The expression of Toll-like receptors 2, 4, 5, 7 and 9 in Merkel cell carcinoma.

PubMed

Jouhi, Lauri; Koljonen, Virve; Böhling, Tom; Haglund, Caj; Hagström, Jaana

2015-04-01

We sought to clarify whether the expression of toll-like receptors (TLR) in Merkel cell carcinoma (MCC) is linked to tumor and patient characteristics, especially the presence of Merkel cell polyoma virus (MCV). The study comprised of 128 patients with data on Merkel cell polyomavirus (MCV) status and clinical features were included in the study. Immunohistochemistry for TLR expression was performed on tissue microarray (TMA) slides. TLR 2, 4, 5, 7 and 9 expression was noted in most of the tumor specimens. Decreased expression of TLR 9 correlated strongly with MCV positivity. Cytoplasmic TLR 2 expression correlated with small tumor size, while nuclear TLR 2 and TLR 5 expressions with larger tumors. Increased nuclear TLR 4 expression and decreased TLR 7 expression were associated with older age. TLR 2, 4, 5, 7 and 9 appear to reflect certain clinicopathological variables and prognostic markers of MCC tumors. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Aurora-A overexpression and aneuploidy predict poor outcome in serous ovarian carcinoma.

PubMed

Lassus, Heini; Staff, Synnöve; Leminen, Arto; Isola, Jorma; Butzow, Ralf

2011-01-01

Aurora-A is a potential oncogene and therapeutic target in ovarian carcinoma. It is involved in mitotic events and overexpression leads to centrosome amplification and chromosomal instability. The objective of this study was to evaluate the clinical significance of Aurora-A and DNA ploidy in serous ovarian carcinoma. Serous ovarian carcinomas were analysed for Aurora-A protein by immunohistochemistry (n=592), Aurora-A copy number by CISH (n=169), Aurora-A mRNA by real-time PCR (n=158) and DNA ploidy by flowcytometry (n=440). Overexpression of Aurora-A was found in 27% of the tumors, cytoplasmic overexpression in 11% and nuclear in 17%. The cytoplasmic and nuclear overexpression were nearly mutually exclusive. Both cytoplasmic and nuclear overexpression were associated with shorter survival, high grade, high proliferation index and aberrant p53. Interestingly, only cytoplasmic expression was associated with aneuploidy and expression of phosphorylated Aurora-A. DNA ploidy was associated with poor patient outcome as well as aggressive clinicopathological parameters. In multivariate analysis, Aurora-A overexpression appeared as an independent prognostic factor for disease-free survival, together with grade, stage and ploidy. Aurora-A protein expression is strongly linked with poor patient outcome and aggressive disease characteristics, which makes Aurora-A a promising biomarker and a potential therapeutic target in ovarian carcinoma. Cytoplasmic and nuclear Aurora-A protein may have different functions. DNA aneuploidy is a strong predictor of poor prognosis in serous ovarian carcinoma. Copyright © 2010 Elsevier Inc. All rights reserved.

Synergistic growth inhibition of squamous cell carcinoma of the head and neck by erlotinib and epigallocatechin-3-gallate: the role of p53-dependent inhibition of nuclear factor-kappaB.

PubMed

Amin, A R M Ruhul; Khuri, Fadlo R; Chen, Zhuo Georgia; Shin, Dong M

2009-06-01

We have previously reported that the green tea polyphenol epigallocatechin-3-gallate (EGCG) and the epidermal growth factor receptor-tyrosine kinase inhibitor erlotinib had synergistic growth-inhibitory effects in cell culture and a nude mouse xenograft model of squamous cell carcinoma of the head and neck. However, the mechanism of their antitumor synergism is not fully understood. In the current study, we investigate the mechanism of their synergistic growth-inhibitory effects. The treatment of squamous cell carcinoma of the head and neck cell lines with erlotinib time-dependently increased the expression of cell cycle regulatory proteins p21 and p27 and apoptosis regulatory protein Bim. EGCG alone had very little or no effect on the expression of these proteins among the cell lines. However, simultaneous treatment with EGCG and erlotinib strongly inhibited erlotinib-induced expression of p21 and p27 without affecting the expression of Bim. Moreover, erlotinib increased the expression of p53 protein, the ablation of which by short hairpin RNA strongly inhibited EGCG- and erlotinib-mediated growth inhibition and the expression of p21, p27, and Bim. In addition, combined treatment with erlotinib and EGCG inhibited the protein level of p65 subunit of nuclear factor-kappaB and its transcriptional target Bcl-2, but failed to do so in cells with ablated p53. Taken together, our results, for the first time, suggest that erlotinib treatment activates p53, which plays a critical role in synergistic growth inhibition by erlotinib and EGCG via inhibiting nuclear factor-kappaB signaling pathway. Characterizing the underlying mechanisms of EGCG and erlotinib synergism will provide an important rationale for chemoprevention or treatment trials using this combination.

Global loss of a nuclear lamina component, lamin A/C, and LINC complex components SUN1, SUN2, and nesprin-2 in breast cancer.

PubMed

Matsumoto, Ayaka; Hieda, Miki; Yokoyama, Yuhki; Nishioka, Yu; Yoshidome, Katsuhide; Tsujimoto, Masahiko; Matsuura, Nariaki

2015-10-01

Cancer cells exhibit a variety of features indicative of atypical nuclei. However, the molecular mechanisms underlying these phenomena remain to be elucidated. The linker of nucleoskeleton and cytoskeleton (LINC) complex, a nuclear envelope protein complex consisting mainly of the SUN and nesprin proteins, connects nuclear lamina and cytoskeletal filaments and helps to regulate the size and shape of the nucleus. Using immunohistology, we found that a nuclear lamina component, lamin A/C and all of the investigated LINC complex components, SUN1, SUN2, and nesprin-2, were downregulated in human breast cancer tissues. In the majority of cases, we observed lower expression levels of these analytes in samples' cancerous regions as compared to their cancer-associated noncancerous regions (in cancerous regions, percentage of tissue samples exhibiting low protein expression: lamin A/C, 85% [n = 73]; SUN1, 88% [n = 43]; SUN2, 74% [n = 43]; and nesprin-2, 79% [n = 53]). Statistical analysis showed that the frequencies of recurrence and HER2 expression were negatively correlated with lamin A/C expression (P < 0.05), and intrinsic subtype and ki-67 level were associated with nesprin-2 expression (P < 0.05). In addition, combinatorial analysis using the above four parameters showed that all patients exhibited reduced expression of at least one of four components despite the tumor's pathological classification. Furthermore, several cultured breast cancer cell lines expressed less SUN1, SUN2, nesprin-2 mRNA, and lamin A/C compared to noncancerous mammary gland cells. Together, these results suggest that the strongly reduced expression of LINC complex and nuclear lamina components may play fundamental pathological functions in breast cancer progression. © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Specific repression of β-globin promoter activity by nuclear ferritin

PubMed Central

Broyles, Robert H.; Belegu, Visar; DeWitt, Christina R.; Shah, Sandeep N.; Stewart, Charles A.; Pye, Quentin N.; Floyd, Robert A.

2001-01-01

Developmental hemoglobin switching involves sequential globin gene activations and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repressor of the adult β-globin gene in embryonic erythroid cells. Our data show that a ferritin-family protein in K562 cell nuclear extracts binds specifically to a highly conserved CAGTGC motif in the β-globin promoter at −153 to −148 bp from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin that is enriched in ferritin-H chains also binds the CAGTGC promoter segment. Expression clones of ferritin-H markedly repress β-globin promoter-driven reporter gene expression in cotransfected CV-1 cells in which the β-promoter has been stimulated with the transcription activator erythroid Krüppel-like factor (EKLF). We have constructed chloramphenicol acetyltransferase reporter plasmids containing either a wild-type or mutant β-globin promoter for the −150 CAGTGC motif and have compared the constructs for susceptibility to repression by ferritin-H in cotransfection assays. We find that stimulation by cotransfected EKLF is retained with the mutant promoter, whereas repression by ferritin-H is lost. Thus, mutation of the −150 CAGTGC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promoter in our cell transfection assay, providing a strong link between DNA binding and function, and strong support for our proposal that nuclear ferritin-H is a repressor of the human β-globin gene. Such a repressor could be helpful in treating sickle cell and other genetic diseases. PMID:11481480

Ferritin L and Ferritin H are differentially located within hepatic and extra hepatic organs under physiological and acute phase conditions.

PubMed

Ahmad, Shakil; Moriconi, Federico; Naz, Naila; Sultan, Sadaf; Sheikh, Nadeem; Ramadori, Giuliano; Malik, Ihtzaz Ahmed

2013-01-01

Ferritin L (FTL) and Ferritin H (FTH) subunits are responsible for intercellular iron storage. We previously reported increasing amounts of liver cytoplasmic and nuclear iron content during acute phase response (APR). Aim of the present study is to demonstrate intracellular localization of ferritin subunits in liver compared with extra hepatic organs of rat under physiological and acute phase conditions. Rats were administered turpentine-oil (TO) intramuscularly to induce a sterile abscess (acute-phase-model) and sacrificed at different time points. Immunohistochemistry was performed utilizing horse-reddish-peroxidise conjugated secondary antibody on 4μm thick section. Liver cytoplasmic and nuclear protein were used for Western blot analysis. By means of immunohistology, FTL was detected in cytoplasm while a strong nuclear positivity for FTH was evident in the liver. Similarly, in heart, spleen and brain FTL was detected mainly in the cytoplasm while FTH demonstrated intense nuclear and a weak cytoplasmic expression. Western blot analysis of cytoplasmic and nuclear fractions from liver, heart, spleen and brain further confirmed mainly cytoplasmic expression of FTL in contrast to the nuclear and cytoplasmic expression of FTH. The data presented demonstrate the differential localization of FTL and FTH within hepatic and extra hepatic organs being FTL predominantly in the cytoplasm while FTH predominantly in nucleus.

Release of Severe Acute Respiratory Syndrome Coronavirus Nuclear Import Block Enhances Host Transcription in Human Lung Cells

PubMed Central

Tilton, Susan C.; Menachery, Vineet D.; Gralinski, Lisa E.; Schäfer, Alexandra; Matzke, Melissa M.; Webb-Robertson, Bobbie-Jo M.; Chang, Jean; Luna, Maria L.; Long, Casey E.; Shukla, Anil K.; Bankhead, Armand R.; Burkett, Susan E.; Zornetzer, Gregory; Tseng, Chien-Te Kent; Metz, Thomas O.; Pickles, Raymond; McWeeney, Shannon; Smith, Richard D.; Katze, Michael G.; Waters, Katrina M.; Baric, Ralph S.

2013-01-01

The severe acute respiratory syndrome coronavirus accessory protein ORF6 antagonizes interferon signaling by blocking karyopherin-mediated nuclear import processes. Viral nuclear import antagonists, expressed by several highly pathogenic RNA viruses, likely mediate pleiotropic effects on host gene expression, presumably interfering with transcription factors, cytokines, hormones, and/or signaling cascades that occur in response to infection. By bioinformatic and systems biology approaches, we evaluated the impact of nuclear import antagonism on host expression networks by using human lung epithelial cells infected with either wild-type virus or a mutant that does not express ORF6 protein. Microarray analysis revealed significant changes in differential gene expression, with approximately twice as many upregulated genes in the mutant virus samples by 48 h postinfection, despite identical viral titers. Our data demonstrated that ORF6 protein expression attenuates the activity of numerous karyopherin-dependent host transcription factors (VDR, CREB1, SMAD4, p53, EpasI, and Oct3/4) that are critical for establishing antiviral responses and regulating key host responses during virus infection. Results were confirmed by proteomic and chromatin immunoprecipitation assay analyses and in parallel microarray studies using infected primary human airway epithelial cell cultures. The data strongly support the hypothesis that viral antagonists of nuclear import actively manipulate host responses in specific hierarchical patterns, contributing to the viral pathogenic potential in vivo. Importantly, these studies and modeling approaches not only provide templates for evaluating virus antagonism of nuclear import processes but also can reveal candidate cellular genes and pathways that may significantly influence disease outcomes following severe acute respiratory syndrome coronavirus infection in vivo. PMID:23365422

The strategy of fusion genes construction determines efficient expression of introduced transcription factors.

PubMed

Adamus, Tomasz; Konieczny, Paweł; Sekuła, Małgorzata; Sułkowski, Maciej; Majka, Marcin

2014-01-01

The main goal in gene therapy and biomedical research is an efficient transcription factors (TFs) delivery system. SNAIL, a zinc finger transcription factor, is strongly involved in tumor, what makes its signaling pathways an interesting research subject. The necessity of tracking activation of intracellular pathways has prompted fluorescent proteins usage as localization markers. Advanced molecular cloning techniques allow to generate fusion proteins from fluorescent markers and transcription factors. Depending on fusion strategy, the protein expression levels and nuclear transport ability are significantly different. The P2A self-cleavage motif through its cleavage ability allows two single proteins to be simultaneously expressed. The aim of this study was to compare two strategies for introducing a pair of genes using expression vector system. We have examined GFP and SNAI1 gene fusions by comprising common nucleotide polylinker (multiple cloning site) or P2A motif in between them, resulting in one fusion or two independent protein expressions respectively. In each case transgene expression levels and translation efficiency as well as nuclear localization of expressed protein have been analyzed. Our data showed that usage of P2A motif provides more effective nuclear transport of SNAIL transcription factor than conventional genes linker. At the same time the fluorescent marker spreads evenly in subcellular space.

5,7-Dimethoxyflavone, an activator of PPARα/γ, inhibits UVB-induced MMP expression in human skin fibroblast cells.

PubMed

Kim, Jae Kyung; Mun, Sukyeong; Kim, Myung-Suk; Kim, Mi-Bo; Sa, Bo-Kyung; Hwang, Jae-Kwan

2012-03-01

Peroxisome proliferator-activated receptors (PPARs), which are members of the nuclear hormone receptor superfamily, are a family of ligand-activated transcription factors that consist of three isotypes (PPAR α, δ and γ). PPAR activity was previously thought to be limited to lipid metabolism and glucose homeostasis; however, intensive studies of PPARα/γ in recent years have revealed their importance in age-related inflammation and photoaging as regulators of cytokines, matrix metalloproteinases (MMPs) and nuclear factor-kappa B (NF-κB). We evaluated the ability of the PPARα/γ activator 5,7-dimethoxyflavone (5,7-DMF) to inhibit ultraviolet B (UVB)-induced MMP expression in Hs68 human skin fibroblasts. Hs68 cells were treated with 5,7-DMF and then exposed to UVB irradiation. MMP expression, production and activity were determined by reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay and gelatin zymography. PPARα/γ expression, catalase expression, and mitogen-activated protein kinase (MAPK), activator protein-1 (AP-1) and NF-κB signalling were evaluated by Western blot analysis. PPARα/γ activity was assessed with the GAL4/PPARα/γ transactivation assay. We found that 5,7-DMF strongly decreased MMP expression, production and activity. In addition, 5,7-DMF significantly increased PPARα/γ activation and catalase expression, thereby downregulating UVB-induced reactive oxygen species (ROS) production, ROS-induced MAPK signalling and downstream transcription factors. Finally, 5,7-DMF reduced IκBα phosphorylation, blocked NF-κB p65 nuclear translocation, strongly suppressed proinflammatory cytokines such as interleukin-6 (IL-6) and IL-8. 5,7-DMF prevents UVB-induced MMP expression by suppressing UVB-induced oxidative stress and age-related inflammation via NF-κB and MAPK/AP-1 pathways. Our findings suggest the usefulness of 5,7-DMF for preventing and treating skin photoaging. © 2011 John Wiley & Sons A/S.

Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning.

PubMed

Tung, Hsuan; Wei, Sung-Chan; Lo, Huei-Ru; Chao, Yu-Chan

2016-01-01

Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and ultimately affect viral replication.

Rare sugar D-allose strongly induces thioredoxin-interacting protein and inhibits osteoclast differentiation in Raw264 cells.

PubMed

Yamada, Kana; Noguchi, Chisato; Kamitori, Kazuyo; Dong, Youyi; Hirata, Yuko; Hossain, Mohammad A; Tsukamoto, Ikuko; Tokuda, Masaaki; Yamaguchi, Fuminori

2012-02-01

Oxidative stress modulates the osteoclast differentiation via redox systems, and thioredoxin 1 (Trx) promotes the osteoclast formation by regulating the activity of transcription factors. The function of Trx is known to be regulated by its binding partner, thioredoxin-interacting protein (TXNIP). We previously reported that the expression of TXNIP gene is strongly induced by a rare sugar D-allose. In this study, we tested the hypothesis that D-allose could inhibit the osteoclast differentiation by regulating the Trx function. We used a murine Raw264 cell line that differentiates to the osteoclast by the receptor activator of nuclear factor-κB ligand (RANKL) treatment. The effect of sugars was evaluated by tartrate-resistant acid phosphatase staining. The expression and localization of TXNIP and Trx protein were examined by Western blotting and immunohistochemisty. The activity of the nuclear factor-κB, nuclear factor of activated T cells, and activator protein 1 transcription factors was measured by the luciferase reporter assay. The addition of D-allose (25 mmol/L) inhibited the osteoclast differentiation down to 9.53% ± 1.27% of a receptor activator of nuclear factor-κB ligand-only treatment. During the osteoclast differentiation, a significant increase of TNXIP was observed by D-allose treatment. The immunohistochemical analysis showed that both Trx and TXNIP existed in the nucleus in preosteoclasts and osteoclasts. Overexpression of TXNIP by plasmid transfection also inhibited the osteoclast formation, indicating the functional importance of TXNIP for the osteoclast differentiation. Transcriptional activity of the activator protein 1, nuclear factor-κB, and nuclear factor of activated T cells, known to be modulated by Trx, were inhibited by D-allose. In conclusion, our data indicate that D-allose is a strong inhibitor of the osteoclast differentiation, and this effect could be caused by TXNIP induction and a resulting inhibition of the Trx function. Copyright © 2012 Elsevier Inc. All rights reserved.

Identification of two functional nuclear localization signals in the capsid protein of duck circovirus

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

Xiang, Qi-Wang; Zou, Jin-Feng; Wang, Xin

The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1-17 and 18-36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNAmore » binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.« less

Nuclear receptor TLX stimulates hippocampal neurogenesis and enhances learning and memory in a transgenic mouse model.

PubMed

Murai, Kiyohito; Qu, Qiuhao; Sun, GuoQiang; Ye, Peng; Li, Wendong; Asuelime, Grace; Sun, Emily; Tsai, Guochuan E; Shi, Yanhong

2014-06-24

The role of the nuclear receptor TLX in hippocampal neurogenesis and cognition has just begun to be explored. In this study, we generated a transgenic mouse model that expresses TLX under the control of the promoter of nestin, a neural precursor marker. Transgenic TLX expression led to mice with enlarged brains with an elongated hippocampal dentate gyrus and increased numbers of newborn neurons. Specific expression of TLX in adult hippocampal dentate gyrus via lentiviral transduction increased the numbers of BrdU(+) cells and BrdU(+)NeuN(+) neurons. Furthermore, the neural precursor-specific expression of the TLX transgene substantially rescued the neurogenic defects of TLX-null mice. Consistent with increased neurogenesis in the hippocampus, the TLX transgenic mice exhibited enhanced cognition with increased learning and memory. These results suggest a strong association between hippocampal neurogenesis and cognition, as well as significant contributions of TLX to hippocampal neurogenesis, learning, and memory.

Ligand protons in a frozen solution of copper histidine relax via a T1e-driven three-spin mechanism

NASA Astrophysics Data System (ADS)

Stoll, S.; Epel, B.; Vega, S.; Goldfarb, D.

2007-10-01

Davies electron-nuclear double resonance spectra can exhibit strong asymmetries for long mixing times, short repetition times, and large thermal polarizations. These asymmetries can be used to determine nuclear relaxation rates in paramagnetic systems. Measurements of frozen solutions of copper(L-histidine)2 reveal a strong field dependence of the relaxation rates of the protons in the histidine ligand, increasing from low (g‖) to high (g⊥) field. It is shown that this can be attributed to a concentration-dependent T1e-driven relaxation process involving strongly mixed states of three spins: the histidine proton, the Cu(II) electron spin of the same complex, and another distant electron spin with a resonance frequency differing from the spectrometer frequency approximately by the proton Larmor frequency. The protons relax more efficiently in the g⊥ region, since the number of distant electrons able to participate in this relaxation mechanism is higher than in the g‖ region. Analytical expressions for the associated nuclear polarization decay rate Teen-1 are developed and Monte Carlo simulations are carried out, reproducing both the field and the concentration dependences of the nuclear relaxation.

Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope.

PubMed

Lamm, Christian E; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

2016-03-10

In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell's nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein.

Stroke Induces Nuclear Shuttling of Histone Deacetylase 4.

PubMed

Kassis, Haifa; Shehadah, Amjad; Chopp, Michael; Roberts, Cynthia; Zhang, Zheng Gang

2015-07-01

Histone deacetylases (HDACs) 4 and 5 are abundantly expressed in the brain and have been implicated in the regulation of neurodegeneration. Under physiological conditions, HDACs 4 and 5 are expressed in the cytoplasm of brain cells where they cannot directly access chromatin. In response to external stimuli, they can shuttle to the nucleus and regulate gene expression. However, the effect of stroke on nuclear shuttling of HDACs 4 and 5 remains unknown. Using a rat model of middle cerebral artery occlusion, we examined the subcellular localization of HDACs 4 and 5 in the peri-infarct cortex during brain repair after stroke. Stroke significantly increased nuclear HDAC4 immunoreactivity in neurons, but not in astrocytes or in oligodendrocytes, of the peri-infarct cortex at 2, 7, and 14 days after middle cerebral artery occlusion. Neurons with nuclear HDAC4 immunoreactivity distributed across all layers of the peri-infarct cortex and were Ctip2+ excitatory and parvalbumin+ inhibitory neurons. These neurons were not TUNEL or BrdU positive. Furthermore, nuclear HDAC4 immunoreactivity was positively and significantly correlated with increased dendritic, axonal, and myelin densities as determined by microtubule-associated protein 2, phosphorylated neurofilament heavy chain, and myelin basic protein, respectively. Unlike HDAC4, stroke did not alter nuclear localization of HDAC5. Our data show that stroke induces nuclear shuttling of HDAC4 in neurons in the peri-infarct cortex, and that increased nuclear HDAC4 is strongly associated with neuronal remodeling but not with neuronal cell death, suggesting a role for nuclear HDAC4 in promoting neuronal recovery after ischemic injury. © 2015 American Heart Association, Inc.

Nuclear localization of foamy virus Gag precursor protein.

PubMed Central

Schliephake, A W; Rethwilm, A

1994-01-01

All foamy viruses give rise to a strong nuclear staining when infected cells are reacted with sera from infected hosts. This nuclear fluorescence distinguishes foamy viruses from all other retroviruses. The experiments reported here indicate that the foamy virus Gag precursor protein is transiently located in the nuclei of infected cells and this is the likely reason for the typical foamy virus nuclear fluorescence. By using the vaccinia virus expression system, a conserved basic sequence motif in the nucleocapsid domain of foamy virus Gag proteins was identified to be responsible for the nuclear transport of the gag precursor molecule. This motif was also found to be able to direct a heterologous protein, the Gag protein of human immunodeficiency virus, into the nucleus. Images PMID:8035493

Nuclear Photosynthetic Gene Expression Is Synergistically Modulated by Rates of Protein Synthesis in Chloroplasts and Mitochondria[W

PubMed Central

Pesaresi, Paolo; Masiero, Simona; Eubel, Holger; Braun, Hans-Peter; Bhushan, Shashi; Glaser, Elzbieta; Salamini, Francesco; Leister, Dario

2006-01-01

Arabidopsis thaliana mutants prors1-1 and -2 were identified on the basis of a decrease in effective photosystem II quantum yield. Mutations were localized to the 5′-untranslated region of the nuclear gene PROLYL-tRNA SYNTHETASE1 (PRORS1), which acts in both plastids and mitochondria. In prors1-1 and -2, PRORS1 expression is reduced, along with protein synthesis in both organelles. PRORS1 null alleles (prors1-3 and -4) result in embryo sac and embryo development arrest. In mutants with the leaky prors1-1 and -2 alleles, transcription of nuclear genes for proteins involved in photosynthetic light reactions is downregulated, whereas genes for other chloroplast proteins are upregulated. Downregulation of nuclear photosynthetic genes is not associated with a marked increase in the level of reactive oxygen species in leaves and persists in the dark, suggesting that the transcriptional response is light and photooxidative stress independent. The mrpl11 and prpl11 mutants are impaired in the mitochondrial and plastid ribosomal L11 proteins, respectively. The prpl11 mrpl11 double mutant, but neither of the single mutants, resulted in strong downregulation of nuclear photosynthetic genes, like that seen in leaky mutants for PRORS1, implying that, when organellar translation is perturbed, signals derived from both types of organelles cooperate in the regulation of nuclear photosynthetic gene expression. PMID:16517761

Dose-dependent difference of nuclear receptors involved in murine liver hypertrophy by piperonyl butoxide.

PubMed

Sakamoto, Yohei; Yoshida, Midori; Tamura, Kei; Takahashi, Miwa; Kodama, Yukio; Inoue, Kaoru

2015-12-01

Nuclear receptors play important roles in chemically induced liver hypertrophy in rodents. To clarify the involvement of constitutive androstane receptor (CAR) and other nuclear receptors in mouse liver hypertrophy induced by different doses of piperonyl butoxide (PBO), wild-type and CAR-knockout mice were administered PBO (200, 1,000, or 5,000 ppm) in the basal diet for 1 week. Increased liver weight and diffuse hepatocellular hypertrophy were observed at 5,000 ppm for both genotypes, accompanied by increased Cyp3a11 mRNA and CYP3A protein expression, suggesting that CAR-independent pathway, possibly pregnane X receptor (PXR), plays a major role in the induction of hypertrophy. Moreover, wild-type mice at 5,000 ppm showed enhanced hepatocellular hypertrophy and strong positive staining for CYP2B in the centrilobular area, suggesting the localized contribution of CAR. At 1,000 ppm, only wild-type mice showed liver weight increase and centrilobular hepatocellular hypertrophy concurrent with elevated Cyp2b10 mRNA expression and strong CYP2B staining, indicating that CAR was essential at 1,000 ppm. We concluded that high-dose PBO induced hypertrophy via CAR and another pathway, while lower dose of PBO induced a pathway mediated predominantly by CAR. The dose-responsiveness on liver hypertrophy is important for understanding the involvement of nuclear receptors.

Expression of nitric oxide synthase in the developing eye of Zebrafish Danio rerio

NASA Astrophysics Data System (ADS)

Wang, Yongjun; Zhang, Shicui; Sawant, M. S.

2004-12-01

Expression of nitric oxide synthase (NOS) in the developing eye of zebrafish was studied by NADPH-diaphorase staining technique. NOS activity was first observed in the optic primordium and the lens placode at 5-somite stage, and remained basically unchanged up to the prim-5 stage. Upon hatching, NOS activity was nearly equally detected in the gangalion cell layer and the photoreceptor layer in the developing retina. However, it began declining in the inner plexiform layer and the inner nuclear layer at this stage. NOS activity disappeared in the lens although the anterior lens epithelium was strongly stained. Two days after hatching, NOS activity was still strong in the photoreceptor layer, but decreased markedly in the gangalion cell layer, the inner plexiform layer and the inner nuclear layer with the retinal patterning. These suggested that nitric oxide (NO), the product of NOS, is not only involved in the modulation of patterning and differentiation of the retinal cells but also in the regulation of proliferation, and differentiation of the lens fibrocytes.

Nuclear overexpression of the overexpressed in lung cancer 1 predicts worse prognosis in gastric adenocarcinoma.

PubMed

Wang, Jue; Shen, Hongchang; Fu, Guobin; Zhao, Dandan; Wang, Weibo

2017-02-07

We have performed this retrospective study to elucidate whether elevated expression of the overexpressed in lung cancer 1 (OLC1) was related to the clinicopathological parameters and prognosis of patients with gastric adenocarcinoma. Additionally, different effects of various subcellular OLC1 expression on gastric adeno-carcinogenesis were focused on in our study. Both overall and subcellular expression of OLC1 was evaluated by immunohistochemistry(IHC) via tissue microarrays from total 393 samples. The Kaplan-Meier method and Cox's proportional hazard model were exerted to further explore the correlation between OLC1 and prognosis. Total overexpression of OLC1 was significantly associated with stage (P = 0.004) and differentiation (P = 0.009), and only the strong total expression could predict a poor prognosis (HR = 1.31, P = 0.04). There were significant associations found between nuclear overexpression and tumor invasion depth(P = 0.002), lymph node (P < 0.001), stage (P = 0.004), differentiation (P < 0.001) and smoking history (P = 0.045). Furthermore, over-expressed nuclear OLC1 protein could be an independent risk factor for gastric adenocarcinoma (univariate: HR = 1.43, P = 0.003; multivariate: HR = 1.39, P = 0.011). In general, both total and nuclear overexpression of OLC1 could be the signs of gastric adeno-carcinogenesis, which might be served as the biomarkers for diagnosis at an early stage, even at the onset of tumorigenesis. Rather than the total expression, nuclear overexpression of OLC1 was correlated with most clinicopathological parameters and could predict a poor overall survival as an independent factor for prognosis, which made it a more effective and sensitive biomarker for gastric adenocarcinoma.

Redox regulation of antioxidant enzymes: post-translational modulation of catalase and glutathione peroxidase activity by resveratrol in diabetic rat liver.

PubMed

Sadi, Gökhan; Bozan, Davut; Yildiz, Huseyin Bekir

2014-08-01

Resveratrol is a strong antioxidant that exhibits blood glucose-lowering effects, which might contribute to its usefulness in preventing complications associated with diabetes. The present study aimed to investigate resveratrol effects on catalase (CAT) and glutathione peroxidase (GPx) gene and protein expression, their phosphorylation states and activities in rat liver of STZ-induced diabetes. Diabetes increased the levels of total protein phosphorylation and p-CAT, while mRNA expression, protein levels, and activity were reduced. Although diabetes induced transcriptional repression over GPx, it did not affect the protein levels and activity. When resveratrol was administered to diabetic rats, an increase in activity was associated with an increase in p-GPx levels. Decrease in Sirtuin1 (SIRT1) and nuclear factor erythroid 2-related factor (Nrf2) and increase in nuclear factor kappa B (NFκB) gene expression in diabetes were associated with a decrease in CAT and GPx mRNA expression. A possible compensatory mechanism for reduced gene expression of antioxidant enzymes is proved to be nuclear translocation of redox-sensitive Nrf2 and NFκB in diabetes which is confirmed by the increase in nuclear and decrease in cytoplasmic protein levels of Nrf2 and NFκB. Taken together, these findings revealed that an increase in the oxidized state in diabetes intricately modified the cellular phosphorylation status and regulation of antioxidant enzymes. Gene regulation of antioxidant enzymes was accompanied by nuclear translocation of Nrf2 and NFκB. Resveratrol administration also activated a coordinated cytoprotective response against diabetes-induced changes in liver tissues.

Inverse expression of estrogen receptor-beta and nuclear factor-kappaB in urinary bladder carcinogenesis.

PubMed

Kontos, Stylianos; Kominea, Athina; Melachrinou, Maria; Balampani, Eleni; Sotiropoulou-Bonikou, Georgia

2010-09-01

To investigate the expression of nuclear factor-kappaB (NF-kappaB) and estrogen receptor-beta (ER-beta) signalling pathways in bladder urothelial carcinoma according to clinicopathological features, in order to elucidate their role during carcinogenesis. Immunohistochemical methodology was carried out on formalin-fixed, paraffin-embedded sections from urinary bladder carcinomas of 140 patients (94 males and 46 females) who underwent transurethral resection of bladder neoplasms. Correlations between ER-beta and NF-kappaB, and tumor grade and T-stage were evaluated, along with demographic data, sex and age. A significant decrease in ER-beta expression in the nucleus of bladder cells during loss of cell differentiation (r(s) = -0.61, P-value < 0.001, test of trend P-value = 0.003) and in muscle invasive carcinomas (T2-T4; test of trend P-value < 0.001) was found. p65 Subunit of NF-kappaB was expressed in the nucleus and in the cytoplasm of bladder epithelial cells. A strong positive association between tumor grade and nuclear expression of NF-kappaB was shown. No correlation between NF-kappaB, nuclear or cytoplasmic staining, with T-stage was observed. An inverse correlation between ER-beta and nuclear p65 immunoreactivity was observed (r(s) = -0.45, P-value < 0.001). There was no correlation with demographic data. Our immunohistochemical study suggests the possible inverse regulation of NF-kappaB and ER-beta transcription factor during bladder carcinogenesis. Selective ER-beta agonists and agents, inhibitors of NF-kappaB, might represent a possible new treatment strategy for bladder urothelial tumors.

Comprehensive analysis of the dynamic structure of nuclear localization signals.

PubMed

Yamagishi, Ryosuke; Okuyama, Takahide; Oba, Shuntaro; Shimada, Jiro; Chaen, Shigeru; Kaneko, Hiroki

2015-12-01

Most transcription and epigenetic factors in eukaryotic cells have nuclear localization signals (NLSs) and are transported to the nucleus by nuclear transport proteins. Understanding the features of NLSs and the mechanisms of nuclear transport might help understand gene expression regulation, somatic cell reprogramming, thus leading to the treatment of diseases associated with abnormal gene expression. Although many studies analyzed the amino acid sequence of NLSs, few studies investigated their three-dimensional structure. Therefore, we conducted a statistical investigation of the dynamic structure of NLSs by extracting the conformation of these sequences from proteins examined by X-ray crystallography and using a quantity defined as conformational determination rate (a ratio between the number of amino acids determining the conformation and the number of all amino acids included in a certain region). We found that determining the conformation of NLSs is more difficult than determining the conformation of other regions and that NLSs may tend to form more heteropolymers than monomers. Therefore, these findings strongly suggest that NLSs are intrinsically disordered regions.

Nuclear C-MYC expression level is associated with disease progression and potentially predictive of two year overall survival in prostate cancer.

PubMed

Zeng, Wen; Sun, Hanying; Meng, Fankai; Liu, Zeming; Xiong, Jing; Zhou, Sheng; Li, Fan; Hu, Jia; Hu, Zhiquan; Liu, Zheng

2015-01-01

Upregulation of nuclear C-MYC protein has been reported to be an early event in prostate cancer (PCa); however, its clinicopathological and prognostic significance remain controversial. We determined the association of nuclear C-MYC protein expression with clinicopathological parameters, prognosis, ETS-related gene (ERG) expression, and TMPRSS2-ERG status in PCa. Nuclear C-MYC and ERG expression by immunohistochemistry and TMPRSS2-ERG status by triple-color probe fluorescence in situ hybridization assay were determined in 50 hormone-naïve PCa patients and 31 radical prostatectomy specimens. Nuclear C-MYC immunostaining was negative, positive, and strong positive in 27.5%, 32.5%, and 40.0% of cases, respectively. C-MYC immunostaining was significantly associated with clinical T stage (P < 0.001), distant metastasis at the time of diagnosis (P < 0.001) and TMPRSS2-ERG status (P = 0.001) but not with ERG immunostaining (P = 0.818). In the Kaplan-Meier analysis, C-MYC positive cases were found to have worse 2-year OS compared with C-MYC negative cases (P = 0.027). However, in the univariate Cox analysis, only TMPRSS2-ERG status (hazard ratio [HR] 0.189, 95% CI 0.057-0.629; P = 0.007) and distant metastasis (HR 3.545, 95% CI 1.056-11.894; P = 0.040) were significantly associated with 2-year OS. After adjusting for these two factors, TMPRSS2-ERG status still impacted 2-year OS (HR 0.196, 95% CI 0.049-0.778; P = 0.020). Nuclear C-MYC overexpression may be associated with disease progression and potentially predictive of 2-year OS in PCa. This is the first study to demonstrate an association between nuclear C-MYC immunostaining and TMPRSS2-ERG status in PCa.

Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome

PubMed Central

Turan, Kadir; Mibayashi, Masaki; Sugiyama, Kenji; Saito, Shoko; Numajiri, Akiko; Nagata, Kyosuke

2004-01-01

Mx proteins belong to the dynamin superfamily of high molecular weight GTPases and interfere with multiplication of a wide variety of viruses. Earlier studies show that nuclear mouse Mx1 and human MxA designed to be localized in the nucleus inhibit the transcription step of the influenza virus genome. Here we set a transient influenza virus transcription system using luciferase as a reporter gene and cells expressing the three RNA polymerase subunits, PB1, PB2 and PA, and NP. We used this reporter assay system and nuclear-localized MxA proteins to get clues for elucidating the anti-influenza virus activity of MxA. Nuclear-localized VP16-MxA and MxA-TAg NLS strongly interfered with the influenza virus transcription. Over-expression of PB2 led to a slight resumption of the transcription inhibition by nuclear MxA, whereas over-expression of PB1 and PA did not affect the MxA activity. Of interest is that the inhibitory activity of the nuclear MxA was markedly neutralized by over-expression of NP. An NP devoid of its C-terminal region, but containing the N-terminal RNA binding domain, also neutralized the VP16-MxA activity in a dose-dependent manner, whereas an NP lacking the N-terminal region did not affect the VP16-MxA activity. Further, not only VP16-MxA but also the wild-type MxA was found to interact with NP in influenza virus-infected cells. This indicates that the nuclear MxA suppresses the influenza virus transcription by interacting with not only PB2 but also NP. PMID:14752052

Impact of Leishmania Infection on Host Macrophage Nuclear Physiology and Nucleopore Complex Integrity

PubMed Central

Isnard, Amandine; Christian, Jan G.; Kodiha, Mohamed; Stochaj, Ursula; McMaster, W. Robert; Olivier, Martin

2015-01-01

The protease GP63 is an important virulence factor of Leishmania parasites. We previously showed that GP63 reaches the perinuclear area of host macrophages and that it directly modifies nuclear translocation of the transcription factors NF-κB and AP-1. Here we describe for the first time, using molecular biology and in-depth proteomic analyses, that GP63 alters the host macrophage nuclear envelope, and impacts on nuclear processes. Our results suggest that GP63 does not appear to use a classical nuclear localization signal common between Leishmania species for import, but degrades nucleoporins, and is responsible for nuclear transport alterations. In the nucleoplasm, GP63 activity accounts for the degradation and mislocalization of proteins involved amongst others in gene expression and in translation. Collectively, our data indicates that Leishmania infection strongly affects nuclear physiology, suggesting that targeting of nuclear physiology may be a strategy beneficial for virulent Leishmania parasites. PMID:25826301

Usefulness of a break-apart FISH assay in the diagnosis of Xp11.2 translocation renal cell carcinoma.

PubMed

Kim, Soo Hee; Choi, Yoomi; Jeong, Hae Yeon; Lee, Kyoungbun; Chae, Ji Youn; Moon, Kyung Chul

2011-09-01

Xp11.2 translocation renal cell carcinoma (RCC) is a rare subtype of RCC predominantly reported in young patients. It results from gene fusions between the transcription factor E3 (TFE3) gene, which is located on chromosome Xp11.2, and various fusion partners. Recently, a dual color, break-apart fluorescence in situ hybridization (FISH) assay to detect Xp11.2 translocation was reported. We performed this study to evaluate the usefulness of the FISH assay in the diagnosis of Xp11.2 translocation RCC using a commercially available TFE3 break-apart probe. We immunohistochemically analyzed TFE3 nuclear expression in 809 cases of RCCs using 14 tissue microarray blocks and selected nine cases those showed moderate to strong positive nuclear immunoreactivity for TFE3. The extent of TFE3 nuclear expression was variable. The TFE3 FISH assay was performed in these 9 selected cases and 44 negative control cases. Only four out of nine selected cases showed the TFE3 break-apart signal. TFE3 FISH-positive cases mainly showed diffuse and strong TFE3 immunopositivity, but one case revealed focal and moderate TFE3 staining. On the contrary, TFE3 FISH-negative cases mainly revealed focal and moderate TFE3 immunoreactivity, however, one FISH-negative case revealed diffuse and strong TFE3 nuclear immunopositivity. All negative control cases revealed normal TFE3 FISH results. Our results reveal that TFE3 immunohistochemistry can show false-positive results, and that the TFE3 break-apart FISH assay is a useful complementary method for confirming the diagnosis of Xp11.2 translocation RCC.

Expression of EphA2 protein is positively associated with age, tumor size and Fuhrman nuclear grade in clear cell renal cell carcinomas.

PubMed

Wang, Longxin; Hu, Haibing; Tian, Feng; Zhou, Wenquan; Zhou, Shuigen; Wang, Jiandong

2015-01-01

The receptor tyrosine kinase of EphA2 has been shown frequently overexpressed in various types of human carcinomas, which implicated that it plays important roles in carcinogenesis. Although EphA2 protein expression has been investigated in many types of human carcinomas, the relationship between the expression of EphA2 protein in clear cell renal cell carcinoma was not well documented. In the present study, using specific anit-EphA2 polyclonal antibody and immunohistochemistry, we evaluated EphA2 protein expression levels in clear cell RCC specimens surgically resected from 90 patients. Our results shows that EphA2 protein was positively expressed in all normal renal tubes of 90 samples (100%, 3+), which was expressed at low levels in renal cortex but high levels in the collecting ducts of the renal medulla and papilla. EphA2 was negatively or weakly expressed in 30 out of 90 samples (33.3%, 0/1+), moderately expressed in 24 samples (26.7%, 2+) and strongly expressed in 36 samples (40%, 3+). Expression of EphA2 was positively associated with age (P=0.029), tumor diameters (P<0.001) and Fuhrman nuclear grade (P<0.001). Our results indicate that EphA2 variably expressed in clear cell renal cell carcinomas. High expression of EphA2 was more often found in big size and high nuclear grade tumors, which indicated EphA2 protein may be used as a new marker for the prognosis of clear cell renal cell carcinoma.

p54nrb is a new regulator of progression of malignant melanoma.

PubMed

Schiffner, Susanne; Zimara, Nicole; Schmid, Rainer; Bosserhoff, Anja-Katrin

2011-08-01

Nuclear RNA-binding protein p54(nrb) and its murine homolog NonO are known to be involved in a variety of nuclear processes including transcription and RNA processing. Melanoma inhibitory activity (MIA) has been shown to play an essential role in the progression of malignant melanoma and to influence melanoma-associated molecules and pathways in the early tumor formation steps. Interestingly, recent studies suggest that MIA is a regulator of p54(nrb). Here, we show that p54(nrb) is strongly expressed and localized in the nucleus of both melanoma cell lines and melanoma tissue samples compared with normal human melanocytes or normal skin, respectively. Furthermore, all tested melanoma cell lines revealed strong p54(nrb) promoter activity. Treatment with MIA-specific small interfering RNAs showed an influence of MIA on p54(nrb) expression on both messenger RNA (mRNA) and protein level. Knockdown of p54(nrb) protein in melanoma cell lines led to reduced proliferation rates and to a strong decrease in their migratory potential. In addition, attachment to laminin and poly-l-lysine was significantly increased. We could identify Connexin-43 (Cx-43) as a downstream target molecule of p54(nrb) as knockdown of p54(nrb) resulted in enhanced Cx-43 mRNA and protein levels. As a confirmation of these findings, melanoma cell lines showed very low Cx-43 expression levels compared with melanocytes. Our results demonstrate that p54(nrb) is highly expressed in malignant melanoma and, as a MIA target molecule, it seems to be involved in the development and progression of malignant melanoma.

The aryl hydrocarbon receptor-mediated disruption of vitellogenin synthesis in the fish liver: Cross-talk between AHR- and ERα-signalling pathways

PubMed Central

Bemanian, Vahid; Male, Rune; Goksøyr, Anders

2004-01-01

Background In the fish liver, the synthesis of egg yolk protein precursor vitellogenin (VTG) is under control of the estrogen receptor alpha (ERα). Environmental contaminants such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) are suspected to have antiestrogenic effects. The aryl hydrocarbon receptor (AHR) is the initial cellular target for TCDD and related compounds. The AHR is a ligand-activated transcription factor that stimulates the expression of the genes encoding xenobiotic metabolizing enzymes, such as cytochrome P450 1A (CYP1A). In this study, the effects of activation of AHR on the hepatic expression of VTG and ERα genes, in primary cultured salmon hepatocytes, have been investigated. Results The expression of the genes encoding VTG and ERα were strongly induced by 17β-estradiol (E2). However, the expression of VTG was disrupted by exposure of the cells to TCDD while CYP1A expression was enhanced. The effect of TCDD on VTG and CYP1A expression was annulled by the AHR-inhibitor α-naphthoflavone. Furthermore, exposure of the cells to TCDD abolished E2-induced accumulation of ERα mRNA. The AHR-mediated inhibitory effects on the expression of the VTG and ERα genes may occur at transcriptional and/or post-transcriptional levels. Nuclear run-off experiments revealed that simultaneous exposure of the cells to E2 and TCDD strongly inhibited the initiation of transcription of the VTG and ERα genes. In addition, inhibition of RNA synthesis by actinomycin D treatment showed that post-transcriptional levels of VTG and ERα mRNAs were not significantly altered upon treatment of the cells with TCDD. These results suggested that activation of AHR may inhibit the transactivation capacity of the ERα. Further, electrophoretic mobility shift assays using nuclear extracts prepared from cells treated for one or two hours with E2, alone or in mixture with TCDD, showed a strong reduction in the DNA binding activities upon TCDD treatment. These results also suggested that activation of the AHR signalling pathway caused a marked decrease in the number of the nuclear ERα or that activated AHR blocked the ability of ERα to bind to its target DNA sequence. Finally, our results from Northern hybridizations indicated that E2 treatment of the cells did not cause any significant effect on the TCDD-induced levels of CYP1A mRNA. Conclusion In fish hepatocytes E2 induces ERα and VTG gene expression. The presence of dioxin (TCDD) abolishes this induction, probably through the action of AHR in complex with AHR nuclear translocator, and possibly by direct interference with the auto-regulatory transcriptional loop of ERα. Furthermore, E2 does not interfere with TCDD induced CYP1A gene expression, suggesting that cross-talk between the ERα- and AHR-signalling pathways is unidirectional. PMID:15119955

Meningeal hemangiopericytoma and solitary fibrous tumors carry the NAB2-STAT6 fusion and can be diagnosed by nuclear expression of STAT6 protein.

PubMed

Schweizer, Leonille; Koelsche, Christian; Sahm, Felix; Piro, Rosario M; Capper, David; Reuss, David E; Pusch, Stefan; Habel, Antje; Meyer, Jochen; Göck, Tanja; Jones, David T W; Mawrin, Christian; Schittenhelm, Jens; Becker, Albert; Heim, Stephanie; Simon, Matthias; Herold-Mende, Christel; Mechtersheimer, Gunhild; Paulus, Werner; König, Rainer; Wiestler, Otmar D; Pfister, Stefan M; von Deimling, Andreas

2013-05-01

Non-central nervous system hemangiopericytoma (HPC) and solitary fibrous tumor (SFT) are considered by pathologists as two variants of a single tumor entity now subsumed under the entity SFT. Recent detection of frequent NAB2-STAT6 fusions in both, HPC and SFT, provided additional support for this view. On the other hand, current neuropathological practice still distinguishes between HPC and SFT. The present study set out to identify genes involved in the formation of meningeal HPC. We performed exome sequencing and detected the NAB2-STAT6 fusion in DNA of 8/10 meningeal HPC thereby providing evidence of close relationship of these tumors with peripheral SFT. Due to the considerable effort required for exome sequencing, we sought to explore surrogate markers for the NAB2-STAT6 fusion protein. We adopted the Duolink proximity ligation assay and demonstrated the presence of NAB2-STAT6 fusion protein in 17/17 HPC and the absence in 15/15 meningiomas. More practical, presence of the NAB2-STAT6 fusion protein resulted in a strong nuclear signal in STAT6 immunohistochemistry. The nuclear reallocation of STAT6 was detected in 35/37 meningeal HPC and 25/25 meningeal SFT but not in 87 meningiomas representing the most important differential diagnosis. Tissues not harboring the NAB2-STAT6 fusion protein presented with nuclear expression of NAB2 and cytoplasmic expression of STAT6 proteins. In conclusion, we provide strong evidence for meningeal HPC and SFT to constitute variants of a single entity which is defined by NAB2-STAT6 fusion. In addition, we demonstrate that this fusion can be rapidly detected by STAT6 immunohistochemistry which shows a consistent nuclear reallocation. This immunohistochemical assay may prove valuable for the differentiation of HPC and SFT from other mesenchymal neoplasms.

Nuclear expression and gain-of-function β-catenin mutation in glomangiopericytoma (sinonasal-type hemangiopericytoma): insight into pathogenesis and a diagnostic marker.

PubMed

Lasota, Jerzy; Felisiak-Golabek, Anna; Aly, F Zahra; Wang, Zeng-Feng; Thompson, Lester D R; Miettinen, Markku

2015-05-01

Glomangiopericytoma (sinonasal-type hemangiopericytoma) is a rare mesenchymal neoplasm with myoid phenotype (smooth muscle actin-positive), which distinguishes this tumor from soft tissue hemangiopericytoma/solitary fibrous tumor. Molecular genetic changes underlying the pathogenesis of glomangiopericytoma are not known. In this study, 13 well-characterized glomangiopericytomas were immunohistochemically evaluated for β-catenin expression. All analyzed tumors showed strong expression and nuclear accumulation of β-catenin. Following this observation, β-catenin glycogen serine kinase-3 beta phosphorylation region, encoded by exon 3, was PCR amplified in all cases and evaluated for mutations using Sanger sequencing. Heterozygous mutations were identified in 12 of 13 tumors. All mutations consisted of single-nucleotide substitutions: three in codon 32 (c.94G>C (n=2) and c.95A>T), four in codon 33 (two each c.98C>G and c.98C>T), two in codon 37 (c.109T>G), one in codon 41 (c.121A>G), and two in codon 45 (c.133T>C). At the protein level, these substitutions would lead to p.D32H, p.D32V, p.S33C, p.S33F, p.S37A, p.T41A, and p.S45L mutations, respectively. Previously, similar mutations have been reported in different types of cancers and shown to trigger activation of β-catenin signaling. All analyzed glomangiopericytomas showed prominent nuclear expression of cyclin D1, as previously shown for tumors with nuclear expression of β-catenin as a sign of oncogenic activation. These results demonstrate that mutational activation of β-catenin and associated cyclin D1 overexpression may be central events in the pathogenesis of glomangiopericytoma. In additon, nuclear accumulation of β-catenin is a diagnostic marker for glomangiopericytoma.

Nuclear Calcium Signaling Controls Expression of a Large Gene Pool: Identification of a Gene Program for Acquired Neuroprotection Induced by Synaptic Activity

PubMed Central

Zhang, Sheng-Jia; Zou, Ming; Lu, Li; Lau, David; Ditzel, Désirée A. W.; Delucinge-Vivier, Celine; Aso, Yoshinori; Descombes, Patrick; Bading, Hilmar

2009-01-01

Synaptic activity can boost neuroprotection through a mechanism that requires synapse-to-nucleus communication and calcium signals in the cell nucleus. Here we show that in hippocampal neurons nuclear calcium is one of the most potent signals in neuronal gene expression. The induction or repression of 185 neuronal activity-regulated genes is dependent upon nuclear calcium signaling. The nuclear calcium-regulated gene pool contains a genomic program that mediates synaptic activity-induced, acquired neuroprotection. The core set of neuroprotective genes consists of 9 principal components, termed Activity-regulated Inhibitor of Death (AID) genes, and includes Atf3, Btg2, GADD45β, GADD45γ, Inhibin β-A, Interferon activated gene 202B, Npas4, Nr4a1, and Serpinb2, which strongly promote survival of cultured hippocampal neurons. Several AID genes provide neuroprotection through a common process that renders mitochondria more resistant to cellular stress and toxic insults. Stereotaxic delivery of AID gene-expressing recombinant adeno-associated viruses to the hippocampus confers protection in vivo against seizure-induced brain damage. Thus, treatments that enhance nuclear calcium signaling or supplement AID genes represent novel therapies to combat neurodegenerative conditions and neuronal cell loss caused by synaptic dysfunction, which may be accompanied by a deregulation of calcium signal initiation and/or propagation to the cell nucleus. PMID:19680447

Phenotypical and molecular distinctness of sinonasal haemangiopericytoma compared to solitary fibrous tumour of the sinonasal tract.

PubMed

Agaimy, Abbas; Barthelmeß, Sarah; Geddert, Helene; Boltze, Carsten; Moskalev, Evgeny A; Koch, Michael; Wiemann, Stefan; Hartmann, Arndt; Haller, Florian

2014-11-01

Sinonasal haemangiopericytoma (SN-HPC) is a rare sinonasal mesenchymal neoplasm of perivascular myoid cell origin. Solitary fibrous tumour (SFT) occurs only very rarely in the sinonasal tract. SFT and soft tissue HPC have been considered a single entity. Recently, recurrent gene fusions involving NAB2-STAT6 resulting in differential expression of STAT6 were characterized as central molecular events in SFT. However, no data exist for NAB2-STAT6 status or STAT6 expression in SN-HPC. We examined six SN-HPCs and two sinonasal SFTs by immunohistochemistry and RT-PCR for NAB2-STAT6 fusions. SN-HPC affected three females and three males (mean age: 72 years). They expressed smooth muscle actin, lacked strong CD34 reactivity and were negative for nuclear STAT6 expression. RT-PCR analysis confirmed the absence of NAB2-STAT6 fusions in all cases. Conversely, both sinonasal SFTs (in males aged 39 and 52 years) displayed classical features of pleuropulmonary and soft-tissue SFTs (uniformly CD34-positive with strong nuclear expression of STAT6). RT-PCR revealed NAB2-STAT6 fusions in both cases. These findings confirm the molecular and phenotypical distinctness of these two entities. While SN-HPC is a site-specific sinonasal neoplasm of as yet unknown molecular pathogenesis, sinonasal SFTs show phenotypical and molecular identity to their pleural/extrapleural counterparts. © 2014 John Wiley & Sons Ltd.

Correlation of cytoplasmic beta-catenin and cyclin D1 overexpression during thyroid carcinogenesis around Semipalatinsk nuclear test site.

PubMed

Meirmanov, Serik; Nakashima, Masahiro; Kondo, Hisayoshi; Matsufuji, Reiko; Takamura, Noboru; Ishigaki, Katsu; Ito, Masahiro; Prouglo, Yuri; Yamashita, Shunichi; Sekine, Ichiro

2003-06-01

The Semipalatinsk nuclear test site (SNTS), the Republic of Kazakhstan, has been contaminated by radioactive fallout. The alteration of oncogenic molecules in thyroid cancer around the SNTS was considered worthy of analysis because it presented the potential to elucidate the relationship between radiation exposure and thyroid cancer. This study aimed to analyze both beta-catenin and cyclin D1 expressions in thyroid carcinomas around the SNTS. We examined nine cases of chronic thyroiditis, eight cases of follicular adenomas, and 23 cases of papillary carcinomas. Immunohistochemically, all carcinomas displayed a strong cytosolic beta-catenin expression, while both chronic thyroiditis and follicular adenomas showed a significantly lower cytoplasmic beta-catenin (22.2% and 37.5%, respectively). No cyclin D1 immunoreactivity was evident in chronic thyroiditis. In contrast, 62.5% of follicular adenomas and 87.0% of papillary carcinoma showed cyclin D1 overexpression. Additionally, a strong correlation between cytoplasmic beta-catenin and cyclin D1 expression was suggested in thyroid tumors. This study revealed a higher prevalence of both aberrant beta-catenin expression and cyclin D1 overexpression in papillary thyroid cancers around the SNTS than sporadic cases. The analysis of the alteration of the Wnt signaling-related molecules in thyroid cancer around the SNTS may be important to gain an insight into radiation-induced thyroid tumorigenesis.

Nuclear receptor TLX stimulates hippocampal neurogenesis and enhances learning and memory in a transgenic mouse model

PubMed Central

Murai, Kiyohito; Qu, Qiuhao; Sun, GuoQiang; Ye, Peng; Li, Wendong; Asuelime, Grace; Sun, Emily; Tsai, Guochuan E.; Shi, Yanhong

2014-01-01

The role of the nuclear receptor TLX in hippocampal neurogenesis and cognition has just begun to be explored. In this study, we generated a transgenic mouse model that expresses TLX under the control of the promoter of nestin, a neural precursor marker. Transgenic TLX expression led to mice with enlarged brains with an elongated hippocampal dentate gyrus and increased numbers of newborn neurons. Specific expression of TLX in adult hippocampal dentate gyrus via lentiviral transduction increased the numbers of BrdU+ cells and BrdU+NeuN+ neurons. Furthermore, the neural precursor-specific expression of the TLX transgene substantially rescued the neurogenic defects of TLX-null mice. Consistent with increased neurogenesis in the hippocampus, the TLX transgenic mice exhibited enhanced cognition with increased learning and memory. These results suggest a strong association between hippocampal neurogenesis and cognition, as well as significant contributions of TLX to hippocampal neurogenesis, learning, and memory. PMID:24927526

The zebrafish orphan nuclear receptor genes nr2e1 and nr2e3 are expressed in developing eye and forebrain.

PubMed

Kitambi, Satish Srinivas; Hauptmann, Giselbert

2007-02-01

Mammalian Nr2e1 (Tailless, Mtll or Tlx) and Nr2e3 (photoreceptor-specific nuclear receptor, Pnr) are highly related orphan nuclear receptors, that are expressed in eye and forebrain-derived structures. In this study, we analyzed the developmental expression patterns of zebrafish nr2e1 and nr2e3. RT-PCR analysis showed that nr2e1 and nr2e3 are both expressed during embryonic and post-embryonic development. To examine the spatial distribution of nr2e1 and nr2e3 during development whole-mount in situ hybridization was performed. At tailbud stage, initial nr2e1 expression was localized to the rostral brain rudiment anterior to pax2.1 and eng2 expression at the prospective midbrain-hindbrain boundary. During subsequent stages, nr2e1 became widely expressed in fore- and midbrain primordia, eye and olfactory placodes. At 24hpf, strong nr2e1 expression was detected in telencephalon, hypothalamus, dorsal thalamus, pretectum, midbrain tectum, and retina. At 2dpf, the initially widespread nr2e1 expression became more restricted to distinct regions within the fore- and midbrain and to the retinal ciliary margin, the germinal zone which gives rise to retina and presumptive iris. Expression of nr2e3 was exclusively found in the developing retina and epiphysis. In both structures, nr2e3 expression was found in photoreceptor cells. The developmental expression profile of zebrafish nr2e1 and nr2e3 is consistent with evolutionary conserved functions in eye and rostral brain structures.

Cloning, expression and nuclear localization of human NPM3, a member of the nucleophosmin/nucleoplasmin family of nuclear chaperones

PubMed Central

Shackleford, Gregory M; Ganguly, Amit; MacArthur, Craig A

2001-01-01

Background Studies suggest that the related proteins nucleoplasmin and nucleophosmin (also called B23, NO38 or numatrin) are nuclear chaperones that mediate the assembly of nucleosomes and ribosomes, respectively, and that these activities are accomplished through the binding of basic proteins via their acidic domains. Recently discovered and less well characterized members of this family of acidic phosphoproteins include mouse nucleophosmin/nucleoplasmin 3 (Npm3) and Xenopus NO29. Here we report the cloning and initial characterization of the human ortholog of Npm3. Results Human genomic and cDNA clones of NPM3 were isolated and sequenced. NPM3 lies 5.5 kb upstream of FGF8 and thus maps to chromosome 10q24-26. In addition to amino acid similarities, NPM3 shares many physical characteristics with the nucleophosmin/nucleoplasmin family, including an acidic domain, multiple potential phosphorylation sites and a putative nuclear localization signal. Comparative analyses of 14 members of this family from various metazoans suggest that Xenopus NO29 is a candidate ortholog of human and mouse NPM3, and they further group both proteins closer with the nucleoplasmins than with the nucleophosmins. Northern blot analysis revealed that NPM3 was strongly expressed in all 16 human tissues examined, with especially robust expression in pancreas and testis; lung displayed the lowest level of expression. An analysis of subcellular fractions of NIH3T3 cells expressing epitope-tagged NPM3 revealed that NPM3 protein was localized solely in the nucleus. Conclusions Human NPM3 is an abundant and widely expressed protein with primarily nuclear localization. These biological activities, together with its physical relationship to the chaparones nucleoplasmin and nucleophosmin, are consistent with the proposed function of NPM3 as a molecular chaperone functioning in the nucleus. PMID:11722795

Nesprin-2 epsilon: A novel nesprin isoform expressed in human ovary and Ntera-2 cells

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

Lam, Le Thanh; Boehm, Sabrina V.; Roberts, Roland G.

2011-08-26

Highlights: {yields} A novel epsilon isoform of nesprin-2 has been discovered. {yields} This 120 kDa protein was predicted by bioinformatic analysis, but has not previously been observed. {yields} It is the main isoform expressed in a teratocarcinoma cell line and is also found in ovary. {yields} Like other nesprins, it is located at the nuclear envelope. {yields} We suggest it may have a role in very early development or in some ovary-specific function. -- Abstract: The nuclear envelope-associated cytoskeletal protein, nesprin-2, is encoded by a large gene containing several internal promoters that produce shorter isoforms. In a study of Ntera-2more » teratocarcinoma cells, a novel isoform, nesprin-2-epsilon, was found to be the major mRNA and protein product of the nesprin-2 gene. Its existence was predicted by bioinformatic analysis, but this is the first direct demonstration of both the mRNA and the 120 kDa protein which is located at the nuclear envelope. In a panel of 21 adult and foetal human tissues, the nesprin-2-epsilon mRNA was strongly expressed in ovary but was a minor isoform elsewhere. The expression pattern suggests a possible link with very early development and a likely physiological role in ovary.« less

Cytoplasmic localization of alteration/deficiency in activation 3 (ADA3) predicts poor clinical outcome in breast cancer patients.

PubMed

Mirza, Sameer; Rakha, Emad A; Alshareeda, Alaa; Mohibi, Shakur; Zhao, Xiangshan; Katafiasz, Bryan J; Wang, Jun; Gurumurthy, Channabasavaiah Basavaraju; Bele, Aditya; Ellis, Ian O; Green, Andrew R; Band, Hamid; Band, Vimla

2013-02-01

Transcriptional activation by estrogen receptor (ER) is a key step to breast oncogenesis. Given previous findings that ADA3 is a critical component of HAT complexes that regulate ER function and evidence that overexpression of other ER coactivators such as SRC-3 is associated with clinical outcomes in breast cancer, the current study was designed to assess the potential significance of ADA3 expression/localization in human breast cancer patients. In this study, we analyzed ADA3 expression in breast cancer tissue specimens and assessed the correlation of ADA3 staining with cancer progression and patient outcome. Tissue microarrays prepared from large series of breast cancer patients with long-term follow-ups were stained with anti-ADA3 monoclonal antibody using immunohistochemistry. Samples were analyzed for ADA3 expression followed by correlation with various clinicopathological parameters and patients' outcomes. We report that breast cancer specimens show predominant nuclear, cytoplasmic, or mixed nuclear + cytoplasmic ADA3 staining patterns. Predominant nuclear ADA3 staining correlated with ER+ status. While predominant cytoplasmic ADA3 staining negatively correlated with ER+ status, but positively correlated with ErbB2, EGFR, and Ki67. Furthermore, a positive correlation of cytoplasmic ADA3 was observed with higher histological grade, mitotic counts, Nottingham Prognostic Index, and positive vascular invasion. Patients with nuclear ADA3 and ER positivity have better breast cancer specific survival and distant metastasis free survival. Significantly, cytoplasmic expression of ADA3 showed a strong positive association with reduced BCSS and DMFS in ErbB2+/EGFR+ patients. Although in multivariate analyses ADA3 expression was not an independent marker of survival, predominant nuclear ADA3 staining in breast cancer tissues correlates with ER+ expression and together serves as a marker of good prognosis, whereas predominant cytoplasmic ADA3 expression correlates with ErbB2+/EGFR+ expression and together is a marker of poor prognosis. Thus, ADA3 cytoplasmic localization together with ErbB2+/EGFR+ status may serve as better prognostic marker than individual proteins to predict survival of patients.

X-inactivation patterns in female Leber`s hereditary optic neuropathy patients do not support a strong X-linked determinant

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

Pegoraro, E.; Hoffman, E.P.; Carelli, V.

1996-02-02

Leber`s hereditary optic neuropathy (LHON) accounts for about 3% of the cases of blindness in young adult males. The underlying mitochondrial pathogenesis of LHON has been well studied, with specific mitochondrial DNA (mtDNA) mutations of structural genes described and well characterized. However, enigmatic aspects of the disease are not explained by mutation data, such as the higher proportion of affected males, the later onset of the disease in females, and the presence of unaffected individuals with a high proportion of mutant mtDNA. A hypothesis which has been put forward to explain the unusual disease expression is a dual model ofmore » mtDNA and X-linked nuclear gene inheritance. If a nuclear X-linked modifier gene influences the expression of the mitochondrial-linked mutant gene then the affected females should be either homozygous for the nuclear determinant, or if heterozygous, lyonization should favor the mutant X. In order to determine if an X-linked gene predisposes to LHON phenotype we studied X-inactivation patterns in 35 females with known mtDNA mutations from 10 LHON pedigrees. Our results do not support a strong X-linked determinant in LHON cause: 2 of the 10 (20%) manifesting carriers showed skewing of X-inactivation, as did 3 of the 25 (12%) nonmanifesting carriers. 39 refs., 2 figs., 1 tab.« less

Nuclear-cytoplasmic localization of acetyl coenzyme A synthetase-1 in the rat brain

PubMed Central

Ariyannur, Prasanth S.; Moffett, John R.; Madhavarao, Chikkathur N; Arun, Peethambaran; Vishnu, Nisha; Jacobowitz, David M.; Hallows, William C.; Denu, John M.; Namboodiri, Aryan M.A.

2011-01-01

Acetyl coenzyme A synthetase 1 (AceCS1) catalyzes the synthesis of acetyl coenzyme A from acetate and coenzyme A, and is thought to play diverse roles ranging from fatty acid synthesis to gene regulation. Using an affinity purified antibody generated against an 18-mer peptide sequence of AceCS1, and a polyclonal antibody directed against recombinant AceCS1 protein, we examined the expression of AceCS1 in the rat brain. AceCS1 immunoreactivity in the adult rat brain was present predominantly in cell nuclei, with only light to moderate cytoplasmic staining in some neurons, axons and oligodendrocytes. Some non-neuronal cell nuclei were very strongly immunoreactive, including those of some oligodendrocytes, whereas neuronal nuclei ranged from unstained to moderately stained. Both antibodies stained some neuronal cell bodies and axons, especially in the hindbrain. AceCS1 immunoreactivity was stronger and more widespread in the brains of 18 day old rats than in adults, with increased expression in oligodendrocytes and neurons, including cortical pyramidal cells. Expression of AceCS1 was substantially upregulated in neurons throughout the brain after controlled cortical impact injury. The strong AceCS1 expression observed in the nuclei of CNS cells during brain development and after injury is consistent with a role in nuclear histone acetylation and therefore the regulation of chromatin structure and gene expression. The cytoplasmic staining observed in some oligodendrocytes, especially during postnatal brain development, suggests an additional role in CNS lipid synthesis and myelination. Neuronal and axonal localization implicates AceCS1 in cytoplasmic acetylation reactions in some neurons. PMID:20533355

Effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells.

PubMed

Minchenko, O H; Riabovol, O O; Tsymbal, D O; Minchenko, D O; Ratushna, O O

2016-01-01

We have studied the effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells under the inhibition of IRE1 (inositol requiring enzyme-1), which controls cell proliferation and tumor growth as a central mediator of endoplasmic reticulum stress. It was shown that hypoxia down-regulated gene expression of malate dehydrogenase 2 (MDH2), malic enzyme 2 (ME2), mitochondrial aspartate aminotransferase (GOT2), and subunit B of succinate dehydrogenase (SDHB) in control (transfected by empty vector) glioma cells in a gene specific manner. At the same time, the expression level of mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) and subunit D of succinate dehydrogenase (SDHD) genes in these cells does not significantly change in hypoxic conditions. It was also shown that the inhibition of ІRE1 signaling enzyme function in U87 glioma cells decreases the effect of hypoxia on the expression of ME2, GOT2, and SDHB genes and introduces the sensitivity of IDH2 gene to hypoxia. Furthermore, the expression of all studied genes depends on IRE1-mediated endoplasmic reticulum stress signaling in gene specific manner, because ІRE1 knockdown significantly decreases their expression in normoxic conditions, except for IDH2 gene, which expression level is strongly up-regulated. Therefore, changes in the expression level of nuclear genes encoding ME2, MDH2, IDH2, SDHB, SDHD, and GOT2 proteins possibly reflect metabolic reprogramming of mitochondria by hypoxia and IRE1-mediated endoplasmic reticulum stress signaling and correlate with suppression of glioma cell proliferation under inhibition of the IRE1 enzyme function.

Silencing and transcriptional properties of the imprinted Airn ncRNA are independent of the endogenous promoter

PubMed Central

Stricker, Stefan H; Steenpass, Laura; Pauler, Florian M; Santoro, Federica; Latos, Paulina A; Huang, Ru; Koerner, Martha V; Sloane, Mathew A; Warczok, Katarzyna E; Barlow, Denise P

2008-01-01

The Airn macro ncRNA is the master regulator of imprinted expression in the Igf2r imprinted gene cluster where it silences three flanking genes in cis. Airn transcription shows unusual features normally viewed as promoter specific, such as impaired post-transcriptional processing and a macro size. The Airn transcript is 108 kb long, predominantly unspliced and nuclear localized, with only a minority being variably spliced and exported. Here, we show by deletion of the Airn ncRNA promoter and replacement with a constitutive strong or weak promoter that splicing suppression and termination, as well as silencing activity, are maintained by strong Airn expression from an exogenous promoter. This indicates that all functional regions are located within the Airn transcript. DNA methylation of the maternal imprint control element (ICE) restricts Airn expression to the paternal allele and we also show that a strong active promoter is required to maintain the unmethylated state of the paternal ICE. Thus, Airn expression not only induces silencing of flanking mRNA genes but also protects the paternal copy of the ICE from de novo methylation. PMID:19008856

Human Homolog of Drosophila Ariadne (HHARI) is a marker of cellular proliferation associated with nuclear bodies

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

Elmehdawi, Fatima; Wheway, Gabrielle; Szymanska, Katarzyna

2013-02-01

HHARI (also known as ARIH1) is an ubiquitin-protein ligase and is the cognate of the E2, UbcH7 (UBE2L3). To establish a functional role for HHARI in cellular proliferation processes, we performed a reverse genetics screen that identified n=86/522 (16.5%) ubiquitin conjugation components that have a statistically significant effect on cell proliferation, which included HHARI as a strong hit. We then produced and validated a panel of specific antibodies that establish HHARI as both a nuclear and cytoplasmic protein that is expressed in all cell types studied. HHARI was expressed at higher levels in nuclei, and co-localized with nuclear bodies includingmore » Cajal bodies (p80 coilin, NOPP140), PML and SC35 bodies. We confirmed reduced cellular proliferation after ARIH1 knockdown with individual siRNA duplexes, in addition to significantly increased levels of apoptosis, an increased proportion of cells in G2 phase of the cell cycle, and significant reductions in total cellular RNA levels. In head and neck squamous cell carcinoma biopsies, there are higher levels of HHARI expression associated with increased levels of proliferation, compared to healthy control tissues. We demonstrate that HHARI is associated with cellular proliferation, which may be mediated through its interaction with UbcH7 and modification of proteins in nuclear bodies. -- Highlights: ► We produce and validate new antibody reagents for the ubiquitin-protein ligase HHARI. ► HHARI colocalizes with nuclear bodies including Cajal, PML and SC35 bodies. ► We establish new functions in cell proliferation regulation for HHARI. ► Increased HHARI expression associates with squamous cell carcinoma and proliferation.« less

Interleukin-33 is expressed in the lesional epidermis in herpes virus infection but not in verruca vulgaris.

PubMed

Jin, Meijuan; Komine, Mayumi; Tsuda, Hidetoshi; Oshio, Tomoyuki; Ohtsuki, Mamitaro

2018-04-25

Interleukin (IL)-33 is released on cell injury and activates the immune reaction. IL-33 is involved in antiviral reaction in herpes virus infection, but the source that secretes IL-33 has not been identified. We speculate that keratinocytes injured in herpes virus infection secrete IL-33. In order to detect IL-33 in the lesional epidermis of patients with herpes virus infection, we immunostained several cutaneous herpes virus infection samples with an anti-IL-33 antibody, and compared them with cutaneous human papilloma virus (HPV) infection samples. We observed strong nuclear and mild cytoplasmic staining in epidermal keratinocytes of the lesional skin samples with herpes simplex virus and varicella zoster virus infections. However, staining was not observed in the epidermis of verruca vulgaris (VV) samples. We assumed that the strong immune reaction to herpes virus infection may depend on strong IL-33 expression in the epidermis, while very weak immune reaction in samples from patients with VV may be due to low or no expression of IL-33 in the lesional epidermis. © 2018 Japanese Dermatological Association.

Possible involvement of nuclear factor erythroid 2-related factor 2 in the gene expression of Cyp2b10 and Cyp2a5.

PubMed

Ashino, Takashi; Ohkubo-Morita, Haruyo; Yamamoto, Masayuki; Yoshida, Takemi; Numazawa, Satoshi

2014-01-01

Cytochrome P450 gene expression is altered by various chemical compounds. In this study, we used nuclear factor erythroid 2-related factor 2 (Nrf2)-deficient (Nrf2(-⧸-)) mice to investigate the involvement of Nrf2 in Cyp2b10 and Cyp2a5 gene expression. Phorone, an Nrf2 activator, strongly increased Cyp2b10 and Cyp2a5 mRNA as well as Nrf2 target genes, including NAD(P)H-quinone oxidoreductase-1 and heme oxygenase-1, in wild-type mouse livers 8 h after treatment. The phorone-induced mRNA levels in Nrf2(-⧸-) mouse livers were lower than that in wild-type mouse livers. Nrf2(-⧸-) mice showed attenuated Cyp2b10 and Cyp2a5 induction by phenobarbital, a classical Cyp2b inducer. These findings suggest that the Nrf2 pathway is involved in Cyp2b10 and Cyp2a5 gene expression.

Possible involvement of nuclear factor erythroid 2-related factor 2 in the gene expression of Cyp2b10 and Cyp2a5☆

PubMed Central

Ashino, Takashi; Ohkubo-Morita, Haruyo; Yamamoto, Masayuki; Yoshida, Takemi; Numazawa, Satoshi

2014-01-01

Cytochrome P450 gene expression is altered by various chemical compounds. In this study, we used nuclear factor erythroid 2-related factor 2 (Nrf2)–deficient (Nrf2−⧸−) mice to investigate the involvement of Nrf2 in Cyp2b10 and Cyp2a5 gene expression. Phorone, an Nrf2 activator, strongly increased Cyp2b10 and Cyp2a5 mRNA as well as Nrf2 target genes, including NAD(P)H-quinone oxidoreductase-1 and heme oxygenase-1, in wild-type mouse livers 8 h after treatment. The phorone-induced mRNA levels in Nrf2−⧸− mouse livers were lower than that in wild-type mouse livers. Nrf2−⧸− mice showed attenuated Cyp2b10 and Cyp2a5 induction by phenobarbital, a classical Cyp2b inducer. These findings suggest that the Nrf2 pathway is involved in Cyp2b10 and Cyp2a5 gene expression. PMID:24494203

Hepatocyte nuclear factor-4alpha induces transdifferentiation of hematopoietic cells into hepatocytes.

PubMed

Khurana, Satish; Jaiswal, Amit K; Mukhopadhyay, Asok

2010-02-12

Hematopoietic stem cells can directly transdifferentiate into hepatocytes because of cellular plasticity, but the molecular basis of transdifferentiation is not known. Here, we show the molecular basis using lineage-depleted oncostatin M receptor beta-expressing (Lin(-)OSMRbeta(+)) mouse bone marrow cells in a hepatic differentiation culture system. Differentiation of the cells was marked by the expression of albumin. Hepatocyte nuclear factor (HNF)-4alpha was expressed and translocated into the nuclei of the differentiating cells. Suppression of its activation in OSM-neutralized culture medium inhibited cellular differentiation. Ectopic expression of full-length HNF4alpha in 32D myeloid cells resulted in decreased myeloid colony-forming potential and increased expression of hepatocyte-specific genes and proteins. Nevertheless, the neohepatocytes produced in culture expressed active P450 enzyme. The obligatory role of HNF4alpha in hepatic differentiation was confirmed by transfecting Lin(-)OSMRbeta(+) cells with dominant negative HNF4alpha in the differentiation culture because its expression inhibited the transcription of the albumin and tyrosine aminotransferase genes. The loss and gain of functional activities strongly suggested that HNF4alpha plays a central role in the transdifferentiation process. For the first time, this report demonstrates the mechanism of transdifferentiation of hematopoietic cells into hepatocytes, in which HNF4alpha serves as a molecular switch.

A nuclear gene for the iron-sulfur subunit of mitochondrial complex II is specifically expressed during Arabidopsis seed development and germination.

PubMed

Elorza, Alvaro; Roschzttardtz, Hannetz; Gómez, Isabel; Mouras, Armand; Holuigue, Loreto; Araya, Alejandro; Jordana, Xavier

2006-01-01

Three nuclear genes, SDH2-1, SDH2-2 and SDH2-3, encode the essential iron-sulfur subunit of mitochondrial complex II in Arabidopsis thaliana. SDH2-1 and SDH2-2 probably arose via a recent duplication event and we reported that both are expressed in all organs from adult plants. In contrast, transcripts from SDH2-3 were not detected. Here we present data demonstrating that SDH2-3 is specifically expressed during seed development. SDH2-3 transcripts appear during seed maturation, persist through desiccation, are abundant in dry seeds and markedly decline during germination. Analysis of transgenic Arabidopsis plants carrying the SDH2-3 promoter fused to the beta-glucuronidase reporter gene shows that the SDH2-3 promoter is activated in the embryo during maturation, from the bent-cotyledon stage. beta-Glucuronidase expression correlates with the appearance of endogenous SDH2-3 transcripts, suggesting that control of this nuclear gene is achieved through transcriptional regulation. Furthermore, progressive deletions of this promoter identified a 159 bp region (-223 to -65) important for SDH2-3 transcriptional activation in seeds. Interestingly, the SDH2-3 promoter remains active in embryonic tissues during germination and post-germinative growth, and is turned off in vegetative tissues (true leaves). In contrast to SDH2-3 transcripts, SDH2-1 and SDH2-2 transcripts are barely detected in dry seeds and increase during germination and post-germinative growth. The opposite expression patterns of SDH2 nuclear genes strongly suggest that during germination the embryo-specific SDH2-3 is replaced by SDH2-1 or SDH2-2 in mitochondrial complex II.

Generation of a transgenic medaka (Oryzias latipes) strain for visualization of nuclear dynamics in early developmental stages.

PubMed

Inoue, Takanobu; Iida, Atsuo; Maegawa, Shingo; Sehara-Fujisawa, Atsuko; Kinoshita, Masato

2016-12-01

In this study, we verified nuclear transport activity of an artificial nuclear localization signal (aNLS) in medaka fish (Oryzias latipes). We generated a transgenic medaka strain expresses the aNLS tagged enhanced green fluorescent protein (EGFP) driven by a medaka beta-actin promoter. The aNLS-EGFP was accumulated in the nuclei of somatic tissues and yolk nuclei of oocytes, but undetectable in the spermatozoa. The fluorescent signal was observed from immediately after fertilization by a maternal contribution. Furthermore, male and female pronuclei were visualized in fertilized eggs, and nuclear dynamics of pronuclear fusion and subsequent cleavage were captured by time-lapse imaging. In contrast, SV40NLS exhibited no activity of nuclear transport in early embryos. In conclusion, the aNLS possesses a strong nuclear localization activity and is a useful probe for fluorescent observation of the pronuclei and nuclei in early developmental stage of medaka. © 2016 Japanese Society of Developmental Biologists.

The reprogramming factor nuclear receptor subfamily 5, group A, member 2 cannot replace octamer-binding transcription factor 4 function in the self-renewal of embryonic stem cells.

PubMed

Choi, Kyeng-Won; Oh, Hye-Rim; Lee, Jaeyoung; Lim, Bobae; Han, Yong-Mahn; Oh, Junseo; Kim, Jungho

2014-02-01

Although octamer-binding transcription factor 4 (Oct-4) is one of the most intensively studied factors in mammalian development, no cellular genes capable of replacing Oct-4 function in embryonic stem (ES) cells have been found. Recent data show that nuclear receptor subfamily 5, group A, member 2 (Nr5a2) is able to replace Oct-4 function in the reprogramming process; however, it is unclear whether Nr5a2 can replace Oct-4 function in ES cells. In this study, the ability of Nr5a2 to maintain self-renewal and pluripotency in ES cells was investigated. Nr5a2 localized to the nucleus in ES cells, similarly to Oct-4. However, expression of Nr5a2 failed to rescue the stem cell phenotype or to maintain the self-renewal ability of ES cells. Furthermore, as compared with Oct-4-expressing ES cells, Nr5a2-expressing ES cells showed a reduced number of cells in S-phase, did not expand normally, and did not remain in an undifferentiated state. Ectopic expression of Nr5a2 in ES cells was not able to activate transcription of ES cell-specific genes, and gene expression profiling demonstrated differences between Nr5a2-expressing and Oct-4-expressing ES cells. In addition, Nr5a2-expressing ES cells were not able to form teratomas in nude mice. Taken together, these results strongly suggest that the gene regulation properties of Nr5a2 and Oct-4 and their abilities to confer self-renewal and pluripotency of ES cells differ. The present study provides strong evidence that Nr5a2 cannot replace Oct-4 function in ES cells. © 2013 FEBS.

Nuclei of Tsuga canadensis: Role of Flavanols in Chromatin Organization

PubMed Central

Feucht, Walter; Schmid, Markus; Treutter, Dieter

2011-01-01

Needle primordia of Tsuga canadensis (hemlock) arising from flank meristems of a shoot apex, form cell lineages consisting of four or eight cells. Within a recently established lineage there is striking uniformity in the pattern of nuclear flavanols. This fact points to an identical transcriptional expression of these flavanols during cell cycling. However two lineages, even if located close together within the same meristem, can be very different in the expression of both cell shape and nuclear flavanol pattern, indicating that epigenetic positional signals are operating in a collective specification of cell lineage development. There is a wide range of nuclear flavanol patterning from a mosaic-like distribution in an activated cell type to a homogenous appearance in silenced cell types. Single cells deriving from lineages are desynchronized because they underlie a signaling network at a higher tissue level which results in stronger epigenetic modifications of their nuclear flavanols. As an extreme case of epigenetic modulation, transient drought conditions caused a drastic reduction of nuclear flavanols. Upon treatment with sucrose or cytokinin, these nuclear flavanols could be fully restored. Analytical determination of the flavanols revealed 3.4 mg/g DW for newly sprouting needles and 19.6 mg/g DW for anthers during meiosis. The roughly 6-fold difference in flavanols is apparently a reflection of the highly diverging organogenetic processes. Collectively, the studies provide strong evidence for combinatorial interplay between cell fate and nuclear flavanols. PMID:22072922

A role for nuclear translocation of tripeptidyl-peptidase II in reactive oxygen species-dependent DNA damage responses

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

Preta, Giulio; Klark, Rainier de; Glas, Rickard, E-mail: rickard.glas@ki.se

2009-11-27

Responses to DNA damage are influenced by cellular metabolism through the continuous production of reactive oxygen species (ROS), of which most are by-products of mitochondrial respiration. ROS have a strong influence on signaling pathways during responses to DNA damage, by relatively unclear mechanisms. Previous reports have shown conflicting data on a possible role for tripeptidyl-peptidase II (TPPII), a large cytosolic peptidase, within the DNA damage response. Here we show that TPPII translocated into the nucleus in a p160-ROCK-dependent fashion in response to {gamma}-irradiation, and that nuclear expression of TPPII was present in most {gamma}-irradiated transformed cell lines. We used amore » panel of nine cell lines of diverse tissue origin, including four lymphoma cell lines (T, B and Hodgkins lymphoma), a melanoma, a sarcoma, a colon and two breast carcinomas, where seven out of nine cell lines showed nuclear TPPII expression after {gamma}-irradiation. Further, this required cellular production of ROS; treatment with either N-acetyl-Cysteine (anti-oxidant) or Rotenone (inhibitor of mitochondrial respiration) inhibited nuclear accumulation of TPPII. The local density of cells was important for nuclear accumulation of TPPII at early time-points following {gamma}-irradiation (at 1-4 h), indicating a bystander effect. Further, we showed that the peptide-based inhibitor Z-Gly-Leu-Ala-OH, but not its analogue Z-Gly-(D)-Leu-Ala-OH, excluded TPPII from the nucleus. This correlated with reduced nuclear expression of p53 as well as caspase-3 and -9 activation in {gamma}-irradiated lymphoma cells. Our data suggest a role for TPPII in ROS-dependent DNA damage responses, through alteration of its localization from the cytosol into the nucleus.« less

A role for nuclear translocation of tripeptidyl-peptidase II in reactive oxygen species-dependent DNA damage responses.

PubMed

Preta, Giulio; de Klark, Rainier; Glas, Rickard

2009-11-27

Responses to DNA damage are influenced by cellular metabolism through the continuous production of reactive oxygen species (ROS), of which most are by-products of mitochondrial respiration. ROS have a strong influence on signaling pathways during responses to DNA damage, by relatively unclear mechanisms. Previous reports have shown conflicting data on a possible role for tripeptidyl-peptidase II (TPPII), a large cytosolic peptidase, within the DNA damage response. Here we show that TPPII translocated into the nucleus in a p160-ROCK-dependent fashion in response to gamma-irradiation, and that nuclear expression of TPPII was present in most gamma-irradiated transformed cell lines. We used a panel of nine cell lines of diverse tissue origin, including four lymphoma cell lines (T, B and Hodgkins lymphoma), a melanoma, a sarcoma, a colon and two breast carcinomas, where seven out of nine cell lines showed nuclear TPPII expression after gamma-irradiation. Further, this required cellular production of ROS; treatment with either N-acetyl-Cysteine (anti-oxidant) or Rotenone (inhibitor of mitochondrial respiration) inhibited nuclear accumulation of TPPII. The local density of cells was important for nuclear accumulation of TPPII at early time-points following gamma-irradiation (at 1-4h), indicating a bystander effect. Further, we showed that the peptide-based inhibitor Z-Gly-Leu-Ala-OH, but not its analogue Z-Gly-(D)-Leu-Ala-OH, excluded TPPII from the nucleus. This correlated with reduced nuclear expression of p53 as well as caspase-3 and -9 activation in gamma-irradiated lymphoma cells. Our data suggest a role for TPPII in ROS-dependent DNA damage responses, through alteration of its localization from the cytosol into the nucleus.

Nuclear Multidrug-Resistance Related Protein 1 Contributes to Multidrug-Resistance of Mucoepidermoid Carcinoma Mainly via Regulating Multidrug-Resistance Protein 1: A Human Mucoepidermoid Carcinoma Cells Model and Spearman's Rank Correlation Analysis

PubMed Central

Liu, Yuan; Xu, Xiaofang; Guan, Sumin; Wu, Junzheng; Liu, Yanpu

2013-01-01

Background Multidrug resistance-related protein 1 (MRP1/ABCC1) and multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1) are both membrane-bound drug transporters. In contrast to MDR1, MRP1 also transports glutathione (GSH) and drugs conjugated to GSH. Due to its extraordinary transport properties, MRP1/ABCC1 contributes to several physiological functions and pathophysiological incidents. We previously found that nuclear translocation of MRP1 contributes to multidrug-resistance (MDR) of mucoepidermoid carcinoma (MEC). The present study investigated how MRP1 contributes to MDR in the nuclei of MEC cells. Methods Western blot and RT-PCR was carried out to investigate the change of multidrug-resistance protein 1 (MDR1) in MC3/5FU cells after MRP1 was downregulated through RNA interference (RNAi). Immunohistochemistry (IHC) staining of 127 cases of MEC tissues was scored with the expression index (EI). The EI of MDR1 and MRP1 (or nuclear MRP1) was analyzed with Spearman's rank correlation analysis. Using multiple tumor tissue assays, the location of MRP1 in other tissues was checked by HIC. Luciferase reporter assays of MDR1 promoter was carried out to check the connection between MRP1 and MDR1 promoter. Results MRP1 downregulation led to a decreased MDR1 expression in MC3/5FU cells which was caused by decreased activity of MDR1 promoter. IHC study of 127 cases of MEC tissues demonstrated a strong positive correlation between nuclear MRP1 expression and MDR1 expression. Furthermore, IHC study of multiple tumor tissue array sections showed that although nuclear MRP1 widely existed in MEC tissues, it was not found in normal tissues or other tumor tissues. Conclusions Our findings indicate that nuclear MRP1 contributes to MDR mainly through regulating MDR1 expression in MEC. And the unique location of MRP1 made it an available target in identifying MEC from other tumors. PMID:24013781

Hepatocyte Ploidy Is a Diversity Factor for Liver Homeostasis

PubMed Central

Kreutz, Clemens; MacNelly, Sabine; Follo, Marie; Wäldin, Astrid; Binninger-Lacour, Petra; Timmer, Jens; Bartolomé-Rodríguez, María M.

