Codon-Resolution Analysis Reveals a Direct and Context-Dependent Impact of Individual Synonymous Mutations on mRNA Level

PubMed Central

Chen, Siyu; Li, Ke; Cao, Wenqing; Wang, Jia; Zhao, Tong; Huan, Qing; Yang, Yu-Fei; Wu, Shaohuan; Qian, Wenfeng

2017-01-01

Abstract Codon usage bias (CUB) refers to the observation that synonymous codons are not used equally frequently in a genome. CUB is stronger in more highly expressed genes, a phenomenon commonly explained by stronger natural selection on translational accuracy and/or efficiency among these genes. Nevertheless, this phenomenon could also occur if CUB regulates gene expression at the mRNA level, a hypothesis that has not been tested until recently. Here, we attempt to quantify the impact of synonymous mutations on mRNA level in yeast using 3,556 synonymous variants of a heterologous gene encoding green fluorescent protein (GFP) and 523 synonymous variants of an endogenous gene TDH3. We found that mRNA level was positively correlated with CUB among these synonymous variants, demonstrating a direct role of CUB in regulating transcript concentration, likely via regulating mRNA degradation rate, as our additional experiments suggested. More importantly, we quantified the effects of individual synonymous mutations on mRNA level and found them dependent on 1) CUB and 2) mRNA secondary structure, both in proximal sequence contexts. Our study reveals the pleiotropic effects of synonymous codon usage and provides an additional explanation for the well-known correlation between CUB and gene expression level. PMID:28961875

Mushroom β-Glucan May Immunomodulate the Tumor-Associated Macrophages in the Lewis Lung Carcinoma

PubMed Central

Wang, Wan-Jhen; Wu, Yu-Sheng; Chen, Sherwin; Liu, Chi-Feng

2015-01-01

The present study showed that oral mushroom beta-glucan treatment significantly increased IFN-γ mRNA expression but significantly reduced COX-2 mRNA expression within the lung. For LLC tumor model, oral Ganoderma lucidum or Antrodia camphorata polysaccharides treatments significantly reduced TGF-β production in serum. In addition, IL-12 and IFN-γ mRNA expression were significantly increased, but IL-6, IL-10, COX-2, and TGF-β mRNA expression were substantially following oral mushroom polysaccharides treatments. The study highlights the efficacious effect of mushroom polysaccharides for ameliorating the immune suppression in the tumor microenvironment. Increased M1 phenotype of tumor-associated macrophages and attenuated M2 phenotype of tumor-associated macrophages could be achieved by ingesting mushroom polysaccharides. PMID:26167490

mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

PubMed

Kropp, Jenna; Khatib, Hasan

2015-01-01

In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

Alkaline phosphatase in osteoblasts is down-regulated by pulsatile fluid flow

NASA Technical Reports Server (NTRS)

Hillsley, M. V.; Frangos, J. A.

1997-01-01

It is our hypothesis that interstitial fluid flow plays a role in the bone remodeling response to mechanical loading. The fluid flow-induced expression of three proteins (collagen, osteopontin, and alkaline phosphatase) involved in bone remodeling was investigated. Rat calvarial osteoblasts subjected to pulsatile fluid flow at an average shear stress of 5 dyne/cm2 showed decreased alkaline phosphatase (AP) mRNA expression after only 1 hour of flow. After 3 hours of flow, AP mRNA levels had decreased to 30% of stationary control levels and remained at this level for an additional 5 hours of flow. Steady flow (4 dyne/cm2 fluid shear stress), in contrast, resulted in a delayed and less dramatic decrease in AP mRNA expression to 63% of control levels after 8 hours of flow. The reduced AP mRNA expression under pulsatile flow conditions was followed by reduced AP enzyme activity after 24 hours. No changes in collagen or osteopontin mRNA expression were detected over 8 hours of pulsatile flow. This is the first time fluid flow has been shown to affect gene expression in osteoblasts.

Minoxidil upregulates the expression of vascular endothelial growth factor in human hair dermal papilla cells.

PubMed

Lachgar, S; Charveron, M; Gall, Y; Bonafe, J L

1998-03-01

The hair follicle dermal papilla which controls hair growth, is characterized in the anagen phase by a highly developed vascular network. We have demonstrated in a previous study that the expression of an angiogenic growth factor called vascular endothelial growth factor (VEGF) mRNA varied during the hair cycle. VEGF mRNA is strongly expressed in dermal papilla cells (DPC) in the anagen phase, but during the catagen and telogen phases. VEGF mRNA is less strongly expressed. This involvement of VEGF during the hair cycle allowed us to determine whether VEGF mRNA expression by DPC was regulated by minoxidil. In addition, the effect of minoxidil on VEGF protein synthesis in both cell extracts and DPC-conditioned medium, was investigated immunoenzymatically. Both VEGF mRNA and protein were significantly elevated in treated DPC compared with controls. DPC incubated with increasing minoxidil concentrations (0.2, 2, 6, 12 and 24 mumol/L) induced a dose-dependent expression of VEGF mRNA. Quantification of transcripts showed that DPC stimulated with 24 mumol/L minoxidil express six times more VEGF mRNA than controls. Similarly, VEGF protein production increases in cell extracts and conditioned media following minoxidil stimulation. These studies strongly support the likely involvement of minoxidil in the development of dermal papilla vascularization via a stimulation of VEGF expression, and support the hypothesis that minoxidil has a physiological role in maintaining a good vascularization of hair follicles in androgenetic alopecia.

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.

Regulation of LPS-induced tissue factor expression in human monocytic THP-1 cells by curcumin

USDA-ARS?s Scientific Manuscript database

Tissue factor (TF) is a transmembrane receptor, which initiates thrombotic episodes associated with various diseases. In addition to membrane-bound TF, we have discovered an alternatively spliced form of human TF mRNA. It was later confirmed that this form of TF mRNA expresses a soluble protein circ...

Stimulatory effect of fibroblast-derived prostaglandin E₂ on keratinocyte stratification in the skin equivalent.

PubMed

Arai, Koji Y; Fujioka, Atsuko; Okamura, Ryoko; Nishiyama, Toshio

2014-01-01

Epidermal-dermal interaction plays important roles in physiological events such as wound healing. In this study, we examined a double paracrine mechanism between keratinocytes and fibroblasts through interleukin-1 (IL-1) and an IL-1-induced inflammatory mediator prostaglandin E₂ (PGE₂) using the skin equivalent. The epidermal layer of the skin equivalent expressed high levels of IL-1α mRNA (IL1A mRNA) and relatively low levels of IL-1β mRNA (IL1B mRNA). IL1A mRNA was not detected in fibroblasts. Fibroblasts also expressed low but not negligible levels of IL1B mRNA only in the presence of keratinocytes. Expression of prostaglandin-endoperoxide synthase 2 mRNA (PTGS2 mRNA) and production of PGE₂ in three-dimensionally cultured fibroblasts were noticeably stimulated by co-culture with keratinocytes, whereas PTGS2 mRNA expression in the epidermal layer was very low. In addition, hydroxyprostaglandin dehydrogenase 15-(NAD) mRNA was highly expressed in keratinocytes but not in fibroblasts, and exogenous IL-1β stimulated PTGS2 mRNA expression in the dermal equivalent. The thickness of the epidermal layer and the number of MKI67-positive keratinocytes in the skin equivalent were decreased by treatment with indomethacin, and the decrease recovered when exogenous PGE₂ was added. These results indicate that keratinocytes stimulate their own proliferation through a double paracrine mechanism mediated by IL-1 and PGE₂. © 2014 by the Wound Healing Society.

hnRNP-Q1 represses nascent axon growth in cortical neurons by inhibiting Gap-43 mRNA translation

PubMed Central

Williams, Kathryn R.; McAninch, Damian S.; Stefanovic, Snezana; Xing, Lei; Allen, Megan; Li, Wenqi; Feng, Yue; Mihailescu, Mihaela Rita; Bassell, Gary J.

2016-01-01

Posttranscriptional regulation of gene expression by mRNA-binding proteins is critical for neuronal development and function. hnRNP-Q1 is an mRNA-binding protein that regulates mRNA processing events, including translational repression. hnRNP-Q1 is highly expressed in brain tissue, suggesting a function in regulating genes critical for neuronal development. In this study, we have identified Growth-associated protein 43 (Gap-43) mRNA as a novel target of hnRNP-Q1 and have demonstrated that hnRNP-Q1 represses Gap-43 mRNA translation and consequently GAP-43 function. GAP-43 is a neuronal protein that regulates actin dynamics in growth cones and facilitates axonal growth. Previous studies have identified factors that regulate Gap-43 mRNA stability and localization, but it remains unclear whether Gap-43 mRNA translation is also regulated. Our results reveal that hnRNP-Q1 knockdown increased nascent axon length, total neurite length, and neurite number in mouse embryonic cortical neurons and enhanced Neuro2a cell process extension; these phenotypes were rescued by GAP-43 knockdown. Additionally, we have identified a G-quadruplex structure in the 5′ untranslated region of Gap-43 mRNA that directly interacts with hnRNP-Q1 as a means to inhibit Gap-43 mRNA translation. Therefore hnRNP-Q1–mediated repression of Gap-43 mRNA translation provides an additional mechanism for regulating GAP-43 expression and function and may be critical for neuronal development. PMID:26658614

Expression of dopamine D2 receptor and choline acetyltransferase mRNA in the dopamine deafferented rat caudate-putamen.

PubMed

Brené, S; Lindefors, N; Herrera-Marschitz, M; Persson, H

1990-01-01

In situ hybridization was used to study dopamine D2 receptor (D2R) and choline acetyltransferase (ChAT) mRNA expression in neurons of the rat forebrain, both on control animals and after a unilateral 6-hydroxydopamine (6-OHDA) lesion of midbrain dopamine neurons. D2R mRNA expressing neurons were seen in regions which are known to be heavily innervated by midbrain dopamine fibers such as caudate-putamen, nucleus accumbens and olfactory tubercle. ChAT mRNA expressing neurons were seen in caudate-putamen, nucleus accumbens and septal regions including vertical limb of the diagonal band. In caudate-putamen, approximately 55% of the medium sized neurons, which is the predominating neuronal cell-size in this region, were specifically labeled with the D2R probe. In addition, approximately 95% of the large size neurons in caudate-putamen were specifically labeled with both the D2R and ChAT probes, suggesting that most cholinergic neurons in the caudate-putamen express D2R mRNA. After a unilateral lesion of midbrain dopamine neurons, no change in the level of either D2R or ChAT mRNA were seen in the large size intrinsic cholinergic neurons in caudate-putamen. Similarly, no evidence was obtained for altered levels of D2R mRNA in medium size neurons in medial caudate-putamen, or nucleus accumbens. However, an increase in the number of medium size neurons expressing D2R mRNA was observed in the lateral part of the dopamine deafferented caudate-putamen. Thus, it appears that midbrain dopamine deafferentation causes an increase in D2R mRNA expression in a subpopulation of medium size neurons in the lateral caudate-putamen.

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

Differential neonatal imprinting and regulation by estrogen of estrogen receptor subtypes alpha and beta and of the truncated estrogen receptor product (TERP-1) mRNA expression in the male rat pituitary.

PubMed

Tena-Sempere, M; Barreiro, M L; González, L C; Pinilla, L; Aguilar, E

2001-11-01

Two distinct nuclear estrogen receptors (ERs) have been identified, the classical one, renamed ERalpha, and the more recently cloned ERbeta. In a variety of tissues, gene expression of both receptor subtypes results in the generation of multiple transcripts encoding the full-length as well as several alternately spliced isoforms. In the rat pituitary, a truncated, tissue-specific variant of ERalpha, called TERP-1, has been identified and found able to modulate ERalpha and ERbeta activity. So far, its pattern of expression and hormonal regulation have been mostly studied in females. The present study was designed to analyze the pattern of expression of TERP-1 mRNA in the male rat pituitary at different stages of postnatal development, and to evaluate the impact of neonatal imprinting and estrogen treatment upon TERP-1 expression in the male pituitary. Assessment of TERP-1 mRNA levels by semi-quantitative RT-PCR, using a variant-specific primer pair, revealed that TERP-1 is also expressed in the male rat pituitary. Relative mRNA expression levels changed markedly during postnatal development, with moderate expression of the TERP-1 transcript at birth, barely detectable levels during the infantile-prepubertal period, and maximal values in adulthood. Expression of TERP-1 was sensitive to neonatal estrogen exposure, which resulted in a significant, persistent increase in mRNA levels from the infantile period until puberty. This phenomenon was not mimicked by neonatal blockade of endogenous GnRH. In addition, estrogen was able to acutely up-regulate pituitary TERP-1 mRNA expression levels in prepubertal (30-day-old) and adult (75-day-old) males. Interestingly, neonatal imprinting as well as acute estrogen treatment resulted in opposite effects on TERP-1 and full-length ERalpha and ERbeta transcripts, the latter being decreased under both conditions. In conclusion, our data indicate that TERP-1 mRNA is expressed in a developmentally regulated manner in the male rat pituitary, and is affected by neonatal estrogen imprinting and acute estrogen treatment. Regulation of TERP-1 expression by neonatal or acute estrogen treatment may thus represent an additional tuning mechanism for estrogen actions in the male rat pituitary. Copyright 2001 S. Karger AG, Basel

Shift from posttranscriptional to predominant transcriptional control of the expression of the GM-CSF gene during activation of human Jurkat cells.

PubMed

Razanajaona, D; Maroc, C; Lopez, M; Mannoni, P; Gabert, J

1992-05-01

The expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is differentially regulated in various cell types. We investigated the mechanisms controlling its expression in 12-O-tetradecanoylphorbol-13-acetate plus phytohemagglutinin-stimulated Jurkat cells, a human T-cell line. In unstimulated cells, GM-CSF mRNA was undetectable by Northern blot. Upon activation, it was detected from 3 h onward, with a progressive increase in the levels of the transcript up to 24 h of stimulation. Whereas cycloheximide treatment at the time of stimulation blocked mRNA induction, its addition at later times resulted in a marked increase in transcript levels. Run-on analysis showed that transcription of the GM-CSF gene was low to undetectable in unstimulated cells; stimulation led to transcriptional activation, which was weak at 6 h but had increased 16-fold at 24 h. In addition, the mRNA half-life decreased during activation, from 2.5 h at 6 h down to 45 min at 24 h. Cycloheximide treatment increased GM-CSF mRNA half-life (3- and 4-fold, respectively). Our results show: (a) both transcriptional and posttranscriptional signals regulate GM-CSF mRNA levels in activated Jurkat cells, (b) de novo protein synthesis is required for mRNA induction, whereas destabilizing labile proteins control the transcript stability, and (c) a shift from a posttranscriptional to a predominant transcriptional control of GM-CSF gene expression occurs during activation.

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.

Testosterone Regulates NUCB2 mRNA Expression in Male Mouse Hypothalamus and Pituitary Gland

PubMed Central

Seon, Sojeong; Jeon, Daun; Kim, Heejeong; Chung, Yiwa; Choi, Narae; Yang, Hyunwon

2017-01-01

ABSTRACT Nesfatin-1/NUCB2 is known to take part in the control of the appetite and energy metabolism. Recently, many reports have shown nesfatin-1/NUCB2 expression and function in various organs. We previously demonstrated that nesfatin-1/NUCB2 expression level is higher in the pituitary gland compared to other organs and its expression is regulated by 17β-estradiol and progesterone secreted from the ovary. However, currently no data exist on the expression of nesfatin-1/NUCB2 and its regulation mechanism in the pituitary of male mouse. Therefore, we examined whether nesfatin-1/NUCB2 is expressed in the male mouse pituitary and if its expression is regulated by testosterone. As a result of PCR and western blotting, we found that a large amount of nesfatin-1/NUCB2 was expressed in the pituitary and hypothalamus. The NUCB2 mRNA expression level in the pituitary was decreased after castration, but not in the hypothalamus. In addition, its mRNA expression level in the pituitary was increased after testosterone treatment in the castrated mice, whereas, the expression level in the hypothalamus was significantly decreased after the treatment with testosterone. The in vitro experiment to elucidate the direct effect of testosterone on NUCB2 mRNA expression showed that NUCB2 mRNA expression was significantly decreased with testosterone in cultured hypothalamus tissue, but increased with testosterone in cultured pituitary gland. The present study demonstrated that nesfatin-1/NUCB2 was highly expressed in the male mouse pituitary and was regulated by testosterone. This data suggests that reproductive-endocrine regulation through hypothalamus-pituitary-testis axis may contribute to NUCB2 mRNA expression in the mouse hypothalamus and pituitary gland. PMID:28484746

Cloning and mRNA expression of guanylin, uroguanylin, and guanylyl cyclase C in the Spinifex hopping mouse, Notomys alexis.

PubMed

Donald, John A; Bartolo, Ray C

2003-06-01

Guanylin and uroguanylin are peptides that activate guanylyl cyclase C (GC-C) receptors in the intestine and kidney, which causes an increase in the excretion of salt and water. The Spinifex hopping mouse, Notomys alexis, is a desert rodent that can survive for extended periods without free access to water and it was hypothesised that to conserve water, the expression of guanylin, uroguanylin, and GC-C would be down-regulated to reduce the excretion of water in urine and faeces. Accordingly, this study examined the expression of guanylin, uroguanylin, and GC-C mRNA in Notomys under normal (access to water) and water-deprived conditions. Initially, guanylin and uroguanylin cDNAs encoding the full open reading frame were cloned and sequenced. A PCR analysis showed guanylin and uroguanylin mRNA expression in the small intestine, caecum, proximal and distal colon, heart, and kidney. In addition, a partial GC-C cDNA was obtained and GC-C mRNA expression was demonstrated in the proximal and distal colon, but not the kidney. Subsequently, a semi-quantitative PCR method showed that water deprivation in Notomys caused a significant increase in guanylin and uroguanylin mRNA expression in the distal colon, and in guanylin and GC-C mRNA expression in the proximal colon. No significant difference in guanylin and uroguanylin mRNA expression was observed in the kidney. The results of this study indicate that there is, in fact, an up-regulation of the colonic guanylin system in Notomys after 7 days of water deprivation.

Correlation analysis of the mRNA and miRNA expression profiles in the nascent synthetic allotetraploid Raphanobrassica

PubMed Central

Ye, Bingyuan; Wang, Ruihua; Wang, Jianbo

2016-01-01

Raphanobrassica is an allopolyploid species derived from inter-generic hybridization that combines the R genome from R. sativus and the C genome from B. oleracea var. alboglabra. In the present study, we used a high-throughput sequencing method to identify the mRNA and miRNA profiles in Raphanobrassica and its parents. A total of 33,561 mRNAs and 283 miRNAs were detected, 9,209 mRNAs and 134 miRNAs were differentially expressed respectively, 7,633 mRNAs and 39 miRNAs showed ELD expression, 5,219 mRNAs and 57 miRNAs were non-additively expressed in Raphanobrassica. Remarkably, differentially expressed genes (DEGs) were up-regulated and maternal bias was detected in Raphanobrassica. In addition, a miRNA-mRNA interaction network was constructed based on reverse regulated miRNA-mRNAs, which included 75 miRNAs and 178 mRNAs, 31 miRNAs were non-additively expressed target by 13 miRNAs. The related target genes were significantly enriched in the GO term ‘metabolic processes’. Non-additive related target genes regulation is involved in a range of biological pathways, like providing a driving force for variation and adaption in this allopolyploid. The integrative analysis of mRNA and miRNA profiling provides more information to elucidate gene expression mechanism and may supply a comprehensive and corresponding method to study genetic and transcription variation of allopolyploid. PMID:27874043

[Effects of Losartan on expression of heme oxygenases in volume-overloaded rats with left-to-right shunt].

PubMed

Yuan, Li-Xing; Liu, Han-Min; Li, Mi; Gao, Ju; Zhou, Tong-Fu

2005-09-01

To study the expression of heme oxygenase-1 mRNA and pulmonary remodeling before and after surgical establishment of left-to-right shunt in volume-overloaded SD rats and rats with Losartan intervention. Left-to-right shunt volume-overloaded SD rat models were established by aortocaval shunt operation. Seven rats with shunt were placed on Losartan (Losartan group), 7 rats with but not given Losartan were included in the operation group, and 4 rats after sham operation served as controls. Pulmonary pressure and right ventricular pressure were measured during catheterization. The relative weights ventricles were determined after execution of the rats. Pulmonary vascular remodeling parameters, including percentage arterial wall thickness and percentage muscularized small arteries, were assessed by morphometry. Heme oxygenase-1 (HO-1) mRNA expression and heme oxygenase-2 (HO-2) mRNA expression were detected RT-PCR method. Pulmonary artery pressure and right ventricular relative weight decreased significantly in the rats of Losartan group; in addition, the percentage arterial wall thickness and percentage of muscularized small arteries in the Losartan group were reduced as compared with those in the operation group. The level 1 mRAN expression in rats with shunt was significantly higher than that in rats without shunt. The level mRNA expression in the Losartan group decreased remarkably as compared against that in the operation The level of HO-1 mRNA expression in lungs was significantly higher than that in ventricles. There statistically significant differences in HO-2 mRNA expression levels between the three rat groups. Losartan intervention can markedly reduce pulmonary pressure, inhibit vascular remodeling in volume-overloaded left-to-right shunt rats, and result in down-regulation of HO-1 mRNA expression.

Effect of age on the expression of Pex (Phex) in the mouse.

PubMed

Meyer, R A; Young, C G; Meyer, M H; Garges, P L; Price, D K

2000-04-01

Pex is a newly discovered gene (also called Phex) whose mutation is the cause of X-linked hypophosphatemia. Other members of this gene family encode endopeptidases that activate or inactivate endocrine and paracrine factors. Though embryonic bone expresses mRNA for the Pex gene at relatively high levels, we have found Pex expression to be widespread in adult organs and to be poorly expressed in adult bone. This led to the hypothesis that Pex mRNA expression changes with age. To test this, genetically normal mice of the B6C3H hybrid strain were studied at 0 (newborn), 2, 3, 10, and 72 weeks of age. Organs known to express Pex were collected, and RNA was extracted from them. Following reverse transcription, cDNA was amplified by the polymerase chain reaction with primers for Pex and G3PDH, a housekeeping gene. The amplimers were separated by electrophoresis, blotted onto nylon membranes, and hybridized with radioactively labeled internal oligonucleotide probes. The radioactivity was quantified, and the data were analyzed as the Pex/G3PDH ratio. The brain samples had high levels of Pex mRNA expression that rose slightly with age. Calvaria, kidney, and lung samples had the highest Pex mRNA expression at birth. In these organs Pex mRNA expression fell with age to undetectable or barely detectable levels. Thymus, heart, and skeletal muscle samples had low Pex mRNA expression at birth that did not change with age. Some organs showed a decline in G3PDH levels with age, but Pex expression decreased more, leading to a reduced Pex/G3PDH ratio. The widespread expression of mRNA for Pex suggests a role beyond that of phosphate homeostasis. The high level of expression in newborn animals suggests a role in growth and development. This seems to occur in addition to its role for the endocrine regulation of phosphate homeostasis by as yet unknown humoral agents that must occur throughout life. In summary, Pex mRNA expression is high in brain and bone at birth. Expression remains high in brain with age but falls with age in bone, kidney, and lung.

Changes in the responsiveness of hypothalamic PK2 and PKR1 gene expression to fasting in developing male rats.

PubMed

Iwasa, Takeshi; Matsuzaki, Toshiya; Tungalagsuvd, Altankhuu; Munkhzaya, Munkhsaikhan; Kawami, Takako; Yamasaki, Mikio; Murakami, Masahiro; Kato, Takeshi; Kuwahara, Akira; Yasui, Toshiyuki; Irahara, Minoru

2014-11-01

Prokineticin (PK2) and its receptors (PKRs) are expressed in several regions of the central nervous system, including the hypothalamus. It has been reported that PK2 inhibits food intake via PKR1 and that the hypothalamic PK2 mRNA levels of adult rodents were reduced by food deprivation. However, some hypothalamic factors do not exhibit sensitivity to undernutrition in the early neonatal period, but subsequently become sensitive to it during the neonatal to pre-pubertal period. In this study, we investigated the changes in the sensitivity of hypothalamic PK2 and PKR1 mRNA expression to fasting during the developmental period in male rats. Under the fed conditions, the rats' hypothalamic PK2 and/or PKR1 mRNA levels were higher on postnatal day (PND) 10 than on PND20 or PND30. In addition, the hypothalamic PK2 and/or PKR1 mRNA levels of the male rats were higher than those of the females at all examined ages (PND10, 20, and 30). Hypothalamic PK2 mRNA expression was decreased by 24h fasting at PND10 and 30, but not at PND20. In addition, hypothalamic PKR1 mRNA expression was decreased by 24h fasting at PND10, but not at PND20 or 30. These results indicate that both PK2 and PKR1 are sensitive to nutritional status in male rats and that this sensitivity has already been established by the early neonatal period. It can be speculated that the PK2 system might compensate for the immaturity of other appetite regulatory factors in the early neonatal period. Copyright © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

Neurotensin expression and release in human colon cancers.

PubMed Central

Evers, B M; Ishizuka, J; Chung, D H; Townsend, C M; Thompson, J C

1992-01-01

Neurotensin (NT), a distal gut peptide released by intraluminal fats, is trophic for normal small bowel and colonic mucosa. In addition, NT stimulates growth of certain colon cancers; the mechanism for this effect is not known. The purpose of this study was to determine whether human colon cancers (HCC) (1) express the mRNA for NT/neuromedin N (N), (2) produce NT peptide, and (3) express the mRNA for a functional NT receptor (NTR). RNA was extracted from four HCC cell lines in culture, nine HCC lines established in athymic nude mice, and from six HCC and adjacent normal mucosa from freshly resected operative specimens; the RNA was analyzed for NT/N mRNA by Northern hybridization with a complementary DNA probe. Neurotensin peptide content, NTR expression, and intracellular Ca++ ([Ca++]i) mobilization in response to NT were evaluated in three HCC cell lines (LoVo, HT29, HCT116). Neurotensin/N mRNA transcripts were identified in all four of the HCC cell lines and in one of nine HCC in nude mice. Neurotensin expression was found in two of six freshly resected HCC and in none of the six corresponding samples of normal mucosa. Neurotensin peptide was identified by RIA in LoVo, HT29, and HCT116. In addition, NTR mRNA was found in HT29 and HCT116. Neurotensin stimulated [Ca++]i mobilization in HCT116 (without serum) and in LoVo (with 0.25% serum). These findings demonstrate the presence of NT/N mRNA and NT peptide and the presence of a functional NTR in certain HCC. Neurotensin, a potent trophic factor for normal gut mucosa, may function as an autocrine growth factor in certain human colon cancers. Images FIG. 1. FIG. 4. PMID:1329682

Phenolic compounds increase the transcription of mouse intestinal maltase-glucoamylase and sucrase-isomaltase.

PubMed

Simsek, Meric; Quezada-Calvillo, Roberto; Nichols, Buford L; Hamaker, Bruce R

2017-05-24

Diverse natural phenolic compounds show inhibition activity of intestinal α-glucosidases, which may constitute the molecular basis for their ability to control systemic glycemia. Additionally, phenolics can modify mRNA expression for proteins involved in nutritional, metabolic or immune processes. To explore the possibility that phenolics can regulate the mRNA expression, enzymatic activity, and protein synthesis/processing of intestinal Maltase-Glucoamylase (MGAM) and Sucrase-Isomaltase (SI), small intestinal explants from Balb/c mice were cultured for 24 h in the presence or absence of gallic acid, caffeic acid, and (+)-catechin at 0.1, 0.5, and 1 mM. We measured the levels of MGAM and SI mRNA expression by qRT-PCR, maltase and sucrase activities by a standard colorimetric method and the molecular size distribution of MGAM and SI proteins by western blotting. mRNA expression for MGAM was induced by the three phenolic compounds at 0.1 mM. mRNA expression for SI was induced by caffeic and gallic acids, but not by (+)-catechin. Caffeic acid was the most effective inducer of mRNA expression of these enzymes. Total maltase and sucrase activities were not affected by treatment with phenolics. The proportion of high molecular size forms of MGAM was significantly increased by two of the three phenolic compounds, but little effect was observed on SI proteins. Thus, changes in the protein synthesis/processing, affecting the proportions of the different molecular forms of MGAM, may account for the lack of correlation between mRNA expression and enzymatic activity.

Transcriptome-Wide Identification of Preferentially Expressed Genes in the Hypothalamus and Pituitary Gland

PubMed Central

St-Amand, Jonny; Yoshioka, Mayumi; Tanaka, Keitaro; Nishida, Yuichiro

2012-01-01

To identify preferentially expressed genes in the central endocrine organs of the hypothalamus and pituitary gland, we generated transcriptome-wide mRNA profiles of the hypothalamus, pituitary gland, and parietal cortex in male mice (12–15 weeks old) using serial analysis of gene expression (SAGE). Total counts of SAGE tags for the hypothalamus, pituitary gland, and parietal cortex were 165824, 126688, and 161045 tags, respectively. This represented 59244, 45151, and 55131 distinct tags, respectively. Comparison of these mRNA profiles revealed that 22 mRNA species, including three potential novel transcripts, were preferentially expressed in the hypothalamus. In addition to well-known hypothalamic transcripts, such as hypocretin, several genes involved in hormone function, intracellular transduction, metabolism, protein transport, steroidogenesis, extracellular matrix, and brain disease were identified as preferentially expressed hypothalamic transcripts. In the pituitary gland, 106 mRNA species, including 60 potential novel transcripts, were preferentially expressed. In addition to well-known pituitary genes, such as growth hormone and thyroid stimulating hormone beta, a number of genes classified to function in transport, amino acid metabolism, intracellular transduction, cell adhesion, disulfide bond formation, stress response, transcription, protein synthesis, and turnover, cell differentiation, the cell cycle, and in the cytoskeleton and extracellular matrix were also preferentially expressed. In conclusion, the current study identified not only well-known hypothalamic and pituitary transcripts but also a number of new candidates likely to be involved in endocrine homeostatic systems regulated by the hypothalamus and pituitary gland. PMID:22649398

Erk-Creb pathway suppresses glutathione-S-transferase pi expression under basal and oxidative stress conditions in zebrafish embryos.

PubMed

Hrubik, Jelena; Glisic, Branka; Fa, Svetlana; Pogrmic-Majkic, Kristina; Andric, Nebojsa

2016-01-05

Transcriptional activation of phase II enzymes including glutathione-S-transferase pi class (Gst Pi) is important for redox regulation and defense from xenobiotics. The role of extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) in regulation of Gst Pi expression has been described using adult mammalian cells. Whether these signaling pathways contribute to Gst Pi expression during embryogenesis is unknown. Using zebrafish embryo model, we provide novel evidence that Erk signaling acts as a specific suppressor of gstp1-2 mRNA during early embryogenesis. Addition of Erk inhibitor U0126 enhanced gstp1-2 mRNA expression during transition from blastula to the segmentation stage and from pharyngula until the hatching stage. Basal Erk activity did not affect gstp1-2 expression in tert-butylhydroquinone-exposed embryos. Addition of phorbol 12-myristate 13-acetate increased Erk activity leading to suppression of gstp1-2 mRNA. Activation of cAMP/Creb pathway by forskolin prevented gstp1-2 expression, whereas U0126 suppressed Creb phosphorylation, thus setting up Creb as a proximal transmitter of Erk inhibitory effect. Collectively, these findings suggest that Erk-Creb pathway exerts suppressive effect on gstp1-2 mRNA in a narrow developmental window. This study also provides a novel link between Erk and gstp1-2 expression, setting apart a possible differential regulation of gstp1-2 in adult and embryonic cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Expression of peroxisome proliferator-activated receptor alpha messenger ribonucleic acid and protein in human and rat testis.

PubMed

Schultz, R; Yan, W; Toppari, J; Völkl, A; Gustafsson, J A; Pelto-Huikko, M

1999-07-01

Peroxisome proliferator-activated receptor a (PPARalpha), a member of the steroid hormone receptor superfamily, has been linked to lipid homeostasis and tumorigenesis in tissues with high expression of receptor protein. On the other hand, the role of PPARalpha in tissues with a lower expression is not well known. Here we demonstrate the localization of PPARalpha messenger RNA (mRNA) and protein in developing and adult rat testis. Additionally, we demonstrate the expression of PPARalpha protein in adult human testis. Our experiments with Northern analysis, in situ hybridization and immunocytochemistry reveal a complex distribution of PPARalpha in tubular and interstitial cells of both adult and developing rat testis. The overall expression is rather low but may be modified by exogenous or endogenous stimuli. An up-regulation of PPARalpha mRNA could be observed after stimulation with FSH. In the developing rat testis, a clear expression of PPARalpha mRNA was present from the first days after birth. Additionally, PPARalpha mRNA and protein increased toward adulthood. In adult human testis PPARalpha immunoreactivity (IR) was present in interstitial Leydig cells and tubular cells. In the seminiferous epithelium of adult human testis the expression of PPARalpha-IR could be seen in meiotic spermatocytes, spermatids and myoid peritubular cells. The findings of our study suggest that PPARalpha may be involved in the regulation of growth and differentiation of tubular and interstitial cells in rat and human testis.

Transcriptome-wide identification of preferentially expressed genes in the hypothalamus and pituitary gland.

PubMed

St-Amand, Jonny; Yoshioka, Mayumi; Tanaka, Keitaro; Nishida, Yuichiro

2011-01-01

To identify preferentially expressed genes in the central endocrine organs of the hypothalamus and pituitary gland, we generated transcriptome-wide mRNA profiles of the hypothalamus, pituitary gland, and parietal cortex in male mice (12-15 weeks old) using serial analysis of gene expression (SAGE). Total counts of SAGE tags for the hypothalamus, pituitary gland, and parietal cortex were 165824, 126688, and 161045 tags, respectively. This represented 59244, 45151, and 55131 distinct tags, respectively. Comparison of these mRNA profiles revealed that 22 mRNA species, including three potential novel transcripts, were preferentially expressed in the hypothalamus. In addition to well-known hypothalamic transcripts, such as hypocretin, several genes involved in hormone function, intracellular transduction, metabolism, protein transport, steroidogenesis, extracellular matrix, and brain disease were identified as preferentially expressed hypothalamic transcripts. In the pituitary gland, 106 mRNA species, including 60 potential novel transcripts, were preferentially expressed. In addition to well-known pituitary genes, such as growth hormone and thyroid stimulating hormone beta, a number of genes classified to function in transport, amino acid metabolism, intracellular transduction, cell adhesion, disulfide bond formation, stress response, transcription, protein synthesis, and turnover, cell differentiation, the cell cycle, and in the cytoskeleton and extracellular matrix were also preferentially expressed. In conclusion, the current study identified not only well-known hypothalamic and pituitary transcripts but also a number of new candidates likely to be involved in endocrine homeostatic systems regulated by the hypothalamus and pituitary gland.

Calcineurin regulates slow myosin, but not fast myosin or metabolic enzymes, during fast-to-slow transformation in rabbit skeletal muscle cell culture

PubMed Central

Meißner, Joachim D; Gros, Gerolf; Scheibe, Renate J; Scholz, Michael; Kubis, Hans-Peter

2001-01-01

The addition of cyclosporin A (500 ng ml−1) - an inhibitor of the Ca2+-calmodulin-regulated serine/threonine phosphatase calcineurin - to primary cultures of rabbit skeletal muscle cells had no influence on the expression of fast myosin heavy chain (MHC) isoforms MHCIIa and MHCIId at the level of protein and mRNA, but reduced the expression of slow MHCI mRNA. In addition, no influence of cyclosporin A on the expression of citrate synthase (CS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was found. The level of enzyme activity of CS was also not affected. When the Ca2+ ionophore A23187 (4 × 10−7m) was added to the medium, a partial fast-to-slow transformation occurred. The level of MHCI mRNA increased, and the level of MHCIId mRNA decreased. Cotreatment with cyclosporin A was able to prevent the upregulation of MHCI at the level of mRNA as well as protein, but did not reverse the decrease in MHCIId expression. The expression of MHCIIa was also not influenced by cyclosporin A. Cyclosporin A was not able to prevent the upregulation of CS mRNA under Ca2+ ionophore treatment and failed to reduce the increased enzyme activity of CS. The expression of GAPDH mRNA was reduced under Ca2+ ionophore treatment and was not altered under cotreatment with cyclosporin A. When the myotubes in the primary muscle culture were electrostimulated at 1 Hz for 15 min periods followed by pauses of 30 min, a partial fast-to-slow transformation was induced. Again, cotreatment with cyclosporin A prevented the upregulation of MHCI at the level of mRNA and protein without affecting MHCIId expression. The nuclear translocation of the calcineurin-regulated transcription factor nuclear factor of activated thymocytes (NFATc1) during treatment with Ca2+ ionophore, and the prevention of the translocation in the presence of cyclosporin A, were demonstrated immunocytochemically in the myotubes of the primary culture. The effects of cyclosporin A demonstrate the involvement of calcineurin-dependent signalling pathways in controlling the expression of MHCI, but not of MHCIIa, MHCIId, CS and GAPDH, during Ca2+ ionophore- and electrostimulation-induced fast-to-slow transformations. The data indicate a differential regulation of MHCI, of MHCII and of metabolism. Calcineurin alone is not sufficient to mediate the complete transformation. PMID:11351029

Expression of three gonadotropin subunits and gonadotropin receptor mRNA during male-to-female sex change in the cinnamon clownfish, Amphiprion melanopus.

PubMed

An, Kwang Wook; Lee, Jehee; Choi, Cheol Young

2010-08-01

To quantify the sex-change progression from male to female in the cinnamon clownfish, Amphiprion melanopus, we divided gonadal development into three stages (I, mature male; II, male at 90 days after removal of the female; and III, mature female), and the expression of GTH subunits and GTH receptors during each of these stages was investigated. The mRNA of the three GTH subunits and their receptors increased with progression from male to female. To understand the effect of gonadotropin-releasing hormone (GnRH) on this progression, we examined expression of genes encoding the GTH subunit mRNA in the pituitary and the GTH-receptor mRNA in the gonads in addition to investigating changes in plasma E(2) levels after GnRH analogue (GnRHa) injection. GnRHa treatment increased mRNA expression levels of these genes, as well as plasma E(2) levels, indicating that GnRH plays an important regulatory role in the brain-pituitary-gonad axis of immature cinnamon clownfish. Copyright (c) 2010 Elsevier Inc. All rights reserved.

mRNA expression of corticotropin-releasing factor and urocortin 1 after restraint and foot shock together with alprazolam administration.

PubMed

Cespedes, Isabel C; de Oliveira, Amanda R; da Silva, Joelcimar M; da Silva, André V; Sita, Luciane V; Bittencourt, Jackson C

2010-12-01

Corticotropin-releasing factor (CRF) is expressed in the paraventricular nucleus of the hypothalamus (PVN), and act centrally to provoke stress-like autonomic and behavioral responses. Urocortins 1-3 are additional ligands to the CRF receptors 1 and 2. Ucn 1 neurons are primarily concentrated in the Edinger-Westphal (EW) nucleus and also have been associated with stress responses. It is also known that UCN 1 respond in different ways depending on the stressor presented. Benzodiazepines can act via the CRF peptidergic system and chronic administration of alprazolam does not interfere with CRF mRNA expression in the PVN, but significantly increase Ucn 1 mRNA expression in the EW. The aim of our study was to investigate the relationship between different stressor stimuli, foot shock (FS) and restraint (R), and the mRNA expression of CRF and Ucn 1 in the PVN and EW using alprazolam (A). We employed fos activation and in situ hybridization. Restraint group presented increased fos-ir and CRF mRNA expression in the PVN compared to FS group. The stress responses of R group were prevented by A. In the EW, fos-ir was higher in the FS group than in the R group, whereas Ucn 1 mRNA expression was higher in the R group than in the FS group. Alprazolam significantly increased fos-ir and Ucn 1 mRNA expression in both groups. Our results show that PVN and EW respond in different ways to the same stressors. Furthermore, EW of stressed animals replies in a complementary way comparing to PVN with the use of Alprazolam. Copyright © 2010 Elsevier Inc. All rights reserved.

Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation

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

Trempe, J.P.; Carter, B.J.

1988-01-01

The authors studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p/sub 40/ promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p/sub 40/. When the rep gene was present in cis or in trans, cat expression from p/sub 40/ was decreased 3- to 10-fold, but there was a 2- to 10-fold increase in the level of p/sub 40/ mRNA. Conversely, cat expression increased and the p/submore » 40/ mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p/sub 40/ mRNA levels. They also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p/sub 5/ promoter in a fashion independent of rep.« less

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

LAST, a c-Myc-inducible long noncoding RNA, cooperates with CNBP to promote CCND1 mRNA stability in human cells

PubMed Central

Li, Jinming

2017-01-01

Cyclin D1 is a critical regulator of cell cycle progression and works at the G1 to S-phase transition. Here, we report the isolation and characterization of the novel c-Myc-regulated lncRNA LAST (LncRNA-Assisted Stabilization of Transcripts), which acts as a CCND1 mRNA stabilizer. Mechanistically, LAST was shown to cooperate with CNBP to bind to the 5′UTR of CCND1 mRNA to protect against possible nuclease targeting. In addition, data from CNBP RIP-seq and LAST RNA-seq showed that CCND1 mRNA might not be the only target of LAST and CNBP; three additional mRNAs were shown to be post-transcriptional targets of LAST and CNBP. In a xenograft model, depletion of LAST diminished and ectopic expression of LAST induced tumor formation, which are suggestive of its oncogenic function. We thus report a previously unknown lncRNA involved in the fine-tuned regulation of CCND1 mRNA stability, without which CCND1 exhibits, at most, partial expression. PMID:29199958

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

Genetic analyses of Per.C6 cell clones producing a therapeutic monoclonal antibody regarding productivity and long-term stability.

PubMed

Tsuruta, Lilian Rumi; Lopes Dos Santos, Mariana; Yeda, Fernanda Perez; Okamoto, Oswaldo Keith; Moro, Ana Maria

2016-12-01

Genetic characterization of protein-producing clones represents additional value to cell line development. In the present study, ten Per.C6 clones producing a Rebmab100 monoclonal antibody were selected using two cloning methods: six clones originated from limiting dilution cloning and four by the automated colony picker ClonePix FL. A stability program was performed for 50 generations, including 4 batches distributed along the timeframe to determine specific productivity (Qp) maintenance. Four stable clones (two from limiting dilution and two from ClonePix FL) were further evaluated. The relative mRNA expression levels of both heavy chain (HC) and light chain (LC) genes were verified at generations 0, 30-35, and 50-55 of the stability program. At generations 0 and 30-35, LC gene expression level was higher than HC gene, whereas at generation 50-55, the opposite prevailed. A high correlation was observed between Qp and HC or LC mRNA expression level for all clones at each generation analyzed along the continuous culture. The mRNA stability study was performed at steady-state culture. The LC gene displayed a higher half-life and lower decay constant than HC gene, accounting for the higher observed expression level of LC mRNA in comparison to HC mRNA. Clone R6 was highlighted due its high Qp, mRNA expression levels, and mRNA stability. Besides the benefits of applying genetic characterization for the selection of stable and high-producing clones, the present study shows for the first time the correlation between Qp and HC or LC expression levels and also mRNA stability in clones derived from human cell line Per.C6(®).

Decidual activin: its role in the apoptotic process and its regulation by prolactin.

PubMed

Tessier, Christian; Prigent-Tessier, Anne; Bao, Lei; Telleria, Carlos M; Ferguson-Gottschall, Susan; Gibori, Gil B; Gu, Yan; Bowen-Shauver, Jennifer M; Horseman, Nelson D; Gibori, Geula

2003-05-01

Successful pregnancy requires profound differentiation and reorganization of the uterine tissues including, as pregnancy progresses, extensive apoptosis of decidual tissue to accommodate the developing conceptus. We have previously shown a positive correlation between expression of activin A and apoptosis in the decidua and have also shown that expression of activin A occurs at the time when prolactin (PRL) receptors disappear from decidual cells. The goals of this study were to examine whether activin A plays a role in decidual apoptosis and whether expression of activin A in the decidua is regulated by PRL and placental lactogens. Studies were carried out using primary rat decidual cells, a decidual cell line (GG-AD), and PRL null mice. Treatment of decidual cells with activin A significantly increased DNA degradation, caspase 3 activity, and caspase 3 mRNA expression. However, this effect was observed only in the absence of endogenous activin production by these cells. Addition of follistatin to decidual cells that were producing activin A decreased both caspase 3 activity and mRNA expression. Similarly, addition of activin-blocking antibodies to cultures of GG-AD cells, which also produce activin A, caused a reduction in both DNA degradation and caspase 3 activity. PRL and placental lactogens caused an inhibition of activin A mRNA expression in primary decidual cells. Even more convincingly, decidua of PRL null mice expressed abundant activin A at a time when no expression of this hormone is detected in wild-type mice and treatment of PRL null mice with PRL caused a profound inhibition of activin A mRNA expression. In summary, our investigations into the role and regulation of decidual activin have revealed that activin A can induce cell death in the decidua and that its expression is under tight regulation by PRL and placental lactogens.

Promoter hypermethylation of the RECK gene is associated with its low expression and poor survival of esophageal squamous cell carcinoma

PubMed Central

Zhu, Jing; Ling, Yang; Xu, Yun; Lu, Mingzhu; Liu, Yongping; Zhang, Changsong

2017-01-01

The present study aimed to investigate the association between the methylation status of the reversion-inducing cysteine-rich protein with kazal motifs (RECK) gene and its mRNA expression levels in patients with esophageal squamous cell carcinoma (ESCC). The methylation status of RECK was analyzed by methylation-specific polymerase chain reaction (PCR), and RECK mRNA expression levels were analyzed by quantitative PCR, in 310 paired ESCC tissues. The mean RECK methylation index (MI) was 0.65 in ESCCs and 0.49 in non-tumor samples. There was a significant association between RECK methylation and the American Joint Committee on Cancer stage and lymph node metastasis in ESCC (P<0.0001; P=0.001). The mRNA expression level of RECK was lower in ESCC tissues (mean-∆Cq=−4.66) compared with non-tumor tissues (mean-∆Cq=−2.79), and decreased RECK mRNA expression levels were associated with lymph node metastasis in ESCC. In addition, RECK mRNA levels were decreased in ESCC patients with hypermethylation of the RECK gene (∆MI >0.16; mean-∆∆Cq=−2.85) compared with those with hypomethylation of the RECK gene (∆MI ≤0.16; mean-∆∆Ct=−0.83), and there was a significant difference in the mRNA expression levels of RECK between those with N0–1 and N2–3 lymph node metastasis (P<0.0001). A significant correlation was observed between RECK mRNA expression levels, the MI of RECK and poor postoperative survival (P=0.0003; P<0.0001). The results of the present study suggested that promoter hypermethylation may be an important factor for loss of RECK mRNA expression and may be an indicator of poor survival in ESCC. PMID:28454343

Inactivation of bone morphogenetic protein 2 may predict clinical outcome and poor overall survival for renal cell carcinoma through epigenetic pathways

PubMed Central

Mitsui, Yozo; Hirata, Hiroshi; Arichi, Naoko; Hiraki, Miho; Yasumoto, Hiroaki; Chang, Inik; Fukuhara, Shinichiro; Yamamura, Soichiro; Shahryari, Varahram; Deng, Guoren; Saini, Sharanjot; Majid, Shahana; Dahiya, Rajvir; Tanaka, Yuichiro; Shiina, Hiroaki

2015-01-01

We investigated whether impaired regulation of bone morphogenetic protein-2 (BMP-2) via epigenetic pathways is associated with renal cell carcinoma (RCC) pathogenesis. Expression and CpG methylation of the BMP-2 gene were analyzed using RCC cell lines, and 96 matched RCC and normal renal tissues. We also performed functional analysis using BMP-2 restored RCC cells. A significant association of BMP-2 mRNA expression was also found with advanced tumor stage and lymph node involvement, while lower BMP-2 mRNA expression was significantly associated with poor overall survival after radical nephrectomy. In RCC cells, BMP-2 restoration significantly inhibited cell proliferation, migration, invasion, and colony formation. In addition, BMP-2 overexpression induced p21WAF1/CIP1 and p27KIP1 expression, and cellular apoptosis in RCC cells. BMP-2 mRNA expression was significantly enhanced in RCC cells by 5-aza-2′-deoxycitidine treatment. The prevalence of BMP-2 promoter methylation was significantly greater and BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples. Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in tumors. Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be a useful molecular marker for designing improved diagnostic and therapeutic strategies for RCC. PMID:25797254

Topographical cone photopigment gene expression in deutan-type red-green color vision defects.

PubMed

Bollinger, Kathryn; Sjoberg, Stacy A; Neitz, Maureen; Neitz, Jay

2004-01-01

Eye donors were identified who had X-chromosome photopigment gene arrays like those of living deuteranomalous men; the arrays contained two genes encoding long-wavelength sensitive (L) pigments as well as genes to encode middle-wavelength sensitive (M) photopigment. Ultrasensitive methods failed to detect the presence of M photopigment mRNA in the retinas of these deutan donors. This provides direct evidence that deuteranomaly is caused by the complete absence of M pigment mRNA. Additionally, for those eyes with mRNA corresponding to two different L-type photopigments, the ratio of mRNA from the first vs. downstream L genes was analyzed across the retinal topography. Results show that the pattern of first relative to downstream L gene expression in the deuteranomalous retina is similar to the pattern of L vs. M gene expression found in normal retinas.

Co-ordinated expression of MMP-2 and its putative activator, MT1-MMP, in human placentation.

PubMed

Bjørn, S F; Hastrup, N; Lund, L R; Danø, K; Larsen, J F; Pyke, C

1997-08-01

The spatial expression of mRNA for matrix metalloproteinase 2 (MMP-2), its putative activator, the membrane-type 1 matrix metalloproteinase (MT1-MMP), and the MMP-2 substrate type IV collagen was investigated in human placentas of both normal and tubal ectopic pregnancies and in cyclic endometrium using in-situ hybridization. Cytokeratin staining applied to adjacent sections was used to identify epithelial and trophoblast cells. In both normal and tubal pregnancies MT1-MMP, MMP-2 and type IV collagen mRNA were highly expressed and co-localized in the extravillous cytotrophoblasts of anchoring villi, in cytotrophoblasts that had penatrated into the placental bed and in cytotrophoblastic cell islands. In addition, the decidual cells of normal pregnancies in some areas co-expressed MT1-MMP and MMP-2 mRNA, with moderate signals for both components. Fibroblast-like stromal cells in tubal pregnancies were positive for MMP-2 mRNA but generally negative for MT1-MMP mRNA. The consistent co-localization of MT1-MMP with MMP-2 and type IV collagen in the same subset of cytotrophoblasts strongly suggests that all three components co-operate in the tightly regulated fetal invasion process. The co-expression of MT1-MMP and MMP-2 mRNA in some of the decidual cells indicates that these cells are also actively involved in the placentation process.

Stimulation of GLUT-1 glucose transporter expression in response to hyperosmolarity.

PubMed

Hwang, D Y; Ismail-Beigi, F

2001-10-01

Glucose transporter isoform-1 (GLUT-1) expression is stimulated in response to stressful conditions. Here we examined the mechanisms mediating the enhanced expression of GLUT-1 by hyperosmolarity. GLUT-1 mRNA, GLUT-1 protein, and glucose transport increased after exposure of Clone 9 cells to 600 mosmol/l (produced by addition of mannitol). The stimulation of glucose transport was biphasic: in the early phase (0-6 h) a approximately 2.5-fold stimulation of glucose uptake was associated with no change in the content of GLUT-1 mRNA, GLUT-1 protein, or GLUT-1 in the plasma membrane, whereas the approximately 17-fold stimulation of glucose transport during the late phase (12-24 h) was associated with increases in both GLUT-1 mRNA (approximately 7.5-fold) and GLUT-1 protein content. Cell sorbitol increased after 3 h of exposure to hyperosmolarity. The increase in GLUT-1 mRNA content was associated with an increase in the half-life of the mRNA from 2 to 8 h. A 44-bp region in the proximal GLUT-1 promoter was necessary for basal activity and for the two- to threefold increases in expression by hyperosmolarity. It is concluded that the increase in GLUT-1 mRNA content is mediated by both enhanced transcription and stabilization of GLUT-1 mRNA and is associated with increases in GLUT-1 content and glucose transport activity.

Olive Leaf Extract Elevates Hepatic PPAR α mRNA Expression and Improves Serum Lipid Profiles in Ovariectomized Rats.

PubMed

Yoon, Leena; Liu, Ya-Nan; Park, Hyunjin; Kim, Hyun-Sook

2015-07-01

We hypothesized that olive leaf extract might alleviate dyslipidemia resulting from estrogen deficiency. Serum lipid profile and mRNA expression of the related genes in the liver and adipose tissue were analyzed after providing olive leaf extract (200 or 400 mg/kg body weight; n=7 for each group) to ovariectomized rats for 10 weeks. After 10 weeks' administration, the rats in the olive leaf extract-administered groups showed significantly lower levels of serum triglyceride and very-low-density lipoprotein (VLDL)-cholesterol compared with the rats in the control group, whereas the administration of olive leaf extract did not significantly change the elevated low-density lipoprotein cholesterol levels. In addition, administration of high dose of olive leaf extract significantly decreased the liver triglyceride and increased serum estradiol levels. mRNA expressions of peroxisome proliferator-activated receptor alpha (PPAR α) and acyl-CoA oxidase (ACO) were not affected by ovariectomy, however, administration of olive leaf extract significantly increased both PPAR α and ACO mRNA expression. Expression of adiponectin mRNA in adipose tissue was significantly decreased in the ovariectomized control group. Rats administered low-dose olive leaf extract showed significantly elevated adiponectin mRNA expression compared with rats in the ovariectomized control group. Even though dose-dependent effects were not observed in most of the measurements, these results suggest that genes involved in lipid metabolism may be regulated by olive leaf extract administration in ovariectomized rats.

Resveratrol post-transcriptionally regulates pro-inflammatory gene expression via regulation of KSRP RNA binding activity

PubMed Central

Bollmann, Franziska; Art, Julia; Henke, Jenny; Schrick, Katharina; Besche, Verena; Bros, Matthias; Li, Huige; Siuda, Daniel; Handler, Norbert; Bauer, Florian; Erker, Thomas; Behnke, Felix; Mönch, Bettina; Härdle, Lorena; Hoffmann, Markus; Chen, Ching-Yi; Förstermann, Ulrich; Dirsch, Verena M.; Werz, Oliver; Kleinert, Hartmut; Pautz, Andrea

2014-01-01

Resveratrol shows beneficial effects in inflammation-based diseases like cancer, cardiovascular and chronic inflammatory diseases. Therefore, the molecular mechanisms of the anti-inflammatory resveratrol effects deserve more attention. In human epithelial DLD-1 and monocytic Mono Mac 6 cells resveratrol decreased the expression of iNOS, IL-8 and TNF-α by reducing mRNA stability without inhibition of the promoter activity. Shown by pharmacological and siRNA-mediated inhibition, the observed effects are SIRT1-independent. Target-fishing and drug responsive target stability experiments showed selective binding of resveratrol to the RNA-binding protein KSRP, a central post-transcriptional regulator of pro-inflammatory gene expression. Knockdown of KSRP expression prevented resveratrol-induced mRNA destabilization in human and murine cells. Resveratrol did not change KSRP expression, but immunoprecipitation experiments indicated that resveratrol reduces the p38 MAPK-related inhibitory KSRP threonine phosphorylation, without blocking p38 MAPK activation or activity. Mutation of the p38 MAPK target site in KSRP blocked the resveratrol effect on pro-inflammatory gene expression. In addition, resveratrol incubation enhanced KSRP-exosome interaction, which is important for mRNA degradation. Finally, resveratrol incubation enhanced its intra-cellular binding to the IL-8, iNOS and TNF-α mRNA. Therefore, modulation of KSRP mRNA binding activity and, thereby, enhancement of mRNA degradation seems to be the common denominator of many anti-inflammatory effects of resveratrol. PMID:25352548

Gene expression analysis in lymphoblasts derived from patients with autism spectrum disorder.

PubMed

Yasuda, Yuka; Hashimoto, Ryota; Yamamori, Hidenaga; Ohi, Kazutaka; Fukumoto, Motoyuki; Umeda-Yano, Satomi; Mohri, Ikuko; Ito, Akira; Taniike, Masako; Takeda, Masatoshi

2011-05-26

The autism spectrum disorders (ASDs) are complex neurodevelopmental disorders that result in severe and pervasive impairment in the development of reciprocal social interaction and verbal and nonverbal communication skills. In addition, individuals with ASD have stereotypical behavior, interests and activities. Rare mutations of some genes, such as neuroligin (NLGN) 3/4, neurexin (NRXN) 1, SHANK3, MeCP2 and NHE9, have been reported to be associated with ASD. In the present study, we investigated whether alterations in mRNA expression levels of these genes could be found in lymphoblastoid cell lines derived from patients with ASD. We measured mRNA expression levels of NLGN3/4, NRXN1, SHANK3, MeCP2, NHE9 and AKT1 in lymphoblastoid cells from 35 patients with ASD and 35 healthy controls, as well as from 45 patients with schizophrenia and 45 healthy controls, using real-time quantitative reverse transcriptase polymerase chain reaction assays. The mRNA expression levels of NLGN3 and SHANK3 normalized by β-actin or TBP were significantly decreased in the individuals with ASD compared to controls, whereas no difference was found in the mRNA expression level of MeCP2, NHE9 or AKT1. However, normalized NLGN3 and SHANK3 gene expression levels were not altered in patients with schizophrenia, and expression levels of NLGN4 and NRXN1 mRNA were not quantitatively measurable in lymphoblastoid cells. Our results provide evidence that the NLGN3 and SHANK3 genes may be differentially expressed in lymphoblastoid cell lines from individuals with ASD compared to those from controls. These findings suggest the possibility that decreased mRNA expression levels of these genes might be involved in the pathophysiology of ASD in a substantial population of ASD patients.

Caffeine modulates glucocorticoid-induced expression of CTGF in lung epithelial cells and fibroblasts.

PubMed

Fehrholz, Markus; Glaser, Kirsten; Speer, Christian P; Seidenspinner, Silvia; Ottensmeier, Barbara; Kunzmann, Steffen

2017-03-23

Although caffeine and glucocorticoids are frequently used to treat chronic lung disease in preterm neonates, potential interactions are largely unknown. While anti-inflammatory effects of glucocorticoids are well defined, their impact on airway remodeling is less characterized. Caffeine has been ascribed to positive effects on airway inflammation as well as remodeling. Connective tissue growth factor (CTGF, CCN2) plays a key role in airway remodeling and has been implicated in the pathogenesis of chronic lung diseases such as bronchopulmonary dysplasia (BPD) in preterm infants. The current study addressed the impact of glucocorticoids on the regulation of CTGF in the presence of caffeine using human lung epithelial and fibroblast cells. The human airway epithelial cell line H441 and the fetal lung fibroblast strain IMR-90 were exposed to different glucocorticoids (dexamethasone, budesonide, betamethasone, prednisolone, hydrocortisone) and caffeine. mRNA and protein expression of CTGF, TGF-β1-3, and TNF-α were determined by means of quantitative real-time PCR and immunoblotting. H441 cells were additionally treated with cAMP, the adenylyl cyclase activator forskolin, and the selective phosphodiesterase (PDE)-4 inhibitor cilomilast to mimic caffeine-mediated PDE inhibition. Treatment with different glucocorticoids (1 μM) significantly increased CTGF mRNA levels in H441 (p < 0.0001) and IMR-90 cells (p < 0.01). Upon simultaneous exposure to caffeine (10 mM), both glucocorticoid-induced mRNA and protein expression were significantly reduced in IMR-90 cells (p < 0.0001). Of note, 24 h exposure to caffeine alone significantly suppressed basal expression of CTGF mRNA and protein in IMR-90 cells. Caffeine-induced reduction of CTGF mRNA expression seemed to be independent of cAMP levels, adenylyl cyclase activation, or PDE-4 inhibition. While dexamethasone or caffeine treatment did not affect TGF-β1 mRNA in H441 cells, increased expression of TGF-β2 and TGF-β3 mRNA was detected upon exposure to dexamethasone or dexamethasone and caffeine, respectively. Moreover, caffeine increased TNF-α mRNA in H441 cells (6.5 ± 2.2-fold, p < 0.05) which has been described as potent inhibitor of CTGF expression. In addition to well-known anti-inflammatory features, glucocorticoids may have adverse effects on long-term remodeling by TGF-β1-independent induction of CTGF in lung cells. Simultaneous treatment with caffeine may attenuate glucocorticoid-induced expression of CTGF, thereby promoting restoration of lung homeostasis.

Nitric Oxide Increases the Decay of Matrix Metalloproteinase 9 mRNA by Inhibiting the Expression of mRNA-Stabilizing Factor HuR

PubMed Central

Akool, El-Sayed; Kleinert, Hartmut; Hamada, Farid M. A.; Abdelwahab, Mohamed H.; Förstermann, Ulrich; Pfeilschifter, Josef; Eberhardt, Wolfgang

2003-01-01

Dysregulation of extracellular matrix turnover is an important feature of many inflammatory processes. Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin-1 beta. We demonstrate that NO does strongly destabilize MMP-9 mRNA, since different luciferase reporter gene constructs containing the MMP-9 3′ untranslated region (UTR) displayed significant reduced luciferase activity in response to the presence of NO. Moreover, by use of an in vitro degradation assay we found that the cytoplasmic fractions of NO-treated cells contained a higher capacity to degrade MMP-9 transcripts than those obtained from control cells. An RNA electrophoretic mobility shift assay demonstrated that three of four putative AU-rich elements present in the 3′ UTR of MMP-9 were constitutively occupied by the mRNA-stabilizing factor HuR and that the RNA binding was strongly attenuated by the presence of NO. The addition of recombinant glutathione transferase-HuR prevented the rapid decay of MMP-9 mRNA, whereas the addition of a neutralizing anti-HuR antibody caused an acceleration of MMP-9 mRNA degradation. Furthermore, the expression of HuR mRNA and protein was significantly reduced by exogenously and endogenously produced NO. These inhibitory effects were mimicked by the cGMP analog 8-bromo-cGMP and reversed by LY-83583, an inhibitor of soluble guanylyl cyclase. These results demonstrate that NO acts in a cGMP-dependent mechanism to inhibit the expression level of HuR, thereby reducing the stability of MMP-9 mRNA. PMID:12832476

Fidelity and enhanced sensitivity of differential transcription profiles following linear amplification of nanogram amounts of endothelial mRNA

NASA Technical Reports Server (NTRS)

Polacek, Denise C.; Passerini, Anthony G.; Shi, Congzhu; Francesco, Nadeene M.; Manduchi, Elisabetta; Grant, Gregory R.; Powell, Steven; Bischof, Helen; Winkler, Hans; Stoeckert, Christian J Jr;

Localization and expression of messenger RNAs for the peroxisome proliferator-activated receptors in ovarian tissue from naturally cycling and pseudopregnant rats.

PubMed

Komar, Carolyn M; Curry, Thomas E

2002-05-01

Structural and functional development of the corpus luteum (CL) involves tissue remodeling, angiogenesis, lipid metabolism, and steroid production. The peroxisome proliferator-activated receptors (PPARs) have been shown to play a role in these as well as in a multitude of other cellular processes. To examine the expression of mRNA corresponding to the PPAR family members (alpha, delta, and gamma) in luteal tissue, ovaries were collected from gonadotropin-treated, immature rats on Days 1, 4, 8, and 14 of pseudopregnancy and from adult, cycling animals on each day of the estrous cycle. Ovaries were processed for in situ hybridization or RNA isolation for analysis by RNase protection assay. The expression of PPARgamma mRNA was abundant in granulosa cells of developing follicles during both pseudopregnancy and the estrous cycle and was low to undetectable in CL from pseudopregnant rats. However, luteal tissue in cycling animals, especially CL remaining from previous cycles, had high levels of PPARgamma mRNA. The PPARalpha mRNA was localized mainly in the theca and stroma, and PPARdelta mRNA was expressed throughout the ovary. Levels of mRNA for PPARgamma decreased between Days 1 and 4 of pseudopregnancy, and PPARalpha mRNA levels were lower on the day of estrus compared to pro- and metestrus (P < 0.05). The PPARdelta mRNA levels remained steady throughout the estrous cycle and pseudopregnancy. These data illustrate a difference in the luteal expression of mRNA for PPARgamma between the adult, cycling rat and the immature, gonadotropin-treated rat. This differential pattern of expression may be related to the difference in timing of the preovulatory prolactin surge, because the gonadotropin-primed animals would not experience a prolactin surge coincident with the LH surge, as occurs in adult, cycling animals. Additionally, the expression pattern of PPARdelta mRNA indicates that it may be involved in cellular functions involved with maintaining basal ovarian function, whereas PPARalpha may play a role in lipid metabolism in the theca and stroma.

Differences in expression of retinal pigment epithelium mRNA between normal canines

PubMed Central

2004-01-01

Abstract A reference database of differences in mRNA expression in normal healthy canine retinal pigment epithelium (RPE) has been established. This database identifies non-informative differences in mRNA expression that can be used in screening canine RPE for mutations associated with clinical effects on vision. Complementary DNA (cDNA) pools were prepared from mRNA harvested from RPE, amplified by PCR, and used in a subtractive hybridization protocol (representational differential analysis) to identify differences in RPE mRNA expression between canines. The effect of relatedness of the test canines on the frequency of occurrence of differences was evaluated by using 2 unrelated canines for comparison with 2 female sibling canines of blue heeler/bull terrier lineage. Differentially expressed cDNA species were cloned, sequenced, and identified by comparison to public database entries. The most frequently observed differentially expressed sequence from the unrelated canine comparison was cDNA with 21 base pairs (bp) identical to the human epithelial membrane protein 1 gene (present in 8 of 20 clones). Different clones from the same-sex sibling RPE contained repetitions of several short sequence motifs including the human epithelial membrane protein 1 (4 of 25 clones). Other prevalent differences between sibling RPE included sequences similar to a chicken genetic marker sequence motif (5 of 25), and 6 clones with homology to porcine major histocompatibility loci. In addition to identifying several repetitively occurring, noninformative, differentially expressed RPE mRNA species, the findings confirm that fewer differences occurred between siblings, highlighting the importance of using closely related subjects in representational difference analysis studies. PMID:15352545

Natural and synthetic STAT3 inhibitors reduce hepcidin expression in differentiated mouse hepatocytes expressing the active phosphorylated STAT3 form.

PubMed

Fatih, Nadia; Camberlein, Emilie; Island, Marie Laure; Corlu, Anne; Abgueguen, Emmanuelle; Détivaud, Lénaïck; Leroyer, Patricia; Brissot, Pierre; Loréal, Olivier

2010-05-01

During the inflammatory process, hepcidin overexpression favours the development of anaemia of chronic diseases which represents the second most common form of anaemia worldwide. The identification of therapeutic agents decreasing hepcidin expression is therefore an important goal. The aim of this study was to target the STAT3 signalling involved in the development of increased hepcidin expression related to chronic inflammation. In a co-culture model associating mouse hepatocytes and rat liver epithelial cells, the mRNA levels of hepcidin1, albumin, aldolase B, Cyp3a4, Stat3, Smad4 and iron regulatory genes were measured by real-time PCR. STAT3 and phosphorylated SMAD1/5/8 proteins were analysed by Western blot. At variance of hepatocyte pure culture, co-culture provided high levels of hepcidin1 mRNA, reaching 400% of the freshly isolated hepatocyte values after 6 days of culture. Hepcidin expression was associated with the maintenance of hepatocyte phenotype, STAT3 phosphorylation and functional BMP/SMAD pathway. Stat3 siRNAs inhibited the hepcidin1 mRNA expression. STAT3 inhibitors, including curcumin, AG490 and a peptide (PpYLKTK), reduced hepcidin1 mRNA expression even when cells were additionally exposed to IL-6. Hepcidin1 mRNA was expressed at high levels by hepatocytes in the co-culture model, and STAT3 pathway activation was controlled through STAT3 inhibitors. Such inhibitors could be useful to prevent anaemia related to hepcidin overexpression during chronic inflammation.

Expression of a serine protease (motopsin PRSS12) mRNA in the mouse brain: in situ hybridization histochemical study.

PubMed

Iijima, N; Tanaka, M; Mitsui, S; Yamamura, Y; Yamaguchi, N; Ibata, Y

1999-03-20

Serine proteases are considered to play several important roles in the brain. In an attempt to find novel brain-specific serine proteases (BSSPs), motopsin (PRSS-12) was cloned from a mouse brain cDNA library by polymerase chain reaction (PCR). Northern blot analysis demonstrated that the postnatal 10-day mouse brain contained the most amount of motopsin mRNA. At this developmental stage, in situ hybridization histochemistry showed that motopsin mRNA was specifically expressed in the following regions: cerebral cortical layers II/III, V and VIb, endopiriform cortex and the limbic system, particularly in the CA1 region of the hippocampal formation. In addition, in the brainstem, the oculomotor nucleus, trochlear nucleus, mecencephalic and motor nuclei of trigeminal nerve (N), abducens nucleus, facial nucleus, nucleus of the raphe pontis, dorsoral motor nucleus of vagal N, hypoglossal nucleus and ambiguus nucleus showed motopsin mRNA expression. Expression was also found in the anterior horn of the spinal cord. The above findings strongly suggest that neurons in almost all motor nuclei, particularly in the brainstem and spinal cord, express motopsin mRNA, and that motopsin seems to have a close relation to the functional role of efferent neurons. Copyright 1999 Elsevier Science B.V.

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.

Molecular characterization of melanin-concentrating hormone (MCH) in Schizothorax prenanti: cloning, tissue distribution and role in food intake regulation.

PubMed

Wang, Tao; Yuan, Dengyue; Zhou, Chaowei; Lin, Fangjun; Wei, Rongbin; Chen, Hu; Wu, Hongwei; Xin, Zhiming; Liu, Ju; Gao, Yundi; Chen, Defang; Yang, Shiyong; Wang, Yan; Pu, Yundan; Li, Zhiqiong

2016-06-01

Melanin-concentrating hormone (MCH) is a crucial neuropeptide involved in various biological functions in both mammals and fish. In this study, the full-length MCH cDNA was obtained from Schizothorax prenanti by rapid amplification of cDNA ends polymerase chain reaction. The full-length MCH cDNA contained 589 nucleotides including an open reading frame of 375 nucleotides encoding 256 amino acids. MCH mRNA was highly expressed in the brain by real-time quantitative PCR analysis. Within the brain, expression of MCH mRNA was preponderantly detected in the hypothalamus. In addition, the MCH mRNA expression in the S. prenanti hypothalamus of fed group was significantly decreased compared with the fasted group at 1 and 3 h post-feeding, respectively. Furthermore, the MCH gene expression presented significant increase in the hypothalamus of fasted group compared with the fed group during long-term fasting. After re-feeding, there was a dramatic decrease in MCH mRNA expression in the hypothalamus of S. prenanti. The results indicate that the expression of MCH is affected by feeding status. Taken together, our results suggest that MCH may be involved in food intake regulation in S. prenanti.

mRNA expression pattern of selected candidate genes differs in bovine oviductal epithelial cells in vitro compared with the in vivo state and during cell culture passages.

PubMed

Danesh Mesgaran, Sadjad; Sharbati, Jutta; Einspanier, Ralf; Gabler, Christoph

2016-08-15

The mammalian oviduct provides the optimal environment for gamete maturation including sperm capacitation, fertilization, and development of the early embryo. Various cell culture models for primary bovine oviductal epithelial cells (BOEC) were established to reveal such physiological events. The aim of this study was to evaluate 17 candidate mRNA expression patterns in oviductal epithelial cells (1) in transition from in vivo cells to in vitro cells; (2) during three consecutive cell culture passages; (3) affected by the impact of LOW or HIGH glucose content media; and (4) influenced by different phases of the estrous cycle in vivo and in vitro. In addition, the release of a metabolite and proteins from BOEC at two distinct cell culture passage numbers was estimated to monitor the functionality. BOEC from 8 animals were isolated and cultured for three consecutive passages. Total RNA was extracted from in vivo and in vitro samples and subjected to reverse transcription quantitative polymerase chain reaction to reveal mRNA expression of selected candidate genes. The release of prostaglandin E2 (PGE2), oviduct-specific glycoprotein 1 (OVGP1) and interleukin 8 (IL8) by BOEC was measured by EIA or ELISA after 24 h. Almost all candidate genes (prostaglandin synthases, enzymes of cellular metabolism and mucins) mRNA expression pattern differed compared in vivo with in vitro state. In addition, transcription of most candidate genes was influenced by the number of cell culture passages. Different glucose medium content did not affect mRNA expression of most candidate genes. The phase of the estrous cycle altered some candidate mRNA expression in BOEC in vitro at later passages. The release of PGE2 and OVGP1 between passages did not differ. However, BOEC in passage 3 released significantly higher amount of IL8 compared with cells in passage 0. This study supports the hypothesis that candidate mRNA expression in BOEC was influenced by transition from the in vivo situation to the new in vitro environment and during consecutive passages. The consequence of cell culture passaging on BOEC ability to release bioactive compounds should be considered.

Expression of aquaporin-3 and -8 mRNAs in the parr and smolt stages of sockeye salmon, Oncorhynchus nerka: effects of cortisol treatment and seawater acclimation.

PubMed

Choi, Young Jae; Shin, Hyun Suk; Kim, Na Na; Cho, Sung Hwoan; Yamamoto, Yuzo; Ueda, Hiroshi; Lee, Jehee; Choi, Cheol Young

2013-06-01

This study aimed to examine the role of 2 aquaporin (AQP) isoforms (AQP3, and -8) in sockeye salmon (Oncorhynchus nerka) in response to a hyperosmotic challenge from freshwater to seawater (SW) during the parr and smoltification (smolt) stages. AQP3 mRNA was primarily detected in the osmoregulatory organs, such as gills, while AQP8 mRNA was primarily found in the intestine. These results suggested that AQP isoforms play a role in osmoregulation in specific osmoregulatory organs. Similarly, AQP3 mRNA expression in the gills (mean values:1.06 ± 0.05 [parr] and 1.29 ± 0.07 [smolt]) was significantly higher than AQP8 mRNA levels (parr: 0.04 ± 0.003; smolt: 0.14 ± 0.004), and in the intestine, AQP8 mRNA expression (parr: 0.89 ± 0.007; smolt: 1.91 ± 0.03) was significantly higher than AQP3 mRNA levels (parr: 0.24 ± 0.006; smolt: 0.83 ± 0.005); these expression patterns were similar in vivo and in vitro. Additionally, AQP mRNA levels were lower in cortisol treated than in control groups. Therefore, these results suggest that AQPs play important roles in the water absorption mechanisms associated with multiple AQP isoforms, and that cortisol enhances the hypo-osmoregulatory capacity of fish in SW, and also controls the expression of AQPs in a hyperosmotic environment. Copyright © 2013 Elsevier Inc. All rights reserved.

Expression and significance of CD44s, CD44v6, and nm23 mRNA in human cancer.

PubMed

Liu, Yong-Jun; Yan, Pei-Song; Li, Jun; Jia, Jing-Fen

2005-11-14

To investigate the relationship between the expression levels of nm23 mRNA, CD44s, and CD44v6, and oncogenesis, development and metastasis of human gastric adenocarcinoma, colorectal adenocarcinoma, intraductal carcinoma of breast, and lung cancer. Using tissue microarray by immuhistochemical (IHC) staining and in situ hybridization (ISH), we examined the expression levels of nm23 mRNA, CD44s, and CD44v6 in 62 specimens of human gastric adenocarcinoma and 62 specimens of colorectal adenocarcinoma; the expression of CD44s and CD44v6 in 120 specimens of intraductal carcinoma of breast and 20 specimens of normal breast tissue; the expression of nm23 mRNA in 72 specimens of human lung cancer and 23 specimens of normal tissue adjacent to cancer. The expression of nm23 mRNA in the tissues of gastric and colorectal adenocarcinoma was not significantly different from that in the normal tissues adjacent to cancer (P>0.05), and was not associated with the invasion of tumor and the pathology grade of adenocarcinoma (P>0.05). However, the expression of nm23 mRNA was correlated negatively to the lymph node metastasis of gastric and colorectal adenocarcinoma (r = -0.49, P<0.01; r = -4.93, P<0.01). The expression of CD44s in the tissues of gastric and colorectal adenocarcinoma was significantly different from that in the normal tissues adjacent to cancer (P<0.05; P<0.01). CD44v6 was expressed in the tissues of gastric and colorectal adenocarcinoma only, the expression of CD44v6 was significantly associated with the lymph node metastasis, invasion and pathological grade of the tumor (r = 0.47, P<0.01; r = 5.04, P<0.01). CD44s and CD44v6 were expressed in intraductal carcinoma of breast, the expression of CD44s and CD44v6 was significantly associated with lymph node metastases and invasion (P<0.01). However, neither of them was expressed in the normal breast tissue. In addition, the expression of CD44v6 was closely related to the degree of cell differentiation of intraductal carcinoma of breast (c2 = 5.68, P<0.05). The expressional level of nm23 mRNA was closely related to the degree of cell differentiation (P<0.05) and lymph node metastasis (P<0.01), but the expression of nm23 gene was not related to sex, age, and type of histological classification (P>0.05). Patients with overexpression of CD44s and CD44v6 and low expression of nm23 mRNA have a higher lymph node metastatic rate and invasion. In addition, overexpression of CD44v6 is closely related to the degree of cell differentiation. Detection of the three genes is able to provide a reliable index to evaluate the invasion and metastasis of tumor cells.

Expression profile and distribution of Efhc1 gene transcript during rodent brain development.

PubMed

Conte, Fábio F; Ribeiro, Patrícia A O; Marchesini, Rafael B; Pascoal, Vinícius D B; Silva, Joelcimar M; Oliveira, Amanda R; Gilioli, Rovílson; Sbragia, Lourenço; Bittencourt, Jackson C; Lopes-Cendes, Iscia

2009-09-01

One of the putative causative genes for juvenile myoclonic epilepsy (JME) is EFHC1. We report here the expression profile and distribution of Efhc1 messenger RNA (mRNA) during mouse and rat brain development. Real-time polymerase chain reaction revealed that there is no difference in the expression of Efhc1 mRNA between right and left hemispheres in both species. In addition, the highest levels of Efhc1 mRNA were found at intra-uterine stages in mouse and in adulthood in rat. In common, there was a progressive decrease in Efhc1 expression from 1-day-old neonates to 14-day-old animals in both species. In situ hybridization studies showed that rat and mouse Efhc1 mRNAs are expressed in ependymal cells of ventricle walls. Our findings suggest that Efhc1 expression is more important during initial phases of brain development and that at this stage it could be involved in key developmental mechanisms underlying JME.

Addition of bone morphogenetic protein type 2 to ascorbate and β-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells.

PubMed

Cruz, Ariadne Cristiane Cabral; Silva, Mariana Lúcia; Caon, Thiago; Simões, Cláudia Maria Oliveira

2012-01-01

Bone morphogenetic protein type 2 (BMP-2) is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and β-glycerophosphate. Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2) or absence (ASCs+OM) of BMP-2. The alkaline phosphatase (ALP) activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II), osteonectin, and osteocalcin were evaluated by qPCR. ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity), intermediate (osteonectin and osteocalcin), or final (calcium deposition) phases, suggesting that the exogenous addition of BMP-2 did not improve the in vitro osteogenesis process of human ASCs.

Periapical cytokine expression in sickle cell disease.

PubMed

Ferreira, Shirlene Barbosa Pimentel; de Brito, Luciana Carla Neves; Oliveira, Michelle Pimenta; Maciel, Kamilla Faria; Martelli Júnior, Hercílio; Vieira, Leda Quercia; Sobrinho, Antônio Paulino Ribeiro

2015-03-01

Sickle cell anemia (SCA) is the most prevalent genetic disease worldwide. Patients with SCA exhibit increased levels of proinflammatory mediators as part of a permanently activated immunoinflammatory status. The aim of this study was to evaluate the mRNA expression levels of the cytokines interferon (IFN-γ), tumor necrosis factor, interleukin (IL-1β, IL-17A, IL-10), receptor activator for nuclear factor kappa B ligand, and the chemokines CCL2/MCP-1 and CCL5 in the periapical interstitial fluid from SCA individuals compared with healthy individuals. Samples were collected from 12 teeth of SCA patients and 12 non-SCA patients with apical periodontitis. In addition, 12 teeth were sampled from the periapical region of healthy patients with vital pulp (control). The expression of cytokine mRNA was detected by using real-time polymerase chain reaction. The expression of mRNA for the Th1-associated cytokines IFN-γ, tumor necrosis factor-α, and IL-1β were significantly higher in SCA individuals than in the control individuals (P < .05). Among Th1-associated cytokines, only IFN-γ was significantly increased in non-SCA compared with control patients (vital pulp). The expression of IL-17A mRNA was significant higher in SCA cases than in control samples (P < .05), whereas the IL-10 mRNA expression was significantly increased in SCA and non-SCA individuals when compared with the control group. Similar levels of receptor activator for nuclear factor kappa B ligand, CCL2, and CCL5 mRNA expression were observed in all samples. However, no significant differences were observed in the expression of cytokine or chemokine mRNA between SCA and non-SCA individuals (P > .05). The results were able to demonstrate that SCA patients presented prone proinflammatory ability, despite the fact that any differences in periapical immune responses between SCA and non-SCA individuals were not observed. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

[Effect of Bushen Huoxue Compound on Retinal Müller Cells in High Glucose or AGEs Conditions].

PubMed

Xie, Xue-jun; Song, Ming-xia; Zhang, Mei; Qin, Wei; Wan, Li; Fang, Yang

2015-06-01

To explore the effect of Bushen Huoxue Compound (BHC) on lactate dehydrogenase (LDH) leakage, expressions of vascular endothelial growth factor (VEGF) and VEGF mRNA in retinal Muller cells under high glucose condition or advanced glycosylation end products (AGEs) condition by using serum pharmacological method. The retinal Müller cells of 5-7 days post-natal Sprague Dawley (SD) rats were cultured with modified enzyme-digestion method. Purified retinal Muller cells were cultured in normal conditions, high glucose condition (50 mmol/L) or AGEs (50 mg/L and 100 mg/L) conditions, and BHC-containing serum was added to culture medium. The LDH leakage and VEGF expressions were measured by enzyme-linked immunosorbent assay (ELISA). In addition, the relative expression of VEGF mRNA was tested by reverse transcription polymerase chain reaction (RT-PCR). Compared with the normal control group, expressions of VEGF and VEGF mRNA were significantly increased in the high glucose group, the low dose AGEs group and the high dose AGEs group (all P < 0.01). The LDH leakage was obviously increased in the high dose AGEs group, when compared with the normal control group and the high glucose group (P < 0.01). The LDH leakage, expressions of VEGF and VEGF mRNA were obviously decreased by BHC-containing serum both in high glucose and AGEs conditions (P < 0.05, P < 0.01). BHC-containing serum had no significant effect on the LDH leakage and expressions of VEGF and VEGF mRNA in normal conditions (P > 0.05). AGEs intervention could obviously lower the stability of Müller cell membrane. Up-regulated expressions of VEGF and VEGF mRNA in cultured Müller cells could be induced by AGEs or high glucose. BHC-containing serum could stabilize the stability of Müller cell membrane, inhibit the transcription of VEGF mRNA and decrease the protein expression of VEGF, which might be one of important mechanisms for preventing and treating diabetic retinopathy.

The Andes hantavirus NSs protein is expressed from the viral small mRNA by a leaky scanning mechanism.

PubMed

Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P; Pino, Karla; Tischler, Nicole D; Ohlmann, Théophile; Darlix, Jean-Luc; López-Lastra, Marcelo

2012-02-01

The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism.

The Andes Hantavirus NSs Protein Is Expressed from the Viral Small mRNA by a Leaky Scanning Mechanism

PubMed Central

Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P.; Pino, Karla; Tischler, Nicole D.; Ohlmann, Théophile; Darlix, Jean-Luc

2012-01-01

The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism. PMID:22156529

Nitrous oxide production and mRNA expression analysis of nitrifying and denitrifying bacterial genes under floodwater disappearance and fertilizer application.

PubMed

Riya, Shohei; Takeuchi, Yuki; Zhou, Sheng; Terada, Akihiko; Hosomi, Masaaki

2017-06-01

A pulse of nitrous oxide (N 2 O) emission has been observed following the disappearance of floodwater by drainage. However, its mechanism is not well understood. We conducted a column study to clarify the mechanism for N 2 O production during floodwater disappearance by using a microsensor and determining the bacterial gene expression. An increase in N 2 O flux was observed following floodwater disappearance after the addition of NH 4 + , with a corresponding increase in the concentrations of NO 3 - and dissolved N 2 O in the oxic and anoxic soil layers, respectively. The transcription level of the bacterial amoA mRNA did not change, while that of nirK mRNA increased sharply after an hour of floodwater disappearance. An additional anoxic soil slurry experiment demonstrated that the addition of NO 3 - induced the expression of nirK gene and caused a concomitant increase in N 2 O production. These findings suggest that NO 3 - production in the oxic layers is important as it provides a substrate and induces the synthesis of denitrification enzymes in the anoxic layer during N 2 O production.

Development of a quantitative assay to measure expression of transforming growth factor ß (TGF-ß) in Lost River sucker (Deltistes luxatus) and shortnose sucker (Chasmistes brevirostris) and evaluation of potential pitfalls in use with field-collected samples

USGS Publications Warehouse

Robertson, Laura S.; Ottinger, Christopher A.; Burdick, Summer M.; VanderKooi, Scott P.

2012-01-01

The Nature Conservancy is in the process of restoring the Williamson River Delta in an attempt to recreate important juvenile habitat for the endangered shortnose sucker Chasmistes brevirostris and the endangered Lost River sucker Deltistes luxatus. Measurement of TGF-β mRNA expression level was one of the indicators chosen to evaluate juvenile sucker health during the restoration process. TGF-β mRNA expression level has been correlated with disease status in several laboratory studies and TGF-β mRNA expression level has been used as a species-specific indicator of immune status in field-based fish health assessments. We describe here the identification of TGF-β and a possible splice variant from shortnose sucker and from Lost River sucker. The performance of a quantitative RT-PCR assay to measure TGF-β mRNA expression level was evaluated in field-collected spleen and kidney tissue samples. The quality of extracted RNA was higher in tissues harvested in September compared to July and higher in tissues harvested at lower temperature compared to higher temperature. In addition, the expression level of both TGF-β and 18S as assessed by qRT-PCR was higher in samples with higher quality RNA. TGF-β mRNA expression was lower in kidney than in spleen in both Lost River sucker and shortnose sucker.

Expression of receptor protein tyrosine kinase tif is regulated during leukemia cell differentiation.

PubMed

Dai, W; Pan, H Q; Ouyang, B; Greenberg, J M; Means, R T; Li, B; Cardie, J

1996-06-01

tif is a recently cloned and characterized cDNA predicting a transmembrane protein with a putative tyrosine kinase structure in its cytoplasmic domain. By analysis of the purified tif cytoplasmic domain expressed in Escherichia coli, we have demonstrated that tif is an active protein tyrosine kinase capable of autophosphorylation on tyrosine residues and this phosphorylation is inhibited by a tyrosine-specific inhibitor genistein. Northern blot analyses of various leukemia cell lines have revealed that tif mRNA expression is primarily confined to those bearing erythroid and megakaryocytic phenotypes. Megakaryocytic differentiation of K562 and HEL cells induced by phorbol 12-myristate 13-acetate is accompanied by down-regulation of tif mRNA expression. In addition, treatment of K562 and HEL with hexamethylene bis-acetamide, but not with hemin, decreases the steady-state level of tif mRNA. These combined results suggest that the receptor tyrosine kinase tif is involved in hematopoietic development.

Cyclic strain increases protease-activated receptor-1 expression in vascular smooth muscle cells

NASA Technical Reports Server (NTRS)

Nguyen, K. T.; Frye, S. R.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

2001-01-01

Cyclic strain regulates many vascular smooth muscle cell (VSMC) functions through changing gene expression. This study investigated the effects of cyclic strain on protease-activated receptor-1 (PAR-1) expression in VSMCs and the possible signaling pathways involved, on the basis of the hypothesis that cyclic strain would enhance PAR-1 expression, reflecting increased thrombin activity. Uniaxial cyclic strain (1 Hz, 20%) of cells cultured on elastic membranes induced a 2-fold increase in both PAR-1 mRNA and protein levels. Functional activity of PAR-1, as assessed by cell proliferation in response to thrombin, was also increased by cyclic strain. In addition, treatment of cells with antioxidants or an NADPH oxidase inhibitor blocked strain-induced PAR-1 expression. Preincubation of cells with protein kinase inhibitors (staurosporine or Ro 31-8220) enhanced strain-increased PAR-1 expression, whereas inhibitors of NO synthase, tyrosine kinase, and mitogen-activated protein kinases had no effect. Cyclic strain in the presence of basic fibroblast growth factor induced PAR-1 mRNA levels beyond the effect of cyclic strain alone, whereas no additive effect was observed between cyclic strain and platelet-derived growth factor-AB. Our findings that cyclic strain upregulates PAR-1 mRNA expression but that shear stress downregulates this gene in VSMCs provide an opportunity to elucidate signaling differences by which VSMCs respond to different mechanical forces.

Gene Expression, DNA Methylation and Prognostic Significance of DNA Repair Genes in Human Bladder Cancer.

