WorldWideScience

Sample records for hypoxia translation regulators

  1. Hypoxia Induces Autophagy through Translational Up-Regulation of Lysosomal Proteins in Human Colon Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Ming-Chih Lai

    Full Text Available Hypoxia occurs in a wide variety of physiological and pathological conditions, including tumorigenesis. Tumor cells have to adapt to hypoxia by altering their gene expression and protein synthesis. Here, we showed that hypoxia inhibits translation through activation of PERK and inactivation of mTOR in human colon cancer HCT116 cells. Prolonged hypoxia (1% O2, 16 h dramatically inhibits general translation in HCT116 cells, yet selected mRNAs remain efficiently translated under such a condition. Using microarray analysis of polysome- associated mRNAs, we identified a large number of hypoxia-regulated genes at the translational level. Efficiently translated mRNAs during hypoxia were validated by polysome profiling and quantitative real-time RT-PCR. Pathway enrichment analysis showed that many of the up-regulated genes are involved in lysosome, glycan and lipid metabolism, antigen presentation, cell adhesion, and remodeling of the extracellular matrix and cytoskeleton. The majority of down-regulated genes are involved in apoptosis, ubiquitin-mediated proteolysis, and oxidative phosphorylation. Further investigation showed that hypoxia induces lysosomal autophagy and mitochondrial dysfunction through translational regulation in HCT116 cells. The abundance of several translation factors and the mTOR kinase activity are involved in hypoxia-induced mitochondrial autophagy in HCT116 cells. Our studies highlight the importance of translational regulation for tumor cell adaptation to hypoxia.

  2. Translational regulation in nutrigenomics.

    Science.gov (United States)

    Liu, Botao; Qian, Shu-Bing

    2011-11-01

    The emergence of genome-wide analysis to interrogate cellular DNA, RNA, and protein content has revolutionized the study of the control network that mediates cellular homeostasis. Nutrigenomics addresses the effect of nutrients on gene expression, which provides a basis for understanding the biological activity of dietary components. Translation of mRNAs represents the last step of genetic flow and primarily defines the proteome. Translational regulation is thus critical for gene expression, in particular, under nutrient excess or deficiency. Until recently, it was unclear how the global effects of translational control are influenced by nutrient signaling. An emerging concept of translational reprogramming addresses how to maintain the expression of specific proteins during pathophysiological conditions by translation of selective mRNAs. Here we describe recent advances in our understanding of translational control, nutrient signaling, and their dysregulation in aging and cancer. The mechanistic understanding of translational regulation in response to different nutrient conditions may help identify potential dietary and therapeutic targets to improve human health.

  3. HypoxamiRs : Regulators of cardiac hypoxia and energy metabolism

    NARCIS (Netherlands)

    Azzouzi, Hamid el; Leptidis, Stefanos; Doevendans, Pieter A.; De Windt, Leon J.

    2015-01-01

    Hypoxia and its intricate regulation are at the epicenter of cardiovascular research. Mediated by hypoxia-inducible factors as well as by several microRNAs, recently termed 'hypoxamiRs', hypoxia affects several cardiac pathophysiological processes. Hypoxia is the driving force behind the regulation

  4. ATR controls cellular adaptation to hypoxia through positive regulation of hypoxia-inducible factor 1 (HIF-1) expression.

    Science.gov (United States)

    Fallone, F; Britton, S; Nieto, L; Salles, B; Muller, C

    2013-09-12

    Tumor cells adaptation to severe oxygen deprivation (hypoxia) plays a major role in tumor progression. The transcription factor HIF-1 (hypoxia-inducible factor 1), whose α-subunit is stabilized under hypoxic conditions is a key component of this process. Recent studies showed that two members of the phosphoinositide 3-kinase-related kinases (PIKKs) family, ATM (ataxia telangiectasia mutated) and DNA-PK (DNA-dependent protein kinase), regulate the hypoxic-dependent accumulation of HIF-1. These proteins initiate cellular stress responses when DNA damage occurs. In addition, it has been demonstrated that extreme hypoxia induces a replicative stress resulting in regions of single-stranded DNA at stalled replication forks and the activation of ATR (ataxia telangiectasia and Rad3 related protein), another member of the PIKKs family. Here, we show that even less severe hypoxia (0.1% O2) also induces activation of ATR through replicative stress. Importantly, in using either transiently silenced ATR cells, cells expressing an inactive form of ATR or cells exposed to an ATR inhibitor (CGK733), we demonstrate that hypoxic ATR activation positively regulates the key transcription factor HIF-1 independently of the checkpoint kinase Chk1. We show that ATR kinase activity regulates HIF-1α at the translational level and we find that the elements necessary for the regulation of HIF-1α translation are located within the coding region of HIF-1α mRNA. Finally, by using three independent cellular models, we clearly show that the loss of ATR expression and/or kinase activity results in the decrease of HIF-1 DNA binding under hypoxia and consequently affects protein expression levels of two HIF-1 target genes, GLUT-1 and CAIX. Taken together, our data show a new function for ATR in cellular adaptation to hypoxia through regulation of HIF-1α translation. Our work offers new prospect for cancer therapy using ATR inhibitors with the potential to decrease cellular adaptation in hypoxic

  5. FGF2 translationally induced by hypoxia is involved in negative and positive feedback loops with HIF-1alpha.

    Directory of Open Access Journals (Sweden)

    Caroline Conte

    Full Text Available BACKGROUND: Fibroblast growth factor 2 (FGF2 is a major angiogenic factor involved in angiogenesis and arteriogenesis, however the regulation of its expression during these processes is poorly documented. FGF2 mRNA contains an internal ribosome entry site (IRES, a translational regulator expected to allow mRNA expression during cellular stress. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we have developed a skin ischemia model in transgenic mice expressing a reporter transgene under the control of the FGF2 IRES. The results reveal that FGF2 is induced at the protein level during ischemia, concomitant with HIF-1alpha induction and a decrease in FGF2 mRNA. In addition, the FGF2 IRES is strongly activated under these ischemic conditions associated with hypoxia, whereas cap-dependent translation is repressed by 4E-BP hypophosphorylation. We also show that up-regulation of FGF2 protein expression in response to hypoxia correlates with the increase of FGF2 IRES activity in vitro, in human retinoblasts 911. The use of siRNAs targeting HIF or FGF2 indicates that FGF2 and HIF-1alpha reciprocally regulate their expression/accumulation, by a negative feedback loop in early hypoxia, followed by a positive feedback loop in late hypoxia. CONCLUSION/SIGNIFICANCE: FGF2 expression is up-regulated in vivo and in vitro in response to hypoxia. Strikingly, this up-regulation is not transcriptional. It seems to occur by an IRES-dependent mechanism, revealing new mechanistic aspects of the hypoxic response. In addition, our data show that FGF2 interacts with HIF-1alpha in a unique crosstalk, with distinct stages in early and late hypoxia. These data reveal the physiological importance of IRES-dependent translation during hypoxic stress and underline the complexity of the cellular response to hypoxia, suggesting a novel role of FGF2 in the regulation of HIF-1alpha during the induction of angiogenesis.

  6. Translational Regulation in Nutrigenomics12

    Science.gov (United States)

    Liu, Botao; Qian, Shu-Bing

    2011-01-01

    The emergence of genome-wide analysis to interrogate cellular DNA, RNA, and protein content has revolutionized the study of the control network that mediates cellular homeostasis. Nutrigenomics addresses the effect of nutrients on gene expression, which provides a basis for understanding the biological activity of dietary components. Translation of mRNAs represents the last step of genetic flow and primarily defines the proteome. Translational regulation is thus critical for gene expression, in particular, under nutrient excess or deficiency. Until recently, it was unclear how the global effects of translational control are influenced by nutrient signaling. An emerging concept of translational reprogramming addresses how to maintain the expression of specific proteins during pathophysiological conditions by translation of selective mRNAs. Here we describe recent advances in our understanding of translational control, nutrient signaling, and their dysregulation in aging and cancer. The mechanistic understanding of translational regulation in response to different nutrient conditions may help identify potential dietary and therapeutic targets to improve human health. PMID:22332093

  7. Hypoxia-regulated MicroRNAs in gastroesophageal cancer

    DEFF Research Database (Denmark)

    Winther, M.; Alsner, J.; Sørensen, B.S.

    2016-01-01

    Background/aim: The present study aimed to identify hypoxia-regulated microRNAs (HRMs) in vitro and investigate the clinical role of candidate HRMs in patients with gastroesophageal cancer (GEC). Materials and Methods: microRNA expression changes induced by hypoxia in human GEC cell lines were...

  8. Hypoxia-regulated MicroRNAs in Gastroesophageal Cancer

    DEFF Research Database (Denmark)

    Winther, Mette; Alsner, Jan; Sørensen, Brita Singers

    2016-01-01

    BACKGROUND/AIM: The present study aimed to identify hypoxia-regulated microRNAs (HRMs) in vitro and investigate the clinical role of candidate HRMs in patients with gastroesophageal cancer (GEC). MATERIALS AND METHODS: microRNA expression changes induced by hypoxia in human GEC cell lines were...... associations of HRMs and clinical outcome in patients with GEC were identified. CONCLUSION: This study supports the involvement of hypoxia on miRNAs in vitro and confirms the role of miR-210 as being a universal HRM....

  9. Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants

    Science.gov (United States)

    Gibbs, Daniel J.; Lee, Seung Cho; Isa, Nurulhikma Md; Gramuglia, Silvia; Fukao, Takeshi; Bassel, George W.; Correia, Cristina Sousa; Corbineau, Françoise; Theodoulou, Frederica L.; Bailey-Serres, Julia; Holdsworth, Michael J.

    2011-01-01

    Plants and animals are obligate aerobes, requiring oxygen for mitochondrial respiration and energy production. In plants, an unanticipated decline in oxygen availability (hypoxia), as caused by root waterlogging or foliage submergence, triggers changes in gene transcription and mRNA translation that promote anaerobic metabolism and thus sustain substrate-level ATP production1. In contrast to animals2, oxygen sensing has not been ascribed to a mechanism of gene regulation in response to oxygen deprivation in plants. Here we show that the N-end rule pathway of targeted proteolysis acts as a homeostatic sensor of severe low oxygen in Arabidopsis, through its regulation of key hypoxia response transcription factors. We found that plants lacking components of the N-end rule pathway constitutively express core hypoxia response genes and are more tolerant of hypoxic stress. We identify the hypoxia-associated Ethylene Response Factor (ERF) Group VII transcription factors of Arabidopsis as substrates of this pathway. Regulation of these proteins by the N-end rule pathway occurs through a characteristic conserved motif at the N-terminus initiating with MetCys- (MC-). Enhanced stability of one of these proteins, HRE2, under low oxygen conditions improves hypoxia survival and reveals a molecular mechanism for oxygen sensing in plants via the evolutionarily conserved N-end rule pathway. SUB1A-1, a major determinant of submergence tolerance in rice3, was shown not to be a substrate for the N-end rule pathway despite containing the N-terminal motif, suggesting that it is uncoupled from N-end rule pathway regulation, and that enhanced stability may relate to the superior tolerance of Sub1 rice varieties to multiple abiotic stresses4. PMID:22020279

  10. Drosophila genome-wide RNAi screen identifies multiple regulators of HIF-dependent transcription in hypoxia.

    Directory of Open Access Journals (Sweden)

    Andrés Dekanty

    2010-06-01

    Full Text Available Hypoxia-inducible factors (HIFs are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1 gene, a central element of the microRNA (miRNA translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF-dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF-related pathologies, including heart attack, cancer, and stroke.

  11. Germinal centre hypoxia and regulation of antibody qualities by a hypoxia response system.

    Science.gov (United States)

    Cho, Sung Hoon; Raybuck, Ariel L; Stengel, Kristy; Wei, Mei; Beck, Thomas C; Volanakis, Emmanuel; Thomas, James W; Hiebert, Scott; Haase, Volker H; Boothby, Mark R

    2016-09-08

    Germinal centres (GCs) promote humoral immunity and vaccine efficacy. In GCs, antigen-activated B cells proliferate, express high-affinity antibodies, promote antibody class switching, and yield B cell memory. Whereas the cytokine milieu has long been known to regulate effector functions that include the choice of immunoglobulin class, both cell-autonomous and extrinsic metabolic programming have emerged as modulators of T-cell-mediated immunity. Here we show in mice that GC light zones are hypoxic, and that low oxygen tension () alters B cell physiology and function. In addition to reduced proliferation and increased B cell death, low impairs antibody class switching to the pro-inflammatory IgG2c antibody isotype by limiting the expression of activation-induced cytosine deaminase (AID). Hypoxia induces HIF transcription factors by restricting the activity of prolyl hydroxyl dioxygenase enzymes, which hydroxylate HIF-1α and HIF-2α to destabilize HIF by binding the von Hippel-Landau tumour suppressor protein (pVHL). B-cell-specific depletion of pVHL leads to constitutive HIF stabilization, decreases antigen-specific GC B cells and undermines the generation of high-affinity IgG, switching to IgG2c, early memory B cells, and recall antibody responses. HIF induction can reprogram metabolic and growth factor gene expression. Sustained hypoxia or HIF induction by pVHL deficiency inhibits mTOR complex 1 (mTORC1) activity in B lymphoblasts, and mTORC1-haploinsufficient B cells have reduced clonal expansion, AID expression, and capacities to yield IgG2c and high-affinity antibodies. Thus, the normal physiology of GCs involves regional variegation of hypoxia, and HIF-dependent oxygen sensing regulates vital functions of B cells. We propose that the restriction of oxygen in lymphoid organs, which can be altered in pathophysiological states, modulates humoral immunity.

  12. Hypoxia-Inducible Factor 3 Is an Oxygen-Dependent Transcription Activator and Regulates a Distinct Transcriptional Response to Hypoxia

    Directory of Open Access Journals (Sweden)

    Peng Zhang

    2014-03-01

    Full Text Available Hypoxia-inducible factors (HIFs play key roles in the cellular response to hypoxia. It is widely accepted that whereas HIF-1 and HIF-2 function as transcriptional activators, HIF-3 inhibits HIF-1/2α action. Contrary to this idea, we show that zebrafish Hif-3α has strong transactivation activity. Hif-3α is degraded under normoxia. Mutation of P393, P493, and L503 inhibits this oxygen-dependent degradation. Transcriptomics and chromatin immunoprecipitation analyses identify genes that are regulated by Hif-3α, Hif-1α, or both. Under hypoxia or when overexpressed, Hif-3α binds to its target gene promoters and upregulates their expression. Dominant-negative inhibition and knockdown of Hif-3α abolish hypoxia-induced Hif-3α-promoter binding and gene expression. Hif-3α not only mediates hypoxia-induced growth and developmental retardation but also possesses hypoxia-independent activities. Importantly, transactivation activity is conserved and human HIF-3α upregulates similar genes in human cells. These findings suggest that Hif-3 is an oxygen-dependent transcription factor and activates a distinct transcriptional response to hypoxia.

  13. Epithelial Barrier Regulation by Hypoxia-Inducible Factor.

    Science.gov (United States)

    Glover, Louise E; Colgan, Sean P

    2017-09-01

    Mucosal tissues represent surfaces that are exposed to the outside world and provide a conduit for internal and external communication. Tissues such as the intestine and the lung are lined by layer(s) of epithelial cells that, when organized in three dimensions, provide a critical barrier to the flux of luminal contents. This selective barrier is provided through the regulated expression of junctional proteins and mucins. Tissue oxygen metabolism is central to the maintenance of homeostasis in the mucosa. In some organs (e.g., the colon), low baseline Po2 determines tissue metabolism and results in basal expression of the transcription factor, hypoxia-inducible factor (HIF), which is enhanced after ischemia/inflammation. Recent studies have indicated that HIF contributes fundamentally to the expression of barrier-related genes and in the regulation of barrier-adaptive responses within the mucosa. Here, we briefly review recent literature on the topic of hypoxia and HIF regulation of barrier in mucosal health and during disease.

  14. Fyn kinase regulates translation in mammalian mitochondria.

    Science.gov (United States)

    Koc, Emine C; Miller-Lee, Jennifer L; Koc, Hasan

    2017-03-01

    Mitochondrial translation machinery solely exists for the synthesis of 13 mitochondrially-encoded subunits of the oxidative phosphorylation (OXPHOS) complexes in mammals. Therefore, it plays a critical role in mitochondrial energy production. However, regulation of the mitochondrial translation machinery is still poorly understood. In comprehensive proteomics studies with normal and diseased tissues and cell lines, we and others have found the majority of mitochondrial ribosomal proteins (MRPs) to be phosphorylated. Neither the kinases for these phosphorylation events nor their specific roles in mitochondrial translation are known. Mitochondrial kinases are responsible for phosphorylation of MRPs enriched from bovine mitoplasts by strong cation-exchange chromatography and identified by mass spectrometry-based proteomics analyses of kinase rich fractions. Phosphorylation of recombinant MRPs and 55S ribosomes was assessed by in vitro phosphorylation assays using the kinase-rich fractions. The effect of identified kinase on OXPHOS and mitochondrial translation was assessed by various cell biological and immunoblotting approaches. Here, we provide the first evidence for the association of Fyn kinase, a Src family kinase, with mitochondrial translation components and its involvement in phosphorylation of 55S ribosomal proteins in vitro. Modulation of Fyn expression in human cell lines has provided a link between mitochondrial translation and energy metabolism, which was evident by the changes in 13 mitochondrially encoded subunits of OXPHOS complexes. Our findings suggest that Fyn kinase is part of a complex mechanism that regulates protein synthesis and OXPHOS possibly by tyrosine phosphorylation of translation components in mammalian mitochondria. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Regulation of cerebral blood flow in mammals during chronic hypoxia: a matter of balance.

    Science.gov (United States)

    Ainslie, Philip N; Ogoh, Shigehiko

    2010-02-01

    Respiratory-induced changes in the partial pressures of arterial carbon dioxide (PaCO2) and oxygen (PaO2) play a major role in cerebral blood flow (CBF) regulation. Elevations in PaCO2 (hypercapnia) lead to vasodilatation and increases in CBF, whereas reductions in PaCO2 (hypocapnia) lead to vasoconstriction and decreases in CBF. A fall in PaO2 (hypoxia) below a certain threshold (balance between the myriad of vasodilators and constrictors derived from the endothelium, neuronal innervations and perfusion pressure. This review examines the extent and mechanisms by which hypoxia regulates CBF. Particular focus will be given to the marked influence of hypoxia associated with exposure to high altitude and chronic lung disease. The associated implications of these hypoxia-induced integrative alterations for the regulation of CBF are discussed, and future avenues for research are proposed.

  16. Eukaryotic translation initiation factor 5A (eIF5A) is essential for HIF-1α activation in hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Tariq, Mohammad [Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Graduate School of Science and Engineering, Saitama University, 645 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan); Ito, Akihiro, E-mail: akihiro-i@riken.jp [Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Japan Agency for Medical Research and Development, AMED-CREST, 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100-0004 (Japan); Ishfaq, Muhammad; Bradshaw, Elliot [Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Graduate School of Science and Engineering, Saitama University, 645 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan); Yoshida, Minoru [Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Graduate School of Science and Engineering, Saitama University, 645 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan); Japan Agency for Medical Research and Development, AMED-CREST, 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100-0004 (Japan)

    2016-02-05

    The eukaryotic initiation factor 5A (eIF5A) is an essential protein involved in translation elongation and cell proliferation. eIF5A undergoes several post-translational modifications including hypusination and acetylation. Hypusination is indispensable for the function of eIF5A. On the other hand, the precise function of acetylation remains unknown, but it may render the protein inactive since hypusination blocks acetylation. Here, we report that acetylation of eIF5A increases under hypoxia. During extended hypoxic periods an increase in the level of eIF5A acetylation correlated with a decrease in HIF-1α, suggesting involvement of eIF5A activity in HIF-1α expression under hypoxia. Indeed, suppression of eIF5A by siRNA oligo-mediated knockdown or treatment with GC7, a deoxyhypusine synthase inhibitor, led to significant reduction of HIF-1α activity. Furthermore, knockdown of eIF5A or GC7 treatment reduced tumor spheroid formation with a concomitant decrease in HIF-1α expression. Our results suggest that functional, hypusinated eIF5A is necessary for HIF-1α expression during hypoxia and that eIF5A is an attractive target for cancer therapy. - Highlights: • Hypoxia induces acetylation of eIF5A. • Active eIF5A is necessary for HIF-1α activation in hypoxia. • Active eIF5A is important for tumor spheroid growth.

  17. Stromal Expression of Hypoxia Regulated Proteins Is an Adverse Prognostic Factor in Colorectal Carcinomas

    Directory of Open Access Journals (Sweden)

    Arjen H. G. Cleven

    2007-01-01

    Full Text Available Background: Hypoxia modifies the phenotype of tumors in a way that promotes tumor aggressiveness and resistance towards chemotherapy and radiotherapy. However, the expression and influence of hypoxia-regulated proteins on tumor biology are not well characterized in colorectal tumors. We studied the role of protein expression of hypoxia-inducible factor (HIF-1α, HIF-2α, carbonic anhydrase 9 (CA9 and glucose transporter 1 (GLUT1 in patients with colorectal adenocarcinomas. Methods: Expression of HIF-1α, HIF-2α, CA9 and GLUT1 was quantified by immunohistochemistry in 133 colorectal adenocarcinomas. The expression of hypoxia markers was correlated with clinicopathological variables and overall patient survival. Results: Expression of these hypoxia markers was detected in the epithelial compartment of the tumor cells as well as in tumor-associated stromal cells. Although tumor cells frequently showed expression of one or more of the investigated hypoxia markers, no correlation among these markers or with clinical response was found. However, within the tumor stroma, positive correlations between the hypoxia markers HIF-2α, CA9 and GLUT1 were observed. Furthermore expression of HIF-2α and CA9 in tumor-associated stroma were both associated with a significantly reduced overall survival. In the Cox proportional hazard model, stromal HIF-2α expression was an independent prognostic factor for survival. Conclusion: These observations show, that expression of hypoxia regulated proteins in tumor-associated stromal cells, as opposed to their expression in epithelial tumor cells, is associated with poor outcome in colorectal cancer. This study suggests that tumor hypoxia may influence tumor-associated stromal cells in a way that ultimately contributes to patient prognosis.