2017-01-01

Polyploidy, the existence of cells containing more than one pair of chromosomes, is a well-known feature of mammalian hepatocytes. Polyploid hepatocytes are found either as cells with a single polyploid nucleus or as multinucleated cells with diploid or even polyploid nuclei. In this study, we evaluate the degree of polyploidy in the murine liver by accounting both DNA content and number of nuclei per cell. We demonstrate that mouse hepatocytes with diploid nuclei have distinct metabolic characteristics compared to cells with polyploid nuclei. In addition to strong differential gene expression, comprising metabolic as well as signaling compounds, we found a strongly decreased insulin binding of nuclear polyploid cells. Our observations were associated with nuclear ploidy but not with total ploidy within a cell. We therefore suggest ploidy of the nuclei as an new diversity factor of hepatocytes and hypothesize that hepatocytes with polyploid nuclei may have distinct biological functions than mono-nuclear ones. This diversity is independent from the well-known heterogeneity related to the cells' position along the porto-central liver-axis. PMID:29163206

cMYC Expression in Infiltrating Gliomas: Associations with IDH1 Mutations, Clinicopathologic Features and Outcome

PubMed Central

Odia, Yazmin; Orr, Brent A.; Bell, W. Robert; Eberhart, Charles G.; Rodriguez, Fausto J.

2013-01-01

Gliomas are among the most frequent adult primary brain tumors. Mutations in IDH1, a metabolic enzyme, strongly correlate with secondary glioblastomas and increased survival. cMYC is an oncogene also implicated in aberrant metabolism, but its prognostic impact remains unclear. Recent genotyping studies also showed SNP variants near the cMYC gene locus, associate with an increased risk for development of IDH1/2 mutant gliomas suggesting a possible interaction between cMYC and IDH1. We evaluated nuclear cMYC protein levels and IDH1 (R132H) by immunohistochemistry in patients with oligodendroglioma/oligoastrocytomas (n=20), astrocytomas (grade II) (n=19), anaplastic astrocytomas (n=21) or glioblastomas (n=111). Of 158 tumors with sufficient tissue, 110 (70%) showed nuclear cMYC immunopositivity – most frequent (95%, χ2 p=0.0248) and intense (mean 1.33, ANOVA p=0.0179) in anaplastic astrocytomas versus glioblastomas (63%) or low grade gliomas (74%). cMYC expression associated with younger age as well as p53 immunopositivity (OR=3.6, p=0.0332) and mutant IDH1 (R132H) (OR=7.4, p=0.06) among malignant gliomas in our cohort. Independent analysis of the publically available TCGA glioblastoma dataset confirmed our strong association between cMYC and mutant IDH1 expression. Both IDH1 (R132H) and cMYC protein expression were associated with improved overall survival by univariate analysis. However, cMYC co-expression associated with shortened time to malignant transformation and overall survival among IDH1 (R132H) mutants in both univariate and multivariate analyses. In summary, our findings suggest that cMYC may be associated with a unique clinicopathologic and biologic group of infiltrating gliomas and help mediate the malignant transformation of IDH1 mutant gliomas. PMID:23934175

Two nuclear life cycle-regulated genes encode interchangeable subunits c of mitochondrial ATP synthase in Podospora anserina.

PubMed

Déquard-Chablat, Michelle; Sellem, Carole H; Golik, Pawel; Bidard, Frédérique; Martos, Alexandre; Bietenhader, Maïlis; di Rago, Jean-Paul; Sainsard-Chanet, Annie; Hermann-Le Denmat, Sylvie; Contamine, Véronique

2011-07-01

An F(1)F(O) ATP synthase in the inner mitochondrial membrane catalyzes the late steps of ATP production via the process of oxidative phosphorylation. A small protein subunit (subunit c or ATP9) of this enzyme shows a substantial genetic diversity, and its gene can be found in both the mitochondrion and/or nucleus. In a representative set of 26 species of fungi for which the genomes have been entirely sequenced, we found five Atp9 gene repartitions. The phylogenetic distribution of nuclear and mitochondrial Atp9 genes suggests that their evolution has included two independent transfers to the nucleus followed by several independent episodes of the loss of the mitochondrial and/or nuclear gene. Interestingly, we found that in Podospora anserina, subunit c is exclusively produced from two nuclear genes (PaAtp9-5 and PaAtp9-7), which display different expression profiles through the life cycle of the fungus. The PaAtp9-5 gene is specifically and strongly expressed in germinating ascospores, whereas PaAtp9-7 is mostly transcribed during sexual reproduction. Consistent with these observations, deletion of PaAtp9-5 is lethal, whereas PaAtp9-7 deletion strongly impairs ascospore production. The P. anserina PaAtp9-5 and PaAtp9-7 genes are therefore nonredundant. By swapping the 5' and 3' flanking regions between genes we demonstrated, however, that the PaAtp9 coding sequences are functionally interchangeable. These findings show that after transfer to the nucleus, the subunit c gene in Podospora became a key target for the modulation of cellular energy metabolism according to the requirements of the life cycle.

Nuclear delivery of recombinant OCT4 by chitosan nanoparticles for transgene-free generation of protein-induced pluripotent stem cells.

PubMed

Tammam, Salma; Malak, Peter; Correa, Daphne; Rothfuss, Oliver; Azzazy, Hassan M E; Lamprecht, Alf; Schulze-Osthoff, Klaus

2016-06-21

Protein-based reprogramming of somatic cells is a non-genetic approach for the generation of induced pluripotent stem cells (iPSCs), whereby reprogramming factors, such as OCT4, SOX2, KLF4 and c-MYC, are delivered as functional proteins. The technique is considered safer than transgenic methods, but, unfortunately, most protein-based protocols provide very low reprogramming efficiencies. In this study, we developed exemplarily a nanoparticle (NP)-based delivery system for the reprogramming factor OCT4. To this end, we expressed human OCT4 in Sf9 insect cells using a baculoviral expression system. Recombinant OCT4 showed nuclear localization in Sf9 cells indicating proper protein folding. In comparison to soluble OCT4 protein, encapsulation of OCT4 in nuclear-targeted chitosan NPs strongly stabilized its DNA-binding activity even under cell culture conditions. OCT4-loaded NPs enabled cell treatment with high micromolar concentrations of OCT4 and successfully delivered active OCT4 into human fibroblasts. Chitosan NPs therefore provide a promising tool for the generation of transgene-free iPSCs.

The expression of bcl-2 and bcl-6 protein in normal and malignant transitional epithelium.

PubMed

Lin, Zhenhua; Kim, Hankyeom; Park, Hongseok; Kim, Youngsik; Cheon, Jun; Kim, Insun

2003-08-01

The bcl-2 proto-oncogene plays a key role in cell longevity by preventing apoptosis. Bcl-2 is important in developing and maintaining the normal function of lymphoid and epithelial tissues. The bcl-6 protein is a 96 kDa nuclear protein selectively expressed in mature B cells within normal germinal centers as well as in their transformed counterparts in diffuse large B cell lymphoma. Recently, the bcl-6 protein has also been reported to be expressed in normal skin and epidermal neoplasms. In this study, 47 cases of transitional cell carcinomas (TCCs) were immunohistochemically studied for bcl-2 and bcl-6 protein expression. The results showed that bcl-2 was expressed only on basal layer cells, whereas bcl-6 expression was restricted to the superficial layers in the normal transitional epithelium. Von Brunn's nests showed strong immunostaining to bcl-2, but were negative to bcl-6. Among 47 TCCs, 15 (32.6%) and 29 (61.7%) cases were positive for bcl-2 and bcl-6, respectively. Compared with the normal transitional epithelium, the expression of bcl-2 was significantly decreased, whereas bcl-6 expression was significantly increased in TCCs. Additionally, the strong expression of bcl-6 had a positive correlation with the histopathologic grade of TCC. In conclusion, bcl-2 and bcl-6 proteins may play a role in the pathogenesis of TCCs, and bcl-6 expression reflects histopathologic grade.

Over-accumulation of nuclear IGF-1 receptor in tumor cells requires elevated expression of the receptor and the SUMO-conjugating enzyme Ubc9

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

Deng, Hua; Lin, Yingbo; Badin, Margherita

2011-01-14

Research highlights: {yields} SUMOylation mediates nuclear translocation of IGF-1R which activates transcription. {yields} Here we show that nuclear IGF-1R over-accumulates in tumor cells. {yields} This requires overexpression of the receptor that is a common feature in tumor cells. {yields} An increased expression of the SUMO ligase Ubc9 seems to be an involved mechanism too. -- Abstract: The insulin-like growth factor 1 receptor (IGF-1R) plays crucial roles in tumor cell growth and is overexpressed in many cancers. IGF-1R's trans-membrane kinase signaling pathways have been well characterized. Very recently, we showed that SUMOylation mediates nuclear translocation of the IGF-1R, and that nuclearmore » IGF-1R (nIGF-1R) binds to enhancer regions and activates transcription. We identified three lysine residues in the {beta}-subunit of the receptor and that mutation of these blocks nuclear translocation and gene activation. Furthermore, accumulation of nIGF-1R was proven strongly dependent on the specific SUMO-conjugating enzyme Ubc9. Here we show that nIGF-1R originates solely from the cell membrane and that phosphorylation of the core tyrosine residues of the receptor kinase is crucial for nuclear accumulation. We also compared the levels of nIGF-1R, measured as nuclear/membrane ratios, in tumor and normal cells. We found that the breast cancer cell line MCF-7 has 13-fold higher amounts of nIGF-1R than breast epithelial cells (IME) which showed only a small amount of nIGF-1R. In comparison, the total expression of IGF-1R was only 3.7- higher in MCF-7. Comparison of several other tumor and normal cell lines showed similar tumor cell over-accumulation of nIGF-1R, exceeding the total receptor expression substantially. Ectopic overexpression (>10-fold) of the receptor increased nIGF-1R in IME cells but not to that high level as in wild type MCF-7. The levels of Ubc9 were higher in all tumor cell lines, compared to the normal cells, and this probably contributes to over-accumulation of nIGF-1R. Over-accumulation of nIGF-1R may contribute to deregulated gene expression and therewith play a pathophysiological role in cancer cells.« less

FHL1B Interacts with Lamin A/C and Emerin at the Nuclear Lamina and is Misregulated in Emery-Dreifuss Muscular Dystrophy.

PubMed

Ziat, Esma; Mamchaoui, Kamel; Beuvin, Maud; Nelson, Isabelle; Azibani, Feriel; Spuler, Simone; Bonne, Gisèle; Bertrand, Anne T

2016-11-29

Emery-Dreifuss muscular dystrophy (EDMD) is associated with mutations in EMD and LMNA genes, encoding for the nuclear envelope proteins emerin and lamin A/C, indicating that EDMD is a nuclear envelope disease. We recently reported mutations in FHL1 gene in X-linked EDMD. FHL1 encodes FHL1A, and the two minor isoforms FHL1B and FHL1C. So far, none have been described at the nuclear envelope. To gain insight into the pathophysiology of EDMD, we focused our attention on the poorly characterized FHL1B isoform. The amount and the localisation of FHL1B were evaluated in control and diseased human primary myoblasts using immunofluorescence and western blotting. We found that in addition to a cytoplasmic localization, this isoform strongly accumulated at the nuclear envelope of primary human myoblasts, like but independently of lamin A/C and emerin. During myoblast differentiation, we observed a major reduction of FHL1B protein expression, especially in the nucleus. Interestingly, we found elevated FHL1B expression level in myoblasts from an FHL1-related EDMD patient where the FHL1 mutation only affects FHL1A, as well as in myoblasts from an LMNA-related EDMD patient. Altogether, the specific localization of FHL1B and its modulation in disease-patient's myoblasts confirmed FHL1-related EDMD as a nuclear envelope disease.

Contrasting genetic structure between mitochondrial and nuclear markers in the dengue fever mosquito from Rio de Janeiro: implications for vector control

PubMed Central

Rašić, Gordana; Schama, Renata; Powell, Rosanna; Maciel-de Freitas, Rafael; Endersby-Harshman, Nancy M; Filipović, Igor; Sylvestre, Gabriel; Máspero, Renato C; Hoffmann, Ary A

2015-01-01

Dengue is the most prevalent global arboviral disease that affects over 300 million people every year. Brazil has the highest number of dengue cases in the world, with the most severe epidemics in the city of Rio de Janeiro (Rio). The effective control of dengue is critically dependent on the knowledge of population genetic structuring in the primary dengue vector, the mosquito Aedes aegypti. We analyzed mitochondrial and nuclear genomewide single nucleotide polymorphism markers generated via Restriction-site Associated DNA sequencing, as well as traditional microsatellite markers in Ae. aegypti from Rio. We found four divergent mitochondrial lineages and a strong spatial structuring of mitochondrial variation, in contrast to the overall nuclear homogeneity across Rio. Despite a low overall differentiation in the nuclear genome, we detected strong spatial structure for variation in over 20 genes that have a significantly altered expression in response to insecticides, xenobiotics, and pathogens, including the novel biocontrol agent Wolbachia. Our results indicate that high genetic diversity, spatially unconstrained admixing likely mediated by male dispersal, along with locally heterogeneous genetic variation that could affect insecticide resistance and mosquito vectorial capacity, set limits to the effectiveness of measures to control dengue fever in Rio. PMID:26495042

Single-cell heterogeneity and cell-cycle-related viral gene bursts in the human leukaemia virus HTLV-1

PubMed Central

Billman, Martin R; Rueda, David; Bangham, Charles R M

2017-01-01

Background: The human leukaemia virus HTLV-1 expresses essential accessory genes that manipulate the expression, splicing and transport of viral mRNAs.  Two of these genes, tax and hbz, also promote proliferation of the infected cell, and both genes are thought to contribute to oncogenesis in adult T-cell leukaemia/lymphoma.  The regulation of HTLV-1 proviral latency is not understood.  tax, on the proviral plus strand, is usually silent in freshly-isolated cells, whereas the minus-strand-encoded hbz gene is persistently expressed at a low level.  However, the persistently activated host immune response to Tax indicates frequent expression of tax in vivo.  Methods: We used single-molecule RNA-FISH to quantify the expression of HTLV-1 transcripts at the single-cell level in a total of >19,000 cells from five T-cell clones, naturally infected with HTLV-1, isolated by limiting dilution from peripheral blood of HTLV-1-infected subjects.  Results: We found strong heterogeneity both within and between clones in the expression of the proviral plus-strand (detected by hybridization to the tax gene) and the minus-strand ( hbz gene). Both genes are transcribed in bursts; tax expression is enhanced in the absence of hbz, while hbz expression increased in cells with high tax expression. Surprisingly, we found that hbz expression is strongly associated with the S and G 2/M phases of the cell cycle, independent of tax expression.  Contrary to current belief, hbz is not expressed in all cells at all times, even within one clone.  In hbz-positive cells, the abundance of hbz transcripts showed a very strong positive linear correlation with nuclear volume. Conclusions: The occurrence of intense, intermittent plus-strand gene bursts in independent primary HTLV-1-infected T-cell clones from unrelated individuals strongly suggests that the HTLV-1 plus-strand is expressed in bursts in vivo.  Our results offer an explanation for the paradoxical correlations observed between the host immune response and HTLV-1 transcription. PMID:29062917

Nuclear Phosphatidylinositol-Phosphate Type I Kinase α-Coupled Star-PAP Polyadenylation Regulates Cell Invasion

PubMed Central

A.P., Sudheesh

2017-01-01

ABSTRACT Star-PAP, a nuclear phosphatidylinositol (PI) signal-regulated poly(A) polymerase (PAP), couples with type I PI phosphate kinase α (PIPKIα) and controls gene expression. We show that Star-PAP and PIPKIα together regulate 3′-end processing and expression of pre-mRNAs encoding key anti-invasive factors (KISS1R, CDH1, NME1, CDH13, FEZ1, and WIF1) in breast cancer. Consistently, the endogenous Star-PAP level is negatively correlated with the cellular invasiveness of breast cancer cells. While silencing Star-PAP or PIPKIα increases cellular invasiveness in low-invasiveness MCF7 cells, Star-PAP overexpression decreases invasiveness in highly invasive MDA-MB-231 cells in a cellular Star-PAP level-dependent manner. However, expression of the PIPKIα-noninteracting Star-PAP mutant or the phosphodeficient Star-PAP (S6A mutant) has no effect on cellular invasiveness. These results strongly indicate that PIPKIα interaction and Star-PAP S6 phosphorylation are required for Star-PAP-mediated regulation of cancer cell invasion and give specificity to target anti-invasive gene expression. Our study establishes Star-PAP–PIPKIα-mediated 3′-end processing as a key anti-invasive mechanism in breast cancer. PMID:29203642

Nuclear Phosphatidylinositol-Phosphate Type I Kinase α-Coupled Star-PAP Polyadenylation Regulates Cell Invasion.

PubMed

A P, Sudheesh; Laishram, Rakesh S

2018-03-01

Star-PAP, a nuclear phosphatidylinositol (PI) signal-regulated poly(A) polymerase (PAP), couples with type I PI phosphate kinase α (PIPKIα) and controls gene expression. We show that Star-PAP and PIPKIα together regulate 3'-end processing and expression of pre-mRNAs encoding key anti-invasive factors ( KISS1R , CDH1 , NME1 , CDH13 , FEZ1 , and WIF1 ) in breast cancer. Consistently, the endogenous Star-PAP level is negatively correlated with the cellular invasiveness of breast cancer cells. While silencing Star-PAP or PIPKIα increases cellular invasiveness in low-invasiveness MCF7 cells, Star-PAP overexpression decreases invasiveness in highly invasive MDA-MB-231 cells in a cellular Star-PAP level-dependent manner. However, expression of the PIPKIα-noninteracting Star-PAP mutant or the phosphodeficient Star-PAP (S6A mutant) has no effect on cellular invasiveness. These results strongly indicate that PIPKIα interaction and Star-PAP S6 phosphorylation are required for Star-PAP-mediated regulation of cancer cell invasion and give specificity to target anti-invasive gene expression. Our study establishes Star-PAP-PIPKIα-mediated 3'-end processing as a key anti-invasive mechanism in breast cancer. Copyright © 2018 A.P. and Laishram.

Functional characterization of the human phosphodiesterase 7A1 promoter.

PubMed Central

Torras-Llort, Mònica; Azorín, Fernando

2003-01-01

In this paper, the human phosphodiesterase 7A1 (h PDE7A1 ) promoter region was identified and functionally characterized. Transient transfection experiments indicated that a 2.9 kb fragment of the h PDE7A1 5'-flanking region, to position -2907, has strong promoter activity in Jurkat T-cells. Deletion analysis showed that the proximal region, up to position -988, contains major cis -regulatory elements of the h PDE7A1 promoter. This minimal promoter region contains a regulatory CpG island which is essential for promoter activity. The CpG island contains three potential cAMP-response-element-binding protein (CREB)-binding sites that, as judged by in vivo dimethyl sulphate (DMS) footprinting, are occupied in Jurkat T-cells. Moreover, over-expression of CREB results in increased promoter activity, but, on the other hand, promoter activity decreases when a dominant-negative form of CREB (KCREB) is over-expressed. In vivo DMS footprinting strongly indicates that other transcription factors, such Ets-2, nuclear factor of activated T-cells 1 (NFAT-1) and nuclear factor kappaB (NF-kappaB), might also contribute to the regulation of h PDE7A1 promoter. Finally, h PDE7A1 promoter was found to be induced by treatment with PMA, but not by treatment with dibutyryl cAMP or forskolin. These results provide insights into the factors and mechanisms that regulate expression of the h PDE7A gene. PMID:12737631

Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA

PubMed Central

Schertzer, Michael; Jouravleva, Karina; Perderiset, Mylene; Dingli, Florent; Loew, Damarys; Le Guen, Tangui; Bardoni, Barbara; de Villartay, Jean-Pierre; Revy, Patrick; Londoño-Vallejo, Arturo

2015-01-01

Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1–HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS. PMID:25628358

Targeting of RNA Polymerase II by a nuclear Legionella pneumophila Dot/Icm effector SnpL.

PubMed

Schuelein, Ralf; Spencer, Hugh; Dagley, Laura F; Li, Peng Fei; Luo, Lin; Stow, Jennifer L; Abraham, Gilu; Naderer, Thomas; Gomez-Valero, Laura; Buchrieser, Carmen; Sugimoto, Chihiro; Yamagishi, Junya; Webb, Andrew I; Pasricha, Shivani; Hartland, Elizabeth L

2018-04-24

The intracellular pathogen Legionella pneumophila influences numerous eukaryotic cellular processes through the Dot/Icm-dependent translocation of more than 300 effector proteins into the host cell. Although many translocated effectors localize to the Legionella replicative vacuole, other effectors can affect remote intracellular sites. Following infection, a subset of effector proteins localizes to the nucleus where they subvert host cell transcriptional responses to infection. Here we identified Lpg2519 (Lpp2587/Lpw27461), as a new nuclear-localized effector that we have termed SnpL. Upon ectopic expression or during L. pneumophila infection, SnpL showed strong nuclear localization by immunofluorescence microscopy but was excluded from nucleoli. Using immunoprecipitation and mass spectrometry, we determined the host-binding partner of SnpL as the eukaryotic transcription elongation factor, SUPT5H/Spt5. SUPT5H is an evolutionarily conserved component of the DRB sensitivity-inducing factor complex (DSIF complex) that regulates RNA polymerase II (Pol II) dependent mRNA processing and transcription elongation. Protein interaction studies showed that SnpL bound to the central KOW motif region of SUPT5H. Ectopic expression of SnpL led to massive upregulation of host gene expression and macrophage cell death. The activity of SnpL further highlights the ability of L. pneumophila to control fundamental eukaryotic processes such as transcription that, in the case of SnpL, leads to global upregulation of host gene expression. This article is protected by copyright. All rights reserved.

The variations in the nuclear proteome reveal new transcription factors and mechanisms involved in UV stress response in Pinus radiata.

PubMed

Pascual, Jesús; Alegre, Sara; Nagler, Matthias; Escandón, Mónica; Annacondia, María Luz; Weckwerth, Wolfram; Valledor, Luis; Cañal, María Jesús

2016-06-30

The importance of UV stress and its side-effects over the loss of plant productivity in forest species demands a deeper understanding of how pine trees respond to UV irradiation. Although the response to UV stress has been characterized at system and cellular levels, the dynamics within the nuclear proteome triggered by UV is still unknown despite that they are essential for gene expression and regulation of plant physiology. To fill this gap this work aims to characterize the variations in the nuclear proteome as a response to UV irradiation by using state-of-the-art mass spectrometry-based methods combined with novel bioinformatics workflows. The combination of SEQUEST, de novo sequencing, and novel annotation pipelines allowed cover sensing and transduction pathways, endoplasmic reticulum-related mechanisms and the regulation of chromatin dynamism and gene expression by histones, histone-like NF-Ys, and other transcription factors previously unrelated to this stress source, as well as the role of alternative splicing and other mechanisms involved in RNA translation and protein synthesis. The determination of 33 transcription factors, including NF-YB13, Pp005698_3 (NF-YB) and Pr009668_2 (WD-40), which are correlated to stress responsive mechanisms like an increased accumulation of photoprotective pigments and reduced photosynthesis, pointing them as strong candidate biomarkers for breeding programs aimed to improve UV resistance of pine trees. The description of the nuclear proteome of Pinus radiata combining a classic approach based on the use of SEQUEST and the use of a mass accuracy precursor alignment (MAPA) allowed an unprecedented protein coverage. This workflow provided the methodological basis for characterizing the changes in the nuclear proteome triggered by UV irradiation, allowing the depiction of the nuclear events involved in stress response and adaption. The relevance of some of the discovered proteins will suppose a major advance in stress biology field, also providing a set of transcription factors that can be considered as strong biomarker candidates to select trees more tolerant to UV radiation in forest upgrade programs. Copyright © 2016 Elsevier B.V. All rights reserved.

Differential expression of upstream stimulatory factor (USF) 2 variants in eutopic endometria from women with endometriosis: estradiol regulation.

PubMed

Castro, Jazmin; Araya, Germán; Inostroza, Pamela; Hidalgo, Paulina; González-Ramos, Reinaldo; Sovino, Hugo; Boric, M Angélica; Fuentes, Ariel; Johnson, M Cecilia

2015-10-09

Endometriosis, pro-inflammatory and invasive benign disease estrogen dependent, abnormally express in endometria the enzyme P450Arom, positively regulated by steroid factor-1 (SF-1). Our objective was to study the nuclear protein contents of upstream stimulating factor 2 (USF2a and USF2b), a positive regulator of SF-1, throughout the menstrual cycle in eutopic endometria from women with and without (control) endometriosis and the involvement of nuclear estrogen receptors (ER) and G-coupled protein estrogen receptor (GPER)-1. Upstream stimulating factor 2 protein contents were higher in mid (USF2b) and late (USF2a and USF2b) secretory phase in eutopic endometria from endometriosis than control (p < 0.05). In isolated control epithelial cells incubated with E2 and PGE2, to resemble the endometriosis condition, the data showed: (a) significant increase of USF2a and USF2b nuclear protein contents when treated with E2, PPT (specific agonist for ERα) or G1 (specific agonist for GPER1); (b) no increase in USF2 binding to SF-1 E-Box/DNA consensus sequence in E2-treated cells; (c) USF2 variants protein contents were not modified by PGE2; (d) SF-1 nuclear protein content was significantly higher than basal when treated with PGE2, E2 or G1, stimulation unaffected by ICI (nuclear ER antagonist); and (e) increased (p < 0.05) cytosolic protein contents of P450Arom when treated with PGE2, E2, PPT or G1 compared to basal, effect that was additive with E2 + PGE2 together. Nevertheless, in endometriosis cells, the high USF2, SF-1 and P450Arom protein contents in basal condition were unmodified. These data strongly suggest that USF2 variants and P450Arom are regulated by E2 through ERα and GPER1, whereas SF-1 through GPER1, visualized by the response of the cells obtained from control endometria, being unaffected the endogenously stimulated cells from endometriosis origin. The lack of E2 stimulation on USF2/SF-1 E-Box/DNA-sequence binding and the absence of PGE2 effect on USF2 variants opposite to the strong induction that they exert on SF1 and P450 proteins suggest different mechanisms and indirect regulations. The sustained USF2 variants protein expression during the secretory phase in eutopic endometria from women with endometriosis may participate in the pathophysiology of this disease strongly associated with infertility and its characteristic endometrial invasion to ectopic sites in the pelvic cavity.

Podophyllum hexandrum (Himalayan mayapple) extract provides radioprotection by modulating the expression of proteins associated with apoptosis.

PubMed

Kumar, Raj; Singh, Pankaj Kumar; Sharma, Ashok; Prasad, Jagdish; Sagar, Ravinder; Singh, Surender; Arora, Rajesh; Sharma, Rakesh Kumar

2005-08-01

Podophyllum hexandrum Royale (Himalayan mayapple), a high-altitude Himalayan plant, has been shown to provide over 80% whole-body radioprotection in mice. To investigate the radioprotective potential of P. hexandrum at the molecular level, expression patterns of various proteins associated with apoptosis were studied in the spleen of male Swiss albino strain A mice by immunoblotting. Treatment with P. hexandrum [200 mg/kg of body weight; an ethanolic 50% (w/v) extract delivered intraperitoneally] 2 h before irradiation resulted in MAPKAP (mitogen-activated protein kinase-activated protein) kinase-2 activation along with HSF-1 (heat-shock transcription factor-1), leading to up-regulation of HSP-70 (heat-shock protein-70) as compared with sham-irradiated (10 Gy) mice. Strong inhibition of AIF (apoptosis-inducing factor) expression was observed in the mice treated with P. hexandrum 2 h before irradiation as compared with the sham-irradiated group. Inhibition in the translocation of free NF-kappaB (nuclear factor kappaB) from cytoplasm to nucleus was observed upon P. hexandrum pretreatment 2 h before irradiation when compared with radiation-treated mice. P. hexandrum pre-treatment (2 h before irradiation) resulted in inhibition of NF-kappaB translocation, and the expression of tumour suppressor protein p53 was observed to be down-regulated as compared with sham-irradiated control. An increase in the expression of proteins responsible for cell proliferation [Bcl-2 (B-cell chronic lymphocytic lymphoma 2), Ras-GAP (Ras-GTPase-activating protein) and PCNA (proliferating cell nuclear antigen)] was observed in the P. hexandrum-pretreated irradiated mice as compared with sham-irradiated controls. Caspase 3 activation resulted PARP [poly(ADP-ribose) DNA polymerase] cleavage, and DNA degradation was strongly inhibited in the mice treated with P. hexandrm (+/-irradiation) as compared with the mice treated with radiation (+/-heat shock). The present study thus clearly demonstrated that P. hexandrum extract provides protection from gamma-radiation by the modulation of expression of proteins associated with cell death.

Nuclear YB-1 expression as a negative prognostic marker in nonsmall cell lung cancer.

PubMed

Gessner, C; Woischwill, C; Schumacher, A; Liebers, U; Kuhn, H; Stiehl, P; Jürchott, K; Royer, H D; Witt, C; Wolff, G

2004-01-01

The human Y-box binding protein, YB-1, is a multifunctional protein that regulates gene expression. Nuclear expression of YB-1 has been associated with chemoresistance and poor prognosis of tumour patients. Representative samples from autopsied material of primary tumours from 77 patients with NSCLC were investigated by immunohistochemistry for subcellular distribution of YB-1 and p53, in order to evaluate the prognostic role of nuclear expression of YB-1. Cytoplasmic YB-1 expression was found in all tumour samples, whereas nuclear expression was only observed in 48%. There was no correlation with histological classification, clinical parameters or tumour size, stage and metastasis status. However, patients with positive nuclear YB-1 expression in tumours showed reduced survival times when compared with patients without nuclear expression. Including information about the histology and mutational status for p53 increased the prognostic value of nuclear YB-1. Patients with nuclear YB-1 expression and p53 mutations had the worst prognosis (median survival 3 months), while best outcome was found in patients with no nuclear YB-1 and wildtype p53 (median survival 15 months). This suggests that the combined analysis of both markers allows a better identification of subgroups with varying prognosis. Nuclear expression of Y-box binding protien seems to be an independent prognostic marker.

SNIP1: a new activator of HSE signaling pathway.

PubMed

Li, Qiang; An, Jian; Liu, Xianghua; Zhang, Mingjun; Ling, Yichen; Wang, Chenji; Zhao, Jing; Yu, Long

2012-03-01

In the last 10 years, more and more attention has been focused on SNIP1 (Smad nuclear interacting protein 1), which functions as a transcriptional coactivator. We report here that through quantitative real-time PCR analysis in 18 different human tissues, SNIP1 was found to be expressed ubiquitously. When overexpressed in HeLa cells, SNIP1-EGFP fused protein exhibited a nuclear localization with a characteristic subnuclear distribution in speckles or formed larger discrete nuclear bodies in some cells. Reporter gene assay showed that overexpression of SNIP1 in HEK 293 cells or H1299 cells strongly activated the HSE signaling pathway. Moreover, SNIP1 could selectively regulate the transcription of HSP70A1A and HSP27. Taken together, our findings suggest that SNIP1 might also be a positive regulator of HSE signaling pathway.

Lectin purified from Musca domestica pupa up-regulates NO and iNOS production via TLR4/NF-κB signaling pathway in macrophages.

PubMed

Cao, Xiaohong; Zhou, Minghui; Wang, Chunling; Hou, Lihua; Zeng, Bin

2011-04-01

The present study reported that nitric oxide (NO) was up-regulated by the induction of lectin purified from Musca domestica pupa (MPL) in macrophages without cytotoxicity. The mRNA expression and protein secretion of inducible nitric oxide synthase (iNOS) were strongly induced by MPL treatments. Subsequent investigation revealed that the nuclear factor-κB (NF-κB) inhibitory κB (IκB) in endochylema was inhibited and NF-κB translocated into the nucleus after MPL treatment. Meanwhile, the IKKβ was strongly induced and the production of the toll-like receptor 4 (TLR4) was significantly up-regulated. Moreover, MPL increased NO production via inducing the expression of iNOS through the activation of NF-κB, which suggested that MPL probably acted as an activating agent of the NF-κB activation. Copyright © 2010 Elsevier B.V. All rights reserved.

Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris.

PubMed

Zepeda, Andrea B; Figueroa, Carolina A; Abdalla, Dulcineia S P; Maranhão, Andrea Q; Ulloa, Patricio H; Pessoa, Adalberto; Farías, Jorge G

2014-01-01

Pichia pastoris is methylotrophic yeast used as an efficient expression system for heterologous protein production. In order to evaluate the effects of temperature (10 and 30 °C) and methanol (1 and 3% (v/v)) on genetically-modified Pichia pastoris, different biomarkers were evaluated: Heat stress (HSF-1 and Hsp70), oxidative stress (OGG1 and TBARS) and antioxidant (GLR). Three yeast cultures were performed: 3X = 3% methanol-10 °C, 4X = 3% methanol-30 °C, and 5X = 1% methanol-10°C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. Ours results show that at 3% methanol -30 °C there is an increase of mitochondrial OGG1 (mtOGG1), Glutathione Reductase (GLR) and TBARS. In addition, there was a cytosolic expression of HSF-1 and HSP-70, which indicates a deprotection against nucleolar fragmentation (apoptosis). On the other hand, at 3% methanol -10 °C and 1% and at methanol -10 °C conditions there was nuclear expression of OGG1, lower levels of TBARS and lower expression of GLR, cytosolic expression of HSF-1 and nuclear expression HSP-70. In conclusion, our results suggest that 3% methanol-30 °C is a condition that induces a strong oxidative stress and risk factors of apoptosis in modified-genetically P. pastoris.

Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris

PubMed Central

Zepeda, Andrea B.; Figueroa, Carolina A.; Abdalla, Dulcineia S.P.; Maranhão, Andrea Q.; Ulloa, Patricio H.; Pessoa, Adalberto; Farías, Jorge G.

2014-01-01

Pichia pastoris is methylotrophic yeast used as an efficient expression system for heterologous protein production. In order to evaluate the effects of temperature (10 and 30 °C) and methanol (1 and 3% (v/v)) on genetically-modified Pichia pastoris, different biomarkers were evaluated: Heat stress (HSF-1 and Hsp70), oxidative stress (OGG1 and TBARS) and antioxidant (GLR). Three yeast cultures were performed: 3X = 3% methanol-10 °C, 4X = 3% methanol-30 °C, and 5X = 1% methanol-10°C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. Ours results show that at 3% methanol −30 °C there is an increase of mitochondrial OGG1 (mtOGG1), Glutathione Reductase (GLR) and TBARS. In addition, there was a cytosolic expression of HSF-1 and HSP-70, which indicates a deprotection against nucleolar fragmentation (apoptosis). On the other hand, at 3% methanol −10 °C and 1% and at methanol −10 °C conditions there was nuclear expression of OGG1, lower levels of TBARS and lower expression of GLR, cytosolic expression of HSF-1 and nuclear expression HSP-70. In conclusion, our results suggest that 3% methanol-30 °C is a condition that induces a strong oxidative stress and risk factors of apoptosis in modified-genetically P. pastoris. PMID:25242930

Expression of COUP-TFII Nuclear Receptor in Restricted GABAergic Neuronal Populations in the Adult Rat Hippocampus

PubMed Central

Fuentealba, Pablo; Klausberger, Thomas; Karayannis, Theofanis; Suen, Wai Yee; Huck, Jojanneke; Tomioka, Ryohei; Rockland, Kathleen; Capogna, Marco; Studer, Michèle; Morales, Marisela; Somogyi, Peter

2015-01-01

The COUP-TFII nuclear receptor, also known as NR2F2, is expressed in the developing ventral telencephalon and modulates the tangential migration of a set of subpallial neuronal progenitors during forebrain development. Little information is available about its expression patterns in the adult brain. We have identified the cell populations expressing COUP-TFII and the contribution of some of them to network activity in vivo. Expression of COUP-TFII by hippocampal pyramidal and dentate granule cells, as well as neurons in the neocortex, formed a gradient increasing from undetectable in the dorsal to very strong in the ventral sectors. In the dorsal hippocampal CA1 area, COUP-TFII was restricted to GABAergic interneurons and expressed in several, largely nonoverlapping neuronal populations. Immunoreactivity was present in calretinin-, neuronal nitric oxide synthase-, and reelin-expressing cells, as well as in subsets of cholecystokinin- or calbindin-expressing or radiatum-retrohippocampally projecting GABAergic cells, but not in parvalbumin-and/or somatostatin-expressing interneurons. In vivo recording and juxtacellular labeling of COUP-TFII-expressing cells revealed neurogliaform cells, basket cells in stratum radiatum and tachykinin-expressing radiatum dentate innervating interneurons, identified by their axodendritic distributions. They showed cell type-selective phase-locked firing to the theta rhythm but no activation during sharp wave/ripple oscillations. These basket cells in stratum radiatum and neurogliaform cells fired at the peak of theta oscillations detected extracellularly in stratum pyramidale, unlike previously reported ivy cells, which fired at the trough. The characterization of COUP-TFII-expressing neurons suggests that this developmentally important transcription factor plays cell type-specific role(s)in the adult hippocampus. PMID:20130170

Ganoderma lucidum Polysaccharides Reduce Lipopolysaccharide-Induced Interleukin-1β Expression in Cultured Smooth Muscle Cells and in Thoracic Aortas in Mice

PubMed Central

Liang, Chan-Jung; Lee, Chiang-Wen; Sung, Hsin-Ching; Chen, Yung-Hsiang; Hsu, Hsien-Yeh; Tseng, Ying-Chin; Li, Chi-Yuan; Wang, Shu-Huei

2014-01-01

The expression of inflammatory cytokines on vascular walls is a critical event in vascular diseases and inflammation. The aim of the present study was to examine the effects of an extract of Ganoderma lucidum (Reishi) polysaccharides (EORPs), which is effective against immunological disorders, on interleukin- (IL-) 1β expression by human aortic smooth muscle cells (HASMCs) and the underlying mechanism. The lipopolysaccharide- (LPS-) induced IL-1β expression was significantly reduced when HASMCs were pretreated with EORP by Western blot and immunofluorescent staining. Pretreatment with 10 μg/mL EORP decreased LPS-induced ERK, p38, JNK, and Akt phosphorylation. But the increase in IL-1β expression with LPS treatment was only inhibited by pretreatment with the ERK1/2 inhibitor, while the JNK and p38 inhibitors had no effect. In addition, EORP reduced the phosphorylation and nuclear translocation of nuclear factor- (NF-) κB p65 in LPS-treated HASMCs. Furthermore, in vivo, IL-1β expression was strongly expressed in thoracic aortas in LPS-treated mice. Oral administration of EORP decreased IL-1β expression. The level of IL-1β expression in LPS-treated or in LPS/EORP-treated group was very low and was similar to that of the saline-treated group in toll-like receptor 4-deficient (TLR4−/−) mice. These findings suggest that EORP has the anti-inflammatory property and could prove useful in the prevention of vascular diseases and inflammatory responses. PMID:24723958

Effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes

PubMed Central

2010-01-01

Background Cryopreservation of oocytes, which is an interesting procedure to conserve female gametes, is an essential part of reproductive biotechnology. The objective of the present study was to investigate the effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes. Methods Immature oocytes (germinal vesicles) isolated from ovaries of normal bitches (> 6 months of age) were either vitrified in open pulled straw (OPS) using 20% ethylene glycol (EG) and 20% dimethyl sulfoxide (DMSO) as vitrification solution or exposed to vitrification solution without subjected to liquid nitrogen. After warming, oocytes were investigated for nuclear maturation following in vitro maturation (IVM), ultrastructural changes using transmission electron microscopy (TEM) and gene expression using RT-PCR. Fresh immature oocytes were used as the control group. Results The rate of resumption of meiosis in vitrified-warmed oocytes (53.4%) was significantly (P < 0.05) lower than those of control (93.8%) and exposure (91.4%) groups. However, there were no statistically significant differences among groups in the rates of GV oocytes reaching the maturation stage (metaphase II, MII). The ultrastructural alterations revealed by TEM showed that cortical granules, mitochondria, lipid droplets and smooth endoplasmic reticulum (SER) were affected by vitrification procedures. RT-PCR analysis for gene expression revealed no differences in HSP70, Dnmt1, SOD1 and BAX genes among groups, whereas Bcl2 was strongly expressed in vitrified-warmed group when compared to the control. Conclusion Immature canine oocytes were successfully cryopreserved, resumed meiosis and developed to the MII stage. The information obtained in this study is crucial for the development of an effective method to cryopreserve canine oocytes for establishment of genetic banks of endangered canid species. PMID:20565987

Increased expression of the retinoic acid-metabolizing enzyme CYP26A1 during the progression of cervical squamous neoplasia and head and neck cancer.

PubMed

Osanai, Makoto; Lee, Gang-Hong

2014-10-07

Retinoic acid (RA) is a critical regulator of cell differentiation, proliferation, and apoptosis in various cell types. Recently, the RA-metabolizing enzyme CYP26A1 (cytochrome P450, family 26, subfamily A, polypeptide 1) has been shown to have an oncogenic function in breast carcinogenesis. However, the relevance of elevated CYP26A1 expression in human cancers remains to be clarified. We immunohistochemically examined the expression of CYP26A1 in cervical squamous cell carcinoma (SCC) and its precursors, including low- and high-grade squamous intraepithelial lesions (LSIL and HSIL, respectively), as well as head and neck cancer (HNC). The association between CYP26A1 expression and a number of clinicopathological parameters was also evaluated. CYP26A1 was not expressed in normal cervical epithelium. CYP26A1 expression was present in LSIL but limited to basal and parabasal cells. HSIL cases exhibited strong nuclear expression of CYP26A1 and mixed cytoplasmic expression patterns with widely distributed expression toward the epithelial surface. Importantly, strong cytoplasmic staining of CYP26A1 was observed in 19 of 50 (38%) patients with cervical SCC. Elevated expression of CYP26A1 was significantly associated with younger age (<50 years) and lymph node involvement (pN). Similarly, CYP26A1 was not expressed in non-neoplastic tissues of the head and neck, but strong cytoplasmic staining of CYP26A1 was observed in 52 of 128 (41%) HNC cases. Such strong CYP26A1 expression was significantly associated with the primary tumor stage of carcinomas (pT) and the pathological tumor-node-metastasis (pTNM) stage in HNC. Our results indicated an elevated CYP26A1 expression in malignant and precancerous dysplastic lesions of the human cervix, which also increased with the progression of cervical squamous neoplasia. In addition, this report is the first to demonstrate the increased expression of CYP26A1 in HNC and its significant correlation with primary tumor growth. These data suggested that CYP26A1 overexpression might contribute to the development and progression of cervical malignancies and squamous neoplasia of the head and neck.

Dual targeting and retrograde translocation: regulators of plant nuclear gene expression can be sequestered by plastids.

PubMed

Krause, Kirsten; Oetke, Svenja; Krupinska, Karin

2012-01-01

Changes in the developmental or metabolic state of plastids can trigger profound changes in the transcript profiles of nuclear genes. Many nuclear transcription factors were shown to be controlled by signals generated in the organelles. In addition to the many different compounds for which an involvement in retrograde signaling is discussed, accumulating evidence suggests a role for proteins in plastid-to-nucleus communication. These proteins might be sequestered in the plastids before they act as transcriptional regulators in the nucleus. Indeed, several proteins exhibiting a dual localization in the plastids and the nucleus are promising candidates for such a direct signal transduction involving regulatory protein storage in the plastids. Among such proteins, the nuclear transcription factor WHIRLY1 stands out as being the only protein for which an export from plastids and translocation to the nucleus has been experimentally demonstrated. Other proteins, however, strongly support the notion that this pathway might be more common than currently believed.

KPNA7, a nuclear transport receptor, promotes malignant properties of pancreatic cancer cells in vitro

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

Laurila, Eeva; Vuorinen, Elisa; Fimlab Laboratories, Biokatu 4, 33520 Tampere

2014-03-10

Pancreatic cancer is an aggressive malignancy and one of the leading causes of cancer deaths. The high mortality rate is mostly due to the lack of appropriate tools for early detection of the disease and a shortage of effective therapies. We have previously shown that karyopherin alpha 7 (KPNA7), the newest member of the alpha karyopherin family of nuclear import receptors, is frequently amplified and overexpressed in pancreatic cancer. Here, we report that KPNA7 expression is absent in practically all normal human adult tissues but elevated in several pancreatic cancer cell lines. Inhibition of KPNA7 expression in AsPC-1 and Hs700Tmore » pancreatic cancer cells led to a reduction in cell growth and decreased anchorage independent growth, as well as increased autophagy. The cell growth effects were accompanied by an induction of the cell cycle regulator p21 and a G1 arrest of the cell cycle. Interestingly, the p21 induction was caused by increased mRNA synthesis and not defective nuclear transport. These data strongly demonstrate that KPNA7 silencing inhibits the malignant properties of pancreatic cancer cells in vitro and thereby provide the first evidence on the functional role for KPNA7 in human cancer. - Highlights: • KPNA7 expression is elevated in several pancreatic cancer cell lines. • KPNA7 silencing in high expressing cancer cells leads to growth inhibition. • The cell growth reduction is associated with p21 induction and G1 arrest. • KPNA7 silencing is also accompanied with increased autophagy.« less

Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA.

PubMed

Schertzer, Michael; Jouravleva, Karina; Perderiset, Mylene; Dingli, Florent; Loew, Damarys; Le Guen, Tangui; Bardoni, Barbara; de Villartay, Jean-Pierre; Revy, Patrick; Londoño-Vallejo, Arturo

2015-02-18

Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1-HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

Iwasaki, Yuko; Iwasaki, Hitoshi; Yatoh, Shigeru

Transgenic mice expressing nuclear sterol regulatory element-binding protein-1a under the control of the insulin promoter were generated to determine the role of SREBP-1a in pancreatic {beta}-cells. Only low expressors could be established, which exhibited mild hyperglycemia, impaired glucose tolerance, and reduced plasma insulin levels compared to C57BL/6 controls. The islets isolated from the transgenic mice were fewer and smaller, and had decreased insulin content and unaltered glucagon staining. Both glucose- and potassium-stimulated insulin secretions were decreased. The transgenic islets consistently expressed genes for fatty acids and cholesterol synthesis, resulting in accumulation of triglycerides but not cholesterol. PDX-1, {beta}{epsilon}{tau}{alpha}2, MafA, andmore » IRS-2 were suppressed, partially explaining the loss and dysfunction of {beta}-cell mass. The transgenic mice on a high fat/high sucrose diet still exhibited impaired insulin secretion and continuous {beta}-cell growth defect. Therefore, nuclear SREBP-1a, even at a low level, strongly disrupts {beta}-cell mass and function.« less

Surface structure of neutron stars with high magnetic fields

NASA Technical Reports Server (NTRS)

Fushiki, I.; Gudmundsson, E. H.; Pethick, C. J.

1989-01-01

The equation of state of cold dense matter in strong magnetic fields is calculated in the Thomas-Fermi and Thomas-Fermi-Dirac approximations. For use in the latter calculation, a new expression is derived for the exchange energy of the uniform electron gas in a strong magnetic field. Detailed calculations of the density profile in the surface region of a neutron star are described for a variety of equations of state, and these show that the surface density profile is strongly affected by the magnetic field, irrespective of whether or not matter in a magnetic field has a condensed state bound with respect to isolated atoms. It is also shown that, as a consequence of the field dependence of the screening potential, magnetic fields can significantly increase nuclear reaction rates.

IκB kinase 2 determines oligodendrocyte loss by non-cell-autonomous activation of NF-κB in the central nervous system

PubMed Central

Raasch, Jenni; Zeller, Nicolas; van Loo, Geert; Merkler, Doron; Mildner, Alexander; Erny, Daniel; Knobeloch, Klaus-Peter; Bethea, John R.; Waisman, Ari; Knust, Markus; Del Turco, Domenico; Deller, Thomas; Blank, Thomas; Priller, Josef; Brück, Wolfgang

2011-01-01

The IκB kinase complex induces nuclear factor kappa B activation and has recently been recognized as a key player of autoimmunity in the central nervous system. Notably, IκB kinase/nuclear factor kappa B signalling regulates peripheral myelin formation by Schwann cells, however, its role in myelin formation in the central nervous system during health and disease is largely unknown. Surprisingly, we found that brain-specific IκB kinase 2 expression is dispensable for proper myelin assembly and repair in the central nervous system, but instead plays a fundamental role for the loss of myelin in the cuprizone model. During toxic demyelination, inhibition of nuclear factor kappa B activation by conditional ablation of IκB kinase 2 resulted in strong preservation of central nervous system myelin, reduced expression of proinflammatory mediators and a significantly attenuated glial response. Importantly, IκB kinase 2 depletion in astrocytes, but not in oligodendrocytes, was sufficient to protect mice from myelin loss. Our results reveal a crucial role of glial cell-specific IκB kinase 2/nuclear factor kappa B signalling for oligodendrocyte damage during toxic demyelination. Thus, therapies targeting IκB kinase 2 function in non-neuronal cells may represent a promising strategy for the treatment of distinct demyelinating central nervous system diseases. PMID:21310728

Importin 8 regulates the transport of mature microRNAs into the cell nucleus.

PubMed

Wei, Yao; Li, Limin; Wang, Dong; Zhang, Chen-Yu; Zen, Ke

2014-04-11

Mature microRNAs (miRNAs), ∼ 22-nucleotide noncoding RNAs regulating target gene expression at the post-transcriptional level, have been recently shown to be transported into the nucleus where they modulate the biogenesis of other miRNAs or their own expression. However, the mechanism that governs the transport of mature miRNAs from cytoplasm to nucleus remains unknown. Here, we report that importin 8 (IPO8), a member of the karyopherin β (also named the protein import receptor importin β) family, plays a critical role in mediating the cytoplasm-to-nucleus transport of mature miRNAs. Specifically knocking down IPO8 but not other karyopherin β family proteins via siRNA significantly decreases the nuclear transport of various known nucleus-enriched miRNAs without affecting their total cellular levels. IPO8-mediated nuclear transport of mature miRNAs is also dependent on the association of IPO8 with the Argonaute 2 (Ago2) complex. Cross-immunoprecipitation and Western blot analysis show that IPO8 is physically associated with Ago2. Knocking down IPO8 via siRNA markedly decreases the nuclear transport of Ago2 but does not affect the total cellular Ago2 level. Furthermore, dissociating the binding of miRNAs with Ago2 by trypaflavine strongly reduces the IPO8-mediated nuclear transport of miRNAs.

Expression of HIWI in human esophageal squamous cell carcinoma is significantly associated with poorer prognosis

PubMed Central

2009-01-01

Background HIWI, the human homologue of Piwi family, is present in CD34+ hematopoietic stem cells and germ cells, but not in well-differentiated cell populations, indicating that HIWI may play an impotent role in determining or maintaining stemness of these cells. That HIWI expression has been detected in several type tumours may suggest its association with clinical outcome in cancer patients. Methods With the methods of real-time PCR, western blot, immunocytochemistry and immunohistochemistry, the expression of HIWI in three esophageal squamous cancer cell lines KYSE70, KYSE140 and KYSE450 has been characterized. Then, we investigated HIWI expression in a series of 153 esophageal squamous cell carcinomas using immunohistochemistry and explored its association with clinicopathological features. Results The expression of HIWI was observed in tumour cell nuclei or/and cytoplasm in 137 (89.5%) cases, 16 (10.5%) cases were negative in both nuclei and cytoplasm. 86 (56.2%) were strongly positive in cytoplasm, while 49 (32.0%) were strongly positive in nuclei. The expression level of HIWI in cytoplasm of esophageal cancer cells was significantly associated with histological grade (P = 0.011), T stage (P = 0.035), and clinic outcome (P < 0.001), while there was no correlation between the nuclear HIWI expression and clinicopathological features. Conclusion The expression of HIWI in the cytoplasm of esophageal cancer cells is significantly associated with higher histological grade, clinical stage and poorer clinical outcome, indicating its possible involvement in cancer development. PMID:19995427

The absence of p53 during Human Cytomegalovirus infection leads to decreased UL53 expression, disrupting UL50 localization to the inner nuclear membrane, and thereby inhibiting capsid nuclear egress

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

Kuan, Man I; O’Dowd, John M.; Fortunato, Elizabeth

Our electron microscopy study (Kuan et al., 2016) found HCMV nuclear capsid egress was significantly reduced in p53 knockout cells (p53KOs), correlating with inhibited formation of infoldings of the inner nuclear membrane (IINMs). Molecular examination of these phenomena has found p53KOs expressed UL97 and phosphorylated lamins, however the lamina failed to remodel. The nuclear egress complex (NEC) protein UL50 was expressed in almost all cells. UL50 re-localized to the inner nuclear membrane (INM) in ~90% of wt cells, but only ~35% of p53KOs. UL53 expression was significantly reduced in p53KOs, and cells lacking UL50 nuclear staining, expressed no UL53. Re-introductionmore » of p53 into p53KOs largely recovered UL53 positivity and UL50 nuclear re-localization. Nuclear rim located UL50/53 puncta, which co-localized with the major capsid protein, were largely absent in p53KOs. We believe these puncta were IINMs. In the absence of p53, UL53 expression was inhibited, disrupting formation of the NEC/IINMs, and reducing functional virion secretion. -- Highlights: •Phosphorylated nuclear lamins were inefficiently remodeled in p53KO cells. •p53KO cells expressed UL50, but it was not efficiently targeted to the nuclear rim. •UL53 was not expressed in the large majority of p53KO cells. •Cells failing to express UL53 did not localize UL50 to the nucleus. •NEC puncta/infoldings of the inner nuclear membrane were scarce in p53KO cells.« less

Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

PubMed

Sugiyama, Y; Sasajima, J; Mizukami, Y; Koizumi, K; Kawamoto, T; Ono, Y; Karasaki, H; Tanabe, H; Fujiya, M; Kohgo, Y

2016-06-01

The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

Dihydroavenanthramide D inhibits human breast cancer cell invasion through suppression of MMP-9 expression

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

Lee, Young-Rae; Noh, Eun-Mi; Oh, Hyun Ju

2011-02-25

Research highlights: {yields} MMP-9 plays a pivotal role in the invasion of MCF-7 breast cancer cells. {yields} TPA stimulates MMP-9 expression through activation of MAPK/NF-{kappa}B and MAPK/AP-1 pathways. {yields} Dihydroavenanthramide D suppresses MMP-9 expression via inhibition of TPA-induced MAPK/NF-{kappa}B and MAPK/AP-1 activations. {yields} Dihydroavenanthramide D blocks cell invasion of MCF-7 breast cancer cells. -- Abstract: Dihydroavenanthramide D (DHAvD) is a synthetic analog to naturally occurring avenanthramide, which is the active component of oat. Previous study demonstrates that DHAvD strongly inhibits activation of nuclear factor-kappa B (NF-{kappa}B), which is a major component in cancer cell invasion. The present study investigated whethermore » DHAvD can modulate MMP-9 expression and cell invasion in MCF-7 human breast cancer cells. MMP-9 expression and cell invasion in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) was increased, whereas these inductions were muted by DHAvD. DHAvD also suppressed activation of mitogen-activated protein kinase (MAPK), and MAPK-mediated nuclear factor-kappa B (NF-{kappa}B) and activator protein-1 (AP-1) activations in TPA-treated MCF-7 cells. The results indicate that DHAvD-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of the MAPK/NF-{kappa}B and MAPK/AP-1 pathways in MCF-7 cells. DHAvD may have potential value in breast cancer metastasis.« less

Long interspersed nuclear elements (LINEs) show tissue-specific, mosaic genome and methylation-unrestricted, widespread expression of noncoding RNAs in somatic tissues of the rat

PubMed Central

Singh, Deepak K.; Rath, Pramod C.

2012-01-01

We report strong somatic and germ line expression of LINE RNAs in eight different tissues of rat by using a novel ~2.8 kb genomic PstI-LINE DNA (P1-LINE) isolated from the rat brain. P1-LINE is present in a 93 kb LINE-SINE-cluster in sub-telomeric region of chromosome 12 (12p12) and as multiple truncated copies interspersed in all rat chromosomes. P1-LINEs occur as inverted repeats at multiple genomic loci in tissue-specific and mosaic patterns. P1-LINE RNAs are strongly expressed in brain, liver, lungs, heart, kidney, testes, spleen and thymus into large to small heterogeneous RNAs (~5.0 to 0.2 kb) in tissue-specific and dynamic patterns in individual rats. P1-LINE DNA is strongly methylated at CpG-dinucleotides in most genomic copies in all the tissues and weakly hypomethylated in few copies in some tissues. Small (700–75 nt) P1-LINE RNAs expressed in all tissues may be possible precursors for small regulatory RNAs (PIWI-interacting/piRNAs) bioinformatically derived from P1-LINE. The strong and dynamic expression of LINE RNAs from multiple chromosomal loci and the putative piRNAs in somatic tissues of rat under normal physiological conditions may define functional chromosomal domains marked by LINE RNAs as long noncoding RNAs (lncRNAs) unrestricted by DNA methylation. The tissue-specific, dynamic RNA expression and mosaic genomic distribution of LINEs representing a steady-state genomic flux of retrotransposon RNAs suggest for biological role of LINE RNAs as long ncRNAs and small piRNAs in mammalian tissues independent of their cellular fate for translation, reverse-transcription and retrotransposition. This may provide evolutionary advantages to LINEs and mammalian genomes. PMID:23064113

Anti-inflammatory function of Withangulatin A by targeted inhibiting COX-2 expression via MAPK and NF-kappaB pathways.

PubMed

Sun, Lijuan; Liu, Jianwen; Cui, Daling; Li, Jiyu; Yu, Youjun; Ma, Lei; Hu, Lihong

2010-02-15

Withangulatin A (WA), an active component isolated from Physalis angulata L., has been reported to possess anti-tumor and trypanocidal activities in model systems via multiple biochemical mechanisms. The aim of this study is to investigate its anti-inflammatory potential and the possible underlying mechanisms. In the current study, WA significantly suppressed mice T lymphocytes proliferation stimulated with LPS in a dose- and time-dependent manner and inhibited pro-inflammation cytokines (IL-2, IFN-gamma, and IL-6) dramatically. Moreover, WA targeted inhibited COX-2 expression mediated by MAPKs and NF-kappaB nuclear translocation pathways in mice T lymphocytes, and this result was further confirmed by the COX-1/2 luciferase reporter assay. Intriguingly, administration of WA inhibited the extent of mice ear swelling and decreased pro-inflammatory cytokines production in mice blood serum. Based on these evidences, WA influences the mice T lymphocytes function through targeted inhibiting COX-2 expression via MAPKs and NF-kappaB nuclear translocation signaling pathways, and this would make WA a strong candidate for further study as an anti-inflammatory agent. (c) 2009 Wiley-Liss, Inc.

Docosahexaenoic acid antagonizes the boosting effect of palmitic acid on LPS inflammatory signaling by inhibiting gene transcription and ceramide synthesis

PubMed Central

Jin, Junfei; Lu, Zhongyang; Li, Yanchun; Cowart, L. Ashley; Lopes-Virella, Maria F.

2018-01-01

It is well known that saturated fatty acids (SFAs) and unsaturated fatty acid, in particular omega-3 polyunsaturated fatty acids (n-3 PUFAs), have different effects on inflammatory signaling: SFAs are pro-inflammatory but n-3 PUFAs have strong anti-inflammatory properties. We have reported that palmitic acid (PA), a saturated fatty acid, robustly amplifies lipopolysaccharide (LPS) signaling to upregulate proinflammatory gene expression in macrophages. We also reported that the increased production of ceramide (CER) via sphingomyelin (SM) hydrolysis and CER de novo synthesis plays a key role in the synergistic effect of LPS and PA on proinflammatory gene expression. However, it remains unclear if n-3 PUFAs are capable of antagonizing the synergistic effect of LPS and PA on gene expression and CER production. In this study, we employed the above macrophage culture system and lipidomical analysis to assess the effect of n-3 PUFAs on proinflammatory gene expression and CER production stimulated by LPS and PA. Results showed that DHA strongly inhibited the synergistic effect of LPS and PA on proinflammatory gene expression by targeting nuclear factor kappa B (NFκB)-dependent gene transcription. Results also showed that DHA inhibited the cooperative effect of LPS and PA on CER production by targeting CER de novo synthesis, but not SM hydrolysis. Furthermore, results showed that myriocin, a specific inhibitor of serine palmitoyltransferase, strongly inhibited both LPS-PA-stimulated CER synthesis and proinflammatory gene expression, indicating that CER synthesis is associated with proinflammatory gene expression and that inhibition of CER synthesis contributes to DHA-inhibited proinflammatory gene expression. Taken together, this study demonstrates that DHA antagonizes the boosting effect of PA on LPS signaling on proinflammatory gene expression by targeting both NFκB-dependent transcription and CER de novo synthesis in macrophages. PMID:29474492

Docosahexaenoic acid antagonizes the boosting effect of palmitic acid on LPS inflammatory signaling by inhibiting gene transcription and ceramide synthesis.

PubMed

Jin, Junfei; Lu, Zhongyang; Li, Yanchun; Cowart, L Ashley; Lopes-Virella, Maria F; Huang, Yan

2018-01-01

It is well known that saturated fatty acids (SFAs) and unsaturated fatty acid, in particular omega-3 polyunsaturated fatty acids (n-3 PUFAs), have different effects on inflammatory signaling: SFAs are pro-inflammatory but n-3 PUFAs have strong anti-inflammatory properties. We have reported that palmitic acid (PA), a saturated fatty acid, robustly amplifies lipopolysaccharide (LPS) signaling to upregulate proinflammatory gene expression in macrophages. We also reported that the increased production of ceramide (CER) via sphingomyelin (SM) hydrolysis and CER de novo synthesis plays a key role in the synergistic effect of LPS and PA on proinflammatory gene expression. However, it remains unclear if n-3 PUFAs are capable of antagonizing the synergistic effect of LPS and PA on gene expression and CER production. In this study, we employed the above macrophage culture system and lipidomical analysis to assess the effect of n-3 PUFAs on proinflammatory gene expression and CER production stimulated by LPS and PA. Results showed that DHA strongly inhibited the synergistic effect of LPS and PA on proinflammatory gene expression by targeting nuclear factor kappa B (NFκB)-dependent gene transcription. Results also showed that DHA inhibited the cooperative effect of LPS and PA on CER production by targeting CER de novo synthesis, but not SM hydrolysis. Furthermore, results showed that myriocin, a specific inhibitor of serine palmitoyltransferase, strongly inhibited both LPS-PA-stimulated CER synthesis and proinflammatory gene expression, indicating that CER synthesis is associated with proinflammatory gene expression and that inhibition of CER synthesis contributes to DHA-inhibited proinflammatory gene expression. Taken together, this study demonstrates that DHA antagonizes the boosting effect of PA on LPS signaling on proinflammatory gene expression by targeting both NFκB-dependent transcription and CER de novo synthesis in macrophages.

p63 protein is essential for the embryonic development of vibrissae and teeth

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

Rufini, Alessandro; Weil, Miguel; McKeon, Frank

2006-02-17

Development of skin appendages strongly depends on epithelial-mesenchymal interactions. One of the genes involved in this process is p63, a member of the p53 family of transcription factors, essential for ectodermal development, as elucidated by the phenotype of p63 knock-out mice. Surprisingly, no information on p63 expression in tooth and hair is yet available. Here, we show p63 expression during teeth and vibrissae morphogenesis in mouse embryos and we also show a correlation with the expression patterns of the epithelial marker keratin 5 and the proliferation marker Ki67. Our results show that p63 colocalizes with both K5 and Ki67 inmore » the epithelium of developing vibrissae, while in teeth p63 is expressed, together with K5, in the undifferentiated ectoderm (enamel organ), and in ameloblasts, a subpopulation of differentiated ectodermal cells. Moreover, p63 expression in tooth seems not to be fully colocalized with nuclear Ki67 expression.« less

Viral mimicry of Cdc2/cyclin-dependent kinase 1 mediates disruption of nuclear lamina during human cytomegalovirus nuclear egress.

PubMed

Hamirally, Sofia; Kamil, Jeremy P; Ndassa-Colday, Yasmine M; Lin, Alison J; Jahng, Wan Jin; Baek, Moon-Chang; Noton, Sarah; Silva, Laurie A; Simpson-Holley, Martha; Knipe, David M; Golan, David E; Marto, Jarrod A; Coen, Donald M

2009-01-01

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser(22)). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser(22) is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle.

Validation of nuclear STAT6 immunostaining as a diagnostic marker of meningeal solitary fibrous tumor (SFT)/hemangiopericytoma.

PubMed

Berghoff, Anna S; Kresl, Philip; Bienkowski, Michal; Koelsche, Christian; Rajky, Ursula; Hainfellner, Johannes A; Preusser, Matthias

NAB2-STAT6 gene fusion is a molecular characteristic of solitary fibrous tumors (SFT) and hemangiopericytoma, underscoring their definition as one diagnostic entity. NAB2-STAT6 fusion is associated with nuclear relocation of STAT6 protein that can be detected by immunohistochemistry. We evaluated the diagnostic value of STAT6 expression in meningeal tumors. 77 meningeal tumors (17/77 (22.0%) SFT/hemangiopericytoma, 11/77 meningothelial meningioma, 10/77 atypical meningioma 8/77 chordoid meningioma, 9/77 fibroblastic meningioma, 10/77 transitional meningioma, 3/77 rhabdoid meningioma and 9/77 anaplastic meningioma) were included. STAT6 immunohistochemistry was performed on FFPE specimens using a fully automated slide-staining system and anti-STAT6 antibody SC-20:sc621. Two independent observers analyzed all specimens blinded to histological diagnoses, and a third observer was consulted in case of discordancy. STAT6 immunohistochemistry yielded an exclusively nuclear immunostaining signal. 16/17 (94%) SFT/hemangiopericytoma specimens presented with clear-cut, wide-spread, and moderate to strong staining in tumor cell nuclei and were rated as STAT6-positive. In only 1 SFT case with weak and focal nuclear STAT6 immunostaining signal, STAT6 expression was rated discordant (observer 1: STAT6-negative, observers 2 and 3: STAT6-positive). All non-SFT/hemangiopericytoma cases were unanimously rated as STAT6-negative. In 76/77 (98.7%) cases the evaluation of STAT6 immunostaining results was in agreement among observers. STAT6 immunohistochemistry is a robust method to verify diagnosis of SFT/hemangiopericytoma and should therefore be included in the diagnostic work-up of meningeal tumors. In singular cases, weak and focal STAT6 expression may lead to false-negative evaluation and may prompt further molecular work-up.
.

Immunohistochemical analysis of S6K1 and S6K2 localization in human breast tumors.

PubMed

Filonenko, Valeriy V; Tytarenko, Ruslana; Azatjan, Sergey K; Savinska, Lilya O; Gaydar, Yuriy A; Gout, Ivan T; Usenko, Vasiliy S; Lyzogubov, Valeriy V

2004-12-01

To perform an immunohistochemical analysis of human breast adenomas and adenocarcinomas as well as normal breast tissues in respect of S6 ribosomal protein kinase (S6K) expression and localization in normal and transformed cells. The expression level and localization of S6K have been detected in formalin fixed, paraffin embedded sections of normal human breast tissues, adenomas and adenocarcinomas with different grade of differentiation. Immunohistochemical detection of S6K1 and S6K2 in normal human breast tissues and breast tumors were performed using specific monoclonal and polyclonal antibodies against S6K1 and S6K2 with following semiquantitative analysis. The increase of S6K content in the cytoplasm of epithelial cells in benign and malignant tumors has been detected. Nuclear accumulation of S6K1 and to a greater extend S6K2 have been found in breast adenocarcinomas. About 80% of breast adenocarcinomas cases revealed S6K2 nuclear staining comparing to normal tissues. In 31% of cases more then 50% of cancer cells had strong nuclear staining. Accumulation of S6K1 in the nucleus of neoplastic cells has been demonstrated in 25% of cases. Nuclear localization of S6K in the epithelial cells in normal breast tissues has not been detected. Immunohistochemical analysis of S6K1 and S6K2 expression in normal human breast tissues, benign and malignant breast tumors clearly indicates that both kinases are overexpressed in breast tumors. Semiquantitative analysis of peculiarities of S6K localization in normal tissues and tumors revealed that nucleoplasmic accumulation of S6K (especially S6K2) is a distinguishing feature of cancer cells.

Nitrogen-responsive Regulation of GATA Protein Family Activators Gln3 and Gat1 Occurs by Two Distinct Pathways, One Inhibited by Rapamycin and the Other by Methionine Sulfoximine*

PubMed Central

Georis, Isabelle; Tate, Jennifer J.; Cooper, Terrance G.; Dubois, Evelyne

2011-01-01

Nitrogen availability regulates the transcription of genes required to degrade non-preferentially utilized nitrogen sources by governing the localization and function of transcription activators, Gln3 and Gat1. TorC1 inhibitor, rapamycin (Rap), and glutamine synthetase inhibitor, methionine sulfoximine (Msx), elicit responses grossly similar to those of limiting nitrogen, implicating both glutamine synthesis and TorC1 in the regulation of Gln3 and Gat1. To better understand this regulation, we compared Msx- versus Rap-elicited Gln3 and Gat1 localization, their DNA binding, nitrogen catabolite repression-sensitive gene expression, and the TorC1 pathway phosphatase requirements for these responses. Using this information we queried whether Rap and Msx inhibit sequential steps in a single, linear cascade connecting glutamine availability to Gln3 and Gat1 control as currently accepted or alternatively inhibit steps in two distinct parallel pathways. We find that Rap most strongly elicits nuclear Gat1 localization and expression of genes whose transcription is most Gat1-dependent. Msx, on the other hand, elicits nuclear Gln3 but not Gat1 localization and expression of genes that are most Gln3-dependent. Importantly, Rap-elicited nuclear Gln3 localization is absolutely Sit4-dependent, but that elicited by Msx is not. PP2A, although not always required for nuclear GATA factor localization, is highly required for GATA factor binding to nitrogen-responsive promoters and subsequent transcription irrespective of the gene GATA factor specificities. Collectively, our data support the existence of two different nitrogen-responsive regulatory pathways, one inhibited by Msx and the other by rapamycin. PMID:22039046

Nitrogen-responsive regulation of GATA protein family activators Gln3 and Gat1 occurs by two distinct pathways, one inhibited by rapamycin and the other by methionine sulfoximine.

PubMed

Georis, Isabelle; Tate, Jennifer J; Cooper, Terrance G; Dubois, Evelyne

2011-12-30

Nitrogen availability regulates the transcription of genes required to degrade non-preferentially utilized nitrogen sources by governing the localization and function of transcription activators, Gln3 and Gat1. TorC1 inhibitor, rapamycin (Rap), and glutamine synthetase inhibitor, methionine sulfoximine (Msx), elicit responses grossly similar to those of limiting nitrogen, implicating both glutamine synthesis and TorC1 in the regulation of Gln3 and Gat1. To better understand this regulation, we compared Msx- versus Rap-elicited Gln3 and Gat1 localization, their DNA binding, nitrogen catabolite repression-sensitive gene expression, and the TorC1 pathway phosphatase requirements for these responses. Using this information we queried whether Rap and Msx inhibit sequential steps in a single, linear cascade connecting glutamine availability to Gln3 and Gat1 control as currently accepted or alternatively inhibit steps in two distinct parallel pathways. We find that Rap most strongly elicits nuclear Gat1 localization and expression of genes whose transcription is most Gat1-dependent. Msx, on the other hand, elicits nuclear Gln3 but not Gat1 localization and expression of genes that are most Gln3-dependent. Importantly, Rap-elicited nuclear Gln3 localization is absolutely Sit4-dependent, but that elicited by Msx is not. PP2A, although not always required for nuclear GATA factor localization, is highly required for GATA factor binding to nitrogen-responsive promoters and subsequent transcription irrespective of the gene GATA factor specificities. Collectively, our data support the existence of two different nitrogen-responsive regulatory pathways, one inhibited by Msx and the other by rapamycin.

Orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) protein negatively regulates bone morphogenetic protein 2-induced osteoblast differentiation through suppressing runt-related gene 2 (Runx2) activity.

PubMed

Lee, Kkot-Nim; Jang, Won-Gu; Kim, Eun-Jung; Oh, Sin-Hye; Son, Hye-Ju; Kim, Sun-Hun; Franceschi, Renny; Zhang, Xiao-Kun; Lee, Shee-Eun; Koh, Jeong-Tae

2012-06-01

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is an orphan nuclear receptor of the steroid-thyroid hormone receptor superfamily. COUP-TFII is widely expressed in multiple tissues and organs throughout embryonic development and has been shown to regulate cellular growth, differentiation, and organ development. However, the role of COUP-TFII in osteoblast differentiation has not been systematically evaluated. In the present study, COUP-TFII was strongly expressed in multipotential mesenchymal cells, and the endogenous expression level decreased during osteoblast differentiation. Overexpression of COUP-TFII inhibited bone morphogenetic protein 2 (BMP2)-induced osteoblastic gene expression. The results of alkaline phosphatase, Alizarin Red staining, and osteocalcin production assay showed that COUP-TFII overexpression blocks BMP2-induced osteoblast differentiation. In contrast, the down-regulation of COUP-TFII synergistically induced the expression of BMP2-induced osteoblastic genes and osteoblast differentiation. Furthermore, the immunoprecipitation assay showed that COUP-TFII and Runx2 physically interacted and COUP-TFII significantly impaired the Runx2-dependent activation of the osteocalcin promoter. From the ChIP assay, we found that COUP-TFII repressed DNA binding of Runx2 to the osteocalcin gene, whereas Runx2 inhibited COUP-TFII expression via direct binding to the COUP-TFII promoter. Taken together, these findings demonstrate that COUP-TFII negatively regulates osteoblast differentiation via interaction with Runx2, and during the differentiation state, BMP2-induced Runx2 represses COUP-TFII expression and promotes osteoblast differentiation.

Expression Levels of DNA Damage Repair Proteins Are Associated With Overall Survival in Platinum-Treated Advanced Urothelial Carcinoma.

PubMed

Mullane, Stephanie A; Werner, Lillian; Guancial, Elizabeth A; Lis, Rosina T; Stack, Edward C; Loda, Massimo; Kantoff, Philip W; Choueiri, Toni K; Rosenberg, Jonathan; Bellmunt, Joaquim

2016-08-01

Combination platinum chemotherapy is standard first-line therapy for metastatic urothelial carcinoma (mUC). Defining the platinum response biomarkers for patients with mUC could establish personalize medicine and provide insights into mUC biology. Although DNA repair mechanisms have been hypothesized to mediate the platinum response, we sought to analyze whether increased expression of DNA damage genes would correlate with worse overall survival (OS) in patients with mUC. We retrospectively identified a clinically annotated cohort of patients with mUC, who had been treated with first-line platinum combination chemotherapy. A tissue microarray was constructed from formalin-fixed paraffin-embedded tissue from the primary tumor before treatment. Immunohistochemical analysis of the following DNA repair proteins was performed: ERCC1, RAD51, BRCA1/2, PAR, and PARP-1. Nuclear and cytoplasmic expression was analyzed using multispectral imaging. Nuclear staining was used for the survival analysis. Cox regression analysis was used to evaluate the associations between the percentage of positive nuclear staining and OS in multivariable analysis, controlling for known prognostic variables. In a cohort of 104 patients with mUC, a greater percentage of nuclear staining of ERCC1 (hazard ratio [HR], 2.7; 95% confidence interval [CI], 1.5-4.9; P = .0007), RAD51 (HR, 5.6; 95% CI, 1.7-18.3; P = .005), and PAR (HR, 2.2; 95% CI, 1.1-4.4; P = .026) was associated with worse OS. BRCA1, BRCA2, and PARP-1 expression was not associated with OS (P = .76, P = .38, and P = .09, respectively). A greater percentage of combined ERCC1 and RAD51 nuclear staining was strongly associated with worse OS (P = .005). A high percentage of nuclear staining of ERCC1, RAD51, and PAR, assessed by immunohistochemistry, correlated with worse OS for patients with mUC treated with first-line platinum combination chemotherapy, supporting the evidence of the DNA repair pathways' role in the prognosis of mUC. We also report new evidence that RAD51 and PAR might play a role in the platinum response. Additional prospective studies are required to determine the prognostic or predictive nature of these biomarkers in mUC. Copyright © 2015 Elsevier Inc. All rights reserved.

Public perspectives on nuclear security. US national security surveys, 1993--1997

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

Herron, K.G.; Jenkins-Smith, H.C.

This is the third report in a series of studies to examine how US attitudes about nuclear security are evolving in the post-Cold War era and to identify trends in public perceptions and preferences relevant to the evolution of US nuclear security policy. It presents findings from three surveys: a nationwide telephone survey of randomly selected members of the US general public; a written survey of randomly selected members of American Men and Women of Science; and a written survey of randomly selected state legislators from all fifty US states. Key areas of investigation included nuclear security, cooperation between USmore » and Russian scientists about nuclear issues, vulnerabilities of critical US infrastructures and responsibilities for their protection, and broad areas of US national science policy. While international and US national security were seen to be slowly improving, the primary nuclear threat to the US was perceived to have shifted from Russia to China. Support was found for nuclear arms control measures, including mutual reductions in stockpiles. However, respondents were pessimistic about eliminating nuclear armaments, and nuclear deterrence continued to be highly values. Participants favored decreasing funding f/or developing and testing new nuclear weapons, but supported increased investments in nuclear weapons infrastructure. Strong concerns were expressed about nuclear proliferation and the potential for nuclear terrorism. Support was evident for US scientific cooperation with Russia to strengthen security of Russian nuclear assets. Elite and general public perceptions of external and domestic nuclear weapons risks and external and domestic nuclear weapons benefits were statistically significantly related to nuclear weapons policy options and investment preferences. Demographic variables and individual belief systems were systematically related both to risk and benefit perceptions and to policy and spending preferences.« less

A Dominant Mutation in Nuclear Receptor Interacting Protein 1 Causes Urinary Tract Malformations via Dysregulation of Retinoic Acid Signaling.

PubMed

Vivante, Asaf; Mann, Nina; Yonath, Hagith; Weiss, Anna-Carina; Getwan, Maike; Kaminski, Michael M; Bohnenpoll, Tobias; Teyssier, Catherine; Chen, Jing; Shril, Shirlee; van der Ven, Amelie T; Ityel, Hadas; Schmidt, Johanna Magdalena; Widmeier, Eugen; Bauer, Stuart B; Sanna-Cherchi, Simone; Gharavi, Ali G; Lu, Weining; Magen, Daniella; Shukrun, Rachel; Lifton, Richard P; Tasic, Velibor; Stanescu, Horia C; Cavaillès, Vincent; Kleta, Robert; Anikster, Yair; Dekel, Benjamin; Kispert, Andreas; Lienkamp, Soeren S; Hildebrandt, Friedhelm

2017-08-01

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of CKD in the first three decades of life. However, for most patients with CAKUT, the causative mutation remains unknown. We identified a kindred with an autosomal dominant form of CAKUT. By whole-exome sequencing, we identified a heterozygous truncating mutation (c.279delG, p.Trp93fs*) of the nuclear receptor interacting protein 1 gene ( NRIP1 ) in all seven affected members. NRIP1 encodes a nuclear receptor transcriptional cofactor that directly interacts with the retinoic acid receptors (RARs) to modulate retinoic acid transcriptional activity. Unlike wild-type NRIP1, the altered NRIP1 protein did not translocate to the nucleus, did not interact with RAR α , and failed to inhibit retinoic acid-dependent transcriptional activity upon expression in HEK293 cells. Notably, we also showed that treatment with retinoic acid enhanced NRIP1 binding to RAR α RNA in situ hybridization confirmed Nrip1 expression in the developing urogenital system of the mouse. In explant cultures of embryonic kidney rudiments, retinoic acid stimulated Nrip1 expression, whereas a pan-RAR antagonist strongly reduced it. Furthermore, mice heterozygous for a null allele of Nrip1 showed a CAKUT-spectrum phenotype. Finally, expression and knockdown experiments in Xenopus laevis confirmed an evolutionarily conserved role for NRIP1 in renal development. These data indicate that dominant NRIP1 mutations can cause CAKUT by interference with retinoic acid transcriptional signaling, shedding light on the well documented association between abnormal vitamin A levels and renal malformations in humans, and suggest a possible gene-environment pathomechanism in this disease. Copyright © 2017 by the American Society of Nephrology.

Characterization of Nrf2 activation and heme oxygenase-1 expression in NIH3T3 cells exposed to aqueous extracts of cigarette smoke.

PubMed

Knörr-Wittmann, Constanze; Hengstermann, Arnd; Gebel, Stephan; Alam, Jawed; Müller, Thomas

2005-12-01

Cigarette smoke (CS) is a complex chemical mixture estimated to be composed of up to 5000 different chemicals, many of which are prooxidant. Here we show that, at least in vitro, the cellular response designed to combat oxidative stress resulting from CS exposure is primarily controlled by the transcription factor Nrf2, a principal inducer of antioxidant and phase II-related genes. The prominent role of Nrf2 in the cellular response to CS is substantiated by the following observations: In NIH3T3 cells exposed to aqueous extracts of CS (i) Nrf2 is strongly stabilized and becomes detectable in nuclear extracts. (ii) Nuclear localization of Nrf2 coincides with increased DNA binding of a putative Nrf2/MafK heterodimer to its cognate cis-regulatory site, i.e., the antioxidant-responsive element (ARE). (iii) Studies on the regulatory elements of the oxidative stress-inducible gene heme oxygenase-1 (hmox1) using various hmox1 promoter/luciferase reporter constructs revealed that the strong CS-dependent expression of this gene is primarily governed by the distal enhancers 1 ("E1") and 2 ("E2"), which both contain three canonical ARE-like stress-responsive elements (StREs). Notably, depletion of Nrf2 levels caused by RNA interference significantly compromised CS-induced hmox1 promoter activation, based on the distinct Nrf2 sensitivity exhibited by E1 and E2. Finally, (iv) siRNA-dependent knock-down of Nrf2 completely abrogated CS-induced expression of phase II-related genes. Taken together, these results confirm the outstanding role of Nrf2 both in sensing (oxidant) stress and in orchestrating an efficient transcriptional response aimed at resolving the stressing conditions.

Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing their proliferation and clonogenicity

PubMed Central

Deep, Gagan; Kumar, Rahul; Jain, Anil K.; Dhar, Deepanshi; Panigrahi, Gati K.; Hussain, Anowar; Agarwal, Chapla; El-Elimat, Tamam; Sica, Vincent P.; Oberlies, Nicholas H.; Agarwal, Rajesh

2016-01-01

Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1–5 μg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47phox). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1α levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity. PMID:26979487

Cytosolic malate dehydrogenase regulates RANKL-mediated osteoclastogenesis via AMPK/c-Fos/NFATc1 signaling

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

Oh, Se Jeong; Gu, Dong Ryun; Center for Metabolic Function Regulation

2016-06-17

Cytosolic malate dehydrogenase (malate dehydrogenase 1, MDH1) plays pivotal roles in the malate/aspartate shuttle that might modulate metabolism between the cytosol and mitochondria. In this study, we investigated the role of MDH1 in osteoclast differentiation and formation. MDH1 expression was induced by receptor activator of nuclear factor kappa-B ligand (RANKL) treatment. Knockdown of MDH1 by infection with retrovirus containing MDH1-specific shRNA (shMDH1) reduced mature osteoclast formation and bone resorption activity. Moreover, the expression of marker genes associated with osteoclast differentiation was downregulated by shMDH1 treatment, suggesting a role of MDH1 in osteoclast differentiation. In addition, intracellular ATP production was reducedmore » following the activation of adenosine 5′ monophosphate-activated protein kinase (AMPK), a cellular energy sensor and negative regulator of RANKL-induced osteoclast differentiation, in shMDH1-infected osteoclasts compared to control cells. In addition, the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a critical transcription factor of osteoclastogenesis, was decreased with MDH1 knockdown during RANKL-mediated osteoclast differentiation. These findings provide strong evidence that MDH1 plays a critical role in osteoclast differentiation and function via modulation of the intracellular energy status, which might affect AMPK activity and NFATc1 expression.« less

Crlz-1 Is Prominently Expressed in Spermatogonia and Sertoli Cells during Early Testis Development and in Spermatids during Late Spermatogenesis

PubMed Central

Lim, Jung-Hyun; Choi, Seong-Young; Yoo, Han-Woong; Cho, Sun-Jung; Son, Youngsook

2013-01-01

The expression of the Crlz-1 gene in mouse testis, where it was found to be expressed most highly among the tested mouse organs, was analyzed spatiotemporally by employing RT-PCR and in situ hybridization techniques with the aid of immunohistochemistry and/or immunofluorescence methods. In 1-week-old neonatal testis, Crlz-1 was strongly expressed in the spermatogonia and Sertoli cells in its seminiferous cord. In 2- to 3-week-old prepubertal testis, where Sertoli cells cease to proliferate, Crlz-1 expression dropped and remained weakly at the rim layer of seminiferous cords and/or tubules, where spermatogonia are present. In the adult testis at 12 weeks after birth, Crlz-1 was expressed mainly in the spermatids near the lumen of seminiferous tubules. In a further in situ hybridization of Crlz-1 in the 12-week-old adult testis with hematoxylin nuclear counterstaining, Crlz-1 was mainly expressed at step 16 of spermatids between stages VII and VIII of seminiferous tubules as well as in their residual bodies at stage IX of seminiferous tubules. PMID:23525569

Mechanistic and Treatment Implications of ΔNp63 Expression in a Rare Case of Metastatic Hidradenocarcinoma

PubMed Central

Driscoll, James J.; Gauerke, Steven; Monahan, Brian C.

2009-01-01

Hidradenocarcinomas are rare, aggressive adnexal tumors of sweat gland origin that demonstrate a high potential for local recurrence, metastasis and poor outcome. These neoplasms can derive from preexisting clear cell hidradenomas, but more commonly appear de novo with the molecular events responsible for the pathogenesis currently unknown. Molecular markers of pathogenesis as well as effective forms of adjuvant chemotherapy are missing due to the lack of accurate diagnosis, paucity of cases and confusion with other visceral solid tumors. Here, we report a 37-year-old man who presented with a rapidly growing, painful palpable mass located in the right inguinal area. The patient was a nonsmoker, did not consume alcohol and had a medical history remarkable only for a lower abdominal superficial skin lesion in the same area that had been excised 11 years earlier. Although initially slow growing, the lesion eventually expanded, was surgically excised and was diagnosed as a hidradenoma. There was no family history of malignancy and the patient had not experienced any constitutional symptoms. We probed the immunohistochemical status and detected negative staining for the estrogen, progesterone and Her2 receptors, while strong, diffuse nuclear staining was seen in the majority of cells consistent with p53 overexpression. Similarly, strong nuclear reactivity was seen with p63 and p73 antibodies. The p63 gene contains 2 separate promoters which express at least 6 major transcripts that lead to 2 fundamentally different classes of proteins; 3 isoforms (TAp63α, β and γ) encode proteins that induce apoptosis, whereas the other 3 isoforms (ΔNp63α, β and γ) may exert inhibitory effects on p53. Interest in p63 stems from this ‘two genes in one’-concept. Importantly, the nuclear presence of ΔNp63 was detected widespread throughout the tumor. We have identified a subtype of hidradenocarcinomas that express ΔNp63 and uncovered an unforeseen commonality with triple-negative breast tumors. To our knowledge, this is the first report of a sweat gland tumor that displayed expression of both ΔNp63 and p73 and demonstrated a triple-negative receptor status. Such a link between 2 seemingly disparate tumor types indicates a mutual pathway of tumorigenesis and suggests the potential for common therapeutic regimens. PMID:20740144

Mechanistic and Treatment Implications of DeltaNp63 Expression in a Rare Case of Metastatic Hidradenocarcinoma.

PubMed

Driscoll, James J; Gauerke, Steven; Monahan, Brian C

2009-03-14

Hidradenocarcinomas are rare, aggressive adnexal tumors of sweat gland origin that demonstrate a high potential for local recurrence, metastasis and poor outcome. These neoplasms can derive from preexisting clear cell hidradenomas, but more commonly appear de novo with the molecular events responsible for the pathogenesis currently unknown. Molecular markers of pathogenesis as well as effective forms of adjuvant chemotherapy are missing due to the lack of accurate diagnosis, paucity of cases and confusion with other visceral solid tumors. Here, we report a 37-year-old man who presented with a rapidly growing, painful palpable mass located in the right inguinal area. The patient was a nonsmoker, did not consume alcohol and had a medical history remarkable only for a lower abdominal superficial skin lesion in the same area that had been excised 11 years earlier. Although initially slow growing, the lesion eventually expanded, was surgically excised and was diagnosed as a hidradenoma. There was no family history of malignancy and the patient had not experienced any constitutional symptoms. We probed the immunohistochemical status and detected negative staining for the estrogen, progesterone and Her2 receptors, while strong, diffuse nuclear staining was seen in the majority of cells consistent with p53 overexpression. Similarly, strong nuclear reactivity was seen with p63 and p73 antibodies. The p63 gene contains 2 separate promoters which express at least 6 major transcripts that lead to 2 fundamentally different classes of proteins; 3 isoforms (TAp63alpha, beta and gamma) encode proteins that induce apoptosis, whereas the other 3 isoforms (DeltaNp63alpha, beta and gamma) may exert inhibitory effects on p53. Interest in p63 stems from this 'two genes in one'-concept. Importantly, the nuclear presence of DeltaNp63 was detected widespread throughout the tumor. We have identified a subtype of hidradenocarcinomas that express DeltaNp63 and uncovered an unforeseen commonality with triple-negative breast tumors. To our knowledge, this is the first report of a sweat gland tumor that displayed expression of both DeltaNp63 and p73 and demonstrated a triple-negative receptor status. Such a link between 2 seemingly disparate tumor types indicates a mutual pathway of tumorigenesis and suggests the potential for common therapeutic regimens.

The β-Defensin Gallinacin-6 Is Expressed in the Chicken Digestive Tract and Has Antimicrobial Activity against Food-Borne Pathogens▿

PubMed Central

van Dijk, Albert; Veldhuizen, Edwin J. A.; Kalkhove, Stefanie I. C.; Tjeerdsma-van Bokhoven, Johanna L. M.; Romijn, Roland A.; Haagsman, Henk P.