PubMed

Wojtczyk-Miaskowska, Anita; Presler, Malgorzata; Michajlowski, Jerzy; Matuszewski, Marcin; Schlichtholz, Beata

2017-01-01

This study investigated the gene expression and DNA methylation of selected DNA repair genes (MBD4, TDG, MLH1, MLH3) and DNMT1 in human bladder cancer in the context of pathophysiological and prognostic significance. To determine the relationship between the gene expression pattern, global methylation and promoter methylation status, we performed real-time PCR to quantify the mRNA of selected genes in 50 samples of bladder cancer and adjacent non-cancerous tissue. The methylation status was analyzed by methylation-specific polymerase chain reaction (MSP) or digestion of genomic DNA with a methylation-sensitive restriction enzyme and PCR with gene-specific primers (MSRE-PCR). The global DNA methylation level was measured using the antibody-based 5-mC detection method. The relative levels of mRNA for MBD4, MLH3, and MLH1 were decreased in 28% (14/50), 34% (17/50) and 36% (18/50) of tumor samples, respectively. The MBD4 mRNA expression was decreased in 46% of non-muscle invasive tumors (Ta/T1) compared with 11% found in muscle invasive tumors (T2-T4) (P<0.003). Analysis of mRNA expression for TDG did not show any significant differences between Ta/T1 and T2-T4 tumors. The frequency of increased DNMT1 mRNA expression was higher in T2-T4 (52%) comparing to Ta/T1 (16%). The overall methylation rates in tumor tissue were 18% for MBD4, 25% for MLH1 and there was no evidence of MLH3 promoter methylation. High grade tumors had significantly lower levels of global DNA methylation (P=0.04). There was a significant association between shorter survival and increased expression of DNMT1 mRNA (P=0.002), decreased expression of MLH1 mRNA (P=0.032) and the presence of MLH1 promoter methylation (P=0.006). This study highlights the importance of DNA repair pathways and provides the first evidence of the role of MBD4 and MLH3 in bladder cancer. In addition, our findings suggest that DNMT1 mRNA and MLH1 mRNA expression, as well as the status of MLH1 promoter methylation, are attractive prognostic markers in this pathology. © 2017 The Author(s). Published by S. Karger AG, Basel.

Expression of beta 3-adrenoceptor mRNA in rat tissues.

PubMed

Evans, B A; Papaioannou, M; Bonazzi, V R; Summers, R J

1996-01-01

1. This study examines the expression of beta 3-adrenoceptor messenger RNA (beta 3-AR mRNA) in rat tissues to allow comparison with atypical beta-adrenoceptors determined by functional and radioligand binding techniques. 2. A reverse transcription/polymerase chain reaction protocol has been developed for determining the relative amounts of beta 3-AR mRNA in rat tissues. 3. Measurement of adipsin and uncoupling protein (UCP) mRNA was used to examine all tissues for the presence of white and brown adipose tissue which may contribute beta 3-AR mRNA. 4. The beta 3-AR mRNA is expressed at high levels in brown and white adipose tissue, stomach fundus, the longitudinal/circular smooth muscle of both colon and ileum, and colon submucosa. There was substantial expression of adipsin in colon submucosa and moderate expression in fundus, suggesting that in these regions at least some of the beta 3-AR signal may be contributed by fat. Pylorus and colon mucosa showed moderate levels of beta 3-AR mRNA with lower levels of adipsin. Ileum mucosa and submucosa showed low but readily detectable levels of beta 3-AR. 5. Expression of adipsin in rat skeletal muscles coupled to very low levels of beta 3-AR mRNA indicates that the observed beta 3-AR may be due to the presence of intrinsic fat. beta 3-AR mRNA was virtually undetectable in heart, lung and liver. These results raise the possibility that the atypical beta-AR demonstrated by functional and/or binding studies in muscle and in heart is not the beta 3-AR. 6. By use of two different sets of primers for amplification of beta 3-AR cDNA, no evidence was found for differential splicing of the mRNA in any of the tissues examined. 7. The detection of beta 3-AR mRNA in the gut mucosa and submucosa suggests that in addition to its established roles in lipolysis, thermogenesis and regulation of gut motility beta 3-AR may subserve other functions in the gastrointestinal tract. The absence of beta 3-AR mRNA in rat heart or its presence with adipsin in skeletal muscle suggests that atypical beta-adrenoceptor responses in heart and skeletal muscle are unlikely to be mediated by beta 3-AR.

Effect of developmental exposure to chlorpyrifos on the expression of neurotrophin growth factors and cell-specific markers in neonatal rat brain.

PubMed

Betancourt, Angela M; Burgess, Shane C; Carr, Russell L

2006-08-01

Chlorpyrifos (CPS), a known neurotoxicant, is a widely used agricultural organophosphorus insecticide. The effects of postnatal exposure to CPS on the expression of mRNA for two factors critical to brain development, nerve growth factor (NGF) and reelin, were investigated in the forebrain of rats. In addition, the expression of mRNA for the muscarinic acetylcholine receptor (mAChR) M(1) subtype and cell-specific markers for developing neurons (beta-III tubulin), astrocytes (glial fibrillary acidic protein, GFAP), and oligodendrocytes (myelin-associated glycoprotein, MAG) was also investigated. Oral administration of CPS (1.5 or 3.0 mg/kg) or the corn oil vehicle was performed daily from postnatal days (PNDs) 1 through 6. No signs of overt toxicity or of cholinergic hyperstimulation were observed after CPS administration. Body weight was significantly different from controls on PND7 in both males and females exposed to 3.0 mg/kg CPS. Quantitative PCR was performed on the forebrain. The expression of NGF, reelin, and M(1) mAChR mRNA was significantly reduced with both dosages of CPS in both sexes. beta-III Tubulin mRNA expression remained unchanged after exposure, whereas MAG mRNA expression was significantly decreased with both dosages of CPS in both sexes, suggesting effects on the developing oligodendrocytes. In contrast, GFAP mRNA levels were significantly increased with both dosages of CPS in both sexes, suggesting increased astrocyte reactivity. Our findings indicate that dosages of CPS which cause significant cholinesterase inhibition but do not exert overt toxicity can adversely affect the expression levels of critical genes involved in brain development during the early postnatal period in the rat.

Quantification of heat shock protein mRNA expression in warm and cold anoxic turtles (Trachemys scripta) using an external RNA control for normalization.

PubMed

Stecyk, Jonathan A W; Couturier, Christine S; Fagernes, Cathrine E; Ellefsen, Stian; Nilsson, Göran E

2012-03-01

The mRNA expression of heat-shock protein 90 (HSP90) and heat-shock cognate 70 (HSC70) was examined in cardiac chambers and telencephalon of warm- (21°C) and cold-acclimated (5°C) turtles (Trachemys scripta) exposed to normoxia, prolonged anoxia or anoxia followed by reoxygenation. Additionally, the suitability of total RNA as well as mRNA from β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cyclophilin A (PPIA) for normalizing gene expression data was assessed, as compared to the use of an external RNA control. Measurements of HSP90 and HSC70 mRNA expression revealed that anoxia and reoxygenation have tissue- and gene-specific effects. By and large, the alterations support previous investigations on HSP protein abundance in the anoxic turtle heart and brain, as well as the hypothesized roles of HSP90 and HSC70 during stress and non-stress conditions. However, more prominent was a substantially increased HSP90 and HSC70 mRNA expression in the cardiac chambers with cold acclimation. The finding provides support for the notion that cold temperature induces a number of adaptations in tissues of anoxia-tolerant vertebrates that precondition them for winter anoxia. β-actin, GAPDH and PPIA mRNA expression and total RNA also varied with oxygenation state and acclimation temperature in a tissue- and gene-specific manner, as well as among tissue types, thus disqualifying them as suitable for real-time RT-PCR normalization. Thus, the present data highlights the advantages of normalizing real-time RT-PCR data to an external RNA control, an approach that also allows inter-tissue and potentially inter-species comparisons of target gene expression. Copyright © 2011 Elsevier Inc. All rights reserved.

Single-cell mRNA profiling reveals transcriptional heterogeneity among pancreatic circulating tumour cells.

PubMed

Lapin, Morten; Tjensvoll, Kjersti; Oltedal, Satu; Javle, Milind; Smaaland, Rune; Gilje, Bjørnar; Nordgård, Oddmund

2017-05-31

Single-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process. In addition, such analyses may reveal new knowledge about the mechanisms underlying chemotherapy resistance and tumour progression in patients with cancer. Single circulating tumour cells were isolated from patients with locally advanced or metastatic pancreatic cancer with immuno-magnetic depletion and immuno-fluorescence microscopy. mRNA expression was analysed with single-cell multiplex RT-qPCR. Hierarchical clustering and principal component analysis were performed to identify expression patterns. Circulating tumour cells were detected in 33 of 56 (59%) examined blood samples. Single-cell mRNA profiling of intact isolated circulating tumour cells revealed both epithelial-like and mesenchymal-like subpopulations, which were distinct from leucocytes. The profiled circulating tumour cells also expressed elevated levels of stem cell markers, and the extracellular matrix protein, SPARC. The expression of SPARC might correspond to an epithelial-mesenchymal transition in pancreatic circulating tumour cells. The analysis of single pancreatic circulating tumour cells identified distinct subpopulations and revealed elevated expression of transcripts relevant to the dissemination of circulating tumour cells to distant organ sites.

Replacement of the yeast TRP4 3' untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail.

PubMed Central

Düvel, Katrin; Valerius, Oliver; Mangus, David A; Jacobson, Allan; Braus, Gerhard H

2002-01-01

The mRNA poly(A) tail serves different purposes, including the facilitation of nuclear export, mRNA stabilization, efficient translation, and, finally, specific degradation. The posttranscriptional addition of a poly(A) tail depends on sequence motifs in the 3' untranslated region (3' UTR) of the mRNA and a complex trans-acting protein machinery. In this study, we have replaced the 3' UTR of the yeast TRP4 gene with sequences encoding a hammerhead ribozyme that efficiently cleaves itself in vivo. Expression of the TRP4-ribozyme allele resulted in the accumulation of a nonpolyadenylated mRNA. Cells expressing the TRP4-ribozyme mRNA showed a reduced growth rate due to a reduction in Trp4p enzyme activity. The reduction in enzyme activity was not caused by inefficient mRNA export from the nucleus or mRNA destabilization. Rather, analyses of mRNA association with polyribosomes indicate that translation of the ribozyme-containing mRNA is impaired. This translational defect allows sufficient synthesis of Trp4p to support growth of trp4 cells, but is, nevertheless, of such magnitude as to activate the general control network of amino acid biosynthesis. PMID:12003493

Final report: FASEB Summer Research Conference on ''Post-transcriptional control of gene expression: Effectors of mRNA decay'' [agenda and attendees list

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

Maquat, Lynne

2002-12-01

The goal of this meeting was to provide an interactive forum for scientists working on prokaryotic and eukaryotic mRNA decay. A special seminar presented by a leader in the field of mRNA decay in S. cerevisiae focused on what is known and what needs to be determined, not only for yeast but for other organisms. The large attendance (110 participants) reflects the awareness that mRNA decay is a key player in gene regulation in a way that is affected by the many steps that precede mRNA formation. Sessions were held on the following topics: mRNA transport and mRNP; multicomponent eukaryoticmore » nucleases; nonsense-mediated mRNA decay and nonsense-associated altered splicing; Cis-acting sequences/Trans-acting factors of mRNA decay; translational accuracy; multicomponent bacterial nucleases; interplay between mRNA polyadenylation, translation and decay in prokaryotes and prokaryotic organelles; and RNA interference and other RNA mediators of gene expression. In addition to the talks and two poster sessions, there were three round tables: (1) Does translation occur in the nucleus? (2) Differences and similarities in the mechanisms of mRNA decay in different eukaryotes, and (3) RNA surveillance in bacteria?« less

[Circadian rhythm variation of the clock genes Per1 and cell cycle related genes in different stages of carcinogenesis of buccal mucosa in animal model].

PubMed

Tan, Xuemei; Ye, Hua; Yang, Kai; Chen, Dan; Tang, Hong

2015-07-01

To investigate the expression and circadian rhythm variation of biological clock gene Per1 and cell cycle genes p53, CyclinD1, cyclin-dependent kinases (CDK1), CyclinB1 in different stages of carcinogenesis in buccal mucosa and its relationship with the development of buccal mucosa carcinoma. Ninety golden hamsters were housed under 12 hours light-12 hours dark cycles, and the model of buccal squamous cell carcinoma was established by using the dimethylbenzanthracene (DMBA) to smear the golden hamster buccal mucosa. Before the DMBA was used and after DMBA was used 6 weeks and 14 weeks respectively, the golden hamsters were sacrificed at 6 different time points (5 rats per time point) within 24 hour, including 4, 8, 12, 16, 20 and 24 hour after lights onset (HALO), and the normal buccal mucosa, precancerous lesions and cancer tissue were obtained, respectively. HE stained sections were prepared to observe the canceration of each tissue. Real time RT-PCR was used to detect the mRNA expression of Per1, p53, CyclinD1, CDK1 and CyclinB1, and a cosine analysis method was applied to determine the circadian rhythm variation of Per1, p53, CyclinD1, CDK1 and CyclinB1 mRNA expression, which were characterized by median, amplitude and acrophase. The expression of Per1, p53, CDK1 and CyclinD1 mRNA in 6 different time points within 24 hours in the tissues of three different stages of carcinogenesis had circadian rhythm, respectively. However, the CyclinB1 mRNA was expressed with circadian rhythm just in normal and cancer tissue (P < 0.05), while in precancerous lesions the circadian rhythm was in disorder (P > 0.05). As the development of carcinoma, the median of Per1 and p53 mRNA expression were significantly decreased (P < 0.05), yet the median of CDK1, CyclinB1 and CyclinD1 mRNA expression were significantly increased (P < 0.05). The amplitude of Per1, p53 and CyclinD1 mRNA expression was significantly decreased as the development of carcinoma (P < 0.05), however the amplitude of CDK1 mRNA expression was significantly increased (P < 0.05). In addition, there was no significant difference in the amplitude of CyclinB1 mRNA expression. The time that the peak expression value of Per1 and CDK1 mRNA appeared (Acrophase) in precancerous lesions was remarkably earlier than that in normal tissues, but the acrophase of p53 and CyclinD1 mRNA expression was remarkably delayed. Moreover, the acrophase of CDK1 and CyclinB1 mRNA expression in cancer tissues was obviously earlier than that in normal tissues, yet there was no significant variation in acrophase of Per1, p53, CyclinD1 mRNA expression between normal tissues and cancer tissues. The circadian rhythm of clock gene Per1 and cell cycle genes p53, CyclinD1, CDK1, CyclinB1 expression remarkably varied with the occurrence and development of carcinoma. Further research into the interaction between circadian and cell cycle of two cycle activity and relationship with the carcinogenesis may providenew ideas and methods of individual treatment and the mechanism of carcinogenesis.

The G protein-coupled estrogen receptor 1 (GPER/GPR30) does not predict survival in patients with ovarian cancer

PubMed Central

2012-01-01

Background Even though ovarian tumors are not generally considered estrogen-sensitive, estrogens may still have an impact on ovarian tumor progression. The recently identified trans-membrane estrogen receptor GPER is involved in rapid estrogen signaling. Furthermore, it binds selective estrogen receptor modulators with agonistic effect, which could explain tamoxifen controversies. Methods GPER mRNA was assayed with quantitative real-time PCR (qPCR) in 42 primary ovarian tumors and 7 ovarian cancer cell lines. ERα and ERβ mRNA were analyzed for comparison. GPER protein was semi-quantified with densitometric scanning of Western blots and its tissue distribution analyzed with immunohistochemistry (IHC) in 40 ovarian tumors. In addition, IHC was evaluated in a tissue microarray (TMA) of 150 primary malignant ovarian tumors. Results All tumor samples contained GPER mRNA. The content of mRNA was not different between benign and malignant tumors, but one third of malignant samples over-expressed GPER mRNA. The content of ERα mRNA was higher in malignant than in benign tumors, whereas ERβ mRNA was higher in benign than in malignant tumors. GPER mRNA was detected in all seven ovarian cancer cell lines with highest levels in TOV21G and TOV112D cells. Similar expression pattern was seen for ERβ mRNA. Western blot demonstrated GPER protein in all tumor samples. Semi-quantification showed no difference between benign and malignant tumors, but about one third of malignant samples over-expressed GPER protein. GPER staining was localized mainly in epithelial cells. In the TMA study we found no correlation between GPER staining and clinical stage, histological grade or patient survival. Conclusions GPER mRNA as well as GPER protein is present in both benign and malignant ovarian tumor tissue. About one third of malignant tumors over-expressed both GPER mRNA and protein. This, however, correlated neither with histological or clinical parameters nor with patient survival. PMID:22424333

Atorvastatin Protects Myocardium Against Ischemia-Reperfusion Injury Through Inhibiting miR-199a-5p.

PubMed

Zuo, YaBei; Wang, YuZhao; Hu, HaiJuan; Cui, Wei

2016-01-01

This study aimed to evaluate the protective effects of atorvastatin against myocardial ischemia/reperfusion (I/R) injury in cardiomyocytes and its possible underlying mechanism. Direct cytotoxic effect of OGD/R on cardiomyocytes with and without atorvastatin pretreatment was evaluated. Effects of atorvastatin on expression of GSK-3β and miR-199a-5p were determined using RT-PCR and Western blot. In addition, GSK-3β expression with miR-199a-5p upregulation and downregulation was detected using RT-PCR, Western blot, and immunohistochemistry. Pretreatment with atorvastatin significantly improved the recovery of cells viability from OGD/R (p<0.05). In addition, the atorvastatin pretreatment significantly increased GSK-3β expression both in mRNA level and protein level and decreased miR-199a-5p expression in mRNA level (p<0.05). Upregulation and downregulation of miR-199a-5p respectively decreased and increased GSK-3β expression both in mRNA level and protein level. These results suggested that atorvastatin provides the cardioprotective effects against I/R injury via increasing GSK-3β through inhibition of miR-199a-5p. © 2016 The Author(s) Published by S. Karger AG, Basel.

Adipose cell differentiation: evidence for a two-step process in the polyamine-dependent Ob1754 clonal line.

PubMed Central

Amri, E Z; Dani, C; Doglio, A; Etienne, J; Grimaldi, P; Ailhaud, G

1986-01-01

A subclone of preadipocyte Ob17 cells has been isolated (Ob1754 clonal line). Confluent Ob1754 cells treated with an inhibitor of spermidine and spermine synthesis, methylglyoxal bis(guanylhydrazone), were totally dependent upon putrescine addition for the expression of glycerol-3-phosphate dehydrogenase which behaved as a late marker of adipose conversion. Under these conditions, the early expression of lipoprotein lipase during growth arrest remained unchanged. Studies at the mRNA level showed that the expression of unidentified pOb24 and pGH3 mRNAs, which was parallel to that of lipoprotein lipase, is independent of polyamine addition whereas the late emergence of glycerol-3-phosphate dehydrogenase mRNA was putrescine-dependent and co-ordinated with the expression of pAL422 mRNA encoding for a myelin-P2 homologue [Bernlohr, Angus, Lane, Bolanowski & Kelly (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5468-5472]. The appearance of lipoprotein lipase preceded DNA synthesis and post-confluent mitoses which were both putrescine-dependent and which took place before the appearance of glycerol-3-phosphate dehydrogenase. Thus the adipose conversion of Ob1754 cells involves the expression of at least two separate sets of markers which are differently regulated. Images Fig. 3. Fig. 6. PMID:3800927

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

Galloway, Chad A.; Smith, Harold C., E-mail: harold.smith@rochester.edu

Apolipoprotein B mRNA is edited at cytidine 6666 in the enterocytes lining the small intestine of all mammals; converting a CAA codon to a UAA stop codon. The conversion is {approx}80% efficient in this tissue and leads to the expression of the truncated protein, ApoB48, essential for secretion of dietary lipid as chylomicrons. Caco-2 cell raft cultures have been used as an in vitro model for the induction of editing activity during human small intestinal cell differentiation. This induction of apoB mRNA editing has been ascribed to the expression of APOBEC-1. In agreement our data demonstrated differentiation-dependent induction of expressionmore » of the editing enzyme APOBEC-1 and in addition we show alternative splicing of the essential auxiliary factor ACF. However, transfection of these editing factors in undifferentiated proliferating Caco-2 cells was not sufficient to induce robust apoB mRNA editing activity. Only differentiation of Caco-2 cells could induce more physiological like levels of apoB mRNA editing. The data suggested that additional regulatory mechanism(s) were induced by differentiation that controlled the functional activity of editing factors.« less

CD200:CD200R Interactions Regulate Osteoblastogenesis and Osteoclastogenesis in Space

NASA Astrophysics Data System (ADS)

Kos, Olha; Lee, Lydia; Gorezynski, Reginald M.

2008-06-01

We report data from studies on a recent FOTON mission, using an eOSTEO cell culture system developed by Systems Technologies Canada Inc., showing that in space overexpression of CD200 (using cell cultures derived from transgenic mice expressing CD200 under control of a doxycycline-inducible promoter) is associated with an attenuation in the suppression of mRNA markers of osteoblastogeneis (including BSP, OPG) with concomitant loss of the preferential increased osteoclastogenesis which is otherwise seen in the absence of CD200. In separate cultures we also explored the additional effect of altered inflammatory cytokines on the perturbation of expression of these bone-related genes, using cells from cytokine-receptor knockout mice. Our data suggest that while exogenous inflammatory cytokines (TNFα+IL1β) increased mRNAs typical for osteoclastogenesis under ground conditions, they appeared to produce no further modification of mRNA expression in flight. We suggest that altered mRNA expression in flight is not primarily driven by altered expression of inflammatory cytokines.

Mechanism of the melanogenesis stimulation activity of (-)-cubebin in murine B16 melanoma cells.

PubMed

Hirata, Noriko; Naruto, Shunsuke; Ohguchi, Kenji; Akao, Yukihiro; Nozawa, Yoshinori; Iinuma, Munekazu; Matsuda, Hideaki

2007-07-15

(-)-Cubebin showed a melanogenesis stimulation activity in a concentration-dependent manner in murine B16 melanoma cells without any significant effects on cell proliferation. Tyrosinase activity was increased at 24-72 h after addition of cubebin to B16 cells, and then intracellular melanin amount was increased at 48-96 h after the treatment. The expression levels of tyrosinase were time-dependently enhanced after the treatment with cubebin. At the same time, the expression levels of tyrosinase mRNA were also increased after addition of cubebin. Furthermore Western blot analysis revealed that cubebin elevated the level of phosphorylation of p38 mitogen-activated protein kinase (MAPK). SB203580, a selective inhibitor of p38 MAPK, completely blocked cubebin-induced expression of tyrosinase mRNA in B16 cells. These results suggested that cubebin increased melanogenesis in B16 cells through the enhancement of tyrosinase expression mediated by activation of p38 MAPK.

Triclosan activates aryl hydrocarbon receptor (AhR)-dependent apoptosis and affects Cyp1a1 and Cyp1b1 expression in mouse neocortical neurons.

PubMed

Szychowski, Konrad A; Wnuk, Agnieszka; Kajta, Małgorzata; Wójtowicz, Anna K

2016-11-01

Triclosan (TCS) is an antimicrobial agent that is used extensively in personal care and in sanitizing products, such as soaps, toothpastes, and hair products. A number of studies have revealed the presence of TCS in human tissues, such as fat, liver and brain, in addition to blood and breast milk. The aim of the present study was to investigate the impact of TCS on AhR and Cyp1a1/Cyp1b1 signaling in mouse neocortical neurons in primary cultures. In addition to the use of selective ligands and siRNAs, expression levels of mRNA and proteins as well as caspase-3 activity, reactive oxygen species (ROS) formation, and lactate dehydrogenase (LDH) release have been measured. We also studied the involvement of the AhR in TCS-induced LDH release and caspase-3 activation as well as the effect of TCS on ROS generation. Cultures of neocortical neurons were prepared from Swiss mouse embryos on day 15/16 of gestation. The cells were cultured in phenol red-free Neurobasal medium with B27 and glutamine, and the neurons were exposed to 1 and 10µM TCS. Our experiments showed that the expression of AhR and Cyp1a1 mRNA decreased in cells exposed to 10µM TCS for 3 or 6h. In the case of Cyp1b1, mRNA expression remained unchanged compared with the control group following 3h of exposure to TCS, but after 6h, the mRNA expression of Cyp1b1 was decreased. Our results confirmed that the AhR is involved in the TCS mechanism of action, and our data demonstrated that after the cells were transfected with AhR siRNA, the cytotoxic and pro-apoptotic properties of TCS were decreased. The decrease in Cyp1a1 mRNA and protein expression levels accompanied by a decrease in its activity. The stimulation of Cyp1a1 activity produced by the application of an AhR agonist (βNF) was attenuated by TCS, whereas the addition of AhR antagonist (αNF) reversed the inhibitory effects of TCS. In our experiments, TCS diminished Cyp1b1 mRNA and enhanced its protein expression. In case of Cyp1a1 we observed paradoxical effect of TCS action, which caused the decrease in activity and protein expression of Cyp1a1 and the increase in protein level of AhR. Therefore, we determined the effects of TCS on the production of ROS. Our results revealed that TCS increased the production of ROS and that this effect of TCS was reversed by 10µM N-acetyl-L-cysteine (NAC), the ROS scavenger. To confirm an involvement of ROS in TCS-induced neurotoxicity we measured AhR, Cyp1a1, and Cyp1b1 mRNA expression levels in cells co-treated with TCS and NAC. In the presence of NAC, TCS enhanced mRNA expression of the cytochromes and AhR at 3 and 6h, respectively. We postulate that TCS exhibits primary and secondary effects. The primary effects such as impairment of Cyp1a1 signaling are mediated by TCS-induced ROS production, whereas secondary effects of TCS are due to transcriptional activity of AhR and estrogenic properties of TCS. Copyright © 2016 Elsevier Inc. All rights reserved.

Role of β1-Integrin in Colorectal Cancer: Case-Control Study

PubMed Central

Oh, Bo-Young; Kim, Kwang Ho; Chung, Soon Sup; Hong, Kyoung Sook

2014-01-01

Purpose In the metastatic process, interactions between circulating tumor cells (CTCs) and the extracellular matrix or surrounding cells are required. β1-Integrin may mediate these interactions. The aim of this study was to investigate whether β1-integrin is associated with the detection of CTCs in colorectal cancer. Methods We enrolled 30 patients with colorectal cancer (experimental group) and 30 patients with benign diseases (control group). Blood samples were obtained from each group, carcinoembryonic antigen (CEA) mRNA for CTCs marker and β1-integrin mRNA levels were estimated by using reverse transcription-polymerase chain reaction, and the results were compared between the two groups. In the experimental group, preoperative results were compared with postoperative results for each marker. In addition, we analyzed the correlation between the expressions of β1-integrin and CEA. Results CEA mRNA was detected more frequently in colorectal cancer patients than in control patients (P = 0.008). CEA mRNA was significantly reduced after surgery in the colorectal cancer patients (P = 0.032). β1-Integrin mRNA was detected more in colorectal cancer patients than in the patients with benign diseases (P < 0.001). In colorectal cancer patients, expression of β1-integrin mRNA was detected more for advanced-stage cancer than for early-stage cancer (P = 0.033) and was significantly decreased after surgery (P < 0.001). In addition, expression of β1-integrin mRNA was significantly associated with that of CEA mRNA in colorectal cancer patients (P = 0.001). Conclusion In conclusion, β1-integrin is a potential factor for forming a prognosis following surgical resection in colorectal cancer patients. β1-Integrin may be a candidate for use as a marker for early detection of micrometastatic tumor cells and for monitoring the therapeutic response in colorectal cancer patients. PMID:24851215

Regulation of Hippocampal α1d Adrenergic Receptor mRNA by Corticosterone in Adrenalectomized Rats

PubMed Central

Day, Heidi E.W.; Kryskow, Elisa M.; Watson, Stanley J.; Akil, Huda; Campeau, Serge

2008-01-01

The hippocampal formation receives extensive noradrenergic projections and expresses high levels of mineralocorticoid (MR) and glucocorticoid (GR) receptors. Considerable evidence suggests that the noradrenergic system influences hippocampal corticosteroid receptors. However, there is relatively little data describing the influence of glucocorticoids on noradrenergic receptors in the hippocampal formation. α1d adrenergic receptor (ADR) mRNA is expressed at high levels in the hippocampal formation, within cells that express MR or GR. In order to determine whether expression of α1d ADR mRNA is influenced by circulating glucocorticoids, male rats underwent bilateral adrenalectomy (ADX) or sham surgery, and were killed after 1, 3, 7 or 14 days. Levels of α1d ADR mRNA were profoundly decreased in hippocampal subfields CA1, CA2 and CA3 and the medial and lateral blades of the dentate gyrus, as early as 1 day after ADX, as determined by in situ hybridization. The effect was specific for the hippocampal formation, with levels of α1d mRNA unaltered by ADX in the lateral amygdala, reticular thalamic nucleus, retrosplenial cortex or primary somatosensory cortex. Additional rats underwent ADX or sham surgery and received a corticosterone pellet (10 or 50 mg) or placebo for 7 days. Corticosterone replacement prevented the ADX-induced decrease in hippocampal α1d ADR mRNA, with the magnitude of effect depending on corticosterone dose and hippocampal subregion. These data indicate that α1d ADR mRNA expression in the hippocampal formation is highly sensitive to circulating levels of corticosterone, and provides further evidence for a close interaction between glucocorticoids and the noradrenergic system in the hippocampus. PMID:18534559

Increased cortical expression of the zinc transporter SLC39A12 suggests a breakdown in zinc cellular homeostasis as part of the pathophysiology of schizophrenia

PubMed Central

Scarr, Elizabeth; Udawela, Madhara; Greenough, Mark A; Neo, Jaclyn; Suk Seo, Myoung; Money, Tammie T; Upadhyay, Aradhana; Bush, Ashley I; Everall, Ian P; Thomas, Elizabeth A; Dean, Brian

2016-01-01

Our expression microarray studies showed messenger RNA (mRNA) for solute carrier family 39 (zinc transporter), member 12 (SLC39A12) was higher in dorsolateral prefrontal cortex from subjects with schizophrenia (Sz) in comparison with controls. To better understand the significance of these data we ascertained whether SLC39A12 mRNA was altered in a number of cortical regions (Brodmann’s area (BA) 8, 9, 44) from subjects with Sz, in BA 9 from subjects with mood disorders and in rats treated with antipsychotic drugs. In addition, we determined whether inducing the expression of SLC39A12 resulted in an increased cellular zinc uptake. SLC39A12 variant 1 and 2 mRNA was measured using quantitative PCR. Zinc uptake was measured in CHO cells transfected with human SLC39A12 variant 1 and 2. In Sz, compared with controls, SLC39A12 variant 1 and 2 mRNA was higher in all cortical regions studied. The were no differences in levels of mRNA for either variant of SLC39A12 in BA 9 from subjects with mood disorders and levels of mRNA for Slc39a12 was not different in the cortex of rats treated with antipsychotic drugs. Finally, expressing both variants in CHO-K1 cells was associated with an increase in radioactive zinc uptake. As increased levels of murine Slc39a12 mRNA has been shown to correlate with increasing cellular zinc uptake, our data would be consistent with the possibility of a dysregulated zinc homeostasis in the cortex of subjects with schizophrenia due to altered expression of SLC39A12. PMID:27336053

Evaluation of gene expression levels for cytokines in ocular toxoplasmosis.

PubMed

Maia, M M; Meira-Strejevitch, C S; Pereira-Chioccola, V L; de Hippólito, D D C; Silva, V O; Brandão de Mattos, C C; Frederico, F B; Siqueira, R C; de Mattos, L C

2017-10-01

This study evaluated levels for mRNA expression of 7 cytokines in ocular toxoplasmosis. Peripheral blood mononuclear cells (PBMC) of patients with ocular toxoplasmosis (OT Group, n = 23) and chronic toxoplasmosis individuals (CHR Group, n = 9) were isolated and stimulated in vitro with T. gondii antigen. Negative controls (NC) were constituted of 7 PBMC samples from individuals seronegative for toxoplasmosis. mRNA expression for cytokines was determined by qPCR. Results showed a significant increase in mRNA levels from antigen stimulated PBMCs derived from OT Group for expressing IL-6 (at P < .005 and P < .0005 for CHR and NC groups, respectively), IL-10 (at P < .0005 and P < .005 for CHR and NC groups, respectively) and TGF-β (at P < .005) for NC group. mRNA levels for TNF-α and IL-12 were also upregulated in patients with OT compared to CHR and NC individuals, although without statistical significance. Additionally, mRNA levels for IL-27 and IFN-γ in PBMC of patients with OT were upregulated in comparison with NC individuals. Differences between OT and NC groups were statistically significant at P < .05 and P < .0005, respectively. © 2017 John Wiley & Sons Ltd.

Effects of copper on growth, metamorphosis and endocrine disruption of Bufo gargarizans larvae.

PubMed

Wang, Chao; Liang, Gang; Chai, Lihong; Wang, Hongyuan

2016-01-01

Chinese toad (Bufo gargarizans) tadpoles were exposed to copper (1, 6.4, 32 and 64μgL(-1) copper) from the beginning of larval period through completion of metamorphosis. We examined the effects of chronic copper exposure on mortality, growth, time to metamorphosis, tail resorption time, body size at the metamorphic climax (Gs 42) and completion of metamorphosis (Gs 46) and thyroid gland histology. In addition, type 2 and 3 iodothyronine deiodinase (Dio2 and Dio3), thyroid hormone receptors (TRα and TRβ) mRNA levels were also measured to assess disruption of TH synthesis. Our result showed that 6.4-64μgL(-1) copper concentration increased the mortality and inhibited the growth of B. gargarizans tadpoles. In addition, significant reduction in size at Gs 42 and a time delay to Gs 42 were observed at 6.4-64μgL(-1) copper treatments. Moreover, histological examinations have clearly revealed that 64μgL(-1) copper caused follicular cell hyperplasia in thyroid gland. According to real-time PCR results, exposure to 32 and 64μgL(-1) copper significantly up-regulated mRNA expression of Dio3, but down-regulated mRNA expression of TRα and TRβ mRNA level. We concluded that copper delayed amphibian metamorphosis through changing mRNA expression of Dio3, TRα and TRβ, which suggests that copper might have the endocrine-disrupting effect. Copyright © 2015 Elsevier B.V. All rights reserved.

Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery.

PubMed

Azimi, Sayyed M; Sheridan, Steven D; Ghannad-Rezaie, Mostafa; Eimon, Peter M; Yanik, Mehmet Fatih

2018-05-01

Identification of optimal transcription-factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription-factor copy-numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH -expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition. © 2018, Azimi et al.

Gene expression analysis in lymphoblasts derived from patients with autism spectrum disorder

PubMed Central

2011-01-01

Background The autism spectrum disorders (ASDs) are complex neurodevelopmental disorders that result in severe and pervasive impairment in the development of reciprocal social interaction and verbal and nonverbal communication skills. In addition, individuals with ASD have stereotypical behavior, interests and activities. Rare mutations of some genes, such as neuroligin (NLGN) 3/4, neurexin (NRXN) 1, SHANK3, MeCP2 and NHE9, have been reported to be associated with ASD. In the present study, we investigated whether alterations in mRNA expression levels of these genes could be found in lymphoblastoid cell lines derived from patients with ASD. Methods We measured mRNA expression levels of NLGN3/4, NRXN1, SHANK3, MeCP2, NHE9 and AKT1 in lymphoblastoid cells from 35 patients with ASD and 35 healthy controls, as well as from 45 patients with schizophrenia and 45 healthy controls, using real-time quantitative reverse transcriptase polymerase chain reaction assays. Results The mRNA expression levels of NLGN3 and SHANK3 normalized by β-actin or TBP were significantly decreased in the individuals with ASD compared to controls, whereas no difference was found in the mRNA expression level of MeCP2, NHE9 or AKT1. However, normalized NLGN3 and SHANK3 gene expression levels were not altered in patients with schizophrenia, and expression levels of NLGN4 and NRXN1 mRNA were not quantitatively measurable in lymphoblastoid cells. Conclusions Our results provide evidence that the NLGN3 and SHANK3 genes may be differentially expressed in lymphoblastoid cell lines from individuals with ASD compared to those from controls. These findings suggest the possibility that decreased mRNA expression levels of these genes might be involved in the pathophysiology of ASD in a substantial population of ASD patients. PMID:21615902

RpoS induces expression of the Vibrio anguillarum quorum-sensing regulator VanT.

PubMed

Weber, Barbara; Croxatto, Antony; Chen, Chang; Milton, Debra L

2008-03-01

In vibrios, regulation of the Vibrio harveyi-like LuxR transcriptional activators occurs post-transcriptionally via small regulatory RNAs (sRNAs) that destabilize the luxR mRNA at a low cell population, eliminating expression of LuxR. Expression of the sRNAs is modulated by the vibrio quorum-sensing phosphorelay systems. However, vanT mRNA, which encodes a LuxR homologue in Vibrio anguillarum, is abundant at low and high cell density, indicating that VanT expression may be regulated via additional mechanisms. In this study, Western analyses showed that VanT was expressed throughout growth with a peak of expression during late exponential growth. VanO induced partial destabilization of vanT mRNA via activation of at least one Qrr sRNA. Interestingly, the sigma factor RpoS significantly stabilized vanT mRNA and induced VanT expression during late exponential growth. This induction was in part due to RpoS repressing expression of Hfq, an RNA chaperone. RpoS is not part of the quorum-sensing regulatory cascade since RpoS did not regulate expression or activity of VanO, and RpoS was not regulated by VanO or VanT. VanT and RpoS were needed for survival following UV irradiation and for pigment and metalloprotease production, suggesting that RpoS works with the quorum-sensing systems to modulate expression of VanT, which regulates survival and stress responses.

Neuroleptic Drugs and PACAP Differentially Affect the mRNA Expression of Genes Encoding PAC1/VPAC Type Receptors.

PubMed

Jóźwiak-Bębenista, Marta; Kowalczyk, Edward

2017-04-01

Several lines of evidence suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide playing an important role as a neuromodulator. It has been indicated that PACAP is associated with mental diseases, and that regulation of the PACAPergic signals could be a potential target for the treatment of such psychiatric states as schizophrenia. Recent studies have suggested that action of neuroleptic drugs is mediated not only by dopaminergic and serotonergic neurotransmission, but also via neuropeptides which may act both as neurotransmitters and as neuromodulators. The present study examines whether currently-used neuroleptics influence the action of PACAP receptors, whose expression is altered in a schizophrenic patient. Real-time polymerase chain reaction (PCR) was used to examine the effects of haloperidol, olanzapine and amisulpride on the expression of genes coding PAC1/VPAC type receptors in the T98G glioblastoma cell line, as an example of an in vitro model of glial cells. PAC1 mRNA expression fell after 24-h incubation with haloperidol or olanzapine; however the effect was not maintained after 72 h, and haloperidol even up-regulated PAC1 mRNA expression in a dose-dependent manner. All the examined drugs decreased VPAC2 mRNA expression, especially after 72-h incubation. Haloperidol (typical neuroleptic) was distinctly more potent than atypical neuroleptic drugs (olanzapine and amisulpride). In addition, PACAP increased PAC1 and VPAC2 mRNA expression. In conclusion, our findings suggest PACAP receptors may be involved in the mechanism of typical and atypical neuroleptic drugs.

Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading

NASA Technical Reports Server (NTRS)

Carlson, C. J.; Booth, F. W.; Gordon, S. E.

1999-01-01

Transgenic mice lacking a functional myostatin (MSTN) gene demonstrate greater skeletal muscle mass resulting from muscle fiber hypertrophy and hyperplasia (McPherron, A. C., A. M. Lawler, and S. -J. Lee. Nature 387: 83-90, 1997). Therefore, we hypothesized that, in normal mice, MSTN may act as a negative regulator of muscle mass. Specifically, we hypothesized that the predominately slow (type I) soleus muscle, which demonstrates greater atrophy than the fast (type II) gastrocnemius-plantaris complex (Gast/PLT), would show more elevation in MSTN mRNA abundance during hindlimb unloading (HU). Surprisingly, MSTN mRNA was not detectable in weight-bearing or HU soleus muscle, which atrophied 42% by the 7th day of HU in female ICR mice. In contrast, MSTN mRNA was present in weight-bearing Gast/PLT muscle and was significantly elevated (67%) at 1 day but not at 3 or 7 days of HU. However, the Gast/PLT muscle had only atrophied 17% by the 7th day of HU. Because the soleus is composed only of type I and IIa fibers, whereas the Gast/PLT expresses type IId/x and IIb in addition to type I and IIa, it was necessary to perform a more careful analysis of the relationship between MSTN mRNA levels and myosin heavy-chain (MHC) isoform expression (as a marker of fiber type). A significant correlation (r = 0.725, P < 0. 0005) was noted between the percentage of MHC isoform IIb expression and MSTN mRNA abundance in several muscles of the mouse hindlimb. These results indicate that MSTN expression is not strongly associated with muscle atrophy induced by HU; however, it is strongly associated with MHC isoform IIb expression in normal muscle.

Distribution of androgen receptor mRNA expression in vocal, auditory, and neuroendocrine circuits in a teleost fish.

PubMed

Forlano, Paul M; Marchaterre, Margaret; Deitcher, David L; Bass, Andrew H

2010-02-15

Across all major vertebrate groups, androgen receptors (ARs) have been identified in neural circuits that shape reproductive-related behaviors, including vocalization. The vocal control network of teleost fishes presents an archetypal example of how a vertebrate nervous system produces social, context-dependent sounds. We cloned a partial cDNA of AR that was used to generate specific probes to localize AR expression throughout the central nervous system of the vocal plainfin midshipman fish (Porichthys notatus). In the forebrain, AR mRNA is abundant in proposed homologs of the mammalian striatum and amygdala, and in anterior and posterior parvocellular and magnocellular nuclei of the preoptic area, nucleus preglomerulosus, and posterior, ventral and anterior tuberal nuclei of the hypothalamus. Many of these nuclei are part of the known vocal and auditory circuitry in midshipman. The midbrain periaqueductal gray, an essential link between forebrain and hindbrain vocal circuitry, and the lateral line recipient nucleus medialis in the rostral hindbrain also express abundant AR mRNA. In the caudal hindbrain-spinal vocal circuit, high AR mRNA is found in the vocal prepacemaker nucleus and along the dorsal periphery of the vocal motor nucleus congruent with the known pattern of expression of aromatase-containing glial cells. Additionally, abundant AR mRNA expression is shown for the first time in the inner ear of a vertebrate. The distribution of AR mRNA strongly supports the role of androgens as modulators of behaviorally defined vocal, auditory, and neuroendocrine circuits in teleost fish and vertebrates in general. 2009 Wiley-Liss, Inc.

Oxytocin Intranasal Administration Affects Neural Networks Upstream of GNRH Neurons.

PubMed

Salehi, Mohammad Saied; Khazali, Homayoun; Mahmoudi, Fariba; Janahmadi, Mahyar

2017-08-01

The last decade has witnessed a surge in studies on the clinical applications of intranasal oxytocin as a method of enhancing social interaction. However, the molecular and cellular mechanisms underlying its function are not completely understood. Since oxytocin is involved in the regulation of hypothalamic-pituitary-gonadal axis by affecting the gonadotropin-releasing hormone (GNRH) system, the present study addressed whether intranasal application of oxytocin has a role in affecting GNRH expression in the male rat hypothalamus. In addition, we assessed expression of two excitatory (kisspeptin and neurokinin B) and two inhibitory (dynorphin and RFamide-related peptide-3) neuropeptides upstream of GNRH neurons as a possible route to relay oxytocin information. Here, adult male rats received 20, 40, or 80 μg oxytocin intranasally once a day for 10 consecutive days, and then, the posterior (PH) and anterior hypothalamus (AH) dissected for evaluation of target genes. Using qRT-PCR, we found that oxytocin treatment increased Gnrh mRNA levels in both the PH and AH. In addition, oxytocin at its highest dose increased kisspeptin expression in the AH by around 400%, whereas treatments, dose dependently decreased kisspeptin mRNA in the PH. The expression of neurokinin B was increased from the basal levels following the intervention. Furthermore, although intranasal-applied oxytocin decreased hypothalamic RFamide-related peptide-3 mRNA level, the dynorphin mRNA was not affected. These observations are consistent with the hypothesis that applications of intranasal oxytocin can affect the GNRH system.

Ovarian kisspeptin expression is related to age and to monocyte chemoattractant protein-1.

PubMed

Merhi, Zaher; Thornton, Kimberley; Bonney, Elizabeth; Cipolla, Marilyn J; Charron, Maureen J; Buyuk, Erkan

2016-04-01

The objective of this study was to test the hypothesis that ovarian kisspeptin (kiss1) and its receptor (kiss1r) expression are affected by age, obesity, and the age- and obesity-related chemokine monocyte chemoattractant protein-1 (MCP-1). Ovaries from reproductive-aged and older C57BL/6J mice fed normal chow (NC) or high-fat (HF) diet, ovaries from age-matched young MCP-1 knockout and young control mice on NC, and finally, cumulus and mural granulosa cells (GCs) from women who underwent in vitro fertilization (IVF) were collected. Kiss1, kiss1r, anti-Mullerian hormone (AMH), and AMH receptor (AMHR-II) messenger RNA (mRNA) expression levels were quantified using real-time polymerase chain reaction (RT-PCR). In mouse ovaries, kiss1 and kiss1r mRNA levels were significantly higher in old compared to reproductive-aged mice, and diet-induced obesity did not alter kiss1 or kiss1r mRNA levels. Compared to young control mice, young MCP-1 knockout mice had significantly lower ovarian kiss1 mRNA but significantly higher AMH and AMHR-II mRNA levels. In human cumulus GCs, kiss1r mRNA levels were positively correlated with age but not with BMI. There was no expression of kiss1 mRNA in either cumulus or mural GCs. These data suggest a possible age-related physiologic role for the kisspeptinergic system in ovarian physiology. Additionally, the inflammatory MCP-1 may be associated with kiss1 and AMH genes, which are important in ovulation and folliculogenesis, respectively.

Regulation of GnRH I receptor gene expression by the GnRH agonist triptorelin, estradiol, and progesterone in the gonadotroph-derived cell line alphaT3-1.

PubMed

Weiss, J M; Polack, S; Treeck, O; Diedrich, K; Ortmann, O

2006-08-01

The secretion of luteinizing hormone (LH) and the GnRH receptor (GnRH-R) concentration are modulated by ovarian steroids and GnRH. To elucidate whether this regulation is due to alterations at the transcriptional level, we examined the GnRH I-R mRNA expression in the gonadotroph-derived cell line alphaT3-1 treated with different estradiol and progesterone paradigms and the GnRH I agonist triptorelin. alphaT3-1 cells were treated with different steroid paradigms: 1 nM estradiol or 100 nM progesterone for 48 h alone or in combination. Cells were exposed to 10 nM or 100 pM triptorelin for 30 min, 3 h, 9 h, or, in pulsatile way, with a 5-min pulse per hour. The GnRH I-R mRNA was determined by Northern blot analysis. GnRH I-R mRNA from cells treated with continuous triptorelin decreased in a time- and concentration-dependent manner. Pulsatile triptorelin increased GnRH I-R gene expression. Progesterone alone further enhanced this effect, whereas estradiol and its combination with progesterone diminished it. Continuous combined treatment with estradiol and progesterone lead to a significant decrease of GnRH I-R mRNA by 30% and by 35% for estradiol alone. The addition of 10 nM triptorelin for 30 min or 3 h could not influence that steroid effect. In conclusion, estradiol and progesterone exclusively decreased GnRH I-R mRNA in alphaT3-1 cells no matter whether they are treated additionally with the GnRH I agonist triptorelin. The enhanced sensitivity of gonadotrophs and GnRH I-R upregulation by estradiol is not due to increased GnRH I gene expression because GnRH I-R mRNA is downregulated by estradiol and progesterone. Other pathways of the GnRH I-R signal transduction might be involved.

Low BMI is correlated with increased TGF-β and IL-10 mRNA levels in the peripheral blood of breast cancer patients.

PubMed

Liu, Chao; Wang, Qian; Sun, Bing; Meng, Xiangying; Li, Lan; Yang, Liuchun; Cong, Yang; Liu, Jiannan; Xuan, Liang; Huang, Yan; Wu, Shikai

2018-03-01

Transforming growth factor-β (TGF-β), interleukin-10 (IL-10), and forkhead box P3 (Foxp3) have important roles in breast cancer development. Previous studies confirmed a correlation between these immune molecules and tumor characteristics, but their association with nutritional status in breast cancer is largely unknown. We aimed to investigate the association between body mass index (BMI), hemoglobin, total protein, albumin, globulin (GLB), albumin/GLB ratio (AGR), pre-albumin, prognostic nutritional index, and TGF-β, IL-10, and Foxp3 mRNA expression in patients with breast cancer. Quantitative real-time PCR was used to detect the mRNA expression of TGF-β, IL-10, and Foxp3 in the peripheral blood of 107 patients with breast cancer and 21 healthy controls. We found that TGF-β mRNA levels were 2.6-fold, 3.2-fold, and 2.3-fold higher in patients with low BMI (<23), low AGR, and high GLB, respectively, than in their counterparts (P < 0.05). In addition, IL-10 mRNA expression levels in patients with normal BMI (<23) were 2.8-fold and 3.5-fold higher than in those who were overweight (23≤ BMI <25) and obese (BMI ≥ 25), respectively (P < 0.05). In addition, TGF-β, IL-10, and Foxp3 mRNA levels were significantly higher in patients with breast cancer than in healthy controls (P < 0.05). In summary, our results suggest that nutritional status, especially BMI, may strongly affect systematic immune function in patients with breast cancer. © 2018 IUBMB Life, 70(3):237-245, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

The evolution and functional characterization of lined seahorse (Hippocampus erectus) CCKs involved in fasting and thermal stress response.

PubMed

Zhang, Huixian; Qin, Geng; Sun, Jinhui; Zhang, Bo; Lin, Qiang

2018-01-01

The peptide cholecystokinin (CCK) plays an important role in the regulation of vertebrate appetite and feeding behaviour. In the present study, the full-length cDNA and genomic DNA sequences of two CCK precursors were cloned and analysed in the Syngnathidae fish, the lined seahorse (Hippocampus erectus). Both CCK1 and CCK2 in the seahorse consist of four exons. The sequence of the octapeptide of seahorse CCK1 (DYMGWMDF) was the same as that of the chicken and human, while the octapeptide of seahorse CCK2 (DYEGWMDF) was unique among vertebrates. According to the phylogenetic analysis, two types of CCKs were produced by teleost-specific genome duplication (TGD). Both CCK1 and CCK2 were highly expressed in the brain, while detectable amounts of CCK1 mRNA in the brood pouch and CCK2 mRNA in the intestine were also found. Both CCK1 and CCK2 mRNA levels significantly increased during the transition from endogenous to exogenous nutrition. Additionally, fasting induced a significant increase in the CCK1 mRNA expression in the brain of juvenile seahorses but had no effect on CCK2 transcript levels. In addition, the CCK1 and CCK2 mRNA levels in the seahorse brain significantly increased after a high-temperature treatment. Thus, the mRNA expression of CCK had obvious tissue specificities and this preliminary study opens new avenues for further functional studies on the endocrine regulations of CCK in the transition from endogenous to exogenous nutrition, food intake regulation and metabolism in the seahorse. Copyright © 2017 Elsevier Inc. All rights reserved.

Low Incidence along with Low mRNA Levels of EGFRvIII in Prostate and Colorectal Cancers Compared to Glioblastoma

PubMed Central

Peciak, Joanna; Stec, Wojciech J; Treda, Cezary; Ksiazkiewicz, Magdalena; Janik, Karolina; Popeda, Marta; Smolarz, Maciej; Rosiak, Kamila; Hulas-Bigoszewska, Krystyna; Och, Waldemar; Rieske, Piotr; Stoczynska-Fidelus, Ewelina

2017-01-01

Background: The presence as well as the potential role of EGFRvIII in tumors other than glioblastoma still remains a controversial subject with many contradictory data published. Previous analyses, however, did not consider the level of EGFRvIII mRNA expression in different tumor types. Methods: Appropriately designed protocol for Real-time quantitative reverse-transcription PCR (Real-time qRT-PCR) was applied to analyze EGFRvIII and EGFRWT mRNA expression in 155 tumor specimens. Additionally, Western Blot (WB) analysis was performed for selected samples. Stable cell lines showing EGFRvIII expression (CAS-1 and DK-MG) were analyzed by means of WB, immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH). Results: Our analyses revealed EGFRvIII expression in 27.59% of glioblastomas (8/29), 8.11% of colorectal cancers (3/37), 6.52% of prostate cancers (3/46) and none of breast cancers (0/43). Despite the average relative expression of EGFRvIII varying greatly among tumors of different tissues (approximately 800-fold) or even within the same tissue group (up to 8000-fold for GB), even the marginal expression of EGFRvIII mRNA can be detrimental to cancer progression, as determined by the analysis of stable cell lines endogenously expressing the oncogene. Conclusion: EGFRvIII plays an unquestionable role in glioblastomas with high expression of this oncogene. Our data suggests that EGFRvIII importance should not be underestimated even in tumors with relatively low expression of this oncogene. PMID:28123609

Peroxisome proliferator-activated receptor (PPAR) isoforms are differentially expressed in peri-implantation porcine conceptuses.

PubMed

Blitek, Agnieszka; Szymanska, Magdalena

2017-10-01

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family of ligand-dependent transcription factors. PPARs are critical regulators of glucose homeostasis and lipid metabolism, and affect cell proliferation and differentiation. In the current study, we examined (1) the profiles of PPARA, PPARD, and PPARG mRNA expression and DNA binding activity in porcine conceptuses collected on Days 10-11 (spherical and tubular conceptuses), 11-12 (filamentous conceptuses), 13-14, and 15-16 (elongated conceptuses) of pregnancy, (2) the presence of PPARA, PPARD, and PPARG proteins in Days 10, 12, and 15 conceptuses. Moreover, we analyzed the abundance of retinoid X receptor (RXR; PPARs heterodimer partner) transcripts as well as the correlation between PPARs mRNA expression and the expression of genes important for and/or associated with elongation of porcine conceptuses: aromatase (CYP19A1), prostaglandin endoperoxide synthase 2 (PTGS2), glucose transporter 1 (SLC2A1), and interleukin 1B (IL1B). PPARA mRNA expression in conceptuses did not change during Days 10-14 of gestation, but was greater on Days 15-16 compared to Days 10-11 (P < 0.05). A considerable increase in PPARD and PPARG mRNA expression was observed in filamentous conceptuses from Days 11-12 compared to spherical and tubular conceptuses from Days 10-11 (P < 0.01), followed by a decrease on Days 13-14 and 15-16 (P < 0.05). PPARA, PPARD, and PPARG proteins were present in conceptus tissue demonstrating nuclear localization clearly visible on Days 12 and 15 of pregnancy. DNA binding activity of the PPARD isoform was greater in filamentous conceptuses from Days 11-12 than in spherical and tubular conceptuses from Days 10-11 (P < 0.01). Moreover, concentrations of active PPARD and PPARG proteins in nuclear fractions of conceptus tissue were greater on Days 11-12 compared to Days 13-14 and 15-16 of pregnancy (P < 0.05). RXRA, RXRD, and RXRG mRNA expression in conceptuses increased on Days 11-12 compared to Days 10-11 (P < 0.05). PPARD and PPARG mRNA expression showed strong positive correlations with PTGS2 mRNA expression (P < 0.0001). Additionally, PPARD gene expression correlated with SLC2A1 and IL1B mRNA expression (P < 0.01). Collectively, these results indicate that among all three PPARs expressed in peri-implantation porcine conceptuses, PPARD and PPARG may be involved in conceptus elongation before implantation. Copyright © 2017 Elsevier Inc. All rights reserved.

[Impacts of the formula of Suoquanwan(SQW) on expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency].

PubMed

Cao, Hong-Ying; Wu, Qing-He; Huang, Ping; He, Jin-Yang

2009-06-01

To observe the impacts of the formula of Suoquanwan (SQW) on the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency. The model rats were induced by adenine (250 mg/kg) for 4 weeks, then treated respectively with SQW or dDAVP. The expression of AQP-2 mRNA and AVPR-V2 mRNA in kidney of Yang-deficiency model by realtime fluorescence quantitative PCR method were investigated. In model rats, the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney decreased, dDAVP and SQW high dose could increased the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney. The others had no influence on the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney. SQW can increase the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency.

Role of the mRNA export factor Sus1 in oxidative stress tolerance in Candida albicans.

PubMed

Xiao, Chenpeng; Yu, Qilin; Zhang, Bing; Li, Jianrong; Zhang, Dan; Li, Mingchun

2018-02-05

In eukaryotes, the nuclear export of mRNAs is essential for gene expression. However, little is known about the role of mRNA nuclear export in the important fungal pathogen, Candida albicans. In this study, we identified C. albicans Sus1, a nucleus-localized protein that is required for mRNA export. Interestingly, the sus1Δ/Δ displayed hyper-sensitivity to extracellular oxidative stress, enhanced ROS accumulation and severe oxidative stress-related cell death. More strikingly, although the mutant exhibited normal activation of the expression of oxidative stress response (OSR) genes, it had attenuated activity of ROS scavenging system, which may be attributed to the defect in OSR mRNA export in this mutant. In addition, the virulence of the sus1Δ/Δ was seriously attenuated. Taken together, our findings provide evidence that the mRNA export factor Sus1 plays an important role in oxidative stress tolerance and pathogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

Bacillus subtilis and yeast cell wall improve the intestinal health of broilers challenged by Clostridium perfringens.

PubMed

Li, Z; Wang, W; Lv, Z; Liu, D; Guo, Y

2017-12-01

1. The objective was to investigate the effects of Bacillus subtilis, yeast cell wall (YCW) and their combination on intestinal health of broilers challenged by Clostridium perfringens over a 21-d period. 2. Using a 5 × 2 factorial arrangement of treatments, 800 1-d-old male Cobb 500 broilers were used to study the effects of feed additives (without additive or with zinc bacitracin, B. subtilis, YCW, and the combination of B. subtilis and YCW), pathogen challenge (without or with Clostridium perfringens challenge), and their interactive effects. 3. C. perfringens infection increased intestinal lesions scores, damaged intestinal histomorphology, increased serum endotoxin concentration, cytokine mRNA expression and intestinal population of C. perfringens and Escherichia coli and decreased ileal bifidobacteria numbers. The 4 additives decreased serum endotoxin. Zinc bacitracin tended to decrease cytokine mRNA expression and the intestinal number of C. perfringens and E. coli. B. subtilis, YCW and their combination increased cytokine mRNA expression. B. subtilis and YCW decreased the number of C. perfringens and E. coli in the ileum, and their combination decreased pathogens numbers in the ileum and caecum. 4. In conclusion, B. subtilis, YCW and their combination improved the intestinal health of NE-infected broilers, and could be potential alternatives to antibiotics.

Exon 2-mediated c-myc mRNA decay in vivo is independent of its translation.

PubMed Central

Pistoi, S; Roland, J; Babinet, C; Morello, D

1996-01-01

We have previously shown that the steady-state level of c-myc mRNA in vivo is primarily controlled by posttranscriptional regulatory mechanisms. To identify the sequences involved in this process, we constructed a series of H-2/myc transgenic lines in which various regions of the human c-MYC gene were placed under the control of the quasi-ubiquitous H-2K class I regulatory sequences. We demonstrated that the presence of one of the two coding exons, exon 2 or exon 3, is sufficient to confer a level of expression of transgene mRNA similar to that of endogenous c-myc in various adult tissues as well as after partial hepatectomy or after protein synthesis inhibition. We now focus on the molecular mechanisms involved in modulation of expression of mRNAs containing c-myc exon 2 sequences, with special emphasis on the coupling between translation and c-myc mRNA turnover. We have undertaken an analysis of expression, both at the mRNA level and at the protein level, of new transgenic constructs in which the translation is impaired either by disruption of the initiation codon or by addition of stop codons upstream of exon 2. Our results show that the translation of c-myc exon 2 is not required for regulated expression of the transgene in the different situations analyzed, and therefore they indicate that the mRNA destabilizing function of exon 2 is independent of translation by ribosomes. Our investigations also reveal that, in the thymus, some H-2/myc transgenes express high levels of mRNA but low levels of protein. Besides the fact that these results suggest the existence of tissue-specific mechanisms that control c-myc translatability in vivo, they also bring another indication of the uncoupling of c-myc mRNA translation and degradation. PMID:8756668

Effects of gold thioglucose treatment on central corticotrophin-releasing hormone systems in mice.

PubMed

Noguchi, T; Makino, S; Shinahara, M; Nishiyama, M; Hashimoto, K; Terada, Y

2013-04-01

Systemic administration of gold thioglucose (GTG) causes a hypothalamic lesion that extends from the ventral part of the ventromedial hypothalamus (VMH) to the dorsal part of the arcuate nucleus (ARC), resulting in hyperphagia and obesity in mice. In the present study, we used in situ hybridisation histochemistry to explore the effects of GTG on the central corticotrophin-releasing hormone (CRH) system, which regulates feeding and energy homeostasis. Type 2 CRH receptor (CRHR-2) mRNA expression decreased by 40% at 8 weeks in the VMH and by 40-60% at 2 and 8 weeks in the ARC after GTG injection. By contrast, CRHR-2 mRNA expression in the hypothalamic paraventricular nucleus (PVN) and lateral septum was unchanged. Urocortin (Ucn) 3 mRNA expression in the perifornical area and medial amygdala decreased, whereas CRH mRNA expression in the PVN increased at 2 and 8 weeks after GTG injection. Ucn 1 mRNA expression in the Edingher-Westphal nucleus and Ucn 2 mRNA expression in the PVN were unchanged. Because Ucn 3 is an anorexigenic and a possible endogenous ligand for VMH CRHR-2, our results suggest that decreased Ucn 3 expression and decreased VMH CRHR-2 expression contribute, in part, to GTG-induced hyperphagia and obesity. To determine whether VMH CRHR-2 mediates the anorexigenic effects of Ucn 3, Ucn 3 was administered i.c.v. and food intake was measured 8 weeks after GTG treatment. Ucn 3 decreased cumulative food intake on days 4-7 after surgery compared to i.c.v. administration of vehicle in control mice. By contrast, the anorexigenic effects of i.c.v. Ucn 3 were abolished in GTG-treated mice. Taken together, our results indicate that the Ucn 3 pathway, which innervates the VMH, is involved in appetite regulation via CRHR-2. It remains to be determined whether CRHR-2 in the ARC has additional roles in appetite regulation by Ucn 3. © 2012 British Society for Neuroendocrinology.

ONTOGENIC EXPRESSION OF HUMAN CARBOXYLESTERASE-2 AND CYTOCHROME P450 3A4 IN LIVER AND DUODENUM: POSTNATAL SURGE AND ORGAN-DEPENDENT REGULATION1

PubMed Central

Chen, Yi-Tzai; Trzoss, Lynnie; Yang, Dongfang; Yan, Bingfang

2015-01-01

Human carboxylesterase-2 (CES2) and cytochrome P450 3A4 (CYP3A4) are two major drug metabolizing enzymes that play critical roles in hydrolytic and oxidative biotransformation, respectively. They share substrates but may have opposite effect on therapeutic potential such as the metabolism of the anticancer prodrug irinotecan. Both CES2 and CYP3A4 are expressed in the liver and the gastrointestinal tract. This study was conducted to determine whether CES2 and CYP3A4 are expressed under developmental regulation and whether the regulation occurs differentially between the liver and duodenum. A large number of tissues (112) were collected with majority of them from donors at 1-198 days of age. In addition, multi-sampling (liver, duodenum and jejunum) was performed in some donors. The expression was determined at mRNA and protein levels. In the liver, CES2 and CYP3A4 mRNA exhibited a postnatal surge (1 versus 2 months of age) by 2.7 and 29 fold, respectively. CYP3A4 but not CES2 mRNA in certain pediatric groups reached or even exceeded the adult level. The duodenal samples, on the other hand, showed a gene-specific expression pattern at mRNA level. CES2 mRNA increased with age but the opposite was true with CYP3A4 mRNA. The levels of CES2 and CYP3A4 protein, on the other hand, increased with age in both liver and duodenum. The multi-sampling study demonstrated significant correlation of CES2 expression between the duodenum and jejunum. However, neither duodenal nor jejunal expression correlated with hepatic expression of CES2. These findings establish that developmental regulation occurs in a gene and organ-dependent manner. PMID:25724353

Comparison of Osteogenic and Chondrogenic Differentiation Ability of Buccal Fat Pad Derived Mesenchymal Stem Cells and Gingival Derived Cells.

PubMed

Ghaderi, Hamid; Razmkhah, Mahboobeh; Kiany, Farin; Chenari, Nooshafarin; Haghshenas, Mohammad Reza; Ghaderi, Abbas

2018-06-01

One major goal of tissue engineering and regenerative medicine is to find an appropriate source of mesenchymal stem cells (MSCs) with higher differentiation ability. In this experimental study, the osteogenic and chondrogenic differentiation ability of buccal fat pad derived MSCs (BFP-MSCs) with gingival derived cells (GDCs) were compared. BFP-MSCs and GDCs were cultured enzymatically and expanded. The expanded cells were analyzed for membrane-associated markers, using flow cytometry. Then the ability of these cells to differentiate into osteocyte and chondrocyte was assessed morphologically and by mRNA expression of collagen I (COLL), BGLA and bone morphogenetic protein 2 (BMP2) using qRT-PCR. Flow cytometry analysis showed that both BFP-MSCs and GDCs expressed the characteristic stem cell markers such as CD73, CD44, and CD90, whereas they did not express hematopoietic markers. Mineralized calcium deposition was observed apparently in BFP-MSCs cultured in osteogenic medium but GDCs showed fewer mineralized nodules. The mRNA expression levels of BGLA and BMP2 showed 7×105 and 733-fold more mRNA expression in BFP-MSCs treated with differentiation media compared to the control group. In chondrogenic differentiation, BFP-MSCs transformed from a spindle to a cuboidal shape while GDCs showed only a slight transformation. In addition, mRNA expression of COLL showed 282-fold higher expression in BFP-MSCs in comparison to the control group. Such significant difference in mRNA expression of BGLA, BMP2, and COLL was not observed in GDCs compared to their corresponding controls. Based on the present results, BFP yields a greater proportion of stem cells compared to gingiva. Therefore, this tissue can be introduced as an easily available source for the treatment of periodontal defects and other maxillofacial injuries.

Profiling of m6A RNA modifications identified an age-associated regulation of AGO2 mRNA stability.

PubMed

Min, Kyung-Won; Zealy, Richard W; Davila, Sylvia; Fomin, Mikhail; Cummings, James C; Makowsky, Daniel; Mcdowell, Catherine H; Thigpen, Haley; Hafner, Markus; Kwon, Sang-Ho; Georgescu, Constantin; Wren, Jonathan D; Yoon, Je-Hyun

2018-06-01

Gene expression is dynamically regulated in a variety of mammalian physiologies. During mammalian aging, there are changes that occur in protein expression that are highly controlled by the regulatory steps in transcription, post-transcription, and post-translation. Although there are global profiles of human transcripts during the aging processes available, the mechanism(s) by which transcripts are differentially expressed between young and old cohorts remains unclear. Here, we report on N6-methyladenosine (m6A) RNA modification profiles of human peripheral blood mononuclear cells (PBMCs) from young and old cohorts. An m6A RNA profile identified a decrease in overall RNA methylation during the aging process as well as the predominant modification on proteincoding mRNAs. The m6A-modified transcripts tend to be more highly expressed than nonmodified ones. Among the many methylated mRNAs, those of DROSHA and AGO2 were heavily methylated in young PBMCs which coincided with a decreased steady-state level of AGO2 mRNA in the old PBMC cohort. Similarly, downregulation of AGO2 in proliferating human diploid fibroblasts (HDFs) also correlated with a decrease in AGO2 mRNA modifications and steady-state levels. In addition, the overexpression of RNA methyltransferases stabilized AGO2 mRNA but not DROSHA and DICER1 mRNA in HDFs. Moreover, the abundance of miRNAs also changed in the young and old PBMCs which are possibly due to a correlation with AGO2 expression as observed in AGO2-depleted HDFs. Taken together, we uncovered the role of mRNA methylation on the abundance of AGO2 mRNA resulting in the repression of miRNA expression during the process of human aging. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Addition of bone morphogenetic protein type 2 to ascorbate and β-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells

PubMed Central

CRUZ, Ariadne Cristiane Cabral; SILVA, Mariana Lúcia; CAON, Thiago; SIMÕES, Cláudia Maria Oliveira

2012-01-01

Bone morphogenetic protein type 2 (BMP-2) is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. Objectives This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and β-glycerophosphate. Material and Methods Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2) or absence (ASCs+OM) of BMP-2. The alkaline phosphatase (ALP) activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II), osteonectin, and osteocalcin were evaluated by qPCR. Results: ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. Conclusions We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity), intermediate (osteonectin and osteocalcin), or final (calcium deposition) phases, suggesting that the exogenous addition of BMP-2 did not improve the in vitro osteogenesis process of human ASCs. PMID:23329244

Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast.

PubMed

Lamping, Erwin; Niimi, Masakazu; Cannon, Richard D

2013-07-29

A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5' UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5' UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = -15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = -4.4 kcal/mol) inhibited Cdr1p expression by ~50%. We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression.

Small, synthetic, GC-rich mRNA stem-loop modules 5′ proximal to the AUG start-codon predictably tune gene expression in yeast

PubMed Central

2013-01-01

Background A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5′ UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Results Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5′ UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = −15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = −4.4 kcal/mol) inhibited Cdr1p expression by ~50%. Conclusion We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression. PMID:23895661

Developmental changes in the hypothalamic mRNA expression levels of brain-derived neurotrophic factor and serum leptin levels: Their responses to fasting in male and female rats.

PubMed

Iwasa, Takeshi; Matsuzaki, Toshiya; Yano, Kiyohito; Munkhzaya, Munkhsaikhan; Tungalagsuvd, Altankhuu; Yiliyasi, Maira; Kuwahara, Akira; Irahara, Minoru

2016-11-01

The actions and responses of hypothalamic appetite regulatory factors change markedly during the neonatal to pre-pubertal period in order to maintain appropriate metabolic and nutritional conditions. In this study, we examined the developmental changes in the hypothalamic mRNA levels of brain-derived neurotrophic factor (BDNF), which is a potent anorectic factor and the changes in the sensitivity of the hypothalamic expression of this factor to fasting during the neonatal to pre-pubertal period. Under fed conditions, hypothalamic BDNF mRNA expression decreased during development in both male and female rats. Similarly, the serum levels of leptin, which is a positive regulator of hypothalamic BDNF expression, also tended to fall during the developmental period. The serum leptin level and the hypothalamic BDNF mRNA level were found to be positively correlated in both sexes under the fed conditions. Hypothalamic BDNF mRNA expression was decreased by 24h fasting (separating the rats from their mothers) in the early neonatal period (postnatal day 10) in both males and females, but no such changes were seen at postnatal day 20. Twenty-four hours' fasting (food deprivation) did not affect hypothalamic BDNF mRNA expression in the pre-pubertal period (postnatal day 30). On the other hand, the rats' serum leptin levels were decreased by 24h fasting (separating the rats from their mothers at postnatal day 10 and 20, and food deprivation at postnatal day 30) throughout the early neonatal to pre-pubertal period. The correlation between serum leptin and hypothalamic BDNF mRNA levels was not significant under the fasted conditions. It can be speculated that leptin partially regulates hypothalamic BDNF mRNA levels, but only in fed conditions. Such changes in hypothalamic BDNF expression might play a role in maintaining appropriate metabolic and nutritional conditions and promoting normal physical development. In addition, because maternal separation induces a negative energy balance and short- and long-term stress responses, it is also possible that reductions in hypothalamic BDNF mRNA levels in the early neonatal period (postnatal day 10) may be partially induced by stress responses of the maternal deprivation. Copyright © 2016 ISDN. Published by Elsevier Ltd. All rights reserved.

Effects of molybdenum and cadmium on the oxidative damage and kidney apoptosis in Duck.

PubMed

Shi, Lele; Cao, Huabin; Luo, Junrong; Liu, Ping; Wang, Tiancheng; Hu, Guoliang; Zhang, Caiying

2017-11-01

Molybdenum (Mo) is an essential element for human beings and animals; however, high dietary intake of Mo can lead to adverse reactions. Cadmium (Cd) is one of the major transitional metals which has toxic effects in animals. To investigate the co-induced toxic effects of Mo and Cd on oxidative damage and kidney apoptosis in duck, 120 ducks were randomly divided into control group and 5 treatment groups which were treated with a commercial diet containing different dosages of Mo and Cd. Kidney samples were collected on the 60th and 120th days to determine the mRNA expression levels of ceruloplasmin (CP), metallothionein (MT), Bak-1, and Caspase-3 by quantitative RT-PCR. Additionally, we also determined the antioxidant activity indexes and contents of Mo, Cd, copper (Cu), iron (Fe), zinc (Zn), and selenium (Se) in serum. Meanwhile, ultrastructural changes of the kidney were observed. The results showed that glutathione reductase (GR) activity and CP level in serum were decreased in combination groups. In addition, the antioxidant indexes were decreased in co-treated groups compared with single treated groups. The mRNA expression levels of Bak-1 and Caspase-3 increased in co-treated groups. The mRNA expression level of CP in high-dose combination group was downregulated, while the mRNA expression of MT was upregulated except for low-dose Mo group. Additionally, in the later period the content of Cu in serum decreased in joint groups while the contents of Mo and Cd increased. In addition, ultrastructural changes showed mitochondrial crest fracture, swelling, deformed nuclei, and karyopyknosis in co-treated groups. Taken together, it was suggested that dietary Mo and Cd might lead to oxidative stress, kidney apoptosis and disturb homeostasis of trace elements in duck, and it showed a possible synergistic relationship between the two elements. Copyright © 2017 Elsevier Inc. All rights reserved.

Deregulated Expression of SRC, LYN and CKB Kinases by DNA Methylation and Its Potential Role in Gastric Cancer Invasiveness and Metastasis

PubMed Central

Rey, Juan Antonio; Pinto, Giovanny Rebouças; Lamarão, Leticia Martins; Montenegro, Raquel Carvalho; Alves, Ana Paula Negreiros Nunes; Assumpção, Paulo Pimentel; Borges, Barbara do Nascimento; Smith, Marília Cardoso; Burbano, Rommel Rodriguez

2015-01-01

Kinases are downstream modulators and effectors of several cellular signaling cascades and play key roles in the development of neoplastic disease. In this study, we aimed to evaluate SRC, LYN and CKB protein and mRNA expression, as well as their promoter methylation, in gastric cancer. We found elevated expression of SRC and LYN kinase mRNA and protein but decreased levels of CKB kinase, alterations that may have a role in the invasiveness and metastasis of gastric tumors. Expression of the three studied kinases was also associated with MYC oncogene expression, a possible biomarker for gastric cancer. To understand the mechanisms that regulate the expression of these genes, we evaluated the DNA promoter methylation of the three kinases. We found that reduced SRC and LYN methylation and increased CKB methylation was associated with gastric cancer. The reduced SRC and LYN methylation was associated with increased levels of mRNA and protein expression, suggesting that DNA methylation is involved in regulating the expression of these kinases. Conversely, reduced CKB methylation was observed in samples with reduced mRNA and protein expression, suggesting CKB expression was found to be only partly regulated by DNA methylation. Additionally, we found that alterations in the DNA methylation pattern of the three studied kinases were also associated with the gastric cancer onset, advanced gastric cancer, deeper tumor invasion and the presence of metastasis. Therefore, SRC, LYN and CKB expression or DNA methylation could be useful markers for predicting tumor progression and targeting in anti-cancer strategies. PMID:26460485

Deregulated Expression of SRC, LYN and CKB Kinases by DNA Methylation and Its Potential Role in Gastric Cancer Invasiveness and Metastasis.

PubMed

Mello, Adriano Azevedo; Leal, Mariana Ferreira; Rey, Juan Antonio; Pinto, Giovanny Rebouças; Lamarão, Leticia Martins; Montenegro, Raquel Carvalho; Alves, Ana Paula Negreiros Nunes; Assumpção, Paulo Pimentel; Borges, Barbara do Nascimento; Smith, Marília Cardoso; Burbano, Rommel Rodriguez

2015-01-01

Kinases are downstream modulators and effectors of several cellular signaling cascades and play key roles in the development of neoplastic disease. In this study, we aimed to evaluate SRC, LYN and CKB protein and mRNA expression, as well as their promoter methylation, in gastric cancer. We found elevated expression of SRC and LYN kinase mRNA and protein but decreased levels of CKB kinase, alterations that may have a role in the invasiveness and metastasis of gastric tumors. Expression of the three studied kinases was also associated with MYC oncogene expression, a possible biomarker for gastric cancer. To understand the mechanisms that regulate the expression of these genes, we evaluated the DNA promoter methylation of the three kinases. We found that reduced SRC and LYN methylation and increased CKB methylation was associated with gastric cancer. The reduced SRC and LYN methylation was associated with increased levels of mRNA and protein expression, suggesting that DNA methylation is involved in regulating the expression of these kinases. Conversely, reduced CKB methylation was observed in samples with reduced mRNA and protein expression, suggesting CKB expression was found to be only partly regulated by DNA methylation. Additionally, we found that alterations in the DNA methylation pattern of the three studied kinases were also associated with the gastric cancer onset, advanced gastric cancer, deeper tumor invasion and the presence of metastasis. Therefore, SRC, LYN and CKB expression or DNA methylation could be useful markers for predicting tumor progression and targeting in anti-cancer strategies.

Leptin receptor mRNA in rat brain astrocytes

PubMed Central

Hsuchou, Hung; Pan, Weihong; Barnes, Maria J.; Kastin, Abba J.

2009-01-01

We recently reported that mouse astrocytes express leptin receptors (ObR), and that obesity induces upregulation of astrocytic ObR. To provide further evidence of the importance of astrocytic ObR expression, we performed double-labeling fluorescent in-situ hybridization (FISH) and immunohistochemistry in the rat hypothalamus. Laser confocal microscopic image analysis showed that ObR mRNA was present in glial fibrillary acidic protein (+) cells that show distinctive astrocytic morphology as well as in neurons. In addition to the presence of ObR mRNA, ObR protein was shown in both astrocytes and neurons in the rat hypothalamus by double-labeling immunohistochemistry. In cultured rat C6 astrocytoma cells treated with different doses of lipopolysaccharide for 6 h, the mRNA for ObRa or ObRb did not show significant changes, as measured by quantitative RT-PCR. However, the protein expression of both ObRa and ObRb, determined by western blotting, was increased after the C6 cells were treated with either lipopolysaccharide or tumor necrosis factor-α. The results indicate that astrocytic ObR expression is present in rats as well as mice, and that it probably plays a role in the neuroinflammatory response. PMID:19747514

miR-106a suppresses tumor cells death in colorectal cancer through targeting ATG7.

PubMed

Hao, Haibin; Xia, Guangfeng; Wang, Chao; Zhong, Fuping; Liu, Laipeng; Zhang, Dong

2017-06-01

Autophagy-related gene 7 (ATG7) and miR-106a play an important role in cancer cell autophagy and apoptosis, but the outcome of ATG7 and miR-106a in colorectal cancer (CRC) still remains not clear. In this study, we found that ATG7 and miR-106a expression were mutually related with cell death and prognosis in CRC patients. In addition, we also showed that ATG7 and miR-106a expression were changeable in colorectal cancer cell lines when compared with normal cell lines, but ATG7 and miR-106a mRNA level was negatively correlated. Furthermore, ATG7 protein and mRNA levels decreased after over-expression of miR-106a, whereas the suppression of ATG7 had the opposite effect. We confirmed that miR-106a down-regulated ATG7 mRNA level by binding the specific sequence of ATG7 mRNA 3'UTR region. Moreover, the over-expression of ATG7 induced CRC cells death both in vitro and in vivo. Taken together, our study data demonstrated that ATG7 aggravated the cell death of CRC, which was inhibited by miR-106a.

Chronic Heat Stress Impairs the Quality of Breast-Muscle Meat in Broilers by Affecting Redox Status and Energy-Substance Metabolism.

PubMed

Lu, Zhuang; He, Xiaofang; Ma, Bingbing; Zhang, Lin; Li, Jiaolong; Jiang, Yun; Zhou, Guanghong; Gao, Feng

2017-12-27

We investigated the molecular mechanisms by which chronic heat stress impairs the breast-meat quality of broilers. Broilers were assigned to three groups: the normal control (NC) group, heat-stress (HS) group, and pair-fed (PF) group. After 7 days of heat exposure (32 °C), the high temperature had caused oxidative stress; elevated the activity of citrate synthase (CS), the mRNA expression of M-CPT1, and the phosphorylation level of AMPKα; and reduced the mRNA expression of avUCP. After 14 days of heat exposure, the heat stress had increased the lightness and drip loss and decreased the pH and shear force of the breast meat. Additionally, the heat exposure had increased the mRNA expressions of FAS, ACC, and PDK4; the content of lipids; and the activities of lactic dehydrogenase and pyruvate kinase, and it had decreased the mRNA expression of M-CPT1 and the activity of CS. In conclusion, chronic heat stress impairs meat quality by causing mitochondria to malfunction and affecting energy-substance aerobic metabolism, resulting in increased glycolysis and intramuscular fat deposition.

Developmental Patterns of Doublecortin Expression and White Matter Neuron Density in the Postnatal Primate Prefrontal Cortex and Schizophrenia

PubMed Central

Fung, Samantha J.; Joshi, Dipesh; Allen, Katherine M.; Sivagnanasundaram, Sinthuja; Rothmond, Debora A.; Saunders, Richard; Noble, Pamela L.; Webster, Maree J.; Shannon Weickert, Cynthia

2011-01-01

Postnatal neurogenesis occurs in the subventricular zone and dentate gyrus, and evidence suggests that new neurons may be present in additional regions of the mature primate brain, including the prefrontal cortex (PFC). Addition of new neurons to the PFC implies local generation of neurons or migration from areas such as the subventricular zone. We examined the putative contribution of new, migrating neurons to postnatal cortical development by determining the density of neurons in white matter subjacent to the cortex and measuring expression of doublecortin (DCX), a microtubule-associated protein involved in neuronal migration, in humans and rhesus macaques. We found a striking decline in DCX expression (human and macaque) and density of white matter neurons (humans) during infancy, consistent with the arrival of new neurons in the early postnatal cortex. Considering the expansion of the brain during this time, the decline in white matter neuron density does not necessarily indicate reduced total numbers of white matter neurons in early postnatal life. Furthermore, numerous cells in the white matter and deep grey matter were positive for the migration-associated glycoprotein polysialiated-neuronal cell adhesion molecule and GAD65/67, suggesting that immature migrating neurons in the adult may be GABAergic. We also examined DCX mRNA in the PFC of adult schizophrenia patients (n = 37) and matched controls (n = 37) and did not find any difference in DCX mRNA expression. However, we report a negative correlation between DCX mRNA expression and white matter neuron density in adult schizophrenia patients, in contrast to a positive correlation in human development where DCX mRNA and white matter neuron density are higher earlier in life. Accumulation of neurons in the white matter in schizophrenia would be congruent with a negative correlation between DCX mRNA and white matter neuron density and support the hypothesis of a migration deficit in schizophrenia. PMID:21966452

A disintegrin and metalloproteinase 17 mRNA and protein expression in esophageal squamous cell carcinoma, as well as its clinicopathological factors and prognosis

PubMed Central

LIU, HONG-BIN; YANG, QI-CHANG; SHEN, YI; ZHU, YAN; ZHANG, XIAO-JUAN; CHEN, HAO

2015-01-01

The aim of the present study was to explore a disintegrin and metalloproteinase 17 (ADAM17) mRNA and protein expression in esophageal squamous cell carcinoma and its association with clinicopathological factors and prognosis. Through semi-quantitative reverse transcription polymerase chain reaction, the ADAM17 mRNA expression in 50 cases of esophageal squamous cell carcinoma and corresponding normal esophageal mucosa were detected. Using streptavidin peroxidase conjugated immunohistochemistry, ADAM17 protein levels were detected in 80 cases of esophageal squamous cell carcinoma and corresponding normal esophageal mucosa. A log rank test and the Cox proportional hazards model were used for the esophageal cancer survival analysis. ADAM17 mRNA expression levels in esophageal squamous cell carcinoma and corresponding normal esophageal mucosa were 0.937±0.241 and 0.225±0.077, respectively (P<0.01). ADAM17 mRNA expression in esophageal squamous cell carcinoma was correlated with lymph node metastasis (P<0.01) and tumor, node and metastasis (TNM) staging (P<0.05), however, it was not correlated with gender, age or histological grade (P>0.05). ADAM17 protein expression rates in esophageal squamous cell carcinoma and corresponding normal esophageal mucosa were 66.25 and 6.25% respectively, a difference that was statistically significant (P<0.01). In addition, ADAM17 protein expression in esophageal squamous cells was correlated with lymph node metastasis and TNM stage (P<0.05), while it was not correlated with gender, age or histological grade (P>0.05). ADAM17 protein expression and epidermal growth factor receptor (EGFR) protein expression were positively correlated (P<0.01). Lymph node metastasis, TNM stage, ADAM17 and EGFR protein expression may be used as independent prognostic indicators of esophageal squamous cell carcinoma (all P<0.05). ADAM17 mRNA and protein were highly expressed in esophageal squamous cell carcinoma; they have important roles in invasion and metastasis and a certain value in judging the prognosis of patients with esophageal squamous cell carcinoma. PMID:25351873

Lower Selenoprotein T Expression and Immune Response in the Immune Organs of Broilers with Exudative Diathesis Due to Selenium Deficiency.

PubMed

Pan, Tingru; Liu, Tianqi; Tan, Siran; Wan, Na; Zhang, Yiming; Li, Shu

2018-04-01

The objective of the present study was to investigate whether dietary selenium (Se) deficiency would affect the expression of selenoprotein T (SelT) and immune response in the immune organs of broilers. Changes in expression of inflammatory cytokines and oxidative stress response caused by Se deficiency can lead to organism damage, which in turn leads to immune response. Sixty (1-day-old) broilers were divided into the control group and Se-deficiency group. Animal models with exudative diathesis were duplicated in the broilers by feeding them Se-deficient diet for 20 days. After the Se-deficient group exhibited symptoms of exudative diathesis, all the broilers were euthanized, and their immune organs were taken for analysis. The tissues including spleen, bursa of Fabricius, and thymus were treated to determine the pathological changes (including microscopic and ultramicroscopic), the messenger RNA (mRNA) expression levels of SelT and its synthetase (SecS and SPS1), cytokine mRNA expression levels, and antioxidant status. The microscopic and ultramicroscopic analyses showed that immune tissues were obviously injured in the Se-deficient group. The mRNA expression of SelT was decreased compared with that in the control group. Meanwhile, the mRNA expression levels of SecS and SPS1 were downregulated. In the Se-deficient group, the mRNA expression levels of IL-1R and IL-1β were higher than those of three control organs. Additionally, the IL-2 and INF-γ mRNA expression levels were lower than those of the control group. The activity of CAT was decreased, and the contents of H 2 O 2 and •OH were increased due to Se deficiency. Pearson method analysis showed that the expression of SelT had a positive correlation with IL-2, INF-γ, SecS, and SPS1 and a negative correlation with IL-1R and IL-1β. In summary, these data indicated that Se-deficient diet decreased the SelT expression and its regulation of oxidative stress, and it inhibited a pleiotropic mechanism of the immune response.

Expression of Glutamine Transporter Slc38a3 (SNAT3) During Acidosis is Mediated by a Different Mechanism than Tissue-Specific Expression

PubMed Central

Balkrishna, Sarojini; Bröer, Angelika; Welford, Scott M.; Hatzoglou, Maria; Bröer, Stefan

2015-01-01

Background Despite homeostatic pH regulation, systemic and cellular pH changes take place and strongly influence metabolic processes. Transcription of the glutamine transporter SNAT3 (Slc38a3) for instance is highly up-regulated in the kidney during metabolic acidosis to provide glutamine for ammonia production. Methods Slc38a3 promoter activity and messenger RNA stability were measured in cultured cells in response to different extracellular pH values. Results Up-regulation of SNAT3 mRNA was mediated both by the stabilization of its mRNA and by the up-regulation of gene transcription. Stabilisation of the mRNA involved a pH-response element, while enhanced transcription made use of a second pH-sensitive Sp1 binding site in addition to a constitutive Sp1 binding site. Transcriptional regulation dominated the early response to acidosis, while mRNA stability was more important for chronic adaptation. Tissue-specific expression of SNAT3, by contrast, appeared to be controlled by promoter methylation and histone modifications. Conclusions Regulation of SNAT3 gene expression by extracellular pH involves post-transcriptional and transcriptional mechanisms, the latter being distinct from the mechanisms that control the tissue-specific expression of the gene. PMID:24854847

Post-Transcriptional Regulation of the Human Mu-Opioid Receptor (MOR) by Morphine-Induced RNA Binding Proteins hnRNP K and PCBP1

PubMed Central

Song, Kyu Young; Choi, Hack Sun; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

2016-01-01

Expression of the mu-opioid receptor (MOR) protein is controlled by extensive transcriptional and post-transcriptional processing. MOR gene expression has previously been shown to be altered by a post-transcriptional mechanism involving the MOR mRNA untranslated region (UTR). Here, we demonstrate for the first time the role of heterogeneous nuclear ribonucleic acids (hnRNA)-binding protein (hnRNP) K and poly(C)-binding protein 1 (PCBP1) as post-transcriptional inducers in MOR gene regulation. In the absence of morphine, a significant level of MOR mRNA is sustained in its resting state and partitions in the translationally inactive polysomal fraction. Morphine stimulation activates the downstream targets hnRNP K and PCPB1 and induces partitioning of the MOR mRNA to the translationally active fraction. Using reporter and ligand binding assays, as well as RNA EMSA, we reveal potential RNP binding sites located in the 5′-untranslated region of human MOR mRNA. In addition, we also found that morphine-induced RNPs could regulate MOR expression. Our results establish the role of hnRNP K and PCPB1 in the translational control of morphine-induced MOR expression in human neuroblastoma (NMB) cells as well as cells stably expressing MOR (NMB1). PMID:27292014

Platelet and intestinal 5-HT2A receptor mRNA in autistic spectrum disorders - results of a pilot study.

