylation of the translation factors, eIF2α and eEF2, and by the concomitant increased translation of a subset of mRNAs, among which the mRNA encoding for the activity regulated cytoskeleton-associated protein, also known as activity regulated gene 3.1 (Arc/Arg3.1). The enrichment of Arc/Arg3.1 mRNA in the polysomal fraction is accompanied by a robust increase of Arc/Arg3.1 protein levels within the striatum. Immunofluorescence analysis revealed that this increase occurred preferentially in D1R-expressing SPNs localized in striosome compartments. Our results suggest that the decreased global protein synthesis following repeated exposure to D-amphetamine favors the translation of a specific subset of mRNAs in the striatum. PMID:28119566

Hepatic ontogeny and tissue distribution of mRNAs of epigenetic modifiers in mice using RNA-sequencing

PubMed Central

Lu, Hong; Cui, Julia; Gunewardena, Sumedha; Yoo, Byunggil; Zhong, Xiao-bo; Klaassen, Curtis

2012-01-01

Developmental regulation of gene expression is controlled by distinct epigenetic signatures catalyzed by various epigenetic modifiers. Little is known about the ontogeny and tissue distribution of these epigenetic modifiers. In the present study, we used a novel approach of RNA-sequencing to elucidate hepatic ontogeny and tissue distribution of mRNA expression of 142 epigenetic modifiers, including enzymes involved in DNA methylation/demethylation, histone acetylation/deacetylation, histone methylation/demethylation, histone phosphorylation and chromosome remodeling factors in male C57BL/6 mice. Livers from male C57BL/6 mice were collected at 12 ages from prenatal to adulthood. Many of these epigenetic modifiers were expressed at much higher levels in perinatal livers than adult livers, such as Dnmt1, Dnmt3a, Dnmt3b, Apobec3, Kat1, Ncoa4, Setd8, Ash2l, Dot1l, Cbx1, Cbx3, Cbx5, Cbx6, Ezh2, Suz12, Eed, Suv39h1, Suv420h2, Dek, Hdac1, Hdac2, Hdac7, Kdm2b, Kdm5c, Kdm7, Prmt1–5, Prmt7, Smarca4, Smarcb1, Chd4 and Ino80e. In contrast, hepatic mRNA expression of a few epigenetic modifiers increased during postnatal liver development, such as Smarca2, Kdm1b, Cbx7 and Chd3. In adult mice (60 d of age), most epigenetic modifiers were expressed at moderately (1–3-fold) higher levels in kidney and/or small intestine than liver. In conclusion, this study, for the first time, unveils developmental changes in mRNA abundance of all major known epigenetic modifiers in mouse liver. These data suggest that ontogenic changes in mRNA expression of epigenetic modifiers may play important roles in determining the addition and/or removal of corresponding epigenetic signatures during liver development. PMID:22772165

Hepatic ontogeny and tissue distribution of mRNAs of epigenetic modifiers in mice using RNA-sequencing.

PubMed

Lu, Hong; Cui, Julia Yue; Gunewardena, Sumedha; Yoo, Byunggil; Zhong, Xiao-bo; Klaassen, Curtis D

2012-08-01

Developmental regulation of gene expression is controlled by distinct epigenetic signatures catalyzed by various epigenetic modifiers. Little is known about the ontogeny and tissue distribution of these epigenetic modifiers. In the present study, we used a novel approach of RNA-sequencing to elucidate hepatic ontogeny and tissue distribution of mRNA expression of 142 epigenetic modifiers, including enzymes involved in DNA methylation/demethylation, histone acetylation/deacetylation, histone methylation/demethylation, histone phosphorylation and chromosome remodeling factors in male C57BL/6 mice. Livers from male C57BL/6 mice were collected at 12 ages from prenatal to adulthood. Many of these epigenetic modifiers were expressed at much higher levels in perinatal livers than adult livers, such as Dnmt1, Dnmt3a, Dnmt3b, Apobec3, Kat1, Ncoa4, Setd8, Ash2l, Dot1l, Cbx1, Cbx3, Cbx5, Cbx6, Ezh2, Suz12, Eed, Suv39h1, Suv420h2, Dek, Hdac1, Hdac2, Hdac7, Kdm2b, Kdm5c, Kdm7, Prmt1-5, Prmt7, Smarca4, Smarcb1, Chd4 and Ino80e. In contrast, hepatic mRNA expression of a few epigenetic modifiers increased during postnatal liver development, such as Smarca2, Kdm1b, Cbx7 and Chd3. In adult mice (60 d of age), most epigenetic modifiers were expressed at moderately (1-3-fold) higher levels in kidney and/or small intestine than liver. In conclusion, this study, for the first time, unveils developmental changes in mRNA abundance of all major known epigenetic modifiers in mouse liver. These data suggest that ontogenic changes in mRNA expression of epigenetic modifiers may play important roles in determining the addition and/or removal of corresponding epigenetic signatures during liver development.

Protective role for ovarian glutathione S-transferase isoform pi during 7,12-dimethylbenz[a]anthracene-induced ovotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhattacharya, Poulomi, E-mail: poulomib@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

2012-04-15

7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles at all developmental stages. This study investigated a role for the glutathione S-transferase (Gst) isoforms alpha (a), mu (m) and pi (p) and the transcription factors, Ahr and Nrf2, during DMBA-induced ovotoxicity, and their regulation by phosphatidylinositol-3 kinase (PI3K) signaling. Negative regulation of JNK by GSTP during DMBA exposure was also studied. Post-natal day (PND) 4 Fischer 344 rat ovaries were exposed to vehicle control (1% DMSO) ± DMBA (1 μM) or vehicle control (1% DMSO) ± LY294002 (PI3K inhibitor; 20 μM) for 1, 2, 4, or 6 days. Total RNA or protein was isolated,more » followed by RT-PCR or Western blotting to determine mRNA or protein level, respectively. Immunoprecipitation using an anti-GSTP antibody was performed to determine interaction between GSTP and JNK, followed by Western blotting to determine JNK and p-c-Jun protein level. DMBA had no impact on Gsta, Gstm or Nrf2 mRNA level, but increased Gstp mRNA and protein after 2 days. Ahr mRNA and protein increased after 2 and 4 days of DMBA exposure, respectively and DMBA increased NRF2 protein level after 4 days. JNK bound to GSTP was increased during DMBA exposure, with a concomitant decrease in unbound JNK and p-c-Jun. Ahr and Gstp mRNA were decreased (2 days) and increased (4 days) by PI3K inhibition, while Gstm mRNA increased (P < 0.05) after both time points, and there was no effect on Nrf2 mRNA. PI3K inhibition increased AHR, NRF2 and GSTP protein level. These findings support involvement of ovarian GSTP during DMBA exposure, and indicate a regulatory role for the PI3K signaling pathway on ovarian xenobiotic metabolism gene expression. -- Highlights: ► Ovarian GSTP is activated in response to DMBA exposure. ► AhR and Nrf2 transcription factors are up-regulated by DMBA. ► PI3K signaling regulates Ahr, Nrf2 and Gstp expression. ► GSTP negatively regulates ovarian JNK in response to DMBA exposure.« less

Expression of prostaglandin E receptor subtype EP4 in conjunctival epithelium of patients with ocular surface disorders: case-control study.

PubMed

Ueta, Mayumi; Sotozono, Chie; Yamada, Keiko; Yokoi, Norihiko; Inatomi, Tsutomu; Kinoshita, Shigeru

2012-01-01

To confirm the downregulation of PTGER4 mRNA in the conjunctiva of Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and ocular cicatricial pemphigoid (OCP) patients and to examine the expression of its EP4 protein in the conjunctival epithelium of patients with various ocular surface disorders. Case-control study. We performed quantitative reverse transcription-PCR (RT-PCR) analysis of PTGER4 mRNA in conjunctival tissue sections from patients with SJS/TEN and OCP to confirm the downregulation of PTGER4 mRNA expression. We also analysed EP4 immunohistologically in other ocular surface disorders. Conjunctival tissues were obtained from patients undergoing surgical reconstruction of the ocular surface due to chemical eye burns, subacute SJS/TEN or chronic SJS/TEN, chronic OCP, severe graft versus host disease (GVHD) and from patients with Mooren's ulcers treated by resection of the inflammatory conjunctiva. The expression of PTGER4 mRNA and EP4 protein assessed by quantitative RT-PCR assay and immunohistological methods. PTGER4 mRNA was significantly lower in conjunctival tissues from SJS and OCP patients than in the control conjunctivochalasis samples. EP4 protein was detected in conjunctival epithelium from patients with chemical eye burn and in control conjunctival epithelium from patients with conjunctivochalasis. Its expression varied in conjunctival epithelium from patients with Mooren's ulcer. We did not detect EP4 immunoreactivity in conjunctival epithelium from patients with subacute SJS/TEN, severe GVHD, chronic SJS/TEN or OCP. The strong downregulation of EP4 expression in conjunctival epithelium from patients with OCP or SJS/TEN may be attributable to ocular surface inflammation.

Expression of prostaglandin E receptor subtype EP4 in conjunctival epithelium of patients with ocular surface disorders: case-control study

PubMed Central

Ueta, Mayumi; Sotozono, Chie; Yamada, Keiko; Yokoi, Norihiko; Inatomi, Tsutomu; Kinoshita, Shigeru

2012-01-01

Objectives To confirm the downregulation of PTGER4 mRNA in the conjunctiva of Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and ocular cicatricial pemphigoid (OCP) patients and to examine the expression of its EP4 protein in the conjunctival epithelium of patients with various ocular surface disorders. Design Case-control study. Setting and participants We performed quantitative reverse transcription-PCR (RT-PCR) analysis of PTGER4 mRNA in conjunctival tissue sections from patients with SJS/TEN and OCP to confirm the downregulation of PTGER4 mRNA expression. We also analysed EP4 immunohistologically in other ocular surface disorders. Conjunctival tissues were obtained from patients undergoing surgical reconstruction of the ocular surface due to chemical eye burns, subacute SJS/TEN or chronic SJS/TEN, chronic OCP, severe graft versus host disease (GVHD) and from patients with Mooren's ulcers treated by resection of the inflammatory conjunctiva. Primary and secondary outcome measures The expression of PTGER4 mRNA and EP4 protein assessed by quantitative RT-PCR assay and immunohistological methods. Results PTGER4 mRNA was significantly lower in conjunctival tissues from SJS and OCP patients than in the control conjunctivochalasis samples. EP4 protein was detected in conjunctival epithelium from patients with chemical eye burn and in control conjunctival epithelium from patients with conjunctivochalasis. Its expression varied in conjunctival epithelium from patients with Mooren's ulcer. We did not detect EP4 immunoreactivity in conjunctival epithelium from patients with subacute SJS/TEN, severe GVHD, chronic SJS/TEN or OCP. Conclusions The strong downregulation of EP4 expression in conjunctival epithelium from patients with OCP or SJS/TEN may be attributable to ocular surface inflammation. PMID:23065448

Shikonin suppresses proliferation and induces cell cycle arrest through the inhibition of hypoxia-inducible factor-1α signaling.

PubMed

Li, Ming Yue; Mi, Chunliu; Wang, Ke Si; Wang, Zhe; Zuo, Hong Xiang; Piao, Lian Xun; Xu, Guang Hua; Li, Xuezheng; Ma, Juan; Jin, Xuejun

2017-08-25

Hypoxia enhances the development of solid tumors. Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that is dominantly expressed under hypoxia in solid tumor cells and is a key factor of tumor regulation. HIF-1α regulates several target genes involved in many aspects of cancer progression, including angiogenesis, metastasis, and cell proliferation, as well as imparting resistance to cancer treatment. In this study, we assessed shikonin, which derives from the traditional medical herb Lithospermum erythrorhizon, for its anti-cancer effects in hypoxia-induced human colon cancer cell lines. Shikonin showed potent inhibitory activity against hypoxia-induced HIF-1α activation in various human cancer cell lines and efficient scavenging activity of hypoxia-induced reactive oxygen species in tumor cells. Further analysis revealed that shikonin inhibited HIF-1α protein synthesis without affecting the expression of HIF-1α mRNA or degrading HIF-1α protein. It was subsequently shown to attenuate the activation of downstream mTOR/p70S6K/4E-BP1/eIF4E kinase. Shikonin also dose-dependently caused the cell cycle arrest of activated HCT116 cells and inhibited the proliferation of HCT116 and SW620 cells. Moreover, it significantly inhibited tumor growth in a xenograft modal. These findings suggest that shikonin could be considered for use as a potential drug in human colon cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulation of Torpor in the Gray Mouse Lemur: Transcriptional and Translational Controls and Role of AMPK Signaling.

PubMed

Zhang, Jing; Tessier, Shannon N; Biggar, Kyle K; Wu, Cheng-Wei; Pifferi, Fabien; Perret, Martine; Storey, Kenneth B

2015-04-01

The gray mouse lemur (Microcebus murinus) is one of few primate species that is able to enter daily torpor or prolonged hibernation in response to environmental stresses. With an emerging significance to human health research, lemurs present an optimal model for exploring molecular adaptations that regulate primate hypometabolism. A fundamental challenge is how to effectively regulate energy expensive cellular processes (e.g., transcription and translation) during transitions to/from torpor without disrupting cellular homeostasis. One such regulatory mechanism is reversible posttranslational modification of selected protein targets that offers fine cellular control without the energetic burden. This study investigates the role of phosphorylation and/or acetylation in regulating key factors involved in energy homeostasis (AMP-activated protein kinase, or AMPK, signaling pathway), mRNA translation (eukaryotic initiation factor 2α or eIF2α, eukaryotic initiation factor 4E or eIF4E, and initiation factor 4E binding protein or 4EBP), and gene transcription (histone H3) in six tissues of torpid and aroused gray mouse lemurs. Our results indicated selective tissue-specific changes of these regulatory proteins. The relative level of Thr172-phosphorylated AMPKα was significantly elevated in the heart but reduced in brown adipose tissue during daily torpor, as compared to the aroused lemurs, implicating the regulation of AMPK activity during daily torpor in these tissues. Interestingly, the levels of the phosphorylated eIFs were largely unaltered between aroused and torpid animals. Phosphorylation and acetylation of histone H3 were examined as a marker for transcriptional regulation. Compared to the aroused lemurs, level of Ser10-phosphorylated histone H3 decreased significantly in white adipose tissue during torpor, suggesting global suppression of gene transcription. However, a significant increase in acetyl-histone H3 in the heart of torpid lemurs indicated a possible stimulation of transcriptional activity of this tissue. Overall, our study demonstrates that AMPK signaling and posttranslational regulation of selected proteins may play crucial roles in the control of transcription/translation during daily torpor in mouse lemurs. Copyright © 2015. Production and hosting by Elsevier Ltd.

Alteration of Motor Network Function Following Injury

DTIC Science & Technology

2013-10-01

neurotransmitters/neuromodulators, suggesting that transient block does not cause a loss of ability to release chemical transmitters .  Targets... neuro - Fig. 5. Channel mRNA correlations in LP neurons. mRNA levels of BK-KCa, Shal, and H are graphed in all pairwise combinations. Each point...www.jn.org on January 4, 2012 jn.physiology.org D ow nloaded from Marder E, Eisen JS. Transmitter identification of pyloric neurons: electrically coupled

Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf

The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10 mg/kg b.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) andmore » Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits–NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. - Highlights: • Aluminium decreases the mRNA levels of mitochondrial and nuclear encoded subunits. • It decreases the mtDNA copy number and mitochondrial content in rat brain. • It down-regulates the mRNA and protein levels of PGC-1α, NRF-1, NRF-2 and Tfam. • It also disturbs the mitochondrial or nuclear architecture of neurons. • Finally it also decreases mitochondrial number in HC and CS regions of rat brain.« less

Fibroblast growth factor-2 expression in the preimplantation equine conceptus and endometrium of pregnant and cyclic mares.

PubMed

de Ruijter-Villani, Marta; van Boxtel, Paula R M; Stout, Tom A E

2013-12-01

Uterine-derived growth factors and cytokines play essential roles in regulating preimplantation conceptus development. In several species, fibroblast growth factor-2 (FGF2) promotes embryogenesis, trophoblast cell migration, and adhesion. This study investigated mRNA expression for FGF2, its receptors (FGFR1-4), the activating factor FGF binding protein (FGF-BP) in equine endometrium and trophectoderm during early pregnancy and the estrous cycle, and localized FGF2 protein in both endometrium and conceptus tissues. FGF2, FGFRs1-4, and FGFBP mRNAs were expressed in endometrium throughout the estrous cycle and early pregnancy, and in days 14 to 28 conceptus membranes. FGF2 transcription was higher during estrus than on days 7 or 14 of diestrus, suggesting estrogen dependency. Endometrial expression of FGF2 mRNA and protein increased as pregnancy progressed from days 21 and day 28; FGF2 protein was localized predominantly in the luminal and glandular epithelium. FGF2 mRNA was detectable in trophectoderm from as early as day 14, and transcription and translation increased in day 21 and 28 allantochorion. FGF2 protein was localized mainly in the trophectoderm up to day 21 but was present in both trophectoderm and endoderm of day 28 allantochorion. FGFR1 mRNA was down-regulated in the endometrium at day 7 of diestrus but increased again by day 14. Gene expression for all of the FGFR2 splice variants, including FGFR2IIIc, was up-regulated during estrus. During early pregnancy, endometrial FGFR1 expression decreased, whereas FGFR2IIIc expression did not change. Conceptus mRNA expression for all FGFRs increased as pregnancy progressed. FGFBP expression remained unchanged in endometrium, but increased in the conceptus between days 14 and 28, suggesting a role in regulating FGF2 activity in the developing conceptus. We conclude that during weeks 3 and 4 of pregnancy, the equine endometrial epithelium produces FGF2, which may play a role in trophoblast development and adhesion. Copyright © 2013 Elsevier Inc. All rights reserved.

Prostaglandin E2 produced by Entamoeba histolytica binds to EP4 receptors and stimulates interleukin-8 production in human colonic cells.

PubMed

Dey, Indranil; Chadee, Kris

2008-11-01

Entamoeba histolytica pathogenesis in the colon occurs in a stepwise fashion. It begins with colonization of the mucin layer, which is followed by stimulation of a proinflammatory response that causes nonspecific tissue damage that may facilitate parasite invasion of the underlying colonic mucosa. Unfortunately, the parasite and/or host factors that stimulate a proinflammatory response in the gut are poorly understood. In this study, we found that live E. histolytica or secretory or proteins (SP) and soluble ameba components (SAP) can markedly increase interleukin-8 (IL-8) mRNA expression and protein production in colonic epithelial cells. The IL-8-stimulating molecule produced by live amebae was identified as prostaglandin E(2) (PGE(2)) as trophozoites treated with cyclooxygenase inhibitors inhibited the biosynthesis of PGE(2) and eliminated IL-8 production induced by live parasites or ameba components. Moreover, using specific prostaglandin EP2 and EP4 receptor agonists and antagonists, we found that PGE(2) binds exclusively through EP4 receptors in colonic epithelial cells to stimulate IL-8 production. Silencing of EP4 receptors with EP4 small interfering RNA completely eliminated SP- and SAP-induced IL-8 production. These studies identified bioactive PGE(2) as a one of the major virulence factors produced by E. histolytica that can stimulate the potent neutrophil chemokine and activator IL-8, which can trigger an acute host inflammatory response. Thus, the induction of IL-8 production in response to E. histolytica-derived PGE(2) may be a mechanism that explains the initiation and amplification of acute inflammation associated with intestinal amebiasis.