  18. Multi-dimensional histone methylations for coordinated regulation of gene expression under hypoxia.

    Science.gov (United States)

    Lee, Seongyeol; Lee, Jieon; Chae, Sehyun; Moon, Yunwon; Lee, Ho-Youl; Park, Bongju; Yang, Eun Gyeong; Hwang, Daehee; Park, Hyunsung

    2017-11-16

    Hypoxia increases both active and repressive histone methylation levels via decreased activity of histone demethylases. However, how such increases coordinately regulate induction or repression of hypoxia-responsive genes is largely unknown. Here, we profiled active and repressive histone tri-methylations (H3K4me3, H3K9me3, and H3K27me3) and analyzed gene expression profiles in human adipocyte-derived stem cells under hypoxia. We identified differentially expressed genes (DEGs) and differentially methylated genes (DMGs) by hypoxia and clustered the DEGs and DMGs into four major groups. We found that each group of DEGs was predominantly associated with alterations in only one type among the three histone tri-methylations. Moreover, the four groups of DEGs were associated with different TFs and localization patterns of their predominant types of H3K4me3, H3K9me3 and H3K27me3. Our results suggest that the association of altered gene expression with prominent single-type histone tri-methylations characterized by different localization patterns and with different sets of TFs contributes to regulation of particular sets of genes, which can serve as a model for coordinated epigenetic regulation of gene expression under hypoxia. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Tumor suppressor p53 negatively regulates glycolysis stimulated by hypoxia through its target RRAD

    Science.gov (United States)

    Wu, Rui; Liang, Yingjian; Lin, Meihua; Liu, Jia; Chan, Chang S.; Hu, Wenwei; Feng, Zhaohui

    2014-01-01

    Cancer cells display enhanced glycolysis to meet their energetic and biosynthetic demands even under normal oxygen concentrations. Recent studies have revealed that tumor suppressor p53 represses glycolysis under normoxia as a novel mechanism for tumor suppression. As the common microenvironmental stress for tumors, hypoxia drives the metabolic switch from the oxidative phosphorylation to glycolysis, which is crucial for survival and proliferation of cancer cells under hypoxia. The p53's role and mechanism in regulating glycolysis under hypoxia is poorly understood. Here, we found that p53 represses hypoxia-stimulated glycolysis in cancer cells through RRAD, a newly-identified p53 target. RRAD expression is frequently decreased in lung cancer. Ectopic expression of RRAD greatly reduces glycolysis whereas knockdown of RRAD promotes glycolysis in lung cancer cells. Furthermore, RRAD represses glycolysis mainly through inhibition of GLUT1 translocation to the plasma membrane. Under hypoxic conditions, p53 induces RRAD, which in turn inhibits the translocation of GLUT1 and represses glycolysis in lung cancer cells. Blocking RRAD by siRNA greatly abolishes p53's function in repressing glycolysis under hypoxia. Taken together, our results revealed an important role and mechanism of p53 in antagonizing the stimulating effect of hypoxia on glycolysis, which contributes to p53's function in tumor suppression. PMID:25114038

  20. Widespread translational control contributes to the regulation of Arabidopsis photomorphogenesis

    Science.gov (United States)

    Liu, Ming-Jung; Wu, Szu-Hsien; Chen, Ho-Ming; Wu, Shu-Hsing

    2012-01-01

    Environmental ‘light' has a vital role in regulating plant growth and development. Transcriptomic profiling has been widely used to examine how light regulates mRNA levels on a genome-wide scale, but the global role of translational regulation in the response to light is unknown. Through a transcriptomic comparison of steady-state and polysome-bound mRNAs, we reveal a clear impact of translational control on thousands of genes, in addition to transcriptomic changes, during photomorphogenesis. Genes encoding ribosomal protein are preferentially regulated at the translational level, which possibly contributes to the enhanced translation efficiency. We also reveal that mRNAs regulated at the translational level share characteristics of longer half-lives and shorter cDNA length, and that transcripts with a cis-element, TAGGGTTT, in their 5′ untranslated region have higher translatability. We report a previously neglected aspect of gene expression regulation during Arabidopsis photomorphogenesis. The identities and molecular signatures associated with mRNAs regulated at the translational level also offer new directions for mechanistic studies of light-triggered translational enhancement in Arabidopsis. PMID:22252389

  1. Regulation of mRNA translation during mitosis.

    Science.gov (United States)

    Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D

    2015-08-25

    Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

  2. Loss of PINK1 attenuates HIF-1α induction by preventing 4E-BP1-dependent switch in protein translation under hypoxia.

    Science.gov (United States)

    Lin, William; Wadlington, Natasha L; Chen, Linan; Zhuang, Xiaoxi; Brorson, James R; Kang, Un Jung

    2014-02-19

    Parkinson's disease (PD) has multiple proposed etiologies with implication of abnormalities in cellular homeostasis ranging from proteostasis to mitochondrial dynamics to energy metabolism. PINK1 mutations are associated with familial PD and here we discover a novel PINK1 mechanism in cellular stress response. Using hypoxia as a physiological trigger of oxidative stress and disruption in energy metabolism, we demonstrate that PINK1(-/-) mouse cells exhibited significantly reduced induction of HIF-1α protein, HIF-1α transcriptional activity, and hypoxia-responsive gene upregulation. Loss of PINK1 impairs both hypoxia-induced 4E-BP1 dephosphorylation and increase in the ratio of internal ribosomal entry site (IRES)-dependent to cap-dependent translation. These data suggest that PINK1 mediates adaptive responses by activating IRES-dependent translation, and the impairments in translation and the HIF-1α pathway may contribute to PINK1-associated PD pathogenesis that manifests under cellular stress.

  3. Hypoxia Up-Regulates Galectin-3 in Mammary Tumor Progression and Metastasis

    Science.gov (United States)

    Barros, Rita; Gomes, Catarina; de Matos, Augusto J.; Reis, Celso A.; Rutteman, Gerard R.; Gärtner, Fátima

    2015-01-01

    The tumor microenvironment encompasses several stressful conditions for cancer cells such as hypoxia, oxidative stress and pH alterations. Galectin-3, a well-studied member of the beta-galactoside-binding animal family of lectins has been implicated in multiple steps of metastasis as cell-cell and cell-ECM adhesion, promotion of angiogenesis, cell proliferation and resistance to apoptosis. However, both its aberrantly up- and down-regulated expression was observed in several types of cancer. Thus, the mechanisms that regulate galectin-3 expression in neoplastic settings are not clear. In order to demonstrate the putative role of hypoxia in regulating galectin-3 expression in canine mammary tumors (CMT), in vitro and in vivo studies were performed. In malignant CMT cells, hypoxia was observed to induce expression of galectin-3, a phenomenon that was almost completely prevented by catalase treatment of CMT-U27 cells. Increased galectin-3 expression was confirmed at the mRNA level. Under hypoxic conditions the expression of galectin-3 shifts from a predominant nuclear location to cytoplasmic and membrane expressions. In in vivo studies, galectin-3 was overexpressed in hypoxic areas of primary tumors and well-established metastases. Tumor hypoxia thus up-regulates the expression of galectin-3, which may in turn increase tumor aggressiveness. PMID:26222311

  4. Hypoxia reduces mature hERG channels through calpain up-regulation.

    Science.gov (United States)

    Lamothe, Shawn M; Song, WonJu; Guo, Jun; Li, Wentao; Yang, Tonghua; Baranchuk, Adrian; Graham, Charles H; Zhang, Shetuan

    2017-11-01

    Human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium current (IKr) potassium channel, which is important for cardiac repolarization. Impairment of hERG function is the primary cause of acquired long QT syndrome, which predisposes individuals to cardiac arrhythmias and sudden death. Patients with hypoxia due to conditions such as cardiac ischemia or obstructive sleep apnea display increased incidence of cardiac arrhythmias and sudden death. We sought to understand the mechanisms that underlie hypoxia-associated cardiac arrhythmias. Using cell biology and electrophysiologic techniques, we found that hypoxic culture of hERG-expressing human embryonic kidney (HEK) cells and neonatal rat cardiomyocytes reduced hERG current/IKr and mature ERG channel expression with a concomitant increase in calpain expression. Calpain was actively released into the extracellular milieu and degraded cell-surface hERG. In contrast to hERG, the ether-a-go-go (EAG) channel was not reduced by hypoxic culture. By making chimeric channels between hERG and EAG, we identified that hypoxia-induced calpain degraded hERG by targeting its extracellular S5-pore linker. The scorpion toxin BeKm-1, which is known to selectively bind to the S5-pore linker of hERG, prevented hypoxia-induced hERG reduction. Our data provide novel information about hypoxia-mediated hERG dysfunction and may have biological and clinical implications in hypoxia-associated diseases.-Lamothe, S. M., Song, W., Guo, J., Li, W., Yang, T., Baranchuk, A., Graham, C. H., Zhang, S. Hypoxia reduces mature hERG channels through calpain up-regulation. © FASEB.

  5. Acute and Chronic Sustained Hypoxia Do Not Substantially Regulate Amyloid-β Peptide Generation In Vivo.

    Directory of Open Access Journals (Sweden)

    Alberto Serrano-Pozo

    Full Text Available Recent epidemiological evidence has linked hypoxia with the development of Alzheimer disease (AD. A number of in vitro and in vivo studies have reported that hypoxia can induce amyloid-β peptide accumulation through various molecular mechanisms including the up-regulation of the amyloid-β precursor protein, the β-secretase Bace1, or the γγ-secretase complex components, as well as the down-regulation of Aβ-degrading enzymes.To investigate the effects of acute and chronic sustained hypoxia in Aβ generation in vivo.2-3 month-old C57/Bl6J wild-type mice were exposed to either normoxia (21% O2 or hypoxia (9% O2 for either 4 to 72 h (acute or 21-30 days (chronic sustained in a hermetic chamber. Brain mRNA levels of Aβ-related genes were measured by quantitative real-time PCR, whereas levels of Bace1 protein, full length AβPP, and its C-terminal fragments (C99/C88 ratio were measured by Western blot. In addition, 8 and 14-month-old APP/PS1 transgenic mice were subjected to 9% O2 for 21 days and levels of Aβ40, Aβ42, full length AβPP, and soluble AβPPα (sAβPPα were measured by ELISA or WB.Hypoxia (either acute or chronic sustained did not impact the transcription of any of the Aβ-related genes in young wild-type mice. A significant reduction of Bace1 protein level was noted with acute hypoxia for 16 h but did not correlate with an increased level of full length AβPP or a decreased C99/C83 ratio. Chronic sustained hypoxia did not significantly alter the levels of Bace1, full length AβPP or the C99/C83 ratio. Last, chronic sustained hypoxia did not significantly change the levels of Aβ40, Aβ42, full length AβPP, or sAβPPα in either young or aged APP/PS1 mice.Our results argue against a hypoxia-induced shift of AβPP proteolysis from the non-amyloidogenic to the amyloidogenic pathways. We discuss the possible methodological caveats of previous in vivo studies.

  6. Hypoxia Regulates mTORC1-Mediated Keratinocyte Motility and Migration via the AMPK Pathway.

    Directory of Open Access Journals (Sweden)

    Tiantian Yan

    Full Text Available Keratinocyte migration, the initial event and rate-limiting step in wound healing, plays a vital role in restoration of the intact skin barrier, also known as re-epithelialization. After acute tissue injury, hypoxic microenvironment gradually develops and acts as an early stimulus to initiate the healing process. Although we have previously found that hypoxia induces keratinocyte migration, the underlying mechanism remains unknown. Here, we first observed that hypoxia increased mTORC1 activity. Recombinant lentivirus vector and Rapamycin were used for silencing mTORC1 in HaCaT cells and primary mouse keratinocytes (MKs. Using cell migration assay and a Zeiss chamber equipped with imaging system, we also demonstrated that mTORC1 downregulation reversed hypoxia-induced keratinocyte motility and lateral migration. Importantly, hypoxia-activated mTORC1 was accompanied by the AMPK downregulation, and we found that the AMPK pathway activators Metformin (Met and 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR decreased the mTORC1 activity, cell motility and lateral migration. Thus, our results suggest that hypoxia regulates mTORC1-mediated keratinocyte motility and migration via the AMPK pathway.

  7. Up-regulation of erythropoietin receptor by nitric oxide mediates hypoxia preconditioning.

    Science.gov (United States)

    Chen, Zhi-Yong; Wang, Li; Asavaritkrai, Pundit; Noguchi, Constance Tom

    2010-11-01

    Erythropoietin (Epo), known to stimulate erythroid progenitor cell survival, proliferation, and differentiation, has been shown to be neuroprotective against brain ischemia in animal models. Both Epo and Epo receptor (EpoR) are expressed in the brain and are up-regulated by hypoxia. Brain Epo signaling can stimulate neural cell survival and prevent neuron apoptosis. Neurons from EpoR null mice exhibit marked increased sensitivity to hypoxia. In endothelial cells, Epo has been shown to stimulate nitric oxide (NO) production, particularly at low pO(2). We found here that the EpoR expression on neural cells and Epo's neuroprotective effect were regulated by NO. Hypoxia increased NO production as well as EpoR expression, and inhibition of NOS activity reduced the proportion of EpoR-expressing neurons induced at low pO(2). Conversely, addition of NO donor to cultures grown under normoxia induced EpoR. Similarly, NO donor increased EpoR promoter activity in a reporter gene assay, suggesting that NO regulates EpoR at the transcription level. Preincubation of neurons with NO results in induction of EpoR, which gives rise to protection against hypoxia even in the absence of exogenous Epo, although at high concentration NO is toxic. These data provide evidence of a role for NO in Epo activity in brain and suggest links between NO production, EpoR expression, and Epo signaling in neuroprotection.

  8. Structure of the chloroplast ribosome: novel domains for translation regulation.

    Directory of Open Access Journals (Sweden)

    Andrea L Manuell

    2007-08-01

    Full Text Available Gene expression in chloroplasts is controlled primarily through the regulation of translation. This regulation allows coordinate expression between the plastid and nuclear genomes, and is responsive to environmental conditions. Despite common ancestry with bacterial translation, chloroplast translation is more complex and involves positive regulatory mRNA elements and a host of requisite protein translation factors that do not have counterparts in bacteria. Previous proteomic analyses of the chloroplast ribosome identified a significant number of chloroplast-unique ribosomal proteins that expand upon a basic bacterial 70S-like composition. In this study, cryo-electron microscopy and single-particle reconstruction were used to calculate the structure of the chloroplast ribosome to a resolution of 15.5 A. Chloroplast-unique proteins are visualized as novel structural additions to a basic bacterial ribosome core. These structures are located at optimal positions on the chloroplast ribosome for interaction with mRNAs during translation initiation. Visualization of these chloroplast-unique structures on the ribosome, combined with mRNA cross-linking, allows us to propose a model for translation initiation in chloroplasts in which chloroplast-unique ribosomal proteins interact with plastid-specific translation factors and RNA elements to facilitate regulated translation of chloroplast mRNAs.

  9. Regulation of Translation Initiation under Biotic and Abiotic Stresses

    Directory of Open Access Journals (Sweden)

    Ana B. Castro-Sanz

    2013-02-01

    Full Text Available Plants have developed versatile strategies to deal with the great variety of challenging conditions they are exposed to. Among them, the regulation of translation is a common target to finely modulate gene expression both under biotic and abiotic stress situations. Upon environmental challenges, translation is regulated to reduce the consumption of energy and to selectively synthesize proteins involved in the proper establishment of the tolerance response. In the case of viral infections, the situation is more complex, as viruses have evolved unconventional mechanisms to regulate translation in order to ensure the production of the viral encoded proteins using the plant machinery. Although the final purpose is different, in some cases, both plants and viruses share common mechanisms to modulate translation. In others, the mechanisms leading to the control of translation are viral- or stress-specific. In this paper, we review the different mechanisms involved in the regulation of translation initiation under virus infection and under environmental stress in plants. In addition, we describe the main features within the viral RNAs and the cellular mRNAs that promote their selective translation in plants undergoing biotic and abiotic stress situations.

  10. 3D culture broadly regulates tumor cell hypoxia response and angiogenesis via pro-inflammatory pathways.

    Science.gov (United States)

    DelNero, Peter; Lane, Maureen; Verbridge, Scott S; Kwee, Brian; Kermani, Pouneh; Hempstead, Barbara; Stroock, Abraham; Fischbach, Claudia

    2015-07-01

    Oxygen status and tissue dimensionality are critical determinants of tumor angiogenesis, a hallmark of cancer and an enduring target for therapeutic intervention. However, it is unclear how these microenvironmental conditions interact to promote neovascularization, due in part to a lack of comprehensive, unbiased data sets describing tumor cell gene expression as a function of oxygen levels within three-dimensional (3D) culture. Here, we utilized alginate-based, oxygen-controlled 3D tumor models to study the interdependence of culture context and the hypoxia response. Microarray gene expression analysis of tumor cells cultured in 2D versus 3D under ambient or hypoxic conditions revealed striking interdependence between culture dimensionality and hypoxia response, which was mediated in part by pro-inflammatory signaling pathways. In particular, interleukin-8 (IL-8) emerged as a major player in the microenvironmental regulation of the hypoxia program. Notably, this interaction between dimensionality and oxygen status via IL-8 increased angiogenic sprouting in a 3D endothelial invasion assay. Taken together, our data suggest that pro-inflammatory pathways are critical regulators of tumor hypoxia response within 3D environments that ultimately impact tumor angiogenesis, potentially providing important therapeutic targets. Furthermore, these results highlight the importance of pathologically relevant tissue culture models to study the complex physical and chemical processes by which the cancer microenvironment mediates new vessel formation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells.

    Directory of Open Access Journals (Sweden)

    Veronica Kalhori

    Full Text Available Sphingosine-1-phosphate (S1P is a bioactive lipid, which regulates several cancer-related processes including migration and angiogenesis. We have previously shown S1P to induce migration of follicular ML-1 thyroid cancer cells. Hypoxia-induced factor-1 (HIF-1 is an oxygen-sensitive transcription factor, which adapts cells to hypoxic conditions through increased survival, motility and angiogenesis. Due to these properties and its increased expression in response to intratumoral hypoxia, HIF-1 is considered a significant regulator of tumor biology. We found S1P to increase expression of the regulatory HIF-1α subunit in normoxic ML-1 cells. S1P also increased HIF-1 activity and expression of HIF-1 target genes. Importantly, inhibition or knockdown of HIF-1α attenuated the S1P-induced migration of ML-1 cells. S1P-induced HIF-1α expression was mediated by S1P receptor 3 (S1P3, Gi proteins and their downstream effectors MEK, PI3K, mTOR and PKCβI. Half-life measurements with cycloheximide indicated that S1P treatment stabilized the HIF-1α protein. On the other hand, S1P activated translational regulators eIF-4E and p70S6K, which are known to control HIF-1α synthesis. In conclusion, we have identified S1P as a non-hypoxic regulator of HIF-1 activity in thyroid cancer cells, studied the signaling involved in S1P-induced HIF-1α expression and shown S1P-induced migration to be mediated by HIF-1.

  12. Proteomic analysis of signaling network regulation in renal cell carcinomas with differential hypoxia-inducible factor-2α expression.

    Directory of Open Access Journals (Sweden)

    Lokesh Dalasanur Nagaprashantha

    Full Text Available BACKGROUND: The loss of von Hippel-Lindau (VHL protein function leads to highly vascular renal tumors characterized by an aggressive course of disease and refractoriness to chemotherapy and radiotherapy. Loss of VHL in renal tumors also differs from tumors of other organs in that the oncogenic cascade is mediated by an increase in the levels of hypoxia-inducible factor-2α (HIF2α instead of hypoxia-inducible factor-1α (HIF1α. METHODS AND PRINCIPAL FINDINGS: We used renal carcinoma cell lines that recapitulate the differences between mutant VHL and wild-type VHL genotypes. Utilizing a method relying on extracted peptide intensities as a label-free approach for quantitation by liquid chromatography-mass spectrometry, our proteomics study revealed regulation of key proteins important for cancer cell survival, proliferation and stress-resistance, and implicated differential regulation of signaling networks in VHL-mutant renal cell carcinoma. We also observed upregulation of cellular energy pathway enzymes and the stress-responsive mitochondrial 60-kDa heat shock protein. Finding reliance on glutaminolysis in VHL-mutant renal cell carcinoma was of particular significance, given the generally predominant dependence of tumors on glycolysis. The data have been deposited to the ProteomeXchange with identifier PXD000335. CONCLUSIONS AND SIGNIFICANCE: Pathway analyses provided corroborative evidence for differential regulation of molecular and cellular functions influencing cancer energetics, metabolism and cell proliferation in renal cell carcinoma with distinct VHL genotype. Collectively, the differentially regulated proteome characterized by this study can potentially guide translational research specifically aimed at effective clinical interventions for advanced VHL-mutant, HIF2α-over-expressing tumors.

  13. Hypoxia regulates microRNA expression in the human carotid body

    Energy Technology Data Exchange (ETDEWEB)

    Mkrtchian, Souren, E-mail: souren.mkrtchian@ki.se [Section for Anesthesiology and Intensive Care Medicine, Department of Physiology and Pharmacology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Lee, Kian Leong, E-mail: csilkl@nus.edu.sg [Cancer Science Institute of Singapore, National University of Singapore, 117599 Singapore (Singapore); Kåhlin, Jessica [Section for Anesthesiology and Intensive Care Medicine, Department of Physiology and Pharmacology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Function Perioperative Medicine and Intensive Care, Karolinska University Hospital, SE-171 76 Stockholm (Sweden); Ebberyd, Anette [Section for Anesthesiology and Intensive Care Medicine, Department of Physiology and Pharmacology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Poellinger, Lorenz [Cancer Science Institute of Singapore, National University of Singapore, 117599 Singapore (Singapore); Department of Cell and Molecular Biology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Fagerlund, Malin Jonsson; Eriksson, Lars I. [Section for Anesthesiology and Intensive Care Medicine, Department of Physiology and Pharmacology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Function Perioperative Medicine and Intensive Care, Karolinska University Hospital, SE-171 76 Stockholm (Sweden)

    2017-03-15

    The carotid body (CB) is the key sensing organ for physiological oxygen levels in the body. Under conditions of low oxygen (hypoxia), the CB plays crucial roles in signaling to the cardiorespiratory center in the medulla oblongata for the restoration of oxygen homeostasis. How hypoxia regulates gene expression in the human CB remains poorly understood. While limited information on transcriptional regulation in animal CBs is available, the identity and impact of important post-transcriptional regulators such as non-coding RNAs, and in particular miRNAs are not known. Here we show using ex vivo experiments that indeed a number of miRNAs are differentially regulated in surgically removed human CB slices when acute hypoxic conditions were applied. Analysis of the hypoxia-regulated miRNAs shows that they target biological pathways with upregulation of functions related to cell proliferation and immune response and downregulation of cell differentiation and cell death functions. Comparative analysis of the human CB miRNAome with the global miRNA expression patterns of a large number of different human tissues showed that the CB miRNAome had a unique profile which reflects its highly specialized functional status. Nevertheless, the human CB miRNAome is most closely related to the miRNA expression pattern of brain tissues indicating that they may have the most similar developmental origins. - Highlights: • Hypoxia triggers differential expression of many miRNAs in the human carotid body. • This can lead to the upregulation of proliferation and immune response functions. • CB expression profile in the carotid body resembles the miRNA expression pattern in the brain. • miRNAs are involved in the regulation of carotid body functions including oxygen sensing.

  14. Hypoxia-inhibited dual-specificity phosphatase-2 expression in endometriotic cells regulates cyclooxygenase-2 expression.