PubMed

Kazek, Beata; Huzarska, Małgorzata; Grzybowska-Chlebowczyk, Urszula; Kajor, Maciej; Ciupińska-Kajor, Monika; Woś, Halina; Marszał, Elzbieta

2010-01-01

The etiology and pathogenesis of autistic spectrum disorders (ASD) are still unknown. Platelet hyperserotonemia has been detected in 25-60% of autistic children. Higher incidence of gastrointestinal problems in people with autism is observed. The aim was compare the expression of platelet 5-HT(2A)r mRNA in autistic and non autistic groups. In a subgroup of patients with gastrointestinal problems an upper gastrointestinal tract endoscopy was performed and additionally the expression of 5-HT(2A) receptor mRNA in the duodenum was assessed. The examination was conducted in 79 children - 51 with ASD and 28 without autistic traits. Statistically significant differences between the study and control groups were proven in gastrointestinal problems. The analyses reveal a significantly higher level of 5-HT(2A)r mRNA in platelets of the study group patients, which could suggest serotonin system dysregulation.

Downregulation of aquaporin 9 decreases catabolic factor expression through nuclear factor‑κB signaling ins chondrocytes.

PubMed

Takeuchi, Kazuhiro; Hayashi, Shinya; Matumoto, Tomoyuki; Hashimoto, Shingo; Takayama, Koji; Chinzei, Nobuaki; Kihara, Shinsuke; Haneda, Masahiko; Kirizuki, Shinsuke; Kuroda, Yuichi; Tsubosaka, Masanori; Nishida, Kotaro; Kuroda, Ryosuke

2018-06-13

Aquaporins (AQPs) are small integral membrane proteins that are essential for water transport across membranes. AQP9, one of the 13 mammalian AQPs (including AQP0 to 12), has been reported to be highly expressed in hydrarthrosis and synovitis patients. Given that several studies have identified signal transduction as an additional function of AQPs, it is hypothesized that AQP9 may modulate inflammatory signal transduction in chondrocytes. Therefore, the present study used a model of interleukin (IL)‑1β‑induced inflammation to determine the mechanisms associated with AQP9 functions in chondrocytes. Osteoarthritis (OA) and normal cartilage samples were subjected to immunohistological analysis. In addition, matrix metalloproteinase (MMP)3, MMP13 and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS‑5) mRNA and protein analysis was conducted in normal human articular chondrocytes from the knee (NHAC‑Kn) stimulated with IL‑1β by reverse transcription‑polymerase chain reaction (RT‑qPCR) and western blotting, respectively. AQP9 knockdown was also performed by transfection of AQP9‑specific small interfering RNA using Lipofectamine. AQP1, 3, 7, 9 and 11 mRNA expression levels were detected in OA human chondrocytes and in IL‑1β‑treated normal human chondrocytes. The levels of AQP9, MMP‑3, MMP‑13 and ADAMTS‑5 mRNA were increased by treatment with 10 ng/ml IL‑1β in a time‑dependent manner, while knockdown of AQP9 expression significantly decreased the mRNA levels of the MMP3, MMP13 and ADAMTS‑5 genes, as well as the phosphorylation of IκB kinase (IKK). Treatment with a specific IKK inhibitor also significantly decreased the expression levels of MMP‑3, MMP‑13 and ADAMTS‑5 in response to IL‑1β stimulation. Furthermore, immunohistochemical analysis demonstrated that AQP9 and inflammatory markers were highly expressed in OA cartilage. In addition, the downregulation of AQP9 in cultured chondrocytes decreased the catabolic gene expression in response to IL‑1β stimulation through nuclear factor‑κB signaling. Therefore, AQP9 may be a promising target for the treatment of OA.

Silencing of Kv4.1 potassium channels inhibits cell proliferation of tumorigenic human mammary epithelial cells

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

Jang, Soo Hwa; Choi, Changsun; Hong, Seong-Geun

2009-06-26

Potassium channel activity has been shown to facilitate cell proliferation in cancer cells. In the present study, the role of Kv4.1 channels in immortal and tumorigenic human mammary epithelial cells was investigated. Kv4.1 protein expression was positively correlated with tumorigenicity. Moreover, transfection with siRNAs targeting Kv4.1 mRNA suppressed proliferation of tumorigenic mammary epithelial cells. Experiments using mRNA isolated from human breast cancer tissues revealed that the level of Kv4.1 mRNA expression varied depending on the stage of the tumor. Kv4.1 protein expression increased during stages T2 and T3 compared to normal tissue. These results demonstrated that Kv4.1 plays a rolemore » in proliferation of tumorigenic human mammary epithelial cells. In addition, elevated Kv4.1 expression may be useful as a diagnostic marker for staging mammary tumors and selective blockers of Kv4.1 may serve to suppress tumor cell proliferation.« less

Blood-Brain Barrier Permeability Is Exacerbated in Experimental Model of Hepatic Encephalopathy via MMP-9 Activation and Downregulation of Tight Junction Proteins.

PubMed

Dhanda, Saurabh; Sandhir, Rajat

2018-05-01

The present study was designed to investigate the mechanisms involved in blood-brain barrier (BBB) permeability in bile duct ligation (BDL) model of chronic hepatic encephalopathy (HE). Four weeks after BDL surgery, a significant increase was observed in serum bilirubin levels. Masson trichrome staining revealed severe hepatic fibrosis in the BDL rats. 99m Tc-mebrofenin retention was increased in the liver of BDL rats suggesting impaired hepatobiliary transport. An increase in permeability to sodium fluorescein, Evans blue, and fluorescein isothiocyanate (FITC)-dextran along with increase in water and electrolyte content was observed in brain regions of BDL rats suggesting disrupted BBB. Increased brain water content can be attributed to increase in aquaporin-4 mRNA and protein expression in BDL rats. Matrix metalloproteinase-9 (MMP-9) mRNA and protein expression was increased in brain regions of BDL rats. Additionally, mRNA and protein expression of tissue inhibitor of matrix metalloproteinases (TIMPs) was also increased in different regions of brain. A significant decrease in mRNA expression and protein levels of tight junction proteins, viz., occludin, claudin-5, and zona occluden-1 (ZO-1) was observed in different brain regions of BDL rats. VCAM-1 mRNA and protein expression was also found to be significantly upregulated in different brain regions of BDL animals. The findings from the study suggest that increased BBB permeability in HE involves activation of MMP-9 and loss of tight junction proteins.

Pnrc2 regulates 3'UTR-mediated decay of segmentation clock-associated transcripts during zebrafish segmentation.

PubMed

Gallagher, Thomas L; Tietz, Kiel T; Morrow, Zachary T; McCammon, Jasmine M; Goldrich, Michael L; Derr, Nicolas L; Amacher, Sharon L

2017-09-01

Vertebrate segmentation is controlled by the segmentation clock, a molecular oscillator that regulates gene expression and cycles rapidly. The expression of many genes oscillates during segmentation, including hairy/Enhancer of split-related (her or Hes) genes, which encode transcriptional repressors that auto-inhibit their own expression, and deltaC (dlc), which encodes a Notch ligand. We previously identified the tortuga (tor) locus in a zebrafish forward genetic screen for genes involved in cyclic transcript regulation and showed that cyclic transcripts accumulate post-splicing in tor mutants. Here we show that cyclic mRNA accumulation in tor mutants is due to loss of pnrc2, which encodes a proline-rich nuclear receptor co-activator implicated in mRNA decay. Using an inducible in vivo reporter system to analyze transcript stability, we find that the her1 3'UTR confers Pnrc2-dependent instability to a heterologous transcript. her1 mRNA decay is Dicer-independent and likely employs a Pnrc2-Upf1-containing mRNA decay complex. Surprisingly, despite accumulation of cyclic transcripts in pnrc2-deficient embryos, we find that cyclic protein is expressed normally. Overall, we show that Pnrc2 promotes 3'UTR-mediated decay of developmentally-regulated segmentation clock transcripts and we uncover an additional post-transcriptional regulatory layer that ensures oscillatory protein expression in the absence of cyclic mRNA decay. Copyright © 2017 Elsevier Inc. All rights reserved.

Modification of N6-methyladenosine RNA methylation on heat shock protein expression.

PubMed

Yu, Jiayao; Li, Yi; Wang, Tian; Zhong, Xiang

2018-01-01

This study was conducted to investigate effect of N6-methyladenosine (m6A) RNA methylation on Heat shock proteins (HSPs) and dissect the profile of HSP RNA methylation. The results showed that m6A methyltransferases METTL3 mRNA was decreased in responses to heat shock stress in HepG2 cells, but m6A-specific binding protein YTHDF2 mRNA was upregulated in a manner similar to HSP70 induction. Immunofluorescence staining showed that the majority of YTHDF2 was present in the cytosol, however, nearly all YTHDF2 translocated from the cytosol into the nucleus after heat shock. METTL3 knockdown significantly changed HSP70, HSP60, and HSP27 mRNA expression in HepG2 cells using siRNA, however, mRNA lifetime was not impacted. Silence of YTHDF2 using siRNA did not change expression of HSP70, but significantly increased HSP90, HSP60, and HSPB1 mRNA expression. In addition, m6A-seq revealed that HSP m6A methylation peaks are mainly enriched on exons and around stop codons, and shows a unique distribution profile in the 5'UTR and 3'UTR. Knockdown of METTL3 changed the methylation patterns of HSPs transcript. In conclusion, m6A RNA methylation regulates HSP gene expression. Differential expression of HSPs modulated by m6A may depend on the m6A site and abundance of the target gene. This finding provides insights into new regulatory mechanisms of HSPs in normal and stress situations.

Translational repression of mouse mu opioid receptor expression via leaky scanning

PubMed Central

Song, Kyu Young; Hwang, Cheol Kyu; Kim, Chun Sung; Choi, Hack Sun; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

2007-01-01

Mu opioid receptor (MOR) expression is under temporal and spatial controls, but expression levels of the MOR gene are relatively low in vivo. In addition to transcriptional regulations, upstream AUGs (uAUGs) and open reading frames (uORFs) profoundly affect the translation of the primary ORF and thus the protein levels in several genes. The 5′-untranslated region (UTR) of mouse MOR mRNA contains three uORFs preceding the MOR main initiation codon. In MOR-fused EGFP or MOR promoter/luciferase reporter constructs, mutating each uAUG individually or in combinations increased MOR transient heterologous expression in neuroblastoma NMB and HEK293 cells significantly. Translation of such constructs increased up to 3-fold without altering the mRNA levels if either the third uAUG or both the second and third AUGs were mutated. Additionally, these uAUG-mediated translational inhibitions were independent of their peptide as confirmed by internal mutation analyses in each uORF. Translational studies indicated that protein syntheses were initiated at these uAUG initiation sites, with the third uAUG initiating the highest translation level. These results support the hypothesis that uORFs in mouse MOR mRNA act as negative regulators through a ribosome leaky scanning mechanism. Such leaky scanning resulted in the suppression of mouse MOR under normal conditions. PMID:17284463

Biomaterials for mRNA Delivery

PubMed Central

Islam, Mohammad Ariful; Reesor, Emma K. G.; Xu, Yingjie; Zope, Harshal R.; Zetter, Bruce R.; Shi, Jinjun

2015-01-01

Messenger RNA (mRNA) has recently emerged with remarkable potential as an effective alternative to DNA-based therapies because of several unique advantages. mRNA does not require nuclear entry for transfection activity and has a negligible chance of integrating into the host genome which excludes the possibility of potentially detrimental genomic alternations. Chemical modification of mRNA has further enhanced its stability and decreased its activation of innate immune responses. Additionally, mRNA has been found to have rapid expression and predictable kinetics. Nevertheless, the ubiquitous application of mRNA remains challenging given its unfavorable attributes, such as large size, negative charge and susceptibility to enzymatic degradation. Further refinement of mRNA delivery modalities is therefore essential for its development as a therapeutic tool. This review provides an exclusive overview of current state-of-the-art biomaterials and nanotechnology platforms for mRNA delivery, and discusses future prospects to bring these exciting technologies into clinical practice. PMID:26280625

Ovarian Expression, Localization, and Function of Tissue Inhibitor of Metalloproteinase 3 (TIMP3) During the Periovulatory Period of the Human Menstrual Cycle1

PubMed Central

Rosewell, Katherine L.; Li, Feixue; Puttabyatappa, Muraly; Akin, James W.; Brännström, Mats; Curry, Thomas E.

2013-01-01

ABSTRACT Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability. PMID:24048576

Ovarian expression, localization, and function of tissue inhibitor of metalloproteinase 3 (TIMP3) during the periovulatory period of the human menstrual cycle.

PubMed

Rosewell, Katherine L; Li, Feixue; Puttabyatappa, Muraly; Akin, James W; Brännström, Mats; Curry, Thomas E

2013-11-01

Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability.

Cellular antioxidant effects of atorvastatin in vitro and in vivo.

PubMed

Wassmann, Sven; Laufs, Ulrich; Müller, Kirsten; Konkol, Christian; Ahlbory, Katja; Bäumer, Anselm T; Linz, Wolfgang; Böhm, Michael; Nickenig, Georg

2002-02-01

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may exert direct effects on vascular cells and beneficially influence endothelial dysfunction. Because reactive oxygen species (ROS) may lead to vascular damage and dysfunction, we investigated the effect of atorvastatin on ROS production and the underlying mechanisms in vitro and in vivo. Cultured rat aortic vascular smooth muscle cells were incubated with 10 micromol/L atorvastatin. Angiotensin II-induced and epidermal growth factor-induced ROS production were significantly reduced by atorvastatin (dichlorofluorescein fluorescence laser microscopy). Atorvastatin downregulated mRNA expression of the NAD(P)H oxidase subunit nox1, whereas p22phox mRNA expression was not significantly altered (reverse transcription-polymerase chain reaction, Northern analysis). Membrane translocation of rac1 GTPase, which is required for the activation of NAD(P)H oxidase, was inhibited by atorvastatin (Western blot). mRNA expression of superoxide dismutase isoforms and glutathione peroxidase was not modified by atorvastatin, whereas catalase expression was upregulated at mRNA and protein levels, resulting in an increased enzymatic activity. Effects of atorvastatin on ROS production and nox1, rac1, and catalase expression were inhibited by L-mevalonate but not by 25-hydroxycholesterol. In addition, spontaneously hypertensive rats were treated with atorvastatin for 30 days. ROS production in aortic segments was significantly reduced in statin-treated rats (lucigenin chemiluminescence). Treatment with atorvastatin reduced vascular mRNA expression of p22phox and nox1 and increased aortic catalase expression. mRNA expression of superoxide dismutases, glutathione peroxidase, and NAD(P)H oxidase subunits gp91phox, p40phox, p47phox, and p67phox remained unchanged. Translocation of rac1 from the cytosol to the cell membrane was also reduced in vivo. Thus, atorvastatin exerts cellular antioxidant effects in cultured rat vascular smooth muscle cells and in the vasculature of spontaneously hypertensive rats mediated by decreased expression of essential NAD(P)H oxidase subunits and by upregulation of catalase expression. These effects of atorvastatin may contribute to the vasoprotective effects of statins.

Amelioration of Cardiac Function and Activation of Anti-Inflammatory Vasoactive Peptides Expression in the Rat Myocardium by Low Level Laser Therapy

PubMed Central

Manchini, Martha Trindade; Serra, Andrey Jorge; Feliciano, Regiane dos Santos; Santana, Eduardo Tadeu; Antônio, Ednei Luis; de Tarso Camillo de Carvalho, Paulo; Montemor, Jairo; Crajoinas, Renato Oliveira; Girardi, Adriana Castello Costa; Tucci, Paulo José Ferreira; Silva, José Antônio

2014-01-01

Low-level laser therapy (LLLT) has been used as an anti-inflammatory treatment in several disease conditions, even when inflammation is a secondary consequence, such as in myocardial infarction (MI). However, the mechanism by which LLLT is able to protect the remaining myocardium remains unclear. The present study tested the hypothesis that LLLT reduces inflammation after acute MI in female rats and ameliorates cardiac function. The potential participation of the Renin-Angiotensin System (RAS) and Kallikrein-Kinin System (KKS) vasoactive peptides was also evaluated. LLLT treatment effectively reduced MI size, attenuated the systolic dysfunction after MI, and decreased the myocardial mRNA expression of interleukin-1 beta and interleukin-6 in comparison to the non-irradiated rat tissue. In addition, LLLT treatment increased protein and mRNA levels of the Mas receptor, the mRNA expression of kinin B2 receptors and the circulating levels of plasma kallikrein compared to non-treated post-MI rats. On the other hand, the kinin B1 receptor mRNA expression decreased after LLLT. No significant changes were found in the expression of vascular endothelial growth factor (VEGF) in the myocardial remote area between laser-irradiated and non-irradiated post-MI rats. Capillaries density also remained similar between these two experimental groups. The mRNA expression of the inducible nitric oxide synthase (iNOS) was increased three days after MI, however, this effect was blunted by LLLT. Moreover, endothelial NOS mRNA content increased after LLLT. Plasma nitric oxide metabolites (NOx) concentration was increased three days after MI in non-treated rats and increased even further by LLLT treatment. Our data suggest that LLLT diminishes the acute inflammation in the myocardium, reduces infarct size and attenuates left ventricle dysfunction post-MI and increases vasoactive peptides expression and nitric oxide (NO) generation. PMID:24991808

[Influence of hepatocyte growth factor on iNOS, NO and IL-1β in the cerebrum during cerebral ischemia/reperfusion in rats].

PubMed

He, Fang; Ye, Bei; Chen, Jianzhen; Sun, Xiaoyan; Li, Chang

2014-01-01

To explore the effect of hepatocyte growth factor (HGF) on inducible nitric oxide synthase (iNOS), NO and interleukin-1β (IL-1β) in the cerebrum of rats subjected to cerebral ischemia/reperfusion (I/R). Sprague-Dawley rats were randomly divided into 5 groups: a sham group, an I/R group,an HGF1 group, an HGF2 group, and an HGF3 group. The latter 3 groups were respectively injected 15, 30 and 60 μg/kg HGF. The focal cerebral I/R model was established by sutureoccluded method. After 1.5 h ischemia followed by 24 h reperfusion, the iNOS activity and NO content in the ischemic cerebral tissue were assessed. The expression of iNOS mRNA and IL-1β mRNA was detected. The level of iNOS protein and IL-1β content were determined. In addition, cultured cerebral cortical neurons in vitro were exposed to I/R. Then the expression of iNOS and IL-1β protein in the neurons was detected, and NO content was assessed. The iNOS activity and NO content in the ischemic cerebral tissue were increased. The expression of iNOS mRNA and IL-1β mRNA was upregulated. The level of iNOS protein and IL- 1β content were increased. Administration of HGF decreased the iNOS activity and NO content, and downregulated the expression of iNOS mRNA, IL-1β mRNA, iNOS protein and IL-1β content in the ischemic cerebral tissue. HGF decreased the expression of IL-1β, iNOS protein and NO content in the cortical neurons exposed to I/R in vitro. HGF can inhibit the expression of IL-1β and decrease the expression of iNOS and content of NO, which is probably one of the mechanisms mediating the protection of HGF against cerebral ischemia injury.

The effects of cyclosporin on the collagenolytic activity of gingival fibroblasts.

PubMed

Hyland, Paula L; Traynor, Patrick S; Myrillas, Theofilos T; Marley, John J; Linden, Gerard J; Winter, Paul; Leadbetter, Nicola; Cawston, Timothy E; Irwin, Chris R

2003-04-01

The immunosuppressive agent cyclosporin is associated with a number of major side-effects including the development of gingival overgrowth. Although the pathogenesis of cyclosporin-induced gingival overgrowth remains unclear, it has been suggested that the finely regulated balance between extracellular matrix synthesis and degradation may be disturbed, resulting in an accumulation of excess connective tissue components within the gingival tissue. The aim of this study was to investigate the effect of cyclosporin on matrix metalloproteinases (MMP)-1 and tissue inhibitors of MMP (TIMP)-1 expression at the mRNA, protein, and enzyme activity levels. Gingival fibroblasts were grown to confluence and then cultured in serum-free medium supplemented with cyclosporin over the concentration range of 0 to 2000 ng/ml. MMP-1 and TIMP-1 mRNA levels in cultures were determined by reverse transcription polymerase chain reaction (RT-PCR), protein levels in whole conditioned medium were assessed by enzyme-linked immunosorbent assay (ELISA), and collagenolytic activity determined using a 3H-acetylated type I collagen degradation assay. Tissue mRNA levels in normal and overgrown gingiva were also determined by RT-PCR. Results indicated that cyclosporin inhibited MMP-1 expression at both the mRNA and protein level in a dose- and time-dependent fashion. The effects on TIMP-1 expression were less clear, cyclosporin inhibiting mRNA expression, but having no effect on TIMP-1 protein levels at any concentration studied. Addition of the drug resulted in reduced levels of collagenolytic activity in the culture medium. MMP-1 mRNA expression was significantly reduced in overgrown compared to normal tissue. These results add support to the hypothesis that the accumulation of collagen seen in gingival overgrowth can be explained by a cyclosporin-induced inhibition of collagenolytic activity within the gingival tissues.

DNA methylation regulates gabrb2 mRNA expression: developmental variations and disruptions in l-methionine-induced zebrafish with schizophrenia-like symptoms.

PubMed

Wang, L; Jiang, W; Lin, Q; Zhang, Y; Zhao, C

2016-11-01

Single nucleotide polymorphisms (SNPs) in the human type A gamma-aminobutyric acid (GABA) receptor β 2 subunit gene (GABRB2) have been associated with schizophrenia and quantitatively correlated with mRNA expression in the postmortem brain tissue of patients with schizophrenia. l-Methionine (MET) administration has been reported to cause a recrudescence of psychotic symptoms in patients with schizophrenia, and similar symptoms have been generated in MET-induced mice. In this study, a zebrafish animal model was used to evaluate the relationship between the gabrb2 mRNA expression and its promoter DNA methylation in developmental and MET-induced schizophrenia-like zebrafish. The results indicated developmental increases in global DNA methylation and decreases in gabrb2 promoter methylation in zebrafish. A significant increase in gabrb2 mRNA levels was observed after GABA was synthesized. Additionally, the MET-triggered schizophrenia-like symptoms in adult zebrafish, involving social withdrawal and cognitive dysfunction analyzed with social interaction and T-maze behavioral tests, were accompanied by significantly increased DNA methylation levels in the global genome and the gabrb2 promoter. Furthermore, the significant correlation between gabrb2 mRNA expression and gabrb2 promoter methylation observed in the developmental stages became non-significant in MET-triggered adult zebrafish. These findings demonstrate that gabrb2 mRNA expression is associated with DNA methylation varies by developmental stage and show that these epigenetic association mechanisms are disrupted in MET-triggered adult zebrafish with schizophrenia-like symptoms. In conclusion, these results provide plausible epigenetic evidence of the GABA A receptor β 2 subunit involvement in the schizophrenia-like behaviors and demonstrate the potential use of zebrafish models in neuropsychiatric research. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

c-fms mRNA is regulated posttranscriptionally by 1,25(OH)2D3 in HL-60 cells.

PubMed

Biskobing, D M; Fan, D; Rubin, J

1997-09-01

Macrophage colony-stimulating factor (MCSF) is required for normal osteoclast and macrophage development. The receptor for MCSF (c-fms) is expressed on the pluripotent precursor and mature osteoclasts and macrophages. We have previously shown in myelomonocytic HL-60 cells that phorbol myristate acetate (PMA) upregulates c-fms mRNA expression. This induction of c-fms is inhibited by 1,25(OH)2D3. The major regulatory control of c-fms mRNA levels by PMA has been identified as posttranscriptional. However, a role of transcript elongation in controlling levels of c-fms mRNA has also been suggested. To better understand the 1,25(OH)2D3 regulation of c-fms mRNA expression we studied nuclear run on, mRNA stability, and transcript elongation in HL-60 cells treated with 10 ng/ml phorbol myristate acetate, 10 nM 1,25(OH)2D3 alone or combined. We demonstrated by nuclear run on that c-fms was constitutively transcribed in 1,25(OH)2D3 as well as control and PMA-treated cells. Transcript elongation was evaluated by RT-PCR for exon 2 or exon 3. Both exons were minimally expressed in control and 1,25(OH)2D3-treated cells, and increased in PMA-treated cells; this increased expression was inhibited by the addition of 1,25(OH)2D3. These results fail to show differential transcript elongation. Measurement of mRNA stability demonstrated decreased mRNA half-life to 5 hours in cells treated with PMA and 1,25(OH)2D3 compared with a half-life of 8 hours in cells treated with PMA alone. Our findings demonstrate that c-fms is regulated by 1,25(OH)2D3 at the posttranscriptional level by changes in mRNA stability. This gives the cell the ability to respond to local signals with rapid changes in c-fms levels altering the ability of the cell to respond to MCSF.

Relationship Between the DPD and TS mRNA Expression and the Response to S-1-Based Chemotherapy and Prognosis in Patients with Advanced Gastric Cancer.

PubMed

Shen, Xiao-Ming; Zhou, Chong; Lian, Lian; Li, Li-Qun; Li, Wei; Tao, Min

2015-04-01

The aim was to determine changes in dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) mRNAs in the blood of advanced gastric cancer (AGC) patients to see whether these enzymes affected the patients' response to S-1-based chemotherapy and prognosis. For this purpose, pretreatment DPD/TS mRNA expressions were determined in 40 AGC patients using RT-PCR. The patients were then administered with S-1-based regimen (S-1 + cisplatin) and toxicities were recorded. The relationship between the DPD/TS mRNA expressions and the chemotherapy response, drug resistance, and prognosis was analyzed. The data show that DPD mRNA expression correlated significantly with Lauren type while TS mRNA expression correlated with distant metastasis. Patients with higher DPD and/or TS mRNA expression(s) showed poor response, while those with low DPD mRNA expression showed better response to the chemotherapy. Pooled analysis showed that the patients with low DPD/TS mRNA expressions had better therapeutic response. The incidence of bone marrow suppression, diarrhea, and oral mucositis was high in patients with low DPD mRNA expression. Median overall survival (OS) in 40 patients was 13.5 months. It was 17 months for low and 10 months for high DPD (P = 0.044) and TS mRNA expression (P = 0.047). Pooled analysis showed that the patients with both low DPD/TS mRNA expressions had longer OS (P = 0.001). In conclusion, the detection of DPD and/or TS mRNA expression can be used to predict the response to S-1-based chemotherapy, drug resistance, and prognosis in AGC patients as well as to help guide the individualized treatment of gastric cancer.

Substance P induced preprotachykinin-a mRNA, neutral endopeptidase mRNA and substance P in cultured normal fibroblasts.

PubMed

Bae, Sang-Jae; Matsunaga, Yoshitaka; Takenaka, Motoi; Tanaka, Yoichi; Hamazaki, Yoichiro; Shimizu, Kazuhiro; Katayama, Ichiro

2002-04-01

In certain skin diseases, stress can modulate the induction and/or progression of cutaneous manifestations. However, little is known about the circuit in neuroendocrine and in the immune systems of the skin. To address this question, we have analyzed the regulatory mechanisms of autocrine induction of substance P (SP) by cultured normal human fibroblasts that compose the major population of the skin and might augment stress-induced skin inflammatory responses. In nonstimulated conditions, normal fibroblasts express a moderate amount of preprotachykinin-A (PPT-A), a precursor of SP mRNA, and exogenous SP significantly upregulated PPT-A mRNA expression. Maximum response of SP peptide and SP mRNA in fibroblasts was observed 1-3 h after stimulation with SP. In contrast, the expression of neutral endopeptidase (NEP), a cell surface peptide with hydrolyzing activity of SP, was increased in fibroblasts stimulated with SP after 24 h. The administration of NEP inhibitor (phosphoramidon) to the fibroblasts induced higher SP production. In addition, the neurokinin (NK) receptor antagonists (spantide, FK224 and FK888) and protein synthesis inhibitor (cycloheximide) inhibited SP production by 30-40% of control response. In immunostaining study, specific cytoplasmic staining of SP was observed in fibroblasts stimulated with SP. Finally, we confirmed that the nucleotide sequence of the PPT-A expressed in fibroblasts perfectly corresponded to the gene bank human PPT-A cDNA. This is the first report that SP mRNA, NEP mRNA and SP peptide can be induced by normal human skin fibroblasts in response to exogenous SP, and that fibroblast-derived SP might play an important role in the induction and acceleration of certain cutaneous diseases. Copyright 2002 S. Karger AG, Basel

Social defeat promotes a reactive endothelium in a brain region-dependent manner with increased expression of key adhesion molecules, selectins and chemokines associated with the recruitment of myeloid cells to the brain.

PubMed

Sawicki, C M; McKim, D B; Wohleb, E S; Jarrett, B L; Reader, B F; Norden, D M; Godbout, J P; Sheridan, J F

2015-08-27

Repeated social defeat (RSD) in mice causes myeloid cell trafficking to the brain that contributes to the development of prolonged anxiety-like behavior. Myeloid cell recruitment following RSD occurs in regions where neuronal and microglia activation is observed. Thus, we hypothesized that crosstalk between neurons, microglia, and endothelial cells contributes to brain myeloid cell trafficking via chemokine signaling and vascular adhesion molecules. Here we show that social defeat caused an exposure- and brain region-dependent increase in several key adhesion molecules and chemokines involved in the recruitment of myeloid cells. For example, RSD induced distinct patterns of adhesion molecule expression that may explain brain region-dependent myeloid cell trafficking. VCAM-1 and ICAM-1 mRNA expression were increased in an exposure-dependent manner. Furthermore, RSD-induced VCAM-1 and ICAM-1 protein expression were localized to the vasculature of brain regions implicated in fear and anxiety responses, which spatially corresponded to previously reported patterns of myeloid cell trafficking. Next, mRNA expression of additional adhesion molecules (E- and P-selectin, PECAM-1) and chemokines (CXCL1, CXCL2, CXCL12, CCL2) were determined in the brain. Social defeat induced an exposure-dependent increase in mRNA levels of E-selectin, CXCL1, and CXCL2 that increased with additional days of social defeat. While CXCL12 was unaffected by RSD, CCL2 expression was increased by six days of social defeat. Last, comparison between enriched CD11b(+) cells (microglia/macrophages) and enriched GLAST-1(+)/CD11b(-) cells (astrocytes) revealed RSD increased mRNA expression of IL-1β, CCL2, and CXCL2 in microglia/macrophages but not in astrocytes. Collectively, these data indicate that key mediators of leukocyte recruitment were increased in the brain vasculature following RSD in an exposure- and brain region-dependent manner. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Social defeat promotes a reactive endothelium in a brain region-dependent manner with increased expression of key adhesion molecules, selectins and chemokines associated with the recruitment of myeloid cells to the brain

PubMed Central

Sawicki, Caroline M.; McKim, Daniel B.; Wohleb, Eric S.; Jarrett, Brant L.; Reader, Brenda F.; Norden, Diana M.; Godbout, Jonathan P.; Sheridan, John F.

2014-01-01

Repeated social defeat (RSD) in mice causes myeloid cell trafficking to the brain that contributes to the development of prolonged anxiety-like behavior. Myeloid cell recruitment following RSD occurs in regions where neuronal and microglia activation is observed. Thus, we hypothesized that crosstalk between neurons, microglia, and endothelial cells contributes to brain-myeloid cell trafficking via chemokine signaling and vascular adhesion molecules. Here we show that social defeat caused an exposure- and brain region-dependent increase in several key adhesion molecules and chemokines involved in the recruitment of myeloid cells. For example, RSD induced distinct patterns of adhesion molecule expression that may explain brain region-dependent myeloid cell trafficking. VCAM-1 and ICAM-1 mRNA expression were increased in an exposure-dependent manner. Furthermore, RSD-induced VCAM-1 and ICAM-1 protein expression were localized to the vasculature of brain regions implicated in fear and anxiety responses, which spatially corresponded to previously reported patterns of myeloid cell trafficking. Next, mRNA expression of additional adhesion molecules (E- and P-selectin, PECAM-1) and chemokines (CXCL1, CXCL2, CXCL12, CCL2) were determined in the brain. Social defeat induced an exposure-dependent increase in mRNA levels of E-selectin, CXCL1, and CXCL2 that increased with additional days of social defeat. While CXCL12 was unaffected by RSD, CCL2 expression was increased by six days of social defeat. Last, comparison between enriched CD11b+ cells (microglia/macrophages) and enriched GLAST-1+/CD11b− cells (astrocytes) revealed RSD increased mRNA expression of IL-1β, CCL2, and CXCL2 in microglia/macrophages but not in astrocytes. Collectively, these data indicate that key mediators of leukocyte recruitment were increased in the brain vasculature following RSD in an exposure- and brain-region dependent manner. PMID:25445193

Expression of β-catenin protein in hepatocellular carcinoma and its relationship with alpha-fetoprotein.

PubMed

Ren, Ya-Jun; Huang, Tao; Yu, Hong-Lu; Zhang, Li; He, Qian-Jin; Xiong, Zhi-Fan; Peng, Hua

2016-12-01

This study aimed to investigate the expression of β-catenin in hepatocellular carcinoma (HCC) tissues and its relationship with α-fetoprotein (AFP) in HCC. Immunohistochemistry was used to determine the expression of β-catenin in normal liver tissues (n=10), liver cirrhosis tissues (n=20), and primary HCC tissues (n=60). The relationship between β-catenin expression and clinical parameters of HCC was investigated. Real-time PCR and Western blotting were used to detect the mRNA and protein expression levels of β-catenin in the liver cancer cell line SMMC-7721 transfected with a plasmid encoding AFP, and also the mRNA and protein expression levels of β-catenin were measured in the liver cancer cell line Huh7 before and after the transfection with AFP shRNA plasmids. The results showed that β-catenin was only expressed on the cell membrane in normal liver tissues. Its localization to the cytoplasm and nucleus of cells was observed in a small proportion of cirrhotic tissues or adjacent HCC tissues, and such ectopic expression of β-catenin was predominant in HCC tissues. The abnormal expression of β-catenin was correlated with serum AFP levels, cancer cell differentiation and vascular invasion (P<0.05). Additionally, the increased expression of AFP resulted in the upregulation of β-catenin mRNA and protein levels, while knockdown of AFP with AFP shRNA led to significantly decreased β-catenin mRNA and protein levels (P<0.05). It was suggested that the abnormal expression of β-catenin is implicated in hepatic carcinogenesis and development. AFP can lead to increased expression of β-catenin, which may account for the poor prognosis of AFP-associated HCC patients.

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

Hu, Zhi; Huang, Ge; Sadanandam, Anguraj

Introduction: HJURP (Holliday Junction Recognition Protein) is a newly discovered gene reported to function at centromeres and to interact with CENPA. However its role in tumor development remains largely unknown. The goal of this study was to investigate the clinical significance of HJURP in breast cancer and its correlation with radiotherapeutic outcome. Methods: We measured HJURP expression level in human breast cancer cell lines and primary breast cancers by Western blot and/or by Affymetrix Microarray; and determined its associations with clinical variables using standard statistical methods. Validation was performed with the use of published microarray data. We assessed cell growthmore » and apoptosis of breast cancer cells after radiation using high-content image analysis. Results: HJURP was expressed at higher level in breast cancer than in normal breast tissue. HJURP mRNA levels were significantly associated with estrogen receptor (ER), progesterone receptor (PR), Scarff-Bloom-Richardson (SBR) grade, age and Ki67 proliferation indices, but not with pathologic stage, ERBB2, tumor size, or lymph node status. Higher HJURP mRNA levels significantly decreased disease-free and overall survival. HJURP mRNA levels predicted the prognosis better than Ki67 proliferation indices. In a multivariate Cox proportional-hazard regression, including clinical variables as covariates, HJURP mRNA levels remained an independent prognostic factor for disease-free and overall survival. In addition HJURP mRNA levels were an independent prognostic factor over molecular subtypes (normal like, luminal, Erbb2 and basal). Poor clinical outcomes among patients with high HJURP expression werevalidated in five additional breast cancer cohorts. Furthermore, the patients with high HJURP levels were much more sensitive to radiotherapy. In vitro studies in breast cancer cell lines showed that cells with high HJURP levels were more sensitive to radiation treatment and had a higher rate of apoptosis than those with low levels. Knock down of HJURP in human breast cancer cells using shRNA reduced the sensitivity to radiation treatment. HJURP mRNA levels were significantly correlated with CENPA mRNA levels. Conclusions: HJURP mRNA level is a prognostic factor for disease-free and overall survival in patients with breast cancer and is a predictive biomarker for sensitivity to radiotherapy.« less

BRCA1: A Novel Prognostic Factor in Resected Non-Small-Cell Lung Cancer

PubMed Central

Rosell, Rafael; Skrzypski, Marcin; Jassem, Ewa; Taron, Miquel; Bartolucci, Roberta; Sanchez, Jose Javier; Mendez, Pedro; Chaib, Imane; Perez-Roca, Laia; Szymanowska, Amelia; Rzyman, Witold; Puma, Francesco; Kobierska-Gulida, Grazyna; Farabi, Raffaele; Jassem, Jacek

2007-01-01

Background Although early-stage non-small-cell lung cancer (NSCLC) is considered a potentially curable disease following complete resection, patients have a wide spectrum of survival according to stage (IB, II, IIIA). Within each stage, gene expression profiles can identify patients with a higher risk of recurrence. We hypothesized that altered mRNA expression in nine genes could help to predict disease outcome: excision repair cross-complementing 1 (ERCC1), myeloid zinc finger 1 (MZF1) and Twist1 (which regulate N-cadherin expression), ribonucleotide reductase subunit M1 (RRM1), thioredoxin-1 (TRX1), tyrosyl-DNA phosphodiesterase (Tdp1), nuclear factor of activated T cells (NFAT), BRCA1, and the human homolog of yeast budding uninhibited by benzimidazole (BubR1). Methodology and Principal Findings We performed real-time quantitative polymerase chain reaction (RT-QPCR) in frozen lung cancer tissue specimens from 126 chemonaive NSCLC patients who had undergone surgical resection and evaluated the association between gene expression levels and survival. For validation, we used paraffin-embedded specimens from 58 other NSCLC patients. A strong inter-gene correlation was observed between expression levels of all genes except NFAT. A Cox proportional hazards model indicated that along with disease stage, BRCA1 mRNA expression significantly correlated with overall survival (hazard ratio [HR], 1.98 [95% confidence interval (CI), 1.11-6]; P = 0.02). In the independent cohort of 58 patients, BRCA1 mRNA expression also significantly correlated with survival (HR, 2.4 [95%CI, 1.01-5.92]; P = 0.04). Conclusions Overexpression of BRCA1 mRNA was strongly associated with poor survival in NSCLC patients, and the validation of this finding in an independent data set further strengthened this association. Since BRCA1 mRNA expression has previously been linked to differential sensitivity to cisplatin and antimicrotubule drugs, BRCA1 mRNA expression may provide additional information for customizing adjuvant antimicrotubule-based chemotherapy, especially in stage IB, where the role of adjuvant chemotherapy has not been clearly demonstrated. PMID:17987116

Estrogen receptor mRNA expression patterns in the liver and ovary of female rainbow trout over a complete reproductive cycle

PubMed Central

Nagler, James J.; Cavileer, Timothy D.; Verducci, Joseph S.; Schultz, Irvin R.; Hook, Sharon E.; Hayton, William L.

2012-01-01

Estrogens are critical hormones involved in reproduction and need to bind to estrogen receptors in target organs for biological activity. Fishes have two distinct estrogen receptor subtypes, alpha (α) and beta (β), with variable combinations of additional isoforms of each subtype dependent on the history of genome duplication within a taxon. The comparative expression patterns of estrogen receptor isoforms during the female reproductive cycle will provide important insights into the unique function and importance of each. The purpose of this study was to measure the mRNAs for the four estrogen receptor isoforms (erα1, erα2, erβ1, erβ2) in the liver and ovary of adult, female rainbow trout over the course of an annual reproductive cycle. The expression of estrogen receptor mRNA isoforms was measured by quantitative real-time RT-PCR. Several reproductive indices (gonadosomatic index, maximum oocyte diameter, plasma estradiol-17β, plasma vitellogenin, and ovulation) were also quantified for comparison and used in a correlation analysis to examine any inter-relationships. Of the four isoforms, the expression of erα1 was highest in the liver, and had a significant positive correlation with liver erβ1 expression. Liver expression of erα2 mRNA was the lowest, but showed a significant positive correlation with maximum oocyte diameter in the ovary. The pattern of the erβ isoforms in liver was one of initially elevated mRNA expression followed by a gradual decrease as reproductive development proceeded. In the ovary the erβ1 isoform had the highest mRNA expression of all estrogen receptor isoforms, at the beginning of the reproductive cycle, but then decreased afterward. Both ovarian erβ isoforms had a significant positive correlation with one another. In contrast, erα2 mRNA expression showed a high maximum level in the ovary near the end of the cycle along with a significant positive correlation with plasma estradiol-17β levels; the highest gonadosomatic indices, maximum oocyte diameter, and vitellogenin levels occurred then too. PMID:22732076

Expression and Regulation of the Fkbp5 Gene in the Adult Mouse Brain

PubMed Central

Scharf, Sebastian H.; Liebl, Claudia; Binder, Elisabeth B.

2011-01-01

Background Chronic stress has been found to be a major risk factor for various human pathologies. Stress activates the hypothalamic-pituitary-adrenal (HPA) axis, which is tightly regulated via, among others, the glucocorticoid receptor (GR). The activity of the GR is modulated by a variety of proteins, including the co-chaperone FK506 binding protein 51 (FKBP5). Although FKBP5 has been associated with risk for affective disorders and has been implicated in GR sensitivity, previous studies focused mainly on peripheral blood, while information about basal distribution and induction in the central nervous system are sparse. Methodology/Principal Findings In the present study, we describe the basal expression pattern of Fkbp5 mRNA in the brain of adult male mice and show the induction of Fkbp5 mRNA via dexamethasone treatment or different stress paradigms. We could show that Fkbp5 is often, but not exclusively, expressed in regions also known for GR expression, for example the hippocampus. Furthermore, we were able to induce Fkbp5 expression via dexamethasone in the CA1 and DG subregions of the hippocampus, the paraventricular nucleus (PVN) and the central amygdala (CeA). Increase of Fkbp5 mRNA was also found after restrained stress and 24 hours of food deprivation in the PVN and the CeA, while in the hippocampus only food deprivation caused an increase in Fkbp5 mRNA. Conclusions/Significance Interestingly, regions with a low basal expression showed higher increase in Fkbp5 mRNA following induction than regions with high basal expression, supporting the hypothesis that GR sensitivity is, at least partly, mediated via Fkbp5. In addition, this also supports the use of Fkbp5 gene expression as a marker for GR sensitivity. In summary, we were able to give an overview of the basal expression of fkbp5 mRNA as well as to extend the findings of induction of Fkbp5 and its regulatory influence on GR sensitivity from peripheral blood to the brain. PMID:21347384

Effects of dietary octacosanol supplementation on laying performance, egg quality, serum hormone levels, and expression of genes related to the reproductive axis in laying hens.

PubMed

Long, L; Wu, S G; Yuan, F; Zhang, H J; Wang, J; Qi, G H

2017-04-01

This experiment was conducted to evaluate the effects of dietary octacosanol supplementation on laying performance, egg quality, serum hormone levels, and gene expression related to reproductive axis in laying hens to confirm the reproduction-promoting function of octacosanol. In total, 360 Hy-Line Brown (67-wk-old) laying hens were randomly assigned to one of three treatments with 0, 5, and 10 mg octacosanol (extracted from rice bran, purity >92%)/kg feed. The feeding trial lasted for 10 weeks. The results showed that the dietary addition of 5 and 10 mg/kg octacosanol improved feed efficiency by 4.9% and 3.4% (P < 0.01), increased the albumen height by 20.5% and 13.3% (P < 0.01), the Haugh unit score by 12.9% and 8.7% (P < 0.01), and the eggshell strength by 39.5% and 24.5% (P < 0.01), respectively, compared with the control diet. Dietary octacosanol addition significantly affected serum triiodothyronine, estradiol, follicle-stimulating hormone levels (P < 0.05), and progesterone and luteinizing hormone level (P < 0.01). Compared with the control, dietary addition of octacosanol at 5 mg/kg promoted the follicle-stimulating hormone receptor (FSHR) mRNA expression in different-sized follicles, and significantly increased the FSHR mRNA expression of granulosa cells from the F2 and F3 follicles (P < 0.05). Dietary supplementation with both 5 and 10 mg/kg octacosanol promoted the mRNA expression of luteinizing hormone receptor and prolactin receptor in different-sized follicles, and significantly up-regulated the expression levels in F1 granulosa cells (P < 0.05). The ovarian weight was significantly increased with the dietary addition of 5 mg/kg octacosanol (P < 0.05). The numbers of small yellow follicles and large white follicles were increased with the addition of dietary 5 and 10 mg/kg octacosanol (P < 0.01). This study provides evidence that octacosanol has the capacity to improve reproductive performance, indicating that it is a potentially effective feed additive in egg production. © 2016 Poultry Science Association Inc.

Functional analysis of CTRP3/cartducin in Meckel's cartilage and developing condylar cartilage in the fetal mouse mandible

PubMed Central

Yokohama-Tamaki, Tamaki; Maeda, Takashi; Tanaka, Tetsuya S; Shibata, Shunichi

2011-01-01

CTRP3/cartducin, a novel C1q family protein, is expressed in proliferating chondrocytes in the growth plate and has an important role in regulating the growth of both chondrogenic precursors and chondrocytes in vitro. We examined the expression of CTRP3/cartducin mRNA in Meckel's cartilage and in condylar cartilage of the fetal mouse mandible. Based on in situ hybridization studies, CTRP3/cartducin mRNA was not expressed in the anlagen of Meckel's cartilage at embryonic day (E)11.5, but it was strongly expressed in Meckel's cartilage at E14.0, and then reduced in the hypertrophic chondrocytes at E16.0. CTRP3/cartducin mRNA was not expressed in the condylar anlagen at E14.0, but was expressed in the upper part of newly formed condylar cartilage at E15.0. At E16.0, CTRP3/cartducin mRNA was expressed from the polymorphic cell zone to the upper part of the hypertrophic cell zone, but was reduced in the lower part of the hypertrophic cell zone. CTRP3/cartducin-antisense oligodeoxynucleotide (AS-ODN) treatment of Meckel's cartilage and condylar anlagen from E14.0 using an organ culture system indicated that, after 4-day culture, CTRP3/cartducin abrogation induced curvature deformation of Meckel's cartilage with loss of the perichondrium and new cartilage formation. Aggrecan, type I collagen, and tenascin-C were simultaneously immunostained in this newly formed cartilage, indicating possible transformation from the perichondrium into cartilage. Further, addition of recombinant mouse CTRP3/cartducin protein to the organ culture medium with AS-ODN tended to reverse the deformation. These results suggest a novel function for CTRP3/cartducin in maintaining the perichondrium. Moreover, AS-ODN induced a deformation of the shape, loss of the perichondrium/fibrous cell zone, and disorder of the distinct architecture of zones in the mandibular condylar cartilage. Additionally, AS-ODN-treated condylar cartilage showed reduced levels of mRNA expression of aggrecan, collagen types I and X, and reduced BrdU-incorporation. These results suggest that CTRP3/cartducin is not only involved in the proliferation and differentiation of chondrocytes, but also contributes to the regulation of mandibular condylar cartilage. PMID:21371032

Trinitrotoluene Induces Endoplasmic Reticulum Stress and Apoptosis in HePG2 Cells.

PubMed

Song, Li; Wang, Yue; Wang, Jun; Yang, Fan; Li, Xiaojun; Wu, Yonghui

2015-11-09

This study aims to describe trinitrotoluene (TNT)-induced endoplasmic reticulum stress (ERS) and apoptosis in HePG2 cells. HePG2 cells were cultured in vitro with 0, 6, 12, or 24 μg/ml TNT solution for 12, 24, and 48 h. Western blotting was performed to detect intracellular ERS-related proteins, including glucose-regulated protein (GRP) 78, GRP94, Caspase 4, p-Jun N-terminal kinase (JNK), and C/EBP homologous protein (CHOP). Real-time PCR was used to measure mRNA expression from the respective genes. The expressions of ERS-related proteins GRP78 and GRP94 as well as mRNA and protein expression of ERS signaling apoptotic CHOP in the TNT treatment group were significantly increased. In addition, the mRNA and protein expression levels of ERS-induced apoptotic protein Caspase-4 were significantly increased. Flow cytometry revealed that after TNT treatment, the apoptosis rate also significantly increased. TNT could increase the expression levels of GRP78, GRP94, Caspase-4, and CHOP in HePG2 cells; this increase in protein expression might be involved in HePG2 apoptosis through the induction of the ERS pathway.

Trinitrotoluene Induces Endoplasmic Reticulum Stress and Apoptosis in HePG2 Cells

PubMed Central

Song, Li; Wang, Yue; Wang, Jun; Yang, Fan; Li, Xiaojun; Wu, Yonghui

2015-01-01

Background This study aims to describe trinitrotoluene (TNT)-induced endoplasmic reticulum stress (ERS) and apoptosis in HePG2 cells. Material/Methods HePG2 cells were cultured in vitro with 0, 6, 12, or 24 μg/ml TNT solution for 12, 24, and 48 h. Western blotting was performed to detect intracellular ERS-related proteins, including glucose-regulated protein (GRP) 78, GRP94, Caspase 4, p-Jun N-terminal kinase (JNK), and C/EBP homologous protein (CHOP). Real-time PCR was used to measure mRNA expression from the respective genes. Results The expressions of ERS-related proteins GRP78 and GRP94 as well as mRNA and protein expression of ERS signaling apoptotic CHOP in the TNT treatment group were significantly increased. In addition, the mRNA and protein expression levels of ERS-induced apoptotic protein Caspase-4 were significantly increased. Flow cytometry revealed that after TNT treatment, the apoptosis rate also significantly increased. Conclusions TNT could increase the expression levels of GRP78, GRP94, Caspase-4, and CHOP in HePG2 cells; this increase in protein expression might be involved in HePG2 apoptosis through the induction of the ERS pathway. PMID:26551326

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

Kinetics of lipid-nanoparticle-mediated intracellular mRNA delivery and function

NASA Astrophysics Data System (ADS)

Zhdanov, Vladimir P.

2017-10-01

mRNA delivery into cells forms the basis for one of the new and promising ways to treat various diseases. Among suitable carriers, lipid nanoparticles (LNPs) with a size of about 100 nm are now often employed. Despite high current interest in this area, the understanding of the basic details of LNP-mediated mRNA delivery and function is limited. To clarify the kinetics of mRNA release from LNPs, the author uses three generic models implying (i) exponential, (ii) diffusion-controlled, and (iii) detachment-controlled kinetic regimes, respectively. Despite the distinct differences in these kinetics, the associated transient kinetics of mRNA translation to the corresponding protein and its degradation are shown to be not too sensitive to the details of the mRNA delivery by LNPs (or other nanocarriers). In addition, the author illustrates how this protein may temporarily influence the expression of one gene or a few equivalent genes. The analysis includes positive or negative regulation of the gene transcription via the attachment of the protein without or with positive or negative feedback in the gene expression. Stable, bistable, and oscillatory schemes have been scrutinized in this context.

Regulation of neuropeptide Y gene expression in rat brain.

PubMed

Lindefors, N; Brené, S; Herrera-Marschitz, M; Persson, H

1990-01-01

NPY mRNA expression was studied in rat brain using in situ hybridization and RNA blot analysis. Transsynaptic regulation of NPY gene expression was specifically studied in caudate-putamen and frontoparietal (somatosensory) cortex of rats with unilateral lesion of midbrain dopamine neurons and in sham-injected animals. NPY mRNA expression in these two brain regions and the regulation of midbrain dopamine neurons were compared with that of SOM, PPT, CCK and GAD mRNA expression. Neurons expressing NPY and SOM mRNA showed a similar distribution and the expression of both NPY and SOM appears to be regulated by dopamine in a similar fashion. Following a unilateral dopamine deafferentation, the numerical density of both NPY and SOM mRNA expressing neurons almost doubled in the lesioned rat caudate-putamen with no change in the average grain density over positive neurons. Hence, in the intact caudate-putamen dopamine appears to normally suppress expression of these two neuropeptide genes. An activation of both NPY and SOM mRNA expression in many non- or low-expressing neurons is seen when the level of dopamine is decreased. In the frontoparietal cortex, on the other hand, dopamine appears to stimulate NPY and SOM gene expression. RNA blot analysis shows clear-cut changes of NPY mRNA levels in both caudate-putamen and frontoparietal cortex consistent with the changes observed using in situ hybridization. No evidence was found for a change in CCK mRNA expression by the dopamine deafferentation, while PPT mRNA expression decreased in the deafferented caudate-putamen. Consequently, dopamine exerts dissimilar effects on the expression of different neuropeptide genes, that in turn do not respond in the same way in different brain regions. Indirect evidence is also presented indicating that dopamine regulates NPY mRNA expression in a subpopulation of neurons that possibly also express GAD mRNA, both in caudate-putamen and in frontoparietal cortex.

Protein-bound polysaccharide-K augments the anticancer effect of fluoropyrimidine derivatives possibly by lowering dihydropyrimidine dehydrogenase expression in gastrointestinal cancers.

PubMed

Mekata, Eiji; Murata, Satoshi; Sonoda, Hiromichi; Shimizu, Tomoharu; Umeda, Tomoko; Shiomi, Hisanori; Naka, Shigeyuki; Yamamoto, Hiroshi; Abe, Hajime; Edamatsu, Takeo; Fujieda, Ayako; Fujioka, Masaki; Wada, Tsutomu; Tani, Tohru

2013-12-01

Protein-bound polysaccharide-K (PSK) enhances the antitumor effect of anticancer drug when used clinically in combination with such drugs. PSK is known to act by immune-mediated mechanisms; however, the relationship between PSK and metabolic enzymes of anticancer drugs is unknown. We used the collagen gel droplet-embedded culture drug sensitivity test (CD-DST) clinically to evaluate the sensitivity of anticancer drugs. In the present study, we modified the CD-DST by adding peripheral blood mononuclear cells (PBMCs) (immuno-CD-DST) and examined the antitumor effect of PSK in combination with anticancer drugs. First, HCT116 human colon cancer cells were cultured with PSK and 5-fluorouracil (5-FU) or 5'-deoxy-5-fluorouridine (5'-DFUR) in the presence or absence of PBMCs, and the antiproliferative effects were compared. In the presence of PBMCs, PSK augmented the inhibitory effects of 5-FU and 5'-DFUR on HCT116 cell proliferation. Next, using human gastric cancer and colon cancer cell lines, the effects of PSK on mRNA expression of various metabolic enzymes of fluoropyrimidines: dihydropyrimidine dehydrogenase (DPD), thymidylate synthase, thymidine phosphorylase and orotate phosphoribosyl transferase, were examined by real-time PCR. PSK significantly enhanced DPD mRNA expression in all of the cancer cell lines tested, but not those of the other enzymes. Addition of IFN-α and TRAIL, cytokines known to inhibit DPD expression, to the cultures reduced DPD mRNA expression in the cancer cells. When PBMC samples collected from healthy volunteers were cultured with PSK, IFN-α mRNA expression increased in 3 of the 5 PBMC samples, while TRAIL mRNA expression was unchanged. The present results propose the possibility that PSK induces PBMCs to express IFN-α which inhibits DPD expression, and consequently augments the antitumor effect of 5-FU or 5'-DFUR. Immuno-CD-DST is useful for evaluating drugs with immunological mechanisms of action.

MCG101-induced cancer anorexia-cachexia features altered expression of hypothalamic Nucb2 and Cartpt and increased plasma levels of cocaine- and amphetamine-regulated transcript peptides.

PubMed

Burgos, Jonathan R; Iresjö, Britt-Marie; Smedh, Ulrika

2016-04-01

The aim of the present study was to explore central and peripheral host responses to an anorexia-cachexia producing tumor. We focused on neuroendocrine anorexigenic signals in the hypothalamus, brainstem, pituitary and from the tumor per se. Expression of mRNA for corticotropin-releasing hormone (CRH), cocaine- and amphetamine-regulated transcript (CART), nesfatin-1, thyrotropin (TSH) and the TSH receptor were explored. In addition, we examined changes in plasma TSH, CART peptides (CARTp) and serum amyloid P component (SAP). C57BL/6 mice were implanted with MCG101 tumors or sham-treated. A sham-implanted, pair‑fed (PF) group was included to delineate between primary tumor and secondary effects from reduced feeding. Food intake and body weight were measured daily. mRNA levels from microdissected mouse brain samples were assayed using qPCR, and plasma levels were determined using ELISA. MCG101 tumors expectedly induced anorexia and loss of body weight. Tumor-bearing (TB) mice exhibited an increase in nesfatin-1 mRNA as well as a decrease in CART mRNA in the paraventricular area (PVN). The CART mRNA response was secondary to reduced caloric intake whereas nesfatin-1 mRNA appeared to be tumor-specifically induced. In the pituitary, CART and TSH mRNA were upregulated in the TB and PF animals compared to the freely fed controls. Plasma levels for CARTp were significantly elevated in TB but not PF mice whereas levels of TSH were unaffected. The plasma CARTp response was correlated to the degree of inflammation represented by SAP. The increase in nesfatin-1 mRNA in the PVN highlights nesfatin-1 as a plausible candidate for causing tumor-induced anorexia. CART mRNA expression in the PVN is likely an adaptation to reduced caloric intake secondary to a cancer anorexia-cachexia syndrome (CACS)‑inducing tumor. The MCG101 tumor did not express CART mRNA, thus the elevation of plasma CARTp is host derived and likely driven by inflammation.

The stress-induced heat shock protein 70.3 expression is regulated by a dual-component mechanism involving alternative polyadenylation and HuR.

PubMed

Kraynik, Stephen M; Gabanic, Andrew; Anthony, Sarah R; Kelley, Melissa; Paulding, Waltke R; Roessler, Anne; McGuinness, Michael; Tranter, Michael

2015-06-01

Heat shock protein 70.3 (Hsp70.3) expression increases in response to cellular stress and plays a cytoprotective role. We have previously shown that Hsp70.3 expression is controlled through coordinated post-transcriptional regulation by miRNAs and alternative polyadenylation (APA), and APA-mediated shortening of the Hsp70.3 3'-UTR facilitates increased protein expression. A stress-induced increase in Hsp70.3 mRNA and protein expression is accompanied by alternative polyadenylation (APA)-mediated truncation of the 3'UTR of the Hsp70.3 mRNA transcript. However, the role that APA plays in stress-induced expression of Hsp70.3 remains unclear. Our results show that APA-mediated truncation of the Hsp70.3 3'UTR increases protein expression through enhanced polyribosome loading. Additionally, we demonstrate that the RNA binding protein HuR, which has been previously shown to play a role in mediating APA, is necessary for heat shock mediated increase in Hsp70.3 mRNA and protein. However, it is somewhat surprising to note that HuR does not play a role in APA of the Hsp70.3 mRNA, and these two regulatory events appear to be mutually exclusive regulators of Hsp70.3 expression. These results not only provide important insight to the regulation of stress response genes following heat shock, but also contribute an enhanced understanding of how alternative polyadenylation contributes to gene regulation. Copyright © 2015 Elsevier B.V. All rights reserved.

Autotaxin Expression Is Regulated at the Post-transcriptional Level by the RNA-binding Proteins HuR and AUF1.

PubMed

Sun, Shuhong; Zhang, Xiaotian; Lyu, Lin; Li, Xixi; Yao, Siliang; Zhang, Junjie

2016-12-09

Autotaxin (ATX) is a key enzyme that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a lysophospholipid mediator that regulates cellular activities through its specific G protein-coupled receptors. The ATX-LPA axis plays an important role in various physiological and pathological processes, especially in inflammation and cancer development. Although the transcriptional regulation of ATX has been widely studied, the post-transcriptional regulation of ATX is largely unknown. In this study, we identified conserved adenylate-uridylate (AU)-rich elements in the ATX mRNA 3'-untranslated region (3'UTR). The RNA-binding proteins HuR and AUF1 directly bound to the ATX mRNA 3'UTR and had antagonistic functions in ATX expression. HuR enhanced ATX expression by increasing ATX mRNA stability, whereas AUF1 suppressed ATX expression by promoting ATX mRNA decay. HuR and AUF1 were involved in ATX regulation in Colo320 human colon cancer cells and the LPS-stimulated human monocytic THP-1 cells. HuR knockdown suppressed ATX expression in B16 mouse melanoma cells, leading to inhibition of cell migration. This effect was reversed by AUF1 knockdown to recover ATX expression or by the addition of LPA. These results suggest that the post-transcriptional regulation of ATX expression by HuR and AUF1 modulates cancer cell migration. In summary, we identified HuR and AUF1 as novel post-transcriptional regulators of ATX expression, thereby elucidating a novel mechanism regulating the ATX-LPA axis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Involvement of hypothalamic cyclooxygenase-2, interleukin-1β and melanocortin in the development of docetaxel-induced anorexia in rats.

PubMed

Yamamoto, Kouichi; Asano, Keiko; Ito, Yui; Matsukawa, Naoki; Kim, Seikou; Yamatodani, Atsushi

2012-12-16

Docetaxel, a taxane derivative, is frequently used for the treatment of advanced breast cancer, non-small cell lung cancer, and metastatic prostate cancer. Clinical reports demonstrated that docetaxel-based chemotherapy often induces anorexia, but the etiology is not completely understood. To elucidate possible mechanisms, we investigated the involvement of central interleukin (IL)-1β, cyclooxygenase (COX)-2, and pro-opiomelanocortin (POMC) in the development of docetaxel-induced anorexia in rats. Rats received docetaxel (10mg/kg, i.p.) with or without pretreatment with selective COX-2 inhibitors, NS-398 (10 and 30 mg/kg, i.g.) or celecoxib (10 and 30 mg/kg, i.g.), and a non-selective COX inhibitor, indomethacin (10mg/kg, i.g.), then food intake was monitored for 24h after administration. We also examined expression of IL-1β, COX-2, and POMC mRNA in hypothalamus of docetaxel-treated rats and the effect of a COX-2 inhibitor on docetaxel-induced POMC mRNA expression. Food consumption in rats was significantly decreased 24h after administration of docetaxel and anorexia was partially reversed by all COX inhibitors. Administration of docetaxel increased IL-1β, COX-2, and POMC mRNA expression in the hypothalamus of rats. The time required to increase these gene expressions was comparable to the latency period of docetaxel-induced anorexia in rats. In addition, pretreatment with COX-2 inhibitors suppressed docetaxel-induced expression of POMC mRNA. These results suggest that IL-1β and COX-2 mRNA expression and subsequent activation of POMC in the hypothalamus may contribute to the development of docetaxel-induced anorexia in rats. Copyright © 2012. Published by Elsevier Ireland Ltd.

Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

PubMed Central

Martins, Rute; Proença, Daniela; Silva, Bruno; Barbosa, Cristina; Silva, Ana Luísa; Faustino, Paula; Romão, Luísa

2012-01-01

Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 3′ untranslated region (UTR), along with exon seven. Therefore, this 3′ UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 3′-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites – two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its protein expression according to the physiological cellular requirements. PMID:22530027

A novel cantharidin analog N-Benzylcantharidinamide reduces the expression of MMP-9 and invasive potentials of Hep3B via inhibiting cytosolic translocation of HuR

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

Lee, Ji-Yeon; Chung, Tae-Wook; Choi, Hee-Jung

2014-05-02

Highlights: • We examined the inhibition of N-Benzylcantharidinamide on MMP-9-mediated invasion. • Unlike cantharidin, N-Benzylcantharidinamide has very low toxicity on Hep3B cells. • The reduced MMP-9 expression was due to HuR-mediated decrease of mRNA stability. • We suggest N-Benzylcantharidinamide as a novel inhibitor of MMP-9-related invasion. - Abstract: Invasion and metastasis are major causes of malignant tumor-associated mortality. The present study aimed to investigate the molecular events underlying inhibitory effect of N-Benzylcantharidinamide, a novel synthetic analog of cantharidin, on matrix metalloproteinase-9 (MMP-9)-mediated invasion in highly metastatic hepatocellular carcinoma Hep3B cells. In this investigation, among six analogs of cantharidin, only N-Benzylcantharidinamidemore » has the inhibitory action on MMP-9 expression at non-toxic dose. The MMP-9 expression and invasion of Hep3B cells were significantly suppressed by treatment of N-Benzylcantharidinamide in a dose-dependent manner. On the other hand, the transcriptional activity of MMP-9 promoter and nuclear levels of NF-κB and AP-1 as the main transcriptional factors inducing MMP-9 expression were not affected by it although the level of MMP-9 mRNA was reduced by treatment of N-Benzylcantharidinamide. Interestingly, the stability of MMP-9 mRNA was significantly reduced by N-Benzylcantharidinamide-treatment. In addition, the cytosolic translocation of human antigen R (HuR), which results in the increase of MMP-9 mRNA stability through interaction of HuR with 3′-untranslated region of MMP-9 mRNA, was suppressed by treatment of N-Benzylcantharidinamide, in a dose-dependent manner. Taken together, it was demonstrated, for the first time, that N-Benzylcantharidinamide suppresses MMP-9 expression by reducing HuR-mediated MMP-9 mRNA stability for the inhibition of invasive potential in highly metastatic Hep3B cells.« less

PAI-1 mRNA expression and plasma level in rheumatoid arthritis: relationship with 4G/5G PAI-1 polymorphism.

PubMed

Muñoz-Valle, José Francisco; Ruiz-Quezada, Sandra Luz; Oregón-Romero, Edith; Navarro-Hernández, Rosa Elena; Castañeda-Saucedo, Eduardo; De la Cruz-Mosso, Ulises; Illades-Aguiar, Berenice; Leyva-Vázquez, Marco Antonio; Castro-Alarcón, Natividad; Parra-Rojas, Isela

2012-12-01

Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting the synovial membrane, cartilage and bone. PAI-1 is a key regulator of the fibrinolytic system through which plasminogen is converted to plasmin. The plasmin activates the matrix metalloproteinase system, which is closely related with the joint damage and bone destruction in RA. The aim of this study was to investigate the relationship between 4G/5G PAI-1 polymorphism with mRNA expression and PAI-1 plasma protein levels in RA patients. 113 RA patients and 123 healthy subjects (HS) were included in the study. The 4G/5G PAI-1 polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism method; the PAI-1 mRNA expression was determined by real-time PCR; and the soluble PAI-1 (sPAI-1) levels were quantified using an ELISA kit. No significant differences in the genotype and allele frequencies of 4G/5G PAI-1 polymorphism were found between RA patients and HS. However, the 5G/5G genotype was the most frequent in both studied groups: RA (42%) and HS (44%). PAI-1 mRNA expression was slightly increased (0.67 fold) in RA patients with respect to HS (P = 0.0001). In addition, in RA patients, the 4G/4G genotype carriers showed increased PAI-1 mRNA expression (3.82 fold) versus 4G/5G and 5G/5G genotypes (P = 0.0001), whereas the sPAI-1 plasma levels did not show significant differences. Our results indicate that the 4G/5G PAI-1 polymorphism is not a marker of susceptibility in the Western Mexico. However, the 4G/4G genotype is associated with high PAI-1 mRNA expression but not with the sPAI-1 levels in RA patients.

Expression of Jagged1/Notch3 Signaling Pathway and their Relationship with the Tumor Angiogenesis in TNBC.

PubMed

Xue, Siliang; He, Lang; Zhang, Xiao; Zhou, Jin; Li, Fanghua; Wang, Xiaoshan

2017-02-01

Jagged1/Notch3 signaling pathway plays a key role in angiogenesis of breast cancer, but little is known in TNBC. This study was designed to investigate the expression of Jagged1/Notch3 mRNA and protein in TNBC, analyze their correlations with clinicopathological characteristics and prognosis. Moreover, the interrelationship among Jagged1/Notch3 and VEGF was initially evaluated. Jagged1/Notch3 mRNA and protein expression levels were determined by Q-RT-PCR and Western blotting. Additionally, Immunohistochemistry for Jagged1/Notch3 was detected by Ventana platform, VEGF and CD34 was performed using the EnVision/HRP technique. mRNA transcriptionof Jagged1/Notch3 was in accord with protein expression. TNBC patients with positive Jagged1 expression had poorer DFS (p = 0.008) and OS (p = 0.004). Jagged1 expression was independent predictors of OS (p = 0.038). The expression of VEGF was positively correlative to MVD (p = 0.018), MVD was significantly associated with Jagged1 (p <0.0001) and Notch3 (p <0.0001). The expression of Jagged1/Notch3 has no correlation with VEGF, only in positive VEGF expression of TNBC patients Jagged1/Notch3 had influence on DFS and OS (p <0.05). Jagged1/Notch3 was -expressed at both the mRNA and protein levels, Jagged1 served as an independent predictor of poor prognosis. We speculate that there is a cross-talk between Jagged1/Notch3 and VEGF in TNBC angiogenesis. Jagged1/Notch3 is expected to be an important signaling pathway for TNBC progression and a potential target for TNBC neovascularization therapy. Copyright © 2017 IMSS. Published by Elsevier Inc. All rights reserved.

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

Susceptibility loci of CNOT6 in the general mRNA degradation pathway and lung cancer risk - a re-analysis of eight GWASs

PubMed Central

Zhou, Fei; Wang, Yanru; Liu, Hongliang; Ready, Neal; Han, Younghun; Hung, Rayjean J.; Brhane, Yonathan; McLaughlin, John; Brennan, Paul; Bickeböller, Heike; Rosenberger, Albert; Houlston, Richard S.; Caporaso, Neil; Landi, Maria Teresa; Brüske, Irene; Risch, Angela; Ye, Yuanqing; Wu, Xifeng; Christiani, David C.; Goodman, Gary; Chen, Chu; Amos, Christopher I.; Qingyi, Wei

2017-01-01

Purpose mRNA degradation is an important regulatory step for controlling gene expression and cell functions. Genetic abnormalities of the genes involved in mRNA degradation were found to be associated with cancer risks. Therefore, we systematically investigated the roles of genetic variants of genes in the general mRNA degradation pathway in lung cancer risk. Experimental design Meta-analyses were conducted in six lung cancer genome-wide association studies (GWASs) from the Transdisciplinary Research in Cancer of the Lung and additional two GWASs from Harvard University and deCODE in the International Lung Cancer Consortium. Expression quantitative trait loci analysis (eQTL) was used for in silico functional validation of the identified significant susceptibility loci. Results This pathway-based analysis included 4,603 single nucleotide polymorphisms (SNP) in 68 genes in 14,463 lung cancer cases and 44,188 controls, of which 20 SNPs were found to be associated with lung cancer risk with a false discovery rate threshold of <0.05. Among the 11 newly identified SNPs in CNOT6, which were in high linkage disequilibrium, the rs2453176 with a RegulomDB score “1f” was chosen as the tag SNP for further analysis. We found that the rs2453176 T allele was significantly associated with lung cancer risk (odds ratio=1.11, 95% confidence interval=1.04–1.18, P=0.001) in the eight GWASs. In the eQTL analysis, we found that levels of CNOT6 mRNA expression were significantly correlated with the rs2453176 T allele, which provided additional biological basis for the observed positive association. Conclusion The CNOT6 rs2453176 SNP may be a new functional susceptible locus for lung cancer risk. PMID:27805284

Susceptibility loci of CNOT6 in the general mRNA degradation pathway and lung cancer risk-A re-analysis of eight GWASs.

PubMed

Zhou, Fei; Wang, Yanru; Liu, Hongliang; Ready, Neal; Han, Younghun; Hung, Rayjean J; Brhane, Yonathan; McLaughlin, John; Brennan, Paul; Bickeböller, Heike; Rosenberger, Albert; Houlston, Richard S; Caporaso, Neil; Landi, Maria Teresa; Brüske, Irene; Risch, Angela; Ye, Yuanqing; Wu, Xifeng; Christiani, David C; Goodman, Gary; Chen, Chu; Amos, Christopher I; Wei, Qingyi

2017-04-01

mRNA degradation is an important regulatory step for controlling gene expression and cell functions. Genetic abnormalities involved in mRNA degradation genes were found to be associated with cancer risks. Therefore, we systematically investigated the roles of genetic variants in the general mRNA degradation pathway in lung cancer risk. Meta-analyses were conducted using summary data from six lung cancer genome-wide association studies (GWASs) from the Transdisciplinary Research in Cancer of the Lung and additional two GWASs from Harvard University and deCODE in the International Lung Cancer Consortium. Expression quantitative trait loci analysis (eQTL) was used for in silico functional validation of the identified significant susceptibility loci. This pathway-based analysis included 6816 single nucleotide polymorphisms (SNP) in 68 genes in 14 463 lung cancer cases and 44 188 controls. In the single-locus analysis, we found that 20 SNPs were associated with lung cancer risk with a false discovery rate threshold of <0.05. Among the 11 newly identified SNPs in CNOT6, which were in high linkage disequilibrium, the rs2453176 with a RegulomDB score "1f" was chosen as the tagSNP for further analysis. We found that the rs2453176 T allele was significantly associated with lung cancer risk (odds ratio = 1.11, 95% confidence interval = 1.04-1.18) in the eight GWASs. In the eQTL analysis, we found that levels of CNOT6 mRNA expression were significantly correlated with the rs2453176 T allele, which provided additional biological basis for the observed positive association. The CNOT6 rs2453176 SNP may be a new functional susceptible locus for lung cancer risk. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Expression of CD30 mRNA, CD30L mRNA and a variant form of CD30 mRNA in restimulated peripheral blood mononuclear cells (PBMC) of patients with helminthic infections resembling a Th2 disease

PubMed Central

Kilwinski, J; Berger, T; Mpalaskas, J; Reuter, S; Flick, W; Kern, P

1999-01-01

It has been proposed that CD30, a member of the tumour necrosis factor (TNF) receptor superfamily, is preferentially up-regulated on Th2-type human T cells. In order to investigate a correlation between infection with Echinococcus multilocularis and CD30 expression, we analysed regulation of CD30 mRNA, a variant form of CD30 mRNA (CD30v) and CD30 ligand (CD30L) mRNA expression on PBMC from patients with alveolar echinococcosis (AE) using reverse transcriptase-polymerase chain reaction (RT-PCR). In PBMC of patients with AE as well as healthy donors, spontaneous expression of CD30L mRNA and the CD30v mRNA could be detected. However, the intact form of CD30 mRNA could be detected neither in freshly isolated PBMC of patients nor in PBMC of healthy individuals. Expression of CD30L mRNA and the variant form of CD30 mRNA was frequently detected at individual time points during 72 h of culture of PBMC stimulated with crude Echinococcus antigen. In contrast to CD30v or CD30L mRNA expression, induction of CD30 mRNA expression was detected only in three out of six (50%) healthy donors and in 10 out of 21 (48%) patients with alveolar echinococcosis after 72 h of incubation. As a control, mitogenic stimulation of PBMC of both healthy individuals and infected patients led to expression of intact CD30 mRNA within 24 h of culture. These data demonstrate the different expression of two different forms of CD30 mRNA in PBMC of human individuals. The specific induction of CD30 expression is correlated only in rare cases with the clinical status of patients with AE, indicating the lack of a general induction of CD30 mRNA in this Th2-type-dominated helminthic disease. The data provide further evidence that the CD30 receptor is not an exclusive marker for a Th2-type response. PMID:9933429

mRNA changes in nucleus accumbens related to methamphetamine addiction in mice

NASA Astrophysics Data System (ADS)

Zhu, Li; Li, Jiaqi; Dong, Nan; Guan, Fanglin; Liu, Yufeng; Ma, Dongliang; Goh, Eyleen L. K.; Chen, Teng

2016-11-01

Methamphetamine (METH) is a highly addictive psychostimulant that elicits aberrant changes in the expression of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) in the nucleus accumbens of mice, indicating a potential role of METH in post-transcriptional regulations. To decipher the potential consequences of these post-transcriptional regulations in response to METH, we performed strand-specific RNA sequencing (ssRNA-Seq) to identify alterations in mRNA expression and their alternative splicing in the nucleus accumbens of mice following exposure to METH. METH-mediated changes in mRNAs were analyzed and correlated with previously reported changes in non-coding RNAs (miRNAs and lncRNAs) to determine the potential functions of these mRNA changes observed here and how non-coding RNAs are involved. A total of 2171 mRNAs were differentially expressed in response to METH with functions involved in synaptic plasticity, mitochondrial energy metabolism and immune response. 309 and 589 of these mRNAs are potential targets of miRNAs and lncRNAs respectively. In addition, METH treatment decreases mRNA alternative splicing, and there are 818 METH-specific events not observed in saline-treated mice. Our results suggest that METH-mediated addiction could be attributed by changes in miRNAs and lncRNAs and consequently, changes in mRNA alternative splicing and expression. In conclusion, our study reported a methamphetamine-modified nucleus accumbens transcriptome and provided non-coding RNA-mRNA interaction networks possibly involved in METH addiction.

Iodine excess exposure during pregnancy and lactation impairs maternal thyroid function in rats

PubMed Central

Salgueiro, Rafael Barrera; Vitzel, Kaio Fernando; Pantaleão, Thiago; Corrêa da Costa, Vânia Maria

2017-01-01

Adequate maternal iodine consumption during pregnancy and lactation guarantees normal thyroid hormones (TH) production, which is crucial to the development of the fetus. Indeed, iodine deficiency is clearly related to maternal hypothyroidism and deleterious effects in the fetal development. Conversely, the effects of iodine excess (IE) consumption on maternal thyroid function are still controversial. Therefore, this study aimed to investigate the impact of IE exposure during pregnancy and lactation periods on maternal hypothalamus–pituitary–thyroid axis. IE-exposed dams presented reduced serum TH concentration and increased serum thyrotropin (TSH) levels. Moreover, maternal IE exposure increased the hypothalamic expression of Trh and the pituitary expression of Trhr, Dio2, Tsha and Tshb mRNA, while reduced the Gh mRNA content. Additionally, IE-exposed dams presented thyroid morphological alterations, increased thyroid oxidative stress and decreased expression of thyroid genes/proteins involved in TH synthesis, secretion and metabolism. Furthermore, Dio1 mRNA expression and D1 activity were reduced in the liver and the kidney of IE-treated animals. Finally, the mRNA expression of Slc5a5 and Slc26a4 were reduced in the mammary gland of IE-exposed rats. The latter results are in accordance with the reduction of prolactin expression and serum levels in IE-treated dams. In summary, our study indicates that the exposure to IE during pregnancy and lactation induces primary hypothyroidism in rat dams and impairs iodide transfer to the milk. PMID:28814477

High Expression of Antiviral and Vitamin D Pathway Genes Are a Natural Characteristic of a Small Cohort of HIV-1-Exposed Seronegative Individuals.

PubMed

Aguilar-Jimenez, Wbeimar; Saulle, Irma; Trabattoni, Daria; Vichi, Francesca; Lo Caputo, Sergio; Mazzotta, Francesco; Rugeles, Maria T; Clerici, Mario; Biasin, Mara

2017-01-01

Natural resistance to HIV-1 infection is influenced by genetics, viral-exposure, and endogenous immunomodulators such as vitamin D (VitD), being a multifactorial phenomenon that characterizes HIV-1-exposed seronegative individuals (HESNs). We compared mRNA expression of 10 antivirals, 5 immunoregulators, and 3 VitD pathway genes by qRT-PCR in cells of a small cohort of 11 HESNs, 16 healthy-controls (HCs), and 11 seropositives (SPs) at baseline, in response to calcidiol (VitD precursor) and/or aldithriol-2-(AT2)-inactivated HIV-1. In addition, the expression of TIM-3 on T and NK cells of six HCs after calcidiol and calcitriol (active VitD) treatments was evaluated by flow cytometry. Calcidiol increased the mRNA expression of HAVCR2 (TIM-3; Th1-cells inhibitor) in HCs and HESNs. AT2-HIV-1 increased the mRNA expression of the activating VitD enzyme CYP27B1 , of the endogenous antiviral proteins MX2, TRIM22, APOBEC3G , and of immunoregulators ERAP2 and HAVCR2 , but reduced the mRNA expression of VitD receptor ( VDR ) and antiviral peptides PI3 and CAMP in all groups. Remarkably, higher mRNA levels of VDR, CYP27B1, PI3, CAMP, SLPI , and of ERAP2 were found in HESNs compared to HCs either at baseline or after stimuli. Furthermore, calcitriol increases the percentage of CD4+ T cells expressing TIM-3 protein compared to EtOH controls. These results suggest that high mRNA expression of antiviral and VitD pathway genes could be genetically determined in HESNs more than viral-induced at least in peripheral blood mononuclear cells. Moreover, the virus could potentiate bio-activation and use of VitD, maintaining the homeostasis of the immune system. Interestingly, VitD-induced TIM-3 on T cells, a T cell inhibitory and anti-HIV-1 molecule, requires further studies to analyze the functional outcomes during HIV-1 infection.

Striatal output markers do not alter in response to circling behaviour in 6-OHDA lesioned rats produced by acute or chronic administration of the monoamine uptake inhibitor BTS 74 398.

PubMed

Lane, E L; Cheetham, S; Jenner, P

2008-01-01

The monoamine uptake inhibitor BTS 74 398 induces ipsilateral circling in 6-hydroxydopamine (6-OHDA) lesioned rats without induction of abnormal motor behaviours associated with L-dopa administration. We examined whether this was reflected in the expression of peptide mRNA in the direct and indirect striatal output pathways.6-OHDA lesioning of the nigrostriatal pathway increased striatal expression of PPE-A mRNA and decreased levels of PPT mRNA with PPE-B mRNA expression remaining unchanged. Acute L-dopa administration normalised PPE-A mRNA and elevated PPT mRNA while PPE-B mRNA expression remained unchanged. Acute administration of BTS 74 398 did not alter striatal peptide mRNA levels. Following chronic treatment with L-dopa, PPE-A mRNA expression in the lesioned striatum continued to be normalised and PPT mRNA was increased compared to the intact side. PPE-B mRNA expression was also markedly increased relative to the non-lesioned striatum. Chronic BTS 74 398 administration did not alter mRNA expression in the 6-OHDA lesioned striatum although small increases in PPT mRNA expression in the intact and sham lesioned striatum were observed. The failure of BTS 74 398 to induce changes in striatal neuropeptide mRNA correlated with its failure to induce abnormal motor behaviours or behavioural sensitisation but does not explain how it produces a reversal of motor deficits. An action in another area of the brain appears likely and may explain the subsequent failure of BTS 74 398 and related compounds to exert anti-parkinsonian actions in man.

Action of specific thyroid hormone receptor α(1) and β(1) antagonists in the central and peripheral regulation of thyroid hormone metabolism in the rat.

PubMed

van Beeren, Hermina C; Kwakkel, Joan; Ackermans, Mariëtte T; Wiersinga, Wilmar M; Fliers, Eric; Boelen, Anita

2012-12-01

The iodine-containing drug amiodarone (Amio) and its noniodine containing analogue dronedarone (Dron) are potent antiarrhythmic drugs. Previous in vivo and in vitro studies have shown that the major metabolite of Amio, desethylamiodarone, acts as a thyroid hormone receptor (TR) α(1) and β(1) antagonist, whereas the major metabolite of Dron debutyldronedarone acts as a selective TRα(1) antagonist. In the present study, Amio and Dron were used as tools to discriminate between TRα(1) or TRβ(1) regulated genes in central and peripheral thyroid hormone metabolism. Three groups of male rats received either Amio, Dron, or vehicle by daily intragastric administration for 2 weeks. We assessed the effects of treatment on triiodothyronine (T(3)) and thyroxine (T(4)) plasma and tissue concentrations, deiodinase type 1, 2, and 3 mRNA expressions and activities, and thyroid hormone transporters monocarboxylate transporter 8 (MCT8), monocarboxylate transporter 10 (MCT10), and organic anion transporter 1C1 (OATP1C1). Amio treatment decreased serum T(3), while serum T(4) and thyrotropin (TSH) increased compared to Dron-treated and control rats. At the central level of the hypothalamus-pituitary-thyroid axis, Amio treatment decreased hypothalamic thyrotropin releasing hormone (TRH) expression, while increasing pituitary TSHβ and MCT10 mRNA expression. Amio decreased the pituitary D2 activity. By contrast, Dron treatment resulted in decreased hypothalamic TRH mRNA expression only. Upon Amio treatment, liver T(3) concentration decreased substantially compared to Dron and control rats (50%, p<0.01), but liver T(4) concentration was unaffected. In addition, liver D1, mRNA, and activity decreased, while the D3 activity and mRNA increased. Liver MCT8, MCT10, and OATP1C1 mRNA expression were similar between groups. Our results suggest an important role for TRα1 in the regulation of hypothalamic TRH mRNA expression, whereas TRβ plays a dominant role in pituitary and liver thyroid hormone metabolism.

Effects of environmental stress on mRNA expression levels of seven genes related to oxidative stress and growth in Atlantic salmon Salmo salar L. of farmed, hybrid and wild origin.

PubMed

Solberg, Monica F; Kvamme, Bjørn Olav; Nilsen, Frank; Glover, Kevin A

2012-12-05

Ten generations of domestication selection has caused farmed Atlantic salmon Salmo salar L. to deviate from wild salmon in a range of traits. Each year hundreds of thousands of farmed salmon escape into the wild. Thus, interbreeding between farmed escapees and wild conspecifics represents a significant threat to the genetic integrity of wild salmon populations. In a previous study we demonstrated how domestication has inadvertently selected for reduced responsiveness to stress in farmed salmon. To complement that study, we have evaluated the expression of seven stress-related genes in head kidney of salmon of farmed, hybrid and wild origin exposed to environmentally induced stress. In general, the crowding stressor used to induce environmental stress did not have a strong impact on mRNA expression levels of the seven genes, except for insulin-like growth factor-1 (IGF-1) that was downregulated in the stress treatment relative to the control treatment. mRNA expression levels of glutathione reductase (GR), Cu/Zn superoxide dismutase (Cu/Zn SOD), Mn superoxide dismutase (Mn SOD), glutathione peroxidase (GP) and IGF-1 were affected by genetic origin, thus expressed significantly different between the salmon of farmed, hybrid or wild origin. A positive relationship was detected between body size of wild salmon and mRNA expression level of the IGF-1 gene, in both environments. No such relationship was observed for the hybrid or farmed salmon. Farmed salmon in this study displayed significantly elevated mRNA levels of the IGF-1 gene relative to the wild salmon, in both treatments, while hybrids displayed a non additive pattern of inheritance. As IGF-1 mRNA levels are positively correlated to growth rate, the observed positive relationship between body size and IGF-1 mRNA levels detected in the wild but neither in the farmed nor the hybrid salmon, could indicate that growth selection has increased IGF-1 levels in farmed salmon to the extent that they may not be limiting growth rate.

Effects of environmental stress on mRNA expression levels of seven genes related to oxidative stress and growth in Atlantic salmon Salmo salar L. of farmed, hybrid and wild origin

PubMed Central

2012-01-01

Background Ten generations of domestication selection has caused farmed Atlantic salmon Salmo salar L. to deviate from wild salmon in a range of traits. Each year hundreds of thousands of farmed salmon escape into the wild. Thus, interbreeding between farmed escapees and wild conspecifics represents a significant threat to the genetic integrity of wild salmon populations. In a previous study we demonstrated how domestication has inadvertently selected for reduced responsiveness to stress in farmed salmon. To complement that study, we have evaluated the expression of seven stress-related genes in head kidney of salmon of farmed, hybrid and wild origin exposed to environmentally induced stress. Results In general, the crowding stressor used to induce environmental stress did not have a strong impact on mRNA expression levels of the seven genes, except for insulin-like growth factor-1 (IGF-1) that was downregulated in the stress treatment relative to the control treatment. mRNA expression levels of glutathione reductase (GR), Cu/Zn superoxide dismutase (Cu/Zn SOD), Mn superoxide dismutase (Mn SOD), glutathione peroxidase (GP) and IGF-1 were affected by genetic origin, thus expressed significantly different between the salmon of farmed, hybrid or wild origin. A positive relationship was detected between body size of wild salmon and mRNA expression level of the IGF-1 gene, in both environments. No such relationship was observed for the hybrid or farmed salmon. Conclusion Farmed salmon in this study displayed significantly elevated mRNA levels of the IGF-1 gene relative to the wild salmon, in both treatments, while hybrids displayed a non additive pattern of inheritance. As IGF-1 mRNA levels are positively correlated to growth rate, the observed positive relationship between body size and IGF-1 mRNA levels detected in the wild but neither in the farmed nor the hybrid salmon, could indicate that growth selection has increased IGF-1 levels in farmed salmon to the extent that they may not be limiting growth rate. PMID:23217180

Toll-Like Receptor and Accessory Molecule mRNA Expression in Humans and Mice as Well as in Murine Autoimmunity, Transient Inflammation, and Progressive Fibrosis

PubMed Central

Ramaiah, Santhosh Kumar Vankayala; Günthner, Roman; Lech, Maciej; Anders, Hans-Joachim

2013-01-01

The cell type-, organ-, and species-specific expression of the Toll-like receptors (TLRs) are well described, but little is known about the respective expression profiles of their accessory molecules. We therefore determined the mRNA expression levels of LBP, MD2, CD36, CD14, granulin, HMGB1, LL37, GRP94, UNC93b1, TRIL, PRAT4A, AP3B1, AEP and the respective TLRs in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. In addition, the expression profiles in transient tissue inflammation upon renal ischemia-reperfusion injury, in spleens and kidneys from mice with lupus-like systemic autoimmunity, and in progressive tissue fibrosis upon unilateral ureteral obstruction were studied. Several TLR co-factors were specifically regulated during the different phases of these disease entities, suggesting a functional involvement in the disease process. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to TLR-mediated innate immunity, which seems to be involved in the tissue injury phase, in the phase of tissue regeneration, and in progressive tissue remodelling. PMID:23803655

Inhibition of COX-2 and PGE2 in LPS-stimulated RAW264.7 cells by lonimacranthoide VI, a chlorogenic acid ester saponin

PubMed Central

GUAN, FUQIN; WANG, HAITING; SHAN, YU; CHEN, YU; WANG, MING; WANG, QIZHI; YIN, MIN; ZHAO, YOUYI; FENG, XU; ZHANG, JIANHUA

2014-01-01

Lonimacranthoide VI, first isolated from the flower buds of Lonicera macranthoides in our previous study, is a rare chlorogenic acid ester acylated at C-23 of hederagenin. In the present study, the anti-inflammatory effects of lonimacranthoide VI were studied. Lipopolysaccharides (LPS) induced an inflammatory response through the production of prostaglandin E2 (PGE2), and these levels were reduced when lonimacranthoide VI was pre-administered. Additionally, the mechanism of the anti-inflammatory effects of lonimacranthoide VI was investigated by measuring cyclooxygenase (COX) activity and mRNA expression. The results showed that lonimacranthoide VI inhibited mRNA expression and in vitro activity of COX-2 in a dose-dependent manner, whereas only the higher lonimacranthoide VI concentration possibly reduced COX-1 expression and in vitro activity. Taken together, these results indicate that lonimacranthoide VI is an important anti-inflammatory constituent of Lonicera macranthoides and that the anti-inflammatory effect is attributed to the inhibition of PGE2 production through COX activity and mRNA expression. PMID:25054024

Calcium-Dependent Protein Kinase Genes in Corn Roots

NASA Technical Reports Server (NTRS)

Takezawa, D.; Patil, S.; Bhatia, A.; Poovaiah, B. W.