2007-01-01

Food-borne pathogens are responsible for most cases of food poisoning in developed countries and are often associated with poultry products, including chicken. Little is known about the role of β-defensins in the chicken digestive tract and their efficacy. In this study, the expression of chicken β-defensin gallinacin-6 (Gal-6) and its antimicrobial activity against food-borne pathogens were investigated. Reverse transcription-PCR analysis showed high expression of Gal-6 mRNA in the esophagus and crop, moderate expression in the glandular stomach, and low expression throughout the intestinal tract. Putative transcription factor binding sites for nuclear factor kappa beta, activator protein 1, and nuclear factor interleukin-6 were found in the Gal-6 gene upstream region, which suggests a possible inducible nature of the Gal-6 gene. In colony-counting assays, strong bactericidal and fungicidal activity was observed, including bactericidal activity against food-borne pathogens Campylobacter jejuni, Salmonella enterica serovar Typhimurium, Clostridium perfringens, and Escherichia coli. Treatment with 16 μg/ml synthetic Gal-6 resulted in a 3 log unit reduction in Clostridium perfringens survival within 60 min, indicating fast killing kinetics. Transmission electron microscopy examination of synthetic-Gal-6-treated Clostridium perfringens cells showed dose-dependent changes in morphology after 30 min, including intracellular granulation, cytoplasm retraction, irregular septum formation in dividing cells, and cell lysis. The high expression in the proximal digestive tract and broad antimicrobial activity suggest that chicken β-defensin gallinacin-6 plays an important role in chicken innate host defense. PMID:17194828

Irradiated fibroblasts promote epithelial–mesenchymal transition and HDGF expression of esophageal squamous cell carcinoma

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

Bao, Ci-Hang; Wang, Xin-Tong; Ma, Wei

2015-03-06

Recent evidence suggested that nonirradiated cancer-associated fibroblasts (CAFs) promoted aggressive phenotypes of cancer cells through epithelial–mesenchymal transition (EMT). Hepatoma-derived growth factor (HDGF) is a radiosensitive gene of esophageal squamous cell carcinoma (ESCC). This study aimed to investigate the effect of irradiated fibroblasts on EMT and HDGF expression of ESCC. Our study demonstrated that coculture with nonirradiated fibroblasts significantly increased the invasive ability of ESCC cells and the increased invasiveness was further accelerated when they were cocultured with irradiated fibroblasts. Scattering of ESCC cells was also accelerated by the supernatant from irradiated fibroblasts. Exposure of ESCC cells to supernatant from irradiatedmore » fibroblasts resulted in decreased E-cadherin, increased vimentin in vitro and β-catenin was demonstrated to localize to the nucleus in tumor cells with irradiated fibroblasts in vivo models. The expression of HDGF and β-catenin were increased in both fibroblasts and ESCC cells of irradiated group in vitro and in vivo models. Interestingly, the tumor cells adjoining the stromal fibroblasts displayed strong nuclear HDGF immunoreactivity, which suggested the occurrence of a paracrine effect of fibroblasts on HDGF expression. These data suggested that irradiated fibroblasts promoted invasion, growth, EMT and HDGF expression of ESCC. - Highlights: • Irradiated CAFs accelerated invasiveness and scattering of ESCC cell lines. • Irradiated CAFs promoted EMT of ESCC cells. • Irradiated fibroblasts induced nuclear β-catenin relocalization in ESCC cells. • Irradiated fibroblasts increased HDGF expression in vitro and in vivo.« less

Silencing by nuclear matrix attachment distinguishes cell-type specificity: association with increased proliferation capacity.

PubMed

Linnemann, Amelia K; Krawetz, Stephen A

2009-05-01

DNA loop organization by nuclear scaffold/matrix attachment is a key regulator of gene expression that may provide a means to modulate phenotype. We have previously shown that attachment of genes to the NaCl-isolated nuclear matrix correlates with their silencing in HeLa cells. In contrast, expressed genes were associated with the lithium 3,5-diiodosalicylate (LIS)-isolated nuclear scaffold. To define their role in determining phenotype matrix attached regions (MARs) on human chromosomes 14-18 were identified as a function of expression in a primary cell line. The locations of MARs in aortic adventitial fibroblast (AoAF) cells were very stable (r = 0.909) and 96% of genes attached at MARs are silent (P < 0.001). Approximately one-third of the genes uniquely expressed in AoAF cells were associated with the HeLa cell nuclear matrix and silenced. Comparatively, 81% were associated with the AoAF cell nuclear scaffold (P < 0.001) and expressed. This suggests that nuclear scaffold/matrix association mediates a portion of cell type-specific gene expression thereby modulating phenotype. Interestingly, nuclear matrix attachment and thus silencing of specific genes that regulate proliferation and maintain the integrity of the HeLa cell genome suggests that transformation may at least in part be achieved through aberrant nuclear matrix attachment.

Left-right axis asymmetry determining human Cryptic gene is transcriptionally repressed by Snail.

PubMed

Gupta, Kartik; Pilli, Vijaya Satish Sekhar; Aradhyam, Gopala Krishna

2016-10-28

Establishment of the left-right axis is important for positioning organs asymmetrically in the developing vertebrate-embryo. A number of factors like maternally deposited molecules have emerged essential in initiating the specification of the axis; the downstream events, however, are regulated by signal-transduction and gene-expression changes identifying which remains a crucial challenge. The EGF-CFC family member Cryptic, that functions as a co-receptor for some TGF-beta ligands, is developmentally expressed in higher mammals and mutations in the gene cause loss or change in left-right axis asymmetry. Despite the strong phenotype, no transcriptional-regulator of this gene is known till date. Using promoter-analyses tools, we found strong evidence that the developmentally essential transcription factor Snail binds to the human Cryptic-promoter. We cloned the promoter-region of human Cryptic in a reporter gene and observed decreased Cryptic-promoter activation upon increasing Snail expression. Further, the expression of Cryptic is down-regulated upon exogenous Snail expression, validating the reporter assays and the previously identified role of Snail as a transcriptional repressor. Finally, we demonstrate using gel-shift assay that Snail in nuclear extract of PANC1 cells interacts with the promoter-construct bearing putative Snail binding sites and confirm this finding using chromatin immunoprecipitation assay. Snail represses the expression of human Cryptic and therefore, might affect the signaling via Nodal that has previously been demonstrated to specify the left-right axis using the EGF-CFC co-receptors.

Human Cytomegalovirus UL50 and UL53 Recruit Viral Protein Kinase UL97, Not Protein Kinase C, for Disruption of Nuclear Lamina and Nuclear Egress in Infected Cells

PubMed Central

Sharma, Mayuri; Kamil, Jeremy P.; Coughlin, Margaret; Reim, Natalia I.

2014-01-01

Herpesvirus nucleocapsids traverse the nuclear envelope into the cytoplasm in a process called nuclear egress that includes disruption of the nuclear lamina. In several herpesviruses, a key player in nuclear egress is a complex of two proteins, whose homologs in human cytomegalovirus (HCMV) are UL50 and UL53. However, their roles in nuclear egress during HCMV infection have not been shown. Based largely on transfection studies, UL50 and UL53 have been proposed to facilitate disruption of the nuclear lamina by recruiting cellular protein kinase C (PKC), as occurs with certain other herpesviruses, and/or the viral protein kinase UL97 to phosphorylate lamins. To investigate these issues during HCMV infection, we generated viral mutants null for UL50 or UL53. Correlative light electron microscopic analysis of null mutant-infected cells showed the presence of intranuclear nucleocapsids and the absence of cytoplasmic nucleocapsids. Confocal immunofluorescence microscopy revealed that UL50 and UL53 are required for disruption of the nuclear lamina. A subpopulation of UL97 colocalized with the nuclear rim, and this was dependent on UL50 and, to a lesser extent, UL53. However, PKC was not recruited to the nuclear rim, and its localization was not affected by the absence of UL50 or UL53. Immunoprecipitation from cells infected with HCMV expressing tagged UL53 detected UL97 but not PKC. In summary, HCMV UL50 and UL53 are required for nuclear egress and disruption of nuclear lamina during HCMV infection, and they recruit UL97, not PKC, for these processes. Thus, despite the strong conservation of herpesvirus nuclear egress complexes, a key function can differ among them. PMID:24155370

Human cytomegalovirus UL50 and UL53 recruit viral protein kinase UL97, not protein kinase C, for disruption of nuclear lamina and nuclear egress in infected cells.

PubMed

Sharma, Mayuri; Kamil, Jeremy P; Coughlin, Margaret; Reim, Natalia I; Coen, Donald M

2014-01-01

Herpesvirus nucleocapsids traverse the nuclear envelope into the cytoplasm in a process called nuclear egress that includes disruption of the nuclear lamina. In several herpesviruses, a key player in nuclear egress is a complex of two proteins, whose homologs in human cytomegalovirus (HCMV) are UL50 and UL53. However, their roles in nuclear egress during HCMV infection have not been shown. Based largely on transfection studies, UL50 and UL53 have been proposed to facilitate disruption of the nuclear lamina by recruiting cellular protein kinase C (PKC), as occurs with certain other herpesviruses, and/or the viral protein kinase UL97 to phosphorylate lamins. To investigate these issues during HCMV infection, we generated viral mutants null for UL50 or UL53. Correlative light electron microscopic analysis of null mutant-infected cells showed the presence of intranuclear nucleocapsids and the absence of cytoplasmic nucleocapsids. Confocal immunofluorescence microscopy revealed that UL50 and UL53 are required for disruption of the nuclear lamina. A subpopulation of UL97 colocalized with the nuclear rim, and this was dependent on UL50 and, to a lesser extent, UL53. However, PKC was not recruited to the nuclear rim, and its localization was not affected by the absence of UL50 or UL53. Immunoprecipitation from cells infected with HCMV expressing tagged UL53 detected UL97 but not PKC. In summary, HCMV UL50 and UL53 are required for nuclear egress and disruption of nuclear lamina during HCMV infection, and they recruit UL97, not PKC, for these processes. Thus, despite the strong conservation of herpesvirus nuclear egress complexes, a key function can differ among them.

Sudan III dye strongly induces CYP1A1 mRNA expression in HepG2 cells.

PubMed

Ohno, Marumi; Ikenaka, Yoshinori; Ishizuka, Mayumi

2012-01-01

Sudan dyes possess a high affinity to the aryl hydrocarbon receptor (AHR) and potently induce its target genes, such as cytochrome P450 (CYP) 1A1, through unknown mechanisms. We investigated a detailed event occurring in cells after binding of Sudan dye to AHR in HepG2 cells. Treatment with 10 µM Sudan III caused rapid translocation of AHR into the nucleus and increased expression levels of human CYP1A1 mRNA by approximately 20-fold after 16 and 24 h. The transactivation was due to the activation of a region located at -1137 to +59 bp from CYP1A1, in particular, four xenobiotic responsive elements (XREs) existing in the region. AHR and the Ah receptor nuclear translocator interacted with XRE sequences in a gel shift assay using nuclear extract from Sudan III--treated HepG2 cells. Moreover, we suggest that constitutive androstane receptor could modify CYP1A1 transactivation by Sudan III. Copyright © 2012 Wiley Periodicals, Inc.

Molecular characterization of DNA repair protein Ku70 from Vitis vinifera and its purification from transgenic tobacco.

PubMed

Tak, Himanshu; Mhatre, Minal

2013-08-01

The DNA double strand break repair in plants is preferentially by non homologous end joining (NHEJ) pathway. A key protein of NHEJ pathway is Ku70. We have identified Ku70 homolog (VvKu70) from grapevine genome database. In this report we characterize a Ku70 homologue from Vitis vinifera cv. Mango. The VvKu70 expression was found to increase strongly in response to gamma radiation. The transcript level of VvKu70 was found to increase up to 36 h in gamma irradiated shoots of grapevine. The expression of VvKu70 was found in many organs like stem, leaves and roots. A GFP fused VvKu70 protein was found to be nuclear localized which indicates that the VvKu70 is a nuclear localized protein. The VvKu70 identified by in silico approaches is present as a single copy number in V. vinifera cv. Mango genome. The VvKu70-GFP fused protein possesses ATPase activity and fails to bind dsDNA but binds ssDNA.

Tau regulates the subcellular localization of calmodulin

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

Barreda, Elena Gomez de; Avila, Jesus, E-mail: javila@cbm.uam.es; CIBER de Enfermedades Neurodegenerativas, 28031 Madrid

Highlights: {yields} In this work we have tried to explain how a cytoplasmic protein could regulate a cell nuclear function. We have tested the role of a cytoplasmic protein (tau) in regulating the expression of calbindin gene. We found that calmodulin, a tau-binding protein with nuclear and cytoplasmic localization, increases its nuclear localization in the absence of tau. Since nuclear calmodulin regulates calbindin expression, a decrease in nuclear calmodulin, due to the presence of tau that retains it at the cytoplasm, results in a change in calbindin expression. -- Abstract: Lack of tau expression in neuronal cells results in amore » change in the expression of few genes. However, little is known about how tau regulates gene expression. Here we show that the presence of tau could alter the subcellular localization of calmodulin, a protein that could be located at the cytoplasm or in the nucleus. Nuclear calmodulin binds to co-transcription factors, regulating the expression of genes like calbindin. In this work, we have found that in neurons containing tau, a higher proportion of calmodulin is present in the cytoplasm compared with neurons lacking tau and that an increase in cytoplasmic calmodulin correlates with a higher expression of calbindin.« less

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

Yang, Zhipan; Lu, Qingtao; Wen, Xiaogang

Highlights: Black-Right-Pointing-Pointer Rice rubisco activase promoter was analyzed in transgenic Arabidopsis system. Black-Right-Pointing-Pointer Region conferring tissue specific and light inducible expression of Rca was identified. Black-Right-Pointing-Pointer -58 to +43 bp region mediates tissue-specific expression of rice Rca. Black-Right-Pointing-Pointer Light inducible expression of rice Rca is mediated by -297 to -58 bp region. Black-Right-Pointing-Pointer Rice nuclear proteins bind specifically with the light inducible region. -- Abstract: To gain a better understanding of the regulatory mechanism of the rice rubisco activase (Rca) gene, variants of the Rca gene promoter (one full-length and four deletion mutants) fused to the coding region of themore » bacterial reporter gene {beta}-glucuronidase (GUS) were introduced into Arabidopsis via Agrobacterium-mediated transformation. Our results show that a 340 bp fragment spanning from -297 to +43 bp relative to the transcription initiation site is enough to promote tissue-specific and light-inducible expression of the rice Rca gene as done by the full-length promoter (-1428 to +43 bp). Further deletion analysis indicated that the region conferring tissue-specificity of Rca expression is localized within a 105 bp fragment from -58 to +43 bp, while light-inducible expression of Rca is mediated by the region from -297 to -58 bp. Gel shift assays and competition experiments demonstrated that rice nuclear proteins bind specifically with the fragment conferring light responsiveness at more than one binding site. This implies that multiple cis-elements may be involved in light-induced expression of the rice Rca gene. These works provide a useful reference for understanding transcriptional regulation mechanism of the rice Rca gene, and lay a strong foundation for further detection of related cis-elements and trans-factors.« less

Differential expression of inwardly rectifying K+ channels and aquaporins 4 and 5 in autoimmune uveitis indicates misbalance in Müller glial cell-dependent ion and water homeostasis.

PubMed

Eberhardt, Christina; Amann, Barbara; Feuchtinger, Annette; Hauck, Stefanie M; Deeg, Cornelia A

2011-05-01

Reactive gliosis is a well-established response to virtually every retinal disease. Autoimmune uveitis, a sight threatening disease, is characterized by recurrent relapses through autoaggressive T-cells. The purpose of this study was to assess retinal Müller glial cell function in equine recurrent uveitis (ERU), a spontaneous disease model resembling the human disease, by investigating membrane proteins implicated in ion and water homeostasis. We found that Kir2.1 was highly expressed in diseased retinas, whereas Kir4.1 was downregulated in comparison to controls. Distribution of Kir2.1 appeared Müller cell associated in controls, whereas staining of cell somata in the inner nuclear layer was observed in uveitis. In contrast to other subunits, Kir4.1 was evenly expressed along equine Müller cells, whereas in ERU, Kir4.1 almost disappeared from Müller cells. Hence, we suggest a different mechanism for potassium buffering in the avascular equine retina and, moreover, an impairment in uveitis. Uveitic retinas showed significantly increased expression of AQP4 as well as a displaced expression from Müller cells in healthy specimens to an intense circular expression pattern in the outer nuclear layer in ERU cases. Most interestingly, we detected the aquaporin family member protein AQP5 to be expressed in Müller cells with strong enrichments in Müller cell secondary processes. This finding indicates that fluid regulation within the equine retina may be achieved by an additional aquaporin. Furthermore, AQP5 was significantly decreased in uveitis. We conclude that the Müller cell response in autoimmune uveitis implies considerable changes in its potassium and water physiology.

Hepatoprotective Effect of Polyphenol-Enriched Fraction from Folium Microcos on Oxidative Stress and Apoptosis in Acetaminophen-Induced Liver Injury in Mice.

PubMed

Wu, Hongtan; Zhang, Gang; Huang, Lisen; Pang, Haiyue; Zhang, Na; Chen, Yupei; Wang, Gueyhorng

2017-01-01

Folium Microcos (FM), the leaves of Microcos paniculata L., shows various biological functions including antioxidant activity and α -glucosidase inhibitory effect. However, its therapeutic potential in acute liver injury is still unknown. This study investigated the hepatoprotective effect and underlying mechanisms of the polyphenol-enriched fraction (FMF) from Folium Microcos . FMF exhibited strong free radical scavenging activities and prevented HepG2/Hepa1-6 cells from hydrogen peroxide- (H 2 O 2 -) induced ROS production and apoptosis in vitro. Antioxidant activity and cytoprotective effects were further verified by alleviating APAP-induced hepatotoxicity in mice. Western blot analysis revealed that FMF pretreatment significantly abrogated APAP-mediated phosphorylation of MAPKs, activation of proapoptotic protein caspase-3/9 and Bax, and restored expression of antiapoptotic protein Bcl2. APAP-intoxicated mice pretreated with FMF showed increased nuclear accumulation of nuclear factor erythroid 2-related factor (Nrf2) and elevated hepatic expression of its target genes, NAD(P)H:quinine oxidoreductase 1 (NQO1) and hemeoxygenase-1(HO-1). HPLC analysis revealed the four predominantly phenolic compounds present in FMF: narcissin, isorhamnetin-3-O- β -D-glucoside, isovitexin, and vitexin. Consequently, these findings indicate that FMF possesses a hepatoprotective effect against APAP-induced hepatotoxicity mainly through dual modification of ROS/MAPKs/apoptosis axis and Nrf2-mediated antioxidant response, which may be attributed to the strong antioxidant activity of phenolic components.

Myostatin is a direct regulator of osteoclast differentiation and its inhibition reduces inflammatory joint destruction in mice.

PubMed

Dankbar, Berno; Fennen, Michelle; Brunert, Daniela; Hayer, Silvia; Frank, Svetlana; Wehmeyer, Corinna; Beckmann, Denise; Paruzel, Peter; Bertrand, Jessica; Redlich, Kurt; Koers-Wunrau, Christina; Stratis, Athanasios; Korb-Pap, Adelheid; Pap, Thomas

2015-09-01

Myostatin (also known as growth and differentiation factor 8) is a secreted member of the transforming growth factor-β (TGF-β) family that is mainly expressed in skeletal muscle, which is also its primary target tissue. Deletion of the myostatin gene (Mstn) in mice leads to muscle hypertrophy, and animal studies support the concept that myostatin is a negative regulator of muscle growth and regeneration. However, myostatin deficiency also increases bone formation, mainly through loading-associated effects on bone. Here we report a previously unknown direct role for myostatin in osteoclastogenesis and in the progressive loss of articular bone in rheumatoid arthritis (RA). We demonstrate that myostatin is highly expressed in the synovial tissues of RA subjects and of human tumor necrosis factor (TNF)-α transgenic (hTNFtg) mice, a model for human RA. Myostatin strongly accelerates receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclast formation in vitro through transcription factor SMAD2-dependent regulation of nuclear factor of activated T-cells (NFATC1). Myostatin deficiency or antibody-mediated inhibition leads to an amelioration of arthritis severity in hTNFtg mice, chiefly reflected by less bone destruction. Consistent with these effects in hTNFtg mice, the lack of myostatin leads to increased grip strength and less bone erosion in the K/BxN serum-induced arthritis model in mice. The results strongly suggest that myostatin is a potent therapeutic target for interfering with osteoclast formation and joint destruction in RA.

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export.

PubMed

Behrens, Ryan T; Aligeti, Mounavya; Pocock, Ginger M; Higgins, Christina A; Sherer, Nathan M

2017-02-01

HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding. HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place. Copyright © 2017 American Society for Microbiology.

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

PubMed Central

Behrens, Ryan T.; Aligeti, Mounavya; Pocock, Ginger M.; Higgins, Christina A.

2016-01-01

ABSTRACT HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding. IMPORTANCE HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place. PMID:27852860

Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-{alpha}

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

Tsukasaki, Masayuki; Yamada, Atsushi, E-mail: yamadaa@dent.showa-u.ac.jp; Suzuki, Dai

2011-07-15

Highlights: {yields} TNF-{alpha} inhibits POEM gene expression. {yields} Inhibition of POEM gene expression is caused by NF-{kappa}B activation by TNF-{alpha}. {yields} Over-expression of POEM recovers inhibition of osteoblast differentiation by TNF-{alpha}. -- Abstract: POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-{alpha} (TNF-{alpha}), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-{alpha}-induced down-regulation of POEM gene expression occurred in both time- andmore » dose-dependent manners through the nuclear factor kappa B (NF-{kappa}B) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-{alpha} in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-{alpha}-induced inhibition of osteoblast differentiation. These results suggest that TNF-{alpha} inhibits POEM expression through the NF-{kappa}B signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-{alpha}.« less

Molecular characterization of tocopherol biosynthetic genes in sweetpotato that respond to stress and activate the tocopherol production in tobacco.

PubMed

Ji, Chang Yoon; Kim, Yun-Hee; Kim, Ho Soo; Ke, Qingbo; Kim, Gun-Woo; Park, Sung-Chul; Lee, Haeng-Soon; Jeong, Jae Cheol; Kwak, Sang-Soo

2016-09-01

Tocopherol (vitamin E) is a chloroplast lipid that is presumed to be involved in the plant response to oxidative stress. In this study, we isolated and characterized five tocopherol biosynthetic genes from sweetpotato (Ipomoea batatas [L.] Lam) plants, including genes encoding 4-hydroxyphenylpyruvate dioxygenase (IbHPPD), homogentisate phytyltransferase (IbHPT), 2-methyl-6-phytylbenzoquinol methyltransferase (IbMPBQ MT), tocopherol cyclase (IbTC) and γ-tocopherol methyltransferase (IbTMT). Fluorescence microscope analysis indicated that four proteins localized into the chloroplast, whereas IbHPPD observed in the nuclear. Quantitative RT-PCR analysis revealed that the expression patterns of the five tocopherol biosynthetic genes varied in different plant tissues and under different stress conditions. All five genes were highly expressed in leaf tissues, whereas IbHPPD and IbHPT were highly expressed in the thick roots. The expression patterns of these five genes significantly differed in response to PEG, NaCl and H2O2-mediated oxidative stress. IbHPPD was strongly induced following PEG and H2O2 treatment and IbHPT was strongly induced following PEG treatment, whereas IbMPBQ MT and IbTC were highly expressed following NaCl treatment. Upon infection of the bacterial pathogen Pectobacterium chrysanthemi, the expression of IbHPPD increased sharply in sweetpotato leaves, whereas the expression of the other genes was reduced or unchanged. Additionally, transient expression of the five tocopherol biosynthetic genes in tobacco (Nicotiana bentamiana) leaves resulted in increased transcript levels of the transgenes expressions and tocopherol production. Therefore, our results suggested that the five tocopherol biosynthetic genes of sweetpotato play roles in the stress defense response as transcriptional regulators of the tocopherol production. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP

PubMed Central

Wickramasinghe, Vihandha O.; Andrews, Robert; Ellis, Peter; Langford, Cordelia; Gurdon, John B.; Stewart, Murray; Venkitaraman, Ashok R.; Laskey, Ronald A.

2014-01-01

The nuclear phase of the gene expression pathway culminates in the export of mature messenger RNAs (mRNAs) to the cytoplasm through nuclear pore complexes. GANP (germinal- centre associated nuclear protein) promotes the transfer of mRNAs bound to the transport factor NXF1 to nuclear pore complexes. Here, we demonstrate that GANP, subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex, promotes selective nuclear export of a specific subset of mRNAs whose transport depends on NXF1. Genome-wide gene expression profiling showed that half of the transcripts whose nuclear export was impaired following NXF1 depletion also showed reduced export when GANP was depleted. GANP-dependent transcripts were highly expressed, yet short-lived, and were highly enriched in those encoding central components of the gene expression machinery such as RNA synthesis and processing factors. After injection into Xenopus oocyte nuclei, representative GANP-dependent transcripts showed faster nuclear export kinetics than representative transcripts that were not influenced by GANP depletion. We propose that GANP promotes the nuclear export of specific classes of mRNAs that may facilitate rapid changes in gene expression. PMID:24510098

Nuclear physics from lattice QCD at strong coupling.

PubMed

de Forcrand, Ph; Fromm, M

2010-03-19

We study numerically the strong coupling limit of lattice QCD with one flavor of massless staggered quarks. We determine the complete phase diagram as a function of temperature and chemical potential, including a tricritical point. We clarify the nature of the low temperature dense phase, which is strongly bound "nuclear" matter. This strong binding is explained by the nuclear potential, which we measure. Finally, we determine, from this first-principles limiting case of QCD, the masses of "atomic nuclei" up to A=12 "carbon".

Role of nuclear factor-kappa B activation in the adverse effects induced by air pollution particulate matter (PM2.5) in human epithelial lung cells (L132) in culture.

PubMed

Dagher, Zeina; Garçon, Guillaume; Billet, Sylvain; Verdin, Anthony; Ledoux, Frédéric; Courcot, Dominique; Aboukais, Antoine; Shirali, Pirouz

2007-01-01

To contribute to improving knowledge on the adverse health effects induced by particulate matter (PM) air pollution, an extensive investigation was undertaken of the underlying mechanisms of action activated by PM(2.5) air pollution collected in Dunkerque, a strongly industrialized French seaside city. Their chemical and physical characteristics have been previously determined, and earlier in vitro short-term studies have shown them to cause dose-dependent and time-dependent oxidative damage, gene expression and protein secretion of inflammatory mediators, and apoptotic events in human lung epithelial cells (L132) in culture. Hence, this work studied the activation of nuclear factor-kappa B (NF-kappaB)/inhibitory kappa B (IkappaB) by Dunkerque city PM(2.5) in these target cells, by determination of phosphorylated p65 and phosphorylated IkappaBalpha protein levels in cytoplasmic extracts, and p65 and p50 DNA binding in nuclear extracts. In PM-exposed L132 cells, there were concentration- and/or time-dependent increases in nuclear p65 and cytoplasmic IkB-alpha phosphorylation, and nuclear p65 and p50 DNA binding. Taken together, these results showed that Dunkerque city PM(2.5) were involved in the activation of the NF-kappaB/IkappaB complex, notably through the occurrence of oxidative stress conditions, and, therefore, in the gene expression and protein secretion of inflammatory mediators in target L132 cells. Hence, these findings suggested that the activation of the NF-kappaB/IkappaB complex preceded cytotoxicity in Dunkerque city PM-exposed L132 cells. (c) 2007 John Wiley & Sons, Ltd.

Intron retention and nuclear loss of SFPQ are molecular hallmarks of ALS.

PubMed

Luisier, Raphaelle; Tyzack, Giulia E; Hall, Claire E; Mitchell, Jamie S; Devine, Helen; Taha, Doaa M; Malik, Bilal; Meyer, Ione; Greensmith, Linda; Newcombe, Jia; Ule, Jernej; Luscombe, Nicholas M; Patani, Rickie

2018-05-22

Mutations causing amyotrophic lateral sclerosis (ALS) strongly implicate ubiquitously expressed regulators of RNA processing. To understand the molecular impact of ALS-causing mutations on neuronal development and disease, we analysed transcriptomes during in vitro differentiation of motor neurons (MNs) from human control and patient-specific VCP mutant induced-pluripotent stem cells (iPSCs). We identify increased intron retention (IR) as a dominant feature of the splicing programme during early neural differentiation. Importantly, IR occurs prematurely in VCP mutant cultures compared with control counterparts. These aberrant IR events are also seen in independent RNAseq data sets from SOD1- and FUS-mutant MNs. The most significant IR is seen in the SFPQ transcript. The SFPQ protein binds extensively to its retained intron, exhibits lower nuclear abundance in VCP mutant cultures and is lost from nuclei of MNs in mouse models and human sporadic ALS. Collectively, we demonstrate SFPQ IR and nuclear loss as molecular hallmarks of familial and sporadic ALS.

Silencing by nuclear matrix attachment distinguishes cell-type specificity: association with increased proliferation capacity

PubMed Central

Linnemann, Amelia K.; Krawetz, Stephen A.

2009-01-01

DNA loop organization by nuclear scaffold/matrix attachment is a key regulator of gene expression that may provide a means to modulate phenotype. We have previously shown that attachment of genes to the NaCl-isolated nuclear matrix correlates with their silencing in HeLa cells. In contrast, expressed genes were associated with the lithium 3,5-diiodosalicylate (LIS)-isolated nuclear scaffold. To define their role in determining phenotype matrix attached regions (MARs) on human chromosomes 14–18 were identified as a function of expression in a primary cell line. The locations of MARs in aortic adventitial fibroblast (AoAF) cells were very stable (r = 0.909) and 96% of genes attached at MARs are silent (P < 0.001). Approximately one-third of the genes uniquely expressed in AoAF cells were associated with the HeLa cell nuclear matrix and silenced. Comparatively, 81% were associated with the AoAF cell nuclear scaffold (P < 0.001) and expressed. This suggests that nuclear scaffold/matrix association mediates a portion of cell type-specific gene expression thereby modulating phenotype. Interestingly, nuclear matrix attachment and thus silencing of specific genes that regulate proliferation and maintain the integrity of the HeLa cell genome suggests that transformation may at least in part be achieved through aberrant nuclear matrix attachment. PMID:19276204

Robust Principal Component Analysis Regularized by Truncated Nuclear Norm for Identifying Differentially Expressed Genes.

PubMed

Wang, Ya-Xuan; Gao, Ying-Lian; Liu, Jin-Xing; Kong, Xiang-Zhen; Li, Hai-Jun

2017-09-01

Identifying differentially expressed genes from the thousands of genes is a challenging task. Robust principal component analysis (RPCA) is an efficient method in the identification of differentially expressed genes. RPCA method uses nuclear norm to approximate the rank function. However, theoretical studies showed that the nuclear norm minimizes all singular values, so it may not be the best solution to approximate the rank function. The truncated nuclear norm is defined as the sum of some smaller singular values, which may achieve a better approximation of the rank function than nuclear norm. In this paper, a novel method is proposed by replacing nuclear norm of RPCA with the truncated nuclear norm, which is named robust principal component analysis regularized by truncated nuclear norm (TRPCA). The method decomposes the observation matrix of genomic data into a low-rank matrix and a sparse matrix. Because the significant genes can be considered as sparse signals, the differentially expressed genes are viewed as the sparse perturbation signals. Thus, the differentially expressed genes can be identified according to the sparse matrix. The experimental results on The Cancer Genome Atlas data illustrate that the TRPCA method outperforms other state-of-the-art methods in the identification of differentially expressed genes.

Clinical validation of nuclear factor kappa B expression in invasive breast cancer.

PubMed

Agrawal, Anil Kumar; Pielka, Ewa; Lipinski, Artur; Jelen, Michal; Kielan, Wojciech; Agrawal, Siddarth

2018-01-01

Breast cancer is the most commonly diagnosed cancer in Polish women. The expression of transcription nuclear factor kappa B, a key inducer of inflammatory response promoting carcinogenesis and cancer progression in breast cancer, is not well-established. We assessed the nuclear factor kappa B expression in a total of 119 invasive breast carcinomas and 25 healthy control samples and correlated this expression pattern with several clinical and pathologic parameters including histologic type and grade, tumor size, lymph node status, estrogen receptor status, and progesterone receptor status. The data used for the analysis were derived from medical records. An immunohistochemical analysis of nuclear factor kappa B, estrogen receptor, and progesterone receptor was carried out and evaluation of stainings was performed. The expression of nuclear factor kappa B was significantly higher than that in the corresponding healthy control samples. No statistical difference was demonstrated in nuclear factor kappa B expression in relation to age, menopausal status, lymph node status, tumor size and location, grade and histologic type of tumor, and hormonal status (estrogen receptor and progesterone receptor). Nuclear factor kappa B is significantly overexpressed in invasive breast cancer tissues. Although nuclear factor kappa B status does not correlate with clinicopathological findings, it might provide important additional information on prognosis and become a promising object for targeted therapy.

Aging impairs transcriptional regulation of vascular endothelial growth factor in human microvascular endothelial cells: implications for angiogenesis and cell survival.

PubMed

Ahluwalia, A; Jones, M K; Szabo, S; Tarnawski, A S

2014-04-01

In some tissues, aging impairs angiogenesis and reduces expression of vascular endothelial growth factor A (VEGF), a fundamental regulator of angiogenesis. We previously examined angiogenesis in aging and young gastric mucosa in vivo and in vitro and showed that an imbalance between expressions of VEGF (pro-angiogenic factor) and endostatin (anti-angiogenic protein) results in an aging-related impairment of angiogenesis in rats. However, the human relevance of these findings, and whether these mechanisms apply to endothelial cells derived from other tissues, is not clear. Since P-STAT3 and P-CREB are transcription factors that, in association with HIF-1α, can activate VEGF gene expression in some cells (e.g., liver cancer cells, vascular smooth muscle cells), we examined the expression of these two proteins in human dermal microvascular endothelial cells (HMVECs) derived from aging and neonatal individuals. We examined and quantified in vitro angiogenesis, expression of VEGF, P-STAT3, P-CREB and importin-α in HMVECs isolated from neonates (neonatal) and a 66 year old subject (aging). We also examined the effects of treatment with exogenous VEGF and endostatin on in vitro angiogenesis in these cells. Endothelial cells isolated from aging individuals had impaired angiogenesis (vs. neonatal endothelial cells) and reduced expression of VEGF mRNA and protein. Aged HMVECs also had reduced importin-α expression, and reduced expression and nuclear translocation of P-STAT3 and P-CREB. Reduced VEGF gene expression in aged HMVECs strongly correlated with the decreased levels of P-STAT3, P-CREB and importin-α in these cells. Our study clearly demonstrates that endothelial cells from aging individuals have impaired angiogenesis and reduced expression of VEGF likely due to impaired nuclear transport of P-STAT3 and P-CREB transcription factors in these cells.

COP1 is required for UV-B–induced nuclear accumulation of the UVR8 photoreceptor

PubMed Central

Skvortsova, Mariya Y.; Loubéry, Sylvain

2016-01-01

The UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) promotes UV-B acclimation and tolerance in Arabidopsis thaliana. UVR8 localizes to both cytosol and nucleus, but its main activity is assumed to be nuclear. UV-B photoreception stimulates nuclear accumulation of UVR8 in a presently unknown manner. Here, we show that CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) is required for UV-B–induced nuclear accumulation of UVR8, but bypassing the COP1 requirement for UVR8 nuclear accumulation did not rescue the cop1 mutant UV-B phenotype. Using a glucocorticoid receptor (GR)-based fusion protein system to conditionally localize GR-UVR8 to the nucleus, we have demonstrated that both photoactivation and nuclear localization of UVR8 are required for UV-B–induced photomorphogenic responses. In contrast, there was no UV-B response when UV-B–activated UVR8 was artificially retained in the cytosol. In agreement with a predominantly nuclear activity, constitutively active UVR8W285A accumulated in the nucleus also in the absence of UV-B. Furthermore, GR-COP1 expression lines suggested that UV-B–activated UVR8 can be coimported into the nucleus by COP1. Our data strongly support localization of UVR8 signaling in the nucleus and a dual role for COP1 in the regulation of UV-B–induced UVR8 nuclear accumulation and in UVR8-mediated UV-B signaling. PMID:27407149

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.

RNA helicase-like protein as an early regulator of transcription factors for plant chilling and freezing tolerance

PubMed Central

Gong, Zhizhong; Lee, Hojoung; Xiong, Liming; Jagendorf, André; Stevenson, Becky; Zhu, Jian-Kang

2002-01-01

Susceptibility to chilling injury prevents the cultivation of many important crops and limits the extended storage of horticultural commodities. Although freezing tolerance is acquired through cold-induced gene expression changes mediated in part by the CBF family of transcriptional activators, whether plant chilling resistance or sensitivity involves the CBF genes is not known. We report here that an Arabidopsis thaliana mutant impaired in the cold-regulated expression of CBF genes and their downstream target genes is sensitive to chilling stress. Expression of CBF3 under a strong constitutive promoter restores chilling resistance to the mutant plants. The mutated gene was cloned and found to encode a nuclear localized RNA helicase. Our results identify a regulator of CBF genes, and demonstrate the importance of gene regulation and the CBF transcriptional activators in plant chilling resistance. PMID:12165572

Evidence for Gating Roles of Protein Kinase A and Protein Kinase C in Estradiol-Induced Luteinizing Hormone Receptor (lhcgr) Expression in Zebrafish Ovarian Follicle Cells

PubMed Central

Liu, Ka-Cheuk; Ge, Wei

2013-01-01

Estradiol (E2) stimulates luteinizing hormone receptor (lhcgr) expression in zebrafish follicle cells via nuclear estrogen receptors (nERs) that are likely expressed on the membrane, and lhcgr responds to E2 in a biphasic manner during 24-h treatment. These observations raise an interesting question on the signaling mechanism underlying E2 regulation, in particular the biphasic response of lhcgr expression. In the present study, we demonstrated that E2 regulation of lhcgr was significantly influenced by the activity of cAMP-PKA pathway. Activation of cAMP-PKA pathway by forskolin or db-cAMP suppressed E2-stimulated lhcgr expression in short-term (3 h) but enhanced its effect in long-term (24 h), suggesting differential roles of PKA at these two phases of lhcgr response. PKA inhibitor H89 showed reversed effects. In contrast, PKC pathway had consistent permissive effect on E2-induced lhcgr expression as evidenced by strong inhibition of E2 effect by PKC inhibitors GF109203X and Ro-31-8220 at both 3 and 24 h. One of the mechanisms by which PKA and PKC gated E2 effect might be through regulating nERs, particularly esr2a. Despite the strong influence of PKA and PKC, our data did not suggest direct mediating roles for these two pathways in E2 stimulation of lhcgr expression; yet they likely play critical gating roles in E2 signal transduction. As a follow-up study to our previous report on E2 regulation of gonadotropin receptors in the zebrafish ovary, the present study provides further evidence for the involvement of classical intracellular signal transduction pathways in E2 stimulation of lhcgr expression in the follicle cells. PMID:23658740

NET23/STING Promotes Chromatin Compaction from the Nuclear Envelope

PubMed Central

de las Heras, Jose I.; Saiz-Ros, Natalia; Makarov, Alexandr A.; Lazou, Vassiliki; Meinke, Peter; Waterfall, Martin; Kelly, David A.; Schirmer, Eric C.

2014-01-01

Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture. PMID:25386906

staggerer phenotype in retinoid-related orphan receptor α-deficient mice

PubMed Central

Steinmayr, Markus; André, Elisabeth; Conquet, François; Rondi-Reig, Laure; Delhaye-Bouchaud, Nicole; Auclair, Nathalie; Daniel, Hervé; Crépel, Francis; Mariani, Jean; Sotelo, Constantino; Becker-André, Michael

1998-01-01

Retinoid-related orphan receptor α (RORα) is a member of the nuclear receptor superfamily. To study its physiological role we generated null-mutant mice by targeted insertion of a lacZ reporter gene encoding the enzyme β-galactosidase. In heterozygous RORα+/− mice we found β-galactosidase activity, indicative of RORα protein expression, confined to the central nervous system, skin and testis. In the central nervous system, the RORα gene is expressed in cerebellar Purkinje cells, the thalamus, the suprachiasmatic nuclei, and retinal ganglion cells. In skin, RORα is strongly expressed in the hair follicle, the epidermis, and the sebaceous gland. Finally, the peritubular cells of the testis and the epithelial cells of the epididymis also strongly express RORα. Recently, it was reported that the ataxic mouse mutant staggerer (sg/sg) is caused by a deletion in the RORα gene. The analysis of the cerebellar and the behavioral phenotype of homozygous RORα−/− mice proves identity to sg/sg mice. Although the absence of RORα causes dramatic developmental effects in the cerebellum, it has no apparent morphological effect on thalamus, hypothalamus, and retina. Similarly, testis and skin of RORα−/− mice display a normal phenotype. However, the pelage hair of both sg/sg and RORα−/− is significantly less dense and when shaved shows reluctance to regrow. PMID:9520475

Ethanol Extract of Cirsium japonicum var. ussuriense Kitamura Exhibits the Activation of Nuclear Factor Erythroid 2-Related Factor 2-dependent Antioxidant Response Element and Protects Human Keratinocyte HaCaT Cells Against Oxidative DNA Damage.

PubMed

Yoo, Ok-Kyung; Choi, Bu Young; Park, Jin-Oh; Lee, Ji-Won; Park, Byoung-Kwon; Joo, Chul Gue; Heo, Hyo-Jung; Keum, Young-Sam

2016-03-01

Keratinocytes are constantly exposed to extracellular insults, such as ultraviolet B, toxic chemicals and mechanical stress, all of which can facilitate the aging of keratinocytes via the generation of intracellular reactive oxygen species (ROS). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays a critical role in protecting keratinocytes against oxidants and xenobiotics by binding to the antioxidant response element (ARE), a cis-acting element existing in the promoter of most phase II cytoprotective genes. In the present study, we have attempted to find novel ethanol extract(s) of indigenous plants of Jeju island, Korea that can activate the Nrf2/ARE-dependent gene expression in human keratinocyte HaCaT cells. As a result, we identified that ethanol extract of Cirsium japonicum var. ussuriense Kitamura (ECJUK) elicited strong stimulatory effect on the ARE-dependent gene expression. Supporting this observation, we found that ECJUK induced the expression of Nrf2, hemoxygenase-1, and quinone oxidoreductase-1 and this event was correlated with Akt1 phosphorylation. We also found that ECJUK increased the intracellular reduced glutathione level and suppressed 12-O-tetradecanoylphorbol acetate-induced 8-hydroxyguanosine formation without affecting the overall viability. Collectively, our results provide evidence that ECJUK can protect against oxidative stress-mediated damages through the activation of Nrf2/ARE-dependent phase II cytoprotective gene expression.

Effects of hypnotic bromovalerylurea on microglial BV2 cells.

PubMed

Kawasaki, Shun; Abe, Naoki; Ohtake, Fumito; Islam, Afsana; Choudhury, Mohammed Emamussalehin; Utsunomiya, Ryo; Kikuchi, Satoshi; Nishihara, Tasuku; Kuwabara, Jun; Yano, Hajime; Watanabe, Yuji; Aibiki, Mayuki; Yorozuya, Toshihiro; Tanaka, Junya

2017-06-01

An old sedative and hypnotic bromovalerylurea (BU) has anti-inflammatory effects. BU suppressed nitric oxide (NO) release and proinflammatory cytokine expression by lipopolysaccharide (LPS)-treated BV2 cells, a murine microglial cell line. However, BU did not inhibit LPS-induced nuclear translocation of nuclear factor-κB and subsequent transcription. BU suppressed LPS-induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) and expression of interferon regulatory factor 1 (IRF1). The Janus kinase 1 (JAK1) inhibitor filgotinib suppressed the NO release much more weakly than that of BU, although filgotinib almost completely prevented LPS-induced STAT1 phosphorylation. Knockdown of JAK1, STAT1, or IRF1 did not affect the suppressive effects of BU on LPS-induced NO release by BV2 cells. A combination of BU and filgotinib synergistically suppressed the NO release. The mitochondrial complex I inhibitor rotenone, which did not prevent STAT1 phosphorylation or IRF1 expression, suppressed proinflammatory mediator expression less significantly than BU. BU and rotenone reduced intracellular ATP (iATP) levels to a similar extent. A combination of rotenone and filgotinib suppressed NO release by LPS-treated BV2 cells as strongly as BU. These results suggest that anti-inflammatory actions of BU may be attributable to the synergism of inhibition of JAK1/STAT1-dependent pathways and reduction in iATP level. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Angiotensin II Receptor Antagonism Reduces Transforming Growth Factor Beta and Smad Signaling in Thoracic Aortic Aneurysm

PubMed Central

Nataatmadja, Maria; West, Jennifer; Prabowo, Sulistiana; West, Malcolm

2013-01-01

ABSTRACT Background The expression of transforming growth factor beta (TGF-β) and Smad3 regulates extracellular matrix homeostasis and inflammation in aortic aneurysms. The expression of Smad3 depends on signaling by angiotensin II (AngII) receptor pathways through TGF-β receptor–dependent and –independent pathways. Methods To determine the expression of AngII type 1 (AT1R) and type 2 receptors (AT2R), TGF-β, and Smad3 in thoracic aortic aneurysms, we performed immunohistochemistry testing on tissue and cultured cells derived from subjects with Marfan syndrome (MFS) and bicuspid aortic valve (BAV) malformation and from normal aortas of subjects who were organ donors. Results MFS and BAV aneurysm tissue showed enhanced accumulation of TGF-β and Smad3 in vascular smooth muscle cells (VSMCs) and in inflammatory cells in the subintimal layer and tunica media. The normal aortic wall exhibited minimal TGF-β and Smad3 staining. Cultured VSMCs from MFS and BAV samples showed nuclear Smad3 and strong cytoplasmic TGF-β expression in the cytoplasmic vesicles. In control cells, Smad3 was located mainly in the cytoplasm, and weak cytoplasmic TGF-β was distributed with a pattern similar to that of the aneurysm-derived cells. Compared to normal aorta cells, AT1R and AT2R expression was increased in both aneurysm types. Treatment of cultured VSMCs with the AT1R antagonist losartan caused both reduced TGF-β vesicle localization and nuclear expression of Smad3. Conclusions Increased TGF-β and Smad3 expression in aneurysm tissue and cultured VSMCs is consistent with aberrant TGF-β expression and the activation of Smad3 signaling. Losartan-mediated reduction in TGF-β expression and the cytoplasmic localization of Smad3 support a role for AT1R antagonism in the inhibition of aneurysm progression. PMID:23532685

Sulforaphane rescues amyloid-β peptide-mediated decrease in MerTK expression through its anti-inflammatory effect in human THP-1 macrophages.

PubMed

Jhang, Kyoung A; Park, Jin-Sun; Kim, Hee-Sun; Chong, Young Hae

2018-03-12

Mer tyrosine kinase (MerTK) activity necessary for amyloid-stimulated phagocytosis strongly implicates that MerTK dysregulation might contribute to chronic inflammation implicated in Alzheimer's disease (AD) pathology. However, the precise mechanism involved in the regulation of MerTK expression by amyloid-β (Aβ) in proinflammatory environment has not yet been ascertained. The objective of this study was to determine the underlying mechanism involved in Aβ-mediated decrease in MerTK expression through Aβ-mediated regulation of MerTK expression and its modulation by sulforaphane in human THP-1 macrophages challenged with Aβ1-42. We used protein preparation, Ca 2+ influx fluorescence imaging, nuclear fractionation, Western blotting techniques, and small interfering RNA (siRNA) knockdown to perform our study. Aβ1-42 elicited a marked decrease in MerTK expression along with increased intracellular Ca 2+ level and induction of proinflammatory cytokines such as IL-1β and TNF-α. Ionomycin A and thapsigargin also increased intracellular Ca 2+ levels and production of IL-1β and TNF-α, mimicking the effect of Aβ1-42. In contrast, the Aβ1-42-evoked responses were attenuated by depletion of Ca 2+ with ethylene glycol tetraacetic acid. Furthermore, recombinant IL-1β or TNF-α elicited a decrease in MerTK expression. However, immunodepletion of IL-1β or TNF-α with neutralizing antibodies significantly inhibited Aβ1-42-mediated downregulation of MerTK expression. Notably, sulforaphane treatment potently inhibited Aβ1-42-induced intracellular Ca 2+ level and rescued the decrease in MerTK expression by blocking nuclear factor-κB (NF-κB) nuclear translocation, thereby decreasing IL-1β and TNF-α production upon Aβ1-42 stimulation. Such adverse effects of sulforaphane were replicated by BAY 11-7082, a NF-κB inhibitor. Moreover, sulforaphane's anti-inflammatory effects on Aβ1-42-induced production of IL-1β and TNF-α were significantly diminished by siRNA-mediated knockdown of MerTK, confirming a critical role of MerTK in suppressing Aβ1-42-induced innate immune response. These findings implicate that targeting of MerTK with phytochemical sulforaphane as a mechanism for preventing Aβ1-42-induced neuroinflammation has potential to be applied in AD therapeutics.

Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Development and Its Functional Implications

PubMed Central

Popken, Jens; Graf, Alexander; Krebs, Stefan; Blum, Helmut; Schmid, Volker J.; Strauss, Axel; Guengoer, Tuna; Zakhartchenko, Valeri; Wolf, Eckhard; Cremer, Thomas

2015-01-01

The present study demonstrates a major remodeling of the nuclear envelope and its underlying lamina during bovine preimplantation development. Up to the onset of major embryonic genome activation (MGA) at the 8-cell stage nuclei showed a non-uniform distribution of nuclear pore complexes (NPCs). NPCs were exclusively present at sites where DNA contacted the nuclear lamina. Extended regions of the lamina, which were not contacted by DNA, lacked NPCs. In post-MGA nuclei the whole lamina was contacted rather uniformly by DNA. Accordingly, NPCs became uniformly distributed throughout the entire nuclear envelope. These findings shed new light on the conditions which control the integration of NPCs into the nuclear envelope. The switch from maternal to embryonic production of mRNAs was accompanied by multiple invaginations covered with NPCs, which may serve the increased demands of mRNA export and protein import. Other invaginations, as well as interior nuclear segments and vesicles without contact to the nuclear envelope, were exclusively positive for lamin B. Since the abundance of these invaginations and vesicles increased in concert with a massive nuclear volume reduction, we suggest that they reflect a mechanism for fitting the nuclear envelope and its lamina to a shrinking nuclear size during bovine preimplantation development. In addition, a deposit of extranuclear clusters of NUP153 (a marker for NPCs) without associated lamin B was frequently observed from the zygote stage up to MGA. Corresponding RNA-Seq data revealed deposits of spliced, maternally provided NUP153 mRNA and little unspliced, newly synthesized RNA prior to MGA, which increased strongly at the initiation of embryonic expression of NUP153 at MGA. PMID:25932910

Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Development and Its Functional Implications.

PubMed

Popken, Jens; Graf, Alexander; Krebs, Stefan; Blum, Helmut; Schmid, Volker J; Strauss, Axel; Guengoer, Tuna; Zakhartchenko, Valeri; Wolf, Eckhard; Cremer, Thomas

2015-01-01

The present study demonstrates a major remodeling of the nuclear envelope and its underlying lamina during bovine preimplantation development. Up to the onset of major embryonic genome activation (MGA) at the 8-cell stage nuclei showed a non-uniform distribution of nuclear pore complexes (NPCs). NPCs were exclusively present at sites where DNA contacted the nuclear lamina. Extended regions of the lamina, which were not contacted by DNA, lacked NPCs. In post-MGA nuclei the whole lamina was contacted rather uniformly by DNA. Accordingly, NPCs became uniformly distributed throughout the entire nuclear envelope. These findings shed new light on the conditions which control the integration of NPCs into the nuclear envelope. The switch from maternal to embryonic production of mRNAs was accompanied by multiple invaginations covered with NPCs, which may serve the increased demands of mRNA export and protein import. Other invaginations, as well as interior nuclear segments and vesicles without contact to the nuclear envelope, were exclusively positive for lamin B. Since the abundance of these invaginations and vesicles increased in concert with a massive nuclear volume reduction, we suggest that they reflect a mechanism for fitting the nuclear envelope and its lamina to a shrinking nuclear size during bovine preimplantation development. In addition, a deposit of extranuclear clusters of NUP153 (a marker for NPCs) without associated lamin B was frequently observed from the zygote stage up to MGA. Corresponding RNA-Seq data revealed deposits of spliced, maternally provided NUP153 mRNA and little unspliced, newly synthesized RNA prior to MGA, which increased strongly at the initiation of embryonic expression of NUP153 at MGA.

The ZO-1–associated Y-box factor ZONAB regulates epithelial cell proliferation and cell density

PubMed Central

Balda, Maria S.; Garrett, Michelle D.; Matter, Karl

2003-01-01

Epithelial tight junctions regulate paracellular permeability, restrict apical/basolateral intramembrane diffusion of lipids, and have been proposed to participate in the control of epithelial cell proliferation and differentiation. Previously, we have identified ZO-1–associated nucleic acid binding proteins (ZONAB), a Y-box transcription factor whose nuclear localization and transcriptional activity is regulated by the tight junction–associated candidate tumor suppressor ZO-1. Now, we found that reduction of ZONAB expression using an antisense approach or by RNA interference strongly reduced proliferation of MDCK cells. Transfection of wild-type or ZONAB-binding fragments of ZO-1 reduced proliferation as well as nuclear ZONAB pools, indicating that promotion of proliferation by ZONAB requires its nuclear accumulation. Overexpression of ZONAB resulted in increased cell density in mature monolayers, and depletion of ZONAB or overexpression of ZO-1 reduced cell density. ZONAB was found to associate with cell division kinase (CDK) 4, and reduction of nuclear ZONAB levels resulted in reduced nuclear CDK4. Thus, our data indicate that tight junctions can regulate epithelial cell proliferation and cell density via a ZONAB/ZO-1–based pathway. Although this regulatory process may also involve regulation of transcription by ZONAB, our data suggest that one mechanism by which ZONAB and ZO-1 influence proliferation is by regulating the nuclear accumulation of CDK4. PMID:12566432

A Novel Fission Yeast Gene, tht1 +, Is Required for the Fusion of Nuclear Envelopes during Karyogamy

PubMed Central

Tange, Yoshie; Horio, Tetsuya; Shimanuki, Mizuki; Ding, Da-Qiao; Hiraoka, Yasushi; Niwa, Osami

1998-01-01

We have isolated a fission yeast karyogamy mutant, tht1, in which nuclear congression and the association of two spindle pole bodies occurs but the subsequent fusion of nuclear envelopes is blocked. The tht1 mutation does not prevent meiosis, so cells execute meiosis with two unfused nuclei, leading to the production of aberrant asci. The tht1 + gene was cloned and sequenced. Predicted amino acid sequence has no significant homology to previously known proteins but strongly suggests that it is a type I membrane protein. The tht1 + gene is dispensable for vegetative growth and expressed only in conjugating cells. Tht1p is a glycoprotein susceptible to endoglycosilase H digestion. Site- directed mutagenesis showed that the N-glycosylation site, as well as the COOH-terminal region of Tht1p, is essential for its function. A protease protection assay indicated that the COOH terminus is cytoplasmic. Immunocytological analysis using a HA-tagged Tht1p suggested that the protein is localized in nuclear envelopes and in the ER during karyogamy and that its levels are reduced in cells containing fused nuclei. PMID:9442101

Nup98 FG domains from diverse species spontaneously phase-separate into particles with nuclear pore-like permselectivity

PubMed Central

Schmidt, Hermann Broder; Görlich, Dirk

2015-01-01

Nuclear pore complexes (NPCs) conduct massive transport mediated by shuttling nuclear transport receptors (NTRs), while keeping nuclear and cytoplasmic contents separated. The NPC barrier in Xenopus relies primarily on the intrinsically disordered FG domain of Nup98. We now observed that Nup98 FG domains of mammals, lancelets, insects, nematodes, fungi, plants, amoebas, ciliates, and excavates spontaneously and rapidly phase-separate from dilute (submicromolar) aqueous solutions into characteristic ‘FG particles’. This required neither sophisticated experimental conditions nor auxiliary eukaryotic factors. Instead, it occurred already during FG domain expression in bacteria. All Nup98 FG phases rejected inert macromolecules and yet allowed far larger NTR cargo complexes to rapidly enter. They even recapitulated the observations that large cargo-domains counteract NPC passage of NTR⋅cargo complexes, while cargo shielding and increased NTR⋅cargo surface-ratios override this inhibition. Their exquisite NPC-typical sorting selectivity and strong intrinsic assembly propensity suggest that Nup98 FG phases can form in authentic NPCs and indeed account for the permeability properties of the pore. DOI: http://dx.doi.org/10.7554/eLife.04251.001 PMID:25562883

Identification of the Photoreceptor Transcriptional Co-Repressor SAMD11 as Novel Cause of Autosomal Recessive Retinitis Pigmentosa

PubMed Central

Corton, M.; Avila-Fernández, A.; Campello, L.; Sánchez, M.; Benavides, B.; López-Molina, M. I.; Fernández-Sánchez, L.; Sánchez-Alcudia, R.; da Silva, L. R. J.; Reyes, N.; Martín-Garrido, E.; Zurita, O.; Fernández-San José, P.; Pérez-Carro, R.; García-García, F.; Dopazo, J.; García-Sandoval, B.; Cuenca, N.; Ayuso, C.

2016-01-01

Retinitis pigmentosa (RP), the most frequent form of inherited retinal dystrophy is characterized by progressive photoreceptor degeneration. Many genes have been implicated in RP development, but several others remain to be identified. Using a combination of homozygosity mapping, whole-exome and targeted next-generation sequencing, we found a novel homozygous nonsense mutation in SAMD11 in five individuals diagnosed with adult-onset RP from two unrelated consanguineous Spanish families. SAMD11 is ortholog to the mouse major retinal SAM domain (mr-s) protein that is implicated in CRX-mediated transcriptional regulation in the retina. Accordingly, protein-protein network analysis revealed a significant interaction of SAMD11 with CRX. Immunoblotting analysis confirmed strong expression of SAMD11 in human retina. Immunolocalization studies revealed SAMD11 was detected in the three nuclear layers of the human retina and interestingly differential expression between cone and rod photoreceptors was observed. Our study strongly implicates SAMD11 as novel cause of RP playing an important role in the pathogenesis of human degeneration of photoreceptors. PMID:27734943

CD30 expression is rare in myeloid leukemia cutis; a study of 55 cases and implications for routine diagnostic algorithms

PubMed Central

Ogunrinade, Olakunle; Terrano, David; Chiu, April; Pulitzer, Melissa

2016-01-01

Expression of CD30 in blastoid cutaneous infiltrates typically signifies a CD30+ lymphoproliferative disorder, often requiring minimal immunohistochemical work-up, if clinically consonant. However, myeloid and other hematologic malignancies often express CD30. We retrospectively examined the prevalence of CD30 expression in 41 patients (median age 59) and 55 biopsies with the diagnosis of leukemia cutis (LC) to determine whether an extensive immunohistochemical workup is warranted in all large, round cell CD30+ cutaneous infiltrates. Each patient had refractory or recurrent disease, the histologic presence of a large mononuclear cell infiltrate, and varied cytogenetics. CD30+ mononuclear cells within the infiltrate ranged from rare to many in twenty-two biopsies (22/55). In eighteen biopsies, CD30+ cells were interpreted as lymphocytic based on morphology, strong cytoplasmic and Golgi staining for CD30, and negative CD34 and CD117 staining. One case showing 3+ staining of lymphocytes was identified as a post-transplant lymphoproliferative disorder. The second 3+ case was favored to represent a subset CD30-positive AML. Three other cases with 1+ membranous and cytoplasmic staining were interpreted as myeloid leukemia. In conclusion, CD30 positivity in myeloid leukemia in the skin is rare and does not often exhibit the strong membranous (2+ or 3+) and/or Golgi staining seen in reactive lymphocytes. AML or myeloid LC may occasionally show 1+ (and rarely 2-3+) cytoplasmic/membranous or non-specific blush nuclear CD30 labeling. Strong diffuse staining for CD30 should prompt consideration of a reactive lymphoid/lymphoproliferative process, and, when the clinical likelihood of CD30+ leukemia cutis is low, may obviate the need for further immunohistochemistry. PMID:27893466

Growth factors FGF8 and FGF2 and their receptor FGFR1, transcriptional factors Msx-1 and MSX-2, and apoptotic factors p19 and RIP5 participate in the early human limb development.

PubMed

Becic, Tina; Kero, Darko; Vukojevic, Katarina; Mardesic, Snjezana; Saraga-Babic, Mirna

2018-04-01

The expression pattern of fibroblast growth factors FGF8 and FGF2 and their receptor FGFR1, transcription factors MSX-1 and MSX-2, as well as cell proliferation (Ki-67) and cell death associated caspase-3, p19 and RIP5 factors were analyzed in histological sections of eight 4th-9th-weeks developing human limbs by immunohistochemistry and semi-thin sectioning. Increasing expression of all analyzed factors (except FGF8) characterized both the multilayered human apical ectodermal ridge (AER), sub-ridge mesenchyme (progress zone) and chondrocytes in developing human limbs. While cytoplasmic co-expression of MSX-1 and MSX-2 was observed in both limb epithelium and mesenchyme, p19 displayed strong cytoplasmic expression in non-proliferating cells. Nuclear expression of Ki-67 proliferating cells, and partly of MSX-1 and MSX-2 was detected in the whole limb primordium. Strong expression of factors p19 and RIP5, both in the AER and mesenchyme of human developing limbs indicates their possible involvement in control of cell senescence and cell death. In contrast to animal studies, expression of FGFR1 in the surface ectoderm and p19 in the whole limb primordium might reflect interspecies differences in limb morphology. Expression of FGF2 and downstream RIP5 gene, and transcription factors Msx-1 and MSX-2 did not show human-specific changes in expression pattern. Based on their spatio-temporal expression during human limb development, our study indicates role of FGFs and Msx genes in stimulation of cell proliferation, limb outgrowth, digit elongation and separation, and additionally MSX-2 in control of vasculogenesis. The cascade of orchestrated gene expressions, including the analyzed developmental factors, jointly contribute to the complex human limb development. Copyright © 2018 Elsevier GmbH. All rights reserved.

Emerin and histone deacetylase 3 (HDAC3) cooperatively regulate expression and nuclear positions of MyoD, Myf5, and Pax7 genes during myogenesis

PubMed Central

Demmerle, Justin; Koch, Adam J.; Holaska, James M.

2016-01-01

The spatial organization of chromatin is critical in establishing cell-type dependent gene expression programs. The inner nuclear membrane protein emerin has been implicated in regulating global chromatin architecture. We show emerin associates with genomic loci of muscle differentiation promoting factors in murine myogenic progenitors, including Myf5 and MyoD. Prior to their transcriptional activation Myf5 and MyoD loci localized to the nuclear lamina in proliferating progenitors and moved to the nucleoplasm upon transcriptional activation during differentiation. The Pax7 locus, which is transcribed in proliferating progenitors, localized to the nucleoplasm and Pax7 moved to the nuclear lamina upon repression during differentiation. Localization of Myf5, MyoD, and Pax7 to the nuclear lamina and proper temporal expression of these genes required emerin and HDAC3. Interestingly, activation of HDAC3 catalytic activity rescued both Myf5 localization to the nuclear lamina and its expression. Collectively, these data support a model whereby emerin facilitates repressive chromatin formation at the nuclear lamina by activating the catalytic activity of HDAC3 to regulate the coordinated spatiotemporal expression of myogenic differentiation genes. PMID:24062260

BLZF1 expression is of prognostic significance in hepatocellular carcinoma

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

Huang, Run-Yue, E-mail: ry_huang@hotmail.com; Su, Shu-Guang; Wu, Dan-Chun

2015-11-20

BLZF1, a member of b-ZIP family, has been implicated in epigenetic regulation and Wnt/β-catenin signaling. Its expression and clinical significance in human cancers remain largely unknown. In this study, we showed that BLZF1 expression was reduced in hepatocellular carcinoma (HCC) tissues, compared to the paracarcinoma tissues, at both mRNA and protein levels. Results of immunohistochemistry revealed that BLZF1 was presented in both nuclear and cytoplasm. Decreased expression of nuclear and cytosolic BLZF1 in HCC was depicted in 68.2% and 79.2% of the 634 cases. Nuclear BLZF1 expression was significantly associated with tumor multiplicity (P = 0.048) and tumor capsule (P = 0.028), while cytosolicmore » BLZF1 expression was correlated with serum AFP level (P = 0.017), tumor differentiation (P = 0.001) and tumor capsule (P = 0.003). Kaplan–Meier analysis indicated both nuclear and cytosolic BLZF1 expression was associated with poor overall survival. Low nuclear BLZF1 also indicated unfavorable disease-free survival and high tendency of tumor recurrence. Furthermore, multiple Cox regression analysis revealed nuclear BLZF1 as an independent factor for overall survival (Hazard Ratio (HR) = 0.827, 95% confident interval (95%CI): 0.697–0.980, P = 0.029). The prognostic value of BLZF1 was further confirmed by stratified analyses. Collectively, our data suggest BLZF1 is a novel unfavorable biomarker for prognosis of patients with HCC. - Highlights: • BLZF1 expression was much lower in HCC tissues. • Low BLZF1 expression was associated with poor outcomes in a cohort of 634 HCC patients. • Multiple Cox regression analysis indicated nuclear BLZF1 as an independent predictor for overall survival.« less

Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues.

PubMed

Wang, Chunyi; Mao, Jinghe; Redfield, Samantha; Mo, Yinyuan; Lage, Janice M; Zhou, Xinchun

2014-10-01

Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data in benign and malignant tissues from the same organ/tissue were then compared using the Student's t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (in either the nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas, skin, soft tissues and uterus) show differences for only one S1PR (cytoplasmic or nuclear), and twenty three organs/tissues show no significant difference in IHC scores for any S1PR (cytoplasmic or nuclear) between benign and malignant changes. This is the first study to evaluate the expression level of all S1PRs in benign and malignant tissues from multiple human organs. This study provides data regarding the systemic distribution, subcellular localization and differences in expression of all five S1PRs in benign and malignant changes for each organ/tissue. Copyright © 2014 Elsevier Inc. All rights reserved.

Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues

PubMed Central

Wang, Chunyi; Mao, Jinghe; Redfield, Samantha; Mo, Yinyuan; Lage, Janice M.; Zhou, Xinchun

2014-01-01

Aims Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. Methods We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data were then compared in benign and malignant tissues from the same organ/tissue using the student t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. Results We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (either in nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas, skin, soft tissues and uterus) show differences for only one S1PR (cytoplasmic or nuclear), and twenty three organs/tissues show no significant difference in IHC score of any S1PR (cytoplasmic or nuclear) between benign and malignant changes. Conclusion This is the first study to evaluate the expression level of all S1PRs in benign and malignant tissues from multiple human organs. This study provides data regarding the systemic distribution, subcellular localization and differences in expression of all five S1PRs in benign and malignant changes for each organ/tissue. PMID:25084322

A role for Hippo/YAP-signaling in FGF-induced lens epithelial cell proliferation and fibre differentiation.

PubMed

Dawes, L J; Shelley, E J; McAvoy, J W; Lovicu, F J

2018-04-01

Recent studies indicate an important role for the transcriptional co-activator Yes-associated protein (YAP), and its regulatory pathway Hippo, in controlling cell growth and fate during lens development; however, the exogenous factors that promote this pathway are yet to be identified. Given that fibroblast growth factor (FGF)-signaling is an established regulator of lens cell behavior, the current study investigates the relationship between this pathway and Hippo/YAP-signaling during lens cell proliferation and fibre differentiation. Rat lens epithelial explants were cultured with FGF2 to induce epithelial cell proliferation or fibre differentiation. Immunolabeling methods were used to detect the expression of Hippo-signaling components, Total and Phosphorylated YAP, as well as fibre cell markers, Prox-1 and β-crystallin. FGF-induced lens cell proliferation was associated with a strong nuclear localisation of Total-YAP and low-level immuno-staining for phosphorylated-YAP. FGF-induced lens fibre differentiation was associated with a significant increase in cytoplasmic phosphorylated YAP (inactive state) and enhanced expression of core Hippo-signaling components. Inhibition of YAP with Verteporfin suppressed FGF-induced lens cell proliferation and ablated cell elongation during lens fibre differentiation. Inhibition of either FGFR- or MEK/ERK-signaling suppressed FGF-promoted YAP nuclear translocation. Here we propose that FGF promotes Hippo/YAP-signaling during lens cell proliferation and differentiation, with FGF-induced nuclear-YAP expression playing an essential role in promoting the proliferation of lens epithelial cells. An FGF-induced switch from proliferation to differentiation, hence regulation of lens growth, may play a key role in mediating Hippo suppression of YAP transcriptional activity. Copyright © 2018 Elsevier Ltd. All rights reserved.

Role of YAP activation in nuclear receptor CAR-mediated proliferation of mouse hepatocytes.

PubMed

Abe, Taiki; Amaike, Yuto; Shizu, Ryota; Takahashi, Miki; Kano, Makoto; Hosaka, Takuomi; Sasaki, Takamitsu; Kodama, Susumu; Matsuzawa, Atsushi; Yoshinari, Kouichi

2018-06-08

Constitutive androstane receptor (CAR) is a xenobiotic-responsive nuclear receptor that is highly expressed in the liver. CAR activation induces hepatocyte proliferation and hepatocarcinogenesis in rodents, but the mechanisms remain unclear. In this study, we investigated the association of CAR-dependent cell proliferation with Yes-associated protein (YAP), which is a transcriptional cofactor controlling organ size and cell growth through the interaction with various transcriptional factors including TEAD. In mouse livers, TCPOBOP (a mouse CAR activator) treatment increased the nuclear YAP accumulation and mRNA levels of YAP target genes as well as cell-cycle related genes along with liver hypertrophy and verteporfin (an inhibitor of YAP/TEAD interaction) cotreatment tended to attenuate them. Furthermore, in cell-based reporter gene assays, CAR activation enhanced the YAP/TEAD-dependent transcription. To investigate the role of YAP/TEAD activation in the CAR-dependent hepatocyte proliferation, we sought to establish an in vitro system completely reproducing CAR-dependent cell proliferation. Since CAR was only slightly expressed in cultured mouse primary hepatocytes compared to mouse livers and no proliferation was observed after treatment with TCPOBOP, we overexpressed CAR using mouse CAR expressing adenovirus (Ad-mCAR-V5) in mouse primary hepatocytes. Ad-mCAR-V5 infection and TCPOBOP treatment induced hepatocyte proliferation. Similar results were obtained with immortalized normal mouse hepatocytes as well. In the established in vitro system, CAR-dependent proliferation was strongly inhibited by Yap knockdown and completely abolished by verteporfin treatment. Our present results obtained in in vivo and in vitro experiments suggest that YAP/TEAD activation plays key roles in CAR-dependent proliferation of murine hepatocytes.

Squamous cell carcinoma antigen (SCCA) is up-regulated during Barrett’s carcinogenesis and predicts esophageal adenocarcinoma resistance to neoadjuvant chemotherapy

PubMed Central

Fassan, Matteo; Realdon, Stefano; Vianello, Luca; Quarta, Santina; Ruol, Alberto; Castoro, Carlo; Scarpa, Marco; Zaninotto, Giovanni; Guzzardo, Vincenza; Sileni, Vanna Chiarion; Pontisso, Patrizia; Rugge, Massimo

2017-01-01

Squamous Cell Carcinoma Antigen (SCCA) is consistently overexpressed in many different solid tumors, and has been associated with both tumor aggressiveness and chemoresistance. No data, however, is currently available on SCCA expression during esophageal Barrett's carcinogenesis, nor on SCCA expression's role on esophageal adenocarcinoma chemoresistance. The SCCA immunohistochemical expression was assessed in a series of 100 biopsy samples covering the whole histological spectrum of Barrett's oncogenesis. Squamous native mucosa was characterized by a moderate to strong cytoplasmic and nuclear SCCA expression in suprabasal, medium, and superficial layers. On the other hand, almost half of the considered lesions did not express SCCA; the other half featured weak to moderate SCCA expression. The relationship between SCCA protein expression and tumor response to neoadjuvant chemotherapy was assessed in 90 esophageal adenocarcinoma specimens (40 biopsy and 50 surgery specimens), stratified according to Mandard tumor regression grade. As observed in other settings, the presence of SCCA expression clustered in the group of tumors characterized by a lower responsiveness to neoadjuvant treatments. The present results suggest an involvement of SCCA in a subset of Barrett-related tumors, and prompt to consider the SCCA-protein expression as response-predictive marker of neoadjuvant therapy in esophageal adenocarcinomas. PMID:28042960

Similar chemokine receptor profiles in lymphomas with central nervous system involvement - possible biomarkers for patient selection for central nervous system prophylaxis, a retrospective study.

PubMed

Lemma, Siria A; Pasanen, Anna Kaisa; Haapasaari, Kirsi-Maria; Sippola, Antti; Sormunen, Raija; Soini, Ylermi; Jantunen, Esa; Koivunen, Petri; Salokorpi, Niina; Bloigu, Risto; Turpeenniemi-Hujanen, Taina; Kuittinen, Outi

2016-05-01

Central nervous system (CNS) relapse occurs in around 5% of diffuse large B-cell lymphoma (DLBCL) cases. No biomarkers to identify high-risk patients have been discovered. We evaluated the expression of lymphocyte-guiding chemokine receptors in systemic and CNS lymphomas. Immunohistochemical staining for CXCR4, CXCR5, CCR7, CXCL12, and CXCL13 was performed on 89 tissue samples, including cases of primary central nervous system lymphoma (PCNSL), secondary CNS lymphoma (sCNSL), and systemic DLBCL. Also, 10 reactive lymph node samples were included. Immunoelectron microscopy was performed on two PCNSLs, one sCNSL, one systemic DLBCL, and one reactive lymph node samples, and staining was performed for CXCR4, CXCR5, CXCL12, and CXCL13. Chi-square test was used to determine correlations between clinical parameters, diagnostic groups, and chemokine receptor expression. Strong nuclear CXCR4 positivity correlated with systemic DLBCL, whereas strong cytoplasmic CXCR5 positivity correlated with CNS involvement (P = 0.003 and P = 0.039). Immunoelectron microscopy revealed a nuclear CXCR4 staining in reactive lymph node, compared with cytoplasmic and membranous localization seen in CNS lymphomas. We found that CNS lymphoma presented a chemokine receptor profile different from systemic disease. Our findings give new information on the CNS tropism of DLBCL and, if confirmed, may contribute to more effective targeting of CNS prophylaxis among patients with DLBCL. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Systematic Analysis of the Functional Relevance of Nuclear Structure and Mechanics in Breast Cancer Progression

DTIC Science & Technology

2013-07-01

epithelial cells; MDA-MB-231 metastatic breast cancer cells) with systematic alterations in the expression of lamins A, B1, B2, C, and lamin B receptor...LBR). We then evaluated the effect of altered lamin expression on nuclear stiffness in these cell lines. While increased expression of lamin A...caused stiffer, less deformable nuclei, reduction of lamins A/C expression by shRNA reduced nuclear stiffness. The effect of alterations in other lamins

Tributyltin engages multiple nuclear receptor pathways and suppresses osteogenesis in bone marrow multipotent stromal cells.

PubMed

Baker, Amelia H; Watt, James; Huang, Cassie K; Gerstenfeld, Louis C; Schlezinger, Jennifer J

2015-06-15

Organotins are members of the environmental obesogen class of contaminants because they activate peroxisome proliferator-activated receptor γ (PPARγ), the essential regulator of adipogenesis. Exposure to thiazolidinediones (PPARγ ligands used to treat type 2 diabetes) is associated with increased fractures. Diminished bone quality likely results from PPARγ's role in promoting adipogenesis while suppressing osteogenesis of bone marrow multipotent mesenchymal stromal cells (BM-MSC). We hypothesized that tributyltin (TBT) would be a potent modifier of BM-MSC differentiation and a negative regulator of bone formation. Organotins interact with both PPARγ and retinoid X receptors (RXR), suggesting that they activate multiple nuclear receptor pathways. To investigate the role of RXR in the actions of TBT, the effects of PPARγ (rosiglitazone) and RXR (bexarotene, LG100268) agonists were compared to the effects of TBT in BMS2 cells and primary mouse BM-MSC cultures. In BMS2 cells, TBT induced the expression of Fabp4, Abca1, and Tgm2 in an RXR-dependent manner. All agonists suppressed osteogenesis in primary mouse BM-MSC cultures, based on decreased alkaline phosphatase activity, mineralization, and expression of osteoblast-related genes. While rosiglitazone and TBT strongly activated adipogenesis, based on lipid accumulation and expression of adipocyte-related genes, the RXR agonists did not. Extending these analyses to other RXR heterodimers showed that TBT and the RXR agonists activated the liver X receptor pathway, whereas rosiglitazone did not. Application of either a PPARγ antagonist (T0070907) or an RXR antagonist (HX531) significantly reduced rosiglitazone-induced suppression of bone nodule formation. Only the RXR antagonist significantly reduced LG100268- and TBT-induced bone suppression. The RXR antagonist also inhibited LG100268- and TBT-induced expression of Abca1, an LXR target gene, in primary BM-MSC cultures. These results provide novel evidence that TBT activates multiple nuclear receptor pathways in BM-MSCs, activation of RXR is sufficient to suppress osteogenesis, and TBT suppresses osteogenesis largely through its direct interaction with RXR.

Andrographolide stimulates p38 mitogen-activated protein kinase-nuclear factor erythroid-2-related factor 2-heme oxygenase 1 signaling in primary cerebral endothelial cells for definite protection against ischemic stroke in rats.

PubMed

Yen, Ting-Lin; Chen, Ray-Jade; Jayakumar, Thanasekaran; Lu, Wan-Jung; Hsieh, Cheng-Ying; Hsu, Ming-Jen; Yang, Chih-Hao; Chang, Chao-Chien; Lin, Yen-Kuang; Lin, Kuan-Hung; Sheu, Joen-Rong

2016-04-01

Stroke pathogenesis involves complex oxidative stress-related pathways. The nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) pathways have been considered molecular targets in pharmacologic intervention for ischemic diseases. Andrographolide, a labdane diterpene, has received increasing attention in recent years because of its various pharmacologic activities. We determined that andrographolide modulates the mitogen-activated protein kinase (MAPK)-Nrf2-HO-1 signaling cascade in primary cerebral endothelial cells (CECs) to provide positive protection against middle cerebral artery occlusion (MCAO)-induced ischemic stroke in rats. In the present study, andrographolide (10 μM) increased HO-1 protein and messenger RNA expressions, Nrf2 phosphorylation, and nuclear translocation in CECs, and these activities were disrupted by a p38 MAPK inhibitor, SB203580, but not by the extracellular signal-regulated kinase inhibitor PD98059 or c-Jun amino-terminal kinase inhibitor SP600125. Similar results were observed in confocal microscopy analysis. Moreover, andrographolide-induced Nrf2 and HO-1 protein expressions were significantly inhibited by Nrf2 small interfering RNA. Moreover, HO-1 knockdown attenuated the protective effect of andrographolide against oxygen-glucose deprivation-induced CEC death. Andrographolide (0.1 mg/kg) significantly suppressed free radical formation, blood-brain barrier disruption, and brain infarction in MCAO-insulted rats, and these effects were reversed by the HO-1 inhibitor zinc protoporphyrin IX. The mechanism is attributable to HO-1 activation, as directly evidenced by andrographolide-induced pronounced HO-1 expression in brain tissues, which was highly localized in the cerebral capillary. In conclusion, andrographolide increased Nrf2-HO-1 expression through p38 MAPK regulation, confirming that it provides protection against MCAO-induced brain injury. These findings provide strong evidence that andrographolide could be a therapeutic agent for treating ischemic stroke or neurodegenerative diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Isolation and characterization of a novel calmodulin-binding protein from potato

NASA Technical Reports Server (NTRS)

Reddy, Anireddy S N.; Day, Irene S.; Narasimhulu, S. B.; Safadi, Farida; Reddy, Vaka S.; Golovkin, Maxim; Harnly, Melissa J.

2002-01-01

Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.

PA28γ is a novel corepressor of HTLV-1 replication and controls viral latency

PubMed Central

Ko, Nga Ling; Taylor, John M.; Bellon, Marcia; Bai, Xue Tao; Shevtsov, Sergey P.; Dundr, Miroslav

2013-01-01

The establishment of a latent reservoir by human tumor viruses is a vital step in initiating cellular transformation and represents a major shortcoming to current therapeutic strategies and the ability to eradicate virus-infected cells. Human T-cell leukemia virus type 1 (HTLV-1) establishes a lifelong infection and is linked to adult T-cell leukemia lymphoma (ATLL). Here, we demonstrate that HTLV-1 p30 recruits the cellular proteasome activator PA28γ onto the viral tax/rex mRNA to prevent its nuclear export and suppress virus replication. Interaction of p30 with a PA28γ retaining fully functional proteasome activity is required for p30's ability to repress HTLV-1. Consistently, HTLV-1 molecular clones replicate better and produce more virus particles in PA28γ-deficient cells. These results define a unique and novel role for the cellular factor PA28γ in the control of nuclear RNA trafficking and HTLV-1–induced latency. Importantly, knockdown of PA28γ expression in ATLL cells latently infected with HTLV-1 reactivates expression of viral tax/rex RNA and the Tax protein. Because Tax is the most immunogenic viral antigen and triggers strong CTL responses, our results suggest that PA28γ-targeted therapy may reactivate virus expression from latently infected cells and allow their eradication from the host. PMID:23104922

Selenoprotein W enhances skeletal muscle differentiation by inhibiting TAZ binding to 14-3-3 protein.

PubMed

Jeon, Yeong Ha; Park, Yong Hwan; Lee, Jea Hwang; Hong, Jeong-Ho; Kim, Ick Young

2014-07-01

Selenoprotein W (SelW) is expressed in various tissues, particularly in skeletal muscle. We have previously reported that SelW is up-regulated during C2C12 skeletal muscle differentiation and inhibits binding of 14-3-3 to its target proteins. 14-3-3 reduces myogenic differentiation by inhibiting nuclear translocation of transcriptional co-activator with PDZ-binding motif (TAZ). Phosphorylation of TAZ at Ser89 is required for binding to 14-3-3, leading to cytoplasmic retention of TAZ and a delay in myogenic differentiation. Here, we show that myogenic differentiation was delayed in SelW-knockdown C2C12 cells. Down-regulation of SelW also increased TAZ binding to 14-3-3, which eventually resulted in decreasing translocation of TAZ to the nucleus. However, phosphorylation of TAZ at Ser89 was not affected. Although phosphorylation of TAZ at Ser89 was sustained by the phosphatase inhibitor okadaic acid, nuclear translocation of TAZ was increased by ectopic expression of SelW. This result was due to decreased binding of TAZ to 14-3-3. We also found that the interaction between TAZ and MyoD was increased by ectopic expression of SelW. Taken together, these findings strongly demonstrate that SelW enhances C2C12 cell differentiation by inhibiting TAZ binding to 14-3-3. Copyright © 2014 Elsevier B.V. All rights reserved.

Potential Role of Rebamipide in Osteoclast Differentiation and Mandibular Condylar Cartilage Homeostasis.

PubMed

Izawa, Takashi; Hutami, Islamy Rahma; Tanaka, Eiji

2018-04-20

Temporomandibular joint osteoarthritis (TMJ-OA) is a degenerative disease that involves changes in subchondral bone and progressive degradation of cartilage. Currently, rebamipide, a gastroprotective drug, is administered to protect gastric mucosa and accelerate ulcer healing. Recent studies have shown that rebamipide also attenuates cartilage degeneration by suppressing oxidative damage and inducing homeostasis of the extracellular matrix of articular chondrocytes. Regarding the latter, reduced expression of cathepsin K, NFATc1, c-Src, and integrin β3, and increased expression of nuclear factor-kappa B, have been found to be mediated by the transcription factor, receptor activator of nuclear factor kappa-B ligand (RANKL). Treatment with rebamipide was also found to activate, mitogen-activated protein kinases such as p38, ERK, and JNK to reduce osteoclast differentiation. Taken together, these results strongly indicate that rebamipide mediates inhibitory effects on cartilage degradation and osteoclastogenesis in TMJ-OA. Here, we highlight recent evidence regarding the potential for rebamipide to affect osteoclast differentiation and TMJ-OA pathogenesis. We also discuss the potential role of rebamipide to serve as a new strategy for the treatment of TMJ-OA. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Myeloid Leukemia Factor Acts in a Chaperone Complex to Regulate Transcription Factor Stability and Gene Expression.

PubMed

Dyer, Jamie O; Dutta, Arnob; Gogol, Madelaine; Weake, Vikki M; Dialynas, George; Wu, Xilan; Seidel, Christopher; Zhang, Ying; Florens, Laurence; Washburn, Michael P; Abmayr, Susan M; Workman, Jerry L

2017-06-30

Mutations that affect myelodysplasia/myeloid leukemia factor (MLF) proteins are associated with leukemia and several other cancers. However, with no strong homology to other proteins of known function, the role of MLF proteins in the cell has remained elusive. Here, we describe a proteomics approach that identifies MLF as a member of a nuclear chaperone complex containing a DnaJ protein, BCL2-associated anthanogene 2, and Hsc70. This complex associates with chromatin and regulates the expression of target genes. The MLF complex is bound to sites of nucleosome depletion and sites containing active chromatin marks (e.g., H3K4me3 and H3K4me1). Hence, MLF binding is enriched at promoters and enhancers. Additionally, the MLF-chaperone complex functions to regulate transcription factor stability, including the RUNX transcription factor involved in hematopoiesis. Although Hsc70 and other co-chaperones have been shown to play a role in nuclear translocation of a variety of proteins including transcription factors, our findings suggest that MLF and the associated co-chaperones play a direct role in modulating gene transcription. Copyright © 2016 Elsevier Ltd. All rights reserved.

Intranuclear DNA release is a determinant of transfection activity for a non-viral vector: biocleavable polyrotaxane as a supramolecularly dissociative condenser for efficient intranuclear DNA release.

PubMed

Yamada, Yuma; Nomura, Taku; Harashima, Hideyoshi; Yamashita, Atsushi; Katoono, Ryo; Yui, Nobuhiko

2010-01-01

It has been believed that nuclear gene delivery is the most important process for gene expression, and various non-viral vectors are currently being developed with this assumption. However, some of our earlier studies revealed a surprising difference in transfection activity between viral and non-viral vectors: this difference is largely due to the result of the intranuclear disposition of DNA rather than its delivery to the nucleus (Hama S. et al. (2006), Quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems. Mol. Ther., 13, 786-794). Here, we report on some direct evidence that demonstrates the importance of the release of intranuclear DNA on transfection activity. The data show that transfection activity can be substantially enhanced by integrating a multifunctional envelope-type nano device (MEND) and a biocleavable polyrotaxane (DMAE-SS-PRX) as an artificial condenser. Our integration system showed significantly higher transfection activity compared to conventional gene delivery system. Moreover, this system provides a strong support for our hypothesis that intranuclear DNA disposition plays a critical role in gene expression for non-viral vectors.

Trichohyalin-like 1 protein, a member of fused S100 proteins, is expressed in normal and pathologic human skin

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

Yamakoshi, Takako; Makino, Teruhiko, E-mail: tmakino@med.u-toyama.ac.jp; Ur Rehman, Mati

2013-03-01

Highlights: ► Trichohyalin-like 1 protein is a member of the fused-type S100 protein gene family. ► Specific antibodies against the C-terminus of the TCHHL1 protein were generated. ► TCHHL1 proteins were expressed in the basal layer of the normal epidermis. ► TCHHL1 proteins were strongly expressed in tumor nests of BCC and SCC. ► The expression of TCHHL1 proteins increased in epidermis of psoriasis vulgaris. - Abstract: Trichohyalin-like 1 (TCHHL1) protein is a novel member of the fused-type S100 protein gene family. The deduced amino acid sequence of TCHHL1 contains an EF-hand domain in the N-terminus, one trans-membrane domain andmore » a nuclear localization signal. We generated specific antibodies against the C-terminus of the TCHHL1 protein and examined the expression of TCHHL1 proteins in normal and pathological human skin. An immunohistochemical study showed that TCHHL1 proteins were expressed in the basal layer of the normal epidermis. In addition, signals of TCHHL1 proteins were observed around the nuclei of cultured growing keratinocytes. Accordingly, TCHHL1 mRNA has been detected in normal skin and cultured growing keratinocytes. Furthermore, TCHHL1 proteins were strongly expressed in the peripheral areas of tumor nests in basal cell carcinomas and squamous cell carcinomas. A dramatic increase in the number of Ki67 positive cells was observed in TCHHL1-expressing areas. The expression of TCHHL1 proteins also increased in non-cancerous hyperproliferative epidermal tissues such as those of psoriasis vulgaris and lichen planus. These findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocytes.« less

Regulation of the heat shock response under anoxia in the turtle, Trachemys scripta elegans.

PubMed

Krivoruchko, Anastasia; Storey, Kenneth B

2010-03-01

The effects of 20 h of anoxic submergence in cold water and 5 h of aerobic recovery on the heat shock response were analyzed in four organs of the anoxia-tolerant turtle Trachemys scripta elegans. Immunoblotting was used to analyze levels of active and inactive forms of the heat shock transcription factor 1 (HSF1), nuclear translocation of HSF1, and the levels of six heat shock proteins (HSPs). PCR was also used to retrieve the turtle HSF1 nucleotide sequence; its deduced amino acid sequence showed 97% identity with chicken HSF1. White skeletal muscle showed a strong fivefold increase in the amount of active HSF1 under anoxic conditions as well as an 80% increase in nuclear localization. This was accompanied by upregulation of five HSPs by 1.8- to 2.9-fold: Hsp25, Hsp40, Hsp70, Hsc70, and Hsp90, the latter two remained elevated after 5 h of aerobic recovery. Kidney and liver showed little change in active HSF1 content during anoxia and recovery, but a significant increase in the nuclear localization of HSF1 during anoxia. This supported enhanced expression of three HSPs in kidney (Hsp40, Hsc70, and Hsp90) and four in liver (Hsp40, Hsp60, Hsp70, Hsc70). Heart displayed a strong increase in active HSF1 during anoxia and recovery (6.6- to 6.8-fold higher than control) and increased nuclear localization but heart HSP levels did not rise. The data demonstrate organ-specific regulation of HSPs during anoxia exposure and aerobic recovery in T. s. elegans and suggest that the heat shock response is an important aspect of cytoprotection during facultative anaerobiosis, particularly with regard to underwater hibernation of turtles in cold water.