In vivo fluctuation of Tax, Foxp3, CTLA-4, and GITR mRNA expression in CD4(+)CD25(+) T cells of patients with human T-lymphotropic virus type 1-associated myelopathy.

PubMed

Ramirez, E; Cartier, L; Rodriguez, L; Alberti, C; Valenzuela, M A

2010-11-01

HTLV-1 Tax expression exerts an inhibitory effect on the Foxp3 transcription factor in CD4(+)CD25(+) T-regulatory cells (Treg). For a better understanding of the role of Tax mRNA in the gene expression of cellular markers we measured Tax, Foxp3, CTLA-4, GITR, TGF-β, and IL-10 mRNA in Treg cells of 50 patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP; 27 women and 23 men; mean age: 56.7 years). The control group consisted of 23 non-infected subjects (12 women and 11 men) with a mean age of 51.3 years. Real-time PCR was used to measure mRNA of Tax proteins and several cellular markers of Treg function. Determinations revealed a high level of Tax mRNA in HAM/TSP (124.35 copies/100 CD4(+)CD25(+) T cells). Foxp3, GITR, and CTLA-4 mRNA levels were lower in HAM/TSP patients (mean ± SD, 22.07 ± 0.78, 9.63 ± 0.36, and 4.54 ± 0.39, respectively) than in non-infected controls (47.15 ± 12.94, 22.14 ± 1.91, and 21.07 ± 2.31). Both groups had similar levels of TGF-β and IL-10. An inverse relationship was found between Tax levels and Foxp3, CTLA-4, and GITR levels. Conversely, there was a direct correlation between levels of Foxp3, GITR, and CTLA-4. Disease severity and evolution time did not correlate with Tax or Foxp3 levels. The present results suggest that Tax and Foxp3 mRNA vary with the same degree of disease severity in HAM/TSP patients. Tax fluctuations may affect CTLA-4 and GITR expression via the Foxp3 pathway, causing virus-induced dysfunction of CD4(+)CD25(+) T cells in HAM/TSP patients.

Localization of the putative precursor of Alzheimer's disease-specific amyloid at nuclear envelopes of adult human muscle.

PubMed Central

Zimmermann, K; Herget, T; Salbaum, J M; Schubert, W; Hilbich, C; Cramer, M; Masters, C L; Multhaup, G; Kang, J; Lemaire, H G

1988-01-01

Cloning and sequence analysis revealed the putative amyloid A4 precursor (pre-A4) of Alzheimer's disease to have characteristics of a membrane-spanning glycoprotein. In addition to brain, pre-A4 mRNA was found in adult human muscle and other tissues. We demonstrate by in situ hybridization that pre-A4 mRNA is present in adult human muscle, in cultured human myoblasts and myotubes. Immunofluorescence with antipeptide antibodies shows the putative pre-A4 protein to be expressed in adult human muscle and associated with some but not all nuclear envelopes. Despite high levels of a single 3.5-kb pre-A4 mRNA species in cultured myoblasts and myotubes, the presence of putative pre-A4 protein could not be detected by immunofluorescence. This suggests that putative pre-A4 protein is stabilized and therefore functioning in the innervated muscle tissue but not in developing, i.e. non-innervated cultured muscle cells. The selective localization of the protein on distinct nuclear envelopes could reflect an interaction with motor endplates. Images PMID:2896589

Curcumin attenuates cardiomyocyte hypertrophy induced by high glucose and insulin via the PPARγ/Akt/NO signaling pathway.

PubMed

Chen, Rongchun; Peng, Xiaofeng; Du, Weimin; Wu, Yang; Huang, Bo; Xue, Lai; Wu, Qin; Qiu, Hongmei; Jiang, Qingsong

2015-05-01

To investigate the potential effect of curcumin on cardiomyocyte hypertrophy and a possible mechanism involving the PPARγ/Akt/NO signaling pathway in diabetes. The cardiomyocyte hypertrophy induced by high glucose (25.5mmol/L) and insulin (0.1μmol/L) (HGI) and the antihypertrophic effect of curcumin were evaluated in primary culture by measuring the cell surface area, protein content and atrial natriuretic factor (ANF) mRNA expression. The mRNA and protein expressions were assayed by reverse transcription PCR and Western blotting, whereas the NO concentration and endothelial NO synthase (eNOS) activity were determined using nitrate reduction and ELISA methods, respectively. The cardiomyocyte hypertrophy induced by HGI was characterized by increasing ANF mRNA expression, total protein content, and cell surface area, with downregulated mRNA and protein expressions of both PPARγ and Akt, which paralleled the declining eNOS mRNA expression, eNOS content, and NO concentration. The effects of HGI were inhibited by curcumin (1, 3, 10μmol/L) in a concentration-dependent manner. GW9662 (10μmol/L), a selective PPARγ antagonist, could abolish the effects of curcumin. LY294002 (20μmol/L), an Akt blocker, and N(G)-nitro-l-arginine-methyl ester (100μmol/L), a NOS inhibitor, could also diminish the effects of curcumin. The results suggested that curcumin supplementation can improve HGI-induced cardiomyocytes hypertrophy in vitro through the activation of PPARγ/Akt/NO signaling pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Chronic administration of methamphetamine promotes atherosclerosis formation in ApoE-/- knockout mice fed normal diet.

PubMed

Gao, Bo; Li, Lun; Zhu, Pengfei; Zhang, Mingjing; Hou, Lingbo; Sun, Yufei; Liu, Xiaoyan; Peng, Xiaohong; Gu, Ye

2015-11-01

Chronic methamphetamine (METH) abuse could induce neurotoxicity due to reactive oxygen species generation and sympathetic activation. Both factors are associated with atherosclerosis, so we tested the hypothesis that chronic METH administration might also promote atherosclerosis formation in Apo E-/- knockout mice fed normal diet. Male ApoE-/- mice (6 weeks-old) were treated with saline (NS) or METH [4 mg/kg/day (M4) or 8 mg/kg/day (M8) through intraperitoneal injection] for 24 weeks. Atherosclerotic lesion area on oil red O stained en face aorta was dose-dependently increased in M4 and M8 groups compared to NS group. Percentage of atherosclerotic lesion area was significantly higher in M8 group compared to NS and M4 groups. Plasma CRP was increased and inflammatory cytokine (ICAM-1, VCAM-1, TNF-α, and INF-γ) expression on aortic root was upregulated in METH groups compared to NS group. Neuropeptide Y (NPY) protein and mRNA expressions in aortic root and myocardial tissue were determined by Western blot and real time PCR, which were significantly upregulated in M4 and M8 groups. Moreover, mRNA expressions of NPY1R, NPY2R and NPY5R in aortic and myocardial tissue were also significantly upregulated in M4 and M8 groups. Raw264.7 cells were treated with NPY, NPY receptor antagonists, METH (10 μM or 100 μM) with or without lipopolysaccharide (LPS), and the expressions of TNF-α, CRP, MCP-1 and reactive oxygen species (ROS) production were significantly increased in METH and LPS + METH groups compared to control and LPS groups. Co-treatment with NPY1R antagonist decreased the expressions of TNF-α, CRP and MCP-1 in NPY and METH treated cells. Chronic METH administration can promote inflammation and atherosclerotic plague formation in ApoE-/- mice fed normal chow. NPY might be involved in the pathogenesis of METH-induced atherogenic effects through NPY Y1 receptor pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Morphoproteomics, E6/E7 in-situ hybridization, and biomedical analytics define the etiopathogenesis of HPV-associated oropharyngeal carcinoma and provide targeted therapeutic options.

PubMed

Brown, Robert E; Naqvi, Syed; McGuire, Mary F; Buryanek, Jamie; Karni, Ron J

2017-08-17

Human papillomavirus (HPV) has been identified as an etiopathogenetic factor in oropharyngeal squamous cell carcinoma. The HPV E6 and E7 oncogenes are instrumental in promoting proliferation and blocking differentiation leading to tumorigenesis. Although surgical intervention can remove such tumors, the potential for an etiologic field effect with recurrent disease is real. A downstream effector of E7 oncoprotein, enhancer of zeste homolog 2 (EZH2), is known to promote proliferation and to pose a block in differentiation and in turn, could lead to HPV-induced malignant transformation. However, the EZH2 pathway is amenable to low toxicity therapies designed to promote differentiation to a more benign state and prevent recurrent disease by inhibiting the incorporation of HPV into the genome. This is the first study using clinical specimens to demonstrate EZH2 protein expression in oropharyngeal carcinoma (OPC). The study included eight patients with oropharyngeal carcinoma, confirmed p16INK4a- positive by immunohistochemistry (IHC). The tissue expression of E6/E7 messenger RNA (mRNA) was measured by RNAscope® in-situ hybridization technology. Expression of EZH2, Ki-67, and mitotic indices were assessed by morphoproteomic analysis. Biomedical analytics expanded the results with data from Ingenuity Pathway Analysis (IPA) and KEGG databases to construct a molecular network pathway for further insights. Expression of E6 and E7 oncogenes in p16INK4a- positive oropharyngeal carcinoma was confirmed. EZH2 and its correlates, including elevated proliferation index (Ki-67) and mitotic progression were also present. Biomedical analytics validated the relationship between HPV- E6 and E7 and the expression of the EZH2 pathway. There is morphoproteomic and mRNA evidence of the association of p16INK4a-HPV infection with the E6 and E7 oncogenes and the expression of EZH2, Ki-67 and mitotic progression in oropharyngeal carcinoma. The molecular network biology was confirmed by biomedical analytics as consistent with published literature. This is significant because the biology lends itself to targeted therapeutic options using metformin, curcumin, celecoxib and sulforaphane as therapeutic strategies to prevent progression or recurrence of disease.

HPV Virus Transcriptional Status Assessment in a Case of Sinonasal Carcinoma.

PubMed

Ilardi, Gennaro; Russo, Daniela; Varricchio, Silvia; Salzano, Giovanni; Dell'Aversana Orabona, Giovanni; Napolitano, Virginia; Di Crescenzo, Rosa Maria; Borzillo, Alessandra; Martino, Francesco; Merolla, Francesco; Mascolo, Massimo; Staibano, Stefania

2018-03-16

Human Papilloma Virus (HPV) can play a causative role in the development of sinonasal tract malignancies. In fact, HPV may be the most significant causative agent implicated in sinonasal tumorigenesis and is implicated in as many as 21% of sinonasal carcinomas. To date, there are no definitive, reliable and cost-effective, diagnostic tests approved by the FDA for the unequivocal determination of HPV status in head and neck cancers. We followed an exhaustive algorithm to correctly test HPV infection, including a sequential approach with p16INK4a IHC, viral DNA genotyping and in situ hybridization for E6/E7 mRNA. Here, we report a case of sinonasal carcinoma with discordant results using HPV test assays. The tumor we describe showed an irregular immunoreactivity for p16INK4a, and it tested positive for HPV DNA; nevertheless, it was negative for HR-HPV mRNA. We discuss the possible meaning of this discrepancy. It would be advisable to test HPV transcriptional status of sinonasal carcinoma on a diagnostic routine basis, not only by p16INK4a IHC assay, but also by HPV DNA genotyping and HR-HPV mRNA assessment.

Effects of thalidomide on the expression of adhesion molecules in rat liver cirrhosis.

PubMed

Lv, Peng; Paul, Shelley Chireyath; Xiao, Yanjv; Liu, Shiquan; Luo, Hesheng

2006-01-01

This study was to evaluate the effects of thalidomide on expression of adhesion molecules in liver cirrhosis. The cirrhosis was induced in Wistar rats by intraperitoneal injection of CCl(4), and thalidomide (10 mg/kg/day or 100 mg/kg/day) was given by intragastric administration for 8 weeks. Liver histopathology and immunohistochemistry were significantly improved and the expressions of ICAM-1, VCAM-1, E-selectin, and TNF-alpha mRNA and protein were decreased significantly in rats treated with a high dose of thalidomide. Close positive correlation was observed in the expression of the TNF-alpha mRNA and that of ICAM-1, VCAM-1, and E-selectin mRNA, respectively. These results indicate that thalidomide exerts its effect on the downregulation of adhesion molecules via TNF-alpha signaling pathway to inhibit liver fibrosis.

Effects of Thalidomide on the Expression of Adhesion Molecules in Rat Liver Cirrhosis

PubMed Central

Lv, Peng; Paul, Shelley Chireyath; Xiao, Yanjv; Liu, Shiquan; Luo, Hesheng

2006-01-01

This study was to evaluate the effects of thalidomide on expression of adhesion molecules in liver cirrhosis. The cirrhosis was induced in Wistar rats by intraperitoneal injection of CCl4, and thalidomide (10 mg/kg/day or 100 mg/kg/day) was given by intragastric administration for 8 weeks. Liver histopathology and immunohistochemistry were significantly improved and the expressions of ICAM-1, VCAM-1, E-selectin, and TNF-α mRNA and protein were decreased significantly in rats treated with a high dose of thalidomide. Close positive correlation was observed in the expression of the TNF-α mRNA and that of ICAM-1, VCAM-1, and E-selectin mRNA, respectively. These results indicate that thalidomide exerts its effect on the downregulation of adhesion molecules via TNF-α signaling pathway to inhibit liver fibrosis. PMID:17047296

Differential expression of decorin and biglycan genes during palatogenesis in normal and retinoic acid-treated mice.

PubMed

Zhang, Yuxiang; Mori, Tetsuji; Iseki, Ken; Hagino, Seita; Takaki, Hiromi; Takeuchi, Mayumi; Hikake, Tsuyoshi; Tase, Choichiro; Murakawa, Masahiro; Yokoya, Sachihiko; Wanaka, Akio

2003-04-01

Proteoglycans are involved in secondary palate formation. In the present study, we focused on two small leucine-rich proteoglycans, decorin and biglycan, because they assembled extracellular matrix molecules such as collagens and modulated signaling pathway of transforming growth factor-beta. To investigate the functions of decorin and biglycan in palatogenesis, we compared their mRNA expression patterns between normal palate and retinoic acid-induced cleft palate in mice by using in situ hybridization analysis during the period of embryonic day 13.5 (E13.5) to E15.5. On E13.5, decorin mRNA was expressed in the epithelia and mesenchyme on the nasal side of the developing secondary palate. During the period the palate shelves were fusing (E14.5), decorin mRNA was strongly expressed in the mesenchyme but its expression pattern was asymmetric; decorin mRNA expression area in the nasal side was broader than that in the oral side. The expression of decorin mRNA was hardly detected in the mesenchyme on either side of the medial edge epithelium. After fusion (E15.5), its expression converged to the mesenchyme just around the palatine bone. Biglycan mRNA was ubiquitously distributed throughout the palatal mesenchyme for the mid-gestation period. Its expression area became limited to the ossification area within the palate after the late gestation period. In the retinoic acid-treated mice, the area of the decorin gene expression expanded to the core region of the palate primordium where little signal was observed in control mice. On the other hand, biglycan in the retinoic acid-treated mice did not show remarkable change in its distribution patterns compared with that in the control mice. These findings suggest that decorin and biglycan play distinct roles in palatogenesis, and decorin was more actively involved in the process of secondary palate formation than biglycan. Up-regulation of decorin gene expression in the retinoic acid-treated mice might influence the pathogenesis of cleft palate. Copyright 2003 Wiley-Liss, Inc.

Human hematopoietic progenitors express erythropoietin.

PubMed

Stopka, T; Zivny, J H; Stopkova, P; Prchal, J F; Prchal, J T

1998-05-15

Erythropoietin (EPO) is a factor essential for erythroid cell proliferation, differentiation, and survival. The production of EPO by the kidneys in response to hypoxia and anemia is well documented. To determine whether EPO is also produced by hematopoietic cells, we analyzed the expression of EPO in normal human hematopoietic progenitors and in their progeny. Undifferentiated CD34(+)lin- hematopoietic progenitors do not have detectable EPO mRNA. Differentiating CD34(+) cells that are stimulated with recombinant human EPO in serum-free liquid cultures express both EPO and EPO receptor (EPOR). Because CD34(+) cells represent a heterogeneous cell population, we analyzed individual burst-forming units-erythroid (BFU-E) and nonerythroid colony-forming unit-granulocyte-macrophage colonies for EPO mRNA. Only BFU-E colonies were positive for EPO mRNA. Lysates from pooled BFU-E colonies stained positively for EPO by immunoblotting. To further confirm the intrinsic nature of erythroid EPO, we replaced extrinsic EPO in erythroid colony cultures with EPO-mimicking peptide (EMP). We show EPO expression in the EMP-stimulated BFU-Es at both mRNA and protein levels. Stimulation of bone marrow mononuclear cells (BMMCs) with EMP upregulated EPO expression. Furthermore, we found EPO and EPOR mRNAs as well as EPO protein in K562 cells, a human erythroleukemia cell line. Stimulation of K562 cells with EMP upregulated EPO expression. We suggest that EPO of erythroid origin may have a role in the regulation of erythropoiesis.

Chicken ovalbumin upstream promoter-transcription factor II regulates nuclear receptor, myogenic, and metabolic gene expression in skeletal muscle cells.

PubMed

Crowther, Lisa M; Wang, Shu-Ching Mary; Eriksson, Natalie A; Myers, Stephen A; Murray, Lauren A; Muscat, George E O

2011-02-24

We demonstrate that chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) mRNA is more abundantly expressed (than COUP-TFI mRNA) in skeletal muscle C2C12 cells and in (type I and II) skeletal muscle tissue from C57BL/10 mice. Consequently, we have utilized the ABI TaqMan Low Density Array (TLDA) platform to analyze gene expression changes specifically attributable to ectopic COUP-TFII (relative to vector only) expression in muscle cells. Utilizing a TLDA-based platform and 5 internal controls, we analyze the entire NR superfamily, 96 critical metabolic genes, and 48 important myogenic regulatory genes on the TLDA platform utilizing 5 internal controls. The low density arrays were analyzed by rigorous statistical analysis (with Genorm normalization, Bioconductor R, and the Empirical Bayes statistic) using the (integromics) statminer software. In addition, we validated the differentially expressed patho-physiologically relevant gene (identified on the TLDA platform) glucose transporter type 4 (Glut4). We demonstrated that COUP-TFII expression increased the steady state levels of Glut4 mRNA and protein, while ectopic expression of truncated COUP-TFII lacking helix 12 (COUP-TFΔH12) reduced Glut4 mRNA expression in C2C12 cells. Moreover, COUP-TFII expression trans-activated the Glut4 promoter (-997/+3), and ChIP analysis identified selective recruitment of COUP-TFII to a region encompassing a highly conserved SP1 binding site (in mouse, rat, and human) at nt positions -131/-118. Mutation of the SpI site ablated COUP-TFII mediated trans-activation of the Glut4 promoter. In conclusion, this study demonstrates that in skeletal muscle cells, COUP-TFII regulates several nuclear hormone receptors, and critical metabolic and muscle specific genes.

Optimization of a nonviral transfection system to evaluate Cox-2 controlled interleukin-4 expression for osteoarthritis gene therapy in vitro.