    Science.gov (United States)

    Wu, Meng-Hsing; Lin, Shih-Chieh; Hsiao, Kuei-Yang; Tsai, Shaw-Jenq

    2011-11-01

    Endometriosis is one of the most common gynaecological diseases that significantly reduces the life qualify of affected women and their families. Aberrant expression of cyclooxygenase-2 (COX-2), and thus over-production of prostaglandin E(2) (PGE(2) ) has been shown to play critical roles in the development of this disease. However, the mechanism responsible for COX-2 over-expression remains obscure. Here, we provide evidence for what we believe is a novel mechanism in regulating COX-2 expression in endometriotic stromal cells. Dual-specificity phosphatase-2 (DUSP2), a nuclear phosphatase that inactivates mitogen-activated protein kinase (MAPK), is markedly down-regulated in stromal cells of ectopic endometriotic tissues, which results in prolonged activation of extracellular signal-regulated kinase (ERK) and p38 MAPK and increased COX-2 expression. Expression of DUSP2 is inhibited by hypoxia inducible factor-1α (HIF-1α) at the transcriptional level. Treatment of normal endometrial stromal cells with hypoxia, or chemicals that cause HIF-1α accumulation, results in DUSP2 down-regulation, prolonged ERK phosphorylation and COX-2 over-expression. In contrast, forced expression of DUSP2 under hypoxia abolishes HIF-1α-induced ERK phosphorylation and COX-2 expression. Furthermore, suppression of DUSP2 by HIF-1α in eutopic endometrial stromal cells increases sensitivity of cox-2 gene to interleukin-1β stimulation, a phenomenon resembling endometriotic stromal cell characteristics. Taken together, these data suggest that DUSP2 is an important molecule in endometrial physiology and that hypoxia-inhibited DUSP2 expression is a critical factor for the development of endometriosis. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  15. Neuroprotection via RNA-binding protein RBM3 expression is regulated by hypothermia but not by hypoxia in human SK-N-SH neurons

    Directory of Open Access Journals (Sweden)

    Rosenthal LM

    2017-05-01

    Full Text Available Lisa-Maria Rosenthal,1 Giang Tong,1 Christoph Walker,1 Sylvia J Wowro,1 Jana Krech,1 Constanze Pfitzer,1,2 Georgia Justus,1 Felix Berger,1,3 Katharina Rose Luise Schmitt1 1Department of Congenital Heart Disease/Pediatric Cardiology, German Heart Institute Berlin, 2Berlin Institute of Health (BIH, 3Department of Pediatric Cardiology, Charité – University Medical Center, Berlin, Germany Objective: Therapeutic hypothermia is an established treatment for perinatal asphyxia. Yet, many term infants continue to die or suffer from neurodevelopmental disability. Several experimental studies have demonstrated a beneficial effect of mild-to-moderate hypothermia after hypoxic injury, but the understanding of hypothermia-induced neuroprotection remains incomplete. In general, global protein synthesis is attenuated by hypothermia, but a small group of RNA-binding proteins including the RNA-binding motif 3 (RBM3 is upregulated in response to cooling. The aim of this study was to establish an in vitro model to investigate the effects of hypoxia and hypothermia on neuronal cell survival, as well as to examine the kinetics of concurrent cold-shock protein RBM3 gene expression. Methods: Experiments were performed by using human SK-N-SH neurons exposed to different oxygen concentrations (21%, 8%, or 0.2% O2 for 24 hours followed by moderate hypothermia (33.5°C or normothermia for 24, 48, or 72 hours. Cell death was determined by quantification of lactate dehydrogenase and neuron-specific enolase releases into the cell cultured medium, and cell morphology was assessed by using immunofluorescence staining. The regulation of RBM3 gene expression was assessed by reverse transcriptase-quantitative polymerase chain reaction and Western blot analysis.Results: Exposure to hypoxia (0.2% O2 for 24 hours resulted in significantly increased cell death in SK-N-SH neurons, whereas exposure to 8% O2 had no significant impact on cell viability. Post-hypoxia treatment with

  16. Ezrin regulating the cytoskeleton remodelling is required for hypoxia-induced Myofibroblast proliferation and migration

    Directory of Open Access Journals (Sweden)

    Bin eYi

    2015-03-01

    Full Text Available Background: Hypoxia pulmonary arterial hypertension (HPAH is a disease of the small vessels characterized by sustained vasoconstriction, thickening of arterial walls, vascular remodelling, and progressive increase in pulmonary vascular resistance, thus leading to right heart failure and finally death. Recent evidence demonstrated that massive Pulmonary Artery Smooth Muscle-like Cells (PASMLCs accumulating in the intima might also be developed from the differentiation of Pulmonary Myofibroblast (PMF of tunica media. And PMF appeared the phenomenon of the cytoskeleton remodeling. So, it would be important in the clarification of the pivotal factors controlling this cytoskeleton structure change. Methods: PMFs were cultured from the normal rats and then divided into three groups and incubated by nomal or hypoxic conditions respectively. mRNA level was evaluated by real time reverse transcription polymerase chain reaction, and protein expression was detected by western blot. Cell proliferation was determined by the MTT and thymidine incorporation assay. Results: Here, we report that the hypoxia increased the expression levels of Ezrin mRNA and protein in PMFs, which might explain the expression of cytoskeletal proteins (Destrin, a1-actin, and a1-tubulin in PMFs was significantly induced by hypoxia. After inhibiting Ezrin in PMFs by siRNA transfection, we found the over-expression of cytoskeletal proteins induced by hypoixa were significantly suppressed at all time points. Additionally, we found that hypoxia or over-expression of Ezrin through Ad-Ezrin transfection significantly increases the proliferation and migration of PMFs, and which could be inverted by the transfection of siRNA. Conclusion: These findings suggest that Ezrin regulating of aberrant dysregulation of cytoskeletal proteins may be the major cause of PMFs' proliferation and migration under the condition of hypoxia and may, therefore, play a fundamental role in the accumulation of

  17. Body temperature regulation during acclimation to cold and hypoxia in rats.

    Science.gov (United States)

    Cadena, V; Tattersall, G J

    2014-12-01

    Extreme environmental conditions present challenges for thermoregulation in homoeothermic organisms such as mammals. Such challenges are exacerbated when two stressors are experienced simultaneously and each stimulus evokes opposing physiological responses. This is the case of cold, which induces an increase in thermogenesis, and hypoxia, which suppresses metabolism conserving oxygen and preventing hypoxaemia. As an initial approach to understanding the thermoregulatory responses to cold and hypoxia in a small mammal, we explored the effects of acclimation to these two stressors on the body temperature (Tb) and the daily and ultradian Tb variations of Sprague-Dawley rats. As Tb is influenced by sleep-wake cycles, these Tb variations reflect underlying adjustments in set-point and thermosensitivity. The Tb of rats decreased precipitously during initial hypoxic exposure which was more pronounced in cold (Tb=33.4 ± 0.13) than in room temperature (Tb=35.74 ± 0.17) conditions. This decline was followed by an increase in Tb stabilising at a new level ~0.5°C and ~1.4°C below normoxic values at room and cold temperatures, respectively. Daily Tb variations were blunted during hypoxia with a greater effect in the cold. Ultradian Tb variations exhibited daily rhythmicity that disappeared under hypoxia, independent of ambient temperature. The adjustments in Tb during hypoxia and/or cold are in agreement with the hypothesis that an initial decrease in the Tb set-point is followed by its partial re-establishment with chronic hypoxia. This rebound of the Tb set-point might reflect cellular adjustments that would allow animals to better deal with low oxygen conditions, diminishing the drive for a lower Tb set-point. Cold and hypoxia are characteristic of high altitude environments. Understanding how mammals cope with changes in oxygen and temperature will shed light into their ability to colonize new environments along altitudinal clines and increase our understanding of how

  18. F11R expression upon hypoxia is regulated by RNA editing.

    Directory of Open Access Journals (Sweden)

    Michal Ben-Zvi

    Full Text Available F11R is a cell adhesion molecule found on the surface of human platelets. It plays a role in platelet aggregation, cell migration and cell proliferation. F11R is subjected to RNA editing, a post-transcriptional modification which affects RNA structure, stability, localization, translation and splicing. RNA editing in the 3'UTR of F11R and RNA levels are increased upon hypoxia. We therefore set to examine if RNA editing plays a role in the increase of F11R RNA seen upon hypoxic conditions. We show that ADAR1, but not ADAR2, takes part in the editing of F11R however editing alone is not sufficient for obtaining an elevation in RNA levels. In addition we show that hyper-edited mature mRNAs are retained in the nucleus and are associated with p54(nrb. We therefore conclude that hypoxia-induced edited RNAs of F11R are preferentially stabilized and accumulate in the nucleus preventing their export to the cytoplasm for translation. This mechanism may be used by additional proteins in the cell as part of the cell's effort to reduce metabolism upon hypoxic stress.

  19. ACLY and ACC1 Regulate Hypoxia-Induced Apoptosis by Modulating ETV4 via α-ketoglutarate.

    Directory of Open Access Journals (Sweden)

    Melissa M Keenan

    2015-10-01

    Full Text Available In order to propagate a solid tumor, cancer cells must adapt to and survive under various tumor microenvironment (TME stresses, such as hypoxia or lactic acidosis. To systematically identify genes that modulate cancer cell survival under stresses, we performed genome-wide shRNA screens under hypoxia or lactic acidosis. We discovered that genetic depletion of acetyl-CoA carboxylase (ACACA or ACC1 or ATP citrate lyase (ACLY protected cancer cells from hypoxia-induced apoptosis. Additionally, the loss of ACLY or ACC1 reduced levels and activities of the oncogenic transcription factor ETV4. Silencing ETV4 also protected cells from hypoxia-induced apoptosis and led to remarkably similar transcriptional responses as with silenced ACLY or ACC1, including an anti-apoptotic program. Metabolomic analysis found that while α-ketoglutarate levels decrease under hypoxia in control cells, α-ketoglutarate is paradoxically increased under hypoxia when ACC1 or ACLY are depleted. Supplementation with α-ketoglutarate rescued the hypoxia-induced apoptosis and recapitulated the decreased expression and activity of ETV4, likely via an epigenetic mechanism. Therefore, ACC1 and ACLY regulate the levels of ETV4 under hypoxia via increased α-ketoglutarate. These results reveal that the ACC1/ACLY-α-ketoglutarate-ETV4 axis is a novel means by which metabolic states regulate transcriptional output for life vs. death decisions under hypoxia. Since many lipogenic inhibitors are under investigation as cancer therapeutics, our findings suggest that the use of these inhibitors will need to be carefully considered with respect to oncogenic drivers, tumor hypoxia, progression and dormancy. More broadly, our screen provides a framework for studying additional tumor cell stress-adaption mechanisms in the future.

  20. Regulation of matrix stiffness on the epithelial-mesenchymal transition of breast cancer cells under hypoxia environment

    Science.gov (United States)

    Lv, Yonggang; Chen, Can; Zhao, Boyuan; Zhang, Xiaomei

    2017-06-01

    Substrate stiffness and hypoxia are associated with tumor development and progression, respectively. However, the synergy of them on the biological behavior of human breast cancer cell is still largely unknown. This study explored how substrate stiffness regulates the cell phenotype, viability, and epithelial-mesenchymal transition (EMT) of human breast cancer cells MCF-7 under hypoxia (1% O2). TRITC-phalloidin staining showed that MCF-7 cells transformed from round to irregular polygon with stiffness increase either in normoxia or hypoxia. While being accompanied with the upward tendency from a 0.5- to a 20-kPa substrate, the percentage of cell apoptosis was significantly higher in hypoxia than that in normoxia, especially on the 20-kPa substrate. Additionally, it was hypoxia, but not normoxia, that promoted the EMT of MCF-7 by upregulating hypoxia-inducible factor-1α (HIF-1α), vimentin, Snail 1, and matrix metalloproteinase 2 (MMP 2) and 9 (MMP 9), and downregulating E-cadherin simultaneously regardless of the change of substrate stiffness. In summary, this study discovered that hypoxia and stiffer substrate (20 kPa) could synergistically induce phenotype change, apoptosis, and EMT of MCF-7 cells. Results of this study have an important significance on further exploring the synergistic effect of stiffness and hypoxia on the EMT of breast cancer cells and its molecular mechanism.

  1. Hypoxia regulates Notch-3 mRNA and receptor activation in prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Armelle Meunier

    2016-05-01

    Full Text Available The Notch-3 receptor is a recognized key regulator of vascular responses and is increasingly associated with tumorigenesis. Hypoxia-inducible factors activate specific signaling pathways such as Notch in a number of cellular models. This study aimed to evaluate the regulation of Notch-3 by hypoxia in prostate cancer cells. Notch-3 gene and protein expression was established in a panel of aerobic and hypoxic prostate cell lines in vitro, the CWR22 xenograft model and RNA extracted from low grade (Gleason score = 7; non-hypoxic (low HIF, low VEGF; hypoxic (high HIF, high VEGF patient FFPE specimens. NOTCH-3 was upregulated in PC3 (3-fold, 22Rv1 (4.1-fold and DU145 (3.8-fold but downregulated in LnCaP (12-fold compared to the normal cell lines. NOTCH-3 expression was modified following hypoxic exposure in these cells. NOTCH-3 was upregulated (2.2-fold in higher grade and hypoxic tumors, when compared to benign and aerobic pools. In the CWR22 xenograft model, Notch-3 expression was restored in castrate resistant tumors. Nuclear translocation of the Notch-3 intracellular domain was no longer detected following exposure of cells to hypoxia but not associated with a change in expression of HES-1. Our data further identifies Notch-3 as a potentially key hypoxic-responsive member of the Notch pathway in prostate tumorigenesis.

  2. Megakaryocytic leukemia 1 (MKL1 regulates hypoxia induced pulmonary hypertension in rats.

    Directory of Open Access Journals (Sweden)

    Zhibin Yuan

    Full Text Available Hypoxia induced pulmonary hypertension (HPH represents a complex pathology that involves active vascular remodeling, loss of vascular tone, enhanced pulmonary inflammation, and increased deposition of extracellular matrix proteins. Megakaryocytic leukemia 1 (MKL1 is a transcriptional regulator known to influence cellular response to stress signals in the vasculature. We report here that in response to chronic hypobaric hypoxia, MKL1 expression was up-regulated in the lungs in rats. Short hairpin RNA (shRNA mediated depletion of MKL1 significantly ameliorated the elevation of pulmonary arterial pressure in vivo with a marked alleviation of vascular remodeling. MKL1 silencing also restored the expression of NO, a key vasoactive molecule necessary for the maintenance of vascular tone. In addition, hypoxia induced pulmonary inflammation was dampened in the absence of MKL1 as evidenced by normalized levels of pro-inflammatory cytokines and chemokines as well as reduced infiltration of pro-inflammatory immune cells in the lungs. Of note, MKL1 knockdown attenuated fibrogenesis in the lungs as indicated by picrosirius red staining. Finally, we demonstrate that MKL1 mediated transcriptional activation of type I collagen genes in smooth muscle cells under hypoxic conditions. In conclusion, we data highlight a previously unidentified role for MKL1 in the pathogenesis of HPH and as such lay down groundwork for future investigation and drug development.

  3. Identification of a hypoxia-regulated miRNA signature in bladder cancer and a role for miR-145 in hypoxia-dependent apoptosis

    Science.gov (United States)

    Blick, C; Ramachandran, A; McCormick, R; Wigfield, S; Cranston, D; Catto, J; Harris, A L

    2015-01-01

    Background: Hypoxia leads to the stabilisation of the hypoxia-inducible factor (HIF) transcription factor that drives the expression of target genes including microRNAs (miRNAs). MicroRNAs are known to regulate many genes involved in tumourigenesis. The aim of this study was to identify hypoxia-regulated miRNAs (HRMs) in bladder cancer and investigate their functional significance. Methods: Bladder cancer cell lines were exposed to normoxic and hypoxic conditions and interrogated for the expression of 384 miRNAs by qPCR. Functional studies were carried out using siRNA-mediated gene knockdown and chromatin immunoprecipitations. Apoptosis was quantified by annexin V staining and flow cytometry. Results: The HRM signature for NMI bladder cancer lines includes miR-210, miR-193b, miR-145, miR-125-3p, miR-708 and miR-517a. The most hypoxia-upregulated miRNA was miR-145. The miR-145 was a direct target of HIF-1α and two hypoxia response elements were identified within the promoter region of the gene. Finally, the hypoxic upregulation of miR-145 contributed to increased apoptosis in RT4 cells. Conclusions: We have demonstrated the hypoxic regulation of a number of miRNAs in bladder cancer. We have shown that miR-145 is a novel, robust and direct HIF target gene that in turn leads to increased cell death in NMI bladder cancer cell lines. PMID:26196183

  4. Hypoxia induced E-cadherin involving regulators of Hippo pathway due to HIF-1α stabilization/nuclear translocation in bone metastasis from breast carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Maroni, Paola [Istituto Ortopedico Galeazzi, IRCCS, Milano (Italy); Matteucci, Emanuela [Dipartiimento di Scienze Biomediche per la Salute, Molecular Pathology Laboratory, Università degli Studi di Milano, Milano (Italy); Drago, Lorenzo; Banfi, Giuseppe [Istituto Ortopedico Galeazzi, IRCCS, Milano (Italy); Dipartiimento di Scienze Biomediche per la Salute, Molecular Pathology Laboratory, Università degli Studi di Milano, Milano (Italy); Bendinelli, Paola [Dipartiimento di Scienze Biomediche per la Salute, Molecular Pathology Laboratory, Università degli Studi di Milano, Milano (Italy); Desiderio, Maria Alfonsina, E-mail: a.desiderio@unimi.it [Dipartiimento di Scienze Biomediche per la Salute, Molecular Pathology Laboratory, Università degli Studi di Milano, Milano (Italy)

    2015-01-15

    The present study deals with the molecular mechanisms involved in the regulation of E-cadherin expression under hypoxia, because the adjustment of the amount of E-cadherin due to physical stimuli of the microenvironment might influence the colonization of metastasis to skeleton. We analyzed the effect of 1% oxygen tension, that is similar to that encountered in the bone marrow by metastatic cells spreading from breast carcinoma. The purpose was to evaluate the hypoxia-orchestrated control of E-cadherin transactivation via hypoxia inducible factor-1 (HIF-1) and peroxisome proliferator activated receptor-γ (PPARγ), and the involvement of Hippo pathway members, as regulators of transcription factors. To give a translational significance to the study, we took into consideration human pair-matched ductal breast carcinoma and bone metastasis: E-cadherin and Wwox were expressed in bone metastasis but not in breast carcinoma, while HIF-1α and TAZ seemed localized principally in nuclei of metastasis and were found in all cell compartments of breast carcinoma. A close examination of the regulatory mechanisms underlying E-cadherin expression in bone metastasis was done in 1833 clone derived from MDA-MB231 cells. Hypoxia induced E-cadherin only in 1833 clone, but not in parental cells, through HIF-1 and PPARγ activities, while Wwox decreased. Since Wwox was highly expressed in bone metastasis, the effect of ectopic Wwox was evaluated, and we showed E-cadherin transactivation and enhanced invasiveness in WWOX transfected 1833 cells. Also, hypoxia was additive with ectopic Wwox remarkably enhancing HIF-1α nuclear shuttle and accumulation due to the lengthening of the half-life of HIF-1α protein; under this experimental condition HIF-1α appeared as a slower migrated band compared with control, in agreement with the phosphorylation state. The in vitro data strongly supported the almost exclusive presence of HIF-1α in nuclei of human-bone metastasis. Thus, we identified

  5. Hypoxia-induced down-regulation of neprilysin by histone modification in mouse primary cortical and hippocampal neurons.

    Directory of Open Access Journals (Sweden)

    Zheng Wang

    Full Text Available Amyloid β-peptide (Aβ accumulation leads to neurodegeneration and Alzheimer's disease (AD. Aβ metabolism is a dynamic process in the Aβ production and clearance that requires neprilysin (NEP and other enzymes to degrade Aβ. It has been reported that NEP expression is significantly decreased in the brain of AD patients. Previously we have documented hypoxia is a risk factor for Aβ generation in vivo and in vitro through increasing Aβ generation by altering β-cleavage and γ-cleavage of APP and down-regulating NEP, and causing tau hyperphosphorylation. Here, we investigated the molecular mechanisms of hypoxia-induced down-regulation of NEP. We found a significant decrease in NEP expression at the mRNA and protein levels after hypoxic treatment in mouse primary cortical and hippocampal neurons. Chromatin immunoprecipitation (ChIP assays and relative quantitative PCR (q-PCR revealed an increase of histone H3-lysine9 demethylation (H3K9me2 and a decrease of H3 acetylation (H3-Ace in the NEP promoter regions following hypoxia. In addition, we found that hypoxia caused up-regulation of histone methyl transferase (HMT G9a and histone deacetylases (HDACs HDAC-1. Decreased expression of NEP during hypoxia can be prevented by application with the epigenetic regulators 5-Aza-2'-deoxycytidine (5-Aza, HDACs inhibitor sodium valproate (VA, and siRNA-mediated knockdown of G9a or HDAC1. DNA methylation PCR data do not support that hypoxia affects the methylation of NEP promoters. This study suggests that hypoxia may down-regulate NEP by increasing H3K9me2 and decreasing H3-Ace modulation.

  6. Hypoxia-Inducible Factor Directs POMC Gene to Mediate Hypothalamic Glucose Sensing and Energy Balance Regulation

    Science.gov (United States)

    Zhang, Hai; Zhang, Guo; Gonzalez, Frank J.; Park, Sung-min; Cai, Dongsheng

    2011-01-01

    Hypoxia-inducible factor (HIF) is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic effects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance. PMID:21814490

  7. Ursodeoxycholic acid protects cardiomyocytes against cobalt chloride induced hypoxia by regulating transcriptional mediator of cells stress hypoxia inducible factor 1α and p53 protein.

    Science.gov (United States)

    Mohamed, Anis Syamimi; Hanafi, Noorul Izzati; Sheikh Abdul Kadir, Siti Hamimah; Md Noor, Julina; Abdul Hamid Hasani, Narimah; Ab Rahim, Sharaniza; Siran, Rosfaiizah

    2017-10-01

    In hepatocytes, ursodeoxycholic acid (UDCA) activates cell signalling pathways such as p53, intracellular calcium ([Ca2+ ]i ), and sphingosine-1-phosphate (S1P)-receptor via Gαi -coupled-receptor. Recently, UDCA has been shown to protect the heart against hypoxia-reoxygenation injury. However, it is not clear whether UDCA cardioprotection against hypoxia acts through a transcriptional mediator of cells stress, HIF-1α and p53. Therefore, in here, we aimed to investigate whether UDCA could protect cardiomyocytes (CMs) against hypoxia by regulating expression of HIF-1α, p53, [Ca2+ ]i , and S1P-Gαi -coupled-receptor. Cardiomyocytes were isolated from newborn rats (0-2 days), and hypoxia was induced by using cobalt chloride (CoCl2 ). Cardiomyocytes were treated with UDCA and cotreated with either FTY720 (S1P-receptor agonist) or pertussis toxin (PTX; Gαi inhibitor). Cells were subjected for proliferation assay, beating frequency, QuantiGene Plex assay, western blot, immunofluorescence, and calcium imaging. Our findings showed that UDCA counteracted the effects of CoCl2 on cell viability, beating frequency, HIF-1α, and p53 protein expression. We found that these cardioprotection effects of UDCA were similar to FTY720, S1P agonist. Furthermore, we observed that UDCA protects CMs against CoCl2 -induced [Ca2+ ]i dynamic alteration. Pharmacological inhibition of the Gαi -sensitive receptor did not abolish the cardioprotection of UDCA against CoCl2 detrimental effects, except for cell viability and [Ca2+ ]i . Pertussis toxin is partially effective in inhibiting UDCA protection against CoCl2 effects on CM cell viability. Interestingly, PTX fully inhibits UDCA cardioprotection on CoCl2 -induced [Ca2+ ]i dynamic changes. We conclude that UDCA cardioprotection against CoCl2 -induced hypoxia is similar to FTY720, and its actions are not fully mediated by the Gαi -coupled protein sensitive pathways. Ursodeoxycholic acid is the most hydrophilic bile acid and is currently

  8. Translational control of human acetyl-CoA carboxylase 1 mRNA is mediated by an internal ribosome entry site in response to ER stress, serum deprivation or hypoxia mimetic CoCl2.

    Science.gov (United States)

    Damiano, Fabrizio; Testini, Mariangela; Tocci, Romina; Gnoni, Gabriele V; Siculella, Luisa

    2018-01-14

    Acetyl-CoA carboxylase 1 (ACC1) is a cytosolic enzyme catalyzing the rate limiting step in de novo fatty acid biosynthesis. There is mounting evidence showing that ACC1 is susceptible to dysregulation and that it is over-expressed in liver diseases associated with lipid accumulation and in several cancers. In the present study, ACC1 regulation at the translational level is reported. Using several experimental approaches, the presence of an internal ribosome entry site (IRES) has been established in the 5' untranslated region (5' UTR) of the ACC1 mRNA. Transfection experiments with the ACC1 5' UTR inserted in a dicistronic reporter vector show a remarkable increase in the downstream cistron translation, through a cap-independent mechanism. The endoplasmic reticulum (ER) stress condition and the related unfolded protein response (UPR), triggered by treatment with thapsigargin and tunicamycin, cause an increase of the cap-independent translation of ACC1 mRNA in HepG2 cells, despite the overall reduction in global protein synthesis. Other stress conditions, such as serum starvation and incubation with hypoxia mimetic agent CoCl2, up-regulate ACC1 expression in HepG2 cells at the translational level. Overall, these findings indicate that the presence of an IRES in the ACC1 5' UTR allows ACC1 mRNA translation in conditions that are inhibitory to cap-dependent translation. A potential involvement of the cap-independent translation of ACC1 in several pathologies, such as obesity and cancer, has been discussed. Copyright © 2018. Published by Elsevier B.V.

  9. Vitamin D Controls Resistance Artery Function through Regulation of Perivascular Adipose Tissue Hypoxia and Inflammation

    Science.gov (United States)

    Pelham, Christopher J.; Drews, Elizabeth M.; Agrawal, Devendra K.