1996-01-01

Two cDNAs encoding Ca-2(+) - Dependent Protein Kinases (CDPKs), Corn Root Protein Kinase 1 and 2 (CRPK 1, CRPK 2) were isolated from the root tip library of corn (Zea mays L., cv. Merit) and their nucleotide sequences were determined. Deduced amino acid sequences of both the clones have features characteristic of plant CDPKS, including all 11 conserved serine/threonine kinase subdomains, a junction domain and a calmodulin-like domain with four Ca-2(+), -binding sites. Northern analysis revealed that CRPKI mRNA is preferentially expressed in roots, especially in the root tip; whereas, the expression of CRPK2 mRNA was very low in all the tissues tested. In situ hybridization experiments revealed that CRPKI mRNA is highly expressed in the root apex, as compared to other parts of the root. Partially purified CDPK from the root tip phosphorylates syntide-2, a common peptide substrate for plant CDPKs, and the phosphorylation was stimulated 7-fold by the addition of Ca-2(+). Our results show that two CDPK isoforms are expressed in corn roots and they may be involved in the Ca-2(+)-dependent signal transduction process.

Rat lung metallothionein and heme oxygenase gene expression following ozone and zinc oxide exposure.

PubMed

Cosma, G; Fulton, H; DeFeo, T; Gordon, T

1992-11-01

We have conducted exposures in rats to determine pulmonary responses following inhalation of two common components of welding fumes, zinc oxide and ozone. To examine their effects on target-inducible gene expression, we measured mRNA levels of two metal-responsive genes, metallothionein (MT) and heme oxygenase (HO), in lung tissue by RNA slot-blot analysis. A 3-hr exposure to ZnO fume via a combustion furnace caused a substantial elevation in lung MT mRNA at all concentrations tested. Exposures to 5 and 2.5 mg/m3 ZnO resulted in peak 8-fold increases in MT mRNA levels (compared to air-exposed control animal values) immediately after exposure, while 1 mg/m3 ZnO exposure caused a 3.5-fold elevation in MT mRNA. These levels returned to approximate control gene expression values 24 hr after exposure. In addition, ZnO exposure caused an immediate elevation in lung HO gene expression levels, with 8-, 11-, and 5-fold increases observed after the same ZnO exposure levels (p < 0.05). Like MT gene induction, HO mRNA values returned to approximate control levels 24 hr after exposure. In striking contrast to the induction of MT and HO gene expression after ZnO exposures, there was no elevation in gene expression following a 6-hr exposure to 0.5 and 1 ppm ozone, even when lungs were examined as late as 72 hr after exposure. Our results demonstrate the induction of target gene expression following the inhalation of ZnO at concentrations equal to, and below, the current recommended threshold limit value of 5 mg/m3 ZnO. Furthermore, the lack of effect of ozone exposure on MT and HO gene expression suggests no involvement of these genes in the acute respiratory response to this oxidant compound.

Regulation of mouse hepatic CYP2D9 mRNA expression by growth and adrenal hormones.

PubMed

Jarukamjorn, Kanokwan; Sakuma, Tsutomu; Jaruchotikamol, Atika; Oguro, Miki; Nemoto, Nobuo

2006-02-01

The constitutive expression of CYP2D9 is sexually dimorphic, namely, strong in males, but diminutive in females. Repetition of mimic growth hormone (GH) secretion pattern impressively returned the mRNA expression level to that in intact mice: the GH secretion pattern's regulation of CYP2D9 mRNA expression has been predominantly disrupted by exogenous GH-administration. The extensive decline of CYP2D9 mRNA expression becoming a sexually non-specific P450 in 9-week-old male mice exposed as neonates to monosodium L-glutamate (MSG) suggested that the male GH secretion pattern is a key to the regulation of male-specific CYP2D9 mRNA expression in adult mice. Dexamethasone (Dex) showed possibility to induce CYP2D9 mRNA expression in adult MSG-neonatally treated mice of either sex. However, the antagonism was observed by co-administration of Dex and GH in the males. Dex-administration in adrenalectomized mice significantly elevated CYP2D9 mRNA expression levels. These findings suggest that an adrenal hormone participates in the regulatory mechanism of CYP2D9 mRNA expression in association with GH.

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.

Prognostic relevance of the expressions of CAV1 and TES genes on 7q31 in melanoma.

PubMed

Vizkeleti, Laura; Ecsedi, Szilvia; Rakosy, Zsuzsa; Begany, Agnes; Emri, Gabriella; Toth, Reka; Orosz, Adrienn; Szollosi, Attila Gabor; Mehes, Gabor; Adany, Roza; Balazs, Margit

2012-01-01

The 7q31 locus contains several genes affected in cancer progression. Although evidences exist regarding its impact on tumorigenesis, the role of genetic alterations and the expressions of locus-related genes are still controversial. Our study aimed to define the 7q31 copy number alterations in primary melanomas, primary-metastatic tumor pairs and cell lines. Data were correlated with clinical-pathological parameters. Genetic data show that 7q31 copy number distribution was heterogeneous in both primary and metastatic tumors. Extra copies were highly accompanied by chromosome 7 polisomy, and significantly increased in primary lesions with poor prognosis. Additionally, we determined the mRNA and protein levels of the locus-related CAV1 and TES genes. TES mRNA level was associated with metastatic location. CAV1 mRNA and protein levels were significantly higher in thicker tumors, however, lack of protein was also observed in a subpopulation of thin lesions. Expressions of CAV1 and TES were not associated with 7q31 alterations. In conclusion, 7q31 amplification can predict unfavorable outcome. Alterations of TES mRNA level may predict the location of metastasis. CAV1 possibly affect the cancer cell invasion.

MicroRNA-200c Modulates the Expression of MUC4 and MUC16 by Directly Targeting Their Coding Sequences in Human Pancreatic Cancer

PubMed Central

Radhakrishnan, Prakash; Mohr, Ashley M.; Grandgenett, Paul M.; Steele, Maria M.; Batra, Surinder K.; Hollingsworth, Michael A.

2013-01-01

Transmembrane mucins, MUC4 and MUC16 are associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. We discovered that miR-200c interacts with specific sequences within the coding sequence of MUC4 and MUC16 mRNAs, and evaluated the regulatory nature of this association. Pancreatic cancer cell lines S2.028 and T3M-4 transfected with miR-200c showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared to mock-transfected cells, respectively. A significant reduction of glycoprotein expression was also observed. These results indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to regulating proteins that modulate EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer. PMID:24204560

MicroRNA-200c modulates the expression of MUC4 and MUC16 by directly targeting their coding sequences in human pancreatic cancer.

PubMed

Radhakrishnan, Prakash; Mohr, Ashley M; Grandgenett, Paul M; Steele, Maria M; Batra, Surinder K; Hollingsworth, Michael A

2013-01-01

Transmembrane mucins, MUC4 and MUC16 are associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. We discovered that miR-200c interacts with specific sequences within the coding sequence of MUC4 and MUC16 mRNAs, and evaluated the regulatory nature of this association. Pancreatic cancer cell lines S2.028 and T3M-4 transfected with miR-200c showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared to mock-transfected cells, respectively. A significant reduction of glycoprotein expression was also observed. These results indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to regulating proteins that modulate EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer.

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

Danno, Hirosuke; Ishii, Kiyo-aki; Nakagawa, Yoshimi

To elucidate the physiological role of CREBH, the hepatic mRNA and protein levels of CREBH were estimated in various feeding states of wild and obesity mice. In the fast state, the expression of CREBH mRNA and nuclear protein were high and profoundly suppressed by refeeding in the wild-type mice. In ob/ob mice, the refeeding suppression was impaired. The diet studies suggested that CREBH expression was activated by fatty acids. CREBH mRNA levels in the mouse primary hepatocytes were elevated by addition of the palmitate, oleate and eicosapenonate. It was also induced by PPAR{alpha} agonist and repressed by PPAR{alpha} antagonist. Luciferasemore » reporter gene assays indicated that the CREBH promoter activity was induced by fatty acids and co-expression of PPAR{alpha}. Deletion studies identified the PPRE for PPAR{alpha} activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) assay confirmed that PPAR{alpha} directly binds to the PPRE. Activation of CREBH at fasting through fatty acids and PPAR{alpha} suggest that CREBH is involved in nutritional regulation.« less

Cloning, Characterization, and Expression of Animal Toxin Genes for Vaccine Development

DTIC Science & Technology

1990-04-27

amino acids have been modified to pyroglutamate . The function of this modification is not known. There are additional venom components that are...been constructed from the messenger ribonucleic acid (mRNA) isolated from venom glands of different poisonous animals such as snakes, scorpions, and...ribonucleic acid (mRNA) isolated from venom glands of different poisonous animals such as snakes, scorpions, and snails. The gene banks thus created con

Human Keratinocytes Are Vanilloid Resistant

PubMed Central

Pecze, László; Szabó, Kornélia; Széll, Márta; Jósvay, Katalin; Kaszás, Krisztián; Kúsz, Erzsébet; Letoha, Tamás; Prorok, János; Koncz, István; Tóth, András; Kemény, Lajos; Vizler, Csaba; Oláh, Zoltán

2008-01-01

Background Use of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca2+-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. Methods To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. Results Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca2+-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1–50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca2+-cytotoxity ([RTX]>15 µM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca2+-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. Conclusion TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials. PMID:18852901

Effects of naringin on the expression of miR-19b and cell apoptosis in human hepatocellular carcinoma

PubMed Central

Xie, Dafei; Yuan, Peiwen; Wang, Dong; Jin, Hua; Chen, Hui

2017-01-01

The effects of naringin on the expression of miR-19b and cell apoptosis were investigated in the human hepatocellular carcinoma cell line HepG2. HepG2 cells were treated with varied concentrations of naringin. The effects of naringin on the proliferation of HepG2 cells were observed by an MTT assay, morphological changes of cells were observed by an inverted microscope, cell apoptosis was detected by DAPI staining, miR-19b mRNA levels were determined with RT-PCR, and the expression of Bax and Bcl-2 proteins was examined by western blot assay. MTT results showed that naringin significantly inhibited the proliferation of HepG2 cells. Apoptotic HepG2 cells showed obvious changes in morphology under inverted microscope. DAPI staining suggested that naringin could induce cell shrinkage and nuclear chromatin condensation. RT-PCR results showed that naringin could upregulate the expression of miR-19b mRNA. Finally, western blot suggested that naringin upregulated the expression of Bax protein, but downregulated the expression of Bcl-2 protein. In conclusion, naringin can upregulate the expression of miR-19b mRNA and induce HepG2 cell apoptosis. In addition, it can also upregulate the expression of Bax protein and downregulate the expression of Bcl-2 protein during the process of apoptosis. PMID:28789364

Effects of naringin on the expression of miR-19b and cell apoptosis in human hepatocellular carcinoma.

PubMed

Xie, Dafei; Yuan, Peiwen; Wang, Dong; Jin, Hua; Chen, Hui

2017-08-01

The effects of naringin on the expression of miR-19b and cell apoptosis were investigated in the human hepatocellular carcinoma cell line HepG2. HepG2 cells were treated with varied concentrations of naringin. The effects of naringin on the proliferation of HepG2 cells were observed by an MTT assay, morphological changes of cells were observed by an inverted microscope, cell apoptosis was detected by DAPI staining, miR-19b mRNA levels were determined with RT-PCR, and the expression of Bax and Bcl-2 proteins was examined by western blot assay. MTT results showed that naringin significantly inhibited the proliferation of HepG2 cells. Apoptotic HepG2 cells showed obvious changes in morphology under inverted microscope. DAPI staining suggested that naringin could induce cell shrinkage and nuclear chromatin condensation. RT-PCR results showed that naringin could upregulate the expression of miR-19b mRNA. Finally, western blot suggested that naringin upregulated the expression of Bax protein, but downregulated the expression of Bcl-2 protein. In conclusion, naringin can upregulate the expression of miR-19b mRNA and induce HepG2 cell apoptosis. In addition, it can also upregulate the expression of Bax protein and downregulate the expression of Bcl-2 protein during the process of apoptosis.

Altered profile of mRNA expression in atrioventricular node of streptozotocin-induced diabetic rats

PubMed Central

Howarth, Frank Christopher; Parekh, Khatija; Jayaprakash, Petrilla; Inbaraj, Edward Samuel; Oz, Murat; Dobrzynski, Halina; Adrian, Thomas Edward

2017-01-01

Prolonged action potential duration, reduced action potential firing rate, upstroke velocity and rate of diastolic depolarization have been demonstrated in atrioventricular node (AVN) cells from streptozotocin (STZ)-induced diabetic rats. To further clarify the molecular basis of these electrical disturbances, the mRNA profiles encoding a variety of proteins associated with the generation and conduction of electrical activity in the AVN, were evaluated in the STZ-induced diabetic rat heart. Expression of mRNA was measured in AVN biopsies using reverse transcription-quantitative polymerase chain reaction techniques. Notable differences in mRNA expression included upregulation of genes encoding membrane and intracellular Ca2+ transport, including solute carrier family 8 member A1, transient receptor potential channel 1, ryanodine receptor 2/3, hyperpolarization-activated cyclic-nucleotide 2 and 3, calcium channel voltage-dependent, β2 subunit and sodium channels 3a, 4a, 7a and 3b. In addition to this, potassium channels potassium voltage-gated channel subfamily A member 4, potassium channel calcium activated intermediate/small conductance subfamily N α member 2, potassium voltage-gated channel subfamily J members 3, 5, and 11, potassium channel subfamily K members 1, 2, 3 and natriuretic peptide B (BNP) were upregulated in AVN of STZ heart, compared with controls. Alterations in gene expression were associated with upregulation of various proteins including the inwardly rectifying, potassium channel Kir3.4, NCX1 and BNP. The present study demonstrated notable differences in the profile of mRNA encoding proteins associated with the generation, conduction and regulation of electrical signals in the AVN of the STZ-induced diabetic rat heart. These data will provide a basis for a substantial range of future studies to investigate whether variations in mRNA translate into alterations in electrophysiological function. PMID:28731153

Molecular genetic mechanisms of allelic specific regulation of murine Comt expression

PubMed Central

Segall, Samantha K.; Shabalina, Svetlana A.; Meloto, Carolina B.; Wen, Xia; Cunningham, Danielle; Tarantino, Lisa M.; Wiltshire, Tim; Gauthier, Josée; Tohyama, Sarasa; Martin, Loren J.; Mogil, Jeffrey S.; Diatchenko, Luda

2015-01-01

Abstract A functional allele of the mouse catechol-O-methyltransferase (Comt) gene is defined by the insertion of a B2 short interspersed repeat element in its 3′-untranslated region (UTR). This allele has been associated with a number of phenotypes, such as pain and anxiety. In comparison with mice carrying the ancestral allele (Comt+), ComtB2i mice show higher Comt mRNA and enzymatic activity levels. Here, we investigated the molecular genetic mechanisms underlying this allelic specific regulation of Comt expression. Insertion of the B2 element introduces an early polyadenylation signal generating a shorter Comt transcript, in addition to the longer ancestral mRNA. Comparative analysis and in silico prediction of Comt mRNA potential targets within the transcript 3′ to the B2 element was performed and allowed choosing microRNA (miRNA) candidates for experimental screening: mmu-miR-3470a, mmu-miR-3470b, and mmu-miR-667. Cell transfection with each miRNA downregulated the expression of the ancestral transcript and COMT enzymatic activity. Our in vivo experiments showed that mmu-miR-667-3p is strongly correlated with decreasing amounts of Comt mRNA in the brain, and lentiviral injections of mmu-miR-3470a, mmu-miR-3470b, and mmu-miR-667 increase hypersensitivity in the mouse formalin model, consistent with reduced COMT activity. In summary, our data demonstrate that the Comt+ transcript contains regulatory miRNA signals in its 3′-untranslated region leading to mRNA degradation; these signals, however, are absent in the shorter transcript, resulting in higher mRNA expression and activity levels. PMID:26067582

Oxidative stress, endocrine disruption, and malformation of Bufo gargarizans embryo exposed to sub-lethal cadmium concentrations.

PubMed

Wu, Chao; Zhang, Yuhui; Chai, Lihong; Wang, Hongyuan

2017-01-01

Thyroid hormone (TH) is critical for vertebrate postembryonic development as well as embryonic development. Chinese toad (Bufo gargarizans) embryos were exposed to different concentrations of cadmium (5, 50, 100, 200 and 500μg Cd L -1 ) for 7days. Malformations were monitored daily, and growth and development of embryos were measured at day 4 and 7, and type 2 and 3 iodothyronine deiodinase (Dio2 and Dio3), thyroid hormone receptors (TRα and TRβ) mRNA levels were also measured to assess disruption of TH synthesis. In addition, superoxide dismutase (SOD), glutathione peroxidase (GPx) and heat shock proteins (HSPs) mRNA expression were examined to evaluate the ability of scavenging ROS. Our results demonstrated a bimodal inhibitory effect of Cd on the embryo growth and development of Bufo gargarizans. Reduced mean stage, total length and weight were observed at 5, 50, 200 and 500, but not at 100μg Cd L -1 . Embryos malformation occurred in all cadmium treatments. Morphological abnormalities of embryos are characterized by axial flexures, abdominal edema, stunted growth and fin flexure. Real-time PCR results show that exposure to cadmium down-regulated TRα and Dio3 mRNA expression and up-regulated Dio2 mRNA level. SOD and GPx mRNA expression was significantly up-regulated after cadmium exposure. We concluded that cadmium could change mRNA expression of TRα, Dio2 and Dio3 leading the inhibition of growth and development of B. gargarizans embryo, which suggests that cadmium might have the endocrine-disrupting effect in embryos. Moreover, the reduced ability of scavenging ROS induced by cadmium might be responsible for the teratogenic effects of cadmium. Copyright © 2016 Elsevier B.V. All rights reserved.

Transcription mapping and expression patterns of genes in the major immediate-early region of Kaposi's sarcoma-associated herpesvirus.

PubMed

Saveliev, Alexei; Zhu, Fan; Yuan, Yan

2002-08-01

Viral immediate-early (IE) genes are the first class of viral genes expressed during primary infection or reactivation from latency. They usually encode regulatory proteins that play crucial roles in viral life cycle. In a previous study, four regions in the KSHV genome were found to be actively transcribed in the immediate-early stage of viral reactivation in primary effusion lymphoma cells. Three immediate-early transcripts were characterized in these regions, as follows: mRNAs for ORF50 (KIE-1), ORF-45 (KIE-2), and ORF K4.2 (KIE-3) (F. X. Zhu, T. Cusano, and Y. Yuan, 1999, J. Virol. 73, 5556-5567). In the present study, we further analyzed the expression of genes in these IE regions in BC-1 and BCBL-1 cells. One of the immediate-early regions (KIE-1) that encompasses ORF50 and other genes was intensively studied to establish a detailed transcription map and expression patterns of genes in this region. This study led to identification of several novel IE transcripts in this region. They include a 2.6-kb mRNA which encodes ORF48/ORF29b, a family of transcripts that are complementary to ORF50 mRNA and a novel K8 IE mRNA of 1.5 kb. Together with the IE mRNA for ORF50 which was identified previously, four immediate-early genes have been mapped to KIE-1 region. Therefore, we would designate KIE-1 the major immediate-early region of KSHV. In addition, we showed that transcription of K8 gene is controlled by two promoters, yielding two transcripts, an immediate-early mRNA of 1.5 kb and a delayed-early mRNA of 1.3 kb.

Expression patterns of nestin and dentin sialoprotein during dentinogenesis in mice.

PubMed

Quispe-Salcedo, Angela; Ida-Yonemochi, Hiroko; Nakatomi, Mitsushiro; Ohshima, Hayato

2012-04-01

Differentiated odontoblasts could not be identified by one unique phenotypic marker, but the combination of expression of dentin phosphoprotein (Dpp), dentin sialoprotein (Dsp), dentin matrix protein 1 (Dmp1), and nestin may be valuable for the assessment of these cells. However, the findings using these proteins remain controversial. This study aimed to compare two odontoblast differentiation markers: nestin and Dsp in the process of dentinogenesis in mice. We performed immunohistochemistry and/or in situ hybridization technique for nestin and Dsp using 3-week-old incisors as well as postnatal 1-day- to 8-week-old molars. Preodontoblasts began to express nestin and Dsp proteins and Dsp mRNA, which increased in their intensity according to the progress of odontoblast differentiation in both incisors and developing molars. Nestin was consistently expressed in the differentiated odontoblasts even after the completion of dentin matrix deposition. The expression of Dsp mRNA coincided with the odontoblast secretory activity for dentin matrix deposition. In contrast, other pulpal cells, predentin matrix and dentinal tubules also showed a positive reaction for Dsp protein in addition to differentiated odontoblasts. In conclusion, nestin is valuable as a differentiation marker for odontoblasts, whereas Dsp mRNA is a functional marker for their secretory activity.

Thymidylate synthase (TS) protein expression as a prognostic factor in advanced colorectal cancer: a comparison with TS mRNA expression.

PubMed

Nakagawa, Tateo; Shimada, Mitsuo; Kurita, Nobuhiro; Iwata, Takashi; Nishioka, Masanori; Yoshikawa, Kozo; Higashijima, Jun; Utsunomiya, Tohru

2012-06-01

The role of intratumoral thymidylate synthase (TS) mRNA or protein expression is still controversial and little has been reported regarding relation of them in colorectal cancer. Forty-six patients with advanced colorectal cancer who underwent surgical resection were included. TS mRNA expression was determined by the Danenberg tumor profile method based on laser-captured micro-dissection of the tumor cells. TS protein expression was evaluated using immunohistochemical staining. TS mRNA expression tended to relate TS protein expression. Statistical significance was not found in overall survival between the TS mRNA high group and low group regardless of performing adjuvant chemotherapy. The overall survival in the TS protein negative group was significantly higher than that in positive group in all and the patients without adjuvant chemotherapy. Multivariate analysis showed TS protein expression was as an independent prognostic factor. TS protein expression tends to be related TS mRNA expression and is an independent prognostic factor in advanced colorectal cancer.

Bexarotene, a Selective RXRα Agonist, Reverses Hypotension Associated with Inflammation and Tissue Injury in a Rat Model of Septic Shock.

PubMed

Tunctan, Bahar; Kucukkavruk, Sefika P; Temiz-Resitoglu, Meryem; Guden, Demet S; Sari, Ayse N; Sahan-Firat, Seyhan

2018-02-01

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that can activate or inhibit the expression of many target genes by forming a heterodimer complex with the retinoid X receptor (RXR). The aim of this study was to investigate effects of bexarotene, a selective RXRα agonist, on the changes in renal, cardiac, hepatic, and pulmonary expression/activity of inducible nitric oxide synthase (iNOS) and cytochrome P450 (CYP) 4F6 in relation to PPARα/β/γ-RXRα heterodimer formation in a rat model of septic shock. Rats were injected with dimethyl sulfoxide or bexarotene 1 h after administration of saline or lipopolysaccharide (LPS). Mean arterial pressure (MAP) and heart rate (HR) were recorded from rats, which had received either saline or LPS before and after 1, 2, 3, and 4 h. Serum iNOS, LTB 4 , myeloperoxidase (MPO), and lactate dehydrogenase (LDH) levels as well as tissue iNOS and CYP4F6 mRNA expression in addition to PPARα/β/γ and RXRα proteins were measured. LPS-induced decrease in MAP and increase in HR were associated with a decrease in PPARα/β/γ-RXRα heterodimer formation and CYP4F6 mRNA expression. LPS also caused an increase in systemic iNOS, LTB 4 , MPO, and LDH levels as well as iNOS mRNA expression. Bexarotene at 0.1 mg/kg (i.p.) prevented the LPS-induced changes, except tachycardia. The results suggest that increased formation of PPARα/β/γ-RXRα heterodimers and CYP4F6 expression/activity in addition to decreased iNOS expression contributes to the beneficial effect of bexarotene to prevent the hypotension associated with inflammation and tissue injury during rat endotoxemia.

A novel homozygous stop-codon mutation in human HFE responsible for nonsense-mediated mRNA decay.

PubMed

Padula, Maria Carmela; Martelli, Giuseppe; Larocca, Marilena; Rossano, Rocco; Olivieri, Attilio

2014-09-01

HFE-hemochromatosis (HH) is an autosomal disease characterized by excessive iron absorption. Homozygotes for H63D variant, and still less H63D heterozygotes, generally do not express HH phenotype. The data collected in our previous study in the province of Matera (Basilicata, Italy) underlined that some H63D carriers showed altered iron metabolism, without additional factors. In this study, we selected a cohort of 10/22 H63D carriers with severe biochemical iron overload (BIO). Additional analysis was performed for studying HFE exons, exon-intron boundaries, and untranslated regions (UTRs) by performing DNA extraction, PCR amplification and sequencing. The results showed a novel substitution (NM_000410.3:c.847C>T) in a patient exon 4 (GenBankJQ478433); it introduces a premature stop-codon (PTC). RNA extraction and reverse-transcription were also performed. Quantitative real-time PCR was carried out for verifying if our aberrant mRNA is targeted for nonsense-mediated mRNA decay (NMD); we observed that patient HFE mRNA was expressed much less than calibrator, suggesting that the mutated HFE protein cannot play its role in iron metabolism regulation, resulting in proband BIO. Our finding is the first evidence of a variation responsible for a PTC in iron cycle genes. The genotype-phenotype correlation observed in our cases could be related to the additional mutation. Copyright © 2014 Elsevier Inc. All rights reserved.

TP53 and ATM mRNA expression in skin and skeletal muscle after low-level laser exposure.

PubMed

Guedes de Almeida, Luciana; Sergio, Luiz Philippe da Silva; de Paoli, Flavia; Mencalha, Andre Luiz; da Fonseca, Adenilson de Souza

2017-08-01

Low-level lasers are widespread in regenerative medicine, but the molecular mechanisms involved in their biological effects are not fully understood, particularly those on DNA stability. Therefore, this study aimed to investigate mRNA expression of genes related to DNA genomic stability in skin and skeletal muscle tissue from Wistar rats exposed to low-level red and infrared lasers. For this, TP53 (Tumor Protein 53) and ATM (Ataxia Telangiectasia Mutated gene) mRNA expressions were evaluated by real-time quantitative PCR (RT-qPCR) technique 24 hours after low-level red and infrared laser exposure. Our data showed that relative TP53 mRNA expression was not significantly altered in both tissues exposed to lasers. For ATM, relative mRNA expression in skin tissue was not significantly altered, but in muscle tissue, laser exposure increased relative ATM mRNA expression. Low-level red and infrared laser radiations alter ATM mRNA expression related to DNA stability in skeletal muscle tissue.

Effect of biliary drainage on inducible nitric oxide synthase, CD14 and TGR5 expression in obstructive jaundice rats

PubMed Central

Wang, Zi-Kai; Xiao, Jian-Guo; Huang, Xue-Fei; Gong, Yi-Chun; Li, Wen

2013-01-01

AIM: To investigate the effect of biliary drainage on inducible nitric oxide synthase (iNOS), CD14 and TGR5 expression in rats with obstructive jaundice (OJ). METHODS: Male adult Sprague-Dawley rats were randomly assigned to four groups: OJ, sham operation (SH), internal biliary drainage (ID) and external biliary drainage (ED). Rat models were successfully established by two operations and succumbed for extraction of Kupffer cells (KCs) and liver tissue collection on the 8th and 15th day. KCs were isolated by in situ hepatic perfusion and digested with collagen IV, density gradient centrifuged by percoll reagent and purified by cell culture attachment. The isolated KCs were cultured with the endotoxin lipopolysaccharide (LPS) with and without the addition of ursodeoxycholic acid (UDCA). The expression of iNOS, CD14 and bile acid receptor-TGR5 protein in rat liver tissues was determined by immunohistochemistry. The expression of iNOS and CD14 messenger RNA (mRNA) on the isolated KCs was detected by reverse transcription polymerase chain reaction (PCR) and the TGR5 mRNA level in KCs was measured by real-time quantitative PCR. RESULTS: The iNOS protein was markedly expressed in the liver of OJ rats, but rare expressed in SH rats. After relief of OJ, the iNOS expression was decidedly suppressed in the ID group (ID vs OJ, P < 0.01), but obviously increased in rats of ED (ED vs OJ, P = 0.004). When interfered only with LPS, the expression of iNOS mRNA by KCs was increased in the OJ group compared with the SH group (P = 0.004). After relief of biliary obstruction, the iNOS mRNA expression showed slight changes in the ED group (ED vs OJ, P = 0.71), but dropped in the ID group (ID vs OJ, P = 0.001). Compared with the simple intervention with LPS, the expressions of iNOS mRNA were significantly inhibited in all four groups after interfered with both LPS and UDCA (P < 0.01, respectively). After bile duct ligation, the CD14 protein expression in rat liver was significantly strengthened (OJ vs SH, P < 0.01), but the CD14 mRNA level by KCs was not up-regulated (OJ vs SH, P = 0.822). After relieving the OJ, the expression of CD14 protein was reduced in the ID group (ID vs OJ, P < 0.01), but not reduced in ED group (ED vs OJ, P = 0.591). And then the CD14 mRNA expression was aggravated by ED (ED vs OJ, P < 0.01), but was not significantly different between the ID group and the SH and OJ groups (ID vs SH, P = 0.944; ID vs OJ, P = 0.513, respectively). The expression of TGR5 protein and mRNA increased significantly in OJ rats (OJ vs SH, P = 0.001, respectively). After relief of OJ, ID could reduce the expression of TGR5 protein and mRNA to the levels of SH group (ID vs SH, P = 0.22 and P = 0.354, respectively), but ED could not (ED vs SH, P = 0.001, respectively). CONCLUSION: ID could be attributed to the regulatory function of activation of KCs and release of inflammatory mediators. PMID:23613625

MYC, FBXW7 and TP53 copy number variation and expression in Gastric Cancer

PubMed Central

2013-01-01

Background MYC deregulation is a common event in gastric carcinogenesis, usually as a consequence of gene amplification, chromosomal translocations, or posttranslational mechanisms. FBXW7 is a p53-controlled tumor-suppressor that plays a role in the regulation of cell cycle exit and reentry via MYC degradation. Methods We evaluated MYC, FBXW7, and TP53 copy number, mRNA levels, and protein expression in gastric cancer and paired non-neoplastic specimens from 33 patients and also in gastric adenocarcinoma cell lines. We also determined the invasion potential of the gastric cancer cell lines. Results MYC amplification was observed in 51.5% of gastric tumor samples. Deletion of one copy of FBXW7 and TP53 was observed in 45.5% and 21.2% of gastric tumors, respectively. MYC mRNA expression was significantly higher in tumors than in non-neoplastic samples. FBXW7 and TP53 mRNA expression was markedly lower in tumors than in paired non-neoplastic specimens. Moreover, deregulated MYC and FBXW7 mRNA expression was associated with the presence of lymph node metastasis and tumor stage III-IV. Additionally, MYC immunostaining was more frequently observed in intestinal-type than diffuse-type gastric cancers and was associated with MYC mRNA expression. In vitro studies showed that increased MYC and reduced FBXW7 expression is associated with a more invasive phenotype in gastric cancer cell lines. This result encouraged us to investigate the activity of the gelatinases MMP-2 and MMP-9 in both cell lines. Both gelatinases are synthesized predominantly by stromal cells rather than cancer cells, and it has been proposed that both contribute to cancer progression. We observed a significant increase in MMP-9 activity in ACP02 compared with ACP03 cells. These results confirmed that ACP02 cells have greater invasion capability than ACP03 cells. Conclusion In conclusion, FBXW7 and MYC mRNA may play a role in aggressive biologic behavior of gastric cancer cells and may be a useful indicator of poor prognosis. Furthermore, MYC is a candidate target for new therapies against gastric cancer. PMID:24053468

Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

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

Liu, Yanlong; Department of Medicine, University of Louisville, Louisville, KY; Wang, Chunhong

2012-10-15

Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl{sub 2}), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physicalmore » hypoxia (1% oxygen) and CoCl{sub 2} treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl{sub 2} administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl{sub 2}-induced reactive oxygen species (ROS) formation and completely negated CoCl{sub 2}-induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl{sub 2} administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl{sub 2} increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl{sub 2}-induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and absorption. -- Graphical abstract: Physical and chemical hypoxia decrease FGF-21 expression, which is inhibited by antioxidant, N-acetyl cysteine (NAC), in Caco-2 cells. Highlights: ► Hypoxia down-regulates FGF21 expression in Caco-2 cells. ► FGF21 down-regulation is HIF-α independent. ► FGF21 down-regulation is modulated by oxidative stress-mediated mRNA stability. ► FGF21 is involved in hypoxia‐induced triglyceride accumulation in Caco-2 cells.« less

Hypomethylation of inflammatory genes (COX2, EGR1, and SOCS3) and increased urinary 8-nitroguanine in arsenic-exposed newborns and children

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

Phookphan, Preeyaphan; Navasumrit, Panida

Early-life exposure to arsenic increases risk of developing a variety of non-malignant and malignant diseases. Arsenic-induced carcinogenesis may be mediated through epigenetic mechanisms and pathways leading to inflammation. Our previous study reported that prenatal arsenic exposure leads to increased mRNA expression of several genes related to inflammation, including COX2, EGR1, and SOCS3. This study aimed to investigate the effects of arsenic exposure on promoter DNA methylation and mRNA expression of these inflammatory genes (COX2, EGR1, and SOCS3), as well as the generation of 8-nitroguanine, which is a mutagenic DNA lesion involved in inflammation-related carcinogenesis. Prenatally arsenic-exposed newborns had promoter hypomethylationmore » of COX2, EGR1, and SOCS3 in cord blood lymphocytes (p < 0.01). A follow-up study in these prenatally arsenic-exposed children showed a significant hypomethylation of these genes in salivary DNA (p < 0.01). In vitro experiments confirmed that arsenite treatment at short-term high doses (10–100 μM) and long-term low doses (0.5–1 μM) in human lymphoblasts (RPMI 1788) caused promoter hypomethylation of these genes, which was in concordance with an increase in their mRNA expression. Additionally, the level of urinary 8-nitroguanine was significantly higher (p < 0.01) in exposed newborns and children, by 1.4- and 1.8-fold, respectively. Arsenic accumulation in toenails was negatively correlated with hypomethylation of these genes and positively correlated with levels of 8-nitroguanine. These results indicated that early-life exposure to arsenic causes hypomethylation of COX2, EGR1, and SOCS3, increases mRNA expression of these genes, and increases 8-nitroguanine formation. These effects may be linked to mechanisms of arsenic-induced inflammation and cancer development later in life. - Highlight: • Early-life arsenic exposure caused promoter hypomethylation of COX2, EGR1 and SOCS3. • Hypomethylation of these genes is associated with increased mRNA expression. • Arsenite treatment in vitro showed hypomethylation and increased mRNA expression. • Arsenic-exposed newborns and children had higher levels of urinary 8-nitroguanine. • Urinary 8-nitroguanine correlated with hypomethylation and mRNA expression.« less

UDP-Glucuronosyltransferase Expression in Mouse Liver Is Increased in Obesity- and Fasting-Induced Steatosis

PubMed Central

Xu, Jialin; Kulkarni, Supriya R.; Li, Liya

2012-01-01

UDP-glucuronosyltransferases (Ugt) catalyze phase II conjugation reactions with glucuronic acid, which enhances chemical polarity and the elimination from the body. Few studies have addressed whether Ugt expression and activity are affected by liver disease, such as steatosis. The purpose of this study was to determine whether steatosis induced by obesity or fasting could affect liver Ugt mRNA expression and activity. Male C57BL/6J and Lepob/ob (ob/ob) mice were fed ad libitum or food was withheld for 24 h. In steatotic livers of ob/ob mice, Ugt1a1, -1a6, -1a9, -2a3, -3a1, and -3a2 mRNA expression increased. Fasting, which also induced steatosis, increased hepatic Ugt1a1, -1a6, -1a7, -1a9, -2b1, -2b5, -2a3, -3a1, and -3a2 mRNA expression in mouse liver. Likewise, acetaminophen glucuronidation increased by 47% in hepatic microsomes from ob/ob mice compared with that in C57BL/6J mice, but not after fasting. In both steatosis models, Ugt induction was accompanied by increased aryl hydrocarbon receptor, constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor (PPAR)-α, pregnane X receptor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and peroxisome proliferator-activated receptor-γ coactivator-1α mRNA expression. In addition, fasting increased CAR, PPAR, and Nrf2 binding activity. The work points to hepatic triglyceride concentrations corresponding with nuclear receptor and Ugt expression. The findings indicate that steatosis significantly alters hepatic Ugt expression and activity, which could have a significant impact on determining circulating hormone levels, drug efficacy, and environmental chemical clearance. PMID:22031624

UDP-glucuronosyltransferase expression in mouse liver is increased in obesity- and fasting-induced steatosis.

PubMed

Xu, Jialin; Kulkarni, Supriya R; Li, Liya; Slitt, Angela L

2012-02-01

UDP-glucuronosyltransferases (Ugt) catalyze phase II conjugation reactions with glucuronic acid, which enhances chemical polarity and the elimination from the body. Few studies have addressed whether Ugt expression and activity are affected by liver disease, such as steatosis. The purpose of this study was to determine whether steatosis induced by obesity or fasting could affect liver Ugt mRNA expression and activity. Male C57BL/6J and Lep(ob/ob) (ob/ob) mice were fed ad libitum or food was withheld for 24 h. In steatotic livers of ob/ob mice, Ugt1a1, -1a6, -1a9, -2a3, -3a1, and -3a2 mRNA expression increased. Fasting, which also induced steatosis, increased hepatic Ugt1a1, -1a6, -1a7, -1a9, -2b1, -2b5, -2a3, -3a1, and -3a2 mRNA expression in mouse liver. Likewise, acetaminophen glucuronidation increased by 47% in hepatic microsomes from ob/ob mice compared with that in C57BL/6J mice, but not after fasting. In both steatosis models, Ugt induction was accompanied by increased aryl hydrocarbon receptor, constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor (PPAR)-α, pregnane X receptor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and peroxisome proliferator-activated receptor-γ coactivator-1α mRNA expression. In addition, fasting increased CAR, PPAR, and Nrf2 binding activity. The work points to hepatic triglyceride concentrations corresponding with nuclear receptor and Ugt expression. The findings indicate that steatosis significantly alters hepatic Ugt expression and activity, which could have a significant impact on determining circulating hormone levels, drug efficacy, and environmental chemical clearance.

Neurotensin gene expression increases during proestrus in the rostral medial preoptic nucleus: potential for direct communication with gonadotropin-releasing hormone neurons.

PubMed

Smith, M J; Wise, P M

2001-07-01

Neurotensin (NT)-containing neurons in the rostral portion of the medial preoptic nucleus (rMPN) of the brain may play a key role in regulating the pattern of secretion of GnRH, thereby influencing the reproductive cycle in females. The major goals of this study were to determine whether NT messenger RNA (mRNA) levels in the rMPN exhibit a unique pattern of expression in temporal association with the preovulatory LH surge and to assess whether NT neurons may communicate directly with GnRH neurons. We analyzed NT gene expression in rats using in situ hybridization over the day of proestrus and compared this with diestrous day 1. We also determined whether the high-affinity NT receptor (NT1) is expressed in GnRH neurons using dual-label in situ hybridization and whether this expression varies over the estrous cycle. We found that NT mRNA levels in the rMPN increase significantly on the day of proestrus, rising before the LH surge. No such change was detected on diestrous day 1, when the LH surge does not occur. Furthermore, we observed that a significant number of GnRH neurons coexpress NT1 mRNA and that the number of GnRH neurons expressing NT1 mRNA peaks on proestrus. Together with previous findings, our results suggest that increased expression of NT in the rMPN may directly stimulate GnRH neurons on proestrus, contributing to the LH surge. In addition, our results suggest that responsiveness of GnRH neurons to NT stimulation is enhanced on proestrus due to increased expression of NT receptors within GnRH neurons.

GDF-15 gene expression alterations in human lymphoblastoid cells and peripheral blood lymphocytes following exposure to ionizing radiation

PubMed Central

Li, Shuang; Zhang, Qing-Zhao; Zhang, De-Qin; Feng, Jiang-Bin; Luo, Qun; Lu, Xue; Wang, Xin-Ru; Li, Kun-Peng; Chen, De-Qing; Mu, Xiao-Feng; Gao, Ling; Liu, Qing-Jie

2017-01-01

The identification of rapid, sensitive and high-throughput biomarkers is imperative in order to identify individuals harmed by radiation accidents, and accurately evaluate the absorbed doses of radiation. DNA microarrays have previously been used to evaluate the alterations in growth/differentiation factor 15 (GDF15) gene expression in AHH-1 human lymphoblastoid cells, following exposure to γ-rays. The present study aimed to characterize the relationship between the dose of ionizing radiation and the produced effects in GDF-15 gene expression in AHH-1 cells and human peripheral blood lymphocytes (HPBLs). GDF-15 mRNA and protein expression levels following exposure to γ-rays and neutron radiation were assessed by reverse transcription-quantitative polymerase chain reaction and western blot analysis in AHH-1 cells. In addition, alterations in GDF-15 gene expression in HPBLs following ex vivo irradiation were evaluated. The present results demonstrated that GDF-15 mRNA and protein expression levels in AHH-1 cells were significantly upregulated following exposure to γ-ray doses ranging between 1 and 10 Gy, regardless of the dose rate. A total of 48 h following exposure to neutron radiation, a dose-response relationship was identified in AHH-1 cells at γ-ray doses between 0.4 and 1.6 Gy. GDF-15 mRNA levels in HPBLs were significantly upregulated following exposure to γ-ray doses between 1 and 8 Gy, within 4–48 h following irradiation. These results suggested that significant time- and dose-dependent alterations in GDF-15 mRNA and protein expression occur in AHH-1 cells and HPBLs in the early phases following exposure to ionizing radiation. In conclusion, alterations in GDF-15 gene expression may have potential as a biomarker to evaluate radiation exposure. PMID:28440431

Roles of Heparan Sulfate Sulfation in Dentinogenesis*

PubMed Central

Hayano, Satoru; Kurosaka, Hiroshi; Yanagita, Takeshi; Kalus, Ina; Milz, Fabian; Ishihara, Yoshihito; Islam, Md. Nurul; Kawanabe, Noriaki; Saito, Masahiro; Kamioka, Hiroshi; Adachi, Taiji; Dierks, Thomas; Yamashiro, Takashi

2012-01-01

Cell surface heparan sulfate (HS) is an essential regulator of cell signaling and development. HS traps signaling molecules, like Wnt in the glycosaminoglycan side chains of HS proteoglycans (HSPGs), and regulates their functions. Endosulfatases Sulf1 and Sulf2 are secreted at the cell surface to selectively remove 6-O-sulfate groups from HSPGs, thereby modifying the affinity of cell surface HSPGs for its ligands. This study provides molecular evidence for the functional roles of HSPG sulfation and desulfation in dentinogenesis. We show that odontogenic cells are highly sulfated on the cell surface and become desulfated during their differentiation to odontoblasts, which produce tooth dentin. Sulf1/Sulf2 double null mutant mice exhibit a thin dentin matrix and short roots combined with reduced expression of dentin sialophosphoprotein (Dspp) mRNA, encoding a dentin-specific extracellular matrix precursor protein, whereas single Sulf mutants do not show such defective phenotypes. In odontoblast cell lines, Dspp mRNA expression is potentiated by the activation of the Wnt canonical signaling pathway. In addition, pharmacological interference with HS sulfation promotes Dspp mRNA expression through activation of Wnt signaling. On the contrary, the silencing of Sulf suppresses the Wnt signaling pathway and subsequently Dspp mRNA expression. We also show that Wnt10a protein binds to cell surface HSPGs in odontoblasts, and interference with HS sulfation decreases the binding affinity of Wnt10a for HSPGs, which facilitates the binding of Wnt10a to its receptor and potentiates the Wnt signaling pathway, thereby up-regulating Dspp mRNA expression. These results demonstrate that Sulf-mediated desulfation of cellular HSPGs is an important modification that is critical for the activation of the Wnt signaling in odontoblasts and for production of the dentin matrix. PMID:22351753

Gibberellic Acid Regulates Chalcone Synthase Gene Transcription in the Corolla of Petunia hybrida 1

PubMed Central

Weiss, David; van Blokland, Rik; Kooter, Jan M.; Mol, Joseph N. M.; van Tunen, Arjen J.

1992-01-01

The pigmentation of Petunia hybrida corollas is regulated by gibberellic acid (GA3). It controls the increase of flavonoid enzyme levels and their corresponding mRNAs. We have used an in vitro culture system for corollas to study the regulatory role of GA3 in the expression of flavonoid genes. By determining steady-state mRNA levels, we show that the accumulation of chalcone synthase (chs) mRNA in young corollas is dependent on the presence of both sucrose and GA3 in the culture medium. Whereas sucrose had a general metabolic effect on gene expression, the stimulatory role of GA3 was specific. Analysis of nascent transcripts in isolated corolla nuclei showed that changes in steady-state chs mRNA levels correlated very well with changes in the transcription rate. We therefore conclude that GA3 controls the expression of chs at the transcriptional level. Preculturing the corollas in sucrose medium without GA3 resulted in a lower chs mRNA level. The expression could be reinduced by the addition of GA3. The hormone is thus required for the induction but also for the maintenance of chs transcription. The delayed reinduction of chs expression, the lag time in the kinetics of chs mRNA accumulation, and the inhibitory effect of cycloheximide on the action of GA3 suggest that GA3 controls chs transcription in an indirect manner. Our data support a model in which GA3 induces the production of a regulatory protein such as a receptor or a trans-acting factor that is directly involved in chs transcription. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6 PMID:16668613

Effects of spaceflight on murine skeletal muscle gene expression

PubMed Central

Allen, David L.; Bandstra, Eric R.; Harrison, Brooke C.; Thorng, Seiha; Stodieck, Louis S.; Kostenuik, Paul J.; Morony, Sean; Lacey, David L.; Hammond, Timothy G.; Leinwand, Leslie L.; Argraves, W. Scott; Bateman, Ted A.; Barth, Jeremy L.

2009-01-01

Spaceflight results in a number of adaptations to skeletal muscle, including atrophy and shifts toward faster muscle fiber types. To identify changes in gene expression that may underlie these adaptations, we used both microarray expression analysis and real-time polymerase chain reaction to quantify shifts in mRNA levels in the gastrocnemius from mice flown on the 11-day, 19-h STS-108 shuttle flight and from normal gravity controls. Spaceflight data also were compared with the ground-based unloading model of hindlimb suspension, with one group of pure suspension and one of suspension followed by 3.5 h of reloading to mimic the time between landing and euthanization of the spaceflight mice. Analysis of microarray data revealed that 272 mRNAs were significantly altered by spaceflight, the majority of which displayed similar responses to hindlimb suspension, whereas reloading tended to counteract these responses. Several mRNAs altered by spaceflight were associated with muscle growth, including the phosphatidylinositol 3-kinase regulatory subunit p85α, insulin response substrate-1, the forkhead box O1 transcription factor, and MAFbx/atrogin1. Moreover, myostatin mRNA expression tended to increase, whereas mRNA levels of the myostatin inhibitor FSTL3 tended to decrease, in response to spaceflight. In addition, mRNA levels of the slow oxidative fiber-associated transcriptional coactivator peroxisome proliferator-associated receptor (PPAR)-γ coactivator-1α and the transcription factor PPAR-α were significantly decreased in spaceflight gastrocnemius. Finally, spaceflight resulted in a significant decrease in levels of the microRNA miR-206. Together these data demonstrate that spaceflight induces significant changes in mRNA expression of genes associated with muscle growth and fiber type. PMID:19074574

Cloning of HSP90, expression and localization of HSP70/90 in different tissues including lactating/non-lactating yak (Bos grunniens) breast tissue.

PubMed

Liu, Penggang; Yu, Sijiu; Cui, Yan; He, Junfeng; Yu, Chuan; Wen, Zexing; Pan, Yangyang; Yang, Kun; Song, Liangli; Yang, Xue

2017-01-01

The aim of this study is to investigate the expression and localization of HSP70/90 in different tissues and explore the regulation effects of HSP70/90 at lactation period of female yaks. HSP90 mRNA was cloned from the heart samples of female yaks, Quantitative real-time (qRT-PCR), Western blotting (WB), immunohistochemistry and immunofluorescence assays were utilized to analyze the expressions of HSP70/90 mRNA and protein in different tissues. Sequence analysis showed that HSP90 is a conserved molecular chaperone of female yaks. The qRT-PCR, WB results showed that the expressions of HSP70/90 mRNA and protein were significantly different in different tissues, and 3-fold higher expression during the lactation period than the non-lactation period of breast tissue (P < 0.01). Immunohistochemistry and immunofluorescence assays results showed that HSP70/90 were located in the cardiac muscle cells, cerebellar medulla, theca cells lining at the reproductive system, and the mammary epithelia of the breasts. In addition, the expression level of HSP70 was higher than those of HSP90 in all examined tissues. Therefore, our results strongly suggest that the expression and localization of HSP70/90 could provide significant evidence to further research in tissue specific expression, and lactation function of female yaks.

Fas expression in renal cell carcinoma accurately predicts patient survival after radical nephrectomy.

PubMed

Sejima, Takehiro; Morizane, Shuichi; Hinata, Nobuyuki; Yao, Akihisa; Isoyama, Tadahiro; Saito, Motoaki; Takenaka, Atsushi

2012-01-01

To investigate Fas, Fas ligand (FasL) and Bcl-2 expression, which are considered to be important apoptotic regulatory factors in renal cell carcinomas (RCCs). mRNA quantification and immunohistochemistry allowed for the determination of the expression of these three factors in surgically resected tumors from 82 patients with RCC. The correlation of protein and gene expression with more than 10 years of survival data following nephrectomy (along with clinical and pathologic parameters) was analyzed using uni- and multivariate statistical models. A significantly poorer outcome was observed in patients with tumors expressing high levels of Fas mRNA in the multivariate analysis (p = 0.0002). In addition, patient survival was significantly worse in FasL mRNA-positive tumor cases when compared with FasL mRNA-negative cases (p = 0.0345). Ten cases relapsed more than 5 years after nephrectomy. Among them, the tumors of 8 cases (80%) did not express FasL mRNA. Analysis of Bcl-2 did not show statistical significance of Bcl-2 expression as a prognostic indicator. The data suggest that pronounced Fas expression is a surrogate biomarker of active cancer cell proliferation. Given the FasL tumor counterattack theory, FasL overexpression in RCC may be one of the host immune deficiencies, consequently leading to poor prognosis. Copyright © 2012 S. Karger AG, Basel.

The phosphotransferase VanU represses expression of four qrr genes antagonizing VanO-mediated quorum-sensing regulation in Vibrio anguillarum

PubMed Central

Weber, Barbara; Lindell, Kristoffer; El Qaidi, Samir; Hjerde, Erik; Willassen, Nils-Peder

2011-01-01

Vibrio anguillarum utilizes quorum sensing to regulate stress responses required for survival in the aquatic environment. Like other Vibrio species, V. anguillarum contains the gene qrr1, which encodes the ancestral quorum regulatory RNA Qrr1, and phosphorelay quorum-sensing systems that modulate the expression of small regulatory RNAs (sRNAs) that destabilize mRNA encoding the transcriptional regulator VanT. In this study, three additional Qrr sRNAs were identified. All four sRNAs were positively regulated by σ54 and the σ54-dependent response regulator VanO, and showed a redundant activity. The Qrr sRNAs, together with the RNA chaperone Hfq, destabilized vanT mRNA and modulated expression of VanT-regulated genes. Unexpectedly, expression of all four qrr genes peaked at high cell density, and exogenously added N-acylhomoserine lactone molecules induced expression of the qrr genes at low cell density. The phosphotransferase VanU, which phosphorylates and activates VanO, repressed expression of the Qrr sRNAs and stabilized vanT mRNA. A model is presented proposing that VanU acts as a branch point, aiding cross-regulation between two independent phosphorelay systems that activate or repress expression of the Qrr sRNAs, giving flexibility and precision in modulating VanT expression and inducing a quorum-sensing response to stresses found in a constantly changing aquatic environment. PMID:21948044

The phosphotransferase VanU represses expression of four qrr genes antagonizing VanO-mediated quorum-sensing regulation in Vibrio anguillarum.

PubMed

Weber, Barbara; Lindell, Kristoffer; El Qaidi, Samir; Hjerde, Erik; Willassen, Nils-Peder; Milton, Debra L

2011-12-01

Vibrio anguillarum utilizes quorum sensing to regulate stress responses required for survival in the aquatic environment. Like other Vibrio species, V. anguillarum contains the gene qrr1, which encodes the ancestral quorum regulatory RNA Qrr1, and phosphorelay quorum-sensing systems that modulate the expression of small regulatory RNAs (sRNAs) that destabilize mRNA encoding the transcriptional regulator VanT. In this study, three additional Qrr sRNAs were identified. All four sRNAs were positively regulated by σ(54) and the σ(54)-dependent response regulator VanO, and showed a redundant activity. The Qrr sRNAs, together with the RNA chaperone Hfq, destabilized vanT mRNA and modulated expression of VanT-regulated genes. Unexpectedly, expression of all four qrr genes peaked at high cell density, and exogenously added N-acylhomoserine lactone molecules induced expression of the qrr genes at low cell density. The phosphotransferase VanU, which phosphorylates and activates VanO, repressed expression of the Qrr sRNAs and stabilized vanT mRNA. A model is presented proposing that VanU acts as a branch point, aiding cross-regulation between two independent phosphorelay systems that activate or repress expression of the Qrr sRNAs, giving flexibility and precision in modulating VanT expression and inducing a quorum-sensing response to stresses found in a constantly changing aquatic environment.

Saikosaponin A Alleviates Symptoms of Attention Deficit Hyperactivity Disorder through Downregulation of DAT and Enhancing BDNF Expression in Spontaneous Hypertensive Rats.

PubMed

Jichao, Sun; Xinmin, Han; Xianguo, Ren; Dongqi, Yin; Rongyi, Zhou; Shuang, Lei; Yue, You; Yuchen, Song; Jingnan, Ying

2017-01-01

The disturbed dopamine availability and brain-derived neurotrophic factor (BDNF) expression are due in part to be associated with attention deficit hyperactivity disorder (ADHD). In this study, we investigated the therapeutical effect of saikosaponin a (SSa) isolated from Bupleurum Chinese DC, against spontaneously hypertensive rat (SHR) model of ADHD. Methylphenidate and SSa were orally administered for 3 weeks. Activity was assessed by open-field test and Morris water maze test. Dopamine (DA) and BDNF were determined in specific brain regions. The mRNA or protein expression of tyrosine hydroxylase (TH), dopamine transporter (DAT), and vesicles monoamine transporter (VMAT) was also studied. Both MPH and SSa reduced hyperactivity and improved the spatial learning memory deficit in SHRs. An increased DA concentration in the prefrontal cortex (PFC) and striatum was also observed after treating with the SSa. The increased DA concentration may partially be attributed to the decreased mRNA and protein expression of DAT in PFC while SSa exhibited no significant effects on the mRNA expression of TH and VMAT in PFC of SHRs. In addition, BDNF expression in SHRs was also increased after treating with SSa or MPH. The obtained result suggested that SSa may be a potential drug for treating ADHD.

Stimulation of EphB2/ephrin-B1 signalling by tumour necrosis factor alpha in human dental pulp stem cells.

PubMed

Zhu, Lifang; Dissanayaka, Waruna Lakmal; Green, David William; Zhang, Chengfei

2015-04-01

The aim of this study was to investigate whether in vitro stimulation of dental pulp stem cells (DPSCs) by tumour necrosis factor alpha (TNF-α) would induce secretion of EphB2/ephrin-B1 signalling. Dental pulp stem cells isolated from human dental pulp were treated with TNF-α (5-100 ng/ml) over 2-48 h. EphB2/ephrin-B1 mRNA and protein levels were measured by real-time polymerase chain reaction (RT-PCR) and western blot analysis respectively. Additionally, DPSCs were pre-incubated with TNF-α receptor neutralizing antibodies or infected with nuclear factor-kappa B (NF-ĸB) inhibitor, p38 MAPK inhibitor, Jun N-terminal kinase (JNK) inhibitor and MEK inhibitor before TNF-α treatment. Results were analysed by one-way ANOVA. Tumour necrosis factor alpha increased EphB2 mRNA expression in DPSCs at concentrations up to 20 ng/ml and ephrin-B1 at concentrations up to 40 ng/ml (P < 0.05). Its mRNA expression reached maximum at 24 h when treated with TNF-α at 20 ng/ml (P < 0.05). EphB2/ephrin-B1 protein expression levels were high at 16 and 24 h as shown by western blotting. Neutralizing antibodies for TNFR1/2 receptors down-regulated EphB2/ephrin-B1 mRNA expression (P < 0.05) and ephrin-B1 protein expression, but not EphB2 protein expression. JNK-inhibitor inhibited EphB2 mRNA expression only (P < 0.05). EphB2/ephrin-B1 were invoked in DPSCs with TNF-α treatment via the JNK-dependent pathway, but not NF-ĸB, p38 MAPK or MEK signalling. © 2015 John Wiley & Sons Ltd.

Stimulatory effect of insulin on 5alpha-reductase type 1 (SRD5A1) expression through an Akt-dependent pathway in ovarian granulosa cells.

PubMed

Kayampilly, Pradeep P; Wanamaker, Brett L; Stewart, James A; Wagner, Carrie L; Menon, K M J

2010-10-01

Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 μg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P<0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P<0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation.

Improved Brain Expression of Anti-Amyloid β scFv by Complexation of mRNA Including a Secretion Sequence with PEG-based Block Catiomer.

PubMed

Perche, Federico; Uchida, Satoshi; Akiba, Hiroki; Lin, Chin-Yu; Ikegami, Masaru; Dirisala, Anjaneyulu; Nakashima, Toshihiro; Itaka, Keiji; Tsumoto, Kohei; Kataoka, Kazunori

2017-01-01

The ever-increasing number of people living with Alzheimer's disease urges to develop more effective therapies. Despite considerable success, anti-Alzheimer immunotherapy still faces the challenge of intracerebral and intracellular delivery. This work introduces in situ production of anti-amyloid beta (Aβ) antibody after intracerebral injection of PEG-PAsp(DET)/mRNA polyplexes as a novel immunotherapy approach and a safer alternative compared to high systemic antibodies doses or administration of adenovirus encoding anti- Aβ antibodies. We used mRNA encoding three different Aβ-specific scFV with a secretion signal for passive immunotherapy. scFv contained a 6xHis-tag for immuno-detection. The secretion signal from IL2 (IL2ss) was added to allow extracellular engagement of senile plaques. Aβ affinity of scFv was measured by surface plasmon resonance. To allow intracellular delivery, scFv were administered as polyplexes formed with our smart copolymer polyethylene glycol-poly[N'-[N-(2-aminoethyl)-2-aminoethyl] aspartamide] [PEG-PAsp (DET)]. We evaluated scFv expression in cellulo by Western blot and ELISA, their ability to disaggregate amyloid aggregates by thioflavine T assay. Moreover, in vivo expression and therapeutic activity were evaluated in a murine amyloidosis model, by anti-6xHis-tag ELISA and anti- Aβ ELISA, respectively. The selected anti-amyloid beta scFv showed affinity towards Aβ and disaggregated Aβ fibers in vitro. Whereas both DNA and mRNA transfection led to scFV expression in cancer cells, only mRNA led to detectable scFv expression in primary neurons. In addition, the use of IL2ss increased by 3.4-fold scFv secretion by primary neurons over mRNA polyplexes devoid of secretion signal. In vivo, a 3 to 11- fold of intracranial scFv levels was measured for mRNA compared to DNA polyplexes and higher in vivo scFv levels were obtained with mRNA containing IL2ss over non-secreted mRNA. Intracranial injection of anti-Aβ mRNA polyplexes with IL2ss resulted in 40 % Aβ decrease in an acute amyloidosis model; with no decrease detected with control scFv mRNA nor DNA polyplexes. However, no Aβ decrease was detected in a more challenging transgenic model of Alzheimer's disease. Our results introduce a concerted approach not only for Alzheimer's disease treatment but also for immunotherapy against neurological diseases. The effectivity of our platform required the intracranial delivery of anti-Aβ scFv as mRNA not DNA, as mRNA with an IL2ss secretion sequence to favor engagement of Aβ in the amyloidosis model, complexation with a smart copolymer for efficient transfection of primary neurons and to achieve detectable mRNA expression in the brain during 48h. Amyloid burden decrease in an acute amyloidosis model was only achieved when these three factors (mRNA coding scFv, smart copolymer, IL2ss) were integrated into a single formulation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Actions of hypoxia on catecholamine synthetic enzyme mRNA expression before and after development of adrenal innervation in the sheep fetus

PubMed Central

Adams, M B; McMillen, I C

2000-01-01

We have investigated adrenal mRNA expression of the catecholamine synthetic enzymes tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) following acute hypoxia in fetal sheep before (< 105 days gestation, n = 20) and after (> 125 days gestation, n = 20) the development of adrenal innervation and following pretreatment with the nicotinic receptor anatgonist hexamethonium (n = 12). Total RNA was extracted from fetal adrenal glands collected at specific time points at 3-20 h after the onset of either hypoxia (∼50% reduction in fetal arterial oxygen saturation (SO2) for 30 min), or normoxia. Before 105 days, there was a decrease in adrenal TH mRNA expression at 20 h after hypoxia and adrenal TH mRNA expression was directly related to the changes in arterial PO2 measured during normoxia and hypoxia. After 125 days, adrenal TH mRNA levels were suppressed for up to 12 h following hypoxia. In both age groups, adrenal PNMT mRNA expression increased at 3-5 h after hypoxia and was inversely related to the changes in fetal arterial PO2 during normoxia or hypoxia. After 125 days, the administration of hexamethonium (25 mg kg−1, I. V.) reduced TH mRNA but not PNMT mRNA expression after normoxia. After hexamethonium pretreatment, there was no significant change in either adrenal TH or PNMT mRNA expression following hypoxia. We conclude that acute hypoxia differentially regulates adrenal TH and PNMT mRNA expression in the fetal sheep both before and after the development of adrenal innervation. After the development of adrenal innervation, however, the effect of acute hypoxia upon adrenal TH and PNMT mRNA expression is dependent upon neurogenic input acting via nicotinic receptors. PMID:11118487

Expression of three isoforms of Na-K-2Cl cotransporter (NKCC2) in the kidney and regulation by dehydration.

PubMed

Itoh, Kazuko; Izumi, Yuichiro; Inoue, Takeaki; Inoue, Hideki; Nakayama, Yushi; Uematsu, Takayuki; Fukuyama, Takashi; Yamazaki, Taiga; Yasuoka, Yukiko; Makino, Takeshi; Nagaba, Yasushi; Tomita, Kimio; Kobayashi, Noritada; Kawahara, Katsumasa; Mukoyama, Masashi; Nonoguchi, Hiroshi

2014-10-24

Sodium reabsorption via Na-K-2Cl cotransporter 2 (NKCC2) in the thick ascending limbs has a major role for medullary osmotic gradient and subsequent water reabsorption in the collecting ducts. We investigated intrarenal localization of three isoforms of NKCC2 mRNA expressions and the effects of dehydration on them in rats. To further examine the mechanisms of dehydration, the effects of hyperosmolality on NKCC2 mRNA expression in microdissected renal tubules was studied. RT-PCR and RT-competitive PCR were employed. The expressions of NKCC2a and b mRNA were observed in the cortical thick ascending limbs (CAL) and the distal convoluted tubules (DCT) but not in the medullary thick ascending limbs (MAL), whereas NKCC2f mRNA expression was seen in MAL and CAL. Two-day dehydration did not affect these mRNA expressions. In contrast, hyperosmolality increased NKCC2 mRNA expression in MAL in vitro. Bradykinin dose-dependently decreased NKCC2 mRNA expression in MAL. However, dehydration did not change NKCC2 protein expression in membrane fraction from cortex and outer medulla and in microdissected MAL. These data show that NKCC2a/b and f types are mainly present in CAL and MAL, respectively. Although NKCC2 mRNA expression was stimulated by hyperosmolality in vitro, NKCC2 mRNA and protein expressions were not stimulated by dehydration in vivo. These data suggest the presence of the inhibitory factors for NKCC2 expression in dehydration. Considering the role of NKCC2 for the countercurrent multiplier system, NKCC2f expressed in MAL might be more important than NKCC2a/b. Copyright © 2014 Elsevier Inc. All rights reserved.

Senescence marker protein 30 (SMP30)/regucalcin (RGN) expression decreases with aging, acute liver injuries and tumors in zebrafish

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

Fujisawa, Koichi; Terai, Shuji, E-mail: terais@yamaguchi-u.ac.jp; Hirose, Yoshikazu

2011-10-22

Highlights: {yields} Zebrafish SMP30/RGN mRNA expression decreases with aging. {yields} Decreased expression was observed in liver tumors as compared to the surrounding area. {yields} SMP30/RGN is important for liver proliferation and tumorigenesis. -- Abstract: Senescence marker protein 30 (SMP30)/regucalcin (RGN) is known to be related to aging, hepatocyte proliferation and tumorigenesis. However, expression and function of non-mammalian SMP30/RGN is poorly understood. We found that zebrafish SMP30/RGN mRNA expression decreases with aging, partial hepatectomy and thioacetamide-induced acute liver injury. SMP30/RGN expression was also greatly decreased in a zebrafish liver cell line. In addition, we induced liver tumors in adult zebrafish bymore » administering diethylnitrosamine. Decreased expression was observed in foci, hepatocellular carcinomas, cholangiocellular carcinomas and mixed tumors as compared to the surrounding area. We thus showed the importance of SMP30/RGN in liver proliferation and tumorigenesis.« less

Response of genes involved in lipid metabolism in rat epididymal white adipose tissue to different fasting conditions after long-term fructose consumption.

PubMed

Li, Jin-Xiu; Ke, Da-Zhi; Yao, Ling; Wang, Shang; Ma, Peng; Liu, Li; Zuo, Guo-Wei; Jiang, Li-Rong; Wang, Jian-Wei

2017-03-04

There has been much concern regarding the dietary fructose contributes to the development of metabolic syndrome. High-fructose diet changes the expression of genes involved in lipid metabolism. Levels of a number of hepatic lipogenic enzymes are increased by a high-carbohydrate diet in fasted-refed model rats/mice. Both the white adipose tissue (WAT) and the liver play a key role in the maintenance of nutrient homeostasis. Here, the aim of this study was to analyze the expression of key genes related to lipid metabolism in epididymal WAT (eWAT) in response to different fasting condition after long-term chronic fructose consumption. Rats were fed standard chow supplemented with 10% w/v fructose solution for 5 weeks, and killed after chow-fasting and fructose withdrawal (fasting) or chow-fasting and continued fructose (fructose alone) for 14 h. Blood parameters and the expression of genes involved in fatty acid synthesis (ChREBP, SREBP-1c, FAS, SCD1), triglyceride biosynthesis (DGAT-1, DGAT-2) and lipid mobilization (ATGL, HSL) in eWAT were analyzed. In addition, mRNA levels of PPAR-γ, CD36 and LPL were also detected. As expected, fructose alone increased the mRNA expression of FAS, SCD1, and correspondingly decreased ATGL and HSL mRNA levels. However, ChREBP, DGAT-2, ATGL and HSL mRNA levels restored near to normal while FAS and SCD1 tend to basic level under fasting condition. The mRNA expression of SREBP-1c, PPAR-γ and LPL did not changed at any situations but CD36 mRNA decreased remarkably in fructose alone group. In conclusion, these findings demonstrate that genes involved in lipid metabolism in rat eWAT are varied in response to different fasting conditions after long-term fructose consumption. Copyright © 2017. Published by Elsevier Inc.

The Shine-Dalgarno sequence of riboswitch-regulated single mRNAs shows ligand-dependent accessibility bursts

NASA Astrophysics Data System (ADS)

Rinaldi, Arlie J.; Lund, Paul E.; Blanco, Mario R.; Walter, Nils G.

2016-01-01

In response to intracellular signals in Gram-negative bacteria, translational riboswitches--commonly embedded in messenger RNAs (mRNAs)--regulate gene expression through inhibition of translation initiation. It is generally thought that this regulation originates from occlusion of the Shine-Dalgarno (SD) sequence upon ligand binding; however, little direct evidence exists. Here we develop Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) to investigate the ligand-dependent accessibility of the SD sequence of an mRNA hosting the 7-aminomethyl-7-deazaguanine (preQ1)-sensing riboswitch. Spike train analysis reveals that individual mRNA molecules alternate between two conformational states, distinguished by `bursts' of probe binding associated with increased SD sequence accessibility. Addition of preQ1 decreases the lifetime of the SD's high-accessibility (bursting) state and prolongs the time between bursts. In addition, ligand-jump experiments reveal imperfect riboswitching of single mRNA molecules. Such complex ligand sensing by individual mRNA molecules rationalizes the nuanced ligand response observed during bulk mRNA translation.

Messenger RNA Delivery for Tissue Engineering and Regenerative Medicine Applications.

PubMed

Patel, Siddharth; Athirasala, Avathamsa; Menezes, Paula P; Ashwanikumar, N; Zou, Ting; Sahay, Gaurav; Bertassoni, Luiz E

2018-06-07

The ability to control cellular processes and precisely direct cellular reprogramming has revolutionized regenerative medicine. Recent advances in in vitro transcribed (IVT) mRNA technology with chemical modifications have led to development of methods that control spatiotemporal gene expression. Additionally, there is a current thrust toward the development of safe, integration-free approaches to gene therapy for translational purposes. In this review, we describe strategies of synthetic IVT mRNA modifications and nonviral technologies for intracellular delivery. We provide insights into the current tissue engineering approaches that use a hydrogel scaffold with genetic material. Furthermore, we discuss the transformative potential of novel mRNA formulations that when embedded in hydrogels can trigger controlled genetic manipulation to regenerate tissues and organs in vitro and in vivo. The role of mRNA delivery in vascularization, cytoprotection, and Cas9-mediated xenotransplantation is additionally highlighted. Harmonizing mRNA delivery vehicle interactions with polymeric scaffolds can be used to present genetic cues that lead to precise command over cellular reprogramming, differentiation, and secretome activity of stem cells-an ultimate goal for tissue engineering.

Murine social stress results in long lasting voiding dysfunction.

PubMed

Butler, Stephan; Luz, Sandra; McFadden, Kile; Fesi, Joanna; Long, Christopher; Spruce, Lynn; Seeholzer, Steven; Canning, Douglas; Valentino, Rita; Zderic, Stephen

2018-01-01

Repeated exposure to social stress shifts the voiding phenotype in male mice leading to bladder wall remodeling and is associated with increased expression of the stress neuropeptide, corticotropin-releasing factor (CRF) in Barrington's nucleus neurons. In these studies, we set out to determine if the voiding phenotype could recover upon removal from the stressor. Male mice were exposed for 1h daily to an aggressor and the voiding phenotype was assessed at one month followed by randomization to three groups. One group underwent immediate sacrifice. Two groups were allowed a one month recovery from the social stress exposure with or without the addition of fluoxetine (1.2mg/ml) in their drinking water and repeat voiding patterns were measured prior to sacrifice. Social stress significantly increased bladder mass, bladder mass corrected for body weight, voided volumes, and decreased urinary frequency. The abnormal voiding phenotype persisted after a 1month recovery with no effect from the addition of fluoxetine. CRF mRNA in Barrington's nucleus was increased by social stress and remained elevated following recovery with no effect from the addition of fluoxetine. The mRNA and protein expression for the alpha 1 chains of type 1 and type III collagen was unchanged across all groups suggesting that changes in the extracellular matrix of the bladder are not responsible for the voiding phenotype. This persisting voiding dysfunction correlates with the persistent elevation of CRF mRNA expression in Barrington's nucleus. Copyright © 2017. Published by Elsevier Inc.

Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol.

PubMed

Lam, L T; Pickeral, O K; Peng, A C; Rosenwald, A; Hurt, E M; Giltnane, J M; Averett, L M; Zhao, H; Davis, R E; Sathyamoorthy, M; Wahl, L M; Harris, E D; Mikovits, J A; Monks, A P; Hollingshead, M G; Sausville, E A; Staudt, L M

2001-01-01

Flavopiridol, a flavonoid currently in cancer clinical trials, inhibits cyclin-dependent kinases (CDKs) by competitively blocking their ATP-binding pocket. However, the mechanism of action of flavopiridol as an anti-cancer agent has not been fully elucidated. Using DNA microarrays, we found that flavopiridol inhibited gene expression broadly, in contrast to two other CDK inhibitors, roscovitine and 9-nitropaullone. The gene expression profile of flavopiridol closely resembled the profiles of two transcription inhibitors, actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), suggesting that flavopiridol inhibits transcription globally. We were therefore able to use flavopiridol to measure mRNA turnover rates comprehensively and we found that different functional classes of genes had distinct distributions of mRNA turnover rates. In particular, genes encoding apoptosis regulators frequently had very short half-lives, as did several genes encoding key cell-cycle regulators. Strikingly, genes that were transcriptionally inducible were disproportionately represented in the class of genes with rapid mRNA turnover. The present genomic-scale measurement of mRNA turnover uncovered a regulatory logic that links gene function with mRNA half-life. The observation that transcriptionally inducible genes often have short mRNA half-lives demonstrates that cells have a coordinated strategy to rapidly modulate the mRNA levels of these genes. In addition, the present results suggest that flavopiridol may be more effective against types of cancer that are highly dependent on genes with unstable mRNAs.

Human pDCs display sex-specific differences in type I interferon subtypes and interferon α/β receptor expression.

PubMed

Ziegler, Susanne M; Beisel, Claudia; Sutter, Kathrin; Griesbeck, Morgane; Hildebrandt, Heike; Hagen, Sven H; Dittmer, Ulf; Altfeld, Marcus

2017-02-01

The outcomes of many diseases differ between women and men, with women experiencing a higher incidence and more severe pathogenesis of autoimmune and some infectious diseases. It has been suggested that this is partially due to activation of plasmacytoid dendritic cells (pDCs), the main producers of interferon (IFN)-α, in response to toll-like receptor (TLR)7 stimulation. We investigated the induction of type I IFN (IFN-I) subtypes upon TLR7 stimulation on isolated pDCs. Our data revealed a sex-specific differential expression of IFN-Is, with pDCs from females showing a significantly higher mRNA expression of all 13 IFN-α subtypes. In addition, pDCs from females had higher levels of IFN-β mRNA after stimulation, indicating that sex differences in IFN-I production by pDCs were mediated by a signaling event upstream of the first loop of IFN-I mRNA transcription. Furthermore, the surface expression levels of the common IFN-α/β receptor subunit 2 were significantly higher on pDCs from females in comparison to males. These data indicate that higher IFN-α production is already established at the mRNA level and propose a contribution of higher IFN-α/β receptor 2 expression on pDCs to the immunological differences in IFN-I production observed between females and males. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interferon-alpha-induced changes in metallothionein expression in liver biopsies from patients with chronic hepatitis C.

PubMed

Nagamine, Takeaki; Suzuki, Keiji; Kondo, Toshihiko; Nakazato, Kyomi; Kakizaki, Satoru; Takagi, Hitoshi; Nakajima, Katuyuki

2005-08-01

An association between reactive oxygen species and liver damage has been postulated in the course of hepatitis C virus (HCV) infection. Metallothionein (MT), induced by HCV core protein and interferon (IFN), plays a role in scavenging free radicals. MT expression in liver biopsies obtained from 21 patients with chronic HCV infection before and after IFN-alpha therapy was investigated. Changes in Knodell histological activity index (HAI) scores, MT protein levels (immunohistochemistry), MT-I and MT-II messenger (m)RNA expression levels (in situ hybridization) and proliferating cell nuclear antigen (PCNA) labelling index were determined and compared in serial liver specimens. MT staining was clustered around the portal tracts with inflammatory cells and fibrosis. The pattern of MT protein before IFN-alpha therapy was similar in all patients, but was higher in IFN-sustained responders than in nonresponders after IFN-alpha therapy. HAI scores and PCNA labelling indexes were significantly reduced after IFN-alpha therapy. MT-II mRNA expression correlated positively with PCNA index before therapy and with HAI scores after therapy (P<0.05). No correlation was found between MT-I mRNA and HAI scores or PCNA index. The findings indicate that IFN-alpha-induced hepatic MT may participate in the therapeutic effects of IFN-alpha for HCV. In addition, MT-II mRNA expression may be involved in cell proliferation in the livers of patients with chronic HCV infection.

Creatine supplementation increases glycogen storage but not GLUT-4 expression in human skeletal muscle.

PubMed

van Loon, Luc J C; Murphy, Robyn; Oosterlaar, Audrey M; Cameron-Smith, David; Hargreaves, Mark; Wagenmakers, Anton J M; Snow, Rodney

2004-01-01

It has been speculated that creatine supplementation affects muscle glucose metabolism in humans by increasing muscle glycogen storage and up-regulating GLUT-4 protein expression. In the present study, we assessed the effects of creatine loading and prolonged supplementation on muscle glycogen storage and GLUT-4 mRNA and protein content in humans. A total of 20 subjects participated in a 6-week supplementation period during which creatine or a placebo was ingested. Muscle biopsies were taken before and after 5 days of creatine loading (20 g.day(-1)) and after 6 weeks of continued supplementation (2 g.day(-1)). Fasting plasma insulin concentrations, muscle creatine, glycogen and GLUT-4 protein content as well as GLUT-4, glycogen synthase-1 (GS-1) and glycogenin-1 (Gln-1) mRNA expression were determined. Creatine loading significantly increased total creatine, free creatine and creatine phosphate content with a concomitant 18 +/- 5% increase in muscle glycogen content (P<0.05). The subsequent use of a 2 g.day(-1) maintenance dose for 37 days did not maintain total creatine, creatine phosphate and glycogen content at the elevated levels. The initial increase in muscle glycogen accumulation could not be explained by an increase in fasting plasma insulin concentration, muscle GLUT-4 mRNA and/or protein content. In addition, neither muscle GS-1 nor Gln-1 mRNA expression was affected. We conclude that creatine ingestion itself stimulates muscle glycogen storage, but does not affect muscle GLUT-4 expression.

Expression of MUC1, MUC2, MUC3 and MUC4 mucin mRNAs in human pancreatic and intestinal tumor cell lines.

PubMed

Hollingsworth, M A; Strawhecker, J M; Caffrey, T C; Mack, D R

1994-04-15

We examined the steady-state expression levels of mRNA for the MUC1, MUC2, MUC3 and MUC4 gene products in 12 pancreatic tumor cell lines, 6 colon tumor cell lines, and one ileocecal tumor cell line. The results showed that 10 of 12 pancreatic tumor cell lines expressed MUC1 mRNA and that 7 of these 12 lines also expressed relatively high levels of MUC4 mRNA. In contrast, MUC2 mRNA was expressed at only low levels and MUC3 was not detected in the pancreatic tumor cell lines. All 7 intestinal tumor cell lines examined expressed MUC2, and 5 of 7 expressed MUC3; however only one expressed significant levels of MUC1 and 2 expressed low levels of MUC4 mRNA. This report of high levels of MUC4 mRNA expression by pancreatic tumor cells raises the possibility that mucin carbohydrate epitopes defined by antibodies such as DuPan 2 may be expressed on a second mucin core protein produced by pancreatic tumor cells.

Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-{alpha}-dependent pathway in human dermal fibroblasts

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

Yamane, Takumi; Kobayashi-Hattori, Kazuo; Oishi, Yuichi, E-mail: y3oishi@nodai.ac.jp

2011-11-18

Highlights: Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. Black-Right-Pointing-Pointer Adiponectin also increases the phosphorylation of AMPK. Black-Right-Pointing-Pointer A pharmacological activator of AMPK increases mRNA levels of PPAR{alpha} and HAS2. Black-Right-Pointing-Pointer Adiponectin-induced HAS2 mRNA expression is blocked by a PPAR{alpha} antagonist. Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis via an AMPK/PPAR{alpha}-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis alongmore » with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1{beta}-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPAR{alpha} antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPAR{alpha}-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.« less

Role of miRNAs and alternative mRNA 3'-end cleavage and polyadenylation of their mRNA targets in cardiomyocyte hypertrophy.

PubMed

Soetanto, R; Hynes, C J; Patel, H R; Humphreys, D T; Evers, M; Duan, G; Parker, B J; Archer, S K; Clancy, J L; Graham, R M; Beilharz, T H; Smith, N J; Preiss, T

2016-05-01

miRNAs play critical roles in heart disease. In addition to differential miRNA expression, miRNA-mediated control is also affected by variable miRNA processing or alternative 3'-end cleavage and polyadenylation (APA) of their mRNA targets. To what extent these phenomena play a role in the heart remains unclear. We sought to explore miRNA processing and mRNA APA in cardiomyocytes, and whether these change during cardiac hypertrophy. Thoracic aortic constriction (TAC) was performed to induce hypertrophy in C57BL/6J mice. RNA extracted from cardiomyocytes of sham-treated, pre-hypertrophic (2 days post-TAC), and hypertrophic (7 days post-TAC) mice was subjected to small RNA- and poly(A)-test sequencing (PAT-Seq). Differential expression analysis matched expectations; nevertheless we identified ~400 mRNAs and hundreds of noncoding RNA loci as altered with hypertrophy for the first time. Although multiple processing variants were observed for many miRNAs, there was little change in their relative proportions during hypertrophy. PAT-Seq mapped ~48,000 mRNA 3'-ends, identifying novel 3' untranslated regions (3'UTRs) for over 7000 genes. Importantly, hypertrophy was associated with marked changes in APA with a net shift from distal to more proximal mRNA 3'-ends, which is predicted to decrease overall miRNA repression strength. We independently validated several examples of 3'UTR proportion change and showed that alternative 3'UTRs associate with differences in mRNA translation. Our work suggests that APA contributes to altered gene expression with the development of cardiomyocyte hypertrophy and provides a rich resource for a systems-level understanding of miRNA-mediated regulation in physiological and pathological states of the heart. Copyright © 2016 Elsevier B.V. All rights reserved.

Ventilation-induced increases in EGFR ligand mRNA are not altered by intra-amniotic LPS or ureaplasma in preterm lambs.

PubMed

Hillman, Noah H; Gisslen, Tate; Polglase, Graeme R; Kallapur, Suhas G; Jobe, Alan H

2014-01-01

Chorioamnionitis and mechanical ventilation are associated with bronchopulmonary dysplasia (BPD) in preterm infants. Mechanical ventilation at birth activates both inflammatory and acute phase responses. These responses can be partially modulated by previous exposure to intra-amniotic (IA) LPS or Ureaplasma parvum (UP). Epidermal growth factor receptor (EGFR) ligands participate in lung development, and angiotensin converting enzyme (ACE) 1 and ACE2 contribute to lung inflammation. We asked whether brief mechanical ventilation at birth altered EGFR and ACE pathways and if antenatal exposure to IA LPS or UP could modulate these effects. Ewes were exposed to IA injections of UP, LPS or saline multiple days prior to preterm delivery at 85% gestation. Lambs were either immediately euthanized or mechanically ventilated for 2 to 3 hr. IA UP and LPS cause modest changes in the EGFR ligands amphiregulin (AREG), epiregulin (EREG), heparin binding epidermal growth factor (HB-EGF), and betacellulin (BTC) mRNA expression. Mechanical ventilation greatly increased mRNA expression of AREG, EREG, and HB-EGF, with no additional increases resulting from IA LPS or UP. With ventilation AREG and EREG mRNA localized to cells in terminal airspace. EGFR mRNA also increased with mechanical ventilation. IA UP and LPS decreased ACE1 mRNA and increased ACE2 mRNA, resulting in a 4 fold change in the ACE1/ACE2 ratio. Mechanical ventilation with large tidal volumes increased both ACE1 and ACE2 expression. The alterations seen in ACE with IA exposures and EGFR pathways with mechanical ventilation may contribute to the development of BPD in preterm infants.

[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.

MCT1 and MCT4 kinetic of mRNA expression in different tissues after aerobic exercise at maximal lactate steady state workload.

PubMed

de Araujo, G G; Gobatto, C A; de Barros Manchado-Gobatto, F; Teixeira, L Fm; Dos Reis, I Gm; Caperuto, L C; Papoti, M; Bordin, S; Cavaglieri, C R; Verlengia, R

2015-01-01

We evaluate the mRNA expression of monocarboxylate transporters 1 and 4 (MCT1 and MCT4) in skeletal muscle (soleus, red and white gastrocnemius), heart and liver tissues in mice submitted to a single bout of swimming exercise at the maximal lactate steady state workload (MLSSw). After 72 h of MLSS test, the animals were submitted to a swimming exercise session for 25 min at individual MLSSw. Tissues and muscle samples were obtained at rest (control, n=5), immediately (n=5), 5 h (n=5) and 10 h (n=5) after exercise for determination of the MCT1 and MCT4 mRNA expression (RT-PCR). The MCT1 mRNA expression in liver increased after 10 h in relation to the control, immediate and 5 h groups, but the MCT4 remained unchanged. The MCT1 mRNA expression in heart increased by 31 % after 10 h when compared to immediate, but no differences were observed in relation to the control group. No significant differences were observed for red gastrocnemius in MCT1 and MCT4 mRNA expression. However, white gastrocnemius increased MCT1 mRNA expression immediately when compared to rest, 5 and 10 h test groups. In soleus muscle, the MCT1 mRNA expression increased immediately, 5 and 10 h after exercise when compared to the control. In relation to MCT4 mRNA expression, the soleus increased immediately and 10 h after acute exercise when compared to the control group. The soleus, liver and heart were the main tissues that showed improved the MCT1 mRNA expression, indicating its important role in controlling MLSS concentration in mice.

Curcumin administration suppress acetylcholinesterase gene expression in cadmium treated rats.

PubMed

Akinyemi, Ayodele Jacob; Oboh, Ganiyu; Fadaka, Adewale Oluwaseun; Olatunji, Babawale Peter; Akomolafe, Seun

2017-09-01

Curcumin, the main polyphenolic component of turmeric (Curcuma longa) rhizomes have been reported to exert anticholinesterase potential with limited information on how they regulate acetylcholinesterase (AChE) gene expression. Hence, this study sought to evaluate the effect of curcumin on cerebral cortex acetylcholinesterase (AChE) activity and their mRNA gene expression level in cadmium (Cd)-treated rats. Furthermore, in vitro effect of different concentrations of curcumin (1-5μg/mL) on rat cerebral cortex AChE activity was assessed. Animals were divided into six groups (n=6): group 1 serve as control (without Cd) and receive saline/vehicle, group 2 receive saline plus curcumin at 25mg/kg, group 3 receive saline plus curcumin 50mg/kg, group 4 receive Cd plus vehicle, group 5 receive Cd plus curcumin at 25mg/kg and group 6 receive Cd plus curcumin at 50mg/kg. Rats received Cd (2.5mg/kg) and curcumin (25 and 50mg/kg, respectively) by oral gavage for 7days. Acetylcholinesterase activity was measured by Ellman's method and AChE expression was carried out by a quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assay. We observed that acute administration of Cd increased acetylcholinesterase activity and in addition caused a significant (P<0.05) increase in AChE mRNA levels in whole cerebral cortex when compared to control group. However, co-treatment with curcumin inhibited AChE activity and alters AChE mRNA levels when compared to Cd-treated group. In addition, curcumin inhibits rat cerebral cortex AChE activity in vitro. In conclusion, curcumin exhibit anti-acetylcholinesterase activity and suppressed AChE mRNA gene expression level in Cd exposed rats, thus providing some biochemical and molecular evidence on the therapeutic effect of this turmeric-derived compound in treating neurological disorders including Alzheimer's disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Limb segment vibration modulates spinal reflex excitability and muscle mRNA expression after spinal cord injury

PubMed Central

Chang, Shuo-Hsiu; Tseng, Shih-Chiao; McHenry, Colleen L.; Littmann, Andrew E.; Suneja, Manish; Shields, Richard K.