PubMed

Lang, Annemarie; Neuhaus, Johannes; Pfeiffenberger, Moritz; Schröder, Erik; Ponomarev, Igor; Weber, Yvonne; Gaber, Timo; Schmidt, Michael F G

2014-01-01

Gene therapy appears to have the potential for achieving a long-term remedy for osteoarthritis (OA). However, there is a risk of adverse reactions, especially when using cytomegalovirus-controlled expression. To provide a safe application, we focused on the expression of therapeutic cytokines [e.g. interleukin (IL)-4] in a disease-responsive manner by use of the previously cloned Cox-2 promoter as 'genetic switch'. In the present study, we report the functionality of a controlled gene therapeutic system in an equine osteoarthritic cell model. Different nonviral transfection reagents were tested for their efficiency on equine chondrocytes stimulated with equine IL-1β or lipopolysaccharide to create an inflammatory environment. To optimize the transfection, we successfully redesigned the vector by excluding the internal ribosomal entry site (IRES). The functionality of our Cox-2 promoter construct with respect to expressing IL-4 was proven at the mRNA and protein levels and the anti-inflammatory potential of IL-4 was confirmed by analyzing the expression of IL-1β, IL-6, IL-8, matrix metalloproteinase (MMP)-1, MMP-3 and tumor necrosis factor (TNF)-α using a quantitative polymerase chain reaction. Nonviral transfection reagents yielded transfection rates from 21% to 44% with control vectors with and without IRES, respectively. Stimulation of equine chondrocytes resulted in a 20-fold increase of mRNA expression of IL-1β. Such exogenous stimulation of chondrocytes transfected with pNCox2-IL4 led to an increase of IL-4 mRNA expression, whereas expression of inflammatory mediators decreased. The timely link between these events confirms the anti-inflammatory potential of synthesized IL-4. We consider that this approach has significant potential for translation into a useful anti-inflammation therapy. Molecular tools such as the described therapeutic plasmid pave the way for a local-controlled, self-limiting gene therapy. Copyright © 2014 John Wiley & Sons, Ltd.

Bilirubin modulated cytokines, growth factors and angiogenesis to improve cutaneous wound healing process in diabetic rats.

PubMed

Ram, Mahendra; Singh, Vishakha; Kumawat, Sanjay; Kant, Vinay; Tandan, Surendra Kumar; Kumar, Dinesh

2016-01-01

Bilirubin has shown cutaneous wound healing potential in some preliminary studies. Here we hypothesize that bilirubin facilitates wound healing in diabetic rats by modulating important healing factors/candidates and antioxidant parameters in a time-dependent manner. Diabetes was induced in male Wistar rats by streptozotocin. In all diabetic rats wounds were created under pentobarbitone anesthesia. All the rats were divided into two groups, of which one (control) was treated with ointment base and other with bilirubin ointment (0.3%). Wound closer measurement and tissue collection were done on days 3, 7, 14 and 19 post-wounding. The relative expressions of hypoxia inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 alpha (SDF-1α), transforming growth factor- beta1 (TGF-β1()), tumor necrosis factor-α (TNF-α) and interlukin-10 (IL-10) mRNA and proteins and the mRNA of interlukin-1 beta (IL-1β) and matrix metalloprteinase-9 (MMP-9) were determined in the wound tissues. CD-31 staining and collagen content were evaluated by immunohistochemistry and picrosirius red staining, respectively. Histopathological changes were assessed by H&E staining. The per cent wound closer was significantly higher from day 7 onwards in bilirubin-treated rats. HIF-1α, VEGF, SDF-1α, TGF-β1, IL-10 mRNA and protein levels were significantly higher on days 3, 7 and 14 in bilirubin-treated rats. The mRNA expression and protein level of TNF-α and the mRNA of IL-1β and MMP-9 were progressively and markedly reduced in bilirubin-treated rats. The collagen deposition and formation of blood vessels were greater in bilirubin-treated rats. Bilirubin markedly facilitated cutaneous wound healing in diabetic rats by modulating growth factors, cytokines, neovasculogenesis and collagen contents to the wound site. Topical application of bilirubin ointment might be of great use in cutaneous wound healing in diabetic patients. Copyright © 2015 Elsevier B.V. All rights reserved.

Transcription of the pst Operon of Clostridium acetobutylicum Is Dependent on Phosphate Concentration and pH

PubMed Central

Fischer, Ralf-Jörg; Oehmcke, Sonja; Meyer, Uta; Mix, Maren; Schwarz, Katrin; Fiedler, Tomas; Bahl, Hubert

2006-01-01

The pst operon of Clostridium acetobutylicum ATCC 824 comprises five genes, pstS, pstC, pstA, pstB, and phoU, and shows a gene architecture identical to that of Escherichia coli. Deduced proteins are predicted to represent a high-affinity phosphate-specific ABC (ATP-binding cassette) transport system (Pst) and a protein homologous to PhoU, a negative phosphate regulon regulator. We analyzed the expression patterns of the pst operon in Pi-limited chemostat cultures during acid production at pH 5.8 or solvent production at pH 4.5 and in response to Pi pulses. Specific mRNA transcripts were found only when external Pi concentrations had dropped below 0.2 mM. Two specific transcripts were detected, a 4.7-kb polycistronic mRNA spanning the whole operon and a quantitatively dominating 1.2-kb mRNA representing the first gene, pstS. The mRNA levels clearly differed depending on the external pH. The amounts of the full-length mRNA detected were about two times higher at pH 5.8 than at pH 4.5. The level of pstS mRNA increased by a factor of at least 8 at pH 5.8 compared to pH 4.5 results. Primer extension experiments revealed only one putative transcription start point 80 nucleotides upstream of pstS. Thus, additional regulatory sites are proposed in the promoter region, integrating two different extracellular signals, namely, depletion of inorganic phosphate and the pH of the environment. After phosphate pulses were applied to a phosphate-limited chemostat we observed faster phosphate consumption at pH 5.8 than at pH 4.5, although higher optical densities were recorded at pH 4.5. PMID:16855236

Region-specific effects of developmental exposure to cocaine on fibroblast growth factor-2 expression in the rat brain.

PubMed

Giannotti, Giuseppe; Caffino, Lucia; Mottarlini, Francesca; Racagni, Giorgio; Fumagalli, Fabio

2016-07-01

Adolescence is a period of high vulnerability to drugs of abuse and alterations of the proper developmental trajectory via psychostimulant exposure might change the physiological brain homeostasis. By microdissection of brain areas via punching, we investigated whether repeated exposure to cocaine during adolescence (from postnatal day 28 [PND28] to PND42) has altered fibroblast growth factor-2 (FGF-2) messenger RNA (mRNA) levels in selected brain subregions critical for the action of cocaine. We found a reduction of FGF-2 mRNA levels in ventral tegmental area (VTA), where mesocortical and mesolimbic pathways originate. The analysis of the trophic factor levels in the distal projecting regions revealed a selective reduction of FGF-2 mRNA levels in infralimbic (IL) subregion of the medial prefrontal cortex (the terminal region of the mesocortical pathway) and in the nucleus accumbens core (cNAc) (the terminal region of the mesolimbic pathway). Last, we found reduced FGF-2 mRNA levels also in brain regions which, although in a different manner, contribute to the reward system, i.e., the central nucleus of amygdala (cAmy) and the ventral portion of hippocampus (vHip). The widespread and coordinated reduction of FGF-2 mRNA levels across the brain's reward neurocircuitry might represent a defensive strategy set in motion to oppose to the psychostimulant properties of cocaine. Moreover, given the role of FGF-2 in modulating mood disorders, the reduced trophic support here observed might sustain the negative emotional state set in motion by repeated exposure to cocaine.

Estradiol Increases Mucus Synthesis in Bronchial Epithelial Cells

PubMed Central

Tam, Anthony; Wadsworth, Samuel; Dorscheid, Delbert; Man, Shu-Fan Paul; Sin, Don D.

2014-01-01

Airway epithelial mucus hypersecretion and mucus plugging are prominent pathologic features of chronic inflammatory conditions of the airway (e.g. asthma and cystic fibrosis) and in most of these conditions, women have worse prognosis compared with male patients. We thus investigated the effects of estradiol on mucus expression in primary normal human bronchial epithelial cells from female donors grown at an air liquid interface (ALI). Treatment with estradiol in physiological ranges for 2 weeks caused a concentration-dependent increase in the number of PAS-positive cells (confirmed to be goblet cells by MUC5AC immunostaining) in ALI cultures, and this action was attenuated by estrogen receptor beta (ER-β) antagonist. Protein microarray data showed that nuclear factor of activated T-cell (NFAT) in the nuclear fraction of NHBE cells was increased with estradiol treatment. Estradiol increased NFATc1 mRNA and protein in ALI cultures. In a human airway epithelial (1HAE0) cell line, NFATc1 was required for the regulation of MUC5AC mRNA and protein. Estradiol also induced post-translational modification of mucins by increasing total fucose residues and fucosyltransferase (FUT-4, -5, -6) mRNA expression. Together, these data indicate a novel mechanism by which estradiol increases mucus synthesis in the human bronchial epithelium. PMID:24964096

Overexpression of pig selenoprotein S blocks OTA-induced promotion of PCV2 replication by inhibiting oxidative stress and p38 phosphorylation in PK15 cells.

PubMed

Gan, Fang; Hu, Zhihua; Huang, Yu; Xue, Hongxia; Huang, Da; Qian, Gang; Hu, Junfa; Chen, Xingxiang; Wang, Tian; Huang, Kehe

2016-04-12

Porcine circovirus type 2 (PCV2) is the primary cause of porcine circovirus disease, and ochratoxin A (OTA)-induced oxidative stress promotes PCV2 replication. In humans, selenoprotein S (SelS) has antioxidant ability, but it is unclear whether SelS affects viral infection. Here, we stably transfected PK15 cells with pig pCDNA3.1-SelS to overexpress SelS. Selenium (Se) at 2 or 4 μM and SelS overexpression blocked the OTA-induced increases of PCV2 DNA copy number and infected cell numbers. SelS overexpression also increased glutathione (GSH), NF-E2-related factor 2 (Nrf2) mRNA, and γ-glutamyl-cysteine synthetase mRNA levels; decreased reactive oxygen species (ROS) levels; and inhibited p38 phosphorylation in PCV2-infected PK15 cells, regardless of OTA treatment. Buthionine sulfoximine reversed all of the above SelS-induced changes. siRNA-mediated SelS knockdown decreased Nrf2 mRNA and GSH levels, increased ROS levels, and promoted PCV2 replication in OTA-treated PK15 cells. These data indicate that pig SelS blocks OTA-induced promotion of PCV2 replication by inhibiting the oxidative stress and p38 phosphorylation in PK15 cells.

Overexpression of pig selenoprotein S blocks OTA-induced promotion of PCV2 replication by inhibiting oxidative stress and p38 phosphorylation in PK15 cells

PubMed Central

Gan, Fang; Hu, Zhihua; Huang, Yu; Xue, Hongxia; Huang, Da; Qian, Gang; Hu, Junfa; Chen, Xingxiang; Wang, Tian; Huang, Kehe

2016-01-01

Porcine circovirus type 2 (PCV2) is the primary cause of porcine circovirus disease, and ochratoxin A (OTA)-induced oxidative stress promotes PCV2 replication. In humans, selenoprotein S (SelS) has antioxidant ability, but it is unclear whether SelS affects viral infection. Here, we stably transfected PK15 cells with pig pCDNA3.1-SelS to overexpress SelS. Selenium (Se) at 2 or 4 μM and SelS overexpression blocked the OTA-induced increases of PCV2 DNA copy number and infected cell numbers. SelS overexpression also increased glutathione (GSH), NF-E2-related factor 2 (Nrf2) mRNA, and γ-glutamyl-cysteine synthetase mRNA levels; decreased reactive oxygen species (ROS) levels; and inhibited p38 phosphorylation in PCV2-infected PK15 cells, regardless of OTA treatment. Buthionine sulfoximine reversed all of the above SelS-induced changes. siRNA-mediated SelS knockdown decreased Nrf2 mRNA and GSH levels, increased ROS levels, and promoted PCV2 replication in OTA-treated PK15 cells. These data indicate that pig SelS blocks OTA-induced promotion of PCV2 replication by inhibiting the oxidative stress and p38 phosphorylation in PK15 cells. PMID:26943035

P2Y receptors and atherosclerosis in apolipoprotein E-deficient mice

PubMed Central

Guns, Pieter-Jan DF; Hendrickx, Jan; Van Assche, Tim; Fransen, Paul; Bult, Hidde

2010-01-01

Background and purpose: P2Y nucleotide receptors are involved in the regulation of vascular tone, smooth muscle cell (SMC) proliferation and inflammatory responses. The present study investigated whether they are involved in atherosclerosis. Experimental approach: mRNA of P2Y receptors was quantified (RT-PCR) in atherosclerotic and plaque-free aorta segments of apolipoprotein E-deficient (apoE–/–) mice. Macrophage activation was assessed in J774 macrophages, and effects of non-selective purinoceptor antagonists on atherosclerosis were evaluated in cholesterol-fed apoE–/– mice. Key results: P2Y6 receptor mRNA was consistently elevated in segments with atherosclerosis, whereas P2Y2 receptor expression remained unchanged. Expression of P2Y1 or P2Y4 receptor mRNA was low or undetectable, and not influenced by atherosclerosis. P2Y6 mRNA expression was higher in cultured J774 macrophages than in cultured aortic SMCs. Furthermore, immunohistochemical staining of plaques demonstrated P2Y6-positive macrophages, but few SMCs, suggesting that macrophage recruitment accounted for the increase in P2Y6 receptor mRNA during atherosclerosis. In contrast to ATP, the P2Y6-selective agonist UDP increased mRNA expression and activity of inducible nitric oxide synthase and interleukin-6 in J774 macrophages; this effect was blocked by suramin (100–300 µM) or pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS, 10–30 µM). Finally, 4-week treatment of cholesterol-fed apoE–/– mice with suramin or PPADS (50 and 25 mg·kg−1·day−1 respectively) reduced plaque size, without changing plaque composition (relative SMC and macrophage content) or cell replication. Conclusions and implications: These results suggest involvement of nucleotide receptors, particularly P2Y6 receptors, during atherosclerosis, and warrant further research with selective purinoceptor antagonists or P2Y6 receptor-deficient mice. PMID:20050854

[Effects of Porphyromonas endodontalis lipopolysaccharides on the expression of matrix metalloproteinase-9 in mouse osteoblasts].

PubMed

Li, X L; Yu, Y Q; Qiu, L H; Yang, D; Wang, X M; Yu, J T

2017-08-09

Objective: To evaluate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on the expression of matrix metalloproteinase-9 (MMP-9) mRNA and protein as well as enzyme activity in MC3T3-E1 cells and the role of nuclear factor-κB (NF-κB) in the process, so as to investigate the expression of MMP-9 dependent signaling pathways in mouse osteoblasts induced by Pe LPS. Methods: The experiment was conducted in 3 sessions: MC3T3-E1 cells were treated with various concentrations of Pe LPS (0-20 mg/L) and 10 mg/L Pe LPS for different time intervals (0-48 h). The expression of MMP-9 mRNA and protein were detected by real-time reverse transcription-PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), while the enzyme activity was detected by gelatin zymography method. The expression of MMP-9 mRNA was also detected in 10 mg/L Pe LPS treated MC3T3-El cells after pretreated with specific NF-κB inhibitor BAY 11-7082 for l h. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. Results: The levels of MMP-9 mRNA and protein increased significantly after the treatment with various concentrations of Pe LPS (0-20 mg/L), which indicated that Pe LPS induced osteoblasts to express MMP-9 in dose dependent manners. The expression of MMP-9 protein increased from (5 395±362) ng/L (blank control group) to (12 684±375) ng/L (20 mg/L group). Maximal induction of MMP-9 mRNA expression was found in the MC3T3-E1 cells treated with 10 mg/L Pe LPS for 24 h. The expression of MMP-9 mRNA in the 20 mg/L group was about 7 times than that in the blank control group. After 24 h, the expression of MMP-9 mRNA decreased. Maximal expression of MMP-9 protein was found in the MC3T3-E1 cells treated with 10 mg/L Pe LPS for 48 h ([35 055±2 346] ng/L) showing the highest enzyme activity. The mRNA of MMP-9 decreased significantly after pretreatment with 10 µmol/L BAY 11-7082 for 1 h. Conclusions: Pe LPS might induce the expression of MMP-9 in MC3T3-E1 cells through the signaling of NF-κB.

Characterization of the endocrine, digestive and morphological adjustments of the intestine in response to food deprivation and torpor in cunner, Tautogolabrus adspersus.

PubMed

Hayes, James; Volkoff, Hélène

2014-04-01

The cunner, Tautogolabrus adspersus, is a marine teleost endemic to the cold waters of the Northwest Atlantic Ocean. The cunner is non-migratory and is known for its remarkable ability to endure the freezing winter months with little to no food by entering a torpid/dormant state. To evaluate the physiological strategies employed by the cunner's intestinal tract to withstand food deprivation, fish were sampled for their gut after a four-week period of acute food deprivation during their summer (active/feeding) state, as well as after 4months of overwinter fasting. Digestive capacity was evaluated by measuring digestive enzyme activity and related mRNA transcript expression for trypsin, alkaline phosphatase, alanine aminopeptidase and lipase. In order to assess how gut hormones affect/are affected by acute fasting and torpor, we examined the intestinal mRNA expression of several putative appetite regulators, i.e. CCK, apelin, orexin and mTOR. Short-term summer fasting induced a reduction in the activity, but not the transcript expression, of all digestive enzymes examined as well as a reduction in gut apelin mRNA. Torpor induced a reduction in the activity of all enzymes with the exception of alanine aminopeptidase, and a decrease in mRNA levels of alanine aminopeptidase, orexin, CCK and mTOR. Our results suggest that both acute fasting and long-term fasting induce a reduction in the intestinal function of cunner, as evidenced by an overall decrease in the activities of digestive enzymes and mRNA expression of several factors involved in feeding and digestion. Copyright © 2014 Elsevier Inc. All rights reserved.

Inflammatory airway responses by nasal inoculation of suspended particulate matter in NC/Nga mice.

PubMed

Ogino, Keiki; Takahashi, Noriko; Kubo, Masayuki; Takeuchi, Akihito; Nakagiri, Motoharu; Fujikura, Yoshihisa

2014-06-01

To evaluate the allergic effect of airborne particulate matter (PM) on the airway, separated soluble supernatant (Sup) and insoluble precipitate (Pre) in suspended PM were inoculated into NC/Nga mice with a high sensitivity for mite allergens. Sup, Pre, or both Sup and Pre with or without pronase treatment were inoculated via the nasal route five times for sensitization and a challenge inoculation on the 11th day in NC/Nga mice. On the 14th day, mice were examined for airway hyperresponsiveness (AHR), bronchoalveolar lavage fluid (BALF) cell count, mRNA expression of Th1 and Th2 cytokines in the lung tissue, and histopathology. Synergistic effects of Sup and Pre were observed as increases in AHR and a histopathological change of Periodic acid-Schiff (PAS) staining. Increases in neutrophils, macrophages, and lymphocytes of BALF cells were dependent on Pre. The expression of IL-4 mRNA was increased by Sup, and those of IL-5 mRNA and Il-13 mRNA was increased by Sup and Pre. Augmented AHR, mRNA expression of IL-4, peribronchial inflammation, and PAS staining by Sup plus Pre were attenuated by treatment of Sup with pronase to digest proteins. These results suggest that some proteins of ambient PM may be important environmental factors for AHR and airway inflammation with the aid of insoluble particulates, although some soluble factors such as endotoxins cannot be ruled out. Copyright © 2012 Wiley Periodicals, Inc.