    2016-01-01

    Vitamin D deficiency in human subjects is associated with hypertension, metabolic syndrome and related risk factors of cardiovascular diseases. Serum 25-hydroxyvitamin D levels correlate inversely with adiposity in obese and lean individuals. Bioactive vitamin D, or calcitriol, exerts anti-inflammatory effects on adipocytes, preadipocytes and macrophages in vitro. We tested the hypothesis that vitamin D deficiency alters the phenotype of perivascular adipose tissue (PVAT) leading to impaired function in resistance artery. To examine the effects of vitamin D and PVAT on vascular reactivity, myograph experiments were performed on arteries, with or without intact PVAT, from mice maintained on vitamin D-deficient, vitamin D-sufficient or vitamin D-supplemented diet. Systolic blood pressure was significantly increased in mice on vitamin D-deficient diet. Importantly, vitamin D deficiency enhanced angiotensin II-induced vasoconstriction and impaired the normal ability of PVAT to suppress contractile responses of the underlying mesenteric resistance artery to angiotensin II and serotonin. Furthermore, vitamin D deficiency caused upregulation of the mRNA expression of tumor necrosis factor-α, hypoxia-inducible factor-1α and its downstream target lysyl oxidase in mesenteric PVAT. Incubation of mesenteric arteries under hypoxic conditions impaired the anti-contractile effects of intact PVAT on those arteries from mice on vitamin D-sufficient diet. Vitamin D supplementation protected arteries against hypoxia-induced impairment of PVAT function. The protective effects of vitamin D against vascular dysfunction, hypertension and cardiovascular diseases may be mediated, at least in part, through regulation of inflammatory and hypoxia signaling pathways in PVAT. PMID:27374117

  10. Plasma levels of hypoxia-regulated factors in patients with age-related macular degeneration.

    Science.gov (United States)

    Ioanna, Zygoula; Christian, Schori; Christian, Grimm; Daniel, Barthelmes

    2018-02-01

    Various hypoxia-related proteins are differentially expressed in the retina and secreted to the vitreous and/or aqueous humor of patients affected by dry or neovascular age-related macular degeneration (nAMD). To determine whether these conditions alter concentrations of cytokines also in the systemic circulation, we measured plasma levels of six hypoxia-related proteins. Plasma was prepared from EDTA blood that was collected from patients affected by dry AMD (n = 5), nAMD (n = 11), proliferative diabetic retinopathy (PDR; n = 9), and patients with an epiretinal membrane (ERM; n = 11). ERM samples served as negative controls, PDR samples as positive controls. Protein concentrations of vascular endothelial growth factor (VEGF), erythropoietin (EPO), angiopoietin-like 4 (ANGPTL4), placental growth factor (PlGF), tumor necrosis factor alpha (TNF-α), and pigment epithelium-derived factor (PEDF) were determined by enzyme-linked immunosorbent assay (ELISA). The concentration of PlGF was significantly increased in plasma of patients affected by nAMD. Although no statistically significant differences were found for EPO, ANGPTL4, PlGF, TNF-α, and PEDF, the mean concentration of VEGF was lowest in the nAMD group. Plasma concentrations of the six factors did not correlate with gender or age of patients. nAMD may increase plasma concentrations of PlGF, making it a candidate as a biomarker for the neovascular form of AMD. Other factors, however, were not differentially regulated, suggesting that their systemic concentrations are not generally increased in hypoxia-related retinal diseases.

  11. Regulation of dynamin family proteins by post-translational modifications.

    Science.gov (United States)

    Kar, Usha P; Dey, Himani; Rahaman, Abdur

    2017-06-01

    Dynamin superfamily proteins comprising classical dynamins and related proteins are membrane remodelling agents involved in several biological processes such as endocytosis, maintenance of organelle morphology and viral resistance. These large GTPases couple GTP hydrolysis with membrane alterations such as fission, fusion or tubulation by undergoing repeated cycles of self-assembly/disassembly. The functions of these proteins are regulated by various post-translational modifications that affect their GTPase activity, multimerization or membrane association. Recently, several reports have demonstrated variety of such modifications providing a better understanding of the mechanisms by which dynamin proteins influence cellular responses to physiological and environmental cues. In this review, we discuss major post-translational modifications along with their roles in the mechanism of dynamin functions and implications in various cellular processes.

  12. Fumarate and Succinate Regulate Expression of Hypoxia-inducible Genes via TET Enzymes.

    Science.gov (United States)

    Laukka, Tuomas; Mariani, Christopher J; Ihantola, Tuukka; Cao, John Z; Hokkanen, Juho; Kaelin, William G; Godley, Lucy A; Koivunen, Peppi

    2016-02-19

    The TET enzymes are members of the 2-oxoglutarate-dependent dioxygenase family and comprise three isoenzymes in humans: TETs 1-3. These TETs convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA, and high 5-hmC levels are associated with active transcription. The importance of the balance in these modified cytosines is emphasized by the fact that TET2 is mutated in several human cancers, including myeloid malignancies such as acute myeloid leukemia (AML). We characterize here the kinetic and inhibitory properties of Tets and show that the Km value of Tets 1 and 2 for O2 is 30 μm, indicating that they retain high activity even under hypoxic conditions. The AML-associated mutations in the Fe(2+) and 2-oxoglutarate-binding residues increased the Km values for these factors 30-80-fold and reduced the Vmax values. Fumarate and succinate, which can accumulate to millimolar levels in succinate dehydrogenase and fumarate hydratase-mutant tumors, were identified as potent Tet inhibitors in vitro, with IC50 values ∼400-500 μm. Fumarate and succinate also down-regulated global 5-hmC levels in neuroblastoma cells and the expression levels of some hypoxia-inducible factor (HIF) target genes via TET inhibition, despite simultaneous HIFα stabilization. The combination of fumarate or succinate treatment with TET1 or TET3 silencing caused differential effects on the expression of specific HIF target genes. Altogether these data show that hypoxia-inducible genes are regulated in a multilayered manner that includes epigenetic regulation via TETs and 5-hmC levels in addition to HIF stabilization. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Fumarate and Succinate Regulate Expression of Hypoxia-inducible Genes via TET Enzymes*

    Science.gov (United States)

    Laukka, Tuomas; Mariani, Christopher J.; Ihantola, Tuukka; Cao, John Z.; Hokkanen, Juho; Kaelin, William G.; Godley, Lucy A.; Koivunen, Peppi

    2016-01-01

    The TET enzymes are members of the 2-oxoglutarate-dependent dioxygenase family and comprise three isoenzymes in humans: TETs 1–3. These TETs convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA, and high 5-hmC levels are associated with active transcription. The importance of the balance in these modified cytosines is emphasized by the fact that TET2 is mutated in several human cancers, including myeloid malignancies such as acute myeloid leukemia (AML). We characterize here the kinetic and inhibitory properties of Tets and show that the Km value of Tets 1 and 2 for O2 is 30 μm, indicating that they retain high activity even under hypoxic conditions. The AML-associated mutations in the Fe2+ and 2-oxoglutarate-binding residues increased the Km values for these factors 30–80-fold and reduced the Vmax values. Fumarate and succinate, which can accumulate to millimolar levels in succinate dehydrogenase and fumarate hydratase-mutant tumors, were identified as potent Tet inhibitors in vitro, with IC50 values ∼400–500 μm. Fumarate and succinate also down-regulated global 5-hmC levels in neuroblastoma cells and the expression levels of some hypoxia-inducible factor (HIF) target genes via TET inhibition, despite simultaneous HIFα stabilization. The combination of fumarate or succinate treatment with TET1 or TET3 silencing caused differential effects on the expression of specific HIF target genes. Altogether these data show that hypoxia-inducible genes are regulated in a multilayered manner that includes epigenetic regulation via TETs and 5-hmC levels in addition to HIF stabilization. PMID:26703470

  14. Hypoxia regulates CD44 and its variant isoforms through HIF-1α in triple negative breast cancer.

    Directory of Open Access Journals (Sweden)

    Balaji Krishnamachary

    Full Text Available BACKGROUND: The CD44 transmembrane glycoproteins play multifaceted roles in tumor progression and metastasis. CD44 expression has also been associated with stem-like breast cancer cells. Hypoxia commonly occurs in tumors and is a major cause of radiation and chemo-resistance. Hypoxia is known to inhibit differentiation and facilitates invasion and metastasis. Here we have investigated the effect of hypoxia on CD44 and two of its isoforms in MDA-MB-231 and SUM-149 triple negative human breast cancer cells and MDA-MB-231 tumors using imaging and molecular characterization. METHODS AND FINDINGS: The roles of hypoxia and hypoxia inducible factor (HIF in regulating the expression of CD44 and its variant isoforms (CD44v6, CD44v7/8 were investigated in human breast cancer cells, by quantitative real-time polymerase chain reaction (qRT-PCR to determine mRNA levels, and fluorescence associated cell sorting (FACS to determine cell surface expression of CD44, under normoxic and hypoxic conditions. In vivo imaging studies with tumor xenografts derived from MDA-MD-231 cells engineered to express tdTomato red fluorescence protein under regulation of hypoxia response elements identified co-localization between hypoxic fluorescent regions and increased concentration of (125I-radiolabeled CD44 antibody. CONCLUSIONS: Our data identified HIF-1α as a regulator of CD44 that increased the number of CD44 molecules and the percentage of CD44 positive cells expressing variant exons v6 and v7/8 in breast cancer cells under hypoxic conditions. Data from these cell studies were further supported by in vivo observations that hypoxic tumor regions contained cells with a higher concentration of CD44 expression.

  15. Hypoxia regulates CD44 and its variant isoforms through HIF-1α in triple negative breast cancer.

    Science.gov (United States)

    Krishnamachary, Balaji; Penet, Marie-France; Nimmagadda, Sridhar; Mironchik, Yelena; Raman, Venu; Solaiyappan, Meiyappan; Semenza, Gregg L; Pomper, Martin G; Bhujwalla, Zaver M

    2012-01-01

    The CD44 transmembrane glycoproteins play multifaceted roles in tumor progression and metastasis. CD44 expression has also been associated with stem-like breast cancer cells. Hypoxia commonly occurs in tumors and is a major cause of radiation and chemo-resistance. Hypoxia is known to inhibit differentiation and facilitates invasion and metastasis. Here we have investigated the effect of hypoxia on CD44 and two of its isoforms in MDA-MB-231 and SUM-149 triple negative human breast cancer cells and MDA-MB-231 tumors using imaging and molecular characterization. The roles of hypoxia and hypoxia inducible factor (HIF) in regulating the expression of CD44 and its variant isoforms (CD44v6, CD44v7/8) were investigated in human breast cancer cells, by quantitative real-time polymerase chain reaction (qRT-PCR) to determine mRNA levels, and fluorescence associated cell sorting (FACS) to determine cell surface expression of CD44, under normoxic and hypoxic conditions. In vivo imaging studies with tumor xenografts derived from MDA-MD-231 cells engineered to express tdTomato red fluorescence protein under regulation of hypoxia response elements identified co-localization between hypoxic fluorescent regions and increased concentration of (125)I-radiolabeled CD44 antibody. Our data identified HIF-1α as a regulator of CD44 that increased the number of CD44 molecules and the percentage of CD44 positive cells expressing variant exons v6 and v7/8 in breast cancer cells under hypoxic conditions. Data from these cell studies were further supported by in vivo observations that hypoxic tumor regions contained cells with a higher concentration of CD44 expression.

  16. Alcohols inhibit translation to regulate morphogenesis in C. albicans

    Science.gov (United States)

    Egbe, Nkechi E.; Paget, Caroline M.; Wang, Hui; Ashe, Mark P.

    2015-01-01

    Many molecules are secreted into the growth media by microorganisms to modulate the metabolic and physiological processes of the organism. For instance, alcohols like butanol, ethanol and isoamyl alcohol are produced by the human pathogenic fungus, Candida albicans and induce morphological differentiation. Here we show that these same alcohols cause a rapid inhibition of protein synthesis. More specifically, the alcohols target translation initiation, a complex stage of the gene expression process. Using molecular techniques, we have identified the likely translational target of these alcohols in C. albicans as the eukaryotic translation initiation factor 2B (eIF2B). eIF2B is the guanine nucleotide exchange factor for eIF2, which supports the exchange reaction where eIF2.GDP is converted to eIF2.GTP. Even minimal regulation at this step will lead to alterations in the levels of specific proteins that may allow the exigencies of the fungus to be realised. Indeed, similar to the effects of alcohols, a minimal inhibition of protein synthesis with cycloheximide also causes an induction of filamentous growth. These results suggest a molecular basis for the effect of various alcohols on morphological differentiation in C. albicans. PMID:25843913

  17. Safflor yellow B reduces hypoxia-mediated vasoconstriction by regulating endothelial micro ribonucleic acid/nitric oxide synthase signaling.

    Science.gov (United States)

    Wang, Chaoyun; Yang, Ying; Li, Miao; Liu, Xin; Wang, Qiaoyun; Xin, Wenyu; Sun, Hongliu; Zheng, Qingyin

    2017-11-07

    Hypoxia-induced generation of vasoconstrictors reduces cerebral blood flow (CBF) while nitric oxide (NO) synthase (NOS) and microRNAs (miRNA) in endothelial cells (ECs) suppress vasoconstriction. Safflor yellow B (SYB), a natural plant compound, previously attenuated angiotensin II-mediated injury of ECs and maintained endothelial function. This study investigated the putative involvement of NOS and miRNAs in SYB-mediated resistance to hypoxia-induced vasoconstriction. In vivo , chronic hypoxia was induced in rats, and SYB was administered intravenously. In vitro , rat primary aortic ECs were cultured under oxygen and glucose deprivation. After treatment with anti-microR-199a, as well as the NOS inhibitor, N(G)-nitro-L-arginine methyl ester, SYB, or both, cell viability, NO and peroxynitrite (ONOO-) levels, NOS expression, and miRNA levels were evaluated. SYB significantly alleviated hypoxia-mediated vasoconstriction and increased CBF endothelium-dependently. SYB upregulated miR-199a, increased EC viability, decreased endothelin-1 (ET-1) levels, inhibited protein kinase C (PKC) activity, and suppressed hypoxia inducible factor-1α (HIF-1α) expression. Furthermore, the SYB-mediated reduction of inducible NOS reduced ONOO- levels. In addition, SYB downregulated miR-138 and, thereby, enhanced S100A1 and endothelial NOS activity. Hypoxia-mediated regulation of miR-138 and miR-199a inhibited endothelial NOS expression and activation, which triggered ET-1 release and vasoconstriction. Therefore, SYB treatment reduced hypoxia-induced vasoconstriction through miR-199a/endothelial NOS signaling.

  18. Regulation of mitochondrial genome replication by hypoxia: The role of DNA oxidation in D-loop region.

    Science.gov (United States)

    Pastukh, Viktor M; Gorodnya, Olena M; Gillespie, Mark N; Ruchko, Mykhaylo V

    2016-07-01

    Mitochondria of mammalian cells contain multiple copies of mitochondrial (mt) DNA. Although mtDNA copy number can fluctuate dramatically depending on physiological and pathophysiologic conditions, the mechanisms regulating mitochondrial genome replication remain obscure. Hypoxia, like many other physiologic stimuli that promote growth, cell proliferation and mitochondrial biogenesis, uses reactive oxygen species as signaling molecules. Emerging evidence suggests that hypoxia-induced transcription of nuclear genes requires controlled DNA damage and repair in specific sequences in the promoter regions. Whether similar mechanisms are operative in mitochondria is unknown. Here we test the hypothesis that controlled oxidative DNA damage and repair in the D-loop region of the mitochondrial genome are required for mitochondrial DNA replication and transcription in hypoxia. We found that hypoxia had little impact on expression of mitochondrial proteins in pulmonary artery endothelial cells, but elevated mtDNA content. The increase in mtDNA copy number was accompanied by oxidative modifications in the D-loop region of the mitochondrial genome. To investigate the role of this sequence-specific oxidation of mitochondrial genome in mtDNA replication, we overexpressed mitochondria-targeted 8-oxoguanine glycosylase Ogg1 in rat pulmonary artery endothelial cells, enhancing the mtDNA repair capacity of transfected cells. Overexpression of Ogg1 resulted in suppression of hypoxia-induced mtDNA oxidation in the D-loop region and attenuation of hypoxia-induced mtDNA replication. Ogg1 overexpression also reduced binding of mitochondrial transcription factor A (TFAM) to both regulatory and coding regions of the mitochondrial genome without altering total abundance of TFAM in either control or hypoxic cells. These observations suggest that oxidative DNA modifications in the D-loop region during hypoxia are important for increased TFAM binding and ensuing replication of the mitochondrial

  19. RegulatING chromatin regulators: post-translational modification of the ING family of epigenetic regulators.

    Science.gov (United States)

    Satpathy, Shankha; Nabbi, Arash; Riabowol, Karl

    2013-03-15

    The five human ING genes encode at least 15 splicing isoforms, most of which affect cell growth, differentiation and apoptosis through their ability to alter gene expression by epigenetic mechanisms. Since their discovery in 1996, ING proteins have been classified as type II tumour suppressors on the basis of reports describing their down-regulation and mislocalization in a variety of cancer types. In addition to their regulation by transcriptional mechanisms, understanding the range of PTMs (post-translational modifications) of INGs is important in understanding how ING functions are fine-tuned in the physiological setting and how they add to the repertoire of activities affected by the INGs. In the present paper we review the different PTMs that have been reported to occur on INGs. We discuss the PTMs that modulate ING function under normal conditions and in response to a variety of stresses. We also describe the ING PTMs that have been identified by several unbiased MS-based PTM enrichment techniques and subsequent proteomic analysis. Among the ING PTMs identified to date, a subset has been characterized for their biological significance and have been shown to affect processes including subcellular localization, interaction with enzymatic complexes and ING protein half-life. The present review aims to highlight the emerging role of PTMs in regulating ING function and to suggest additional pathways and functions where PTMs may effect ING function.

  20. Post-translational regulation and modifications of flavivirus structural proteins.

    Science.gov (United States)

    Roby, Justin A; Setoh, Yin Xiang; Hall, Roy A; Khromykh, Alexander A

    2015-07-01

    Flaviviruses are a group of single-stranded, positive-sense RNA viruses that generally circulate between arthropod vectors and susceptible vertebrate hosts, producing significant human and veterinary disease burdens. Intensive research efforts have broadened our scientific understanding of the replication cycles of these viruses and have revealed several elegant and tightly co-ordinated post-translational modifications that regulate the activity of viral proteins. The three structural proteins in particular - capsid (C), pre-membrane (prM) and envelope (E) - are subjected to strict regulatory modifications as they progress from translation through virus particle assembly and egress. The timing of proteolytic cleavage events at the C-prM junction directly influences the degree of genomic RNA packaging into nascent virions. Proteolytic maturation of prM by host furin during Golgi transit facilitates rearrangement of the E proteins at the virion surface, exposing the fusion loop and thus increasing particle infectivity. Specific interactions between the prM and E proteins are also important for particle assembly, as prM acts as a chaperone, facilitating correct conformational folding of E. It is only once prM/E heterodimers form that these proteins can be secreted efficiently. The addition of branched glycans to the prM and E proteins during virion transit also plays a key role in modulating the rate of secretion, pH sensitivity and infectivity of flavivirus particles. The insights gained from research into post-translational regulation of structural proteins are beginning to be applied in the rational design of improved flavivirus vaccine candidates and make attractive targets for the development of novel therapeutics.

  1. MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia.

    Directory of Open Access Journals (Sweden)

    Zhong Hua

    Full Text Available MicroRNAs (miRNAs are a class of 20-24 nt non-coding RNAs that regulate gene expression primarily through post-transcriptional repression or mRNA degradation in a sequence-specific manner. The roles of miRNAs are just beginning to be understood, but the study of miRNA function has been limited by poor understanding of the general principles of gene regulation by miRNAs. Here we used CNE cells from a human nasopharyngeal carcinoma cell line as a cellular system to investigate miRNA-directed regulation of VEGF and other angiogenic factors under hypoxia, and to explore the principles of gene regulation by miRNAs. Through computational analysis, 96 miRNAs were predicted as putative regulators of VEGF. But when we analyzed the miRNA expression profile of CNE and four other VEGF-expressing cell lines, we found that only some of these miRNAs could be involved in VEGF regulation, and that VEGF may be regulated by different miRNAs that were differentially chosen from 96 putative regulatory miRNAs of VEGF in different cells. Some of these miRNAs also co-regulate other angiogenic factors (differential regulation and co-regulation principle. We also found that VEGF was regulated by multiple miRNAs using different combinations, including both coordinate and competitive interactions. The coordinate principle states that miRNAs with independent binding sites in a gene can produce coordinate action to increase the repressive effect of miRNAs on this gene. By contrast, the competitive principle states when multiple miRNAs compete with each other for a common binding site, or when a functional miRNA competes with a false positive miRNA for the same binding site, the repressive effects of miRNAs may be decreased. Through the competitive principle, false positive miRNAs, which cannot directly repress gene expression, can sometimes play a role in miRNA-mediated gene regulation. The competitive principle, differential regulation, multi-miRNA binding sites, and false

  2. Invited review: decoding the microRNA response to hypoxia

    DEFF Research Database (Denmark)

    Pocock, Roger

    2011-01-01

    into how these tiny RNA molecules function. miRNAs are 20-24 nucleotide non-coding RNA molecules that predominantly regulate transcripts of target genes through translational inhibition. Much recent interest has focused on the influence of miRNAs on homeostatic regulation, and in particular, hypoxic...... of hypoxia and discuss where future breakthroughs in this area may be made....

  3. Translational Challenge Models in Support of Efficacy Studies: Effect of Cerebral Hypoxia on Cognitive Performances in Rodents.

    Science.gov (United States)

    Deguil, Julie; Ravasi, Laura; Lanteaume, Laura; Lamberty, Yves; Bordet, Regis

    2016-01-01

    Empirical evidence currently supports the idea that neurovascular dysfunction is involved in the neurodegenerative process of Alzheimer's disease (AD). In fact, epidemiological studies report that i) vascular risk factors are directly associated with an increased incidence of AD and ii) vascular lesions are frequently co-existent with AD. The neurovascular unit is a key control system for oxygen and nutrients exchange between neurons and microvessels so the integrity of this system is essential for neuronal activity and cell survival. This suggests that hypoxia arising from various vascular injuries may participate in the pathogenesis of AD and aggravate cognitive deficit. Moreover, hypoxia appears to have a direct effect on cognitive functions, in particular memory, by inducing a transient or definitive dysfunction of synaptic transmission. The interplay of hypoxic phenomenon and the development of AD-related pathologies support the use of hypoxia as a challenge model to assess symptomatic (i.e. cognitive enhancers) AD-treatment. Such challenge should be characterized and validated with current symptomatic drugs based on different mechanisms of action before being offered as alternative models for testing new drugs. To date, symptomatic treatments of AD including anticholinesterasic- (donepezil, rivastigmine and galantamine) and antiglutamatergic- (memantine) drugs target various neurotransmission impairments occurring at different stages of the disease. The first aim of the present review is to provide an overview of the methods used to achieve experimental hypoxia in rodents and to characterize the cognitive alterations induced by each method. The second objective is to summarize the main results from studies that have tested the effect of acetylcholinesterase inhibitors on hypoxiainduced cognitive impairment. Overall, the literature research yielded only a small number of studies investigating the effect of hypoxia on cognition in rodents and the different

  4. Intermittent Hypoxia Regulates Stem-like Characteristics and Differentiation of Neuroblastoma Cells

    Science.gov (United States)

    Bhaskara, Vasantha Kumar; Mohanam, Indra; Rao, Jasti S.; Mohanam, Sanjeeva

    2012-01-01

    Background Neuroblastomas are the most common extracranial solid tumors in children. Neuroblastomas are derived from immature cells of the sympathetic nervous system and are characterized by clinical and biological heterogeneity. Hypoxia has been linked to tumor progression and increased malignancy. Intermittent hypoxia or repeated episodes of hypoxia followed by re-oxygenation is a common phenomenon in solid tumors including neuroblastoma and it has a significant influence on the outcome of therapies. The present study focuses on how intermittent hypoxia modulates the stem-like properties and differentiation in neuroblastoma cells. Methods and Findings Cell survival was assessed by clonogenic assay and cell differentiation was determined by morphological characterization. Hypoxia-inducible genes were analyzed by real-time PCR and Western blotting. Immunofluorescence, real-time PCR and Western blotting were utilized to study stem cell markers. Analysis of neural crest / sympathetic nervous system (SNS) markers and neuronal differentiation markers were done by real-time PCR and Western blotting, respectively. Intermittent hypoxia stimulated the levels of HIF-1α and HIF-2 α proteins and enhanced stem-like properties of neuroblastoma cells. In intermittent hypoxia-conditioned cells, downregulation of SNS marker genes and upregulation of genes expressed in the neural crest were observed. Intermittent hypoxia suppressed the retinoic acid-induced differentiation of neuroblastoma cells. Conclusions Our results suggest that intermittent hypoxia enhances stem-like characteristics and suppresses differentiation propensities in neuroblastoma cells. PMID:22363512

  5. The Nrf1 CNC-bZIP protein is regulated by the proteasome and activated by hypoxia.

    Science.gov (United States)

    Chepelev, Nikolai L; Bennitz, Joshua D; Huang, Ting; McBride, Skye; Willmore, William G

    2011-01-01

    Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is a transcription factor mediating cellular responses to xenobiotic and pro-oxidant stress. Nrf1 regulates the transcription of many stress-related genes through the electrophile response elements (EpREs) located in their promoter regions. Despite its potential importance in human health, the mechanisms controlling Nrf1 have not been addressed fully. We found that proteasomal inhibitors MG-132 and clasto-lactacystin-β-lactone stabilized the protein expression of full-length Nrf1 in both COS7 and WFF2002 cells. Concomitantly, proteasomal inhibition decreased the expression of a smaller, N-terminal Nrf1 fragment, with an approximate molecular weight of 23 kDa. The EpRE-luciferase reporter assays revealed that proteasomal inhibition markedly inhibited the Nrf1 transactivational activity. These results support earlier hypotheses that the 26 S proteasome processes Nrf1 into its active form by removing its inhibitory N-terminal domain anchoring Nrf1 to the endoplasmic reticulum. Immunoprecipitation demonstrated that Nrf1 is ubiquitinated and that proteasomal inhibition increased the degree of Nrf1 ubiquitination. Furthermore, Nrf1 protein had a half-life of approximately 5 hours in COS7 cells. In contrast, hypoxia (1% O(2)) significantly increased the luciferase reporter activity of exogenous Nrf1 protein, while decreasing the protein expression of p65, a shorter form of Nrf1, known to act as a repressor of EpRE-controlled gene expression. Finally, the protein phosphatase inhibitor okadaic acid activated Nrf1 reporter activity, while the latter was repressed by the PKC inhibitor staurosporine. Collectively, our data suggests that Nrf1 is controlled by several post-translational mechanisms, including ubiquitination, proteolytic processing and proteasomal-mediated degradation as well as by its phosphorylation status. © 2011 Chepelev et al.