2012-01-01

Objective We investigated the effect of various doses of vertical oscillation (vibration) on soleus H-reflex amplitude and post-activation depression in individuals with and without SCI. We also explored the acute effect of short-term limb vibration on skeletal muscle mRNA expression of genes associated with spinal plasticity. Methods Six healthy adults and five chronic complete SCI subjects received vibratory stimulation of their tibia over three different gravitational accelerations (0.3g, 0.6g, and 1.2g) at a fixed frequency (30 Hz). Soleus H-reflexes were measured before, during, and after vibration. Two additional chronic complete SCI subjects had soleus muscle biopsies 3 h following a single bout of vibration. Results H-reflex amplitude was depressed over 83% in both groups during vibration. This vibratory-induced inhibition lasted over 2 min in the control group, but not in the SCI group. Post-activation depression was modulated during the long-lasting vibratory inhibition. A single bout of mechanical oscillation altered mRNA expression from selected genes associated with synaptic plasticity. Conclusions Vibration of the lower leg inhibits the H-reflex amplitude, influences post-activation depression, and alters skeletal muscle mRNA expression of genes associated with synaptic plasticity. Significance Limb segment vibration may offer a long term method to reduce spinal reflex excitability after SCI. PMID:21963319

Dopaminergic Modulation of Risky Decision-Making

PubMed Central

Simon, Nicholas W.; Montgomery, Karienn S.; Beas, Blanca S.; Mitchell, Marci R.; LaSarge, Candi L.; Mendez, Ian A.; Bañuelos, Cristina; Vokes, Colin M.; Taylor, Aaron B.; Haberman, Rebecca P.; Bizon, Jennifer L.; Setlow, Barry

2012-01-01

Many psychiatric disorders are characterized by abnormal risky decision-making and dysregulated dopamine receptor expression. The current study was designed to determine how different dopamine receptor subtypes modulate risk-taking in young adult rats, using a “Risky Decision-making Task” that involves choices between small “safe” rewards and large “risky” rewards accompanied by adverse consequences. Rats showed considerable, stable individual differences in risk preference in the task, which were not related to multiple measures of reward motivation, anxiety, or pain sensitivity. Systemic activation of D2-like receptors robustly attenuated risk-taking, whereas drugs acting on D1-like receptors had no effect. Systemic amphetamine also reduced risk-taking, an effect which was attenuated by D2-like (but not D1-like) receptor blockade. Dopamine receptor mRNA expression was evaluated in a separate cohort of drug-naive rats characterized in the task. D1 mRNA expression in both nucleus accumbens shell and insular cortex was positively associated with risk-taking, while D2 mRNA expression in orbitofrontal and medial prefrontal cortex predicted risk preference in opposing nonlinear patterns. Additionally, lower levels of D2 mRNA in dorsal striatum were associated with greater risk-taking. These data strongly implicate dopamine signaling in prefrontal corticalstriatal circuitry in modulating decision-making processes involving integration of reward information with risks of adverse consequences. PMID:22131407

RNA-Binding Protein Dnd1 Promotes Breast Cancer Apoptosis by Stabilizing the Bim mRNA in a miR-221 Binding Site.

PubMed

Cheng, Feng; Pan, Ying; Lu, Yi-Min; Zhu, Lei; Chen, Shuzheng

2017-01-01

RNA-binding proteins (RBPs) and miRNAs are capable of controlling processes in normal development and cancer. Both of them could determine RNA transcripts fate from synthesis to decay. One such RBP, Dead end (Dnd1), is essential for regulating germ-cell viability and suppresses the germ-cell tumors development, yet how it exerts its functions in breast cancer has remained unresolved. The level of Dnd1 was detected in 21 cancerous tissues paired with neighboring normal tissues by qRT-PCR. We further annotated TCGA (The Cancer Genome Atlas) mRNA expression profiles and found that the expression of Dnd1 and Bim is positively correlated ( p = 0.04). Patients with higher Dnd1 expression level had longer overall survival ( p = 0.0014) by KM Plotter tool. Dnd1 knockdown in MCF-7 cells decreased Bim expression levels and inhibited apoptosis. While knockdown of Dnd1 promoted the decay of Bim mRNA 3'UTR, the stability of Bim-5'UTR was not affected. In addition, mutation of miR-221-binding site in Bim-3'UTR canceled the effect of Dnd1 on Bim mRNA. Knockdown of Dnd1 in MCF-7 cells confirmed that Dnd1 antagonized miR-221-inhibitory effects on Bim expression. Overall, our findings indicate that Dnd1 facilitates apoptosis by increasing the expression of Bim via its competitive combining with miR-221 in Bim-3'UTR. The new function of Dnd1 may contribute to a vital role in breast cancer development.

l-DOPA Decarboxylase (DDC) Expression Status as a Novel Molecular Tumor Marker for Diagnostic and Prognostic Purposes in Laryngeal Cancer.

PubMed

Patsis, Christos; Glyka, Vasiliki; Yiotakis, Ioannis; Fragoulis, Emmanuel G; Scorilas, Andreas

2012-08-01

l-DOPA decarboxylase (DDC) plays an essential role in the enzymatic synthesis of dopamine and alterations in its gene expression have been reported in several malignancies. Our objective was to analyze DDC messenger RNA (mRNA) and protein expression in laryngeal tissues and to evaluate the clinical implication of this molecule in laryngeal cancer. In this study, total RNA was isolated from 157 tissue samples surgically removed from 100 laryngeal cancer patients. A highly sensitive real-time polymerase chain reaction methodology based on SYBR Green I fluorescent dye was developed for the quantification of DDC mRNA levels. In addition, Western blot analysis was performed for the detection of DDC protein. DDC mRNA expression was revealed to be significantly downregulated in primary laryngeal cancer samples compared with their nonmalignant counterparts (P = .001). A significant negative association was also disclosed between DDC mRNA levels and TNM staging (P = .034). Univariate analysis showed that patients bearing DDC-positive tumors had a significantly decreased risk of death (hazard ratio = 0.23, P = .012) and local recurrence (hazard ratio = 0.32, P =.006), whereas DDC expression retained its favorable prognostic significance in the multivariate analysis. Kaplan-Meier curves further demonstrated that DDC-positive patients experienced longer overall and disease-free survival periods (P = .006 and P = .004, respectively). Moreover, DDC protein was detected in both neoplastic and noncancerous tissues. Therefore, our results suggest that DDC expression status could qualify as a promising biomarker for the future clinical management of laryngeal cancer patients.

l-DOPA Decarboxylase (DDC) Expression Status as a Novel Molecular Tumor Marker for Diagnostic and Prognostic Purposes in Laryngeal Cancer1

PubMed Central

Patsis, Christos; Glyka, Vasiliki; Yiotakis, Ioannis; Fragoulis, Emmanuel G; Scorilas, Andreas

2012-01-01

l-DOPA decarboxylase (DDC) plays an essential role in the enzymatic synthesis of dopamine and alterations in its gene expression have been reported in several malignancies. Our objective was to analyze DDC messenger RNA (mRNA) and protein expression in laryngeal tissues and to evaluate the clinical implication of this molecule in laryngeal cancer. In this study, total RNA was isolated from 157 tissue samples surgically removed from 100 laryngeal cancer patients. A highly sensitive real-time polymerase chain reaction methodology based on SYBR Green I fluorescent dye was developed for the quantification of DDC mRNA levels. In addition, Western blot analysis was performed for the detection of DDC protein. DDC mRNA expression was revealed to be significantly downregulated in primary laryngeal cancer samples compared with their nonmalignant counterparts (P = .001). A significant negative association was also disclosed between DDC mRNA levels and TNM staging (P = .034). Univariate analysis showed that patients bearing DDC-positive tumors had a significantly decreased risk of death (hazard ratio = 0.23, P = .012) and local recurrence (hazard ratio = 0.32, P =.006), whereas DDC expression retained its favorable prognostic significance in the multivariate analysis. Kaplan-Meier curves further demonstrated that DDC-positive patients experienced longer overall and disease-free survival periods (P = .006 and P = .004, respectively). Moreover, DDC protein was detected in both neoplastic and noncancerous tissues. Therefore, our results suggest that DDC expression status could qualify as a promising biomarker for the future clinical management of laryngeal cancer patients. PMID:22937181

Expression of VGF mRNA in the adult rat central nervous system.

PubMed

Snyder, S E; Salton, S R

1998-04-27

VGF is a secretory peptide precursor that is expressed and processed by neuronal cells in a cell type-specific fashion. In addition, VGF transcription and secretion are rapidly and relatively selectively induced by neurotrophins and depolarization in vitro. To gain insight into the possible function(s) of VGF in the nervous system, we have carried out a detailed examination of the distribution of VGF mRNA in the adult rat central nervous system by using in situ hybridization. Robust expression was detected in many neurons throughout the brain and spinal cord, in several types of neurons in the retina, and in presumptive chromaffin cells of the adrenal medulla. In the brain, prominent expression of VGF mRNA was observed in neurons of the main and accessory olfactory bulbs; in the anterior olfactory nucleus; in the induseum griseum and taenia tecta; in the olfactory tubercle; in CA1-CA3, the hilus of the dentate gyrus, and the subicular complex of the hippocampal formation; in the piriform, periamygdaloid, transitional, and lateral entorhinal cortices; in the endopiriform nucleus; in the hypothalamus, particularly the preoptic, periventricular, supraoptic, suprachiasmatic, and arcuate nuclei; and in a number of septal, thalamic, amygdaloid, and brainstem nuclei. Labeling was also seen in neurons of the neocortex and transitional cortical areas, particularly in layer V, and in basal ganglia and cerebellum. These data demonstrate that VGF mRNA is expressed much more extensively in the brain than has been described in previous RNA or immunohistochemical studies, and, furthermore, that VGF is widely expressed in the spinal cord and retina.

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

Cheng, Xingguo, E-mail: chengx@stjohns.edu; Vispute, Saurabh G.; Liu, Jie

The toxic effects of dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), mainly through activation of the aryl hydrocarbon receptor (AhR) are well documented. Fibroblast growth factor (Fgf) 21 plays critical roles in metabolic adaptation to fasting by increasing lipid oxidation and ketogenesis in the liver. The present study was performed to determine whether activation of the AhR induces Fgf21 expression. In mouse liver, TCDD increased Fgf21 mRNA in both dose- and time-dependent manners. In addition, TCDD markedly increased Fgf21 mRNA expression in cultured mouse and human hepatocytes. Moreover, TCDD increased mRNA (in liver) and protein levels (in both liver and serum) ofmore » Fgf21 in wild-type mice, but not in AhR-null mice. Chromatin immunoprecipitation assays showed that TCDD increased AhR protein binding to the Fgf21 promoter (− 105/+ 1 base pair). Fgf21-null mice administered 200 μg/kg of TCDD died within 20 days, whereas wild-type mice receiving the same treatment were still alive at one month after administration. This indicates that TCDD-induced Fgf21 expression protects against TCDD toxicity. Diethylhexylphthalate (DEHP) pretreatment attenuated TCDD-induced Fgf21 expression in mouse liver and white adipose tissue, which may explain a previous report that DEHP pretreatment decreases TCDD-induced wasting. In conclusion, Fgf21 appears to be a target gene of AhR-signaling pathway in mouse and human liver. - Highlights: • TCDD induced Fgf21 expression at both mRNA and protein levels. • Fgf21 induction by TCDD is AhR-dependent. • DEHP attenuated TCDD-induced Fgf21 expression.« less

Translation of vph mRNA in Streptomyces lividans and Escherichia coli after removal of the 5' untranslated leader.

PubMed

Wu, C J; Janssen, G R

1996-10-01

The Streptomyces vinaceus viomycin phosphotransferase (vph) mRNA contains an untranslated leader with a conventional Shine-Dalgarno homology. The vph leader was removed by ligation of the vph coding sequence to the transcriptional start site of a Streptomyces or an Escherichia coli promoter, such that transcription would initiate at the first position of the vph start codon. Analysis of mRNA demonstrated that transcription initiated primarily at the A of the vph AUG translational start codon in both Streptomyces lividans and E. coli; cells expressing the unleadered vph mRNA were resistant to viomycin indicating that the Shine-Dalgarno sequence, or other features contained within the leader, was not necessary for vph translation. Addition of four nucleotides (5'-AUGC-3') onto the 5' end of the unleadered vph mRNA resulted in translation initiation from the vph start codon and the AUG triplet contained within the added sequence. Translational fusions of vph sequence to a Tn5 neo reporter gene indicated that the first 16 codons of vph coding sequence were sufficient to specify the translational start site and reading frame for expression of neomycin resistance in both E. coli and S. lividans.

Cellular localization of thrombopoietin mRNA in the liver by in situ hybridization.

PubMed

Nomura, S; Ogami, K; Kawamura, K; Tsukamoto, I; Kudo, Y; Kanakura, Y; Kitamura, Y; Miyazaki, H; Kato, T

1997-07-01

The expression of thrombopoietin (TPO) mRNA is observed in several tissues, including liver, kidney, brain, skeletal muscle, intestine, spleen, and bone marrow. Among these organs, the highest expression of TPO mRNA is detected in the liver. We identified cells producing TPO by means of in situ hybridization of adult rat liver using digoxigenin-11-UTP-labeled cRNA probes. We found that the cells expressing TPO mRNA also expressed serum albumin mRNA. TPO mRNA was detected in parenchymal cells (hepatocytes) but not in non-parenchymal cells (including endothelial cells, epithelial cells, and so forth). To determine the location of TPO expression in embryogenesis, sections of fetal mice were further analyzed by in situ hybridization. TPO mRNA was detected only in hepatocytes of fetal liver, which was also the major site of hematopoiesis. The expression of TPO mRNA in fetal liver was observed from 12.5 days postcoitus. Northern blot analysis showed that mouse liver transcribed the same size of TPO mRNA in the fetus and in the adult. These results clearly demonstrate that hepatocytes are the primary site of TPO production in the liver from fetus to adult.

Comparative Analysis of mRNA Isoform Expression in Cardiac Hypertrophy and Development Reveals Multiple Post-Transcriptional Regulatory Modules

PubMed Central

Park, Ji Yeon; Li, Wencheng; Zheng, Dinghai; Zhai, Peiyong; Zhao, Yun; Matsuda, Takahisa; Vatner, Stephen F.; Sadoshima, Junichi; Tian, Bin

2011-01-01

Cardiac hypertrophy is enlargement of the heart in response to physiological or pathological stimuli, chiefly involving growth of myocytes in size rather than in number. Previous studies have shown that the expression pattern of a group of genes in hypertrophied heart induced by pressure overload resembles that at the embryonic stage of heart development, a phenomenon known as activation of the “fetal gene program”. Here, using a genome-wide approach we systematically defined genes and pathways regulated in short- and long-term cardiac hypertrophy conditions using mice with transverse aortic constriction (TAC), and compared them with those regulated at different stages of embryonic and postnatal development. In addition, exon-level analysis revealed widespread mRNA isoform changes during cardiac hypertrophy resulting from alternative usage of terminal or internal exons, some of which are also developmentally regulated and may be attributable to decreased expression of Fox-1 protein in cardiac hypertrophy. Genes with functions in certain pathways, such as cell adhesion and cell morphology, are more likely to be regulated by alternative splicing. Moreover, we found 3′UTRs of mRNAs were generally shortened through alternative cleavage and polyadenylation in hypertrophy, and microRNA target genes were generally de-repressed, suggesting coordinated mechanisms to increase mRNA stability and protein production during hypertrophy. Taken together, our results comprehensively delineated gene and mRNA isoform regulation events in cardiac hypertrophy and revealed their relations to those in development, and suggested that modulation of mRNA isoform expression plays an importance role in heart remodeling under pressure overload. PMID:21799842

Elevated expression of the proto-oncogene c-kit in patients with mastocytosis.

PubMed

Nagata, H; Worobec, A S; Semere, T; Metcalfe, D D

1998-02-01

The stem cell factor (SCF)c-kit receptor interaction plays a critical role in the development and survival of mast cells. Several studies have also associated c-kit receptor mutations with the human diseases, mastocytosis and piebaldism. Overexpression of c-kit has been reported to be associated with myeloproliferative disorders and myelodysplastic syndromes. Using peripheral blood mononuclear cells (PBMCs) from 11 patients with indolent mastocytosis (category I), mastocytosis with an associated hematologic disorder (category II), or aggressive mastocytosis (category III); a patient with CMML unassociated with mastocytosis, and PBMCs from 13 normal subjects, we examined the level of expression of c-kit mRNA along with other c-kit isoforms to determine if overexpression of the c-kit receptor was associated with mastocytosis. Using quantitative competitive PCR, c-kit mRNA levels on average were found to be statistically elevated in the five patients with mastocytosis with an associated hematologic disorder and in the patient with aggressive mastocytosis as compared with controls, but not elevated in patients with indolent mastocytosis. The relative mRNA expression for the two c-kit isoforms was not significantly different in the mastocytosis patients compared with controls. This demonstration of the overexpression of c-kit mRNA in mastocytosis, and particularly those patients with clinical evidence of myelodysplastic syndrome, adds evidence to support the conclusion that mastocytosis, at least in some patients, is a feature of myelodysplasia; and suggests that determination of c-kit mRNA expression in PBMCs may provide an additional approach to assessing prognosis.

Detection of MMP-9 and TIMP-3 mRNA expression in the villi of patients undergoing early spontaneous abortion: A report of 30 cases.

PubMed

Jiang, Guangli; Qi, Yuxia

2015-05-01

The aim of the present study was to investigate the correlation of matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase inhibitor (TIMP)-3 expression with spontaneous abortion (SA) during early pregnancy. The villus tissues of 30 SA cases and 20 requested abortion cases were collected during surgery and constituted the SA and normal abortion (NA) groups, respectively. The total villous RNA was extracted and the expression levels of MMP -9 and TIMP-3 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR) assay to calculate the MMP-9/TIMP-3 mRNA ratio. The MMP-9 mRNA expression level and MMP-9/TIMP-3 mRNA ratio of the SA group were significantly higher than those of the NA group (P<0.01), while the TIMP-3 mRNA levels of the two groups were similar (P>0.05). The MMP-9 mRNA expression level of the SA group was higher than that of the NA group; thus, the MMP-9/TIMP-3 mRNA ratio was higher. These results suggest that the expression level of MMP-9 mRNA and the MMP-9/TIMP-3 mRNA ratio are associated with SA.

Non-Smad TGF-β signaling components are possible biomarkers of tamoxifen resistance

NASA Astrophysics Data System (ADS)

Babyshkina, N.; Zavyalova, M.; Patalyak, S.; Dronova, T.; Slonimskaya, E.; Cherdyntseva, N.

2017-09-01

A crosstalk between the estrogen receptor alpha (ERα) and tyrosine kinase receptors contribute to endocrine resistance. We investigated the effect of the four Smad-independent TGF-β signaling components and the distribution pattern of ERα expression on the response to adjuvant tamoxifen treatment in 122 estrogen positive breast cancer patients. We identified a low mRNA expression of TGF-βR1 in tamoxifen resistant group patients (TR) in contrast to tamoxifen sensitive group (TS). Similarly, negative TGF-βR1 expression was significantly higher in TR patients than in TS patients. The expression of TGF-βR1 was strongly correlated with the distribution pattern of ERα expression, level of CD44+/CD24-/low cells and Akt (pS473) expression. The patients with a low mRNA expression of TGF-βR1 as well as with a negative TGF-βR1 expression had an unfavorable prognosis concerning progression-free survival. The expression of TGF-βR1 and the distribution pattern of ERα expression can be considered as additional molecular predictive markers for estrogen positive breast cancer patients treated with adjuvant tamoxifen.

Prognostic impact of MYC protein expression in central nervous system diffuse large B-cell lymphoma: comparison with MYC rearrangement and MYC mRNA expression.

PubMed

Son, Seung-Myoung; Ha, Sang-Yun; Yoo, Hae-Yong; Oh, Dongryul; Kim, Seok-Jin; Kim, Won-Seog; Ko, Young-Hyeh

2017-01-01

The prognostic role of MYC has been well documented in non-central nervous system diffuse large B-cell lymphoma; however, it remains controversial in central nervous system diffuse large B-cell lymphoma. To investigate the prognostic value of MYC, we analyzed the MYC protein expression by immunohistochemistry, mRNA expression by RNA in situ hybridization, and gene status by fluorescence in situ hybridization in 74 cases of central nervous system diffuse large B-cell lymphoma. Moreover, we examined the correlation between MYC translocation, mRNA expression, and protein expression. The mean percentage of MYC immunopositive cells was 49%. Using a 44% cutoff value, 49 (66%) cases showed MYC protein overexpression. The result of mRNA in situ hybridization using the RNA scope technology was obtained using the H-scoring system; the median value was 34.2. Using the cutoff value of 63.5, 16 (22%) cases showed MYC mRNA overexpression. MYC gene rearrangement was detected in five out of 68 (7%) cases. MYC translocation showed no statistically significant correlation with mRNA expression; however, all MYC translocation-positive cases showed MYC protein overexpression, with a higher mean percentage of MYC protein expression than that of translocation-negative cases (78 vs 48%, P=0.001). The level of MYC mRNA expression was moderately correlated with the level of MYC protein expression (P<0.001). The mean percentage of MYC protein expression in the high MYC mRNA group was higher than that in the low MYC mRNA group (70 vs 47%, P<0.001). A univariate analysis showed that age over 60 years, Eastern Cooperative Oncology Group (ECOG) performance status ≥2 and MYC protein overexpression were significantly associated with an increased risk of death. MYC translocation and MYC mRNA expression had no prognostic significance. On multivariate analysis, MYC protein overexpression and ECOG score retained prognostic significance.

Age-related changes in cyclic phosphatidic acid-induced hyaluronic acid synthesis in human fibroblasts.

PubMed

Sano, Katsura; Gotoh, Mari; Dodo, Kyoko; Tajima, Noriaki; Shimizu, Yoshibumi; Murakami-Murofushi, Kimiko

2018-01-01

Hyaluronic acid is a major component of the extracellular matrix, which is important for skin hydration. As aging brings skin dehydration, we aimed to clarify the mRNA expression of hyaluronic acid-related proteins in human skin fibroblasts from donors of various ages (range 0.7-69 years). Previously, we reported that cyclic phosphatidic acid (cPA), a unique phospholipid mediator, stimulated the expression of HAS2 and increased hyaluronic acid synthesis in human skin fibroblasts (donor age: 3 days). In this study, we measured the mRNA expression of hyaluronic acid-related proteins: hyaluronan synthase (HAS) 1-3, hyaluronidase-1, -2, and hyaluronic acid-binding protein (versican). In addition, we tested whether cPA could increase hyaluronic acid synthesis in skin fibroblasts derived from donors of various ages. The expression of HAS1, 3, hyaluronidase-1, and -2 did not change with aging. However, the mRNA expression of versican decreased with aging. Although it is thought that the amount of hyaluronic acid in the dermis decreases with aging, the mRNA expression of HAS2 was increased. But the amount of hyaluronic acid secreted by fibroblasts did not increase with aging. This suggests that the activity and/or protein expression of HAS2 decrease with aging. Furthermore, we observed that cPA caused the increase of hyaluronic acid synthesis at any age, and this effect was increased with aging. These results suggest that aging made the fibroblasts more sensitive to cPA treatment. Therefore, cPA represents a suitable candidate for the health maintenance and improvement of the skin by increasing the level of hyaluronic acid in the dermis.

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

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.

Methylation of the tryptophan hydroxylase‑2 gene is associated with mRNA expression in patients with major depression with suicide attempts.

PubMed

Zhang, Yuqi; Chang, Zaohuo; Chen, Jionghua; Ling, Yang; Liu, Xiaowei; Feng, Zhang; Chen, Caixia; Xia, Minghua; Zhao, Xingfu; Ying, Wang; Qing, Xu; Li, Guilin; Zhang, Changsong

2015-08-01

Tryptophan hydroxylase-2 (TPH2) contributes to alterations in the function of neuronal serotonin (5-HT), which are associated with various psychopathologies, including major depressive disorder (MDD) or suicidal behavior. The methylation of a single CpG site in the promoter region of TPH2 affects gene expression. Suicide and MDD are strongly associated and genetic factors are at least partially responsible for the variability in suicide risk. The aim of the present study was to investigate whether variations in TPH2 methylation in peripheral blood samples may predispose patients with MDD to suicide attempts. TPH2 mRNA expression levels differed significantly between 50 patients with MDD who had attempted suicide (MDD + suicide group) and 75 control patients with MDD (MDD group); TPH2 expression levels were significantly decreased (P=0.0005) in the patients who had attempted suicide. Furthermore, the frequency of TPH2 methylation was 36.0% in the MDD + suicide group, while it was 13.0% in the MDD group. The results of the present study demonstrated that methylation in the promoter region of TPH2 significantly affected the mRNA expression levels of TPH2, thus suggesting that methylation of the TPH2 promoter may silence TPH2 mRNA expression in MDD patients with or without suicidal behavior. In addition, there was a significant correlation between the methylation status of the TPH2 promoter and depression, hopelessness and cognitive impairment in the MDD + suicide group. In conclusion, the present study demonstrated that TPH2 expression was regulated by DNA methylation of the TPH2 promoter region in patients with MDD.

Expression of BMI-1 and Mel-18 in breast tissue - a diagnostic marker in patients with breast cancer

PubMed Central

2010-01-01

Background Polycomb Group (PcG) proteins are epigenetic silencers involved in maintaining cellular identity, and their deregulation can result in cancer. Expression of Mel-18 and Bmi-1 has been studied in tumor tissue, but not in adjacent non-cancerous breast epithelium. Our study compares the expression of the two genes in normal breast epithelium of cancer patients and relates it to the level of expression in the corresponding tumors as well as in breast epithelium of healthy women. Methods A total of 79 tumors, of which 71 malignant tumors of the breast, 6 fibroadenomas, and 2 DCIS were studied and compared to the reduction mammoplastic specimens of 11 healthy women. In addition there was available adjacent cancer free tissue for 23 of the malignant tumors. The tissue samples were stored in RNAlater, RNA was isolated to create expression microarray profile. These two genes were then studied more closely first on mRNA transcription level by microarrays (Agilent 44 K) and quantitative RT-PCR (TaqMan) and then on protein expression level using immunohistochemistry. Results Bmi-1 mRNA is significantly up-regulated in adjacent normal breast tissue in breast cancer patients compared to normal breast tissue from noncancerous patients. Conversely, mRNA transcription level of Mel-18 is lower in normal breast from patients operated for breast cancer compared to breast tissue from mammoplasty. When protein expression of these two genes was evaluated, we observed that most of the epithelial cells were positive for Bmi-1 in both groups of tissue samples, although the expression intensity was stronger in normal tissue from cancer patients compared to mammoplasty tissue samples. Protein expression of Mel-18 showed inversely stronger intensity in tissue samples from mammoplasty compared to normal breast tissue from patients operated for breast cancer. Conclusion Bmi-1 mRNA level is consistently increased and Mel-18 mRNA level is consistently decreased in adjacent normal breast tissue of cancer patients as compared to normal breast tissue in women having had reduction mammoplasties. Bmi-1/Mel-18 ratio can be potentially used as a tool for stratifying women at risk of developing malignancy. PMID:21162745

DAPK1 as an independent prognostic marker in liver cancer.

PubMed

Li, Ling; Guo, Libin; Wang, Qingshui; Liu, Xiaolong; Zeng, Yongyi; Wen, Qing; Zhang, Shudong; Kwok, Hang Fai; Lin, Yao; Liu, Jingfeng

2017-01-01

The death-associated protein kinase 1 (DAPK1) can act as an oncogene or a tumor suppressor gene depending on the cellular context as well as external stimuli. Our study aims to investigate the prognostic significance of DAPK1 in liver cancer in both mRNA and protein levels. The mRNA expression of DAPK1 was extracted from the Gene Expression Omnibus database in three independent liver cancer datasets while protein expression of DAPK1 was detected by immunohistochemistry in our Chinese liver cancer patient cohort. The associations between DAPK1 expression and clinical characteristics were tested. DAPK1 mRNA expression was down-regulated in liver cancer. Low levels of DAPK1 mRNA were associated with shorter survival in a liver cancer patient cohort ( n  = 115;  p  = 0.041), while negative staining of DAPK1 protein was significantly correlated with shorter time to progression ( p  = 0.002) and overall survival ( p  = 0.02). DAPK1 was an independent prognostic marker for both time to progression and overall survival by multivariate analysis. Liver cancer with the b-catenin mutation has a lower DAPK1 expression, suggesting that DAPK1 may be regulated under the b-catenin pathway. In addition, we also identified genes that are co-regulated with DAPK1. DAPK1 expression was positively correlated with IRF2, IL7R, PCOLCE and ZBTB16, and negatively correlated with SLC16A3 in both liver cancer datasets. Among these genes, PCOLCE and ZBTB16 were significantly down-regulated, while SLC16A3 was significantly upregulated in liver cancer. By using connectivity mapping of these co-regulated genes, we have identified amcinonide and sulpiride as potential small molecules that could potentially reverse DAPK1/PCOLCE/ZBTB16/SLC16A3 expression. Our study demonstrated for the first time that both DAPK1 mRNA and protein expression levels are important prognostic markers in liver cancer, and have identified genes that may contribute to DAPK1-mediated liver carcinogenesis.

A preliminary study on the role of the complement regulatory protein, cluster of differentiation 55, in mice with diabetic neuropathic pain.

PubMed

Nie, Fachuan; Su, Dong; Shi, Ying; Chen, Jinmei; Wang, Haihui; Qin, Wanxiang; Chen, Yaohua; Wang, Suxia; Li, Lei

2015-03-01

The aim of this study was to investigate the role of the complement regulatory protein cluster of differentiation 55 (CD55) in the pathogenesis of diabetic neuropathic pain (DNP). Healthy adult male C57BL/6J mice were intraperitoneally injected with streptozotocin (STZ) in order to induce DNP. Peripheral blood glucose and protein, and the mRNA expression levels of C3 and CD55 in the spinal cord were determined. In addition, the behaviors of these mice were observed. The results showed that STZ‑treated mice displayed the clinical manifestations of diabetes mellitus, and that their peripheral blood glucose was markedly increased. On the 21st and 28th days following the STZ injection, the mechanical pain threshold and thermal pain threshold of the mice were dramatically reduced (P<0.05). |Additionally, 14 days post‑STZ injection, the mRNA expression of C3 in the spinal cord was significantly increased, which continued for 28 days. On the 21st and 28th days, the number of C3 positive cells in the spinal cord was markedly increased. Seven days after the STZ injection, the number of cells positive for CD55 was markedly reduced in the spinal dorsal horn and subsequently remained at a low level. The mRNA expression of CD55 also was significantly reduced (P<0.05) and remained so for 28 days. The reduction in the expression levels of CD55 occurred earlier than the changes in the expression of C3, suggesting that the downregulation of CD55 expression precedes, and has an important role regarding, the activation of C3 in the occurrence and development of DNP.

Schizothorax prenanti corticotropin-releasing hormone (CRH): molecular cloning, tissue expression, and the function of feeding regulation.

PubMed

Wang, Tao; Zhou, Chaowei; Yuan, Dengyue; Lin, Fangjun; Chen, Hu; Wu, Hongwei; Wei, Rongbin; Xin, Zhiming; Liu, Ju; Gao, Yundi; Li, Zhiqiong

2014-10-01

Corticotropin-releasing hormone (CRH) is a potent mediator of endocrine, autonomic, behavioral, and immune responses to stress. For a better understanding of the structure and function of the CRH gene and to study its effect on feeding regulation in cyprinid fish, the cDNA of the CRH gene from the brain of Schizothorax prenanti was cloned and sequenced. The full-length CRH cDNA consisted of 1,046 bp with an open reading frame of 489 bp encoding a protein of 162 amino acids. Real-time quantitative PCR analyses revealed that CRH was widely expressed in central and peripheral tissues. In particular, high expression level of CRH was detected in brain. Furthermore, CRH mRNA expression was examined in different brain regions, especially high in hypothalamus. In addition, there was no significant change in CRH mRNA expression in fed group compared with the fasted group in the S. prenanti hypothalamus during short-term fasting. However, CRH gene expression presented significant decrease in the hypothalamus in fasted group compared with the fed group (P < 0.05) on day 7; thereafter, re-feeding could lead to a significant increase in CRH mRNA expression in fasted group on day 9. The results suggest that the CRH may play a critical role in feeding regulation in S. prenanti.

Cloning, Expression Analysis and Enzyme Activity Assays of the α-Carbonic Anhydrase Gene from Chlamydomonas sp. ICE-L.

PubMed

Qu, Changfeng; He, Yingying; Zheng, Zhou; An, Meiling; Li, Lulu; Wang, Xixi; He, Xiaodong; Wang, Yibin; Liu, Fangming; Miao, Jinlai

2018-01-01

The α-carbonic anhydrase (α-CA) is a zinc ion-containing enzyme that catalyzes the hydration of carbon dioxide. In this paper, a full-length α-CA gene was cloned from Chlamydomonas sp. ICE-L using RT-PCR and RACE-PCR for bioinformatic analysis. The α-CA open reading frame obtained by PCR was cloned into a vector and transformed into Escherichia coli to generate α-CA-producing bacteria. The α-CA was highly expressed upon induction with isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0.8 mM. A single band with a molecular weight of approximate 40 kDa expressed in the recombinant E. coli strain harboring the α-CA vector was observed in SDS-PAGE analysis. The carbon dioxide hydration activity and esterase activity of α-CA expressed by the recombinant strain were 0.404 U/mg and 0.319 U, respectively. In addition, three conditions, temperature, salinity and UVB radiation exposure, were selected to analyze α-CA transcription levels by qRT-PCR. The results suggested UVB exposure increased the expression of relative mRNA; meanwhile, the α-CA mRNA expression was rapidly induced by temperature and salinity stress, indicating that Chlamydomonas sp. ICE-L might modulate the α-CA mRNA expression to adapt to the extreme environments.

Piwil1 mediates meiosis during spermatogenesis in chicken.

PubMed

Xu, Lu; Chang, Guobin; Ma, Teng; Wang, Hongzhi; Chen, Jing; Li, Zhiteng; Guo, Xiaomin; Wan, Fang; Ren, Lichen; Lu, Wei; Chen, Guohong

2016-03-01

Piwil1 mediates spermatogenesis and ensures stable cell division rates in germline cells in mammals. However, the involvement of Piwil1 in poultry spermatogenesis and meiosis is poorly understood. In the present study, we used TaqMan RT-qPCR to characterize Piwil1 mRNA expression in different types of spermatogenic cells, including primordial germ cells (PGCs), spermatogonial stem cells (SSCs), spermatogonia cells (Sa), tetraploid cells (Tp), round sperm cells (Rs), mature sperm, and in PGCs treated with retinoic acid. Our results revealed that Piwil1 is differentially expressed during spermatogenesis in chicken. Compared to PGCs, SSCs, Tp, and Sa, Rs cells presented the highest Piwil1 mRNA expression levels. Retinoic acid significantly upregulated Piwil1 and Stra8 mRNA expression as well as Piwil1 levels in chicken PGCs. In addition, retinoic acid induced PGCs to progress through all the meiotic stages, eventually leading to haploid cell formation, which was determined using flow cytometry and western blot analysis. Taken together, our results showed that during spermatogenesis, Piwil1 was first expressed at low levels in germ stem cells, PGCs, and SSCs. Its expression levels increased during later meiosis stages. Finally, no expression was detected in mature sperm after meiosis. Treatment of PGCs with retinoic acid further demonstrated that Piwil1 plays a key role in meiosis during chicken spermatogenesis. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

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.

Characterization of glucagon-like peptide 2 pathway member expression in bovine gastrointestinal tract.

PubMed

Connor, E E; Baldwin, R L; Capuco, A V; Evock-Clover, C M; Ellis, S E; Sciabica, K S

2010-11-01

Glucagon-like peptide 2 (GLP-2), secreted by enteroendocrine cells, has several physiological effects on the intestine of monogastric species, including promotion of growth of intestinal epithelium, reduction of epithelial cell apoptosis, and enhancement of intestinal blood flow, nutrient absorption, and epithelial barrier function. The regulatory functions of GLP-2 in the ruminant gastrointestinal tract (GIT) have not been well studied. The objectives of this investigation were to characterize the mRNA expression of 4 members of the GLP-2 pathway throughout the bovine GIT, including (1) proglucagon (GCG), the parent peptide from which GLP-2 is derived through cleavage by prohormone convertase; (2) prohormone convertase (PCSK1); (3) GLP-2 receptor (GLP2R); and (4) dipeptidyl peptidase IV (DPP4), the enzyme that inactivates GLP-2. Gene expression was evaluated in rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, and rectum collected at slaughter from prepubertal heifers, mature cows in early, mid, and late lactation, and nonlactating cows (n=3 per stage) by a gene expression profiling assay. In addition, mRNA expression of 14 genes involved in nutrient transport, enzyme activity, blood flow, apoptosis, and proliferation were evaluated in the 9 GIT tissues for their association with GCG and GLP2R mRNA expression. Immunohistochemistry was used to localize GLP2R protein in tissues of the lower GIT. Results indicated that mRNA expression of GCG, PCSK1, GLP2R, and DPP4 varies across the 9 GIT tissues, with greatest expression in small and large intestines, and generally nondetectable levels in forestomachs. Expression of DPP4 and GLP2R mRNA varied by developmental stage or lactational state in intestinal tissues. Expression of GCG or GLP2R mRNA was correlated with molecular markers of proliferation, apoptosis, blood flow, enzyme activity, and urea transport, depending on the tissue examined, which suggests a potential for involvement of GLP-2 in these physiological processes in the ruminant GIT. The GLP2R protein was expressed in intestinal crypts of the bovine GIT, which is consistent with the distribution in monogastric species. Our findings support a functional role of the GLP-2 pathway in bovine GIT and the potential for use of GLP-2 as a therapy to improve intestinal function and nutrient absorption in ruminants. Copyright © 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The regulated in development and DNA damage response 2 (REDD2) gene mediates human monocyte cell death through a reduction in thioredoxin-1 expression.

PubMed

Imen, Jguirim-Souissi; Billiet, Ludivine; Cuaz-Pérolin, Clarisse; Michaud, Nadège; Rouis, Mustapha

2009-05-15

In a previous study, we identified the regulated in development and DNA damage response 2 (REDD2) gene as a highly expressed gene in human atherosclerotic lesions in comparison to normal artery, as well as in cultured human macrophages, and showed its implication in oxidized low-density lipoprotein (LDL)-induced macrophage death sensitivity. In this article, we attempt to identify the mechanism by which REDD2 induces such a phenomenon. Transient transfection of U-937 monocytic cells with a pCI.CMV.REDD2 expression vector increased by approximately twofold the mRNA levels of REDD2 in comparison to control cells transfected with pCI.CMV.GFP. Reactive oxygen species (ROS) production was significantly induced in REDD2-transfected cells compared with control cells (157+/-48 and 100+/-8 arbitrary units/mg cell protein, respectively; p<0.05). Moreover, a significant increase in parameters known to reflect the oxidative modifications of LDL was observed. Among enzymes involved in ROS production or degradation, we found a specific reduction in thioredoxin-1 (Trx-1) mRNA ( approximately 52+/-7% decrease, p<0.01 vs control cells) and protein ( approximately 60+/-4% decrease, p<0.001 vs control cells) levels in cells overexpressing REDD2 in comparison to control cells. In contrast, transfection of U-937 cells with siRNA against REDD2 decreased the mRNA levels of REDD2 by approximately 60% and increased Trx-1 mRNA and protein levels. Moreover, we observed no or a moderate increase in Bax (proapoptotic) and a significant decrease in Bcl2 (antiapoptotic) gene expression in cells that overexpress REDD2 compared to control cells. In addition, we showed that Trx-1 mRNA and protein levels were increased at low H(2)O(2) doses and decreased at higher doses. Interestingly, macrophages isolated from human atherosclerotic lesions differentially express REDD2 and Trx-1. Indeed, in certain patients, levels of REDD2 mRNA were low and those of Trx-1 mRNA were high. In contrast, in other patients, levels of REDD2 were high and levels of Trx-1 mRNA were low.

Effect of human vascular endothelial growth factor gene transfer on endogenous vascular endothelial growth factor mRNA expression in a rat fibroblast and osteoblast culture model.

PubMed

Li, Ru; Li, Claire H; Nauth, Aaron; McKee, Michael D; Schemitsch, Emil H

2010-09-01

Vascular endothelial growth factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy enhances bone healing of a rabbit tibia segmental bone defect in vivo. The aim of this project was to examine the effect of exogenous human VEGF on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model. Rat fibroblasts and osteoblasts were harvested from the dermal tissue and periosteum, respectively, of Fisher 344 rats. The cells were then cultured and transfected with pcDNA-human VEGF using Superfect reagent (Qiagen). Four experimental groups were created: 1) fibroblast-VEGF; 2) osteoblast-VEGF; 3) nontransfected fibroblast controls; and 4) nontransfected osteoblast controls. The cultured cells were harvested at 1, 3, and 7 days after the gene transfection. The total mRNA was extracted (Trizol; Invitrogen); both human VEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. The human VEGF165 mRNA was detected by reverse transcriptase-polymerase chain reaction from transfected fibroblasts and osteoblasts at 1, 3, and 7 days after gene transfection. The human VEGF165 levels peaked at Day 1 and then gradually reduced expression in both transfected fibroblasts and osteoblasts. Two endogenous rat VEGF isoforms were detected in this cell culture model: rat VEGF120 and rat VEGF164. We compared the rat VEGF120 and rat VEGF164 expression level of the fibroblasts or osteoblasts that were transfected with human VEGF165, with nontransfected control cells. Both the transfected fibroblasts and osteoblasts showed greater expression of rat VEGF164 than nontransfected controls at Day 1 (peak level) and Day 3, but not at Day 7. The expression of rat VEGF120 was lower in transfected fibroblasts, but higher in transfected osteoblasts, than the relevant control groups at any time point after transfection. In addition, human VEGF gene transfection increased osteoblast cell proliferation after 3 days. These in vitro results suggest that cell-based human VEGF gene therapy is not only effective at causing human VEGF expression, but also enhances endogenous rat VEGF mRNA expression in both fibroblasts and osteoblasts, particularly the rat VEGF164 isoform.

Angiotensin II promotes the proliferation of activated pancreatic stellate cells by Smad7 induction through a protein kinase C pathway

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

Hama, Kouji; Ohnishi, Hirohide; Aoki, Hiroyoshi

2006-02-17

Activated pancreatic stellate cells (PSCs) play major roles in promoting pancreatic fibrosis. We previously reported that angiotensin II (Ang II) enhances activated PSC proliferation through EGF receptor transactivation. In the present study, we elucidated a novel intracellular mechanism by which Ang II stimulates cellular proliferation. TGF-{beta}{sub 1} inhibits activated PSC proliferation via a Smad3 and Smad4-dependent pathway in an autocrine manner. We demonstrated that Ang II inhibited TGF-{beta}{sub 1}-induced nuclear accumulation of Smad3 and Smad4. Furthermore, Ang II rapidly induced inhibitory Smad7 mRNA expression. Adenovirus-mediated Smad7 overexpression inhibited TGF-{beta}{sub 1}-induced nuclear accumulation of Smad3 and Smad4, and potentiated activated PSCmore » proliferation. PKC inhibitor Go6983 blocked the induction of Smad7 mRNA expression by Ang II. In addition, 12-O-tetradecanoyl-phorbol 13-acetate, a PKC activator, increased Smad7 mRNA expression. These results suggest that Ang II enhances activated PSC proliferation by blocking autocrine TGF-{beta}{sub 1}-mediated growth inhibition by inducing Smad7 expression via a PKC-dependent pathway.« less

Comparative effects of conjugated linoleic acid (CLA) and linoleic acid (LA) on the oxidoreduction status in THP-1 macrophages.

PubMed

Rybicka, Marta; Stachowska, Ewa; Gutowska, Izabela; Parczewski, Miłosz; Baśkiewicz, Magdalena; Machaliński, Bogusław; Boroń-Kaczmarska, Anna; Chlubek, Dariusz

2011-04-27

The aim of this study was to investigate the effect of conjugated linoleic acids (CLAs) on macrophage reactive oxygen species synthesis and the activity and expression of antioxidant enzymes, catalase (Cat), glutathione peroxidase (GPx), and superoxide dismutase (SOD). The macrophages were obtained from the THP-1 monocytic cell line. Cells were incubated with the addition of cis-9,trans-11 CLA or trans-10,cis-12 CLA or linoleic acid. Reactive oxygen species (ROS) formation was estimated by flow cytometry. Enzymes activity was measured spectrophotometrically. The antioxidant enzyme mRNA expression was estimated by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Statistical analysis was based on nonparametric statistical tests [Friedman analysis of variation (ANOVA) and Wilcoxon signed-rank test]. cis-9,trans-11 CLA significantly increased the activity of Cat, while trans-10,cis-12 CLA notably influenced GPx activity. Both isomers significantly decreased mRNA expression for Cat. Only trans-10,cis-12 significantly influenced mRNA for SOD-2 expression. The CLAs activate processes of the ROS formation in macrophages. Adverse metabolic effects of each isomer action were observed.

Expression of calmodulin mRNA in rat olfactory neuroepithelium.

PubMed

Biffo, S; Goren, T; Khew-Goodall, Y S; Miara, J; Margolis, F L

1991-04-01

A calmodulin (CaM) cDNA was isolated by differential hybridization screening of a lambda gt10 library prepared from rat olfactory mucosa. This cDNA fragment, containing most of the open reading frame of the rat CaMI gene, was subcloned and used to characterize steady-state expression of CaM mRNA in rat olfactory neuroepithelium and bulb. Within the bulb mitral cells are the primary neuronal population expressing CaM mRNA. The major CaM mRNA expressed in the olfactory mucosa is 1.7 kb with smaller contributions from mRNAs of 4.0 and 1.4 kb. CaM mRNA was primarily associated with the olfactory neurons and, despite the cellular complexity of the tissue and the known involvement of CaM in diverse cellular processes, was only minimally evident in sustentacular cells, gland cells or respiratory epithelium. Following bulbectomy CaM mRNA declines in the olfactory neuroepithelium as does olfactory marker protein (OMP) mRNA. In contrast to the latter, CaM mRNA makes a partial recovery by one month after surgery. These results, coupled with those from in situ hybridization, indicate that CaM mRNA is expressed in both mature and immature olfactory neurons. The program regulating CaM gene expression in olfactory neurons is distinct from those controlling expression of B50/GAP43 in immature, or OMP in mature, neurons respectively.

Stimulation of nuclear receptor REV-ERBs regulates tumor necrosis factor-induced expression of proinflammatory molecules in C6 astroglial cells

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

Morioka, Norimitsu, E-mail: mnori@hiroshima-u.ac.jp; Tomori, Mizuki; Zhang, Fang Fang

Under physiological conditions, astrocytes maintain homeostasis in the CNS. Following inflammation and injury to the CNS, however, activated astrocytes produce neurotoxic molecules such as cytokines and chemokines, amplifying the initial molecular-cellular events evoked by inflammation and injury. Nuclear receptors REV-ERBα and REV-ERBβ (REV-ERBs) are crucial in the regulation of inflammation- and metabolism-related gene transcription. The current study sought to elucidate a role of REV-ERBs in rat C6 astroglial cells on the expression of inflammatory molecules following stimulation with the neuroinflammatory cytokine tumor necrosis factor (TNF). Stimulation of C6 cells with TNF (10 ng/ml) significantly increased the mRNA expression of CCL2, interleukin-6more » (IL-6), inducible nitric oxide synthase (iNOS), and matrix metalloprotease (MMP)-9, but not fibroblast growth factor-2 (FGF-2), cyclooxygenase-2 (COX-2) and MMP-2. Treatment with either REV-ERB agonists GSK4112 or SR9009 significantly blocked TNF-induced upregulation of CCL2 mRNA and MMP-9 mRNA, but not IL-6 mRNA and iNOS mRNA expression. Furthermore, treatment with RGFP966, a selective histone deacetylase 3 (HDAC3) inhibitor, potently reversed the inhibitory effects of GSK4112 on TNF-induced expression of MMP-9 mRNA, but not CCL2 mRNA. Expression of Rev-erbs mRNA in C6 astroglial cells, primary cultured rat cortical and spinal astrocytes was confirmed by reverse transcription polymerase chain reaction. Together, the findings demonstrate an anti-inflammatory effect, downregulating of MMP-9 and CCL2 transcription, of astroglial REV-ERBs activation through HDAC3-dependent and HDAC3-independent mechanisms. - Highlights: • Rev-erbα mRNA and Rev-erbβ mRNA are expressed in C6 astroglial cells. • TNF increases the expression of CCL2, IL-6, MMP-9 and iNOS mRNA. • REV-ERB activation inhibits CCL2 mRNA and MMP-9 mRNA expression. • HDAC3 activity is involved in the inhibitory effect of REV-ERB on MMP-9 induction.« less

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

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.

Metformin reduces morphine tolerance by inhibiting microglial-mediated neuroinflammation.

PubMed

Pan, Yinbing; Sun, Xiaodi; Jiang, Lai; Hu, Liang; Kong, Hong; Han, Yuan; Qian, Cheng; Song, Chao; Qian, Yanning; Liu, Wentao

2016-11-17

Tolerance seriously impedes the application of morphine in clinical medicine. Thus, it is necessary to investigate the exact mechanisms and efficient treatment. Microglial activation and neuroinflammation in the spinal cord are thought to play pivotal roles on the genesis and maintaining of morphine tolerance. Activation of adenosine monophosphate-activated kinase (AMPK) has been associated with the inhibition of inflammatory nociception. Metformin, a biguanide class of antidiabetic drugs and activator of AMPK, has a potential anti-inflammatory effect. The present study evaluated the effects and potential mechanisms of metformin in inhibiting microglial activation and alleviating the antinociceptive tolerance of morphine. The microglial cell line BV-2 cells and mouse brain-derived endothelial cell line bEnd3 cells were used. Cytokine expression was measured using quantitative polymerase chain reaction. Cell signaling was assayed by western blot and immunohistochemistry. The antinociception and morphine tolerance were assessed in CD-1 mice using tail-flick tests. We found that morphine-activated BV-2 cells, including the upregulation of p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation, pro-inflammatory cytokines, and Toll-like receptor-4 (TLR-4) mRNA expression, which was inhibited by metformin. Metformin suppressed morphine-induced BV-2 cells activation through increasing AMPK phosphorylation, which was reversed by the AMPK inhibitor compound C. Additionally, in BV-2 cells, morphine did not affect the cell viability and the mRNA expression of anti-inflammatory cytokines. In bEnd3 cells, morphine did not affect the mRNA expression of interleukin-1β (IL-1β), but increased IL-6 and tumor necrosis factor-α (TNF-α) mRNA expression; the effect was inhibited by metformin. Morphine also did not affect the mRNA expression of TLR-4 and chemokine ligand 2 (CCL2). Furthermore, systemic administration of metformin significantly blocked morphine-induced microglial activation in the spinal cord and then attenuated the development of chronic morphine tolerance in mice. Metformin significantly attenuated morphine antinociceptive tolerance by suppressing morphine-induced microglial activation through increasing AMPK phosphorylation.

Chronic intermittent ethanol exposure selectively alters the expression of Gα subunit isoforms and RGS subtypes in rat prefrontal cortex.

PubMed

Luessen, D J; Sun, H; McGinnis, M M; McCool, B A; Chen, R

2017-10-01

Chronic alcohol exposure induces pronounced changes in GPCR-mediated G-protein signaling. Recent microarray and RNA-seq analyses suggest associations between alcohol abuse and the expression of genes involved in G-protein signaling. The activity of G-proteins (e.g. Gαi/o and Gαq) is negatively modulated by regulator of G-protein signaling (RGS) proteins which are implicated in drugs of abuse including alcohol. The present study used 7days of chronic intermittent ethanol exposure followed by 24h withdrawal (CIE) to investigate changes in mRNA and protein levels of G-protein subunit isoforms and RGS protein subtypes in rat prefrontal cortex, a region associated with cognitive deficit attributed to excessive alcohol drinking. We found that this ethanol paradigm induced differential expression of Gα subunits and RGS subtypes. For example, there were increased mRNA and protein levels of Gαi1/3 subunits and no changes in the expression of Gαs and Gαq subunits in ethanol-treated animals. Moreover, CIE increased the mRNA but not the protein levels of Gαo. Additionally, a modest increase in Gαi2 mRNA level by CIE was accompanied by a pronounced increase in its protein level. Interestingly, we found that CIE increased mRNA and protein levels of RGS2, RGS4, RGS7 and RGS19 but had no effect on the expression of RGS5, RGS6, RGS8, RGS12 or RGS17. Changes in the expression of Gα subunits and RGS subtypes could contribute to the functional alterations of certain GPCRs following chronic ethanol exposure. The present study suggests that RGS proteins may be potential new targets for intervention of alcohol abuse via modification of Gα-mediated GPCR function. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of Novel Androgen-Regulated Pathways and mRNA Isoforms through Genome-Wide Exon-Specific Profiling of the LNCaP Transcriptome

PubMed Central

Carling, Phillippa J.; Buist, Thomas; Zhang, Chaolin; Grellscheid, Sushma N.; Armstrong, Kelly; Stockley, Jacqueline; Simillion, Cedric; Gaughan, Luke; Kalna, Gabriela; Zhang, Michael Q.; Robson, Craig N.; Leung, Hing Y.; Elliott, David J.

2011-01-01

Androgens drive the onset and progression of prostate cancer (PCa) by modulating androgen receptor (AR) transcriptional activity. Although several microarray-based studies have identified androgen-regulated genes, here we identify in-parallel global androgen-dependent changes in both gene and alternative mRNA isoform expression by exon-level analyses of the LNCaP transcriptome. While genome-wide gene expression changes correlated well with previously-published studies, we additionally uncovered a subset of 226 novel androgen-regulated genes. Gene expression pathway analysis of this subset revealed gene clusters associated with, and including the tyrosine kinase LYN, as well as components of the mTOR (mammalian target of rapamycin) pathway, which is commonly dysregulated in cancer. We also identified 1279 putative androgen-regulated alternative events, of which 325 (∼25%) mapped to known alternative splicing events or alternative first/last exons. We selected 30 androgen-dependent alternative events for RT-PCR validation, including mRNAs derived from genes encoding tumour suppressors and cell cycle regulators. Of seven positively-validating events (∼23%), five events involved transcripts derived from alternative promoters of known AR gene targets. In particular, we found a novel androgen-dependent mRNA isoform derived from an alternative internal promoter within the TSC2 tumour suppressor gene, which is predicted to encode a protein lacking an interaction domain required for mTOR inhibition. We confirmed that expression of this alternative TSC2 mRNA isoform was directly regulated by androgens, and chromatin immunoprecipitation indicated recruitment of AR to the alternative promoter region at early timepoints following androgen stimulation, which correlated with expression of alternative transcripts. Together, our data suggest that alternative mRNA isoform expression might mediate the cellular response to androgens, and may have roles in clinical PCa. PMID:22194994

Distinct expression patterns of the E3 ligase SIAH-1 and its partner Kid/KIF22 in normal tissues and in the breast tumoral processes.

PubMed

Bruzzoni-Giovanelli, Heriberto; Fernandez, Plinio; Veiga, Lucía; Podgorniak, Marie-Pierre; Powell, Darren J; Candeias, Marco M; Mourah, Samia; Calvo, Fabien; Marín, Mónica

2010-02-09

SIAH proteins are the human members of an highly conserved family of E3 ubiquitin ligases. Several data suggest that SIAH proteins may have a role in tumor suppression and apoptosis. Previously, we reported that SIAH-1 induces the degradation of Kid (KIF22), a chromokinesin protein implicated in the normal progression of mitosis and meiosis, by the ubiquitin proteasome pathway. In human breast cancer cells stably transfected with SIAH-1, Kid/KIF22 protein level was markedly reduced whereas, the Kid/KIF22 mRNA level was increased. This interaction has been further elucidated through analyzing SIAH and Kid/KIF22 expression in both paired normal and tumor tissues and cell lines. It was observed that SIAH-1 protein is widely expressed in different normal tissues, and in cells lines but showing some differences in western blotting profiles. Immunofluorescence microscopy shows that the intracellular distribution of SIAH-1 and Kid/KIF22 appears to be modified in human tumor tissues compared to normal controls. When mRNA expression of SIAH-1 and Kid/KIF22 was analyzed by real-time PCR in normal and cancer breast tissues from the same patient, a large variation in the number of mRNA copies was detected between the different samples. In most cases, SIAH-1 mRNA is decreased in tumor tissues compared to their normal counterparts. Interestingly, in all breast tumor tissues analyzed, variations in the Kid/KIF22 mRNA levels mirrored those seen with SIAH-1 mRNAs. This concerted variation of SIAH-1 and Kid/KIF22 messengers suggests the existence of an additional level of control than the previously described protein-protein interaction and protein stability regulation. Our observations also underline the need to re-evaluate the results of gene expression obtained by qRT-PCR and relate it to the protein expression and cellular localization when matched normal and tumoral tissues are analyzed.

Regulation of expression of collagenase-3 in normal, differentiating rat osteoblasts

NASA Technical Reports Server (NTRS)

Winchester, S. K.; Bloch, S. R.; Fiacco, G. J.; Partridge, N. C.

1999-01-01

We investigated the regulation of collagenase-3 expression in normal, differentiating rat osteoblasts. Fetal rat calvarial cell cultures showed an increase in alkaline phosphatase activity reaching maximal levels between 7-14 days post-confluence, then declining with the onset of mineralization. Collagenase-3 mRNA was just detectable after proliferation ceased at day 7, increased up to day 21, and declined at later ages. Postconfluent cells maintained in non-mineralizing medium expressed collagenase-3 but did not show the developmental increase exhibited by cells switched to mineralization medium. Cells maintained in non-mineralizing medium continued to proliferate; cells in mineralization medium ceased proliferation. In addition, collagenase-3 mRNA was not detected in subcultured cells allowed to remineralize. These results suggest that enhanced accumulation of collagenase-3 mRNA is triggered by cessation of proliferation or acquisition of a mineralized extracellular matrix and that other factors may also be required. After initiation of basal expression, parathyroid hormone (PTH) caused a dose-dependent increase in collagenase-3 mRNA. Both the cyclic adenosine monophosphate (cAMP) analogue, 8-bromo-cAMP (8-Br-cAMP), and the protein kinase C (PKC) activator, phorbol myristate acetate, increased collagenase-3 expression, while the calcium ionophore, ionomycin, did not, suggesting that PTH was acting through the protein kinase A (PKA) and PKC pathways. Inhibition of protein synthesis with cycloheximide caused an increase in basal collagenase-3 expression but blocked the effect of PTH, suggesting that an inhibitory factor prevents basal expression while an inductive factor is involved with PTH action. In summary, collagenase-3 is expressed in mineralized osteoblasts and cessation of proliferation and initiation of mineralization are triggers for collagenase-3 expression. PTH also stimulates expression of the enzyme through both PKA and PKC pathways in the mineralizing osteoblast. Copyright 1999 Wiley-Liss, Inc.

Neuropilin-1 and neuropilin-2 are differentially expressed in human proteinuric nephropathies and cytokine-stimulated proximal tubular cells.

PubMed

Schramek, Herbert; Sarközi, Rita; Lauterberg, Christina; Kronbichler, Andreas; Pirklbauer, Markus; Albrecht, Rudolf; Noppert, Susie-Jane; Perco, Paul; Rudnicki, Michael; Strutz, Frank M; Mayer, Gert

2009-11-01

Neuropilin-1 (NRP1) and neuropilin-2 (NRP2) are transmembrane glycoproteins with large extracellular domains that interact with class 3 semaphorins, vascular endothelial growth factor (VEGF) family members, and ligands, such as hepatocyte growth factor, platelet-derived growth factor BB, transforming growth factor-beta1 (TGF-beta1), and fibroblast growth factor2 (FGF2). Neuropilins (NRPs) have been implicated in tumor growth and vascularization, as novel mediators of the primary immune response and in regeneration and repair; however, their role in renal pathophysiology is largely unknown. Here, we report upregulation of tubular and interstitial NRP2 protein expression in patients with focal segmental glomerulosclerosis (FSGS). In an additional cohort of patients with minimal change disease (MCD), membranous nephropathy (MN), and FSGS, elevated NRP2 mRNA expression in kidney biopsies inversely correlated with estimated glomerular filtration rate (eGFR) at the time of biopsy. Furthermore, upregulation of NRP2 mRNA correlated with post-bioptic decline of kidney function. Expression of NRP1 and NRP2 in human proximal tubular cells (PTCs) was differentially affected after stimulation with TGF-beta1, interleukin-1beta (IL-1beta), and oncostatin M (OSM). Although the pro-fibrotic mediators, TGF-beta1 and IL-1beta, induced upregulation of NRP2 expression but downregulation of NRP1 expression, OSM stimulated the expression of both NRP1 and NRP2. Basal and OSM-induced NRP1 mRNA expression, as well as TGF-beta1-induced NRP2 mRNA and protein expression were partially mediated by MEK1/2-ERK1/2 signaling. This is the first report suggesting a differential role of NRP1 and NRP2 in renal fibrogenesis, and TGF-beta1, IL-1beta, and OSM represent the first ligands known to stimulate NRP2 expression in mammalian cells.

Triptolide Upregulates Myocardial Forkhead Helix Transcription Factor p3 Expression and Attenuates Cardiac Hypertrophy

PubMed Central

Ding, Yuan-Yuan; Li, Jing-Mei; Guo, Feng-Jie; Liu, Ya; Tong, Yang-Fei; Pan, Xi-Chun; Lu, Xiao-Lan; Ye, Wen; Chen, Xiao-Hong; Zhang, Hai-Gang

2016-01-01

The forkhead/winged helix transcription factor (Fox) p3 can regulate the expression of various genes, and it has been reported that the transfer of Foxp3-positive T cells could ameliorate cardiac hypertrophy and fibrosis. Triptolide (TP) can elevate the expression of Foxp3, but its effects on cardiac hypertrophy remain unclear. In the present study, neonatal rat ventricular myocytes (NRVM) were isolated and stimulated with angiotensin II (1 μmol/L) to induce hypertrophic response. The expression of Foxp3 in NRVM was observed by using immunofluorescence assay. Fifty mice were randomly divided into five groups and received vehicle (control), isoproterenol (Iso, 5 mg/kg, s.c.), one of three doses of TP (10, 30, or 90 μg/kg, i.p.) for 14 days, respectively. The pathological morphology changes were observed after Hematoxylin and eosin, lectin and Masson’s trichrome staining. The levels of serum brain natriuretic peptide (BNP) and troponin I were determined by enzyme-linked immunosorbent assay and chemiluminescence, respectively. The mRNA and protein expressions of α- myosin heavy chain (MHC), β-MHC and Foxp3 were determined using real-time PCR and immunohistochemistry, respectively. It was shown that TP (1, 3, 10 μg/L) treatment significantly decreased cell size, mRNA and protein expression of β-MHC, and upregulated Foxp3 expression in NRVM. TP also decreased heart weight index, left ventricular weight index and, improved myocardial injury and fibrosis; and decreased the cross-scetional area of the myocardium, serum cardiac troponin and BNP. Additionally, TP markedly reduced the mRNA and protein expression of myocardial β-MHC and elevated the mRNA and protein expression of α-MHC and Foxp3 in a dose-dependent manner. In conclusion, TP can effectively ameliorate myocardial damage and inhibit cardiac hypertrophy, which is at least partly related to the elevation of Foxp3 expression in cardiomyocytes. PMID:27965581

Truncated midkine as a marker of diagnosis and detection of nodal metastases in gastrointestinal carcinomas.

PubMed Central

Aridome, K.; Takao, S.; Kaname, T.; Kadomatsu, K.; Natsugoe, S.; Kijima, F.; Aikou, T.; Muramatsu, T.

1998-01-01

Midkine (MK) is a growth factor identified as a product of a retinoic acid-responsive gene. A truncated form of MK mRNA, which lacks a sequence encoding the N-terminally located domain, was recently found in cancer cells. We investigated the expression of the truncated MK mRNA in specimens of 47 surgically removed human gastrointestinal organs using polymerase chain reaction. Truncated MK was not detected in all of the 46 corresponding non-cancerous regions. On the other hand, this short MK mRNA was expressed in the primary tumours in 12 of 16 gastric cancers, 8 of 13 colorectal carcinomas, five of nine hepatocellular carcinomas, two of two oesophageal carcinomas and one ampullary duodenal cancer. In addition, truncated MK was detectable in all of the 14 lymph node metastases but in none of three metastatic sites in the liver, suggesting that truncated MK mRNA could become a good marker of nodal metastases in gastrointestinal tract. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9716029

Characterization of a novel rice gene OsATX and modulation of its expression by components of the stress signalling pathways.

PubMed

Agrawal, Ganesh K; Rakwal, Randeep; Jwa, N-S; Agrawal, Vishwanath P

2002-09-01

In our search to identify gene(s) involved in the rice self-defense responses, we cloned a novel rice (Oryza sativa L. cv. Nipponbare) gene, OsATX, a single copy gene, from the JA treated rice seedling leaves cDNA library. This gene encodes a 69 amino acid polypeptide with a predicted molecular mass of 7649.7 and a pI of 5.6. OsATX was responsive to cutting (wounding by cutting the excised leaf), over its weak constitutive expression in the healthy leaves. The critical signalling molecules, jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), and hydrogen peroxide, together with protein phosphatase inhibitors, effectively up-regulated the OsATX expression with time, over the excised leaf cut control, whereas ethylene had no affect. Furthermore, copper, a heavy metal, also up-regulated OsATX expression. Moreover, induced expression of OsATX mRNA was influenced by light signal(s), and showed a requirement for de novo synthesized protein factors. Additionally, co-application of either JA or ABA with SA drastically suppressed the induced OsATX mRNA level. Finally, the blast pathogen, Magnaporthe grisea, triggered OsATX mRNA accumulation. These results strongly suggest a function/role(s) for OsATX in defense/stress responses in rice.

Protease-activated receptor 2 agonist increases cell proliferation and invasion of human pancreatic cancer cells

PubMed Central

XIE, LIQUN; DUAN, ZEXING; LIU, CAIJU; ZHENG, YANMIN; ZHOU, JING

2015-01-01

The aim of this study was to determine the expression of protease-activated receptor 2 (PAR-2) in the human pancreatic cancer cell line SW1990, and to evaluate its effect on cell proliferation and invasion. The expression of PAR-2 protein and mRNA in SW1990 cells was determined by immunocytochemistry and reverse transcription polymerase chain reaction (PCR), respectively. MTT and cell invasion and migration assays, as well as semi-quantitative PCR and zymography analysis, were additionally performed. PAR-2 mRNA was significantly upregulated in the cells treated with trypsin or the PAR-2 activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) (P<0.01), but not in the Val-Lys-Gly-Ile-Leu-Ser group (P>0.05). Trypsin and SLIGKV significantly promoted SW1990 cell proliferation in a dose- and time-dependent manner (P<0.05). Compared with the control group, trypsin and SLIGKV significantly increased the mRNA expression (P<0.01) and gelatinolytic activity (P<0.01) of matrix metalloproteinase (MMP)-2. In conclusion, PAR-2 is expressed in SW1990 cells. PAR-2 activation may promote the invasion and migration of human pancreatic cancer cells by increasing MMP-2 expression. PMID:25452809

Neurotrophins and tonsillar hypertrophy in children with obstructive sleep apnea.

PubMed

Goldbart, Aviv D; Mager, Edward; Veling, Maria C; Goldman, Julie L; Kheirandish-Gozal, Leila; Serpero, Laura D; Piedimonte, Giovanni; Gozal, David

2007-10-01

Enlarged adenotonsillar tissue (AT) is a major determinant of obstructive sleep apnea (OSA) severity in children; however, mechanisms of AT proliferation are poorly understood. We hypothesized that early exposure to respiratory syncytial virus (RSV) may modify AT proliferation through up-regulation of nerve growth factor (NGF)-neurokinin 1 (NK1) receptor dependent pathways. AT harvested from 34 children with OSA and 25 children with recurrent tonsillitis (RI) were examined for mRNA expression of multiple growth factors and their receptors. In addition, NK1 receptor expression and location, and substance P tissue concentrations were compared in AT from OSA and RI children. NGF mRNA and its high-affinity tyrosine kinase receptor (trkA) expression were selectively increased in OSA (p<0.001). NK1 receptor mRNA and protein expression were also enhanced in OSA (p<0.01), and substance P concentrations in OSA patients were higher than in RI (p<0.0001). AT from OSA children exhibit distinct differences in the expression of NGF and trkA receptors, NK1 receptors, and substance P. The homology between these changes and those observed in the lower airways following RSV infection suggests that RSV may have induced neuro-immunomodulatory changes within AT, predisposing them to increased proliferation, and ultimately contribute to emergence of OSA.

Neurotrophins and Tonsillar Hypertrophy in Children With Obstructive Sleep Apnea

PubMed Central

GOLDBART, AVIV D.; MAGER, EDWARD; VELING, MARIA C.; GOLDMAN, JULIE L.; KHEIRANDISH-GOZAL, LEILA; SERPERO, LAURA D.; PIEDIMONTE, GIOVANNI; GOZAL, DAVID

2013-01-01

Enlarged adenotonsillar tissue (AT) is a major determinant of obstructive sleep apnea (OSA) severity in children; however, mechanisms of AT proliferation are poorly understood. We hypothesized that early exposure to respiratory syncytial virus (RSV) may modify AT proliferation through up-regulation of nerve growth factor (NGF)-neurokinin 1 (NK1) receptor dependent pathways. AT harvested from 34 children with OSA and 25 children with recurrent tonsillitis (RI) were examined for mRNA expression of multiple growth factors and their receptors. In addition, NK1 receptor expression and location, and substance P tissue concentrations were compared in AT from OSA and RI children. NGF mRNA and its high-affinity tyrosine kinase receptor (trkA) expression were selectively increased in OSA (p < 0.001). NK1 receptor mRNA and protein expression were also enhanced in OSA (p < 0.01), and substance P concentrations in OSA patients were higher than in RI (p < 0.0001). AT from OSA children exhibit distinct differences in the expression of NGF and trkA receptors, NK1 receptors, and substance P. The homology between these changes and those observed in the lower airways following RSV infection suggests that RSV may have induced neuro-immunomodulatory changes within AT, predisposing them to increased proliferation, and ultimately contribute to emergence of OSA. PMID:17667845

Integrative analysis of multi-omics data for identifying multi-markers for diagnosing pancreatic cancer

PubMed Central

2015-01-01

Background microRNA (miRNA) expression plays an influential role in cancer classification and malignancy, and miRNAs are feasible as alternative diagnostic markers for pancreatic cancer, a highly aggressive neoplasm with silent early symptoms, high metastatic potential, and resistance to conventional therapies. Methods In this study, we evaluated the benefits of multi-omics data analysis by integrating miRNA and mRNA expression data in pancreatic cancer. Using support vector machine (SVM) modelling and leave-one-out cross validation (LOOCV), we evaluated the diagnostic performance of single- or multi-markers based on miRNA and mRNA expression profiles from 104 PDAC tissues and 17 benign pancreatic tissues. For selecting even more reliable and robust markers, we performed validation by independent datasets from the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) data depositories. For validation, miRNA activity was estimated by miRNA-target gene interaction and mRNA expression datasets in pancreatic cancer. Results Using a comprehensive identification approach, we successfully identified 705 multi-markers having powerful diagnostic performance for PDAC. In addition, these marker candidates annotated with cancer pathways using gene ontology analysis. Conclusions Our prediction models have strong potential for the diagnosis of pancreatic cancer. PMID:26328610

Regulation of Bovine Leukemia Virus tax and pol mRNA Levels by Interleukin-2 and -10

PubMed Central

Pyeon, Dohun; Splitter, Gary A.

1999-01-01

Recently, particular cytokines have been identified to affect progression of a variety of diseases and retrovirus infections. Previously, we demonstrated that interleukin-2 (IL-2), IL-12, and gamma interferon increased in peripheral blood mononuclear cells (PBMCs) from animals with early disease and decreased in PBMCs from animals with late disease stages of bovine leukemia virus (BLV) infection. In contrast, IL-10 increased with disease progression. To examine the effects of these cytokines on BLV expression, BLV tax and pol mRNA and p24 protein were quantified by competitive PCR and immunoblotting, respectively. IL-10 inhibited BLV tax and pol mRNA levels in BLV-infected PBMCs; however, the inhibitory effect of IL-10 was prevented in PBMCs depleted of monocytes and/or macrophages (monocyte/macrophages). To determine whether these factors were secreted or monocyte/macrophage associated, monocyte/macrophage-depleted PBMCs were cultured with isolated monocyte/macrophages in transwells where contact between monocyte/macrophages and nonadherent PBMCs was blocked. BLV tax and pol mRNA levels increased in transwell cultures similar to cultures containing nonseparated cells, and IL-10 addition inhibited the increase of BLV tax and pol mRNA. These results suggest that monocyte/macrophages secrete soluble factor(s) that increases BLV mRNA levels and that secretion of these soluble factor(s) could be inhibited by IL-10. In contrast, IL-2 increased BLV tax and pol mRNA and p24 protein production. Thus, IL-10 production by BLV-infected animals with late stage disease may serve to control BLV mRNA levels, while IL-2 may increase BLV mRNA in the early disease stage. To determine a correlation between cell proliferation and BLV expression, the effect of IL-2 and IL-10 on PBMC proliferation was tested. As anticipated, IL-2 stimulated while IL-10 suppressed antigen-specific PBMC proliferation. The present study, combined with our previous findings, suggests that increased IL-10 production in late disease stages suppresses BLV mRNA levels, while IL-2-activated immune responses stimulate BLV expression by BLV-infected B cells. PMID:10482594

Quantitative assessment of CYP11B1 and CYP11B2 expression in aldosterone-producing adenomas.

PubMed

Fallo, F; Pezzi, V; Barzon, L; Mulatero, P; Veglio, F; Sonino, N; Mathis, J M

2002-12-01

The presence and pathophysiological role of CYP11B1 (11beta-hydroxylase) gene in the zona glomerulosa of human adrenal cortex is still controversial. In order to specifically quantify CYP11B1, CYP11B2 (aldosterone synthase) and CYP17(17alpha-hydroxylase) mRNA levels, we developed a real-time RT-PCR assay and examined the expression in a series of adrenal tIssues, including six normal adrenals from patients adrenalectomized for renal cancer and twelve aldosterone-producing adenomas (APA) from patients with primary aldosteronism. CYP11B1 mRNA levels were clearly detected in normal adrenals, which comprised both zona glomerulosa and fasciculata/reticularis cells, but were also measured at a lower range (P<0.05) in APA. The levels of CYP11B2 mRNA were lower (P<0.005) in normal adrenals than in APA. CYP17 mRNAlevels were similar in normal adrenals and in APA. In patients with APA, CYP11B2 and CYP11B1 mRNA levels were not correlated either with basal aldosterone or with the change from basal aldosterone in response to posture or to dexamethasone. No correlation between CYP11B1 mRNA or CYP11B2 mRNA and the percentage of zona fasciculata-like cells was observed in APA. Real-time RT-PCR can be reliably used to quantify CYP11B1 and CYP11B2 mRNA levels in adrenal tIssues. Expression of CYP11B1 in hyperfunctioning zona glomerulosa suggests an additional formation of corticosterone via 11beta-hydroxylase, providing further substrate for aldosterone biosynthesis. CYP11B1 and CYP11B2 mRNA levels in APA are not related to the in vivo secretory activity of glomerulosa cells, where post-transcriptional factors might ultimately regulate aldosterone production.

1,25-Dihydroxyvitamin D3 and its analogues increase catalase at the mRNA, protein and activity level in a canine transitional carcinoma cell line.

PubMed

Middleton, R P; Nelson, R; Li, Q; Blanton, A; Labuda, J A; Vitt, J; Inpanbutr, N

2015-12-01

Antioxidant enzymes, such as catalase, superoxide dismutases (SOD), MnSOD and Cu/ZnSOD, protect cells by scavenging reactive oxygen species (ROS). Numerous studies have reported the anti-cancer effects of 1,25-dihydroxyvitamin D3 (calcitriol) and its related analogues, seocalcitol and analogue V. In this study, canine bladder transitional cell carcinoma (cbTCC) cells were used to determine effects of calcitriol and its related analogues on antioxidant enzyme gene expression, protein expression and activity. Catalase mRNA was increased in response to calcitriol (10(-7) M), and seocalcitol (10(-7) and 10(-9) M). MnSOD mRNA was decreased in response to calcitriol at 10(-7) M. Catalase was significantly increased in response to calcitriol (10(-7) and 10(-9) M), and seocalcitol (10(-9) M). Catalase enzymatic activity increased in response to calcitriol, seocalcitol and analogue V (10(-9) M). In addition, global gene expression analysis identified the involvement of mitogen-activated protein kinase (MAPK) signalling in cbTCC's response to calcitriol and seocalcitol treatment.

The Nuclear Pore-Associated TREX-2 Complex Employs Mediator to Regulate Gene Expression

PubMed Central

Schneider, Maren; Hellerschmied, Doris; Schubert, Tobias; Amlacher, Stefan; Vinayachandran, Vinesh; Reja, Rohit; Pugh, B. Franklin; Clausen, Tim; Köhler, Alwin

2015-01-01

Summary Nuclear pore complexes (NPCs) influence gene expression besides their established function in nuclear transport. The TREX-2 complex localizes to the NPC basket and affects gene-NPC interactions, transcription, and mRNA export. How TREX-2 regulates the gene expression machinery is unknown. Here, we show that TREX-2 interacts with the Mediator complex, an essential regulator of RNA Polymerase (Pol) II. Structural and biochemical studies identify a conserved region on TREX-2, which directly binds the Mediator Med31/Med7N submodule. TREX-2 regulates assembly of Mediator with the Cdk8 kinase and is required for recruitment and site-specific phosphorylation of Pol II. Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent at specific genes. TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing. Our data provide mechanistic insight into how an NPC-associated adaptor complex accesses the core transcription machinery. PMID:26317468

Cafeteria feeding induces interleukin-1beta mRNA expression in rat liver and brain.

PubMed

Hansen, M K; Taishi, P; Chen, Z; Krueger, J M

1998-06-01

intake affects gut-immune function and can provide a strong intestinal antigen challenge resulting in activation of host defense mechanisms in the digestive system. Previously, we showed that feeding rats a cafeteria diet increases non-rapid eye movement sleep by a subdiaphragmatic mechanism. Food intake and sleep regulation and the immune system share the regulatory molecule interleukin-1beta (IL-1beta). Thus this study examined the effects of a cafeteria diet on IL-1beta mRNA and IL-1 receptor accessory protein (IL-1RAP) mRNA expression in rat liver and brain. Rats were fed normal rat chow or a palatable diet consisting of bread, chocolate, and shortbread cookies (cafeteria diet). After 3 days, midway between the light period of the light-dark cycle, rats were killed by decapitation. Feeding rats a cafeteria diet resulted in increased IL-1beta mRNA expression in the liver and hypothalamus compared with rats fed only the normal rat chow. In addition, cafeteria feeding decreased IL-1RAP mRNA levels in the liver and brain stem. These results indicate that feeding has direct effects on cytokine production and together with other data suggest that the increased sleep that accompanies increased feeding may be the result of increased brain IL-1beta. These results further suggest that cytokine-to-brain communication may be important in normal physiological conditions, such as feeding, as well as being important during inflammatory responses.

Concerted elevation of acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) activity through independent stimulation of mRNA expression of DGAT1 and DGAT2 by carbohydrate and insulin.

PubMed

Meegalla, Rupalie L; Billheimer, Jeffrey T; Cheng, Dong

2002-11-01

Glucose and insulin are anabolic signals which upregulate the transcriptions of a series of lipogenic enzymes to convert excess carbohydrate into triglycerides for efficient energy storage. These enzymes include ATP-citrate lyase (ACL), acetyl-coenzyme A carboxylase (ACC), fatty acid synthase (FAS), and glycerol-3-phosphate acyltransferase (G3PA). Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) is important to synthesize fatty acids into triglycerides. Two DGATs from different gene families have recently been identified. In the current study, we report that glucose preferentially enhances DGAT1 mRNA expression, whereas insulin specifically increases the level of DGAT2 mRNA. Treatment of adipocytes with glucose and insulin together results in higher DGAT activity in the membrane than cells treated with either of the agents alone, indicating that glucose and insulin have additive effect on DGAT activation. In mice treated with fast/refeeding protocol, DGAT2 mRNA decreased upon fasting and was replenished upon refeeding in adipose tissue and liver. This pattern of change was not observed for DGAT1. Inasmuch as DGAT1 mRNA is less abundant in liver, we suggest that DGAT1 is more involved in fat absorption in the intestine and in basal level triglyceride synthesis in adipose tissue where it is more highly expressed. In contrast, DGAT2 is more likely to play important roles in assembly of de novo synthesized fatty acids into VLDL particles in the liver.

Upregulation of the immune protein gene hemolin in the epidermis during the wandering larval stage of the Indian meal moth, Plodia interpunctella.

PubMed

Aye, Tin Tin; Shim, Jae-Kyoung; Rhee, In-Koo; Lee, Kyeong-Yeoll

2008-08-01

Expression of hemolin, which generates an immune protein, was up-regulated in wandering fifth instar larval stage of Plodia interpunctella. The mRNA level peaked in the middle of the wandering stage. Major expression was in the epidermis, rather than in the fat body or gut. To test a possible ecdysteroid effect on hemolin induction we treated with RH-5992, an ecdysteroid agonist, and KK-42, which inhibits ecdysteroid biosynthesis in both feeding and wandering fifth instar larvae. When feeding larvae were treated with RH-5992 the hemolin mRNA level was increased. When wandering larvae were treated with KK-42 its level was reduced. In addition, when KK-42-treated larvae were subsequently treated with RH-5992 the hemolin mRNA level was recovered. These results strongly suggest that ecdysteroid up-regulates the expression of hemolin mRNA. Hormonal and bacterial effects on hemolin induction were further analyzed at the tissue level. Major induction of hemolin mRNA was detected following both RH-5992 treatment and bacterial injection in the epidermis of both feeding and wandering larvae. Minor induction of hemolin was detected in the fat body following a bacterial injection, but not RH-5992 treatment. We infer that in P. interpunctella larvae, the epidermis is the major tissue for hemolin induction in naïve insects and in insects manipulated with bacterial and hormonal treatments.

O6-Methylguanine-DNA Methyltransferase (MGMT) mRNA Expression Predicts Outcome in Malignant Glioma Independent of MGMT Promoter Methylation

PubMed Central

Kreth, Simone; Thon, Niklas; Eigenbrod, Sabina; Lutz, Juergen; Ledderose, Carola; Egensperger, Rupert; Tonn, Joerg C.; Kretzschmar, Hans A.; Hinske, Ludwig C.; Kreth, Friedrich W.

2011-01-01

Background We analyzed prospectively whether MGMT (O6-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression. Methodology/Principal Findings Adult patients with a histologically proven malignant astrocytoma (glioblastoma: N = 53, anaplastic astrocytoma: N = 10) were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP) and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA) database (N = 229 glioblastomas). Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001); the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001); however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6) did significantly worse than those with low transcriptional activity (p<0.01). Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6) did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001). Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT mRNA expression. Conclusions/Significance MGMT mRNA expression plays a direct role for mediating tumor sensitivity to alkylating agents. Discordant findings indicate methylation-independent pathways of MGMT expression regulation. DNMT1 and DNMT3b are likely to be involved in CGI methylation. However, their exact role yet has to be defined. PMID:21365007

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).

Molecular cloning and gene expression analysis of Ercc6l in Sika deer (Cervus nippon hortulorum).

PubMed

Yin, Yupeng; Tang, Lina; Zhang, Jiabao; Tang, Bo; Li, Ziyi

2011-01-01

One important protein family that functions in nucleotide excision repair (NER) factors is the SNF2 family. A newly identified mouse ERCC6-like gene, Ercc6l (excision repair cross-complementing rodent repair deficiency, complementation group 6-like), has been shown to be another developmentally related member of the SNF2 family. In this study, Sika deer Ercc6l cDNA was first cloned and then sequenced. The full-length cDNA of the Sika deer Ercc6l gene is 4197 bp and contains a 3732 bp open reading frame that encodes a putative protein of 1243 amino acids. The similarity of Sika deer Ercc6l to Bos taurus Ercc6l is 94.05% at the amino acid sequence level. The similarity, however, is reduced to 68.42-82.21% when compared to Ercc6l orthologs in other mammals and to less than 50% compared to orthologs in Gallus gallus and Xenopus. Additionally, the expression of Ercc6l mRNA was investigated in the organs of fetal and adult Sika deer (FSD and ASD, respectively) by quantitative RT-PCR. The common expression level of Ercc6l mRNA in the heart, liver, spleen, lung, kidney, and stomach from six different developmental stages of 18 Sika deer were examined, though the expression levels in each organ varied among individual Sika deer. During development, there was a slight trend toward decreased Ercc61 mRNA expression. The highest Ercc6l expression levels were seen at 3 months old in every organ and showed the highest level of detection in the spleen of FSD. The lowest Ercc6l expression levels were seen at 3 years old. We are the first to successfully clone Sika deer Ercc6l mRNA. Ercc6l transcript is present in almost every organ. During Sika deer development, there is a slight trend toward decreased Ercc61 mRNA expression. It is possible that Ercc6l has other roles in embryonic development and in maintaining the growth of animals.

Molecular Cloning and Gene Expression Analysis of Ercc6l in Sika Deer (Cervus nippon hortulorum)

PubMed Central

Zhang, Jiabao; Tang, Bo; Li, Ziyi

2011-01-01

Background One important protein family that functions in nucleotide excision repair (NER) factors is the SNF2 family. A newly identified mouse ERCC6-like gene, Ercc6l (excision repair cross-complementing rodent repair deficiency, complementation group 6-like), has been shown to be another developmentally related member of the SNF2 family. Methodology/Principal Findings In this study, Sika deer Ercc6l cDNA was first cloned and then sequenced. The full-length cDNA of the Sika deer Ercc6l gene is 4197 bp and contains a 3732 bp open reading frame that encodes a putative protein of 1243 amino acids. The similarity of Sika deer Ercc6l to Bos taurus Ercc6l is 94.05% at the amino acid sequence level. The similarity, however, is reduced to 68.42–82.21% when compared to Ercc6l orthologs in other mammals and to less than 50% compared to orthologs in Gallus gallus and Xenopus. Additionally, the expression of Ercc6l mRNA was investigated in the organs of fetal and adult Sika deer (FSD and ASD, respectively) by quantitative RT-PCR. The common expression level of Ercc6l mRNA in the heart, liver, spleen, lung, kidney, and stomach from six different developmental stages of 18 Sika deer were examined, though the expression levels in each organ varied among individual Sika deer. During development, there was a slight trend toward decreased Ercc61 mRNA expression. The highest Ercc6l expression levels were seen at 3 months old in every organ and showed the highest level of detection in the spleen of FSD. The lowest Ercc6l expression levels were seen at 3 years old. Conclusions/Significance We are the first to successfully clone Sika deer Ercc6l mRNA. Ercc6l transcript is present in almost every organ. During Sika deer development, there is a slight trend toward decreased Ercc61 mRNA expression. It is possible that Ercc6l has other roles in embryonic development and in maintaining the growth of animals. PMID:21695076

Improving the Transduction of Bone Marrow–Derived Cells with an Integrase-Defective Lentiviral Vector

PubMed Central

Pay, S. Louise; Qi, Xiaoping; Willard, Jeffrey F.; Godoy, Juliana; Sankhavaram, Kavya; Horton, Ranier; Mitter, Sayak K.; Quigley, Judith L.; Chang, Lung-Ji; Grant, Maria B.; Boulton, Michael E.

2018-01-01

In lentiviral vector (LV) applications where transient transgene expression is sufficient, integrase-defective lentiviral vectors (IDLVs) are beneficial for reducing the potential for off-target effects associated with insertional mutagenesis. It was previously demonstrated that human RPE65 mRNA expression from an integrating lentiviral vector (ILV) induces endogenous Rpe65 and Cralbp mRNA expression in murine bone marrow–derived cells (BMDCs), initiating programming of the cells to retinal pigment epithelium (RPE)-like cells. These cells regenerate RPE in retinal degeneration models when injected systemically. As transient expression of RPE65 is sufficient to activate endogenous RPE-associated genes for programming BMDCs, use of an ILV is an unnecessary risk. In this study, an IDLV expressing RPE65 (IDLV3-RPE65) was generated. Transduction with IDLV3-RPE65 is less efficient than the integrating vector (ILV3-RPE65). Therefore, IDLV3-RPE65 transduction was enhanced with a combination of preloading 20 × -concentrated viral supernatant on RetroNectin at a multiplicity of infection of 50 and transduction of BMDCs by low-speed centrifugation. RPE65 mRNA levels increased from ∼12-fold to ∼25-fold (p < 0.05) after modification of the IDLV3-RPE65 transduction protocol, achieving expression similar to the ∼27-fold (p < 0.05) increase observed with ILV3-RPE65. Additionally, the study shows that the same preparation of RetroNectin can be used to coat up to three wells with no reduction in transduction. Critically, IDLV3-RPE65 transduction initiates endogenous Rpe65 mRNA expression in murine BMDCs and Cralbp/CRALBP mRNA in both murine and human BMDCs, similar to expression observed in ILV3-RPE65-transduced cells. Systemic administration of ILV3-RPE65 or IDLV3-RPE65 programmed BMDCs in a mouse model of retinal degeneration is sufficient to retain visual function and reduce retinal degeneration compared to mice receiving no treatment or naïve BMDC. It is concluded that IDLV3-RPE65 is appropriate for programming BMDCs to RPE-like cells. PMID:29160102

Nuclear Proteomics Reveals the Role of Protein Synthesis and Chromatin Structure in Root Tip of Soybean during the Initial Stage of Flooding Stress.