Effect of mitomycin on normal dermal fibroblast and HaCat cell: an in vitro study

PubMed Central

Wang, Yao-wen; Ren, Ji-hao; Xia, Kun; Wang, Shu-hui; Yin, Tuan-fang; Xie, Ding-hua; Li, Li-hua

2012-01-01

Objective: To evaluate the effects of mitomycin on the growth of human dermal fibroblast and immortalized human keratinocyte line (HaCat cell), particularly the effect of mitomycin on intracellular messenger RNA (mRNA) synthesis of collagen and growth factors of fibroblast. Methods: The normal dermal fibroblast and HaCat cell were cultured in vitro. Cell cultures were exposed to 0.4 and 0.04 mg/ml of mitomycin solution, and serum-free culture medium was used as control. The cellular morphology change, growth characteristics, cell proliferation, and apoptosis were observed at different intervals. For the fibroblasts, the mRNA expression changes of transforming growth factor (TGF)-β1, basic fibroblast growth factor (bFGF), procollagen I, and III were detected by reverse transcription polymerase chain reaction (RT-PCR). Results: The cultured normal human skin fibroblast and HaCat cell grew exponentially. A 5-min exposure to mitomycin at either 0.4 or 0.04 mg/ml caused marked dose-dependent cell proliferation inhibition on both fibroblasts and HaCat cells. Cell morphology changed, cell density decreased, and the growth curves were without an exponential phase. The fibroblast proliferated on the 5th day after the 5-min exposure of mitomycin at 0.04 mg/ml. Meanwhile, 5-min application of mitomycin at either 0.04 or 0.4 mg/ml induced fibroblast apoptosis but not necrosis. The apoptosis rate of the fibroblast increased with a higher concentration of mytomycin (p<0.05). A 5-min exposure to mitomycin at 0.4 mg/ml resulted in a marked decrease in the mRNA production of TGF-β1, procollagen I and III, and a marked increase in the mRNA production of bFGF. Conclusions: Mitomycin can inhibit fibroblast proliferation, induce fibroblast apoptosis, and regulate intracellular protein expression on mRNA levels. In additon, mitomycin can inhibit HaCat cell proliferation, so epithelial cell needs more protecting to avoid mitomycin’s side effect when it is applied clinically. PMID:23225855

E-cadherin and beta-catenin are down-regulated in prostatic bone metastases.

PubMed

Bryden, A A G; Hoyland, J A; Freemont, A J; Clarke, N W; Schembri Wismayer, D; George, N J R

2002-03-01

To determine the E-cadherin and beta-catenin expression phenotype in untreated primary prostate cancer and corresponding bone metastases. Paired bone metastasis and primary prostate specimens were obtained from 14 men with untreated metastatic prostate carcinoma. The tumours were histologically graded by an independent pathologist. Expression of mRNA for E-cadherin and beta-catenin was detected within the tumour cells using in-situ hybridization with a 35S-labelled cDNA probe. The expression of E-cadherin and beta-catenin were graded as uniform, heterogeneous or negative. The mRNA for E-cadherin was expressed in 13 of 14 primary carcinomas and 11 bone metastases; beta-catenin was expressed by 13 and nine, respectively. Of the primary tumours, nine expressed E-cadherin and beta-catenin uniformly; in contrast, all metastases had down-regulated E-cadherin and/or beta-catenin. The down-regulation of E-cadherin and beta-catenin are a feature of the metastatic phenotype, which may be a significant factor in the genesis of bone metastases. However, this does not appear to be reflected in the expression of these molecules in the primary tumours.

GLD-4-Mediated Translational Activation Regulates the Size of the Proliferative Germ Cell Pool in the Adult C. elegans Germ Line

PubMed Central

Millonigg, Sophia; Eckmann, Christian R.

2014-01-01

To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult Caenorhabditis elegans reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A) polymerase (cytoPAP) GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that gld-4 activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with glp-1 mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of glp-1 mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant translational circuit with GLD-2 cytoPAP and the translational repressor GLD-1 to restrict proliferation. Together with previous findings, our combined data reveals two interconnected translational activation/repression circuitries of broadly conserved RNA regulators that maintain the balance between adult germ cell proliferation and differentiation. PMID:25254367

Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

PubMed Central

Małecki, Jędrzej; Nilges, Benedikt S.; Moen, Anders; Leidel, Sebastian A.

2017-01-01

Abstract Many cellular proteins are methylated on lysine residues and this has been most intensively studied for histone proteins. Lysine methylations on non-histone proteins are also frequent, but in most cases the functional significance of the methylation event, as well as the identity of the responsible lysine (K) specific methyltransferase (KMT), remain unknown. Several recently discovered KMTs belong to the so-called seven-β-strand (7BS) class of MTases and we have here investigated an uncharacterized human 7BS MTase currently annotated as part of the endothelin converting enzyme 2, but which should be considered a separate enzyme. Combining in vitro enzymology and analyzes of knockout cells, we demonstrate that this MTase efficiently methylates K36 in eukaryotic translation elongation factor 1 alpha (eEF1A) in vitro and in vivo. We suggest that this novel KMT is named eEF1A-KMT4 (gene name EEF1AKMT4), in agreement with the recently established nomenclature. Furthermore, by ribosome profiling we show that the absence of K36 methylation affects translation dynamics and changes translation speed of distinct codons. Finally, we show that eEF1A-KMT4 is part of a novel family of human KMTs, defined by a shared sequence motif in the active site and we demonstrate the importance of this motif for catalytic activity. PMID:28520920

Membrane-association of mRNA decapping factors is independent of stress in budding yeast

PubMed Central

Huch, Susanne; Gommlich, Jessie; Muppavarapu, Mridula; Beckham, Carla; Nissan, Tracy

2016-01-01

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation. PMID:27146487

Membrane-association of mRNA decapping factors is independent of stress in budding yeast.

PubMed

Huch, Susanne; Gommlich, Jessie; Muppavarapu, Mridula; Beckham, Carla; Nissan, Tracy

2016-05-05

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation.

Cytology and human papillomavirus testing 6 to 12 months after ASCUS or LSIL cytology in organized screening to predict high-grade cervical neoplasia between screening rounds.

PubMed

Tropé, Ameli; Sjøborg, Katrine D; Nygård, Mari; Røysland, Kjetil; Campbell, Suzanne; Alfsen, G Cecilie; Jonassen, Christine M

2012-06-01

We carried out a prospective study comparing the performance of human papillomavirus (HPV) E6/E7 mRNA (PreTect HPV-Proofer; NorChip, Klokkarstua, Norway) and DNA (Amplicor HPV test; Roche Diagnostics, Basel, Switzerland) triage testing of women 6 to 12 months after atypical-squamous-cells-of-undetermined-significance (ASCUS) or low-grade-squamous-intraepithelial-lesion (LSIL) cytology in organized screening to predict high-grade cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) between screening rounds. Between January 2005 and April 2008, 692 study women with screening-detected ASCUS/LSIL cytology 6 to 12 months earlier returned for HPV mRNA and DNA testing and repeat cytology. The median follow-up time was 3 years, using existing health care facilities. Follow-up test results were available for 625 women. Of the 145 CIN2+ cases detected during the study period, 95 (65.5%) were HPV mRNA positive 6 to 12 months after screening-detected ASCUS/LSIL, 44 (30.4%) were HPV mRNA negative, and 6 (4.1%) were invalid. The corresponding HPV DNA results were 139 (95.9%), 5 (3.4%), and 1 (0.7%), respectively. The cumulative incidences of CIN2+ 3 years after a negative HPV mRNA and DNA test were 10.3% (95% confidence interval [CI], 7.2 to 13.3%) and 1.8% (95% CI, 0.0 to 3.6%), respectively. The cumulative incidences of CIN2+ 3 years after positive HPV mRNA and DNA tests were 52.8% (95% CI, 40.1 to 60.1%) and 41.3% (95% CI, 35.5 to 46.6%), respectively. In conclusion, both positive HPV mRNA and DNA test results have a high enough long-term prediction of CIN2+ risk to consider referral to colposcopy as good practice when performed in delayed triage of women with ASCUS/LSIL cytology. In addition, the low CIN2+ risk among women with a negative Amplicor HPV test in our study confirms its safe use in a clinical setting.

Cytology and Human Papillomavirus Testing 6 to 12 Months after ASCUS or LSIL Cytology in Organized Screening To Predict High-Grade Cervical Neoplasia between Screening Rounds

PubMed Central

Sjøborg, Katrine D.; Nygård, Mari; Røysland, Kjetil; Campbell, Suzanne; Alfsen, G. Cecilie; Jonassen, Christine M.

2012-01-01

We carried out a prospective study comparing the performance of human papillomavirus (HPV) E6/E7 mRNA (PreTect HPV-Proofer; NorChip, Klokkarstua, Norway) and DNA (Amplicor HPV test; Roche Diagnostics, Basel, Switzerland) triage testing of women 6 to 12 months after atypical-squamous-cells-of-undetermined-significance (ASCUS) or low-grade-squamous-intraepithelial-lesion (LSIL) cytology in organized screening to predict high-grade cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) between screening rounds. Between January 2005 and April 2008, 692 study women with screening-detected ASCUS/LSIL cytology 6 to 12 months earlier returned for HPV mRNA and DNA testing and repeat cytology. The median follow-up time was 3 years, using existing health care facilities. Follow-up test results were available for 625 women. Of the 145 CIN2+ cases detected during the study period, 95 (65.5%) were HPV mRNA positive 6 to 12 months after screening-detected ASCUS/LSIL, 44 (30.4%) were HPV mRNA negative, and 6 (4.1%) were invalid. The corresponding HPV DNA results were 139 (95.9%), 5 (3.4%), and 1 (0.7%), respectively. The cumulative incidences of CIN2+ 3 years after a negative HPV mRNA and DNA test were 10.3% (95% confidence interval [CI], 7.2 to 13.3%) and 1.8% (95% CI, 0.0 to 3.6%), respectively. The cumulative incidences of CIN2+ 3 years after positive HPV mRNA and DNA tests were 52.8% (95% CI, 40.1 to 60.1%) and 41.3% (95% CI, 35.5 to 46.6%), respectively. In conclusion, both positive HPV mRNA and DNA test results have a high enough long-term prediction of CIN2+ risk to consider referral to colposcopy as good practice when performed in delayed triage of women with ASCUS/LSIL cytology. In addition, the low CIN2+ risk among women with a negative Amplicor HPV test in our study confirms its safe use in a clinical setting. PMID:22518869

Unphosphorylated HSP27 (HSPB1) regulates the translation initiation process via a direct association with eIF4E in osteoblasts.

PubMed

Kuroyanagi, Gen; Tokuda, Haruhiko; Yamamoto, Naohiro; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Otsuka, Takanobu

2015-09-01

Heat-shock protein 27 (HSP27/HSPB1) and its phosphorylation are implicated in multiple physiological and pathophysiological cell functions. Our previous study reported that unphosphorylated HSP27 has an inhibitory role in triiodothyronine (T(3))‑induced osteocalcin (OC) synthesis in osteoblasts. However, the mechanisms behind the HSP27‑mediated effects on osteoblasts remain to be clarified. In the present study, to investigate the exact mechanism of HSP27 and its phosphorylation in osteoblasts, the molecular targets of HSP27 were explored using osteoblast‑like MC3T3‑E1 cells. The levels of OC mRNA induced by T(3) in the HSP27‑overexpressing cells did not show any significant differences compared with those in the control empty vector‑transfected cells. Therefore, the interactions between HSP27 and translational molecules were focused on, including eukaryotic translation initiation factor 4E (eIF4E), eIF4G and 4E‑binding protein 1 (4E‑BP1). The HSP27 protein in the unstimulated cells co‑immunoprecipitated with eIF4E, but not eIF4G or 4E‑BP1. In addition, the association of eIF4E with 4E‑BP1 was observed in the HSP27‑overexpressing cells, as well as in the control cells. Under T(3) stimulation, the binding of eIF4E to eIF4G was markedly attenuated in the HSP27‑overexpressing cells compared with the control cells. In addition, the binding of HSP27 to eIF4E in the unstimulated cells was diminished by the phosphorylation of HSP27. In response to T(3) stimulation, the association of eIF4E with eIF4G in the unphosphorylatable HSP27‑overexpressing cells was markedly reduced compared with the phospho‑mimic HSP27‑overexpressing cells. Taken together, these findings strongly suggest that unphosphorylated HSP27 associates with eIF4E in osteoblasts and suppresses the translation initiation process.

Overlapping and differential localization of Bmp-2, Bmp-4, Msx-2 and apoptosis in the endocardial cushion and adjacent tissues of the developing mouse heart.

PubMed

Abdelwahid, E; Rice, D; Pelliniemi, L J; Jokinen, E

2001-07-01

The bone morphogenetic proteins BMP-2 and BMP-4 and the homeobox gene MSX-2 are required for normal development of many embryonic tissues. To elucidate their possible roles during the remodeling of the tubular heart into a fully septated four-chambered heart, we have localized the mRNA of Bmp-2, Bmp-4, Msx-2 and apoptotic cells in the developing mouse heart from embryonic day (E)11 to E17. mRNA was localized by in situ hybridization, and apoptotic cells by TUNEL (TDT-mediated dUTP-biotin nick end-labeling) as well as by transmission electron microscopy. By analyzing adjacent serial sections, we demonstrated that the expression of Msx-2 and Bmp-2 strikingly overlapped in the atrioventricular canal myocardium, in the atrioventricular junctional myocardium, and in the maturing myocardium of the atrioventricular valves. Bmp-4 was expressed in the outflow tract myocardium and in the endocardial cushion of the outflow tract ridges from E12 to E14. Msx-2 appeared in the mesenchyme of the atrioventricular endocardial cushion from E11 to E14, while Bmp-2 and Bmp-4 were detected between E11 and E14. Apoptotic cells were also detected in the mesenchyme of the endocardial cushion between E12 and E14. Our results suggest that BMP-2 and MSX-2 are tightly linked to the formation of the atrioventricular junction and valves and that BMP-4 is involved in the development of the outflow tract myocardium and of the endocardial cushion. In addition, BMP-2, BMP-4 and MSX-2 and apoptosis seem to be associated with differentiation of the endocardial cushion.

Cytokine expression in response to root canal infection in gnotobiotic mice.

PubMed

Maciel, K F; Neves de Brito, L C; Tavares, W L F; Moreira, G; Nicoli, J R; Vieira, L Q; Ribeiro Sobrinho, A P

2012-04-01

To examine cytokine expression profiles during periapical lesion development in response to synergetic human pathogens in a gnotobiotic mouse model. Human strains of Fusobacterium nucleatum and Peptostreptococcus prevotii were inoculated into the root canals of germ-free mice in either mono- or bi-association. Animals were killed 7 and 14 days after infection, and periapical tissues were collected. mRNA expression of the cytokines IFN-γ, TNF-α, Receptor activator of nuclear factor kappa-B ligand (RANKL), IL-10, IL-4 and transforming growth factor β (TGF-β) was assessed using real-time PCR. Levene's test was used to assess the equality of variance of the data, whereas a t-test for independent samples was used to evaluate the significance of the differences between groups (P < 0.05). The mRNA expression of IFN-γ and TNF-α was up-regulated by F. nucleatum during the acute (day 7) and chronic phase (day 14) of periapical lesion development. However, in bi-infection the expression of IFN-γ and TNF-α were effectively absent at both time-points. RANKL mRNA expression was down-regulated during dual infection at the chronic phase. As IL-4 expression was similar at both time-points, IL-4 does not appear to be involved in the periapical response to these bacterial strains. IL-10 was up-regulated during the chronic phase by mono-infection with either F. nucleatum or P. prevotii. Dual infection increased TGF-β mRNA expression on day 7, which paralleled the decrease in IFN-γ and TNF-α mRNA levels at the same time-point. F. nucleatum increased TGF-β mRNA expression during the chronic phase. Cytokine profiles depend on the nature of the bacterial challenge. Both TGF-β and IL-10 appeared to be regulating the proinflammatory cytokine responses at both time-points of the periapical immune response. © 2012 International Endodontic Journal.

Picornavirus 2A protease regulates stress granule formation to facilitate viral translation

PubMed Central

Yang, Xiaodan; Hu, Zhulong; Fan, Shanshan; Zhang, Qiang; Zhong, Yi; Guo, Dong; Qin, Yali

2018-01-01

Stress granules (SGs) contain stalled messenger ribonucleoprotein complexes and are related to the regulation of mRNA translation. Picornavirus infection can interfere with the formation of SGs. However, the detailed molecular mechanisms and functions of picornavirus-mediated regulation of SG formation are not clear. Here, we found that the 2A protease of a picornavirus, EV71, induced atypical stress granule (aSG), but not typical stress granule (tSG), formation via cleavage of eIF4GI. Furthermore, 2A was required and sufficient to inhibit tSGs induced by EV71 infection, sodium arsenite, or heat shock. Infection of 2A protease activity-inactivated recombinant EV71 (EV71-2AC110S) failed to induce aSG formation and only induced tSG formation, which is PKR and eIF2α phosphorylation-dependent. By using a Renilla luciferase mRNA reporter system and RNA fluorescence in situ hybridization assay, we found that EV71-induced aSGs were beneficial to viral translation through sequestering only cellular mRNAs, but not viral mRNAs. In addition, we found that the 2A protease of other picornaviruses such as poliovirus and coxsackievirus also induced aSG formation and blocked tSG formation. Taken together, our results demonstrate that, on one hand, EV71 infection induces tSG formation via the PKR-eIF2α pathway, and on the other hand, 2A, but not 3C, blocks tSG formation. Instead, 2A induces aSG formation by cleaving eIF4GI to sequester cellular mRNA but release viral mRNA, thereby facilitating viral translation. PMID:29415027

Association between macrophage migration inhibitory factor in the endometrium and estrogen in endometriosis

PubMed Central

ZHANG, XIAO; MU, LIN

2015-01-01

Recent studies have shown that macrophage migration inhibitory factor (MIF) has a possible role in endometriosis-related pain and infertility, yet it has not been explored whether the mRNA level of MIF is altered in endometrial tissues from patients with endometriosis. The aim of the present study was to compare the expression of MIF in endometrial tissues from women with and without endometriosis, and to analyze the association between endometrial MIF expression and 17β-estradiol (E2). The protein and mRNA expression of MIF in the human endometrial tissue was assessed by western blotting and reverse transcription-polymerase chain reaction analysis, respectively. The MIF expression of women with endometriosis was found to be significantly higher than that of the controls. A positive correlation was noted between the serum E2 level and MIF expression. In endometrial cells from women with endometriosis, the level of E2-induced MIF upregulation was significantly higher than that in cells from women without endometriosis. In conclusion, this study demonstrated a significant increase in MIF expression in the endometrial tissues of women with endometriosis and an association between MIF expression and E2 level. MIF expression in endometrial cells from patients with endometriosis showed an increased sensitivity to stimulation by E2. PMID:26622394

Decreased expression of GATA4 in the diaphragm of nitrofen-induced congenital diaphragmatic hernia.