0610009K11Rik, a testis-specific and germ cell nuclear receptor-interacting protein

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

Zhang Heng; Denhard, Leslie A.; Zhou Huaxin

Using an in silico approach, a putative nuclear receptor-interacting protein 0610009K11Rik was identified in mouse testis. We named this gene testis-specific nuclear receptor-interacting protein-1 (Tnrip-1). Tnrip-1 was predominantly expressed in the testis of adult mouse tissues. Expression of Tnrip-1 in the testis was regulated during postnatal development, with robust expression in 14-day-old or older testes. In situ hybridization analyses showed that Tnrip-1 is highly expressed in pachytene spermatocytes and spermatids. Consistent with its mRNA expression, Tnrip-1 protein was detected in adult mouse testes. Immunohistochemical studies showed that Tnrip-1 is a nuclear protein and mainly expressed in pachytene spermatocytes and roundmore » spermatids. Moreover, co-immunoprecipitation analyses showed that endogenous Tnrip-1 protein can interact with germ cell nuclear receptor (GCNF) in adult mouse testes. Our results suggest that Tnrip-1 is a testis-specific and GCNF-interacting protein which may be involved in the modulation of GCNF-mediated gene transcription in spermatogenic cells within the testis.« less

A gain-of-function mutation of plastidic invertase alters nuclear gene expression with sucrose treatment partially via GENOMES UNCOUPLED1-mediated signaling.

PubMed

Maruta, Takanori; Miyazaki, Nozomi; Nosaka, Ryota; Tanaka, Hiroyuki; Padilla-Chacon, Daniel; Otori, Kumi; Kimura, Ayako; Tanabe, Noriaki; Yoshimura, Kazuya; Tamoi, Masahiro; Shigeoka, Shigeru

2015-05-01

Plastid gene expression (PGE) is one of the signals that regulate the expression of photosynthesis-associated nuclear genes (PhANGs) via GENOMES UNCOUPLED1 (GUN1)-dependent retrograde signaling. We recently isolated Arabidopsis sugar-inducible cotyledon yellow-192 (sicy-192), a gain-of-function mutant of plastidic invertase, and showed that following the treatment of this mutant with sucrose, the expression of PhANGs as well as PGE decreased, suggesting that the sicy-192 mutation activates a PGE-evoked and GUN1-mediated retrograde pathway. To clarify the relationship between the sicy-192 mutation, PGE, and GUN1-mediated pathway, plastid and nuclear gene expression in a double mutant of sicy-192 and gun1-101, a null mutant of GUN1 was studied. Plastid-encoded RNA polymerase (PEP)-dependent PGE was markedly suppressed in the sicy-192 mutant by the sucrose treatment, but the suppression as well as cotyledon yellow phenotype was not mitigated by GUN1 disruption. Microarray analysis revealed that the altered expression of nuclear genes such as PhANG in the sucrose-treated sicy-192 mutant was largely dependent on GUN1. The present findings demonstrated that the sicy-192 mutation alters nuclear gene expression with sucrose treatment via GUN1, which is possibly followed by inhibiting PEP-dependent PGE, providing a new insight into the role of plastid sugar metabolism in nuclear gene expression. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Expression and subcellular localization of a novel nuclear acetylcholinesterase protein.

PubMed

Santos, Susana Constantino Rosa; Vala, Inês; Miguel, Cláudia; Barata, João T; Garção, Pedro; Agostinho, Paula; Mendes, Marta; Coelho, Ana V; Calado, Angelo; Oliveira, Catarina R; e Silva, João Martins; Saldanha, Carlota

2007-08-31

Acetylcholine is found in the nervous system and also in other cell types (endothelium, lymphocytes, and epithelial and blood cells), which are globally termed the non-neuronal cholinergic system. In this study we investigated the expression and subcellular localization of acetylcholinesterase (AChE) in endothelial cells. Our results show the expression of the 70-kDa AChE in both cytoplasmic and nuclear compartments. We also describe, for the first time, a nuclear and cytoskeleton-bound AChE isoform with approximately 55 kDa detected in endothelial cells. This novel isoform is decreased in response to vascular endothelial growth factor via the proteosomes pathway, and it is down-regulated in human leukemic T-cells as compared with normal T-cells, suggesting that the decreased expression of the 55-kDa AChE protein may contribute to an angiogenic response and associate with tumorigenesis. Importantly, we show that its nuclear expression is not endothelial cell-specific but also evidenced in non-neuronal and neuronal cells. Concerning neuronal cells, we can distinguish an exclusively nuclear expression in postnatal neurons in contrast to a cytoplasmic and nuclear expression in embryonic neurons, suggesting that the cell compartmentalization of this new AChE isoform is changed during the development of nervous system. Overall, our studies suggest that the 55-kDa AChE may be involved in different biological processes such as neural development, tumor progression, and angiogenesis.

Geiger-Nuttall Law for Nuclei in Strong Electromagnetic Fields.

PubMed

Delion, D S; Ghinescu, S A

2017-11-17

We investigate the influence of a strong laser electromagnetic field on the α-decay rate by using the Hennenberger frame of reference. We introduce an adimensional parameter D=S_{0}/R_{0}, where R_{0} is the geometrical nuclear radius and S_{0}∼sqrt[I]/ω^{2} is a length parameter depending on the laser intensity I and frequency ω. We show that the barrier penetrability has a strong increase for intensities corresponding to D>D_{crit}=1, due to the fact that the resulting Coulomb potential becomes strongly anisotropic even for spherical nuclei. As a consequence, the contribution of the monopole term increases the barrier penetrability by 2 orders of magnitude, while the total contribution has an effect of 6 orders of magnitude at D∼3D_{crit}. In the case of deformed nuclei, the electromagnetic field increases the penetrability by an additional order of magnitude for a quadrupole deformation β_{2}∼0.3. The influence of the electromagnetic field can be expressed in terms of a shifted Geiger-Nuttal law by a term depending on S_{0} and deformation.

Geiger-Nuttall Law for Nuclei in Strong Electromagnetic Fields

NASA Astrophysics Data System (ADS)

Delion, D. S.; Ghinescu, S. A.

2017-11-01

We investigate the influence of a strong laser electromagnetic field on the α -decay rate by using the Hennenberger frame of reference. We introduce an adimensional parameter D =S0/R0, where R0 is the geometrical nuclear radius and S0˜√{I }/ω2 is a length parameter depending on the laser intensity I and frequency ω . We show that the barrier penetrability has a strong increase for intensities corresponding to D >Dcrit=1 , due to the fact that the resulting Coulomb potential becomes strongly anisotropic even for spherical nuclei. As a consequence, the contribution of the monopole term increases the barrier penetrability by 2 orders of magnitude, while the total contribution has an effect of 6 orders of magnitude at D ˜3 Dcrit. In the case of deformed nuclei, the electromagnetic field increases the penetrability by an additional order of magnitude for a quadrupole deformation β2˜0.3 . The influence of the electromagnetic field can be expressed in terms of a shifted Geiger-Nuttal law by a term depending on S0 and deformation.

Einstein's 1919 View

NASA Astrophysics Data System (ADS)

Goradia, Shantilal

2012-10-01

When Rutherford discovered the nuclear force in 1919, he felt the force he discovered reflected some deviation of Newtonian gravity. Einstein too in his 1919 paper published the failure of the general relativity and Newtonian gravity to explain nuclear force and, in his concluding remarks, he retracted his earlier introduction of the cosmological constant. Consistent with his genius, we modify Newtonian gravity as probabilistic gravity using natural Planck units for a realistic study of nature. The result is capable of expressing both (1) nuclear force [strong coupling], and (2) Newtonian gravity in one equation, implying in general, in layman's words, that gravity is the cumulative effect of all quantum mechanical forces which are impossible to measure at long distances. Non discovery of graviton and quantum gravity silently support our findings. Continuing to climb on the shoulders of the giants enables us to see horizons otherwise unseen, as reflected in our book: ``Quantum Consciousness - The Road to Reality,'' and physics/0210040, where we derive the fine structure constant as a function of the age of the universe in Planck times consistent with Gamow's hint, using natural logarithm consistent with Feynman's hint.

Nuclear fear and children: the impact of parental nuclear activism, responsivity, and fear

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

LaGuardia, M.R.

1986-01-01

This study examines the extent to which parental nuclear fear, parental activism, and parental responsivity is associated with children's (age 10) nuclear fear. Other associated variables investigated include: nuclear denial, general anxiety and fear, and the personal characteristics of sex, socio-economic status, and academic aptitude. Findings indicate that children attend to nuclear issues when their parents attend to a significant degree. Children's hopelessness about the arms race is increased as parents' worry about nuclear war increases. Children's fear about not surviving a nuclear war increases as parents' worry about survivability decreases. Children who have more general fears also indicated thatmore » they have a high level of hopelessness, pervasive worry, and much concern about being able to survive a nuclear war. Children with a high degree of general anxiety did not indicate high degrees of nuclear fears. Children with high academic aptitude were more knowledgeable about nuclear issues and expressed more fears about the nuclear threat. Boys demonstrated more knowledge about nuclear issues than girls, and girls expressed much more frequent fear and worry about the nuclear threat than boys. Parents of lower socio-economic statues (SES) expressed more denial about the nuclear threat and were more pro-military than the higher SES parents.« less

CDKL5 expression is modulated during neuronal development and its subcellular distribution is tightly regulated by the C-terminal tail.

PubMed

Rusconi, Laura; Salvatoni, Lisa; Giudici, Laura; Bertani, Ilaria; Kilstrup-Nielsen, Charlotte; Broccoli, Vania; Landsberger, Nicoletta

2008-10-31

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome (RTT), West syndrome, and X-linked infantile spasms, sharing the common feature of mental retardation and early seizures. CDKL5 is a rather uncharacterized kinase, but its involvement in RTT seems to be explained by the fact that it works upstream of MeCP2, the main cause of Rett syndrome. To understand the role of this kinase for nervous system functions and to address if molecular mechanisms are involved in regulating its distribution and activity, we studied the ontogeny of CDKL5 expression in developing mouse brains by immunostaining and Western blotting. The expression profile of CDKL5 was compared with that of MeCP2. The two proteins share a general expression profile in the adult mouse brain, but CDKL5 levels appear to be highly modulated at the regional level. Its expression is strongly induced in early postnatal stages, and in the adult brain CDKL5 is present in mature neurons, but not in astroglia. Interestingly, the presence of CDKL5 in the cell nucleus varies at the regional level of the adult brain and is developmentally regulated. CDKL5 shuttles between the cytoplasm and the nucleus and the C-terminal tail is involved in localizing the protein to the cytoplasm in a mechanism depending on active nuclear export. Accordingly, Rett derivatives containing disease-causing truncations of the C terminus are constitutively nuclear, suggesting that they might act as gain of function mutations in this cellular compartment.

Clinical significance of MUC13 in pancreatic ductal adenocarcinoma.

PubMed

Khan, Sheema; Zafar, Nadeem; Khan, Shabia S; Setua, Saini; Behrman, Stephen W; Stiles, Zachary E; Yallapu, Murali M; Sahay, Peeyush; Ghimire, Hemendra; Ise, Tomoko; Nagata, Satoshi; Wang, Lei; Wan, Jim Y; Pradhan, Prabhakar; Jaggi, Meena; Chauhan, Subhash C

2018-01-15

Poor prognosis of pancreatic cancer (PanCa) is associated with lack of an effective early diagnostic biomarker. This study elucidates significance of MUC13, as a diagnostic/prognostic marker of PanCa. MUC13 was assessed in tissues using our in-house generated anti-MUC13 mouse monoclonal antibody and analyzed for clinical correlation by immunohistochemistry, immunoblotting, RT-PCR, computational and submicron scale mass-density fluctuation analyses, ROC and Kaplan Meir curve analyses. MUC13 expression was detected in 100% pancreatic intraepithelial neoplasia (PanIN) lesions (Mean composite score: MCS = 5.8; AUC >0.8, P < 0.0001), 94.6% of pancreatic ductal adenocarcinoma (PDAC) samples (MCS = 9.7, P < 0.0001) as compared to low expression in tumor adjacent tissues (MCS = 4, P < 0.001) along with faint or no expression in normal pancreatic tissues (MCS = 0.8; AUC >0.8; P < 0.0001). Nuclear MUC13 expression positively correlated with nodal metastasis (P < 0.05), invasion of cancer to peripheral tissues (P < 0.5) and poor patient survival (P < 0.05; prognostic AUC = 0.9). Submicron scale mass density and artificial intelligence based algorithm analyses also elucidated association of MUC13 with greater morphological disorder (P < 0.001) and nuclear MUC13 as strong predictor for cancer aggressiveness and poor patient survival. This study provides significant information regarding MUC13 expression/subcellular localization in PanCa samples and supporting the use anti-MUC13 MAb for the development of PanCa diagnostic/prognostic test. Copyright © 2018 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.

Proliferating cellular nuclear antigen expression as a marker of perivascular macrophages in simian immunodeficiency virus encephalitis.

PubMed

Williams, Kenneth; Schwartz, Annette; Corey, Sarah; Orandle, Marlene; Kennedy, William; Thompson, Brendon; Alvarez, Xavier; Brown, Charlie; Gartner, Suzanne; Lackner, Andrew

2002-08-01

Brain perivascular macrophages are a major target of simian immunodeficiency virus (SIV) infection in rhesus macaques and HIV infection in humans. Perivascular macrophages are distinct from parenchymal microglia in their location, morphology, expression of myeloid markers, and turnover in the CNS. In contrast to parenchymal microglia, perivascular macrophages are continuously repopulated by blood monocytes, which undergo maturation to macrophages on entering the central nervous system (CNS). We studied differences in monocyte/macrophages in vivo that might account for preferential infection of perivascular macrophages by SIV. In situ hybridization for SIV and proliferating cellular nuclear antigen (PCNA) immunohistochemistry demonstrated that SIV-infected and PCNA-positive cells were predominantly found in perivascular cuffs of viremic animals and in histopathological lesions that characterize SIV encephalitis (SIVE) in animals with AIDS. Multilabel techniques including double-label immunohistochemistry and combined in situ hybridization and immunofluorescence confocal microscopy revealed numerous infected perivascular macrophages that were PCNA-positive. Outside the CNS, SIV-infected, PCNA-expressing macrophage subpopulations were found in the small intestine and lung of animals with AIDS. While PCNA is used as a marker of cell proliferation it is also strongly expressed in non-dividing cells undergoing DNA synthesis and repair. Therefore, more specific markers for cell proliferation including Ki-67, topoisomerase IIalpha, and bromodeoxyuridine (BrdU) incorporation were used which indicated that PCNA-positive cells within SIVE lesions were not proliferating. These observations are consistent with perivascular macrophages as terminally differentiated, non-dividing cells and underscores biological differences that could potentially define mechanisms of preferential, productive infection of perivascular macrophages in the rhesus macaque model of neuroAIDS. These studies suggest that within CNS and non-CNS tissues there exist subpopulations of macrophages that are SIV-infected and express PCNA.

Distinction between papillary thyroid hyperplasia and papillary thyroid carcinoma by immunohistochemical staining for cytokeratin 19, galectin-3, and HBME-1.

PubMed

Casey, Mary B; Lohse, Christine M; Lloyd, Ricardo V

2003-01-01

The histopathology of papillary thyroid hyperplasia and papillary thyroid carcinoma is similar enough to cause a diagnostic dilemma in a few cases. Both lesions may have papillary fronds with fibrovascular cores, nuclear crowding, and nuclear anisocytosis. Formalin- fixed paraffin-embedded tissues from 30 randomly selected patients with papillary thyroid hyperplasia and an equal number from patients with papillary thyroid carcinoma were analyzed for expression of cytokeratin 19 (CK19), galectin-3, and HBME-1. Cases of papillary thyroid carcinoma had moderate to strong CK19, galectin-3, and HBME-1 reactivity although both CK19 and galectin-3 showed positive staining in a significant number of nonneoplastic thyroid cases. HBME-1 was uncommon in the nonneoplastic cases. These results indicate that HBME-1 may be useful in helping to distinguish papillary thyroid carcinoma from hyperplasia in diagnostically difficult cases.

A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export.

PubMed

Gong, Danyang; Kim, Yong Hoon; Xiao, Yuchen; Du, Yushen; Xie, Yafang; Lee, Kevin K; Feng, Jun; Farhat, Nisar; Zhao, Dawei; Shu, Sara; Dai, Xinghong; Chanda, Sumit K; Rana, Tariq M; Krogan, Nevan J; Sun, Ren; Wu, Ting-Ting

2016-11-09

Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication. Published by Elsevier Inc.

Aberrant Promoter Methylation and Expression of UTF1 during Cervical Carcinogenesis

PubMed Central

Deplus, Rachel; Lampe, Xavier; Krusy, Nathalie; Calonne, Emilie; Delbecque, Katty; Kridelka, Frederic; Fuks, François; Ennaji, My Mustapha; Delvenne, Philippe

2012-01-01

Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC) markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-2′-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression. PMID:22880087

Anti-inflammatory effect of ethanolic extract from Myagropsis myagroides on murine macrophages and mouse ear edema

PubMed Central

2012-01-01

Background This study aims to investigate anti-inflammatory effect of ethanolic extract of Myagropsis myagroides (EMM) in the lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and the phorbol 12-myristate 13-acetate (PMA)-induced ear edema in mice, and to clarify its underlying molecular mechanisms. Methods The levels of nitric oxide (NO), prostaglandin E2 (PGE2), and pro-inflammatory cytokines were measured by Griess assay and enzyme linked immunosorbent assay. The expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), mitogen-activated protein kinases (MAPKs), and Akt were measured using Western blotting. Nuclear translocation and transcriptional activation of nuclear factor-κB (NF-κB) were determined by immunocytochemistry and reporter gene assay, respectively. PMA-induced mouse ear edema was used as the animal model of inflammation. Anti-inflammatory compounds in EMM were isolated using high-performance liquid chromatography and identified by nuclear magnetic resonance. Results EMM significantly inhibited the production of NO, PGE2, and pro-inflammatory cytokines in a dose-dependent manner and suppressed the expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells. EMM strongly suppressed nuclear translocation of NF-κB by preventing degradation of inhibitor of κB-α as well as by inhibiting phosphorylation of Akt and MAPKs. EMM reduced ear edema in PMA-induced mice. One of the anti-inflammatory compounds in EMM was identified as 6,6’-bieckol. Conclusions These results suggest that the anti-inflammatory properties of EMM are associated with the down-regulation of iNOS, COX-2, and pro-inflammatory cytokines through the inhibition of NF-κB pathway in LPS-stimulated macrophages. PMID:23031211

Nuclear apoJ: A low dose radiation inducible regulator of cell death. Final report for period September 15, 1998 - September 14, 2001

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

Aronow, Bruce J.

2002-04-19

This project was based on preliminary data that was published by Dr. Boothman (Yang et al. 2000) which indicated a strong induction of apoJ gene expression, increased secretion of the protein, and accumulation of an apparently somewhat different form of the apoJ protein in the nucleus of MCF-7 breast carcinoma cells undergoing response to DNA damage. A clone expressing apoJ protein was isolated that was capable of interacting with Ku80, a component of the double strand break repair complex that is essential for the successful repair of rearranging immunoglobulin and T-cell receptor genes as evidenced by failure to produce maturemore » B and T cells in the absence of Ku70. ApoJ clones isolated and characterized by Dr. Boothman bound strongly to a Ku-70 ''bait'' protein. Over-expression of these same clones in a cell line was capable of killing the cell. ApoJ is very strongly induced in many instances of programmed cell death and has been proposed repeatedly to play some sort of effector role in the process. Our principle hypothesis for this study was that the strong induction of the apoJ gene and the particular expression of a nuclear form of the protein was potentially a causal factor in the decision point made by the cell as it attempts to repair double-strand breakage based DNA damage. The hypothesis was that if sufficiently high damage occurred, it would be deleterious to maintain the cell's viability through continued DNA repair. One method to inhibit DNA repair might be by inhibiting proteins such as Ku-70 that are necessary for double-strand break repair. If apoJ does play a critical role in tipping the decision balance over to cell death, we reasoned that deficiency of apoJ would cause increased accumulation of cells with DNA damage and that this might decrease cell death in response to DNA damage and increase tumor occurrence rates. To test this hypothesis and its potential implications, we exposed wildtype and apoJ deficient animals that we constructed through gene targeting to increasing levels of ionizing radiation from a Cesium source. Data gathered under the support of this grant application initially indicated that apoJ deficient animals were more resistant to radiation, but as we accumulated more and more data points and covered a tighter exposure range, the genotype-based differences became insignificant. However, the possibility existed that because mortality based radiation-resistance could be attributable to mechanism for which nuclear apoJ was not rate determining, we maintained a very large of colony of apoJ knockout and wildtype animals in both the C57/B16 and Cv129 strain backgrounds that were exposed to sub-lethal levels of ionizing radiation to monitor for the occurrence of tumors. These animals were allowed to fully recover and age normally in either germ free or normal animal housing. Our results demonstrated no significant differences between wildtype and apoJ knockout animals over a period that extended up to 30 months for individual animals. We recorded similar weight gain, a relatively low mortality rate, and a similar mixture and rate of sarcoma and adenocarcinomas after surviving the initial ionizing radiation exposures. Thus we conclude that apoJ gene function, which was totally eliminated by our gene targeting, did not influence radiation sensitivity or serve as a tumor suppressor in response to DNA damage.« less

Gender-Specific Potential Inhibitory Role of Ca2+/Calmodulin Dependent Protein Kinase Phosphatase (CaMKP) in Pressure-Overloaded Mouse Heart

PubMed Central

Prévilon, Miresta; Pezet, Mylène; Vinet, Laurent; Mercadier, Jean-Jacques; Rouet-Benzineb, Patricia

2014-01-01

Background Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) has been proposed as a potent regulator of multifunctional Ca2+/calmodulin-dependent protein kinases (i.e., CaMKII). The CaMKII-dependent activation of myocyte enhancer factor 2 (MEF2) disrupts interactions between MEF2-histone deacetylases (HDACs), thereby de-repressing downstream gene transcription. Whether CaMKP modulates the CaMKII- MEF2 pathway in the heart is unknown. Here, we investigated the molecular and functional consequences of left ventricular (LV) pressure overload in the mouse of both genders, and in particular we evaluated the expression levels and localization of CaMKP and its association with CaMKII-MEF2 signaling. Methodology and Principal Findings Five week-old B6D1/F1 mice of both genders underwent a sham-operation or thoracic aortic constriction (TAC). Thirty days later, TAC was associated with pathological LV hypertrophy characterized by systolic and diastolic dysfunction. Gene expression was assessed by real-time PCR. Fetal gene program re-expression comprised increased RNA levels of brain natriuretic peptide and alpha-skeletal actin. Mouse hearts of both genders expressed both CaMKP transcript and protein. Activation of signalling pathways was studied by Western blot in LV lysates or subcellular fractions (nuclear and cytoplasmic). TAC was associated with increased CaMKP expression in male LVs whereas it tended to be decreased in females. The DNA binding activity of MEF2 was determined by spectrophotometry. CaMKP compartmentalization differed according to gender. In male TAC mice, nuclear CaMKP was associated with inactive CaMKII resulting in less MEF2 activation. In female TAC mice, active CaMKII (phospho-CaMKII) detected in the nuclear fraction, was associated with a strong MEF2 transcription factor-binding activity. Conclusions/Significance Gender-specific CaMKP compartmentalization is associated with CaMKII-mediated MEF2 activation in pressure-overloaded hearts. Therefore, CaMKP could be considered as an important novel cellular target for the development of new therapeutic strategies for heart diseases. PMID:24608696

Gender-specific potential inhibitory role of Ca2+/calmodulin dependent protein kinase phosphatase (CaMKP) in pressure-overloaded mouse heart.

PubMed

Prévilon, Miresta; Pezet, Mylène; Vinet, Laurent; Mercadier, Jean-Jacques; Rouet-Benzineb, Patricia

2014-01-01

Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) has been proposed as a potent regulator of multifunctional Ca2+/calmodulin-dependent protein kinases (i.e., CaMKII). The CaMKII-dependent activation of myocyte enhancer factor 2 (MEF2) disrupts interactions between MEF2-histone deacetylases (HDACs), thereby de-repressing downstream gene transcription. Whether CaMKP modulates the CaMKII- MEF2 pathway in the heart is unknown. Here, we investigated the molecular and functional consequences of left ventricular (LV) pressure overload in the mouse of both genders, and in particular we evaluated the expression levels and localization of CaMKP and its association with CaMKII-MEF2 signaling. Five week-old B6D1/F1 mice of both genders underwent a sham-operation or thoracic aortic constriction (TAC). Thirty days later, TAC was associated with pathological LV hypertrophy characterized by systolic and diastolic dysfunction. Gene expression was assessed by real-time PCR. Fetal gene program re-expression comprised increased RNA levels of brain natriuretic peptide and alpha-skeletal actin. Mouse hearts of both genders expressed both CaMKP transcript and protein. Activation of signalling pathways was studied by Western blot in LV lysates or subcellular fractions (nuclear and cytoplasmic). TAC was associated with increased CaMKP expression in male LVs whereas it tended to be decreased in females. The DNA binding activity of MEF2 was determined by spectrophotometry. CaMKP compartmentalization differed according to gender. In male TAC mice, nuclear CaMKP was associated with inactive CaMKII resulting in less MEF2 activation. In female TAC mice, active CaMKII (phospho-CaMKII) detected in the nuclear fraction, was associated with a strong MEF2 transcription factor-binding activity. Gender-specific CaMKP compartmentalization is associated with CaMKII-mediated MEF2 activation in pressure-overloaded hearts. Therefore, CaMKP could be considered as an important novel cellular target for the development of new therapeutic strategies for heart diseases.

Modulation of Nrf2 by Olive Oil and Wine Polyphenols and Neuroprotection

PubMed Central

Martínez-Huélamo, Miriam; Boronat, Anna; de la Torre, Rafael

2017-01-01

Strong adherence to a Mediterranean diet is associated with improved cognitive function and a lower prevalence of mild cognitive impairment. Olive oil and red wine are rich sources of polyphenols which are responsible in part for the beneficial effects on cognitive functioning. Polyphenols induce endogenous antioxidant defense mechanisms by modulating transcription factors such as the nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This review discusses the scientific data supporting the modulating effect of olive oil and red wine polyphenols on Nrf2 expression, and the potential health benefits associated with cognitive functioning. PMID:28954417

Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A).

PubMed

Oi, Ami; Katayama, Syouichi; Hatano, Naoya; Sugiyama, Yasunori; Kameshita, Isamu; Sueyoshi, Noriyuki

2017-01-08

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization. Copyright © 2016 Elsevier Inc. All rights reserved.

Protective Effect of Flavonoids from Ziziphus jujuba cv. Jinsixiaozao against Acetaminophen-Induced Liver Injury by Inhibiting Oxidative Stress and Inflammation in Mice.

PubMed

Huang, Weizhen; Wang, Yongjie; Jiang, Xiaoyan; Sun, Yueyue; Zhao, Zhongxi; Li, Siying

2017-10-20

This study was aimed to investigate the chemical composition, antioxidant activities and hepatoprotective effect of flavonoids from Ziziphus jujub a cv. Jinsixiaozao (ZJF). The composition of ZJF was analyzed by high performance liquid chromatography (HPLC) and Liquid chromatography-mass spectrometry (LC-MS), and antioxidant properties were investigated by biological assays in vitro. The hepatoprotective activity of ZJF was evaluated in acetaminophen (APAP)-treated BALB/c mice. Results indicate that ZJF displayed significant antioxidant capacity. Pretreatment with ZJF significantly decreased APAP-elevated serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin (TB). Activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were enhanced with ZJF administration, while malondialdehyde (MDA) level and glutathione (GSH) depletion were reduced. Meanwhile, ZJF reversed the suppression of nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, and up-regulated the protein expression of NAD(P)H: quinone oxidoreductase 1(NQO1) in liver damage mice. Furthermore, ZJF attenuated APAP-induced inflammatory mediator production, such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β). Expression of p65 showed that ZJF dampened nuclear factor-κB (NF-κB) activation. The results strongly indicate that the hepatoprotective role of ZJF in APAP-induced hepatotoxicity might result from its induction of antioxidant defense via activation of Nrf2 and reduction of inflammation via inhibition of NF-κB.

6-Shogaol is more effective than 6-gingerol and curcumin in inhibiting 12-O-tetradecanoylphorbol 13-acetate-induced tumor promotion in mice.

PubMed

Wu, Hou; Hsieh, Min-Chi; Lo, Chih-Yu; Liu, Cheng Bin; Sang, Shengmin; Ho, Chi-Tang; Pan, Min-Hsiung

2010-09-01

We previously reported that 6-shogaol strongly suppressed lipopolysaccharide-induced overexpression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in murine macrophages. In this study, we further compared curcumin, 6-gingerol, and 6-shogaol's molecular mechanism of action and their anti-tumor properties. We demonstrate that topical application of 6-shogaol more effectively inhibited 12-O-tetradecanoylphorbol 13-acetate (TPA)-stimulated transcription of iNOS and COX-2 mRNA expression in mouse skin than curcumin and 6-gingerol. Pretreatment with 6-shogaol has resulted in the reduction of TPA-induced nuclear translocation of the nuclear factor-kappaB subunits. 6-Shogaol also reduced TPA-induced phosphorylation of IkappaBalpha and p65, and caused subsequent degradation of IkappaBalpha. Moreover, 6-shogaol markedly suppressed TPA-induced activation of extracellular signal-regulate kinase1/2, p38 mitogen-activated protein kinase, JNK1/2, and phosphatidylinositol 3-kinase/Akt, which are upstream of nuclear factor-kappaB and AP-1. Furthermore, 6-shogaol significantly inhibited 7,12-dimethylbenz[a]anthracene/TPA-induced skin tumor formation measured by the tumor multiplicity of papillomas at 20 wk. Presented data reveal for the first time that 6-shogaol is an effective anti-tumor agent that functions by down-regulating inflammatory iNOS and COX-2 gene expression in mouse skin. It is suggested that 6-shogaol is a novel functional agent capable of preventing inflammation-associated tumorigenesis.

Abnormal expression of p27kip1 protein in levator ani muscle of aging women with pelvic floor disorders – a relationship to the cellular differentiation and degeneration

PubMed Central

Bukovsky, Antonin; Copas, Pleas; Caudle, Michael R; Cekanova, Maria; Dassanayake, Tamara; Asbury, Bridgett; Van Meter, Stuart E; Elder, Robert F; Brown, Jeffrey B; Cross, Stephanie B

2001-01-01

Background Pelvic floor disorders affect almost 50% of aging women. An important role in the pelvic floor support belongs to the levator ani muscle. The p27/kip1 (p27) protein, multifunctional cyclin-dependent kinase inhibitor, shows changing expression in differentiating skeletal muscle cells during development, and relatively high levels of p27 RNA were detected in the normal human skeletal muscles. Methods Biopsy samples of levator ani muscle were obtained from 22 symptomatic patients with stress urinary incontinence, pelvic organ prolapse, and overlaps (age range 38–74), and nine asymptomatic women (age 31–49). Cryostat sections were investigated for p27 protein expression and type I (slow twitch) and type II (fast twitch) fibers. Results All fibers exhibited strong plasma membrane (and nuclear) p27 protein expression. cytoplasmic p27 expression was virtually absent in asymptomatic women. In perimenopausal symptomatic patients (ages 38–55), muscle fibers showed hypertrophy and moderate cytoplasmic p27 staining accompanied by diminution of type II fibers. Older symptomatic patients (ages 57–74) showed cytoplasmic p27 overexpression accompanied by shrinking, cytoplasmic vacuolization and fragmentation of muscle cells. The plasma membrane and cytoplasmic p27 expression was not unique to the muscle cells. Under certain circumstances, it was also detected in other cell types (epithelium of ectocervix and luteal cells). Conclusions This is the first report on the unusual (plasma membrane and cytoplasmic) expression of p27 protein in normal and abnormal human striated muscle cells in vivo. Our data indicate that pelvic floor disorders are in perimenopausal patients associated with an appearance of moderate cytoplasmic p27 expression, accompanying hypertrophy and transition of type II into type I fibers. The patients in advanced postmenopause show shrinking and fragmentation of muscle fibers associated with strong cytoplasmic p27 expression. PMID:11696252

Silencing the epigenetic silencer KDM4A for TRAIL and DR5 simultaneous induction and antitumor therapy.

PubMed

Wang, Junjian; Wang, Haibin; Wang, Ling-Yu; Cai, Demin; Duan, Zhijian; Zhang, Yanhong; Chen, Peng; Zou, June X; Xu, Jianzhen; Chen, Xinbin; Kung, Hsing-Jien; Chen, Hong-Wu

2016-11-01

Recombinant TRAIL and agonistic antibodies to death receptors (DRs) have been in clinical trial but displayed limited anti-cancer efficacy. Lack of functional DR expression in tumors is a major limiting factor. We report here that chromatin regulator KDM4A/JMJD2A, not KDM4B, has a pivotal role in silencing tumor cell expression of both TRAIL and its receptor DR5. In TRAIL-sensitive and -resistant cancer cells of lung, breast and prostate, KDM4A small-molecule inhibitor compound-4 (C-4) or gene silencing strongly induces TRAIL and DR5 expression, and causes TRAIL-dependent apoptotic cell death. KDM4A inhibition also strongly sensitizes cells to TRAIL. C-4 alone potently inhibits tumor growth with marked induction of TRAIL and DR5 expression in the treated tumors and effectively sensitizes them to the newly developed TRAIL-inducer ONC201. Mechanistically, C-4 does not appear to act through the Akt-ERK-FOXO3a pathway. Instead, it switches histone modifying enzyme complexes at promoters of TRAIL and DR5 transcriptional activator CHOP gene by dissociating KDM4A and nuclear receptor corepressor (NCoR)-HDAC complex and inducing the recruitment of histone acetylase CBP. Thus, our results reveal KDM4A as a key epigenetic silencer of TRAIL and DR5 in tumors and establish inhibitors of KDM4A as a novel strategy for effectively sensitizing tumors to TRAIL pathway-based therapeutics.

Silencing the epigenetic silencer KDM4A for TRAIL and DR5 simultaneous induction and antitumor therapy

PubMed Central

Wang, Junjian; Wang, Haibin; Wang, Ling-Yu; Cai, Demin; Duan, Zhijian; Zhang, Yanhong; Chen, Peng; Zou, June X; Xu, Jianzhen; Chen, Xinbin; Kung, Hsing-Jien; Chen, Hong-Wu

2016-01-01

Recombinant TRAIL and agonistic antibodies to death receptors (DRs) have been in clinical trial but displayed limited anti-cancer efficacy. Lack of functional DR expression in tumors is a major limiting factor. We report here that chromatin regulator KDM4A/JMJD2A, not KDM4B, has a pivotal role in silencing tumor cell expression of both TRAIL and its receptor DR5. In TRAIL-sensitive and -resistant cancer cells of lung, breast and prostate, KDM4A small-molecule inhibitor compound-4 (C-4) or gene silencing strongly induces TRAIL and DR5 expression, and causes TRAIL-dependent apoptotic cell death. KDM4A inhibition also strongly sensitizes cells to TRAIL. C-4 alone potently inhibits tumor growth with marked induction of TRAIL and DR5 expression in the treated tumors and effectively sensitizes them to the newly developed TRAIL-inducer ONC201. Mechanistically, C-4 does not appear to act through the Akt-ERK-FOXO3a pathway. Instead, it switches histone modifying enzyme complexes at promoters of TRAIL and DR5 transcriptional activator CHOP gene by dissociating KDM4A and nuclear receptor corepressor (NCoR)-HDAC complex and inducing the recruitment of histone acetylase CBP. Thus, our results reveal KDM4A as a key epigenetic silencer of TRAIL and DR5 in tumors and establish inhibitors of KDM4A as a novel strategy for effectively sensitizing tumors to TRAIL pathway-based therapeutics. PMID:27612013

LEF1 is preferentially expressed in the tubal-peritoneal junctions and is a reliable marker of tubal intraepithelial lesions

PubMed Central

Schmoeckel, Elisa; Odai-Afotey, Ashley A.; Schleiβheimer, Michael; Rottmann, Miriam; Flesken-Nikitin, Andrea; Ellenson, Lora H.; Kirchner, Thomas; Mayr, Doris; Nikitin, Alexander Yu.

2017-01-01

Recently it has been reported that serous tubal intraepithelial carcinoma (STIC), the likely precursor of ovarian/extra-uterine high-grade serous carcinoma, are frequently located in the vicinity of tubal-peritoneal junctions, consistent with the cancer-prone features of many epithelial transitional regions. To test if p53 (aka TP53)-signatures and secretory cell outgrowths (SCOUTs) also localize to tubal-peritoneal junctions, we examined these lesions in the fallopian tubes of patients undergoing salpingo-oophorectomy for sporadic high-grade serous carcinomas or as a prophylactic procedure for carriers of familial BRCA1 or 2 mutations. STICs were located closest to the tubal-peritoneal junctions with an average distance of 1.31 mm, while SCOUTs were not detected in the fimbriated end of the fallopian tube. Since many epithelial transitional regions contain stem cells, we also determined the expression of stem cell markers in the normal fallopian tube, tubal intraepithelial lesions and high-grade serous carcinomas. Of those, LEF1 was consistently expressed in the tubal-peritoneal junctions and all lesions, independent of p53 status. All SCOUTs demonstrated strong nuclear expression of β-catenin consistent with the LEF1 participation in the canonical WNT pathway. However, β-catenin was preferentially located in the cytoplasm of cells comprising STICs and p53 signatures, suggesting WNT-independent function of LEF1 in those lesions. Both frequency of LEF1 expression and β-catenin nuclear expression correlated with the worst 5 year patient survival, supporting important role of both proteins in high-grade serous carcinoma. Taken together, our findings suggest the existence of stem cell niche within the tubal-peritoneal junctions. Furthermore, they support the notion that the pathogenesis of SCOUTs is distinct from that of STICs and p53 signatures. The location and discrete patterns of LEF1 and β-catenin expression may serve as highly sensitive and reliable ancillary markers for the detection and differential diagnosis of tubal intraepithelial lesions. PMID:28664938

Differential expression of steroid 5alpha-reductase isozymes and association with disease severity and angiogenic genes predict their biological role in prostate cancer.

PubMed

Das, Kakoli; Lorena, Pia D N; Ng, Lai Kuan; Lim, Diana; Shen, Liang; Siow, Woei Yun; Teh, Ming; Reichardt, Juergen K V; Salto-Tellez, Manuel

2010-09-01

The biological role of steroid 5alpha-reductase isozymes (encoded by the SRD5A1 and SRD5A2 genes) and angiogenic factors that play important roles in the pathogenesis and vascularization of prostate cancer (PC) is poorly understood. The sub-cellular expression of these isozymes and vascular endothelial growth factor (VEGF) in PC tissue microarrays (n=62) was examined using immunohistochemistry. The effect of SRD5A inhibition on the angiogenesis pathway genes in PC was also examined in prostate cell lines, LNCaP, PC3, and RWPE-1, by treating them with the SRD5A inhibitors finasteride and dutasteride, followed by western blot, quantitative PCR, and ELISA chip array techniques. In PC tissues, nuclear SRD5A1 expression was strongly associated with higher cancer Gleason scores (P=0.02), higher cancer stage (P=0.01), and higher serum prostate specific antigen (PSA) levels (P=0.01), whereas nuclear SRD5A2 expression was correlated with VEGF expression (P=0.01). Prostate tumor cell viability was significantly reduced in dutasteride-treated PC3 and RWPE-1 cells compared with finasteride-treated groups. Expression of the angiogenesis pathway genes transforming growth factor beta 1 (TGFB1), endothelin (EDN1), TGFalpha (TGFA), and VEGFR1 was upregulated in LNCaP cells, and at least 7 out of 21 genes were upregulated in PC3 cells treated with finasteride (25 muM). Our findings suggest that SRD5A1 expression predominates in advanced PC, and that inhibition of SRD5A1 and SRD5A2 together was more effective in reducing cell numbers than inhibition of SRD5A2 alone. However, these inhibitors did not show any significant difference in prostate cell angiogenic response. Interestingly, some angiogenic genes remained activated after treatment, possibly due to the duration of treatment and tumor resistance to inhibitors.

Chemopreventive effects of a curcumin-like diarylpentanoid [2,6-bis(2,5-dimethoxybenzylidene)cyclohexanone] in cellular targets of rheumatoid arthritis in vitro.

PubMed

Lee, Ka-Heng; Abas, Faridah; Mohamed Alitheen, Noorjahan Banu; Shaari, Khozirah; Lajis, Nordin Haji; Israf, Daud Ahmad; Syahida, Ahmad

2015-07-01

Synovial fibroblast has emerged as a potential cellular target in progressive joint destruction in rheumatoid arthritis development. In this study, BDMC33 (2,6-bis[2,5-dimethoxybenzylidene]cyclohexanone), a curcumin analogue with enhanced anti-inflammatory activity has been synthesized and the potency of BDMC33 on molecular and cellular basis of synovial fibroblasts (SF) were evaluated in vitro. Synovial fibroblast cells (HIG-82) were cultured in vitro and induced by phorbol-12-myristate acetate (PMA) to stimulate the expression of matrix metalloproteinase (MMPs) and pro-inflammatory cytokines. The protective effects of BDMC33 were evaluated toward MMP activities, pro-inflammatory cytokine expression and nuclear factor kappa-B (NF-κB) activation by using various bioassay methods, including zymography, Western blotting, reverse transcription polymerase chain reaction, immunofluorescense microscopy and electrophoretic mobility shift assay. The results showed that BDMC33 significantly inhibited the pro-gelatinase B (pro-MMP-9) and collagenase activities via suppression of MMP-1 in activated SF. In addition, BDMC33 strongly suppressed MMP-3 gene expression as well as inhibited COX-2 and IL-6 pro-inflammatory gene expression. We also demonstrated that BDMC33 abolished the p65 NF-κB nuclear translocation and NF-κB DNA binding activity in PMA-stimulated SF. BDMC33 represents an effective chemopreventive agent and could be used as a promising lead compound for further development of rheumatoid arthritis therapeutic intervention. © 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.

Cross-linked matrix rigidity and soluble retinoids synergize in nuclear lamina regulation of stem cell differentiation.

PubMed

Ivanovska, Irena L; Swift, Joe; Spinler, Kyle; Dingal, Dave; Cho, Sangkyun; Discher, Dennis E

2017-07-07

Synergistic cues from extracellular matrix and soluble factors are often obscure in differentiation. Here the rigidity of cross-linked collagen synergizes with retinoids in the osteogenesis of human marrow mesenchymal stem cells (MSCs). Collagen nanofilms serve as a model matrix that MSCs can easily deform unless the film is enzymatically cross-linked, which promotes the spreading of cells and the stiffening of nuclei as both actomyosin assembly and nucleoskeletal lamin-A increase. Expression of lamin-A is known to be controlled by retinoic acid receptor (RAR) transcription factors, but soft matrix prevents any response to any retinoids. Rigid matrix is needed to induce rapid nuclear accumulation of the RARG isoform and for RARG-specific antagonist to increase or maintain expression of lamin-A as well as for RARG-agonist to repress expression. A progerin allele of lamin-A is regulated in the same manner in iPSC-derived MSCs. Rigid matrices are further required for eventual expression of osteogenic markers, and RARG-antagonist strongly drives lamin-A-dependent osteogenesis on rigid substrates, with pretreated xenografts calcifying in vivo to a similar extent as native bone. Proteomics-detected targets of mechanosensitive lamin-A and retinoids underscore the convergent synergy of insoluble and soluble cues in differentiation. © 2017 Ivanovska 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).

Epigallocatechin-3-gallate prevents lipid peroxidation and enhances antioxidant defense system via modulating hepatic nuclear transcription factors in heat-stressed quails.

PubMed

Sahin, K; Orhan, C; Tuzcu, M; Ali, S; Sahin, N; Hayirli, A

2010-10-01

Epigallocatechin-3-gallate (EGCG), a polyphenol derived from green tea, exerts antioxidant effects. Oxidative stress is one of the consequences of heat stress (HS), which also depresses performance in poultry. This experiment was conducted to elucidate the action mode of EGCG in alleviation of oxidative stress in heat-stressed quail (Coturnix coturnix japonica). A total of 180 five-week-old female Japanese quails were reared either at 22°C for 24 h/d (thermoneutral, TN) or 34°C for 8 h/d (HS) for 12 wk. Birds in both environments were randomly fed 1 of 3 diets: basal diet and basal diet added with 200 or 400 mg of EGCG/kg of diet. Each of the 2×3 factorially arranged groups was replicated in 10 cages, each containing 3 quails. Performance variables [feed intake (FI) and egg production (EP)], oxidative stress biomarkers [malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px)] and hepatic transcription factors [nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2)] were analyzed using 2-way ANOVA. Exposure to HS caused reductions in FI by 9.7% and EP by 14.4%, increased hepatic MDA level by 84.8%, and decreased hepatic SOD, CAT, and GSH-Px activities by 25.8, 52.3, and 45.5%, respectively (P<0.0001 for all). The hepatic NF-κB expression was greater (156 vs. 82%) and Nrf2 expression was lower (84 vs. 118%) for quails reared under the HS environment than for those reared under the TN environment (P<0.0001 for both). In response to increasing supplemental EGCG level, there were linear increases in FI from 29.6 to 30.9 g/d and EP from 84.3 to 90.1%/d, linear decreases in hepatic MDA level from 2.82 to 1.72 nmol/g and Nrf2 expression from 77.5 to 123.3%, and linear increases in hepatic SOD (146.4 to 182.2), CAT (36.2 to 47.1), and GSH-Px (13.5 to 18.5) activities (U/mg of protein) and NF-κB expression (149.7 to 87.3%) (P<0.0001 for all). Two-way treatment interactions revealed that the degree of restorations in all response variables was more notable under the HS environment than under the TN environment as supplemental EGCG level was increased. Moreover, levels of oxidative biomarkers were strongly correlated with expressions of hepatic nuclear transcription factors. In conclusion, supplemental EGCG alleviates oxidative stress through modulating the hepatic nuclear transcription factors in heat-stressed quails.

Immunohistochemistry panel segregates molecular types of hepatocellular carcinoma in Brazilian autopsy cases

PubMed Central

Felipe-Silva, Aloísio; Wakamatsu, Alda; dos Santos Cirqueira, Cinthya; Alves, Venâncio Avancini Ferreira

2016-01-01

AIM: To assess the distribution of proteins coded by genes reported as relevant for the molecular classification of hepatocellular carcinoma (HCC). METHODS: In this retrospective cross-sectional study, the following clinicopathological data were analyzed in 80 autopsied HCC patients: sex, age, ethnicity, alcohol intake, infection with hepatitis B and/or C virus, infection with human immunodeficiency virus, prior treatment, basic and immediate causes of death, liver weight, presence of cirrhosis, number and size of nodules, gross pattern, histological grade and variants, architectural pattern, invasion of large veins, and presence and location of extrahepatic metastases. The protein products of genes known to be involved in molecular pathogenesis of HCC, including epidermal growth factor receptor (EGFR), MET, keratin 19 (K19), vimentin, beta-catenin, mechanistic target of rapamycin (mTOR), extracellular signaling-related kinase (ERK)1, ERK2, Ki67, cyclin D1, caspase 3 and p53, were detected by immunohistochemistry on tissue microarrays. The expression levels were scored and statistically assessed for correlation with HCC parameters. RESULTS: Infection with hepatitis C virus was identified in 49% of the 80 autopsy patients, cirrhosis in 90%, advanced tumors in 95%, and extrahepatic metastases in 38%. Expression of K19, p53 and ERK1 correlated to high-grade lesions. Expression of ERK1, nuclear beta-catenin, cyclin D1 and ERK2 correlated to higher rates of cell proliferation as determined by Ki67. Expression of MET, EGFR (> 0) and caspase 3 correlated with lower histological grades. Expression of EGFR correlated to that of caspase 3, and overexpression of EGFR (≥ 200/300) was observed in low-grade tumors more frequently (grades 1 and 2: 67% vs grade 3: 27% and grade 4: 30%). Expression of ERK1 was associated with that of K19 and vimentin, whereas expression of ERK2 was associated with that of cyclin D1, MET and membrane beta-catenin. Expression of vimentin was strongly correlated with that of K19. CONCLUSION: Expression of K19, p53, ERK1, ERK2, vimentin and nuclear beta-catenin was related to higher-grade markers, as opposed to expression/overexpression of EGFR, MET and caspase 3. PMID:27468214

Expression profile of human tissue kallikrein 15 provides preliminary insights into its roles in the prostate and testis.

PubMed

Filippou, Panagiota S; Ren, Annie H; Soosaipillai, Antoninus; Papaioannou, Michail-Dimitrios; Korbakis, Dimitrios; Safar, Roaa; Diamandis, Eleftherios P; Conner, James

2018-06-26

Human tissue kallikrein 15 (KLK15) is the latest member of the kallikrein-related peptidase family. Little is known about the pathophysiological roles of KLK15. Previous studies implied a role of KLK15 in prostate cancer. In the present study, we examined KLK15 protein expression using a new immunoassay (ELISA) and immunohistochemistry (IHC). Highest KLK15 levels were detected in the testis and seminal fluid, whereas lower levels were observed in prostate and other tissues. Immunohistochemical analysis of testis suggests that KLK15 is strongly expressed in mature spermatids, but not in immature germ cells. KLK15 displayed predominantly nuclear localization in the basal cell layer of the prostatic epithelium. We also measured KLK15 in supernatants of various cell lines. Highest KLK15 levels were primarily detected in prostate cancer cell lines and KLK15 expression was hormone-independent, in contrast to KLK3. Collectively, our data provide insights into the localization and possible role of KLK15 in human physiology. Copyright © 2018. Published by Elsevier Inc.

Prognostic impact of c-Rel nuclear expression and REL amplification and crosstalk between c-Rel and the p53 pathway in diffuse large B-cell lymphoma

PubMed Central

Ok, Chi Young; Tzankov, Alexandar; Manyam, Ganiraju C.; Sun, Ruifan; Visco, Carlo; Zhang, Mingzhi; Montes-Moreno, Santiago; Dybkaer, Karen; Chiu, April; Orazi, Attilio; Zu, Youli; Bhagat, Govind; Richards, Kristy L.; Hsi, Eric D.; Choi, William W.L.; van Krieken, J. Han; Huh, Jooryung; Ponzoni, Maurilio; Ferreri, Andrés J.M.; Møller, Michael B.; Wang, Jinfeng; Parsons, Ben M.; Winter, Jane N.; Piris, Miguel A.; Pham, Lan V.; Medeiros, L. Jeffrey; Young, Ken H.

2015-01-01

Dysregulated NF-κB signaling is critical for lymphomagenesis. The regulation, function, and clinical relevance of c-Rel/NF-κB activation in diffuse large B-cell lymphoma (DLBCL) have not been well studied. In this study we analyzed the prognostic significance and gene-expression signature of c-Rel nuclear expression as surrogate of c-Rel activation in 460 patients with de novo DLBCL. Nuclear c-Rel expression, observed in 137 (26.3%) DLBCL patients frequently associated with extranoal origin, did not show significantly prognostic impact in the overall- or germinal center B-like-DLBCL cohort, likely due to decreased pAKT and Myc levels, up-regulation of FOXP3, FOXO3, MEG3 and other tumor suppressors coincided with c-Rel nuclear expression, as well as the complicated relationships between NF-κB members and their overlapping function. However, c-Rel nuclear expression correlated with significantly poorer survival in p63+ and BCL-2− activated B-cell-like-DLBCL, and in DLBCL patients with TP53 mutations. Multivariate analysis indicated that after adjusting clinical parameters, c-Rel positivity was a significantly adverse prognostic factor in DLBCL patients with wild type TP53. Gene expression profiling suggested dysregulations of cell cycle, metabolism, adhesion, and migration associated with c-Rel activation. In contrast, REL amplification did not correlate with c-Rel nuclear expression and patient survival, likely due to co-amplification of genes that negatively regulate NF-κB activation. These insights into the expression, prognostic impact, regulation and function of c-Rel as well as its crosstalk with the p53 pathway underscore the importance of c-Rel and have significant therapeutic implications. PMID:26324762

A Novel Nuclear Trafficking Module Regulates the Nucleocytoplasmic Localization of the Rabies Virus Interferon Antagonist, P Protein*

PubMed Central

Oksayan, Sibil; Wiltzer, Linda; Rowe, Caitlin L.; Blondel, Danielle; Jans, David A.; Moseley, Gregory W.

2012-01-01

Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1–P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3–P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection. PMID:22700958

Tlx acts as a proangiogenic switch by regulating extracellular assembly of fibronectin matrices in retinal astrocytes.

PubMed

Uemura, Akiyoshi; Kusuhara, Sentaro; Wiegand, Stanley J; Yu, Ruth T; Nishikawa, Shin-ichi

2006-02-01

In response to hypoxia, hypoxia-inducible factors act as the primary proangiogenic triggers by regulating transcription levels of target genes, including VEGF. However, little is known about the specific factors that control other components of the angiogenic process, particularly formation of matrix scaffolds that promote adhesion and migration of endothelial cells. We show that in the postnatal mouse retina, the orphan nuclear receptor tailless (Tlx) is strongly expressed in the proangiogenic astrocytes, which secrete VEGF and fibronectin. Tlx expression by retinal astrocytes is controlled by oxygen concentration and rapidly downregulated upon contact with blood vessels. In mice null for Tlx, retinal astrocytes maintain VEGF expression; however, the extracellular assembly of fibronectin matrices by astrocytes is severely impaired, leading to defective scaffold formation and a complete failure of normal retinal vascular development. This work identifies Tlx as an essential component of the molecular network involved in the hypoxia-inducible proangiogenic switch in retinal astrocytes.

Tlx acts as a proangiogenic switch by regulating extracellular assembly of fibronectin matrices in retinal astrocytes

PubMed Central

Uemura, Akiyoshi; Kusuhara, Sentaro; Wiegand, Stanley J.; Yu, Ruth T.; Nishikawa, Shin-Ichi

2006-01-01

In response to hypoxia, hypoxia-inducible factors act as the primary proangiogenic triggers by regulating transcription levels of target genes, including VEGF. However, little is known about the specific factors that control other components of the angiogenic process, particularly formation of matrix scaffolds that promote adhesion and migration of endothelial cells. We show that in the postnatal mouse retina, the orphan nuclear receptor tailless (Tlx) is strongly expressed in the proangiogenic astrocytes, which secrete VEGF and fibronectin. Tlx expression by retinal astrocytes is controlled by oxygen concentration and rapidly downregulated upon contact with blood vessels. In mice null for Tlx, retinal astrocytes maintain VEGF expression; however, the extracellular assembly of fibronectin matrices by astrocytes is severely impaired, leading to defective scaffold formation and a complete failure of normal retinal vascular development. This work identifies Tlx as an essential component of the molecular network involved in the hypoxia-inducible proangiogenic switch in retinal astrocytes. PMID:16424942

RIP4 is a target of multiple signal transduction pathways in keratinocytes: Implications for epidermal differentiation and cutaneous wound repair

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

Adams, Stephanie; Munz, Barbara, E-mail: barbara.munz@charite.de

2010-01-01

Receptor interacting protein 4 (RIP4) is an important regulator of epidermal morphogenesis during embryonic development. We could previously show that expression of the rip4 gene is strongly downregulated in cutaneous wound repair, which might be initiated by a broad variety of growth factors and cytokines. Here, we demonstrate that in keratinocytes, rip4 expression is controlled by a multitude of different signal transduction pathways, such as the p38 mitogen-activated protein kinase (MAPK) and the nuclear factor kappa B (NF-{kappa}B) cascade, in a unique and specific manner. Furthermore, we show that the steroid dexamethasone abolishes the physiological rip4 downregulation after injury andmore » might thus contribute to the phenotype of reduced and delayed wound reepithelialization seen in glucocorticoid-treated patients. As a whole, our data indicate that rip4 expression is regulated in a complex manner, which might have therapeutic implications.« less

RNA interference can target pre-mRNA: consequences for gene expression in a Caenorhabditis elegans operon.

PubMed Central

Bosher, J M; Dufourcq, P; Sookhareea, S; Labouesse, M

1999-01-01

In nematodes, flies, trypanosomes, and planarians, introduction of double-stranded RNA results in sequence-specific inactivation of gene function, a process termed RNA interference (RNAi). We demonstrate that RNAi against the Caenorhabditis elegans gene lir-1, which is part of the lir-1/lin-26 operon, induced phenotypes very different from a newly isolated lir-1 null mutation. Specifically, lir-1(RNAi) induced embryonic lethality reminiscent of moderately strong lin-26 alleles, whereas the lir-1 null mutant was viable. We show that the lir-1(RNAi) phenotypes resulted from a severe loss of lin-26 gene expression. In addition, we found that RNAi directed against lir-1 or lin-26 introns induced similar phenotypes, so we conclude that lir-1(RNAi) targets the lir-1/lin-26 pre-mRNA. This provides direct evidence that RNA interference can prevent gene expression by targeting nuclear transcripts. Our results highlight that caution may be necessary when interpreting RNA interference without the benefit of mutant alleles. PMID:10545456

Nuclear Expression of GS28 Protein: A Novel Biomarker that Predicts Prognosis in Colorectal Cancers

PubMed Central

Lee, Sung Hak; Yoo, Hyung Jae; Rim, Do Eun; Cui, Yinji; Lee, Ahwon; Jung, Eun Sun; Oh, Seung Taek; Kim, Jun Gi; Kwon, Oh-Joo; Kim, Su Young; Jeong, Seong-Whan

2017-01-01

Aims: GS28 (Golgi SNARE protein, 28 kDa), a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) protein family, plays a critical role in mammalian endoplasmic reticulum (ER)-Golgi or intra-Golgi vesicle transport. To date, few researches on the GS28 protein in human cancer tissues have been reported. In this study, we assessed the prognostic value of GS28 in patients with colorectal cancer (CRC). Methods and results: We screened for GS28 expression using immunohistochemistry in 230 surgical CRC specimens. The CRCs were right-sided and left-sided in 28.3% (65/230) and 71.3% (164/230) of patients, respectively. GS28 staining results were available in 214 cases. Among these, there were 26 nuclear predominant cases and 188 non-nuclear predominant cases. Stromal GS28 expression was noted in 152 cases of CRC. GS28 nuclear predominant immunoreactivity was significantly associated with advanced tumour stage (p = 0.045) and marginally associated with perineural invasion (p = 0.064). Decreased GS28 expression in the stromal cells was significantly associated with lymph node metastasis (N stage; p = 0.036). GS28 expression was not associated with epidermal growth factor receptor (EGFR) immunohistochemical positivity or KRAS mutation status. Investigation of the prognostic value of GS28 with Kaplan-Meier analysis revealed a correlation with overall survival (p = 0.004). Cases with GS28 nuclear predominant expression had significantly poorer overall survival than those with a non-nuclear predominant pattern. Conclusions: Taken together, these results indicate that GS28 nuclear predominant expression could serve as a prognostic marker for CRC and may help in identifying aggressive forms of CRC. PMID:28638266

Correlated non-nuclear COX2 and low HER2 expression confers a good prognosis in colorectal cancer.

PubMed

Zhou, Fei-Fei; Huang, Rong; Jiang, Jun; Zeng, Xiao-Hong; Zou, Shu-Qian

2018-06-05

COX2 and HER2 are shown to be critical in the regulation of cancer progression. However, the prognostic value of nuclear COX2 in colorectal cancer (CRC) and its relationship with HER2 still remains unknown. In this study, the expression and biological significance of COX2 and HER2 were evaluated in CRC at mRNA and protein levels. RNA-Seq data of CRC were downloaded from TCGA, and 229 CRC and 50 non-cancerous subjects were enrolled in this study. Bioinformatics and immunohistochemistry analysis was performed based on the obtained data. Survival analysis was conducted to identify factors associated with overall survival of CRC patients. We showed that mRNA and protein levels of COX2 and HER2 were upregulated in CRC compared with the adjacent tissues. COX2 protein levels and nuclear COX2 expression were correlated with a poor prognosis of CRC patients. In addition, we also revealed that nuclear COX2 expression was positively associated with HER2 expression. Non-nuclear COX2 combined with low HER2 expression, was negatively correlated with Duke's stage and lymph node metastasis, predicting the best outcomes for CRC patients. In addition, our data indicated that non-nuclear COX2 combined with low HER2 expression is an independent prognostic factor for CRC after surgical resection. The study suggests that nuclear COX2 in combination with HER2 can serve as potential biomarkers for the clinical diagnosis and prognosis of CRC, and targeted inhibition of COX2 and HER2 might be an alternative strategy for the management of CRC.

LEM4/ANKLE-2 deficiency impairs post-mitotic re-localization of BAF, LAP2α and LaminA to the nucleus, causes nuclear envelope instability in telophase and leads to hyperploidy in HeLa cells.

PubMed

Snyers, Luc; Erhart, Renate; Laffer, Sylvia; Pusch, Oliver; Weipoltshammer, Klara; Schöfer, Christian

2018-01-01

The human LEM-domain protein family is involved in fundamental aspects of nuclear biology. The LEM-domain interacts with the barrier-to-autointegration factor (BAF), which itself binds DNA. LEM-domain proteins LAP2, emerin and MAN1 are proteins of the inner nuclear membrane; they have important functions: maintaining the integrity of the nuclear lamina and regulating gene expression at the nuclear periphery. LEM4/ANKLE-2 has been proposed to participate in nuclear envelope reassembly after mitosis and to mediate dephosphorylation of BAF through binding to phosphatase PP2A. Here, we used CRISPR/Cas9 to create several cell lines deficient in LEM4/ANKLE-2. By using time-lapse video microscopy, we show that absence of this protein severely compromises the post mitotic re-association of the nuclear proteins BAF, LAP2α and LaminA to chromosomes. These defects give rise to a strong mechanical instability of the nuclear envelope in telophase and to a chromosomal instability leading to increased number of hyperploid cells. Reintroducing LEM4/ANKLE-2 in the cells by transfection could efficiently restore the telophase association of BAF and LAP2α to the chromosomes. This rescue phenotype was abolished for N- or C-terminally truncated mutants that had lost the capacity to bind PP2A. We demonstrate also that, in addition to binding to PP2A, LEM4/ANKLE-2 binds BAF through its LEM-domain, providing further evidence for a generic function of this domain as a principal interactor of BAF. Copyright © 2017 Elsevier GmbH. All rights reserved.

The Strong Nuclear Force

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

Lincoln, Don

Scientists are aware of four fundamental forces- gravity, electromagnetism, and the strong and weak nuclear forces. Most people have at least some familiarity with gravity and electromagnetism, but not the other two. How is it that scientists are so certain that two additional forces exist? In this video, Fermilab’s Dr. Don Lincoln explains why scientists are so certain that the strong force exists.

The Strong Nuclear Force

ScienceCinema

Lincoln, Don

2018-01-16

Scientists are aware of four fundamental forces- gravity, electromagnetism, and the strong and weak nuclear forces. Most people have at least some familiarity with gravity and electromagnetism, but not the other two. How is it that scientists are so certain that two additional forces exist? In this video, Fermilab’s Dr. Don Lincoln explains why scientists are so certain that the strong force exists.

Rat PPAR delta contains a CGG triplet repeat and is prominently expressed in the thalamic nuclei.

PubMed

Xing, G; Zhang, L; Zhang, L; Heynen, T; Yoshikawa, T; Smith, M; Weiss, S; Detera-Wadleigh, S

1995-12-26

We have isolated a new rat sequence containing motifs of a nuclear hormone receptor from a brain cDNA library. The deduced amino acid sequence encoded by the cDNA clone showed a strong homology to the human NUCI and the mouse peroxisome proliferator activated receptor delta (PPAR delta). We therefore refer to this new clone as rat PPAR delta (rPPAR delta). The new feature of rPPAR delta is a 14 CGG triplet repeat on the 5' untranslated region, not previously reported in either NUCI or mPPAR delta. We found that rPPAR delta was expressed as a 3.5-kb transcript which showed a wide distribution in adult rat tissues. Abundant expression was detected in brain, heart, skeletal muscle, kidney and lung. Weaker expression was noted in the liver, spleen and testis. To determine the specific brain localization of rPPAR delta we performed in situ hybridization analysis. Prominent expression was observed in the thalamus, particularly in the posterior part of the ventral medial nucleus, a site responsive to pain and cold stress. These results raise the possibility that PPAR delta might play a role in modulating response to thermal and pain sensations.

Diagnostic implications of IDH1-R132H and OLIG2 expression patterns in rare and challenging glioblastoma variants.

PubMed

Joseph, Nancy M; Phillips, Joanna; Dahiya, Sonika; M Felicella, Michelle; Tihan, Tarik; Brat, Daniel J; Perry, Arie

2013-03-01

Recent work has demonstrated that nearly all diffuse gliomas display nuclear immunoreactivity for the bHLH transcription factor OLIG2, and the R132H mutant isocitrate dehydrogenase 1 (IDH1) protein is expressed in the majority of diffuse gliomas other than primary glioblastoma. However, these antibodies have not been widely applied to rarer glioblastoma variants, which can be diagnostically challenging when the astrocytic features are subtle. We therefore surveyed the expression patterns of OLIG2 and IDH1 in 167 non-conventional glioblastomas, including 45 small cell glioblastomas, 45 gliosarcomas, 34 glioblastomas with primitive neuroectodermal tumor-like foci (PNET-like foci), 23 with an oligodendroglial component, 11 granular cell glioblastomas, and 9 giant cell glioblastomas. OLIG2 was strongly expressed in all glioblastomas with oligodendroglial component, 98% of small cell glioblastomas, and all granular cell glioblastomas, the latter being particularly helpful in ruling out macrophage-rich lesions. In 74% of glioblastomas with PNET-like foci, OLIG2 expression was retained in the PNET-like foci, providing a useful distinction from central nervous system PNETs. The glial component of gliosarcomas was OLIG2 positive in 93% of cases, but only 14% retained focal expression in the sarcomatous component; as such this marker would not reliably distinguish these from pure sarcoma in most cases. OLIG2 was expressed in 67% of giant cell glioblastomas. IDH1 was expressed in 55% of glioblastomas with oligodendroglial component, 15% of glioblastomas with PNET-like foci, 7% of gliosarcomas, and none of the small cell, granular cell, or giant cell glioblastomas. This provides further support for the notion that most glioblastomas with oligodendroglial component are secondary, while small cell glioblastomas, granular cell glioblastomas, and giant cell glioblastomas are primary variants. Therefore, in one of the most challenging differential diagnoses, IDH1 positivity could provide strong support for glioblastoma with oligodendroglial component, while essentially excluding small cell glioblastoma.

Momordica charantia Inhibits Inflammatory Responses in Murine Macrophages via Suppression of TAK1.

PubMed

Yang, Woo Seok; Yang, Eunju; Kim, Min-Jeong; Jeong, Deok; Yoon, Deok Hyo; Sung, Gi-Ho; Lee, Seungihm; Yoo, Byong Chul; Yeo, Seung-Gu; Cho, Jae Youl

2018-01-01

Momordica charantia known as bitter melon is a representative medicinal plant reported to exhibit numerous pharmacological activities such as antibacterial, antidiabetic, anti-inflammatory, anti-oxidant, antitumor, and hypoglycemic actions. Although this plant has high ethnopharmacological value for treating inflammatory diseases, the molecular mechanisms by which it inhibits the inflammatory response are not fully understood. In this study, we aim to identify the anti-inflammatory mechanism of this plant. To this end, we studied the effects of its methanol extract (Mc-ME) on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Specifically, we evaluated nitric oxide (NO) production, mRNA expression of inflammatory genes, luciferase reporter gene activity, and putative molecular targets. Mc-ME blocked NO production in a dose-dependent manner in RAW264.7 cells; importantly, no cytotoxicity was observed. Moreover, the mRNA expression levels of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 were decreased by Mc-ME treatment in a dose-dependent manner. Luciferase assays and nuclear lysate immunoblotting analyses strongly indicated that Mc-ME decreases the levels of p65 [a nuclear factor (NF)-[Formula: see text]B subunit] and c-Fos [an activator protein (AP)-1 subunit]. Whole lysate immunoblotting assays, luciferase assays, and overexpression experiments suggested that transforming growth factor [Formula: see text]-activated kinase 1 (TAK1) is targeted by Mc-ME, thereby suppressing NF-[Formula: see text]B and AP-1 activity via downregulation of extracellular signal-regulated kinases (ERKs) and AKT. These results strongly suggest that Mc-ME exerts its anti-inflammatory activity by reducing the action of TAK1, which also affects the activation of NF-[Formula: see text]B and AP-1.

Prognostic Significance of Nuclear β-Catenin Expression in Patients with Colorectal Cancer from Iran

PubMed Central

Nazemalhosseini Mojarad, Ehsan; Kashfi, Seyed Mohammad Hossein; Mirtalebi, Hanieh; Almasi, Shohre; Chaleshi, Vahid; Kishani Farahani, Roya; Tarban, Peyman; Molaei, Mahsa; Zali, Mohammad Reza; J.K. Kuppen, Peter

2015-01-01

Background: Beta catenin plays a key role in cancer tumorigenesis. However, its prognostic significance in patients with colorectal cancer (CRC) remains controversial. It has been demonstrated that 90% of all tumors have a mutation in individual components of multiple oncogenes in Wnt/β-catenin pathway. Accumulation of nuclear β-catenin in cytoplasm leads to uncontrolled cell proliferation. Thus, nuclear β-catenin accumulation may be a valuable biomarker associated with invasion, metastasis and poor prognosis of CRC. Objectives: In this study the prognostic value of beta catenin expression in 165 Iranian CRC patients was evaluated. Patients and Methods: In this cross sectional retrospective study immunohistochemistry analyses of formalin-fixed paraffin-embedded (FFPE) tumor tissues were performed to characterize the expression of nuclear β-catenin in a series of 165 Iranian patients with colorectal carcinoma. Heat-induced antigen retrieval using the microwave method was applied for all staining procedures. Staining was scored independently by two observers, and a high level of concordance (90%) was achieved. Statistical analysis was done using the SPSS software for Windows, version 13.0.0 (SPSS Inc., Chicago, IL). Two-tailed P < 0.05 was considered statistically significant. Results: The patients consisted of 85 males and 80 females. Eighty-eight patients had primary tumor of the rectum and sigmoid, while 77 patients had primary tumor of the colon. The mean period of follow-up was 47.2 ± 10 months and the median period of follow-up was 38 months (range 6 - 58) for each patient. Of 165 tumors, 32 tumors (19.39 %) showed expression of β-catenin and 133 (80.6 %) were negative for β-catenin expression. Based on our findings the distribution of Microsatellite Instability (MSI) status differed between patients with nuclear β-catenin positive and negative tumors and this difference was significant (P = 0.001). Patients with nuclear β-catenin positive expression profile were found to be younger than patients with negative nuclear β-catenin expression (P = 0.010). Univariate and multivariate analysis showed that tumors with β-catenin expression had a poorer prognosis compared to tumors without β-catenin expression. Conclusions: According to our findings, the distribution of nuclear b-catenin expression is a poor prognostic marker in patients with colon cancer. PMID:26421170

Nuclear trafficking of the HIV-1 pre-integration complex depends on the ADAM10 intracellular domain

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

Endsley, Mark A., E-mail: maendsle@utmb.edu; Somasunderam, Anoma D., E-mail: asomasun@utmb.edu; Li, Guangyu, E-mail: LIG001@mail.etsu.edu

Previously, we showed that ADAM10 is necessary for HIV-1 replication in primary human macrophages and immortalized cell lines. Silencing ADAM10 expression interrupted the HIV-1 life cycle prior to nuclear translocation of viral cDNA. Furthermore, our data indicated that HIV-1 replication depends on the expression of ADAM15 and γ-secretase, which proteolytically processes ADAM10. Silencing ADAM15 or γ-secretase expression inhibits HIV-1 replication between reverse transcription and nuclear entry. Here, we show that ADAM10 expression also supports replication in CD4{sup +} T lymphocytes. The intracellular domain (ICD) of ADAM10 associates with the HIV-1 pre-integration complex (PIC) in the cytoplasm and immunoprecipitates and co-localizesmore » with HIV-1 integrase, a key component of PIC. Taken together, our data support a model whereby ADAM15/γ-secretase processing of ADAM10 releases the ICD, which then incorporates into HIV-1 PIC to facilitate nuclear trafficking. Thus, these studies suggest ADAM10 as a novel therapeutic target for inhibiting HIV-1 prior to nuclear entry. - Highlights: • Nuclear trafficking of the HIV-1 pre-integration complex depends on ADAM10. • ADAM10 associates with HIV-1 integrase in the pre-integration complex. • HIV-1 replication depends on the expression of ADAM15 and γ-secretase. • Silencing ADAM15 or γ-secretase expression inhibits nuclear import of viral cDNA. • ADAM10 is important for HIV-1 replication in human macrophages and CD4{sup +} T lymphocytes.« less

STAT6 is amplified in a subset of dedifferentiated liposarcoma.

PubMed

Doyle, Leona A; Tao, Derrick; Mariño-Enríquez, Adrián

2014-09-01

A recurrent intrachromosomal rearrangement on chromosome 12q in solitary fibrous tumor leads to the formation of a NAB2-STAT6 fusion oncogene. As a result, nuclear expression of the cytoplasmic transcription factor STAT6 is found in solitary fibrous tumor and serves as a useful diagnostic marker. STAT6 is located in 12q13, a region containing well-characterized oncogenes that are commonly amplified in dedifferentiated liposarcoma; we have previously reported that STAT6 is expressed in a subset of dedifferentiated liposarcoma. The aim of this study was to determine the frequency of STAT6 expression in dedifferentiated liposarcoma and the underlying genetic mechanism. STAT6 protein expression was analyzed by immunohistochemistry in a well-characterized series of 35 previously unpublished cases of dedifferentiated liposarcoma, all with nuclear MDM2 and/or CDK4 expression by immunohistochemistry and/or cytogenetic features of dedifferentiated liposarcoma. FISH for STAT6 was performed in all cases with STAT6 expression, and a subset of control cases without STAT6 expression. In total 4/35 cases (11%) showed STAT6 expression (three with multifocal staining of moderate to strong intensity and one with weak focal staining). FISH demonstrated amplification of STAT6 in all cases positive for STAT6 by immunohistochemistry; in contrast, FISH performed on four STAT6-negative dedifferentiated liposarcomas demonstrated no STAT6 amplification (P=0.0286). Of the four STAT6 amplified cases, three patients were male and one was female, ranging in age from 51 to 76 years. Tumors were located in the mediastinum (n=2), paratesticular soft tissue (n=1), and perirenal soft tissue (n=1). Three patients received pre-operative chemotherapy +/- radiation therapy. In conclusion, STAT6 is amplified in a subset of dedifferentiated liposarcoma, resulting in STAT6 protein expression that can be detected by immunohistochemistry and may be a potential pitfall in the differential diagnosis of dedifferentiated liposarcoma and solitary fibrous tumor. These findings suggest a role for STAT6-mediated transcriptional activity in some cases of dedifferentiated liposarcoma and highlight the genomic complexity and heterogeneity of dedifferentiated liposarcoma.

Diffusion profiling of tumor volumes using a histogram approach can predict proliferation and further microarchitectural features in medulloblastoma.

PubMed

Schob, Stefan; Beeskow, Anne; Dieckow, Julia; Meyer, Hans-Jonas; Krause, Matthias; Frydrychowicz, Clara; Hirsch, Franz-Wolfgang; Surov, Alexey

2018-05-31

Medulloblastomas are the most common central nervous system tumors in childhood. Treatment and prognosis strongly depend on histology and transcriptomic profiling. However, the proliferative potential also has prognostical value. Our study aimed to investigate correlations between histogram profiling of diffusion-weighted images and further microarchitectural features. Seven patients (age median 14.6 years, minimum 2 years, maximum 20 years; 5 male, 2 female) were included in this retrospective study. Using a Matlab-based analysis tool, histogram analysis of whole apparent diffusion coefficient (ADC) volumes was performed. ADC entropy revealed a strong inverse correlation with the expression of the proliferation marker Ki67 (r = - 0.962, p = 0.009) and with total nuclear area (r = - 0.888, p = 0.044). Furthermore, ADC percentiles, most of all ADCp90, showed significant correlations with Ki67 expression (r = 0.902, p = 0.036). Diffusion histogram profiling of medulloblastomas provides valuable in vivo information which potentially can be used for risk stratification and prognostication. First of all, entropy revealed to be the most promising imaging biomarker. However, further studies are warranted.

Ki-67 protein is associated with ribosomal RNA transcription in quiescent and proliferating cells.

PubMed

Bullwinkel, Jörn; Baron-Lühr, Bettina; Lüdemann, Anja; Wohlenberg, Claudia; Gerdes, Johannes; Scholzen, Thomas

2006-03-01

The nuclear Ki-67 protein (pKi-67) has previously been shown to be exclusively expressed in proliferating cells. As a result, antibodies against this protein are widely used as prognostic tools in cancer diagnostics. Here we show, that despite the strong downregulation of pKi-67 expression in non-proliferating cells, the protein can nevertheless be detected at sites linked to ribosomal RNA (rRNA) synthesis. Although this finding does not argue against the use of pKi-67 as a proliferation marker, it has wide ranging implications for the elucidation of pKi-67 function. Employing the novel antibody TuBB-9, we could further demonstrate that also in proliferating cells, a fraction of pKi-67 is found at sites linked to the rRNA transcription machinery during interphase and mitosis. Moreover, chromatin immunoprecipitation (ChIP) assays provided evidence for a physical association of pKi-67 with chromatin of the promoter and transcribed region of the rRNA gene cluster. These data strongly suggest a role for pKi-67 in the early steps of rRNA synthesis. Copyright 2005 Wiley-Liss, Inc.

Loss of nuclear NOTCH1, but not its negative regulator NUMB, is an independent predictor of cervical malignancy

PubMed Central

Vázquez-Ulloa, Elenaé; Ramos-Cruz, Ana Clara; Prada, Diddier; Avilés-Salas, Alejandro; Chávez-Blanco, Alma Delia; Herrera, Luis A.; Lizano, Marcela; Contreras-Paredes, Adriana

2018-01-01

The participation of NOTCH signaling in invasive cervical cancer (ICC) remains controversial since both tumor suppressive and oncogenic properties have been described. Additionally, the role of NUMB, a negative regulator of NOTCH, remains unclear in ICC. We aimed to investigate the role of NOTCH1 and NUMB expression and their localization in cervical intraepithelial neoplasia (CIN) and ICC samples. A total of 144 biopsies were obtained from the Instituto Nacional de Cancerología, México from 2004 to 2017, and were subjected to immunohistochemistry for NOTCH1 and NUMB. We found that nuclear NOTCH1 expression was more frequently found in CIN samples compared with ICC (77.55% vs. 15.79%, p = 0.001). NUMB was almost exclusively found in the nucleus of CIN samples (32.65% vs. 6.32%, p = 0.001). Cytoplasmic expression of NOTCH1 (44.21%) and NUMB (35.79%) was the most frequent localization in ICC. Multivariable-adjusted analysis showed that the loss of nuclear NOTCH1 expression was an independent predictor of malignancy (β = –3.428, 95% confidence interval [95% CI] = –5.127, –1.728, p = 0.001). In contrast, the association between cytoplasmic NUMB expression and cervical cancer was lost after adjusting for nuclear NOTCH1 expression (β = 2.074, 95% [CI] = –0.358, 4.506, P = 0.094). Additionally, patients with cytoplasmic NOTCH1 expression showed a borderline association with longer overall survival (OS) than those with nuclear NOTCH1 expression (P = 0.08). Our data suggest that the loss of nuclear NOTCH1 but not NUMB might be an independent predictor of malignancy in cervical cancer. PMID:29721172

Loss of nuclear NOTCH1, but not its negative regulator NUMB, is an independent predictor of cervical malignancy.

PubMed

Vázquez-Ulloa, Elenaé; Ramos-Cruz, Ana Clara; Prada, Diddier; Avilés-Salas, Alejandro; Chávez-Blanco, Alma Delia; Herrera, Luis A; Lizano, Marcela; Contreras-Paredes, Adriana

2018-04-10

The participation of NOTCH signaling in invasive cervical cancer (ICC) remains controversial since both tumor suppressive and oncogenic properties have been described. Additionally, the role of NUMB, a negative regulator of NOTCH, remains unclear in ICC. We aimed to investigate the role of NOTCH1 and NUMB expression and their localization in cervical intraepithelial neoplasia (CIN) and ICC samples. A total of 144 biopsies were obtained from the Instituto Nacional de Cancerología, México from 2004 to 2017, and were subjected to immunohistochemistry for NOTCH1 and NUMB. We found that nuclear NOTCH1 expression was more frequently found in CIN samples compared with ICC (77.55% vs. 15.79%, p = 0.001). NUMB was almost exclusively found in the nucleus of CIN samples (32.65% vs. 6.32%, p = 0.001). Cytoplasmic expression of NOTCH1 (44.21%) and NUMB (35.79%) was the most frequent localization in ICC. Multivariable-adjusted analysis showed that the loss of nuclear NOTCH1 expression was an independent predictor of malignancy (β = -3.428, 95% confidence interval [95% CI] = -5.127, -1.728, p = 0.001). In contrast, the association between cytoplasmic NUMB expression and cervical cancer was lost after adjusting for nuclear NOTCH1 expression (β = 2.074, 95% [CI] = -0.358, 4.506, P = 0.094). Additionally, patients with cytoplasmic NOTCH1 expression showed a borderline association with longer overall survival (OS) than those with nuclear NOTCH1 expression ( P = 0.08). Our data suggest that the loss of nuclear NOTCH1 but not NUMB might be an independent predictor of malignancy in cervical cancer.

Elevated nuclear expression of the SMRT corepressor in breast cancer is associated with earlier tumor recurrence

PubMed Central

Migliaccio, Ilenia; Chaubal, Vaishali; Wu, Meng-Fen; Pace, Margaret C.; Hartmaier, Ryan; Jiang, Shiming; Edwards, Dean P.; Gutiérrez, M. Carolina; Hilsenbeck, Susan G.; Oesterreich, Steffi

2012-01-01

Silencing mediator of retinoic acid and thyroid hormone receptor (SMRT), also known as nuclear corepressor 2 (NCOR2) is a transcriptional corepressor for multiple members of the nuclear receptor superfamily of transcription factors, including estrogen receptor-α (ERα). In the classical model of corepressor action, SMRT binds to antiestrogen-bound ERα at target promoters and represses ERα transcriptional activity and gene expression. Herein SMRT mRNA and protein expression was examined in a panel of 30 breast cancer cell lines. Expression of both parameters was found to vary considerably amongst lines and the correlation between protein and mRNA expression was very poor (R2 = 0.0775). Therefore, SMRT protein levels were examined by immunohistochemical staining of a tissue microarray of 866 patients with stage I–II breast cancer. Nuclear and cytoplasmic SMRT were scored separately according to the Allred score. The majority of tumors (67 %) were negative for cytoplasmic SMRT, which when detected was found at very low levels. In contrast, nuclear SMRT was broadly detected. There was no significant difference in time to recurrence (TTR) according to SMRT expression levels in the ERα-positive tamoxifen-treated patients (P = 0.297) but the difference was significant in the untreated patients (P = 0.01). In multivariate analysis, ERα-positive tamoxifen-untreated patients with high nuclear SMRT expression (SMRT 5-8, i.e., 2nd to 4th quartile) had a shorter TTR (HR = 1.94, 95 % CI, 1.24–3.04; P = 0.004) while there was no association with SMRT expression for ERα-positive tamoxifen-treated patients. There was no association between SMRT expression and overall survival for patients, regardless of whether they received tamoxifen. Thus while SMRT protein expression was not predictive of outcome after antiestrogen therapy, it may have value in predicting tumor recurrence in patients not receiving adjuvant tamoxifen therapy. PMID:23015261

Cooperative activity of GABP with PU.1 or C/EBPε regulates lamin B receptor gene expression, implicating their roles in granulocyte nuclear maturation1

PubMed Central

Malu, Krishnakumar; Garhwal, Rahul; Pelletier, Margery G. H.; Gotur, Deepali; Halene, Stephanie; Zwerger, Monika; Yang, Zhong-Fa; Rosmarin, Alan G.; Gaines, Peter

2016-01-01

Nuclear segmentation is a hallmark feature of mammalian neutrophil differentiation, but the mechanisms that control this process are poorly understood. Gene expression in maturing neutrophils requires combinatorial actions of lineage-restricted and more widely expressed transcriptional regulators. Examples include interactions of the widely expressed ETS transcription factor, GA-binding protein (GABP), with the relatively lineage-restricted ETS factor, PU.1, and with CCAAT enhancer binding proteins, C/EBPα and C/EBPε. Whether such cooperative interactions between these transcription factors also regulate the expression of genes encoding proteins that control nuclear segmentation is unclear. We investigated the roles of ETS and C/EBP family transcription factors in regulating the gene encoding the lamin B receptor (LBR), an inner nuclear membrane protein whose expression is required for neutrophil nuclear segmentation. Although C/EBPε was previously shown to bind the Lbr promoter, surprisingly, we found that neutrophils derived from Cebpe null mice exhibited normal Lbr gene and protein expression. Instead, GABP provided transcriptional activation through the Lbr promoter in the absence of C/EBPε, and activities supported by GABP were greatly enhanced by either C/EBPε or PU.1. Both GABP and PU.1 bound Ets sites in the Lbr promoter in vitro, and in vivo within both early myeloid progenitors and differentiating neutrophils. These findings demonstrate that GABP, PU.1, and C/EBPε cooperate to control transcription of the gene encoding LBR, a nuclear envelope protein that is required for the characteristic lobulated morphology of mature neutrophils. PMID:27342846

CD147 promotes the formation of functional osteoclasts through NFATc1 signalling

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

Nishioku, Tsuyoshi, E-mail: nishiokut@niu.ac.jp; Department of Pharmaceutical Care and Health Sciences, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180; Terasawa, Mariko

CD147, a membrane glycoprotein of the immunoglobulin superfamily, is highly upregulated during dynamic cellular events including tissue remodelling. Elevated CD147 expression is present in the joint of rheumatoid arthritis patients. However, the role of CD147 in bone destruction remains unclear. To determine whether CD147 is involved in osteoclastogenesis, we studied its expression in mouse osteoclasts and its role in osteoclast differentiation and function. CD147 expression was markedly upregulated during osteoclast differentiation. To investigate the role of CD147 in receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption activity, osteoclast precursor cells were transfected with CD147 siRNA. Decreasedmore » CD147 expression inhibited osteoclast formation and bone resorption, inhibited RANKL-induced nuclear translocation of the nuclear factor of activated T cells (NFAT) c1 and decreased the expression of the d2 isoform of vacuolar ATPase Vo domain and cathepsin K. Therefore, CD147 plays a critical role in the differentiation and function of osteoclasts by upregulating NFATc1 through the autoamplification of its expression in osteoclastogenesis. - Highlights: • CD147 expression was markedly upregulated during osteoclast differentiation. • Downregulation of CD147 expression inhibited osteoclastgenesis and bone resorption. • Decreased CD147 expression inhibited RANKL-induced nuclear translocation of NFATc1.« less

Extracellular IL-33 cytokine, but not endogenous nuclear IL-33, regulates protein expression in endothelial cells.

PubMed

Gautier, Violette; Cayrol, Corinne; Farache, Dorian; Roga, Stéphane; Monsarrat, Bernard; Burlet-Schiltz, Odile; Gonzalez de Peredo, Anne; Girard, Jean-Philippe

2016-10-03

IL-33 is a nuclear cytokine from the IL-1 family that plays important roles in health and disease. Extracellular IL-33 activates a growing number of target cells, including group 2 innate lymphoid cells, mast cells and regulatory T cells, but it remains unclear whether intracellular nuclear IL-33 has additional functions in the nucleus. Here, we used a global proteomic approach based on high-resolution mass spectrometry to compare the extracellular and intracellular roles of IL-33 in primary human endothelial cells, a major source of IL-33 protein in human tissues. We found that exogenous extracellular IL-33 cytokine induced expression of a distinct set of proteins associated with inflammatory responses in endothelial cells. In contrast, knockdown of endogenous nuclear IL-33 expression using two independent RNA silencing strategies had no reproducible effect on the endothelial cell proteome. These results suggest that IL-33 acts as a cytokine but not as a nuclear factor regulating gene expression in endothelial cells.

The metastasis suppressor, NDRG1, inhibits “stemness” of colorectal cancer via down-regulation of nuclear β-catenin and CD44

PubMed Central

Wangpu, Xiongzhi; Yang, Xiao; Zhao, Jingkun; Lu, Jiaoyang; Guan, Shaopei; Lu, Jun; Kovacevic, Zaklina; Liu, Wensheng; Mi, Lan; Jin, Runsen; Sun, Jing; Yue, Fei; Ma, Junjun; Lu, Aiguo; Richardson, Des R.; Wang, Lishun; Zheng, Minhua

2015-01-01

N-myc downstream-regulated gene 1 (NDRG1), has been identified as an important metastasis suppressor for colorectal cancer (CRC). In this study, we investigated: (1) the effects of NDRG1 on CRC stemness and tumorigenesis; (2) the molecular mechanisms involved; and (3) the relationship between NDRG1 expression and colorectal cancer prognosis. Our investigation demonstrated that CRC cells with silenced NDRG1 showed more tumorigenic ability and stem cell-like properties, such as: colony and sphere formation, chemoresistance, cell invasion, high expression of CD44, and tumorigenicity in vivo. Moreover, NDRG1 silencing reduced β-catenin expression on the cell membrane, while increasing its nuclear expression. The anti-tumor activity of NDRG1 was demonstrated to be mediated by preventing β-catenin nuclear translocation, as silencing of this latter molecule could reverse the effects of silencing NDRG1 expression. NDRG1 expression was also demonstrated to be negatively correlated to CRC prognosis. In addition, there was a negative correlation between NDRG1 and nuclear β-catenin and also NDRG1 and CD44 expression in clinical CRC specimens. Taken together, our investigation demonstrates that the anti-metastatic activity of NDRG1 in CRC occurs through the down-regulation of nuclear β-catenin and suggests that NDRG1 is a significant therapeutic target. PMID:26418878

Septin 7 immunoexpression in papillary thyroid carcinoma: a preliminary study.