  6. Hypoxia regulates human lung fibroblast proliferation via p53-dependent and -independent pathways

    Directory of Open Access Journals (Sweden)

    Ameshima Shingo

    2009-03-01

    Full Text Available Abstract Background Hypoxia induces the proliferation of lung fibroblasts in vivo and in vitro. However, the subcellular interactions between hypoxia and expression of tumor suppressor p53 and cyclin-dependent kinase inhibitors p21 and p27 remain unclear. Methods Normal human lung fibroblasts (NHLF were cultured in a hypoxic chamber or exposed to desferroxamine (DFX. DNA synthesis was measured using bromodeoxyuridine incorporation, and expression of p53, p21 and p27 was measured using real-time RT-PCR and Western blot analysis. Results DNA synthesis was increased by moderate hypoxia (2% oxygen but was decreased by severe hypoxia (0.1% oxygen and DFX. Moderate hypoxia decreased p21 synthesis without affecting p53 synthesis, whereas severe hypoxia and DFX increased synthesis of both p21 and p53. p27 protein expression was decreased by severe hypoxia and DFX. Gene silencing of p21 and p27 promoted DNA synthesis at ambient oxygen concentrations. p21 and p53 gene silencing lessened the decrease in DNA synthesis due to severe hypoxia or DFX exposure. p21 gene silencing prevented increased DNA synthesis in moderate hypoxia. p27 protein expression was significantly increased by p53 gene silencing, and was decreased by wild-type p53 gene transfection. Conclusion These results indicate that in NHLF, severe hypoxia leads to cell cycle arrest via the p53-p21 pathway, but that moderate hypoxia enhances cell proliferation via the p21 pathway in a p53-independent manner. In addition, our results suggest that p27 may be involved in compensating for p53 in cultured NHLF proliferation.

  7. Post-translational modification-regulated leukocyte adhesion and migration.

    Science.gov (United States)

    Loh, Jia Tong; Su, I-Hsin

    2016-06-14

    Leukocytes undergo frequent phenotypic changes and rapidly infiltrate peripheral and lymphoid tissues in order to carry out immune responses. The recruitment of circulating leukocytes into inflamed tissues depends on integrin-mediated tethering and rolling of these cells on the vascular endothelium, followed by transmigration into the tissues. This dynamic process of migration requires the coordination of large numbers of cytosolic and transmembrane proteins whose functional activities are typically regulated by post-translational modifications (PTMs). Our recent studies have shown that the lysine methyltransferase, Ezh2, critically regulates integrin signalling and governs the adhesion dynamics of leukocytes via direct methylation of talin, a key molecule that controls these processes by linking integrins to the actin cytoskeleton. In this review, we will discuss the various modes of leukocyte migration and examine how PTMs of cytoskeletal/adhesion associated proteins play fundamental roles in the dynamic regulation of leukocyte migration. Furthermore, we will discuss molecular details of the adhesion dynamics controlled by Ezh2-mediated talin methylation and the potential implications of this novel regulatory mechanism for leukocyte migration, immune responses, and pathogenic processes, such as allergic contact dermatitis and tumorigenesis.

  8. Hypoxia-inducible factors - regulation, role and comparative aspects in tumourigenesis

    DEFF Research Database (Denmark)

    Hansen, A E; Kristensen, A T; Law, I

    2011-01-01

    Hypoxia-inducible factors (HIFs) play a key role in the cellular response experienced in hypoxic tumours, mediating adaptive responses that allow hypoxic cells to survive in the hostile environment. Identification and understanding of tumour hypoxia and the influence on cellular processes carries...

  9. Hypoxia-inducible factor-1α regulates β cell function in mouse and human islets

    Science.gov (United States)

    Cheng, Kim; Ho, Kenneth; Stokes, Rebecca; Scott, Christopher; Lau, Sue Mei; Hawthorne, Wayne J.; O’Connell, Philip J.; Loudovaris, Thomas; Kay, Thomas W.; Kulkarni, Rohit N.; Okada, Terumasa; Wang, Xiaohui L.; Yim, Sun Hee; Shah, Yatrik; Grey, Shane T.; Biankin, Andrew V.; Kench, James G.; Laybutt, D. Ross; Gonzalez, Frank J.; Kahn, C. Ronald; Gunton, Jenny E.

    2010-01-01

    Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that regulates cellular stress responses. While the levels of HIF-1α protein are tightly regulated, recent studies suggest that it can be active under normoxic conditions. We hypothesized that HIF-1α is required for normal β cell function and reserve and that dysregulation may contribute to the pathogenesis of type 2 diabetes (T2D). Here we show that HIF-1α protein is present at low levels in mouse and human normoxic β cells and islets. Decreased levels of HIF-1α impaired glucose-stimulated ATP generation and β cell function. C57BL/6 mice with β cell–specific Hif1a disruption (referred to herein as β-Hif1a-null mice) exhibited glucose intolerance, β cell dysfunction, and developed severe glucose intolerance on a high-fat diet. Increasing HIF-1α levels by inhibiting its degradation through iron chelation markedly improved insulin secretion and glucose tolerance in control mice fed a high-fat diet but not in β-Hif1a-null mice. Increasing HIF-1α levels markedly increased expression of ARNT and other genes in human T2D islets and improved their function. Further analysis indicated that HIF-1α was bound to the Arnt promoter in a mouse β cell line, suggesting direct regulation. Taken together, these findings suggest an important role for HIF-1α in β cell reserve and regulation of ARNT expression and demonstrate that HIF-1α is a potential therapeutic target for the β cell dysfunction of T2D. PMID:20440072

  10. MiRNA-486 regulates angiogenic activity and survival of mesenchymal stem cells under hypoxia through modulating Akt signal

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Xue-Feng [High Altitude Medicine of Ministry of Chinese Education and Research Center for High Altitude Medicine, Qinghai University, Xining 810001 (China); Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Department of Respiration, Qinghai Provincial People' s Hospital, Xining (China); Wang, Hua; Xiao, Feng-Jun [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Yin, Yue [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Department of Hematology, Peking University First Hospital, Beijing (China); Xu, Qin-Qin [High Altitude Medicine of Ministry of Chinese Education and Research Center for High Altitude Medicine, Qinghai University, Xining 810001 (China); Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Ge, Ri-Li, E-mail: geriligao@hotmail.com [High Altitude Medicine of Ministry of Chinese Education and Research Center for High Altitude Medicine, Qinghai University, Xining 810001 (China); Wang, Li-Sheng, E-mail: wangls@bmi.ac.cn [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China)

    2016-02-12

    MicroRNA-486 (miR-486) was first identified from human fetal liver cDNA library and validated as a regulator of hematopoiesis. Its roles in regulating the biological function of bone marrow-derived mesnechymal stem cells (BM-MSCs) under hypoxia have not been explored yet. In this study, we demonstrated that exposure to hypoxia upregulates miR-486 expression in BM-MSCs. Lentivirus-mediated overexpression of miR-486 resulted in increase of hepatocyte growth factor (HGF) and vascular endothelial growth factor(VEGF) in both mRNA and protein levels. MiR-486 expression also promotes proliferation and reduces apoptosis of BM-MSCs. Whereas MiR-486 knockdown downregulated the secretion of HGF and VEGF and induced apoptosis of BM-MSCs. Furthermore, PTEN-PI3K/AKT signaling was validated to be involved in changes of BM-MSC biological functions regulated by miR-486. These results suggested that MiR-486 mediated the hypoxia-induced angiogenic activity and promoted the proliferation and survival of BM-MSCs through regulating PTEN-PI3K/AKT signaling. These findings might provide a novel understanding of effective therapeutic strategy for hypoxic-ischemic diseases. - Highlights: • miR-486 is a hypoxia-induced miRNA. • miR-486 regulates the secretion of HGF and VEGF, promotes proliferation, and inhibits apoptosis of BM-MSCs. • miR-486 enhances PI3K/AKT activity signaling by targeting PTEN molecule.

  11. Transforming Growth Factor-beta 1 Involved in the Pathogenesis of Endometriosis through Regulating Expression of Vascular Endothelial Growth Factor under Hypoxia

    Directory of Open Access Journals (Sweden)

    Yue-Xin Yu

    2017-01-01

    Conclusions: The data support the hypothesis that TGF-β1 is involved in the pathogenesis of EMs through regulating VEGF expression. An additive effect of TGF-β1 and hypoxia is taking place at the transcriptional level.

  12. Integrated analysis of microRNA and mRNA expression and association with HIF binding reveals the complexity of microRNA expression regulation under hypoxia

    National Research Council Canada - National Science Library

    Camps, Carme; Saini, Harpreet K; Mole, David R; Choudhry, Hani; Reczko, Martin; Guerra-Assunção, José Afonso; Tian, Ya-Min; Buffa, Francesca M; Harris, Adrian L; Hatzigeorgiou, Artemis G; Enright, Anton J; Ragoussis, Jiannis

    2014-01-01

    .... Our aim is to investigate in depth the regulation of microRNA expression by hypoxia in the breast cancer cell line MCF-7, establish the relationship between microRNA expression and HIF binding sites...

  13. Immune-Mediated and Hypoxia-Regulated Programs: Accomplices in Resistance to Anti-angiogenic Therapies.

    Science.gov (United States)

    Croci, Diego O; Mendez-Huergo, Santiago P; Cerliani, Juan P; Rabinovich, Gabriel A

    2017-04-13

    In contrast to mechanisms taking place during resistance to chemotherapies or other targeted therapies, compensatory adaptation to angiogenesis blockade does not imply a mutational alteration of genes encoding drug targets or multidrug resistance mechanisms but instead involves intrinsic or acquired activation of compensatory angiogenic pathways. In this article we highlight hypoxia-regulated and immune-mediated mechanisms that converge in endothelial cell programs and preserve angiogenesis in settings of vascular endothelial growth factor (VEGF) blockade. These mechanisms involve mobilization of myeloid cell populations and activation of cytokine- and chemokine-driven circuits operating during intrinsic and acquired resistance to anti-angiogenic therapies. Particularly, we focus on findings underscoring a role for galectins and glycosylated ligands in promoting resistance to anti-VEGF therapies and discuss possible strategies to overcome or attenuate this compensatory pathway. Finally, we highlight emerging evidence demonstrating the interplay between immunosuppressive and pro-angiogenic programs in the tumor microenvironment (TME) and discuss emerging combinatorial anticancer strategies aimed at simultaneously potentiating antitumor immune responses and counteracting aberrant angiogenesis.

  14. Hypoxia-regulated therapeutic gene as a preemptive treatment strategy against ischemia/reperfusion tissue injury.

    Science.gov (United States)

    Pachori, Alok S; Melo, Luis G; Hart, Melanie L; Noiseux, Nicholas; Zhang, Lunan; Morello, Fulvio; Solomon, Scott D; Stahl, Gregory L; Pratt, Richard E; Dzau, Victor J

    2004-08-17

    Ischemia and reperfusion represent major mechanisms of tissue injury and organ failure. The timing of administration and the duration of action limit current treatment approaches using pharmacological agents. In this study, we have successfully developed a preemptive strategy for tissue protection using an adenoassociated vector system containing erythropoietin hypoxia response elements for ischemia-regulated expression of the therapeutic gene human heme-oxygenase-1 (hHO-1). We demonstrate that a single administration of this vector several weeks in advance of ischemia/reperfusion injury to multiple tissues such as heart, liver, and skeletal muscle yields rapid and timely induction of hHO-1 during ischemia that resulted in dramatic reduction in tissue damage. In addition, overexpression of therapeutic transgene prevented long-term pathological tissue remodeling and normalized tissue function. Application of this regulatable system using an endogenous physiological stimulus for expression of a therapeutic gene may be a feasible strategy for protecting tissues at risk of ischemia/reperfusion injury.

  15. Ribosome stalling regulates IRES-mediated translation in eukaryotes, a parallel to prokaryotic attenuation

    NARCIS (Netherlands)

    Fernandez, James; Yaman, Ibrahim; Huang, Charles; Liu, Haiyan; Lopez, Alex B.; Komar, Anton A.; Caprara, Mark G.; Merrick, William C.; Snider, Martin D.; Kaufman, Randal J.; Lamers, Wouter H.; Hatzoglou, Maria

    2005-01-01

    It was previously shown that the mRNA for the cat-1 Arg/Lys transporter is translated from an internal ribosome entry site (IRES) that is regulated by cellular stress. Amino acid starvation stimulated cat-1 translation via a mechanism that requires translation of an ORF in the mRNA leader and

  16. Regulation of rat intrapulmonary arterial tone by arachidonic acid and prostaglandin E2 during hypoxia.

    Directory of Open Access Journals (Sweden)

    Gaoliang Yan

    Full Text Available Arachidonic acid (AA and its metabolites, prostaglandins (PG are known to be involved in regulation of vascular homeostasis including vascular tone and vessel wall tension, but their potential role in Hypoxic pulmonary vasoconstriction (HPV remains unclear. In this study, we examined the effects of AA and PGE2 on the hypoxic response in isolated rat intrapulmonary arteries (IPAs.We carried out the investigation on IPAs by vessel tension measurement. Isotetrandrine (20 µM significantly inhibited phase I, phase IIb and phase IIc of hypoxic vasoconstriction. Both indomethacin (100 µM and NS398 attenuated KPSS-induced vessel contraction and phase I, phase IIb and phase IIc of HPV, implying that COX-2 plays a primary role in the hypoxic response of rat IPAs. PGE2 alone caused a significant vasoconstriction in isolated rat IPAs. This constriction is mediated by EP4. Blockage of EP4 by L-161982 (1 µM significantly inhibited phase I, phase IIb and phase IIc of hypoxic vasoconstriction. However, AH6809 (3 µM, an antagonist of EP1, EP2, EP3 and DP1 receptors, exerted no effect on KPSS or hypoxia induced vessel contraction. Increase of cellular cAMP by forskolin could significantly reduce KPSS-induced vessel contraction and abolish phase I, phase II b and phase II c of HPV.Our results demonstrated a vasoconstrictive effect of PGE2 on rat IPAs and this effect is via activation of EP4. Furthermore, our results suggest that intracellular cAMP plays dual roles in regulation of vascular tone, depending on the spatial distribution of cAMP and its coupling with EP receptor and Ca(2+ channels.

  17. Posttranscriptional and transcriptional regulation of endothelial nitric-oxide synthase during hypoxia: the role of microRNAs.

    Science.gov (United States)

    Kalinowski, Leszek; Janaszak-Jasiecka, Anna; Siekierzycka, Anna; Bartoszewska, Sylwia; Woźniak, Marcin; Lejnowski, Dawid; Collawn, James F; Bartoszewski, Rafal

    2016-01-01

    Understanding the cellular pathways that regulate endothelial nitric oxide (eNOS, NOS3 ) expression and consequently nitric oxide (NO) bioavailability during hypoxia is a necessary aspect in the development of novel treatments for cardiovascular disorders. eNOS expression and eNOS-dependent NO cellular signaling during hypoxia promote an equilibrium of transcriptional and posttranscriptional molecular mechanisms that belong to both proapoptotic and survival pathways. Furthermore, NO bioavailability results not only from eNOS levels, but also relies on the presence of eNOS substrate and cofactors, the phosphorylation status of eNOS, and the presence of reactive oxygen species (ROS) that can inactivate eNOS. Since both NOS3 levels and these signaling pathways can also be a subject of posttranscriptional modulation by microRNAs (miRNAs), this class of short noncoding RNAs contribute another level of regulation for NO bioavailability. As miRNA antagomirs or specific target protectors could be used in therapeutic approaches to regulate NO levels, either by changing NOS3 mRNA stability or through factors governing eNOS activity, it is critical to understand their role in governing eNOS activity during hypoxa. In contrast to a large number of miRNAs reported to the change eNOS expression during hypoxia, only a few miRNAs modulate eNOS activity. Furthermore, impaired miRNA biogenesis leads to NOS3 mRNA stabilization under hypoxia. Here we discuss the recent studies that define miRNAs' role in maintaining endothelial NO bioavailability emphasizing those miRNAs that directly modulate NOS3 expression or eNOS activity.

  18. Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Yonghui; Kato, Tomohisa; Furu, Moritoshi [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University (Japan); Nasu, Akira [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University (Japan); Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University (Japan); Kajita, Yoichiro [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University (Japan); Department of Urology, Graduate School of Medicine, Kyoto University (Japan); Mitsui, Hiroto [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University (Japan); Department of Musculoskeletal Medicine, Graduate School of Medical Sciences, Nagoya City University (Japan); Ueda, Michiko [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University (Japan); Aoyama, Tomoki [Human Health Sciences, Graduate School of Medicine, Kyoto University (Japan); Nakayama, Tomitaka; Nakamura, Takashi [Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University (Japan); Toguchida, Junya, E-mail: togjun@frontier.kyoto-u.ac.jp [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University (Japan); Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University (Japan); Center for iPS Cell Research and Application, Institute for Integrated Cell - Material Sciences, Kyoto University (Japan)

    2010-01-15

    Hypoxia has been considered to affect the properties of tissue stem cells including mesenchymal stem cells (MSCs). Effects of long periods of exposure to hypoxia on human MSCs, however, have not been clearly demonstrated. MSCs cultured under normoxic conditions (20% pO{sub 2}) ceased to proliferate after 15-25 population doublings, while MSCs cultured under hypoxic conditions (1% pO{sub 2}) retained the ability to proliferate with an additional 8-20 population doublings. Most of the MSCs cultured under normoxic conditions were in a senescent state after 100 days, while few senescent cells were found in the hypoxic culture, which was associated with a down-regulation of p16 gene expression. MSCs cultured for 100 days under hypoxic conditions were superior to those cultured under normoxic conditions in the ability to differentiate into the chondro- and adipogenic, but not osteogenic, lineage. Among the molecules related to mitogen-activated protein kinase (MAPK) signaling pathways, extracellular signal regulated kinase (ERK) was significantly down-regulated by hypoxia, which helped to inhibit the up-regulation of p16 gene expression. Therefore, the hypoxic culture retained MSCs in an undifferentiated and senescence-free state through the down-regulation of p16 and ERK.

  19. Effect of chronic hypoxia on K+ channels: regulation in human pulmonary vascular smooth muscle cells.

    Science.gov (United States)

    Peng, W; Hoidal, J R; Karwande, S V; Farrukh, I S

    1997-04-01

    We investigated the effects of chronic hypoxia on the major outward K+ currents in early cultured human main pulmonary arterial smooth muscle cells (HPSMC). Unitary currents were measured from inside-out, outside-out, and cell-attached patches of HPSMC. Chronic hypoxia depolarized resting membrane potential (Em) and reduced the activity of a charybdotoxin (CTX)- and iberiotoxin-sensitive, Ca2+-dependent K+ channel (KCa). The 4-aminopyridine-sensitive and CTX-insensitive channel or the delayed rectifier K+ channel was unaffected by chronic hypoxia. Chronic hypoxia caused a +33- to +53-mV right shift in voltage-dependent activation of K(Ca) and a decrease in K(Ca) activity at all cytosolic Ca2+ concentrations ([Ca2+]i) in the range of 0.1-10 microM. Thus the hypoxia-induced decrease in K(Ca) activity was most likely due to a decrease in K(Ca) sensitivity to Em and [Ca2+]i. Chronic hypoxia reduced the ability of nitric oxide (NO.) and guanosine 3',5'-cyclic monophosphate (cGMP) to activate K(Ca). The cGMP-dependent protein kinase-induced activation of K(Ca) was also significantly inhibited by chronic hypoxia. In addition, inhibiting channel dephosphorylation with calyculin A caused significantly less increase in K(Ca) activity in membrane patches excised from chronically hypoxic HPSMC compared with normoxic controls. This suggests that the mechanism by which hypoxia modulates NO.-induced K(Ca) activation is by decreasing the NO./cGMP-mediated phosphorylation of the channel.

  20. Hypoxia-Inducible Regulation of a Prodrug-Activating Enzyme for Tumor-Specific Gene Therapy

    Directory of Open Access Journals (Sweden)

    Toru Shibata

    2002-01-01

    Full Text Available Previous studies have suggested that tumor hypoxia could be exploited for cancer gene therapy. Using hypoxia-responsive elements derived from the human vascular endothelial growth factor gene, we have generated vectors expressing a bacterial nitroreductase. (20NTR gene that can activate the anticancer prodrug CB1954. Stable transfectants of human HT1080 tumor cells with hypoxia-inducible vectors were established with G418 selection. Hypoxic induction of NTR protein correlated with increased sensitivity to in vitro exposure of HT 1080 cells to the prodrug. Growth delay assays were performed with established tumor xenografts derived from the same cells to detect the in vivo efficacy of CB1954 conversion to its cytotoxic form. Significant antitumor effects were achieved with intraperitoneal injections of CB1954 both in tumors that express NTR constitutively or with a hypoxia-inducible promoter. In addition, respiration of 10% O2 increased tumor hypoxia in vivo and enhanced the antitumor effects. Taken together, these results demonstrate that hypoxia-inducible vectors may be useful for tumor-selective gene therapy, although the problem of delivery of the vector to the tumors, particularly to the hypoxic cells in the tumors, is not addressed by these studies.

  1. Alternative polyadenylation can regulate post-translational membrane localization

    Science.gov (United States)

    Mitra, Mithun; Johnson, Elizabeth L.; Coller, Hilary A.