PubMed

Yin, Xiaojian; Komatsu, Setsuko

2016-07-01

To identify the upstream events controlling the regulation of flooding-responsive proteins in soybean, proteomic analysis of nuclear proteins in root tip was performed. By using nuclear fractions, which were highly enriched, a total of 365 nuclear proteins were changed in soybean root tip at initial stage of flooding stress. Four exon-junction complex-related proteins and NOP1/NOP56, which function in upstream of 60S preribosome biogenesis, were decreased in flooded soybean. Furthermore, proteomic analysis of crude protein extract revealed that the protein translation was suppressed by continuous flooding stress. Seventeen chromatin structure-related nuclear proteins were decreased in response to flooding stress. Out of them, histone H3 was clearly decreased with protein abundance and mRNA expression levels at the initial flooding stress. Additionally, a number of protein synthesis-, RNA-, and DNA-related nuclear proteins were decreased in a time-dependent manner. mRNA expressions of genes encoding the significantly changed flooding-responsive nuclear proteins were inhibited by the transcriptional inhibitor, actinomycin D. These results suggest that protein translation is suppressed through inhibition of preribosome biogenesis- and mRNA processing-related proteins in nuclei of soybean root tip at initial flooding stress. In addition, flooding stress may regulate histone variants with gene expression in root tip.

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

Emerging functions of alternative splicing coupled with nonsense-mediated decay.

PubMed

Hamid, Fursham M; Makeyev, Eugene V

2014-08-01

Higher eukaryotes rely on AS (alternative splicing) of pre-mRNAs (mRNA precursors) to generate more than one protein product from a single gene and to regulate mRNA stability and translational activity. An important example of the latter function involves an interplay between AS and NMD (nonsense-mediated decay), a cytoplasmic quality control mechanism eliminating mRNAs containing PTCs (premature translation termination codons). Although originally identified as an error surveillance process, AS-NMD additionally provides an efficient strategy for deterministic regulation of gene expression outputs. In this review, we discuss recently published examples of AS-NMD and delineate functional contexts where recurrent use of this mechanism orchestrates expression of important genes.

Embryo-Specific Gene Expression in Microspore-Derived Embryos of Brassica napus. An Interaction between Abscisic Acid and Jasmonic Acid1, 2

PubMed Central

Hays, Dirk B.; Wilen, Ronald W.; Sheng, Chuxing; Moloney, Maurice M.; Pharis, Richard P.

1999-01-01

The induction of napin and oleosin gene expression in Brassica napus microspore-derived embryos (MDEs) was studied to assess the possible interaction between abscisic acid (ABA) and jasmonic acid (JA). Napin and oleosin transcripts were detected sooner following treatment with ABA than JA. Treatment of MDEs with ABA plus JA gave an additive accumulation of both napin and oleosin mRNA, the absolute amount being dependent on the concentration of each hormone. Endogenous ABA levels were reduced by 10-fold after treatment with JA, negating the possibility that the observed additive interaction was due to JA-induced ABA biosynthesis. Also, JA did not significantly increase the uptake of [3H-ABA] from the medium into MDEs. This suggests that the additive interaction was not due to an enhanced carrier-mediated ABA uptake by JA. Finally, when JA was added to MDEs that had been treated with the ABA biosynthesis inhibitor fluridone, napin mRNA did not increase. Based on these results with the MDE system, it is possible that embryos of B. napus use endogenous JA to modulate ABA effects on expression of both napin and oleosin. In addition, JA could play a causal role in the reduction of ABA that occurs during late stages of seed development. PMID:10069845

Expression and clinical significance of ATM and PUMA gene in patients with colorectal cancer.

PubMed

Xiong, Hui; Zhang, Jiangnan

2017-12-01

The expression of ataxia-telangiectasia mutated (ATM) and p53 upregulated modulator of apoptosis (PUMA) genes in patients with colorectal cancer were investigated, to explore the correlation between the expression of ATM and PUMA and tumor development, to evaluate the clinical significance of ATM and PUMA in the treatment of colorectal cancer. Quantitative real-time PCR was used to detect the expression of ATM and PUMA in tumor tissue and adjacent healthy tissue of 67 patients with colorectal cancer and in normal colorectal tissue of 33 patients with colorectal polyps at mRNA level. The expression level of ATM mRNA in colorectal cancer tissues was significantly higher than that in normal mucosa tissues and adjacent non-cancerous tissue (P≤0.05), while no significant differences in expression level of ATM mRNA were found between normal mucosa tissues and adjacent noncancerous tissue (P=0.07). There was a negative correlation between the expression of ATM mRNA and the degree of differentiation of colorectal cancer (r= -0.312, P=0.013), while expression level of ATM mRNA was not significantly correlated with the age, sex, tumor invasion, lymph node metastasis or clinical stage (P>0.05). Expression levels of PUMA mRNA in colorectal cancer tissues, adjacent noncancerous tissue and normal tissues were 0.68±0.07, 0.88±0.04 and 1.76±0.06, respectively. Expression level of PUMA mRNA in colorectal cancer tissues and adjacent noncancerous tissue was significantly lower than that in normal colorectal tissues (P<0.05). The results showed that ATM mRNA is expressed abnormally in colorectal cancer tissues. Expression of PUMA gene in colorectal carcinoma is downregulated, and is negatively correlated with the occurrence of cancer.

Estradiol In Females May Negate Skeletal Muscle Myostatin Mrna Expression And Serum Myostatin Propeptide Levels After Eccentric Muscle Contractions

PubMed Central

Willoughby, Darryn S.; Wilborn, Colin D.

2006-01-01

Eccentric contractions produce a significant degree of inflammation and muscle injury that may increase the expression of myostatin. Due to its anti- oxidant and anti-flammatory effects, circulating 17-β estradiol (E2) may attenuate myostatin expression. Eight males and eight females performed 7 sets of 10 reps of eccentric contractions of the knee extensors at 150% 1-RM. Each female performed the eccentric exercise bout on a day that fell within her mid-luteal phase (d 21-23 of her 28-d cycle). Blood and muscle samples were obtained before and 6 and 24 h after exercise, while additional blood samples were obtained at 48 and 72 h after exercise. Serum E2 and myostatin LAP/propeptide (LAP/pro) levels were determined with ELISA, and myostatin mRNA expression determined using RT-PCR. Data were analyzed with two-way ANOVA and bivariate correlations (p < 0.05). Females had greater levels of serum E2 throughout the 72- h sampling period (p < 0.05). While males had greater body mass and fat-free mass, neither was correlated to the pre-exercise levels of myostatin mRNA and LAP/pro for either gender (p > 0.05). Compared to pre-exercise, males had significant increases (p < 0.05) in LAP/propetide and mRNA of 78% and 28%, respectively, at 24 h post-exercise, whereas females underwent respective decreases of 10% and 21%. E2 and LAP/propeptide were correlated at 6 h (r = -0.804, p = 0.016) and 24 h post- exercise (r = -0.841, p = 0.009) in males, whereas in females E2 levels were correlated to myostatin mRNA at 6 h (r =0.739, p = 0.036) and 24 h (r = 0.813, p = 0.014) post-exercise and LAP/propeptide at 6 h (r = 0.713, p = 0.047) and 24 h (r = 0.735, p = 0.038). In females, myostatin mRNA expression and serum LAP/propeptide levels do not appear to be significantly up-regulated following eccentric exercise, and may be due to higher levels of circulating E2. Key Points The pre-exercise levels of myostatin mRNA and propeptide were not significantly different between genders, and even though the total body mass and fat-free mass of males were significantly greater than females, neither was correlated to myostatin mRNA or LAP/propeptide. Myostatin mRNA expression in females is less than in males 24 h after a single bout of eccentric exercise. Myostatin LAP/propeptide levels in females are lower in females than in males 24 h after a single bout of eccentric exercise, thereby suggesting a gender-specific mechanism in which females may be less responsive to eccentric exercise than males. Myostatin mRNA expression in females is attenuated, possibly due to inhibition in myostatin signaling, and appears to be more related to the presence of a higher level of circulating E2 rather than body composition. Due to their higher level of E2, females seem to be less susceptible to the mechanism by which eccentric exercise apparently up-regulates myostatin mRNA expression in males. PMID:24357964

Visualization of RNA–protein interactions in living cells: FMRP and IMP1 interact on mRNAs

PubMed Central

Rackham, Oliver; Brown, Chris M

2004-01-01

Protein expression depends significantly on the stability, translation efficiency and localization of mRNA. These qualities are largely dictated by the RNA-binding proteins associated with an mRNA. Here, we report a method to visualize and localize RNA–protein interactions in living mammalian cells. Using this method, we found that the fragile X mental retardation protein (FMRP) isoform 18 and the human zipcode-binding protein 1 ortholog IMP1, an RNA transport factor, were present on common mRNAs. These interactions occurred predominantly in the cytoplasm, in granular structures. In addition, FMRP and IMP1 interacted independently of RNA. Tethering of FMRP to an mRNA caused IMP1 to be recruited to the same mRNA and resulted in granule formation. The intimate association of FMRP and IMP1 suggests a link between mRNA transport and translational repression in mammalian cells. PMID:15282548

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

Short communication: Growth hormone receptor expression in two dairy breeds during the periparturient period.

PubMed

Okamura, C S; Bader, J F; Keisler, D H; Lucy, M C

2009-06-01

The growth hormone receptor (GHR) 1A mRNA decreases after calving in the liver of Holstein dairy cows and may coordinate nutrient partitioning. The hypothesis that the decrease in GHR1A mRNA around the time of calving was characteristic of a second dairy breed was tested by examining Guernsey cows in addition to Holstein cows. Holstein and Guernsey cows were housed together and paired by parity and expected calving date. Liver biopsies and blood samples were collected prepartum (d -20 +/- 1) and postpartum on d 3, and d 14 +/- 1. The amounts of GHR1A, GHR1B, GHR1C, and insulin-like growth factor (IGF)1 mRNA were determined by quantitative reverse transcription polymerase chain reaction. Blood concentrations of growth hormone (GH) and IGF1 were measured by RIA. Both breeds underwent a decrease in GHR1A mRNA, a decrease in liver IGF1 mRNA, a decrease in blood IGF1, and an increase in blood GH after calving. The decrease in liver GHR1A and IGF1 mRNA after calving may be an inherent characteristic of dairy breeds that enables nutrient partitioning for greater milk production. Independent genetic selection in 2 dairy breeds seemingly exploited a similar mechanism, reduced GHR1A expression, to decrease blood IGF1 and increase blood GH, a key hormone involved in nutrient partitioning.

A novel RNA binding protein affects rbcL gene expression and is specific to bundle sheath chloroplasts in C4 plants

PubMed Central

2013-01-01

Background Plants that utilize the highly efficient C4 pathway of photosynthesis typically possess kranz-type leaf anatomy that consists of two morphologically and functionally distinct photosynthetic cell types, the bundle sheath (BS) and mesophyll (M) cells. These two cell types differentially express many genes that are required for C4 capability and function. In mature C4 leaves, the plastidic rbcL gene, encoding the large subunit of the primary CO2 fixation enzyme Rubisco, is expressed specifically within BS cells. Numerous studies have demonstrated that BS-specific rbcL gene expression is regulated predominantly at post-transcriptional levels, through the control of translation and mRNA stability. The identification of regulatory factors associated with C4 patterns of rbcL gene expression has been an elusive goal for many years. Results RLSB, encoded by the nuclear RLSB gene, is an S1-domain RNA binding protein purified from C4 chloroplasts based on its specific binding to plastid-encoded rbcL mRNA in vitro. Co-localized with LSU to chloroplasts, RLSB is highly conserved across many plant species. Most significantly, RLSB localizes specifically to leaf bundle sheath (BS) cells in C4 plants. Comparative analysis using maize (C4) and Arabidopsis (C3) reveals its tight association with rbcL gene expression in both plants. Reduced RLSB expression (through insertion mutation or RNA silencing, respectively) led to reductions in rbcL mRNA accumulation and LSU production. Additional developmental effects, such as virescent/yellow leaves, were likely associated with decreased photosynthetic function and disruption of associated signaling networks. Conclusions Reductions in RLSB expression, due to insertion mutation or gene silencing, are strictly correlated with reductions in rbcL gene expression in both maize and Arabidopsis. In both plants, accumulation of rbcL mRNA as well as synthesis of LSU protein were affected. These findings suggest that specific accumulation and binding of the RLSB binding protein to rbcL mRNA within BS chloroplasts may be one determinant leading to the characteristic cell type-specific localization of Rubisco in C4 plants. Evolutionary modification of RLSB expression, from a C3 “default” state to BS cell-specificity, could represent one mechanism by which rbcL expression has become restricted to only one cell type in C4 plants. PMID:24053212

p300 expression repression by hypermethylation associated with tumour invasion and metastasis in oesophageal squamous cell carcinoma

PubMed Central

Zhang, Changsong; Li, Ke; Wei, Lixin; Li, Zhengyou; Yu, Ping; Teng, Lijuan; Wu, Kusheng; Zhu, Jin

2007-01-01

Background Aberrant promoter methylation is an important mechanism for gene silencing. Aims To evaluate the promoter methylation status of p300 gene in patients with oesophageal squamous cell carcinoma (OSCC). Methods The methylation status of p300 promoter was analysed by methylation‐specific PCR (MSP) in 50 OSCC tissues and the matching non‐cancerous tissues. Oesophageal cancer cell lines (ECa‐109 and TE‐10) were treated with the demethylation agent 5‐aza‐2′‐deoxycytidine (5‐Aza‐CdR), and p300 mRNA expression was detected by RT‐PCR. Results p300 methylation was found in 42% (21/50) of the OSCC tissues, but in only 20% (10/50) of the corresponding non‐cancerous tissues (p = 0.017). In OSCC samples, 65% of those with deep tumour invasion (adventitia) and 63% samples with metastasis revealed p300 promoter methylation (p<0.05). p300 mRNA expression was observed in 19.0% (4/21) of methylated tumours and 58.6% (17/29) of unmethylated tumours (p = 0.005). In addition, p300 mRNA expression was observed in 40% (4/10) of methylated non‐neoplastic tissues and 87.5% (35/40) of unmethylated non‐tumours (p = 0.001). The demethylation caused by 5‐Aza‐CdR increased the p300 mRNA expression levels in oesophageal cancer cell lines. Conclusions p300 transcription silenced by promoter hypermethylation could play a role in the pathogenesis of oesophageal squamous cell carcinoma. PMID:17965222

Differential Gene Expression Associated with Honey Bee Grooming Behavior in Response to Varroa Mites.

PubMed

Hamiduzzaman, Mollah Md; Emsen, Berna; Hunt, Greg J; Subramanyam, Subhashree; Williams, Christie E; Tsuruda, Jennifer M; Guzman-Novoa, Ernesto

2017-05-01

Honey bee (Apis mellifera) grooming behavior is an important mechanism of resistance against the parasitic mite Varroa destructor. This research was conducted to study associations between grooming behavior and the expression of selected immune, neural, detoxification, developmental and health-related genes. Individual bees tested in a laboratory assay for various levels of grooming behavior in response to V. destructor were also analyzed for gene expression. Intense groomers (IG) were most efficient in that they needed significantly less time to start grooming and fewer grooming attempts to successfully remove mites from their bodies than did light groomers (LG). In addition, the relative abundance of the neurexin-1 mRNA, was significantly higher in IG than in LG, no groomers (NG) or control (bees without mite). The abundance of poly U binding factor kd 68 and cytochrome p450 mRNAs were significantly higher in IG than in control bees. The abundance of hymenoptaecin mRNA was significantly higher in IG than in NG, but it was not different from that of control bees. The abundance of vitellogenin mRNA was not changed by grooming activity. However, the abundance of blue cheese mRNA was significantly reduced in IG compared to LG or NG, but not to control bees. Efficient removal of mites by IG correlated with different gene expression patterns in bees. These results suggest that the level of grooming behavior may be related to the expression pattern of vital honey bee genes. Neurexin-1, in particular, might be useful as a bio-marker for behavioral traits in bees.

[The expressions of HSP 70 mRNA and c-fos mRNA in the skeletal muscle and cardiac muscle of rabbits by electrocuted].

PubMed

Wang, Ye; Liu, Min; Cheng, Wei-bo; He, Gui-qiong; Li, Fan; Liao, Zhi-gang

2008-08-01

To study the changes of HSP 70 mRNA and c-fos mRNA expression and to find a method to differentiate antemortem from postmortem electrocution. Fifteen New Zealand rabbits were randomly divided into three groups, the antemortem electrocution group, the postmortem electrocution group, and the control group. Each group consists of five rabbits. The levels of HSP 70 mRNA and c-fos mRNA in skeletal muscle and cardiac muscle were examined with quantitative fluorescent RT-PCR. The levels of HSP 70 mRNA and c-fos mRNA in the antemortem electrocution group increased significantly (P<0.05), compared with that of the postmortem electrocution group. The changes of HSP 70 mRNA and c-fos mRNA expression in skeletal muscle and cardiac muscle can be used as an indicator to distinguish antemortem from postmortem electrocution.

Developmental Considerations of Sperm Protein 17 Gene Expression in Rheumatoid Arthritis Synoviocytes

PubMed Central

Takeoka, Yuichi; Kenny, Thomas P.; Yago, Hisashi; Naiki, Mitsuru; Gershwin, M. Eric; Robbins, Dick L.

2002-01-01

Rheumatoid arthritis (RA) is an autoimmune disease characterized by proliferative synovial tissue. We used mRNA differential display and library subtraction to compare mRNA expression in RA and osteoarthritis (OA) synoviocytes. We initially compared the mRNA expression patterns in 1 female RA and 1 OA synovia and found a differentially expressed 350 bp transcript in the RA synoviocytes which was, by sequence analysis, 100% homologous to sperm protein 17 (Sp17). Moreover, the Sp17 transcript was found differentially expressed in a RA synovial library that was subtracted with an OA synovial library. Using specific primers for full length Sp17, a 1.1 kb transcript was amplified from the synoviocytes of 7 additional female RA patients, sequenced and found to 100% homologous to Sp17. Thus, we found the unexpected expression of Sp17, a thought to be gamete-specific protein, in the synoviocytes of 8/8 female RA patients in contrast to control OA synoviocytes. Interestingly, Sp17's structural relationship with cell-binding and recognition proteins, suggests that Sp17 may function in cell-cell recognition and signaling in the RA synoviocyte. Further, Sp17 could have a significant regulatory role in RA synoviocyte gene transcription and/or signal transduction. Thus, Sp17 could have an important role in RA synoviocyte proliferation or defective apoptosis. Finally, the presence of Sp17 in synoviocytes has interesting developmental considerations. PMID:12739786

Common variants of OPA1 conferring genetic susceptibility to leprosy in Han Chinese from Southwest China.

PubMed

Xiang, Yang-Lin; Zhang, Deng-Feng; Wang, Dong; Li, Yu-Ye; Yao, Yong-Gang

2015-11-01

Leprosy is an ancient chronic infection caused by Mycobacterium leprae. Onset of leprosy was highly affected by host nutritional condition and energy production, (partially) due to genomic loss and parasitic life style of M. leprae. The optic atrophy 1 (OPA1) gene plays an essential role in mitochondria, which function in cellular energy supply and innate immunity. To investigate the potential involvement of OPA1 in leprosy. We analyzed 7 common genetic variants of OPA1 in 1110 Han Chinese subjects with and without leprosy, followed by mRNA expression profiling and protein-protein interaction (PPI) network analysis. We observed positive associations between OPA1 variants rs9838374 (Pgenotypic=0.003) and rs414237 (Pgenotypic=0.002) with lepromatous leprosy. expression quantitative trait loci (eQTL) analysis showed that the leprosy-related risk allele C of rs414237 is correlated with lower OPA1 mRNA expression level. Indeed, we identified a decrease of OPA1 mRNA expression in both with patients and cellular model of leprosy. In addition, the PPI analysis showed that OPA1 protein was actively involved in the interaction network of M. leprae induced differentially expressed genes. Our results indicated that OPA1 variants confer risk of leprosy and may affect OPA1 expression, mitochondrial function and antimicrobial pathways. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Expression of uncoupling protein 3 is upregulated in skeletal muscle during sepsis.

PubMed

Sun, Xiaoyan; Wray, Curtis; Tian, Xintian; Hasselgren, Per-Olof; Lu, James

2003-09-01

Uncoupling protein 3 (UCP3) is a member of the mitochondrial transporter superfamily that is expressed primarily in skeletal muscle. UCP3 is upregulated in various conditions characterized by skeletal muscle atrophy, including hyperthyroidism, fasting, denervation, diabetes, cancer, lipopolysaccharide (LPS), and treatment with glucocorticoids (GCs). The influence of sepsis, another condition characterized by muscle cachexia, on UCP3 expression and activity is not known. We examined UCP3 gene and protein expression in skeletal muscles from rats after cecal ligation and puncture and from sham-operated control rats. Sepsis resulted in a two- to threefold increase in both mRNA and protein levels of UCP3 in skeletal muscle. Treatment of rats with the glucocorticoid receptor antagonist RU-38486 prevented the sepsis-induced increase in gene and protein expression of UCP3. The UCP3 mRNA and protein levels were increased 2.4- to 3.6-fold when incubated muscles from normal rats were treated with dexamethasone (DEX) and/or free fatty acids (FFA) ex vivo. In addition, UCP3 mRNA and protein levels were significantly increased in normal rat muscles in vivo with treatment of either DEX or FFA. The results suggest that sepsis upregulates the gene and protein expression of UCP3 in skeletal muscle, which may at least in part be mediated by GCs and FFA.

Both cell substratum regulation and hormonal regulation of milk protein gene expression are exerted primarily at the posttranscriptional level

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

Eisenstein, R.S.; Rosen, J.M.

The mechanism by which individual peptide and steroid hormones and cell-substratum interactions regulate milk protein gene expression has been studied in the COMMA-D mammary epithelial cell line. In the presence of insulin, hydrocortisone, and prolactin, growth of COMMA-D cells on floating collagen gels in comparison with that on a plastic substratum resulted in a 2.5- to 3-fold increase in the relative rate of ..beta..-casein gene transcription but a 37-fold increase in ..beta..-casein mRNA accumulation. In contrast, whey acidic protein gene transcription was constitutive in COMMA-D cells grown on either substratum, but its mRNA was unstable and little intact mature mRNAmore » was detected. Culturing COMMA-D cells on collagen also promoted increased expression of other genes expressed in differentiated mammary epithelial cells, including those encoding ..cap alpha..- and ..gamma..-casein, transferrin, malic enzyme, and phosphoenolpyruvate carboxykinase but decreased the expression of actin and histone genes. Using COMMA-D cells, the authors defined further the role of individual hormones in influencing ..beta..-casein gene transcription. With insulin alone, a basal level of ..beta..-casein gene transcription was detected in COMMA-D cells grown on floating collagen gels. Addition of prolactin but not hydrocortisone resulted in a 2.5- to 3.0-fold increase in ..beta..-casein gene transcription, but both hormones were required to elicit the maximal 73-fold induction in mRNA accumulation. The posttranscriptional effect of hormones on casein mRNA accummulation preceded any detectable changes in the relative rate of transcription. Thus, regulation by both hormones and cell substratum of casein gene expression is exerted primarily at the post transcriptional level.« less

[Effect of Epimedium extract on osteoprotegerin and RANKL mRNA expressions in glucocorticoid-induced femoral head necrosis in rats].

PubMed

Wang, Jian-zhong; Gao, Hong-yan; Wang, Kun-zheng; Zhou, Rong-xing; Li, Xiao-dong; Guo, Jun; Lv, Hui-cheng

2011-10-01

To investigate the effect of glucocorticoid on the expression levels of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) mRNAs in rat femoral head and the antagonistic effect of Epimedium, and explore the mechanism of Epimedium in preventing glucocorticoid-induced femoral head necrosis. Forty-eight adult SD rats were randomized into glucocorticoid group, Epimedium group and control group. In the former two groups, the rats received intramuscular injection of 12.5 mg prednisolone twice a week, and in Epimedium group, additional 1 ml/100 g aqueous Epimedium extract (equivalent to 0.1 g/ml of the crude drug) was administered intragastrically once daily. The control group received only intramuscular saline injection. After 4 weeks of treatment, osteonecrosis of the left femoral head was detected by HE staining, and the right femoral head was sampled for detection of OPG and RANKL mRNA expressions using real-time quantitative PCR. In glucocorticoid, Epimedium and control groups, the mortality rate of the rats was 12.5% (2/16), 6.25% (1/16), 0 (0/16), and femoral head necrosis occurred at a rate of 71.43% (10/14), 26.67% (4/15), and 0 (0/16), respectively. In glucocorticoid group, the expression level of OPG mRNA was significantly lower, RANKL expression significantly higher, and OPG/RANKL ratio significantly lower than those in Epimedium and control groups (P<0.05). OPG, RANKL and their ratios showed no significant differences between Epimedium group and the control group. Epimedium can prevent glucocorticoid-induced femoral head necrosis probably by antagonizing glucocorticiod-induced abnormal expressions of OPG and RANKL mRNA.

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

Molecular cloning of heat shock protein 60 (PtHSP60) from Portunus trituberculatus and its expression response to salinity stress.

PubMed

Xu, Qianghua; Qin, Ye

2012-09-01

Heat shock protein 60 (HSP60) is a highly conserved and multi-functional molecular chaperone that plays an essential role in both cellular metabolism and stress response. Portunus trituberculatus is an important marine fishery and aquaculture species, and water salinity condition influenced its artificial propagations significantly. In order to investigate the function of P. trituberculatus HSP60 against osmotic stress, P. trituberculatus HSP60 gene was firstly cloned. The full-length cDNA of PtHSP60 contains 1,743 nucleotides encoding 577 amino acids with a calculated molecular weight of 61.25 kDa. Multiple alignments indicated that the deduced amino acid sequences of PtHSP60 shared a high level of identity with invertebrate and vertebrate HSP60 sequence including shrimp, fruit fly, zebrafish, and human. The expression profiles of PtHSP60 at mRNA and protein levels under salinity treatment were investigated by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. It was found that the mRNA transcripts of PtHSP60 gene varied among different tissues under normal salinity conditions, and the antennal gland showed the highest expression level among the tissues tested. As for low salinity challenge, the mRNA expression of PtHSP60 gene was higher in the gill and appendicular muscle compared with other tissues, and gill and hypodermis represented the higher gene expressions during the hyperosmotic stress, which indicated that those tissues were salinity-sensitive tissues. In addition, salinity challenges significantly altered the expression of PtHSP60 at mRNA and protein level in a salinity- and time-dependent manner in P. trituberculatus gill tissue. The results indicate that PtHSP60 played important roles in mediating the salinity stress in P. trituberculatus.

Hypothyroidism and oxidative stress: differential effect on the heart of virgin and pregnant rats.

PubMed

Carmona, Y V; Coria, M J; Oliveros, L B; Gimenez, M S

2014-01-01

The present study investigates the effects of hypothyroidism on both the redox state and the thyroid hormone receptors expression in the heart ventricle of virgin and pregnant rats.Hypothyroid state was induced by 6-n-propyl-2-thiouracil in drinking water given to Wistar rats starting 8 days before mating until day 21 of pregnancy or for 30 days in virgin rats. Serum paraoxonase-1 (PON-1) activity, serum and heart nitrites, and thiobarbituric acid-reactive substances (TBARS) were analyzed. Heart protein oxidation, as carbonyls, and copper-zinc superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx), and catalase (CAT) activities, were determined. In addition, heart expressions of NADPH oxidase (NOX-2), CAT, SOD, GPx, and thyroid receptors (TRα and TRβ) mRNA were assessed by RT-PCR. Inducible and endothelial Nitric Oxide Synthase (iNOS and eNOS) were determined by Western blot. Hypothyroidism in the heart of virgin rats decreased TRα and TRβ expressions, and induced oxidative stress, leading to a decrease of nitrites and an increase of carbonyls, NOX-2 mRNA, and GPx activity. A decreased PON-1 activity suggested low protection against oxidative stress in blood circulation. Pregnancy reduced TRα and TRβ mRNA expressions and induced oxidative stress by increasing nitrite and TBARS levels, SOD and CAT activities and NOX-2, eNOS and iNOS expressions, while hypothyroidism, emphasized the decreases of TRα mRNA levels and did not alter the redox state in the heart. TR expressions and redox balance of rat hearts depend on the physiological state. Pregnancy per se seems to protect the heart against oxidative stress induced by hypothyroidism. Supporting Information for this article is available online at http://www.thieme-connect.de/ejournals/toc/hmr. © Georg Thieme Verlag KG Stuttgart · New York.

Sodium Meta-Arsenite Ameliorates Hyperglycemia in Obese Diabetic db/db Mice by Inhibition of Hepatic Gluconeogenesis

PubMed Central

Lee, Eun-Kyu; Oh, Hyun-Hee; Choi, Cheol Soo; Kim, Sujong; Jun, Hee-Sook

2014-01-01

Sodium meta-arsenite (SA) is implicated in the regulation of hepatic gluconeogenesis-related genes in vitro; however, the effects in vivo have not been studied. We investigated whether SA has antidiabetic effects in a type 2 diabetic mouse model. Diabetic db/db mice were orally intubated with SA (10 mg kg−1 body weight/day) for 8 weeks. We examined hemoglobin A1c (HbA1c), blood glucose levels, food intake, and body weight. We performed glucose, insulin, and pyruvate tolerance tests and analyzed glucose production and the expression of gluconeogenesis-related genes in hepatocytes. We analyzed energy metabolism using a comprehensive animal metabolic monitoring system. SA-treated diabetic db/db mice had reduced concentrations of HbA1c and blood glucose levels. Exogenous glucose was quickly cleared in glucose tolerance tests. The mRNA expressions of genes for gluconeogenesis-related enzymes, glucose 6-phosphatase (G6Pase), and phosphoenolpyruvate carboxykinase (PEPCK) were significantly reduced in the liver of SA-treated diabetic db/db mice. In primary hepatocytes, SA treatment decreased glucose production and the expression of G6Pase, PEPCK, and hepatocyte nuclear factor 4 alpha (HNF-4α) mRNA. Small heterodimer partner (SHP) mRNA expression was increased in hepatocytes dependent upon the SA concentration. The expression of Sirt1 mRNA and protein was reduced, and acetylated forkhead box protein O1 (FoxO1) was induced by SA treatment in hepatocytes. In addition, SA-treated diabetic db/db mice showed reduced energy expenditure. Oral intubation of SA ameliorates hyperglycemia in db/db mice by reducing hepatic gluconeogenesis through the decrease of Sirt1 expression and increase in acetylated FoxO1. PMID:25610880

Mycobacterium paratuberculosis, Mycobacterium smegmatis, and lipopolysaccharide induce different transcriptional and post-transcriptional regulation of the IRG1 gene in murine macrophages.

PubMed

Basler, Tina; Jeckstadt, Sabine; Valentin-Weigand, Peter; Goethe, Ralph

2006-03-01

Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic enteritis in ruminants. In addition, MAP is presently the most favored pathogen linked to Crohn's disease. In this study, we were interested in dissecting the molecular mechanisms of macrophage activation or deactivation after infection with MAP. By subtractive hybridization of cDNAs, we identified the immune-responsive gene 1 (IRG1), which was expressed substantially higher in lipopolysaccharide (LPS)-stimulated than in MAP-infected murine macrophage cell lines. A nuclear run-on transcription assay revealed that the IRG1 gene was activated transcriptionally in LPS-stimulated and MAP-infected macrophages with higher expression in LPS-stimulated cells. Analysis of post-transcriptional regulation demonstrated that IRG1 mRNA stability was increased in LPS-stimulated but not in MAP-infected macrophages. Furthermore, IRG1 gene expression of macrophages infected with the nonpathogenic Mycobacterium smegmatis differed from those of LPS-stimulated and MAP-infected macrophages. At 2 h postinfection, M. smegmatis-induced IRG1 gene expression was as low as in MAP-infected, and 8 h postinfection, it increased nearly to the level in LPS-stimulated macrophages. Transient transfection experiments revealed similar IRG1 promoter activities in MAP- and M. smegmatis-infected cells. Northern analysis demonstrated increased IRG1 mRNA stability in M. smegmatis-infected macrophages. IRG1 mRNA stabilization was p38 mitogen-activated protein kinase-independent. Inhibition of protein synthesis revealed that constitutively expressed factors seemed to be responsible for IRG1 mRNA destabilization. Thus, our data demonstrate that transcriptional and post-transcriptional mechanisms are responsible for a differential IRG1 gene expression in murine macrophages treated with LPS, MAP, and M. smegmatis.

Perinatal asphyxia alters neuregulin-1 and COMT gene expression in the medial prefrontal cortex in rats.

PubMed

Wakuda, Tomoyasu; Iwata, Keiko; Iwata, Yasuhide; Anitha, Ayyappan; Takahashi, Taro; Yamada, Kohei; Vasu, Mahesh Mundalil; Matsuzaki, Hideo; Suzuki, Katsuaki; Mori, Norio

2015-01-02

Epidemiological studies suggest that perinatal complications, particularly hypoxia-related ones, increase the risk of schizophrenia. Recent genetic studies of the disorder have identified several putative susceptibility genes, some of which are known to be regulated by hypoxia. It can be postulated therefore that birth complications that cause hypoxia in the fetal brain may be associated with a dysregulation in the expression of some of the schizophrenia candidate genes. To test this, we used an animal model of perinatal asphyxia, in which rat pups were exposed to 15 min of intrauterine anoxia during Caesarean section birth, and examined the expression of mRNA of five of the putative susceptibility genes (NRG1, ErbB4, AKT1, COMT and BDNF) by real-time quantitative PCR in the medial prefrontal cortex (mPFC) and the hippocampus at 6 and 12 weeks after birth. The expression of NRG1 mRNA was significantly decreased in the mPFC, but not in the hippocampus, at 6 and 12 weeks after birth. In addition, a significant increase in COMT mRNA expression was observed in the mPFC at 12 weeks. The alteration in mRNA levels of NRG1 and COMT was not associated with a change in their protein levels. These results suggest that perinatal asphyxia may lead to disturbances in the PFC, which in turn may exert a long-lasting influence on the expression of specific genes, such as NRG1 and COMT. Our results also suggest that translational interruption may occur in this model of perinatal asphyxia. Copyright © 2014 Elsevier Inc. All rights reserved.

Cytochrome P450 system expression and DNA adduct formation in the liver of Zacco platypus following waterborne benzo(a)pyrene exposure: implications for biomarker determination.

PubMed

Lee, Jin Wuk; Kim, Yong Hwa; Yoon, Seokjoo; Lee, Sung Kyu

2014-09-01

Benzo(a)pyrene (BaP) is a polycyclic aromatic hydrocarbon that causes mutations and tumor formation. Zacco platypus is a sentinel species that is suitable for monitoring aquatic environments. We studied cytochrome P450 system (CYP system) expression and DNA adduct formation in the liver of Z. platypus following waterborne exposure to BaP. The results showed both dose and time dependency. The significant induction levels of CYP system mRNA and protein reached maximums at 2 days and 14 days, respectively, and hepatosomatic index was maximally induced at 4 days during 14 days BaP exposure. DNA adduct formation was significantly induced compared to corresponding controls (t-test, p < 0.01) after 4 days of exposure in 100 μg/L BaP. These results indicate that the only use of mRNA expression level of CYP system as a biomarker make us underestimate prolonged toxicity (4-14 days) of BaP and the only use of protein expression level of CYP system make us underestimate acute toxicity (1-2 days) of BaP. Therefore, we suggests that a combinational use of the mRNA expression level and protein expression level of CYP system, hepatosomatic index is a useful biomarker in risk assessment of waterborne BaP exposure. In addition, DNA adduct formation was a useful biomarker in risk assessment of waterborne BaP exposure at 4 days. CYP1A was a more sensitive biomarker than CYP reductase for BaP exposure when considering both the mRNA and protein level. Furthermore, our results show that Z. platypus is a useful species for assessing the risk of waterborne BaP exposure. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

Contributions of testosterone and territory ownership to sexually-motivated behaviors and mRNA expression in the medial preoptic area of male European starlings

PubMed Central

Spool, Jeremy A.; Stevenson, Sharon A.; Angyal, Caroline S.; Riters, Lauren V.

2016-01-01

Animals integrate social information with their internal endocrine state to control the timing of behavior, but how these signals are integrated in the brain is not understood. The medial preoptic area (mPOA) may play an integrative role in the control of courtship behavior, as it receives projections from multiple sensory systems, and is central to the hormonal control of courtship behavior across vertebrates. Additionally, data from many species implicate opioid and dopaminergic systems in the mPOA in the control of male courtship behavior. We used European starlings to test the hypothesis that testosterone (T) and social status (in the form of territory possession) interact to control the timing of courtship behavior by modulating steroid hormone-, opioid- and dopaminergic-related gene expression in the mPOA. We found that only males given both T and a nesting territory produced high rates of courtship behavior in response to a female. T treatment altered patterns of gene expression in the mPOA by increasing androgen receptor, aromatase, mu-opioid receptor and preproenkephalin mRNA and decreasing tyrosine hydroxylase mRNA expression. Territory possession did not alter mRNA expression in the mPOA, despite the finding that only birds with both T and a nesting territory produced courtship behavior. We propose that T prepares the mPOA to respond to the presence of a female with high rates of courtship song by altering gene expression, but that activity in the mPOA is under a continuous (i.e. tonic) inhibition until a male starling obtains a nesting territory. PMID:27633459

Contributions of testosterone and territory ownership to sexually-motivated behaviors and mRNA expression in the medial preoptic area of male European starlings.

PubMed

Spool, Jeremy A; Stevenson, Sharon A; Angyal, Caroline S; Riters, Lauren V

2016-11-01

Animals integrate social information with their internal endocrine state to control the timing of behavior, but how these signals are integrated in the brain is not understood. The medial preoptic area (mPOA) may play an integrative role in the control of courtship behavior, as it receives projections from multiple sensory systems, and is central to the hormonal control of courtship behavior across vertebrates. Additionally, data from many species implicate opioid and dopaminergic systems in the mPOA in the control of male courtship behavior. We used European starlings to test the hypothesis that testosterone (T) and social status (in the form of territory possession) interact to control the timing of courtship behavior by modulating steroid hormone-, opioid- and dopaminergic-related gene expression in the mPOA. We found that only males given both T and a nesting territory produced high rates of courtship behavior in response to a female. T treatment altered patterns of gene expression in the mPOA by increasing androgen receptor, aromatase, mu-opioid receptor and preproenkephalin mRNA and decreasing tyrosine hydroxylase mRNA expression. Territory possession did not alter mRNA expression in the mPOA, despite the finding that only birds with both T and a nesting territory produced courtship behavior. We propose that T prepares the mPOA to respond to the presence of a female with high rates of courtship song by altering gene expression, but that activity in the mPOA is under a continuous (i.e. tonic) inhibition until a male starling obtains a nesting territory. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of betaine on lipopolysaccharide-induced memory impairment in mice and the involvement of GABA transporter 2

PubMed Central

2011-01-01

Background Betaine (glycine betaine or trimethylglycine) plays important roles as an osmolyte and a methyl donor in animals. While betaine is reported to suppress expression of proinflammatory molecules and reduce oxidative stress in aged rat kidney, the effects of betaine on the central nervous system are not well known. In this study, we investigated the effects of betaine on lipopolysaccharide (LPS)-induced memory impairment and on mRNA expression levels of proinflammatory molecules, glial markers, and GABA transporter 2 (GAT2), a betaine/GABA transporter. Methods Mice were continuously treated with betaine for 13 days starting 1 day before they were injected with LPS, or received subacute or acute administration of betaine shortly before or after LPS injection. Then, their memory function was evaluated using Y-maze and novel object recognition tests 7 and 10-12 days after LPS injection (30 μg/mouse, i.c.v.), respectively. In addition, mRNA expression levels in hippocampus were measured by real-time RT-PCR at different time points. Results Repeated administration of betaine (0.163 mmol/kg, s.c.) prevented LPS-induced memory impairment. GAT2 mRNA levels were significantly increased in hippocampus 24 hr after LPS injection, and administration of betaine blocked this increase. However, betaine did not affect LPS-induced increases in levels of mRNA related to inflammatory responses. Both subacute administration (1 hr before, and 1 and 24 hr after LPS injection) and acute administration (1 hr after LPS injection) of betaine also prevented LPS-induced memory impairment in the Y-maze test. Conclusions These data suggest that betaine has protective effects against LPS-induced memory impairment and that prevention of LPS-induced changes in GAT2 mRNA expression is crucial to this ameliorating effect. PMID:22053950

Macrophage colony-stimulating factor induces prolactin expression in rat pituitary gland.

PubMed

Hoshino, Satoya; Kurotani, Reiko; Miyano, Yuki; Sakahara, Satoshi; Koike, Kanako; Maruyama, Minoru; Ishikawa, Fumio; Sakatai, Ichiro; Abe, Hiroyuki; Sakai, Takafumi

2014-06-01

We investigated the role of macrophage colony-stimulating factor (M-CSF) in the pituitary gland to understand the effect of M-CSF on pituitary hormones and the relationship between the endocrine and immune systems. When we attempted to establish pituitary cell lines from a thyrotropic pituitary tumor (TtT), a macrophage cell line, TtT/M-87, was established. We evaluated M-CSF-like activity in conditioned media (CM) from seven pituitary cell lines using TtT/M-87 cells. TtT/M-87 proliferation significantly increased in the presence of CM from TtT/GF cells, a pituitary folliculostellate (FS) cell line. M-CSF mRNA was detected in TtT/GF and MtT/E cells by reverse transcriptase-polymerase chain reaction (RT-PCR), and its expression in TtT/GF cells was increased in a lipopolysaccharide (LPS) dose-dependent manner. M-CSF mRNA expression was also increased in rat anterior pituitary glands by LPS. M-CSF receptor (M-CSFR) mRNA was only detected in TtT/ M-87 cells and increased in the LPS-stimulated rat pituitary glands. In rat pituitary glands, M-CSF and M-CSFR were found to be localized in FS cells and prolactin (PRL)-secreting cells, respectively, by immunohistochemistry. The PRL concentration in rat sera was significantly increased at 24 h after M-CSF administration, and mRNA levels significantly increased in primary culture cells of rat anterior pituitary glands. In addition, TNF-α mRNA was increased in the primary culture cells by M-CSF. These results revealed that M-CSF was secreted from FS cells and M-CSF regulated PRL expression in rat pituitary glands.

An NXF1 mRNA with a retained intron is expressed in hippocampal and neocortical neurons and is translated into a protein that functions as an Nxf1 cofactor.

PubMed

Li, Ying; Bor, Yeou-Cherng; Fitzgerald, Mark P; Lee, Kevin S; Rekosh, David; Hammarskjold, Marie-Louise

2016-12-01

The Nxf1 protein is a major nuclear export receptor for the transport of mRNA, and it also is essential for export of retroviral mRNAs with retained introns. In the latter case, it binds to RNA elements known as constitutive transport elements (CTEs) and functions in conjunction with a cofactor known as Nxt1. The NXF1 gene also regulates expression of its own intron-containing RNA through the use of a functional CTE within intron 10. mRNA containing this intron is exported to the cytoplasm, where it can be translated into the 356-amino acid short Nxf1(sNxf1) protein, despite the fact that it is a prime candidate for nonsense-mediated decay (NMD). Here we demonstrate that sNxf1 is highly expressed in nuclei and dendrites of hippocampal and neocortical neurons in rodent brain. Additionally, we show that sNxf1 localizes in RNA granules in neurites of differentiated N2a mouse neuroblastoma cells, where it shows partial colocalization with Staufen2 isoform SS, a protein known to play a role in dendritic mRNA trafficking. We also show that sNxf1 forms heterodimers in conjunction with the full-length Nxf1 and that sNxf1 can replace Nxt1 to enhance the expression of CTE-containing mRNA and promote its association with polyribosomes. © 2016 Li et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Insulin stimulates the expression of the SHARP-1 gene via multiple signaling pathways.

PubMed

Takagi, K; Asano, K; Haneishi, A; Ono, M; Komatsu, Y; Yamamoto, T; Tanaka, T; Ueno, H; Ogawa, W; Tomita, K; Noguchi, T; Yamada, K

2014-06-01

The rat enhancer of split- and hairy-related protein-1 (SHARP-1) is a basic helix-loop-helix transcription factor. An issue of whether SHARP-1 is an insulin-inducible transcription factor was examined. Insulin rapidly increased the level of SHARP-1 mRNA both in vivo and in vitro. Then, signaling pathways involved with the increase of SHARP-1 mRNA by insulin were determined in H4IIE rat hepatoma cells. Pretreatments with LY294002, wortmannin, and staurosporine completely blocked the induction effect, suggesting the involvement of both phosphoinositide 3-kinase (PI 3-K) and protein kinase C (PKC) pathways. In fact, overexpression of a dominant negative form of atypical protein kinase C lambda (aPKCλ) significantly decreased the induction of the SHARP-1 mRNA. In addition, inhibitors for the small GTPase Rac or Jun N-terminal kinase (JNK) also blocked the induction of SHARP-1 mRNA by insulin. Overexpression of a dominant negative form of Rac1 prevented the activation by insulin. Furthermore, actinomycin D and cycloheximide completely blocked the induction of SHARP-1 mRNA by insulin. Finally, when a SHARP-1 expression plasmid was transiently transfected with various reporter plasmids into H4IIE cells, the promoter activity of PEPCK reporter plasmid was specifically decreased. Thus, we conclude that insulin induces the SHARP-1 gene expression at the transcription level via a both PI 3-K/aPKCλ/JNK- and a PI 3-K/Rac/JNK-signaling pathway; protein synthesis is required for this induction; and that SHARP-1 is a potential repressor of the PEPCK gene expression. © Georg Thieme Verlag KG Stuttgart · New York.

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

The increased mucosal mRNA expressions of complement C3 and interleukin-17 in inflammatory bowel disease

PubMed Central

Sugihara, T; Kobori, A; Imaeda, H; Tsujikawa, T; Amagase, K; Takeuchi, K; Fujiyama, Y; Andoh, A

2010-01-01

Recent studies have demonstrated that the complement system participates in the regulation of T cell functions. To address the local biosynthesis of complement components in inflammatory bowel disease (IBD) mucosa, we investigated C3 and interleukin (IL)-17 mRNA expression in mucosal samples obtained from patients with IBD. The molecular mechanisms underlying C3 induction were investigated in human colonic subepithelial myofibroblasts (SEMFs). IL-17 and C3 mRNA expressions in the IBD mucosa were evaluated by real-time polymerase chain reaction. The C3 levels in the supernatant were determined by enzyme-linked immunosorbent assay. IL-17 and C3 mRNA expressions were elevated significantly in the active lesions from ulcerative colitis (UC) and Crohn's disease (CD) patients. There was a significant positive correlation between IL-17 and C3 mRNA expression in the IBD mucosa. IL-17 stimulated a dose- and time-dependent increase in C3 mRNA expression and C3 secretion in colonic SEMFs. The C3 molecules secreted by colonic SEMFs were a 115-kDa α-chain linked to a 70-kDa β-chain by disulphide bonds, which was identical to serum C3. The IL-17-induced C3 mRNA expression was blocked by p42/44 mitogen-activated protein kinase (MAPK) inhibitors (PD98059 and U0216) and a p38 MAPK inhibitor (SB203580). Furthermore, IL-17-induced C3 mRNA expression was inhibited by an adenovirus containing a stable mutant form of IκBα. C3 and IL-17 mRNA expressions are enhanced, with a strong correlation, in the inflamed mucosa of IBD patients. Part of these clinical findings was considered to be mediated by the colonic SEMF response to IL-17. PMID:20089077

Time-course of 5-HT(6) receptor mRNA expression during memory consolidation and amnesia.

PubMed

Huerta-Rivas, A; Pérez-García, G; González-Espinosa, C; Meneses, A

2010-01-01

Growing evidence indicates that antagonists of the 5-hydroxytryptamine (serotonin) receptor(6) (5-HT(6)) improve memory and reverse amnesia although the mechanisms involved are poorly understood. Hence, in this paper RT-PCR was used to evaluate changes in mRNA expression of 5-HT(6) receptor in trained and untrained rats treated with the 5-HT(6) receptor antagonist SB-399885 and amnesic drugs scopolamine or dizocilpine. Changes in mRNA expression of 5-HT(6) receptor were investigated at different times in prefrontal cortex, hippocampus and striatum. Data indicated that memory in the Pavlovian/instrumental autoshaping task was a progressive process associated to reduced mRNA expression of 5-HT(6) receptor in the three structures examined. SB-399885 improved long-term memory at 48h, while the muscarinic receptor antagonist scopolamine or the non-competitive NMDA receptor antagonist dizocilpine impaired it at 24h. Autoshaping training and treatment with SB-399885 increased 5-HT(6) receptor mRNA expression in (maximum increase) prefrontal cortex and striatum, 24 or 48h. The scopolamine-induced amnesia suppressed 5-HT(6) receptor mRNA expression while the dizocilpine-induced amnesia did not modify 5-HT(6) receptor mRNA expression. SB-399885 and scopolamine or dizocilpine were able to reestablish memory and 5-HT(6) receptor mRNA expression. These data confirmed previous memory evidence and of more interest is the observation that training, SB-399885 and amnesic drugs modulated 5-HT(6) receptor mRNA expression in prefrontal cortex, hippocampus and striatum. Further investigation in different memory tasks, times and amnesia models together with more complex control groups might provide further clues. Copyright 2009 Elsevier Inc. All rights reserved.

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.

Inactivation of parkin by promoter methylation correlated with lymph node metastasis and genomic instability in nasopharyngeal carcinoma.

PubMed

Ni, Haifeng; Zhou, Zhen; Jiang, Bo; Yuan, Xiaoyang; Cao, Xiaolin; Huang, Guangwu; Li, Yong

2017-03-01

This study aimed to investigate the inactivation of the parkin gene by promoter methylation and its relationship with genome instability in nasopharyngeal carcinoma. Parkin was considered as a tumor suppressor gene in various types of cancers. However, its role in nasopharyngeal carcinoma is unexplored. Genomic instabilities were detected in nasopharyngeal carcinoma tissues by the random amplified polymorphic DNA. The methylation-specific polymerase chain reaction, semi-quantitative reverse transcription polymerase chain reaction, and immunohistochemical analysis were used to detect methylation and mRNA and protein expression of parkin in 54 cases of nasopharyngeal carcinoma tissues and 16 cases of normal nasopharyngeal epithelia tissues, and in 5 nasopharyngeal carcinoma cell lines (CNE1, CNE2, TWO3, C666, and HONE1) and 1 normal nasopharyngeal epithelia cell line (NP69). mRNA expression of parkin in CNE1 and CNE2 was analyzed before and after methyltransferase inhibitor 5-aza-2-deoxycytidine treatment. The relationship between promoter methylation and mRNA expression, demethylation and mRNA expression, and mRNA and protein expression of the gene and clinical factors and genomic instabilities were analyzed. The mRNA and protein expression levels were significantly reduced in 54 cases of human nasopharyngeal carcinoma compared with 16 cases of normal nasopharyngeal epithelia. Parkin-methylated cases showed significantly lower mRNA and protein expression levels compared with unmethylated cases. After 5-aza-2-deoxycytidine treatment, parkin mRNA expression was restored in CNE1 and CNE2; 92.59% (50/54) of nasopharyngeal carcinoma demonstrated genomic instability. Parkin is frequently inactivated by promoter methylation, and its mRNA and protein expression correlate with lymph node metastasis and genomic instability. Parkin deficiency probably promotes tumorigenesis in nasopharyngeal carcinoma.

Profiles of mRNA expression of related genes in the duck hypothalamus-pituitary growth axis during embryonic and early post-hatch development.

PubMed

Hu, Yan; Liu, Hongxiang; Song, Chi; Xu, Wenjuan; Ji, Gaige; Zhu, Chunhong; Shu, Jingting; Li, Huifang

2015-03-15

In this study, the ontogeny of body and liver weight and the pattern of related gene mRNA expression in the hypothalamus-pituitary growth axis (HPGA) of two different duck breeds (Anas platyrhynchos domestica) were compared during embryonic and post-hatch development. Duck hypothalamic growth hormone release hormone (GHRH), somatostatin (SS), pituitary growth hormone (GH), liver growth hormone receptor (GHR) and insulin-like growth factor-I (IGF-1) mRNA were first detected on the 13th embryonic day. During early duck development, SS maintained a lower expression status, whereas the other four genes exhibited highly significant variations in an age-specific manner. Highly significant breed specificity was observed with respect to hepatic IGF-1 mRNA expression, which showed a significant breed-age interaction effect. Compared with previous studies on chickens, significant species differences were observed regarding the mRNA expression of bird embryonic HPGA-related genes. During early development, highly significant breed and age specificity were observed with respect to developmental changes in body and liver weight, and varying degrees of significant linear correlation were found between these performances and the mRNA expression of HPGA-related genes in the duck HPGA. These results suggest that different genetic backgrounds may lead to differences in duck growth and HPGA-related gene mRNA expression, and the differential mRNA expression of related genes in the duck HPGA may be particularly important in the early growth of ducks. Furthermore, hepatic IGF-1 mRNA expression presented highly significant breed specificity, and evidence suggests the involvement of hepatic IGF-1 in mediating genetic effects on embryo and offspring growth in ducks. Copyright © 2015 Elsevier B.V. All rights reserved.

Cloning of Russian sturgeon (Acipenser gueldenstaedtii) growth hormone and insulin-like growth factor I and their expression in male and female fish during the first period of growth.

PubMed

Yom Din, S; Hurvitz, A; Goldberg, D; Jackson, K; Levavi-Sivan, B; Degani, G

2008-03-01

In this study, the GH and IGF-I of the Russian sturgeon (rs), Acipenser gueldenstaedtii, were cloned and sequenced, and their mRNA gene expression determined. In addition, to improve our understanding of the GH function, the expression of this hormone was assessed in young males and females. Moreover, IGF-I expression was quantified in young males and compared to that in older ones. The nucleotide sequence of the rsGH cDNA was 980 bp long and had an open reading frame of 642 bp, beginning with the first ATG codon at position 39 and ending with the stop codon at position 683. A putative polyadenylation signal, AATAAA, was recognized 42 bp upstream of the poly (A) tail. The position of the signal- peptide cleavage site was predicted to be at position 111, yielding a signal peptide of 24 amino-acids (aa) and a mature peptide of 190 aa. When the rsGH aa sequence was compared with other species, the highest degree of identity was found to be with mammalians (66-70% identity), followed by anguilliformes and amphibia (61%) and other fish (39-47%). The level of rsGH mRNA was discovered to be similar in pituitaries of females and males of 5 age groups (1, 2, 3, 4, and 5- yr-old). In females and males, the levels did not change dramatically during the first 5 yr of growth. The partial nucleotide sequence of the rsIGF-I was 445 bp long and had an open reading frame of 396 bp, beginning with the ATG codon at position 50. The position of the signal-peptide cleavage site was predicted to be at position 187, yielding a signal peptide of 44 aa. The highest level of IGF-I mRNA expression was recorded in the kidney of adult sturgeons. The IGF-I mRNA expression levels in the intestine, pituitary gland, and liver were not significantly different. Low levels of expression were found in the brain, heart, and muscle. In most tissues, there was no significant difference between mRNA levels of one and 5-yr-old fish. In conclusion, based on the GH-sequence analysis, A. gueldenstaedtii is genetically distant from other teleosts. The expression of the GH mRNA was similar in males and females, and its level remained constant during the first 5 yr of growth. While the IGF-I mRNA expression differed amongst various tissues, the level in each tissue was similar in 1 and 5-yr-old fish.

Extent, Causes, and Consequences of Small RNA Expression Variation in Human Adipose Tissue

PubMed Central

Knights, Andrew J.; Abreu-Goodger, Cei; van de Bunt, Martijn; Guerra-Assunção, José Afonso; Bartonicek, Nenad; van Dongen, Stijn; Mägi, Reedik; Nisbet, James; Barrett, Amy; Rantalainen, Mattias; Nica, Alexandra C.; Quail, Michael A.; Small, Kerrin S.; Glass, Daniel; Enright, Anton J.; Winn, John; Deloukas, Panos; Dermitzakis, Emmanouil T.; McCarthy, Mark I.; Spector, Timothy D.; Durbin, Richard; Lindgren, Cecilia M.

2012-01-01

Small RNAs are functional molecules that modulate mRNA transcripts and have been implicated in the aetiology of several common diseases. However, little is known about the extent of their variability within the human population. Here, we characterise the extent, causes, and effects of naturally occurring variation in expression and sequence of small RNAs from adipose tissue in relation to genotype, gene expression, and metabolic traits in the MuTHER reference cohort. We profiled the expression of 15 to 30 base pair RNA molecules in subcutaneous adipose tissue from 131 individuals using high-throughput sequencing, and quantified levels of 591 microRNAs and small nucleolar RNAs. We identified three genetic variants and three RNA editing events. Highly expressed small RNAs are more conserved within mammals than average, as are those with highly variable expression. We identified 14 genetic loci significantly associated with nearby small RNA expression levels, seven of which also regulate an mRNA transcript level in the same region. In addition, these loci are enriched for variants significant in genome-wide association studies for body mass index. Contrary to expectation, we found no evidence for negative correlation between expression level of a microRNA and its target mRNAs. Trunk fat mass, body mass index, and fasting insulin were associated with more than twenty small RNA expression levels each, while fasting glucose had no significant associations. This study highlights the similar genetic complexity and shared genetic control of small RNA and mRNA transcripts, and gives a quantitative picture of small RNA expression variation in the human population. PMID:22589741

Single step production of Cas9 mRNA for zygote injection.

PubMed

Redel, Bethany K; Beaton, Benjamin P; Spate, Lee D; Benne, Joshua A; Murphy, Stephanie L; O'Gorman, Chad W; Spate, Anna M; Prather, Randall S; Wells, Kevin D

2018-03-01

Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

p53, Bcl-2 and cox-2 are involved in berberine hydrochloride-induced apoptosis of HeLa229 cells.

PubMed

Wang, Hai-Yan; Yu, Hai-Zhong; Huang, Sheng-Mou; Zheng, Yu-Lan

2016-10-01

The present study aimed to investigate the effects of berberine hydrochloride on the proliferation and apoptosis of HeLa229 human cervical cancer cells. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to examine the cytotoxicity of berberine hydrochloride against HeLa229 cells. The effects of berberine hydrochloride on the apoptosis of HeLa229 cells was detected by immunofluorescence and flow cytometry, and the mRNA expression levels of p53, B‑cell lymphoma 2 (Bcl‑2) and cyclooxygenase‑2 (cox‑2) were analyzed by reverse transcription-quantitative polymerase chain reaction. Berberine hydrochloride inhibited the proliferation of HeLa229 cells in a dose‑dependent manner; minimum cell viability (3.61%) was detected following treatment with 215.164 µmol/l berberine hydrochloride and the half maximal inhibitory concentration value was 42.93 µmol/l following treatment for 72 h. In addition, berberine hydrochloride induced apoptosis in HeLa229 cells in a dose‑ and time‑dependent manner. Berberine hydrochloride upregulated the mRNA expression levels of p53, and downregulated mRNA expression levels of Bcl‑2 and cox‑2, in a dose‑dependent manner. In conclusion, berberine hydrochloride inhibited the proliferation and induced apoptosis of HeLa229 cells, potentially via the upregulation of p53 and the downregulation of Bcl‑2 and cox‑2 mRNA expression levels.

Imbalance of tumor necrosis factor receptors during progression in bovine leukemia virus infection

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

Konnai, Satoru; Usui, Tatsufumi; Ikeda, Manabu

2005-09-01

Previously, we found an up-regulation of tumor necrosis factor alpha (TNF)-{alpha} and an imbalance of TNF receptors in sheep experimentally infected with bovine leukemia virus (BLV). In order to investigate the different TNF-{alpha}-induced responses, in this study we examined the TNF-{alpha}-induced proliferative response and the expression levels of two distinct TNF receptors on peripheral blood mononuclear cells (PBMC) derived from BLV-uninfected cattle and BLV-infected cattle that were aleukemic (AL) or had persistent lymphocytosis (PL). The proliferative response of PBMC isolated from those cattle with PL in the presence of recombinant bovine TNF-{alpha} (rTNF-{alpha}) was significantly higher than those from ALmore » cattle and uninfected cattle and the cells from PL cattle expressed significantly higher mRNA levels of TNF receptor type II (TNF-RII) than those from AL and BLV-uninfected cattle. No difference was found in TNF-RI mRNA levels. Most cells expressing TNF-RII in PL cattle were CD5{sup +} or sIgM{sup +} cells and these cells showed resistance to TNF-{alpha}-induced apoptosis. Additionally, there were significant positive correlations between the changes in provirus load and TNF-RII mRNA levels, and TNF-{alpha}-induced proliferation and TNF-RII mRNA levels. These data suggest that imbalance in the expression of TNF receptors could at least in part contribute to the progression of lymphocytosis in BLV infection.« less

Diabetes Diminishes the Portal-Systemic Collateral Vascular Response to Vasopressin via Vasopressin Receptor and Gα Proteins Regulations in Cirrhotic Rats

PubMed Central

Lee, Jing-Yi; Huo, Teh-Ia; Wang, Sun-Sang; Lin, Han-Chieh; Chuang, Chiao-Lin; Lee, Shou-Dong

2013-01-01

Liver cirrhosis may lead to portal-systemic collateral formation and bleeding. The hemostatic effect is influenced by the response of collateral vessels to vasoconstrictors. Diabetes and glucose also influence vasoresponsiveness, but their net effect on collaterals remains unexplored. This study investigated the impact of diabetes or glucose application on portal-systemic collateral vasoresponsiveness to arginine vasopressin (AVP) in cirrhosis. Spraque-Dawley rats with bile duct ligation (BDL)-induced cirrhosis received vehicle (citrate buffer) or streptozotocin (diabetic, BDL/STZ). The in situ collateral perfusion was done after hemodynamic measurements: Both were perfused with Krebs solution, D-glucose, or D-glucose and NaF, with additional OPC-31260 for the BDL/STZ group. Splenorenal shunt vasopressin receptors and Gα proteins mRNA expressions were evaluated. The survival rate of cirrhotic rats was decreased by STZ injection. The collateral perfusion pressure changes to AVP were lower in STZ-injected groups, which were reversed by OPC-31260 (a V2R antagonist) and overcome by NaF (a G protein activator). The splenorenal shunt V2R mRNA expression was increased while Gα proteins mRNA expressions were decreased in BDL/STZ rats compared to BDL rats. The Gαq and Gα11 mRNA expressions also correlated with the maximal perfusion pressure changes to AVP. Diabetes diminished the portal-systemic collateral vascular response to AVP in rats with BDL-induced cirrhosis, probably via V2 receptor up-regulation and Gα proteins down-regulation. PMID:23874439

Diabetes diminishes the portal-systemic collateral vascular response to vasopressin via vasopressin receptor and Gα proteins regulations in cirrhotic rats.

PubMed

Lee, Jing-Yi; Huo, Teh-Ia; Wang, Sun-Sang; Huang, Hui-Chun; Lee, Fa-Yauh; Lin, Han-Chieh; Chuang, Chiao-Lin; Lee, Shou-Dong

2013-01-01

Liver cirrhosis may lead to portal-systemic collateral formation and bleeding. The hemostatic effect is influenced by the response of collateral vessels to vasoconstrictors. Diabetes and glucose also influence vasoresponsiveness, but their net effect on collaterals remains unexplored. This study investigated the impact of diabetes or glucose application on portal-systemic collateral vasoresponsiveness to arginine vasopressin (AVP) in cirrhosis. Spraque-Dawley rats with bile duct ligation (BDL)-induced cirrhosis received vehicle (citrate buffer) or streptozotocin (diabetic, BDL/STZ). The in situ collateral perfusion was done after hemodynamic measurements: Both were perfused with Krebs solution, D-glucose, or D-glucose and NaF, with additional OPC-31260 for the BDL/STZ group. Splenorenal shunt vasopressin receptors and Gα proteins mRNA expressions were evaluated. The survival rate of cirrhotic rats was decreased by STZ injection. The collateral perfusion pressure changes to AVP were lower in STZ-injected groups, which were reversed by OPC-31260 (a V2R antagonist) and overcome by NaF (a G protein activator). The splenorenal shunt V2R mRNA expression was increased while Gα proteins mRNA expressions were decreased in BDL/STZ rats compared to BDL rats. The Gαq and Gα11 mRNA expressions also correlated with the maximal perfusion pressure changes to AVP. Diabetes diminished the portal-systemic collateral vascular response to AVP in rats with BDL-induced cirrhosis, probably via V2 receptor up-regulation and Gα proteins down-regulation.

The TAM-family receptor Mer mediates production of HGF through the RhoA-dependent pathway in response to apoptotic cells.

PubMed

Park, Hyun-Jung; Baen, Ji-Yeon; Lee, Ye-Ji; Choi, Youn-Hee; Kang, Jihee Lee

2012-08-01

The TAM receptor protein tyrosine kinases Tyro3, Axl, and Mer play important roles in macrophage function. We investigated the roles of the TAM receptors in mediating the induction of hepatocyte growth factor (HGF) during the interaction of macrophages with apoptotic cells. Mer-specific neutralizing antibody, small interfering RNA (siRNA), and a recombinant Mer protein (Mer/Fc) inhibited HGF mRNA and protein expression, as well as activation of RhoA, Akt, and specific mitogen-activated protein (MAP) kinases in response to apoptotic cells. Inhibition of Axl or Tyro3 with specific antibodies, siRNA, or Fc-fusion proteins did not prevent apoptotic cell-induced HGF mRNA and protein expression and did not inhibit activation of the postreceptor signaling molecules RhoA and certain MAP kinases, including extracellular signal-regulated protein kinase and c-Jun NH(2)-terminal kinase. However, Axl- and Tyro3-specific blockers did inhibit the activation of Akt and p38 MAP kinase in response to apoptotic cells. In addition, none of the TAM receptors mediated the effects of apoptotic cells on transforming growth factor-β or epidermal growth factor mRNA expression. However, they were involved in the induction of vascular endothelial growth factor mRNA expression. Our data provide evidence that when macrophages interact with apoptotic cells, only Mer of the TAM-family receptors is responsible for mediating transcriptional HGF production through a RhoA-dependent pathway.

Changes in FKBP5 expression and memory functions during cognitive-behavioral therapy in posttraumatic stress disorder: a preliminary study.

PubMed

Szabó, Csilla; Kelemen, Oguz; Kéri, Szabolcs

2014-05-21

Posttraumatic stress disorder (PTSD) is characterized by hyperarousal, flashbacks, avoidance, and memory dysfunctions. Although psychotherapy improves the clinical symptoms, its effect on memory has not been explored. In addition, there is no information about gene expression changes related to hippocampal functions. We assessed PTSD patients (n=20) using the Wechsler Memory Scale-Revised (WAIS-R) and a paired associates learning (PAL) test, as well as changes in blood FK506 binding protein (FKBP5) mRNA expression before and after cognitive behavioral therapy (CBT). Results revealed that before CBT PTSD patients were impaired on WAIS-R delayed recall, attention/concentration, and PAL compared with trauma-exposed control subjects (n=20). These memory dysfunctions showed a significant improvement after CBT. Better performance on the PAL test correlated with enhanced blood FKBP5 mRNA expression. These results suggest that elevated FKBP5 expression during CBT is related to improved associative memory linked to the hippocampal formation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Molecular changes associated with heat-shock treatment in avian mononuclear and lymphoid lineage cells.

PubMed

Miller, L; Qureshi, M A

1992-03-01

The induction of heat-shock protein (HSP) synthesis in avian cells of the mononuclear phagocytic system (MPS) and lymphoid system (LS) lineage was investigated by exposure to in vitro heat-shock conditions. In addition, the kinetics of HSP90 mRNA expression was examined in chicken peritoneal macrophages (PM) as well as heat-shock-induced HSP synthesis in PM from chickens, turkeys, quail, and ducks. Each MPS and LS cell type expressed three major (23, 70, and 90 kDa) HSP following a 1-h heat shock at 45 C. However, a unique heat-induced 32-kDa protein (P32) was expressed only by cells of MPS lineage. The expression of HSP90 mRNA in chicken PM was temperature- and time-dependent. These findings imply that avian PM undergo molecular changes in response to elevated environmental temperatures and that the pattern of HSP expression appears to be distinct for cells of the MPS and LS lineages in chickens.

Expression of hypoxia-inducible factor 1α mRNA in hearts and lungs of broiler chickens with ascites syndrome induced by excess salt in drinking water.

PubMed

Zhang, Jianjun; Feng, Xuejian; Zhao, Lihong; Wang, Wei; Gao, Mingyu; Wu, Boning; Qiao, Jian

2013-08-01

Hypoxia-inducible factor 1 (HIF-1) is a ubiquitously expressed heterodimeric transcription factor that mediates adaptive responses to hypoxia in all nucleated cells of metazoan organisms. Hypoxia-inducible factor 1α is involved in the pathogenesis of pulmonary hypertension in humans and animals, but whether HIF-1α is associated with the development of pulmonary hypertension syndrome (also known as ascites syndrome, AS) in broiler chickens has not been determined. In the present paper we addressed this issue by measuring the expression of HIF-1α mRNA in hearts and lungs of broiler chickens with AS induced by excess salt in drinking water. We conducted 2 experiments. The first experiment was used to observe the effects of excess salt on AS incidence. The results indicated that total incidence (20%) of AS in excess salt group (receiving 0.3% NaCl in drinking water) was much higher compared with the control group (receiving tap water) over a 43-d time course (P < 0.05). In the second experiment, we determined mean pulmonary arterial pressure (mPAP), ascites heart index (AHI), and expression of HIF-1α mRNA in lungs and hearts of broiler chickens after the excess salt treatment. Our results showed that excess salt induced pulmonary hypertension (indicated by higher mPAP) and right ventricular hypertrophy (greater ascites heart index) in broiler chickens. Meanwhile, the expression levels of HIF-1α mRNA in lungs and hearts were significantly increased at different time points in the excess salt group compared with the control group. Linear correlation analysis showed that the expression of HIF-1α mRNA in lungs was significantly positively correlated with mPAP (correlation coefficient = 0.79, P < 0.001), demonstrating that expression of HIF-1α mRNA was gradually increased in the excess salt group with the increase of pulmonary arterial pressure. In addition, the ascitic chickens showed significantly higher transcriptional levels of HIF-1α in hearts and lungs, compared with the age-matched healthy chickens, respectively. Our findings hinted that HIF-1α might be associated with the development of AS induced by excess salt in drinking water in broiler chickens.

Adaptative decrease in expression of the mRNA for uncoupling protein and subunit II of cytochrome c oxidase in rat brown adipose tissue during pregnancy and lactation.

PubMed Central

Martin, I; Giralt, M; Viñas, O; Iglesias, R; Mampel, T; Villarroya, F

1989-01-01

Uncoupling-protein (UCP) mRNA expression is decreased to 15% of virgin control levels between days 10 and 15 of pregnancy, and remains at these low values during late pregnancy and lactation. Abrupt weaning of mid-lactating rats causes a slight but significant increase in UCP mRNA. Expression of mRNA for subunit II of cytochrome c oxidase (COII) decreased to half that of virgin control in late pregnancy and during lactation. Whereas COII mRNA expression is in step with the known modifications of brown-fat mitochondria content during the breeding cycle of the rat, UCP mRNA expression appears to be diminished much earlier than the mitochondrial proton-conductance-pathway activity. On the other hand, the reactivity of brown fat to increase expression of UCP and COII mRNAs in response to acute cold or noradrenaline treatment is not impaired during lactation. Images Fig. 1. Fig. 2. Fig. 3. PMID:2557014

Rapid Induction of Hypothalamic Iodothyronine Deiodinase Expression by Photoperiod and Melatonin in Juvenile Siberian Hamsters (Phodopus sungorus)

PubMed Central

Prendergast, Brian J.; Pyter, Leah M.; Kampf-Lassin, August; Patel, Priyesh N.

2013-01-01

Production of T3 in the mediobasal hypothalamus is critical for regulation of seasonal reproductive physiology. Type 2 iodothyronine deiodinase (DIO2) and DIO3 enzymes catalyze the prohormone T4 into biologically-active T3 and biologically-inactive rT3, respectively. In several seasonally-breeding vertebrates, DIO2 and DIO3 expression is implicated in photoperiod signal transduction in adulthood. These experiments tested the hypothesis that juvenile Siberian hamsters, which are highly responsive to photoperiod at weaning (postnatal day [PND]18), exhibit rapid and sustained changes in hypothalamic dio3 mRNA expression during photoperiod-induced and photoperiod-inhibited puberty. Hypothalamic dio2 and dio3 expression was measured via quantitative PCR in hamsters born and reared in a long-day photoperiod (15L:9D) and weaned on PND18 into short-day photoperiods (9L:15D). In SD males, hypothalamic dio3 mRNA was elevated 2.5-fold within 3 days (PND21) and continued to increase (>20-fold) through PND32; changes in dio3 mRNA preceded inhibition of gonadotropin (FSH) secretion and gonadal regression in SD. Females exhibited comparable dio3 responses to SD. In LD males, dio3 remained low and invariant from PND18–PND32. In contrast, dio2 mRNA rose conspicuously on PND21, independent of photoperiod, returning to basal levels thereafter. In LD, a single afternoon melatonin (MEL) injection on PND18 or PND20 was sufficient to increase hypothalamic dio3 mRNA, and dio3 increased in proportion to the number of successive days of MEL treatment. SD photoperiods and MEL exert rapid, sustained, and additive effects on hypothalamic dio3 mRNA, which may play a central role in inhibiting maturation of the peripubertal hypothalamo-pituitary-gonadal axis. PMID:23295738

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.

Identification of Differentially Expressed K-Ras Transcript Variants in Patients With Leiomyoma.

PubMed

Zolfaghari, Nooshin; Shahbazi, Shirin; Torfeh, Mahnaz; Khorasani, Maryam; Hashemi, Mehrdad; Mahdian, Reza

2017-10-01

Molecular studies have demonstrated a wide range of gene expression variations in uterine leiomyoma. The rat sarcoma virus/rapidly accelerated fibrosarcoma/mitogen-activated protein kinase (RAS/RAF/MAPK) is the crucial cellular pathway in transmitting external signals into nucleus. Deregulation of this pathway contributes to excessive cell proliferation and tumorigenesis. The present study aims to investigate the expression profile of the K-Ras transcripts in tissue samples from patients with leiomyoma. The patients were leiomyoma cases who had no mutation in mediator complex subunit 12 ( MED12) gene. A quantitative approach has been applied to determine the difference in the expression of the 2 main K-Ras messenger RNA (mRNA) variants. The comparison between gene expression levels in leiomyoma and normal myometrium group was performed using relative expression software tool. The expression of K-Ras4B gene was upregulated in leiomyoma group ( P = .016), suggesting the involvement of K-Ras4B in the disease pathogenesis. Pairwise comparison of the K-Ras4B expression between each leiomyoma tissue and its matched adjacent normal myometrium revealed gene upregulation in 68% of the cases. The expression of K-Ras4A mRNA was relatively upregulated in leiomyoma group ( P = .030). In addition, the mean expression of K-Ras4A gene in leiomyoma tissues relative to normal samples was 4.475 (95% confidence interval: 0.10-20.42; standard error: 0.53-12.67). In total, 58% of the cases showed more than 2-fold increase in K-Ras4A gene expression. Our results demonstrated increased expression of both K-Ras mRNA splicing variants in leiomyoma tissue. However, the ultimate result of KRAS expression on leiomyoma development depends on the overall KRAS isoform balance and, consequently, on activated signaling pathways.

Dietary arginine supplementation alleviates intestinal mucosal disruption induced by Escherichia coli lipopolysaccharide in weaned pigs.

PubMed

Liu, Yulan; Huang, Jingjing; Hou, Yongqing; Zhu, Huiling; Zhao, Shengjun; Ding, Binying; Yin, Yulong; Yi, Ganfeng; Shi, Junxia; Fan, Wei

2008-09-01

This study evaluated whether arginine (Arg) supplementation could attenuate gut injury induced by Escherichia coli lipopolysaccharide (LPS) challenge through an anti-inflammatory role in weaned pigs. Pigs were allotted to four treatments including: (1) non-challenged control; (2) LPS-challenged control; (3) LPS+0.5 % Arg; (4) LPS+1.0 % Arg. On day 16, pigs were injected with LPS or sterile saline. At 6 h post-injection, pigs were killed for evaluation of small intestinal morphology and intestinal gene expression. Within 48 h of challenge, 0.5 % Arg alleviated the weight loss induced by LPS challenge (P = 0.025). In all three intestinal segments, 0.5 or 1.0 % Arg mitigated intestinal morphology impairment (e.g. lower villus height and higher crypt depth) induced by LPS challenge (P < 0.05), and alleviated the decrease of crypt cell proliferation and the increase of villus cell apoptosis after LPS challenge (P < 0.01). The 0.5 % Arg prevented the elevation of jejunal IL-6 mRNA abundance (P = 0.082), and jejunal (P = 0.030) and ileal (P = 0.039) TNF-alpha mRNA abundance induced by LPS challenge. The 1.0 % Arg alleviated the elevation of jejunal IL-6 mRNA abundance (P = 0.053) and jejunal TNF-alpha mRNA abundance (P = 0.003) induced by LPS challenge. The 0.5 % Arg increased PPARgamma mRNA abundance in all three intestinal segments (P < 0.10), and 1.0 % Arg increased duodenal PPARgamma mRNA abundance (P = 0.094). These results indicate that Arg supplementation has beneficial effects in alleviating gut mucosal injury induced by LPS challenge. Additionally, it is possible that the protective effects of Arg on the intestine are associated with decreasing the expression of intestinal pro-inflammatory cytokines through activating PPARgamma expression.

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.

Decreased Expression of Arginine-Phenylalanine-Amide-Related Peptide-3 Gene in Dorsomedial Hypothalamic Nucleus of Constant Light Exposure Model of Polycystic Ovarian Syndrome

PubMed Central

Shaaban, Zahra; Jafarzadeh Shirazi, Mohammad Reza; Nooranizadeh, Mohammad Hossein; Tamadon, Amin; Rahmanifar, Farhad; Ahmadloo, Somayeh; Ramezani, Amin; Zamiri, Mohammad Javad; Razeghian Jahromi, Iman; Sabet Sarvestani, Fatemeh; Hosseinabadi, Omid Koohi

2018-01-01

Background An abnormality in pulse amplitude and frequency of gonadotropin releasing hormone (GnRH) secretion is the most characteristics of polycystic ovarian syndrome (PCOS). On the other hand, arginine-phenylalanine-amide (RFamide)-related peptide-3 (RFRP3) inhibits the secretion of GnRH in mammalian hypothalamus. The current study performed in order to investigate the expression of RFRP3 mRNA in the dorsomedial hypothalamic nucleus (DMH) after the induction of PCOS in a rat model of constant light exposure, and the possible role of parity on occurrence of PCOS. Materials and Methods In the experimental study, female nulliparous (n=12) and primiparous (n=12) rats were randomly subdivided into control and PCOS subgroups (n=6). PCOS were induced by 90 days exposure to constant light. After 90 days, blood, brain, and ovaries were sampled. Serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone were evaluated. In addition, six adult female ovariectomized rats as a control of real-time polymerase chain reaction (PCR) tests were prepared and in the DMH of all rats, the relative mRNA expression of RFRP3 was assessed. Results Histological evaluation of ovaries represented the polycystic features. In addition, serum concentrations of testosterone in the PCOS subgroups were more than the controls (P<0.05). Furthermore, the relative expression of RFRP3 mRNA in PCOS subgroups was lower than the controls (P<0.05). Conclusion Constant light model of the PCOS-induced rats decreased the gene expression of RFRP3 in the DMH that suggests the decrease of RFRP3 may reduce its inhibitory effect on GnRH during the PCOS pathogenesis. This effect was stronger in the nulliparous rats than the primiparous. PMID:29334206

Low-level lasers on microRNA and uncoupling protein 2 mRNA levels in human breast cancer cells

NASA Astrophysics Data System (ADS)

Canuto, K. S.; Teixeira, A. F.; Rodrigues, J. A.; Paoli, F.; Nogueira, E. M.; Mencalha, A. L.; Fonseca, A. S.

2017-06-01

MicroRNA is short non-coding RNA and is a mediator of post-transcriptional regulation of gene expression. In addition, uncoupling proteins (UCPs) regulate thermogenesis, metabolic and energy balance, and decrease reactive oxygen species production. Both microRNA and UCP2 expression can be altered in cancer cells. At low power, laser wavelength, frequency, fluence and emission mode deternube photobiological responses, which are the basis of low-level laser therapy. There are few studies on miRNA and UCP mRNA levels after low-level laser exposure on cancer cells. In this work, we evaluate the micrRNA (mir-106b and mir-15a) and UCP2 mRNA levels in human breast cancer cells exposed to low-level lasers. MDA-MB-231 human breast cancer cells were exposed to low-level red and infrared lasers, total RNA was extracted for cDNA synthesis and mRNA levels by real time quantitative polymerase chain reaction were evaluated. Data show that mir-106b and mir-15a relative levels are not altered, but UCP2 mRNA relative levels are increased in MDA-MB-231 human breast cancer cells exposed to low-level red and infrared lasers at fluences used in therapeutic protocols.

Levetiracetam attenuates hippocampal expression of synaptic plasticity-related immediate early and late response genes in amygdala-kindled rats

PubMed Central

2010-01-01

Background The amygdala-kindled rat is a model for human temporal lobe epilepsy and activity-dependent synaptic plasticity. Hippocampal RNA isolated from amygdala-kindled rats at different kindling stages was analyzed to identify kindling-induced genes. Furthermore, effects of the anti-epileptic drug levetiracetam on kindling-induced gene expression were examined. Results Cyclooxygenase-2 (Cox-2), Protocadherin-8 (Pcdh8) and TGF-beta-inducible early response gene-1 (TIEG1) were identified and verified as differentially expressed transcripts in the hippocampus of kindled rats by in situ hybridization and quantitative RT-PCR. In addition, we identified a panel of 16 additional transcripts which included Arc, Egr3/Pilot, Homer1a, Ania-3, MMP9, Narp, c-fos, NGF, BDNF, NT-3, Synaptopodin, Pim1 kinase, TNF-α, RGS2, Egr2/krox-20 and β-A activin that were differentially expressed in the hippocampus of amygdala-kindled rats. The list consists of many synaptic plasticity-related immediate early genes (IEGs) as well as some late response genes encoding transcription factors, neurotrophic factors and proteins that are known to regulate synaptic remodelling. In the hippocampus, induction of IEG expression was dependent on the afterdischarge (AD) duration. Levetiracetam, 40 mg/kg, suppressed the development of kindling measured as severity of seizures and AD duration. In addition, single animal profiling also showed that levetiracetam attenuated the observed kindling-induced IEG expression; an effect that paralleled the anti-epileptic effect of the drug on AD duration. Conclusions The present study provides mRNA expression data that suggest that levetiracetam attenuates expression of genes known to regulate synaptic remodelling. In the kindled rat, levetiracetam does so by shortening the AD duration thereby reducing the seizure-induced changes in mRNA expression in the hippocampus. PMID:20105316

Effects of nitrite stress on mRNA expression of antioxidant enzymes, immune-related genes and apoptosis-related proteins in Marsupenaeus japonicus.

PubMed

Zheng, Jinbin; Mao, Yong; Su, Yongquan; Wang, Jun

2016-11-01

Nitrite accumulation in aquaculture systems is a potential risk factor that may trigger stress responses in aquatic organisms. However, the mechanisms regulating the responses of shrimp to nitrite stress remain unclear. In this study, full-length cDNA sequences of two apoptosis-related genes, caspase-3 and defender against apoptotic death (DAD-1), were cloned from Marsupenaeus japonicus for the first time, and their expression levels and tissue distribution were analyzed by quantitative real-time PCR (qRT-PCR). The full lengths of Mjcaspase-3 and MjDAD-1 were 1203 bp and 640 bp respectively, with deduced amino acid (AA) sequences of 321 and 114 AA. Mjcaspase-3 was predominantly expressed in haemocytes and weakly expressed in the seven other tissues tested. MjDAD-1 was mainly expressed in the defense and digestive tissues, especially in the hepatopancreas and hemocytes. To explore the influence of nitrite stress on the genetic response of antioxidant enzymes, immune-related genes and apoptosis-related proteins, the mRNA expression profiles of MjCAT, MjMnSOD, Mj-ilys, Mj-sty, Mjcaspase-3 and MjDAD-1 in response to nitrite stress were analyzed by qRT-PCR. The mRNA levels of MjCAT, MjMnSOD, Mj-ilys, Mj-sty, Mjcaspase-3 and MjDAD-1 show both time- and dose-dependent changes in response to nitrite stress. The mRNA expression levels of MjCAT and MjSOD peaked at 6 h for all nitrite concentrations tested (p < 0.05) and the up-regulated of MjCAT and MjSOD exhibited a positive correlation with the nitrite concentration. The mRNA expression levels of Mj-ilys and Mj-sty gradually decreased during the experiment period. Mjcaspase-3 mRNA level reached a maximum at 6 h (p < 0.05), and MjDAD-1 reached its peak at 12 h and 48 h in 10 mg/L and 20 mg/L nitrite, respectively. In addition, CAT and SOD activity showed changes in response to nitrite stress that mirrored the induced expression of MjCAT and MjMnSOD, and prolonged nitrite exposure reduced the activity of CAT. This study provided basic data for further elucidating the responses of shrimp to nitrite stress at the molecular level. Copyright © 2016. Published by Elsevier Ltd.

THE M-RNA, EXPRESSION OF SERCA2 AND NCX1 IN THE PROCESS OF PHARMACOLOGICAL CELL PROTECTION IN EXPERIMENTAL ACUTE PANCREATITIS INDUCED BY TAUROCHOLATE.

PubMed

Vasques, Enio Rodrigues; Cunha, José Eduardo Monteiro; Kubrusly, Marcia Saldanha; Coelho, Ana Maria; Sanpietri, Sandra N; Nader, Helena B; Tersariol, Ivarne L S; Lima, Marcelo A; Chaib, Eleazar; D'Albuquerque, Luiz Augusto Carneiro

2018-06-21

Intracellular calcium overload is known to be a precipitating factor of pancreatic cell injury in acute pancreatitis (AP). Intracellular calcium homeostasis depends of Plasmatic Membrane Calcium ATPase (PMCA), Sarcoplasmic Endothelial Reticulum Calcium ATPase 2 (SERCA 2) and the Sodium Calcium Exchanger (NCX1). The antioxidant melatonin (Mel) and Trisulfate Disaccharide (TD) that accelerates NCX1 action could reduce the cell damage determined by the AP. To evaluate m-RNA expressions of SERCA2 and NCX1 in acute pancreatitis induced by sodium taurocholate in Wistar rats pre-treated with melatonin and/or TD. Wistar rats were divided in groups: 1) without AP; 2) AP without pre-treatment; 3) AP and Melatonin; 4) AP and TD; 5) AP and Melatonin associated to TD. Pancreatic tissue samples were collected for detection of SERCA2 and NCX1 m-R NA levels by polymerase chain reaction (PCR). Increased m-RNA expression of SERCA2 in the melatonin treated group, without increase of m-RNA expression of the NCX1. The TD did not affect levels of SERCA2 and NCX1 m-RNA expressions. The combined melatonin and TD treatment reduced the m-RNA expression of SERCA2. The effect of melatonin is restricted to increased m-RNA expression of SERCA2. Although TD does not affect gene expression, its action in accelerating calcium exchanger function can explain the slightest expression of SERCA2 m-RNA when associated with Melatonin, perhaps by a joint action of drugs with different and but possibly complementary mechanisms.

Neuropeptide gene expression in brain is differentially regulated by midbrain dopamine neurons.

PubMed

Lindefors, N; Brené, S; Herrera-Marschitz, M; Persson, H

1990-01-01

In situ hybridization was used to study the expression of prepro-neuropeptide Y (NPY), preprosomatostatin (SOM), preprotachykinin (PPT) and preprocholecystokinin (CCK) mRNA in caudate-putamen and frontoparietal cortex of rat brain with unilateral lesion of midbrain dopamine neurons. Neurons expressing NPY and SOM mRNA showed a similar distribution and the expression of both NPY and SOM appears to be regulated by dopamine in a similar fashion. Following a dopamine deafferentation, the numerical density of both NPY and SOM mRNA producing neurons almost doubled in the lesioned caudate-putamen with no change in the average grain density over positive neurons. Hence, in the intact caudate-putamen dopamine appears to suppress expression of these two neuropeptide genes leading to an activation of both NPY and SOM mRNA expression in many non- or low-expressing neurons when the level of dopamine is decreased. In the fronto-parietal cortex, on the other hand, dopamine appears to stimulate NPY and SOM gene expression. Thus, in the absence of dopamine about half of the NPY positive neurons disappeared. However, for SOM the number of positive neurons did not change, but rather most positive neurons appeared to have down-regulated their SOM mRNA expression. No evidence was found for a change in CCK mRNA expression by the dopamine deafferentation, while PPT mRNA expression decreased in the deafferented caudate-putamen. Consequently, dopamine exerts dissimilar effects on the expression of different neuropeptide genes, that in turn do not respond in the same way in different brain regions.