PubMed

Igci, Yusuf Ziya; Erkilic, Suna; Arslan, Ahmet

2014-07-01

Papillary thyroid carcinoma (PTC) is the most common type among thyroid cancers. The diagnosis of PTC may be challenging when follicular variant (FVPTC) of this disease is present due to the resemblance of nuclear properties of the classical type (CVPTC). However, making use of ancillary molecular markers in the diagnosis of PTC may help. In our study, we aimed to evaluate the SEPT7 protein expression in PTC. A total of 55 paraffin block tissue samples comprising encapsulated FVPTC (FVPTC(e), n=25), and CVPTC (n=15), and benign hyperfunctioning thyroid nodules (HypN, n=15) were used in this study. Nuclear, cytoplasmic, and overall (total) SEPT7 protein expression levels were determined by using immunohistochemistry. Nuclear, cytoplasmic, and overall SEPT7 expressions (p=0.02, p=0.001, p=0.002, respectively) were significantly lower in FVPTC(e) tissues when compared to HypN. In CVPTC group, nuclear expression was significantly lower (p=0.004) while overall and cytoplasmic expressions were not changed (p>0.05). In HypN group, highest nuclear (mean=2.73), cytoplasmic (mean=2.86), and overall (mean=2.86) expression scores were detected. Significantly lower SEPT7 expression in all expressional categories in FVPTC(e) group may be a sign of different molecular signature in this type of tissue. Copyright © 2014 Elsevier GmbH. All rights reserved.

Expression of MLH1 and MSH2 in urothelial carcinoma of the renal pelvis.

PubMed

Ehsani, Laleh; Osunkoya, Adeboye O

2014-09-01

In this study, we investigated microsatellite instability in urothelial carcinoma of the renal pelvis by lack of immunohistochemical staining for MLH1 and MSH2. The study included 44 cases of urothelial carcinoma of the renal pelvis obtained from radical nephroureterectomy specimens at our institution. We evaluated the loss of nuclear immunohistochemical staining of MLH1 and MSH2. Eight of 44 (18 %) patients had negative MLH1 expression and 25/44 (57 %) patients had negative MSH2 expression. Six of 8 (75 %) patients with negative MLH1 expression were male and 2/8 (25 %) patients were female. Nineteen of 25 (75 %) patients with negative MSH2 expression were male, and 6/25 (24 %) patients were female. Seven of 8 (88 %) cases with negative MLH1 expression were high-grade urothelial carcinoma, and 21/25 (84 %) cases with negative MSH2 expression were high-grade urothelial carcinoma. Twenty-one of 44 (48 %) cases had an inverted growth pattern, of which 3/21 (14 %) cases had negative MLH1 expression and 14/21 (67 %) cases had negative MSH2 expression. Our study showed that microsatellite instability based on negative expression of MLH1 and MSH2 was more common in male patients with high-grade urothelial carcinoma. There is a strong correlation between inverted growth pattern and negative MSH2 expression. Microsatellite instability testing should be performed in patients with upper urinary tract carcinoma and may have prognostic value.

The mouse Gtl2 gene is differentially expressed during embryonic development, encodes multiple alternatively spliced transcripts, and may act as an RNA.

PubMed

Schuster-Gossler, K; Bilinski, P; Sado, T; Ferguson-Smith, A; Gossler, A

1998-06-01

We have isolated a novel mouse gene (Gtl2) from the site of a gene trap integration (Gtl2lacZ) that gave rise to developmentally regulated lacZ expression, and a dominant parental-origin-dependent phenotype. Heterozygous Gtl2lacZ mice that inherited the transgene from the father showed a proportionate dwarfism phenotype, whereas the penetrance and expressivity of the phenotype was strongly reduced in Gtl2lacZ mice that inherited the transgene from the mother. Gtl2 expression is highly similar to the beta-galactosidase staining pattern, and is down-regulated but not abolished in mice carrying the Gtl2lacZ insertion. In early postimplantation embryos, Gtl2 is expressed in the visceral yolk sac and embryonic ectoderm. During subsequent development and organogenesis, Gtl2 transcripts are abundant in the paraxial mesoderm closely correlated with myogenic differentiation, in parts of the central nervous system, and in the epithelial ducts of developing excretory organs. The Gtl2 gene gives rise to various differentially spliced transcripts, which contain multiple small open reading frames (ORF). However, none of the ATG codons of these ORFs is in the context of a strong Kozak consensus sequence for initiation of translation, suggesting that Gtl2 might function as an RNA. Nuclear Gtl2 RNA was detected in a temporally and spatially regulated manner, and partially processed Gtl2 transcripts were readily detected in Northern blot hybridizations of polyadenylated RNA, suggesting that primary Gtl2 transcripts are differently processed in various cell types during development. Gtl2 transcript levels are present in parthenogenic embryos but may be reduced, consistent with the pattern of inheritance of the Gtl2lacZ phenotype.

Dyskerin and TERC expression may condition survival in lung cancer patients

PubMed Central

Penzo, Marianna; Ludovini, Vienna; Treré, Davide; Siggillino, Annamaria; Vannucci, Jacopo; Bellezza, Guido; Crinò, Lucio; Montanaro, Lorenzo

2015-01-01

Dyskerin mediates both the modification of uridine on ribosomal and small nuclear RNAs and the stabilization of the telomerase RNA component (TERC). In human tumors dyskerin expression was found to be associated with both rRNA modification and TERC levels. Moreover, dyskerin overexpression has been linked to unfavorable prognosis in a variety of tumor types, however an explanation for the latter association is not available. To clarify this point, we analyzed the connection between dyskerin expression, TERC levels and clinical outcome in two series of primary lung cancers, differing for the presence of TERC gene amplification, a genetic alteration inducing strong TERC overexpression. TERC levels were significantly higher in tumors bearing TERC gene amplification (P = 0.017). In addition, the well-established association between dyskerin expression and TERC levels was observed only in the series without TERC gene amplification (P = 0.003), while it was not present in TERC amplified tumors (P = 0.929). Similarly, the association between dyskerin expression and survival was found in cases not bearing TERC gene amplification (P = 0.009) and was not observed in TERC amplified tumors (P = 0.584). These results indicate that the influence of dyskerin expression on tumor clinical outcome is linked to its role on the maintenance of high levels of TERC. PMID:26301749

Immunohistochemical expression of vascular endothelial growth factor in canine oral squamous cell carcinomas.

PubMed

Martano, Manuela; Restucci, Brunella; Ceccarelli, Dora Maria; Lo Muzio, Lorenzo; Maiolino, Paola

2016-01-01

Angiogenesis is crucial for the growth and metastasis of malignant tumours, and various proangiogenic factors promote this process. One of these factors is vascular endothelial growth factor (VEGF), which appears to play a key role in tumour angiogenesis. The aim of the present study was to assess whether VEGF expression is associated with angiogenesis, disease progression and neoplastic proliferation in canine oral squamous cell carcinoma (OSCC) tissue. VEGF immunoreactivity was quantified by immunohistochemistry in 30 specimens, including normal oral mucosa and OSCC tissues graded as well, moderately or poorly differentiated. VEGF expression was correlated with tumour cell proliferation, as assessed using the proliferating cell nuclear antigen (PCNA) marker and microvessel density (data already published). The present results revealed that VEGF and PCNA expression increased significantly between normal oral tissue and neoplastic tissue, and between well and moderately/poorly differentiated tumours. In addition, VEGF expression was strongly correlated with PCNA expression and microvessel density. It was concluded that VEGF may promote angiogenesis through a paracrine pathway, stimulating endothelial cell proliferation and, similarly, may induce tumour cell proliferation through an autocrine pathway. The present results suggest that the evaluation of VEGF may be a useful additional criterion for estimating malignancy and growth potential in canine OSCCs.

Cell-type-specific regulation of the retinoic acid receptor mediated by the orphan nuclear receptor TLX.

PubMed

Kobayashi, M; Yu, R T; Yasuda, K; Umesono, K

2000-12-01

Malformations in the eye can be caused by either an excess or deficiency of retinoids. An early target gene of the retinoid metabolite, retinoic acid (RA), is that encoding one of its own receptors, the retinoic acid receptor beta (RARbeta). To better understand the mechanisms underlying this autologous regulation, we characterized the chick RARbeta2 promoter. The region surrounding the transcription start site of the avian RARbeta2 promoter is over 90% conserved with the corresponding region in mammals and confers strong RA-dependent transactivation in primary cultured embryonic retina cells. This response is selective for RAR but not retinoid X receptor-specific agonists, demonstrating a principal role for RAR(s) in retina cells. Retina cells exhibit a far higher sensitivity to RA than do fibroblasts or osteoblasts, a property we found likely due to expression of the orphan nuclear receptor TLX. Ectopic expression of TLX in fibroblasts resulted in increased sensitivity to RA induction, an effect that is conserved between chick and mammals. We have identified a cis element, the silencing element relieved by TLX (SET), within the RARbeta2 promoter region which confers TLX- and RA-dependent transactivation. These results indicate an important role for TLX in autologous regulation of the RARbeta gene in the eye.

Cell-Type-Specific Regulation of the Retinoic Acid Receptor Mediated by the Orphan Nuclear Receptor TLX†

PubMed Central

Kobayashi, Mime; Yu, Ruth T.; Yasuda, Kunio; Umesono, Kazuhiko

2000-01-01

Malformations in the eye can be caused by either an excess or deficiency of retinoids. An early target gene of the retinoid metabolite, retinoic acid (RA), is that encoding one of its own receptors, the retinoic acid receptor β (RARβ). To better understand the mechanisms underlying this autologous regulation, we characterized the chick RARβ2 promoter. The region surrounding the transcription start site of the avian RARβ2 promoter is over 90% conserved with the corresponding region in mammals and confers strong RA-dependent transactivation in primary cultured embryonic retina cells. This response is selective for RAR but not retinoid X receptor-specific agonists, demonstrating a principal role for RAR(s) in retina cells. Retina cells exhibit a far higher sensitivity to RA than do fibroblasts or osteoblasts, a property we found likely due to expression of the orphan nuclear receptor TLX. Ectopic expression of TLX in fibroblasts resulted in increased sensitivity to RA induction, an effect that is conserved between chick and mammals. We have identified a cis element, the silencing element relieved by TLX (SET), within the RARβ2 promoter region which confers TLX- and RA-dependent transactivation. These results indicate an important role for TLX in autologous regulation of the RARβ gene in the eye. PMID:11073974

LAF1, a MYB transcription activator for phytochrome A signaling

PubMed Central

Ballesteros, María L.; Bolle, Cordelia; Lois, Luisa M.; Moore, James M.; Vielle-Calzada, Jean-Philippe; Grossniklaus, Ueli; Chua, Nam-Hai

2001-01-01

The photoreceptor phytochrome (phy) A has a well-defined role in regulating gene expression in response to specific light signals. Here, we describe a new Arabidopsis mutant, laf1 (long after far-red light 1) that has an elongated hypocotyl specifically under far-red light. Gene expression studies showed that laf1 has reduced responsiveness to continuous far-red light but retains wild-type responses to other light wavelengths. As far-red light is only perceived by phyA, our results suggest that LAF1 is specifically involved in phyA signal transduction. Further analyses revealed that laf1 is affected in a subset of phyA-dependent responses and the phenotype is more severe at low far-red fluence rates. LAF1 encodes a nuclear protein with strong homology with the R2R3–MYB family of DNA-binding proteins. Experiments using yeast cells identified a transactivation domain in the C-terminal portion of the protein. LAF1 is constitutively targeted to the nucleus by signals in its N-terminal portion, and the full-length protein accumulates in distinct nuclear speckles. This accumulation in speckles is abolished by a point mutation in a lysine residue (K258R), which might serve as a modification site by a small ubiquitin-like protein (SUMO). PMID:11581165

Nuclear Calcium Buffering Capacity Shapes Neuronal Architecture*

PubMed Central

Mauceri, Daniela; Hagenston, Anna M.; Schramm, Kathrin; Weiss, Ursula; Bading, Hilmar

2015-01-01

Calcium-binding proteins (CaBPs) such as parvalbumin are part of the cellular calcium buffering system that determines intracellular calcium diffusion and influences the spatiotemporal dynamics of calcium signals. In neurons, CaBPs are primarily localized to the cytosol and function, for example, in nerve terminals in short-term synaptic plasticity. However, CaBPs are also expressed in the cell nucleus, suggesting that they modulate nuclear calcium signals, which are key regulators of neuronal gene expression. Here we show that the calcium buffering capacity of the cell nucleus in mouse hippocampal neurons regulates neuronal architecture by modulating the expression levels of VEGFD and the complement factor C1q-c, two nuclear calcium-regulated genes that control dendrite geometry and spine density, respectively. Increasing the levels of nuclear calcium buffers by means of expression of a nuclearly targeted form of parvalbumin fused to mCherry (PV.NLS-mC) led to a reduction in VEGFD expression and, as a result, to a decrease in total dendritic length and complexity. In contrast, mRNA levels of the synapse pruning factor C1q-c were increased in neurons expressing PV.NLS-mC, causing a reduction in the density and size of dendritic spines. Our results establish a close link between nuclear calcium buffering capacity and the transcription of genes that determine neuronal structure. They suggest that the development of cognitive deficits observed in neurological conditions associated with CaBP deregulation may reflect the loss of necessary structural features of dendrites and spines. PMID:26231212

Concentration-dependent Effects of Nuclear Lamins on Nuclear Size in Xenopus and Mammalian Cells*

PubMed Central

Jevtić, Predrag; Edens, Lisa J.; Li, Xiaoyang; Nguyen, Thang; Chen, Pan; Levy, Daniel L.

2015-01-01

A fundamental question in cell biology concerns the regulation of organelle size. While nuclear size is exquisitely controlled in different cell types, inappropriate nuclear enlargement is used to diagnose and stage cancer. Clarifying the functional significance of nuclear size necessitates an understanding of the mechanisms and proteins that control nuclear size. One structural component implicated in the regulation of nuclear morphology is the nuclear lamina, a meshwork of intermediate lamin filaments that lines the inner nuclear membrane. However, there has not been a systematic investigation of how the level and type of lamin expression influences nuclear size, in part due to difficulties in precisely controlling lamin expression levels in vivo. In this study, we circumvent this limitation by studying nuclei in Xenopus laevis egg and embryo extracts, open biochemical systems that allow for precise manipulation of lamin levels by the addition of recombinant proteins. We find that nuclear growth and size are sensitive to the levels of nuclear lamins, with low and high concentrations increasing and decreasing nuclear size, respectively. Interestingly, each type of lamin that we tested (lamins B1, B2, B3, and A) similarly affected nuclear size whether added alone or in combination, suggesting that total lamin concentration, and not lamin type, is more critical to determining nuclear size. Furthermore, we show that altering lamin levels in vivo, both in Xenopus embryos and mammalian tissue culture cells, also impacts nuclear size. These results have implications for normal development and carcinogenesis where both nuclear size and lamin expression levels change. PMID:26429910

A Role for Caenorhabditis elegans Importin IMA-2 in Germ Line and Embryonic Mitosis

PubMed Central

Geles, Kenneth G.; Johnson, Jeffrey J.; Jong, Sena; Adam, Stephen A.

2002-01-01

The importin α family of nuclear-cytoplasmic transport factors mediates the nuclear localization of proteins containing classical nuclear localization signals. Metazoan animals express multiple importin α proteins, suggesting their possible roles in cell differentiation and development. Adult Caenorhabditis elegans hermaphrodites express three importin α proteins, IMA-1, IMA-2, and IMA-3, each with a distinct expression and localization pattern. IMA-2 was expressed exclusively in germ line cells from the early embryonic through adult stages. The protein has a dynamic pattern of localization dependent on the stage of the cell cycle. In interphase germ cells and embryonic cells, IMA-2 is cytoplasmic and nuclear envelope associated, whereas in developing oocytes, the protein is cytoplasmic and intranuclear. During mitosis in germ line cells and embryos, IMA-2 surrounded the condensed chromosomes but was not directly associated with the mitotic spindle. The timing of IMA-2 nuclear localization suggested that the protein surrounded the chromosomes after fenestration of the nuclear envelope in prometaphase. Depletion of IMA-2 by RNA-mediated gene interference (RNAi) resulted in embryonic lethality and a terminal aneuploid phenotype. ima-2(RNAi) embryos have severe defects in nuclear envelope formation, accumulating nucleoporins and lamin in the cytoplasm. We conclude that IMA-2 is required for proper chromosome dynamics in germ line and early embryonic mitosis and is involved in nuclear envelope assembly at the conclusion of mitosis. PMID:12221121

Tissue specificity of the hormonal response in sex accessory tissues is associated with nuclear matrix protein patterns.

PubMed

Getzenberg, R H; Coffey, D S

1990-09-01

The DNA of interphase nuclei have very specific three-dimensional organizations that are different in different cell types, and it is possible that this varying DNA organization is responsible for the tissue specificity of gene expression. The nuclear matrix organizes the three-dimensional structure of the DNA and is believed to be involved in the control of gene expression. This study compares the nuclear structural proteins between two sex accessory tissues in the same animal responding to the same androgen stimulation by the differential expression of major tissue-specific secretory proteins. We demonstrate here that the nuclear matrix is tissue specific in the rat ventral prostate and seminal vesicle, and undergoes characteristic alterations in its protein composition upon androgen withdrawal. Three types of nuclear matrix proteins were observed: 1) nuclear matrix proteins that are different and tissue specific in the rat ventral prostate and seminal vesicle, 2) a set of nuclear matrix proteins that either appear or disappear upon androgen withdrawal, and 3) a set of proteins that are common to both the ventral prostate and seminal vesicle and do not change with the hormonal state of the animal. Since the nuclear matrix is known to bind androgen receptors in a tissue- and steroid-specific manner, we propose that the tissue specificity of the nuclear matrix arranges the DNA in a unique conformation, which may be involved in the specific interaction of transcription factors with DNA sequences, resulting in tissue-specific patterns of secretory protein expression.

A Transcriptional Regulatory Network Containing Nuclear Receptors and Long Noncoding RNAs Controls Basal and Drug-Induced Expression of Cytochrome P450s in HepaRG Cells.

PubMed

Chen, Liming; Bao, Yifan; Piekos, Stephanie C; Zhu, Kexin; Zhang, Lirong; Zhong, Xiao-Bo

2018-07-01

Cytochrome P450 (P450) enzymes are responsible for metabolizing drugs. Expression of P450s can directly affect drug metabolism, resulting in various outcomes in therapeutic efficacy and adverse effects. Several nuclear receptors are transcription factors that can regulate expression of P450s at both basal and drug-induced levels. Some long noncoding RNAs (lncRNAs) near a transcription factor are found to participate in the regulatory functions of the transcription factors. The aim of this study is to determine whether there is a transcriptional regulatory network containing nuclear receptors and lncRNAs controlling both basal and drug-induced expression of P450s in HepaRG cells. Small interfering RNAs or small hairpin RNAs were applied to knock down four nuclear receptors [hepatocyte nuclear factor 1 α (HNF1 α ), hepatocyte nuclear factor 4 α (HNF4 α ), pregnane X receptor (PXR), and constitutive androstane receptor (CAR)] as well as two lncRNAs [HNF1 α antisense RNA 1 (HNF1 α -AS1) and HNF4 α antisense RNA 1 (HNF4 α -AS1)] in HepaRG cells with or without treatment of phenobarbital or rifampicin. Expression of eight P450 enzymes was examined in both basal and drug-induced levels. CAR and PXR mainly regulated expression of specific P450s. HNF1 α and HNF4 α affected expression of a wide range of P450s as well as other transcription factors. HNF1 α and HNF4 α controlled the expression of their neighborhood lncRNAs, HNF1 α -AS1 and HNF4 α -AS1, respectively. HNF1 α -AS1 and HNF4 α -AS1 was also involved in the regulation of P450s and transcription factors in diverse manners. Altogether, our study concludes that a transcription regulatory network containing the nuclear receptors and lncRNAs controls both basal and drug-induced expression of P450s in HepaRG cells. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Adolescents' Knowledge of Nuclear Issues and the Effects of Nuclear War.

ERIC Educational Resources Information Center

Roscoe, Bruce; Goodwin, Megan P.

1987-01-01

Surveyed 357 college students to assess awareness of the status of nuclear arms development and possible effects of nuclear war on people and environment. Results suggest that older adolescents are extremely uninformed regarding the current status of nuclear issues and consequences of nuclear war. Indicates a strong need to educate young people…

Epigenetic regulation of the expression of genes involved in steroid hormone biosynthesis and action

PubMed Central

Martinez-Arguelles, Daniel B.; Papadopoulos, Vassilios

2010-01-01

Steroid hormones participate in organ development, reproduction, body homeostasis, and stress responses. The steroid machinery is expressed in a development- and tissue-specific manner, with the expression of these factors being tightly regulated by an array of transcription factors (TFs). Epigenetics provides an additional layer of gene regulation through DNA methylation and histone tail modifications. Evidence of epigenetic regulation of key steroidogenic enzymes is increasing, though this does not seem to be a predominant regulatory pathway. Steroid hormones exert their action in target tissues through steroid nuclear receptors belonging to the NR3A and NR3C families. Nuclear receptor expression levels and post-translational modifications regulate their function and dictate their sensitivity to steroid ligands. Nuclear receptors and TFs are more likely to be epigenetically regulated than proteins involved in steroidogenesis and have secondary impact on the expression of these steroidogenic enzymes. Here we review evidence for epigenetic regulation of enzymes, transcription factors, and nuclear receptors related to steroid biogenesis and action. PMID:20156469

Quark Matter and Nuclear Collisions a Brief History of Strong Interaction Thermodynamics

NASA Astrophysics Data System (ADS)

Satz, Helmut

2012-08-01

The past 50 years have seen the emergence of a new field of research in physics, the study of matter at extreme temperatures and densities. The theory of strong interactions, quantum chromodynamics (QCD), predicts that in this limit, matter will become a plasma of deconfined quarks and gluons — the medium which made up the early universe in the first 10 microseconds after the Big Bang. High energy nuclear collisions are expected to produce short-lived bubbles of such a medium in the laboratory. I survey the merger of statistical QCD and nuclear collision studies for the analysis of strongly interacting matter in theory and experiment.

PGC-1α buffers ROS-mediated removal of mitochondria during myogenesis.

PubMed

Baldelli, S; Aquilano, K; Ciriolo, M R

2014-11-06

Mitochondrial biogenesis and mitophagy are recognized as critical processes underlying mitochondrial homeostasis. However, the molecular pathway(s) coordinating the balance between these cellular programs is still poorly investigated. Here, we show an induction of the nuclear and mitochondrial peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α) during myogenesis, which in turn co-activates the transcription of nuclear and mtDNA-encoded mitochondrial genes. We demonstrate that PGC-1α also buffers oxidative stress occurring during differentiation by promoting the expression of antioxidant enzymes. Indeed, by downregulating PGC-1α, we observed an impairment of antioxidants expression, which was accompanied by a significant reactive oxygen species (ROS) burst and increase of oxidative damage to proteins. In parallel, we detected a decrease of mitochondrial mass and function as well as increased mitophagy through the ROS/FOXO1 pathway. Upon PGC-1α downregulation, we found ROS-dependent nuclear translocation of FOXO1 and transcription of its downstream targets including mitophagic genes such as LC3 and PINK1. Such events were significantly reverted after treatment with the antioxidant Trolox, suggesting that PGC-1α assures mitochondrial integrity by indirectly buffering ROS. Finally, the lack of PGC-1α gave rise to a decrease in MYOG and a strong induction of atrophy-related ubiquitin ligases FBXO32 (FBXO32), indicative of a degenerative process. Overall, our results reveal that in myotubes, PGC-1α takes center place in mitochondrial homeostasis during differentiation because of its ability to avoid ROS-mediated removal of mitochondria.

Expression of an Intestine-Specific Transcription Factor (CDX1) in Intestinal Metaplasia and in Subsequently Developed Intestinal Type of Cholangiocarcinoma in Rat Liver

PubMed Central

Ren, Ping; Silberg, Debra G.; Sirica, Alphonse E.

2000-01-01

CDX1 is a caudal-type homeobox intestine-specific transcription factor that has been shown to be selectively expressed in epithelial cells in intestinal metaplasia of the human stomach and esophagus and variably expressed in human gastric and esophageal adenocarcinomas (Silberg DG, Furth EE, Taylor JK, Schuck T, Chiou T, Traber PG: Gastroenterology 1997, 113: 478–486). Through the use of immunohistochemistry and Western blotting, we investigated whether CDX1 is also uniquely associated with the intestinal metaplasia associated with putative precancerous cholangiofibrosis induced in rat liver during furan cholangiocarcinogenesis, as well as expressed in neoplastic glands in a subsequently developed intestinal type of cholangiocarcinoma. In normal, control adult rat small intestine, specific nuclear immunoreactivity for CDX1 was most prominent in enterocytes lining the crypts. In comparison, epithelium from intestinal metaplastic glands within furan-induced hepatic cholangiofibrosis and neoplastic epithelium from later developed primary intestinal-type cholangiocarcinoma each demonstrated strong nuclear immunoreactivity for CDX1. CDX1-positive cells were detected in hepatic cholangiofibrotic tissue as early as 3 weeks after the start of chronic furan treatment. We further determined that the percentages of CDX1-positive neoplastic glands and glandular nuclei are significantly higher in primary tumors than in a derived, transplantable cholangiocarcinoma serially-propagated in vivo. Western blotting confirmed our immunohistochemical results, and no CDX1 immunoreactivity was detected in normal adult rat liver or in hyperplastic biliary epithelial cells. These findings indicate that CDX1 is specifically associated with early intestinal metaplasia and a later developed intestinal-type of cholangiocarcinoma induced in the liver of furan-treated rats. PMID:10666391

Expression of an intestine-specific transcription factor (CDX1) in intestinal metaplasia and in subsequently developed intestinal type of cholangiocarcinoma in rat liver.

PubMed

Ren, P; Silberg, D G; Sirica, A E

2000-02-01

CDX1 is a caudal-type homeobox intestine-specific transcription factor that has been shown to be selectively expressed in epithelial cells in intestinal metaplasia of the human stomach and esophagus and variably expressed in human gastric and esophageal adenocarcinomas (Silberg DG, Furth EE, Taylor JK, Schuck T, Chiou T, Traber PG: Gastroenterology 1997, 113: 478-486). Through the use of immunohistochemistry and Western blotting, we investigated whether CDX1 is also uniquely associated with the intestinal metaplasia associated with putative precancerous cholangiofibrosis induced in rat liver during furan cholangiocarcinogenesis, as well as expressed in neoplastic glands in a subsequently developed intestinal type of cholangiocarcinoma. In normal, control adult rat small intestine, specific nuclear immunoreactivity for CDX1 was most prominent in enterocytes lining the crypts. In comparison, epithelium from intestinal metaplastic glands within furan-induced hepatic cholangiofibrosis and neoplastic epithelium from later developed primary intestinal-type cholangiocarcinoma each demonstrated strong nuclear immunoreactivity for CDX1. CDX1-positive cells were detected in hepatic cholangiofibrotic tissue as early as 3 weeks after the start of chronic furan treatment. We further determined that the percentages of CDX1-positive neoplastic glands and glandular nuclei are significantly higher in primary tumors than in a derived, transplantable cholangiocarcinoma serially-propagated in vivo. Western blotting confirmed our immunohistochemical results, and no CDX1 immunoreactivity was detected in normal adult rat liver or in hyperplastic biliary epithelial cells. These findings indicate that CDX1 is specifically associated with early intestinal metaplasia and a later developed intestinal-type of cholangiocarcinoma induced in the liver of furan-treated rats.

Single-cell analysis of Daxx and ATRX-dependent transcriptional repression

PubMed Central

Newhart, Alyshia; Rafalska-Metcalf, Ilona U.; Yang, Tian; Negorev, Dmitri G.; Janicki, Susan M.

2012-01-01

Summary Histone H3.3 is a constitutively expressed H3 variant implicated in the epigenetic inheritance of chromatin structures. Recently, the PML-nuclear body (PML-NB)/Nuclear Domain 10 (ND10) proteins, Daxx and ATRX, were found to regulate replication-independent histone H3.3 chromatin assembly at telomeres and pericentric heterochromatin. As it is not completely understood how PML-NBs/ND10s regulate transcription and resistance to viral infection, we have used a CMV-promoter-regulated inducible transgene array, at which Daxx and ATRX are enriched, to delineate the mechanisms through which they regulate transcription. When integrated into HeLa cells, which express both Daxx and ATRX, the array is refractory to activation. However, transcription can be induced when ICP0, the HSV-1 E3 ubiquitin ligase required to reverse latency, is expressed. As ATRX and Daxx are depleted from the activated array in ICP0-expressing HeLa cells, this suggests that they are required to maintain a repressed chromatin environment. As histone H3.3 is strongly recruited to the ICP0-activated array but does not co-localize with the DNA, this also suggests that chromatin assembly is blocked during activation. The conclusion that the Daxx and ATRX pathway is required for transcriptional repression and chromatin assembly at this site is further supported by the finding that an array integrated into the ATRX-negative U2OS cell line can be robustly activated and that histone H3.3 is similarly recruited and unincorporated into the chromatin. Therefore, this study has important implications for understanding gene silencing, viral latency and PML-NB/ND10 function. PMID:22976303

Comparative study of Hippo pathway genes in cellular conveyor belts of a ctenophore and a cnidarian.

PubMed

Coste, Alicia; Jager, Muriel; Chambon, Jean-Philippe; Manuel, Michaël

2016-01-01

The Hippo pathway regulates growth rate and organ size in fly and mouse, notably through control of cell proliferation. Molecular interactions at the heart of this pathway are known to have originated in the unicellular ancestry of metazoans. They notably involve a cascade of phosphorylations triggered by the kinase Hippo, with subsequent nuclear to cytoplasmic shift of Yorkie localisation, preventing its binding to the transcription factor Scalloped, thereby silencing proliferation genes. There are few comparative expression data of Hippo pathway genes in non-model animal species and notably none in non-bilaterian phyla. All core Hippo pathway genes could be retrieved from the ctenophore Pleurobrachia pileus and the hydrozoan cnidarian Clytia hemisphaerica, with the important exception of Yorkie in ctenophore. Expression study of the Hippo, Salvador and Scalloped genes in tentacle "cellular conveyor belts" of these two organisms revealed striking differences. In P. pileus, their transcripts were detected in areas where undifferentiated progenitors intensely proliferate and where expression of cyclins B and D was also seen. In C. hemisphaerica, these three genes and Yorkie are expressed not only in the proliferating but also in the differentiation zone of the tentacle bulb and in mature tentacle cells. However, using an antibody designed against the C. hemiphaerica Yorkie protein, we show in two distinct cell lineages of the medusa that Yorkie localisation is predominantly nuclear in areas of active cell proliferation and mainly cytoplasmic elsewhere. This is the first evidence of nucleocytoplasmic Yorkie shift in association with the arrest of cell proliferation in a cnidarian, strongly evoking the cell division-promoting role of this protein and its inhibition by the activated Hippo pathway in bilaterian models. Our results furthermore highlight important differences in terms of deployment and regulation of Hippo pathway genes between cnidarians and ctenophores.

Evaluation of the osteoclastogenic process associated with RANK / RANK-L / OPG in odontogenic myxomas

PubMed Central

González-Galván, María del Carmen; Mosqueda-Taylor, Adalberto; Bologna-Molina, Ronell; Setien-Olarra, Amaia; Marichalar-Mendia, Xabier; Aguirre-Urizar, José-Manuel

2018-01-01

Background Odontogenic myxoma (OM) is a benign intraosseous neoplasm that exhibits local aggressiveness and high recurrence rates. Osteoclastogenesis is an important phenomenon in the tumor growth of maxillary neoplasms. RANK (Receptor Activator of Nuclear Factor κappa B) is the signaling receptor of RANK-L (Receptor activator of nuclear factor kappa-Β ligand) that activates the osteoclasts. OPG (osteoprotegerin) is a decoy receptor for RANK-L that inhibits pro-osteoclastogenesis. The RANK / RANKL / OPG system participates in the regulation of osteolytic activity under normal conditions, and its alteration has been associated with greater bone destruction, and also with tumor growth. Objectives To analyze the immunohistochemical expression of OPG, RANK and RANK-L proteins in odontogenic myxomas (OMs) and their relationship with the tumor size. Material and Methods Eighteen OMs, 4 small (<3 cm) and 14 large (> 3cm) and 18 dental follicles (DF) that were included as control were studied by means of standard immunohistochemical procedure with RANK, RANKL and OPG antibodies. For the evaluation, 5 fields (40x) of representative areas of OM and DF were selected where the expression of each antibody was determined. Descriptive and comparative statistical analyses were performed with the obtained data. Results There are significant differences in the expression of RANK in OM samples as compared to DF (p = 0.022) and among the OMSs and OMLs (p = 0.032). Also a strong association is recognized in the expression of RANK-L and OPG in OM samples. Conclusions Activation of the RANK / RANK-L / OPG triad seems to be involved in the mechanisms of bone balance and destruction, as well as associated with tumor growth in odontogenic myxomas. Key words:Odontogenic myxoma, dental follicle, RANK, RANK-L, OPG, osteoclastogenesis. PMID:29680857

Long non-coding RNA repertoire and targeting by nuclear exosome, cytoplasmic exonuclease and RNAi in fission yeast.

PubMed

Atkinson, Sophie; Marguerat, Samuel; Bitton, Danny; Bachand, Francois; Rodriguez-Lopez, Maria; Rallis, Charalampos; Lemay, Jean-Francois; Cotobal, Cristina; Malecki, Michal; Smialowski, Pawel; Mata, Juan; Korber, Philipp; Bahler, Jurg

2018-06-18

Long non-coding RNAs (lncRNAs), which are longer than 200 nucleotides but often unstable, contribute a substantial and diverse portion to pervasive non-coding transcriptomes. Most lncRNAs are poorly annotated and understood, although several play important roles in gene regulation and diseases. Here we systematically uncover and analyse lncRNAs in Schizosaccharomyces pombe. Based on RNA-seq data from twelve RNA-processing mutants and nine physiological conditions, we identify 5775 novel lncRNAs, nearly 4-times the previously annotated lncRNAs. The expression of most lncRNAs becomes strongly induced under the genetic and physiological perturbations, most notably during late meiosis. Most lncRNAs are cryptic and suppressed by three RNA-processing pathways: the nuclear exosome, cytoplasmic exonuclease, and RNAi. Double-mutant analyses reveal substantial coordination and redundancy among these pathways. We classify lncRNAs by their dominant pathway into cryptic unstable transcripts (CUTs), Xrn1-sensitive unstable transcripts (XUTs), and Dicer-sensitive unstable transcripts (DUTs). XUTs and DUTs are enriched for antisense lncRNAs, while CUTs are often bidirectional and actively translated. The cytoplasmic exonuclease, along with RNAi, dampens the expression of thousands of lncRNAs and mRNAs that become induced during meiosis. Antisense lncRNA expression mostly negatively correlates with sense mRNA expression in the physiological, but not the genetic conditions. Intergenic and bidirectional lncRNAs emerge from nucleosome-depleted regions, upstream of positioned nucleosomes. Our results highlight both similarities and differences to lncRNA regulation in budding yeast. This broad survey of the lncRNA repertoire and characteristics in S. pombe, and the interwoven regulatory pathways that target lncRNAs, provides a rich framework for their further functional analyses. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Overexpression and localization of heat shock proteins mRNA in pancreatic carcinoma.

PubMed

Ogata, M; Naito, Z; Tanaka, S; Moriyama, Y; Asano, G

2000-06-01

In the present study we examined the localization and overexpression of heat shock proteins (hsps), mainly hsp90, in pancreatic carcinoma tissue compared with control tissue (including chronic pancreatitis and normal pancreas tissue), with the aid of immunohistochemical staining, in situ hybridization and reverse transcriptase polymerase chain reaction. Hsp90 alpha mRNA was overexpressed more highly in pancreatic carcinoma than in the control tissue. The proliferating-cell-nuclear-antigen labeling index was also high in pancreatic carcinoma tissue compared with the other tissue. These findings suggest that the overexpression of hsp90 alpha mRNA in carcinomas may be correlated with cell proliferation. However, hsp90 beta was constitutively overexpressed almost equally in all groups of pancreatic tissue including pancreatic carcinoma, chronic pancreatitis and normal pancreas tissue. Immunohistochemical staining demonstrated a differentiation in the expression of hsp90 between histological types of pancreatic carcinoma. These findings suggest that hsp90 alpha is involved in carcinogenesis and that hsp90 beta is correlated to structural conformation. Hsp90 alpha and hsp90 beta seem to perform different functions in tissue containing malignant cells. P53, MDM2 and WAF1, that were cell-cycle-related oncogene product were more strongly expressed in the nuclei of the cancer cells of the cancer tissue. Especially, MDM2 was more strongly expressed in mucinous carcinoma and the mucin secreting tissues surrounding pancreatic carcinoma tissue. The expression of MDM2 protein might also be correlated to secretion systems during structural conformation and be correlated to hsp90 beta.

TP53 Mutation Status of Tubo-ovarian and Peritoneal High-grade Serous Carcinoma with a Wild-type p53 Immunostaining Pattern.

PubMed

Na, Kiyong; Sung, Ji-Youn; Kim, Hyun-Soo

2017-12-01

Diffuse and strong nuclear p53 immunoreactivity and a complete lack of p53 expression are regarded as indicative of missense and nonsense mutations, respectively, of the TP53 gene. Tubo-ovarian and peritoneal high-grade serous carcinoma (HGSC) is characterized by aberrant p53 expression induced by a TP53 mutation. However, our experience with some HGSC cases with a wild-type p53 immunostaining pattern led us to comprehensively review previous cases and investigate the TP53 mutational status of the exceptional cases. We analyzed the immunophenotype of 153 cases of HGSC and performed TP53 gene sequencing analysis in those with a wild-type p53 immunostaining pattern. Immunostaining revealed that 109 (71.3%) cases displayed diffuse and strong p53 expression (missense mutation pattern), while 39 (25.5%) had no p53 expression (nonsense mutation pattern). The remaining five cases of HGSC showed a wild-type p53 immunostaining pattern. Direct sequencing analysis revealed that three of these cases harbored nonsense TP53 mutations and two had novel splice site deletions. TP53 mutation is almost invariably present in HGSC, and p53 immunostaining can be used as a surrogate marker of TP53 mutation. In cases with a wild-type p53 immunostaining pattern, direct sequencing for TP53 mutational status can be helpful to confirm the presence of a TP53 mutation. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Commensal Microbiota Enhance Both Osteoclast and Osteoblast Activities.

PubMed

Uchida, Yoko; Irie, Koichiro; Fukuhara, Daiki; Kataoka, Kota; Hattori, Takako; Ono, Mitsuaki; Ekuni, Daisuke; Kubota, Satoshi; Morita, Manabu

2018-06-23

Recent studies suggest that the commensal microbiota affects not only host energy metabolism and development of immunity but also bone remodeling by positive regulation of osteoclast activity. However, the mechanism of regulation of bone cells by the commensal microbiota has not been elucidated. In this study, 8-week-old specific pathogen-free (SPF) and germ-free (GF) mice were compared in terms of alveolar bones and primary osteoblasts isolated from calvarias. Micro-CT analysis showed that SPF mice had larger body size associated with lower bone mineral density and bone volume fraction in alveolar bones compared with GF mice. Greater numbers of osteoclasts in alveolar bone and higher serum levels of tartrate-resistant acid phosphatase 5b were observed in SPF mice. Tissue extracts from SPF alveolar bone showed higher levels of cathepsin K, indicating higher osteoclast activity. SPF alveolar extracts also showed elevated levels of γ-carboxylated glutamic acid⁻osteocalcin as a marker of mature osteoblasts compared with GF mice. Polymerase chain reaction (PCR) array analysis of RNA directly isolated from alveolar bone showed that in SPF mice, expression of mRNA of osteocalcin , which also acts as an inhibitor of bone mineralization, was strongly enhanced compared with GF mice. Cultured calvarial osteoblasts from SPF mice showed reduced mineralization but significantly enhanced expression of mRNAs of osteocalcin, alkaline phosphatase, insulin-like growth factor-I/II , and decreased ratio of osteoprotegerin/receptor activator of nuclear factor-kappa B ligand compared with GF mice. Furthermore, PCR array analyses of transcription factors in cultured calvarial osteoblasts showed strongly upregulated expression of Forkhead box g1 . In contrast, Gata-binding protein 3 was strongly downregulated in SPF osteoblasts. These results suggest that the commensal microbiota prevents excessive mineralization possibly by stimulating osteocalcin expression in osteoblasts, and enhances both osteoblast and osteoclast activity by regulating specific transcription factors.

Retrograde movement of tRNAs from the cytoplasm to the nucleus in Saccharomyces cerevisiae

PubMed Central

Shaheen, Hussam H.; Hopper, Anita K.

2005-01-01

In eukaryotes, tRNAs transcribed in the nucleus function in cytoplasmic protein synthesis. The Ran-GTP-binding exportin, Los1p/Xpo-t, and additional pathway(s) mediate tRNA transport to the cytoplasm. Although tRNA movement was thought to be unidirectional, recent reports that yeast precursor tRNA splicing occurs in the cytoplasm, whereas fully spliced tRNAs can reside in the nucleus, require that either the precursor tRNA splicing machinery or mature tRNAs move from the cytoplasm to the nucleus. Our data argue against the first possibility and strongly support the second. Combining heterokaryon analysis with fluorescence in situ hybridization, we show that a foreign tRNA encoded by one nucleus can move from the cytoplasm to a second nucleus that does not encode the tRNA. We also discovered nuclear accumulation of endogenous cytoplasmic tRNAs in haploid yeast cells in response to nutritional deprivation. Nuclear accumulation of cytoplasmic tRNA requires Ran and the Mtr10/Kap111 member of the importin-β family. Retrograde tRNA nuclear import may provide a novel mechanism to regulate gene expression in eukaryotes. PMID:16040803

Syndecan-4 Regulates Muscle Differentiation and Is Internalized from the Plasma Membrane during Myogenesis.

PubMed

Rønning, Sissel B; Carlson, Cathrine R; Stang, Espen; Kolset, Svein O; Hollung, Kristin; Pedersen, Mona E

2015-01-01

The cell surface proteoglycan syndecan-4 has been reported to be crucial for muscle differentiation, but the molecular mechanisms still remain to be fully understood. During in vitro differentiation of bovine muscle cells immunocytochemical analyses showed strong labelling of syndecan-4 intracellularly, in close proximity with Golgi structures, in membranes of intracellular vesicles and finally, in the nuclear area including the nuclear envelope. Chase experiments showed that syndecan-4 was internalized from the plasma membrane during this process. Furthermore, when syndecan-4 was knocked down by siRNA more myotubes were formed, and the expression of myogenic transcription factors, β1-integrin and actin was influenced. However, when bovine muscle cells were treated with a cell-penetrating peptide containing the cytoplasmic region of syndecan-4, myoblast fusion and thus myotube formation was blocked, both in normal cells and in syndecan-4 knock down cells. Altogether this suggests that the cytoplasmic domain of syndecan-4 is important in regulation of myogenesis. The internalization of syndecan-4 from the plasma membrane during muscle differentiation and the nuclear localization of syndecan-4 in differentiated muscle cells may be part of this regulation, and is a novel aspect of syndecan biology which merits further studies.

Retrograde movement of tRNAs from the cytoplasm to the nucleus in Saccharomyces cerevisiae.

PubMed

Shaheen, Hussam H; Hopper, Anita K

2005-08-09

In eukaryotes, tRNAs transcribed in the nucleus function in cytoplasmic protein synthesis. The Ran-GTP-binding exportin, Los1p/Xpo-t, and additional pathway(s) mediate tRNA transport to the cytoplasm. Although tRNA movement was thought to be unidirectional, recent reports that yeast precursor tRNA splicing occurs in the cytoplasm, whereas fully spliced tRNAs can reside in the nucleus, require that either the precursor tRNA splicing machinery or mature tRNAs move from the cytoplasm to the nucleus. Our data argue against the first possibility and strongly support the second. Combining heterokaryon analysis with fluorescence in situ hybridization, we show that a foreign tRNA encoded by one nucleus can move from the cytoplasm to a second nucleus that does not encode the tRNA. We also discovered nuclear accumulation of endogenous cytoplasmic tRNAs in haploid yeast cells in response to nutritional deprivation. Nuclear accumulation of cytoplasmic tRNA requires Ran and the Mtr10/Kap111 member of the importin-beta family. Retrograde tRNA nuclear import may provide a novel mechanism to regulate gene expression in eukaryotes.

Signal-dependent repression of DUSP5 by class I HDACs controls nuclear ERK activity and cardiomyocyte hypertrophy.

PubMed

Ferguson, Bradley S; Harrison, Brooke C; Jeong, Mark Y; Reid, Brian G; Wempe, Michael F; Wagner, Florence F; Holson, Edward B; McKinsey, Timothy A

2013-06-11

Cardiac hypertrophy is a strong predictor of morbidity and mortality in patients with heart failure. Small molecule histone deacetylase (HDAC) inhibitors have been shown to suppress cardiac hypertrophy through mechanisms that remain poorly understood. We report that class I HDACs function as signal-dependent repressors of cardiac hypertrophy via inhibition of the gene encoding dual-specificity phosphatase 5 (DUSP5) DUSP5, a nuclear phosphatase that negatively regulates prohypertrophic signaling by ERK1/2. Inhibition of DUSP5 by class I HDACs requires activity of the ERK kinase, mitogen-activated protein kinase kinase (MEK), revealing a self-reinforcing mechanism for promotion of cardiac ERK signaling. In cardiac myocytes treated with highly selective class I HDAC inhibitors, nuclear ERK1/2 signaling is suppressed in a manner that is absolutely dependent on DUSP5. In contrast, cytosolic ERK1/2 activation is maintained under these same conditions. Ectopic expression of DUSP5 in cardiomyocytes results in potent inhibition of agonist-dependent hypertrophy through a mechanism involving suppression of the gene program for hypertrophic growth. These findings define unique roles for class I HDACs and DUSP5 as integral components of a regulatory signaling circuit that controls cardiac hypertrophy.

The MCM-associated protein MCM-BP is important for human nuclear morphology.

PubMed

Jagannathan, Madhav; Sakwe, Amos M; Nguyen, Tin; Frappier, Lori

2012-01-01

Mini-chromosome maintenance complex-binding protein (MCM-BP) was discovered as a protein that is strongly associated with human MCM proteins, known to be crucial for DNA replication in providing DNA helicase activity. The Xenopus MCM-BP homologue appears to play a role in unloading MCM complexes from chromatin after DNA synthesis; however, the importance of MCM-BP and its functional contribution to human cells has been unclear. Here we show that depletion of MCM-BP by sustained expression of short hairpin RNA (shRNA) results in highly abnormal nuclear morphology and centrosome amplification. The abnormal nuclear morphology was not seen with depletion of other MCM proteins and was rescued with shRNA-resistant MCM-BP. MCM-BP depletion was also found to result in transient activation of the G2 checkpoint, slowed progression through G2 and increased replication protein A foci, indicative of replication stress. In addition, MCM-BP depletion led to increased cellular levels of MCM proteins throughout the cell cycle including soluble MCM pools. The results suggest that MCM-BP makes multiple contributions to human cells that are not limited to unloading of the MCM complex.

The Epstein–Barr virus nuclear protein SM is both a post-transcriptional inhibitor and activator of gene expression

PubMed Central

Ruvolo, Vivian; Wang, Eryu; Boyle, Sarah; Swaminathan, Sankar

1998-01-01

The Epstein–Barr virus (EBV) nuclear protein BS-MLF1 (SM) is expressed early after entry of EBV into the lytic cycle. SM transactivates reporter gene constructs driven by a wide variety of promoters, but the mechanism of SM action is poorly understood. In this study, we demonstrate that the SM protein inhibits expression of intron-containing genes and activates expression of intron-less genes. We demonstrate that SM has the predicted inhibitory effect on expression of a spliced EBV gene but activates an unspliced early EBV gene. SM inhibited gene expression at the post-transcriptional level by preventing the accumulation of nuclear and cytoplasmic RNA transcripts. Conversely, SM led to increased accumulation of nuclear mRNA from intron-less genes without affecting the rate of transcription, indicating that SM enhances nuclear RNA stability. The ratio of cytoplasmic to nuclear polyadenylated mRNA was increased in the presence of SM, suggesting that SM also enhances nucleo-cytoplasmic mRNA transport. The degree of transactivation by SM was dependent on the sequence of the 3′-untranslated region of the target mRNA. Finally, we demonstrate that the amino-terminal portion of SM fused to glutathione-S-transferase binds radioactively labeled RNA in vitro, indicating that SM is a single-stranded RNA binding protein. Importantly, the latent and immediate-early genes of EBV contain introns whereas many early and late genes do not. Thus, SM may down-regulate synthesis of host cell proteins and latent EBV proteins while simultaneously enhancing expression of specific lytic EBV genes by binding to mRNA and modulating its stability and transport. PMID:9671768

The Epstein-Barr virus nuclear protein SM is both a post-transcriptional inhibitor and activator of gene expression.

PubMed

Ruvolo, V; Wang, E; Boyle, S; Swaminathan, S

1998-07-21

The Epstein-Barr virus (EBV) nuclear protein BS-MLF1 (SM) is expressed early after entry of EBV into the lytic cycle. SM transactivates reporter gene constructs driven by a wide variety of promoters, but the mechanism of SM action is poorly understood. In this study, we demonstrate that the SM protein inhibits expression of intron-containing genes and activates expression of intron-less genes. We demonstrate that SM has the predicted inhibitory effect on expression of a spliced EBV gene but activates an unspliced early EBV gene. SM inhibited gene expression at the post-transcriptional level by preventing the accumulation of nuclear and cytoplasmic RNA transcripts. Conversely, SM led to increased accumulation of nuclear mRNA from intron-less genes without affecting the rate of transcription, indicating that SM enhances nuclear RNA stability. The ratio of cytoplasmic to nuclear polyadenylated mRNA was increased in the presence of SM, suggesting that SM also enhances nucleo-cytoplasmic mRNA transport. The degree of transactivation by SM was dependent on the sequence of the 3'-untranslated region of the target mRNA. Finally, we demonstrate that the amino-terminal portion of SM fused to glutathione-S-transferase binds radioactively labeled RNA in vitro, indicating that SM is a single-stranded RNA binding protein. Importantly, the latent and immediate-early genes of EBV contain introns whereas many early and late genes do not. Thus, SM may down-regulate synthesis of host cell proteins and latent EBV proteins while simultaneously enhancing expression of specific lytic EBV genes by binding to mRNA and modulating its stability and transport.

Space nuclear power: Key to outer solar system exploration

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

Bennett, G.L.; Allen, D.M.

1998-07-01

In 1995, in response to threatened budget cuts, the American Institute of Aeronautics and Astronautics (AIAA) approved a position paper supporting the maintenance of the technology base for space nuclear power. The position paper contained four recomemndations: (1) DOE, NASA, and DoD should develop and support an integrated program that maintains the nuclear option and develops the needed high-payoff technologies; (2) Congress should provide strong, continuing financial and political support for the agencies' program; (3) Government and industry leaders should voice their advocacy for a strong space nuclear power program to support future system requirements; and (4) The US shouldmore » continue to maintain its cooperation and technical interchanges with other countries to advance nuclear power source technology and to promote nuclear safety.« less

Tyrosine kinase receptor B (TrkB) expression in colorectal cancers highlights anoikis resistance as a survival mechanism of tumour budding cells.

PubMed

Dawson, Heather; Grundmann, Sandra; Koelzer, Viktor H; Galván, José A; Kirsch, Richard; Karamitopoulou, Eva; Lugli, Alessandro; Inderbitzin, Daniel; Zlobec, Inti

2015-04-01

Tumour buds in colorectal cancer represent an aggressive subgroup of non-proliferating and non-apoptotic tumour cells. We hypothesize that the survival of tumour buds is dependent upon anoikis resistance. The role of tyrosine kinase receptor B (TrkB), a promoter of epithelial-mesenchymal transition and anoikis resistance, in facilitating budding was investigated. Tyrosine kinase receptor B immunohistochemistry was performed on a multiple-punch tissue microarray of 211 colorectal cancer resections. Membranous/cytoplasmic and nuclear expression was evaluated in tumour and buds. Tumour budding was assessed on corresponding whole tissue slides. Relationship to Ki-67 and caspase-3 was investigated. Analysis of Kirsten Ras (KRAS), proto-oncogene B-RAF (BRAF) and cytosine-phosphate-guanosine island methylator phenotype (CIMP) was performed. Membranous/cytoplasmic and nuclear TrkB were strongly, inversely correlated (P < 0.0001; r = -0.41). Membranous/cytoplasmic TrkB was overexpressed in buds compared to the main tumour body (P < 0.0001), associated with larger tumours (P = 0.0236), high-grade budding (P = 0.0011) and KRAS mutation (P = 0.0008). Nuclear TrkB was absent in buds (P <0.0001) and in high-grade budding cancers (P =0.0073). Among patients with membranous/cytoplasmic TrkB-positive buds, high tumour membranous/cytoplasmic TrkB expression was a significant, independent adverse prognostic factor [P = 0.033; 1.79, 95% confidence interval (CI) 1.05-3.05]. Inverse correlations between membranous/cytoplasmic TrkB and Ki-67 (r = -0.41; P < 0.0001) and caspase-3 (r =-0.19; P < 0.05) were observed. Membranous/cytoplasmic TrkB may promote an epithelial-mesenchymal transition (EMT)-like phenotype with high-grade budding and maintain viability of buds themselves. © 2014 John Wiley & Sons Ltd.

Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A)

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

Oi, Ami; Katayama, Syouichi; Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Shiga, 525-8577

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed amore » typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization. - Highlights: • We investigated the mechanism regulating subcellular localization of CDKL5. • DYRK1A was identified as an enzyme that bound to and phosphorylated CDKL5. • The phosphorylation site of CDKL5 was Ser-308, in the vicinity of the NLS. • When DYRK1A was co-expressed, the cytosolic CDKL5 was significantly increased. • In conclusion, DYRK1A regulates CDKL5 localization via phosphorylation on Ser-308.« less

Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle.

PubMed

Pasion, S G; Brown, G W; Brown, L M; Ray, D S

1994-12-01

In trypanosomatids, DNA replication in the nucleus and in the single mitochondrion (or kinetoplast) initiates nearly simultaneously, suggesting that the DNA synthesis (S) phases of the nucleus and the mitochondrion are coordinately regulated. To investigate the basis for the temporal link between nuclear and mitochondrial DNA synthesis phases the expression of the genes encoding DNA ligase I, the 51 and 28 kDa subunits of replication protein A, dihydrofolate reductase and the mitochondrial type II topoisomerase were analyzed during the cell cycle progression of synchronous cultures of Crithidia fasciculata. These DNA replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone (pdN-1) in which TOP2, the gene encoding the mitochondrial topoisomerase, was disrupted by the insertion of a NEO drug-resistance cassette was found to express both a truncated TOP2 mRNA and a truncated topoisomerase polypeptide. The truncated mRNA was also expressed periodically coordinate with the expression of the endogenous TOP2 mRNA indicating that cis elements necessary for periodic expression are contained within cloned sequences. The expression of both TOP2 and nuclear DNA replication genes at the G1/S boundary suggests that regulated expression of these genes may play a role in coordinating nuclear and mitochondrial S phases in trypanosomatids.

The Yeast Nuclear Pore Complex and Transport Through It

PubMed Central

Aitchison, John D.; Rout, Michael P.

2012-01-01

Exchange of macromolecules between the nucleus and cytoplasm is a key regulatory event in the expression of a cell’s genome. This exchange requires a dedicated transport system: (1) nuclear pore complexes (NPCs), embedded in the nuclear envelope and composed of proteins termed nucleoporins (or “Nups”), and (2) nuclear transport factors that recognize the cargoes to be transported and ferry them across the NPCs. This transport is regulated at multiple levels, and the NPC itself also plays a key regulatory role in gene expression by influencing nuclear architecture and acting as a point of control for various nuclear processes. Here we summarize how the yeast Saccharomyces has been used extensively as a model system to understand the fundamental and highly conserved features of this transport system, revealing the structure and function of the NPC; the NPC’s role in the regulation of gene expression; and the interactions of transport factors with their cargoes, regulatory factors, and specific nucleoporins. PMID:22419078

Nuclear Calcium Buffering Capacity Shapes Neuronal Architecture.

PubMed

Mauceri, Daniela; Hagenston, Anna M; Schramm, Kathrin; Weiss, Ursula; Bading, Hilmar

2015-09-18

Calcium-binding proteins (CaBPs) such as parvalbumin are part of the cellular calcium buffering system that determines intracellular calcium diffusion and influences the spatiotemporal dynamics of calcium signals. In neurons, CaBPs are primarily localized to the cytosol and function, for example, in nerve terminals in short-term synaptic plasticity. However, CaBPs are also expressed in the cell nucleus, suggesting that they modulate nuclear calcium signals, which are key regulators of neuronal gene expression. Here we show that the calcium buffering capacity of the cell nucleus in mouse hippocampal neurons regulates neuronal architecture by modulating the expression levels of VEGFD and the complement factor C1q-c, two nuclear calcium-regulated genes that control dendrite geometry and spine density, respectively. Increasing the levels of nuclear calcium buffers by means of expression of a nuclearly targeted form of parvalbumin fused to mCherry (PV.NLS-mC) led to a reduction in VEGFD expression and, as a result, to a decrease in total dendritic length and complexity. In contrast, mRNA levels of the synapse pruning factor C1q-c were increased in neurons expressing PV.NLS-mC, causing a reduction in the density and size of dendritic spines. Our results establish a close link between nuclear calcium buffering capacity and the transcription of genes that determine neuronal structure. They suggest that the development of cognitive deficits observed in neurological conditions associated with CaBP deregulation may reflect the loss of necessary structural features of dendrites and spines. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Production of ABA responses requires both the nuclear and cytoplasmic functional involvement of PYR1

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

Park, EunJoo; Kim, Tae-Houn

Abscisic acid (ABA) enhances stress tolerant responses in plants against unfavorable environmental conditions. In Arabidopsis, ABA promotes interactions between PYR/PYL/RCARs and PP2C, thereby allowing SnRK2s to phosphorylate downstream components required for the regulation of gene expression or for gating ion channels. Because PYR1 is known to localize to nucleus and cytoplasm it is a question whether nuclear or cytoplasmic PYR1 confer different functions to the ABA signaling pathway, as has been previously shown for regulatory proteins. In order to answer this question, transgenic lines expressing nuclear PYR1 were generated in an ABA insensitive mutant background. Enforced nuclear expression of PYR1more » was examined by confocal microscopy and western blot analysis. Physiological analyses of the transgenic lines demonstrated that nuclear PYR1 is sufficient to generate ABA responses, such as, the inhibition of seed germination, root growth inhibition, the induction of gene expression, and stomatal closing movement. However, for the full recovery of ABA responses in the mutant background cytoplasmic PYR1 was required. The study suggests both nuclear and cytoplasmic PYR1 participate in the control of ABA signal transduction. - Highlights: • Nuclear and cytoplasmic functions of PYR1 were studied in the mutant which lacked majority of ABA responses. • Nuclear PYR1 reconstituted partially the ABA responses during seed germination, root growth, and guard cell movement. • Both the nuclear and cytoplasmic functions of PYR1 were required for the full generation of ABA responses.« less

Nucleus-specific expression in the multinuclear mushroom-forming fungus Agaricus bisporus reveals different nuclear regulatory programs.

PubMed

Gehrmann, Thies; Pelkmans, Jordi F; Ohm, Robin A; Vos, Aurin M; Sonnenberg, Anton S M; Baars, Johan J P; Wösten, Han A B; Reinders, Marcel J T; Abeel, Thomas

2018-04-24

Many fungi are polykaryotic, containing multiple nuclei per cell. In the case of heterokaryons, there are different nuclear types within a single cell. It is unknown what the different nuclear types contribute in terms of mRNA expression levels in fungal heterokaryons. Each cell of the mushroom Agaricus bisporus contains two to 25 nuclei of two nuclear types originating from two parental strains. Using RNA-sequencing data, we assess the differential mRNA contribution of individual nuclear types and its functional impact. We studied differential expression between genes of the two nuclear types, P1 and P2, throughout mushroom development in various tissue types. P1 and P2 produced specific mRNA profiles that changed through mushroom development. Differential regulation occurred at the gene level, rather than at the locus, chromosomal, or nuclear level. P1 dominated mRNA production throughout development, and P2 showed more differentially up-regulated genes in important functional groups. In the vegetative mycelium, P2 up-regulated almost threefold more metabolism genes and carbohydrate active enzymes (cazymes) than P1, suggesting phenotypic differences in growth. We identified widespread transcriptomic variation between the nuclear types of A. bisporus Our method enables studying nucleus-specific expression, which likely influences the phenotype of a fungus in a polykaryotic stage. Our findings have a wider impact to better understand gene regulation in fungi in a heterokaryotic state. This work provides insight into the transcriptomic variation introduced by genomic nuclear separation. Copyright © 2018 the Author(s). Published by PNAS.

Negative regulation of parathyroid hormone-related protein expression by steroid hormones

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

Kajitani, Takashi; Tamamori-Adachi, Mimi; Okinaga, Hiroko

Highlights: {yields} Steroid hormones repress expression of PTHrP in the cell lines where the corresponding nuclear receptors are expressed. {yields} Nuclear receptors are required for suppression of PTHrP expression by steroid hormones, except for androgen receptor. {yields} Androgen-induced suppression of PTHrP expression appears to be mediated by estrogen receptor. -- Abstract: Elevated parathyroid hormone-related protein (PTHrP) is responsible for humoral hypercalcemia of malignancy (HHM), which is of clinical significance in treatment of terminal patients with malignancies. Steroid hormones were known to cause suppression of PTHrP expression. However, detailed studies linking multiple steroid hormones to PTHrP expression are lacking. Here wemore » studied PTHrP expression in response to steroid hormones in four cell lines with excessive PTHrP production. Our study established that steroid hormones negatively regulate PTHrP expression. Vitamin D receptor, estrogen receptor {alpha}, glucocorticoid receptor, and progesterone receptor, were required for repression of PTHrP expression by the cognate ligands. A notable exception was the androgen receptor, which was dispensable for suppression of PTHrP expression in androgen-treated cells. We propose a pathway(s) involving nuclear receptors to suppress PTHrP expression.« less

AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells.

PubMed

Lorenzato, Annalisa; Biolatti, Marta; Delogu, Giuseppe; Capobianco, Giampiero; Farace, Cristiano; Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco; Madeddu, Roberto; Olivero, Martina; Di Renzo, Maria Flavia

2013-10-15

The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. © 2013 Elsevier Inc. All rights reserved.

Nuclear translocation of Acinetobacter baumannii transposase induces DNA methylation of CpG regions in the promoters of E-cadherin gene.

PubMed

Moon, Dong Chan; Choi, Chul Hee; Lee, Su Man; Lee, Jung Hwa; Kim, Seung Il; Kim, Dong Sun; Lee, Je Chul

2012-01-01

Nuclear targeting of bacterial proteins has emerged as a pathogenic mechanism whereby bacterial proteins induce host cell pathology. In this study, we examined nuclear targeting of Acinetobacter baumannii transposase (Tnp) and subsequent epigenetic changes in host cells. Tnp of A. baumannii ATCC 17978 possesses nuclear localization signals (NLSs), (225)RKRKRK(230). Transient expression of A. baumannii Tnp fused with green fluorescent protein (GFP) resulted in the nuclear localization of these proteins in COS-7 cells, whereas the truncated Tnp without NLSs fused with GFP were exclusively localized in the cytoplasm. A. baumannii Tnp was found in outer membrane vesicles, which delivered this protein to the nucleus of host cells. Nuclear expression of A. baumannii Tnp fused with GFP in A549 cells induced DNA methylation of CpG regions in the promoters of E-cadherin (CDH1) gene, whereas the cytoplasmic localization of the truncated Tnp without NLSs fused with GFP did not induce DNA methylation. DNA methylation in the promoters of E-cadherin gene induced by nuclear targeting of A. baumannii Tnp resulted in down-regulation of gene expression. In conclusion, our data show that nuclear traffic of A. baumannii Tnp induces DNA methylation of CpG regions in the promoters of E-cadherin gene, which subsequently down-regulates gene expression. This study provides a new insight into the epigenetic control of host genes by bacterial proteins.

Negative transcriptional regulation of mitochondrial transcription factor A (TFAM) by nuclear TFAM

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

Lee, Eun Jin; Kang, Young Cheol; Park, Wook-Ha

2014-07-18

Highlights: • TFAM localizes in nuclei and mitochondria of neuronal cells. • Nuclear TFAM does not bind the Tfam promoter. • Nuclear TFAM reduced the Tfam promoter activity via suppressing NRF-1 activity. • A novel self-negative feedback regulation of Tfam gene expression is explored. • FAM may play different roles depending on its subcellular localizations. - Abstract: The nuclear DNA-encoded mitochondrial transcription factor A (TFAM) is synthesized in cytoplasm and transported into mitochondria. TFAM enhances both transcription and replication of mitochondrial DNA. It is unclear, however, whether TFAM plays a role in regulating nuclear gene expression. Here, we demonstrated thatmore » TFAM was localized to the nucleus and mitochondria by immunostaining, subcellular fractionation, and TFAM-green fluorescent protein hybrid protein studies. In HT22 hippocampal neuronal cells, human TFAM (hTFAM) overexpression suppressed human Tfam promoter-mediated luciferase activity in a dose-dependent manner. The mitochondria targeting sequence-deficient hTFAM also repressed Tfam promoter activity to the same degree as hTFAM. It indicated that nuclear hTFAM suppressed Tfam expression without modulating mitochondrial activity. The repression required for nuclear respiratory factor-1 (NRF-1), but hTFAM did not bind to the NRF-1 binding site of its promoter. TFAM was co-immunoprecipitated with NRF-1. Taken together, we suggest that nuclear TFAM down-regulate its own gene expression as a NRF-1 repressor, showing that TFAM may play different roles depending on its subcellular localizations.« less

Cloning of the transgenic pigs expressing human decay accelerating factor and N-acetylglucosaminyltransferase III.

PubMed

Fujimura, Tatsuya; Kurome, Mayuko; Murakami, Hiroshi; Takahagi, Yoichi; Matsunami, Katsuyoshi; Shimanuki, Shinichi; Suzuki, Kohei; Miyagawa, Shuji; Shirakura, Ryota; Shigehisa, Tamotsu; Nagashima, Hiroshi

2004-01-01

The present paper describes production of cloned pigs from fibroblast cells of transgenic pigs expressing human decay accelerating factor (DAF, CD55) and N-acetylglucosaminyltransferase III (GnT-III) that remodels sugar-chain biosynthesis. Two nuclear transfer protocols were used: a two-step activation (TA) method and a delayed activation (DA) method. Enucleated in vitro-matured oocytes and donor cells were electrically fused in a calcium-containing medium by TA method or in a calcium-free medium by DA method, followed by electrical activation 1-1.5 h later, respectively. In vitro blastocyst formation rates of nuclear transferred embryos reconstructed by TA and DA method were 8% and 14%, respectively. As a result of embryo transfer of the reconstructed embryos made by each method into recipient pigs, both gave rise to cloned piglets. These cloned pigs expressed transgene as much as their nuclear donor cells. In conclusions, (1) pig cloning can be carried out by TA or DA nuclear transfer methods, (2) expression of transgenes can be maintained to cloned pigs from the nuclear donor cells derived from transgenic animals.

Serine/Threonine kinase dependent transcription from the polyhedrin promoter of SpltNPV-I

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

Mishra, Gourav; Gautam, Hemant K.; Das, Rakha H.

2007-07-06

Polyhedrin (polh) and p10 are the two hyper-expressed very late genes of nucleopolyhedroviruses. Alpha amanitin resistant transcription from Spodoptera litura nucleopolyhedrovirus (SpltNPV-I) polyhedrin promoter was observed with virus infected nuclear extract of NIV-HA-197 cells but not with that from uninfected nuclear extract. Anti-protein kinase-1 (pk1) antibody inhibited the transcription and the inhibition reversed on addition of pk1, however, pk1 mutant protein, K50M having no phosphorylation activity did not overcome the transcription inhibition. Chromatin immuno-precipitation assays with viral anti-pk1 antibody showed the interaction of pk1 with the polh while electrophoretic mobility shift assays indicated the strong binding affinity (K {sub d}more » {approx} 5.5 x 10{sup -11}) of purified pk1 with the polh promoter. These results suggested that the viral coded pk1 acts as a transcription factor in transcribing baculovirus very late genes.« less

Probing crustal structures from neutron star compactness

NASA Astrophysics Data System (ADS)

Sotani, Hajime; Iida, Kei; Oyamatsu, Kazuhiro

2017-10-01

With various sets of the parameters that characterize the equation of state (EOS) of nuclear matter, we systematically examine the thickness of a neutron star crust and of the pasta phases contained therein. Then, with respect to the thickness of the phase of spherical nuclei, the thickness of the cylindrical phase and the crust thickness, we successfully derive fitting formulas that express the ratio of each thickness to the star's radius as a function of the star's compactness, the incompressibility of symmetric nuclear matter and the density dependence of the symmetry energy. In particular, we find that the thickness of the phase of spherical nuclei has such a strong dependence on the stellar compactness as the crust thickness, but both of them show a much weaker dependence on the EOS parameters. Thus, via determination of the compactness, the thickness of the phase of spherical nuclei as well as the crust thickness can be constrained reasonably, even if the EOS parameters remain to be well-determined.

OPG/membranous--RANKL complex is internalized via the clathrin pathway before a lysosomal and a proteasomal degradation.

PubMed

Tat, Steeve Kwan; Padrines, Marc; Theoleyre, Sandrine; Couillaud-Battaglia, Severine; Heymann, Dominique; Redini, Françoise; Fortun, Yannick

2006-10-01

The members of the OPG/RANK/RANKL (osteoprotegerin/receptor activator of nuclear factor kappaB/RANK ligand) triad are involved in various osteolytic pathologies such as bone tumors. Although many studies described the use of OPG during the treatment of bone diseases, its bioavailability and the mechanism by which the cells control the extracellular OPG remains blurred. The present work uses a strongly RANKL expressing cellular model to assess the becoming and the bioavailability of exogenous OPG in the context of its interactions with RANKL. The human kidney cell line 293, which initially expresses neither OPG nor RANKL, was stably transfected by the full length of mouse transmembranous form of RANKL (293RL). When OPG is incubated with 293RL cells, the extracellular concentration of OPG was strongly decreased in a time-dependent manner. The OPG disappearance was not inhibited by the addition of several proteases inhibitors, thus excluding any extracellular protease degradation. Contrary to previous results obtained on myeloma cells, which strongly express syndecan-1, the OPG disappearance was unaffected by the use of an antibody against syndecan-1. However, this event was abolished by an antibody against RANKL. These results, not necessarily conflicting, could be in relation with the expression level of the receptors in the two cellular models. In this context, an internalization process was put forward. Confocal microscopy demonstrated via the clathrin pathway an internalization of OPG mediated by RANKL. After being internalized, OPG was then degraded by the proteasome and the lysosome. A similar internalization phenomenon was also observed in osteoblast cells expressing physiologically RANKL, thus validating our data observed on 293RL cells. Western blotting analysis revealed that the half-life of RANKL was greatly reduced in the presence of OPG, pointing out that OPG binding to RANKL induces an enhancement of the ligand internalization. By the light of these results, the inhibitory effect of OPG on bone resorption can be explained not only by a decoy receptor function, competitor inhibitor of the RANK/RANKL binding, but also by the modulation of the RANKL half-life induced by OPG. Reciprocally, this modulation contributes to reduce the bioavailability of OPG.

Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins.

PubMed

Maric, Martina; Haugo, Alison C; Dauer, William; Johnson, David; Roller, Richard J

2014-07-01

Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

Prognostic Significance of Human Apurinic/Apyrimidinic Endonuclease (APE/Ref-1) Expression in Rectal Cancer Treated With Preoperative Radiochemotherapy

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

Kim, Jun-Sang, E-mail: k423j@cnu.ac.kr; Cancer Research Institute, Chungnam National University, Daejeon; Kim, Jin-Man

Purpose: Human apurinic endonuclease/redox factor 1 (APE/Ref-1) mediates repair of radiation-induced DNA lesions and regulates transcription via redox-based activation. We investigated the predictive and prognostic significance of APE/Ref-1 expression in pretreatment biopsy specimens in locally advanced rectal cancer (LARC) (cT3-T4 or N+). Methods and Materials: APE/Ref-1 expression was analyzed by immunohistochemistry in pretreatment biopsy specimens obtained from 83 patients with LARC. Patients received preoperative radiotherapy of 50.4 Gy in 28 fractions, combined with oral capecitabine and leucovorin chemotherapy, followed by curative surgery. The prognostic significance of various clinicopathologic characteristics, including APE/Ref-1 protein expression, was evaluated. Results: APE/Ref-1 was expressed inmore » 97% of patient samples. Exclusive APE/Ref-1 nuclear staining was observed in 49 of 83 samples (59%), and mixed nuclear and cytoplasmic staining was observed in 31 samples (37%). APE/Ref-1 nuclear expression levels were low in 49 patients (59%) and high in 34 patients (41%). The level of APE/Ref-1 nuclear expression was not a prognostic factor for overall and disease-free survival. Cytoplasmic expression of APE/Ref-1 was a borderline-significant predictive factor for pathologic tumor response (p = 0.08) and a significant prognostic factor for disease-free survival, as shown by univariate analysis (p = 0.037). Multivariate analysis confirmed that cytoplasmic localization of APE/Ref-1 is a significant predictor of disease-free survival (hazard ratio, 0.45; p = 0.046). Conclusions: APE/Ref-1 was expressed in a majority of pretreatment biopsy specimens from patients with LARC. The level of APE/Ref-1 nuclear expression was not a significant predictive and prognostic factor; however, cytoplasmic localization of the protein was negatively associated with disease-free survival. These results indicate that cytoplasmic expression of APE/Ref-1 represents an adverse prognostic factor for LARC patients who receive preoperative radiochemotherapy.« 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

Novel scintillators and silicon photomultipliers for nuclear physics and applications

NASA Astrophysics Data System (ADS)

Jenkins, David

2015-06-01

Until comparatively recently, scintillator detectors were seen as an old-fashioned tool of nuclear physics with more attention being given to areas such as gamma-ray tracking using high-purity germanium detectors. Next-generation scintillator detectors, such as lanthanum bromide, which were developed for the demands of space science and gamma- ray telescopes, are found to have strong applicability to low energy nuclear physics. Their excellent timing resolution makes them very suitable for fast timing measurements and their much improved energy resolution compared to conventional scintillators promises to open up new avenues in nuclear physics research which were presently hard to access. Such "medium-resolution" spectroscopy has broad interest across several areas of contemporary interest such as the study of nuclear giant resonances. In addition to the connections to space science, it is striking that the demands of contemporary medical imaging have strong overlap with those of experimental nuclear physics. An example is the interest in PET-MRI combined imaging which requires putting scintillator detectors in a high magnetic field environment. This has led to strong advances in the area of silicon photomultipliers, a solid-state replacement for photomultiplier tubes, which are insensitive to magnetic fields. Broad application to nuclear physics of this technology may be foreseen.

Induction of apoptosis in cells expressing exogenous Hippi, a molecular partner of huntingtin-interacting protein Hip1.

PubMed

Majumder, Pritha; Chattopadhyay, Biswanath; Mazumder, Arindam; Das, Pradeep; Bhattacharyya, Nitai P

2006-05-01

To decipher the pathway of apoptosis induction downstream to caspase-8 activation by exogenous expression of Hippi, an interactor of huntingtin-interacting protein Hip1, we studied apoptosis in HeLa and Neuro2A cells expressing GFP-tagged Hippi. Nuclear fragmentation, caspase-1, caspase-8, caspase-9/caspase-6 and caspase-3 activation were increased significantly in Hippi expressing cells. Cleavage of Bid, release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria were also increased in GFP-Hippi expressing cells. It was observed that caspase-1 and caspase-8 activation was earlier than caspase-3 activation and nuclear fragmentation. Expression of caspase-1, caspase-3 and caspase-7 was increased while anti-apoptotic gene Bcl-2 and mitochondrial genes ND1 and ND4 were reduced in Hippi expressing cells. Besides, the expression SDHA and SDHB, nuclear genes, subunits of mitochondrial complex II were decreased in GFP-Hippi expressing cells. Taken together, we concluded that Hippi expression induced apoptosis by releasing AIF and cytochrome c from mitochondria, activation of caspase-1 and caspase-3, and altering the expression of apoptotic genes and genes involved in mitochondrial complex I and II.

Clonorchis sinensis excretory-secretory products regulate migration and invasion in cholangiocarcinoma cells via extracellular signal-regulated kinase 1/2/nuclear factor-κB-dependent matrix metalloproteinase-9 expression.

PubMed

Pak, Jhang Ho; Shin, Jimin; Song, In-Sung; Shim, Sungbo; Jang, Sung-Wuk

2017-01-01

Matrix metalloproteinase-9 plays an important role in the invasion and metastasis of various types of cancer cells. We have previously reported that excretory-secretory products from Clonorchis sinensis increases matrix metalloproteinase-9 expression. However, the regulatory mechanisms through which matrix metalloproteinase-9 expression affects cholangiocarcinoma development remain unclear. In the current study, we examined the potential role of excretory-secretory products in regulating the migration and invasion of various cholangiocarcinoma cell lines. We demonstrated that excretory-secretory products significantly induced matrix metalloproteinase-9 expression and activity in a concentration-dependent manner. Reporter gene and chromatin immunoprecipitation assays showed that excretory-secretory products induced matrix metalloproteinase-9 expression by enhancing the activity of nuclear factor-kappa B. Moreover, excretory-secretory products induced the degradation and phosphorylation of IκBα and stimulated nuclear factor-kappa B p65 nuclear translocation, which was regulated by extracellular signal-regulated kinase 1/2. Taken together, our findings indicated that the excretory-secretory product-dependent enhancement of matrix metalloproteinase-9 activity and subsequent induction of IκBα and nuclear factor-kappa B activities may contribute to the progression of cholangiocarcinoma. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Dynamic nuclear polarisation via the integrated solid effect II: experiments on naphthalene-h8 doped with pentacene-d14

NASA Astrophysics Data System (ADS)

Eichhorn, T. R.; van den Brandt, B.; Hautle, P.; Henstra, A.; Wenckebach, W. Th.

2014-07-01

In dynamic nuclear polarisation (DNP), also called hyperpolarisation, a small amount of unpaired electron spins is added to the sample containing the nuclear spins, and the polarisation of these unpaired electron spins is transferred to the nuclear spins by means of a microwave field. Traditional DNP polarises the electron spin of stable paramagnetic centres by cooling down to low temperature and applying a strong magnetic field. Then weak continuous wave microwave fields are used to induce the polarisation transfer. Complicated cryogenic equipment and strong magnets can be avoided using short-lived photo-excited triplet states that are strongly aligned in the optical excitation process. However, a much faster transfer of the electron spin polarisation is needed and pulsed DNP methods like nuclear orientation via electron spin locking (NOVEL) and the integrated solid effect (ISE) are used. To describe the polarisation transfer with the strong microwave fields in NOVEL and ISE, the usual perturbation methods cannot be used anymore. In the previous paper, we presented a theoretical approach to calculate the polarisation transfer in ISE. In the present paper, the theory is applied to the system naphthalene-h8 doped with pentacene-d14 yielding the photo-excited triplet states and compared with experimental results.

Effect of nuclear factor kappa B on intercellular adhesion molecule-1 expression and neutrophil infiltration in lung injury induced by intestinal ischemia/reperfusion in rats

PubMed Central

Tian, Xiao-Feng; Yao, Ji-Hong; Li, Ying-Hua; Zhang, Xue-Song; Feng, Bing-An; Yang, Chun-Ming; Zheng, Shu-Sen

2006-01-01

AIM: To investigate the role of nuclear factor kappa B (NF-κB) in the pathogenesis of lung injury induced by intestinal ischemia/reperfusion (I/R), and its effect on intercellular adhesion molecule-1 (ICAM-1) expression and neutrophil infiltration. METHODS: Twenty-four Wistar rats were divided randomly into control, I/R and pyrrolidine dithiocarbamate (PDTC) treatment groups, n = 8 in each. I/R group and PDTC treatment group received superior mysenteric artery (SMA) occluding for 1 h and reperfusion for 2 h. PDTC group was administrated with intraperitoneal injection of 2% 100 mg/kg PDTC 1 h before surgery. Lung histology and bronchia alveolus lung fluid (BALF) protein were assayed. Serum IL-6, lung malondialdehyde (MDA) and myeloperoxidase (MPO) as well as the expression level of NF-κB and ICAM-1 were measured. RESULTS: Lung injury induced by intestinal I/R, was characterized by edema, hemorrhage and neutrophil infiltration as well as by the significant rising of BALF protein. Compared to control group, the levels of serum IL-6 and lung MDA and MPO increased significantly in I/R group (P = 0.001). Strong positive expression of NF-κB p65 and ICAM-1 was observed. After the administration of PDTC, the level of serum IL-6, lung MDA and MPO as well as NF-κB and ICAM-1 decreased significantly (P < 0.05) when compared to I/R group. CONCLUSION: The activation of NF-κB plays an important role in the pathogenesis of lung injury induced by intestinal I/R through upregulating the neutrophil infiltration and lung ICAM-1 expression. PDTC as an inhibitor of NF-κB can prevent lung injury induced by intestinal I/R through inhibiting the activity of NF-κB. PMID:16489637

Inflamed In Vitro Retina: Cytotoxic Neuroinflammation and Galectin-3 Expression

PubMed Central

Bauer, Patrik Maximilian; Zalis, Marina Castro; Abdshill, Hodan; Deierborg, Tomas; Johansson, Fredrik; Englund-Johansson, Ulrica

2016-01-01

Background Disease progression in retinal neurodegeneration is strongly correlated to immune cell activation, which may have either a neuroprotective or neurotoxic effect. Increased knowledge about the immune response profile and retinal neurodegeneration may lead to candidate targets for treatments. Therefore, we have used the explanted retina as a model to explore the immune response and expression of the immune modulator galectin-3 (Gal-3), induced by the cultivation per se and after additional immune stimulation with lipopolysaccharide (LPS), and how this correlates with retinal neurotoxicity. Methods Post-natal mouse retinas were cultured in a defined medium. One group was stimulated with LPS (100 ng/ml, 24 h). Retinal architecture, apoptotic cell death, and micro- and macroglial activity were studied at the time of cultivation (0 days in vitro (DIV)) and at 3, 4 and 7 DIV using morphological staining, biochemical- and immunohistochemical techniques. Results Our results show that sustained activation of macro- and microglia, characterized by no detectable cytokine release and limited expression of Gal-3, is not further inducing apoptosis additional to the axotomy-induced apoptosis in innermost nuclear layer. An elevated immune response was detected after LPS stimulation, as demonstrated primarily by release of immune mediators (i.e. interleukin 2 (IL-2), IL-6, KC/GRO (also known as CLCX1) and tumour necrosis factor-α (TNF-α)), increased numbers of microglia displaying morphologies of late activation stages as well as Gal-3 expression. This was accompanied with increased apoptosis in the two additional nuclear layers, and damage to retinal gross architecture. Conclusion We demonstrate that an immune response characterized by sustained and increased release of cytokines, along with an increase in Gal-3 expression, is accompanied by significant increased neurotoxicity in the explanted retina. Further investigations using the current setting may lead to increased understanding on the mechanisms involved in neuronal loss in retinal neurodegenerations. PMID:27612287

A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection

NASA Astrophysics Data System (ADS)

Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.

2000-05-01

RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

BMI1 Is Expressed in Canine Osteosarcoma and Contributes to Cell Growth and Chemotherapy Resistance

PubMed Central

Gandour-Edwards, Regina; Withers, Sita S.; Holt, Roseline; Rebhun, Robert B.

2015-01-01

BMI1, a stem cell factor and member of the polycomb group of genes, has been shown to contribute to growth and chemoresistance of several human malignancies including primary osteosarcoma (OSA). Naturally occurring OSA in the dog represents a large animal model of human OSA, however the potential role of BMI1 in canine primary and metastatic OSA has not been examined. Immunohistochemical staining of canine primary and metastatic OSA tumors revealed strong nuclear expression of BMI1. An identical staining pattern was found in both primary and metastatic human OSA tissues. Canine OSA cell lines (Abrams, Moresco, and D17) expressed high levels of BMI1 compared with canine osteoblasts and knockdown or inhibition of BMI1 by siRNA or by small molecule BMI1-inhibitor PTC-209 demonstrated a role for BMI1 in canine OSA cell growth and resistance to carboplatin and doxorubicin chemotherapy. These findings suggest that inhibition of BMI1 in primary or metastatic OSA may improve response to chemotherapy and that the dog may serve as a large animal model to evaluate such therapy. PMID:26110620

Saccharomyces cerevisiae possesses a stress-inducible glycyl-tRNA synthetase gene.

PubMed

Chen, Shun-Jia; Wu, Yi-Hua; Huang, Hsiao-Yun; Wang, Chien-Chia

2012-01-01

Aminoacyl-tRNA synthetases are a large family of housekeeping enzymes that are pivotal in protein translation and other vital cellular processes. Saccharomyces cerevisiae possesses two distinct nuclear glycyl-tRNA synthetase (GlyRS) genes, GRS1 and GRS2. GRS1 encodes both cytoplasmic and mitochondrial activities, while GRS2 is essentially silent and dispensable under normal conditions. We herein present evidence that expression of GRS2 was drastically induced upon heat shock, ethanol or hydrogen peroxide addition, and high pH, while expression of GRS1 was somewhat repressed under those conditions. In addition, GlyRS2 (the enzyme encoded by GRS2) had a higher protein stability and a lower K(M) value for yeast tRNA(Gly) under heat shock conditions than under normal conditions. Moreover, GRS2 rescued the growth defect of a GRS1 knockout strain when highly expressed by a strong promoter at 37 °C, but not at the optimal temperature of 30 °C. These results suggest that GRS2 is actually an inducible gene that may function to rescue the activity of GRS1 under stress conditions.

BMI1 is expressed in canine osteosarcoma and contributes to cell growth and chemotherapy resistance.

PubMed

Shahi, Mehdi Hayat; York, Daniel; Gandour-Edwards, Regina; Withers, Sita S; Holt, Roseline; Rebhun, Robert B

2015-01-01

BMI1, a stem cell factor and member of the polycomb group of genes, has been shown to contribute to growth and chemoresistance of several human malignancies including primary osteosarcoma (OSA). Naturally occurring OSA in the dog represents a large animal model of human OSA, however the potential role of BMI1 in canine primary and metastatic OSA has not been examined. Immunohistochemical staining of canine primary and metastatic OSA tumors revealed strong nuclear expression of BMI1. An identical staining pattern was found in both primary and metastatic human OSA tissues. Canine OSA cell lines (Abrams, Moresco, and D17) expressed high levels of BMI1 compared with canine osteoblasts and knockdown or inhibition of BMI1 by siRNA or by small molecule BMI1-inhibitor PTC-209 demonstrated a role for BMI1 in canine OSA cell growth and resistance to carboplatin and doxorubicin chemotherapy. These findings suggest that inhibition of BMI1 in primary or metastatic OSA may improve response to chemotherapy and that the dog may serve as a large animal model to evaluate such therapy.

Molecular cloning, structural analysis, and expression of a human IRLB, MYC promoter-binding protein: new DENN domain-containing protein family emerges.

PubMed

Semova, Natalia; Kapanadze, Bagrat; Corcoran, Martin; Kutsenko, Alexei; Baranova, Ancha; Semov, Alexandre

2003-09-01

IRLB was originally identified as a partial cDNA clone, encoding a 191-aa protein binding the interferon-stimulated response element (ISRE) in the P2 promoter of human MYC. Here, we cloned the full-size IRLB using different bioinformatics tools and an RT-PCR approach. The full-size gene encompasses 131 kb within chromosome 15q22 and consists of 32 exons. IRLB is transcribed as a 6.6-kb mRNA encoding a protein of 1865 aa. IRLB is ubiquitously expressed and its expression is regulated in a growth- and cell cycle-dependent manner. In addition to the ISRE-binding domain IRLB contains a tripartite DENN domain, a nuclear localization signal, two PPRs, and a calmodulin-binding domain. The presence of DENN domains predicts possible interactions of IRLB with GTPases from the Rab family or regulation of growth-induced MAPKs. Strongly homologous proteins were identified in all available vertebrate genomes as well as in Caenorhabditis elegans and Drosophila melanogaster. In human and mouse a family of IRLB proteins exists, consisting of at least three members.

Correlation analysis between expression of PCNA, Ki-67 and COX-2 and X-ray features in mammography in breast cancer.