    2016-01-01

    For many genomic loci, there are more than one potential cleavage and polyadenylation site, resulting in the generation of multiple distinct transcripts. When the proximal polyadenylation site is present within the coding region of the transcript, alternative polyadenylation can result in proteins with distinct amino acid sequences and potentially distinct functions. In most cases, the different possible polyadenylation sites are all present within the 3′ untranslated regions (UTRs), and the amino acid sequence of the encoded proteins are not affected by polyadenylation site selection. In individual instances, the selection of the proximal versus distal polyadenylation site in the 3′UTR can dramatically affect transcript stability and translatability. In some instances, UTR alternative polyadenylation generates RNA isoforms that have distinct subcellular localization patterns, and that can regulate the location of the encoded protein in an RNA-guided manner. In a recent paper, the laboratory of Christine Mayr demonstrated that alternative polyadenylation of the transmembrane protein CD47 results in transcripts with the same localization pattern, but the encoded protein localizes to the endoplasmic reticulum when it is encoded by the transcript generated by using the proximal polyadenylation site in 3′UTR, and the identical protein localizes to the plasma membrane when the transcript is encoded by the distal polyadenylation site, also in the 3′ UTR. Unlike previous studies, the mechanism of localization does not rely on differential trafficking of the mRNA and is instead, based on RNA-mediated recruitment of proteins to the cytoplasmic side of CD47 that support its plasma membrane localization. Other transmembrane proteins were discovered to be regulated similarly. The results demonstrate that the choice of polyadenylation site can affect protein localization and function, even when the sequence of the protein is unaffected. Further, the transcript encoding

  2. Compound C prevents Hypoxia-Inducible Factor-1α protein stabilization by regulating the cellular oxygen availability via interaction with Mitochondrial Complex I

    Directory of Open Access Journals (Sweden)

    Hagen Thilo

    2011-04-01

    Full Text Available Abstract The transcription factor Hypoxia-Inducible Factor-1α is a master regulator of the cellular response to low oxygen concentration. Compound C, an inhibitor of AMP-activated kinase, has been reported to inhibit hypoxia dependent Hypoxia-Inducible Factor-1α activation via a mechanism that is independent of AMP-activated kinase but dependent on its interaction with the mitochondrial electron transport chain. The objective of this study is to characterize the interaction of Compound C with the mitochondrial electron transport chain and to determine the mechanism through which the drug influences the stability of the Hypoxia-Inducible Factor-1α protein. We found that Compound C functions as an inhibitor of complex I of the mitochondrial electron transport chain as demonstrated by its effect on mitochondrial respiration. It also prevents hypoxia-induced Hypoxia-Inducible Factor-1α stabilization in a dose dependent manner. In addition, Compound C does not have significant effects on reactive oxygen species production from complex I via both forward and reverse electron flux. This study provides evidence that similar to other mitochondrial electron transport chain inhibitors, Compound C regulates Hypoxia-Inducible Factor-1α stability by controlling the cellular oxygen concentration.

  3. The Regulation of Translation in Alphavirus-Infected Cells

    Directory of Open Access Journals (Sweden)

    Luis Carrasco

    2018-02-01

    Full Text Available Sindbis virus (SINV contains an RNA genome of positive polarity with two open reading frames (ORFs. The first ORF is translated from the genomic RNA (gRNA, rendering the viral non-structural proteins, whereas the second ORF is translated from a subgenomic mRNA (sgRNA, which directs the synthesis of viral structural proteins. SINV infection strongly inhibits host cell translation through a variety of different mechanisms, including the phosphorylation of the eukaryotic initiation factor eIF2α and the redistribution of cellular proteins from the nucleus to the cytoplasm. A number of motifs have been identified in SINV sgRNA, including a hairpin downstream of the AUG initiation codon, which is involved in the translatability of the viral sgRNA when eIF2 is inactivated. Moreover, a 3′-UTR motif containing three stem-loop structures is involved in the enhancement of translation in insect cells, but not in mammalian cells. Accordingly, SINV sgRNA has evolved several structures to efficiently compete for the cellular translational machinery. Mechanistically, sgRNA translation involves scanning of the 5′-UTR following a non-canonical mode and without the requirement for several initiation factors. Indeed, sgRNA-directed polypeptide synthesis occurs even after eIF4G cleavage or inactivation of eIF4A by selective inhibitors. Remarkably, eIF2α phosphorylation does not hamper sgRNA translation during the late phase of SINV infection. SINV sgRNA thus constitutes a unique model of a capped viral mRNA that is efficiently translated in the absence of several canonical initiation factors. The present review will mainly focus in the non-canonical mechanism of translation of SINV sgRNA.

  4. Hypoxia Inducible Factor (HIF Hydroxylases as Regulators of Intestinal Epithelial Barrier FunctionSummary

    Directory of Open Access Journals (Sweden)

    Mario C. Manresa

    2017-05-01

    Full Text Available Human health is dependent on the ability of the body to extract nutrients, fluids, and oxygen from the external environment while at the same time maintaining a state of internal sterility. Therefore, the cell layers that cover the surface areas of the body such as the lung, skin, and gastrointestinal mucosa provide vital semipermeable barriers that allow the transport of essential nutrients, fluid, and waste products, while at the same time keeping the internal compartments free of microbial organisms. These epithelial surfaces are highly specialized and differ in their anatomic structure depending on their location to provide appropriate and effective site-specific barrier function. Given this important role, it is not surprising that significant disease often is associated with alterations in epithelial barrier function. Examples of such diseases include inflammatory bowel disease, chronic obstructive pulmonary disease, and atopic dermatitis. These chronic inflammatory disorders often are characterized by diminished tissue oxygen levels (hypoxia. Hypoxia triggers an adaptive transcriptional response governed by hypoxia-inducible factors (HIFs, which are repressed by a family of oxygen-sensing HIF hydroxylases. Here, we review recent evidence suggesting that pharmacologic hydroxylase inhibition may be of therapeutic benefit in inflammatory bowel disease through the promotion of intestinal epithelial barrier function through both HIF-dependent and HIF-independent mechanisms. Keywords: Epithelial Barrier, Inflammatory Bowel Disease, Hypoxia, Hypoxia-Inducible Factor (HIF Hydroxylases

  5. Functional interaction between responses to lactic acidosis and hypoxia regulates genomic transcriptional outputs

    Science.gov (United States)

    Tang, Xiaohu; Lucas, Joseph E.; Chen, Julia Ling-Yu; LaMonte, Gregory; Wu, Jianli; Wang, Michael Changsheng; Koumenis, Constantinos; Chi, Jen-Tsan

    2011-01-01

    Within solid tumor microenvironments, lactic acidosis and hypoxia each have powerful effects on cancer pathophysiology. However, the influence that these processes exert on each other is unknown. Here we report that a significant portion of the transcriptional response to hypoxia elicited in cancer cells is abolished by simultaneous exposure to lactic acidosis. In particular, lactic acidosis abolished stabilization of HIF-1α protein which occurs normally under hypoxic conditions. In contrast, lactic acidosis strongly synergized with hypoxia to activate the unfolded protein response (UPR) and an inflammatory response, displaying a strong similarity to ATF4-driven amino acid deprivation responses (AAR). In certain breast tumors and breast tumor cells examined, an integrative analysis of gene expression and array CGH data revealed DNA copy number alterations at the ATF4 locus, an important activator of the UPR/AAR pathway. In this setting, varying ATF4 levels influenced the survival of cells after exposure to hypoxia and lactic acidosis. Our findings reveal that the condition of lactic acidosis present in solid tumors inhibits canonical hypoxia responses and activates UPR and inflammation responses. Further, they suggest that ATF4 status may be a critical determinant of the ability of cancer cells to adapt to oxygen and acidity fluctuations in the tumor microenvironment, perhaps linking short-term transcriptional responses to long-term selection for copy number alterations in cancer cells. PMID:22135092

  6. Regulating the Regulators: The Post-Translational Code of Class I HDAC1 and HDAC2

    Directory of Open Access Journals (Sweden)

    Chiara V. Segré

    2011-01-01

    Full Text Available Class I histone deacetylases (HDACs are cellular enzymes expressed in many tissues and play crucial roles in differentiation, proliferation, and cancer. HDAC1 and HDAC2 in particular are highly homologous proteins that show redundant or specific roles in different cell types or in response to different stimuli and signaling pathways. The molecular details of this dual regulation are largely unknown. HDAC1 and HDAC2 are not only protein modifiers, but are in turn regulated by post-translational modifications (PTMs: phosphorylation, acetylation, ubiquitination, SUMOylation, nitrosylation, and carbonylation. Some of these PTMs occur and crosstalk specifically on HDAC1 or HDAC2, creating a rational “code” for a differential, context-related regulation. The global comprehension of this PTM code is central for dissecting the role of single HDAC1 and HDAC2 in physiology and pathology.

  7. Regulation of human autoimmune regulator (AIRE) gene translation by miR-220b.

    Science.gov (United States)

    Matsuo, Tomohito; Noguchi, Yukiko; Shindo, Mieko; Morita, Yoshifumi; Oda, Yoshie; Yoshida, Eiko; Hamada, Hiroko; Harada, Mine; Shiokawa, Yuichi; Nishida, Takahiro; Tominaga, Ryuji; Kikushige, Yoshikane; Akashi, Koichi; Kudoh, Jun; Shimizu, Nobuyoshi; Tanaka, Yuka; Umemura, Tsukuru; Taniguchi, Taketoshi; Yoshimura, Akihiko; Kobayashi, Takashi; Mitsuyama, Masao; Kurisaki, Hironori; Katsuta, Hitoshi; Nagafuchi, Seiho

    2013-11-01

    Although mutations of autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), presenting a wide spectrum of many characteristic and non-characteristic clinical features, some patients lack AIRE gene mutations. Therefore, something other than a mutation, such as dysregulation of AIRE gene, may be a causal factor for APECED or its related diseases. However, regulatory mechanisms for AIRE gene expression and/or translation have still remained elusive. We found that IL-2-stimulated CD4(+) T (IL-2T) cells showed a high expression of AIRE gene, but very low AIRE protein production, while Epstein-Barr virus-transformed B (EBV-B) cells express both AIRE gene and AIRE protein. By using microarray analysis, we could identify miR-220b as a possible regulatory mechanism for AIRE gene translation in IL-2T cells. Here we report that miR-220b significantly reduced the expression of AIRE protein in AIRE gene with 3'UTR region transfected 293T cells, whereas no alteration of AIRE protein production was observed in the open reading frame of AIRE gene alone transfected cells. In addition, anti-miR-220b reversed the inhibitory function of miR-220b for the expression of AIRE protein in AIRE gene with 3'UTR region transfected cells. Moreover, when AIRE gene transfected cells with mutated 3'UTR were transfected with miR-220b, no reduction of AIRE protein production was observed. Taken together, it was concluded that miR-220b inhibited the AIRE gene translation through the 3'UTR region of AIRE gene, indicating that miR-220b could serve as a regulator for human AIRE gene translation. © 2013.

  8. Considering Protonation as a Post-translational Modification Regulating Protein Structure and Function

    Science.gov (United States)

    Schönichen, André; Webb, Bradley A.; Jacobson, Matthew P.; Barber, Diane L.

    2014-01-01

    Post-translational modification of proteins is an evolutionarily conserved mechanism for regulating activity, binding affinities and stability. Compared with established post-translational modifications such as phosphorylation or uniquitination, post-translational modification by protons within physiological pH ranges is a less recognized mechanism for regulating protein function. By changing the charge of amino acid side chains, post-translational modification by protons can drive dynamical changes in protein conformation and function. Addition and removal of a proton is rapid and reversible and in contrast to most other post-translational modifications does not require an enzyme. Signaling specificity is achieved by only a minority of sites in proteins titrating within the physiological pH range. Here, we examine the structural mechanisms and functional consequences of proton post-translational modification of pH-sensing proteins regulating different cellular processes. PMID:23451893

  9. Oxidant-redox regulation of pulmonary vascular responses to hypoxia and nitric oxide-cGMP signaling.

    Science.gov (United States)

    Wolin, Michael S; Gupte, Sachin A; Neo, Boon Hwa; Gao, Qun; Ahmad, Mansoor

    2010-01-01

    Most current theories for the mechanism of hypoxic pulmonary vasoconstriction (HPV) include a role for reactive oxygen species and/or changes in redox regulation, but extreme controversy exists regarding which systems and redox changes mediate the HPV response. Nitric oxide (NO) appears to help to maintain low pulmonary arterial pressure, suppress HPV, and prevent the development of pulmonary hypertension. Our studies have found a key role for glucose-6-phosphate dehydrogenase in bovine pulmonary arterial smooth muscle functioning to maintain elevated levels of cytosolic NADPH which fuels the generation of vasodilator levels of hydrogen peroxide. HPV results from hypoxia removing vasodilation by peroxide. Decreased superoxide generation by Nox4 oxidase and its conversion to peroxide by Cu,Zn-SOD appear to be potential factors in sensing hypoxia, and decreased cGMP-associated vasodilation and removal of redox controlled vasodilator mechanisms by increased cytosolic NADPH may be key coordinators of the HPV response. Oxidant generation associated with vascular disease processes, including the removal of NO by superoxide, and attenuation of its ability to stimulate cGMP production by oxidation of the heme and thiols of soluble guanylate cyclase attenuate potential beneficial actions of NO on pulmonary arterial function. While pulmonary hypertension appears to have multiple poorly understood effects on redox-associated processes, potentially influencing responses to hypoxia and NO-cGMP signaling, much remains to be elucidated regarding how these processes may be important factors in the progression, expression and therapeutic treatment of pulmonary hypertension.

  10. Regulation of autophagy by protein post-translational modification

    National Research Council Canada - National Science Library

    Wani, Willayat Yousuf; Boyer-Guittaut, Michaël; Dodson, Matthew; Chatham, John; Darley-Usmar, Victor; Zhang, Jianhua

    2015-01-01

    ... status, and has an important role in quality control of macromolecules and organelles. As with other major cellular pathways, autophagy proteins are subjected to regulatory post-translational modification...

  11. Cell cycle regulation and apoptosis mediated by p53 in response to hypoxia in hepatopancreas of the white shrimp Litopenaeus vannamei.

    Science.gov (United States)

    Nuñez-Hernandez, Dahlia M; Felix-Portillo, Monserrath; Peregrino-Uriarte, Alma B; Yepiz-Plascencia, Gloria

    2018-01-01

    Although hypoxic aquatic environments cause negative effects on shrimp, these animals can withstand somewhat hypoxia, but the cellular mechanisms underlying this capacity are still poorly understood. In humans, mild hypoxia causes the induction of many proteins to allow cell survival. In contrast, apoptosis is induced during severe hypoxia leading to cell death. p53 is a key transcription factor that determines cells fate towards cell cycle arrest or induction of apoptosis in humans. The aim of this work was to study the role of p53 in cell cycle regulation and apoptosis in response to hypoxia in hepatopancreas of the white shrimp Litopenaeus vannamei. p53 was silenced by RNAi and afterwards the shrimp were exposed to hypoxia. Cdk-2 was used as indicator of cell cycle progression while caspase-3 expression and caspase activity were analyzed as indicators of apoptosis. p53 levels in hepatopancreas were significantly higher at 48 h after hypoxic treatment. Increased expression levels of Cdk-2 were found in p53-silenced shrimp after 24 and 48 h in the normoxic treatments as well as 48 h after hypoxia, indicating a possible role of p53 in cell cycle regulation. In response to hypoxia, unsilenced shrimp showed an increase in caspase-3 expression levels, however an increase was also observed in caspase activity at 24 h of normoxic conditions in p53-silenced shrimps. Taken together these results indicate the involvement of p53 in regulation of cell cycle and apoptosis in the white shrimp in response to hypoxia. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Hypoxia and HIFs in regulating the development of the hematopoietic system

    NARCIS (Netherlands)

    P. Imanirad (Parisa); E.A. Dzierzak (Elaine)

    2013-01-01

    textabstractMany physiologic processes during the early stages of mammalian ontogeny, particularly placental and vascular development, take place in the low oxygen environment of the uterus. Organogenesis is affected by hypoxia inducible factor (HIF) transcription factors that are sensors of

  13. The clinical impact of hypoxia-regulated gene expression in loco-regional gastroesophageal cancer

    DEFF Research Database (Denmark)

    Winther, M.; Alsner, J.; Tramm, T.

    2015-01-01

    patients with GC treated with neoadjuvant or definitive chemoradiotherapy or perioperative chemotherapy. Based on formalin-fixed, paraffin-embedded, diagnostic biopsies, gene expression of the 15 hypoxia-induced and pH-independent genes was obtained with qPCR. Sufficient amounts of RNA for gene analyses...

  14. Regulation of human skeletal muscle perfusion and its heterogeneity during exercise in moderate hypoxia

    DEFF Research Database (Denmark)

    Heinonen, Ilkka H; Kemppainen, Jukka; Kaskinoro, Kimmo

    2010-01-01

    inhibition had no effect on capillary BF during exercise in either normoxia or hypoxia. Finally, one-leg exercise increased muscle BF heterogeneity both in the resting posterior hamstring part of the exercising leg and in the resting contralateral leg, whereas mean BF was unchanged. In conclusion...

  15. HIF-independent regulation of thioredoxin reductase 1 contributes to the high levels of reactive oxygen species induced by hypoxia.

    Directory of Open Access Journals (Sweden)

    Salvador Naranjo-Suarez

    Full Text Available Cellular adaptation to hypoxic conditions mainly involves transcriptional changes in which hypoxia inducible factors (HIFs play a critical role. Under hypoxic conditions, HIF protein is stabilized due to inhibition of the activity of prolyl hydroxylases (EGLNs. Because the reaction carried out by these enzymes uses oxygen as a co-substrate it is generally accepted that the hypoxic inhibition of EGLNs is due to the reduction in oxygen levels. However, several studies have reported that hypoxic generation of mitochondrial reactive oxygen species (ROS is required for HIF stabilization. Here, we show that hypoxia downregulates thioredoxin reductase 1 (TR1 mRNA and protein levels. This hypoxic TR1 regulation is HIF independent, as HIF stabilization by EGLNs inhibitors does not affect TR1 expression and HIF deficiency does not block TR1 hypoxic-regulation, and it has an effect on TR1 function, as hypoxic conditions also reduce TR1 activity. We found that, when cultured under hypoxic conditions, TR1 deficient cells showed a larger accumulation of ROS compared to control cells, whereas TR1 over-expression was able to block the hypoxic generation of ROS. Furthermore, the changes in ROS levels observed in TR1 deficient or TR1 over-expressing cells did not affect HIF stabilization or function. These results indicate that hypoxic TR1 down-regulation is important in maintaining high levels of ROS under hypoxic conditions and that HIF stabilization and activity do not require hypoxic generation of ROS.

  16. Impact of exercise and moderate hypoxia on glycemic regulation and substrate oxidation pattern.

    Directory of Open Access Journals (Sweden)

    Takuma Morishima

    Full Text Available We examined metabolic and endocrine responses during rest and exercise in moderate hypoxia over a 7.5 h time courses during daytime.Eight sedentary, overweight men (28.6 ± 0.8 kg/m2 completed four experimental trials: a rest trial in normoxia (FiO2 = 20.9%, NOR-Rest, an exercise trial in normoxia (NOR-Ex, a rest trial in hypoxia (FiO2 = 15.0%, HYP-Rest, and an exercise trial in hypoxia (HYP-Ex. Experimental trials were performed from 8:00 to 15:30 in an environmental chamber. Blood and respiratory gas samples were collected over 7.5 h. In the exercise trials, subjects performed 30 min of pedaling exercise at 60% of VO2max at 8:00, 10:30, and 13:00, and rested during the remaining period in each environment. Standard meals were provided at 8:30, 11:00, and 13:30.The areas under the curves for blood glucose and serum insulin concentrations over 7.5 h did not differ among the four trials. At baseline, %carbohydrate contribution was significantly higher in the hypoxic trials than in the normoxic trials (P<0.05. Although exercise promoted carbohydrate oxidation in the NOR-Ex and HYP-Ex trials, %carbohydrate contribution during each exercise and post-exercise period were significantly higher in the HYP-Ex trial than in the NOR-Ex trial (P<0.05.Three sessions of 30 min exercise (60% of VO2max in moderate hypoxia over 7.5 h did not attenuate postprandial glucose and insulin responses in young, overweight men. However, carbohydrate oxidation was significantly enhanced when the exercise was conducted in moderate hypoxia.

  17. Interactive effect of hypoxia and otolith organ engagement on cardiovascular regulation in humans

    Science.gov (United States)

    Monahan, Kevin D.; Ray, Chester A.

    2002-01-01

    We determined the interaction between the vestibulosympathetic reflex and the arterial chemoreflex in 12 healthy subjects. Subjects performed three trials in which continuous recordings of muscle sympathetic nerve activity (MSNA), mean arterial blood pressure (MAP), heart rate (HR), and arterial oxygen saturation were obtained. First, in prone subjects the otolith organs were engaged by use of head-down rotation (HDR). Second, the arterial chemoreflex was activated by inspiration of hypoxic gas (10% O2 and 90% N2) for 7 min with HDR being performed during minute 6. Third, hypoxia was repeated (15 min) with HDR being performed during minute 14. HDR [means +/- SE; increase (Delta)7 +/- 1 bursts/min and Delta50 +/- 11% for burst frequency and total MSNA, respectively; P < 0.05] and hypoxia (Delta6 +/- 2 bursts/min and Delta62 +/- 29%; P < 0.05) increased MSNA. Additionally, MSNA increased when HDR was performed during hypoxia (Delta11 +/- 2 bursts/min and Delta127 +/- 57% change from normoxia; P < 0.05). These increases in MSNA were similar to the algebraic sum of the individual increase in MSNA elicited by HDR and hypoxia (Delta13 +/- 1 bursts/min and Delta115 +/- 36%). Increases in MAP (Delta3 +/- 1 mmHg) and HR (Delta19 +/- 1 beats/min) during combined HDR and hypoxia generally were smaller (P < 0.05) than the algebraic sum of the individual responses (Delta5 +/- 1 mmHg and Delta24 +/- 2 beats/min for MAP and HR, respectively; P < 0.05). These findings indicate an additive interaction between the vestibulosympathetic reflex and arterial chemoreflex for MSNA. Therefore, it appears that MSNA outputs between the vestibulosympathetic reflex and arterial chemoreflex are independent of one another in humans.

  18. Decoding sORF translation - from small proteins to gene regulation.

    Science.gov (United States)

    Cabrera-Quio, Luis Enrique; Herberg, Sarah; Pauli, Andrea

    2016-11-01

    Translation is best known as the fundamental mechanism by which the ribosome converts a sequence of nucleotides into a string of amino acids. Extensive research over many years has elucidated the key principles of translation, and the majority of translated regions were thought to be known. The recent discovery of wide-spread translation outside of annotated protein-coding open reading frames (ORFs) came therefore as a surprise, raising the intriguing possibility that these newly discovered translated regions might have unrecognized protein-coding or gene-regulatory functions. Here, we highlight recent findings that provide evidence that some of these newly discovered translated short ORFs (sORFs) encode functional, previously missed small proteins, while others have regulatory roles. Based on known examples we will also speculate about putative additional roles and the potentially much wider impact that these translated regions might have on cellular homeostasis and gene regulation.