Tissue-specific changes in OGG1 and SOD mRNA expression caused by NaOCl exposure in black seabream ( Acanthopagrus schlegelii)

NASA Astrophysics Data System (ADS)

Park, Ho-Ra; Kim, Yong; Yeo, Won-Jun; Kim, Ji-Hye; Han, Kyung-Nam

2017-09-01

The DNA-damage defense mechanism was studied in black seabreams after oxidative stress caused by exposure to sodium hypochlorite (NaOCl). Liver, muscle, and brain tissues were obtained after different NaOCl-exposure times (0, 24, 48, 72, and 96 h) and concentrations (0.5, 1, 1.5, 2, and 3 mg/L), after which oxoguanine glycosylase (OGG1) and superoxide dismutase (SOD) mRNA-expression levels were analyzed. At all NaOCl concentrations tested, liver OGG1 expression increased to a maximum in a time-dependent manner after NaOCl exposure and then decreased. In muscles, OGG1 expression increased over time following exposure to a low concentration of NaOCl (0.5, 1, and 1.5 mg/L), whereas it showed a mixed pattern (both increases and decreases observed) in the high-concentration groups (2 and 3 mg/L). SOD mRNA expression increased over time, both in the liver and muscles. In the brain, both OGG1 and SOD mRNA expression levels were highest after exposure to the lowest NaOCl concentration (0.5 mg/L), whereas basal levels were maintained over time at higher concentrations. These results indicate that OGG1 and SOD provide resistance to oxidative stress in black seabreams. In addition, continuous exposure to oxidative stress can suppress enzyme expression, suggesting a risk for long-term exposure to NaOCl.

MEK-dependent IL-8 induction regulates the invasiveness of triple-negative breast cancer cells.

PubMed

Kim, Sangmin; Lee, Jeongmin; Jeon, Myeongjin; Lee, Jeong Eon; Nam, Seok Jin

2016-04-01

Interleukin-8 (IL-8) serves as a prognostic marker for breast cancer, and its expression level correlates with metastatic breast cancer and poor prognosis. Here, we investigated the levels of IL-8 expression in a variety of breast cancer cells and the regulatory mechanism of IL-8 in triple-negative breast cancer (TNBC) cells. Our results showed that IL-8 expression correlated positively with overall survival in basal-type breast cancer patients. The levels of IL-8 mRNA expression and protein secretion were significantly increased in TNBC cells compared with non-TNBC cells. In addition, the invasiveness of the TNBC cells was dramatically increased by IL-8 treatment and then augmented invasion-related proteins such as matrix metalloproteinase (MMP)-2 or MMP-9. We observed that elevated IL-8 mRNA expression and protein secretion were suppressed by a specific MEK1/2 inhibitor, UO126. In contrast, the overexpression of constitutively active MEK significantly increased the level of IL-8 mRNA expression in BT474 non-TNBC cells. Finally, we investigated the effect of UO126 on the tumorigenecity of TNBC cells. Our results showed that anchorage-independent growth, cell invasion, and cell migration were also decreased by UO126 in TNBC cells. As such, we demonstrated that IL-8 expression is regulated through MEK/ERK-dependent pathways in TNBC cells. A diversity of MEK blockers, including UO126, may be promising for treating TNBC patients.

EPA and DHA increased PPARγ expression and deceased integrin-linked kinase and integrin β1 expression in rat glomerular mesangial cells treated with lipopolysaccharide.

PubMed

Han, Wenchao; Zhao, Hui; Jiao, Bo; Liu, Fange

2014-04-01

Fish oil containing n-3 polyunsaturated fatty acids (n-3 PUFAs) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is known to prevent the progression of nephropathy and retard the progression of kidney disease. This study sought to investigate the underlying mechanisms of EPA and DHA in terms of peroxisome proliferator-activated receptor γ (PPARγ), integrin-linked kinase (ILK), and integrin β1 expression in glomerular mesangial cells (GMCs) because of their critical roles in the development and progression of nephropathy. Lipopolysaccharide (LPS) significantly reduced the expression of PPARγand increased the expression of ILK at the mRNA level and at the protein level in GMCs as indicated by real-time PCR and Western blotting. In addition, LPS increased integrin β1 expression in GMCs at the mRNA level. Treatment with EPA and DHA significantly increased the expression of PPARγ and decreased the expression of ILK and integrin β1 in GMCs. These data suggest that the renoprotective effects of EPA and DHA may be related to their potential to increase the expression of PPARγ and decrease the expression of ILK and integrin β1.

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.

T-lymphocyte cytokine mRNA expression in cystic echinococcosis.

PubMed

Fauser, S; Kern, P

1997-04-01

In the present study we investigated cytokine mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with cystic echinococcosis (CE) after stimulation with different antigens. By using reverse transcriptase polymerase chain reaction (RT-PCR) we could demonstrate that restimulation with crude Echinococcus granulosus antigen (Eg-Ag) induced or enhanced Th2 cytokine mRNA expression, especially IL-5 (by using antigen from sheep cyst fluid) in 23 out of 26 investigated CE patients and IL-10 (by using antigen from camel cyst fluid) in 10 out of 10 investigated CE patients. In contrast, IL-5 mRNA expression was absent in PBMC of healthy controls after Eg-Ag stimulation. To determine the specificity of this reaction we stimulated PBMC from 11 CE patients with crude Echinococcus multilocularis antigen (Em-Ag) and PBMC from 8 CE patients with Toxocara canis antigen (Tc-Ag). We found that the PBMC of patients showed a similar mRNA cytokine pattern on stimulation with Em-Ag when compared with Eg-Ag stimulation. The cytokine mRNA pattern on stimulation with Tc-Ag, however, resembled the cytokine mRNA pattern of unstimulated PBMC. Furthermore, the stimulation of PBMC with crude Mycobacterium tuberculosis antigen (H37Ra) and purified protein derivative (PPD) of M. tuberculosis revealed distinct IL-5 mRNA expression in all investigated CE patients, whereas in healthy controls IL-5 mRNA expression was very weak or totally absent. Thus, our results indicate an induction of Th2 cytokine mRNA expression in CE patients, which is frequently observed in parasite infections. Interestingly, this response persists after stimulation with tuberculosis antigens, which normally induce Th1 response.

Effects of Feed Contaminant Deoxynivalenol on Plasma Cytokines and mRNA Expression of Immune Genes in the Intestine of Broiler Chickens

PubMed Central

Soodoi, Chimidtseren; Sasgary, Soleman; Strasser, Alois; Böhm, Josef

2013-01-01

An experiment was conducted to investigate the individual and combined effects of dietary deoxynivalenol (DON) and a microbial feed additive on plasma cytokine level and on the expression of immune relevant genes in jejunal tissues of broilers. A total of 40 broiler chicks were obtained from a commercial hatchery and divided randomly into four groups (10 birds per group). Birds were reared in battery cages from one day old for 5 weeks. The dietary groups were 1) control birds fed basal diet; 2) DON group fed basal diet contaminated with 10 mg DON/ kg feed; 3) DON + Mycofix group fed basal diet contaminated with 10 mg DON/ kg feed and supplemented with a commercial feed additive, Mycofix® Select (MS) (2.5 kg/ton of feed); 4) Mycofix group fed basal diet supplemented with MS (2.5 kg/ton of feed). At 35 days, the plasma levels of tumor necrosis factor alpha (TNF-α) and interleukin 8 (IL-8) were quantified by ELISA test kits. Furthermore, the mRNA expression of TNF-α, IL-8, IL-1β, interferon gamma (IFNγ), transforming growth factor beta receptor I (TGFBR1) and nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NF-κβ1) in jejunum were quantified by qRT-PCR. The results showed that the plasma TNF-α decreased in response to DON, while in combination with MS, the effect of DON was reduced. DON down-regulated the relative gene expression of IL-1β, TGFBR1 and IFN-γ, and addition of MS to the DON contaminated diet compensates these effects on IL-1β, TGFBR1 but not for IFN-γ. Furthermore, supplementation of MS to either DON contaminated or control diet up-regulated the mRNA expression of NF-κβ1. In conclusion, DON has the potential to provoke and modulate immunological reactions of broilers and subsequently could increase their susceptibility to disease. The additive seemed to have almost as much of an effect as DON, albeit on different genes. PMID:23977054

Effects of feed contaminant deoxynivalenol on plasma cytokines and mRNA expression of immune genes in the intestine of broiler chickens.

PubMed

Ghareeb, Khaled; Awad, Wageha A; Soodoi, Chimidtseren; Sasgary, Soleman; Strasser, Alois; Böhm, Josef

2013-01-01

An experiment was conducted to investigate the individual and combined effects of dietary deoxynivalenol (DON) and a microbial feed additive on plasma cytokine level and on the expression of immune relevant genes in jejunal tissues of broilers. A total of 40 broiler chicks were obtained from a commercial hatchery and divided randomly into four groups (10 birds per group). Birds were reared in battery cages from one day old for 5 weeks. The dietary groups were 1) control birds fed basal diet; 2) DON group fed basal diet contaminated with 10 mg DON/ kg feed; 3) DON + Mycofix group fed basal diet contaminated with 10 mg DON/ kg feed and supplemented with a commercial feed additive, Mycofix® Select (MS) (2.5 kg/ton of feed); 4) Mycofix group fed basal diet supplemented with MS (2.5 kg/ton of feed). At 35 days, the plasma levels of tumor necrosis factor alpha (TNF-α) and interleukin 8 (IL-8) were quantified by ELISA test kits. Furthermore, the mRNA expression of TNF-α, IL-8, IL-1β, interferon gamma (IFNγ), transforming growth factor beta receptor I (TGFBR1) and nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NF-κβ1) in jejunum were quantified by qRT-PCR. The results showed that the plasma TNF-α decreased in response to DON, while in combination with MS, the effect of DON was reduced. DON down-regulated the relative gene expression of IL-1β, TGFBR1 and IFN-γ, and addition of MS to the DON contaminated diet compensates these effects on IL-1β, TGFBR1 but not for IFN-γ. Furthermore, supplementation of MS to either DON contaminated or control diet up-regulated the mRNA expression of NF-κβ1. In conclusion, DON has the potential to provoke and modulate immunological reactions of broilers and subsequently could increase their susceptibility to disease. The additive seemed to have almost as much of an effect as DON, albeit on different genes.

Advanced colorectal adenoma related gene expression signature may predict prognostic for colorectal cancer patients with adenoma-carcinoma sequence.

PubMed

Li, Bing; Shi, Xiao-Yu; Liao, Dai-Xiang; Cao, Bang-Rong; Luo, Cheng-Hua; Cheng, Shu-Jun

2015-01-01

There are still no absolute parameters predicting progression of adenoma into cancer. The present study aimed to characterize functional differences on the multistep carcinogenetic process from the adenoma-carcinoma sequence. All samples were collected and mRNA expression profiling was performed by using Agilent Microarray high-throughput gene-chip technology. Then, the characteristics of mRNA expression profiles of adenoma-carcinoma sequence were described with bioinformatics software, and we analyzed the relationship between gene expression profiles of adenoma-adenocarcinoma sequence and clinical prognosis of colorectal cancer. The mRNA expressions of adenoma-carcinoma sequence were significantly different between high-grade intraepithelial neoplasia group and adenocarcinoma group. The biological process of gene ontology function enrichment analysis on differentially expressed genes between high-grade intraepithelial neoplasia group and adenocarcinoma group showed that genes enriched in the extracellular structure organization, skeletal system development, biological adhesion and itself regulated growth regulation, with the P value after FDR correction of less than 0.05. In addition, IPR-related protein mainly focused on the insulin-like growth factor binding proteins. The variable trends of gene expression profiles for adenoma-carcinoma sequence were mainly concentrated in high-grade intraepithelial neoplasia and adenocarcinoma. The differentially expressed genes are significantly correlated between high-grade intraepithelial neoplasia group and adenocarcinoma group. Bioinformatics analysis is an effective way to study the gene expression profiles in the adenoma-carcinoma sequence, and may provide an effective tool to involve colorectal cancer research strategy into colorectal adenoma or advanced adenoma.

Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells

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

Jalava, A.M.; Heikkilae, J.E.; Akerman, K.E.O.

1988-11-01

The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc RNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC{sub 8}) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA andmore » DiC{sub 8} it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.« less

Impact of a high-cholesterol diet on expression levels of Niemann-Pick C1-like 1 and intestinal transporters in rats and mice.

PubMed

Kawase, Atsushi; Araki, Yasuha; Ueda, Yukiko; Nakazaki, Sayaka; Iwaki, Masahiro

2016-08-01

Niemann-Pick C1-like 1 (NPC1L1), ATP-binding cassette (ABC)G5, and ABCG8 are all involved in intestinal cholesterol absorption. It is unclear whether a high-cholesterol (HC) diet affects the expression of these transporters in rats and mice as well as humans. We examined the effects of an HC diet on their expression in small intestine and the differences between rats and mice in the responsive of this expression to an HC diet. In addition to these transporters, alterations in six representative drug and nutrient transporters (multidrug resistance-associated protein, breast cancer resistance protein, peptide transporter, sodium-glucose linked transporter, glucose transporter, and L-type amino acid transporter) and transcriptional factors such as hepatocyte nuclear factor (HNF)4α, sterol regulatory element-binding protein (SREBP)2, and liver X receptor (LXR)α were determined. In rats and mice fed an HC diet for 7 days, the mRNA and protein levels of NPC1L1 in the small intestine were determined by real-time reverse transcription polymerase chain reaction and western blotting, respectively. The mRNA levels of ABCG5 and ABCG8, six representative transporters, and transcriptional factors such as HNF4α, SREBP2, and LXR were examined. Significant decreases in the expression levels of NPC1L1 were observed in mice, but not rats, fed the HC diet. The mRNA levels of ABCG5 and ABCG8 were significantly increased in HC rats but not in mice. Only minor changes in the mRNA levels of the other transporters were seen in HC rats and mice. Decreased mRNA levels of HNF4α and SREBP2 in mice could be involved in the reduction in NPC1L1 expression observed upon the introduction of an HC diet. These results indicate that the effects of an HC diet on the expression levels of NPC1L1, ABCG5, and ABCG8 differ between mice and rats.

Stimulatory Effect of Insulin on 5α-Reductase Type 1 (SRD5A1) Expression through an Akt-Dependent Pathway in Ovarian Granulosa Cells

PubMed Central

Kayampilly, Pradeep P.; Wanamaker, Brett L.; Stewart, James A.; Wagner, Carrie L.; Menon, K. M. J.

2010-01-01

Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 μg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P < 0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P < 0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation. PMID:20810561

(+/-)-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.

A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

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

Toki, Yasumichi; Sasaki, Katsunori, E-mail: k-sasaki@asahikawa-med.ac.jp; Tanaka, Hiroki

2016-08-05

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncatedmore » peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.« less

Gene expression levels of heat shock proteins in the soleus and plantaris muscles of rats after hindlimb suspension or spaceflight.

PubMed

Ishihara, Akihiko; Fujino, Hidemi; Nagatomo, Fumiko; Takeda, Isao; Ohira, Yoshinobu

2008-12-01

Gene expression levels of heat shock proteins (HSPs) in the slow-twitch soleus and fast-twitch plantaris muscles of rats were determined after hindlimb suspension or spaceflight. Male rats were hindlimb-suspended for 14 d or exposed to microgravity for 9 d. The mRNA expression levels of HSP27, HSP70, and HSP84 in the hindlimb-suspended and microgravity-exposed groups were compared with those in the controls. The mRNA expression levels of the 3 HSPs in the soleus muscle under normal conditions were higher compared with those in the plantaris muscle. The mRNA expression levels of the 3 HSPs in the soleus muscle were inhibited by hindlimb suspension and spaceflight. The mRNA expression levels of the 3 HSPs in the plantaris muscle did not change after hindlimb suspension. It is suggested that the mRNA expression levels of the 3 HSPs are regulated by the mechanical and neural activity levels, and therefore the decreased mRNA expression levels of HSPs in the slow-twitch muscle following hindlimb suspension and spaceflight are related to a reduction in the mechanical and neural activity levels.

The differential expression of alternatively polyadenylated transcripts is a common stress-induced response mechanism that modulates mammalian mRNA expression in a quantitative and qualitative fashion.

PubMed

Hollerer, Ina; Curk, Tomaz; Haase, Bettina; Benes, Vladimir; Hauer, Christian; Neu-Yilik, Gabriele; Bhuvanagiri, Madhuri; Hentze, Matthias W; Kulozik, Andreas E

2016-09-01

Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells. © 2016 Hollerer et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export

PubMed Central

Zhang, Liang; Das, Priyabrata; Schmolke, Mirco; Manicassamy, Balaji; Wang, Yaming; Deng, Xiaoyi; Cai, Ling; Tu, Benjamin P.; Forst, Christian V.; Roth, Michael G.; Levy, David E.; García-Sastre, Adolfo; de Brabander, Jef; Phillips, Margaret A.

2012-01-01

The NS1 protein of influenza virus is a major virulence factor essential for virus replication, as it redirects the host cell to promote viral protein expression. NS1 inhibits cellular messenger ribonucleic acid (mRNA) processing and export, down-regulating host gene expression and enhancing viral gene expression. We report in this paper the identification of a nontoxic quinoline carboxylic acid that reverts the inhibition of mRNA nuclear export by NS1, in the absence or presence of the virus. This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for de novo pyrimidine biosynthesis, and partially reduced pyrimidine levels. This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1. The release of NS1-mediated mRNA export block by DHODH inhibition also occurred in the presence of vesicular stomatitis virus M (matrix) protein, another viral inhibitor of mRNA export. This reversal of mRNA export block allowed expression of antiviral factors. Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors. PMID:22312003

Aryl‐hydrocarbon receptor activity modulates prolactin expression in the pituitary

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

Moran, Tyler B.; Brannick, Katherine E.; Raetzman, Lori T., E-mail: raetzman@life.illinois.edu

2012-11-15

Pituitary tumors account for 15% of intracranial neoplasms, however the extent to which environmental toxicants contribute to the proliferation and hormone expression of pituitary cells is unknown. Aryl-hydrocarbon receptor (AhR) interacting protein (AIP) loss of function mutations cause somatotrope and lactotrope adenomas in humans. AIP sequesters AhR and inhibits its transcriptional function. Because of the link between AIP and pituitary tumors, we hypothesize that exposure to dioxins, potent exogenous ligands for AhR that are persistent in the environment, may predispose to pituitary dysfunction through activation of AhR. In the present study, we examined the effect of AhR activation on proliferationmore » and endogenous pituitary hormone expression in the GH3 rat somatolactotrope tumor cell line and the effect of loss of AhR action in knockout mice. GH3 cells respond to nM doses of the reversible AhR agonist β-naphthoflavone with a robust induction of Cyp1a1. Although mRNA levels of the anti-proliferative signaling cytokine TGFbeta1 are suppressed upon β-naphthoflavone treatment, we did not observe an alteration in cell proliferation. AhR activation with β-naphthoflavone suppresses Ahr expression and impairs expression of prolactin (PRL), but not growth hormone (GH) mRNA in GH3 cells. In mice, loss of Ahr similarly leads to a reduction in Prl mRNA at P3, while Gh is unaffected. Additionally, there is a significant reduction in pituitary hormones Lhb and Fshb in the absence of Ahr. Overall, these results demonstrate that AhR is important for pituitary hormone expression and suggest that environmental dioxins can exert endocrine disrupting effects at the pituitary. -- Highlights: ► AhR signaling suppresses Prl mRNA expression. ► AhR signaling does not influence pituitary proliferation in culture. ► AhR is necessary for Prl, Lhb and Fshb expression at postnatal day 3.« less

Role of nuclear factor-erythroid 2-related factor 2 (Nrf2) in the transcriptional regulation of brain ABC transporters during acute acetaminophen (APAP) intoxication in mice.

PubMed

Ghanem, Carolina I; Rudraiah, Swetha; Bataille, Amy M; Vigo, María B; Goedken, Michael J; Manautou, José E

2015-04-01

Changes in expression of liver ABC transporters have been described during acute APAP intoxication. However, the effect of APAP on brain ABC transporters is poorly understood. The aim of this study was to evaluate the effect of APAP on brain ABC transporters expression and the role of the oxidative stress sensor Nrf2. Male C57BL/6J mice were administered APAP (400mg/kg) for analysis of brain mRNA and protein expression of Mrp1-6, Bcrp and P-gp. The results show induction of P-gp, Mrp2 and Mrp4 proteins, with no changes in Bcrp, Mrp1 or Mrp5-6. The protein values were accompanied by corresponding changes in mRNA levels. Additionally, brain Nrf2 nuclear translocation and expression of two Nrf2 target genes, quinone oxidoreductase 1 (Nqo1) and Hemoxygenase 1 (Ho-1), was evaluated at 6, 12 and 24h after APAP treatment. Nrf2 nuclear content increased by 58% at 12h after APAP along with significant increments in mRNA and protein expression of Nqo1 and Ho-1. Furthermore, APAP treated Nrf2 knockout mice did not increase mRNA or protein expression of Mrp2 and Mrp4 as observed in wildtypes. In contrast, P-gp induction by APAP was observed in both genotypes. In conclusion, acute APAP intoxication induces protein expression of brain P-gp, Mrp2 and Mrp4. This study also suggests that brain changes in Mrp2 and Mrp4 expression may be due to in situ Nrf2 activation by APAP, while P-gp induction is independent of Nrf2 function. The functional consequences of these changes in brain ABC transporters by APAP deserve further attention. Copyright © 2015 Elsevier Inc. All rights reserved.

Suppressive effects of RXR agonist PA024 on adrenal CYP11B2 expression, aldosterone secretion and blood pressure.

PubMed

Suzuki, Dai; Saito-Hakoda, Akiko; Ito, Ryo; Shimizu, Kyoko; Parvin, Rehana; Shimada, Hiroki; Noro, Erika; Suzuki, Susumu; Fujiwara, Ikuma; Kagechika, Hiroyuki; Rainey, William E; Kure, Shigeo; Ito, Sadayoshi; Yokoyama, Atsushi; Sugawara, Akira

2017-01-01

The effects of retinoids on adrenal aldosterone synthase gene (CYP11B2) expression and aldosterone secretion are still unknown. We therefore examined the effects of nuclear retinoid X receptor (RXR) pan-agonist PA024 on CYP11B2 expression, aldosterone secretion and blood pressure, to elucidate its potential as a novel anti-hypertensive drug. We demonstrated that PA024 significantly suppressed angiotensin II (Ang II)-induced CYP11B2 mRNA expression, promoter activity and aldosterone secretion in human adrenocortical H295R cells. Human CYP11B2 promoter functional analyses using its deletion and point mutants indicated that the suppression of CYP11B2 promoter activity by PA024 was in the region from -1521 (full length) to -106 including the NBRE-1 and the Ad5 elements, and the Ad5 element may be mainly involved in the PA024-mediated suppression. PA024 also significantly suppressed the Ang II-induced mRNA expression of transcription factors NURR1 and NGFIB that bind to and activate the Ad5 element. NURR1 overexpression demonstrated that the decrease of NURR1 expression may contribute to the PA024-mediated suppression of CYP11B2 transcription. PA024 also suppressed the Ang II-induced mRNA expression of StAR, HSD3β2 and CYP21A2, a steroidogenic enzyme group involved in aldosterone biosynthesis. Additionally, the PA024-mediated CYP11B2 transcription suppression was shown to be exerted via RXRα. Moreover, the combination of PPARγ agonist pioglitazone and PA024 caused synergistic suppressive effects on CYP11B2 mRNA expression. Finally, PA024 treatment significantly lowered both the systolic and diastolic blood pressure in Tsukuba hypertensive mice (hRN8-12 x hAG2-5). Thus, RXR pan-agonist PA024 may be a candidate anti-hypertensive drugs that acts via the suppression of aldosterone synthesis and secretion.

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.

Airways in smooth muscle α-actin null mice experience a compensatory mechanism that modulates their contractile response.

PubMed

Shardonofsky, Felix R; Moore, Joan; Schwartz, Robert J; Boriek, Aladin M

2012-03-01

We hypothesized that ablation of smooth muscle α-actin (SM α-A), a contractile-cytoskeletal protein expressed in airway smooth muscle (ASM) cells, abolishes ASM shortening capacity and decreases lung stiffness. In both SM α-A knockout and wild-type (WT) mice, airway resistance (Raw) determined by the forced oscillation technique rose in response to intravenous methacholine (Mch). However, the slope of Raw (cmH(2)O·ml(-1)·s) vs. log(2) Mch dose (μg·kg(-1)·min(-1)) was lower (P = 0.007) in mutant (0.54 ± 0.14) than in WT mice (1.23 ± 0.19). RT-PCR analysis performed on lung tissues confirmed that mutant mice lacked SM α-A mRNA and showed that these mice had robust expressions of both SM γ-A mRNA and skeletal muscle (SKM) α-A mRNA, which were not expressed in WT mice, and an enhanced SM22 mRNA expression relative to that in WT mice. Compared with corresponding spontaneously breathing mice, mechanical ventilation-induced lung mechanical strain increased the expression of SM α-A mRNA in WT lungs; in mutant mice, it augmented the expressions of SM γ-A mRNA and SM22 mRNA and did not alter that of SKM α-A mRNA. In mutant mice, the expression of SM γ-A mRNA in the lung during spontaneous breathing and its enhanced expression following mechanical ventilation are consistent with the likely possibility that in the absence of SM α-A, SM γ-A underwent polymerization and interacted with smooth muscle myosin to produce ASM shortening during cholinergic stimulation. Thus our data are consistent with ASM in mutant mice experiencing compensatory mechanisms that modulated its contractile muscle capacity.

Lower FOXO3 mRNA expression in granulosa cells is involved in unexplained infertility.

PubMed

Yamamoto, Hikaru; Yamashita, Yoshiki; Saito, Natsuho; Hayashi, Atsushi; Hayashi, Masami; Terai, Yoshito; Ohmichi, Masahide

2017-06-01

The aim of this study was to investigate whether FOXO1 and FOXO3 mRNA expression in granulosa cells is the cause of unexplained infertility. Thirty-one patients aged <40 years (13 with unexplained infertility and 18 with male partner infertility as a control group) whose serum anti-Müllerian hormone level was >0.5 ng/μL were enrolled in the study. All patients underwent oocyte retrieval under a short protocol from June 2012 to October 2013. Real-time PCR was carried out using mRNA extracted from granulosa cells retrieved from mature follicles. We compared FOXO1 and FOXO3 mRNA expression ratios in granulosa cells between the unexplained infertility group and the male infertility group. The relation between FOXO1 and FOXO3 mRNA expression ratios in granulosa cells and assisted reproduction technology clinical outcome was also examined. FOXO3 mRNA expression ratio was significantly lower in the unexplained infertility group than in the male infertility group. Moreover, FOXO3 mRNA expression ratio showed a positive correlation with both the number of retrieved oocytes and serum anti-Müllerian hormone level. A positive correlation was also identified between FOXO1 mRNA expression and total dose of hMG. As well, the number of retrieved oocytes in the unexplained infertility group was statistically lower than that in the male infertility group. A lower FOXO3 mRNA expression in granulosa cells leads to poor oocyte development in patients with unexplained infertility undergoing controlled ovarian stimulation for in vitro fertilization-embryo transfer. © 2017 Japan Society of Obstetrics and Gynecology.

Peroxisome Proliferator-Activated Receptors Alpha, Beta, and Gamma mRNA and Protein Expression in Human Fetal Tissues

PubMed Central

Abbott, Barbara D.; Wood, Carmen R.; Watkins, Andrew M.; Das, Kaberi P.; Lau, Christopher S.

2010-01-01

Peroxisome proliferator-activated receptors (PPARs) regulate lipid and glucose homeostasis, are targets of pharmaceuticals, and are also activated by environmental contaminants. Almost nothing is known about expression of PPARs during human fetal development. This study examines expression of PPARα, β, and γ mRNA and protein in human fetal tissues. With increasing fetal age, mRNA expression of PPARα and β increased in liver, but PPARβ decreased in heart and intestine, and PPARγ decreased in adrenal. Adult and fetal mean expression of PPARα, β, and γ mRNA did not differ in intestine, but expression was lower in fetal stomach and heart. PPARα and β mRNA in kidney and spleen, and PPARγ mRNA in lung and adrenal were lower in fetal versus adult. PPARγ in liver and PPARβ mRNA in thymus were higher in fetal versus adult. PPARα protein increased with fetal age in intestine and decreased in lung, kidney, and adrenal. PPARβ protein in adrenal and PPARγ in kidney decreased with fetal age. This study provides new information on expression of PPAR subtypes during human development and will be important in evaluating the potential for the developing human to respond to PPAR environmental or pharmaceutical agonists. PMID:20706641

Induction of cysteine-rich motor neuron 1 mRNA expression in vascular endothelial cells.

PubMed

Nakashima, Yukiko; Takahashi, Satoru

2014-08-22

Cysteine-rich motor neuron 1 (CRIM1) is expressed in vascular endothelial cells and plays a crucial role in angiogenesis. In this study, we investigated the expression of CRIM1 mRNA in human umbilical vein endothelial cells (HUVECs). CRIM1 mRNA levels were not altered in vascular endothelial growth factor (VEGF)-stimulated monolayer HUVECs or in cells in collagen gels without VEGF. In contrast, the expression of CRIM1 mRNA was elevated in VEGF-stimulated cells in collagen gels. The increase in CRIM1 mRNA expression was observed even at 2h when HUVECs did not form tubular structures in collagen gels. Extracellular signal-regulated kinase (Erk) 1/2, Akt and focal adhesion kinase (FAK) were activated by VEGF in HUVECs. The VEGF-induced expression of CRIM1 mRNA was significantly abrogated by PD98059 or PF562271, but was not affected by LY294002. These results demonstrate that CRIM1 is an early response gene in the presence of both angiogenic stimulation (VEGF) and environmental (extracellular matrix) factors, and Erk and FAK might be involved in the upregulation of CRIM1 mRNA expression in vascular endothelial cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Two novel LRR-only proteins in Chlamys farreri: Similar in structure, yet different in expression profile and pattern recognition.

PubMed

Wang, Mengqiang; Wang, Lingling; Xin, Lusheng; Wang, Xiudan; Wang, Lin; Xu, Jianchao; Jia, Zhihao; Yue, Feng; Wang, Hao; Song, Linsheng

2016-06-01

Leucine-rich repeat (LRR)-only proteins could mediate protein-ligand and protein-protein interactions and be involved in the immune response. In the present study, two novel LRR-only proteins, CfLRRop-2 and CfLRRop-3, were identified and characterized from scallop Chlamys farreri. They both contained nine LRR motifs with the consensus signature sequence LxxLxLxxNxL and formed typical horseshoe structure. The CfLRRop-2 and CfLRRop-3 mRNA transcripts were constitutively expressed in haemocytes, muscle, mantle, gill, haepatopancreas and gonad, with the highest expression level in haepatopancreas and gill, respectively. During the ontogenesis of scallop, the mRNA transcripts of CfLRRop-2 were kept at a high level in oocytes and embryos, while those of CfLRRop-3 were expressed at a rather low level from oocytes to blastula. Their mRNA transcripts were significantly increased after the stimulation of lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polyinosinic-polycytidylic acid (poly I:C), and the mRNA expression of CfLRRop-2 rose more intensely than that of CfLRRop-3. After the suppression of CfTLR (previously identified Toll-like receptor in C. farreri) via RNA interference (RNAi), CfLRRop-3 mRNA transcripts increased more intensely and lastingly than those of CfLRRop-2. The rCfLRRop-3 protein could bind LPS, PGN, GLU and poly I:C, while rCfLRRop-2 exhibited no significant binding activity to them. Additionally, rCfLRRop-2 could significantly induce the release of TNF-α from the mixed primary cultured scallop haemocytes, but rCfLRRop-3 failed. These results collectively indicated that CfLRRop-2 might act as an immune effector or pro-inflammatory factor, while CfLRRop-3 would function as a pattern recognition receptor (PRR), suggesting the function of LRR-only protein family has differentiated in scallop. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Co-Induced Effects of Molybdenum and Cadmium on the Trace Elements and the mRNA Expression Levels of CP and MT in Duck Testicles.

PubMed

Xia, Bing; Chen, Hua; Hu, Guoliang; Wang, Liqi; Cao, Huabin; Zhang, Caiying

2016-02-01

To investigate the chronic toxicity of molybdenum (Mo) and cadmium (Cd) on the trace elements and the mRNA expression levels of ceruloplasmin (CP) and metallothionein (MT) in duck testicles, 120 healthy 11-day-old male ducks were randomly divided into six groups with 20 ducks in each group. Ducks were treated with the diet containing different dosages of Mo or Cd. The source of Mo and Cd was hexaammonium molybdate ([(NH4)6Mo7O24·4H2O]) and cadmium sulfate (3CdSO4·8H2O), respectively, in this study. After being treated for 60 and 120 days, ten male birds in each group were randomly selected and euthanized and then testicles were aseptically collected for determining the mRNA expression levels of MT and CP, antioxidant indexes, and contents of trace elements in the testicle. In addition, testicle tissues at 120 days were subjected to histopathological analysis with the optical microscope. The results showed that co-exposure to Mo and Cd resulted in an increase in malondialdehyde (MDA) level while decrease in xanthine oxidase (XOD) and catalase (CAT) activities. The mRNA expression level of MT gene was upregulated while CP was decreased in combination groups. Contents of Mo, copper (Cu), iron (Fe), and zinc (Zn) decreased in combined groups while Cd increased in Cd and combined groups at 120 days. Furthermore, severe congestion, low sperm count, and malformation were observed in low dietary of Mo combined with Cd group and high dietary of Mo combined with Cd group. Our results suggested that Mo and Cd might aggravate testicular degeneration synergistically through altering the mRNA expression levels of MT and CP, increasing lipid peroxidation through inhibiting related enzyme activities and disturbing homeostasis of trace elements in testicles. Interaction of Mo and Cd may have a synergistic effect on the testicular toxicity.

Induction of CXC chemokines in human mesenchymal stem cells by stimulation with secreted frizzled-related proteins through non-canonical Wnt signaling.

PubMed

Bischoff, David S; Zhu, Jian-Hua; Makhijani, Nalini S; Yamaguchi, Dean T

2015-12-26

To investigate the effect of secreted frizzled-related proteins (sFRPs) on CXC chemokine expression in human mesenchymal stem cells (hMSCs). CXC chemokines such as CXCL5 and CXCL8 are induced in hMSCs during differentiation with osteogenic differentiation medium (OGM) and may be involved in angiogenic stimulation during bone repair. hMSCs were treated with conditioned medium (CM) from L-cells expressing non-canonical Wnt5a protein, or with control CM from wild type L-cells, or directly with sFRPs for up to 10 d in culture. mRNA expression levels of both CXCL5 and CXCL8 were quantitated by real-time reverse transcriptase-polymerase chain reaction and secreted protein levels of these proteins determined by ELISA. Dose- (0-500 ng/mL) and time-response curves were generated for treatment with sFRP1. Signal transduction pathways were explored by western blot analysis with pan- or phosphorylation-specific antibodies, through use of specific pathway inhibitors, and through use of siRNAs targeting specific frizzled receptors (Fzd)-2 and 5 or the receptor tyrosine kinase-like orphan receptor-2 (RoR2) prior to treatment with sFRPs. CM from L-cells expressing Wnt5a, a non-canonical Wnt, stimulated an increase in CXCL5 mRNA expression and protein secretion in comparison to control L-cell CM. sFRP1, which should inhibit both canonical and non-canonical Wnt signaling, surprisingly enhanced the expression of CXCL5 at 7 and 10 d. Dickkopf1, an inhibitor of canonical Wnt signaling prevented the sFRP-stimulated induction of CXCL5 and actually inhibited basal levels of CXCL5 expression at 7 but not at 10 d post treatment. In addition, all four sFRPs isoforms induced CXCL8 expression in a dose- and time-dependent manner with maximum expression at 7 d with treatment at 150 ng/mL. The largest increases in CXCL5 expression were seen from stimulation with sFRP1 or sFRP2. Analysis of mitogen-activated protein kinase signaling pathways in the presence of OGM showed sFRP1-induced phosphorylation of extracellular signal-regulated kinase (ERK) (p44/42) maximally at 5 min after sFRP1 addition, earlier than that found in OGM alone. Addition of a phospholipase C (PLC) inhibitor also prevented sFRP-stimulated increases in CXCL8 mRNA. siRNA technology targeting the Fzd-2 and 5 and the non-canonical Fzd co-receptor RoR2 also significantly decreased sFRP1/2-stimulated CXCL8 mRNA levels. CXC chemokine expression in hMSCs is controlled in part by sFRPs signaling through non-canonical Wnt involving Fzd2/5 and the ERK and PLC pathways.

Induction of CXC chemokines in human mesenchymal stem cells by stimulation with secreted frizzled-related proteins through non-canonical Wnt signaling

PubMed Central

Bischoff, David S; Zhu, Jian-Hua; Makhijani, Nalini S; Yamaguchi, Dean T

2015-01-01

AIM: To investigate the effect of secreted frizzled-related proteins (sFRPs) on CXC chemokine expression in human mesenchymal stem cells (hMSCs). METHODS: CXC chemokines such as CXCL5 and CXCL8 are induced in hMSCs during differentiation with osteogenic differentiation medium (OGM) and may be involved in angiogenic stimulation during bone repair. hMSCs were treated with conditioned medium (CM) from L-cells expressing non-canonical Wnt5a protein, or with control CM from wild type L-cells, or directly with sFRPs for up to 10 d in culture. mRNA expression levels of both CXCL5 and CXCL8 were quantitated by real-time reverse transcriptase-polymerase chain reaction and secreted protein levels of these proteins determined by ELISA. Dose- (0-500 ng/mL) and time-response curves were generated for treatment with sFRP1. Signal transduction pathways were explored by western blot analysis with pan- or phosphorylation-specific antibodies, through use of specific pathway inhibitors, and through use of siRNAs targeting specific frizzled receptors (Fzd)-2 and 5 or the receptor tyrosine kinase-like orphan receptor-2 (RoR2) prior to treatment with sFRPs. RESULTS: CM from L-cells expressing Wnt5a, a non-canonical Wnt, stimulated an increase in CXCL5 mRNA expression and protein secretion in comparison to control L-cell CM. sFRP1, which should inhibit both canonical and non-canonical Wnt signaling, surprisingly enhanced the expression of CXCL5 at 7 and 10 d. Dickkopf1, an inhibitor of canonical Wnt signaling prevented the sFRP-stimulated induction of CXCL5 and actually inhibited basal levels of CXCL5 expression at 7 but not at 10 d post treatment. In addition, all four sFRPs isoforms induced CXCL8 expression in a dose- and time-dependent manner with maximum expression at 7 d with treatment at 150 ng/mL. The largest increases in CXCL5 expression were seen from stimulation with sFRP1 or sFRP2. Analysis of mitogen-activated protein kinase signaling pathways in the presence of OGM showed sFRP1-induced phosphorylation of extracellular signal-regulated kinase (ERK) (p44/42) maximally at 5 min after sFRP1 addition, earlier than that found in OGM alone. Addition of a phospholipase C (PLC) inhibitor also prevented sFRP-stimulated increases in CXCL8 mRNA. siRNA technology targeting the Fzd-2 and 5 and the non-canonical Fzd co-receptor RoR2 also significantly decreased sFRP1/2-stimulated CXCL8 mRNA levels. CONCLUSION: CXC chemokine expression in hMSCs is controlled in part by sFRPs signaling through non-canonical Wnt involving Fzd2/5 and the ERK and PLC pathways. PMID:26730270

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.

AKR1C1 and SRD5A1 messenger RNA expression at term in the human myometrium and chorioamniotic membranes.

PubMed

Lee, Richard H; Stanczyk, Frank Z; Stolz, Andrew; Ji, Qing; Yang, Gloria; Goodwin, T Murphy

2008-10-01

We sought to determine relative mRNA expression of AKR1C1 and SRD5A1, which respectively encode for the key progesterone metabolizing enzymes, 20alpha-hydroxysteroid dehydrogenase and 5alpha-reductase type 1, in the myometrium and chorioamniotic membranes during human spontaneous or induced labor and nonlabor. Quantitative real-time reverse-transcriptase polymerase chain reaction was used to compare relative mRNA expression of AKR1C1 and SRD5A1 in the myometrium and chorioamniotic membranes from 20 subjects during three different states of labor: not in labor ( N = 10), spontaneous labor ( N = 5), or induced labor ( N = 5). Labor was defined as regular uterine contractions that resulted in cervical dilation. Myometrial AKR1C1 mRNA expression was significantly greater in spontaneously laboring subjects compared with those not in labor (2.4-fold [1.97 to 2.98], P = 0.02). There was no difference in myometrial AKR1C1 mRNA expression between those with induced labor compared with those not in labor. Regardless of labor status, no differences were observed in the chorioamniotic membrane AKR1C1 mRNA expression between the groups. SRD5A1 mRNA expression was significantly lower in the membranes of both laboring groups when compared with those not in labor (spontaneous: 0.10-fold [0.06 to 0.18], P = 0.007; induced: 0.09-fold [0.03 to 0.25], P = 0.013). Regardless of labor status, there was no difference in SRD5A1 mRNA expression in the myometrium. Our study demonstrated tissue-specific changes in progesterone metabolizing enzyme mRNA expression in human intrauterine tissue at term associated with labor status. These observed changes in mRNA expression may have important implications for progesterone metabolism at those specific sites and thereby may differentially regulate the tissue-specific progesterone concentration and/or the level of specific progesterone metabolites.

Embryonic exposure to the fungicide vinclozolin causes virilization of females and alteration of progesterone receptor expression in vivo: an experimental study in mice

PubMed Central

Buckley, Jill; Willingham, Emily; Agras, Koray; Baskin, Laurence S

2006-01-01

Background Vinclozolin is a fungicide that has been reported to have anti-androgenic effects in rats. We have found that in utero exposure to natural or synthetic progesterones can induce hypospadias in mice, and that the synthetic progesterone medroxyprogesterone acetate (MPA) feminizes male and virilizes female genital tubercles. In the current work, we selected a relatively low dose of vinclozolin to examine its in utero effects on the development of the genital tubercle, both at the morphological and molecular levels. Methods We gave pregnant dams vinclozolin by oral gavage from gestational days 13 through 17. We assessed the fetal genital tubercles from exposed fetuses at E19 to determine location of the urethral opening. After determination of gonadal sex, either genital tubercles were harvested for mRNA quantitation, or urethras were injected with a plastic resin for casting. We analyzed quantified mRNA levels between treated and untreated animals for mRNA levels of estrogen receptors α and β, progesterone receptor, and androgen receptor using nonparametric tests or ANOVA. To determine effects on urethral length (males have long urethras compared to females), we measured the lengths of the casts and performed ANOVA analysis on these data. Results Our morphological results indicated that vinclozolin has morphological effects similar to those of MPA, feminizing males (hypospadias) and masculinizing females (longer urethras). Because these results reflected our MPA results, we investigated the effects of in utero vinclozolin exposure on the mRNA expression levels of androgen, estrogen α and β, and progesterone receptors. At the molecular level, vinclozolin down-regulated estrogen receptor α mRNA in females and up-regulated progesterone receptor mRNA. Vinclozolin-exposed males exhibited up-regulated estrogen receptor α and progesterone receptor mRNA, effects we have also seen with exposure to the synthetic estrogen, ethinyl estradiol. Conclusion The results suggest that vinclozolin virilizes females and directly or indirectly affects progesterone receptor expression. It also affects estrogen receptor expression in a sex-based manner. We found no in vivo effect of vinclozolin on androgen receptor expression. We propose that vinclozolin, which has been designated an anti-androgen, may also exert its effects by involving additional steroid-signaling pathways. PMID:16504050

L-leucine, beta-hydroxy-beta-methylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy.

PubMed

Mobley, Christopher Brooks; Fox, Carlton D; Ferguson, Brian S; Amin, Rajesh H; Dalbo, Vincent J; Baier, Shawn; Rathmacher, John A; Wilson, Jacob M; Roberts, Michael D

2014-01-01

The purpose of this study was to examine if L-leucine (Leu), β-hydroxy-β-methylbutyrate (HMB), or creatine monohydrate (Crea) prevented potential atrophic effects of myostatin (MSTN) on differentiated C2C12 myotubes. After four days of differentiation, myotubes were treated with MSTN (10 ng/ml) for two additional days and four treatment groups were studied: 1) 3x per day 10 mM Leu, 2) 3x per day 10 mM HMB, 3) 3x per day 10 mM Crea, 4) DM only. Myotubes treated with DM without MSTN were analyzed as the control condition (DM/CTL). Following treatment, cells were analyzed for total protein, DNA content, RNA content, muscle protein synthesis (MPS, SUnSET method), and fiber diameter. Separate batch treatments were analyzed for mRNA expression patterns of myostatin-related genes (Akirin-1/Mighty, Notch-1, Ski, MyoD) as well as atrogenes (MuRF-1, and MAFbx/Atrogin-1). MSTN decreased fiber diameter approximately 30% compared to DM/CTL myotubes (p < 0.001). Leu, HMB and Crea prevented MSTN-induced atrophy. MSTN did not decrease MPS levels compared to DM/CTL myotubes, but MSTN treatment decreased the mRNA expression of Akirin-1/Mighty by 27% (p < 0.001) and MyoD by 26% (p < 0.01) compared to DM/CTL myotubes. shRNA experiments confirmed that Mighty mRNA knockdown reduced myotube size, linking MSTN treatment to atrophy independent of MPS. Remarkably, MSTN + Leu and MSTN + HMB myotubes had similar Akirin-1/Mighty and MyoD mRNA levels compared to DM/CTL myotubes. Furthermore, MSTN + Crea myotubes exhibited a 36% (p < 0.05) and 86% (p < 0.001) increase in Akirin-1/Mighty mRNA compared to DM/CTL and MSTN-only treated myotubes, respectively. Leu, HMB and Crea may reduce MSTN-induced muscle fiber atrophy by influencing Akirin-1/Mighty mRNA expression patterns. Future studies are needed to examine if Leu, HMB and Crea independently or synergistically affect Akirin-1/Mighty expression, and how Akirin-1/Mighty expression mechanistically relates to skeletal muscle hypertrophy in vivo.

L-leucine, beta-hydroxy-beta-methylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy

PubMed Central

2014-01-01

Background The purpose of this study was to examine if L-leucine (Leu), β-hydroxy-β-methylbutyrate (HMB), or creatine monohydrate (Crea) prevented potential atrophic effects of myostatin (MSTN) on differentiated C2C12 myotubes. Methods After four days of differentiation, myotubes were treated with MSTN (10 ng/ml) for two additional days and four treatment groups were studied: 1) 3x per day 10 mM Leu, 2) 3x per day 10 mM HMB, 3) 3x per day 10 mM Crea, 4) DM only. Myotubes treated with DM without MSTN were analyzed as the control condition (DM/CTL). Following treatment, cells were analyzed for total protein, DNA content, RNA content, muscle protein synthesis (MPS, SUnSET method), and fiber diameter. Separate batch treatments were analyzed for mRNA expression patterns of myostatin-related genes (Akirin-1/Mighty, Notch-1, Ski, MyoD) as well as atrogenes (MuRF-1, and MAFbx/Atrogin-1). Results MSTN decreased fiber diameter approximately 30% compared to DM/CTL myotubes (p < 0.001). Leu, HMB and Crea prevented MSTN-induced atrophy. MSTN did not decrease MPS levels compared to DM/CTL myotubes, but MSTN treatment decreased the mRNA expression of Akirin-1/Mighty by 27% (p < 0.001) and MyoD by 26% (p < 0.01) compared to DM/CTL myotubes. shRNA experiments confirmed that Mighty mRNA knockdown reduced myotube size, linking MSTN treatment to atrophy independent of MPS. Remarkably, MSTN + Leu and MSTN + HMB myotubes had similar Akirin-1/Mighty and MyoD mRNA levels compared to DM/CTL myotubes. Furthermore, MSTN + Crea myotubes exhibited a 36% (p < 0.05) and 86% (p < 0.001) increase in Akirin-1/Mighty mRNA compared to DM/CTL and MSTN-only treated myotubes, respectively. Conclusions Leu, HMB and Crea may reduce MSTN-induced muscle fiber atrophy by influencing Akirin-1/Mighty mRNA expression patterns. Future studies are needed to examine if Leu, HMB and Crea independently or synergistically affect Akirin-1/Mighty expression, and how Akirin-1/Mighty expression mechanistically relates to skeletal muscle hypertrophy in vivo. PMID:25132809

Embryonic exposure to the fungicide vinclozolin causes virilization of females and alteration of progesterone receptor expression in vivo: an experimental study in mice.

PubMed

Buckley, Jill; Willingham, Emily; Agras, Koray; Baskin, Laurence S

2006-02-21

Vinclozolin is a fungicide that has been reported to have anti-androgenic effects in rats. We have found that in utero exposure to natural or synthetic progesterones can induce hypospadias in mice, and that the synthetic progesterone medroxyprogesterone acetate (MPA) feminizes male and virilizes female genital tubercles. In the current work, we selected a relatively low dose of vinclozolin to examine its in utero effects on the development of the genital tubercle, both at the morphological and molecular levels. We gave pregnant dams vinclozolin by oral gavage from gestational days 13 through 17. We assessed the fetal genital tubercles from exposed fetuses at E19 to determine location of the urethral opening. After determination of gonadal sex, either genital tubercles were harvested for mRNA quantitation, or urethras were injected with a plastic resin for casting. We analyzed quantified mRNA levels between treated and untreated animals for mRNA levels of estrogen receptors alpha and beta, progesterone receptor, and androgen receptor using nonparametric tests or ANOVA. To determine effects on urethral length (males have long urethras compared to females), we measured the lengths of the casts and performed ANOVA analysis on these data. Our morphological results indicated that vinclozolin has morphological effects similar to those of MPA, feminizing males (hypospadias) and masculinizing females (longer urethras). Because these results reflected our MPA results, we investigated the effects of in utero vinclozolin exposure on the mRNA expression levels of androgen, estrogen alpha and beta, and progesterone receptors. At the molecular level, vinclozolin down-regulated estrogen receptor alpha mRNA in females and up-regulated progesterone receptor mRNA. Vinclozolin-exposed males exhibited up-regulated estrogen receptor alpha and progesterone receptor mRNA, effects we have also seen with exposure to the synthetic estrogen, ethinyl estradiol. The results suggest that vinclozolin virilizes females and directly or indirectly affects progesterone receptor expression. It also affects estrogen receptor expression in a sex-based manner. We found no in vivo effect of vinclozolin on androgen receptor expression. We propose that vinclozolin, which has been designated an anti-androgen, may also exert its effects by involving additional steroid-signaling pathways.

Gestational choline supplementation normalized fetal alcohol-induced alterations in histone modifications, DNA methylation and POMC gene expression in β-endorphin-producing POMC neurons of the hypothalamus

PubMed Central

Bekdash, Rola A.; Zhang, Changqing; Sarkar, Dipak K.

2013-01-01

Background Prenatal exposure to ethanol reduces the expression of hypothalamic proopiomelanocortin (POMC) gene, known to control various physiological functions including the organismal stress response. In this study, we determined whether the changes in POMC neuronal functions are associated with altered expressions of histone-modifying and DNA-methylating enzymes in POMC-producing neurons, since these enzymes are known to be involved in regulation of gene expression. In addition, we tested whether gestational choline supplementation prevents the adverse effects of ethanol on these neurons. Methods Pregnant rat dams were fed with alcohol-containing liquid diet or control diet during gestational days 7 and 21 with or without choline, and their male offspring rats were used during the adult period. Using double-immunohistochemistry, real-time reverse transcription polymerase chain reaction (RT-PCR) and methylation specific RT-PCR, we determined protein and mRNA levels of histone-modifying and DNA-methylating enzymes, and the changes in POMC gene methylation and expression in the hypothalamus of adult male offspring rats. Additionally, we measured the basal and lipopolysaccharide (LPS)-induced corticosterone levels in plasma by enzyme-linked immunoabsorbent assay. Results Prenatal ethanol treatment suppressed hypothalamic levels of protein and mRNA of histone activation marks (H3K4me3, Set7/9, acetylated H3K9, phosphorylated H3S10) increased the repressive marks (H3K9me2, G9a, Setdb1) and DNA methylating enzyme (Dnmt1) and the methyl-CpG-binding protein (MeCP2). The treatment also elevated the level of POMC gene methylation, while it reduced levels of POMC mRNA and β-EP, and elevated corticosterone response to LPS. Gestational choline normalized the ethanol-altered protein and the mRNA levels of H3K4me3, Set7/9, H3K9me2, G9a, Setdb1, Dnmt1 and MeCP2. It also normalizes the changes in POMC gene methylation and gene expression, β-EP production and the corticosterone response to LPS. Conclusions These data suggest that prenatal ethanol modulates histone and DNA methylation in POMC neurons that may be resulting in hypermethylation of POMC gene and reduction of POMC gene expression. Gestational choline supplementation prevents the adverse effects of ethanol on these neurons. PMID:23413810

Programmed Cell Death Ligand 1 Expression in Primary Central Nervous System Lymphomas: A Clinicopathological Study.

PubMed

Hayano, Azusa; Komohara, Yoshihiro; Takashima, Yasuo; Takeya, Hiroto; Homma, Jumpei; Fukai, Junya; Iwadate, Yasuo; Kajiwara, Koji; Ishizawa, Shin; Hondoh, Hiroaki; Yamanaka, Ryuya

2017-10-01

Programmed cell death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) have been shown to predict response to PD-L1/PD-1-targeted therapy. We analyzed PD-L1 expression in primary central nervous system lymphomas (PCNSLs). PD-L1 protein and mRNA expression were evaluated in 64 PCNSL tissue samples. IFN-γ, IL-10, CD4, and CD8 mRNA expression was also evaluated. PD-L1 protein was detected in tumor cells in 2 (4.1%) cases and in tumor microenvironments in 25 (52%) cases. PD-L1 mRNA positively correlated with IFN-γ (p=0.0024) and CD4 (p=0.0005) mRNA expression. IFN-γ mRNA positively correlated with CD8 mRNA expression (p=0.0001). Furthermore, tumor cell PD-L1 expression correlated positively with overall survival (p=0.0177), whereas microenvironmental PD-L1 expression exhibited an insignificant negative trend with overall survival (p=0.188). PD-L1 was expressed on both tumor and/or tumor-infiltrating immune cells in PCNSL. The biological roles of this marker warrant further investigation. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

PubMed

Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

2016-04-26

Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

Platelet-activating factor mediates monocyte chemoattractant protein-1 expression in glomerular immune injury.

PubMed

Jocks, T; Freudenberg, J; Zahner, G; Stahl, R A

1998-01-01

These studies were designed to determine the possible role of platelet-activating factor (PAF) in the production of monocyte chemoattractant protein-1 (MCP-1) in glomerular immune injury. The glomerular lesion was induced in isolated perfused rat kidneys by a rabbit anti-rat-thymocyte serum (ATS) and rat serum (RS) as a complement source. Perfusion of kidneys with ATS and RS results in the selective binding of the antiserum to the glomerular mesangium with consecutive intraglomerular activation of complement. Antibody binding and complement activation induced a significant increase in glomerular MCP-1 mRNA levels when assessed by Northern blotting or RT-PCR. Decomplemented RS or non antibody rabbit IgG had only moderate effects on glomerular MCP-1 mRNA levels. The PAF receptor antagonist WEB 2170 almost completely blocked the ATS and RS induced MCP-1 mRNA levels. Perfusion of control kidneys with PAF increased MCP-1 mRNA expression, an effect which was blocked by WEB 2170. Glomerular MCP-1 protein formation, assessed by Western blotting, was stimulated following ATS and RS and PAF, respectively, was blocked by WEB 2170. These data show that PAF, derived from glomerular resident cells following antibody and complement induced injury, stimulates MCP-1 expression. In addition to the direct effects on leukocyte adhesion and activation PAF may mediate inflammatory cell influx in glomerular injuries due to the release of MCP-1.

The Effects of Exercise on Expression of CYP19 and StAR mRNA in Steroid-Induced Polycystic Ovaries of Female Rats.

PubMed

Aghaie, Fatemeh; Khazali, Homayoun; Hedayati, Mehdi; Akbarnejad, Ali

2018-01-01

Polycystic ovarian syndrome (PCOS) is the most frequent female endocrine disorder that affects 5-10% of women. PCOS is characterized by hyperandrogenism, oligo-/anovulation, and polycystic ovaries. The aim of the present research is to evaluate the expression of steroidogenic acute regulatory protein (StAR) and aromatase (CYP19) mRNA in the ovaries of an estradiol valerate (EV)-induced PCOS rat model, and the effect of treadmill and running wheel (voluntary) exercise on these parameters. In this experimental study, we divided adult female Wistar rats that weighed approximately 220 ± 20 g initially into control (n=10) and PCOS (n=30). Subsequently, PCOS group were divided to PCOS, PCOS with treadmill exercise (P-ExT), and PCOS with running wheel exercise (P-ExR) groups (n=10 per group). The expressions of StAR and CYP19 mRNA in the ovaries were determined by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). Data were analyzed by one-way ANOVA using SPSS software, version 16. The data were assessed at α=0.05. There was significantly lower mRNA expression of CYP19 in the EV-induced PCOS, running wheel and treadmill exercise rats compared to the control group (P<0.001). Treadmill exercise (P=0.972) and running wheel exercise (P=0.839) had no significant effects on CYP19 mRNA expression compared to the PCOS group. mRNA expression of StAR in the ovaries of the PCOS group indicated an increasing trend compared to the control group, however this was not statistically significant (P=0.810). We observed that 8 weeks of running wheel and treadmill exercises could not statistically decrease StAR mRNA expression compared to the PCOS group (P=0.632). EV-induced PCOS in rats decreased CYP19 mRNA expression, but had no effect on StAR mRNA expression. We demonstrated that running wheel and moderate treadmill exercise could not modify CYP19 and StAR mRNA expressions. Copyright© by Royan Institute. All rights reserved.

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.

Fluoride-Induced Autophagy via the Regulation of Phosphorylation of Mammalian Targets of Rapamycin in Mice Leydig Cells.

PubMed

Zhang, Jianhai; Zhu, Yuchen; Shi, Yan; Han, Yongli; Liang, Chen; Feng, Zhiyuan; Zheng, Heping; Eng, Michelle; Wang, Jundong

2017-10-11

Fluoride is known to impair testicular function and decrease testosterone levels, yet the underlying mechanisms remain inconclusive. The objective of this study is to investigate the roles of autophagy in fluoride-induced male reproductive toxicity using both in vivo and in vitro Leydig cell models. Using transmission electron microscopy and monodansylcadaverine staining, we observed increasing numbers of autophagosomes in testicular tissue, especially in Leydig cells of fluoride-exposed mice. Further study revealed that fluoride increased the levels of mRNA and protein expression of autophagy markers LC3, Beclin1, and Atg 5 in primary Leydig cells. Furthermore, fluoride inhibited the phosphorylation of mammalian targets of rapamycin and 4EBP1, which in turn resulted in a decrease in the levels of AKT and PI3K mRNA expression, as well as an elevation of the level of AMPK expression in both testes and primary Leydig cells. Additionally, fluoride exposure significantly changed the mRNA expression of the PDK1, TSC, and Atg13 regulator genes in primary Leydig cells but not in testicular cells. Taken together, our findings highlight the roles of autophagy in fluoride-induced testicular and Leydig cell damage and contribute to the elucidation of the underlying mechanisms of fluoride-induced male reproductive toxicity.

Expression of the Wilms' tumor gene WT1 in the murine urogenital system.

PubMed

Pelletier, J; Schalling, M; Buckler, A J; Rogers, A; Haber, D A; Housman, D

1991-08-01

The Wilms' tumor gene WT1 is a recessive oncogene that encodes a putative transcription factor implicated in nephrogenesis during kidney development. In this report we analyze expression of WT1 in the murine urogenital system. WT1 is expressed in non-germ-cell components of the testis and ovaries in both young and adult mice. In situ mRNA hybridization studies demonstrate that WT1 is expressed in the granulosa and epithelial cells of ovaries, the Sertoli cells of the testis, and in the uterine wall. In addition to the 3.1-kb WT1 transcript detected by Northern blotting of RNA from kidney, uterus, and gonads, there is an approximately 2.5-kb WT1-related mRNA species in testis. The levels of WT1 mRNA in the gonads are among the highest observed, surpassing amounts detected in the embryonic kidney. During development, these levels are differentially regulated, depending on the sexual differentiation of the gonad. Expression of WT1 mRNA in the female reproductive system does not fluctuate significantly from days 4 to 40 postpartum. In contrast, WT1 mRNA levels in the tesis increase steadily after birth, reaching their highest expression levels at day 8 postpartum and decreasing slightly as the animal matures. Expression of WT1 in the gonads is detectable as early as 12.5 days postcoitum (p.c.). As an initial step toward exploring the tissue-specific expression of WT1, DNA elements upstream of WT1 were cloned and sequenced. Three putative transcription initiation sites, utilized in testis, ovaries, and uterus, were mapped by S1 nuclease protection assays. The sequences surrounding these sites have a high G + C content, and typical upstream CCAAT and TATAA boxes are not present. These studies allowed us to identify the translation initiation site for WT1 protein synthesis. We have also used an epitope-tagging protocol to demonstrate that WT1 is a nuclear protein, consistent with its role as a transcription factor. Our results demonstrate regulation of WT1 expression during development of the gonads, implicate WT1 in genitourinary development, and provide a molecular framework toward understanding genitourinary defects observed among hereditary cases of Wilms' tumor.

Cytochrome P450-2C11 mRNA is not expressed in endothelial cells dissected from rat renal arterioles.

PubMed

Heil, Sandra G; De Vriese, An S; Kluijtmans, Leo A J; Dijkman, Henry; van Strien, Denise; Akkers, Robert; Blom, Henk J

2005-01-01

Cytochrome P450 (CYP) isoenzymes (CYP2C and CYP2J) are involved in the production of epoxyeicosatrienoic acids, which are postulated as endothelium-derived hyperpolarizing factors (EDHFs). We hypothesized that if CYP2C11 is involved in the EDHF-mediated responses, its mRNA should be expressed in endothelial cells. We, therefore, examined the mRNA expression of CYP2C11 in endothelial cells of renal arterioles. Laser microdissection was applied to isolate endothelial cells from the renal arterioles of 4 male and 4 female Wistar rats. As a positive control of CYP2C11 expression, hepatocytes were also dissected from these rats. RNA was isolated and real-time quantitative polymerase chain reaction (Q-PCR) analysis was applied. Q-PCR analysis showed that CYP2C11 mRNA was not expressed in laser microdissected endothelial cells of renal arterioles of male and female rats. CYP2C11 mRNA expression was highly abundant in hepatocytes dissected from male livers, but in female livers hardly any CYP2C11 mRNA was detected. We have shown that endothelial cells can be dissected from small renal arterioles by laser microdissection to study the mRNA expression of specific genes by Q-PCR. Using this novel tool, we demonstrated that the CYP2C11 mRNA was not expressed in the endothelial cells of renal arterioles. Therefore, we speculate that CYP2C11 does not contribute to the EDHF-mediated responses in renal arterioles. Copyright (c) 2005 S. Karger AG, Basel.

FLT3-ITD and MLL-PTD influence the expression of MDR-1, MRP-1, and BCRP mRNA but not LRP mRNA assessed with RQ-PCR method in adult acute myeloid leukemia.

PubMed

Nasilowska-Adamska, Barbara; Solarska, Iwona; Paluszewska, Monika; Malinowska, Iwona; Jedrzejczak, Wieslaw W; Warzocha, Krzysztof

2014-04-01

Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene-partial tandem duplication (MLL-PTD) are aberrations associated with leukemia which indicate unsatisfactory prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined. We have analyzed the expression of MDR-1, multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) messenger RNA (mRNA) in relation to the mutational status of FLT3-ITD and MLL-PTD in 185 acute myeloid leukemia (AML) adult patients. The real-time quantitative polymerase chain reaction method was performed to assess the expression of the MDR-1, MRP-1, BCRP, and LRP mRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl's rule. Significantly higher expressions of MDR-1 mRNA were found in patients who did not harbor FLT3-ITD (0.20 vs. 0.05; p = 0.0001) and MRP-1 mRNA in patients with this mutation (0.96 vs. 0.70; p = 0.002) and of BCRP mRNA in patients with MLL-PTD (0.61 vs. 0.38; p = 0.03). In univariate analysis, the high expression of MDR-1 mRNA (≥0.1317) negatively influenced the outcome of induction therapy (p = 0.05), whereas the high expression of BCRP mRNA (≥1.1487) was associated with a high relapse rate (RR) (p = 0.013). We found that the high expression of MDR-1 (≥0.1317), MRP-1 (≥0.8409), and BCRP mRNA (≥1.1487) significantly influenced disease-free survival (DFS; p = 0.059, 0.032, and 0.009, respectively) and overall survival (0.048, 0.014, and 0.059, respectively). Moreover, a high expression of BCRP mRNA (≥1.1487) proved to be an independent prognostic factor for RR (p = 0.01) and DFS (p = 0.002) in multivariate analysis. The significant correlation between the expression of MDR-1, MRP-1, and BCRP mRNA and FLT3-ITD or MLL-PTD in AML patients requires further investigation.

The effect of water deprivation on the expression of atrial natriuretic peptide and its receptors in the spinifex hopping mouse, Notomys alexis.

PubMed

Heimeier, Rachel A; Davis, Belinda J; Donald, John A

2002-08-01

This study investigated the mRNA expression of the atrial natriuretic peptide (ANP) system (peptide and receptors) during water deprivation in the spinifex hopping mouse, Notomys alexis, a native of central and western Australia that is well adapted to survive in arid environments. Initially, ANP, NPR-A and NPR-C cDNAs (partial for receptors) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. Using a semi-quantitative multiplex PCR technique, the expression of cardiac ANP mRNA and renal ANP, NPR-A, and NPR-C mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control mice (access to water). The levels of ANP mRNA expression in the heart remained unchanged, but in the kidney ANP mRNA levels were increased in the 7-day water-deprived mice, and were significantly decreased in the 14-day water-deprived mice. NPR-A mRNA levels were significantly higher in 7-day water-deprived mice while no change for NPR-A mRNA expression was observed in 14-day water-deprived mice. No variation in NPR-C mRNA levels was observed. This study shows that water deprivation differentially affects the expression of the ANP system, and that renal ANP expression is more important than cardiac ANP in the physiological adjustment to water deprivation.

Expression of Allene Oxide Synthase Determines Defense Gene Activation in Tomato1

PubMed Central

Sivasankar, Sobhana; Sheldrick, Bay; Rothstein, Steven J.

2000-01-01

Allene oxide synthase (AOS; hydroperoxide dehydratase; EC 4.2.1.92) catalyzes the first step in the biosynthesis of jasmonic acid from lipoxygenase-derived hydroperoxides of free fatty acids. Using the AOS cDNA from tomato (Lycopersicon esculentum), in which the role of jasmonic acid in wound-induced defense gene activation has been best described, we examined the kinetics of AOS induction in response to wounding and elicitors, in parallel with that of the wound-inducible PIN II (proteinase inhibitor II) gene. AOS was induced in leaves by wounding, systemin, 12-oxophytodienoic acid, and methyl jasmonate. The levels of AOS mRNA started declining by 4 h after induction, whereas the levels of PIN II mRNA continued to increase up to 20 h after induction. Salicylic acid inhibited AOS and PIN II expression, and the addition of 12-oxophytodienoic acid or methyl jasmonate did not prevent the inhibition of PIN II expression in the presence of salicylic acid. Ethylene induced the expression of AOS, but the presence of ethylene alone did not produce an optimal induction of PIN II. The addition of silver thiosulfate, an ethylene action inhibitor, prevented the wound-induced expression of both AOS and PIN II. Products of hydroperoxide lyase affected neither AOS nor PIN II, but induced expression of prosystemin. Based on these results, we propose an updated model for defense gene activation in tomato. PMID:10759530

β1- and β2-adrenergic receptor stimulation differ in their effects on PGC-1α and atrogin-1/MAFbx gene expression in chick skeletal muscle.

PubMed

Shimamoto, Saki; Ijiri, Daichi; Kawaguchi, Mana; Nakashima, Kazuki; Tada, Osamu; Inoue, Hiroki; Ohtsuka, Akira

2017-09-01

Adrenaline changes expression of the genes encoding peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α), which is known as a regulator of muscle size, and atrogin-1/muscle atrophy F-box (MAFbx), which is a muscle-specific ubiquitin ligase. However, the subtype of β-adrenergic receptor (β-AR) involved in regulating these genes in skeletal muscle is not yet well defined. In this study, the effects of intraperitoneal injection of adrenaline and three β 1-3 -AR selective agonists on chick skeletal muscle metabolism were examined, to evaluate the functions of β-AR subtypes. Adrenaline decreased atrogin-1/MAFbx mRNA levels accompanied by an increase in PGC-1α mRNA and protein levels. However, among the three selective agonists, only the β 1 -AR agonist, dobutamine, increased PGC-1α mRNA and protein levels, while the β 2 -AR agonist, clenbuterol, suppressed atrogin-1/MAFbx mRNA levels. In addition, preinjection of the β 1 -AR antagonist, acebutolol, and the β 2 -AR antagonist, butoxamine, inhibited the adrenaline-induced increase in PGC-1α mRNA levels and the decrease in atrogin-1/MAFbx mRNA levels, respectively. Compared with adrenaline administration, the β 3 -AR agonist, BRL37344, decreased PGC-1α mRNA levels and increased atrogin-1/MAFbx mRNA levels. These results suggest that, in chick skeletal muscle, PGC-1α is induced via the β 1 -AR, while atrogin-1/MAFbx is suppressed via the β 2 -AR. Copyright © 2017. Published by Elsevier Inc.

Overexpression of dihydrofolate reductase is a factor of poor survival in acute lymphoblastic leukemia.

PubMed

Organista-Nava, Jorge; Gómez-Gómez, Yazmín; Illades-Aguiar, Berenice; Rivera-Ramírez, Ana Bertha; Saavedra-Herrera, Mónica Virginia; Leyva-Vázquez, Marco Antonio

2018-06-01

Dihydrofolate reductase (DHFR) has an important function in DNA synthesis and is a target of methotrexate, which is a crucial treatment option for acute lymphoblastic leukemia (ALL). However, the number of studies conducted to date on DHFR expression in childhood ALL is limited. The aim of the present study was to determine whether the expression of DHFR is associated with survival in childhood ALL. The expression of DHFR in 96 children with ALL and 100 control individuals was determined using reverse transcription-quantitative polymerase chain reaction. The results of the present study demonstrated that the expression of DHFR mRNA in children with ALL was significantly increased (P<0.001), compared with that in the control group. In addition, increased levels of DHFR mRNA were observed in patients with B-cell lineage, compared with patients with T-cell lineage ALL (P<0.05). The Kaplan-Meier estimator analysis revealed that children with ALL who exhibited increased levels of DHFR mRNA had a decreased overall survival time (P<0.05). It was observed that certain patient prognostic features (including age, sex, white blood cell count and high DHFR expression), are associated with poor survival (log-rank test, P<0.05). Therefore, the results of the present study indicated that DHFR upregulation is a factor for poor survival in ALL.

Selenium regulates gene expression of selenoprotein W in chicken skeletal muscle system.

PubMed

Ruan, Hongfeng; Zhang, Ziwei; Wu, Qiong; Yao, Haidong; Li, Jinlong; Li, Shu; Xu, Shiwen

2012-01-01

Selenoprotein W (SelW) is abundantly expressed in skeletal muscles of mammals and necessary for the metabolism of skeletal muscles. However, its expression pattern in skeletal muscle system of birds is still uncovered. Herein, to investigate the distribution of SelW mRNA in chicken skeletal muscle system and its response to different selenium (Se) status, 1-day-old chickens were exposed to various concentrations of Se as sodium selenite in the feed for 35 days. In addition, myoblasts were treated with different concentrations of Se in the medium for 72 h. Then the levels of SelW mRNA in skeletal muscles (wing muscle, pectoral muscle, thigh muscle) and myoblasts were determined on days 1, 15, 25, and 35 and at 0, 24, 48, and 72 h, respectively. The results showed that SelW was detected in all these muscle components and it increased both along with the growth of organism and the differentiation process of myoblasts. The thigh muscle is more responsive to Se intake than the other two skeletal muscle tissues while the optimal Se supplementation for SelW mRNA expression in chicken myoblasts was 10(-7) M. In summary, Se plays important roles in the development of chicken skeletal muscles. To effect optimal SelW gene expression, Se must be provided in the diet and the media in adequate amounts and neither at excessive nor deficient levels.

Soluble soy protein peptic hydrolysate stimulates adipocyte differentiation in 3T3-L1 cells.

PubMed

Goto, Tsuyoshi; Mori, Ayaka; Nagaoka, Satoshi

2013-08-01

The molecular mechanisms underlying the potential health benefit effects of soybean proteins on obesity-associated metabolic disorders have not been fully clarified. In this study, we investigated the effects of soluble soybean protein peptic hydrolysate (SPH) on adipocyte differentiation by using 3T3-L1 murine preadipocytes. The addition of SPH increased lipid accumulation during adipocyte differentiation. SPH increased the mRNA expression levels of an adipogenic marker gene and decreased that of a preadipocyte marker gene, suggesting that SPH promotes adipocyte differentiation. SPH induced antidiabetic and antiatherogenic adiponectin mRNA expression and secretion. Moreover, SPH increased the mRNA expression levels of insulin-responsive glucose transporter 4 and insulin-stimulated glucose uptake. The expression levels of peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation, during adipocyte differentiation were up-regulated in 3T3-L1 cells treated with SPH, and lipid accumulation during adipocyte differentiation induced by SPH was inhibited in the presence of a PPARγ antagonist. However, SPH did not exhibit PPARγ ligand activity. These findings indicate that SPH stimulates adipocyte differentiation, at least in part, via the up-regulation of PPARγ expression levels. These effects of SPH might be important for the health benefit effects of soybean proteins on obesity-associated metabolic disorders. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication.

PubMed

Courtney, David G; Kennedy, Edward M; Dumm, Rebekah E; Bogerd, Hal P; Tsai, Kevin; Heaton, Nicholas S; Cullen, Bryan R

2017-09-13

Many viral RNAs are modified by methylation of the N 6 position of adenosine (m 6 A). m 6 A is thought to regulate RNA splicing, stability, translation, and secondary structure. Influenza A virus (IAV) expresses m 6 A-modified RNAs, but the effects of m 6 A on this segmented RNA virus remain unclear. We demonstrate that global inhibition of m 6 A addition inhibits IAV gene expression and replication. In contrast, overexpression of the cellular m 6 A "reader" protein YTHDF2 increases IAV gene expression and replication. To address whether m 6 A residues modulate IAV RNA function in cis, we mapped m 6 A residues on the IAV plus (mRNA) and minus (vRNA) strands and used synonymous mutations to ablate m 6 A on both strands of the hemagglutinin (HA) segment. These mutations inhibited HA mRNA and protein expression while leaving other IAV mRNAs and proteins unaffected, and they also resulted in reduced IAV pathogenicity in mice. Thus, m 6 A residues in IAV transcripts enhance viral gene expression. Copyright © 2017 Elsevier Inc. All rights reserved.

[The Effect of TALENs-mediated Downregulation Expression of Nanog on Malignant Behavior of Cervical Cancer HeLa Cells].

PubMed

Yu, Ai-qing; Li, Cheng-lin; Yang, Yi; Yan, Shi-rong

2016-01-01

To study the effect of downregulation expression of Nanog on malignant behavior of cervical cancer HeLa cells. Gene editing tool TALENs was employed to induce downregulation expression of Nanog, and Nanog mutation was evaluated by sequencing. RT-PCR and Western blot was used to detect the mRNA and protein expression level, respectively. Colony-formation assay, Transwell invasion assay, and chemotherapy sensibility assay was carried out to assess the capacity of colony-formation, invasion, and chemoresistance, respectively. TALENs successfully induced Nanog mutation and downregulated Nanog expression. Nanog mRNA and protein expression of Nanog-mutated monoclonal HeLa cells downregulated 3 times compared to thoses of wild-type HeLa cells (P < 0.05). Additionally, significant weakened abilities of colony-formation, invasion, and chemoresistance in monoclonal HeLa cells were observed when compared to those of wild-type HeLa cells (P < 0.05). Nanog mutation attenuates the malignant behavior of HeLa cells. Importantly, downregulation or silencing of Nanog is promising to be a novel strategy for the treatment of cervical carcinoma.

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

Waltman, Peter H.; Guo, Jian; Reistetter, Emily Nahas

Micromonas is a unicellular green alga that belongs to the prasinophytes, a sister lineage to land plants. This picoeukaryotic (<2 μm diameter) alga is widespread in the marine environment but still not understood at the cellular level. Here, we examine the mRNA and protein level changes that take place over the course of the day-night cycle using mid-exponential nutrient replete cultures of Micromonas pusilla CCMP1545 grown and analyzed in biological triplicate. During the experiment, samples were collected at key transition points during the diel for evaluation using high-throughput LC-MS proteomics. We also sequenced matched mRNA samples from the same timemore » points, using pair-ended directional Illumina RNA-Seq to investigate the dynamics and relationship between the mRNA and protein expression programs of M. pusilla. Similar to a prior study of the marine cyanobacterium Prochlorococcus, we found significant divergence in the mRNA and proteomics expression dynamics in response to the light:dark cycle. Additionally, expressional responses of genes and the proteins they encoded could also be variable within the same metabolic pathway, such as the oxygenic photosynthesis pathway. A regression framework was used to predict protein levels using both mRNA expression and gene-specific sequence-based features. Several features in the genome sequence were found to influence protein abundance including the codon usage and the length of the 3’ UTR. Collectively, our studies provide insights into the regulation of the proteome over a diel as relationships between the transcriptional and translational programs in the widespread marine green alga Micromonas.« less

Stress induction of Bm1 RNA in silkworm larvae: SINEs, an unusual class of stress genes

PubMed Central

Kimura, Richard H.; Choudary, Prabhakara V.; Stone, Koni K.; Schmid, Carl W.

2001-01-01

This study surveys the induction of RNA polymerase III (Pol III)–directed expression of short interspersed element (SINE) transcripts by various stresses in an animal model, silkworm larvae. Sublethal heat shock and exposure to several toxic compounds increase the level of Bm1 RNA, the silkworm SINE transcript, while also transiently increasing expression of a well-characterized stress-induced transcript, Hsp70 messenger RNA (mRNA). In certain cases, the Bm1 RNA response coincides with that of Hsp70 mRNA, but more often Bm1 RNA responds later in recovery. Baculovirus infection and exposure to certain toxic compounds increase Bm1 RNA but not Hsp70 mRNA, showing that SINE induction is not necessarily coupled to transcription of this particular heat shock gene. SINEs behave as an additional class of stress-inducible genes in living animals but are unusual as stress genes because of their high copy number, genomic dispersion, and Pol III–directed transcription. PMID:11599568

Lipopolysaccharide-binding protein and leptin are associated with stress-induced interleukin-6 cytokine expression ex vivo in obesity.

PubMed

Huang, Chun-Jung; Stewart, Jennifer K; Shibata, Yoshimi; Slusher, Aaron L; Acevedo, Edmund O

2015-05-01

Obesity is associated with enhanced inflammation and mental stress, but limited information has addressed the potential additive effect of psychological stress on obesity-associated inflammation. This study examined whether obese subjects would elicit a greater host immune response (IL-6 mRNA and cytokine) to lipopolysaccharide (LPS) in response to mental stress. Blood samples for LPS-stimulated IL-6 mRNA and cytokine were collected prior to and following mental stress. Results showed that obese subjects elicited a greater LPS-induced IL-6 along with its mRNA expression following mental stress compared to normal-weight subjects. Stress-induced IL-6 cytokine response to LPS was correlated with the baseline levels of plasma LPS binding protein (LBP) and leptin. These findings are consistent with the idea that endogenous inflammatory agents (e.g., LBP and leptin), often elevated with obesity, enhance inflammatory responses to psychological stress. © 2014 Society for Psychophysiological Research.

Andrographis Paniculata shows anti-nociceptive effects in an animal model of sensory hypersensitivity associated with migraine

PubMed Central

Greco, Rosaria; Siani, Francesca; Demartini, Chiara; Zanaboni, Annamaria; Nappi, Giuseppe; Davinelli, Sergio; Scapagnini, Giovanni; Tassorelli, Cristina

2016-01-01

Administration of nitroglycerin (NTG) to rats induces a hyperalgesic condition and neuronal activation of central structures involved in migraine pain. In order to identify therapeutic strategies for migraine pain, we evaluated the anti-nociceptive activity of Andrographis Paniculata (AP), a herbaceous plant, in the hyperalgesia induced by NTG administration in the formalin test. We also analyzed mRNA expression of cytokines in specific brain areas after AP treatment. Male Sprague-Dawley rats were pre-treated with AP extract 30 minutes before NTG or vehicle injection. The data show that AP extract significantly reduced NTG-induced hyperalgesia in phase II of the test, 4 hours after NTG injection. In addition, AP extract reduced IL-6 mRNA expression in the medulla and mesencephalon and also mRNA levels of TNF-alpha in the mesencephalic region. These findings suggest that AP extract may be a potential therapeutic approach in the treatment of general pain, and possibly of migraine. PMID:27027895

Andrographis Paniculata shows anti-nociceptive effects in an animal model of sensory hypersensitivity associated with migraine.

PubMed

Greco, Rosaria; Siani, Francesca; Demartini, Chiara; Zanaboni, Annamaria; Nappi, Giuseppe; Davinelli, Sergio; Scapagnini, Giovanni; Tassorelli, Cristina

2016-01-01

Administration of nitroglycerin (NTG) to rats induces a hyperalgesic condition and neuronal activation of central structures involved in migraine pain. In order to identify therapeutic strategies for migraine pain, we evaluated the anti-nociceptive activity of Andrographis Paniculata (AP), a herbaceous plant, in the hyperalgesia induced by NTG administration in the formalin test. We also analyzed mRNA expression of cytokines in specific brain areas after AP treatment. Male Sprague-Dawley rats were pre-treated with AP extract 30 minutes before NTG or vehicle injection. The data show that AP extract significantly reduced NTG-induced hyperalgesia in phase II of the test, 4 hours after NTG injection. In addition, AP extract reduced IL-6 mRNA expression in the medulla and mesencephalon and also mRNA levels of TNFalpha in the mesencephalic region. These findings suggest that AP extract may be a potential therapeutic approach in the treatment of general pain, and possibly of migraine.

Increasing cellular level of phosphatidic acid enhances FGF-1 production in long term-cultured rat astrocytes.

PubMed

Nagayasu, Yuko; Morita, Shin-Ya; Hayashi, Hideki; Miura, Yutaka; Yokoyama, Kazuki; Michikawa, Makoto; Ito, Jin-Ichi

2014-05-14

We found in a previous study that both mRNA expression and release of fibroblast growth factor 1 (FGF-1) are greater in rat astrocytes that are long term-cultured for one month (W/M cells) than in the cells cultured for one week (W/W cells). However, FGF-1 does not enhance phosphorylation of Akt, MEK, and ERK in W/M cells, while it does in W/W cells. In this work we studied the mechanism to cause these differences between W/W and W/M cells in culture. As it is known that long term culture generates oxidative stress, we characterized the stresses which W/M cells undergo in comparison with W/W cells. The levels of superoxide dismutase 1 (SOD1) and mitochondrial Bax were higher in W/M cells than in W/W cells. W/M cells recovered their ability to respond to FGF-1 to enhance phosphorylation of Akt, MEK, and ERK in the presence of antioxidants. Oxidative stress induced by hydrogen peroxide (H2O2) had no effect on mRNA expression of FGF-1 in W/W cells, although H2O2 enhances release of FGF-1 from W/W cells without inducing apoptosis. The influence of cell density was studied on mRNA expression of FGF-1 and cellular response to FGF-1, as an increasing cell density is observed in W/M cells. The increasing cell density enhanced mRNA expression of FGF-1 in W/W cells without suppression of responses to FGF-1. The decrease in cell density lowered the FGF-1 mRNA expression in W/M cells without recovery of the response to FGF-1 to enhance phosphorylation of Akt, MEK, and ERK. These findings suggest that oxidative stress attenuate sensitivity to FGF-1 and higher cell density may enhance FGF-1 expression in W/M cells. In addition, we found that the cellular level of phosphatidic acid (PA) increased in H2O2-treated W/W and W/M cells and decreased by the treatment with antioxidants, and that PA enhances the mRNA expression of FGF-1 in the W/W cells. These findings suggest that the increasing PA production may enhance FGF-1 expression to protect astrocytes against oxidative stress induced by long-term culture. Copyright © 2014 Elsevier B.V. All rights reserved.

Pattern-Recognition Receptor Signaling Regulator mRNA Expression in Humans and Mice, and in Transient Inflammation or Progressive Fibrosis

PubMed Central

Günthner, Roman; Kumar, Vankayala Ramaiah Santhosh; Lorenz, Georg; Anders, Hans-Joachim; Lech, Maciej

2013-01-01

The cell type-, organ-, and species-specific expression of the pattern-recognition receptors (PRRs) are well described but little is known about the respective expression profiles of their negative regulators. We therefore determined the mRNA expression levels of A20, CYLD, DUBA, ST2, CD180, SIGIRR, TANK, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, SHP1, SHP2, TOLLIP, IRF4, SIKE, NLRX1, ERBIN, CENTB1, and Clec4a2 in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. Additionally, we characterized their expression profiles in mononuclear blood cells upon bacterial endotoxin, which showed a consistent induction of A20, SOCS3, IRAK-M, and Clec4a2 in human and murine cells. Furthermore, we studied the expression pattern in transient kidney ischemia-reperfusion injury versus post-ischemic atrophy and fibrosis in mice. A20, CD180, ST2, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, IRF4, CENTB1, and Clec4a2 were all induced, albeit at different times of injury and repair. Progressive fibrosis was associated with a persistent induction of these factors. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to PRR-mediated innate immunity, which seems to be involved in tissue injury, tissue regeneration and in progressive tissue scarring. PMID:24009023

Impact of STAT/SOCS mRNA Expression Levels after Major Injury

PubMed Central

Brumann, M.; Matz, M.; Kusmenkov, T.; Stegmaier, J.; Biberthaler, P.; Kanz, K.-G.; Mutschler, W.; Bogner, V.

2014-01-01

Background. Fulminant changes in cytokine receptor signalling might provoke severe pathological alterations after multiple trauma. The aim of this study was to evaluate the posttraumatic imbalance of the innate immune system with a special focus on the STAT/SOCS family. Methods. 20 polytraumatized patients were included. Blood samples were drawn 0 h–72 h after trauma; mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3 were quantified by qPCR. Results. IL-10 mRNA expression increased significantly in the early posttraumatic period. STAT 3 mRNA expressions showed a significant maximum at 6 h after trauma. SOCS 1 levels significantly decreased 6 h–72 h after trauma. SOCS 3 levels were significantly higher in nonsurvivors 6 h after trauma. Conclusion. We present a serial, sequential investigation in human neutrophil granulocytes of major trauma patients evaluating mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3. Posttraumatically, immune disorder was accompanied by a significant increase of IL-10 and STAT 3 mRNA expression, whereas SOCS 1 mRNA levels decreased after injury. We could demonstrate that death after trauma was associated with higher SOCS 3 mRNA levels already at 6 h after trauma. To support our results, further investigations have to evaluate protein levels of STAT/SOCS family in terms of posttraumatic immune imbalance. PMID:24648661

Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

PubMed Central

Larsson, S; Hotchkiss, G; Andäng, M; Nyholm, T; Inzunza, J; Jansson, I; Ahrlund-Richter, L

1994-01-01

We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels. Images PMID:8036151

Overexpression of early growth response-1 as a metastasis-regulatory factor in gastric cancer.

PubMed

Kobayashi, Daisuke; Yamada, Mikako; Kamagata, Chinatsu; Kaneko, Reiko; Tsuji, Naoki; Nakamura, Masashi; Yagihashi, Atsuhito; Watanabe, Naoki

2002-01-01

To investigate the potential role of a nuclear transcription factor, early growth response-1 (Egr-1), in formation and progression of gastric cancer, we compared its expression in gastric cancers with that in non-cancerous tissues. Egr-1 mRNA expression was measured using TaqMan RT-PCR. The corresponding protein expression was examined immunohistochemically. Egr-1 mRNA expression was significantly higher in gastric cancer tissues than in normal mucosa (p < 0.0005). These differences were also reflected by protein product expression. Moreover, Egr-1 mRNA expression was higher in cases with metastasis to lymph nodes or remote organs. In cultured gastric cancer cells known to have a high metastatic potential, expression of this mRNA was higher than that of parental cells. It was suggested that Egr-1 has a significant role in carcinogenesis and in cancer progression, especially metastasis. Measurement of this mRNA should be useful for evaluation of the metastatic potential of gastric cancer.

Promoter methylation, mRNA expression of goat tumor‑associated genes and mRNA expression of DNA methyltransferase in enzootic nasal tumors.