PubMed

Qiu, Xiaoming; Mei, Jixin; Yin, Jianjun; Wang, Hong; Wang, Jinqi; Xie, Ming

2017-09-01

This study investigated expression of proliferating cell nuclear antigen (PCNA), proliferation-associated nuclear antigen (Ki-67) and cyclooxygenase-2 (COX-2) in tissues of breast invasive ductal carcinoma, and analyzed the correlations between these indexes and X-ray features in mammography. A total of 90 patients who were admitted to Huangshi Central Hospital and diagnosed as breast invasive ductal carcinoma from January 2014 to January 2016 were selected. The expression of PCNA, Ki-67 and COX-2 in cancer tissues and cancer-adjacent normal tissues of patients were detected by immunohistochemical staining, and X-ray features in mammography of patients were observed. By using Spearman correlation analysis, the correlations between expression of PCNA, Ki-67 and COX-2 and X-ray features in mammography in breast cancer were investigated. As a result, the positive expression rates of PCNA, Ki-67 and COX-2 in cancer tissues of the patient groups were respectively 42.2, 45.6 and 51.1%, which were significantly higher than those in cancer-adjacent normal tissues of the control group (p<0.05). PCNA, Ki-67 and COX-2 expression in cancer tissues of the patient group was associated with clinical staging and lymphatic metastasis (p<0.05), but had no correlation with age and tumor size (p>0.05). PCNA, Ki-67 and COX-2 expression in cancer tissues of the patient group had no correlation with the existence of lumps and localized density-increased shadows (p>0.05), but were associated with manifestations of architectural distortion, calcification as well as skin and nipple depression (p<0.05). Spearman correlation analysis revealed that there was a significantly positive correlation between the expression of PCNA and COX-2 in cancer tissues of the patient group (r=0.676, p<0.05); there was a significantly positive correlation between the expression of Ki-67 and COX-2 (r=0.724, p<0.05); PCNA expression had no obvious correlation with the expression of Ki-67 (p>0.05). In conclusion, PCNA, Ki-67 and COX-2 expression is of great significance in the occurrence, invasion and metastasis of breast invasive ductal carcinoma. There is a strong correlation between PCNA, Ki-67 and COX-2 expression levels and X-ray features in mammography in breast invasive ductal carcinoma. The application of X-ray features in mammography can evaluate the expression levels of PCNA, Ki-67 and COX-2 in tissues of breast invasive ductal carcinoma, thereby prospectively predicting biological behavior of these cancer cells and patient prognosis.

Retention of prolyl hydroxylase PHD2 in the cytoplasm prevents PHD2-induced anchorage-independent carcinoma cell growth

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

Jokilehto, Terhi; Turku Graduate School of Biomedical Sciences, Turku; Hoegel, Heidi

2010-04-15

Cellular oxygen tension is sensed by a family of prolyl hydroxylases (PHD1-3) that regulate the degradation of hypoxia-inducible factors (HIF-1{alpha} and -2{alpha}). The PHD2 isoform is considered as the main downregulator of HIF in normoxia. Our previous results have shown that nuclear translocation of PHD2 associates with poorly differentiated tumor phenotype implying that nuclear PHD2 expression is advantageous for tumor growth. Here we show that a pool of PHD2 is shuttled between the nucleus and the cytoplasm. In line with this, accumulation of wild type PHD2 in the nucleus was detected in human colon adenocarcinomas and in cultured carcinoma cells.more » The PHD2 isoforms showing high nuclear expression increased anchorage-independent carcinoma cell growth. However, retention of PHD2 in the cytoplasm inhibited the anchorage-independent cell growth. A region that inhibits the nuclear localization of PHD2 was identified and the deletion of the region promoted anchorage-independent growth of carcinoma cells. Finally, the cytoplasmic PHD2, as compared with the nuclear PHD2, less efficiently downregulated HIF expression. Forced HIF-1{alpha} or -2{alpha} expression decreased and attenuation of HIF expression increased the anchorage-independent cell growth. However, hydroxylase-inactivating mutations in PHD2 had no effect on cell growth. The data imply that nuclear PHD2 localization promotes malignant cancer phenotype.« less

Lamin A expression in circulating osteoprogenitors as a potential biomarker for frailty: The Nepean Osteoporosis and Frailty (NOF) Study.

PubMed

Al Saedi, Ahmed; Gunawardene, Piumali; Bermeo, Sandra; Vogrin, Sara; Boersma, Derek; Phu, Steven; Singh, Lakshman; Suriyaarachchi, Pushpa; Duque, Gustavo

2018-02-01

Lamin A is a protein of the nuclear lamina. Low levels of lamin A expression are associated with osteosarcopenia in mice. In this study, we hypothesized that low lamin A expression is also associated with frailty in humans. We aimed to develop a non-invasive method to quantify lamin A expression in epithelial and circulating osteoprogenitor (COP) cells, and to determine the relationship between lamin A expression and frailty in older individuals. COP cells and buccal swabs were obtained from 66 subjects (median age 74; 60% female; 26 non-frail, 23 pre-frail, and 17 frail) participating at the Nepean Osteoporosis and Frailty (NOF) Study. We quantified physical performance and disability, and stratified frailty in this population. Lamin A expression in epithelial and COP cells was quantified by flow cytometry. Linear regression models estimated the relationship between lamin A expression in buccal and COP cells, and prevalent disability and frailty. Lamin A expression in buccal cells showed no association with either disability or frailty. Low lamin A expression values in COP cells were associated with frailty. Frail individuals showed 60% lower levels of lamin A compared to non-frail (95% CI -36 to -74%, p<0.001) and 62% lower levels compared to pre-frail (95%CI -40 to -76%, p<0.001). In summary, we have identified lamin A expression in COP cells as a strong indicator of frailty. Further work is needed to understand lamin A expression as a risk stratifier, biomarker, or therapeutic target in frail older persons. Copyright © 2017 Elsevier Inc. All rights reserved.

Oxidative stress-induced miR-27a targets the redox gene nuclear factor erythroid 2-related factor 2 in diabetic embryopathy.

PubMed

Zhao, Yang; Dong, Daoyin; Reece, E Albert; Wang, Ashley R; Yang, Peixin

2018-01-01

Maternal diabetes induces neural tube defects, and oxidative stress is a causal factor for maternal diabetes-induced neural tube defects. The redox gene nuclear factor erythroid 2-related factor 2 is the master regulator of the cellular antioxidant system. In this study, we aimed to determine whether maternal diabetes inhibits nuclear factor erythroid 2-related factor 2 expression and nuclear factor erythroid 2-related factor 2-controlled antioxidant genes through the redox-sensitive miR-27a. We used a well-established type 1 diabetic embryopathy mouse model induced by streptozotocin for our in vivo studies. Embryos at embryonic day 8.5 were harvested for analysis of nuclear factor erythroid 2-related factor 2, nuclear factor erythroid 2-related factor 2-controlled antioxidant genes, and miR-27a expression. To determine if mitigating oxidative stress inhibits the increase of miR-27a and the decrease of nuclear factor erythroid 2-related factor 2 expression, we induced diabetic embryopathy in superoxide dismutase 2 (mitochondrial-associated antioxidant gene)-overexpressing mice. This model exhibits reduced mitochondria reactive oxygen species even in the presence of hyperglycemia. To investigate the causal relationship between miR-27a and nuclear factor erythroid 2-related factor 2 in vitro, we examined C17.2 neural stem cells under normal and high-glucose conditions. We observed that the messenger RNA and protein levels of nuclear factor erythroid 2-related factor 2 were significantly decreased in embryos on embryonic day 8.5 from diabetic dams compared to those from nondiabetic dams. High-glucose also significantly decreased nuclear factor erythroid 2-related factor 2 expression in a dose- and time-dependent manner in cultured neural stem cells. Our data revealed that miR-27a was up-regulated in embryos on embryonic day 8.5 exposed to diabetes, and that high glucose increased miR-27a levels in a dose- and time-dependent manner in cultured neural stem cells. In addition, we found that a miR-27a inhibitor abrogated the inhibitory effect of high glucose on nuclear factor erythroid 2-related factor 2 expression, and a miR-27a mimic suppressed nuclear factor erythroid 2-related factor 2 expression in cultured neural stem cells. Furthermore, our data indicated that the nuclear factor erythroid 2-related factor 2-controlled antioxidant enzymes glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, and glutathione S-transferase A1 were down-regulated by maternal diabetes in embryos on embryonic day 8.5 and high glucose in cultured neural stem cells. Inhibiting miR-27a restored expression of glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, and glutathione S-transferase A1. Overexpressing superoxide dismutase 2 reversed the maternal diabetes-induced increase of miR-27a and suppression of nuclear factor erythroid 2-related factor 2 and nuclear factor erythroid 2-related factor 2-controlled antioxidant enzymes. Our study demonstrates that maternal diabetes-induced oxidative stress increases miR-27a, which, in turn, suppresses nuclear factor erythroid 2-related factor 2 and its responsive antioxidant enzymes, resulting in diabetic embryopathy. Copyright © 2017 Elsevier Inc. All rights reserved.

Differential Subcellular Localization of the Glucocorticoid Receptor in Distinct Neural Stem and Progenitor Populations of the Mouse Telencephalon In Vivo

PubMed Central

Tsiarli, Maria A.; Monaghan, A. Paula; DeFranco, Donald B.

2013-01-01

Glucocorticoids are given to pregnant women at risk for premature delivery to promote lung maturation. Despite reports of detrimental effects of glucocorticoids on telencephalic neural stem/progenitor cells (NSPCs), the regional and cellular expression of the glucocorticoid receptor (GR) in various NSPC populations in the intact brain has not been thoroughly assessed. Therefore in this study we performed a detailed analysis of GR protein expression in the developing mouse ventral and dorsal telencephalon in vivo. At embryonic day 11.5 (E11.5), the majority of Pax6-positive radial glial cells (RGCs) and Tbr2-positive intermediate progenitor cells (IPCs) expressed nuclear GR, while a small number of RGCs on the apical ventricular zone (aVZ), expressed cytoplasmic GR. However, on E13.5, the latter population of RGCs increased in size, whereas abventricular NSPCs and especially neurons of the cortical plate, expressed nuclear GR. In IPCs, GR was always nuclear. A similar expression profile was observed throughout the ventral telencephalon, hippocampus and olfactory bulb, with NSPCs of the aVZ primarily expressing cytoplasmic GR, while abventricular NSPCs and mature cells primarily expressed nuclear GR. Close to birth, nuclear GR accumulated within specific cortical areas such as layer V, the subplate and CA1 area of the hippocampus. In summary, our data show that GR protein is present in early NSPCs of the dorsal and ventral telencephalon at E11.5 and primarily occupies the nucleus. Moreover, our study suggests that the subcellular localization of the receptor may be subjected to region and neurodevelopmental stage-specific regulation. PMID:23751362

Differential subcellular localization of the glucocorticoid receptor in distinct neural stem and progenitor populations of the mouse telencephalon in vivo.

PubMed

Tsiarli, Maria A; Paula Monaghan, A; Defranco, Donald B

2013-07-26

Glucocorticoids are given to pregnant women at risk for premature delivery to promote lung maturation. Despite reports of detrimental effects of glucocorticoids on telencephalic neural stem/progenitor cells (NSPCs), the regional and cellular expressions of the glucocorticoid receptor (GR) in various NSPC populations in the intact brain have not been thoroughly assessed. Therefore in this study we performed a detailed analysis of GR protein expression in the developing mouse ventral and dorsal telencephalon in vivo. At embryonic day 11.5 (E11.5), the majority of Pax6-positive radial glial cells (RGCs) and Tbr2-positive intermediate progenitor cells (IPCs) expressed nuclear GR, while a small number of RGCs on the apical ventricular zone (aVZ), expressed cytoplasmic GR. However, on E13.5, the latter population of RGCs increased in size, whereas abventricular NSPCs and especially neurons of the cortical plate, expressed nuclear GR. In IPCs, GR was always nuclear. A similar expression profile was observed throughout the ventral telencephalon, hippocampus and olfactory bulb, with NSPCs of the aVZ primarily expressing cytoplasmic GR, while abventricular NSPCs and mature cells primarily expressed nuclear GR. Close to birth, nuclear GR accumulated within specific cortical areas such as layer V, the subplate and CA1 area of the hippocampus. In summary, our data show that GR protein is present in early NSPCs of the dorsal and ventral telencephalon at E11.5 and primarily occupies the nucleus. Moreover, our study suggests that the subcellular localization of the receptor may be subjected to region and neurodevelopmental stage-specific regulation. Copyright © 2013 Elsevier B.V. All rights reserved.

Plastid-to-Nucleus Retrograde Signals Are Essential for the Expression of Nuclear Starch Biosynthesis Genes during Amyloplast Differentiation in Tobacco BY-2 Cultured Cells1[W][OA

PubMed Central

Enami, Kazuhiko; Ozawa, Tomoki; Motohashi, Noriko; Nakamura, Masayuki; Tanaka, Kan; Hanaoka, Mitsumasa

2011-01-01

Amyloplasts, a subtype of plastid, are found in nonphotosynthetic tissues responsible for starch synthesis and storage. When tobacco (Nicotiana tabacum) Bright Yellow-2 cells are cultured in the presence of cytokinin instead of auxin, their plastids differentiate from proplastids to amyloplasts. In this program, it is well known that the expression of nucleus-encoded starch biosynthesis genes, such as ADP-Glucose Pyrophosphorylase (AgpS) and Granule-Bound Starch Synthase (GBSS), is specifically induced. In this study, we investigated the roles of plastid gene expression in amyloplast differentiation. Microarray analysis of plastid genes revealed that no specific transcripts were induced in amyloplasts. Nevertheless, amyloplast development accompanied with starch biosynthesis was drastically inhibited in the presence of plastid transcription/translation inhibitors. Surprisingly, the expression of nuclear AgpS and GBSS was significantly repressed by the addition of these inhibitors, suggesting that a plastid-derived signal(s) that reflects normal plastid gene expression was essential for nuclear gene expression. A series of experiments was performed to examine the effects of intermediates and inhibitors of tetrapyrrole biosynthesis, since some of the intermediates have been characterized as candidates for plastid-to-nucleus retrograde signals. Addition of levulinic acid, an inhibitor of tetrapyrrole biosynthesis, resulted in the up-regulation of nuclear AgpS and GBSS gene expression as well as starch accumulation, while the addition of heme showed opposite effects. Thus, these results indicate that plastid transcription and/or translation are required for normal amyloplast differentiation, regulating the expression of specific nuclear genes by unknown signaling mechanisms that can be partly mediated by tetrapyrrole intermediates. PMID:21771917

cAMP inhibits inducible nitric oxide synthase expression and NF-kappaB-binding activity in cultured rat hepatocytes.

PubMed

Harbrecht, B G; Taylor, B S; Xu, Z; Ramalakshmi, S; Ganster, R W; Geller, D A

2001-08-01

The inducible nitric oxide synthase (iNOS) is strongly expressed following inflammatory stimuli. Adenosine 3',5'-cyclic monophosphate (cAMP) increases iNOS expression and activity in a number of cell types but decreases cytokine-stimulated iNOS expression in hepatocytes. The mechanisms for this effect are unknown. Rat hepatocytes were stimulated with cytokines to induce iNOS and cultured with cAMP agonists dibutyryl-cAMP (dbcAMP), 8-bromo-cAMP, and forskolin (FSK). Nitric oxide synthesis was assessed by supernatant nitrite levels and iNOS expression was measured by Northern and Western blot analyses. Nuclear factor kappaB binding was assessed by electromobility shift assay. Cyclic AMP dose dependently decreased NO synthesis in response to a combination of proinflammatory cytokines or interleukin-1beta (IL-1beta) alone. The adenylate cyclase inhibitor SQ 22,536 increased cytokine- or IL-1beta-stimulated NO synthesis. dbcAMP decreased iNOS mRNA expression and iNOS protein expression. Both dbcAMP and glucagon decreased iNOS promoter activity in rat hepatocytes transfected with the murine iNOS promoter and decreased DNA binding of the transcription factor NF-kappaB. These data suggest that cAMP is important in hepatocyte iNOS expression and agents that alter cAMP levels may profoundly alter the response of hepatocytes to inflammatory stimuli through effects onthe iNOS promoter region and NF-kappaB. Copyright 2001 Academic Press.

NURR1 Downregulation Favors Osteoblastic Differentiation of MSCs

PubMed Central

Di Benedetto, Adriana; Cantore, Stefania; Centonze, Matteo; Grano, Maria; Cavalcanti-Adam, Elisabetta A.

2017-01-01

Mesenchymal stem cells (MSCs) have been identified in human dental tissues. Dental pulp stem cells (DPSCs) were classified within MSC family, are multipotent, can be isolated from adult teeth, and have been shown to differentiate, under particular conditions, into various cell types including osteoblasts. In this work, we investigated how the differentiation process of DPSCs toward osteoblasts is controlled. Recent literature data attributed to the nuclear receptor related 1 (NURR1), a still unclarified role in osteoblast differentiation, while NURR1 is primarily involved in dopaminergic neuron differentiation and activity. Thus, in order to verify if NURR1 had a role in DPSC osteoblastic differentiation, we silenced it during all the processes and compared the expression of the main osteoblastic markers with control cultures. Our results showed that the inhibition of NURR1 significantly increased the expression of osteoblast markers collagen I and alkaline phosphatase. Further, in long time cultures, the mineral matrix deposition was strongly enhanced in NURR1-silenced cultures. These results suggest that NURR1 plays a key role in switching DPSC differentiation toward osteoblasts rather than neuronal or even other cell lines. In conclusion, DPSCs represent a source of osteoblast-like cells and downregulation of NURR1 strongly prompted their differentiation toward the osteoblastogenesis process. PMID:28769982

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

PubMed Central

Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

1990-01-01

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors. Images PMID:2404285

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

PubMed

Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

1990-01-01

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.

Matrix metalloproteinase-9 expression in the nuclear compartment of neurons and glial cells in aging and stroke.

PubMed

Pirici, Daniel; Pirici, Ionica; Mogoanta, Laurentiu; Margaritescu, Otilia; Tudorica, Valerica; Margaritescu, Claudiu; Ion, Daniela A; Simionescu, Cristiana; Coconu, Marieta

2012-10-01

Matrix metalloproteinases (MMPs) are well-recognized denominators for extracellular matrix remodeling in the pathology of both ischemic and hemorrhagic strokes. Recent data on non-nervous system tissue showed intracellular and even intranuclear localizations for different MMPs, and together with this, a plethora of new functions have been proposed for these intracellular active enzymes, but are mostly related to apoptosis induction and malign transformation. In neurons and glial cells, on human tissue, animal models and cell cultures, different active MMPs have been also proven to be located in the intra-cytoplasmic or intra-nuclear compartments, with no clear-cut function. In the present study we show for the first time on human tissue the nuclear expression of MMP-9, mainly in neurons and to a lesser extent in astrocytes. We have studied ischemic and hemorrhagic stroke patients, as well as aged control patients. Age and ischemic suffering seemed to be the best predictors for an elevated MMP-9 nuclear expression, and there was no evidence of a clear-cut extracellular proteolytic activity for this compartment, as revealed by intact vascular basement membranes and assessment of vascular densities. More, the majority of the cells expressing MMP-9 in the nuclear compartment also co-expressed activated-caspase 3, indicating a possible link between nuclear MMP-9 localization and apoptosis in neuronal and glial cells following an ischemic or hemorrhagic event. These results, besides showing for the first time the nuclear localization of MMP-9 on a large series of human stroke and aged brain tissues, raise new questions regarding the unknown spectrum of the functions MMPs in human CNS pathology. © 2011 Japanese Society of Neuropathology.

Nuclear translocation of glutathione S-transferase π is mediated by a non-classical localization signal.

PubMed

Kawakatsu, Miho; Goto, Shinji; Yoshida, Takako; Urata, Yoshishige; Li, Tao-Sheng

2011-08-12

Glutathione S-transferase π (GSTπ), a member of the GST family of multifunctional enzymes, is highly expressed in human placenta and involved in the protection of cellular components against electrophilic compounds or oxidative stress. We have recently found that GSTπ is expressed in the cytoplasm, mitochondria, and nucleus in some cancer cells, and that the nuclear expression of GSTπ appears to correlate with resistance to anti-cancer drugs. Although the mitochondrial targeting signal of GSTπ was previously identified in the amino-terminal region, the mechanism of nuclear translocation remains completely unknown. In this study, we find that the region of GSTπ195-208 is critical for nuclear translocation, which is mediated by a novel and non-classical nuclear localization signal. In addition, using an in vitro transport assay, we demonstrate that the nuclear translocation of GSTπ depends on the cytosolic extract and ATP. Although further experiments are needed to understand in depth the precise mechanism of nuclear translocation of GSTπ, our results may help to establish more efficient anti-cancer therapy, especially with respect to resistance to anti-cancer drugs. Copyright © 2011 Elsevier Inc. All rights reserved.

NR4A nuclear receptors support memory enhancement by histone deacetylase inhibitors

PubMed Central

Hawk, Joshua D.; Bookout, Angie L.; Poplawski, Shane G.; Bridi, Morgan; Rao, Allison J.; Sulewski, Michael E.; Kroener, Brian T.; Manglesdorf, David J.; Abel, Ted

2012-01-01

The formation of a long-lasting memory requires a transcription-dependent consolidation period that converts a short-term memory into a long-term memory. Nuclear receptors compose a class of transcription factors that regulate diverse biological processes, and several nuclear receptors have been implicated in memory formation. Here, we examined the potential contribution of nuclear receptors to memory consolidation by measuring the expression of all 49 murine nuclear receptors after learning. We identified 13 nuclear receptors with increased expression after learning, including all 3 members of the Nr4a subfamily. These CREB-regulated Nr4a genes encode ligand-independent “orphan” nuclear receptors. We found that blocking NR4A activity in memory-supporting brain regions impaired long-term memory but did not impact short-term memory in mice. Further, expression of Nr4a genes increased following the memory-enhancing effects of histone deacetylase (HDAC) inhibitors. Blocking NR4A signaling interfered with the ability of HDAC inhibitors to enhance memory. These results demonstrate that the Nr4a gene family contributes to memory formation and is a promising target for improving cognitive function. PMID:22996661

Chiral discrimination in NMR spectroscopy: computation of the relevant molecular pseudoscalars

NASA Astrophysics Data System (ADS)

Buckingham, A. David; Lazzeretti, Paolo; Pelloni, Stefano

2015-07-01

Nuclear magnetic resonance (NMR) is normally blind to chirality but it has been predicted that precessing nuclear spins in a strong magnetic field induce a rotating electric polarisation that is of opposite sign for enantiomers. The polarisation is determined by two pseudoscalars, ? and ?. The former arises from the distortion of the electronic structure by the nuclear magnetic moment in the presence of the strong magnetic field and is equivalent to the linear effect of an electric field on the nuclear shielding tensor. ? determines the temperature-dependent partial orientation of the permanent electric dipole moment of the molecule by the antisymmetric part of the nuclear shielding tensor. Computations of these two contributions are reported for the nuclei in the chiral molecules N-methyloxaziridine, 2-methyloxirane, 1,3-dimethylallene, 1-fluoroethanol, 2-fluoroazirine, 1,2-M-dioxin, 1,2-M-dithiin, 1,2-M-diselenin and 1,2-M-ditellurin. For strongly dipolar molecules, ? is typically two to three orders of magnitude greater than ?, raising hopes for the detection of chirality in NMR spectroscopy. This paper is dedicated to the memory of Prof. Nicholas Handy, F.R.S.

Leupaxin acts as a mediator in prostate carcinoma progression through deregulation of p120catenin expression.

PubMed

Kaulfuss, S; von Hardenberg, S; Schweyer, S; Herr, A M; Laccone, F; Wolf, S; Burfeind, P

2009-11-12

Recently, we could show that the focal adhesion protein leupaxin (LPXN) is expressed in human prostate carcinomas (PCa) and induces invasiveness of androgen-independent PCa cells. In this study we show that LPXN enhanced the progression of existing PCa in vivo by breeding transgenic mice with prostate-specific LPXN expression and TRAMP mice (transgenic adenocarcinoma of mouse prostate). Double transgenic LPXN/TRAMP mice showed a significant increase in poorly differentiated PCa and distant metastases as compared with control TRAMP mice. Additional studies on primary PCa cells generated from both transgenic backgrounds confirmed the connection regarding LPXN overexpression and increased motility and invasiveness of PCa cells. One mediator of LPXN-induced invasion was found to be the cell-cell adhesion protein p120catenin (p120CTN). Both in vitro and in vivo experiments revealed that p120CTN expression negatively correlates with LPXN expression, followed by a redistribution of beta-catenin. Downregulation of LPXN using small interfering RNAs (siRNAs) resulted in a membranous localization of beta-catenin, whereas strong nuclear accumulation of beta-catenin was observed in p120CTN knockdown cells leading to enhanced transcription of the beta-catenin target gene matrix metalloprotease-7. In conclusion, the present results indicate that LPXN enhances the progression of PCa through downregulation of p120CTN expression and that LPXN could function as a marker for aggressive PCa in the future.

Nup153 and Nup98 bind the HIV-1 core and contribute to the early steps of HIV-1 replication

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

Di Nunzio, Francesca, E-mail: francesca.di-nunzio@pasteur.fr; Fricke, Thomas; Miccio, Annarita

The early steps of HIV-1 replication involve the entry of HIV-1 into the nucleus, which is characterized by viral interactions with nuclear pore components. HIV-1 developed an evolutionary strategy to usurp the nuclear pore machinery and chromatin in order to integrate and efficiently express viral genes. In the current work, we studied the role of nucleoporins 153 and 98 (Nup153 and Nup98) in infection of human Jurkat lymphocytes by HIV-1. We showed that Nup153-depleted cells exhibited a defect in nuclear import, while depletion of Nup 98 caused a slight defect in HIV integration. To explore the biochemical viral determinants formore » the requirement of Nup153 and Nup98 during HIV-1 infection, we tested the ability of these nucleoporins to interact with HIV-1 cores. Our findings showed that both nucleoporins bind HIV-1 cores suggesting that this interaction is important for HIV-1 nuclear import and/or integration. Distribution analysis of integration sites in Nup153-depleted cells revealed a reduced tendency of HIV-1 to integrate in intragenic sites, which in part could account for the large infectivity defect observed in Nup153-depleted cells. Our work strongly supports a role for Nup153 in HIV-1 nuclear import and integration. - Highlights: ► We studied the role of Nup98 and Nup153 in HIV-1 infection. ► Nup98 binds the HIV-1 core and is involved in HIV-1 integration. ► Nup153 binds the HIV-1 core and is involved in HIV-1 nuclear import. ► Depletion of Nup153 decreased the integration of HIV-1 in transcriptionally active sites.« less

Body Mass Index Influences the Prognostic Impact of Combined Nuclear Insulin Receptor and Estrogen Receptor Expression in Primary Breast Cancer.

PubMed

Björner, Sofie; Rosendahl, Ann H; Simonsson, Maria; Markkula, Andrea; Jirström, Karin; Borgquist, Signe; Rose, Carsten; Ingvar, Christian; Jernström, Helena

2017-01-01

The prognostic importance of tumor-specific nuclear insulin receptor (InsR) expression in breast cancer is unclear, while membrane and cytoplasmic localization of InsR is better characterized. The insulin signaling network is influenced by obesity and may interact with the estrogen receptor α (ERα) signaling. The purpose was to investigate the interplay between nuclear InsR, ER, body mass index (BMI), and prognosis. Tumor-specific expression of nuclear InsR was evaluated by immunohistochemistry in tissue microarrays from 900 patients with primary invasive breast cancer without preoperative treatment, included in a population-based cohort in Sweden (2002-2012) in relation to prognosis. Patients were followed for up to 11 years during which 107 recurrences were observed. Nuclear InsR + expression was present in 214 patients (23.8%) and increased with longer time between surgery and staining ( P  < 0.001). There were significant effect modifications by ER status and BMI in relation to clinical outcomes. Nuclear InsR + conferred higher recurrence-risk in patients with ER + tumors, but lower risk in patients with ER - tumors ( P interaction  = 0.003). Normal-weight patients with nuclear InsR + tumors had higher recurrence-risk, while overweight or obese patients had half the recurrence-risk compared to patients with nuclear InsR - tumors ( P interaction  = 0.007). Normal-weight patients with a nuclear InsR - /ER + tumor had the lowest risk for recurrence compared to all other nuclear InsR/ER combinations [HR adj 0.50, 95% confidence interval (CI): 0.25-0.97], while overweight or obese patients with nuclear InsR - /ER - tumors had the worst prognosis (HR adj 7.75, 95% CI: 2.04-29.48). Nuclear InsR was more prognostic than ER among chemotherapy-treated patients. In summary, nuclear InsR may have prognostic impact among normal-weight patients with ER + tumors and in overweight or obese patients with ER - tumors. Normal-weight patients with nuclear InsR - /ER + tumors may benefit from less treatment than normal-weight patients with other nuclear InsR/ER combinations. Overweight or obese patients with nuclear InsR - /ER - tumors may benefit from more tailored treatment or weight management.

Nuclear size is sensitive to NTF2 protein levels in a manner dependent on Ran binding

PubMed Central

Vuković, Lidija D.; Jevtić, Predrag; Zhang, Zhaojie; Stohr, Bradley A.; Levy, Daniel L.

2016-01-01

ABSTRACT Altered nuclear size is associated with many cancers, and determining whether cancer-associated changes in nuclear size contribute to carcinogenesis necessitates an understanding of mechanisms of nuclear size regulation. Although nuclear import rates generally positively correlate with nuclear size, NTF2 levels negatively affect nuclear size, despite the role of NTF2 (also known as NUTF2) in nuclear recycling of the import factor Ran. We show that binding of Ran to NTF2 is required for NTF2 to inhibit nuclear expansion and import of large cargo molecules in Xenopus laevis egg and embryo extracts, consistent with our observation that NTF2 reduces the diameter of the nuclear pore complex (NPC) in a Ran-binding-dependent manner. Furthermore, we demonstrate that ectopic NTF2 expression in Xenopus embryos and mammalian tissue culture cells alters nuclear size. Finally, we show that increases in nuclear size during melanoma progression correlate with reduced NTF2 expression, and increasing NTF2 levels in melanoma cells is sufficient to reduce nuclear size. These results show a conserved capacity for NTF2 to impact on nuclear size, and we propose that NTF2 might be a new cancer biomarker. PMID:26823604

Human Foamy Virus Capsid Formation Requires an Interaction Domain in the N Terminus of Gag

PubMed Central

Tobaly-Tapiero, Joelle; Bittoun, Patricia; Giron, Marie-Lou; Neves, Manuel; Koken, Marcel; Saïb, Ali; de Thé, Hugues

2001-01-01

Retroviral Gag expression is sufficient for capsid assembly, which occurs through interaction between distinct Gag domains. Human foamy virus (HFV) capsids assemble within the cytoplasm, although their budding, which mainly occurs in the endoplasmic reticulum, requires the presence of homologous Env. Yet little is known about the molecular basis of HFV Gag precursor assembly. Using fusions between HFV Gag and a nuclear reporter protein, we have identified a strong interaction domain in the N terminus of HFV Gag which is predicted to contain a conserved coiled-coil motif. Deletion within this region in an HFV provirus abolishes viral production through inhibition of capsid assembly. PMID:11287585

Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes

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

Sun, Zhichao; Yu, Xuemei; Wu, Weibin

2012-07-13

Highlights: Black-Right-Pointing-Pointer FGF7 strongly and rapidly down-regulates the expression of CYP7A1 in hepatocytes. Black-Right-Pointing-Pointer FGF7 suppresses the expression of CYP7A1 via FGFR2 and downstream JNK activation. Black-Right-Pointing-Pointer Blocking FGF7 abrogates HSC-induced inhibition of CYP7A1 expression in hepatocytes. -- Abstract: Cholesterol 7{alpha}-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl{sub 4})-induced fibrotic mouse liver, we demonstrated thatmore » the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.« less

Berry Phenolic Compounds Increase Expression of Hepatocyte Nuclear Factor-1α (HNF-1α) in Caco-2 and Normal Colon Cells Due to High Affinities with Transcription and Dimerization Domains of HNF-1α

PubMed Central

Real Hernandez, Luis M.; Fan, Junfeng; Johnson, Michelle H.; Gonzalez de Mejia, Elvira

2015-01-01

Hepatocyte nuclear factor-1α (HNF-1α) is found in the kidneys, spleen, thymus, testis, skin, and throughout the digestive organs. It has been found to promote the transcription of various proteins involved in the management of type II diabetes, including dipeptidyl peptidase-IV (DPP-IV). Phenolic compounds from berries and citrus fruits are known to inhibit DPP-IV, but have not been tested for their interactions with wild-type HNF-1α. By studying the interactions of compounds from berries and citrus fruits have with HNF-1α, pre-transcriptional mechanisms that inhibit the expression of proteins such as DPP-IV may be elucidated. In this study, the interactions of berry phenolic compounds and citrus flavonoids with the dimerization and transcriptional domains of HNF-1α were characterized using the molecular docking program AutoDock Vina. The anthocyanin delphinidin-3-O-arabinoside had the highest binding affinity for the dimerization domain as a homodimer (-7.2 kcal/mol) and transcription domain (-8.3 kcal/mol) of HNF-1α. Anthocyanins and anthocyanidins had relatively higher affinities than resveratrol and citrus flavonoids for both, the transcription domain and the dimerization domain as a homodimer. The flavonoid flavone had the highest affinity for a single unit of the dimerization domain (-6.5 kcal/mol). Nuclear expression of HNF-1α was measured in Caco-2 and human normal colon cells treated with blueberry and blackberry anthocyanin extracts. All extracts tested increased significantly (P < 0.05) the nuclear expression of HNF-1α in Caco-2 cells by 85.2 to 260% compared to a control. The extracts tested increased significantly (P < 0.02) the nuclear expression of HNF-1α in normal colon cells by 48.6 to 243%. It was confirmed that delphinidin-3-O-glucoside increased by 3-fold nuclear HNF-1α expression in Caco-2 cells (P < 0.05). Anthocyanins significantly increased nuclear HNF-1α expression, suggesting that these compounds might regulate the genes HNF-1α promotes. PMID:26413797

p53 nuclear accumulation and ERα expression in ductal hyperplasia of breast in a cohort of 215 Chinese women

PubMed Central

2010-01-01

Introduction Women with ductal hyperplasia including usual ductal hyperplasia (UDH) and atypical ductal hyperplasia (ADH) have an increased risk of developing invasive ductal carcinoma (IDC) of breast. The importance of several molecular markers in breast cancer has been of considerable interest during recent years such as p53 and estrogen receptor alpha (ERα). However, p53 nuclear accumulation and ERα expression have not been assessed in ductal hyperplasia co-existing with ductal carcinoma in situ (DCIS) or IDC versus pure ductal hyperplasia without DCIS or IDC. Materials and methods We investigated p53 nuclear accumulation and ERα expression in breast ductal hyperplasia in a cohort of 215 Chinese women by immunohistochemistry (IHC), which included 129 cases of pure ductal hyperplasia, 86 cases of ductal hyperplasia co-existing with DCIS (41 cases) or IDC (45 cases). Results Nuclear p53 accumulation was identified in 22.8% of ADH (31/136), 41.5% of DCIS (17/41) and 42.2% of IDC (19/45), and no case of UDH (0/79). No difference in nuclear p53 accumulation was observed between pure ADH and ADH co-existing with DCIS (ADH/DCIS) or IDC (ADH/IDC) (P > 0.05). The positive rate of ERα expression was lower in ADH (118/136, 86.8%) than that in UDH (79/79, 100%) (P < 0.001), but higher than that in DCIS (28/41, 68.3%) or IDC (26/45, 57.8%) respectively (P < 0.001). The frequency of ERα expression was lower in ADH/DCIS (23/29, 79.31%) and ADH/IDC (23/30, 76.67%) than that in pure ADH (72/77, 93.51%) respectively (P < 0.05). There was a negative weak correlation between p53 nuclear accumulation and ERα expression as for ADH (coefficient correlation -0.51; P < 0.001). Conclusions Different pathological types of ductal hyperplasia of breast are accompanied by diversity in patterns of nuclear p53 accumulation and ERα expression. At least some pure ADH is molecularly distinct from ADH/CIS or ADH/IDC which indicated the two types of ADH are molecularly distinct entities although they have the same morphological appearance. PMID:20712900

Procyanidins from wild grape (Vitis amurensis) seeds regulate ARE-mediated enzyme expression via Nrf2 coupled with p38 and PI3K/Akt pathway in HepG2 cells.

PubMed

Bak, Min-Ji; Jun, Mira; Jeong, Woo-Sik

2012-01-01

Procyanidins, polymers of flavan-3-ol units, have been reported to exhibit many beneficial health effects such as antioxidant and anti-carcinogenic effects. In this study, we investigated the cancer chemopreventive properties of procyanidins from wild grape (Vitis amurensis) seeds in particular their roles in inducing phase II detoxifying/antioxidant enzymes as well as in modulating the upstream kinases. Ethanolic extract of V. amurensis seeds was fractionated with a series of organic solvents and finally separated into six fractions, F1-F6. Chemical properties of the procyanidins were analyzed by vanillin assay, BuOH-HCl test, and depolymerization with phloroglucinol followed by LC/MS analysis. The F5 had the highest procyanidin content among all the fractions and strongly induced the reporter activity of antioxidant response element as well as the protein expression of nuclear factor E2-related factor (Nrf2) in HepG2 human hepatocarcinoma cells. The procyanidin-rich F5 also strongly induced the expression of the phase II detoxifying and antioxidant enzymes such as NAD(P)H:quinone oxidoreductase1 and hemeoxygenase1. Phosphorylations of the upstream kinases such as MAPKs and PI3K/Akt were significantly increased by treatment with procyanidin fraction. In addition, the procyanidin-mediated Nrf2 expression was partly attenuated by PI3K inhibitor LY294002, and almost completely by p38 inhibitor SB202190, but neither by JNK inhibitor SP600125 nor by MEK1/2 inhibitor U0126. Taken together, the procyanidins from wild grape seeds could be used as a potential natural chemopreventive agent through Nrf2/ARE-mediated phase II detoxifying/antioxidant enzymes induction via p38 and PI3K/Akt pathway.

A Rice cis-Natural Antisense RNA Acts as a Translational Enhancer for Its Cognate mRNA and Contributes to Phosphate Homeostasis and Plant Fitness[C][W

PubMed Central

Jabnoune, Mehdi; Secco, David; Lecampion, Cécile; Robaglia, Christophe; Shu, Qingyao; Poirier, Yves

2013-01-01

cis-natural antisense transcripts (cis-NATs) are widespread in plants and are often associated with downregulation of their associated sense genes. We found that a cis-NAT positively regulates the level of a protein critical for phosphate homeostasis in rice (Oryza sativa). PHOSPHATE1;2 (PHO1;2), a gene involved in phosphate loading into the xylem in rice, and its associated cis-NATPHO1;2 are both controlled by promoters active in the vascular cylinder of roots and leaves. While the PHO1;2 promoter is unresponsive to the plant phosphate status, the cis-NATPHO1;2 promoter is strongly upregulated under phosphate deficiency. Expression of both cis-NATPHO1;2 and the PHO1;2 protein increased in phosphate-deficient plants, while the PHO1;2 mRNA level remained stable. Downregulation of cis-NATPHO1;2 expression by RNA interference resulted in a decrease in PHO1;2 protein, impaired the transfer of phosphate from root to shoot, and decreased seed yield. Constitutive overexpression of NATPHO1;2 in trans led to a strong increase of PHO1;2, even under phosphate-sufficient conditions. Under all conditions, no changes occurred in the level of expression, sequence, or nuclear export of PHO1;2 mRNA. However, expression of cis-NATPHO1;2 was associated with a shift of both PHO1;2 and cis-NATPHO1;2 toward the polysomes. These findings reveal an unexpected role for cis-NATPHO1;2 in promoting PHO1;2 translation and affecting phosphate homeostasis and plant fitness. PMID:24096344

Aberrant ligand-induced activation of G protein-coupled estrogen receptor 1 (GPER) results in developmental malformations during vertebrate embryogenesis.

PubMed

Jayasinghe, B Sumith; Volz, David C

2012-01-01

G protein-coupled estrogen receptor 1 (GPER) is a G protein-coupled receptor (GPCR) unrelated to nuclear estrogen receptors but strongly activated by 17β-estradiol in both mammals and fish. To date, the distribution and functional characterization of GPER within reproductive and nonreproductive vertebrate organs have been restricted to juvenile and adult animals. In contrast, virtually nothing is known about the spatiotemporal distribution and function of GPER during vertebrate embryogenesis. Using zebrafish as an animal model, we investigated the potential functional role and expression of GPER during embryogenesis. Based on real-time PCR and whole-mount in situ hybridization, gper was expressed as early as 1 h postfertilization (hpf) and exhibited strong stage-dependent expression patterns during embryogenesis. At 26 and 38 hpf, gper mRNA was broadly distributed throughout the body, whereas from 50 to 98 hpf, gper expression was increasingly localized to the heart, brain, neuromasts, craniofacial region, and somite boundaries of developing zebrafish. Continuous exposure to a selective GPER agonist (G-1)-but not continuous exposure to a selective GPER antagonist (G-15)-from 5 to 96 hpf, or within three developmental windows ranging from 10 to 72 hpf, resulted in adverse concentration-dependent effects on survival, gross morphology, and somite formation within the trunk of developing zebrafish embryos. Importantly, based on co-exposure studies, G-15 blocked severe G-1-induced developmental toxicity, suggesting that G-1 toxicity is mediated via aberrant activation of GPER. Overall, our findings suggest that xenobiotic-induced GPER activation represents a potentially novel and understudied mechanism of toxicity for environmentally relevant chemicals that affect vertebrate embryogenesis.

Deconstructing sub-clinical psychosis into latent-state and trait variables over a 30-year time span.

PubMed

Rössler, Wulf; Hengartner, Michael P; Ajdacic-Gross, Vladeta; Haker, Helene; Angst, Jules

2013-10-01

Our aim was to deconstruct the variance underlying the expression of sub-clinical psychosis symptoms into portions associated with latent time-dependent states and time-invariant traits. We analyzed data of 335 subjects from the general population of Zurich, Switzerland, who had been repeatedly measured between 1979 (age 20/21) and 2008 (age 49/50). We applied two measures of sub-clinical psychosis derived from the SCL-90-R, namely schizotypal signs (STS) and schizophrenia nuclear symptoms (SNS). Variance was decomposed with latent state-trait analysis and associations with covariates were examined with generalized linear models. At ages 19/20 and 49/50, the latent states underlying STS accounted for 48% and 51% of variance, whereas for SNS those estimates were 62% and 50%. Between those age classes, however, expression of sub-clinical psychosis was strongly associated with stable traits (75% and 89% of total variance in STS and SNS, respectively, at age 27/28). Latent states underlying variance in STS and SNS were particularly related to partnership problems over almost the entire observation period. STS was additionally related to employment problems, whereas drug-use was a strong predictor of states underlying both syndromes at age 19/20. The latent trait underlying expression of STS and SNS was particularly related to low sense of mastery and self-esteem and to high depressiveness. Although most psychosis symptoms are transient and episodic in nature, the variability in their expression is predominantly caused by stable traits. Those time-invariant and rather consistent effects are particularly influential around age 30, whereas the occasion-specific states appear to be particularly influential at ages 20 and 50. © 2013.

Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay.

PubMed

Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan

2016-10-01

This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF‑7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot‑based molecular targeted imaging techniques (which stained pan‑cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF‑7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot‑based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology.

Functional organization of DNA elements regulating SM30alpha, a spicule matrix gene of sea urchin embryos.

PubMed

Yamasu, K; Wilt, F H

1999-02-01

The SM30a gene encodes a protein in the embryonic endoskeleton of the sea urchin Strongylocentrotus purpuratus, and is specifically expressed in the skeletogenic primary mesenchyme cell lineage. To clarify the mechanism for the differentiation of this cell lineage, which proceeds rather autonomously in the embryo, regulation of the SM30alpha gene was investigated previously and it was shown that the distal DNA region upstream of this gene from - 1.6 to - 1.0 kb contained numerous negative regulatory elements that suppressed the ectopic expression of the gene in the gut. Here we study the influence of the proximal region from - 303 to + 104 bp. Analysis of the expression of reporter constructs indicated that a strong positive enhancer element existed in the region from -142 to -105bp. This element worked both in forward and reverse orientations and additively when placed tandemly upstream to the reporter gene. In addition, other weaker positive and negative regulatory sites were also detected throughout the proximal region. Electrophoretic gel mobility shift analyses showed that multiple nuclear proteins were bound to the putative strong enhancer region. One of the proteins binding to this region was present in ear y blastulae, a time when the SM30 gene was still silent, but it was not in prism embryos actively expressing the gene. The binding region for this blastula-specific protein was narrowed down to the region from - 132 to -122 bp, which included the consensus binding site for the mammalian proto-oncogene product, Ets. Two possible SpGCF1 binding sites were identified in the vicinity of the enhancer region. This information was used to make a comparison of the general regulatory architecture of genes that contribute to the formation of the skeletal spicule.

Nuclear domain 10 components promyelocytic leukemia protein and hDaxx independently contribute to an intrinsic antiviral defense against human cytomegalovirus infection.

PubMed

Tavalai, Nina; Papior, Peer; Rechter, Sabine; Stamminger, Thomas

2008-01-01

Infection with DNA viruses commonly results in the association of viral genomes with a cellular subnuclear structure known as nuclear domain 10 (ND10). Recent studies demonstrated that individual ND10 components, like hDaxx or promyelocytic leukemia protein (PML), mediate an intrinsic immune response against human cytomegalovirus (HCMV) infection, strengthening the assumption that ND10 components are part of a cellular antiviral defense mechanism. In order to further define the role of hDaxx and PML for HCMV replication, we generated either primary human fibroblasts with a stable, individual knockdown of PML or hDaxx (PML-kd and hDaxx-kd, respectively) or cells exhibiting a double knockdown. Comparative analysis of HCMV replication in PML-kd or hDaxx-kd cells revealed that immediate-early (IE) gene expression increased to a similar extent, regardless of which ND10 constituent was depleted. Since a loss of PML, the defining component of ND10, results in a dispersal of the entire nuclear substructure, the increased replication efficacy of HCMV in PML-kd cells could be a consequence of the dissociation of the repressor protein hDaxx from its optimal subnuclear localization. However, experiments using three different recombinant HCMVs revealed a differential growth complementation in PML-kd versus hDaxx-kd cells, strongly arguing for an independent involvement in suppressing HCMV replication. Furthermore, infection experiments using double-knockdown cells devoid of both PML and hDaxx illustrated an additional enhancement in the replication efficacy of HCMV compared to the single-knockdown cells. Taken together, our data indicate that both proteins, PML and hDaxx, mediate an intrinsic immune response against HCMV infection by contributing independently to the silencing of HCMV IE gene expression.

Depletion of the cellular levels of Bag-1 proteins attenuates phorbol ester-induced downregulation of I{kappa}B{alpha} and nuclear accumulation of NF-{kappa}B

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

Maier, Jana V., E-mail: Jana.maier@kit.edu; Volz, Yvonne; Berger, Caroline

2010-10-22

Research highlights: {yields}Bag-1 depletion only marginally affects the action of the glucocorticoid receptor but strongly regulates the activity of NF-{kappa}B. {yields}Bag-1 depletion attenuates phosphorylation and degradation of I{kappa}B{alpha} and nuclear accumulation of NF-{kappa}B p65 and p50. {yields}Bag-1 interacts with I{kappa}B{alpha} and partially restores I{kappa}B{alpha} and NF-{kappa}B activation in Bag-1 depleted cells. -- Abstract: Bag-1 consists in humans of four isoforms generated from the same RNA by alternative translation. Overexpression of single Bag-1 isoforms has identified Bag-1 as a negative regulator of action of many proteins including the glucocorticoid receptor (GR). Here we have analysed the ability of Bag-1 to regulatemore » the transrepression function of the GR. Silencing Bag-1 expression only marginally affects the transrepression action of the GR but decreased the action of the transcription factor NF-{kappa}B. Furthermore phosphorylation and degradation of the inhibitor protein I{kappa}B{alpha} and nuclear accumulation of p65 and p50 NF-{kappa}B proteins in response to phorbol ester was attenuated following Bag-1 depletion in HeLa cells. Reconstitution of Bag-1 in depleted cells partially restored I{kappa}B{alpha} and NF-{kappa}B activation. Knock-down of Bag-1 expression also did not significantly alter GR-mediated transactivation but affected the basal transcription of some of the target genes. Thus Bag-1 proteins function as regulators of the action of selective transcription factors.« less

Evolutionary changes in lamin expression in the vertebrate lineage

PubMed Central

Stick, Reimer; Peter, Annette

2017-01-01

ABSTRACT The nuclear lamina is involved in fundamental nuclear functions and provides mechanical stability to the nucleus. Lamin filaments form a meshwork closely apposed to the inner nuclear membrane and a small fraction of lamins exist in the nuclear interior. Mutations in lamin genes cause severe hereditary diseases, the laminopathies. During vertebrate evolution the lamin protein family has expanded. While most vertebrate genomes contain 4 lamin genes, encoding the lamins A, B1, B2, and LIII, the majority of non-vertebrate genomes harbor only a single lamin gene. We have collected lamin gene and cDNA sequence information for representatives of the major vertebrate lineages. With the help of RNA-seq data we have determined relative lamin expression levels for representative tissues for species of 9 different gnathostome lineages. Here we report that the level of lamin A expression is low in cartilaginous fishes and ancient fishes and increases toward the mammals. Lamin B1 expression shows an inverse tendency to that of lamin A. Possible implications for the change in the lamin A to B ratio is discussed in the light of its role in nuclear mechanics. PMID:28430006

Atypical protein kinase C induces cell transformation by disrupting Hippo/Yap signaling

PubMed Central

Archibald, Andrew; Al-Masri, Maia; Liew-Spilger, Alyson; McCaffrey, Luke

2015-01-01

Epithelial cells are major sites of malignant transformation. Atypical protein kinase C (aPKC) isoforms are overexpressed and activated in many cancer types. Using normal, highly polarized epithelial cells (MDCK and NMuMG), we report that aPKC gain of function overcomes contact inhibited growth and is sufficient for a transformed epithelial phenotype. In 2D cultures, aPKC induced cells to grow as stratified epithelia, whereas cells grew as solid spheres of nonpolarized cells in 3D culture. aPKC associated with Mst1/2, which uncoupled Mst1/2 from Lats1/2 and promoted nuclear accumulation of Yap1. Of importance, Yap1 was necessary for aPKC-mediated overgrowth but did not restore cell polarity defects, indicating that the two are separable events. In MDCK cells, Yap1 was sequestered to cell–cell junctions by Amot, and aPKC overexpression resulted in loss of Amot expression and a spindle-like cell phenotype. Reexpression of Amot was sufficient to restore an epithelial cobblestone appearance, Yap1 localization, and growth control. In contrast, the effect of aPKC on Hippo/Yap signaling and overgrowth in NMuMG cells was independent of Amot. Finally, increased expression of aPKC in human cancers strongly correlated with increased nuclear accumulation of Yap1, indicating that the effect of aPKC on transformed growth by deregulating Hippo/Yap1 signaling may be clinically relevant. PMID:26269582

NEAT1 long noncoding RNA regulates transcription via protein sequestration within subnuclear bodies

PubMed Central

Hirose, Tetsuro; Virnicchi, Giorgio; Tanigawa, Akie; Naganuma, Takao; Li, Ruohan; Kimura, Hiroshi; Yokoi, Takahide; Nakagawa, Shinichi; Bénard, Marianne; Fox, Archa H.; Pierron, Gérard

2014-01-01

Paraspeckles are subnuclear structures formed around nuclear paraspeckle assembly transcript 1 (NEAT1)/MENε/β long noncoding RNA (lncRNA). Here we show that paraspeckles become dramatically enlarged after proteasome inhibition. This enlargement is mainly caused by NEAT1 transcriptional up-regulation rather than accumulation of undegraded paraspeckle proteins. Of interest, however, using immuno–electron microscopy, we find that key paraspeckle proteins become effectively depleted from the nucleoplasm by 50% when paraspeckle assembly is enhanced, suggesting a sequestration mechanism. We also perform microarrays from NEAT1-knockdown cells and find that NEAT1 represses transcription of several genes, including the RNA-specific adenosine deaminase B2 (ADARB2) gene. In contrast, the NEAT1-binding paraspeckle protein splicing factor proline/glutamine-rich (SFPQ) is required for ADARB2 transcription. This leads us to hypothesize that ADARB2 expression is controlled by NEAT1-dependent sequestration of SFPQ. Accordingly, we find that ADARB2 expression is strongly reduced upon enhanced SFPQ sequestration by proteasome inhibition, with concomitant reduction in SFPQ binding to the ADARB2 promoter. Finally, NEAT1−/− fibroblasts are more sensitive to proteasome inhibition, which triggers cell death, suggesting that paraspeckles/NEAT1 attenuates the cell death pathway. These data further confirm that paraspeckles are stress-responsive nuclear bodies and provide a model in which induced NEAT1 controls target gene transcription by protein sequestration into paraspeckles. PMID:24173718

Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays

PubMed Central

Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Sethi, Himanshu; Liang, Shoudan; Nelson, David C.; Hegeman, Adrian; Nelson, Clark; Rancour, David; Bednarek, Sebastian; Ulrich, Eldon L.; Zhao, Qin; Wrobel, Russell L.; Newman, Craig S.; Fox, Brian G.; Phillips, George N.; Markley, John L.; Sussman, Michael R.

2005-01-01

Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intron–exon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions “antisense” to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage. PMID:15755812

Genome-wide expression analysis of soybean NF-Y genes reveals potential function in development and drought response.

PubMed

Quach, Truyen N; Nguyen, Hanh T M; Valliyodan, Babu; Joshi, Trupti; Xu, Dong; Nguyen, Henry T

2015-06-01

Nuclear factor-Y (NF-Y), a heterotrimeric transcription factor, is composed of NF-YA, NF-YB and NF-YC proteins. In plants, there are usually more than 10 genes for each family and their members have been identified to be key regulators in many developmental and physiological processes controlling gametogenesis, embryogenesis, nodule development, seed development, abscisic acid (ABA) signaling, flowering time, primary root elongation, blue light responses, endoplasmic reticulum (ER) stress response and drought tolerance. Taking the advantages of the recent soybean genome draft and information on functional characterizations of nuclear factor Y (NF-Y) transcription factor family in plants, we identified 21 GmNF-YA, 32 GmNF-YB, and 15 GmNF-YC genes in the soybean (Glycine max) genome. Phylogenetic analyses show that soybean's proteins share strong homology to Arabidopsis and many of them are closely related to functionally characterized NF-Y in plants. Expression analysis in various tissues of flower, leaf, root, seeds of different developmental stages, root hairs under rhizobium inoculation, and drought-treated roots and leaves revealed that certain groups of soybean NF-Y are likely involved in specific developmental and stress responses. This study provides extensive evaluation of the soybean NF-Y family and is particularly useful for further functional characterization of GmNF-Y proteins in seed development, nodulation and drought adaptation of soybean.

High-resolution telomere fluorescence in situ hybridization reveals intriguing anomalies in germ cell tumors.

PubMed

Shekhani, Mohammed Talha; Barber, John R; Bezerra, Stephania M; Heaphy, Christopher M; Gonzalez Roibon, Nilda Diana; Taheri, Diana; Reis, Leonardo O; Guner, Gunes; Joshu, Corinne E; Netto, George J; Meeker, Alan K

2016-08-01

Testicular germ cell tumor (TGCT) is the most common malignancy of young men. Most patients are completely cured, which distinguishes these from most other malignancies. Orchiectomy specimens (n=76) were evaluated using high-resolution (single-cell discriminative) telomere-specific fluorescence in situ hybridization (FISH) with simultaneous Oct4 immunofluorescence to describe telomere length phenotype in TGCT neoplastic cells. For the first time, the TGCT precursor lesion, germ cell neoplasia in situ (GCNIS) is also evaluated in depth. The intensity of the signals from cancerous cells was compared to the same patient's reference cells-namely, healthy germ cells (defined as "medium" length) and interstitial/somatic cells (defined as "short" telomere length). We observed short telomeres in most GCNIS and pure seminomas (P=.006 and P=.0005, respectively). In contrast, nonseminomas displayed longer telomeres. Lesion-specific telomere lengths were documented in mixed tumor cases. Embryonal carcinoma (EC) demonstrated the longest telomeres. A fraction of EC displays the telomerase-independent alternative lengthening of telomeres (ALT) phenotype (24% of cases). Loss of ATRX or DAXX nuclear expression was strongly associated with ALT; however, nuclear expression of both proteins was retained in half of ALT-positive ECs. The particular distribution of telomere lengths among TGCT and GCNIS precursors implicate telomeres anomalies in pathogenesis. These results may advise management decisions as well. Copyright © 2016. Published by Elsevier Inc.

DNAJC17 is localized in nuclear speckles and interacts with splicing machinery components.

PubMed

Pascarella, A; Ferrandino, G; Credendino, S C; Moccia, C; D'Angelo, F; Miranda, B; D'Ambrosio, C; Bielli, P; Spadaro, O; Ceccarelli, M; Scaloni, A; Sette, C; De Felice, M; De Vita, G; Amendola, E

2018-05-17

DNAJC17 is a heat shock protein (HSP40) family member, identified in mouse as susceptibility gene for congenital hypothyroidism. DNAJC17 knockout mouse embryos die prior to implantation. In humans, germline homozygous mutations in DNAJC17 have been found in syndromic retinal dystrophy patients, while heterozygous mutations represent candidate pathogenic events for myeloproliferative disorders. Despite widespread expression and involvement in human diseases, DNAJC17 function is still poorly understood. Herein, we have investigated its function through high-throughput transcriptomic and proteomic approaches. DNAJC17-depleted cells transcriptome highlighted genes involved in general functional categories, mainly related to gene expression. Conversely, DNAJC17 interactome can be classified in very specific functional networks, with the most enriched one including proteins involved in splicing. Furthermore, several splicing-related interactors, were independently validated by co-immunoprecipitation and in vivo co-localization. Accordingly, co-localization of DNAJC17 with SC35, a marker of nuclear speckles, further supported its interaction with spliceosomal components. Lastly, DNAJC17 up-regulation enhanced splicing efficiency of minigene reporter in live cells, while its knockdown induced perturbations of splicing efficiency at whole genome level, as demonstrated by specific analysis of RNAseq data. In conclusion, our study strongly suggests a role of DNAJC17 in splicing-related processes and provides support to its recognized essential function in early development.

Defining the SUMO System in Maize: SUMOylation Is Up-Regulated during Endosperm Development and Rapidly Induced by Stress1[OPEN

PubMed Central

Augustine, Robert C.; Rytz, Thérèse C.

2016-01-01

In response to abiotic and biotic challenges, plants rapidly attach small ubiquitin-related modifier (SUMO) to a large collection of nuclear proteins, with studies in Arabidopsis (Arabidopsis thaliana) linking SUMOylation to stress tolerance via its modification of factors involved in chromatin and RNA dynamics. Despite this importance, little is known about SUMOylation in crop species. Here, we describe the plant SUMO system at the phylogenetic, biochemical, and transcriptional levels with a focus on maize (Zea mays). In addition to canonical SUMOs, land plants encode a loosely constrained noncanonical isoform and a variant containing a long extension upstream of the signature β-grasp fold, with cereals also expressing a novel diSUMO polypeptide bearing two SUMO β-grasp domains in tandem. Maize and other cereals also synthesize a unique SUMO-conjugating enzyme variant with more restricted expression patterns that is enzymatically active despite a distinct electrostatic surface. Maize SUMOylation primarily impacts nuclear substrates, is strongly induced by high temperatures, and displays a memory that suppresses subsequent conjugation. Both in-depth transcript and conjugate profiles in various maize organs point to tissue/cell-specific functions for SUMOylation, with potentially significant roles during embryo and endosperm maturation. Collectively, these studies define the organization of the maize SUMO system and imply important functions during seed development and stress defense. PMID:27208252

Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays

NASA Technical Reports Server (NTRS)

Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Sethi, Himanshu; Liang, Shoudan; Nelson, David C.; Hegeman, Adrian; Nelson, Clark; Rancour, David; Bednarek, Sebastian;

AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

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

Lorenzato, Annalisa; Biolatti, Marta; Institute for Cancer Research at Candiolo, Candiolo, Torino

The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancermore » cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. - highlights: • CSE1L is a key player in nucleocytoplasmic traffic by forming complex with Ran. • AKT phosphorylates RanBP3 that regulates the nucleocytoplasmic gradient of Ran. • The activated oncogenic AKT drives the nuclear accumulation of CSE1L. • CSE1L in the nucleus up-regulates genes conveying pro-oncogenic signals. • CSE1L might contribute to tumor progression driven by the activated oncogenic AKT.« less

Co-dependence between trypanosome nuclear lamina components in nuclear stability and control of gene expression.

PubMed

Maishman, Luke; Obado, Samson O; Alsford, Sam; Bart, Jean-Mathieu; Chen, Wei-Ming; Ratushny, Alexander V; Navarro, Miguel; Horn, David; Aitchison, John D; Chait, Brian T; Rout, Michael P; Field, Mark C

2016-12-15

The nuclear lamina is a filamentous structure subtending the nuclear envelope and required for chromatin organization, transcriptional regulation and maintaining nuclear structure. The trypanosomatid coiled-coil NUP-1 protein is a lamina component functionally analogous to lamins, the major lamina proteins of metazoa. There is little evidence for shared ancestry, suggesting the presence of a distinct lamina system in trypanosomes. To find additional trypanosomatid lamina components we identified NUP-1 interacting proteins by affinity capture and mass-spectrometry. Multiple components of the nuclear pore complex (NPC) and a second coiled-coil protein, which we termed NUP-2, were found. NUP-2 has a punctate distribution at the nuclear periphery throughout the cell cycle and is in close proximity to NUP-1, the NPCs and telomeric chromosomal regions. RNAi-mediated silencing of NUP-2 leads to severe proliferation defects, gross alterations to nuclear structure, chromosomal organization and nuclear envelope architecture. Further, transcription is altered at telomere-proximal variant surface glycoprotein (VSG) expression sites (ESs), suggesting a role in controlling ES expression, although NUP-2 silencing does not increase VSG switching. Transcriptome analysis suggests specific alterations to Pol I-dependent transcription. NUP-1 is mislocalized in NUP-2 knockdown cells and vice versa, implying that NUP-1 and NUP-2 form a co-dependent network and identifying NUP-2 as a second trypanosomatid nuclear lamina component. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Aberrant Expression of Xist in Aborted Porcine Fetuses Derived from Somatic Cell Nuclear Transfer Embryos

PubMed Central

Yuan, Lin; Wang, Anfeng; Yao, Chaogang; Huang, Yongye; Duan, Feifei; Lv, Qinyan; Wang, Dongxu; Ouyang, Hongsheng; Li, Zhanjun; Lai, Liangxue

2014-01-01

Cloned pigs generated by somatic cell nuclear transfer (SCNT) show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR) and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP). q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos. PMID:25429426

Nuclear Physics in a biological context

NASA Astrophysics Data System (ADS)

Discher, Dennis

2012-02-01

A solid tissue can be soft like fat or brain, stiff like striated muscle and heart, or rigid like bone -- and of course every cell has a nucleus that contributes in some way small or large to tissue mechanics. Indeed, nuclei generally exhibit rheology and plasticity that reflects both the chromatin and the nuclear envelope proteins called lamins, all of which change in differentiation. Profiling of tissue nuclei shows that the nuclear intermediate filament protein Lamin-A/C varies over 30-fold between adult tissues and scales strongly with micro-elasticity of tissue, while other nuclear envelope components such as Lamin-B exhibit small variations. Lamin-A/C has been implicated in aging syndromes that affect muscle and fat but not brain, and we find nuclei in brain-derived cells are indeed dominated by Lamin-B and are much softer than nuclei derived from muscle cells with predominantly Lamin-A/C. In vitro, matrix elasticity can affect expression of nuclear envelope components in adult stem cells, and major changes in Lamin-A/C are indeed shown to direct lineage with lower levels favoring soft tissue and higher levels promoting rigid tissue lineage. Further molecular studies provide evidence that the nucleus transduces physical stress. References: (1) J.D. Pajerowski, K.N. Dahl, F.L. Zhong, P.J. Sammak, and D.E. Discher. Physical plasticity of the nucleus in stem cell differentiation. PNAS 104: 15619-15624 (2007). (2) A. Buxboim, I. Ivanova, and D.E. Discher. Matrix Elasticity, Cytoskeletal Forces, and Physics of the Nucleus: how deeply do cells `feel' outside and in? Journal of Cell Science 123: 297-308 (2010).

Experimental evidence supports a sex-specific selective sieve in mitochondrial genome evolution.

PubMed

Innocenti, Paolo; Morrow, Edward H; Dowling, Damian K

2011-05-13

Mitochondria are maternally transmitted; hence, their genome can only make a direct and adaptive response to selection through females, whereas males represent an evolutionary dead end. In theory, this creates a sex-specific selective sieve, enabling deleterious mutations to accumulate in mitochondrial genomes if they exert male-specific effects. We tested this hypothesis, expressing five mitochondrial variants alongside a standard nuclear genome in Drosophila melanogaster, and found striking sexual asymmetry in patterns of nuclear gene expression. Mitochondrial polymorphism had few effects on nuclear gene expression in females but major effects in males, modifying nearly 10% of transcripts. These were mostly male-biased in expression, with enrichment hotspots in the testes and accessory glands. Our results suggest an evolutionary mechanism that results in mitochondrial genomes harboring male-specific mutation loads.

Mechanisms of Cables 1 gene inactivation in human ovarian cancer development.

PubMed

Sakamoto, Hideo; Friel, Anne M; Wood, Antony W; Guo, Lankai; Ilic, Ana; Seiden, Michael V; Chung, Daniel C; Lynch, Maureen P; Serikawa, Takehiro; Munro, Elizabeth; Oliva, Esther; Orsulic, Sandra; Kirley, Sandra D; Foster, Rosemary; Zukerberg, Lawrence R; Rueda, Bo R

2008-02-01

Cables 1, a cyclin-dependent kinase binding protein, is primarily involved in cell cycle regulation. Loss of nuclear Cables 1 expression is observed in human colon, lung and endometrial cancers. We previously reported that loss of nuclear Cables 1 expression was also observed with high frequency in a limited sample set of human ovarian carcinomas, although the mechanisms underlying loss of nuclear Cables 1 expression remained unknown. Our present objective was to examine Cables 1 expression in ovarian cancer in greater detail, and determine the predominant mechanisms of Cables 1 loss. We assessed potential genetic and epigenetic modifications of the Cables 1 locus through analyses of mutation, polymorphisms, loss of heterozygosity and DNA methylation. We observed a marked loss of nuclear Cables 1 expression in serous and endometrioid ovarian carcinomas that correlated with decreased Cables 1 mRNA levels. Although we detected no Cables 1 mutations, there was evidence of LOH at the Cables 1 locus and epigenetic modification of the Cables 1 promoter region in a subset of ovarian carcinomas and established cancer cell lines. From a functional perspective, over-expression of Cables 1 induced apoptosis, whereas, knockdown of Cables 1 negated this effect. Together these findings suggest that multiple mechanisms underlie the loss of Cables 1 expression in ovarian cancer cells, supporting the hypothesis that Cables 1 is a tumor suppressor in human ovarian cancer.

Communication: Adiabatic and non-adiabatic electron-nuclear motion: Quantum and classical dynamics

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

Albert, Julian; Kaiser, Dustin; Engel, Volker

2016-05-07

Using a model for coupled electronic-nuclear motion we investigate the range from negligible to strong non-adiabatic coupling. In the adiabatic case, the quantum dynamics proceeds in a single electronic state, whereas for strong coupling a complete transition between two adiabatic electronic states takes place. It is shown that in all coupling regimes the short-time wave-packet dynamics can be described using ensembles of classical trajectories in the phase space spanned by electronic and nuclear degrees of freedom. We thus provide an example which documents that the quantum concept of non-adiabatic transitions is not necessarily needed if electronic and nuclear motion ismore » treated on the same footing.« less

Association between high expression of phosphorylated Akt and mammalian target of rapamycin and improved survival in salivary gland adenoid cystic carcinoma.

PubMed

Ouyang, Dai-Qiao; Liang, Li-Zhong; Ke, Zun-Fu; Zheng, Guang-Sen; Weng, De-Sheng; Yang, Wei-Fa; Su, Yu-Xiong; Liao, Gui-Qing

2017-06-01

Previous genomic studies revealed phosphotidylinositol-3-kinase (PI3K)/Akt pathway mutation in human salivary gland adenoid cystic carcinoma (ACC). No validation of its prognostic value has been reported. P-Akt, pan-Akt, phosphorylated-mammalian target of rapamycin (p-mTOR), PI3K, and insulin-like growth factor-1 receptor beta (IGF-1Rβ) were detected on 120 salivary gland ACC/adjacent salivary gland pairs immunohistochemically and were correlated with clinicopathological data. Expression of cytoplasmic and nuclear p-Akt, cytoplasmic p-mTOR, nuclear pan-Akt, and nuclear IGF-1Rβ were higher in ACC than in adjacent salivary glands. P-Akt, p-mTOR, PI3K, and IGF-1Rβ expression were correlated with one another in both cytoplasm and nucleus. Low p-mTOR expression in both subcellular compartments was associated with locoregional recurrence, poor disease-free survival (DFS), and overall survival (OS). Low nuclear p-Akt (Ser473) and p-mTOR expression were independent predictors for poor OS and DFS, respectively. High level of Akt/mTOR activation in ACC is correlated with a significantly improved survival. P-mTOR and nuclear p-Akt are prognostic biomarkers of salivary gland ACC. © 2017 Wiley Periodicals, Inc. Head Neck 39: 1145-1154, 2017. © 2017 Wiley Periodicals, Inc.

PML clastosomes prevent nuclear accumulation of mutant ataxin-7 and other polyglutamine proteins

PubMed Central

Janer, Alexandre; Martin, Elodie; Muriel, Marie-Paule; Latouche, Morwena; Fujigasaki, Hiroto; Ruberg, Merle; Brice, Alexis; Trottier, Yvon; Sittler, Annie

2006-01-01

The pathogenesis of spinocerebellar ataxia type 7 and other neurodegenerative polyglutamine (polyQ) disorders correlates with the aberrant accumulation of toxic polyQ-expanded proteins in the nucleus. Promyelocytic leukemia protein (PML) nuclear bodies are often present in polyQ aggregates, but their relation to pathogenesis is unclear. We show that expression of PML isoform IV leads to the formation of distinct nuclear bodies enriched in components of the ubiquitin-proteasome system. These bodies recruit soluble mutant ataxin-7 and promote its degradation by proteasome-dependent proteolysis, thus preventing the aggregate formation. Inversely, disruption of the endogenous nuclear bodies with cadmium increases the nuclear accumulation and aggregation of mutant ataxin-7, demonstrating their role in ataxin-7 turnover. Interestingly, β-interferon treatment, which induces the expression of endogenous PML IV, prevents the accumulation of transiently expressed mutant ataxin-7 without affecting the level of the endogenous wild-type protein. Therefore, clastosomes represent a potential therapeutic target for preventing polyQ disorders. PMID:16818720

Nuclear localization of Merkel cell polyomavirus large T antigen in Merkel cell carcinoma

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

Nakamura, Tomoyuki; Sato, Yuko; Watanabe, Daisuke

2010-03-15

To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal inmore » any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.« less

Proteomic analysis of the nuclear matrix in the early stages of rat liver carcinogenesis: Identification of differentially expressed and MAR-binding proteins

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

Barboro, Paola; D'Arrigo, Cristina; Repaci, Erica

Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH). In PHN, the NM undergoes changes both in morphology and in protein composition. We detected over 500 protein spots in each two dimensional map and 44 spots were identified. Twenty-three proteins were differentiallymore » expressed; among these, 15 spots were under-expressed and 8 spots were over-expressed in PHN compared to NH. These changes were synchronous with several modifications in both NM morphology and the ability of NM proteins to bind nuclear RNA and/or DNA containing MARs sequences. In PHN, we observed a general decrease in the expression of the basic proteins that bound nuclear RNA and the over-expression of two species of Mw 135 kDa and 81 kDa and pI 6.7-7.0 and 6.2-7.4, respectively, which exclusively bind to MARs. These results suggest that the deregulated expression of these species might be related to large-scale chromatin reorganization observed in the process of carcinogenesis by modulating the interaction between MARs and the scaffold structure.« less

Use of expression constructs to dissect the functional domains of the CHS/beige protein: identification of multiple phenotypes.

PubMed

Ward, Diane McVey; Shiflett, Shelly L; Huynh, Dinh; Vaughn, Michael B; Prestwich, Glenn; Kaplan, Jerry

2003-06-01

The Chediak-Higashi Syndrome (CHS) and the orthologous murine disorder beige are characterized at the cellular level by the presence of giant lysosomes. The CHS1/Beige protein is a 3787 amino acid protein of unknown function. To determine functional domains of the CHS1/Beige protein, we generated truncated constructs of the gene/protein. These truncated proteins were transiently expressed in Cos-7 or HeLa cells and their effect on membrane trafficking was examined. Beige is apparently a cytosolic protein, as are most transiently expressed truncated Beige constructs. Expression of the Beige construct FM (amino acids 1-2037) in wild-type cells led to enlarged lysosomes. Similarly, expression of a 5.5-kb region (amino acids 2035-3787) of the carboxyl terminal of Beige (22B) also resulted in enlarged lysosomes. Expression of FM solely affected lysosome size, whereas expression of 22B led to alterations in lysosome size, changes in the Golgi and eventually cell death. The two constructs could be used to further dissect phenotypes resulting from loss of the Beige protein. CHS or beigej fibroblasts show an absence of nuclear staining using a monoclonal antibody directed against phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5) P2]. Transformation of beige j fibroblasts with a YAC containing the full-length Beige gene resulted in the normalization of lysosome size and nuclear PtdIns(4,5)P2 staining. Expression of the carboxyl dominant negative construct 22B led to loss of nuclear PtdIns(4,5)P2 staining. Expression of the FM dominant negative clone did not alter nuclear PtdIns(4,5) P2 localization. These results suggest that the Beige protein interacts with at least two different partners and that the Beige protein affects cellular events, such as nuclear PtdIns(4,5)P2 localization, in addition to lysosome size.

76 FR 72008 - Entergy Nuclear Operations, Inc., Entergy Operations, Inc., Entergy Nuclear Vermont Yankee, LLC...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-21

... expressed his belief that Entergy deliberately mismanaged its decommissioning trust funds and knowingly provided false financial documentation supporting filings before the Commission and that the NRC staff was... that amended the original petition, the Petitioner expressed his belief that, because the NRC's...

Features of cues and processes during chloroplast-mediated retrograde signaling in the alga Chlamydomonas

USDA-ARS?s Scientific Manuscript database

Retrograde signalling is a selective process defined by cues generated in chloroplast/mitochondria which traverse membranes and end up regulating nuclear gene expression and protein synthesis. The coding and encoding of organellar message(s) that alter nuclear gene expression and/or cellular metabo...

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

Star-PAP Control of BIK Expression and Apoptosis Is Regulated by Nuclear PIPKIα and PKCδ Signaling

PubMed Central

Li, Weimin; Laishram, Rakesh S.; Ji, Zhe; Barlow, Christy A.; Tian, Bin; Anderson, Richard A.

2012-01-01

SUMMARY BIK protein is an initiator of mitochondrial apoptosis and BIK expression is induced by pro-apoptotic signals including DNA damage. Here we demonstrate that 3′-end processing and expression of BIK mRNA are controlled by the nuclear PI4,5P2-regulated poly(A) polymerase Star-PAP downstream of DNA damage. Nuclear PKCδ is a key mediator of apoptosis and DNA damage stimulates PKCδ association with the Star-PAP complex where PKCδ is required for Star-PAP-dependent BIK expression. PKCδ binds the PI4,5P2-generating enzyme PIPKIα, which is essential for PKCδ interaction with the Star-PAP complex and PKCδ activity is directly stimulated by PI4,5P2. Features in the BIK 3′-UTR uniquely define Star-PAP specificity and may block canonical PAP activity toward BIK mRNA. This reveals a nuclear phosphoinositide signaling nexus where PIPKIα, PI4,5P2 and PKCδ regulate Star-PAP control of BIK expression and induction of apoptosis. This pathway is distinct from the Star-PAP-mediated oxidative stress pathway indicating signal-specific regulation of mRNA 3′-end processing. PMID:22244330

Expression of ionotropic glutamate receptors, AMPA, kainite and NMDA, in the pigeon retina.

PubMed

Atoji, Yasuro

2015-07-01

Glutamate is an excitatory neurotransmitter in the vertebrate retina. A previous study found vesicular glutamate transporter 2 (vGluT2) mRNA in the pigeon retina, suggesting that bipolar and ganglion cells are glutamatergic. The present study examined the localization of ionotropic glutamate receptors to identify receptor cells in the pigeon retina using in situ hybridization histochemistry. Nine subunits of AMPA receptor (GluA1, GluA2, GluA3, and GluA4), kainate receptor (GluK1, GluK2, and GluK4), and NMDA receptor (GluN1 and GluN2A) were found to be expressed in the inner nuclear layer (INL) and ganglion cell layers. GluA1, GluA2, GluA3, and GluA4 were primarily expressed in the inner half of INL, and the signal intensity was strong for GluA2, GluA3, and GluA4. GluK1 was intensely expressed in the outer half of INL, whereas GluK2 and GluK4 were mainly localized in the inner half of INL. GluN1 and GluN2A were moderately expressed in the inner half of INL. Horizontal cells expressed GluA3 and GluA4, and ganglion cells expressed all subunits examined. These results suggest that the glutamatergic neurotransmission in the pigeon retina is similar to that in mammals. Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression of the hMLH1 and hMSH2 proteins in normal tissues: relationship to cancer predisposition in hereditary non-polyposis colon cancer.

PubMed

Plevová, Pavlína; Sedláková, Eva; Zapletalová, Jana; Krepelová, Anna; Skýpalová, Petra; Kolár, Zdenek

2005-02-01

The majority of tumours in patients with hereditary non-polyposis colon cancer (HNPCC) occur in large intestine and endometrium; also, other tissues are at increased risk. We studied expression of hMLH1 and hMSH2 proteins in 148 normal samples of various tissues from non-HNPCC patients and in 14 normal colon tissues from HNPCC patients. Immunohistochemical technique was used. Intensity of nuclear staining, percentage of stained cells and H-scores were calculated. Tissues were divided into groups. Groups A, B and C included tissues with increased risk of cancer in HNPCC A) stomach, small and large bowel; (B) endometrium; (C) ovary, ureter, urinary bladder, kidney and liver. Group D tissues were without increased risk. Expression of the proteins was significantly higher in groups A, B and C compared with group D (P<0.0001, P=0.0004 for hMSH2 in C versus D). The expression was highest in testis. In colons of HNPCC patients, expression of the mutated gene product was significantly lower than in non-HNPCC patients. In conclusion, hMLH1/hMSH2 protein expression is constitutively higher in certain cell types of certain tissues, including the majority of tissues that are at increased risk of cancer in HNPCC. However, association of strong hMLH1/hMSH2 expression with cancer risk is not strictly valid.

Sirtuin 1 regulates matrix metalloproteinase-13 expression induced by Porphyromonas endodontalis lipopolysaccharide via targeting nuclear factor–κB in osteoblasts

PubMed Central

Qu, Liu; Yu, Yaqiong; Qiu, Lihong; Yang, Di; Yan, Lu; Guo, Jiajie; Jahan, Rabita

2017-01-01

ABSTRACT Porphyromonas endodontalis lipopolysaccharide (P.e LPS) is an important initiating factor for periapical inflammation and bone destruction. Matrix metalloproteinase-13 (MMP-13) has been shown to participate in the formation and diffusion of periapical bone lesion in chronic apical periodontitis. Sirtuin 1 (SIRT1) is a key regulator of inflammation in mammalian cells which suppresses the release of inflammatory mediators. This study aimed to explore the role of SIRT1 in regulating MMP-13 expression induced by P.e LPS in osteoblasts. P.e LPS stimulated MMP-13 expression in MC3T3-E1 cells. Knockdown of SIRT1 reinforced the increase of MMP-13mRNA expression induced by P.e LPS. SIRT1 activator resveratrol significantly reduced the expression of MMP-13 and SIRT1 inhibitor EX-527 enhanced the expression of MMP-13. Moreover, SIRT1 activation with resveratrol inhibited acetylation of NF-κB p65 and NF-κB transcriptional activity, which were enhanced by P.e LPS. In addition, NF-κB p65 was involved in P.e LPS-induced MMP-13 expression via directly binding to the MMP-13 promoter. However, SIRT1 activation significantly interfered with this binding. These findings strongly suggest that P.e LPS induces MMP-13 expression in osteoblasts, and SIRT1 suppresses this expression of MMP-13 through targeting NF-κB p65. This provides new insights into understanding the actions of SIRT1 on anti-inflammatory and anti-bone resorption activity. PMID:28473882

Sirtuin 1 regulates matrix metalloproteinase-13 expression induced by Porphyromonas endodontalis lipopolysaccharide via targeting nuclear factor-κB in osteoblasts.

PubMed

Qu, Liu; Yu, Yaqiong; Qiu, Lihong; Yang, Di; Yan, Lu; Guo, Jiajie; Jahan, Rabita

2017-01-01

Porphyromonas endodontalis lipopolysaccharide (P.e LPS) is an important initiating factor for periapical inflammation and bone destruction. Matrix metalloproteinase-13 (MMP-13) has been shown to participate in the formation and diffusion of periapical bone lesion in chronic apical periodontitis. Sirtuin 1 (SIRT1) is a key regulator of inflammation in mammalian cells which suppresses the release of inflammatory mediators. This study aimed to explore the role of SIRT1 in regulating MMP-13 expression induced by P.e LPS in osteoblasts. P.e LPS stimulated MMP-13 expression in MC3T3-E1 cells. Knockdown of SIRT1 reinforced the increase of MMP-13mRNA expression induced by P.e LPS. SIRT1 activator resveratrol significantly reduced the expression of MMP-13 and SIRT1 inhibitor EX-527 enhanced the expression of MMP-13. Moreover, SIRT1 activation with resveratrol inhibited acetylation of NF-κB p65 and NF-κB transcriptional activity, which were enhanced by P.e LPS. In addition, NF-κB p65 was involved in P.e LPS-induced MMP-13 expression via directly binding to the MMP-13 promoter. However, SIRT1 activation significantly interfered with this binding. These findings strongly suggest that P.e LPS induces MMP-13 expression in osteoblasts, and SIRT1 suppresses this expression of MMP-13 through targeting NF-κB p65. This provides new insights into understanding the actions of SIRT1 on anti-inflammatory and anti-bone resorption activity.

Signal-dependent repression of DUSP5 by class I HDACs controls nuclear ERK activity and cardiomyocyte hypertrophy

PubMed Central

Ferguson, Bradley S.; Harrison, Brooke C.; Jeong, Mark Y.; Reid, Brian G.; Wempe, Michael F.; Wagner, Florence F.; Holson, Edward B.; McKinsey, Timothy A.

2013-01-01

Cardiac hypertrophy is a strong predictor of morbidity and mortality in patients with heart failure. Small molecule histone deacetylase (HDAC) inhibitors have been shown to suppress cardiac hypertrophy through mechanisms that remain poorly understood. We report that class I HDACs function as signal-dependent repressors of cardiac hypertrophy via inhibition of the gene encoding dual-specificity phosphatase 5 (DUSP5) DUSP5, a nuclear phosphatase that negatively regulates prohypertrophic signaling by ERK1/2. Inhibition of DUSP5 by class I HDACs requires activity of the ERK kinase, mitogen-activated protein kinase kinase (MEK), revealing a self-reinforcing mechanism for promotion of cardiac ERK signaling. In cardiac myocytes treated with highly selective class I HDAC inhibitors, nuclear ERK1/2 signaling is suppressed in a manner that is absolutely dependent on DUSP5. In contrast, cytosolic ERK1/2 activation is maintained under these same conditions. Ectopic expression of DUSP5 in cardiomyocytes results in potent inhibition of agonist-dependent hypertrophy through a mechanism involving suppression of the gene program for hypertrophic growth. These findings define unique roles for class I HDACs and DUSP5 as integral components of a regulatory signaling circuit that controls cardiac hypertrophy. PMID:23720316

Role of nuclear charge change and nuclear recoil on shaking processes and their possible implication on physical processes

NASA Astrophysics Data System (ADS)

Sharma, Prashant

2017-12-01

The probable role of the sudden nuclear charge change and nuclear recoil in the shaking processes during the neutron- or heavy-ion-induced nuclear reactions and weakly interacting massive particle-nucleus scattering has been investigated in the present work. Using hydrogenic wavefunctions, general analytical expressions of survival, shakeup/shakedown, and shakeoff probability have been derived for various subshells of hydrogen-like atomic systems. These expressions are employed to calculate the shaking, shakeup/shakedown, and shakeoff probabilities in some important cases of interest in the nuclear astrophysics and the dark matter search experiments. The results underline that the shaking processes are one of the probable channels of electronic transitions during the weakly interacting massive particle-nucleus scattering, which can be used to probe the dark matter in the sub-GeV regime. Further, it is found that the shaking processes initiating due to nuclear charge change and nuclear recoil during the nuclear reactions may influence the electronic configuration of the participating atomic systems and thus may affect the nuclear reaction measurements at astrophysically relevant energies.

Improved Dual-Luciferase Reporter Assays for Nuclear Receptors

PubMed Central

Paguio, Aileen; Stecha, Pete; Wood, Keith V; Fan, Frank

2010-01-01

Nuclear receptors play important roles in many cellular functions through control of gene transcription. It is also a large target class for drug discovery. Luciferase reporter assays are frequently used to study nuclear receptor function because of their wide dynamic range, low endogenous activity, and ease of use. Recent improvements of luciferase genes and vectors have further enhanced their utilities. Here we applied these improvements to two reporter formats for studying nuclear receptors. The first assay contains a Murine Mammary Tumor Virus promoter upstream of a destabilized luciferase. The presence of response elements for nuclear hormone receptor in this promoter allows the studies of endogenous and/or exogenous full length receptors. The second assay contains a ligand binding domain (LBD) of a nuclear receptor fused to the GAL4 DNA binding domain (DBD) on one vector and multiple Gal4 Upstream Activator Sequences (UAS) upstream of luciferase reporter on another vector. We showed that codon optimization of luciferase reporter genes increased expression levels in conjunction with the incorporation of protein destabilizing sequences into luciferase led to a larger assay dynamic range in both formats. The optimum number of UAS to generate the best response was determined. The expression vector for nuclear receptor LBD/GAL4 DBD fusion also constitutively expresses a Renilla luciferase-neoR fusion protein, which provides selection capability (G418 resistance, neoR) as well as an internal control (Renilla luciferase). This dual-luciferase format allowed detecting compound cytotoxicity or off-target change in expression during drug screening, therefore improved data quality. These luciferase reporter assays provided better research and drug discovery tools for studying the functions of full length nuclear receptors and ligand binding domains. PMID:21687560

MAP kinase-signaling controls nuclear translocation of tripeptidyl-peptidase II in response to DNA damage and oxidative stress

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

Preta, Giulio; Klark, Rainier de; Chakraborti, Shankhamala

2010-08-27

Research highlights: {yields} Nuclear translocation of TPPII occurs in response to different DNA damage inducers. {yields} Nuclear accumulation of TPPII is linked to ROS and anti-oxidant enzyme levels. {yields} MAPKs control nuclear accumulation of TPPII. {yields} Inhibited nuclear accumulation of TPPII decreases DNA damage-induced {gamma}-H2AX expression. -- Abstract: Reactive oxygen species (ROS) are a continuous hazard in eukaroytic cells by their ability to cause damage to biomolecules, in particular to DNA. Previous data indicated that the cytosolic serine peptidase tripeptidyl-peptidase II (TPPII) translocates into the nucleus of most tumor cell lines in response to {gamma}-irradiation and ROS production; an eventmore » that promoted p53 expression as well as caspase-activation. We here observed that nuclear translocation of TPPII was dependent on signaling by MAP kinases, including p38MAPK. Further, this was caused by several types of DNA-damaging drugs, a DNA cross-linker (cisplatinum), an inhibitor of topoisomerase II (etoposide), and to some extent also by nucleoside-analogues (5-fluorouracil, hydroxyurea). In the minority of tumor cell lines where TPPII was not translocated into the nucleus in response to DNA damage we observed reduced intracellular ROS levels, and the expression levels of redox defense systems were increased. Further, treatment with the ROS-inducer {gamma}-hexa-chloro-cyclohexane ({gamma}-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon {gamma}-irradiation. Moreover, blocking nuclear translocation of TPPII in etoposide-treated cells, by using a peptide-derived inhibitor (Z-Gly-Leu-Ala-OH), attenuated expression of {gamma}-H2AX in {gamma}-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling.« less

Nuclear translocation of glutathione S-transferase {pi} is mediated by a non-classical localization signal

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

Kawakatsu, Miho; Goto, Shinji, E-mail: sgoto@nagasaki-u.ac.jp; Yoshida, Takako

2011-08-12

Highlights: {yields} Nuclear translocation of GST{pi} is abrogated by the deletion of the last 16 amino acid residues in the carboxy-terminal region, indicating that residues 195-208 of GST{pi} are required for nuclear translocation. {yields} The lack of a contiguous stretch of positively charged amino acid residues within the carboxy-terminal region of GST{pi}, suggests that the nuclear translocation of GST{pi} is mediated by a non-classical nuclear localization signal. {yields} An in vitro transport assay shows that the nuclear translocation of GST{pi} is dependent on cytosolic factors and ATP. -- Abstract: Glutathione S-transferase {pi} (GST{pi}), a member of the GST family ofmore » multifunctional enzymes, is highly expressed in human placenta and involved in the protection of cellular components against electrophilic compounds or oxidative stress. We have recently found that GST{pi} is expressed in the cytoplasm, mitochondria, and nucleus in some cancer cells, and that the nuclear expression of GST{pi} appears to correlate with resistance to anti-cancer drugs. Although the mitochondrial targeting signal of GST{pi} was previously identified in the amino-terminal region, the mechanism of nuclear translocation remains completely unknown. In this study, we find that the region of GST{pi}195-208 is critical for nuclear translocation, which is mediated by a novel and non-classical nuclear localization signal. In addition, using an in vitro transport assay, we demonstrate that the nuclear translocation of GST{pi} depends on the cytosolic extract and ATP. Although further experiments are needed to understand in depth the precise mechanism of nuclear translocation of GST{pi}, our results may help to establish more efficient anti-cancer therapy, especially with respect to resistance to anti-cancer drugs.« less

Inhibition of IL-6 Signaling Pathway by Curcumin in Uterine Decidual Cells

PubMed Central

Devi, Y. Sangeeta; DeVine, Majesta; DeKuiper, Justin; Ferguson, Susan; Fazleabas, Asgerally T.