  19. Codon usage regulates protein structure and function by affecting translation elongation speed in Drosophila cells.

    Science.gov (United States)

    Zhao, Fangzhou; Yu, Chien-Hung; Liu, Yi

    2017-08-21

    Codon usage biases are found in all eukaryotic and prokaryotic genomes and have been proposed to regulate different aspects of translation process. Codon optimality has been shown to regulate translation elongation speed in fungal systems, but its effect on translation elongation speed in animal systems is not clear. In this study, we used a Drosophila cell-free translation system to directly compare the velocity of mRNA translation elongation. Our results demonstrate that optimal synonymous codons speed up translation elongation while non-optimal codons slow down translation. In addition, codon usage regulates ribosome movement and stalling on mRNA during translation. Finally, we show that codon usage affects protein structure and function in vitro and in Drosophila cells. Together, these results suggest that the effect of codon usage on translation elongation speed is a conserved mechanism from fungi to animals that can affect protein folding in eukaryotic organisms. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Regulation of Translational Efficiency by Disparate 5′-UTRs of PPARγ Splice Variants

    Directory of Open Access Journals (Sweden)

    Shawn McClelland

    2009-01-01

    Full Text Available The PPAR-γ gene encodes for at least 7 unique transcripts due to alternative splicing of five exons in the 5′-untranslated region (UTR. The translated region is encoded by exons 1–6, which are identical in all isoforms. This study investigated the role of the 5′-UTR in regulating the efficiency with which the message is translated to protein. A coupled in vitro transcription-translation assay demonstrated that PPAR-γ1, -γ2, and -γ5 are efficiently translated, whereas PPAR-γ4 and -γ7 are poorly translated. An in vivo reporter gene assay using each 5′-UTR upstream of the firefly luciferase gene showed that the 5′-UTRs for PPAR-γ1, -γ2, and -γ4 enhanced translation, whereas the 5′-UTRs for PPAR-γ5 and -γ7 inhibited translation. Models of RNA secondary structure, obtained by the mfold software, were used to explain the mechanism of regulation by each 5′-UTR. In general, it was found that the translational efficiency was inversely correlated with the stability of the mRNA secondary structure, the presence of base-pairing in the consensus Kozak sequence, the number of start codons in the 5′-UTR, and the length of the 5′-UTR. A better understanding of posttranscriptional regulation of translation will allow modulation of protein levels without altering transcription.

  1. 47 CFR 74.780 - Broadcast regulations applicable to translators, low power, and booster stations.

    Science.gov (United States)

    2010-10-01

    ... 47 Telecommunication 4 2010-10-01 2010-10-01 false Broadcast regulations applicable to translators... COMMISSION (CONTINUED) BROADCAST RADIO SERVICES EXPERIMENTAL RADIO, AUXILIARY, SPECIAL BROADCAST AND OTHER PROGRAM DISTRIBUTIONAL SERVICES Low Power TV, TV Translator, and TV Booster Stations § 74.780 Broadcast...

  2. Proteomic analysis of polyribosomes identifies splicing factors as potential regulators of translation during mitosis.

    Science.gov (United States)

    Aviner, Ranen; Hofmann, Sarah; Elman, Tamar; Shenoy, Anjana; Geiger, Tamar; Elkon, Ran; Ehrlich, Marcelo; Elroy-Stein, Orna

    2017-06-02

    Precise regulation of mRNA translation is critical for proper cell division, but little is known about the factors that mediate it. To identify mRNA-binding proteins that regulate translation during mitosis, we analyzed the composition of polysomes from interphase and mitotic cells using unbiased quantitative mass-spectrometry (LC-MS/MS). We found that mitotic polysomes are enriched with a subset of proteins involved in RNA processing, including alternative splicing and RNA export. To demonstrate that these may indeed be regulators of translation, we focused on heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a test case and confirmed that it is recruited to elongating ribosomes during mitosis. Then, using a combination of pulsed SILAC, metabolic labeling and ribosome profiling, we showed that knockdown of hnRNP C affects both global and transcript-specific translation rates and found that hnRNP C is specifically important for translation of mRNAs that encode ribosomal proteins and translation factors. Taken together, our results demonstrate how proteomic analysis of polysomes can provide insight into translation regulation under various cellular conditions of interest and suggest that hnRNP C facilitates production of translation machinery components during mitosis to provide daughter cells with the ability to efficiently synthesize proteins as they enter G1 phase. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Managing the complexity of communication: regulation of gap junctions by post-translational modification

    DEFF Research Database (Denmark)

    Axelsen, Lene Nygaard; Callø, Kirstine; von Holstein-Rathlou, Niels-Henrik

    2013-01-01

    expression by transcription and translation is of great importance, the trafficking, channel activity and degradation are also under tight control. The function of connexins can be regulated by several post translational modifications, which affect numerous parameters; including number of channels, open...

  4. Amplified in Breast Cancer Regulates Transcription and Translation in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Aleksandra M. Ochnik

    2016-02-01

    Conclusion: The oncogenic transcription factor AIB1 has a novel role in the regulation of polyribosome recruitment and formation of the translational complex. Combinatorial therapies targeting IGF signaling and mRNA translation in AIB1 expressing breast cancers may have clinical benefit and warrants further investigation.

  5. Differential effects of chronic hypoxia and feed restriction on the expression of leptin and its receptor, food intake regulation and the endocrine stress response in common carp.

    Science.gov (United States)

    Bernier, Nicholas J; Gorissen, Marnix; Flik, Gert

    2012-07-01

    Appetite suppression is a common response to hypoxia in fish that confers significant energy savings. Yet little is known about the endocrine signals involved in the regulation of food intake during chronic hypoxia. Thus, we assessed the impact of chronic hypoxia on food intake, the expression of the potent anorexigenic signal leptin and its receptor (lepr), the mRNA levels of key hypothalamic appetite-regulating genes, and the activity of the hypothalamic-pituitary-interrenal (HPI) axis in common carp, Cyprinus carpio. Fish exposed to 10% O(2) saturation for 8 days were chronically anorexic and consumed on average 79% less food than normoxic controls. Hypoxia also elicited gradual and parallel increases in the expression of liver leptin-a-I, leptin-a-II, lepr and erythropoietin, a known hypoxia-responsive gene. In contrast, the liver mRNA levels of all four genes remained unchanged in normoxic fish pair-fed to the hypoxia treatment. In the hypothalamus, expression of the appetite-regulating genes were consistent with an inhibition and stimulation of hunger in the hypoxic and pair-fed fish, respectively, and reduced feed intake led to a decrease in lepr. Although both treatments elicited similar delayed increases in plasma cortisol, they were characterized by distinct HPI axis effector transcript levels and a marked differential increase in pituitary lepr expression. Together, these results show that a reduction in O(2) availability, and not feed intake, stimulates liver leptin-a expression in common carp and suggest that this pleiotropic cytokine is involved in the regulation of appetite and the endocrine stress response during chronic hypoxia.

  6. Translation regulation in plants: an interesting past, an exciting present and a promising future.

    Science.gov (United States)

    Merchante, Catharina; Stepanova, Anna N; Alonso, Jose M

    2017-05-01

    Changes in gene expression are at the core of most biological processes, from cell differentiation to organ development, including the adaptation of the whole organism to the ever-changing environment. Although the central role of transcriptional regulation is solidly established and the general mechanisms involved in this type of regulation are relatively well understood, it is clear that regulation at a translational level also plays an essential role in modulating gene expression. Despite the large number of examples illustrating the critical role played by translational regulation in determining the expression levels of a gene, our understanding of the molecular mechanisms behind such types of regulation has been slow to emerge. With the recent development of high-throughput approaches to map and quantify different critical parameters affecting translation, such as RNA structure, protein-RNA interactions and ribosome occupancy at the genome level, a renewed enthusiasm toward studying translation regulation is warranted. The use of these new powerful technologies in well-established and uncharacterized translation-dependent processes holds the promise to decipher the likely complex and diverse, but also fascinating, mechanisms behind the regulation of translation. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  7. Molecular characterization of factors involved in regulation of archaeal translation

    NARCIS (Netherlands)

    Blombach, F.

    2010-01-01

    The three domains of life – Bacteria, Archaea, and Eukaryotes – can be easily distinguished based on how the genetic information is processed during transcription, translation, and (DNA) replication. Generally, Eukaryotes turned out to employ machineries for these processes that are in their essence

  8. Roles and Regulation of Ketogenesis in Cultured Astroglia and Neurons Under Hypoxia and Hypoglycemia

    Directory of Open Access Journals (Sweden)

    Shinichi Takahashi

    2014-09-01

    Full Text Available Exogenous ketone bodies (KBs, acetoacetate (AA, and β-hydroxybutyrate (BHB act as alternative energy substrates in neural cells under starvation. The present study examined the endogenous ketogenic capacity of astroglia under hypoxia with/without glucose and the possible roles of KBs in neuronal energy metabolism. Cultured neurons and astroglia were prepared from Sprague-Dawley rats. Palmitic acid (PAL and l-carnitine (LC were added to the assay medium. The 4- to 24-hr production of AA and BHB was measured using the cyclic thio-NADH method. 14C-labeled acid-soluble products (KBs and 14CO2 produced from [1-14C]PAL were also measured. l-[U-14C]lactic acid ([14C]LAC, [1-14C]pyruvic acid ([14C]PYR, or β-[1-14C]hydroxybutyric acid ([14C]BHB was used to compare the oxidative metabolism of the glycolysis end products with that of the KBs. Some cells were placed in a hypoxic chamber (1% O2. PAL and LC induced a higher production of KBs in astroglia than in neurons, while the CO2 production from PAL was less than 5% of the KB production in both astroglia and neurons. KB production in astroglia was augmented by the AMP-activated protein kinase activators, AICAR and metformin, as well as hypoxia with/without glucose. Neuronal KB production increased under hypoxia in the absence of PAL and LC. In neurons, [14C]LAC and [14C]PYR oxidation decreased after 24 hr of hypoxia, while [14C]BHB oxidation was preserved. Astroglia responds to ischemia in vitro by enhancing KB production, and astroglia-produced KBs derived from fatty acid might serve as a neuronal energy substrate for the tricarboxylic acid cycle instead of lactate, as pyruvate dehydrogenase is susceptible to ischemia.

  9. Crosstalk between nitrite, myoglobin and reactive oxygen species to regulate vasodilation under hypoxia.

    Directory of Open Access Journals (Sweden)

    Matthias Totzeck

    Full Text Available The systemic response to decreasing oxygen levels is hypoxic vasodilation. While this mechanism has been known for more than a century, the underlying cellular events have remained incompletely understood. Nitrite signaling is critically involved in vessel relaxation under hypoxia. This can be attributed to the presence of myoglobin in the vessel wall together with other potential nitrite reductases, which generate nitric oxide, one of the most potent vasodilatory signaling molecules. Questions remain relating to the precise concentration of nitrite and the exact dose-response relations between nitrite and myoglobin under hypoxia. It is furthermore unclear whether regulatory mechanisms exist which balance this interaction. Nitrite tissue levels were similar across all species investigated. We then investigated the exact fractional myoglobin desaturation in an ex vivo approach when gassing with 1% oxygen. Within a short time frame myoglobin desaturated to 58±12%. Given that myoglobin significantly contributes to nitrite reduction under hypoxia, dose-response experiments using physiological to pharmacological nitrite concentrations were conducted. Along all concentrations, abrogation of myoglobin in mice impaired vasodilation. As reactive oxygen species may counteract the vasodilatory response, we used superoxide dismutase and its mimic tempol as well as catalase and ebselen to reduce the levels of reactive oxygen species during hypoxic vasodilation. Incubation of tempol in conjunction with catalase alone and catalase/ebselen increased the vasodilatory response to nitrite. Our study shows that modest hypoxia leads to a significant nitrite-dependent vessel relaxation. This requires the presence of vascular myoglobin for both physiological and pharmacological nitrite levels. Reactive oxygen species, in turn, modulate this vasodilation response.

  10. Dynamic regulation of metabolic efficiency explains tolerance to acute hypoxia in humans.

    Science.gov (United States)

    Schiffer, Tomas A; Ekblom, Björn; Lundberg, Jon O; Weitzberg, Eddie; Larsen, Filip J

    2014-10-01

    The maximum power principle dictates that open biological systems tend to self-organize to a level of efficiency that allows maximal power production. Applying this principle to cellular energetics and whole-body physiology would suggest that for every metabolic challenge, an optimal efficiency exists that maximizes power production. On exposure to hypoxia, it would be favorable if metabolic efficiency would rapidly adjust so as to better preserve work performance. We tested this idea in humans by measuring metabolic efficiency and exercise tolerance under normoxic (Fio2=20.9%) and hypoxic (Fio2=16%) conditions, where Fio2 is fraction of inhaled oxygen. The results were compared with respirometric analyses of skeletal muscle mitochondria from the same individuals. We found that among healthy trained subjects (n=14) with a wide range of metabolic efficiency (ME), those with a high ME during normoxic exercise were able to better maintain exercise capacity (Wmax) in hypoxia. On hypoxic exposure, these subjects acutely decreased their efficiency from 19.2 to 17.4%, thereby likely shifting it closer to a degree of efficiency where maximal power production is achieved. In addition, mitochondria from these subjects had a lower intrinsic respiration compared to subjects that showed a large drop in Wmax in hypoxia An acute shift in efficiency was also demonstrated in isolated mitochondria exposed to physiological levels of hypoxia as P/O ratio increased from 0.9 to 1.3 with hypoxic exposure. These findings suggest the existence of a physiological adaptive response by which metabolic efficiency is dynamically optimized to maximize power production. © FASEB.

  11. The Atypical Response Regulator AtvR Is a New Player in Pseudomonas aeruginosa Response to Hypoxia and Virulence.

    Science.gov (United States)

    Kaihami, Gilberto Hideo; Breda, Leandro Carvalho Dantas; de Almeida, José Roberto Fogaça; de Oliveira Pereira, Thays; Nicastro, Gianlucca Gonçalves; Boechat, Ana Laura; de Almeida, Sandro Rogério; Baldini, Regina Lúcia

    2017-08-01

    Two-component systems are widespread in bacteria, allowing adaptation to environmental changes. The classical pathway is composed of a histidine kinase that phosphorylates an aspartate residue in the cognate response regulator (RR). RRs lacking the phosphorylatable aspartate also occur, but their function and contribution during host-pathogen interactions are poorly characterized. AtvR (PA14_26570) is the only atypical response regulator with a DNA-binding domain in the opportunistic pathogen Pseudomonas aeruginosa Macrophage infection with the atvR mutant strain resulted in higher levels of tumor necrosis factor alpha secretion as well as increased bacterial clearance compared to those for macrophages infected with the wild-type strain. In an acute pneumonia model, mice infected with the atvR mutant presented increased amounts of proinflammatory cytokines, increased neutrophil recruitment to the lungs, reductions in bacterial burdens, and higher survival rates in comparison with the findings for mice infected with the wild-type strain. Further, several genes involved in hypoxia/anoxia adaptation were upregulated upon atvR overexpression, as seen by high-throughput transcriptome sequencing (RNA-Seq) analysis. In addition, atvR was more expressed in hypoxia in the presence of nitrate and required for full expression of nitrate reductase genes, promoting bacterial growth under this condition. Thus, AtvR would be crucial for successful infection, aiding P. aeruginosa survival under conditions of low oxygen tension in the host. Taken together, our data demonstrate that the atypical response regulator AtvR is part of the repertoire of transcriptional regulators involved in the lifestyle switch from aerobic to anaerobic conditions. This finding increases the complexity of regulation of one of the central metabolic pathways that contributes to Pseudomonas ubiquity and versatility. Copyright © 2017 American Society for Microbiology.

  12. Regulation of Stress Responses and Translational Control by Coronavirus.

    Science.gov (United States)

    Fung, To Sing; Liao, Ying; Liu, Ding Xiang

    2016-07-04

    Similar to other viruses, coronavirus infection triggers cellular stress responses in infected host cells. The close association of coronavirus replication with the endoplasmic reticulum (ER) results in the ER stress responses, which impose a challenge to the viruses. Viruses, in turn, have come up with various mechanisms to block or subvert these responses. One of the ER stress responses is inhibition of the global protein synthesis to reduce the amount of unfolded proteins inside the ER lumen. Viruses have evolved the capacity to overcome the protein translation shutoff to ensure viral protein production. Here, we review the strategies exploited by coronavirus to modulate cellular stress response pathways. The involvement of coronavirus-induced stress responses and translational control in viral pathogenesis will also be briefly discussed.

  13. Translation is actively regulated during the differentiation of CD8(+) effector T cells.

    Science.gov (United States)

    Araki, Koichi; Morita, Masahiro; Bederman, Annelise G; Konieczny, Bogumila T; Kissick, Haydn T; Sonenberg, Nahum; Ahmed, Rafi

    2017-09-01

    Translation is a critical process in protein synthesis, but translational regulation in antigen-specific T cells in vivo has not been well defined. Here we have characterized the translatome of virus-specific CD8(+) effector T cells (Teff cells) during acute infection of mice with lymphocytic choriomeningitis virus (LCMV). Antigen-specific T cells exerted dynamic translational control of gene expression that correlated with cell proliferation and stimulation via the T cell antigen receptor (TCR). The translation of mRNAs that encode translation machinery, including ribosomal proteins, was upregulated during the T cell clonal-expansion phase, followed by inhibition of the translation of those transcripts when the CD8(+) Teff cells stopped dividing just before the contraction phase. That translational suppression was more pronounced in terminal effector cells than in memory precursor cells and was regulated by antigenic stimulation and signals from the kinase mTOR. Our studies show that translation of transcripts encoding ribosomal proteins is regulated during the differentiation of CD8(+) Teff cells and might have a role in fate 'decisions' involved in the formation of memory cells.

  14. Acupuncture reduced apoptosis and up-regulated BDNF and GDNF expression in hippocampus following hypoxia-ischemia in neonatal rats.

    Science.gov (United States)

    Zhang, Yong; Lan, Rui; Wang, Jun; Li, Xiang-Yun; Zhu, Deng-Na; Ma, Yun-Zhi; Wu, Ji-Tao; Liu, Zhen-Huan

    2015-08-22

    Acupuncture attenuates neuronal damages following ischemia. The purpose of the present study was to investigate the beneficial effects of acupuncture on hypoxia-ischemia induced brain damages in neonatal rats. Male postnatal 7 days rats were randomly divided into 3 groups: sham control (sham), hypoxia-ischemia (HI), and HI plus acupuncture treatment (HI+Acu). The rats in HI and HI+Acu groups were submitted to model of neonatal HI, established by occluding the left common carotid artery followed by a 3.5h period of hypoxia (8% O2-92% N2). At 24h after HI, animals were stimulated by acupuncture treatment once a day and the treatment continued during 4 weeks, 5days/week. Behavioral functions, learning and memory ability, and body weight were observed at different time-points after HI. DNA fragmentation assay were performed with TUNEL staining to evaluate apoptosis and expression levels of mitochondrial Bcl-2, mitochondrial Bax, Cleaved caspase 3, Cleaved caspase 9 in the damaged hippocampus were detected by western blotting 28 days following HI. GDNF, BDNF levels in hippocampus were also determined. The results showed that acupuncture significantly promoted growth and development, improved neurobehavioral function, learning and memory ability after 20 days' treatment. Furthermore, we obtained one interesting finding that acupuncture attenuated cellular apoptosis and up-regulated GDNF and BDNF levels in hippocampus. All of these results suggest that acupuncture as a potential treatment may exert neuroprotective effects via inhibiting cellular apoptosis, increased GDNF and BDNF expression levels in rat hippocampus experiencing HI. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. AEG-1 is associated with hypoxia-induced hepatocellular carcinoma chemoresistance via regulating PI3K/AKT/HIF-1alpha/MDR-1 pathway.

    Science.gov (United States)

    Xie, Yong; Zhong, De-Wu

    2016-01-01

    Hypoxia is a common characteristic of hepatocellular carcinoma (HCC) associated with reduced response to chemotherapy, thus increasing the probability of tumor recurrence. Astrocyte elevated gene-1 (AEG-1) has been involved in a wide array of cancer progression including proliferation, chemoresistance, angiogenesis and metastasis, but its effect on HCC chemoresistance induced by hypoxia is unclear. In this study, expression of AEG-1 and multiple drug resistance (MDR-1) were examined in HCC using immunohistochemical staining and RT-PCR. Furthermore, their expression levels were detected in HCC HepG2 cells in normoxia or hypoxia via RT-PCR and Western blot assays. Specific shRNAs were used to silence AEG-1 expression in HepG2 cells. Results showed AEG-1 and MDR-1 expression were higher in HCC tissues than in adjacent normal tissues. Incubation of HepG2 cells in hypoxia increased expression of AEG-1 and MDR-1, compared to incubation in normoxia. Exposure to hypoxia blunted sensitivity of HepG2 cells to Adriamycin, 5-fluorouracil and cis-platinum, as evidenced by modest alterations in cell viability and apoptosis rate, however the sensitivity was elevated with AEG-1 knockdown. PI3K/AKT/HIF-1/MDR-1 pathway was attenuated following AEG-1 knockdown in hypoxia. Based on these data, it was suggested that AEG-1 is associated with hypoxia-induced hepatocellular carcinoma chemoresistance via regulating PI3K/AKT/HIF-1/MDR-1 pathway. This study uncovered a novel potential target for development of an effective therapy against hypoxia-induced HCC chemoresistance.

  16. Trichostatin A enhances estrogen receptor-alpha repression in MCF-7 breast cancer cells under hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Noh, Hyunggyun; Park, Joonwoo; Shim, Myeongguk; Lee, YoungJoo, E-mail: yjlee@sejong.ac.kr

    2016-02-12

    Estrogen receptor (ER) is a crucial determinant of resistance to endocrine therapy, which may change during the progression of breast cancer. We previously showed that hypoxia induces ESR1 gene repression and ERα protein degradation via proteasome-mediated pathway in breast cancer cells. HDAC plays important roles in the regulation of histone and non-histone protein post-translational modification. HDAC inhibitors can induce epigenetic changes and have therapeutic potential for targeting various cancers. Trichostatin A exerts potent antitumor activities against breast cancer cells in vitro and in vivo. In this report, we show that TSA augments ESR1 gene repression at the transcriptional level and downregulates ERα protein expression under hypoxic conditions through a proteasome-mediated pathway. TSA-induced estrogen response element-driven reporter activity in the absence of estrogen was synergistically enhanced under hypoxia; however, TSA inhibited cell proliferation under both normoxia and hypoxia. Our data show that the hypoxia-induced repression of ESR1 and degradation of ERα are enhanced by concomitant treatment with TSA. These findings expand our understanding of hormone responsiveness in the tumor microenvironment; however, additional in-depth studies are required to elucidate the detailed mechanisms of TSA-induced ERα regulation under hypoxia. - Highlights: • TSA augments ESR1 gene repression at the transcriptional level under hypoxia. • TSA downregulates ERα protein expression under hypoxia. • TSA-induced ERα regulation under hypoxia is essential for understanding the behavior and progression of breast cancer.