PubMed

Dingemann, Jens; Doi, Takashi; Gosemann, Jan-Hendrik; Ruttenstock, Elke Maria; Nakazawa, Nana; Puri, Prem

2013-04-01

The molecular mechanisms underlying the diaphragmatic defect in congenital diaphragmatic hernia (CDH) are still poorly understood. The transcription factor GATA4 is essential for normal development of the diaphragm. Recently, mutations in the GATA4 gene have been linked to human and rodent CDH. We hypothesized that diaphragmatic GATA4 expression is downregulated in the nitrofen CDH model. Pregnant rats received Nitrofen or vehicle on day 9 of gestation (D9). Fetuses were sacrificed on D13, D18, or D21. Pleuroperitoneal folds (n=20) and fetal diaphragms (n=40) were (micro) dissected and divided into CDH group and controls. RNA and protein were extracted. GATA4 mRNA levels were determined by real-time PCR. Protein levels were determined by ELISA and Immunohistochemistry. mRNA levels and Protein levels were significantly decreased in the CDH group compared to controls on D13 (mRNA 15.96±6.99 vs. 38.10±5.01, p<0.05), D18 (mRNA 10.45±1.84 vs. 17.68±2.11, Protein 2.59±0.06 vs. 4.58±0.35 p<0.05) and D21 (mRNA 4.31±0.83 vs. 6.87±0.88, Protein 0.16±0.08 vs. 1.26±0.49, p<0.05). Immunoreactivity of GATA4 was markedly decreased in CDH-diaphragms on D13, D18, and D21. We provide evidence for the first time that diaphragmatic expression of GATA4 is downregulated in the nitrofen model, suggesting that decreased expression of GATA4 may impair diaphragmatic development in nitrofen-induced CDH. © 2013 Wiley Periodicals, Inc.

Effects of fenbendazole on the murine humoral immune system.

PubMed

Landin, Ana Marie; Frasca, Daniela; Zaias, Julia; Van der Put, Elaine; Riley, Richard L; Altman, Norman H; Blomberg, Bonnie B

2009-05-01

Pinworms are highly contagious parasites that have been effectively treated in laboratory rodents with fenbendazole (FBZ). Whether FBZ has any detrimental side effects that may compromise experimental results is unknown. Here we asked whether the immune systems from young and aged mice are altered under FBZ treatment. We compared control and FBZ-treated groups of young (age, 2 to 4 mo) and old (age, 22 to 24 mo) BALB/cN mice. The treated mice received a total of 4 wk (alternating-week treatment regimen) of FBZ-medicated feed. Spleen and bone marrow were collected for immunologic assays, and heart, stomach, intestines, kidneys, and liver were evaluated by histopathology. Our results indicate that FBZ treatment has significant effects on the immune systems of mice; these effects are greater in aged mice. FBZ treatment adversely affected mRNA and protein expression of E2A (a transcription factor crucial for B lymphocytes) in activated precursor B lymphocytes obtained from the bone marrow of young and old mice. These effects were reversed by 6 wk on regular feed after the end of treatment. Activated B lymphocytes from the spleens of young and old mice showed decreased function (cell proliferation, E2A mRNA and protein expression) through the last time point of FBZ treatment but recovered by 2 to 4 wk after treatment. Our findings suggest that FBZ treatment may alter sensitive immune and molecular measures as presented here, and postponing the experimental use of mice until at least 6 wk after treatment should be considered.

Effects of Fenbendazole on the Murine Humoral Immune System

PubMed Central

Landin, Ana Marie; Frasca, Daniela; Zaias, Julia; Van der Put, Elaine; Riley, Richard L; Altman, Norman H; Blomberg, Bonnie B

2009-01-01

Pinworms are highly contagious parasites that have been effectively treated in laboratory rodents with fenbendazole (FBZ). Whether FBZ has any detrimental side effects that may compromise experimental results is unknown. Here we asked whether the immune systems from young and aged mice are altered under FBZ treatment. We compared control and FBZ-treated groups of young (age, 2 to 4 mo) and old (age, 22 to 24 mo) BALB/cN mice. The treated mice received a total of 4 wk (alternating-week treatment regimen) of FBZ-medicated feed. Spleen and bone marrow were collected for immunologic assays, and heart, stomach, intestines, kidneys, and liver were evaluated by histopathology. Our results indicate that FBZ treatment has significant effects on the immune systems of mice; these effects are greater in aged mice. FBZ treatment adversely affected mRNA and protein expression of E2A (a transcription factor crucial for B lymphocytes) in activated precursor B lymphocytes obtained from the bone marrow of young and old mice. These effects were reversed by 6 wk on regular feed after the end of treatment. Activated B lymphocytes from the spleens of young and old mice showed decreased function (cell proliferation, E2A mRNA and protein expression) through the last time point of FBZ treatment but recovered by 2 to 4 wk after treatment. Our findings suggest that FBZ treatment may alter sensitive immune and molecular measures as presented here, and postponing the experimental use of mice until at least 6 wk after treatment should be considered. PMID:19476712

Role of glucose utilization in the restoration of hypophysectomy-induced hepatic cytochrome P450 2E1 by growth hormone in rats.

PubMed

Son, M H; Kang, K W; Kim, E J; Ryu, J H; Cho, H; Kim, S H; Kim, W B; Kim, S G

2000-06-15

Growth hormone and insulin are the primary determinants for cytochrome P450 2E1 (CYP2E1) expression. The role of glucose on the induction of CYP2E1 by hypophysectomy and on the restorative effect by growth hormone was investigated in the rat liver. Western and Northern blot analyses revealed that hypophysectomy induced CYP2E1 by 5-fold at 1-4 weeks, relative to control, with a concomitant increase in CYP2E1 mRNA. Hypophysectomized rats (HXR) showed a 20% reduction in the plasma glucose level. Hypophysectomy-induced increase in the CYP2E1 mRNA was completely abolished by glucose feeding in drinking water (10%) for 7 days. Treatment of HXR with hGH (2 I.U./kg, twice a day, for 7 days) inhibited the increases in CYP2E1 protein and mRNA levels with restoration of the plasma glucose level. In contrast to the effect of human growth hormone (hGH) on CYP2E1 in HXR with free access to foods, CYP2E1 expression failed to be restored by hGH in starving HXR. However, glucose feeding of starving HXR abolished the induction of CYP2E1. Effects of hypophysectomy and hGH treatment were studied in streptozotocin-induced diabetic rats. Insulin, but not hGH, prevented an increase in CYP2E1 mRNA in diabetic rats. The hepatic CYP2E1 induction in hypophysectomized diabetic rats was inhibited by hGH treatment, indicating that the hGH effect on CYP2E1 expression did not involve insulin production. These results provide evidence that the induction of hepatic CYP2E1 by hypophysectomy may result from reduced glucose utilization, and that the effect of hGH on CYP2E1 expression may be mediated with enhanced glucose utilization, but not with insulin production.

Myeloid Cell Prostaglandin E2 Receptor EP4 Modulates Cytokine Production but Not Atherogenesis in a Mouse Model of Type 1 Diabetes.

PubMed

Vallerie, Sara N; Kramer, Farah; Barnhart, Shelley; Kanter, Jenny E; Breyer, Richard M; Andreasson, Katrin I; Bornfeldt, Karin E

2016-01-01

Type 1 diabetes mellitus (T1DM) is associated with cardiovascular complications induced by atherosclerosis. Prostaglandin E2 (PGE2) is often raised in states of inflammation, including diabetes, and regulates inflammatory processes. In myeloid cells, a key cell type in atherosclerosis, PGE2 acts predominately through its Prostaglandin E Receptor 4 (EP4; Ptger4) to modulate inflammation. The effect of PGE2-mediated EP4 signaling specifically in myeloid cells on atherosclerosis in the presence and absence of diabetes is unknown. Because diabetes promotes atherosclerosis through increased arterial myeloid cell accumulation, we generated a myeloid cell-targeted EP4-deficient mouse model (EP4M-/-) of T1DM-accelerated atherogenesis to investigate the relationship between myeloid cell EP4, inflammatory phenotypes of myeloid cells, and atherogenesis. Diabetic mice exhibited elevated plasma PGE metabolite levels and elevated Ptger4 mRNA in macrophages, as compared with non-diabetic littermates. PGE2 increased Il6, Il1b, Il23 and Ccr7 mRNA while reducing Tnfa mRNA through EP4 in isolated myeloid cells. Consistently, the stimulatory effect of diabetes on peritoneal macrophage Il6 was mediated by PGE2-EP4, while PGE2-EP4 suppressed the effect of diabetes on Tnfa in these cells. In addition, diabetes exerted effects independent of myeloid cell EP4, including a reduction in macrophage Ccr7 levels and increased early atherogenesis characterized by relative lesional macrophage accumulation. These studies suggest that this mouse model of T1DM is associated with increased myeloid cell PGE2-EP4 signaling, which is required for the stimulatory effect of diabetes on IL-6, markedly blunts the effect of diabetes on TNF-α and does not modulate diabetes-accelerated atherogenesis.

Myeloid Cell Prostaglandin E2 Receptor EP4 Modulates Cytokine Production but Not Atherogenesis in a Mouse Model of Type 1 Diabetes

PubMed Central

Vallerie, Sara N.; Kramer, Farah; Barnhart, Shelley; Kanter, Jenny E.; Breyer, Richard M.; Andreasson, Katrin I.; Bornfeldt, Karin E.

2016-01-01

Type 1 diabetes mellitus (T1DM) is associated with cardiovascular complications induced by atherosclerosis. Prostaglandin E2 (PGE2) is often raised in states of inflammation, including diabetes, and regulates inflammatory processes. In myeloid cells, a key cell type in atherosclerosis, PGE2 acts predominately through its Prostaglandin E Receptor 4 (EP4; Ptger4) to modulate inflammation. The effect of PGE2-mediated EP4 signaling specifically in myeloid cells on atherosclerosis in the presence and absence of diabetes is unknown. Because diabetes promotes atherosclerosis through increased arterial myeloid cell accumulation, we generated a myeloid cell-targeted EP4-deficient mouse model (EP4M-/-) of T1DM-accelerated atherogenesis to investigate the relationship between myeloid cell EP4, inflammatory phenotypes of myeloid cells, and atherogenesis. Diabetic mice exhibited elevated plasma PGE metabolite levels and elevated Ptger4 mRNA in macrophages, as compared with non-diabetic littermates. PGE2 increased Il6, Il1b, Il23 and Ccr7 mRNA while reducing Tnfa mRNA through EP4 in isolated myeloid cells. Consistently, the stimulatory effect of diabetes on peritoneal macrophage Il6 was mediated by PGE2-EP4, while PGE2-EP4 suppressed the effect of diabetes on Tnfa in these cells. In addition, diabetes exerted effects independent of myeloid cell EP4, including a reduction in macrophage Ccr7 levels and increased early atherogenesis characterized by relative lesional macrophage accumulation. These studies suggest that this mouse model of T1DM is associated with increased myeloid cell PGE2-EP4 signaling, which is required for the stimulatory effect of diabetes on IL-6, markedly blunts the effect of diabetes on TNF-α and does not modulate diabetes-accelerated atherogenesis. PMID:27351842

One-Week Exposure to a Free-Choice High-Fat High-Sugar Diet Does Not Interfere With the Lipopolysaccharide-Induced Acute Phase Response in the Hypothalamus of Male Rats.

PubMed

Belegri, Evita; Eggels, Leslie; la Fleur, Susanne E; Boelen, Anita

2018-01-01

Obesity has been associated with increased susceptibility to infection in humans and rodents. Obesity is also associated with low-grade hypothalamic inflammation that depends not only on body weight but also on diet. In the present study, we investigated if the bacterial endotoxin [lipopolysaccharide (LPS)]-induced acute phase response is aggravated in rats on a 1-week free-choice high-fat high-sugar (fcHFHS) diet and explained by diet-induced hypothalamic inflammation. Male Wistar rats were on an fcHFHS diet or chow for 1 week and afterwards intraperitoneally injected with LPS or saline. Hypothalamic inflammatory intermediates and plasma cytokines were measured after LPS. Both LPS and the fcHFHS diet altered hypothalamic Nfkbia mRNA and nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha (NFKBIA) protein levels, whereas Il1 β, Il6 , and Tnf α mRNA expression was solely induced upon LPS. We observed an interaction in hypothalamic Nfkbia and suppressor of cytokine signaling (SOCS) 3 mRNA upon LPS; both were higher in rats on a fcHFHS diet compared with chow animals. Despite this, plasma cytokine levels between fcHFHS diet-fed and chow-fed rats were similar after LPS administration. Consuming a fcHFHS diet but not LPS injections increased hypothalamic Atf4 (a cellular stress marker) mRNA expression, whereas Tlr4 mRNA was decreased only upon LPS. Our study does not support a role for diet-induced mild hypothalamic inflammation in the increased susceptibility to infection despite altered Nfkbia and Socs3 mRNA expression after the diet. Additional factors, related to increased fat mass, might be involved.

Chemical and structural characterization of a model Post-Termination Complex (PoTC) for the ribosome recycling reaction: Evidence for the release of the mRNA by RRF and EF-G

PubMed Central

Iwakura, Nobuhiro; Yokoyama, Takeshi; Quaglia, Fabio; Mitsuoka, Kaoru; Mio, Kazuhiro; Shigematsu, Hideki; Shirouzu, Mikako; Kaji, Akira; Kaji, Hideko

2017-01-01

A model Post-Termination Complex (PoTC) used for the discovery of Ribosome Recycling Factor (RRF) was purified and characterized by cryo-electron microscopic analysis and biochemical methods. We established that the model PoTC has mostly one tRNA, at the P/E or P/P position, together with one mRNA. The structural studies were supported by the biochemical measurement of bound tRNA and mRNA. Using this substrate, we establish that the release of tRNA, release of mRNA and splitting of ribosomal subunits occur during the recycling reaction. Order of these events is tRNA release first followed by mRNA release and splitting almost simultaneously. Moreover, we demonstrate that IF3 is not involved in any of the recycling reactions but simply prevents the re-association of split ribosomal subunits. Our finding demonstrates that the important function of RRF includes the release of mRNA, which is often missed by the use of a short ORF with the Shine-Dalgarno sequence near the termination site. PMID:28542628

Epidermal growth factor (EGF) receptor-ligand based molecular staging predicts prognosis in head and neck squamous cell carcinoma partly due to deregulated EGF- induced amphiregulin expression.

PubMed

Gao, Jian; Ulekleiv, Camilla H; Halstensen, Trond S

2016-09-26

Increased expression of epidermal growth factor receptor (EGFR) and its ligands is associated with poor prognosis and chemoresistance in many carcinoma types, but its role in head and neck squamous cell carcinoma (HNSCC) is unclear. Our aim was to clarify whether mRNA expression of EGFR-ligands was linked to prognosis and cisplatin resistance, and if so, which ligand was most important and how was the expression regulated. To examine the prognostic effect of EGFR-ligand expression, we analyzed tumorous mRNA expression in 399 HNSCC patients. The intracellular signaling pathways controlling epidermal growth factor (EGF)-induced amphiregulin (AREG) expression were examined in three oral squamous cell carcinoma (OSCC) cell lines. Effect of AREG on cisplatin resistance was examined by viability assays in four-, and by association in 11 OSCC cell lines. The patients were divided into five groups according to the median mRNA expression levels of four EGFR ligands, i.e. AREG, EGF, heparin-binding EGF-like growth factor (HBEGF) and beta-cellulin (BTC). The number of increased-expressed EGFR-ligands were progressively correlated to five-year survival, even in advanced TNM-stage IV patients, where five-year mortality increased from 26 % if tumor expressed none to one EGFR-ligand, to 45 % in three to four ligand expressing tumors. Thus, staging the tumor according to these EGFR-ligand mRNA expression pattern completely out performed TNM staging in predicting prognosis. Multivariate analysis identified AREG as the dominating predictor, and AREG was overexpressed in OSCC compared to tumors from other sites. Both EGF and HBEGF stimulation induced strong AREG increase in OSCC cell lines, which was partially mediated by the extracellular signal-regulated kinase 1/2 pathway, and negatively regulated by p38, c-Jun N-terminal kinase, and phosphoinositide-3 kinase. Although increased AREG mRNA expression predicted unfavorable prognosis in platinum treated HNSCC patients, AREG did not mediate cisplatin resistance in the OSCC cell lines. Increased tumorous mRNA expression of four EGFR ligands was progressively associated with poor prognosis in HNSCC. Thus, EGFR-ligands mRNA expression pattern may be a new prognostic biomarker. The tightly regulated EGF-induced AREG mRNA expression was partly lost in the OSCC cell lines and restoring its regulation may be a new target in cancer treatment. Not applicable as the clinical data of the 498 HNSCC patients and their mRNA expression profiles were collected from the open TCGA database: http://cancergenome.nih.gov/cancersselected/headandneck .

Selenium Deficiency Affects the mRNA Expression of Inflammatory Factors and Selenoprotein Genes in the Kidneys of Broiler Chicks.

PubMed

Zhang, Jiu-Li; Xu, Bo; Huang, Xiao-Dan; Gao, Yu-Hong; Chen, Yu; Shan, An-Shan

2016-05-01

The aim of this study was to investigate the influence of Se deficiency on the transcription of inflammatory factors and selenoprotein genes in the kidneys of broiler chicks. One hundred fifty 1-day-old broiler chicks were randomly assigned to two groups fed with either a low-Se diet (L group, 0.033 mg/kg Se) or an adequate Se diet (C group, 0.2 mg/kg Se). The levels of uric acid (UA) and creatinine (Cr) in the serum and the mRNA levels of 6 inflammatory factors and 25 selenoprotein genes in the kidneys were measured as the clinical signs of Se deficiency occurred at 20 days old. The results indicated that the contents of UA and Cr in the serum increased in L group (p < 0.05), and the mRNA levels of the inflammatory factors (NF-κB, iNOS, COX-2, and TNF-α) increased in L group (p < 0.05). Meanwhile, the mRNA levels of PTGEs and HO-1 were not changed. In addition, 25 selenoprotein transcripts displayed ubiquitous expression in the kidneys of the chicks. The mRNA levels of 14 selenoprotein genes (Dio1, Dio2, GPx3, Sepp1, SelH, SelI, SelK, Sepn1, SelO, SelW, Sep15, SelT, SelU, and SelS) decreased, and 9 selenoprotein genes (GPx1, GPx2, GPx4, SelPb, Txnrd1, Txnrd2, Txnrd3, SPS2, and SelM) increased in L group (p < 0.05), but the Dio3 and Sepx1 mRNA levels did not change. The results indicated that Se deficiency resulted in kidney dysfunction, activation of the NF-κB pathway, and a change in selenoprotein gene expression. The changes of inflammatory factor and selenoprotein gene expression levels were directly related to the abnormal renal functions induced by Se deficiency.

Expression of FSH receptor in the hamster ovary during perinatal development

PubMed Central

Chakraborty, Prabuddha; Roy, Shyamal K.

2014-01-01

FSH plays an important role in ovarian follicular development, and it functions via the G-protein coupled FSH receptor. The objectives of the present study were to determine if full-length FSHR mRNA and corresponding protein were expressed in fetal through postnatal hamster ovaries to explain the FSH-induced primordial follicle formation, and if FSH or estrogen (E) would affect the expression. A full-length and two alternately spliced FSHR transcripts were expressed from E14 through P20. The level of the full-length FSHR mRNA increased markedly through P7 before stabilizing at a lower level with the formation and activation of primordial follicles. A predicted 87kDa FSHR protein band was detected in fetal through P4 ovaries, but additional bands appeared as ovary developed. FSHR immunosignal was present in undifferentiated somatic cells and oocytes in early postnatal ovaries, but was granulosa cells specific after follicles formed. Both eCG and E significantly up-regulated full-length FSHR mRNA levels. Therefore, FSHR is expressed in the hamster ovary from the fetal life to account for FSH-induced primordial follicle formation and cAMP production. Further, FSH or E regulates the receptor expression. PMID:25462586

Activation of translation complex eIF4F is essential for the genesis and maintenance of the malignant phenotype in human mammary epithelial cells.