2015-01-01

IL-6 is a multifunctional pro-inflammatory cytokine and has been implicated in many gestational disorders including preterm birth. Currently, there are no appropriate therapeutic interventions available to circumvent inflammatory-mediated gestational disorders. Therefore, the goal of this study was to identify a safe and effective pharmacological compound to counterbalance inflammatory responses in the uterus. Curcumin, a naturally-occuring polyphenolic compound, has been widely used in alternative medicine to treat inflammatory diseases. However, the anti-inflammatory effect of curcumin has not been explored in uterine decidual cells, a major source of IL-6. Therefore, we examined the effect of curcumin on IL-6 expression using two types of uterine decidual cells 1) HuF cells, primary human fibroblast cells obtained from the decidua parietalis; 2) UIII cells, a rodent non-transformed decidual cell line. Curcumin treatment completely abrogated the expression of IL-1β-induced IL-6 in these cells. Curcumin also strongly inhibited the expression of gp130, a critical molecule in IL-6 signaling, whereas expression of IL-6R and sIL-6R was not affected. Curcumin also inhibited phosphorylation and nuclear localization of STAT3, a well-known downstream mediator of IL-6 signaling. Furthermore, curcumin attenuated IL-1β-induced IL-6 promoter reporter activity suggesting transcriptional regulation. To further understand whether NF-ҡB is involved in this inhibition, we examined the effect of curcumin on the expression of p50 and p65 subunits of NF-ҡB in decidual cells. Expression of IL-1β-induced p50 mRNA was repressed by curcumin while p65 mRNA was not affected. However, curcumin treatment dramatically inhibited both p50 and p65 protein levels and prevented its nuclear localization. This effect is at least partly mediated through the deactivation of IKK, since IL-1β-induced IKKα/β phosphorylation is decreased upon curcumin treatment. Our results not only revealed molecular mechanisms underlying curcumin action in uterine decidual cells but also suggest that this compound may have therapeutic potential for the prevention of inflammation-mediated preterm birth and other gestational disorders. PMID:25961579

Genome-Wide Analysis of SREBP1 Activity around the Clock Reveals Its Combined Dependency on Nutrient and Circadian Signals

PubMed Central

Naldi, Aurélien; Baruchet, Michaël; Canella, Donatella; Le Martelot, Gwendal; Guex, Nicolas; Desvergne, Béatrice; Delorenzi, Mauro; Deplancke, Bart; Desvergne, Béatrice; Guex, Nicolas; Herr, Winship; Naef, Felix; Rougemont, Jacques; Schibler, Ueli; Deplancke, Bart; Guex, Nicolas; Herr, Winship; Guex, Nicolas; Andersin, Teemu; Cousin, Pascal; Gilardi, Federica; Gos, Pascal; Martelot, Gwendal Le; Lammers, Fabienne; Canella, Donatella; Gilardi, Federica; Raghav, Sunil; Fabbretti, Roberto; Fortier, Arnaud; Long, Li; Vlegel, Volker; Xenarios, Ioannis; Migliavacca, Eugenia; Praz, Viviane; Guex, Nicolas; Naef, Felix; Rougemont, Jacques; David, Fabrice; Jarosz, Yohan; Kuznetsov, Dmitry; Liechti, Robin; Martin, Olivier; Delafontaine, Julien; Sinclair, Lucas; Cajan, Julia; Krier, Irina; Leleu, Marion; Migliavacca, Eugenia; Molina, Nacho; Naldi, Aurélien; Rey, Guillaume; Symul, Laura; Guex, Nicolas; Naef, Felix; Rougemont, Jacques; Bernasconi, David; Delorenzi, Mauro; Andersin, Teemu; Canella, Donatella; Gilardi, Federica; Martelot, Gwendal Le; Lammers, Fabienne; Baruchet, Michaël; Raghav, Sunil

2014-01-01

In mammals, the circadian clock allows them to anticipate and adapt physiology around the 24 hours. Conversely, metabolism and food consumption regulate the internal clock, pointing the existence of an intricate relationship between nutrient state and circadian homeostasis that is far from being understood. The Sterol Regulatory Element Binding Protein 1 (SREBP1) is a key regulator of lipid homeostasis. Hepatic SREBP1 function is influenced by the nutrient-response cycle, but also by the circadian machinery. To systematically understand how the interplay of circadian clock and nutrient-driven rhythm regulates SREBP1 activity, we evaluated the genome-wide binding of SREBP1 to its targets throughout the day in C57BL/6 mice. The recruitment of SREBP1 to the DNA showed a highly circadian behaviour, with a maximum during the fed status. However, the temporal expression of SREBP1 targets was not always synchronized with its binding pattern. In particular, different expression phases were observed for SREBP1 target genes depending on their function, suggesting the involvement of other transcription factors in their regulation. Binding sites for Hepatocyte Nuclear Factor 4 (HNF4) were specifically enriched in the close proximity of SREBP1 peaks of genes, whose expression was shifted by about 8 hours with respect to SREBP1 binding. Thus, the cross-talk between hepatic HNF4 and SREBP1 may underlie the expression timing of this subgroup of SREBP1 targets. Interestingly, the proper temporal expression profile of these genes was dramatically changed in Bmal1 −/− mice upon time-restricted feeding, for which a rhythmic, but slightly delayed, binding of SREBP1 was maintained. Collectively, our results show that besides the nutrient-driven regulation of SREBP1 nuclear translocation, a second layer of modulation of SREBP1 transcriptional activity, strongly dependent from the circadian clock, exists. This system allows us to fine tune the expression timing of SREBP1 target genes, thus helping to temporally separate the different physiological processes in which these genes are involved. PMID:24603613

Planar and non-planar nucleus-acoustic shock structures in self-gravitating degenerate quantum plasma systems

NASA Astrophysics Data System (ADS)

Zaman, D. M. S.; Amina, M.; Dip, P. R.; Mamun, A. A.

2017-11-01

The basic properties of planar and non-planar (spherical and cylindrical) nucleus-acoustic (NA) shock structures (SSs) in a strongly coupled self-gravitating degenerate quantum plasma system (containing strongly coupled non-relativistically degenerate heavy nuclear species, weakly coupled non-relativistically degenerate light nuclear species, and inertialess non-/ultra-relativistically degenerate electrons) have been investigated. The generalized quantum hydrodynamic model and the reductive perturbation method have been used to derive the modified Burgers equation. It is shown that the strong correlation among heavy nuclear species acts as the source of dissipation and is responsible for the formation of the NA SSs with positive (negative) electrostatic (self-gravitational) potential. It is also observed that the effects of non-/ultra-relativistically degenerate electron pressure, dynamics of non-relativistically degenerate light nuclear species, spherical geometry, etc., significantly modify the basic features of the NA SSs. The applications of our results in astrophysical compact objects like white dwarfs and neutron stars are briefly discussed.

Histone-poly(A) hybrid molecules as tools to block nuclear pores.

PubMed

Cremer, G; Wojtech, E; Kalbas, M; Agutter, P S; Prochnow, D

1995-04-01

Histone-poly(A) hybrid molecules were used for transport experiments with resealed nuclear envelopes and after attachment of a cleavable cross-linker (SASD) to identify nuclear proteins. In contrast to histones, the hybrid molecules cannot be accumulated in resealed nuclear envelopes, and in contrast to poly(A), the export of hybrids from preloaded nuclear envelopes is completely impaired. The experiments strongly confirm the existence of poly(A) as an export signal in mRNA which counteracts the nuclear location signals (NLS) in histones. The contradicting transport signals in the hybrid molecules impair translocation through the nuclear pore complex. The failure to accumulate hybrid molecules into resealed nuclear envelopes results from the covalent attachment of polyadenylic acid to histones in a strict 1:1 molar ratio. This was demonstrated in control transport experiments where radiolabeled histones were simply mixed with nonlabeled poly(A) or radiolabeled poly(A) mixed with nonlabeled histones. In comparison, control uptake experiments with histones covalently linked to a single UMP-mononucleotide are strongly enhanced. Such controls exclude the conceivable possibility of a simple masking of the nuclear location signal in the histones by the covalent attached poly(A) moiety. Photoreactive histone-poly(A) hybrid analogs serve to identify nuclear envelope proteins--presumably in the nuclear pore--with molecular weights of 110, 80, and 71.4 kDa.

Ethnopharmacological in vitro studies on Austria's folk medicine—An unexplored lore in vitro anti-inflammatory activities of 71 Austrian traditional herbal drugs☆

PubMed Central

Vogl, Sylvia; Picker, Paolo; Mihaly-Bison, Judit; Fakhrudin, Nanang; Atanasov, Atanas G.; Heiss, Elke H.; Wawrosch, Christoph; Reznicek, Gottfried; Dirsch, Verena M.; Saukel, Johannes; Kopp, Brigitte

2013-01-01

Ethnopharmacological relevance In Austria, like in most Western countries, knowledge about traditional medicinal plants is becoming scarce. Searching the literature concerning Austria's ethnomedicine reveals its scant scientific exploration. Aiming to substantiate the potential of medicinal plants traditionally used in Austria, 63 plant species or genera with claimed anti-inflammatory properties listed in the VOLKSMED database were assessed for their in vitro anti-inflammatory activity. Material and methods 71 herbal drugs from 63 plant species or genera were extracted using solvents of varying polarities and subsequently depleted from the bulk constituents, chlorophylls and tannins to avoid possible interferences with the assays. The obtained 257 extracts were assessed for their in vitro anti-inflammatory activity. The expression of the inflammatory mediators E-selectin and interleukin-8 (IL-8), induced by the inflammatory stimuli tumor necrosis factor alpha (TNF-α) and the bacterial product lipopolysaccharide (LPS) was measured in endothelial cells. The potential of the extracts to activate the nuclear factors PPARα and PPARγ and to inhibit TNF-α-induced activation of the nuclear factor-kappa B (NF-κB) in HEK293 cells was determined by luciferase reporter gene assays. Results In total, extracts from 67 of the 71 assessed herbal drugs revealed anti-inflammatory activity in the applied in vitro test systems. Thereby, 30 could downregulate E-selectin or IL-8 gene expression, 28 were strong activators of PPARα or PPARγ (inducing activation of more than 2-fold at a concentration of 10 µg/mL) and 21 evoked a strong inhibition of NF-κB (inhibition of more than 80% at 10 µg/mL). Conclusion Our research supports the efficacy of herbal drugs reported in Austrian folk medicine used for ailments associated with inflammatory processes. Hence, an ethnopharmacological screening approach is a useful tool for the discovery of new drug leads. PMID:23770053

The leukemia associated ETO nuclear repressor gene is regulated by the GATA-1 transcription factor in erythroid/megakaryocytic cells

PubMed Central

2010-01-01

Background The Eight-Twenty-One (ETO) nuclear co-repressor gene belongs to the ETO homologue family also containing Myeloid Translocation Gene on chromosome 16 (MTG16) and myeloid translocation Gene-Related protein 1 (MTGR1). By chromosomal translocations ETO and MTG16 become parts of fusion proteins characteristic of morphological variants of acute myeloid leukemia. Normal functions of ETO homologues have as yet not been examined. The goal of this work was to identify structural and functional promoter elements upstream of the coding sequence of the ETO gene in order to explore lineage-specific hematopoietic expression and get hints to function. Results A putative proximal ETO promoter was identified within 411 bp upstream of the transcription start site. Strong ETO promoter activity was specifically observed upon transfection of a promoter reporter construct into erythroid/megakaryocytic cells, which have endogeneous ETO gene activity. An evolutionary conserved region of 228 bp revealed potential cis-elements involved in transcription of ETO. Disruption of the evolutionary conserved GATA -636 consensus binding site repressed transactivation and disruption of the ETS1 -705 consensus binding site enhanced activity of the ETO promoter. The promoter was stimulated by overexpression of GATA-1 into erythroid/megakaryocytic cells. Electrophoretic mobility shift assay with erythroid/megakaryocytic cells showed specific binding of GATA-1 to the GATA -636 site. Furthermore, results from chromatin immunoprecipitation showed GATA-1 binding in vivo to the conserved region of the ETO promoter containing the -636 site. The results suggest that the GATA -636 site may have a role in activation of the ETO gene activity in cells with erythroid/megakaryocytic potential. Leukemia associated AML1-ETO strongly suppressed an ETO promoter reporter in erythroid/megakaryocytic cells. Conclusions We demonstrate that the GATA-1 transcription factor binds and transactivates the ETO proximal promoter in an erythroid/megakaryocytic-specific manner. Thus, trans-acting factors that are essential in erythroid/megakaryocytic differentiation govern ETO expression. PMID:20487545

The Gpn3 Q279* cancer-associated mutant inhibits Gpn1 nuclear export and is deficient in RNA polymerase II nuclear targeting.

PubMed

Barbosa-Camacho, Angel A; Méndez-Hernández, Lucía E; Lara-Chacón, Bárbara; Peña-Gómez, Sonia G; Romero, Violeta; González-González, Rogelio; Guerra-Moreno, José A; Robledo-Rivera, Angélica Y; Sánchez-Olea, Roberto; Calera, Mónica R

2017-11-01

Gpn3 is required for RNA polymerase II (RNAPII) nuclear targeting. Here, we investigated the effect of a cancer-associated Q279* nonsense mutation in Gpn3 cellular function. Employing RNAi, we replaced endogenous Gpn3 by wt or Q279* RNAi-resistant Gpn3R in epithelial model cells. RNAPII nuclear accumulation and transcriptional activity were markedly decreased in cells expressing only Gpn3R Q279*. Wild-type Gpn3R localized to the cytoplasm but a fraction of Gpn3R Q279* entered the cell nucleus and inhibited Gpn1-EYFP nuclear export. This property and the transcriptional deficit in Gpn3R Q279*-expressing cells required a PDZ-binding motif generated by the Q279* mutation. We conclude that an acquired PDZ-binding motif in Gpn3 Q279* caused Gpn3 nuclear entry, and inhibited Gpn1 nuclear export and Gpn3-mediated RNAPII nuclear targeting. © 2017 Federation of European Biochemical Societies.

Nuclear organization mediates cancer-compromised genetic and epigenetic control.

PubMed

Zaidi, Sayyed K; Fritz, Andrew; Tracy, Kirsten; Gordon, Jonathan; Tye, Coralee; Boyd, Joseph; Van Wijnen, Andre; Nickerson, Jeffrey; Imbalzano, Anthony; Lian, Jane; Stein, Janet; Stein, Gary

2018-05-09

Nuclear organization is functionally linked to genetic and epigenetic regulation of gene expression for biological control and is modified in cancer. Nuclear organization supports cell growth and phenotypic properties of normal and cancer cells by facilitating physiologically responsive interactions of chromosomes, genes and regulatory complexes at dynamic three-dimensional microenvironments. We will review nuclear structure/function relationships that include: 1. Epigenetic bookmarking of genes by phenotypic transcription factors to control fidelity and plasticity of gene expression as cells enter and exit mitosis; 2. Contributions of chromatin remodeling to breast cancer nuclear morphology, metabolism and effectiveness of chemotherapy; 3. Relationships between fidelity of nuclear organization and metastasis of breast cancer to bone; 4. Dynamic modifications of higher-order inter- and intra-chromosomal interactions in breast cancer cells; 5. Coordinate control of cell growth and phenotype by tissue-specific transcription factors; 6. Oncofetal epigenetic control by bivalent histone modifications that are functionally related to sustaining the stem cell phenotype; and 7. Noncoding RNA-mediated regulation in the onset and progression of breast cancer. The discovery of components to nuclear organization that are functionally related to cancer and compromise gene expression have the potential for translation to innovative cancer diagnosis and targeted therapy. Copyright © 2018 Elsevier Ltd. All rights reserved.

Nuclear size is sensitive to NTF2 protein levels in a manner dependent on Ran binding.

PubMed

Vuković, Lidija D; Jevtić, Predrag; Zhang, Zhaojie; Stohr, Bradley A; Levy, Daniel L

2016-03-15

Altered nuclear size is associated with many cancers, and determining whether cancer-associated changes in nuclear size contribute to carcinogenesis necessitates an understanding of mechanisms of nuclear size regulation. Although nuclear import rates generally positively correlate with nuclear size, NTF2 levels negatively affect nuclear size, despite the role of NTF2 (also known as NUTF2) in nuclear recycling of the import factor Ran. We show that binding of Ran to NTF2 is required for NTF2 to inhibit nuclear expansion and import of large cargo molecules in Xenopus laevis egg and embryo extracts, consistent with our observation that NTF2 reduces the diameter of the nuclear pore complex (NPC) in a Ran-binding-dependent manner. Furthermore, we demonstrate that ectopic NTF2 expression in Xenopus embryos and mammalian tissue culture cells alters nuclear size. Finally, we show that increases in nuclear size during melanoma progression correlate with reduced NTF2 expression, and increasing NTF2 levels in melanoma cells is sufficient to reduce nuclear size. These results show a conserved capacity for NTF2 to impact on nuclear size, and we propose that NTF2 might be a new cancer biomarker. © 2016. Published by The Company of Biologists Ltd.

Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins

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

Maric, Martina; Haugo, Alison C.; Dauer, William

2014-07-15

Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but ismore » enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway. - Highlights: • We show that wild-type HSV can induce breakdown of the nuclear envelope in a specific cell system. • The viral fusion proteins gB and gH are required for induction of nuclear envelope breakdown. • Nuclear envelope breakdown cannot compensate for deletion of the HSV UL34 gene.« less

The G protein-coupled estrogen receptor (GPER) is expressed in two different subcellular localizations reflecting distinct tumor properties in breast cancer.

PubMed

Samartzis, Eleftherios P; Noske, Aurelia; Meisel, Alexander; Varga, Zsuzsanna; Fink, Daniel; Imesch, Patrick

2014-01-01

The G protein-coupled estrogen receptor (GPER) is a novel estrogen receptor that mediates proliferative effects induced by estrogen but also by tamoxifen. The aim of our study was to analyze the frequency of GPER in a large collective of primary invasive breast carcinomas, with special emphasis on the subcellular expression and to evaluate the association with clinicopathological parameters and patient overall survival. The tissue microarrays from formalin-fixed, paraffin embedded samples of primary invasive breast carcinomas (n = 981) were analyzed for GPER expression using immunohistochemistry. Expression data were compared to the clinicopathological parameters and overall survival. GPER localization was also analyzed in two immortalized breast cancer cell lines T47D and MCF7 by confocal immunofluorescence microscopy. A predominantly cytoplasmic GPER expression was found in 189 carcinomas (19.3%), whereas a predominantly nuclear expression was observed in 529 cases (53.9%). A simultaneous comparable positive expression of both patterns was found in 32 of 981 cases (3.2%), and negative staining was detected in 295 cases (30%). Confocal microscopy confirmed the occurrence of cytoplasmic and nuclear GPER expression in T47D and MCF7. Cytoplasmic GPER expression was significantly associated with non-ductal histologic subtypes, low tumor stage, better histologic differentiation, as well as Luminal A and B subtypes. In contrast, nuclear GPER expression was significantly associated with poorly differentiated carcinomas and the triple-negative subtype. In univariate analysis, cytoplasmic GPER expression was associated with better overall survival (p = 0.012). Our data suggest that predominantly cytoplasmic and/or nuclear GPER expression are two distinct immunohistochemical patterns in breast carcinomas and may reflect different biological features, reason why these patterns should be clearly distinguished in histological evaluations. Prospective studies will be needed to assess whether the expression status of GPER in breast carcinomas should be routinely observed by clinicians, for instance, before implementing endocrine breast cancer treatment.

The G Protein-Coupled Estrogen Receptor (GPER) Is Expressed in Two Different Subcellular Localizations Reflecting Distinct Tumor Properties in Breast Cancer

PubMed Central

Samartzis, Eleftherios P.; Noske, Aurelia; Meisel, Alexander; Varga, Zsuzsanna; Fink, Daniel; Imesch, Patrick

2014-01-01

Introduction The G protein-coupled estrogen receptor (GPER) is a novel estrogen receptor that mediates proliferative effects induced by estrogen but also by tamoxifen. The aim of our study was to analyze the frequency of GPER in a large collective of primary invasive breast carcinomas, with special emphasis on the subcellular expression and to evaluate the association with clinicopathological parameters and patient overall survival. Methods The tissue microarrays from formalin-fixed, paraffin embedded samples of primary invasive breast carcinomas (n = 981) were analyzed for GPER expression using immunohistochemistry. Expression data were compared to the clinicopathological parameters and overall survival. GPER localization was also analyzed in two immortalized breast cancer cell lines T47D and MCF7 by confocal immunofluorescence microscopy. Results A predominantly cytoplasmic GPER expression was found in 189 carcinomas (19.3%), whereas a predominantly nuclear expression was observed in 529 cases (53.9%). A simultaneous comparable positive expression of both patterns was found in 32 of 981 cases (3.2%), and negative staining was detected in 295 cases (30%). Confocal microscopy confirmed the occurrence of cytoplasmic and nuclear GPER expression in T47D and MCF7. Cytoplasmic GPER expression was significantly associated with non-ductal histologic subtypes, low tumor stage, better histologic differentiation, as well as Luminal A and B subtypes. In contrast, nuclear GPER expression was significantly associated with poorly differentiated carcinomas and the triple-negative subtype. In univariate analysis, cytoplasmic GPER expression was associated with better overall survival (p = 0.012). Conclusion Our data suggest that predominantly cytoplasmic and/or nuclear GPER expression are two distinct immunohistochemical patterns in breast carcinomas and may reflect different biological features, reason why these patterns should be clearly distinguished in histological evaluations. Prospective studies will be needed to assess whether the expression status of GPER in breast carcinomas should be routinely observed by clinicians, for instance, before implementing endocrine breast cancer treatment. PMID:24421881

The role of MAP kinases in the induction of iNOS expression in neutrophils exposed to NDMA: the involvement transcription factors.

PubMed

Ratajczak-Wrona, W; Jablonska, E; Garley, M; Jablonski, J; Radziwon, P; Iwaniuk, A

2013-01-01

The role of MAP kinases in the activation of AP-1 (c-Jun, c-Fos) and NF-κB p65 engaged in the regulation of iNOS expression in human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was analyzed in the study. The study included a group of 20 healthy individuals. Isolated human PMN were incubated in the presence of NDMA. Selective MAP kinases inhibitors were used. The expression of proteins in the cytoplasmic and nuclear fractions was assessed using Western blot method. The results show that NDMA intensifies iNOS, c-Jun, NF-κB p65 and IκB-α expression in the analyzed PMNs. The blocking of the p38 pathway led to lower iNOS expression, and higher expression of c-Jun and c-Fos in the cytoplasmic fraction, and also lower c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. A decrease in iNOS expression in the cytoplasmic fraction, and also c-Jun in both fractions of the examined cells, was observed as a result of JNK pathway inhibition. The blocking of the ERK5 pathway led to higher iNOS, c-Jun and c-Fos expression in the cytoplasmic fraction, and higher c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. The study also demonstrated that blocking of the p38 and JNK pathways resulted in higher expression of NF-κB p65 and IκB-α in the cytoplasmic fraction and their lower expression in the nuclear fraction of these cells. Our data indicate the role of MAP kinases p38 and JNK in the activation of c-Jun and NF-κB p65 transcription factors engaged in the regulation of iNOS expression in human neutrophils exposed to NDMA. However ERK5 kinase is not involved in the regulation of iNOS and NO production by those cells.

Upregulation of B23 promotes tumor cell proliferation and predicts poor prognosis in glioma

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

Chen, Jianguo; Department of Neurosurgery, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong, 226001, Jiangsu Province; Sun, Jie

B23 (also known as Nucleophosmin, NPM, numatrin or NO38) is a ubiquitously expressed phosphoprotein belonging to the nucleoplasmin family of chaperones. In this study we intended to investigate the clinical significance of B23 expression in human glioma and its biological function in glioma cells. Western blot and immunohistochemistry analysis showed that B23 was overexpressed in glioma tissues and glioma cell lines. In addition, the expression level of B23 was positively correlated with glioma pathological grade and Ki-67 expression. Kaplan–Meier analysis revealed that a higher B23 expression in patients with glioma was associated with a poorer prognosis. In vitro, after the releasemore » of glioma cell lines from serum starvation, the expression of B23 was upregulated, as well as PCNA (Proliferating Cell Nuclear Antigen) and cyclin A. In addition, knockdown of B23 by small interfering RNA transfection diminished the expression of PCNA, cyclin D1 and arrested cell growth at G1 phase. Taken together, our results implied that B23 could be a candidate prognostic biomarker as well as a potential therapeutical target of glioma. - Highlights: • B23 expression increased as the malignant degree of glioma increased, which was consistent with Ki-67 expression. • High expression of B23 could be a strong determinant of poor prognosis in glioma. • B23 may be involved in the proliferation of glioma in a cell-cycle-dependent pathway. • Knockdown of B23 expression by siRNA could affect the progression of glioma. • B23 may be a potential prognosis biomarker and a possible therapeutic target for glioma.« less

A role for chromatin topology in imprinted domain regulation.

PubMed

MacDonald, William A; Sachani, Saqib S; White, Carlee R; Mann, Mellissa R W

2016-02-01

Recently, many advancements in genome-wide chromatin topology and nuclear architecture have unveiled the complex and hidden world of the nucleus, where chromatin is organized into discrete neighbourhoods with coordinated gene expression. This includes the active and inactive X chromosomes. Using X chromosome inactivation as a working model, we utilized publicly available datasets together with a literature review to gain insight into topologically associated domains, lamin-associated domains, nucleolar-associating domains, scaffold/matrix attachment regions, and nucleoporin-associated chromatin and their role in regulating monoallelic expression. Furthermore, we comprehensively review for the first time the role of chromatin topology and nuclear architecture in the regulation of genomic imprinting. We propose that chromatin topology and nuclear architecture are important regulatory mechanisms for directing gene expression within imprinted domains. Furthermore, we predict that dynamic changes in chromatin topology and nuclear architecture play roles in tissue-specific imprint domain regulation during early development and differentiation.

Developing molecular tools for Chlamydomonas reinhardtii

NASA Astrophysics Data System (ADS)

Noor-Mohammadi, Samaneh

Microalgae have garnered increasing interest over the years for their ability to produce compounds ranging from biofuels to neutraceuticals. A main focus of researchers has been to use microalgae as a natural bioreactor for the production of valuable and complex compounds. Recombinant protein expression in the chloroplasts of green algae has recently become more routine; however, the heterologous expression of multiple proteins or complete biosynthetic pathways remains a significant challenge. To take full advantage of these organisms' natural abilities, sophisticated molecular tools are needed to be able to introduce and functionally express multiple gene biosynthetic pathways in its genome. To achieve the above objective, we have sought to establish a method to construct, integrate and express multigene operons in the chloroplast and nuclear genome of the model microalgae Chlamydomonas reinhardtii. Here we show that a modified DNA Assembler approach can be used to rapidly assemble multiple-gene biosynthetic pathways in yeast and then integrate these assembled pathways at a site-specific location in the chloroplast, or by random integration in the nuclear genome of C. reinhardtii. As a proof of concept, this method was used to successfully integrate and functionally express up to three reporter proteins (AphA6, AadA, and GFP) in the chloroplast of C. reinhardtii and up to three reporter proteins (Ble, AphVIII, and GFP) in its nuclear genome. An analysis of the relative gene expression of the engineered strains showed significant differences in the mRNA expression levels of the reporter genes and thus highlights the importance of proper promoter/untranslated-region selection when constructing a target pathway. In addition, this work focuses on expressing the cofactor regeneration enzyme phosphite dehydrogenase (PTDH) in the chloroplast and nuclear genomes of C. reinhardtii. The PTDH enzyme converts phosphite into phosphate and NAD(P)+ into NAD(P)H. The reduced nicotinamide cofactor NAD(P)H plays a pivotal role in many biochemical oxidation and reduction reactions, thus this enzyme would allow regeneration of NAD(P)H in a microalgae strain over-expressing a NAD(P)H-dependent oxidoreductase. A phosphite dehydrogenase gene was introduced into the chloroplast genome (codon optimized) and nuclear genome of C. reinhardtii by biolistic transformation and electroporation in separate events, respectively. Successful expression of the heterologous protein was confirmed by transcript analysis and protein analysis. In conclusion, this new method represents a useful genetic tool in the construction and integration of complex biochemical pathways into the chloroplast or nuclear genome of microalgae, and this should aid current efforts to engineer algae for recombinant protein expression, biofuels production and production of other desirable natural products.

Airway epithelial cell response to human metapneumovirus infection

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

Bao, X.; Liu, T.; Spetch, L.

2007-11-10

Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections (LRTIs) in infants, elderly and immunocompromised patients. In this study, we show that hMPV can infect in a similar manner epithelial cells representative of different tracts of the airways. hMPV-induced expression of chemokines IL-8 and RANTES in primary small alveolar epithelial cells (SAE) and in a human alveolar type II-like epithelial cell line (A549) was similar, suggesting that A549 cells can be used as a model to study lower airway epithelial cell responses to hMPV infection. A549 secreted a variety of CXC and CC chemokines, cytokines and typemore » I interferons, following hMPV infection. hMPV was also a strong inducer of transcription factors belonging to nuclear factor (NF)-{kappa}B, interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) families, which are known to orchestrate the expression of inflammatory and immunomodulatory mediators.« less

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

Alvarez, Enrique, E-mail: ealvarez@cbm.uam.es; Castello, Alfredo; Carrasco, Luis

Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of proteasemore » fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.« less

Synonymous codon choices in the extremely GC-poor genome of Plasmodium falciparum: compositional constraints and translational selection.

PubMed

Musto, H; Romero, H; Zavala, A; Jabbari, K; Bernardi, G

1999-07-01

We have analyzed the patterns of synonymous codon preferences of the nuclear genes of Plasmodium falciparum, a unicellular parasite characterized by an extremely GC-poor genome. When all genes are considered, codon usage is strongly biased toward A and T in third codon positions, as expected, but multivariate statistical analysis detects a major trend among genes. At one end genes display codon choices determined mainly by the extreme genome composition of this parasite, and very probably their expression level is low. At the other end a few genes exhibit an increased relative usage of a particular subset of codons, many of which are C-ending. Since the majority of these few genes is putatively highly expressed, we postulate that the increased C-ending codons are translationally optimal. In conclusion, while codon usage of the majority of P. falciparum genes is determined mainly by compositional constraints, a small number of genes exhibit translational selection.

Diagnostic utility of NCOA2 fluorescence in situ hybridization and Stat6 immunohistochemistry staining for soft tissue angiofibroma and morphologically similar fibrovascular tumors.

PubMed

Sugita, Shintaro; Aoyama, Tomoyuki; Kondo, Kei; Keira, Yoshiko; Ogino, Jiro; Nakanishi, Katsuya; Kaya, Mitsunori; Emori, Makoto; Tsukahara, Tomohide; Nakajima, Hisaya; Takagi, Masayuki; Hasegawa, Tadashi

2014-08-01

Soft tissue angiofibroma (STA), a recently suggested new histologic entity, is a benign fibrovascular soft tissue tumor composed of bland spindle-shaped tumor cells with abundant collagenous to myxoid stroma and branching small vessels. The lesion has a characteristic AHRR-NCOA2 fusion gene derived from chromosomal translocation of t(5;8)(p15;q13). However, morphologically similar tumors containing abundant fibrovascular and myxoid stroma can complicate diagnosis. We designed an original DNA probe for detecting NCOA2 split signals on fluorescence in situ hybridization (FISH) and estimated its utility with 20 fibrovascular tumors: 4 each of STAs, solitary fibrous tumors (SFTs), and cellular angiofibromas and 3 each of low-grade myxofibrosarcomas, myxoid liposarcomas, and low-grade fibromyxoid sarcomas. We also performed FISH for 13q14 deletion and immunohistochemistry (IHC) staining for estrogen receptor, progesterone receptor, retinoblastoma protein, and MUC-4 expression. Furthermore, IHC for Stat6 was conducted in the 20 cases analyzed by FISH and in an additional 26 SFTs. We found moderate to strong nuclear Stat6 expression in all SFTs but no expression in the other tumors. Both estrogen receptor and progesterone receptor expressions were observed in STAs, SFTs, and cellular angiofibromas. Expression of retinoblastoma protein was found in less than 10% of cells in all tumor types except myxoid liposarcoma. The low-grade fibromyxoid sarcomas were strongly positive for MUC-4. All STAs showed NCOA2 split signals on FISH. All tumors, regardless of histologic type, had 13q14 deletion. The NCOA2 FISH technique is a practical method for confirming STA diagnosis. The combination of NCOA2 FISH and Stat6 IHC proved effective for the differential diagnosis of STA, even when using small biopsy specimens. Copyright © 2014 Elsevier Inc. All rights reserved.

Nuclear-encoded mitochondrial complex I gene expression is restored to normal levels by inhibition of unedited ATP9 transgene expression in Arabidopsis thaliana.

PubMed

Busi, María V; Gómez-Casati, Diego F; Perales, Mariano; Araya, Alejandro; Zabaleta, Eduardo

2006-01-01

Mitochondria play an important role during sporogenesis in plants. The steady state levels of the nuclear-encoded mitochondrial complex I (nCI), PSST, TYKY and NADHBP transcripts increase in flowers of male-sterile plants with impairment of mitochondrial function generated by the expression of the unedited version of ATP9 (u-ATP9). This suggests a nuclear control of nCI genes in response to the mitochondrial flaw. To evaluate this hypothesis, transgenic plants carrying the GUS reporter gene, under the control of the PSST, TYKY and NADHBP promoters, were constructed. We present evidence that suppression by antisense strategy of the expression of u-ATP9 restores the normal levels of three nCI transcripts, indicating that the increase in PSST, TYKY and NADHBP in plants with a mitochondrial flaw occurs at the transcriptional level. The data presented here support the hypothesis that a mitochondrial dysfunction triggers a retrograde signaling which induce some nuclear-encoded mitochondrial genes. Moreover, these results demonstrate that this is a valuable experimental model for studying nucleus-mitochondria cross-talk events.

Prolonged, brain-wide expression of nuclear-localized GCaMP3 for functional circuit mapping

PubMed Central

Kim, Christina K.; Miri, Andrew; Leung, Louis C.; Berndt, Andre; Mourrain, Philippe; Tank, David W.; Burdine, Rebecca D.

2014-01-01

Larval zebrafish offer the potential for large-scale optical imaging of neural activity throughout the central nervous system; however, several barriers challenge their utility. First, ~panneuronal probe expression has to date only been demonstrated at early larval stages up to 7 days post-fertilization (dpf), precluding imaging at later time points when circuits are more mature. Second, nuclear exclusion of genetically-encoded calcium indicators (GECIs) limits the resolution of functional fluorescence signals collected during imaging. Here, we report the creation of transgenic zebrafish strains exhibiting robust, nuclearly targeted expression of GCaMP3 across the brain up to at least 14 dpf utilizing a previously described optimized Gal4-UAS system. We confirmed both nuclear targeting and functionality of the modified probe in vitro and measured its kinetics in response to action potentials (APs). We then demonstrated in vivo functionality of nuclear-localized GCaMP3 in transgenic zebrafish strains by identifying eye position-sensitive fluorescence fluctuations in caudal hindbrain neurons during spontaneous eye movements. Our methodological approach will facilitate studies of larval zebrafish circuitry by both improving resolution of functional Ca2+ signals and by allowing brain-wide expression of improved GECIs, or potentially any probe, further into development. PMID:25505384

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

Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M.

Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associatedmore » proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.« less

Aloe emodin inhibits colon cancer cell migration/angiogenesis by downregulating MMP-2/9, RhoB and VEGF via reduced DNA binding activity of NF-κB.

PubMed

Suboj, Priya; Babykutty, Suboj; Valiyaparambil Gopi, Deepak Roshan; Nair, Rakesh S; Srinivas, Priya; Gopala, Srinivas

2012-04-11

Aloe emodin (AE), a natural anthraquinone, is reported to have antiproliferative activity in various cancer cell lines. In this study we analyzed molecular mechanisms involved in the antimigratory and antiangiogenic activity of this hydroxy anthraquinone in colon cancer cell, WiDr. Our results show that a relatively non toxic concentration of AE suppressed the phorbol-12-myristyl-13-acetate (PMA) induced migration and invasion of tumor cells. On analysis for the molecules involved in the migration/invasion, we found AE downregulated mRNA expression and promoter/gelatinolytic activity of Matrix Metalloproteinase (MMP)-2/9, as well as the RhoB expression at gene and protein level. It was also a strong inhibitor of Vascular Endothelial Growth Factor (VEGF) expression, promoter activity and endothelial cell migration/invasion and in vitro angiogenesis. AE suppressed the nuclear translocation and DNA binding of NF-κB, which is an important transcription factor for controlling MMP-2/9 and VEGF gene expression. Taken together these data indicate that AE target multiple molecules responsible for cellular invasion, migration and angiogenesis. Inhibitory effect on angiogenic and metastatic regulatory processes make AE a sensible candidate as a specific blocker of tumor associated events. Copyright © 2011 Elsevier B.V. All rights reserved.

Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

PubMed Central

Macdonald, Patrick J.; Chen, Yan; Mueller, Joachim D.

2012-01-01

Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37 °C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction. PMID:22093611

Nuclear translocation of β-catenin and decreased expression of epithelial cadherin in human papillomavirus-positive tonsillar cancer: an early event in human papillomavirus-related tumour progression?

PubMed

Stenner, Markus; Yosef, Basima; Huebbers, Christian U; Preuss, Simon F; Dienes, Hans-Peter; Speel, Ernst-Jan M; Odenthal, Margarete; Klussmann, Jens P

2011-06-01

High-risk human papillomaviruses (HPVs) constitute an important risk factor for tonsillar cancer. This study describes changes in cell adhesion molecules during metastasis of HPV-related and HPV-unrelated tonsillar carcinomas. We examined 48 primary tonsillar carcinoma samples (25 HPV-16 DNA-positive, 23 HPV-16 DNA-negative) and their respective lymph node metastases for their HPV status and for the expression of p16, epithelial cadherin (E-cadherin), β-catenin, and vimentin. A positive HPV-specific polymerase chain reaction finding correlated significantly with p16 overexpression in both primary tumours and their metastases (P<0.0001 for both). In HPV-unrelated carcinomas, the expression of E-cadherin was significantly lower in metastases than in primary tumours (P<0.001). In contrast, the expression of nuclear β-catenin was significantly higher in metastases than in primary tumours (P=0.016). In HPV-related carcinomas, nuclear localization of β-catenin expression was already apparent in primary tumours (P=0.030). The expression of vimentin significantly correlated with the grading of the primary tumour (P=0.021). Our data indicate that the down-regulation of E-cadherin and the up-regulation of nuclear β-catenin expression might be crucial steps during tumour progression of tonsillar carcinomas, being already present in primary tumours in HPV-driven carcinomas, but becoming apparent in HPV-unrelated tumours later in the process of metastasis. © 2011 Blackwell Publishing Limited.

Intertwined electron-nuclear motion in frustrated double ionization in driven heteronuclear molecules

NASA Astrophysics Data System (ADS)

Vilà, A.; Zhu, J.; Scrinzi, A.; Emmanouilidou, A.

2018-03-01

We study frustrated double ionization (FDI) in a strongly-driven heteronuclear molecule HeH+ and compare with H2. We compute the probability distribution of the sum of the final kinetic energies of the nuclei for strongly-driven HeH+. We find that this distribution has more than one peak for strongly-driven HeH+, a feature we do not find to be present for strongly-driven H2. Moreover, we compute the probability distribution of the principal quantum number n of FDI. We find that this distribution has several peaks for strongly-driven HeH+, while the respective distribution has one main peak and a ‘shoulder’ at lower principal quantum numbers n for strongly-driven H2. Surprisingly, we find this feature to be a clear signature of the intertwined electron-nuclear motion.

PDE4 and mAKAPβ are nodal organizers of β2-ARs nuclear PKA signaling in cardiac myocytes.

PubMed

Bedioune, Ibrahim; Lefebvre, Florence; Lechêne, Patrick; Varin, Audrey; Domergue, Valérie; Kapiloff, Michael S; Fischmeister, Rodolphe; Vandecasteele, Grégoire

2018-05-03

β1- and β2-adrenergic receptors (β-ARs) produce different acute contractile effects on the heart partly because they impact on different cytosolic pools of cAMP-dependent protein kinase (PKA). They also exert different effects on gene expression but the underlying mechanisms remain unknown. The aim of this study was to understand the mechanisms by which β1- and β2-ARs regulate nuclear PKA activity in cardiomyocytes. We used cytoplasmic and nuclear targeted biosensors to examine cAMP signals and PKA activity in adult rat ventricular myocytes upon selective β1- or β2-ARs stimulation. Both β1- and β2-AR stimulation increased cAMP and activated PKA in the cytoplasm. While the two receptors also increased cAMP in the nucleus, only β1-ARs increased nuclear PKA activity and up-regulated the PKA target gene and pro-apoptotic factor, inducible cAMP element repressor (ICER). Inhibition of PDE4, but not Gi, PDE3, GRK2 nor caveolae disruption disclosed nuclear PKA activation and ICER induction by β2-ARs. Both nuclear and cytoplasmic PKI prevented nuclear PKA activation and ICER induction by β1-ARs, indicating that PKA activation outside the nucleus is required for subsequent nuclear PKA activation and ICER mRNA expression. Cytoplasmic PKI also blocked ICER induction by β2-AR stimulation (with concomitant PDE4 inhibition). However, in this case nuclear PKI decreased ICER up-regulation by only 30%, indicating that other mechanisms are involved. Down-regulation of mAKAPβ partially inhibited nuclear PKA activation upon β1-AR stimulation, and drastically decreased nuclear PKA activation upon β2-AR stimulation in the presence of PDE4 inhibition. β1- and β2-ARs differentially regulate nuclear PKA activity and ICER expression in cardiomyocytes. PDE4 insulates a mAKAPβ-targeted PKA pool at the nuclear envelope that prevents nuclear PKA activation upon β2-AR stimulation.

Nuclear IL-33 is a transcriptional regulator of NF-{kappa}B p65 and induces endothelial cell activation

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

Choi, Yeon-Sook; Park, Jeong Ae; Kim, Jihye

2012-05-04

Highlights: Black-Right-Pointing-Pointer IL-33 as nuclear factor regulated expression of ICAM-1 and VCAM-1. Black-Right-Pointing-Pointer Nuclear IL-33 increased the transcription of NF-{kappa}B p65 by binding to the p65 promoter. Black-Right-Pointing-Pointer Nuclear IL-33 controls NF-{kappa}B-dependent inflammatory responses. -- Abstract: Interleukin (IL)-33, an IL-1 family member, acts as an extracellular cytokine by binding its cognate receptor, ST2. IL-33 is also a chromatin-binding transcriptional regulator highly expressed in the nuclei of endothelial cells. However, the function of IL-33 as a nuclear factor is poorly defined. Here, we show that IL-33 is a novel transcriptional regulator of the p65 subunit of the NF-{kappa}B complex and ismore » involved in endothelial cell activation. Quantitative reverse transcriptase PCR and Western blot analyses indicated that IL-33 mediates the expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in endothelial cells basally and in response to tumor necrosis factor-{alpha}-treatment. IL-33-induced ICAM-1/VCAM-1 expression was dependent on the regulatory effect of IL-33 on the nuclear factor (NF)-{kappa}B pathway; NF-{kappa}B p65 expression was enhanced by IL-33 overexpression and, conversely, reduced by IL-33 knockdown. Moreover, NF-{kappa}B p65 promoter activity and chromatin immunoprecipitation analysis revealed that IL-33 binds to the p65 promoter region in the nucleus. Our data provide the first evidence that IL-33 in the nucleus of endothelial cells participates in inflammatory reactions as a transcriptional regulator of NF-{kappa}B p65.« less

RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

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

López, Claudia S., E-mail: lopezcl@ohsu.edu; Sloan, Rachel; Cylinder, Isabel

The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag proteinmore » expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export.« less

SOBP Is Mutated in Syndromic and Nonsyndromic Intellectual Disability and Is Highly Expressed in the Brain Limbic System

PubMed Central

Birk, Efrat; Har-Zahav, Adi; Manzini, Chiara M.; Pasmanik-Chor, Metsada; Kornreich, Liora; Walsh, Christopher A.; Noben-Trauth, Konrad; Albin, Adi; Simon, Amos J.; Colleaux, Laurence; Morad, Yair; Rainshtein, Limor; Tischfield, David J.; Wang, Peter; Magal, Nurit; Maya, Idit; Shoshani, Noa; Rechavi, Gideon; Gothelf, Doron; Maydan, Gal; Shohat, Mordechai; Basel-Vanagaite, Lina

2010-01-01

Intellectual disability (ID) affects 1%–3% of the general population. We recently reported on a family with autosomal-recessive mental retardation with anterior maxillary protrusion and strabismus (MRAMS) syndrome. One of the reported patients with ID did not have dysmorphic features but did have temporal lobe epilepsy and psychosis. We report on the identification of a truncating mutation in the SOBP that is responsible for causing both syndromic and nonsyndromic ID in the same family. The protein encoded by the SOBP, sine oculis binding protein ortholog, is a nuclear zinc finger protein. In mice, Sobp (also known as Jxc1) is critical for patterning of the organ of Corti; one of our patients has a subclinical cochlear hearing loss but no gross cochlear abnormalities. In situ RNA expression studies in postnatal mouse brain showed strong expression in the limbic system at the time interval of active synaptogenesis. The limbic system regulates learning, memory, and affective behavior, but limbic circuitry expression of other genes mutated in ID is unusual. By comparing the protein content of the +/jc to jc/jc mice brains with the use of proteomics, we detected 24 proteins with greater than 1.5-fold differences in expression, including two interacting proteins, dynamin and pacsin1. This study shows mutated SOBP involvement in syndromic and nonsyndromic ID with psychosis in humans. PMID:21035105

Expression of peroxisome proliferator-activated receptor gamma (PPAR-gamma) in canine nasal carcinomas.

PubMed

Paciello, O; Borzacchiello, G; Varricchio, E; Papparella, S

2007-10-01

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a ligand-activated transcriptional factor belonging to the steroid receptor superfamily. PPAR-gamma is expressed in multiple normal and neoplastic tissues, such as the breast, colon, lung, ovary and placenta. In addition to adipogenic and anti-inflammatory effects, PPAR-gamma activation has been shown to be anti-proliferative by its differentiation-promoting effect, suggesting that activation of PPAR-gamma may be useful in slowing or arresting the proliferation of de-differentiated tumour cells. In this study, we investigated the expression of PPAR-gamma in normal and neoplastic canine nasal epithelium. Twenty-five samples composed of five normal nasal epithelia and 20 canine nasal carcinomas, were immunohistochemically stained for PPAR-gamma. The specificity of the antibody was verified by Western Blot analysis. Confocal laser scanning microscopical investigation was also performed. In normal epithelium, the staining pattern was cytoplasmic and polarized at the cellular free edge. In carcinomas, the neoplastic cells showed mainly strong cytoplasmatic PPAR-gamma expression; moreover, perinuclear immunoreactivity was also detected and few neoplastic cells exhibited a nuclear positivity. Our results demonstrate different patterns of PPAR-gamma expression in normal canine nasal epithelium when compared with canine nasal carcinoma. The importance of this transcription factor in the pathophysiology of several different tumours has stimulated much research in this field and has opened new opportunities for the treatment of the tumours.

A nuclear factor kappa B-derived inhibitor tripeptide inhibits UVB-induced photoaging process.

PubMed

Oh, Jee Eun; Kim, Min Seo; Jeon, Woo-Kwang; Seo, Young Kwon; Kim, Byung-Chul; Hahn, Jang Hee; Park, Chang Seo

2014-12-01

Ultraviolet (UV) irradiation on the skin induces photoaging which is characterized by keratinocyte hyperproliferation, generation of coarse wrinkles, worse of laxity and roughness. Upon UV irradiation, nuclear factor kappa B (NF-κB) is activated which plays a key role in signaling pathway leading to inflammation cascade and this activation stimulates expression of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1alpha (IL-1α) and a stress response gene cyclooxygenase-2 (COX-2). In addition, activation of NF-κB up-regulates the expression of matrix metalloprotease-1 (MMP-1) and consequently collagen in dermis is degraded. In this study, the effects of a NF-κB-derived inhibitor tripeptide on the UVB-induced photoaging and inflammation were investigated in vitro and in vivo. A NF-κB-derived inhibitor tripeptide (NF-κB-DVH) was synthesized based on the sequence of dimerization region of the subunit p65 of NF-κB. Its inhibitory activity was confirmed using chromatin immunoprecipitation assay and in situ proximate ligation assay. The effects of anti-photoaging and anti-inflammation were analyzed by Enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunochemistry. NF-κB-DVH significantly decreased UV-induced expression of TNF-α, IL-1α, MMP-1 and COX-2 while increased production of type I procollagen. Results showed NF-κB-DVH had strong anti-inflammatory activity probably by inhibiting NF-κB activation pathway and suggested to be used as a novel agent for anti-photoaging. Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

The alpha1-fetoprotein locus is activated by a nuclear receptor of the Drosophila FTZ-F1 family.

PubMed

Galarneau, L; Paré, J F; Allard, D; Hamel, D; Levesque, L; Tugwood, J D; Green, S; Bélanger, L

1996-07-01

The alpha1-fetoprotein (AFP) gene is located between the albumin and alpha-albumin genes and is activated by transcription factor FTF (fetoprotein transcription factor), presumed to transduce early developmental signals to the albumin gene cluster. We have identified FTF as an orphan nuclear receptor of the Drosophila FTZ-F1 family. FTF recognizes the DNA sequence 5'-TCAAGGTCA-3', the canonical recognition motif for FTZ-F1 receptors. cDNA sequence homologies indicate that rat FTF is the ortholog of mouse LRH-1 and Xenopus xFF1rA. Rodent FTF is encoded by a single-copy gene, related to the gene encoding steroidogenic factor 1 (SF-1). The 5.2-kb FTF transcript is translated from several in-frame initiator codons into FTF isoforms (54 to 64 kDa) which appear to bind DNA as monomers, with no need for a specific ligand, similar KdS (approximately equal 3 x 10(-10) M), and similar transcriptional effects. FTF activates the AFP promoter without the use of an amino-terminal activation domain; carboxy-terminus-truncated FTF exerts strong dominant negative effects. In the AFP promoter, FTF recruits an accessory trans-activator which imparts glucocorticoid reactivity upon the AFP gene. FTF binding sites are found in the promoters of other liver-expressed genes, some encoding liver transcription factors; FTF, liver alpha1-antitrypsin promoter factor LFB2, and HNF-3beta promoter factor UF2-H3beta are probably the same factor. FTF is also abundantly expressed in the pancreas and may exert differentiation functions in endodermal sublineages, similar to SF-1 in steroidogenic tissues. HepG2 hepatoma cells seem to express a mutated form of FTF.

Concurrent nuclear ERG and MYC protein overexpression defines a subset of locally advanced prostate cancer: Potential opportunities for synergistic targeted therapeutics.

PubMed

Udager, Aaron M; DeMarzo, Angelo M; Shi, Yang; Hicks, Jessica L; Cao, Xuhong; Siddiqui, Javed; Jiang, Hui; Chinnaiyan, Arul M; Mehra, Rohit

2016-06-01

Recurrent ERG gene fusions, the most common genetic alterations in prostate cancer, drive overexpression of the nuclear transcription factor ERG, and are early clonal events in prostate cancer progression. The nuclear transcription factor MYC is also frequently overexpressed in prostate cancer and may play a role in tumor initiation and/or progression. The relationship between nuclear ERG and MYC protein overexpression in prostate cancer, as well as the clinicopathologic characteristics and prognosis of ERG-positive/MYC high tumors, is not well understood. Immunohistochemistry (IHC) for ERG and MYC was performed on formalin-fixed, paraffin-embedded tissue from prostate cancer tissue microarrays (TMAs), and nuclear staining was scored semi-quantitatively (IHC product score range = 0-300). Correlation between nuclear ERG and MYC protein expression and association with clinicopathologic parameters and biochemical recurrence after radical prostatectomy was assessed. 29.1% of all tumor nodules showed concurrent nuclear ERG and MYC protein overexpression (i.e., ERG-positive/MYC high), including 35.0% of secondary nodules. Overall, there was weak positive correlation between ERG and MYC expression across all tumor nodules (rpb  = 0.149, P = 0.045), although this correlation was strongest in secondary nodules (rpb  = 0.520, P = 0.019). In radical prostatectomy specimens, ERG-positive/MYC high tumors were positively associated with the presence of extraprostatic extension (EPE), relative to all other ERG/MYC expression subgroups, however, there was no significant association between concurrent nuclear ERG and MYC protein overexpression and time to biochemical recurrence. Concurrent nuclear ERG and MYC protein overexpression is common in prostate cancer and defines a subset of locally advanced tumors. Recent data indicates that BET bromodomain proteins regulate ERG gene fusion and MYC gene expression in prostate cancer, suggesting possible synergistic targeted therapeutics in ERG-positive/MYC high tumors. Prostate 76:845-853, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The relationship of quantitative nuclear morphology to molecular genetic alterations in the adenoma-carcinoma sequence of the large bowel.

PubMed Central

Mulder, J. W.; Offerhaus, G. J.; de Feyter, E. P.; Floyd, J. J.; Kern, S. E.; Vogelstein, B.; Hamilton, S. R.

1992-01-01

The relationship of abnormal nuclear morphology to molecular genetic alterations that are important in colorectal tumorigenesis is unknown. Therefore, Feulgen-stained isolated nuclei from 22 adenomas and 42 carcinomas that had been analyzed for ras gene mutations and allelic deletions on chromosomes 5q, 18q, and 17p were characterized by computerized image analysis. Both nuclear area and the nuclear shape factor representing irregularity correlated with adenoma-carcinoma progression (r = 0.57 and r = 0.52, P < 0.0001), whereas standard nuclear texture, a parameter of chromatin homogeneity, was inversely correlated with progression (r = -0.80, P < 0.0001). The nuclear parameters were strongly interrelated (P < 0.0005). In multivariate analysis, the nuclear parameters were predominantly associated with adenoma-carcinoma progression (P < or = 0.0001) and were not influenced significantly by the individual molecular genetic alterations. Nuclear texture, however, was inversely correlated with fractional allelic loss, a global measure of genetic changes, in carcinomas (r = -0.39, P = 0.011). The findings indicate that nuclear morphology in colorectal neoplasms is strongly related to tumor progression. Nuclear morphology and biologic behavior appear to be influenced by accumulated alterations in cancer-associated genes. Images Figure 1 PMID:1357973

The Flavone Luteolin Suppresses SREBP-2 Expression and Post-Translational Activation in Hepatic Cells

PubMed Central

Wong, Tsz Yan; Lin, Shu-mei; Leung, Lai K.

2015-01-01

High blood cholesterol has been associated with cardiovascular diseases. The enzyme HMG CoA reductase (HMGCR) is responsible for cholesterol synthesis, and inhibitors of this enzyme (statins) have been used clinically to control blood cholesterol. Sterol regulatory element binding protein (SREBP) -2 is a key transcription factor in cholesterol metabolism, and HMGCR is a target gene of SREBP-2. Attenuating SREBP-2 activity could potentially minimize the expression of HMGCR. Luteolin is a flavone that is commonly detected in plant foods. In the present study, Luteolin suppressed the expression of SREBP-2 at concentrations as low as 1 μM in the hepatic cell lines WRL and HepG2. This flavone also prevented the nuclear translocation of SREBP-2. Post-translational processing of SREBP-2 protein was required for nuclear translocation. Luteolin partially blocked this activation route through increased AMP kinase (AMPK) activation. At the transcriptional level, the mRNA and protein expression of SREBP-2 were reduced through luteolin. A reporter gene assay also verified that the transcription of SREBF2 was weakened in response to this flavone. The reduced expression and protein processing of SREBP-2 resulted in decreased nuclear translocation. Thus, the transcription of HMGCR was also decreased after luteolin treatment. In summary, the results of the present study showed that luteolin modulates HMGCR transcription by decreasing the expression and nuclear translocation of SREBP-2. PMID:26302339

Kelch-like ECH-associated Protein 1-dependent Nuclear Factor-E2-related Factor 2 Activation in Relation to Antioxidation Induced by Sevoflurane Preconditioning.

PubMed

Cai, Min; Tong, Li; Dong, Beibei; Hou, Wugang; Shi, Likai; Dong, Hailong

2017-03-01

The authors have reported that antioxidative effects play a crucial role in the volatile anesthetic-induced neuroprotection. Accumulated evidence shows that endogenous antioxidation could be up-regulated by nuclear factor-E2-related factor 2 through multiple pathways. However, whether nuclear factor-E2-related factor 2 activation is modulated by sevoflurane preconditioning and, if so, what is the signaling cascade underlying upstream of this activation are still unknown. Sevoflurane preconditioning in mice was performed with sevoflurane (2.5%) 1 h per day for five consecutive days. Focal cerebral ischemia/reperfusion injury was induced by middle cerebral artery occlusion. Expression of nuclear factor-E2-related factor 2, kelch-like ECH-associated protein 1, manganese superoxide dismutase, thioredoxin-1, and nicotinamide adenine dinucleotide phosphate quinolone oxidoreductase-1 was detected (n = 6). The antioxidant activities and oxidative product expression were also examined. To determine the role of kelch-like ECH-associated protein 1 inhibition-dependent nuclear factor-E2-related factor 2 activation in sevoflurane preconditioning-induced neuroprotection, the kelch-like ECH-associated protein 1-nuclear factor-E2-related factor 2 signal was modulated by nuclear factor-E2-related factor 2 knockout, kelch-like ECH-associated protein 1 overexpression lentivirus, and kelch-like ECH-associated protein 1 deficiency small interfering RNA (n = 8). The infarct volume, neurologic scores, and cellular apoptosis were assessed. Sevoflurane preconditioning elicited neuroprotection and increased nuclear factor-E2-related factor 2 nuclear translocation, which in turn up-regulated endogenous antioxidation and reduced oxidative injury. Sevoflurane preconditioning reduced kelch-like ECH-associated protein 1 expression. Nuclear factor-E2-related factor 2 ablation abolished neuroprotection and reversed sevoflurane preconditioning by mediating the up-regulation of antioxidants. Kelch-like ECH-associated protein 1 overexpression reversed nuclear factor-E2-related factor 2 up-regulation and abolished the neuroprotection induced by sevoflurane preconditioning. Kelch-like ECH-associated protein 1 small interfering RNA administration improved nuclear factor-E2-related factor 2 expression and the outcome of mice subjected to ischemia/reperfusion injury. Kelch-like ECH-associated protein 1 down-regulation-dependent nuclear factor-E2-related factor 2 activation underlies the ability of sevoflurane preconditioning to activate the endogenous antioxidant response, which elicits its neuroprotection.

Diagnostic utility of hepatocyte nuclear factor 1-beta immunoreactivity in endometrial carcinomas: lack of specificity for endometrial clear cell carcinoma.

PubMed

Fadare, Oluwole; Liang, Sharon X

2012-12-01

Hepatocyte nuclear factor 1-beta (HNF1β) has recently emerged as a relatively sensitive and specific marker for ovarian clear cell carcinoma. The purpose of this study is to assess the diagnostic utility of this marker for endometrial clear cell carcinoma. Immunohistochemical analysis was performed on 75 endometrial tissues using a goat polyclonal antibody raised against a peptide mapping at the C-terminus of human HNF1β protein. The 75 cases included 15 clear cell carcinomas, 20 endometrioid carcinomas, 15 endometrial serous carcinomas/uterine papillary serous carcinomas, 20 cases of normal endometrium, 2 cases of clear cell metaplasia, and 3 cases of Arias Stella reaction. Staining interpretations were based on a semiquantitative scoring system, a 0 to 12+ continuous numerical scale that was derived by multiplying the extent of staining (0 to 4+ scale) by the intensity of staining (0 to 3+ scale) for each case. HNF1β expression was found to be present in a wide spectrum of tissues. Twenty-seven (54%) of the 50 carcinomas displayed at least focal nuclear HNF1β expression, including 11 (73%) of 15, 9 (60%) of 15, and 7 (35%) of 20 clear cell, serous, and endometrioid carcinomas, respectively. The average nuclear staining scores for clear cell carcinomas, endometrioid carcinomas, and serous carcinomas were 5.2, 1.4, and 4.1, respectively. Clear cell carcinomas and endometrioid carcinomas displayed statistically significant differences regarding their nuclear staining scores (P = 0.0027), but clear cell carcinomas and endometrial serous carcinomas did not (P = 0.45). The calculated sensitivity of any nuclear HNF1β expression in classifying a carcinoma as being of the clear cell histotype was 73%, whereas the specificity was 54%. Nineteen of 20 normal endometrium samples displayed at least focal nuclear expression of HNF1β, and this expression was often diffuse. The 5 cases of benign histologic mimics of clear cell carcinomas (Arias Stella reaction and clear cell metaplasia) displayed some degree of HNF1β immunoreactivity, with an average nuclear staining score of 7.3. We conclude that although HNF1β is frequently expressed in clear cell carcinomas, it should be used with caution as a diagnostic marker because of its lack of specificity. It neither distinguishes endometrial serous carcinomas from clear cell carcinomas nor clear cell carcinomas from its benign mimics. The greatest diagnostic utility of HNF1β expression may be in a supportive evidentiary role favoring clear cell carcinoma when the principal differential diagnostic consideration is endometrioid carcinoma.

Expression and Functional Pathway Analysis of Nuclear Receptor NR2F2 in Ovarian Cancer

PubMed Central

Hawkins, Shannon M.; Loomans, Holli A.; Wan, Ying-Wooi; Ghosh-Choudhury, Triparna; Coffey, Donna; Xiao, Weimin; Liu, Zhandong; Sangi-Haghpeykar, Haleh

2013-01-01

Context: Recent evidence implicates the orphan nuclear receptor, nuclear receptor subfamily 2, group F, member 2 (NR2F2; chicken ovalbumin upstream promoter-transcription factor II) as both a master regulator of angiogenesis and an oncogene in prostate and other human cancers. Objective: The objective of the study was to determine whether NR2F2 plays a role in ovarian cancer and dissect its potential mechanisms of action. Design, Setting, and Patients: We examined NR2F2 expression in healthy ovary and ovarian cancers using quantitative PCR and immunohistochemistry. NR2F2 expression was targeted in established ovarian cancer cell lines to assess the impact of dysregulated NR2F2 expression in the epithelial compartment of ovarian cancers. Results: Our results indicate that NR2F2 is robustly expressed in the stroma of healthy ovary with little or no expression in epithelia lining the ovarian surface, clefts, or crypts. This pattern of NR2F2 expression was markedly disrupted in ovarian cancers, in which decreased levels of stromal expression and ectopic epithelial expression were frequently observed. Ovarian cancers with the most disrupted patterns of NR2F2 were associated with significantly shorter disease-free interval by Kaplan-Meier analysis. Targeting NR2F2 expression in established ovarian cancer cell lines enhanced apoptosis and increased proliferation. In addition, we found that NR2F2 regulates the expression of NEK2, RAI14, and multiple other genes involved in the cell cycle, suggesting potential pathways by which dysregulated expression of NR2F2 impacts ovarian cancer. Conclusions: These results uncover novel roles for NR2F2 in ovarian cancer and point to a unique scenario in which a single nuclear receptor plays potentially distinct roles in the stromal and epithelial compartments of the same tissue. PMID:23690307

Induction of polyploidy by nuclear fusion mechanism upon decreased expression of the nuclear envelope protein LAP2β in the human osteosarcoma cell line U2OS.

PubMed

Ben-Shoshan, Shirley Oren; Simon, Amos J; Jacob-Hirsch, Jasmine; Shaklai, Sigal; Paz-Yaacov, Nurit; Amariglio, Ninette; Rechavi, Gideon; Trakhtenbrot, Luba

2014-01-28

Polyploidy has been recognized for many years as an important hallmark of cancer cells. Polyploid cells can arise through cell fusion, endoreplication and abortive cell cycle. The inner nuclear membrane protein LAP2β plays key roles in nuclear envelope breakdown and reassembly during mitosis, initiation of replication and transcriptional repression. Here we studied the function of LAP2β in the maintenance of cell ploidy state, a role which has not yet been assigned to this protein. By knocking down the expression of LAP2β, using both viral and non-viral RNAi approaches in osteosarcoma derived U2OS cells, we detected enlarged nuclear size, nearly doubling of DNA content and chromosomal duplications, as analyzed by fluorescent in situ hybridization and spectral karyotyping methodologies. Spectral karyotyping analyses revealed that near-hexaploid karyotypes of LAP2β knocked down cells consisted of not only seven duplicated chromosomal markers, as could be anticipated by genome duplication mechanism, but also of four single chromosomal markers. Furthermore, spectral karyotyping analysis revealed that both of two near-triploid U2OS sub-clones contained the seven markers that were duplicated in LAP2β knocked down cells, whereas the four single chromosomal markers were detected only in one of them. Gene expression profiling of LAP2β knocked down cells revealed that up to a third of the genes exhibiting significant changes in their expression are involved in cancer progression. Our results suggest that nuclear fusion mechanism underlies the polyploidization induction upon LAP2β reduced expression. Our study implies on a novel role of LAP2β in the maintenance of cell ploidy status. LAP2β depleted U2OS cells can serve as a model to investigate polyploidy and aneuploidy formation by nuclear fusion mechanism and its involvement in cancerogenesis.

Characterization of docosahexaenoic acid (DHA)-induced heme oxygenase-1 (HO-1) expression in human cancer cells: the importance of enhanced BTB and CNC homology 1 (Bach1) degradation.

PubMed

Wang, Shuai; Hannafon, Bethany N; Wolf, Roman F; Zhou, Jundong; Avery, Jori E; Wu, Jinchang; Lind, Stuart E; Ding, Wei-Qun

2014-05-01

The effect of docosahexaenoic acid (DHA) on heme oxygenase-1 (HO-1) expression in cancer cells has never been characterized. This study examines DHA-induced HO-1 expression in human cancer cell model systems. DHA enhanced HO-1 gene expression in a time- and concentration-dependent manner, with maximal induction at 21 h of treatment. This induction of HO-1 expression was confirmed in vivo using a xenograft nude mouse model fed a fish-oil-enriched diet. The increase in HO-1 gene transcription induced by DHA was significantly attenuated by the antioxidant N-acetyl cysteine, suggesting the involvement of oxidative stress. This was supported by direct measurement of lipid peroxide levels after DHA treatment. Using a human HO-1 gene promoter reporter construct, we identified two antioxidant response elements (AREs) that mediate the DHA-induced increase in HO-1 gene transcription. Knockdown of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression compromised the DHA-induced increase in HO-1 gene transcription, indicating the importance of the Nrf2 pathway in this event. However, the nuclear protein levels of Nrf2 remained unchanged upon DHA treatment. Further studies demonstrated that DHA reduces nuclear Bach1 protein expression by promoting its degradation and attenuates Bach1 binding to the AREs in the HO-1 gene promoter. In contrast, DHA enhanced Nrf2 binding to the AREs without affecting nuclear Nrf2 expression levels, indicating a new cellular mechanism that mediates DHA's induction of HO-1 gene transcription. To our knowledge, this is the first characterization of DHA-induced HO-1 expression in human malignant cells. Copyright © 2014 Elsevier Inc. All rights reserved.

p16 Protein and Gigaxonin Are Associated with the Ubiquitination of NFκB in Cisplatin-induced Senescence of Cancer Cells*

PubMed Central

Veena, Mysore S.; Wilken, Reason; Zheng, Jun-Ying; Gholkar, Ankur; Venkatesan, Natarajan; Vira, Darshni; Ahmed, Sameer; Basak, Saroj K.; Dalgard, Clifton L.; Ravichandran, Sandhiya; Batra, Raj K.; Kasahara, Noriyuki; Elashoff, David; Fishbein, Michael C.; Whitelegge, Julian P.; Torres, Jorge Z.; Wang, Marilene B.; Srivatsan, Eri S.

2014-01-01

The molecular mechanism of p16-mediated senescence in cisplatin-treated cancer cells is not fully understood. Here we show that cisplatin treatment of head and neck cancer cells results in nuclear transport of p16 leading to a molecular modification of NFκB. Chromatin immunoprecipitation assays show that this modification is associated with the inhibition of NFκB interacting with its DNA binding sequences, leading to decreased expression of NFκB-transcribed proteins. LCMS proteomic analysis of LAP-TAP-purified proteins from HeLa cells containing a tetracycline-inducible GFP-S peptide-NFκB expression system identified gigaxonin, an ubiquitin E3 ligase adaptor, as an NFκB-interacting protein. Immunoblotting and siRNA studies confirmed the NFκB-gigaxonin interaction and the dependence of this binding on p16-NFκB binding. Using gel shift assays, we have confirmed p16-NFκB and gigaxonin-NFκB interactions. Furthermore, we have observed increased NFκB ubiquitination with cisplatin treatment that is abolished in the absence of p16 and gigaxonin expression. Analysis of 103 primary tumors has shown that increased nuclear p16 expression correlates with enhanced survival of head and neck cancer patients (p < 0.0000542), indicating the importance of nuclear p16 expression in prognosis. Finally, p16 expression is associated with reduced cytokine expression and the presence of human papilloma virus in chemoradiation-sensitive basaloid tumors. However, the absence of p16 expression is associated with enhanced cytokine expression and the absence of human papilloma virus in aggressive tumors. These results clearly demonstrate that nuclear p16 and gigaxonin play an important role in chemosensitivity of head and neck cancers through ubiquitination of NFκB. PMID:25331947

CD147 promotes the formation of functional osteoclasts through NFATc1 signalling.

PubMed

Nishioku, Tsuyoshi; Terasawa, Mariko; Baba, Misaki; Yamauchi, Atsushi; Kataoka, Yasufumi

2016-04-29

CD147, a membrane glycoprotein of the immunoglobulin superfamily, is highly upregulated during dynamic cellular events including tissue remodelling. Elevated CD147 expression is present in the joint of rheumatoid arthritis patients. However, the role of CD147 in bone destruction remains unclear. To determine whether CD147 is involved in osteoclastogenesis, we studied its expression in mouse osteoclasts and its role in osteoclast differentiation and function. CD147 expression was markedly upregulated during osteoclast differentiation. To investigate the role of CD147 in receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption activity, osteoclast precursor cells were transfected with CD147 siRNA. Decreased CD147 expression inhibited osteoclast formation and bone resorption, inhibited RANKL-induced nuclear translocation of the nuclear factor of activated T cells (NFAT) c1 and decreased the expression of the d2 isoform of vacuolar ATPase Vo domain and cathepsin K. Therefore, CD147 plays a critical role in the differentiation and function of osteoclasts by upregulating NFATc1 through the autoamplification of its expression in osteoclastogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Analysis of PI3K/mTOR Pathway Biomarkers and Their Prognostic Value in Women with Hormone Receptor–Positive, HER2-Negative Early Breast Cancer1

PubMed Central

Azim, Hamdy A.; Kassem, Loay; Treilleux, Isabelle; Wang, Qing; El Enein, Mona Abu; Anis, Shady E.; Bachelot, Thomas

2016-01-01

BACKGROUND: The PI3K/AKT/mTOR pathway alterations have been shown to play significant roles in the development, progression, and metastatic spread of breast cancer. Furthermore, they have been implicated in the process of drug resistance, especially endocrinal therapies. In this study, we aimed to define the correlation between the PI3K mutations and the expression of the phosphorylated forms of different downstream molecules in women with estrogen receptor (ER)–positive, human epidermal growth factor receptor 2–negative (luminal) early breast cancer treated at Cairo university hospitals. METHODS: Next-generation sequencing was used to detect mutations in the PIK3CA hotspots (in exons 9 and 20). Immunohistochemistry was performed on tissue microarray blocks prepared from samples of 35 Egyptian luminal breast cancer patients in the pathology department of Centre Léon Bérard (CLB). The intensity and the percentage of stained tumor cells were integrated to define high versus low biomarker expression. The cytoplasmic and nuclear stainings were graded separately. Patients were followed for a median of 4.7 years (2.1 to 6.9 years). Correlation was done between PI3K mutations and the immunohistochemistry expression of pAKT, LKB1, p4EBP1, and pS6 ribosomal protein (pS6RP) with the clinicopathologic features and disease free survival (DFS) of the patients. RESULTS: Median age at diagnosis was 51.3 years (range, 25 to 82 years). Tumors were larger than 20 mm in 79.2% of the cases, whereas 57.9% had axillary lymph node deposits. Only 12.3% of the patients had SBR grade I tumors, 50.8% had grade II, and 36.8% had grade III. ERs were negative in 6 patients (17%) after pathology review. Thirty-two cases were assessable for LKB1 and pAKT, 33 for p4EBP1 and pS6RP, and 24 for PI3K mutations. Nuclear LKB1, cytoplasmic LKB1, nuclear pAKT, cytoplasmic pAKT, nuclear p4EBP1, and cytoplasmic pS6RP expression was high in 65.6%, 62.5%, 62.5%, 68.8%, 42.4%, and 57.6%, respectively. PIK3CA mutations were found in 7 patients (29.2%). PI3K mutations were correlated with nuclear localization of pAKT (i.e., decreased cytoplasmic pAKT, P = .04; and increased nuclear pAKT, P = .10). There was a tendency toward an inverse correlation between PI3K mutations and the expression of pS6RP (P = .10) and p4EBP1 (P = .19). Nuclear LKB1 expression was a marker of good prognosis. It was associated with smaller tumors (P = .05), more ER (P = .08) and progesteron receptor (PgR) positivity (P = .002). In the Kaplan Meier (KM) model, patients with high nuclear LKB1 had longer DFS (hazard ratio = 0.36; 95% confidence interval, 0.15-1.10; P = .08). Nuclear pAKT high expression also carried a tendency toward longer DFS (hazard ratio = 0.51; 95% confidence interval, 0.11-1.16; P = .13). The expression of p4EBP1, pS6RP, and the PI3K mutational status did not show any prognostic significance in our cohort. CONCLUSION: Among the studied biomarkers, only nuclear expression of LKB1 and pAKT tended to predict better survival in breast cancer patients. PI3K mutation was correlated with the expression of nuclear pAKT but not pS6RP or p4EBP1. PMID:27084427

Transmutation of Isotopes --- Ecological and Energy Production Aspects

NASA Astrophysics Data System (ADS)

Gudowski, Waclaw

2000-01-01

This paper describes principles of Accelerator-Driven Transmutation of Nuclear Wastes (ATW) and gives some flavour of the most important topics which are today under investigations in many countries. An assessment of the potential impact of ATW on a future of nuclear energy is also given. Nuclear reactors based on self-sustained fission reactions --- after spectacular development in fifties and sixties, that resulted in deployment of over 400 power reactors --- are wrestling today more with public acceptance than with irresolvable technological problems. In a whole spectrum of reasons which resulted in today's opposition against nuclear power few of them are very relevant for the nuclear physics community and they arose from the fact that development of nuclear power had been handed over to the nuclear engineers and technicians with some generically unresolved problems, which should have been solved properly by nuclear scientists. In a certain degree of simplification one can say, that most of the problems originate from very specific features of a fission phenomenon: self-sustained chain reaction in fissile materials and very strong radioactivity of fission products and very long half-life of some of the fission and activation products. And just this enormous concentration of radioactive fission products in the reactor core is the main problem of managing nuclear reactors: it requires unconditional guarantee for the reactor core integrity in order to avoid radioactive contamination of the environment; it creates problems to handle decay heat in the reactor core and finally it makes handling and/or disposal of spent fuel almost a philosophical issue, due to unimaginable long time scales of radioactive decay of some isotopes. A lot can be done to improve the design of conventional nuclear reactors (like Light Water Reactors); new, better reactors can be designed but it seems today very improbable to expect any radical change in the public perception of conventional nuclear power. In this context a lot of hopes and expectations have been expressed for novel systems called Accelerator-Driven Systems, Accelerator-Driven Transmutation of Waste or just Hybrid Reactors. All these names are used for description of the same nuclear system combining a powerful particle accelerator with a subcritical reactor. A careful analysis of possible environmental impact of ATW together with limitation of this technology is presented also in this paper.

PPKs mediate direct signal transfer from phytochrome photoreceptors to transcription factor PIF3

DOE PAGES

Ni, Weimin; Xu, Shou-Ling; González-Grandío, Eduardo; ...

2017-05-11

Upon light-induced nuclear translocation, phytochrome (phy) sensory photoreceptors interact with, and induce rapid phosphorylation and consequent ubiquitin-mediated degradation of, transcription factors, called PIFs, thereby regulating target gene expression and plant development. Nevertheless, the biochemical mechanism of phy-induced PIF phosphorylation has remained ill-defined. Here in this paper we identify a family of nuclear protein kinases, designated Photoregulatory Protein Kinases (PPK1–4; formerly called MUT9-Like Kinases (MLKs)), that interact with PIF3 and phyB in a light-induced manner in vivo. Genetic analyses demonstrate that the PPKs are collectively necessary for the normal light-induced phosphorylation and degradation of PIF3. PPK1 directly phosphorylates PIF3 in vitro,more » with a phosphosite pattern that strongly mimics the light-induced pattern in vivo. These data establish that the PPKs are directly involved in catalysing the photoactivated-phy-induced phosphorylation of PIF3 in vivo, and thereby are critical components of a transcriptionally centred signalling hub that pleiotropically regulates plant growth and development in response to multiple signalling pathways.« less

Increased arylhydrocarbon receptor expression offers a potential therapeutic target for pancreatic cancer.

PubMed

Koliopanos, Alexander; Kleeff, Jörg; Xiao, Yi; Safe, Stephen; Zimmermann, Arthur; Büchler, Markus W; Friess, Helmut

2002-09-05

The arylhydrocarbon receptor (AhR) was initially identified as a member of the adaptive metabolic and toxic response pathway to polycyclic aromatic hydrocarbons and to halogenated dibenzo-p-dioxins and dibenzofurans. In the present study, we sought to determine the functional significance of the AhR pathway in pancreatic carcinogenesis. AhR expression was analysed by Northern blotting. The exact site of AhR expression was analysed by in situ hybridization and immunohistochemistry. The effects of TCDD and four selective AhR agonists on pancreatic cancer cell lines were investigated by growth assays, apoptosis assays, and induction of the cyclin-dependent kinase inhibitor p21. There was strong AhR mRNA expression in 14 out of 15 pancreatic cancer samples, weak expression in chronic pancreatitis tissues, and faint expression in all normal pancreata. In pancreatic cancer tissues, AhR mRNA and protein expression were localized in the cytoplasm of pancreatic cancer cells. TCDD and the four AhR agonists inhibited pancreatic cancer cell growth in a dose-dependent manner, and decreased anchorage-independent cell growth. DAPI staining did not reveal nuclear fragmentation and CYP1A1 and was not induced by TCDD and AhR agonists. In contrast, TCDD and AhR agonists induced the expression of the cyclin-dependent kinase inhibitor p21. In conclusion, the relatively non-toxic AhR agonists caused growth inhibition in pancreatic cancer cells with high AhR expression levels via cell cycle arrest. In addition, almost all human pancreatic cancer tissues expressed this receptor at high levels, suggesting that these or related compounds may play a role in the therapy of pancreatic cancer in the future.

Telomere-surrounding regions are transcription-permissive 3D nuclear compartments in human cells

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

Quina, Ana Sofia; Instituto Gulbenkian de Ciencia, 2781-901 Oeiras; Parreira, Leonor

2005-07-01

Positioning of genes relative to nuclear heterochromatic compartments is thought to help regulate their transcriptional activity. Given that human subtelomeric regions are rich in highly expressed genes, we asked whether human telomeres are related to transcription-permissive nuclear compartments. To address this question, we investigated in the nuclei of normal human lymphocytes the spatial relations of two constitutively expressed genes (ACTB and RARA) and three nuclear transcripts (ACTB, IL2RA and TCRB) to telomeres and centromeres, as a function of gene activity and transcription levels. We observed that genes and gene transcripts locate close to telomere clusters and away from chromocenters uponmore » activation of transcription. These findings, together with the observation that SC35 domains, which are enriched in pre-mRNA processing factors, are in close proximity to telomeres, indicate that telomere-neighboring regions are permissive to gene expression in human cells. Therefore, the associations of telomeres observed in the interphase nucleus might contribute, as opposed to chromocenters, for the establishment of transcription-permissive 3D nuclear compartments.« less

Interactions between the nuclear matrix and an enhancer of the tryptophan oxygenase gene

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

Kaneoka, Hidenori; Miyake, Katsuhide, E-mail: miyake@nubio.nagoya-u.ac.jp; Iijima, Shinji

2009-10-02

The gene for tryptophan oxygenase (TO) is expressed in adult hepatocytes in a tissue- and differentiation-specific manner. The TO promoter has two glucocorticoid-responsive elements (GREs), and its expression is regulated by glucocorticoid hormone in the liver. We found a novel GRE in close proximity to a scaffold/matrix attachment region (S/MAR) that was located around -8.5 kb from the transcriptional start site of the TO gene by electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) assays. A combination of nuclear fractionation and quantitative PCR analysis showed that the S/MAR was tethered to the nuclear matrix in both fetal and adult hepatocytes. ChIPmore » assay showed that, in adult hepatocytes, the S/MAR-GRE and the promoter proximal regions interacted with lamin and heterogeneous nuclear ribonucleoprotein U in a dexamethasone dependent manner, but this was not the case in fetal cells, suggesting that developmental stage-specific expression of the TO gene might rely on the binding of the enhancer (the -8.5 kb S/MAR-GRE) and the promoter to the inner nuclear matrix.« less

Nuclear Glycolytic Enzyme Enolase of Toxoplasma gondii Functions as a Transcriptional Regulator

PubMed Central

Mouveaux, Thomas; Oria, Gabrielle; Werkmeister, Elisabeth; Slomianny, Christian; Fox, Barbara A.; Bzik, David J.; Tomavo, Stanislas

2014-01-01

Apicomplexan parasites including Toxoplasma gondii have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that T. gondii enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin in vivo. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5′ untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in brain cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in T. gondii. PMID:25153525

Altered expression and activation of signal transducers and activators of transcription (STATs) in hepatitis C virus infection: in vivo and in vitro studies.

PubMed

Larrea, E; Aldabe, R; Molano, E; Fernandez-Rodriguez, C M; Ametzazurra, A; Civeira, M P; Prieto, J

2006-08-01

Signal transducers and activators of transcription (STATs) play a critical role in antiviral defence. STAT3 is also important in cell protection against inflammatory damage. STAT proteins are activated by interferons and by hepatoprotective cytokines of the interleukin 6 superfamily, including cardiotrophin 1. We analysed the status of STATs in hepatitis C virus (HCV) infected livers and the relationship between expression and activation of STATs and HCV replication in Huh7 cells transfected with HCV genomic replicon. STAT3alpha expression was reduced in HCV infected livers showing an inverse correlation with serum alanine aminotransferase. In patients with HCV infection, nuclear staining for phosphorylated STAT3 was faint in parenchymal cells (although conspicuous in infiltrating leucocytes), in contrast with strong nuclear staining in hepatocytes from control livers. Expression and activation of STAT1 (a factor activated by both interferon (IFN)-alpha and IFN-gamma) were increased in HCV infected livers, particularly in those with high inflammatory activity. Conversely, phosphorylated STAT2 (a factor selectively activated by IFN-alpha) was undetectable in livers with HCV infection, a finding that was associated with marked downregulation of the two functional subunits of the IFN-alpha receptor. HCV replication in Huh7 cells caused STAT3alpha downregulation and blocked STAT3 phosphorylation by either IFN-alpha or cardiotrophin 1. HCV replication in Huh7 cells also inhibited STAT1 and STAT2 activation by IFN-alpha while there was no impairment of STAT1 phosphorylation by the proinflammatory cytokine IFN-gamma. STAT3 is downregulated in HCV infected livers and in Huh7 cells bearing the full length HCV replicon. HCV replication is associated with impaired Jak-STAT signalling by antiviral and cytoprotective cytokines. These effects may favour viral replication while facilitating the progression of liver disease.

Dual Role of Cyanidin-3-glucoside on the Differentiation of Bone Cells.

PubMed

Park, K H; Gu, D R; So, H S; Kim, K J; Lee, S H

2015-12-01

Cyanidin-3-glucoside (C3G) is one of the major components of anthocyanin, a water-soluble phytochemical. Recent studies demonstrated the chemopreventive and chemotherapeutic activities of C3G in various conditions, including cancer, although the precise effects of C3G on osteoclast and osteoblast differentiation remain unclear. Here, we investigated the role of C3G in the differentiation of bone-associated cells and its underlying mechanism. C3G inhibited receptor activator of nuclear factor kappa-B ligand (RANKL)-mediated osteoclast differentiation and formation in a dose-dependent manner and downregulated the expression of osteoclast differentiation marker genes. Pretreatment with C3G considerably reduced the induction of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated kinases activation by RANKL in osteoclast precursor cells. Furthermore, C3G dramatically inhibited the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1, which are important transcription factors for osteoclast differentiation and activation. The formation of osteoclasts in coculture of bone marrow cells and calvaria-derived osteoblasts was also inhibited by C3G treatment, although the expression of macrophage colony-stimulating factor and RANKL (master factors for osteoclast differentiation and formation) and osteoprotegerin (a decoy receptor for RANKL) on osteoblasts was unaffected. The inhibitory effect of C3G on osteoclastogenesis is therefore targeted specifically to osteoclasts but not osteoblasts. Moreover, analysis of the expression levels of osteoblast differentiation marker genes and alizarin red staining showed that osteoblast differentiation and matrix formation increased after C3G treatment. Taken together, these results strongly suggest that C3G has a dual role in bone metabolism, as an effective inhibitor of osteoclast differentiation but also as an activator of osteoblast differentiation. Therefore, C3G may be used as a potent preventive or therapeutic agent for bone-related diseases, such as osteoporosis, rheumatoid arthritis, and periodontitis. © International & American Associations for Dental Research 2015.

p53 expression and mutation analysis of odontogenic cysts with and without dysplasia.

PubMed

Cox, Darren P

2012-01-01

Overexpression of p53 protein is well described in odontogenic cystic lesions (OCLs), including those with epithelial dysplasia; however, most p53 antibodies stain both wild-type and mutated p53 protein and may not reflect genotype. Direct sequencing of the p53 gene has not identified mutations in OCLs with dysplasia. The purpose of this study was to determine the molecular basis of p53 expression in several types of OCLs with and without dysplasia. The study material comprised 13 OCLs: odontogenic keratocyst (n = 5), orthokeratinized odontogenic cyst (n = 5), dentigerous cyst (n = 2), lateral periodontal cyst (n = 1), and unspecified developmental odontogenic cyst (UDOC) (n = 1). Five of these had features of mild or moderate epithelial dysplasia. One intraosseous squamous cell carcinoma (SCC) that was believed to have arisen from an antecedent dysplastic orthokeratinized OC was also included. Immunohistochemistry was performed using the DO7 monoclonal antibody that recognizes wild-type and mutated p53. DNA was extracted from microdissected tissue for all samples and exons 4 to 8 of the p53 gene direct sequenced. In 4 of 5 OCLs with dysplasia there was strong nuclear staining of basal and suprabasal cells. In all cases without dysplasia, nuclear expression in basal cells was either negative or weak and was absent in suprabasal cell nuclei. A mutation in exon 6 of the p53 gene (E224D) was identified in both the dysplastic orthokeratinized OC and the subsequent intraosseous SCC. OCLs with features of dysplasia show increased expression of p53 protein that does not reflect p53 mutational status. One dysplastic OC shared the same p53 mutation with a subsequent intraosseous SCC, indicating that p53 mutation may be associated with malignant transformation in this case. Copyright © 2012 Elsevier Inc. All rights reserved.

Cell Cycle-Dependent Expression of Dub3, Nanog and the p160 Family of Nuclear Receptor Coactivators (NCoAs) in Mouse Embryonic Stem Cells

PubMed Central

van der Laan, Siem; Golfetto, Eleonora; Vanacker, Jean-Marc; Maiorano, Domenico

2014-01-01

Pluripotency of embryonic stem cells (ESC) is tightly regulated by a network of transcription factors among which the estrogen-related receptor β (Esrrb). Esrrb contributes to the relaxation of the G1 to S-phase (G1/S) checkpoint in mouse ESCs by transcriptional control of the deubiquitylase Dub3 gene, contributing to Cdc25A persistence after DNA damage. We show that in mESCs, Dub3 gene expression is cell cycle regulated and is maximal prior G1/S transition. In addition, following UV-induced DNA damage in G1, Dub3 expression markedly increases in S-phase also suggesting a role in checkpoint recovery. Unexpectedly, we also observed cell cycle-regulation of Nanog expression, and not Oct4, reaching high levels prior to G1/S transition, finely mirroring Cyclin E1 fluctuations. Curiously, while Esrrb showed only limited cell-cycle oscillations, transcript levels of the p160 family of nuclear receptor coactivators (NCoAs) displayed strong cell cycle-dependent fluctuations. Since NCoAs function in concert with Esrrb in transcriptional activation, we focussed on NCoA1 whose levels specifically increase prior onset of Dub3 transcription. Using a reporter assay, we show that NCoA1 potentiates Esrrb-mediated transcription of Dub3 and we present evidence of protein interaction between the SRC1 splice variant NCoA1 and Esrrb. Finally, we show a differential developmental regulation of all members of the p160 family during neural conversion of mESCs. These findings suggest that in mouse ESCs, changes in the relative concentration of a coactivator at a given cell cycle phase, may contribute to modulation of the transcriptional activity of the core transcription factors of the pluripotent network and be implicated in cell fate decisions upon onset of differentiation. PMID:24695638

Cell cycle-dependent expression of Dub3, Nanog and the p160 family of nuclear receptor coactivators (NCoAs) in mouse embryonic stem cells.