PubMed

Quan, Zifang; Ye, Ni; Hao, Zhongxiang; Wen, Caifang; Liao, Hong; Zhang, Manli; Luo, Lu; Cao, Sanjie; Wen, Xintian; Wu, Rui; Yan, Qigui

2015-10-01

The aim of the present study was to investigate the promoter methylation status and mRNA expression of goat tumor‑associated genes, in addition to the mRNA expression of DNA methyltransferase genes in enzootic nasal tumors (ENT). Methylation‑specific polymerase chain reaction and SYBR Green reverse transcription‑quantitative polymerase chain reaction were used to detect the methylation status and the mRNA expression levels of DNA methyltransferases (DNMTs), O6‑methylguanine‑DNA methyltransferase (MGMT), the tumor suppressor genes P73, P53, GADD45G, CHFR and THBS1, the transcription factor CEBPA, the proto‑oncogenes KRAS, NRAS and C‑myc and EGFR in 24 nasal tumor tissue samples and 20 normal nasal epithelia tissue samples. The associations between promoter methylation and DNMT, and promoter methylation and mRNA expression of the genes were analyzed. The results indicated that the expression levels of DNMT1 increased by 56% compared with those in normal nasal epithelial tissues, while MGMT, DNMT3a and DNMT3b had similar expression levels in the two tissue types. The expression levels of P53 decreased by 36.8% and those of THBS1 by 43%, while C‑myc increased by 2.9‑fold and CEBPA by 2‑fold compared with that in normal nasal epithelial tissues. GADD45G, P73, CHFR and NRAS were observed to have similar expression levels in the two tissue types. However, no expression was observed for EGFR and KRAS. CHFR, GADD45G and THBS1 were identified to be methylated in tumor suppressor genes. The methylation expression rate of the CHFR gene was ~60% in the two tissue types and for THBS1 it was 100% in the nasal tumor tissues as opposed to 20% in the normal nasal epithelial tissues. The exhaustive methylation expression rate of GADD45G was 62.5% and the partial methylation expression rate was 37.5% in nasal tumor tissue, while no methylation was observed in normal nasal epithelial tissues. C‑myc was the only gene identified to be methylated amongst proto‑oncogenes. The methylation expression rate of C‑myc was 87.5% in nasal tumor tissues and 15% in normal nasal epithelial tissues. The methylation expression rate of CEBPA was 100% in nasal tumor tissues and 40% in normal nasal epithelial tissues. The methylation expression rate of the EGFR gene was ~80% in the two tissues. In summary, the present study identified abnormal methylation of the C‑myc, CEBPA, GADD45G and THBS1 genes in nasal tumor tissues. The expression levels of DNMT1, C‑myc and CEBPA were upregulated and the expression of P53 and THBSI were downregulated in nasal tumor tissues, with a significant difference between the two groups (P<0.05). Therefore, it is suggested that these six genes may be used as diagnostic marker candidates for ENT. The results may serve as a foundation for screening of tumor‑specific markers for early diagnosis of ENT and further investigate the epigenetic mechanisms of enzootic nasal tumor virus (ENTV)‑induced nasal epithelium cell carcinoma.

Expression of very low density lipoprotein receptor mRNA in circulating human monocytes: its up-regulation by hypoxia.

PubMed

Nakazato, K; Ishibashi, T; Nagata, K; Seino, Y; Wada, Y; Sakamoto, T; Matsuoka, R; Teramoto, T; Sekimata, M; Homma, Y; Maruyama, Y

2001-04-01

Although very low density lipoprotein (VLDL) receptor expression by macrophages has been shown in the vascular wall, it is not clear whether or not circulating monocytes express the VLDL receptor. We investigated the expression of VLDL receptor mRNA in human peripheral blood monocytes and monocyte-derived macrophages by reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequencing after subcloning of PCR product. VLDL receptor mRNA was detected both in peripheral blood monocytes and monocyte-derived macrophages. Expression of VLDL receptor mRNA was upregulated by hypoxia in monocytes, whereas treatment with oxidized LDL, interleukin-1beta or monocyte chemoattractant protein-1 did not affect the levels of VLDL receptor mRNA in monocytes and macrophages. The present study shows a novel response of VLDL receptor mRNA to hypoxia, suggesting a role for VLDL receptor in the metabolism of lipoproteins in the vascular wall and the development of atherosclerosis.

Modulation of tumor necrosis factor (TNF) receptor expression during monocytic differentiation by glucocorticoids.

PubMed

Goppelt-Struebe, M; Reiser, C O; Schneider, N; Grell, M

1996-10-01

Regulation of tumor necrosis factor receptors by glucocorticoids was investigated during phorbol ester-induced monocytic differentiation. As model system the human monocytic cell lines U937 and THP-1, which express both types of TNF receptors (TNF-R60 and TNF-R80), were differentiated with tetradecanoyl phorbol-13-acetate (TPA, 5 x 10(-9) M) in the presence or absence of dexamethasone (10(-9) - 10(-6) M). Expression of TNF receptors was determined at the mRNA level by Northern blot analysis and at the protein level by FACS analysis. During differentiation, TNF-R60 mRNA was down-regulated, whereas TNF-R80 mRNA levels were increased. Dexamethasone had no effect on TNF-R60 mRNA expression but attenuated TNF-R80 mRNA expression in both cell lines. Cell surface expression of TNF-R60 protein remained essentially unchanged during differentiation of THP-1 cells, whereas a rapid down-regulation of TNF-R80 was observed that was followed by a slow recovery. Surface expression of TNF-R80 was not affected by dexamethasone, whereas TNF-R60 expression was reduced by about 25%. These results indicate differential regulation of the two types of TNF receptors at the mRNA and protein level during monocytic differentiation. Glucocorticoids interfered with mRNA expression of TNF-R80 and protein expression of TNF-R60, but the rather limited effect leaves the question of its functional relevance open. In contrast to other cytokine systems, TNF receptors do not appear to be major targets of glucocorticoid action.

Notch Signaling Is Involved in Neurogenic Commitment of Human Periodontal Ligament-Derived Mesenchymal Stem Cells

PubMed Central

Osathanon, Thanaphum; Manokawinchoke, Jeeranan; Nowwarote, Nunthawan; Aguilar, Panuroot; Palaga, Tanapat

2013-01-01

Notch signaling plays critical roles in stem cells by regulating cell fate determination and differentiation. The aim of this study was to evaluate the participation of Notch signaling in neurogenic commitment of human periodontal ligament-derived mesenchymal stem cells (hPDLSCs) and to examine the ability to control differentiation of these cells using modified surfaces containing affinity immobilized Notch ligands. Neurogenic induction of hPDLSCs was performed via neurosphere formation. Cells were aggregated and form spheres as early 1 day in culture. In addition, the induced cells exhibited increased mRNA and protein expression of neuronal markers that is, β3-tubulin and neurofilament. During neuronal differentiation, a significant increase of Hes1 and Hey1 mRNA expression was noted. Using pharmacological inhibition (γ-secretase inhibitor) or genetic manipulation (overexpression of dominant negative mastermind-like transcription co-activators), neurosphere formation was attenuated and a marked decrease in neurogenic mRNA expression was observed. To confirm the role of Notch signaling in neuronal differentiation of hPDLSCs, the Notch ligand, Jagged-1, is bound to the surface using an affinity immobilization technique. The hPDLSC cultured on a Jagged-1-modified surface had increased expression of Notch signaling target genes, Hes-1 and Hey-1, confirming the activity and potency of surface-bound Jagged-1. Further, hPDLSC on surface-bound Jagged-1 under serum-free conditions showed multiple long and thin neurite-like extensions, and an increase in the expression of neurogenic mRNA markers was observed. Pretreatment of the cells with γ-secretase inhibitor, DAPT, before seeding on the Jagged-1-modified surface blocked development of the neurite-like morphology. Together, the results in this study suggest the involvement of Notch signaling in neurogenic commitment of hPDLSCs. PMID:23379739

Herpes Simplex Virus Is Equipped with RNA- and Protein-Based Mechanisms To Repress Expression of ATRX, an Effector of Intrinsic Immunity

PubMed Central

Jurak, Igor; Silverstein, Leah B.; Sharma, Mayuri

2012-01-01

Intrinsic immunity is a first-line intracellular defense against virus infection, and viruses have evolved mechanisms to counteract it. During herpes simplex virus (HSV) infection, nuclear domain 10 (ND10) components localize adjacent to incoming viral genomes and generate a repressive environment for viral gene expression. Here, we found that the ND10 component, alpha-thalassemia/mental retardation syndrome X-linked (ATRX) protein, is predicted to be a target of HSV-1 miR-H1 and HSV-2 miR-H6. These microRNAs (miRNAs) share a seed sequence and are abundant during lytic infection. Mimics of both miRNAs could deplete endogenous ATRX, and an miR-H1 mimic could repress the expression of a reporter linked to the 3′ untranslated region of ATRX mRNA, identifying a cellular mRNA targeted by an HSV miRNA. Interestingly, ATRX protein and its mRNA were depleted in cells lytically infected with HSV, and ATRX protein was also depleted in cells infected with human cytomegalovirus. However, infection with an HSV-1 mutant lacking miR-H1 still resulted in ATRX depletion. This depletion was sensitive to a proteasome inhibitor and was largely ablated by a deletion of the gene encoding the immediate-early ICP0 protein. Additionally, a deletion of the gene encoding the tegument protein Vhs ablated most of the depletion of ATRX mRNA. Thus, HSV is equipped with multiple mechanisms to limit the expression of ATRX. As ATRX is implicated in repression of lytic viral gene expression, our results suggest roles for these different mechanisms during various phases of HSV infection. PMID:22787211

Atomic hydrogen surrounded by water molecules, H(H2O)m, modulates basal and UV-induced gene expressions in human skin in vivo.

PubMed

Shin, Mi Hee; Park, Raeeun; Nojima, Hideo; Kim, Hyung-Chel; Kim, Yeon Kyung; Chung, Jin Ho

2013-01-01

Recently, there has been much effort to find effective ingredients which can prevent or retard cutaneous skin aging after topical or systemic use. Here, we investigated the effects of the atomic hydrogen surrounded by water molecules, H(H2O)m, on acute UV-induced responses and as well as skin aging. Interestingly, we observed that H(H2O)m application to human skin prevented UV-induced erythema and DNA damage. And H(H2O)m significantly prevented UV-induced MMP-1, COX-2, IL-6 and IL-1β mRNA expressions in human skin in vivo. We found that H(H2O)m prevented UV-induced ROS generation and inhibited UV-induced MMP-1, COX-2 and IL-6 expressions, and UV-induced JNK and c-Jun phosphorylation in HaCaT cells. Next, we investigated the effects of H(H2O)m on intrinsically aged or photoaged skin of elderly subjects. In intrinsically aged skin, H(H2O)m application significantly reduced constitutive expressions of MMP-1, IL-6, and IL-1β mRNA. Additionally, H(H2O)m significantly increased procollagen mRNA and also decreased MMP-1 and IL-6 mRNA expressions in photoaged facial skin. These results demonstrated that local application of H(H2O)m may prevent UV-induced skin inflammation and can modulate intrinsic skin aging and photoaging processes. Therefore, we suggest that modifying the atmospheric gas environment within a room may be a new way to regulate skin functions or skin aging.

Injury downregulates the expression of the human cathelicidin protein hCAP18/LL-37 in atopic dermatitis.

PubMed

Mallbris, Lotus; Carlén, Lina; Wei, Tianling; Heilborn, Johan; Nilsson, Margareta Frohm; Granath, Fredrik; Ståhle, Mona

2010-05-01

Reduced production of antimicrobial peptides was proposed to contribute to susceptibility for skin infections in atopic dermatitis (AD). Focusing on the human cathelicidin protein, hCAP18, the aim of the present study was to explore whether reduced hCAP18 expression is a constitutive trait in AD and if established inducers affect the expression of hCAP18 in the skin of AD. First, we compared levels of hCAP18 mRNA between lesional skin in AD and psoriasis and verified significantly lower expression of hCAP18 mRNA in AD. In non-lesional skin, however, there was no difference between AD, psoriasis and healthy, indicating that there is no constitutive defect in the production of hCAP18 in AD patients. In healthy skin, hCAP18 was reported to be rapidly induced following wounding and here we verified this pattern in healthy controls and in psoriasis. In AD lesions, however, the expression of hCAP18 mRNA was markedly suppressed following wounding. Obviously, the inflammation in AD lesions neutralizes the expected induction of hCAP18 and even induces suppression. Notably, the mechanism to upregulate hCAP18 following vitamin D treatment was functional in lesional as well as in non-lesional AD indicating that the CAMP gene is normally regulated in this respect. In addition, cultured primary keratinocytes from non-lesional skin of psoriasis, AD and healthy skin, upregulated hCAP18mRNA following treatment with vitamin D. Itching is a hallmark of AD and scratching inevitably injures the skin. Failure to upregulate hCAP18 in eczema following injury is likely to affect antimicrobial protection and tissue repair in AD.

Alogliptin alleviates hepatic steatosis in a mouse model of nonalcoholic fatty liver disease by promoting CPT1a expression via Thr172 phosphorylation of AMPKα in the liver.

PubMed

Tobita, Hiroshi; Sato, Shuichi; Yazaki, Tomotaka; Mishiro, Tsuyoshi; Ishimura, Norihisa; Ishihara, Shunnji; Kinoshita, Yoshikazu

2018-05-01

Pioglitazone (PIO) has been reported to be effective for nonalcoholic fatty liver disease (NAFLD) and alogliptin (ALO) may have efficacy against NAFLD progression in patients with type 2 diabetes mellitus (T2DM). The present study examined the effectiveness of ALO in a rodent model of NAFLD and diabetes mellitus. KK‑Ay mice were used to produce an NAFLD model via administration of a choline‑deficient (CD) diet. To examine the effects of alogliptin, KK‑Ay mice were provided with a CD diet with 0.03% ALO and/or 0.02% PIO orally for 8 weeks. Biochemical parameters, pathological alterations and hepatic mRNA levels associated with fatty acid metabolism were assessed. Severe hepatic steatosis was observed in KK‑Ay mice fed with a CD diet, which was alleviated by the administration of ALO and/or PIO. ALO administration increased the hepatic carnitine palmitoyltransferase 1a (CPT1a) mRNA expression level and enhanced the Thr172 phosphorylation of AMP‑activated protein kinase α (AMPKα) in the liver. PIO administration tended to decrease the hepatic fatty acid synthase mRNA expression level and increase the serum adiponectin level. Homeostasis model of assessment‑insulin resistance values tended to improve with ALO and PIO administration. ALO and PIO alleviated hepatic steatosis in KK‑Ay mice fed with a CD diet. ALO increased hepatic mRNA expression levels associated with fatty acid oxidation. In addition, the results of the present study suggested that ALO promotes CPT1a expression via Thr172 phosphorylation of AMPKα.

Herpes simplex virus is equipped with RNA- and protein-based mechanisms to repress expression of ATRX, an effector of intrinsic immunity.

PubMed

Jurak, Igor; Silverstein, Leah B; Sharma, Mayuri; Coen, Donald M

2012-09-01

Intrinsic immunity is a first-line intracellular defense against virus infection, and viruses have evolved mechanisms to counteract it. During herpes simplex virus (HSV) infection, nuclear domain 10 (ND10) components localize adjacent to incoming viral genomes and generate a repressive environment for viral gene expression. Here, we found that the ND10 component, alpha-thalassemia/mental retardation syndrome X-linked (ATRX) protein, is predicted to be a target of HSV-1 miR-H1 and HSV-2 miR-H6. These microRNAs (miRNAs) share a seed sequence and are abundant during lytic infection. Mimics of both miRNAs could deplete endogenous ATRX, and an miR-H1 mimic could repress the expression of a reporter linked to the 3' untranslated region of ATRX mRNA, identifying a cellular mRNA targeted by an HSV miRNA. Interestingly, ATRX protein and its mRNA were depleted in cells lytically infected with HSV, and ATRX protein was also depleted in cells infected with human cytomegalovirus. However, infection with an HSV-1 mutant lacking miR-H1 still resulted in ATRX depletion. This depletion was sensitive to a proteasome inhibitor and was largely ablated by a deletion of the gene encoding the immediate-early ICP0 protein. Additionally, a deletion of the gene encoding the tegument protein Vhs ablated most of the depletion of ATRX mRNA. Thus, HSV is equipped with multiple mechanisms to limit the expression of ATRX. As ATRX is implicated in repression of lytic viral gene expression, our results suggest roles for these different mechanisms during various phases of HSV infection.

Atomic Hydrogen Surrounded by Water Molecules, H(H2O)m, Modulates Basal and UV-Induced Gene Expressions in Human Skin In Vivo

PubMed Central

Shin, Mi Hee; Park, Raeeun; Nojima, Hideo; Kim, Hyung-Chel; Kim, Yeon Kyung; Chung, Jin Ho

2013-01-01

Recently, there has been much effort to find effective ingredients which can prevent or retard cutaneous skin aging after topical or systemic use. Here, we investigated the effects of the atomic hydrogen surrounded by water molecules, H(H2O)m, on acute UV-induced responses and as well as skin aging. Interestingly, we observed that H(H2O)m application to human skin prevented UV-induced erythema and DNA damage. And H(H2O)m significantly prevented UV-induced MMP-1, COX-2, IL-6 and IL-1β mRNA expressions in human skin in vivo. We found that H(H2O)m prevented UV-induced ROS generation and inhibited UV-induced MMP-1, COX-2 and IL-6 expressions, and UV-induced JNK and c-Jun phosphorylation in HaCaT cells. Next, we investigated the effects of H(H2O)m on intrinsically aged or photoaged skin of elderly subjects. In intrinsically aged skin, H(H2O)m application significantly reduced constitutive expressions of MMP-1, IL-6, and IL-1β mRNA. Additionally, H(H2O)m significantly increased procollagen mRNA and also decreased MMP-1 and IL-6 mRNA expressions in photoaged facial skin. These results demonstrated that local application of H(H2O)m may prevent UV-induced skin inflammation and can modulate intrinsic skin aging and photoaging processes. Therefore, we suggest that modifying the atmospheric gas environment within a room may be a new way to regulate skin functions or skin aging. PMID:23637886

Influence of trichloroacetic acid peeling on the skin stress response system.

PubMed

Kimura, Ayako; Kanazawa, Nobuo; Li, Hong-Jin; Yonei, Nozomi; Yamamoto, Yuki; Furukawa, Fukumi

2011-08-01

Although trichloroacetic acid (TCA) peeling is widely applied for cosmetic treatment of photodamaged skin, the entire biological mechanisms have yet to be determined. The skin stress response system (SSRS) involves corticotropin-releasing hormone (CRH) and proopiomelanocortin (POMC) products that are locally-generated in response to locally-provided stressors or pro-inflammatory cytokines. This system would restrict tissue damage and restore local homeostasis. To determine the influence of TCA peeling on the SSRS in vitro and in vivo, expressions of POMC, melanocortin receptor 1 (MC1R), CRH and CRH receptor 1 (CRHR1) mRNA were examined by reverse transcription polymerase chain reaction in Pam212 murine keratinocytes, murine plantar and healthy human abdominal skin specimens after TCA treatment. In addition, their protein expressions as well as those of POMC-derived peptides were examined immunohistochemically. After TCA treatment, transient upregulation of POMC and MC1R mRNA expressions was observed in both murine and human skin, as well as in Pam212. Enhanced POMC protein, recovery of once-impaired MC1R protein, and no enhancement of POMC-derived peptide productions were revealed immunohistochemically in both murine and human epidermis. In contrast, neither expression levels of CRH and CRHR1 mRNA nor epidermal protein were enhanced after TCA application in murine and human skin, except for induction of human CRH mRNA expression. These results suggest that TCA activates the SSRS by inducing POMC and MC1R productions of keratinocytes in the CRH-independent manner, and that the biological effects of POMC itself are responsible for the TCA-induced epidermal SSRS activation. © 2010 Japanese Dermatological Association.

L-carnitine protects against testicular dysfunction caused by gamma irradiation in mice.

PubMed

Ahmed, Mohamed Mohamed; Ibrahim, Zein Shaban; Alkafafy, Mohamed; El-Shazly, Samir Ahmed

2014-07-01

This study was conducted on mice to evaluate the radioprotective role of L-carnitine against γ-ray irradiation-induced testicular damage. Adult male mice were exposed to whole body irradiation at a total dose of 1 Gy. Radiation exposure was continued 24 h a day (0.1 Gy/day) throughout the 10 days exposure period either in the absence and/or presence of L-carnitine at an i.p. dose of 10 mg/kg body weight/day. Results revealed that γ-rays irradiation suppressed the expression of ABP and CYP450SCC mRNA, whereas treatment with L-carnitine prior and throughout γ-rays irradiation exposure inhibited this suppression. Treatment with γ-ray irradiation or L-carnitine down-regulated expression of aromatase mRNA. With combined treatment, L-carnitine significantly normalized aromatase expression. γ-Ray irradiation up-regulated expression of FasL and Cyclin D2 mRNA, while L-carnitine inhibited these up-regulations. Results also showed that γ-ray-irradiation up-regulated TNF-α, IL1-β and IFN-γ mRNA expressions compared to either controls or the L-carnitine treated group. Moreover, γ-irradiation greatly reduced serum testosterone levels, while L-carnitine, either alone or in combination with irradiation, significantly increased serum testosterone levels compared to controls. In addition, γ-irradiation induced high levels of sperm abnormalities (43%) which were decreased to 12% in the presence of L-carnitine. In parallel with these findings, histological examination showed that γ-irradiation induced severe tubular degenerative changes, which were reduced by L-carnitine pre-treatment. These results clarified the immunostimulatory effects of L-carnitine and its radioprotective role against testicular injury. Copyright © 2014 Elsevier GmbH. All rights reserved.

The effect of ZnO nanoparticles on liver function in rats

PubMed Central

Tang, Hua-Qiao; Xu, Min; Rong, Qian; Jin, Ru-Wen; Liu, Qi-Ji; Li, Ying-Lun

2016-01-01

Zinc oxide (ZnO) is widely incorporated as a food additive in animal diets. In order to optimize the beneficial effects of ZnO and minimize any resultant environmental pollution, ZnO nanoparticles are often used for delivery of the zinc. However, the possible toxic effects of ZnO nanoparticles, including effects on cytochrome P450 (CYP450) enzymes, have not been evaluated. In this study, we investigated the effect of ZnO nanoparticles, in doses used in animal feeds, on CYP450 enzymes, liver and intestinal enzymes, liver and kidney histopathology, and hematologic indices in rats. We found that liver and kidney injury occurred when the concentrations of ZnO nanoparticles in feed were 300–600 mg/kg. Also, liver mRNA expression for constitutive androstane receptor was suppressed and mRNA expression for pregnane X receptor was induced when feed containing ZnO nanoparticles was given at a concentration of 600 mg/kg. Although the expression of mRNA for CYP 2C11 and 3A2 enzymes was induced by ZnO nanoparticles, the activities of CYP 2C11 and 3A2 were suppressed. While liver CYP 1A2 mRNA expression was suppressed, CYP 1A2 activity remained unchanged at all ZnO nanoparticle doses. Therefore, it has been concluded that ZnO nanoparticles, in the doses customarily added to animal feed, changed the indices of hematology and blood chemistry, altered the expression and activity of hepatic CYP enzymes, and induced pathological changes in liver and kidney tissues of rats. These findings suggest that greater attention needs to be paid to the toxic effects of ZnO nanoparticles in animal feed, with the possibility that the doses of ZnO should be reduced. PMID:27621621

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

Myogenin, MyoD and IGF-I regulate muscle mass but not fiber-type conversion during resistance training in rats.

PubMed

Aguiar, A F; Vechetti-Júnior, I J; Alves de Souza, R W; Castan, E P; Milanezi-Aguiar, R C; Padovani, C R; Carvalho, R F; Silva, M D P

2013-04-01

The purpose of this study was to test the hypothesis that skeletal muscle adaptations induced by long-term resistance training (RT) are associated with increased myogenic regulatory factors (MRF) and insulin-like growth factor-I (IGF-I) mRNA expression in rats skeletal muscle. Male Wistar rats were divided into 4 groups: 8-week control (C8), 8-week trained (T8), 12-week control (C12) and 12-week trained (T12). Trained rats were submitted to a progressive RT program (4 sets of 10-12 repetitions at 65-75% of the 1RM, 3 day/week), using a squat-training apparatus with electric stimulation. Muscle hypertrophy was determined by measurement of muscle fiber cross-sectional area (CSA) of the muscle fibers, and myogenin, MyoD and IGF-I mRNA expression were measured by RT-qPCR. A hypertrophic stabilization occurred between 8 and 12 weeks of RT (control-relative % area increase, T8: 29% vs. T12: 35%; p>0.05) and was accompanied by the stabilization of myogenin (control-relative % increase, T8: 44.8% vs. T12: 37.7%, p>0.05) and MyoD (control-relative % increase, T8: 22.9% vs. T12: 22.3%, p>0.05) mRNA expression and the return of IGF-I mRNA levels to the baseline (control-relative % increase, T8: 30.1% vs. T12: 1.5%, p<0.05). Moreover, there were significant positive correlations between the muscle fiber CSA and mRNA expression for MyoD (r=0.85, p=0.0001), myogenin (r=0.87, p=0.0001), and IGF-I (r=0.88, p=0.0001). The significant (p<0.05) increase in myogenin, MyoD and IGF-I mRNA expression after 8 weeks was not associated with changes in the fiber-type frequency. In addition, there was a type IIX/D-to-IIA fiber conversion at 12 weeks, even with the stabilization of MyoD and myogenin expression and the return of IGF-I levels to baseline. These results indicate a possible interaction between MRFs and IGF-I in the control of muscle hypertrophy during long-term RT and suggest that these factors are involved more in the regulation of muscle mass than in fiber-type conversion. © Georg Thieme Verlag KG Stuttgart · New York.

Effects of branched-chain volatile fatty acids on lactation performance and mRNA expression of genes related to fatty acid synthesis in mammary gland of dairy cows.

PubMed

Liu, Q; Wang, C; Guo, G; Huo, W J; Zhang, S L; Pei, C X; Zhang, Y L; Wang, H

2018-02-12

Branched-chain volatile fatty acids (BCVFA) supplements could promote lactation performance and milk quality by improving ruminal fermentation and milk fatty acid synthesis. This study was conducted to evaluate the effects of BCVFA supplementation on milk performance, ruminal fermentation, nutrient digestibility and mRNA expression of genes related to fatty acid synthesis in mammary gland of dairy cows. A total of 36 multiparous Chinese Holstein cows averaging 606±4.7 kg of BW, 65±5.2 day in milk (DIM) with daily milk production of 30.6±0.72 kg were assigned to one of four groups blocked by lactation number, milk yield and DIM. The treatments were control, low-BCVFA (LBCVFA), medium-BCVFA (MBCVFA) and high-BCVFA (HBCVFA) with 0, 30, 60 and 90 g BCVFA per cow per day, respectively. Experimental periods were 105 days with 15 days of adaptation and 90 days of data collection. Dry matter (DM) intake tended to increase, but BW changes were similar among treatments. Yields of actual milk, 4% fat corrected milk, milk fat and true protein linearly increased, but feed conversion ratio (FCR) linearly decreased with increasing BCVFA supplementation. Milk fat content linearly increased, but true protein content tended to increase. Contents of C4:0, C6:0, C8:0, C10:0, C12:0, C14:0 and C15:0 fatty acids in milk fat linearly increased, whereas other fatty acids were not affected with increasing BCVFA supplementation. Ruminal pH, ammonia N concentration and propionate molar proportion linearly decreased, but total VFA production and molar proportions of acetate and butyrate linearly increased with increasing BCVFA supplementation. Consequently, acetate to propionate ratios linearly increased. Digestibilities of DM, organic matter, CP, NDF and ADF also linearly increased. In addition, mRNA expressions of peroxisome proliferator-activated receptor γ, sterol regulatory element-binding factor 1 and fatty acid-binding protein 3 linearly increased, mRNA expressions of acetyl-coenzyme A carboxylase-α, fatty acid synthase and stearoyl-CoA desaturase quadratically increased. However, lipoprotein lipase mRNA expression was not affected by treatments. The results indicated that lactation performance and milk fat synthesis increased with BCVFA supplementation by improving ruminal fermentation, nutrient digestibility and mRNA expressions of genes related to milk fat synthesis.

Cloning and functional expression of novel cholesterol transporters ABCG1 and ABCG4 in gonadotropin-releasing hormone neurons of the tilapia.

PubMed

Phang, Y L; Soga, T; Kitahashi, T; Parhar, I S

2012-02-17

In addition to reproduction, gonadotropin-releasing hormone (GnRH) has been postulated to control cholesterol metabolism via cholesterol transport, which is carried out partly by the members of ATP-binding cassette (ABC) transporters G1 (ABCG1) and G4 (ABCG4). However, there is yet to be evidence demonstrating the relationship between these transporters with reference to GnRH neurons. In the present study, we cloned two ABCG1 messenger RNA (mRNA) variants and one ABCG4 mRNA and examined their expression in the brain including GnRH neurons (GnRH1, GnRH2, and GnRH3) in the cichlid tilapia (Oreochromis niloticus). Comparison of nucleotide sequences of the tilapia ABCG1 and ABCG4 with that of other fish species showed that both of these genes are evolutionarily conserved among fishes. ABCG1 and ABCG4 were shown to have high mRNA expressions in the CNS, pituitary, and gonads. In the brain, real-time polymerase chain reaction (PCR) showed that ABCG4 mRNA was higher than ABCG1a in all brain regions including the olfactory bulb (ABCG1=13.34, ABCG4=6796.35; P<0.001), dorsal telencephalon (ABCG1=8.64, ABCG4=10149.13; P=0.001), optic tectum (ABCG1=22.12, ABCG4=13931.04; P<0.01), cerebellum (ABCG1=8.68, ABCG4=12382.90; P<0.01), and preoptic area-midbrain-hypothalamus (ABCG1=21.36, ABCG4=13255.41; P=0.001). Similarly, although ABCG1 mRNA level is much higher in the pituitary compared with the brain, it was still significantly lower compared with ABCG4 (ABCG1=337.73, ABCG4=1157.87; P=0.01). The differential pattern of expression of ABCG1 and ABCG4 in the brain versus pituitary suggests that the two transporters are regulated by different mechanisms. Furthermore, ABCG1 and ABCG4 mRNA expressions were found in all three types of laser-captured GnRH neurons with highly similar percentage of expressions, suggesting that cholesterol efflux from GnRH neurons may require heterodimerization of both ABCG1 and ABCG4. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells.

PubMed

Akide-Ndunge, Oscar Bate; Tambini, Elisa; Giribaldi, Giuliana; McMillan, Paul J; Müller, Sylke; Arese, Paolo; Turrini, Francesco

2009-05-29

Plasmodium falciparum-parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy. Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70-2/70-3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane. In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of antioxidant enzymes was also increased in oxidatively-stressed trophozoites. Results indicated that mRNA expression of parasite antioxidant enzymes and HSPs was co-ordinated and stage-dependent. Secondly, both systems were redox-responsive and showed remarkably increased and co-ordinated expression in oxidatively-stressed parasites and in parasites growing in antioxidant blunted G6PD-deficient RBCs. Lastly, as important anti-malarials either increase oxidant stress or impair antioxidant defense, results may encourage the inclusion of anti-HSP molecules in anti-malarial combined drugs.

Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

PubMed

Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

1995-02-10

Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

Seasonal Relationship between Gonadotropin, Growth Hormone, and Estrogen Receptor mRNA Expression in the Pituitary Gland of Largemouth Bass

PubMed Central

Martyniuk, Christopher J; Kroll, Kevin J.; Porak, Wesley F.; Steward, Cheree; Grier, Harry J.; Denslow, Nancy D.

2011-01-01

The objectives of this study were to investigate the seasonal changes in pituitary gonadotropins, growth hormone (GH), and estrogen receptor (ER) isoform mRNA in wild female and male largemouth bass (LMB) (Micropterus salmoides) from an unpolluted habitat to better understand reproductive physiology in this ecologically important species. Female pituitary luteinizing hormone (LH) β subunit and follicle-stimulating hormone (FSH) β subunit mRNA showed significant seasonal variation with levels peaking from January to April and were lowest from May through August. Male LMB showed more variation in gonadotropin subunit expression from month to month. Females had approximately 2–3 times higher gonadotropin mRNA levels in the pituitary when compared to males. All three gonadotropin mRNAs in females were positively correlated to gonadosomatic index (GSI), but only LHβ mRNA was correlated to GSI in males. Gonadotropin mRNA expression also increased with increasing oocyte and sperm maturation. Gonadotropin β subunit mRNA expression was positively correlated to GH mRNA in both sexes. The expression of all three ER isoforms was significantly correlated to each other in both sexes. The concurrent increase in all three ER mRNA isoforms with increasing gonadotropin mRNA in females and males suggests a prominent role for E2 feedback on pituitary gonadotropin synthesis in both sexes and that each of the three ER isoforms are likely to play a role in the pituitary during teleost reproduction. PMID:19416730

Seasonal relationship between gonadotropin, growth hormone, and estrogen receptor mRNA expression in the pituitary gland of largemouth bass.

PubMed

Martyniuk, Christopher J; Kroll, Kevin J; Porak, Wesley F; Steward, Cheree; Grier, Harry J; Denslow, Nancy D

2009-09-15

The objectives of this study were to investigate the seasonal changes in pituitary gonadotropins, growth hormone (GH), and estrogen receptor (ER) isoform mRNA in wild female and male largemouth bass (LMB) (Micropterus salmoides) from an unpolluted habitat to better understand reproductive physiology in this ecologically important species. Female pituitary luteinizing hormone (LH) beta subunit and follicle stimulating hormone (FSH) beta subunit mRNA showed significant seasonal variation with levels peaking from January to April and were lowest from May to August. Male LMB showed more variation in gonadotropin subunit expression from month to month. Females had approximately 2-3 times higher gonadotropin mRNA levels in the pituitary when compared to males. All three gonadotropin mRNAs in females were positively correlated to gonadosomatic index (GSI), but only LHbeta mRNA was correlated to GSI in males. Gonadotropin mRNA expression also increased with increasing oocyte and sperm maturation. Gonadotropin beta subunit mRNA expression was positively correlated to GH mRNA in both sexes. The expression of all three ER isoforms was significantly correlated to each other in both sexes. The concurrent increase in all three ER mRNA isoforms with increasing gonadotropin mRNA in females and males suggests a prominent role for E2 feedback on pituitary gonadotropin synthesis in both sexes and that each of the three ER isoforms are likely to play a role in the pituitary during teleost reproduction.

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.

Aromatase, steroid-5-alpha-reductase type 1 and type 2 mRNA expression in gonads and in brain of Xenopus laevis during ontogeny.

PubMed

Urbatzka, R; Lutz, I; Kloas, W

2007-01-01

The key enzymes involved in the production of endogenous sex steroids are steroid-5-alpha-reductase and aromatase converting testosterone (T) into dihydrotestosterone (DHT) and into estradiol (E2), respectively. To gain more insights into the molecular mechanisms of sexual differentiation of amphibians, we determined the mRNA expression of steroid-5-alpha-reductase type1 (Srd5a1), type2 (Srd5a2) and aromatase (Aro) during ontogeny starting from the egg and ending after completion of metamorphosis in Xenopus laevis. Expression of all three enzymes was measured by means of semi-quantitative RT-PCR, determining for the first time Srd5a1 and Srd5a2 mRNA expression in amphibians. mRNA was analyzed in whole body homogenates from stage 12 to 48, while brain and gonads with kidney were studied separately from stage 48 to 66. Different ontogenetic mRNA expression patterns were observed for all genes analyzed, revealing early mRNA expression of Srd5a1 already in the egg at stage 12 whereas Srd5a2 and Aro was detected at stage 39. Sex-specific mRNA expressions of Srd5a2 and of Aro were determined in the gonads with kidney but not in brain. Srd5a2 was two-fold higher expressed in testes than in ovaries while Aro mRNA was ten-fold higher in ovaries. No gender-specific mRNA expression was observed for Srd5a1 in gonads and in brain. The ontogenetic patterns of Aro, Srd5a1 and Srd5a2 suggest that these genes are involved in sexual differentiation of gonads and brain already in early developmental stages. Especially in gonads Srd5a2 seems to be important for physiological regulation of testis development while Aro is associated with the development of ovaries.

TS mRNA levels can predict pemetrexed and raltitrexed sensitivity in colorectal cancer.

PubMed

Zhang, Qun; Shen, Jie; Wang, Hao; Hu, Jing; Yu, Lixia; Xie, Li; Wei, Jia; Liu, Baorui; Guan, Wenxian; Qian, Xiaoping

2014-02-01

The purpose of the study is to analyze the relationship between tumor thymidylate synthase (TS) mRNA expression levels and raltitrexed/pemetrexed/5-FU sensitivity. We collected freshly removed colorectal tumor specimens from 50 patients. Chemosensitivities to anticancer drugs were evaluated by histoculture drug response assay. We adopted quantitative reverse transcription polymerase chain reaction for TS mRNA detection and immunohistochemical staining for assessing TS expression in tumor tissues. There is a significant relationship between TS mRNA expression levels and in vitro chemosensitivity of freshly removed colorectal tumor specimens to pemetrexed (P < 0.001)/raltitrexed (P = 0.004)/5-FU (P = 0.007). TS mRNA expression levels can predict pemetrexed and raltitrexed sensitivity in colorectal cancer.

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.

Comparison of cytokine mRNA expression in peripheral CD4(+) , CD8(+) and γδ T cells between healthy Holstein and Japanese Black calves.

PubMed

Ohtsuka, Hiromichi; Kobayashi, Hiroki; Kinouchi, Kumi; Kiyono, Miki; Maeda, Yousuke

2014-05-01

Japanese Black (JB) calves are more susceptible to infectious diseases compared to Holstein (Hol) calves. To clarify the immunological differences between JB and Hol calves, expression of cytokine messenger RNA (mRNA) was examined using peripheral CD4(+) , CD8(+) and γδ T cells. Healthy calves, 24 from each species, were examined. Blood samples were obtained from calves at 1 week, 1 month and 3 months old, eight calves for each age of each species. Peripheral blood mononuclear cells were stimulated with phytohemagglutin (PHA), and T cell subsets were isolated by positive selection using magnetic cell sorting (MACS). Levels of interlekin (IL)-2, IL-4, IL-10 and interferon (IFN)-γ mRNA in three T cell subsets were analyzed. WC1-N1(+) γδ T cell percentages were significantly lower in JB calves at 1 week and 1 month of age compared to Hol calves. In addition JB calves had significantly lower IL-2, IL-10 and IFN-γ mRNA in WC1-N1(+) γδ T cells at 1 and 3 months of age, whereas there were no significant differences in cytokine mRNA of CD4(+) and CD8(+) cells between the two groups. Decreased cytokine mRNA and cell number of peripheral γδ T cells may affect negatively on the immune system of JB calves. © 2014 Japanese Society of Animal Science.

AMPKα2 regulates expression of estrogen-related receptor alpha, a metabolic transcription factor related to heart failure development

PubMed Central

Hu, Xinli; Xu, Xin; Lu, Zhongbing; Zhang, Ping; Fassett, John; Zhang, Ying; Xin, Yi; Hall, Jennifer L.; Viollet, Benoit; Bache, Robert J.; Huang, Yimin; Chen, Yingjie

2011-01-01

The normal expression of myocardial mitochondrial enzymes is essential to maintain the cardiac energy reserve and facilitate responses to stress, but the molecular mechanisms to maintain myocardial mitochondrial enzyme expression have been elusive. Here we report that congestive heart failure is associated with a significant decrease of myocardial Estrogen-Related Receptor alpha (ERRα), but not PPAR gamma coactivator-1 alpha (PGC1α), in human heart failure samples. In addition, chronic pressure overload in mice caused a decrease of ERRα expression that was significantly correlated to the degree of LV dysfunction, pulmonary congestion and decreases of a group of myocardial energy metabolism related genes. We found that the metabolic sensor AMP activated protein kinase (AMPK) regulates ERRα expression in vivo and in vitro. AMPKα2 KO decreased myocardial ERRα (both mRNA and protein) and its downstream targets under basal conditions, with no change in myocardial PGC1α expression. Using cultured rat neonatal cardiac myocytes, we found that overexpression of constitutively active AMPKα significantly induced ERRα mRNA, protein and promoter activity. Conversely, selective gene silencing of AMPKα2 repressed ERRα and its target gene levels, indicating that AMPKα2 is involved in the regulation of ERRα expression. In addition, over-expression of ERRα in AMPKα2 KO neonatal cardiac myocytes partially rescued the repressed expression of some energy metabolism related genes. These data support an important role for AMPKα2 in regulating the expression of myocardial ERRα and its downstream mitochondrial enzymes. PMID:21825219

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.

Inhibition of Gαs/cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4(+) T Helper Cells.

PubMed

Hynes, Thomas R; Yost, Evan A; Yost, Stacy M; Hartle, Cassandra M; Ott, Braden J; Berlot, Catherine H

2015-07-06

The role of cAMP in regulating T cell activation and function has been controversial. cAMP is generally known as an immunosuppressant, but it is also required for generating optimal immune responses. As the effect of cAMP is likely to depend on its cellular context, the current study investigated whether the mechanism of activation of Gαs and adenylyl cyclase influences their effect on T cell receptor (TCR)-stimulated interleukin-2 (IL-2) mRNA levels. The effect of blocking Gs-coupled receptor (GsPCR)-mediated Gs activation on TCR-stimulated IL-2 mRNA levels in CD4(+) T cells was compared with that of knocking down Gαs expression or inhibiting adenylyl cyclase activity. The effect of knocking down Gαs expression on TCR-stimulated cAMP accumulation was compared with that of blocking GsPCR signaling. ZM-241385, an antagonist to the Gs-coupled A2A adenosine receptor (A2AR), enhanced TCR-stimulated IL-2 mRNA levels in primary human CD4(+) T helper cells and in Jurkat T cells. A dominant negative Gαs construct, GαsDN3, also enhanced TCR-stimulated IL-2 mRNA levels. Similar to GsPCR antagonists, GαsDN3 blocked GsPCR-dependent activation of both Gαs and Gβγ. In contrast, Gαs siRNA and 2',5'-dideoxyadenosine (ddA), an adenylyl cyclase inhibitor, decreased TCR-stimulated IL-2 mRNA levels. Gαs siRNA, but not GαsDN3, decreased TCR-stimulated cAMP synthesis. Potentiation of IL-2 mRNA levels by ZM-241385 required at least two days of TCR stimulation, and addition of ddA after three days of TCR stimulation enhanced IL-2 mRNA levels. GsPCRs play an inhibitory role in the regulation of TCR-stimulated IL-2 mRNA levels whereas Gαs and cAMP can play a stimulatory one. Additionally, TCR-dependent activation of Gαs does not appear to involve GsPCRs. These results suggest that the context of Gαs/cAMP activation and the stage of T cell activation and differentiation determine the effect on TCR-stimulated IL-2 mRNA levels.

Up-regulation of cyclooxygenase-2 by product-prostaglandin E2

NASA Technical Reports Server (NTRS)

Tjandrawinata, R. R.; Hughes-Fulford, M.

1997-01-01

The development of prostate cancer has been linked to high level of dietary fat intake. Our laboratory investigates the connection between cancer cell growth and fatty acid products. Studying human prostatic carcinoma PC-3 cells, we found that prostaglandin E2 (PGE2) increased cell growth and up-regulated the gene expression of its own synthesizing enzyme, cyclooxygenase-2 (COX-2). PGE2 increased COX-2 mRNA expression dose-dependently with the highest levels of stimulation seen at the 3-hour period following PGE2 addition. The NSAID flurbiprofen (5 microM), in the presence of exogenous PGE2, inhibited the up-regulation of COX-2 mRNA and cell growth. These data suggest that the levels of local intracellular PGE2 play a major role in the growth of prostate cancer cells through an activation of COX-2 gene expression.

Anti-proliferative and apoptotic effects of the novel taspine derivative tas41 in the Caco-2 cell line.

PubMed

Zhang, Yanmin; Zhang, Jie; Dai, Bingling; Wang, Nan; He, Langchong

2011-05-01

Taspine was screened and isolated for the first time from Radix et Rhizoma Leonticis. Tas41 is a novel taspine derivative. We investigated the effects of tas41 on proliferation of the Caco-2 cell line using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a fluorescence-activated cell sorter (FACS), enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR) and western blotting (WB). Changes in the cell cycle, apoptosis, activation of caspase-3, caspase-8 and caspase-9, and expressions of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) were investigated after Caco-2 cells were treated with tas41. At the same time, expressions of apoptosis protein bcl-2 and bax were determined. Tas41 was found to induce apoptosis in a concentration-dependent manner as confirmed by DNA fragmentation analysis, TUNEL assay and flow cytometry. Protein and mRNA expressions of EGF, VEGF, CDK2, bcl-2 and bax were evaluated by ELISA, WB and RT-PCR. Tas41 had a better anti-proliferative effect than taspine on Caco-2 cells. A DNA ladder and apoptosis was observed, and the increased apoptotic activity by tas41 was accompanied by a decrease in the expression of VEGF protein and mRNA. The activities of caspase-3, caspase-8 and caspase-9 were significantly increased in cells treated with tas41 compared with those in the control group. In addition, protein and mRNA expressions of bcl-2 were decreased, and protein and mRNA expressions of bax were increased. These findings demonstrate that tas41 can inhibit the proliferation of, and induce apoptosis in, Caco-2 cells by activating caspase-3, caspase-8 and caspase-9, downregulating the expressions of VEGF, upregulating the ratio of bax/bcl-2. Copyright © 2011 Elsevier B.V. All rights reserved.

Differential expression of cytokeratin mRNA and protein in normal prostate, prostatic intraepithelial neoplasia, and invasive carcinoma.

PubMed Central

Yang, Y.; Hao, J.; Liu, X.; Dalkin, B.; Nagle, R. B.

1997-01-01

The expression of cytokeratin (CK) mRNA for CK5, -8, -14, -16, and -19 was investigated in normal prostate, prostatic intraepithelial neoplasia (PIN) lesions, and invasive carcinoma using in situ hybridization. Protein localization was carried out in adjacent sections using immunohistochemistry and correlated with mRNA expression. Snap-frozen human prostate samples including 22 examples of normal glands, 20 cases of PIN lesions, and 12 cases of invasive carcinoma were examined. CK5 and -14 mRNA and protein were prominently expressed only in the basal cells of normal glands and PIN lesions. CK14 mRNA was absent in the luminal cells of the most of the PIN lesions but was seen at a low level in some PIN lesions. CK14 protein was not detected in any PIN lesion, suggesting that, if the cell that makes up the PIN lesions is derived from a basal cell, CK14 translation is depressed although a low level of CK14 mRNA may persist. CK8 mRNA and protein were constitutively expressed in all epithelia of normal and abnormal prostate tissues. CK19 mRNA and protein were persistently expressed in both basal and luminal cells of the tubular portion of normal glands as well as PIN lesions, but were expressed heterogeneously in both basal and luminal cells of normal alveoli. CK16 mRNA was expressed in a similar pattern as CK19, but CK16 protein was not detected either in normal or in abnormal prostate tissues. In conclusion, the expression of CK19 in PIN lesions is similar to its tubular expression and would support an origin of PIN lesions from this structure rather than the alveolar portion of the glands. The similar cytokeratin expression between PIN lesions and invasive carcinoma further supports the concept that PIN is a precursor lesion of invasive carcinoma. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:9033282

Expression and methylation of BDNF in the human brain in schizophrenia.

PubMed

Cheah, Sern-Yih; McLeay, Robert; Wockner, Leesa F; Lawford, Bruce R; Young, Ross McD; Morris, Charles P; Voisey, Joanne

2017-08-01

To examine the combined effect of the BDNF Val66Met (rs6265) polymorphism and BDNF DNA methylation on transcriptional regulation of the BDNF gene. DNA methylation profiles were generated for CpG sites proximal to Val66Met, within BDNF promoter I and exon V for prefrontal cortex samples from 25 schizophrenia and 25 control subjects. Val66Met genotypes and BDNF mRNA expression data were generated by transcriptome sequencing. Expression, methylation and genotype data were correlated and examined for association with schizophrenia. There was 43% more of the BDNF V-VIII-IX transcript in schizophrenia samples. BDNF mRNA expression and DNA methylation of seven CpG sites were not associated with schizophrenia after accounting for age and PMI effects. BDNF mRNA expression and DNA methylation were not altered by Val66Met after accounting for age and PMI effects. DNA methylation of one CpG site had a marginally significant positive correlation with mRNA expression in schizophrenia subjects. Schizophrenia risk was not associated with differential BDNF mRNA expression and DNA methylation. A larger age-matched cohort with comprehensive clinical history is required to accurately identify the effects of genotype, mRNA expression and DNA methylation on schizophrenia risk.

Increased expression of ADAM 9 and ADAM 15 mRNA in pancreatic cancer.

PubMed

Yamada, Daisuke; Ohuchida, Kenoki; Mizumoto, Kazuhiro; Ohhashi, Seiji; Yu, Jun; Egami, Takuya; Fujita, Hayato; Nagai, Eishi; Tanaka, Masao

2007-01-01

A disintegrin and metalloproteases (ADAMs) comprise a multifunctional family of membrane-anchored proteins. ADAM 9 and ADAM 15 are involved in cell migration and invasion. Expression of ADAM 9 and ADAM 15 was reported to be altered in several types of cancer. Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure the expression of ADAM 9 mRNA in bulk pancreatic tissues. Results showed no significant difference in the expression of ADAM 9 mRNA between pancreatic cancer and non-neoplastic pancreas. Primary cultured pancreatic fibroblasts also expressed ADAM 9 mRNA. Therefore, a laser microdissection and pressure catapulting technique was employed to isolate cancer cells from tumor tissues. The expression of ADAM 9 and ADAM 15 mRNA was measured in microdissected samples (cancer cells, n = 11; normal epithelial cells, n = 13 for ADAM 9; cancer cells, n = 9; normal epithelial cells, n = 9 for ADAM 15). Pancreatic cancer cells expressed significantly higher levels of ADAM 9 and ADAM 15 mRNA than did normal pancreatic epithelial cells (p = 0.016 for ADAM 9; p = 0.004 for ADAM 15). ADAM 9 and ADAM 15 are involved in pancreatic cancer. Microdissection-based analysis appears to be indispensable for the accurate analysis of the expression of certain ADAM family members in pancreatic cancer.

Hepcidin regulation in wild-type and Hfe knockout mice in response to alcohol consumption: evidence for an alcohol-induced hypoxic response.

PubMed

Heritage, Mandy L; Murphy, Therese L; Bridle, Kim R; Anderson, Gregory J; Crawford, Darrell H G; Fletcher, Linda M

2009-08-01

Expression of Hamp1, the gene encoding the iron regulatory peptide hepcidin, is inappropriately low in HFE-associated hereditary hemochromatosis and Hfe knockout mice (Hfe(-/-)). Since chronic alcohol consumption is also associated with disturbances in iron metabolism, we investigated the effects of alcohol consumption on hepcidin mRNA expression in Hfe(-/-) mice. Hfe(-/-) and C57BL/6 (wild-type) mice were pair-fed either an alcohol liquid diet or control diet for up to 8 weeks. The mRNA levels of hepcidin and ferroportin were measured at the mRNA level by RT-PCR and protein expression of hypoxia inducible factor-1 alpha (HIF-1alpha) was measured by western blot. Hamp1 mRNA expression was significantly decreased and duodenal ferroportin expression was increased in alcohol-fed wild-type mice at 8 weeks. Time course experiments showed that the decrease in hepcidin mRNA was not immediate, but was significant by 4 weeks. Consistent with the genetic defect, Hamp1 mRNA was decreased and duodenal ferroportin mRNA expression was increased in Hfe(-/-) mice fed on the control diet compared with wild-type animals and alcohol further exacerbated these effects. HIF-1alpha protein levels were elevated in alcohol-fed wild-type animals compared with controls. Alcohol may decrease Hamp1 gene expression independently of the HFE pathway possibly via alcohol-induced hypoxia.

Midbrain dopamine neurons regulate preprotachykinin-A mRNA expression in the rat forebrain during development.

PubMed

Brené, S; Lindefors, N; Persson, H

1992-06-01

Intracerebroventricular 6-hydroxydopamine injections were performed at postnatal days 3 and 6 in animals pretreated with the norepinephrine uptakeblocker desimipramine in order to generate a selective lesion of dopamine neurons. In situ hybridization was then used to analyze preprotachykinin-A (PPT-A) mRNA expression in the lesioned as well as in saline-injected control animals. The midbrain dopaminergic lesion caused a 22-25% increase in the level of PPT-A mRNA in cingulate cortex and frontoparietal cortex when analysed at 2 weeks of age, compared to saline-injected control animals. In contrast, the lesion caused no change in PPT-A mRNA expression in the neonatal caudate-putamen. These results indicate that dopamine neurons downregulate the expression of PPT-A mRNA specifically in cingulate cortex and frontoparietal cortex during early postnatal brain development. In the adult rat forebrain, lesioned at P3 and P6, no change in the level of PPT-A mRNA was seen in cingulate cortex and frontoparietal cortex. However, a 29% decrease in PPT-A mRNA was seen in the lateral caudate-putamen with no significant change in neurons of medial caudate-putamen. Thus, dopamine neurons appears to exert a region specific influence on PPT-A mRNA expression during brain development.

Cytokine stimulation of MUC4 expression in human female reproductive tissue carcinoma cell lines and endometrial cancer.

PubMed

Chapela, Patricia J; Broaddus, Russell R; Hawkins, Shannon M; Lessey, Bruce A; Carson, Daniel D

2015-11-01

MUC4, a transmembrane glycoprotein, interferes with cell adhesion, and promotes EGFR signaling in cancer. Studies in rat models have demonstrated steroid hormonal regulation of endometrial MUC4 expression. In this study, qRT-PCR screening of mouse tissues determined that Muc4 mRNA also was robustly expressed in mouse uteri. Previous studies from our labs have demonstrated MUC4 mRNA was expressed at levels <1% of MUC1 mRNA in human endometrium and endometriotic tissue. Multiple human endometrial adenocarcinoma cell lines were assayed for MUC4 mRNA expression revealing extremely low basal expression in the Ishikawa, RL-95-2, AN3CA, and KLE lines. Moderate to high expression was observed in HEC50 and HEC-1A cells. MUC4 mRNA expression was not affected by progesterone and/or estrogen treatment, but was greatly stimulated at both mRNA and protein levels by proinflammatory cytokines (IFN-γ and TNF-α), particularly when used in combination. In endometrial tissue, MUC4 mRNA levels did not change significantly between normal or cancerous samples; although, a subset of patients with grade 1 and 2 tumors displayed substantially higher expression. Likewise, immunostaining of human endometrial adenocarcinoma tissues revealed little to no staining in many patients (low MUC4), but strong staining in some patients (high MUC4) independent of cancer grade. In cases where staining was observed, it was heterogeneous with some cells displaying robust MUC4 expression and others displaying little or no staining. Collectively, these observations demonstrate that while MUC4 is highly expressed in the mouse uterus, it is not a major mucin in normal human endometrium. Rather, MUC4 is a potential marker of endometrial adenocarcinoma in a subset of patients. © 2015 Wiley Periodicals, Inc.

In vivo hair growth-promoting effect of rice bran extract prepared by supercritical carbon dioxide fluid.

PubMed

Choi, Jae-Suk; Jeon, Min-Hee; Moon, Woi-Sook; Moon, Jin-Nam; Cheon, Eun Jin; Kim, Joo-Wan; Jung, Sung Kyu; Ji, Yi-Hwa; Son, Sang Wook; Kim, Mi-Ryung

2014-01-01

The potential hair growth-promoting activity of rice bran supercritical CO2 extract (RB-SCE) and major components of RB-SCE, linoleic acid, policosanol, γ-oryzanol, and γ-tocotrienol, were evaluated with the histological morphology and mRNA expression levels of cell growth factors using real-time reverse transcriptase-polymerase chain reaction (PCR) in C57BL/6 mice. RB-SCE showed hair growth-promoting potential to a similar extent as 3% minoxidil, showing that the hair follicles were induced to be in the anagen stage. The numbers of the hair follicles were significantly increased. In addition, mRNA expression levels of vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), and keratinocyte growth factor (KGF) were also significantly increased and that of transforming growth factor-β (TGF-β) decreased in RB-SCE-treated groups. Among the major components of RB-SCE, linoleic acid and γ-oryzanol induced the formation of hair follicles according to examination of histological morphology and mRNA expression levels of cell growth factors. In conclusion, our results demonstrate that RB-SCE, particularly linoleic acid and γ-oryzanol, promotes hair growth and suggests RB-SCE can be applied as hair loss treatment.

Preventive effect of dietary astaxanthin on UVA-induced skin photoaging in hairless mice.

PubMed

Komatsu, Toshiyuki; Sasaki, Suguru; Manabe, Yuki; Hirata, Takashi; Sugawara, Tatsuya

2017-01-01

Astaxanthin, a carotenoid found mainly in seafood, has potential clinical applications due to its antioxidant activity. In this study, we evaluated the effect of dietary astaxanthin derived from Haematococcus pluvialis on skin photoaging in UVA-irradiated hairless mice by assessing various parameters of photoaging. After chronic ultraviolet A (UVA) exposure, a significant increase in transepidermal water loss (TEWL) and wrinkle formation in the dorsal skin caused by UVA was observed, and dietary astaxanthin significantly suppressed these photoaging features. We found that the mRNA expression of lympho-epithelial Kazal-type-related inhibitor, steroid sulfatase, and aquaporin 3 in the epidermis was significantly increased by UVA irradiation for 70 days, and dietary astaxanthin significantly suppressed these increases in mRNA expression to be comparable to control levels. In the dermis, the mRNA expression of matrix metalloprotease 13 was increased by UVA irradiation and significantly suppressed by dietary astaxanthin. In addition, HPLC-PDA analysis confirmed that dietary astaxanthin reached not only the dermis but also the epidermis. Our results indicate that dietary astaxanthin accumulates in the skin and appears to prevent the effects of UVA irradiation on filaggrin metabolism and desquamation in the epidermis and the extracellular matrix in the dermis.

Preventive effect of dietary astaxanthin on UVA-induced skin photoaging in hairless mice

PubMed Central

Komatsu, Toshiyuki; Sasaki, Suguru; Manabe, Yuki; Hirata, Takashi

2017-01-01

Astaxanthin, a carotenoid found mainly in seafood, has potential clinical applications due to its antioxidant activity. In this study, we evaluated the effect of dietary astaxanthin derived from Haematococcus pluvialis on skin photoaging in UVA-irradiated hairless mice by assessing various parameters of photoaging. After chronic ultraviolet A (UVA) exposure, a significant increase in transepidermal water loss (TEWL) and wrinkle formation in the dorsal skin caused by UVA was observed, and dietary astaxanthin significantly suppressed these photoaging features. We found that the mRNA expression of lympho-epithelial Kazal-type-related inhibitor, steroid sulfatase, and aquaporin 3 in the epidermis was significantly increased by UVA irradiation for 70 days, and dietary astaxanthin significantly suppressed these increases in mRNA expression to be comparable to control levels. In the dermis, the mRNA expression of matrix metalloprotease 13 was increased by UVA irradiation and significantly suppressed by dietary astaxanthin. In addition, HPLC-PDA analysis confirmed that dietary astaxanthin reached not only the dermis but also the epidermis. Our results indicate that dietary astaxanthin accumulates in the skin and appears to prevent the effects of UVA irradiation on filaggrin metabolism and desquamation in the epidermis and the extracellular matrix in the dermis. PMID:28170435

Reversible Ca2+-induced fast-to-slow transition in primary skeletal muscle culture cells at the mRNA level

PubMed Central

Meißner, Joachim D; Kubis, Hans-Peter; Scheibe, Renate J; Gros, Gerolf

2000-01-01

The adult fast character and a Ca2+-inducible reversible transition from a fast to a slow type of rabbit myotube in a primary culture were demonstrated at the mRNA level by Northern blot analysis with probes specific for different myosin heavy chain (MyHC) isoforms and enzymes of energy metabolism. No non-adult MyHC isoform mRNA was detected after 22 days of culture. After 4 weeks of culture the fast MyHCIId mRNA was strongly expressed while MyHCI mRNA was virtually absent, indicating the fast adult character of the myotubes in the primary skeletal muscle culture. The data show that a fast-to-slow transition occurred in the myotubes at the level of MyHC isoform gene expression after treatment with the Ca2+ ionophore A23187. The effects of ionophore treatment were decreased levels of fast MyHCII mRNA and an augmented expression of the slow MyHCI gene. Changes in gene expression started very rapidly 1 day after the onset of ionophore treatment. Levels of citrate synthase mRNA increased and levels of glyceraldehyde 3-phosphate dehydrogenase mRNA decreased during ionophore treatment. This points to a shift from anaerobic to oxidative energy metabolism in the primary skeletal muscle culture cells at the level of gene expression. Withdrawal of the Ca2+ ionophore led to a return to increased levels of MyHCII mRNA and decreased levels of MyHCI mRNA, indicating a slow-to-fast transition in the myotubes and the reversibility of the effect of ionophore on MyHC isoform gene expression. PMID:10673542

Expression profiles and associations of adiponectin and adiponectin receptors with intramuscular fat in Tibetan chicken.

PubMed

Zhang, R; Lin, Y; Zhi, L; Liao, H; Zuo, L; Li, Z; Xu, Y

2017-04-01

1. Adiponectin and its receptors (ADIPOR1 and ADIPOR2) are novel endocrine systems that act at various levels to modulate glucose and lipid metabolism. This study was designed to investigate the spatial expression of adiponectin, ADIPOR1 and ADIPOR2 genes in various tissues in Tibetan chicken. The temporal expression of adiponectin and its receptor mRNAs were also studied in adipose tissue, breast muscle and thigh muscle and the correlations of the levels of adiponectin, ADIPOR1 and ADIPOR2 mRNA with the contents of intramuscular fat in breast muscle and thigh muscle of Tibetan chicken were determined. 2. Quantitative real-time PCR detected chicken adiponectin, ADIPOR1 and ADIPOR2 mRNA transcripts in heart, liver, spleen, lung, kidney, skeletal muscle and adipose tissue. 3. Adipose tissue contained the highest amount of adiponectin mRNA followed by the kidney and liver. The expression levels of ADIPOR1 mRNA were significantly higher in adipose tissue, lung and spleen, and adipose tissue exhibited significantly higher levels of ADIPOR2 mRNA followed by the spleen and lung compared with other tissues. 4. Temporal expression profiles of adiponectin, ADIPOR1 and ADIPOR2 mRNA showed gender differences in adipose tissue and skeletal muscle at certain ages. In adipose tissue, adiponectin mRNA was higher in 154-d-old females and ADIPOR1 mRNA was higher in 154-d-old males: Adiponectin and ADIPOR2 mRNA were higher, and ADIPOR1 mRNA was lower, in thigh muscle in female compared with male chickens. 5. The correlation data showed that, except for adiponectin mRNA, the levels of ADIPOR1 and ADIPOR2 mRNA in thigh muscle of males were significantly positively correlated with IMF (r = 0.206 for the ADIPOR1 gene and r = 0.676 for the ADIPOR2 gene). 6. Taken together, it was concluded that adiponectin and the ADIPOR1 and ADIPOR2 genes are ubiquitously expressed in various tissues of Tibetan chicken and the expression of the adiponectin system is gender-dependant at certain ages in adipose tissue and skeletal muscle.

Cellular distribution and regulation of ghrelin messenger ribonucleic acid in the rat pituitary gland.

PubMed

Caminos, J E; Nogueiras, R; Blanco, M; Seoane, L M; Bravo, S; Alvarez, C V; García-Caballero, T; Casanueva, F F; Diéguez, C

2003-11-01

Ghrelin, a 28-amino-acid acylated peptide, strongly stimulates GH release and food intake. In the present study, we found that ghrelin is expressed in somatotrophs, lactotrophs, and thyrotrophs but not in corticotrophs or gonadotrophs of rat pituitary. Persistent expression of the ghrelin gene is found during postnatal development in male and female rats, although the levels significantly decrease in both cases from pituitaries of 20-d-old rats onward, but at 60 d old, the levels were higher in male than female rats. This sexually dimorphic pattern appears to be mediated by estrogens because ovariectomy, but not orchidectomy, increases pituitary ghrelin mRNA levels. Taking into account that somatotroph cell function is markedly influenced by thyroid hormones, glucocorticoids, GH, and metabolic status, we also assessed such influence. We found that ghrelin mRNA levels decrease in hypothyroid- and glucocorticoid-treated rats, increase in GH-deficient rats (dwarf rats), and remain unaffected by food deprivation. In conclusion, we have defined the specific cell types that express ghrelin in the rat anterior pituitary gland. These data provide direct morphological evidence that ghrelin may well be acting in a paracrine-like fashion in the regulation of anterior pituitary cell function. In addition, we clearly demonstrate that pituitary ghrelin mRNA levels are age and gender dependent. Finally, we show that pituitary ghrelin mRNA levels are influenced by alteration on thyroid hormone, glucocorticoids, and GH levels but not by fasting, which indicates that the regulation of ghrelin gene expression is tissue specific.

Astragalus polysaccharides attenuate PCV2 infection by inhibiting endoplasmic reticulum stress in vivo and in vitro.

PubMed

Xue, Hongxia; Gan, Fang; Qian, Gang; Hu, Junfa; Hao, Shu; Xu, Jing; Chen, Xingxiang; Huang, Kehe

2017-01-10

This study explored the effects of Astragalus polysaccharide (APS) on porcine circovirus type 2 (PCV2) infections and its mechanism in vivo and vitro. First, fifty 2-week-old mice were randomly divided into five groups: a group without PCV2 infection and groups with PCV2 infections at 0, 100, 200 or 400 mg/kg APS treatments. The trial lasted for 28 days. The results showed that APS treatments at 200 and 400 mg/kg reduced the pathological injury of tissues, inhibited PCV2 infection and decreased glucose-regulated protein 78 (GRP78) and GADD153/CHOP gene mRNA and protein expression significantly (P < 0.05). Second, a study on endoplasmic reticulum stress mechanism was carried out in PK15 cells. APS treatments at 15 and 45 μg/mL significantly reduced PCV2 infection and GRP78 mRNA and protein expression (P < 0.05). Tunicamycin supplementation increased GRP78 mRNA and protein expression and significantly attenuated the APS-induced inhibition of PCV2 infection (P < 0.05). Tauroursodeoxycholic acid supplementation decreased GRP78 mRNA and protein expression and significantly inhibited PCV2 infection (P < 0.05). In addition, fifty 2-week-old mice were randomly divided into five groups: Con, PCV2, APS + PCV2, TM + PCV2 and TM + APS + PCV2. The results were similar to those in PK15 cells. Taken together, it could be concluded that APS suppresses PCV2 infection by inhibiting endoplasmic reticulum stress.

Astragalus polysaccharides attenuate PCV2 infection by inhibiting endoplasmic reticulum stress in vivo and in vitro

PubMed Central

Xue, Hongxia; Gan, Fang; Qian, Gang; Hu, Junfa; Hao, Shu; Xu, Jing; Chen, Xingxiang; Huang, Kehe

2017-01-01

This study explored the effects of Astragalus polysaccharide (APS) on porcine circovirus type 2 (PCV2) infections and its mechanism in vivo and vitro. First, fifty 2-week-old mice were randomly divided into five groups: a group without PCV2 infection and groups with PCV2 infections at 0, 100, 200 or 400 mg/kg APS treatments. The trial lasted for 28 days. The results showed that APS treatments at 200 and 400 mg/kg reduced the pathological injury of tissues, inhibited PCV2 infection and decreased glucose-regulated protein 78 (GRP78) and GADD153/CHOP gene mRNA and protein expression significantly (P < 0.05). Second, a study on endoplasmic reticulum stress mechanism was carried out in PK15 cells. APS treatments at 15 and 45 μg/mL significantly reduced PCV2 infection and GRP78 mRNA and protein expression (P < 0.05). Tunicamycin supplementation increased GRP78 mRNA and protein expression and significantly attenuated the APS-induced inhibition of PCV2 infection (P < 0.05). Tauroursodeoxycholic acid supplementation decreased GRP78 mRNA and protein expression and significantly inhibited PCV2 infection (P < 0.05). In addition, fifty 2-week-old mice were randomly divided into five groups: Con, PCV2, APS + PCV2, TM + PCV2 and TM + APS + PCV2. The results were similar to those in PK15 cells. Taken together, it could be concluded that APS suppresses PCV2 infection by inhibiting endoplasmic reticulum stress. PMID:28071725

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.

Endometrial IL-1beta, IL-6 and TNF-alpha, mRNA expression in mares resistant or susceptible to post-breeding endometritis. Effects of estrous cycle, artificial insemination and immunomodulation.

PubMed

Fumuso, Elida; Giguère, Steeve; Wade, José; Rogan, Dragan; Videla-Dorna, Ignacio; Bowden, Raúl A

2003-11-15

Endometrial mRNA expression of the pro-inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) was assessed in mares resistant (RM) or susceptible (SM) to persistent post-breeding endometritis (PPBE). Eight RM and eight SM, were selected based on reproductive records and functional tests out of a herd of 2,000 light cross-type mares. Three experiments were done to study transcription patterns in (i) basal conditions; (ii) after artificial insemination (AI); and (iii) after administration of an immunomodulator at time of artificial insemination. Endometrial biopsies were taken during consecutive cycles: (i) at estrus, when follicles reached 35 mm and at diestrus (7 +/- 1 days after ovulation); (ii) at 24 h post-AI, with dead semen (estrus) and in diestrus; (iii) at 24 h after treatment with a Mycobacterium phlei cell-wall extract (MCWE) preparation and AI (with dead semen), and at diestrus. mRNA expression was quantitated by real time PCR. Under basal conditions, SM had significantly higher mRNA expression of all cytokines in estrus and of IL-1beta and TNF-alpha in diestrus, compared to RM. After AI, there were no differences between RM and SM in estrus; however, mRNA expression for all three pro-inflammatory cytokines was higher than under basal conditions. In diestrus, RM showed significantly lower IL-1beta and TNF-alpha mRNA expression than SM. When MCWE was administered at time of AI, no differences between cytokine induction from RM and SM were found. Globally, mRNA expression for all three cytokines correlated well among themselves when expression was high. The present study showed that (i) in basal conditions RM had lower mRNA expression of pro-inflammatory cytokines than SM with no effect of estrous cycle; (ii) AI upregulated mRNA expression for all three cytokines in both RM and SM, with persistance in diestrus in the latter; (iii) treatment with MCWE at time of AI down-regulated mRNA expression of IL-1 with significant effects in SM which behaved like RM. Immunomodulation with MCWE could be of help in restoring homeostatic local inflammatory mechanisms, thus assisting in the prophylaxis of post-breeding endometritis in mares.

Effects of forced swimming stress on thyroid function, pituitary thyroid-stimulating hormone and hypothalamus thyrotropin releasing hormone expression in adrenalectomy Wistar rats.

PubMed

Sun, Qiuyan; Liu, Aihua; Ma, Yanan; Wang, Anyi; Guo, Xinhong; Teng, Weiping; Jiang, Yaqiu

2016-11-01

In order to study the impact that is imposed on the hypothalamic-pituitary-thyroid (HPT) axis of adrenalectomy male Wistar rats by stress caused by swimming, the blood level of triiodothyronine (T3), thyroxine (T4) and thyroid-stimulating hormone (TSH), the expression of TSHβ mRNA at the pituitary and thyrotropin releasing hormone (TRH) expression at the paraventricular nucleus (PVN) were measured. A total of 50 male Wistar rats of 6-8 weeks of age and with an average weight of 190-210 grams were randomly divided into the following two groups: The surgical (without adrenal glands) and non-surgical (adrenalectomy) group. These two groups were then divided into the following five groups, according to the time delay of sacrifice following forced swim (10 min, 2 h, 12 h and 24 h) and control (not subjected to swimming) groups. A bilateral adrenalectomy animal model was established. Serum TSH in the blood was measurement by chemiluminescent immunoassay, and cerebrum tissue were excised for the measurement of TRH expression using an immunohistochemistry assay. In addition, pituitaries were excised for the extraction of total RNA. Finally, reverse transcription-quantitative polymerase chain reaction was performed for quantitation of TSHβ. Following swimming, the serum T3, T4 and TSH, the TSHβ mRNA expression levels in the pituitary and the TRH expression in the PVN of the surgical group were gradually increased. In the non-surgical group, no significant differences were observed in the serum T3, T4 and TSH levels compared with the control group. The TSHβ mRNA expression at the pituitary showed a similar result. Furthermore, the TRH expression at PVN was gradually increased and stress from swimming could increase the blood T4, T3 and TSH levels, TSHβ mRNA expression at the pituitary and TRH expression at the PVN in adrenalectomy Wistar rats. Moreover, the index in the surgical group changed significantly compared with the non-surgical group. In conclusion, the results suggest that there is a positive correlation between stress from forced swimming and the variation of the HPT axis.

Suppression of hepatic stellate cell activation by microRNA-29b

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

Sekiya, Yumiko; Ogawa, Tomohiro; Liver Research Center, Graduate School of Medicine, Osaka City University, Osaka

Highlights: {yields} Expression of miR-29b was found to be down-regulated during the activation of hepatic stellate cells in primary culture. {yields} Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs. {yields} It blunted the increased expression of {alpha}-SMA, DDR2, FN1, ITGB1, and PDGFR-b mRNAs essential for stellate cell activation. {yields} miR-29b overexpression led stellate cells to remain in a quiescent state, as evidenced by their star-like morphology. {yields} miR-29b overexpression suppressed the expression of c-fos mRNA. -- Abstract: MicroRNAs (miRNAs) participate in the regulation of cellular functions including proliferation, apoptosis, and migration. It has beenmore » previously shown that the miR-29 family is involved in regulating type I collagen expression by interacting with the 3'UTR of its mRNA. Here, we investigated the roles of miR-29b in the activation of mouse primary-cultured hepatic stellate cells (HSCs), a principal collagen-producing cell in the liver. Expression of miR-29b was found to be down-regulated during HSC activation in primary culture. Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs and additionally blunted the increased expression of {alpha}-SMA, DDR2, FN1, ITGB1, and PDGFR-{beta}, which are key genes involved in the activation of HSCs. Further, overexpression of miR-29b led HSCs to remain in a quiescent state, as evidenced by their quiescent star-like cell morphology. Although phosphorylation of FAK, ERK, and Akt, and the mRNA expression of c-jun was unaffected, miR-29b overexpression suppressed the expression of c-fos mRNA. These results suggested that miR-29b is involved in the activation of HSCs and could be a candidate molecule for suppressing their activation and consequent liver fibrosis.« less

Effect of limb ischemic preconditioning on myocardial apoptosis-related proteins in ischemia-reperfusion injury

PubMed Central

GAO, JIANZHI; ZHAO, LINJING; WANG, YONGLING; TENG, QINGLEI; LIANG, LIDONG; ZHANG, JINYING

2013-01-01

The aim of this study was to investigate the effect of limb ischemic preconditioning (LIPC) on myocardial apoptosis in myocardial ischemia-reperfusion injury (MIRI), as well as the regulation of caspase-3 and the B cell lymphoma 2 (Bcl-2) gene in LIPC. A total of 50 rats were divided randomly into 5 groups (n=10). Four rats in each group were drawn out for detection of apoptosis. The sham, MIRI and LIPC groups underwent surgery without additional treatment. In the LY294002 group, LY294002 preconditioning was administered 15 min before reperfusion. In the LY294002+LIPC group, following LIPC, LY294002 was administered 15 min before reperfusion. The relative expression of myocardial Bcl-2 and caspase-3 mRNA and the apoptotic index for each group were determined by reverse transcription-polymerase chain reaction (RT-PCR) and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), respectively. The ultrastructure of the cardiac muscle tissues was observed by election microscopy. Compared with the sham group, the expression of caspase-3 mRNA in the MIRI group significantly increased (P<0.05) and the expression of Bcl-2 mRNA clearly decreased. Compared with the MIRI group, LIPC reduced the expression of caspase-3 and increased the expression of Bcl-2 mRNA (P<0.05). There were no significant differences between the LY294002+LIPC group and the MIRI group. Compared with the sham group, the apoptotic index of myocardial cells in the MIRI group significantly increased (P<0.05). Compared with the MIRI group, LIPC significantly decreased the apoptotic index of myocardial cells (P<0.05) and LY294002 increased the apoptotic index of myocardial cells. Compared with the LIPC group, LY294002+LIPC significantly increased the apoptotic index of myocardial cells (P<0.05). There were no significant differences between the LY294002+LIPC and MIRI groups. In conclusion, LIPC increased the expression of Bcl-2 and decreased caspase-3 mRNA and apoptosis in myocardial tissue following MIRI. Therefore, LIPC plays a protective role in myocardial tissue. PMID:23737869

Distinct expression patterns of the E3 ligase SIAH-1 and its partner Kid/KIF22 in normal tissues and in the breast tumoral processes

PubMed Central

2010-01-01

SIAH proteins are the human members of an highly conserved family of E3 ubiquitin ligases. Several data suggest that SIAH proteins may have a role in tumor suppression and apoptosis. Previously, we reported that SIAH-1 induces the degradation of Kid (KIF22), a chromokinesin protein implicated in the normal progression of mitosis and meiosis, by the ubiquitin proteasome pathway. In human breast cancer cells stably transfected with SIAH-1, Kid/KIF22 protein level was markedly reduced whereas, the Kid/KIF22 mRNA level was increased. This interaction has been further elucidated through analyzing SIAH and Kid/KIF22 expression in both paired normal and tumor tissues and cell lines. It was observed that SIAH-1 protein is widely expressed in different normal tissues, and in cells lines but showing some differences in western blotting profiles. Immunofluorescence microscopy shows that the intracellular distribution of SIAH-1 and Kid/KIF22 appears to be modified in human tumor tissues compared to normal controls. When mRNA expression of SIAH-1 and Kid/KIF22 was analyzed by real-time PCR in normal and cancer breast tissues from the same patient, a large variation in the number of mRNA copies was detected between the different samples. In most cases, SIAH-1 mRNA is decreased in tumor tissues compared to their normal counterparts. Interestingly, in all breast tumor tissues analyzed, variations in the Kid/KIF22 mRNA levels mirrored those seen with SIAH-1 mRNAs. This concerted variation of SIAH-1 and Kid/KIF22 messengers suggests the existence of an additional level of control than the previously described protein-protein interaction and protein stability regulation. Our observations also underline the need to re-evaluate the results of gene expression obtained by qRT-PCR and relate it to the protein expression and cellular localization when matched normal and tumoral tissues are analyzed. PMID:20144232

Lactococcus lactis and Lactobacillus salivarius differently modulate early immunological response of Wistar rats co-administered with Listeria monocytogenes.

PubMed

Lukic, J; Jancic, I; Mirkovic, N; Bufan, B; Djokic, J; Milenkovic, M; Begovic, J; Strahinic, I; Lozo, J

2017-10-13

In the light of the increasing resistance of bacterial pathogens to antibiotics, one of the main global strategies in applied science is development of alternative treatments, which would be safe both for the host and from the environmental perspective. Accordingly, the aim of this study was to test whether two lactic acid bacteria (LAB) strains, Lactococcus lactis BGBU1-4 and Lactobacillus salivarius BGHO1, could be applied as safe supplements for Listeria infection. Two major research objectives were set: to compare the effects of BGBU1-4 and BGHO1 on early immune response in gut tissue of Wistar rats co-administered with Listeria monocytogenes ATCC19111 and next, to test how this applies to their usage as therapeutics in acute ATCC19111 infection. Intestinal villi (IV), Peyer's patches (PP) and mesenteric lymph nodes (MLN) were used for the analysis. The results showed that BGHO1 increased the mRNA expression of innate immune markers CD14, interleukin (IL)-1β and tumour necrosis factor (TNF)-α in PP and IV, and, in parallel, caused a decrease of listeriolysin O (LLO) mRNA expression in same tissues. In MLN of BGHO1 treated rats, LLO expression was increased, along with an increase of the expression of OX-62 mRNA and CD69, pointing to the activation of adaptive immunity. On the other hand, in BGBU1-4 treated rats, there was no reduction of LLO mRNA expression and no induction of innate immunity markers in intestinal tissue. Additionally, CD14 and IL-1β, as well as LLO, but not OX-62 mRNA and CD69 expression, were elevated in MLN of BGBU1-4 treated rats. However, when applied therapeutically, both, BGBU1-4 and BGHO1, lowered Listeria count in spleens of infected rats. Our results not only reveal the potential of LAB to ameliorate Listeria infections, but suggest different immunological effects of two different LAB strains, both of which could be effective in Listeria elimination.

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 .

Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples

PubMed Central

Zubakov, Dmitry; Hanekamp, Eline; Kokshoorn, Mieke; van IJcken, Wilfred

2007-01-01

Human body fluids such as blood and saliva represent the most common source of biological material found at a crime scene. Reliable tissue identification in forensic science can reveal significant insights into crime scene reconstruction and can thus contribute toward solving crimes. Limitations of existing presumptive tests for body fluid identification in forensics, which are usually based on chemoluminescence or protein analysis, are expected to be overcome by RNA-based methods, provided that stable RNA markers with tissue-specific expression patterns are available. To generate sets of stable RNA markers for reliable identification of blood and saliva stains we (1) performed whole-genome gene expression analyses on a series of time-wise degraded blood and saliva stain samples using the Affymetrix U133 plus2 GeneChip, (2) consulted expression databases to obtain additional information on tissue specificity, and (3) confirmed expression patterns of the most promising candidate genes by quantitative real-time polymerase chain reaction including additional forensically relevant tissues such as semen and vaginal secretion. Overall, we identified nine stable mRNA markers for blood and five stable mRNA markers for saliva detection showing tissue-specific expression signals in stains aged up to 180 days of age, expectedly older. Although, all of the markers were able to differentiate blood/saliva from semen samples, none of them could differentiate vaginal secretion because of the complex nature of vaginal secretion and the biological similarity of buccal and vaginal mucosa. We propose the use of these 14 stable mRNA markers for identification of blood and saliva stains in future forensic practice. Electronic supplementary material The online version of this article (doi:10.1007/s00414-007-0182-6) contains supplementary material, which is available to authorized users. PMID:17579879

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

Cytokine mRNA expression in normal skin of various age populations before and after engraftment onto nude mice.

PubMed

Gilhar, A; Ullmann, Y; Shalagino, R; Weisinger, G

1998-01-01

Whether the impact of skin biological age on cytokine expression is a result of this tissue's proliferation potential or not is an important issue in dermatology. We investigated these questions by monitoring cytokine marker mRNA expression from human skin samples from healthy groups of individuals. The skin samples studied represented three age groups: fetal (17-21 weeks), young (18-35 years) and aged (76-88 years). Furthermore, upon skin transplantation of tissue from different age groups onto nude mice, we investigated whether cytokine marker RNA levels would change or normalize. Interestingly, both TNF-alpha and P53 mRNA showed a similar pattern of expression. Both were significantly higher in fetal skin (p < 0.0001 and p < 0.05, respectively), and no difference was noted between aged versus young skin. In contrast to this, IL1-alpha mRNA was expressed at its lowest and highest levels in fetal and young skin, respectively. Following skin transplantation, cytokines and P53 mRNA expression were normalized to similar levels in all age groups. This study implies that when cytokine expression was determined directly at the mRNA level, post-natal expression was not significantly different at either age group. Furthermore, it seems that the environmental conditions surrounding the grafted human skin found on nude mice encouraged normalization of donor cytokine expression.

Differential Expression Patterns of occ1-Related Genes in Adult Monkey Visual Cortex

PubMed Central

Takahata, Toru; Komatsu, Yusuke; Watakabe, Akiya; Hashikawa, Tsutomu; Tochitani, Shiro

2009-01-01

We have previously revealed that occ1 is preferentially expressed in the primary visual area (V1) of the monkey neocortex. In our attempt to identify more area-selective genes in the macaque neocortex, we found that testican-1, an occ1-related gene, and its family members also exhibit characteristic expression patterns along the visual pathway. The expression levels of testican-1 and testican-2 mRNAs as well as that of occ1 mRNA start of high in V1, progressively decrease along the ventral visual pathway, and end of low in the temporal areas. Complementary to them, the neuronal expression of SPARC mRNA is abundant in the association areas and scarce in V1. Whereas occ1, testican-1, and testican-2 mRNAs are preferentially distributed in thalamorecipient layers including “blobs,” SPARC mRNA expression avoids these layers. Neither SC1 nor testican-3 mRNA expression is selective to particular areas, but SC1 mRNA is abundantly observed in blobs. The expressions of occ1, testican-1, testican-2, and SC1 mRNA were downregulated after monocular tetrodotoxin injection. These results resonate with previous works on chemical and functional gradients along the primate occipitotemporal visual pathway and raise the possibility that these gradients and functional architecture may be related to the visual activity–dependent expression of these extracellular matrix glycoproteins. PMID:19073625

Clinical values of AFP, GPC3 mRNA in peripheral blood for prediction of hepatocellular carcinoma recurrence following OLT: AFP, GPC3 mRNA for prediction of HCC.

PubMed

Wang, Yuliang; Shen, Zhongyang; Zhu, Zhijun; Han, Ruifa; Huai, Mingsheng

2011-03-01

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Annually, about 200,000 patients died of HCC in China. Liver transplantation (LT) holds great theoretical appeal in treating HCC. However, the high recurrence rate after transplantation is the most important limiting factor for long-term survival. To assess the value of alpha-fetoprotein (AFP) messenger RNA (mRNA), Glypican-3 (GPC3) mRNA-expressing cells in the peripheral blood (PB) for prediction of HCC recurrence following orthotopic liver transplantation (OLT). 29 patients with HCC who underwent OLT with a minimum clinical follow-up of 12 months were included in this retrospective study. We detected AFP mRNA, GPC3 mRNA-expressing cells in the PB by TaqMan real-time reverse transcriptase-polymerase chain reaction (RT-PCR), pre-, intra- and post-operatively. The early recurrence of patients was evaluated. 8 (28%), 15 (52%), and 9 (31%) patients had AFP mRNA detected pre-, intra-, and post-operatively, respectively. With 12 months of follow-up, HCC recurred in 7 (24%) patients. Univariate analysis revealed that positive pre- and post-operative AFP mRNA, TNM stage as well as vascular invasion were significant predictors for the HCC recurrence. Multivariate analysis revealed that being positive for AFP mRNA pre-operatively remained a significant risk factor for HCC recurrence after OLT. GPC3 mRNA was expressed in all PB samples. There was no significant difference in the expression levels of GPC3 mRNA between the HCC and control groups. There were no significant differences in GPC3 mRNA expression values between those patients with and without tumor recurrence. The pre-operative detection of circulating AFP mRNA-expressing cells could be a useful predictor for HCC recurrence following OLT. GPC3 mRNA-expressing cells in PB seem to have no diagnostic value.

The Inhibitory Effect of C-phycocyanin Containing Protein Extract (C-PC Extract) on Human Matrix Metalloproteinases (MMP-2 and MMP-9) in Hepatocellular Cancer Cell Line (HepG2).

PubMed

Kunte, Mugdha; Desai, Krutika

2017-06-01

Spirulina platensis :have been studied for several biological activities. In the current study C-phycocyanin containing protein extract (C-PC extract) of Spirulina platensis have been studied for its effect on human matrix metalloproteinases (MMP-1, MMP-2 and MMP-9) and tissue inhibitors of MMPs (TIMP-1 and TIMP-2). In the present study, breast cancer cell line (MDA-MB 231) and hepatocellular cancer cell line (HepG2) were examined for inhibition of MMPs at different levels of expression after C-PC extract treatment. Herein, we have demonstrated that C-PC extract significantly reduced activity of MMP-2 by 55.13% and MMP-9 by 57.9% in HepG2 cells at 15 μg concentration. Additionally, the treatment has reduced mRNA expression of MMP-2 and MMP-9 at 20 μg concentration by 1.65-folds and 1.66-folds respectively. The C-PC extract treatment have also downregulated a mRNA expression of TIMP-2 by 1.12 folds at 20 μg concentration in HepG2 cells. Together, these results indicate that C-PC, extract successfully inhibited MMP-2 and -9 at different levels of expression and TIMP-2 at a mRNA expression level; however, extract did not have any effect on MMP-1 expressed in MDA-MB231 and TIMP-1 expressed in HepG2 cells as well as the exact mechanism of inhibition of MMP-2, MMP-9 and TIMP-2 remained unclear.

In Situ Hybridization Method Reveals (Pro)renin Receptor Expressing Cells in the Pituitary Gland of Rats: Correlation with Anterior Pituitary Hormones.

PubMed

Takahashi, Kazuhiro; Yatabe, Megumi; Fujiwara, Ken; Hirose, Takuo; Totsune, Kazuhito; Yashiro, Takashi

2013-02-28

Expression of (pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, was studied in rat pituitary gland. In situ hybridization showed that cells expressing (P)RR mRNA were widely distributed in the anterior lobe and intermediate lobe of the pituitary gland. Double-staining using in situ hybridization for (P)RR mRNA and immunohistochemistry for the pituitary hormones showed that (P)RR mRNA was expressed in most of the GH cells and ACTH cells in the anterior lobe. (P)RR mRNA was also expressed in a few prolactin cells and TSH cells, but not in LH cells. The present study has shown for the first time the distribution of (P)RR mRNA expressing cells in the rat pituitary gland. These findings suggest that (P)RR plays physiological roles in the pituitary gland, such as the modulation of the pituitary hormone secretion.

Effect of electroacupuncture on the expression of interlukin-1beta mRNA after transient focal cerebral ischemia.

PubMed

Xu, Zhen-Feng; Wu, Gen-Cheng; Cao, Xiao-Ding

2002-01-01

It has been reported that interleukin-1beta (IL-1beta ) play a key role in the pathogenesis of cerebral ischemia. Acupuncture is an effective traditional medical therapy in China. The aim of present study was to evaluate the effect of electroacupuncture (EA) on IL-1beta mRNA expression after middle cerebral artery occlusion (MCAO) in rats. Using in situ hybridization technique, it was found that in the MCAO group the expression of IL-1beta mRNA was significantly increased at 2h, 6h, 12h after reperfusion in cerebral ischemic cortex compared with normal group. In EA+ MCAO group the expression of IL-1beta mRNA was significantly decreased at 2h, 6h and 12h in ischemic cortex compared with MCAO group. The results indicated that EA might decrease the IL-1beta protein expression by reducing the IL-beta mRNA expression in ischemic cortex.