PubMed

Avdulov, Svetlana; Li, Shunan; Michalek, Van; Burrichter, David; Peterson, Mark; Perlman, David M; Manivel, J Carlos; Sonenberg, Nahum; Yee, Douglas; Bitterman, Peter B; Polunovsky, Vitaly A

2004-06-01

Common human malignancies acquire derangements of the translation initiation complex, eIF4F, but their functional significance is unknown. Hypophosphorylated 4E-BP proteins negatively regulate eIF4F assembly by sequestering its mRNA cap binding component eIF4E, whereas hyperphosphorylation abrogates this function. We found that breast carcinoma cells harbor increases in the eIF4F constituent eIF4GI and hyperphosphorylation of 4E-BP1 which are two alterations that activate eIF4F assembly. Ectopic expression of eIF4E in human mammary epithelial cells enabled clonal expansion and anchorage-independent growth. Transfer of 4E-BP1 phosphorylation site mutants into breast carcinoma cells suppressed their tumorigenicity, whereas loss of these 4E-BP1 phosphorylation site mutants accompanied spontaneous reversion to a malignant phenotype. Thus, eIF4F activation is an essential component of the malignant phenotype in breast carcinoma.

A prospective study of women with ASCUS or LSIL pap smears at baseline and HPV E6/E7 mRNA positive: a 3-year follow-up.

PubMed

Bruno, M T; Ferrara, M; Fava, V; Barrasso, G; Panella, M M

2018-04-01

Human papillomavirus (HPV) testing is used in the triage of women with a borderline smear result. The efficiency of testing women with a low-grade squamous intraepithelial lesion (LSIL) and atypical squamous cells of undetermined significance (ASCUS) is less clear. For this reason we used a new HPV test that detects E6/E7 messenger RNA (mRNA), which might have a higher specificity. The objective of this prospective study was to assess whether HPV E6/E7 mRNA positivity in women with ASCUS and LSIL at baseline, is able to predict those women who have a high risk of developing a histological cervical intraepithelial neoplasia (CIN2) or worse lesion. We took into consideration the women's age and HPV DNA genotype and followed them up for 3 years. Cervical samples from women with high-risk HPV (HR-HPV) DNA-positive ASCUS (n = 90) or LSIL (n = 222) were tested for the presence of HR-HPV E6/E7 mRNA and the women were monitored for the development of histopathologically verified CIN2+. Thirteen patients with ASCUS and 17 with LSIL did not complete follow-up. All patients with LSIL and ASCUS, enrolled in this study, had confirmed lesions at the colposcopic examination. Follow-up was available for 312 women, 193 were positive in the HR-HPV DNA test and 93 had a HPV E6/E7 mRNA positive test. Finally, 22 women positive in the HPV DNA test for high-risk genotypes and with positive E6/E7 mRNA had a histologically confirmed CIN2+. Only two cases with negative HPV E6/E7 mRNA had CIN2+. The study shows that women positive in the HPV E6/E7 mRNA test have a greater risk of malignant progression of cervical lesions and therefore deserve greater attention and earlier check-ups.

A Transcript-Specific eIF3 Complex Mediates Global Translational Control of Energy Metabolism.

PubMed

Shah, Meera; Su, Dan; Scheliga, Judith S; Pluskal, Tomáš; Boronat, Susanna; Motamedchaboki, Khatereh; Campos, Alexandre Rosa; Qi, Feng; Hidalgo, Elena; Yanagida, Mitsuhiro; Wolf, Dieter A

2016-08-16

The multi-subunit eukaryotic translation initiation factor eIF3 is thought to assist in the recruitment of ribosomes to mRNA. The expression of eIF3 subunits is frequently disrupted in human cancers, but the specific roles of individual subunits in mRNA translation and cancer remain elusive. Using global transcriptomic, proteomic, and metabolomic profiling, we found a striking failure of Schizosaccharomyces pombe cells lacking eIF3e and eIF3d to synthesize components of the mitochondrial electron transport chain, leading to a defect in respiration, endogenous oxidative stress, and premature aging. Energy balance was maintained, however, by a switch to glycolysis with increased glucose uptake, upregulation of glycolytic enzymes, and strict dependence on a fermentable carbon source. This metabolic regulatory function appears to be conserved in human cells where eIF3e binds metabolic mRNAs and promotes their translation. Thus, via its eIF3d-eIF3e module, eIF3 orchestrates an mRNA-specific translational mechanism controlling energy metabolism that may be disrupted in cancer. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Fibroblast growth factor 10 upregulates the expression of mucins in rat conjunctival epithelial cells

PubMed Central

Ma, Mingming; Zhang, Zhengwei; Niu, Weiran; Zheng, Wenjing; Kelimu, Jiang

2011-01-01

Purpose This in vitro study aimed to gain insight into the function of fibroblast growth factor 10 (FGF10) on the ocular surface, especially its effect on mRNA expression of the mucins Muc1, Muc4, and Muc5ac, and mucin protein synthesis. Methods We isolated primary cultured rat conjunctival epithelial cells (Cj-ECs) and treated them with FGF10 (1 ng/ml, 10 ng/ml, 100 ng/ml, and 200 ng/ml) and basic fibroblast growth factor 2 (FGF2; 10 ng/ml) for 24 h or 48 h. The proliferation of Cj-ECs was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). mRNA levels of Muc1, Muc4, and Muc5ac were determined by real-time PCR. Synthesis levels of MUC1 and MUC4 were measured by western blot. Flow cytometry and Annexin V/PI double staining revealed degrees of apoptosis. Results In primary culture, the epithelial cells were compact and cobblestone pavement in shape. Most of the cells were positive for cytokeratin (CK). FGF10 and FGF2 significantly stimulated Muc1, Muc4, and Muc5ac mRNA expression, cell proliferation, and synthesis of MUC1 and MUC4 proteins. FGF10 was more potent than FGF2 in these regards. FGF10 did not restrain the apoptosis of Cj-ECs. Conclusions The results of this study demonstrated that FGF10 is associated with the promotion of Cj-EC proliferation and mucin production. The effects of FGF10 on Cj-ECs support a rationale to investigate its therapeutic potential for ocular surface diseases. PMID:22065934

Identification of Smoking-Associated Differentially Methylated Regions Using Reduced Representation Bisulfite Sequencing and Cell type-Specific Enhancer Activation and Gene Expression.

PubMed

Wan, Ma; Bennett, Brian D; Pittman, Gary S; Campbell, Michelle R; Reynolds, Lindsay M; Porter, Devin K; Crowl, Christopher L; Wang, Xuting; Su, Dan; Englert, Neal A; Thompson, Isabel J; Liu, Yongmei; Bell, Douglas A

2018-04-27

Cigarette smoke is a causal factor in cancers and cardiovascular disease. Smoking-associated differentially methylated regions (SM-DMRs) have been observed in disease studies, but the causal link between altered DNA methylation and transcriptional change is obscure. Our objectives were to finely resolve SM-DMRs and to interrogate the mechanistic link between SM-DMRs and altered transcription of enhancer noncoding RNA (eRNA) and mRNA in human circulating monocytes. We integrated SM-DMRs identified by reduced representation bisulfite sequencing (RRBS) of circulating CD14+ monocyte DNA collected from two independent human studies [ n =38 from Clinical Research Unit (CRU) and n =55 from the Multi-Ethnic Study of Atherosclerosis (MESA), about half of whom were active smokers] with gene expression for protein-coding genes and noncoding RNAs measured by RT-PCR or RNA sequencing. Candidate SM-DMRs were compared with RRBS of purified CD4+ T cells, CD8+ T cells, CD15+ granulocytes, CD19+ B cells, and CD56+ NK cells ( n =19 females, CRU). DMRs were validated using pyrosequencing or bisulfite amplicon sequencing in up to 85 CRU volunteers, who also provided saliva DNA. RRBS identified monocyte SM-DMRs frequently located in putative gene regulatory regions. The most significant monocyte DMR occurred at a poised enhancer in the aryl-hydrocarbon receptor repressor gene ( AHRR ) and it was also detected in both granulocytes and saliva DNA. To our knowledge, we identify for the first time that SM-DMRs in or near AHRR , C5orf55-EXOC-AS , and SASH1 were associated with increased noncoding eRNA as well as mRNA in monocytes. Functionally, the AHRR SM-DMR appeared to up-regulate AHRR mRNA through activating the AHRR enhancer, as suggested by increased eRNA in the monocytes, but not granulocytes, from smokers compared with nonsmokers. Our findings suggest that AHRR SM-DMR up-regulates AHRR mRNA in a monocyte-specific manner by activating the AHRR enhancer. Cell type-specific activation of enhancers at SM-DMRs may represent a mechanism driving smoking-related disease. https://doi.org/10.1289/EHP2395.

In vivo and in vitro studies of MUC1 regulation in sheep endometrium.

PubMed

Raheem, Kabir A; Marei, Waleed F A; Campbell, Bruce K; Fouladi-Nashta, Ali A

2016-06-01

In this study, we investigated the expression of mucin 1 (MUC1) mRNA and protein in sheep endometrium at different time points during follicular and luteal phases of estrous cycle, and also determined the effect of steroid hormone treatments and interferon tau (IFNτ) on MUC1 mRNA expression in endometrial cell culture in vitro. In experiment one, 15 Welsh mountain ewes were synchronized to a common estrus and killed at precise stages of estrous cycle corresponding to (1) pre-LH peak, (2) LH peak, (3) post-LH peak, (4) early luteal, and (5) mid-luteal. Reproductive tracts were harvested and mRNA was extracted from the endometrial tissues. Parts of the uterine horns were fixed for immunohistochemistry. In experiment two, mixed populations of ovine endometrial cells (from slaughterhouse material collected at the postovulatory stage of the estrous cycle) were cultured to 70% confluence before treatment with (1) progesterone (P4, 10 ng/mL, for 48 hours), (2) estradiol (E2, 100 pg/mL, for 48 hours), or with (3) E2 priming for 12 hours (100 pg/mL) followed by P4 (10 ng/mL) for 36 hours. These were compared with: (4) IFNτ (10 ng/mL, for 48 hours), and (5) basic medium (Dulbecco Modified Eagle Medium /F12) as control. The results showed that MUC1 mRNA and protein expression in sheep endometrium were highest during the midluteal stage and very low during the post-LH period compared with the other stages (P < 0.05). MUC1 immunostaining in the luminal epithelium was apically restricted and was not significantly different across all stages of estrous cycle except at the post-LH peak where it was significantly low. In cell culture, MUC1 mRNA expression was significantly upregulated by both steroids either singly or in combination (P < 0.05), and downregulated in the presence of IFNτ. In conclusion, endometrial MUC1 expression is cyclically regulated by both E2 and P4in vivo and in vitro, and directly downregulated by IFNτ treatment in vitro. Copyright © 2016 Elsevier Inc. All rights reserved.

LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing

PubMed Central

James, Victoria; Zhang, Yining; Foxler, Daniel E.; de Moor, Cornelia H.; Kong, Yi Wen; Webb, Thomas M.; Self, Tim J.; Feng, Yungfeng; Lagos, Dimitrios; Chu, Chia-Ying; Rana, Tariq M.; Morley, Simon J.; Longmore, Gregory D.; Bushell, Martin; Sharp, Tyson V.

2010-01-01

In recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5′ m7GTP cap–protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m7GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m7GTP-5′cap and Ago1/2 within the miRISC complex attached to the 3′-UTR of mRNA, creating an inhibitory closed-loop complex. PMID:20616046

Effects of added fermentable carbohydrates in the diet on intestinal proinflammatory cytokine-specific mRNA content in weaning piglets.

PubMed

Pié, S; Awati, A; Vida, S; Falluel, I; Williams, B A; Oswald, I P

2007-03-01

There is increasing evidence showing that dietary supplementation with prebiotics can be effective in the treatment of intestinal inflammation. Because weaning time is characterized by rapid intestinal inflammation, this study investigated the effect of a diet supplemented with a combination of 4 fermentable carbohydrates (lactulose, inulin, sugarbeet pulp, and wheat starch) on the mRNA content of proinflammatory cytokines in newly weaned piglets. Cytokines (IL-1beta, IL-6, IL-8, IL-12p40, IL-18, and tumor necrosis factor-alpha) were analyzed using a semiquantitative reverse-transcription PCR technique on d 1, 4, and 10 in the ileum and colon of piglets fed either a test diet (CHO) or a control diet. In addition to the diet, the effect of enforced fasting on cytokine mRNA content was also evaluated. No effect of fasting was observed on the pro-inflammatory cytokine mRNA content. Our results showed that the CHO diet induced an up-regulation of IL-6 mRNA content in the colon of piglets 4 d postweaning. This up-regulation was specific for the animals fed the CHO diet and was not observed in animals fed the control diet. An increase in IL-1beta mRNA content was also observed on d 4 postweaning in all of the piglets. Correlations between proinflammatory cytokines and the end-products of fermentation indicated that the regulation of cytokines may be linked with some of the fermentation end-products such as branched-chain fatty acids, which are in turn end-products of protein fermentation.

Endometrial expression of the insulin-like growth factor system during uterine involution in the postpartum dairy cow.

PubMed

Llewellyn, S; Fitzpatrick, R; Kenny, D A; Patton, J; Wathes, D C

2008-05-01

Rapid uterine involution in the postpartum period of dairy cows is important to achieve a short interval to conception. Expression patterns for members of the insulin-like growth factor (IGF) family were determined by in situ hybridisation at day 14+/-0.4 postpartum (n=12 cows) to investigate a potential role for IGFs in modulating uterine involution. Expression in each uterine tissue region was measured as optical density units and data were analysed according to region and horn. IGF-I mRNA was localized to the sub-epithelial stroma (SES) of inter-caruncular and caruncular endometrium. Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma (DES), the caruncular stroma and myometrium. IGFBP-2, IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue, and in the DES and caruncular stroma, with IGFBP-4 mRNA additionally expressed in myometrium. IGFBP-3 mRNA was only detectable in luminal epithelium. IGFBP-5 mRNA was found in myometrium, inter-caruncular and caruncular SES and caruncular stroma. These data support a role for IGF-I and IGF-II in the extensive tissue remodelling and repair which the postpartum uterus undergoes to return to its non-pregnant state. The differential expression of binding proteins between tissues (IGFBP-3 in epithelium, IGFBP-2, -4, -5 and -6 in stroma and IGFBP-4 and -5 in myometrium) suggest tight control of IGF activity within each compartment. Differential expression of many members of the IGF family between the significantly larger previously gravid horn and the previously non-gravid horn may relate to differences in their rate of tissue remodelling.

Endometrial expression of the insulin-like growth factor system during uterine involution in the postpartum dairy cow☆

PubMed Central

Llewellyn, S.; Fitzpatrick, R.; Kenny, D.A.; Patton, J.; Wathes, D.C.

2008-01-01

Rapid uterine involution in the postpartum period of dairy cows is important to achieve a short interval to conception. Expression patterns for members of the insulin-like growth factor (IGF) family were determined by in situ hybridisation at day 14 ± 0.4 postpartum (n = 12 cows) to investigate a potential role for IGFs in modulating uterine involution. Expression in each uterine tissue region was measured as optical density units and data were analysed according to region and horn. IGF-I mRNA was localized to the sub-epithelial stroma (SES) of inter-caruncular and caruncular endometrium. Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma (DES), the caruncular stroma and myometrium. IGFBP-2, IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue, and in the DES and caruncular stroma, with IGFBP-4 mRNA additionally expressed in myometrium. IGFBP-3 mRNA was only detectable in luminal epithelium. IGFBP-5 mRNA was found in myometrium, inter-caruncular and caruncular SES and caruncular stroma. These data support a role for IGF-I and IGF-II in the extensive tissue remodelling and repair which the postpartum uterus undergoes to return to its non-pregnant state. The differential expression of binding proteins between tissues (IGFBP-3 in epithelium, IGFBP-2, -4, -5 and -6 in stroma and IGFBP-4 and -5 in myometrium) suggest tight control of IGF activity within each compartment. Differential expression of many members of the IGF family between the significantly larger previously gravid horn and the previously non-gravid horn may relate to differences in their rate of tissue remodelling. PMID:18258405

Stromal-epithelial interactions modulate the effect of ovarian steroids on goat uterine epithelial cell interleukin-18 release.

PubMed

Qi, X F; Nan, Z C; Jin, Y P; Qu, Y Y; Zhao, X J; Wang, A H

2012-05-01

A primary role of epithelial-stromal interactions in mediating steroid hormone action in the uterus has been established. The present study was undertaken to determine the mode of ovarian steroid action in regulating IL-18 release by goat endometrial epithelial cells (EECs) in the presence and absence of endometrial stromal cells (ESCs). Primary and telomerase-immortalized goat EECs grown alone or cocultured with ESCs were treated with two ovarian steroids, 17β-estradiol (E(2)) and progesterone (P(4)). The IL-18 mRNA and protein expression in EECs were studied by reverse transcript (RT) PCR, ELISA, and Western blot assay. The E(2) and/or P(4) treatment of EECs led to a significant increase in both IL-18 mRNA and protein expression either in the primary or in the immortalized EECs compared with that in EECs without the steroid treatment. However, in the presence of ESCs, IL-18 expression by EECs treated with steroids was significantly decreased compared with cells untreated with E(2) and/or P(4). In addition, significantly high abundance of IL-18 mRNA and protein expression by primary and telomerase-immortalized goat EECs was observed in the presence of ESCs compared with those cells without ESCs. These findings suggest that steroids are important for the control of IL-18 expression in goat EECs. Underlying ESCs are needed to mediate the inhibitory effects of steroids on the IL-18 secretory activity of goat EECs in vitro. The IL-18 abundance expressed by goat EECs in vitro are enhanced by underlying ESCs without the treatment of E(2) and/or P(4). Copyright © 2012 Elsevier Inc. All rights reserved.

Chemokine receptors and cortical interneuron dysfunction in schizophrenia.

PubMed

Volk, David W; Chitrapu, Anjani; Edelson, Jessica R; Lewis, David A

2015-09-01

Alterations in inhibitory (GABA) neurons, including deficiencies in the GABA synthesizing enzyme GAD67, in the prefrontal cortex in schizophrenia are pronounced in the subpopulations of neurons that contain the calcium-binding protein parvalbumin or the neuropeptide somatostatin. The presence of similar illness-related deficits in the transcription factor Lhx6, which regulates prenatal development of parvalbumin and somatostatin neurons, suggests that cortical GABA neuron dysfunction may be related to disturbances in utero. Since the chemokine receptors CXCR4 and CXCR7 guide the migration of cortical parvalbumin and somatostatin neurons from their birthplace in the medial ganglionic eminence to their final destination in the neocortex, we sought to determine whether altered CXCR4 and/or CXCR7 mRNA levels were associated with disturbances in GABA-related markers in schizophrenia. Quantitative PCR was used to quantify CXCR4 and CXCR7 mRNA levels in the prefrontal cortex of 62 schizophrenia and 62 healthy comparison subjects that were previously characterized for markers of parvalbumin and somatostatin neurons and in antipsychotic-exposed monkeys. We found elevated mRNA levels for CXCR7 (+29%; p<.0001) and CXCR4 (+14%, p=.052) in schizophrenia subjects but not in antipsychotic-exposed monkeys. CXCR7 mRNA levels were inversely correlated with mRNA levels for GAD67, parvalbumin, somatostatin, and Lhx6 in schizophrenia but not in healthy subjects. These findings suggest that higher mRNA levels for CXCR7, and possibly CXCR4, may represent a compensatory mechanism to sustain the migration and correct positioning of cortical parvalbumin and somatostatin neurons in the face of other insults that disrupt the prenatal development of cortical GABA neurons in schizophrenia. Copyright © 2014 Elsevier B.V. All rights reserved.