PubMed

van der Laan, Siem; Golfetto, Eleonora; Vanacker, Jean-Marc; Maiorano, Domenico

2014-01-01

Pluripotency of embryonic stem cells (ESC) is tightly regulated by a network of transcription factors among which the estrogen-related receptor β (Esrrb). Esrrb contributes to the relaxation of the G1 to S-phase (G1/S) checkpoint in mouse ESCs by transcriptional control of the deubiquitylase Dub3 gene, contributing to Cdc25A persistence after DNA damage. We show that in mESCs, Dub3 gene expression is cell cycle regulated and is maximal prior G1/S transition. In addition, following UV-induced DNA damage in G1, Dub3 expression markedly increases in S-phase also suggesting a role in checkpoint recovery. Unexpectedly, we also observed cell cycle-regulation of Nanog expression, and not Oct4, reaching high levels prior to G1/S transition, finely mirroring Cyclin E1 fluctuations. Curiously, while Esrrb showed only limited cell-cycle oscillations, transcript levels of the p160 family of nuclear receptor coactivators (NCoAs) displayed strong cell cycle-dependent fluctuations. Since NCoAs function in concert with Esrrb in transcriptional activation, we focussed on NCoA1 whose levels specifically increase prior onset of Dub3 transcription. Using a reporter assay, we show that NCoA1 potentiates Esrrb-mediated transcription of Dub3 and we present evidence of protein interaction between the SRC1 splice variant NCoA1 and Esrrb. Finally, we show a differential developmental regulation of all members of the p160 family during neural conversion of mESCs. These findings suggest that in mouse ESCs, changes in the relative concentration of a coactivator at a given cell cycle phase, may contribute to modulation of the transcriptional activity of the core transcription factors of the pluripotent network and be implicated in cell fate decisions upon onset of differentiation.

Early embryonic expression of FGF4/6/9 gene and its role in the induction of mesenchyme and notochord in Ciona savignyi embryos.

PubMed

Imai, Kaoru S; Satoh, Nori; Satou, Yutaka

2002-04-01

In early Ciona savignyi embryos, nuclear localization of beta-catenin is the first step of endodermal cell specification, and triggers the activation of various target genes. A cDNA for Cs-FGF4/6/9, a gene activated downstream of beta-catenin signaling, was isolated and shown to encode an FGF protein with features of both FGF4/6 and FGF9/20. The early embryonic expression of Cs-FGF4/6/9 was transient and the transcript was seen in endodermal cells at the 16- and 32-cell stages, in notochord and muscle cells at the 64-cell stage, and in nerve cord and muscle cells at the 110-cell stage; the gene was then expressed again in cells of the nervous system after neurulation. When the gene function was suppressed with a specific antisense morpholino oligo, the differentiation of mesenchyme cells was completely blocked, and the fate of presumptive mesenchyme cells appeared to change into that of muscle cells. The inhibition of mesenchyme differentiation was abrogated by coinjection of the morpholino oligo and synthetic Cs-FGF4/6/9 mRNA. Downregulation of beta-catenin nuclear localization resulted in the absence of mesenchyme cell differentiation due to failure of the formation of signal-producing endodermal cells. Injection of synthetic Cs-FGF4/6/9 mRNA in beta-catenin-downregulated embryos evoked mesenchyme cell differentiation. These results strongly suggest that Cs-FGF4/6/9 produced by endodermal cells acts an inductive signal for the differentiation of mesenchyme cells. On the other hand, the role of Cs-FGF4/6/9 in the induction of notochord cells is partial; the initial process of the induction was inhibited by Cs-FGF4/6/9 morpholino oligo, but notochord-specific genes were expressed later to form a partial notochord.

Whole blood genome-wide expression profiling and network analysis suggest MELAS master regulators.

PubMed

Mende, Susanne; Royer, Loic; Herr, Alexander; Schmiedel, Janet; Deschauer, Marcus; Klopstock, Thomas; Kostic, Vladimir S; Schroeder, Michael; Reichmann, Heinz; Storch, Alexander

2011-07-01

The heteroplasmic mitochondrial DNA (mtDNA) mutation A3243G causes the mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome as one of the most frequent mitochondrial diseases. The process of reconfiguration of nuclear gene expression profile to accommodate cellular processes to the functional status of mitochondria might be a key to MELAS disease manifestation and could contribute to its diverse phenotypic presentation. To determine master regulatory protein networks and disease-modifying genes in MELAS syndrome. Analyses of whole blood transcriptomes from 10 MELAS patients using a novel strategy by combining classic Affymetrix oligonucleotide microarray profiling with regulatory and protein interaction network analyses. Hierarchical cluster analysis elucidated that the relative abundance of mutant mtDNA molecules is decisive for the nuclear gene expression response. Further analyses confirmed not only transcription factors already known to be involved in mitochondrial diseases (such as TFAM), but also detected the hypoxia-inducible factor 1 complex, nuclear factor Y and cAMP responsive element-binding protein-related transcription factors as novel master regulators for reconfiguration of nuclear gene expression in response to the MELAS mutation. Correlation analyses of gene alterations and clinico-genetic data detected significant correlations between A3243G-induced nuclear gene expression changes and mutant mtDNA load as well as disease characteristics. These potential disease-modifying genes influencing the expression of the MELAS phenotype are mainly related to clusters primarily unrelated to cellular energy metabolism, but important for nucleic acid and protein metabolism, and signal transduction. Our data thus provide a framework to search for new pathogenetic concepts and potential therapeutic approaches to treat the MELAS syndrome.

Overexpression of YAP1 in EGFR mutant lung adenocarcinoma prior to tyrosine kinase inhibitor therapy is associated with poor survival.

PubMed

Hong, Soon Auck; Jang, Si-Hyong; Oh, Mee-Hye; Kim, Sung Joon; Kang, Jin-Hyung; Hong, Sook-Hee

2018-03-01

EGFR tyrosine kinase inhibitor (EGFR TKI) is approved as first-line treatment for advanced-stage EGFR mutant lung adenocarcinoma (LADC). Yes-associated protein 1 (YAP1), a main effector of the Hippo pathway, is associated with adverse prognosis and disruption of EGFR TKI modulation of non-small cell lung cancer. In this study, we demonstrated a prognostic role of YAP1 in EGFR mutant LADC and efficacy of EGFR TKI therapy. A total of 63 patients, including 41 with paired lung cancer specimens before and after EGFR TKI therapy and 22 with non-paired lung cancer specimens prior to EGFR TKI, were enrolled for examination. Expression of YAP1 protein was evaluated using immunohistochemistry. Fifteen paired cases (36.6%) with high nuclear YAP1 expression were detected in the pre-EGFR TKI LADC group and 21 paired cases (52.5%) after treatment with EGFR TKI. Nuclear YAP1 expression was significantly upregulated after EGFR TKI therapy (P = .002). Fifteen paired cases with high nuclear YAP1 expression before EGFR TKI LADCs showed poorer overall survival (OS) (P = .023) and progression-free survival (PFS) (P = .041). Among the 63 patients under study, those with high nuclear YAP1 expression before EGFR TKI showed shorter OS (P = .038) and PFS (P < .001). High nuclear YAP1 expression in cases with acquired EGFR exon 20 T790 M mutant LADCs showed poorer OS (P < .001). We demonstrated that YAP1 burden before clinical application of EGFR TKI plays a crucial role in prognosis of EGFR mutant LADC treated using EGFR TKI. Copyright © 2018 Elsevier GmbH. All rights reserved.

Java Web Start based software for automated quantitative nuclear analysis of prostate cancer and benign prostate hyperplasia.

PubMed

Singh, Swaroop S; Kim, Desok; Mohler, James L

2005-05-11

Androgen acts via androgen receptor (AR) and accurate measurement of the levels of AR protein expression is critical for prostate research. The expression of AR in paired specimens of benign prostate and prostate cancer from 20 African and 20 Caucasian Americans was compared to demonstrate an application of this system. A set of 200 immunopositive and 200 immunonegative nuclei were collected from the images using a macro developed in Image Pro Plus. Linear Discriminant and Logistic Regression analyses were performed on the data to generate classification coefficients. Classification coefficients render the automated image analysis software independent of the type of immunostaining or image acquisition system used. The image analysis software performs local segmentation and uses nuclear shape and size to detect prostatic epithelial nuclei. AR expression is described by (a) percentage of immunopositive nuclei; (b) percentage of immunopositive nuclear area; and (c) intensity of AR expression among immunopositive nuclei or areas. The percent positive nuclei and percent nuclear area were similar by race in both benign prostate hyperplasia and prostate cancer. In prostate cancer epithelial nuclei, African Americans exhibited 38% higher levels of AR immunostaining than Caucasian Americans (two sided Student's t-tests; P < 0.05). Intensity of AR immunostaining was similar between races in benign prostate. The differences measured in the intensity of AR expression in prostate cancer were consistent with previous studies. Classification coefficients are required due to non-standardized immunostaining and image collection methods across medical institutions and research laboratories and helps customize the software for the specimen under study. The availability of a free, automated system creates new opportunities for testing, evaluation and use of this image analysis system by many research groups who study nuclear protein expression.

Non-tuberculous Mycobacteriosis with T-cell Lymphoma in a Red Panda (Ailurus fulgens).

PubMed

Fuke, N; Hirai, T; Makimura, N; Goto, Y; Habibi, W A; Ito, S; Trang, N T; Koshino, K; Takeda, M; Yamaguchi, R

2016-01-01

A 9-year-old male red panda (Ailurus fulgens) became emaciated and died. Necropsy examination revealed systemic lymphadenomegaly. The liver, lungs and left kidney contained multifocal yellow nodules. Microscopical examination revealed granulomatous inflammation in the liver, lungs, kidney, spleen and lymph nodes, with numerous acid-fast bacilli. Sequencing of genetic material isolated from the tissues classified the pathogen as Mycobacterium gastri. Lymphoma was found in the liver, lungs, kidney and lymph nodes. The neoplastic cells were strongly labelled for expression of CD3, Ki67 and proliferating cell nuclear antigen by immunohistochemistry. This is the first report of M. gastri infection with T-cell lymphoma in a red panda. Copyright © 2016 Elsevier Ltd. All rights reserved.

CHD5, a brain-specific paralog of Mi2 chromatin remodeling enzymes, regulates expression of neuronal genes.

PubMed

Potts, Rebecca Casaday; Zhang, Peisu; Wurster, Andrea L; Precht, Patricia; Mughal, Mohamed R; Wood, William H; Zhang, Yonqing; Becker, Kevin G; Mattson, Mark P; Pazin, Michael J

2011-01-01

CHD5 is frequently deleted in neuroblastoma and is a tumor suppressor gene. However, little is known about the role of CHD5 other than it is homologous to chromatin remodeling ATPases. We found CHD5 mRNA was restricted to the brain; by contrast, most remodeling ATPases were broadly expressed. CHD5 protein isolated from mouse brain was associated with HDAC2, p66ß, MTA3 and RbAp46 in a megadalton complex. CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons. CHD5 protein was predominantly nuclear in primary rat neurons and brain sections. Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons. CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme. Expression of gene sets linked to aging and Alzheimer's disease were strongly altered by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct. Together, these results indicate that CHD5 protein is found in a NuRD-like multi-protein complex. CHD5 expression is restricted to the brain, unlike the closely related family members CHD3 and CHD4. CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes. CHD5 is linked to regulation of genes implicated in aging and Alzheimer's disease.

CHD5, a Brain-Specific Paralog of Mi2 Chromatin Remodeling Enzymes, Regulates Expression of Neuronal Genes

PubMed Central

Potts, Rebecca Casaday; Zhang, Peisu; Wurster, Andrea L.; Precht, Patricia; Mughal, Mohamed R.; Wood, William H.; Zhang, Yonqing; Becker, Kevin G.; Mattson, Mark P.; Pazin, Michael J.

2011-01-01

CHD5 is frequently deleted in neuroblastoma and is a tumor suppressor gene. However, little is known about the role of CHD5 other than it is homologous to chromatin remodeling ATPases. We found CHD5 mRNA was restricted to the brain; by contrast, most remodeling ATPases were broadly expressed. CHD5 protein isolated from mouse brain was associated with HDAC2, p66ß, MTA3 and RbAp46 in a megadalton complex. CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons. CHD5 protein was predominantly nuclear in primary rat neurons and brain sections. Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons. CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme. Expression of gene sets linked to aging and Alzheimer's disease were strongly altered by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct. Together, these results indicate that CHD5 protein is found in a NuRD-like multi-protein complex. CHD5 expression is restricted to the brain, unlike the closely related family members CHD3 and CHD4. CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes. CHD5 is linked to regulation of genes implicated in aging and Alzheimer's disease. PMID:21931736

In-Vitro Analysis of Glucose and Quercetin Effects on m-TOR and Nrf-2 Expression in HepG2 Cell Line (Diabetes and Cancer Connection).

PubMed

Yarahmadi, Amir; Khademi, Fatemeh; Mostafavi-Pour, Zohreh; Zal, Fatemeh

2018-05-21

Some types of cancers show a strong relationship with diabetes and play a central role in mortality in the patient population suffering from diabetes mellitus. In this study, HepG2 cells have been used to investigate the toxic effects of hyperglycemia and/or quercetin (Q) on mammalian target of rapamycin (m-TOR) and nuclear factor erythroid 2-related factor 2 (Nrf-2) expression as central molecules involved in cancer. HepG2 cells were cultured with different concentrations of glucose (5.5, 30, and 50 mM) and/or Q (25 µM) for 48 and 72 h. Effects of glucose and/or Q on m-TOR and Nrf-2 expression were assayed by quantitative real-time PCR (qRT-PCR). qRT-PCR results revealed that 30 and 50 mM of glucose increased m-TOR expression at 48 h, although after 72 h, only 30 mM had an increasing effect. At 50 mM, glucose-induced Nrf-2 gene expression after both 48 and 72 h. The results also showed that 25 µM of Q reduced m-TOR and Nrf-2 expression at both 30 and 50 mM after 48 and 72 h incubation. Q has potential effects on reducing oxidative stress caused by hyperglycemia and during diabetes may be able to modulate some carcinogenic signaling pathways.

Protective effect of Disporum sessile D.Don extract against UVB-induced photoaging via suppressing MMP-1 expression and collagen degradation in human skin cells.

PubMed

Mohamed, Mohamed Antar Aziz; Jung, Mira; Lee, Sang Min; Lee, Tae Hoon; Kim, Jiyoung

2014-04-05

In the present study, we report that Disporum sessile D.Don herbal extract (DDE) possesses anti-skin photoaging effect through inhibition of MMP-1 mRNA and protein expression levels and increase collagen production in UVB-irradiated human dermal fibroblast cells (NHDF). To delineate the molecular mechanism by which DDE inhibited MMP-1 expression, immortal human keratinocytes cells (HaCaT) have been used. We have found that DDE inhibited UVB-induced MMP-1 mRNA and protein expression levels in HaCaT cells through inhibition of UVB-induced activation of NF-κB in HaCaT cells. Inhibitors of NF-κB (Bay11-7082), and mitogen-activated protein kinases such as extracellular regulated kinase (PD98059), c-Jun N-terminal kinase (SP600125), and p38 (SB203580) suppressed expression of MMP-1, and phosphorylation of these signaling molecules were attenuated by DDE. DDE also inhibited phosphorylation of IKKα and IκBα, and reduced nuclear translocation of NF-κB. Our results also demonstrated that DDE inhibited NF-κB driven expression of luciferase reporter gene and the DNA binding of NF-κB to its cognate binding site in UV-irradiated cells. Therefore, these results strongly suggest that DDE can be utilized as a potential agent for prevention and treatment of skin photoaging. Copyright © 2014 Elsevier B.V. All rights reserved.

TS expression predicts postoperative recurrence in adenocarcinoma of the lung.

PubMed

Shimokawa, Hidehiko; Uramoto, Hidetaka; Onitsuka, Takamitsu; Iwata, Teruo; Nakagawa, Makoto; Ono, Kenji; Hanagiri, Takeshi

2011-06-01

Not all patients with lung cancer require postoperative adjuvant chemotherapy after a complete resection. However, no useful markers for either selecting appropriate candidates or for predicting clinical recurrence exist. Tumor specimens were collected from 183 consecutive patients who underwent a complete resection for lung adenocarcinoma from 2003 to 2007 in our department. We analyzed the thymidylate synthase (TS) and dihydrofolate reductase (DHFR) expressions in the primary lung adenocarcinoma by immunohistochemisty. The strong expression of TS and DHFR was identified in 39 (21.3%) and 120 (65.6%) patients, respectively. The strong TS expression was identified in 11 (39.3%) of 28 patients and 28 (18.1%) of 155 patients in patients with and without recurrence, respectively (p=0.012). The strong DHFR expression was also identified in 23 (82.1%) and 97 (62.6%) of the patients with and without recurrence, respectively (p=0.045). Logistic regression models indicated the strong TS expression to be an independent factor for tumor recurrence. The strong TS and DHFR expression was associated with a poorer disease-free survival (DFS) according to the survival analysis. A multivariate analysis demonstrated the strong TS expression to be independently associated with an increased risk for poor DFS. The strong TS expression may be a useful marker for predicting postoperative recurrence in patients with lung adenocarcinoma following surgery. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Nuclear factor I-A represses expression of the cell adhesion molecule L1

PubMed Central

2009-01-01

Background The neural cell adhesion molecule L1 plays a crucial role in development and plasticity of the nervous system. Neural cells thus require precise control of L1 expression. Results We identified a full binding site for nuclear factor I (NFI) transcription factors in the regulatory region of the mouse L1 gene. Electrophoretic mobility shift assay (EMSA) showed binding of nuclear factor I-A (NFI-A) to this site. Moreover, for a brain-specific isoform of NFI-A (NFI-A bs), we confirmed the interaction in vivo using chromatin immunoprecipitation (ChIP). Reporter gene assays showed that in neuroblastoma cells, overexpression of NFI-A bs repressed L1 expression threefold. Conclusion Our findings suggest that NFI-A, in particular its brain-specific isoform, represses L1 gene expression, and might act as a second silencer of L1 in addition to the neural restrictive silencer factor (NRSF). PMID:20003413

[Expression of negative emotional responses to the 2011 Great East Japan Earthquake: Analysis of big data from social media].

PubMed

Miura, Asako; Komori, Masashi; Matsumura, Naohiro; Maeda, Kazutoshi

2015-06-01

In this article, we investigated the expression of emotional responses to the 2011 Great East Japan Earthquake by analyzing the frequency of negative emotional terms in tweets posted on Twitter, one of the most popular social media platforms. We focused on differences in time-series variations and diurnal changes between two kinds of disasters: natural disasters (earthquakes and tsunamis) and nuclear accidents. The number of tweets containing negative emotional responses increased sharply shortly after the first huge earthquake and decreased over time, whereas tweets about nuclear accidents showed no correlation with elapsed time. Expressions of anxiety about natural disasters had a circadian rhythm, with a peak at midnight, whereas expressions of anger about the nuclear accident were highly sensitive to critical events related to the accident. These findings were discussed in terms of similarities and differences compared to earlier studies on emotional responses in social media.

Arctigenin Inhibits Osteoclast Differentiation and Function by Suppressing Both Calcineurin-Dependent and Osteoblastic Cell-Dependent NFATc1 Pathways

PubMed Central

Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki; Li, Feng; Kadota, Shigetoshi; Esumi, Hiroyasu; Kobayashi, Yasuhiro; Takahashi, Naoyuki

2014-01-01

Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM) cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA), a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the pit-forming activity of osteoclast-like cells cultured on dentin slices. These results suggest that arctigenin induces a dominant negative species of NFATc1, which inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways. PMID:24465763

Arctigenin inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

PubMed

Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki; Li, Feng; Kadota, Shigetoshi; Esumi, Hiroyasu; Kobayashi, Yasuhiro; Takahashi, Naoyuki

2014-01-01

Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM) cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA), a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the pit-forming activity of osteoclast-like cells cultured on dentin slices. These results suggest that arctigenin induces a dominant negative species of NFATc1, which inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

Nuclear BK Channels Regulate Gene Expression via the Control of Nuclear Calcium Signaling

PubMed Central

Li, Boxing; Jie, Wei; Huang, Lianyan; Wei, Peng; Li, Shuji; Luo, Zhengyi; Friedman, Allyson K.; Meredith, Andrea L.; Han, Ming-Hu; Zhu, Xin-Hong; Gao, Tian-Ming

2014-01-01

Ion channels are essential for the regulation of neuronal functions. The significance of plasma membrane, mitochondrial, endoplasmic reticulum, and lysosomal ion channels in the regulation of Ca2+ is well established. In contrast, surprisingly less is known about the function of ion channels on the nuclear envelope (NE). Here we demonstrate the presence of functional large-conductance, calcium-activated potassium channels (BK channels) on the NE of rodent hippocampal neurons. Functionally blockade of nuclear BK channels (nBK channels) induces NE-derived Ca2+ release, nucleoplasmic Ca2+ elevation, and cAMP response element binding protein (CREB)-dependent transcription. More importantly, blockade of nBK channels regulates nuclear Ca2+-sensitive gene expression and promotes dendritic arborization in a nuclear Ca2+-dependent manner. These results suggest that nBK channel functions as a molecular linker between neuronal activity and nuclear Ca2+ to convey the signals from synapse to nucleus and is a new modulator for synaptic activity-dependent neuronal functions at the NE level. PMID:24952642

Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction.

PubMed

Poleshko, Andrey; Shah, Parisha P; Gupta, Mudit; Babu, Apoorva; Morley, Michael P; Manderfield, Lauren J; Ifkovits, Jamie L; Calderon, Damelys; Aghajanian, Haig; Sierra-Pagán, Javier E; Sun, Zheng; Wang, Qiaohong; Li, Li; Dubois, Nicole C; Morrisey, Edward E; Lazar, Mitchell A; Smith, Cheryl L; Epstein, Jonathan A; Jain, Rajan

2017-10-19

Progenitor cells differentiate into specialized cell types through coordinated expression of lineage-specific genes and modification of complex chromatin configurations. We demonstrate that a histone deacetylase (Hdac3) organizes heterochromatin at the nuclear lamina during cardiac progenitor lineage restriction. Specification of cardiomyocytes is associated with reorganization of peripheral heterochromatin, and independent of deacetylase activity, Hdac3 tethers peripheral heterochromatin containing lineage-relevant genes to the nuclear lamina. Deletion of Hdac3 in cardiac progenitor cells releases genomic regions from the nuclear periphery, leading to precocious cardiac gene expression and differentiation into cardiomyocytes; in contrast, restricting Hdac3 to the nuclear periphery rescues myogenesis in progenitors otherwise lacking Hdac3. Our results suggest that availability of genomic regions for activation by lineage-specific factors is regulated in part through dynamic chromatin-nuclear lamina interactions and that competence of a progenitor cell to respond to differentiation signals may depend upon coordinated movement of responding gene loci away from the nuclear periphery. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification, cloning, and expression analysis of three putative Lymantria dispar nuclear polyhedrosis virus immediate early genes

Treesearch

James M. Slavicek; Nancy Hayes-Plazolles

1991-01-01

Viral immediate early gene products are usually regulatory proteins that control expression of other viral genes at the transcriptional level or are proteins that are part of the viral DNA replication complex. The identification and functional characterization of the immediate early gene products of Lymantria dispar nuclear polyhedrosis virus (LdNPV...

Temporal analysis and spatial mapping of Lymantria dispar nuclear polyhedrosis virus transcripts and in vitro translation polypeptides

Treesearch

James M. Slavicek

1991-01-01

Genomic expression of the Lymantriu dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) was studied. Viral specific transcripts expressed in cell culture at various times from 2 through 72 h postinfection were identified and their genomic origins mapped through Northern analysis. Sixty-five distinct transcripts were identified in this...

Cloning and functional analysis of 5'-upstream region of the Pokemon gene.

PubMed

Yang, Yutao; Zhou, Xiaowei; Zhu, Xudong; Zhang, Chuanfu; Yang, Zhixin; Xu, Long; Huang, Peitang

2008-04-01

Pokemon, the POK erythroid myeloid ontogenic factor, not only regulates the expression of many genes, but also plays an important role in cell tumorigenesis. To investigate the molecular mechanism regulating expression of the Pokemon gene in humans, its 5'-upstream region was cloned and analyzed. Transient analysis revealed that the Pokemon promoter is constitutive. Deletion analysis and a DNA decoy assay indicated that the NEG-U and NEG-D elements were involved in negative regulation of the Pokemon promoter, whereas the POS-D element was mainly responsible for its strong activity. Electrophoretic mobility shift assays suggested that the NEG-U, NEG-D and POS-D elements were specifically bound by the nuclear extract from A549 cells in vitro. Mutation analysis demonstrated that cooperation of the NEG-U and NEG-D elements led to negative regulation of the Pokemon promoter. Moreover, the NEG-U and NEG-D elements needed to be an appropriate distance apart in the Pokemon promoter in order to cooperate. Taken together, our results elucidate the mechanism underlying the regulation of Pokemon gene transcription, and also define a novel regulatory sequence that may be used to decrease expression of the Pokemon gene in cancer gene therapy.

The RB-related gene Rb2/p130 in neuroblastoma differentiation and in B-myb promoter down-regulation.

PubMed

Raschellà, G; Tanno, B; Bonetto, F; Negroni, A; Claudio, P P; Baldi, A; Amendola, R; Calabretta, B; Giordano, A; Paggi, M G

1998-05-01

The retinoblastoma family of nuclear factors is composed of RB, the prototype of the tumour suppressor genes and of the strictly related genes p107 and Rb2/p130. The three genes code for proteins, namely pRb, p107 and pRb2/p130, that share similar structures and functions. These proteins are expressed, often simultaneously, in many cell types and are involved in the regulation of proliferation and differentiation. We determined the expression and the phosphorylation of the RB family gene products during the DMSO-induced differentiation of the N1E-115 murine neuroblastoma cells. In this system, pRb2/p130 was strongly up-regulated during mid-late differentiation stages, while, on the contrary, pRb and p107 resulted markedly decreased at late stages. Differentiating N1E-115 cells also showed a progressive decrease in B-myb levels, a proliferation-related protein whose constitutive expression inhibits neuronal differentiation. Transfection of each of the RB family genes in these cells was able, at different degrees, to induce neuronal differentiation, to inhibit [3H]thymidine incorporation and to down-regulate the activity of the B-myb promoter.

Casein kinase II induces c-fos expression via the serum response element pathway and p67SRF phosphorylation in living fibroblasts.

PubMed Central

Gauthier-Rouvière, C; Basset, M; Blanchard, J M; Cavadore, J C; Fernandez, A; Lamb, N J

1991-01-01

Elevation of intracellular casein kinase II (CKII) levels through microinjection of purified CKII results in the rapid and transient induction of c-fos in quiescent rat embryo fibroblasts, and activation of quiescent cells by serum is accompanied by the nuclear relocation of endogenous CKII. The induction of c-fos by CKII is inhibited by coinjection of oligonucleotides corresponding to the sequence of the serum response element (SRE) present in the c-fos promoter, indicating that competitive displacement of positive factors from the endogenous c-fos SRE prevents c-fos induction by CKII. Furthermore, the expression of c-fos induced by either CKII injection or serum activation is also inhibited by microinjection of antibodies against the 67 kDa serum response factor (p67SRF) indicating the absolute requirement of p67SRF in this process. Finally, we show the specific phosphorylation of p67SRF in vivo following microinjection of CKII into quiescent cells. Together, these data strongly support that CKII induces c-fos expression through binding/activation of the phosphorylated p67SRF at the SRE sequence. Images PMID:1915270

Addiction, Adolescence, and Innate Immune Gene Induction

PubMed Central

Crews, Fulton T.; Vetreno, Ryan Peter

2011-01-01

Repeated drug use/abuse amplifies psychopathology, progressively reducing frontal lobe behavioral control, and cognitive flexibility while simultaneously increasing limbic temporal lobe negative emotionality. The period of adolescence is a neurodevelopmental stage characterized by poor behavioral control as well as strong limbic reward and thrill seeking. Repeated drug abuse and/or stress during this stage increase the risk of addiction and elevate activator innate immune signaling in the brain. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a key glial transcription factor that regulates proinflammatory chemokines, cytokines, oxidases, proteases, and other innate immune genes. Induction of innate brain immune gene expression (e.g., NF-κB) facilitates negative affect, depression-like behaviors, and inhibits hippocampal neurogenesis. In addition, innate immune gene induction alters cortical neurotransmission consistent with loss of behavioral control. Studies with anti-oxidant, anti-inflammatory, and anti-depressant drugs as well as opiate antagonists link persistent innate immune gene expression to key behavioral components of addiction, e.g., negative affect-anxiety and loss of frontal–cortical behavioral control. This review suggests that persistent and progressive changes in innate immune gene expression contribute to the development of addiction. Innate immune genes may represent a novel new target for addiction therapy. PMID:21629837

Light and temperature shape nuclear architecture and gene expression.

PubMed

Kaiserli, Eirini; Perrella, Giorgio; Davidson, Mhairi Lh

2018-06-14

Environmental stimuli play a major role in modulating growth and development throughout the life-cycle of a plant. Quantitative and qualitative variations in light and temperature trigger changes in gene expression that ultimately shape plant morphology for adaptation and survival. Although the phenotypic and transcriptomic basis of plant responses to the constantly changing environment have been examined for decades, the relationship between global changes in nuclear architecture and adaption to environmental stimuli is just being uncovered. This review presents recent discoveries investigating how changes in light and temperature trigger changes in chromatin structure and nuclear organization with a focus on the role of gene repositioning and chromatin accessibility in regulating gene expression. Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.

PGE2 mediates EGFR internalization and nuclear translocation via caveolin endocytosis promoting its transcriptional activity and proliferation in human NSCLC cells

PubMed Central

Bazzani, Lorenzo; Donnini, Sandra; Giachetti, Antonio; Christofori, Gerhard; Ziche, Marina

2018-01-01

Prostaglandin E2 (PGE2) contributes to tumor progression by promoting cancer cell growth, invasion and by creating a favorable pro-tumor microenvironment. PGE2 has been reported to transactivate and internalize into the nucleus receptor tyrosine kinases such as Epidermal growth factor receptor (EGFR), thereby supporting tumor progression. Here we demonstrate that in non-small cell lung carcinoma (NSCLC) cells, PGE2 induces EGFR nuclear translocation via different dynamin-dependent endocytic pathways, promotes the formation of an EGFR-STAT3 complex, affects nuclear EGFR target gene expression and mediates tumor cell proliferation. Indeed, we find that PGE2 induces EGFR internalization and consequent nuclear import through Clathrin- and Caveolin-mediated endocytosis and through the interaction of EGFR with Importin β1. Within the nucleus, EGFR forms a complex with STAT3, an event blocked by ablation of Clathrin Heavy Chain or Caveolin-1. The combination of EGF and PGE2 prolongs nuclear EGFR transcriptional activity manifested by the upregulation of CCND1, PTGS2, MYC and NOS2 mRNA levels and potentiates nuclear EGFR-induced NSCLC cell proliferation. Additionally, NSCLC patients with high expression of a nuclear EGFR gene signature display shorter survival times than those with low expression, thus showing a putative correlation between nuclear EGFR and poor prognosis in NSCLC. Together, our findings indicate a complex mechanism underlying PGE2-induced EGF/EGFR signaling and transcriptional control, which plays a key role in cancer progression. PMID:29599917

PGE2 mediates EGFR internalization and nuclear translocation via caveolin endocytosis promoting its transcriptional activity and proliferation in human NSCLC cells.

PubMed

Bazzani, Lorenzo; Donnini, Sandra; Giachetti, Antonio; Christofori, Gerhard; Ziche, Marina

2018-03-13

Prostaglandin E 2 (PGE 2 ) contributes to tumor progression by promoting cancer cell growth, invasion and by creating a favorable pro-tumor microenvironment. PGE 2 has been reported to transactivate and internalize into the nucleus receptor tyrosine kinases such as Epidermal growth factor receptor (EGFR), thereby supporting tumor progression. Here we demonstrate that in non-small cell lung carcinoma (NSCLC) cells, PGE 2 induces EGFR nuclear translocation via different dynamin-dependent endocytic pathways, promotes the formation of an EGFR-STAT3 complex, affects nuclear EGFR target gene expression and mediates tumor cell proliferation. Indeed, we find that PGE 2 induces EGFR internalization and consequent nuclear import through Clathrin- and Caveolin-mediated endocytosis and through the interaction of EGFR with Importin β1. Within the nucleus, EGFR forms a complex with STAT3, an event blocked by ablation of Clathrin Heavy Chain or Caveolin-1. The combination of EGF and PGE 2 prolongs nuclear EGFR transcriptional activity manifested by the upregulation of CCND1 , PTGS2 , MYC and NOS2 mRNA levels and potentiates nuclear EGFR-induced NSCLC cell proliferation. Additionally, NSCLC patients with high expression of a nuclear EGFR gene signature display shorter survival times than those with low expression, thus showing a putative correlation between nuclear EGFR and poor prognosis in NSCLC. Together, our findings indicate a complex mechanism underlying PGE 2 -induced EGF/EGFR signaling and transcriptional control, which plays a key role in cancer progression.

Expression of the extracellular matrix protein periostin in liver tumours and bile duct carcinomas.

PubMed

Riener, Marc-Oliver; Fritzsche, Florian R; Soll, Christopher; Pestalozzi, Bernhard C; Probst-Hensch, Nicole; Clavien, Pierre-Alain; Jochum, Wolfram; Soltermann, Alex; Moch, Holger; Kristiansen, Glen

2010-04-01

To study the relevance of periostin, known to be involved in epithelial-mesenchymal transition (EMT), in hepatocellular and bile duct cancer. Immunohistochemical periostin expression was semiquantitatively analysed in normal liver tissue (n = 20), hepatocellular carcinoma (HCC; n = 91), liver-cell adenoma (n = 9), focal nodular hyperplasia (n = 13) and bile duct carcinomas (BDC; n = 116) using tissue microarrays. Normal bile ducts, gallbladder epithelium and hepatocytes showed weak cytoplasmic periostin expression. In HCC, there was strong epithelial periostin expression in 19/91 (20.9%) and strong stromal periostin expression in 10/91 cases (11%). Epithelial expression in tumour cells was significantly associated with a higher tumour grade (P < 0.05) and hepatitis B virus infection (P = 0.007). Importantly, there was no strong periostin expression in benign liver tumours. Strong stromal periostin expression was detected in 78/116 (67.2%) BDC and strong epithelial expression in 39/116 (33.6%) BDC. pT stage, differentiation grade and proliferation rate in primary BDC were independent of periostin expression. Epithelial periostin expression was associated with reduced overall survival on univariate and multivariate analysis. The EMT protein periostin is expressed in the stroma and epithelium of a subset of BDC and HCC. Epithelial periostin expression is a marker for malignant transformation of hepatocytes and a novel prognostic marker in BDC.

Nuclear quiescence and histone hyper-acetylation jointly improve protamine-mediated nuclear remodeling in sheep fibroblasts

PubMed Central

Palazzese, Luca; Czernik, Marta; Iuso, Domenico; Toschi, Paola

2018-01-01

Recently we have demonstrated the possibility to replace histones with protamine, through the heterologous expression of human protamine 1 (hPrm1) gene in sheep fibroblasts. Here we have optimized protaminization of somatic nucleus by adjusting the best concentration and exposure time to trichostatin A (TSA) in serum-starved fibroblasts (nuclear quiescence), before expressing Prm1 gene. To stop cell proliferation, we starved cells in 0.5% FBS in MEM (“starved”—ST group), whereas in the Control group (CTR) the cells were cultured in 10% FBS in MEM. To find the most effective TSA concentration, we treated the cells with increasing concentrations of TSA in MEM + 10% FBS. Our results show that combination of cell culture conditions in 50 nM TSA, is more effective in terminating cell proliferation than ST and CTR groups (respectively 8%, 17.8% and 90.2% p<0.0001). Moreover, nuclear quiescence marker genes expression (Dicer1, Smarca 2, Ezh1 and Ddx39) confirmed that our culture conditions kept the cells in a nuclear quiescent state. Finally, ST and 50 nM TSA jointly increased the number of spermatid-like cell (39.4%) at higher rate compared to 25 nM TSA (20.4%, p<0.05) and 100 nM TSA (13.7%, p<0.05). To conclude, we have demonstrated that nuclear quiescence in ST cells and the open nuclear structure conferred by TSA resulted in an improved Prm1-mediated conversion of somatic nuclei into spermatid-like structures. This finding might improve nuclear reprogramming of somatic cells following nuclear transfer. PMID:29543876

The effects of dexamethasone on rat brain cortical nuclear factor kappa B (NF-{kappa}B) in endotoxic shock

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

Wang Zhi; Kang Jinsong; Li Yang

2006-08-01

To explore the molecular mechanism of brain tissue injury induced by lipopolysaccharide (LPS), we studied the effects of endotoxic shock on rat brain cortex NF-{kappa}B and the effects of dexamethasone on these changes. Rats were randomly divided into LPS, LPS + dexamethasone, and control groups. The DNA-binding activity of NF-{kappa}B was observed using electrophoretic mobility shift assay (EMSA). Protein expression in nuclear extracts was studied using Western blots, and nuclear translocation was observed using immunohistochemistry. These indices were assayed at 1 h and 4 h after intravenous injection of LPS (4 mg.kg{sup -1}). EMSA showed significantly increased NF-{kappa}B DNA-binding activitymore » in nuclear extracts from the LPS group at both 1 h and 4 h after LPS injection, compared with the control group (P < 0.01). For the LPS group, the NF-{kappa}B DNA-binding activity was greater at 1 h than at 4 h (P < 0.05). The expression of p65 and p50 protein in the nuclear extracts was also increased, as compared with the control group. However, the expression of p65 and p50 protein from cytosolic extracts did not show any significant change. Dexamethasone down-regulated not only NF-{kappa}B DNA-binding activity but also the expression of p65 protein in the nuclear extracts. From these data, we have concluded that NF-{kappa}B activation and nuclear translocation of NF-{kappa}B play a key role in the molecular mechanism of brain tissue injury in endotoxic shock. Dexamethasone may alleviate brain injury by inhibiting NF-{kappa}B activation.« less

Nuclear NF-κB Expression Correlates With Outcome Among Patients With Head and Neck Squamous Cell Carcinoma Treated With Primary Chemoradiation Therapy

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

Balermpas, Panagiotis; Michel, Yvonne; Wagenblast, Jens

2013-07-15

Background: To examine whether nuclear NF-κB expression correlates with outcome in patients with head and neck squamous cell carcinoma (HNSCC) treated with primary chemoradiation therapy (CRT). Methods and Materials: Between 2007 and 2010, 101 patients with locally advanced primary HNSCC were treated with definitive simultaneous CRT. Pretreatment biopsy specimens were analyzed for NF-κB p65 (RelA) nuclear immunoreactivity. A sample was assigned to be positive with more than 5% positive nuclear expression. The predictive relevance of NF-κB and clinicopathologic factors for overall survival (OS), progression-free survival (PFS), local progression-free survival (LPFS), and metastasis-free survival (DMFS) was examined by univariate and multivariatemore » analysis. Results: No significant differences between the groups were observed with regard to age, sex, total radiation dose, fractionation mode, total chemotherapy applied, T stage or grading. Patients with p65 nuclear positive biopsy specimens showed significantly a higher rate of lymph node metastasis (cN2c or cN3 status, P=.034). Within a mean follow-up time of 25 months (range, 2.33-62.96 months) OS, PFS, and DMFS were significantly poorer in the p65 nuclear positive group (P=.008, P=.027, and P=.008, respectively). These correlations remained significant in multivariate analysis. Conclusion: NF-κB/p65 nuclear expression is associated with increased lymphatic and hematogenous tumor dissemination and decreased survival in HNSCC patients treated with primary CRT. Our results may foster further investigation of a predictive relevance of NF-κB/p65 and its role as a suitable target for a molecular-based targeted therapy in HNSCC cancer.« less

NF-κB Transcriptional Activity Is Modulated by FK506-binding Proteins FKBP51 and FKBP52

PubMed Central

Erlejman, Alejandra G.; De Leo, Sonia A.; Mazaira, Gisela I.; Molinari, Alejandro M.; Camisay, María Fernanda; Fontana, Vanina; Cox, Marc B.; Piwien-Pilipuk, Graciela; Galigniana, Mario D.

2014-01-01

Hsp90 binding immunophilins FKBP51 and FKBP52 modulate steroid receptor trafficking and hormone-dependent biological responses. With the purpose to expand this model to other nuclear factors that are also subject to nuclear-cytoplasmic shuttling, we analyzed whether these immunophilins modulate NF-κB signaling. It is demonstrated that FKBP51 impairs both the nuclear translocation rate of NF-κB and its transcriptional activity. The inhibitory action of FKBP51 requires neither the peptidylprolyl-isomerase activity of the immunophilin nor its association with Hsp90. The TPR domain of FKBP51 is essential. On the other hand, FKBP52 favors the nuclear retention time of RelA, its association to a DNA consensus binding sequence, and NF-κB transcriptional activity, the latter effect being strongly dependent on the peptidylprolyl-isomerase activity and also on the TPR domain of FKBP52, but its interaction with Hsp90 is not required. In unstimulated cells, FKBP51 forms endogenous complexes with cytoplasmic RelA. Upon cell stimulation with phorbol ester, the NF-κB soluble complex exchanges FKBP51 for FKBP52, and the NF-κB biological effect is triggered. Importantly, FKBP52 is functionally recruited to the promoter region of NF-κB target genes, whereas FKBP51 is released. Competition assays demonstrated that both immunophilins antagonize one another, and binding assays with purified proteins suggest that the association of RelA and immunophilins could be direct. These observations suggest that the biological action of NF-κB in different cell types could be positively regulated by a high FKBP52/FKBP51 expression ratio by favoring NF-κB nuclear retention, recruitment to the promoter regions of target genes, and transcriptional activity. PMID:25104352

GATA-3 immunohistochemistry in the differential diagnosis of adenocarcinoma of the urinary bladder.

PubMed

Ellis, Carla L; Chang, Alex G; Cimino-Mathews, Ashley; Argani, Pedram; Youssef, Ramy F; Kapur, Payal; Montgomery, Elizabeth A; Epstein, Jonathan I

2013-11-01

GATA-3 is a newly described marker that labels urothelial and breast carcinoma. However, no prior study has evaluated the expression of GATA-3 in primary bladder adenocarcinoma. Tissue microarrays (TMAs) containing 46 primary bladder adenocarcinomas were constructed. They contained 19 signet ring cell (SRC) and 27 conventional adenocarcinomas. Three additional cases of SRC using routine sections were included resulting in a total of 22 SRCs. In addition, TMAs containing 32 primary gastric signet ring adenocarcinomas and 36 primary lobular breast carcinomas were evaluated. The TMAs were subjected to immunohistochemical analysis for GATA-3, with nuclear labeling scored by intensity and percentage labeling. Breast and urothelial TMAs were also labeled for estrogen receptor, progesterone receptor, and gross cystic duct fluid protein. Diffuse nuclear GATA-3 labeling was seen in 9/22 (41.0%) SRCs and in 2/27 (7.0%) conventional adenocarcinomas (P=0.01). Extracellular mucin production was seen in 12 SRCs. One of 12 (8.0%) SRCs with extracellular mucin was GATA-3 positive, and 8/10 SRCs without extracellular mucin was GATA-3 positive (P=0.005). No nuclear GATA-3 labeling was seen in any gastric signet ring carcinoma. Diffuse, moderate to strong nuclear GATA-3 labeling was seen in 36/36 (100%) primary lobular breast carcinomas. Nuclear GATA-3 labeling is a useful marker for primary adenocarcinomas of the urinary bladder with signet ring features and can be helpful in distinguishing primary signet ring carcinomas of the urinary bladder from gastric signet ring carcinomas. GATA-3 is rarely positive in bladder adenocarcinomas that lack signet ring features and in SRCs displaying extracellular mucin production.

Anti-inflammatory effect of intravenous immunoglobulin in comparison with dexamethasone in vitro: implication for treatment of Kawasaki disease.

PubMed

Makata, Haruyuki; Ichiyama, Takashi; Uchi, Ryutaro; Takekawa, Tsuyoshi; Matsubara, Tomoyo; Furukawa, Susumu

2006-08-01

High-dose intravenous immunoglobulin (IVIG) is a well-established standard therapy for Kawasaki disease (KD) that reduces the risk of developing coronary artery aneurysms. On the other hand, some reports have recommended an alternative therapy with steroids for KD patients. In this study we investigated the anti-inflammatory effect of IVIG in comparison with dexamethasone at clinical doses in vitro. High-dose IVIG inhibited tumor necrosis factor-alpha (TNF-alpha)-induced activation of nuclear factor-kappaB (NF-kappaB) to a greater degree than dexamethasone in human monocytic U937 cells and human coronary arterial endothelial cells (HCAEC), but not in human T lymphocytic Jurkat cells. IVIG was more potent than dexamethasone in reducing the expression of CD16 (FcgammaRIII) in human monocytic THP-1 cells stimulated with lipopolysaccharide and in Jurkat cells stimulated with dimethyl sulfoxide. In HCAEC exposed to TNF-alpha, IVIG and dexamethasone inhibited interleukin-6 production to a similar degree, whereas the expression of E-selectin was inhibited more strongly by IVIG. Our results show that high-dose IVIG inhibits the activation of monocytes/macrophages and coronary arterial endothelial cells more strongly than that of T cells, whereas dexamethasone inhibits the activation of all three cell types. These findings suggest that IVIG or dexamethasone therapy should be chosen to match the types of cells that are activated during acute KD.

A Novel Marker for Purkinje Cells, Ribosomal Protein MPS1/S27: Expression of MPS1 in Human Cerebellum.

PubMed

Fernandez-Pol, J Alberto

2016-01-01

The ribosomal protein metallopanstimulin-1 (MPS1/S27) serves critical survival purposes in cell division, in normal and cancerous cells; for this reason, selective pressures of evolution have conserved the DNA sequences encoding MPS1/S27 in Archaea and Eukariotic cells. The expression of MPS1/S27 protein in human adult cerebellum has not been established. The presence of MPS1/S27, was screened in paraffin-embedded human adult brain specimens processed for tissue inmunohistochemistry. Affinity-purified specific antibodies were directed against the N-terminus of MPS1. The antibodies to MPS1 detected Purkinje cells (PC) and their dendrites. In PC, MPS1 antigen-positive staining was found in: the nucleolus, which was strongly stained; ribosomes attached to the external nuclear membrane; cytoplasm of PC, with strong staining in a punctuate fashion; the soma-attached large dendrite trunks of PC, which were MPS1 antigen-positive; and the granular cell layer, where cellular staining in a few cells that appeared to resemble smaller PC was observed. Since MPS1 is involved in cell division, DNA repair, and ribosomal biogenesis, it may be a useful antigen for studying processes such as protein synthesis, oncogenesis, regeneration, aging, and perhaps diseases of the human cerebellum. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

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.

The Exploration of Hot Nuclear Matter

NASA Astrophysics Data System (ADS)

Jacak, Barbara V.; Müller, Berndt

2012-07-01

When nuclear matter is heated beyond 2 trillion degrees, it becomes a strongly coupled plasma of quarks and gluons. Experiments using highly energetic collisions between heavy nuclei have revealed that this new state of matter is a nearly ideal, highly opaque liquid. A description based on string theory and black holes in five dimensions has made the quark-gluon plasma an archetypical strongly coupled quantum system. Open questions about the structure and theory of the quark-gluon plasma are under active investigation. Many of the insights are also relevant to ultracold fermionic atoms and strongly correlated condensed matter.

JAB1 regulates unphosphorylated STAT3 DNA-binding activity through protein–protein interaction in human colon cancer cells

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

Nishimoto, Arata, E-mail: anishimo@yamaguchi-u.ac.jp; Kugimiya, Naruji; Hosoyama, Toru

2013-08-30

Highlights: •JAB1 interacted with unphosphorylated STAT3 in the nucleus. •JAB1 knockdown tended to increase nuclear STAT3 expression. •JAB1 knockdown significantly decreased unphosphorylated STAT3 DNA-binding activity. •JAB1 knockdown significantly decreased MDR1, NANOG, and VEGF expressions. •Nuclear JAB1, but not nuclear STAT3, correlated with STAT3 DNA-binding activity. -- Abstract: Recent studies have revealed that unphosphorylated STAT3 forms a dimer, translocates to the nucleus, binds to the STAT3 binding site, and activates the transcription of STAT3 target genes, thereby playing an important role in oncogenesis in addition to phosphorylated STAT3. Among signaling steps of unphosphorylated STAT3, nuclear translocation and target DNA-binding are themore » critical steps for its activation. Therefore, elucidating the regulatory mechanism of these signaling steps of unphosphorylated STAT3 is a potential step in the discovery of a novel cancer drug. However, the mechanism of unphosphorylated STAT3 binding to the promoter of target genes remains unclear. In this study, we focused on Jun activation domain-binding protein 1 (JAB1) as a candidate protein that regulates unphosphorylated STAT3 DNA-binding activity. Initially, we observed that both unphosphorylated STAT3 and JAB1 existed in the nucleus of human colon cancer cell line COLO205 at the basal state (no cytokine stimulation). On the other hand, phosphorylated STAT3 did not exist in the nucleus of COLO205 cells at the basal state. Immunoprecipitation using nuclear extract of COLO205 cells revealed that JAB1 interacted with unphosphorylated STAT3. To investigate the effect of JAB1 on unphosphorylated STAT3 activity, RNAi studies were performed. Although JAB1 knockdown tended to increase nuclear STAT3 expression, it significantly decreased unphosphorylated STAT3 DNA-binding activity. Subsequently, JAB1 knockdown significantly decreased the expression levels of MDR1, NANOG, and VEGF, which are STAT3 target genes. Furthermore, the expression level of nuclear JAB1, but not nuclear STAT3, correlated with unphosphorylated STAT3 DNA-binding activity between COLO205 and LoVo cells. Taken together, these results suggest that nuclear JAB1 positively regulates unphosphorylated STAT3 DNA-binding activity through protein–protein interaction in human colon cancer cell line COLO205.« less

Materials for Children about Nuclear War.

ERIC Educational Resources Information Center

Eiss, Harry

President Reagan's Fiscal Year 1987 budget was an attempt to increase dramatically spending on national defense, on nuclear weapons, while cutting back on social programs. The increases for almost all nuclear weapons indicate the Administration of the United States saw its major responsibility as one of providing a strong military, one centered on…

TRAF2 regulates the cytoplasmic/nuclear distribution of TRAF4 and its biological function in breast cancer cells

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

Zhang, Xiaoli; Wen, Zhifeng; Sun, Limei

2013-06-28

Highlights: •TRAF2 appears to interact with TRAF4 in breast cancer cell lines. •TRAF2 affects the localization and function of TRAF4 in breast cancer cell lines. •TRAF4 may play an important role in the activation of NF-κB via TRAF2. -- Abstract: Although numerous studies have shown that tumor necrosis factor receptor-associated factor 4 (TRAF4) plays an important role in the carcinogenesis of many tumor types, its exact molecular mechanism remains elusive. In this study, we examined the regulation function of TRAF2 to the cytoplasmic/nuclear distribution of TRAF4 in the breast cancer cell line. Using cell immunofluorescent staining, we found that TRAF2more » and TRAF4 were co-localized to the cytoplasm in MCF-7 cells. Co-immunoprecipitation showed that TRAF2 could interact with TRAF4 in MCF-10A, MCF-7 and MDA-MB-231 cell lines. Western blotting showed TRAF2 depletion by targeted siRNA in MDA-MB-231 cells led to reduced TRAF4 expression in the cytoplasm and augmented TRAF4 expression in the nucleus. Cytoplasmic expression of TRAF4 was augmented and nuclear expression was reduced when MCF-7 cells were transfected with hTRAF2pLPCX-HA-Flag/P874. MCF-7 cells expressing hTRAF2pLPCX-HA-Flag/P874 had enhanced cell proliferation rates. The nuclear expression of NF-κB significantly increased after TNF-α treatment. When hTRAF2pLPCX-HA-Flag/P874 and the siRNA-TRAF4 plasmid were cotransfected, the nuclear expression of NF-κB was significantly reduced compared with cells transfected with hTRAF2pLPCX-HA-Flag/P874 only. In conclusion, TRAF2 appears to interact with TRAF4 and affect the localization of TRAF4 in breast cancer cell lines. The overexpression of TRAF2 augmented the cytoplasmic expression of TRAF4 which promoted cell proliferation and inhibited cell apoptosis by activating NF-κB nuclear transcription. TRAF4 may play an important role in the activation of NF-κB via TRAF2.« less

Analytical Validation and Clinical Qualification of a New Immunohistochemical Assay for Androgen Receptor Splice Variant-7 Protein Expression in Metastatic Castration-resistant Prostate Cancer.

PubMed

Welti, Jonathan; Rodrigues, Daniel Nava; Sharp, Adam; Sun, Shihua; Lorente, David; Riisnaes, Ruth; Figueiredo, Ines; Zafeiriou, Zafeiris; Rescigno, Pasquale; de Bono, Johann S; Plymate, Stephen R

2016-10-01

The androgen receptor splice variant-7 (AR-V7) has been implicated in the development of castration-resistant prostate cancer (CRPC) and resistance to abiraterone and enzalutamide. To develop a validated assay for detection of AR-V7 protein in tumour tissue and determine its expression and clinical significance as patients progress from hormone-sensitive prostate cancer (HSPC) to CRPC. Following monoclonal antibody generation and validation, we retrospectively identified patients who had HSPC and CRPC tissue available for AR-V7 immunohistochemical (IHC) analysis. Nuclear AR-V7 expression was determined using IHC H score (HS) data. The change in nuclear AR-V7 expression from HSPC to CRPC and the association between nuclear AR-V7 expression and overall survival (OS) was determined. Nuclear AR-V7 expression was significantly lower in HSPC (median HS 50, interquartile range [IQR] 17.5-90) compared to CRPC (HS 135, IQR 80-157.5; p<0.0001), and in biopsy tissue taken before (HS 80, IQR 30-136.3) compared to after (HS 140, IQR 105-167.5; p=0.007) abiraterone or enzalutamide treatment. Lower nuclear AR-V7 expression at CRPC biopsy was associated with longer OS (hazard ratio 1.012, 95% confidence interval 1.004-1.020; p=0.003). While this monoclonal antibody primarily binds to AR-V7 in PC biopsy tissue, it may also bind to other proteins. We provide the first evidence that nuclear AR-V7 expression increases with emerging CRPC and is prognostic for OS, unlike antibody staining for the AR N-terminal domain. These data indicate that AR-V7 is important in CRPC disease biology; agents targeting AR splice variants are needed to test this hypothesis and further improve patient outcome from CRPC. In this study we found that levels of the protein AR-V7 were higher in patients with advanced prostate cancer. A higher level of AR-V7 identifies a group of patients who respond less well to certain prostate cancer treatments and live for a shorter period of time. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Nuclear Transcription Factor Kappa B Downregulation Reduces Chemoresistance in Bone Marrow-derived Cells Through P-glycoprotein Modulation.

PubMed

Loaiza, Brenda; Hernández-Gutierrez, Salomon; Montesinos, Juan Jose; Valverde, Mahara; Rojas, Emilio

2016-02-01

Nuclear transcription factor kappa B (NF-κB) is associated with many types of refractory cancer. However, despite multiple strategies to treat cancer and novel target drugs, multidrug resistance still causes relapses. The best-characterized mechanism responsible for multidrug resistance involves the expression of the MDR-1 gene product, P-glycoprotein (P-gp). Because the direct inhibition of this protein is very toxic, other methods of multidrug resistance (MDR) regulation have been proposed. The MDR-1 promoter sequence contains a κB site, which is recognized by NF-κB. The aim of this work was to characterize whether NF-κB modulation changes the response of bone marrow-derived cells (BMDCs) to chemotherapy. We exposed BMDCs to etoposide and doxorubicin, two of the most used antineoplastic drugs. BMDCs presented high tolerance to these drugs, which correlated with high intrinsic P-gp activity and strong protein expression of NF-κB. To determine the mechanism behind the poor sensitivity of BMDCs to chemotherapy, we blocked the activity of the heterodimer protein NF-κB using the pharmacological inhibitor Bay 11-7085 and through the transfection of an adenovirus negative mutant of I kappa B alpha. The multidrug resistance phenotype of BMDCs was reversed by inhibiting the NF-κB pathway, and this change was accompanied by a decrease in P-gp activity. NF-κB is a possible target for improving the antineoplastic response. Copyright © 2016 IMSS. Published by Elsevier Inc. All rights reserved.

The nuclear protein Poly(ADP-ribose) polymerase 3 (AtPARP3) is required for seed storability in Arabidopsis thaliana.

PubMed

Rissel, D; Losch, J; Peiter, E

2014-11-01

The deterioration of seeds during prolonged storage results in a reduction of viability and germination rate. DNA damage is one of the major cellular defects associated with seed deterioration. It is provoked by the formation of reactive oxygen species (ROS) even in the quiescent state of the desiccated seed. In contrast to other stages of seed life, DNA repair during storage is hindered through the low seed water content; thereby DNA lesions can accumulate. To allow subsequent seedling development, DNA repair has thus to be initiated immediately upon imbibition. Poly(ADP-ribose) polymerases (PARPs) are important components in the DNA damage response in humans. Arabidopsis thaliana contains three homologues to the human HsPARP1 protein. Of these three, only AtPARP3 was very highly expressed in seeds. Histochemical GUS staining of embryos and endosperm layers revealed strong promoter activity of AtPARP3 during all steps of germination. This coincided with high ROS activity and indicated a role of the nuclear-localised AtPARP3 in DNA repair during germination. Accordingly, stored parp3-1 mutant seeds lacking AtPARP3 expression displayed a delay in germination as compared to Col-0 wild-type seeds. A controlled deterioration test showed that the mutant seeds were hypersensitive to unfavourable storage conditions. The results demonstrate that AtPARP3 is an important component of seed storability and viability. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Suppression of cell division by pKi-67 antisense-RNA and recombinant protein.

PubMed

Duchrow, M; Schmidt, M H; Zingler, M; Anemüller, S; Bruch, H P; Broll, R

2001-01-01

The human antigen defined by the monoclonal antibody Ki-67 (pKi-67) is a human nuclear protein strongly associated with cell proliferation and found in all tissues studied. It is widely used as a marker of proliferating cells, yet its function is unknown. To investigate its function we suppressed pKi-67 expression by antisense RNA and overexpressed a partial structure of pKi-67 in HeLa cells. A BrdU-incorporation assay showed a significant decrease in DNA synthesis after antisense inhibition. Cell cycle analysis indicated a higher proportion of cells in G1 phase and a lower proportion of cells in S phase while the number of G(2)/M phase cells remained constant. Overexpression of a recombinant protein encoding three of the repetitive elements from exon 13 of pKi-67 had a similar effect to that obtained by antisense inhibition. The similarity of the effect of expressing 'Ki-67 repeats' and pKi-67 antisense RNA could be explained by a negative effect on the folding of the endogenous protein in the endoplasmatic reticulum. Furthermore excessive self-association of pKi-67 via the repeat structure could inhibit its nuclear transport, preventing it from getting to its presumptive site of action. We conclude that the Ki-67 protein has an important role in the regulation of the cell cycle, which is mediated in part by its repetitive elements. Copyright 2001 S. Karger AG, Basel

Tangeretin Inhibits IL-12 Expression and NF-κB Activation in Dendritic Cells and Attenuates Colitis in Mice.

PubMed

Eun, Su-Hyeon; Woo, Je-Te; Kim, Dong-Hyun

2017-04-01

In the preliminary study, tangeretin (5,6,7,8,4'-pentamethoxy flavone), a major constituent of the pericarp of Citrus sp., inhibited TNF- α , IL-12, and IL-23 expression and nuclear factor kappa-B activation in lipopolysaccharide-stimulated dendritic cells; however, it did not affect IL-10 expression. Furthermore, tangeretin (5, 10, and 20 µM) suppressed the activation and translocation of nuclear factor kappa-B (p65) into the nuclei in vitro by inhibiting the binding of lipopolysaccharide on dendritic cells. Oral administration of tangeretin (10 and 20 mg/kg) suppressed the inflammatory responses, such as nuclear factor kappa-B and mitogen-activated protein kinase activation and myeloperoxidase activity, in the colon of mice with 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Tangeretin increased 2,4,6-trinitrobenzene sulfonic acid-suppressed expression of tight junction proteins occludin, claudin-1, and ZO-1. Tangeretin also inhibited 2,4,6-trinitrobenzene sulfonic acid-induced differentiation of Th1 and Th17 cells as well as the expression of T-bet, ROR γ t, interferon- γ , IL-12, IL-17, and TNF- α . However, tangeretin increased 2,4,6-trinitrobenzene sulfonic acid-suppressed differentiation of regulatory T cells as well as the expression of Foxp3 and IL-10. These results suggest that oral administration of tangeretin may attenuate colitis by suppressing IL-12 and TNF- α expression and nuclear factor kappa-B activation through the inhibition of lipopolysaccharide binding on immune cells such as dendritic cells. Georg Thieme Verlag KG Stuttgart · New York.

Distinct nuclear body components, PML and SMRT, regulate the trans-acting function of HTLV-1 Tax oncoprotein.

PubMed

Ariumi, Yasuo; Ego, Takeshi; Kaida, Atsushi; Matsumoto, Mikiko; Pandolfi, Pier Paolo; Shimotohno, Kunitada

2003-03-20

Several viruses target cellular promyelocytic leukemia (PML)-nuclear bodies (PML-NBs) to induce their disruption, marked morphological changes in these structures or the relocation to PML-NB components to the cytoplasm of infected cells. PML conversely interferes with viral replication. We demonstrate that PML acts as a coactivator for the human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein without direct binding. Tax was identified within interchromatin granule clusters (IGCs)/RNA splicing bodies (SBs), not PML-NBs; Tax expression did not affect PML-NB formation. Moreover, PML and CBP/p300 cooperatively activated Tax-mediated HTLV-1-LTR-dependent gene expression. Interestingly, two PML mutants, PML-RAR and PMLDelta216-331, which fail to form PML-NBs, could also coactivate Tax-mediated trans-acting function but had no effect on retinoic acid receptor (RAR)- or p53-dependent gene expression. In contrast, SMRT (silencing mediator for retinoic acid and thyroid hormone receptors), a nuclear corepressor found within the matrix-associated deacetylase (MAD) nuclear body, relocalized into Tax-associated nuclear bodies upon coexpression with Tax. SMRT coactivated the trans-acting function of Tax through direct binding. Coexpression of SMRT and PML resulted in an additive activation of Tax trans-acting function. Thus, crosstalk between distinct nuclear bodies may control Tax function.

Isolation, sequencing and expression of RED, a novel human gene encoding an acidic-basic dipeptide repeat.

PubMed

Assier, E; Bouzinba-Segard, H; Stolzenberg, M C; Stephens, R; Bardos, J; Freemont, P; Charron, D; Trowsdale, J; Rich, T

1999-04-16

A novel human gene RED, and the murine homologue, MuRED, were cloned. These genes were named after the extensive stretch of alternating arginine (R) and glutamic acid (E) or aspartic acid (D) residues that they contain. We term this the 'RED' repeat. The genes of both species were expressed in a wide range of tissues and we have mapped the human gene to chromosome 5q22-24. MuRED and RED shared 98% sequence identity at the amino acid level. The open reading frame of both genes encodes a 557 amino acid protein. RED fused to a fluorescent tag was expressed in nuclei of transfected cells and localised to nuclear dots. Co-localisation studies showed that these nuclear dots did not contain either PML or Coilin, which are commonly found in the POD or coiled body nuclear compartments. Deletion of the amino terminal 265 amino acids resulted in a failure to sort efficiently to the nucleus, though nuclear dots were formed. Deletion of a further 50 amino acids from the amino terminus generates a protein that can sort to the nucleus but is unable to generate nuclear dots. Neither construct localised to the nucleolus. The characteristics of RED and its nuclear localisation implicate it as a regulatory protein, possibly involved in transcription.

Mitochondrial-nuclear crosstalk, haplotype and copy number variation distinct in muscle fiber type, mitochondrial respiratory and metabolic enzyme activities.

PubMed

Liu, Xuan; Trakooljul, Nares; Hadlich, Frieder; Murani, Eduard; Wimmers, Klaus; Ponsuksili, Siriluck

2017-10-25

Genes expressed in mitochondria work in concert with those expressed in the nucleus to mediate oxidative phosphorylation (OXPHOS), a process that is relevant for muscle metabolism and meat quality. Mitochondrial genome activity can be efficiently studied and compared in Duroc and Pietrain pigs, which harbor different mitochondrial haplotypes and distinct muscle fiber types, mitochondrial respiratory activities, and fat content. Pietrain pigs homozygous-positive for malignant hyperthermia susceptibility (PiPP) carried only haplotype 8 and showed the lowest absolute mtDNA copy number accompanied by a decrease transcript abundance of mitochondrial-encoded subunits ND1, ND6, and ATP6 and nuclear-encoded subunits NDUFA11 and NDUFB8. In contrast, we found that haplotype 4 of Duroc pigs had significantly higher mitochondrial DNA (mtDNA) copy numbers and an increase transcript abundance of mitochondrial-encoded subunits ND1, ND6, and ATP6. These results suggest that the variation in mitochondrial and nuclear genetic background among these animals has an effect on mitochondrial content and OXPHOS system subunit expression. We observed the co-expression pattern of mitochondrial and nuclear encoded OXPHOS subunits suggesting that the mitochondrial-nuclear crosstalk functionally involves in muscle metabolism. The findings provide valuable information for understanding muscle biology processes and energy metabolism, and may direct use for breeding strategies to improve meat quality and animal health.

Stability of HTLV-2 antisense protein is controlled by PML nuclear bodies in a SUMO-dependent manner.

PubMed

Dubuisson, Louise; Lormières, Florence; Fochi, Stefania; Turpin, Jocelyn; Pasquier, Amandine; Douceron, Estelle; Oliva, Anaïs; Bazarbachi, Ali; Lallemand-Breitenbach, Valérie; De Thé, Hugues; Journo, Chloé; Mahieux, Renaud

2018-05-01

Since the identification of the antisense protein of HTLV-2 (APH-2) and the demonstration that APH-2 mRNA is expressed in vivo in most HTLV-2 carriers, much effort has been dedicated to the elucidation of similarities and/or differences between APH-2 and HBZ, the antisense protein of HTLV-1. Similar to HBZ, APH-2 negatively regulates HTLV-2 transcription. However, it does not promote cell proliferation. In contrast to HBZ, APH-2 half-life is very short. Here, we show that APH-2 is addressed to PML nuclear bodies in T-cells, as well as in different cell types. Covalent SUMOylation of APH-2 is readily detected, indicating that APH-2 might be addressed to the PML nuclear bodies in a SUMO-dependent manner. We further show that silencing of PML increases expression of APH-2, while expression of HBZ is unaffected. On the other hand, SUMO-1 overexpression leads to a specific loss of APH-2 expression that is restored upon proteasome inhibition. Furthermore, the carboxy-terminal LAGLL motif of APH-2 is responsible for both the targeting of the protein to PML nuclear bodies and its short half-life. Taken together, these observations indicate that natural APH-2 targeting to PML nuclear bodies induces proteasomal degradation of the viral protein in a SUMO-dependent manner. Hence, this study deciphers the molecular and cellular bases of APH-2 short half-life in comparison to HBZ and highlights key differences in the post-translational mechanisms that control the expression of both proteins.

Japan’s Rearmament Dilemma: Obstacles to Rearmament.

DTIC Science & Technology

1980-01-01

aircraft carrier Enterprise, and the problems with the first Japanese nuclear -powered ship , the Mutsu , have continued to keep the issue of nuclear weapons...4 Formation of the Psychology. .. ............... 4 Nuclear Allergy .. .................... 7 Self-Defense Force .. ................... 10 ARTICLE 9... nuclear weapons, would elicit a strong reaction from the U.S. This specter of the U.S. reaction is another obstacle to rearmament. While most scholars

Estrogen signaling in colorectal carcinoma microenvironment: expression of ERbeta1, AIB-1, and TIF-2 is upregulated in cancer-associated myofibroblasts and correlates with disease progression.

PubMed

Tzelepi, Vassiliki; Grivas, Petros; Kefalopoulou, Zinovia; Kalofonos, Haralabos; Varakis, John N; Melachrinou, Maria; Sotiropoulou-Bonikou, Georgia

2009-04-01

Epidemiological and molecular data suggest the involvement of estrogen signaling in colorectal tissue, mediated mainly through estrogen receptor beta (ERbeta). Estrogens may mediate their effects in epithelial cells indirectly by acting on stromal cells. Expression of ERalpha, ERbeta1, and the ER coregulators, amplified in breast cancer-1 (AIB-1) and transcriptional intermediary factor 2 (TIF-2), was evaluated in myofibroblasts of 107 colorectal carcinomas, 77 paired samples of normal mucosa, and 29 adenomas by immunohistochemistry. Double immunostaining with a-SMA was used to identify the myofibroblasts of normal tissue, adenomas, and cancer microenvironment. ERalpha was not expressed in stromal cells. Nuclear expression of ERbeta1, AIB-1, and TIF-2 in myofibroblasts gradually increased from normal mucosa, through adenomas, to carcinomas. Cytoplasmic ERbeta1 and TIF-2 expression was enhanced in carcinomas compared to normal mucosa and adenomas. Enhanced nuclear and cytoplasmic ERbeta1 expression and elevated nuclear AIB-1 expression were more frequently noted in myofibroblasts of carcinomas of advanced stage. ERbeta1 expression in cancer-associated myofibroblasts correlated to AIB-1 and TIF-2 expression. None of the markers correlated with patients' prognosis. Our findings imply that ERbeta1-dependent (genomic and non-genomic) and ER-coregulator-dependent (AIB-1, TIF-2) signal transductions in myofibroblasts may be involved in the initiation and progression of colorectal carcinomas.

Nuclear lamina builds tissues from the stem cell niche.

PubMed

Chen, Haiyang; Zheng, Yixian

2014-01-01

Recent studies show that nuclear lamins, the type V intermediate filament proteins, are required for proper building of at least some organs. As the major structural components of the nuclear lamina found underneath the inner nuclear membranes, lamins are ubiquitously expressed in all animal cells. How the broadly expressed lamins support the building of specific tissues is not understood. By studying Drosophila testis, we have uncovered a mechanism by which lamin-B functions in the cyst stem cell (CySC) and its differentiated cyst cell, the cell types known to form the niche/microenvironment for the germline stem cells (GSC) and the developing germ line, to ensure testis organogenesis (1). In this extra view, we discuss some remaining questions and the implications of our findings in the understanding of how the ubiquitous nuclear lamina regulates tissue building in a context-dependent manner.

Strongly-Interacting Fermi Gases in Reduced Dimensions

DTIC Science & Technology

2015-11-16

one spin state is surrounded by a particle- hole cloud of the other 1. REPORT DATE (DD-MM-YYYY) 4. TITLE AND SUBTITLE 13. SUPPLEMENTARY NOTES 12...explained in part by a polaron model, in which an atom of one spin state is surrounded by a particle- hole cloud of the other spin state. However, a...superconductivity), nuclear physics (nuclear matter), high-energy physics (effective theories of the strong interactions), astrophysics (compact stellar objects

Polydatin promotes Nrf2-ARE anti-oxidative pathway through activating Sirt1 to resist AGEs-induced upregulation of fibronetin and transforming growth factor-β1 in rat glomerular messangial cells.

PubMed

Huang, Kaipeng; Chen, Cheng; Hao, Jie; Huang, Junying; Wang, Shaogui; Liu, Peiqing; Huang, Heqing

2015-01-05

Sirt1 and nuclear factor-E2 related factor 2 (Nrf2)-anti-oxidant response element (ARE) anti-oxidative pathway play important regulatory roles in the pathological progression of diabetic nephropathy (DN) induced by advanced glycation-end products (AGEs). Polydatin (PD), a glucoside of resveratrol, has been shown to possess strong anti-oxidative bioactivity. Our previous study demonstrated that PD markedly resists the progression of diabetic renal fibrosis and thus, inhibits the development of DN. Whereas, whether PD could resist DN through regulating Sirt1 and consequently promoting Nrf2-ARE pathway needs further investigation. Here, we found that concomitant with decreasing RAGE (the specific receptor for AGEs) expression, PD significantly reversed the downregulation of Sirt1 in terms of protein expression and deacetylase activity and attenuated FN and TGF-β1 expression in GMCs exposed to AGEs. Under AGEs-treatment condition, PD could decrease Keap1 expression and promote the nuclear content, ARE-binding ability, and transcriptional activity of Nrf2. In addition, PD increased the protein levels of heme oxygenase 1 (HO-1) and superoxide dismutase 1 (SOD1), two target genes of Nrf2. The activation of Nrf2-ARE pathway by PD eventually led to the quenching of ROS overproduction sharply boosted by AGEs. Depletion of Sirt1 blocked Nrf2-ARE pathway activation and reversed FN and TGF-β1 downregulation induced by PD in GMCs challenged with AGEs. Along with reducing HO-1 and SOD1 expression, silencing of Nrf2 increased FN and TGF-β1 levels. PD treatment elevated Sirt1 and Nrf2 levels in the kidney tissues of diabetic rats, then improved the anti-oxidative capacity and renal dysfunction of diabetic models, and finally reversed the upregulation of FN and TGF-β1. Taken together, the resistance of PD on upregulated FN and TGF-β1 induced by AGEs via oxidative stress in GMCs is closely associated with its activation of Sirt1-Nrf2-ARE pathway. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Nuclear EGFRvIII resists hypoxic microenvironment induced apoptosis via recruiting ERK1/2 nuclear translocation

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

Xie, Hui; Yang, Jinfeng; Xing, Wenjing

2016-02-05

Glioblastoma (GBM) is the most aggressive type of primary brain tumor. Its interaction with the tumor microenvironment promotes tumor progression. Furthermore, GBM bearing expression of EGFRvIII displays more adaptation to tumor microenvironment related stress. But the mechanisms were poorly understood. Here, we presented evidence that in the human U87MG glioblastoma tumor model, EGFRvIII overexpression led aberrant kinase activation and nuclear translocation of EGFRvIII/ERK1/2 under hypoxia, which induced growth advantage by resisting apoptosis. Additionally, EGFRvIII defective in nuclear entry impaired this capacity in hypoxia adaptation, and partially interrupted ERK1/2 nuclear translocation. Pharmacology or genetic interference ERK1/2 decreased hypoxia resistance triggered bymore » EGFRvIII expression, but not EGFRvIII nuclear translocation. In summary, this study identified a novel role for EGFRvIII in hypoxia tolerance, supporting an important link between hypoxia and subcellular localization alterations of the receptor. - Highlights: • Nuclear translocation of EGFRvIII contributes to GBM cell apoptotic resistance by hypoxia. • Nuclear ERK1/2 facilitates EGFRvIII in hypoxia resistance. • EGFRvIII nuclear translocation is not dependent on ERK1/2.« less

SRSF1-3 contributes to diversification of the immunoglobulin variable region gene by promoting accumulation of AID in the nucleus.

PubMed

Kawaguchi, Yuka; Nariki, Hiroaki; Kawamoto, Naoko; Kanehiro, Yuichi; Miyazaki, Satoshi; Suzuki, Mari; Magari, Masaki; Tokumitsu, Hiroshi; Kanayama, Naoki

2017-04-01

Activation-induced cytidine deaminase (AID) is essential for diversification of the Ig variable region (IgV). AID is excluded from the nucleus, where it normally functions. However, the molecular mechanisms responsible for regulating AID localization remain to be elucidated. The SR-protein splicing factor SRSF1 is a nucleocytoplasmic shuttling protein, a splicing isoform of which called SRSF1-3, has previously been shown to contribute to IgV diversification in chicken DT40 cells. In this study, we examined whether SRSF1-3 functions in IgV diversification by promoting nuclear localization of AID. AID expressed alone was localized predominantly in the cytoplasm. In contrast, co-expression of AID with SRSF1-3 led to the nuclear accumulation of both AID and SRSF1-3 and the formation of a protein complex that contained them both, although SRSF1-3 was dispensable for nuclear import of AID. Expression of either SRSF1-3 or a C-terminally-truncated AID mutant increased IgV diversification in DT40 cells. However, overexpression of exogenous SRSF1-3 was unable to further enhance IgV diversification in DT40 cells expressing the truncated AID mutant, although SRSF1-3 was able to form a protein complex with the AID mutant. These results suggest that SRSF1-3 promotes nuclear localization of AID probably by forming a nuclear protein complex, which might stabilize nuclear AID and induce IgV diversification in an AID C-terminus-dependent manner. Copyright © 2017 Elsevier Inc. All rights reserved.

Redox Protein Expression Predicts Radiotherapeutic Response in Early-Stage Invasive Breast Cancer Patients

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

Woolston, Caroline M.; Al-Attar, Ahmad; Storr, Sarah J.

2011-04-01

Purpose: Early-stage invasive breast cancer patients have commonly undergone breast-conserving surgery and radiotherapy. In a large majority of these patients, the treatment is effective; however, a proportion will develop local recurrence. Deregulated redox systems provide cancer cells protection from increased oxidative stress, such as that induced by ionizing radiation. Therefore, the expression of redox proteins was examined in tumor specimens from this defined cohort to determine whether such expression could predict response. Methods and Materials: The nuclear and cytoplasmic expression of nine redox proteins (glutathione, glutathione reductase, glutaredoxin, glutathione peroxidase 1, 3, and 4, and glutathione S-transferase-{theta}, -{pi}, and -{alpha})more » was assessed using conventional immunohistochemistry on a tissue microarray of 224 tumors. Results: A high cytoplasmic expression of glutathione S-transferase-{theta} significantly correlated with a greater risk of local recurrence (p = .008) and, when combined with a low nuclear expression (p = .009), became an independent predictive factor (p = .002) for local recurrence. High cytoplasmic expression of glutathione S-transferase-{theta} also correlated with a worse overall survival (p = .009). Low nuclear and cytoplasmic expression of glutathione peroxidase 3 (p = .002) correlated with a greater risk of local recurrence and was an independent predictive factor (p = .005). These proteins did not correlate with tumor grade, suggesting their function might be specific to the regulation of oxidative stress rather than alterations of tumor phenotype. Only nuclear (p = .005) and cytoplasmic (p = .001) expression of glutathione peroxidase 4 correlated with the tumor grade. Conclusions: Our results support the use of redox protein expression, namely glutathione S-transferase-{theta} and glutathione peroxidase 3, to predict the response to radiotherapy in early-stage breast cancer patients. If incorporated into routine diagnostic tests, they have the potential to aid clinicians in their stratification of patients into more tailored treatment regimens. Future targeted therapies to these systems might improve the efficacy of reactive oxygen species-inducing therapies, such as radiotherapy.« less

Simultaneous live imaging of the transcription and nuclear position of specific genes

PubMed Central

Ochiai, Hiroshi; Sugawara, Takeshi; Yamamoto, Takashi

2015-01-01

The relationship between genome organization and gene expression has recently been established. However, the relationships between spatial organization, dynamics, and transcriptional regulation of the genome remain unknown. In this study, we developed a live-imaging method for simultaneous measurements of the transcriptional activity and nuclear position of endogenous genes, which we termed the ‘Real-time Observation of Localization and EXpression (ROLEX)’ system. We demonstrated that ROLEX is highly specific and does not affect the expression level of the target gene. ROLEX enabled detection of sub-genome-wide mobility changes that depended on the state of Nanog transactivation in embryonic stem cells. We believe that the ROLEX system will become a powerful tool for exploring the relationship between transcription and nuclear dynamics in living cells. PMID:26092696

Expression profiling of nuclear receptors in breast cancer identifies TLX as a mediator of growth and invasion in triple-negative breast cancer.

PubMed

Lin, Meng-Lay; Patel, Hetal; Remenyi, Judit; Banerji, Christopher R S; Lai, Chun-Fui; Periyasamy, Manikandan; Lombardo, Ylenia; Busonero, Claudia; Ottaviani, Silvia; Passey, Alun; Quinlan, Philip R; Purdie, Colin A; Jordan, Lee B; Thompson, Alastair M; Finn, Richard S; Rueda, Oscar M; Caldas, Carlos; Gil, Jesus; Coombes, R Charles; Fuller-Pace, Frances V; Teschendorff, Andrew E; Buluwela, Laki; Ali, Simak

2015-08-28

The Nuclear Receptor (NR) superfamily of transcription factors comprises 48 members, several of which have been implicated in breast cancer. Most important is estrogen receptor-α (ERα), which is a key therapeutic target. ERα action is facilitated by co-operativity with other NR and there is evidence that ERα function may be recapitulated by other NRs in ERα-negative breast cancer. In order to examine the inter-relationships between nuclear receptors, and to obtain evidence for previously unsuspected roles for any NRs, we undertook quantitative RT-PCR and bioinformatics analysis to examine their expression in breast cancer. While most NRs were expressed, bioinformatic analyses differentiated tumours into distinct prognostic groups that were validated by analyzing public microarray data sets. Although ERα and progesterone receptor were dominant in distinguishing prognostic groups, other NR strengthened these groups. Clustering analysis identified several family members with potential importance in breast cancer. Specifically, RORγ is identified as being co-expressed with ERα, whilst several NRs are preferentially expressed in ERα-negative disease, with TLX expression being prognostic in this subtype. Functional studies demonstrated the importance of TLX in regulating growth and invasion in ERα-negative breast cancer cells.

γ-Oryzanol reduces adhesion molecule expression in vascular endothelial cells via suppression of nuclear factor-κB activation.

PubMed

Sakai, Satoshi; Murata, Takahisa; Tsubosaka, Yoshiki; Ushio, Hideki; Hori, Masatoshi; Ozaki, Hiroshi

2012-04-04

γ-Oryzanol (γ-ORZ) is a mixture of phytosteryl ferulates purified from rice bran oil. In this study, we examined whether γ-ORZ represents a suppressive effect on the lipopolysaccharide (LPS)-induced adhesion molecule expression on vascular endothelium. Treatment with LPS elevated the mRNA expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin in bovine aortic endothelial cells (BAECs). Pretreatment with γ-ORZ dose-dependently decreased the LPS-mediated expression of these genes. Western blotting also revealed that pretreatment with γ-ORZ dose-dependently inhibited LPS-induced VCAM-1 expression in human umbilical vein endothelial cells. Consistently, pretreatment with γ-ORZ dose-dependently reduced LPS-induced U937 monocyte adhesion to BAECs. In immunofluorescence, LPS caused nuclear factor-κB (NF-κB) nuclear translocation in 40% of BAECs, which indicates NF-κB activation. Pretreatment with γ-ORZ, as well as its components (cycloartenyl ferulate, ferulic acid, or cycloartenol), dose-dependently inhibited LPS-mediated NF-κB activation. Collectively, our results suggested that γ-ORZ reduced LPS-mediated adhesion molecule expression through NF-κB inhibition in vascular endothelium.

High-content screening identifies Src family kinases as potential regulators of AR-V7 expression and androgen-independent cell growth

PubMed Central

Szafran, Adam T.; Stephan, Cliff; Bolt, Michael; Mancini, Maureen G.; Marcelli, Marco; Mancini, Michael A.

2018-01-01

Background AR-V7 is an androgen receptor (AR) splice variant that lacks the ligand-binding domain and is isolated from prostate cancer cell lines. Increased expression of AR-V7 is associated with the transition from hormone-sensitive prostate cancer to more advanced castration-resistant prostate cancer (CRPC). Due to the loss of the ligand-binding domain, AR-V7 is not responsive to traditional AR-targeted therapies, and the mechanisms that regulate AR-V7 are still incompletely understood. Therefore, we aimed to explore existing classes of small molecules that may regulate AR-V7 expression and intracellular localization and their potential therapeutic role in CRPC. Methods We used AR high-content analysis (AR-HCA) to characterize the effects of a focused library of well-characterized clinical compounds on AR-V7 expression at the single-cell level in PC3 prostate cancer cells stably expressing green fluorescent protein (GFP)-AR-V7 (GFP-AR-V7:PC3). In parallel, an orthogonal AR-HCA screen of a small interfering (si)RNA library targeting 635 protein kinases was performed in GFP-AR-V7:PC3. The effect of the Src-Abl inhibitor PD 180970 was further characterized using cell-proliferation assays, quantitative PCR, and western blot analysis in multiple hormone-sensitive and CRPC cell lines. Results Compounds that tended to target Akt, Abl, and Src family kinases (SFKs) decreased overall AR-V7 expression, nuclear translocation, absolute nuclear level, and/or altered nuclear distribution. We identified 20 protein kinases that, when knocked down, either decreased nuclear GFP-AR-V7 levels or altered AR-V7 nuclear distribution, a set that included the SFKs Src and Fyn. The Src-Abl dual kinase inhibitor PD180970 decreased expression of AR-V7 by greater than 46% and decreased ligand-independent transcription of AR target genes in the 22RV1 human prostate carcinoma cell line. Further, PD180970 inhibited androgen-independent cell proliferation in endogenous–AR-V7–expressing prostate cancer cell lines and also overcame bicalutamide resistance observed in the 22RV1 cell line. Conclusions SFKs, especially Src and Fyn, may be important upstream regulators of AR-V7 expression and represent promising targets in a subset of CRPCs expressing high levels of AR-V7. PMID:27699828

The Defective Nuclear Lamina in Hutchinson-Gilford Progeria Syndrome Disrupts the Nucleocytoplasmic Ran Gradient and Inhibits Nuclear Localization of Ubc9▿

PubMed Central

Kelley, Joshua B.; Datta, Sutirtha; Snow, Chelsi J.; Chatterjee, Mandovi; Ni, Li; Spencer, Adam; Yang, Chun-Song; Cubeñas-Potts, Caelin; Matunis, Michael J.; Paschal, Bryce M.

2011-01-01

The mutant form of lamin A responsible for the premature aging disease Hutchinson-Gilford progeria syndrome (termed progerin) acts as a dominant negative protein that changes the structure of the nuclear lamina. How the perturbation of the nuclear lamina in progeria is transduced into cellular changes is undefined. Using patient fibroblasts and a variety of cell-based assays, we determined that progerin expression in Hutchinson-Gilford progeria syndrome inhibits the nucleocytoplasmic transport of several factors with key roles in nuclear function. We found that progerin reduces the nuclear/cytoplasmic concentration of the Ran GTPase and inhibits the nuclear localization of Ubc9, the sole E2 for SUMOylation, and of TPR, the nucleoporin that forms the basket on the nuclear side of the nuclear pore complex. Forcing the nuclear localization of Ubc9 in progerin-expressing cells rescues the Ran gradient and TPR import, indicating that these pathways are linked. Reducing nuclear SUMOylation decreases the nuclear mobility of the Ran nucleotide exchange factor RCC1 in vivo, and the addition of SUMO E1 and E2 promotes the dissociation of RCC1 and Ran from chromatin in vitro. Our data suggest that the cellular effects of progerin are transduced, at least in part, through reduced function of the Ran GTPase and SUMOylation pathways. PMID:21670151

The defective nuclear lamina in Hutchinson-gilford progeria syndrome disrupts the nucleocytoplasmic Ran gradient and inhibits nuclear localization of Ubc9.

PubMed

Kelley, Joshua B; Datta, Sutirtha; Snow, Chelsi J; Chatterjee, Mandovi; Ni, Li; Spencer, Adam; Yang, Chun-Song; Cubeñas-Potts, Caelin; Matunis, Michael J; Paschal, Bryce M

2011-08-01

The mutant form of lamin A responsible for the premature aging disease Hutchinson-Gilford progeria syndrome (termed progerin) acts as a dominant negative protein that changes the structure of the nuclear lamina. How the perturbation of the nuclear lamina in progeria is transduced into cellular changes is undefined. Using patient fibroblasts and a variety of cell-based assays, we determined that progerin expression in Hutchinson-Gilford progeria syndrome inhibits the nucleocytoplasmic transport of several factors with key roles in nuclear function. We found that progerin reduces the nuclear/cytoplasmic concentration of the Ran GTPase and inhibits the nuclear localization of Ubc9, the sole E2 for SUMOylation, and of TPR, the nucleoporin that forms the basket on the nuclear side of the nuclear pore complex. Forcing the nuclear localization of Ubc9 in progerin-expressing cells rescues the Ran gradient and TPR import, indicating that these pathways are linked. Reducing nuclear SUMOylation decreases the nuclear mobility of the Ran nucleotide exchange factor RCC1 in vivo, and the addition of SUMO E1 and E2 promotes the dissociation of RCC1 and Ran from chromatin in vitro. Our data suggest that the cellular effects of progerin are transduced, at least in part, through reduced function of the Ran GTPase and SUMOylation pathways.

Attracting students to nuclear careers: INPO educational assistance program

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

Dunkle, M.

1981-10-01

The utility industry is responding to a manpower shortage of 2000 at nuclear plants with a concerted analysis of regional training centers and educational assistance programs through the Institute of Nuclear Power Operations (INPO). University support and cooperation are generally strong. The INPO program includes undergraduate- and graduate-level scholarships and fellowships. (DCK)

Galaxy interactions and the stimulation of nuclear activity

NASA Technical Reports Server (NTRS)

Heckman, Timothy M.

1990-01-01

The author discusses the idea that interactions between galaxies can lead to enhanced galactic activity. He discusses whether, apart from the observational evidence, there is a strong theoretical or heuristic motivation for investigating galaxy interactions as stimulators of nuclear activity in galaxies. Galactic interactions as mechanisms for triggering nuclear starbursts are covered.