BDE-99 (2,2',4,4',5-pentabromodiphenyl ether) triggers epithelial-mesenchymal transition in colorectal cancer cells via PI3K/Akt/Snail signaling pathway.

PubMed

Wang, Fei; Ruan, Xin-Jian; Zhang, Hong-Yan

2015-01-01

The gut is in direct contact with BDE-99 (2,2',4,4',5-pentabromodiphenyl ether), one of the most abundant PBDE congeners in the environment and in human tissues. The objective of the present study was to investigate the effects of BDE-99 on colorectal cancer (CRC) cells. The effects of BDE-99 on cell proliferation were measured by CCK-8 assay in the CRC cell line HCT-116. Wound healing and transwell migration/invasion assays were used to test the migration and invasion of CRC cells. Factors related to epithelial-to-mesenchymal transition (EMT) were measured by real-time PCR and Western blot analysis for mRNA and protein levels, respectively. BDE-99 was found to increase migration and invasion and trigger EMT in HCT-116 cells; EMT was characterized by cells acquiring mesenchymal spindle-like morphology and by increased expression of N-cadherin with a concomitant decrease in E-cadherin. BDE-99 treatment also increased the protein and mRNA levels of the transcription factor Snail, but not Slug, Twist, and ZEB1. Knockdown of Snail by siRNA significantly attenuated BDE-99-induced EMT in HCT-116 cells, suggesting that Snail plays a crucial role in BDE-99-induced EMT. The PI3K/Akt inhibitor LY294002 completely blocked BDE-99-induced Snail and invasion of HCT-116 cells. Our results revealed that BDE-99 can trigger the EMT of colon cancer cells via the PI3K/AKT/Snail signaling pathway. This study provides new insight into the tumorigenesis and metastasis of CRC stimulated by BDE-99 and possibly other PBDE congeners.

PD-1 (PDCD1) Promoter Methylation Is a Prognostic Factor in Patients With Diffuse Lower-Grade Gliomas Harboring Isocitrate Dehydrogenase (IDH) Mutations.

PubMed

Röver, Lea Kristin; Gevensleben, Heidrun; Dietrich, Jörn; Bootz, Friedrich; Landsberg, Jennifer; Goltz, Diane; Dietrich, Dimo

2018-02-01

Immune checkpoints are important targets for immunotherapies. However, knowledge on the epigenetic modification of immune checkpoint genes is sparse. In the present study, we investigated promoter methylation of CTLA4, PD-L1, PD-L2, and PD-1 in diffuse lower-grade gliomas (LGG) harboring isocitrate dehydrogenase (IDH) mutations with regard to mRNA expression levels, clinicopathological parameters, previously established methylation subtypes, immune cell infiltrates, and survival in a cohort of 419 patients with IDH-mutated LGG provided by The Cancer Genome Atlas. PD-L1, PD-L2, and CTLA-4 mRNA expression levels showed a significant inverse correlation with promoter methylation (PD-L1: p=0.005; PD-L2: p<0.001; CTLA-4: p<0.001). Furthermore, immune checkpoint methylation was significantly associated with age (PD-L2: p=0.003; PD-1: p=0.015), molecular alterations, i.e. MGMT methylation (PD-L1: p<0.001; PD-L2: p<0.001), ATRX mutations (PD-L2: p<0.001, PD-1: p=0.001), and TERT mutations (PD-L1: p=0.035, PD-L2: p<0.001, PD-1: p<0.001, CTLA4: p<0.001) as well as methylation subgroups and immune cell infiltrates. In multivariate Cox proportional hazard analysis, PD-1 methylation qualified as strong prognostic factor (HR=0.51 [0.34-0.76], p=0.001). Our findings suggest an epigenetic regulation of immune checkpoint genes via DNA methylation in LGG. PD-1 methylation may assist the identification of patients that might benefit from an alternative treatment, particularly in the context of emerging immunotherapies. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

The Vitis vinifera C-repeat binding protein 4 (VvCBF4) transcriptional factor enhances freezing tolerance in wine grape

PubMed Central

Tillett, Richard L.; Wheatley, Matthew D.; Tattersall, Elizabeth A.R.; Schlauch, Karen A.; Cramer, Grant R.; Cushman, John C.

2014-01-01

Summary Chilling and freezing can reduce significantly vine survival and fruit set in Vitis vinifera wine grape. To overcome such production losses, a recently identified grapevine C-repeat binding factor (CBF) gene, VvCBF4, was overexpressed in grape vine cv. “Freedom” and found to improve freezing survival and reduced freezing-induced electrolyte leakage by up to 2°C in non-cold-acclimated vines. In addition, overexpression of this transgene caused a reduced growth phenotype similar to that observed for CBF overexpression in Arabidopsis and other species. Both freezing tolerance and reduced growth phenotypes were manifested in a transgene dose-dependent manner. To understand the mechanistic basis of VvCBF4 transgene action, one transgenic line (9–12) was genotyped using microarray-based mRNA expression profiling. Forty-seven and 12 genes were identified in unstressed transgenic shoots with either a greater than 1.5-fold increase or decrease in mRNA abundance, respectively. Comparison of mRNA changes with characterized CBF regulons in woody and herbaceous species revealed partial overlaps suggesting that CBF-mediated cold acclimation responses are widely conserved. Putative VvCBF4-regulon targets included genes with functions in cell wall structure, lipid metabolism, epicuticular wax formation, and stress-responses suggesting that the observed cold tolerance and dwarf phenotypes are the result of a complex network of diverse functional determinants. PMID:21914113

The HPV-16 E7 oncoprotein induces centriole multiplication through deregulation of Polo-like kinase 4 expression

PubMed Central

2011-01-01

Background Infection with high-risk human papillomaviruses (HPVs) such as HPV-16 is intimately associated with squamous cell carcinomas (SCCs) of the anogenital tract and a subset of oropharyngeal carcinomas. Such lesions, including pre-invasive precursors, frequently show multipolar mitoses and aneuploidy. The high-risk HPV-16-encoded E7 oncoprotein has been shown to rapidly induce centrosome abnormalities thereby causing the formation of supernumerary mitotic spindle poles and increasing the risk for chromosome missegregation. HPV-16 E7 has been found to rapidly induce centriole overduplication, in part, through the simultaneous formation of more than one daughter centriole at single maternal centrioles (centriole multiplication). The precise molecular mechanism that underlies HPV-16 E7-induced centriole multiplication, however, remains poorly understood. Findings Here, we show that human keratinocytes engineered to stably express the HPV-16 E7 oncoprotein exhibit aberrant Polo-like kinase 4 (PLK4) protein expression at maternal centrioles. Real-time quantitative reverse transcriptase (qRT-PCR) analysis of these cells revealed an increase of PLK4 mRNA levels compared to control cells. Importantly, the ability of the HPV-16 E7 oncoprotein to induce centriole multiplication was found to correlate with its ability to activate the PLK4 promoter and to up-regulate PLK4 mRNA. Conclusions These results highlight the critical role of PLK4 transcriptional deregulation in centriole multiplication in HPV-16 E7-expressing cells. Our findings encourage further experiments to test transcriptional inhibitors or small molecules targeting PLK4 to prevent centriole abnormalities, mitotic infidelity and malignant progression in HPV-associated neoplasms and other tumors in which PLK4 regulation is disrupted. PMID:21609466

Complexities and sequence similarities of mRNA populations of cholinergic (NS20-Y) and adrenergic (N1E-115) murine neuroblastoma cell lines.

PubMed

Strauss, W L

1990-07-01

The clonal murine neuroblastoma cell lines NS20-Y and N1E-115 have been proposed as models for examining the commitment of neural crest cells to either the cholinergic or adrenergic phenotype, respectively. The validity of this model depends in part on the extent to which these two cell lines have diverged as a result of their transformed, rather than neuronal properties. In order to quantitate differences in gene expression between NS20-Y and N1E-115 cells, the mRNA complexity of each cell type was determined. An analysis of the kinetics of hybridization of NS20-Y cell mRNA with cDNA prepared from NS20-Y cell mRNA demonstrated the presence of approximately 11,700 mRNA species assuming an average length of 1900 nucleotides. A similar analysis using mRNA isolated from N1E-115 cells and cDNA prepared from N1E-115 cell mRNA demonstrated that the adrenergic cell line expressed approximately 11,600 mRNA species. The species of mRNA expressed by each cell line were resolved into high, intermediate, and low abundance populations. In order to determine whether mRNAs were expressed by the cholinergic, but not by the adrenergic cell line, NS20-Y cDNA was hybridized to an excess of N1E-115 cell mRNA. An analysis of the solution hybridization kinetics from this procedure demonstrated that the two cell lines do not differ significantly in the nucleotide complexity of their mRNA populations. The extensive similarity between the two mRNA populations suggests that only a small number of genes are expressed differentially between the two cell lines and supports their use as models for the differentiation of cholinergic and adrenergic neurons.

Genome-wide association study of aggressive behaviour in chicken.

PubMed

Li, Zhenhui; Zheng, Ming; Abdalla, Bahareldin Ali; Zhang, Zhe; Xu, Zhenqiang; Ye, Qiao; Xu, Haiping; Luo, Wei; Nie, Qinghua; Zhang, Xiquan

2016-08-03

In the poultry industry, aggressive behaviour is a large animal welfare issue all over the world. To date, little is known about the underlying genetics of the aggressive behaviour. Here, we performed a genome-wide association study (GWAS) to explore the genetic mechanism associated with aggressive behaviour in chickens. The GWAS results showed that a total of 33 SNPs were associated with aggressive behaviour traits (P < 4.6E-6). rs312463697 on chromosome 4 was significantly associated with aggression (P = 2.10905E-07), and it was in the intron region of the sortilin-related VPS10 domain containing receptor 2 (SORCS2) gene. In addition, biological function analysis of the nearest 26 genes around the significant SNPs was performed with Ingenuity Pathway Analysis. An interaction network contained 17 genes was obtained and SORCS2 was involved in this network, interacted with nerve growth factor (NGF), nerve growth factor receptor (NGFR), dopa decarboxylase (L-dopa) and dopamine. After knockdown of SORCS2, the mRNA levels of NGF, L-dopa and dopamine receptor genes DRD1, DRD2, DRD3 and DRD4 were significantly decreased (P < 0.05). In summary, our data indicated that SORCS2 might play an important role in chicken aggressive behaviour through the regulation of dopaminergic pathways and NGF.

mRNA stability in mammalian cells.

PubMed Central

Ross, J

1995-01-01

This review concerns how cytoplasmic mRNA half-lives are regulated and how mRNA decay rates influence gene expression. mRNA stability influences gene expression in virtually all organisms, from bacteria to mammals, and the abundance of a particular mRNA can fluctuate manyfold following a change in the mRNA half-life, without any change in transcription. The processes that regulate mRNA half-lives can, in turn, affect how cells grow, differentiate, and respond to their environment. Three major questions are addressed. Which sequences in mRNAs determine their half-lives? Which enzymes degrade mRNAs? Which (trans-acting) factors regulate mRNA stability, and how do they function? The following specific topics are discussed: techniques for measuring eukaryotic mRNA stability and for calculating decay constants, mRNA decay pathways, mRNases, proteins that bind to sequences shared among many mRNAs [like poly(A)- and AU-rich-binding proteins] and proteins that bind to specific mRNAs (like the c-myc coding-region determinant-binding protein), how environmental factors like hormones and growth factors affect mRNA stability, and how translation and mRNA stability are linked. Some perspectives and predictions for future research directions are summarized at the end. PMID:7565413

Oxaloacetate activates brain mitochondrial biogenesis, enhances the insulin pathway, reduces inflammation and stimulates neurogenesis.

PubMed

Wilkins, Heather M; Harris, Janna L; Carl, Steven M; E, Lezi; Lu, Jianghua; Eva Selfridge, J; Roy, Nairita; Hutfles, Lewis; Koppel, Scott; Morris, Jill; Burns, Jeffrey M; Michaelis, Mary L; Michaelis, Elias K; Brooks, William M; Swerdlow, Russell H

2014-12-15

Brain bioenergetic function declines in some neurodegenerative diseases, this may influence other pathologies and administering bioenergetic intermediates could have therapeutic value. To test how one intermediate, oxaloacetate (OAA) affects brain bioenergetics, insulin signaling, inflammation and neurogenesis, we administered intraperitoneal OAA, 1-2 g/kg once per day for 1-2 weeks, to C57Bl/6 mice. OAA altered levels, distributions or post-translational modifications of mRNA and proteins (proliferator-activated receptor-gamma coactivator 1α, PGC1 related co-activator, nuclear respiratory factor 1, transcription factor A of the mitochondria, cytochrome oxidase subunit 4 isoform 1, cAMP-response element binding, p38 MAPK and adenosine monophosphate-activated protein kinase) in ways that should promote mitochondrial biogenesis. OAA increased Akt, mammalian target of rapamycin and P70S6K phosphorylation. OAA lowered nuclear factor κB nucleus-to-cytoplasm ratios and CCL11 mRNA. Hippocampal vascular endothelial growth factor mRNA, doublecortin mRNA, doublecortin protein, doublecortin-positive neuron counts and neurite length increased in OAA-treated mice. (1)H-MRS showed OAA increased brain lactate, GABA and glutathione thereby demonstrating metabolic changes are detectable in vivo. In mice, OAA promotes brain mitochondrial biogenesis, activates the insulin signaling pathway, reduces neuroinflammation and activates hippocampal neurogenesis. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

HTLV-1 bZIP factor protein targets the Rb/E2F-1 pathway to promote proliferation and apoptosis of primary CD4+ T cells

PubMed Central

Kawatsuki, A; Yasunaga, J-i; Mitobe, Y; Green, PL; Matsuoka, M

2016-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that induces a fatal T-cell malignancy, adult T-cell leukemia (ATL). Among several regulatory/accessory genes in HTLV-1, HTLV-1 bZIP factor (HBZ) is the only viral gene constitutively expressed in infected cells. Our previous study showed that HBZ functions in two different molecular forms, HBZ protein and HBZ RNA. In this study, we show that HBZ protein targets retinoblastoma protein (Rb), which is a critical tumor suppressor in many types of cancers. HBZ protein interacts with the Rb/E2F-1 complex and activates the transcription of E2F-target genes associated with cell cycle progression and apoptosis. Mouse primary CD4+ T cells transduced with HBZ show accelerated G1/S transition and apoptosis, and importantly, T cells from HBZ transgenic (HBZ-Tg) mice also demonstrate enhanced cell proliferation and apoptosis. To evaluate the functions of HBZ protein alone in vivo, we generated a new transgenic mouse strain that expresses HBZ mRNA altered by silent mutations but encoding intact protein. In these mice, the numbers of effector/memory and Foxp3+ T cells were increased, and genes associated with proliferation and apoptosis were upregulated. This study shows that HBZ protein promotes cell proliferation and apoptosis in primary CD4+ T cells through activation of the Rb/E2F pathway, and that HBZ protein also confers onto CD4+ T-cell immunophenotype similar to those of ATL cells, suggesting that HBZ protein has important roles in dysregulation of CD4+ T cells infected with HTLV-1. PMID:26804169

MicroRNA-16 suppresses the activation of inflammatory macrophages in atherosclerosis by targeting PDCD4

PubMed Central

LIANG, XUE; XU, ZHAO; YUAN, MENG; ZHANG, YUE; ZHAO, BO; WANG, JUNQIAN; ZHANG, AIXUE; LI, GUANGPING

2016-01-01

Programmed cell death 4 (PDCD4) is involved in a number of bioprocesses, such as apoptosis and inflammation. However, its regulatory mechanisms in atherosclerosis remain unclear. In this study, we investigated the role and mechanisms of action of PDCD4 in high-fat diet-induced atherosclerosis in mice and in foam cells (characteristic pathological cells in atherosclerotic lesions) derived from ox-LDL-stimulated macrophages. MicroRNA (miR)-16 was predicted to bind PDCD4 by bioinformatics analysis. In the mice with atherosclerosis and in the foam cells, PDCD4 protein expression (but not the mRNA expression) was enhanced, while that of miR-16 was reduced. Transfection with miR-16 mimic decreased the activity of a luciferase reporter containing the 3′ untranslated region (3′UTR) of PDCD4 in the macrophage-derived foam cells. Conversely, treatment with miR-16 inhibitor enhanced the luciferase activity. However, by introducing mutations in the predicted binding site located in the 3′UTR of PDCD4, the miR-16 mimic and inhibitor were unable to alter the level of PDCD4, suggesting that miR-16 is a direct negative regulator of PDCD4 in atherosclerosis. Furthermore, transfection wtih miR-16 mimic and siRNA targeting PDCD4 suppressed the secretion and mRNA expression of pro-inflammatory factors, such as interleukin (IL)-6 and tumor necrosis factor-α (TNF-α), whereas it enhanced the secretion and mRNA expression of the anti-inflammatory factor, IL-10. Treatment with miR-16 inhibitor exerted the opposite effects. In addition, the phosphorylation of p38 and extracellular signal-regulated kinase (ERK), and nuclear factor-κB (NF-κB) expression were altered by miR-16. In conclusion, our data demonstrate that the targeting of PDCD4 by miR-16 may suppress the activation of inflammatory macrophages though mitogen-activated protein kinase (MAPK) and NF-κB signaling in atherosclerosis; thus, PDCD4 may prove to be a potential therapeutic target in the treatment of atherosclerosis. PMID:26936421

Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells.

PubMed

Hacker, David L; Bertschinger, Martin; Baldi, Lucia; Wurm, Florian M

2004-10-27

Human embryonic kidney 293 (HEK293) cells, a widely used host for large-scale transient expression of recombinant proteins, are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 EBNA (HEK293E) cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNA interference (RNAi). The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1alpha (EF-1alpha) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to twofold. E1A mRNA depletion also resulted in a twofold increase in transient expression of a recombinant IgG in both small- and large-scale suspension cultures when the IgG light and heavy chain genes were controlled by the EF-1alpha promoter. Finally, transient IgG expression was enhanced 2.5-fold when the anti-E1A shRNA was expressed from the same vector as the IgG light chain gene. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells, and they established that RNAi can be used to enhance recombinant protein expression in mammalian cells.

Deficiency of the protein-tyrosine phosphatase DEP-1/PTPRJ promotes matrix metalloproteinase-9 expression in meningioma cells.

PubMed

Petermann, Astrid; Stampnik, Yvonn; Cui, Yan; Morrison, Helen; Pachow, Doreen; Kliese, Nadine; Mawrin, Christian; Böhmer, Frank-D

2015-05-01

Brain-invasive growth of a subset of meningiomas is associated with less favorable prognosis. The molecular mechanisms causing invasiveness are only partially understood, however, the expression of matrix metalloproteinases (MMPs) has been identified as a contributing factor. We have previously found that loss of density enhanced phosphatase-1 (DEP-1, also designated PTPRJ), a transmembrane protein-tyrosine phosphatase, promotes meningioma cell motility and invasive growth in an orthotopic xenotransplantation model. We have now analyzed potential alterations of the expression of genes involved in motility control, caused by DEP-1 loss in meningioma cell lines. DEP-1 depleted cells exhibited increased expression of mRNA encoding MMP-9, and the growth factors EGF and FGF-2. The increase of MMP-9 expression in DEP-1 depleted cells was also readily detectable at the protein level by zymography. MMP-9 upregulation was sensitive to chemical inhibitors of growth factor signal transduction. Conversely, MMP-9 mRNA levels could be stimulated with growth factors (e.g. EGF) and inflammatory cytokines (e.g. TNFα). Increase of MMP-9 expression by DEP-1 depletion, or growth factor/cytokine stimulation qualitatively correlated with increased invasiveness in vitro scored as transmigration through matrigel-coated membranes. The studies suggest induction of MMP-9 expression promoted by DEP-1 deficiency, or potentially by growth factors and inflammatory cytokines, as a mechanism contributing to meningioma brain invasiveness.

Effects of fasting on growth hormone, growth hormone receptor, and insulin-like growth factor-I axis in seawater-acclimated tilapia, Oreochromis mossambicus.

PubMed

Fox, B K; Riley, L G; Hirano, T; Grau, E G

2006-09-15

Effects of fasting on the growth hormone (GH)--growth hormone receptor (GHR)-insulin-like growth factor-I (IGF-I) axis were characterized in seawater-acclimated tilapia (Oreochromis mossambicus). Fasting for 4 weeks resulted in significant reductions in body weight and specific growth rate. Plasma GH and pituitary GH mRNA levels were significantly elevated in fasted fish, whereas significant reductions were observed in plasma IGF-I and hepatic IGF-I mRNA levels. There was a significant negative correlation between plasma levels of GH and IGF-I in the fasted fish. No effect of fasting was observed on hepatic GHR mRNA levels. Plasma glucose levels were reduced significantly in fasted fish. The fact that fasting elicited increases in GH and decreases in IGF-I production without affecting GHR expression indicates a possible development of GH resistance.

HPV Involvement in Head and Neck Cancers: Comprehensive Assessment of Biomarkers in 3680 Patients.

PubMed

Castellsagué, Xavier; Alemany, Laia; Quer, Miquel; Halec, Gordana; Quirós, Beatriz; Tous, Sara; Clavero, Omar; Alòs, Llúcia; Biegner, Thorsten; Szafarowski, Tomasz; Alejo, Maria; Holzinger, Dana; Cadena, Enrique; Claros, Edith; Hall, Gillian; Laco, Jan; Poljak, Mario; Benevolo, Maria; Kasamatsu, Elena; Mehanna, Hisham; Ndiaye, Cathy; Guimerà, Núria; Lloveras, Belen; León, Xavier; Ruiz-Cabezas, Juan C; Alvarado-Cabrero, Isabel; Kang, Chang-Suk; Oh, Jin-Kyoung; Garcia-Rojo, Marcial; Iljazovic, Ermina; Ajayi, Oluseyi F; Duarte, Flora; Nessa, Ashrafun; Tinoco, Leopoldo; Duran-Padilla, Marco A; Pirog, Edyta C; Viarheichyk, Halina; Morales, Hesler; Costes, Valérie; Félix, Ana; Germar, Maria Julieta V; Mena, Marisa; Ruacan, Arzu; Jain, Asha; Mehrotra, Ravi; Goodman, Marc T; Lombardi, Luis Estuardo; Ferrera, Annabelle; Malami, Sani; Albanesi, Estela I; Dabed, Pablo; Molina, Carla; López-Revilla, Rubén; Mandys, Václav; González, Manuel E; Velasco, Julio; Bravo, Ignacio G; Quint, Wim; Pawlita, Michael; Muñoz, Nubia; de Sanjosé, Silvia; Xavier Bosch, F

2016-06-01

We conducted a large international study to estimate fractions of head and neck cancers (HNCs) attributable to human papillomavirus (HPV-AFs) using six HPV-related biomarkers of viral detection, transcription, and cellular transformation. Formalin-fixed, paraffin-embedded cancer tissues of the oral cavity (OC), pharynx, and larynx were collected from pathology archives in 29 countries. All samples were subject to histopathological evaluation, DNA quality control, and HPV-DNA detection. Samples containing HPV-DNA were further subject to HPV E6*I mRNA detection and to p16(INK4a), pRb, p53, and Cyclin D1 immunohistochemistry. Final estimates of HPV-AFs were based on HPV-DNA, HPV E6*I mRNA, and/or p16(INK4a) results. A total of 3680 samples yielded valid results: 1374 pharyngeal, 1264 OC, and 1042 laryngeal cancers. HPV-AF estimates based on positivity for HPV-DNA, and for either HPV E6*I mRNA or p16(INK4a), were 22.4%, 4.4%, and 3.5% for cancers of the oropharynx, OC, and larynx, respectively, and 18.5%, 3.0%, and 1.5% when requiring simultaneous positivity for all three markers. HPV16 was largely the most common type. Estimates of HPV-AF in the oropharynx were highest in South America, Central and Eastern Europe, and Northern Europe, and lowest in Southern Europe. Women showed higher HPV-AFs than men for cancers of the oropharynx in Europe and for the larynx in Central-South America. HPV contribution to HNCs is substantial but highly heterogeneous by cancer site, region, and sex. This study, the largest exploring HPV attribution in HNCs, confirms the important role of HPVs in oropharyngeal cancer and drastically downplays the previously reported involvement of HPVs in the other HNCs. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Estradiol stimulates glycogen synthesis whereas progesterone promotes glycogen catabolism in the uterus of the American mink (Neovison vison).

PubMed

Bowman, Kole; Rose, Jack

2017-01-01

Glycogen synthesis by mink uterine glandular and luminal epithelia (GE and LE) is stimulated by estradiol (E 2 ) during estrus. Subsequently, the glycogen deposits are mobilized to near completion to meet the energy requirements of pre-embryonic development and implantation by as yet undetermined mechanisms. We hypothesized that progesterone (P 4 ) was responsible for catabolism of uterine glycogen reserves as one of its actions to ensure reproductive success. Mink were treated with E 2 , P 4 or vehicle (controls) for 3 days and uteri collected 24 h (E 2 , P 4 and vehicle) and 96 h (E 2 ) later. To evaluate E 2 priming, mink were treated with E 2 for 3 days, then P 4 for an additional 3 days (E 2 →P 4 ) and uteri collected 24 h later. Percent glycogen content of uterine epithelia was greater at E 2 + 96 h (GE = 5.71 ± 0.55; LE = 11.54 ± 2.32) than E 2 +24 h (GE = 3.63 ± 0.71; LE = 2.82 ± 1.03), and both were higher than controls (GE = 0.27 ± 0.15; LE = 0.54 ± 0.30; P < 0.05). Treatment as E 2 →P 4 reduced glycogen content (GE = 0.61 ± 0.16; LE = 0.51 ± 0.13), to levels not different from controls, while concomitantly increasing catabolic enzyme (glycogen phosphorylase m and glucose-6-phosphatase) gene expression and amount of phospho-glycogen synthase protein (inactive) in uterine homogenates. Interestingly, E 2 →P 4 increased glycogen synthase 1 messenger RNA (mRNA) and hexokinase 1mRNA and protein. Our findings suggest to us that while E 2 promotes glycogen accumulation by the mink uterus during estrus and pregnancy, it is P 4 that induces uterine glycogen catabolism, releasing the glucose that is essential to support pre-embryonic survival and implantation. © 2016 Japanese Society of Animal Science.

Effects of different magnitudes of mechanical strain on Osteoblasts in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang Lin; Lin Zhu; Li Yongming

2006-05-26

In addition to systemic and local factors, mechanical strain plays a crucial role in bone remodeling during growth, development, and fracture healing, and especially in orthodontic tooth movement. Although many papers have been published on the effects of mechanical stress on osteoblasts or osteoblastic cells, little is known about the effects of different magnitudes of mechanical strain on such cells. In the present study, we investigated how different magnitudes of cyclic tensile strain affected osteoblasts. MC3T3-E1 osteoblastic cells were subjected to 0%, 6%, 12% or 18% elongation for 24 h using a Flexercell Strain Unit, and then the mRNA andmore » protein expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-{kappa}B ligand (RANKL) were examined. The results showed that cyclic tensile strain induced a magnitude-dependent increase (0%, 6%, 12%, and 18%) in OPG synthesis and a concomitant decrease in RANKL mRNA expression and sRANKL release from the osteoblasts. Furthermore, the induction of OPG mRNA expression by stretching was inhibited by indomethacin or genistein, and the stretch-induced reduction of RANKL mRNA was inhibited by PD098059. These results indicate that different magnitudes of cyclic tensile strain influence the biological behavior of osteoblasts, which profoundly affects bone remodeling.« less

Effects of Environmental Enrichment on Doublecortin and BDNF Expression along the Dorso-Ventral Axis of the Dentate Gyrus.

PubMed

Gualtieri, Fabio; Brégère, Catherine; Laws, Grace C; Armstrong, Elena A; Wylie, Nicholas J; Moxham, Theo T; Guzman, Raphael; Boswell, Timothy; Smulders, Tom V

2017-01-01

Adult hippocampal neurogenesis (AHN) in the dentate gyrus is known to respond to environmental enrichment, chronic stress, and many other factors. The function of AHN may vary across the septo-temporal axis of the hippocampus, as different subdivisions are responsible for different functions. The dorsal pole regulates cognitive-related behaviors, while the ventral pole mediates mood-related responses through the hypothalamic-pituitary-adrenal (HPA) axis. In this study, we investigate different methods of quantifying the effect of environmental enrichment on AHN in the dorsal and ventral parts of the dentate gyrus (dDG and vDG). To this purpose, 11-week-old female CD-1 mice were assigned for 8 days to one of two conditions: the Environmental Enrichment (E) group received (i) running wheels, (ii) larger cages, (iii) plastic tunnels, and (iv) bedding with male urine, while the Control (C) group received standard housing. Dorsal CA ( Cornu Ammonis ) and DG regions were larger in the E than the C animals. Distance run linearly predicted the volume of the dorsal hippocampus, as well as of the intermediate and ventral CA regions. In the dDG, the amount of Doublecortin (DCX) immunoreactivity was significantly higher in E than in C mice. Surprisingly, this pattern was the opposite in the vDG (C > E). Real-time PCR measurement of Dcx mRNA and DCX protein analysis using ELISA showed the same pattern. Brain Derived Neurotrophic Factor (BDNF) immunoreactivity and mRNA displayed no difference between E and C, suggesting that upregulation of DCX was not caused by changes in BDNF levels. BDNF levels were higher in vDG than in dDG, as measured by both methods. Bdnf expression in vDG correlated positively with the distance run by individual E mice. The similarity in the patterns of immunoreactivity, mRNA and protein for differential DCX expression and for BDNF distribution suggests that the latter two methods might be effective tools for more rapid quantification of AHN.

Identification of differentially expressed genes in human breast cancer cells induced by 4-hydroxyltamoxifen and elucidation of their pathophysiological relevance and mechanisms

PubMed Central

Fang, Qi; Yao, Shuang; Luo, Guanghua; Zhang, Xiaoying

2018-01-01

While tamoxifen (TAM) is used for treating estrogen receptor (ER)a-positive breast cancer patients, its anti-breast cancer mechanisms are not completely elucidated. This study aimed to examine effects of 4-hydroxyltamoxifen (4-OH-TAM) on ER-positive (ER+) breast cancer MCF-7 cell growth and gene expression profiles. MCF-7 cell growth was inhibited by 4-OH-TAM dose-dependently with IC50 of 29 μM. 332 genes were up-regulated while 320 genes were down-regulated. The mRNA levels of up-regulated genes including STAT1, STAT2, EIF2AK2, TGM2, DDX58, PARP9, SASH1, RBL2 and USP18 as well as down-regulated genes including CCDN1, S100A9, S100A8, ANXA1 and PGR were confirmed by quantitative real-time PCR (qRT-PCR). In human breast tumor tissues, mRNA levels of EIF2Ak2, USP18, DDX58, RBL2, STAT2, PGR, S1000A9, and CCND1 were significantly higher in ER+- than in ER--breast cancer tissues. The mRNA levels of EIF2AK2, TGM2, USP18, DDX58, PARP9, STAT2, STAT1, PGR and CCND1 were all significantly higher in ER+-tumor tissues than in their corresponding tumor-adjacent tissues. These genes, except PGR and CCND1 which were down-regulated, were also up-regulated in ER+ MCF-7 cells by 4-OH-TAM. Total 14 genes mentioned above are involved in regulation of cell proliferation, apoptosis, cell cycles, and estrogen and interferon signal pathways. Bioinformatics analysis also revealed other novel and important regulatory factors that are associated with these genes and involved in the mentioned functional processes. This study has paved a foundation for elucidating TAM anti-breast cancer mechanisms in E2/ER-dependent and independent pathways. PMID:29416786

Identification of differentially expressed genes in human breast cancer cells induced by 4-hydroxyltamoxifen and elucidation of their pathophysiological relevance and mechanisms.

PubMed

Fang, Qi; Yao, Shuang; Luo, Guanghua; Zhang, Xiaoying

2018-01-05

While tamoxifen (TAM) is used for treating estrogen receptor (ER)a-positive breast cancer patients, its anti-breast cancer mechanisms are not completely elucidated. This study aimed to examine effects of 4-hydroxyltamoxifen (4-OH-TAM) on ER-positive (ER + ) breast cancer MCF-7 cell growth and gene expression profiles. MCF-7 cell growth was inhibited by 4-OH-TAM dose-dependently with IC 50 of 29 μM. 332 genes were up-regulated while 320 genes were down-regulated. The mRNA levels of up-regulated genes including STAT1, STAT2, EIF2AK2, TGM2, DDX58, PARP9, SASH1, RBL2 and USP18 as well as down-regulated genes including CCDN1, S100A9, S100A8, ANXA1 and PGR were confirmed by quantitative real-time PCR (qRT-PCR). In human breast tumor tissues, mRNA levels of EIF2Ak2, USP18, DDX58, RBL2, STAT2, PGR, S1000A9, and CCND1 were significantly higher in ER + - than in ER - -breast cancer tissues. The mRNA levels of EIF2AK2, TGM2, USP18, DDX58, PARP9, STAT2, STAT1, PGR and CCND1 were all significantly higher in ER + -tumor tissues than in their corresponding tumor-adjacent tissues. These genes, except PGR and CCND1 which were down-regulated, were also up-regulated in ER + MCF-7 cells by 4-OH-TAM. Total 14 genes mentioned above are involved in regulation of cell proliferation, apoptosis, cell cycles, and estrogen and interferon signal pathways. Bioinformatics analysis also revealed other novel and important regulatory factors that are associated with these genes and involved in the mentioned functional processes. This study has paved a foundation for elucidating TAM anti-breast cancer mechanisms in E2/ER-dependent and independent pathways.

Estrogen Sensitivity of Target Genes and Expression of Nuclear Receptor Co-Regulators in Rat Prostate after Pre- and Postnatal Exposure to the Ultraviolet Filter 4-Methylbenzylidene Camphor

PubMed Central

Durrer, Stefan; Ehnes, Colin; Fuetsch, Michaela; Maerkel, Kirsten; Schlumpf, Margret; Lichtensteiger, Walter

2007-01-01

Background and objectives In previous studies, we found that the ultraviolet filter 4-methyl-benzylidene camphor (4-MBC) exhibits estrogenic activity, is a preferential estrogen receptor (ER)-β ligand, and interferes with development of female reproductive organs and brain of both sexes in rats. Here, we report effects on male development. Methods 4-MBC (0.7, 7, 24, 47 mg/kg/day) was administered in chow to the parent generation before mating, during gestation and lactation, and to offspring until adulthood. mRNA was determined in prostate lobes by real-time reverse transcription–polymerase chain reaction and protein was determined by Western blot analysis. Results 4-MBC delayed male puberty, decreased adult prostate weight, and slightly increased testis weight. Androgen receptor (AR), insulin-like growth factor-1 (IGF-1), ER-α, and ER-β expression in prostate were altered at mRNA and protein levels, with stronger effects in dorsolateral than ventral prostate. To assess sensitivity of target genes to estrogens, offspring were castrated on postnatal day 70, injected with 17β-estradiol (E2; 10 or 50 μg/kg, sc) or vehicle on postnatal day 84, and sacrificed 6 hr later. Acute repression of AR and IGF-1 mRNAs by E2, studied in ventral prostate, was reduced by 4-MBC exposure. This was accompanied by reduced co-repressor N-CoR (nuclear receptor co-repressor) protein in ventral and dorsolateral prostate, whereas steroid receptor coactivator-1 (SRC-1) protein levels were unaffected. Conclusions Our data indicate that 4-MBC affects development of male reproductive functions and organs, with a lowest observed adverse effect level of 0.7 mg/kg. Nuclear receptor coregulators were revealed as targets for endocrine disruptors, as shown for N-CoR in prostate and SRC-1 in uterus. This may have widespread effects on gene regulation. PMID:18174949

Lambda-cyhalothrin disrupts the up-regulation effect of 17β-estradiol on post-synaptic density 95 protein expression via estrogen receptor α-dependent Akt pathway.

PubMed

Wang, Qunan; Xia, Xin; Deng, Xiaomei; Li, Nian; Wu, Daji; Zhang, Long; Yang, Chengwei; Tao, Fangbiao; Zhou, Jiangning

2016-03-01

Lambda-cyhalothrin (LCT), one of the type II pyrethroids, has been widely used throughout the world. The estrogenic effect of LCT to increase cell proliferation has been well established. However, whether the estrogenic effect of LCT will influence neurodevelopment has not been investigated. In addition, 17β-Estradiol (E2) plays a crucial role in neurodevelopment and induces an increase in synaptic proteins. The post-synaptic density 95 (PSD95) protein, which is involved in the development of the structure and function of new spines and localized with estrogen receptor α (ERα) at the post-synaptic density (PSD), was detected in our study by using hippocampal neuron cell line HT22. We found that LCT up-regulated PSD95 and ERα expression, estrogen receptor (ER) antagonist ICI182,780 and phosphatidylinositol-4; 5-bisphosphate 3-kinase (PI3K) inhibitor LY294,002 blocked this effect. In addition, LCT disrupted the promotion effect of E2 on PSD95. To investigate whether the observed changes are caused by ERα-dependent signaling activation, we next detected the effects of LCT on the ERα-mediated PI3K-Protein kinase B (PKB/Akt)-eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) pathway. There existed an activation of Akt and the downstream factor 4E-BP1 after LCT treatment. In addition, LCT could disrupt the activation effect of E2 on the Akt pathway. However, no changes in cAMP response element-binding protein (CREB) activation and PSD95 messenger ribonucleic acid (mRNA) were observed. Our findings demonstrated that LCT could increase the PSD95 protein level via the ERα-dependent Akt pathway, and LCT might disrupt the up-regulation effect of E2 on PSD95 protein expression via this signaling pathway. Copyright © 2015. Published by Elsevier B.V.

Arsenite and its metabolites, MMA(III) and DMA(III), modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice.

PubMed

Medina-Díaz, I M; Estrada-Muñiz, E; Reyes-Hernández, O D; Ramírez, P; Vega, L; Elizondo, G

2009-09-01

Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half the drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA(III) induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA(III) increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA(III) induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.

Arsenite and its metabolites, MMA{sup III} and DMA{sup III}, modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medina-Diaz, I.M.; Estrada-Muniz, E.; Reyes-Hernandez, O.D.

Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half themore » drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA{sup III} induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA{sup III} increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA{sup III} induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.« less