  17. Detecting translational regulation by change point analysis of ribosome profiling data sets.

    Science.gov (United States)

    Zupanic, Anze; Meplan, Catherine; Grellscheid, Sushma N; Mathers, John C; Kirkwood, Tom B L; Hesketh, John E; Shanley, Daryl P

    2014-10-01

    Ribo-Seq maps the location of translating ribosomes on mature mRNA transcripts. While during normal translation, ribosome density is constant along the length of the mRNA coding region, this can be altered in response to translational regulatory events. In the present study, we developed a method to detect translational regulation of individual mRNAs from their ribosome profiles, utilizing changes in ribosome density. We used mathematical modeling to show that changes in ribosome density should occur along the mRNA at the point of regulation. We analyzed a Ribo-Seq data set obtained for mouse embryonic stem cells and showed that normalization by corresponding RNA-Seq can be used to improve the Ribo-Seq quality by removing bias introduced by deep-sequencing and alignment artifacts. After normalization, we applied a change point algorithm to detect changes in ribosome density present in individual mRNA ribosome profiles. Additional sequence and gene isoform information obtained from the UCSC Genome Browser allowed us to further categorize the detected changes into different mechanisms of regulation. In particular, we detected several mRNAs with known post-transcriptional regulation, e.g., premature termination for selenoprotein mRNAs and translational control of Atf4, but also several more mRNAs with hitherto unknown translational regulation. Additionally, our approach proved useful for identification of new transcript isoforms. © 2014 Zupanic et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  18. Regulation of transcriptome, translation, and proteome in response to environmental stress in fission yeast

    Science.gov (United States)

    2012-01-01

    Background Gene expression is controlled globally and at multiple levels in response to environmental stress, but the relationships among these dynamic regulatory changes are not clear. Here we analyzed global regulation during different stress conditions in fission yeast, Schizosaccharomyces pombe, combining dynamic genome-wide data on mRNA, translation, and protein profiles. Results We observed a strong overall concordance between changes in mRNAs and co-directional changes in translation, for both induced and repressed genes, in response to three conditions: oxidative stress, heat shock, and DNA damage. However, approximately 200 genes each under oxidative and heat stress conditions showed discordant regulation with respect to mRNA and translation profiles, with genes and patterns of regulation being stress-specific. For oxidative stress, we also measured dynamic profiles for 2,147 proteins, comprising 43% of the proteome. The mRNAs induced during oxidative stress strongly correlated with increased protein expression, while repressed mRNAs did not relate to the corresponding protein profiles. Overall changes in relative protein expression correlated better with changes in mRNA expression than with changes in translational efficiency. Conclusions These data highlight a global coordination and fine-tuning of gene regulation during stress that mostly acts in the same direction at the levels of transcription and translation. In the oxidative stress condition analyzed, transcription dominates translation to control protein abundance. The concordant regulation of transcription and translation leads to the expected adjustment in protein expression only for up-regulated mRNAs. These patterns of control might reflect the need to balance protein production for stress survival given a limited translational capacity. PMID:22512868

  19. Regulation of host translational machinery by African swine fever virus.

    Science.gov (United States)

    Castelló, Alfredo; Quintas, Ana; Sánchez, Elena G; Sabina, Prado; Nogal, Marisa; Carrasco, Luis; Revilla, Yolanda

    2009-08-01

    African swine fever virus (ASFV), like other complex DNA viruses, deploys a variety of strategies to evade the host's defence systems, such as inflammatory and immune responses and cell death. Here, we analyse the modifications in the translational machinery induced by ASFV. During ASFV infection, eIF4G and eIF4E are phosphorylated (Ser1108 and Ser209, respectively), whereas 4E-BP1 is hyperphosphorylated at early times post infection and hypophosphorylated after 18 h. Indeed, a potent increase in eIF4F assembly is observed in ASFV-infected cells, which is prevented by rapamycin treatment. Phosphorylation of eIF4E, eIF4GI and 4E-BP1 is important to enhance viral protein production, but is not essential for ASFV infection as observed in rapamycin- or CGP57380-treated cells. Nevertheless, eIF4F components are indispensable for ASFV protein synthesis and virus spread, since eIF4E or eIF4G depletion in COS-7 or Vero cells strongly prevents accumulation of viral proteins and decreases virus titre. In addition, eIF4F is not only activated but also redistributed within the viral factories at early times of infection, while eIF4G and eIF4E are surrounding these areas at late times. In fact, other components of translational machinery such as eIF2alpha, eIF3b, eIF4E, eEF2 and ribosomal P protein are enriched in areas surrounding ASFV factories. Notably, the mitochondrial network is polarized in ASFV-infected cells co-localizing with ribosomes. Thus, translation and ATP synthesis seem to be coupled and compartmentalized at the periphery of viral factories. At later times after ASFV infection, polyadenylated mRNAs disappear from the cytoplasm of Vero cells, except within the viral factories. The distribution of these pools of mRNAs is similar to the localization of viral late mRNAs. Therefore, degradation of cellular polyadenylated mRNAs and recruitment of the translation machinery to viral factories may contribute to the inhibition of host protein synthesis, facilitating ASFV

  20. Regulation of host translational machinery by African swine fever virus.

    Directory of Open Access Journals (Sweden)

    Alfredo Castelló

    2009-08-01

    Full Text Available African swine fever virus (ASFV, like other complex DNA viruses, deploys a variety of strategies to evade the host's defence systems, such as inflammatory and immune responses and cell death. Here, we analyse the modifications in the translational machinery induced by ASFV. During ASFV infection, eIF4G and eIF4E are phosphorylated (Ser1108 and Ser209, respectively, whereas 4E-BP1 is hyperphosphorylated at early times post infection and hypophosphorylated after 18 h. Indeed, a potent increase in eIF4F assembly is observed in ASFV-infected cells, which is prevented by rapamycin treatment. Phosphorylation of eIF4E, eIF4GI and 4E-BP1 is important to enhance viral protein production, but is not essential for ASFV infection as observed in rapamycin- or CGP57380-treated cells. Nevertheless, eIF4F components are indispensable for ASFV protein synthesis and virus spread, since eIF4E or eIF4G depletion in COS-7 or Vero cells strongly prevents accumulation of viral proteins and decreases virus titre. In addition, eIF4F is not only activated but also redistributed within the viral factories at early times of infection, while eIF4G and eIF4E are surrounding these areas at late times. In fact, other components of translational machinery such as eIF2alpha, eIF3b, eIF4E, eEF2 and ribosomal P protein are enriched in areas surrounding ASFV factories. Notably, the mitochondrial network is polarized in ASFV-infected cells co-localizing with ribosomes. Thus, translation and ATP synthesis seem to be coupled and compartmentalized at the periphery of viral factories. At later times after ASFV infection, polyadenylated mRNAs disappear from the cytoplasm of Vero cells, except within the viral factories. The distribution of these pools of mRNAs is similar to the localization of viral late mRNAs. Therefore, degradation of cellular polyadenylated mRNAs and recruitment of the translation machinery to viral factories may contribute to the inhibition of host protein synthesis

  1. ERK1/2 and Akt phosphorylation were essential for MGF E peptide regulating cell morphology and mobility but not proangiogenic capacity of BMSCs under severe hypoxia.

    Science.gov (United States)

    Sha, Yongqiang; Yang, Li; Lv, Yonggang

    2018-02-13

    Severe hypoxia inhibits the adhesion and mobility of bone marrow-derived mesenchymal stem cells (BMSCs) and limits their application in bone tissue engineering. In this study, CoCl 2 was used to simulate severe hypoxia and the effects of mechano-growth factor (MGF) E peptide on the morphology, adhesion, migration, and proangiogenic capacity of BMSCs under hypoxia were measured. It was demonstrated that severe hypoxia (500-μM CoCl 2 ) significantly caused cell contraction and reduced cell area, roundness, adhesion, and migration of BMSCs. RhoA and ROCK1 expression levels were upregulated by severe hypoxia, but p-RhoA and mobility-relevant protein (integrin β1, p-FAK and fibronectin) expression levels in BMSCs were inhibited. Fortunately, MGF E peptide could restore all abovementioned indexes except RhoA expression. MEK-ERK1/2 pathway was involved in MGF E peptide regulating cell morphological changes, mobility, and relevant proteins (except p-FAK). PI3K-Akt pathway was involved in MGF E peptide regulating cell area, mobility, and relevant proteins. Besides, severe hypoxia upregulated vascular endothelial growth factor α expression but was harmful for proangiogenic capacity of BMSCs. Our study suggested that MGF E peptide might be helpful for the clinical application of tissue engineering strategy in bone defect repair. Sever hypoxia impairs bone defect repair with bone marrow-derived mesenchymal stem cells (BMSCs). This study proved that mechano-growth factor E (MGF E) peptide could improve the severe hypoxia-induced cell contraction and decline of cell adhesion and migration of BMSCs. Besides, MGF E peptide weakened the effects of severe hypoxia on the cytoskeleton arrangement- and mobility-relevant protein expression levels in BMSCs. The underlying molecular mechanism was also verified. Finally, it was confirmed that MGF E peptide showed an adverse effect on the expression level of vascular endothelial growth factor α in BMSCs under severe hypoxia but could

  2. Mass spectrometry analysis of adipose-derived stem cells reveals a significant effect of hypoxia on pathways regulating extracellular matrix.

    Science.gov (United States)

    Riis, Simone; Stensballe, Allan; Emmersen, Jeppe; Pennisi, Cristian Pablo; Birkelund, Svend; Zachar, Vladimir; Fink, Trine

    2016-04-14

    Adipose-derived stem cells (ASCs) are being increasingly recognized for their potential to promote tissue regeneration and wound healing. These effects appear to be partly mediated by paracrine signaling pathways, and are enhanced during hypoxia. Mass spectrometry (MS) is a valuable tool for proteomic profiling of cultured ASCs, which may help to reveal the identity of the factors secreted by the cells under different conditions. However, serum starvation which is essentially required to obtain samples compatible with secretome analysis by MS can have a significant influence on ASCs. Here, we present a novel and optimized culturing approach based on the use of a clinically relevant serum-free formulation, which was used to assess the effects of hypoxia on the ASC proteomic profile. Human ASCs from three human donors were expanded in StemPro® MSC SFM XenoFree medium. Cells were cultured for 24 h in serum- and albumin-free supplements in either normoxic (20 %) or hypoxic (1 %) atmospheres, after which the cells and conditioned medium were collected, subfractionated, and analyzed using MS. Prior to analysis, the secreted proteins were further subdivided into a secretome (>30 kDa) and a peptidome (3-30 kDa) fraction. MS analysis revealed the presence of 342, 98, and 3228 proteins in the normoxic ASC secretome, peptidome, and proteome, respectively. A relatively small fraction of the proteome (9.6 %) was significantly affected by hypoxia, and the most regulated proteins were those involved in extracellular matrix (ECM) synthesis and cell metabolism. No proteins were found to be significantly modulated by hypoxic treatment across all cultures for the secretome and peptidome samples. This study highlights ECM remodeling as a significant mechanism contributing to the ASC regenerative effect after hypoxic preconditioning, and further underscores considerable inter-individual differences in ASC response to hypoxia. The novel culture paradigm provides a basis for future

  3. Post-translational regulation of Oct4 transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Jonathan P Saxe

    Full Text Available Oct4 is a key component of the molecular circuitry which regulates embryonic stem cell proliferation and differentiation. It is essential for maintenance of undifferentiated, pluripotent cell populations, and accomplishes these tasks by binding DNA in multiple heterodimer and homodimer configurations. Very little is known about how formation of these complexes is regulated, or the mechanisms through which Oct4 proteins respond to complex extracellular stimuli which regulate pluripotency. Here, we provide evidence for a phosphorylation-based mechanism which regulates specific Oct4 homodimer conformations. Point mutations of a putative phosphorylation site can specifically abrogate transcriptional activity of a specific homodimer assembly, with little effect on other configurations. Moreover, we performed bioinformatic predictions to identify a subset of Oct4 target genes which may be regulated by this specific assembly, and show that altering Oct4 protein levels affects transcription of Oct4 target genes which are regulated by this assembly but not others. Finally, we identified several signaling pathways which may mediate this phosphorylation and act in combination to regulate Oct4 transcriptional activity and protein stability. These results provide a mechanism for rapid and reversible alteration of Oct4 transactivation potential in response to extracellular signals.

  4. Acute regulation of hematocrit and acid-base balance in chicken embryos in response to severe intrinsic hypercapnic hypoxia.

    Science.gov (United States)

    Andrewartha, Sarah J; Tazawa, Hiroshi; Burggren, Warren W

    2014-05-01

    The regulation of blood acid-base balance and hematology in day 15 chicken embryos in response to partial water submersion (with egg's air cell in air) and complete submersion producing severe intrinsic hypercapnic hypoxia and recovery in air was studied. The acid-base disturbance during submersion was characterized by initial rapid respiratory changes and then superseded by metabolic processes, resulting in a large progressive hysteresis. Throughout submersion and recovery, blood lactate concentration changed swiftly along with the changes in bicarbonate concentration ([HCO3(-)]), indicating that anaerobic glycolysis determined overall acid-base disturbances. Both partial and complete submersion produced large, rapid increases in hematocrit through proportional increases in mean corpuscular volume and red blood cell concentration. Death ensued once the internal pool of O2 was exhausted and/or the acid-base disturbance became too severe for survival (i.e., [HCO3(-)]a<∼10mmolL(-1)). However, embryos recovered from acid-base and hematological disturbances within 120min recovery in air after short bouts of complete (20min) or partial (60min) submersion, suggesting that shorter severe intrinsic hypercapnic hypoxia does not compromise viability of embryos. Copyright © 2014. Published by Elsevier B.V.

  5. Hypoxia and Mucosal Inflammation

    Science.gov (United States)

    Colgan, Sean P.; Campbell, Eric L.; Kominsky, Douglas J.

    2016-01-01

    Sites of inflammation are defined by significant changes in metabolic activity. Recent studies have suggested that O2 metabolism and hypoxia play a prominent role in inflammation so-called “inflammatory hypoxia,” which results from a combination of recruited inflammatory cells (e.g., neutrophils and monocytes), the local proliferation of multiple cell types, and the activation of multiple O2-consuming enzymes during inflammation. These shifts in energy supply and demand result in localized regions of hypoxia and have revealed the important function off the transcription factor HIF (hypoxia-inducible factor) in the regulation of key target genes that promote inflammatory resolution. Analysis of these pathways has provided multiple opportunities for understanding basic mechanisms of inflammation and has defined new targets for intervention. Here, we review recent work addressing tissue hypoxia and metabolic control of inflammation and immunity. PMID:27193451

  6. The tyrosine phosphatase SHP-1 regulates hypoxia inducible factor-1α (HIF-1α protein levels in endothelial cells under hypoxia.

    Directory of Open Access Journals (Sweden)

    Stefan K Alig

    Full Text Available The tyrosine phosphatase SHP-1 negatively influences endothelial function, such as VEGF signaling and reactive oxygen species (ROS formation, and has been shown to influence angiogenesis during tissue ischemia. In ischemic tissues, hypoxia induced angiogenesis is crucial for restoring oxygen supply. However, the exact mechanism how SHP-1 affects endothelial function during ischemia or hypoxia remains unclear. We performed in vitro endothelial cell culture experiments to characterize the role of SHP-1 during hypoxia.SHP-1 knock-down by specific antisense oligodesoxynucleotides (AS-Odn increased cell growth as well as VEGF synthesis and secretion during 24 hours of hypoxia compared to control AS-Odn. This was prevented by HIF-1α inhibition (echinomycin and apigenin. SHP-1 knock-down as well as overexpression of a catalytically inactive SHP-1 (SHP-1 CS further enhanced HIF-1α protein levels, whereas overexpression of a constitutively active SHP-1 (SHP-1 E74A resulted in decreased HIF-1α levels during hypoxia, compared to wildtype SHP-1. Proteasome inhibition (MG132 returned HIF-1α levels to control or wildtype levels respectively in these cells. SHP-1 silencing did not alter HIF-1α mRNA levels. Finally, under hypoxic conditions SHP-1 knock-down enhanced intracellular endothelial reactive oxygen species (ROS formation, as measured by oxidation of H2-DCF and DHE fluorescence.SHP-1 decreases half-life of HIF-1α under hypoxic conditions resulting in decreased cell growth due to diminished VEGF synthesis and secretion. The regulatory effect of SHP-1 on HIF-1α stability may be mediated by inhibition of endothelial ROS formation stabilizing HIF-1α protein. These findings highlight the importance of SHP-1 in hypoxic signaling and its potential as therapeutic target in ischemic diseases.

  7. DOWN-REGULATION OF THE HYPOTHALAMO-PITUITARY-ADRENAL AXIS REDUCES BRAIN-DAMAGE AND NUMBER OF SEIZURES FOLLOWING HYPOXIA/ISCHAEMIA IN RATS

    NARCIS (Netherlands)

    KRUGERS, HJ; KNOLLEMA, S; KEMPER, RHA; TERHORST, GJ; KORF, J

    1995-01-01

    Several reports suggest that the activity of the hypothalamo-pituitary-adrenal axis (HPA-axis) is increased following hypoxia/ischaemia and that this might be associated with increased neuronal vulnerability. The main goal of this study was to examine the effects of down-regulation of the HPA-axis

  8. mTOR Signaling in Protein Translation Regulation: Implications in Cancer Genesis and Therapeutic Interventions

    Directory of Open Access Journals (Sweden)

    Mehvish Showkat

    2014-01-01

    Full Text Available mTOR is a central nutrient sensor that signals a cell to grow and proliferate. Through distinct protein complexes it regulates different levels of available cellular energy substrates required for cell growth. One of the important functions of the complex is to maintain available amino acid pool by regulating protein translation. Dysregulation of mTOR pathway leads to aberrant protein translation which manifests into various pathological states. Our review focuses on the role mTOR signaling plays in protein translation and its physiological role. It also throws some light on available data that show translation dysregulation as a cause of pathological complexities like cancer and the available drugs that target the pathway for cancer treatment.

  9. SF2/ASF regulates proteomic diversity by affecting the balance between translation initiation mechanisms.

    Science.gov (United States)

    Blaustein, Matías; Quadrana, Leandro; Risso, Guillermo; Mata, Manuel de la; Pelisch, Federico; Srebrow, Anabella

    2009-07-01

    Post-splicing activities have been described for a subset of shuttling serine/arginine-rich splicing regulatory proteins, among them SF2/ASF. We showed that growth factors activate a Ras-PI 3-kinase-Akt/PKB signaling pathway that not only modifies alternative splicing of the fibronectin EDA exon, but also alters in vivo translation of reporter mRNAs containing the EDA binding motif for SF2/ASF, providing two co-regulated levels of isoform-specific amplification. Translation of most eukaryotic mRNAs is initiated via the scanning mechanism, which implicates recognition of the m7G cap at the mRNA 5'-terminus by the eIF4F protein complex. Several viral and cellular mRNAs are translated in a cap-independent manner by the action of cis-acting mRNA elements named internal ribosome entry sites that direct internal ribosome binding to the mRNA. Here we use bicistronic reporters that generate mRNAs carrying two open reading frames, one translated in a cap-dependent manner while the other by internal ribosome entry site-dependent initiation, to show that in vivo over-expression of SF2/ASF increases the ratio between cap-dependent and internal ribosome entry site-dependent translation. Consistently, knocking-down of SF2/ASF causes the opposite effect. Changes in expression levels of SF2/ASF also affect alternative translation of an endogenous mRNA, that one coding for fibroblast growth factor-2. These results strongly suggest a role for SF2/ASF as a regulator of alternative translation, meaning the generation of different proteins by the balance among these two translation initiation mechanisms, and expand the known potential of SF2/ASF to regulate proteomic diversity to the translation field. 2009 Wiley-Liss, Inc.

  10. Sumoylation of hypoxia-inducible factor-1α ameliorates failure of brain stem cardiovascular regulation in experimental brain death.

    Directory of Open Access Journals (Sweden)

    Julie Y H Chan

    2011-03-01

    Full Text Available One aspect of brain death is cardiovascular deregulation because asystole invariably occurs shortly after its diagnosis. A suitable neural substrate for mechanistic delineation of this aspect of brain death resides in the rostral ventrolateral medulla (RVLM. RVLM is the origin of a life-and-death signal that our laboratory detected from blood pressure of comatose patients that disappears before brain death ensues. At the same time, transcriptional upregulation of heme oxygenase-1 in RVLM by hypoxia-inducible factor-1α (HIF-1α plays a pro-life role in experimental brain death, and HIF-1α is subject to sumoylation activated by transient cerebral ischemia. It follows that sumoylation of HIF-1α in RVLM in response to hypoxia may play a modulatory role on brain stem cardiovascular regulation during experimental brain death.A clinically relevant animal model that employed mevinphos as the experimental insult in Sprague-Dawley rat was used. Biochemical changes in RVLM during distinct phenotypes in systemic arterial pressure spectrum that reflect maintained or defunct brain stem cardiovascular regulation were studied. Western blot analysis, EMSA, ELISA, confocal microscopy and immunoprecipitation demonstrated that drastic tissue hypoxia, elevated levels of proteins conjugated by small ubiquitin-related modifier-1 (SUMO-1, Ubc9 (the only known conjugating enzyme for the sumoylation pathway or HIF-1α, augmented sumoylation of HIF-1α, nucleus-bound translocation and enhanced transcriptional activity of HIF-1α in RVLM neurons took place preferentially during the pro-life phase of experimental brain death. Furthermore, loss-of-function manipulations by immunoneutralization of SUMO-1, Ubc9 or HIF-1α in RVLM blunted the upregulated nitric oxide synthase I/protein kinase G signaling cascade, which sustains the brain stem cardiovascular regulatory machinery during the pro-life phase.We conclude that sumoylation of HIF-1α in RVLM ameliorates brain stem

  11. Hypoxia Room

    Data.gov (United States)

    Federal Laboratory Consortium — The Hypoxia Room is a 8x8x8 ft. clear vinyl plastic and aluminum frame construction enclosure located within USAREIM laboratory 028. The Hypoxia Room (manufactured...

  12. Angiotensin-(1-7) attenuated long-term hypoxia-stimulated cardiomyocyte apoptosis by inhibiting HIF-1α nuclear translocation via Mas receptor regulation.

    Science.gov (United States)

    Chang, Ruey-Lin; Lin, Jing-Wei; Kuo, Wei-Wen; Hsieh, Dennis Jine-Yuan; Yeh, Yu-Lan; Shen, Chia-Yao; Day, Cecilia-Hsuan; Ho, Tsung-Jung; Viswanadha, Vijaya Padma; Huang, Chih-Yang

    2016-02-01

    Extreme hypoxia often leads to myocardial apoptosis and causes heart failure. Angiotensin-(1-7)Ang-(1-7) is well known for its cardio-protective effects. However, the effects of Ang-(1-7) on long-term hypoxia (LTH)-induced apoptosis remain unknown. In this study, we found that Ang-(1-7) reduced myocardial apoptosis caused by hypoxia through the Mas receptor. Activation of the Ang-(1-7)/Mas axis down-regulated the hypoxia pro-apoptotic signaling cascade by decreasing the protein levels of hypoxia-inducible factor 1α (HIF-1α) and insulin-like growth factor binding protein-3 (IGFBP3). Moreover, the Ang-(1-7)/Mas axis further inhibited HIF-1α nuclear translocation. On the other hand, Ang-(1-7) activated the IGF1R/PI3K/Akt signaling pathways, which mediate cell survival. However, the above effects were abolished by A779 treatment or silencing of Mas expression. Taken together, our findings indicate that the Ang-(1-7)/Mas axis protects cardiomyocytes from LTH-stimulated apoptosis. The protective effect of Ang-(1-7) is associated with the inhibition of HIF-1α nuclear translocation and the induction of IGF1R and Akt phosphorylation.

  13. Hypoxia-inducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells.

    Science.gov (United States)

    Zhang, Chuanzhao; Zhi, Wanqing Iris; Lu, Haiquan; Samanta, Debangshu; Chen, Ivan; Gabrielson, Edward; Semenza, Gregg L

    2016-10-04

    Exposure of breast cancer cells to hypoxia increases the percentage of breast cancer stem cells (BCSCs), which are required for tumor initiation and metastasis, and this response is dependent on the activity of hypoxia-inducible factors (HIFs). We previously reported that exposure of breast cancer cells to hypoxia induces the ALKBH5-mediated demethylation of N6-methyladenosine (m6A) in NANOG mRNA leading to increased expression of NANOG, which is a pluripotency factor that promotes BCSC specification. Here we report that exposure of breast cancer cells to hypoxia also induces ZNF217-dependent inhibition of m6A methylation of mRNAs encoding NANOG and KLF4, which is another pluripotency factor that mediates BCSC specification. Although hypoxia induced the BCSC phenotype in all breast-cancer cell lines analyzed, it did so through variable induction of pluripotency factors and ALKBH5 or ZNF217. However, in every breast cancer line, the hypoxic induction of pluripotency factor and ALKBH5 or ZNF217 expression was HIF-dependent. Immunohistochemistry revealed that expression of HIF-1α and ALKBH5 was concordant in all human breast cancer biopsies analyzed. ALKBH5 knockdown in MDA-MB-231 breast cancer cells significantly decreased metastasis from breast to lungs in immunodeficient mice. Thus, HIFs stimulate pluripotency factor expression and BCSC specification by negative regulation of RNA methylation.

  14. Strong iron demand during hypoxia-induced erythropoiesis is associated with down-regulation of iron-related proteins and myoglobin in human skeletal muscle

    DEFF Research Database (Denmark)

    Robach, Paul; Cairo, Gaetano; Gelfi, Cecilia

    2007-01-01

    to increase. This study gives new insights into the changes in iron content and iron-oxygen interactions during enhanced erythropoiesis by simultaneously analyzing blood and muscle samples in humans exposed to 7 to 9 days of high altitude hypoxia (HA). HA up-regulates iron acquisition by erythroid cells......Iron is essential for oxygen transport because it is incorporated in the heme of the oxygen-binding proteins hemoglobin and myoglobin. An interaction between iron homeostasis and oxygen regulation is further suggested during hypoxia, in which hemoglobin and myoglobin syntheses have been reported......, mobilizes body iron, and increases hemoglobin concentration. However, contrary to our hypothesis that muscle iron proteins and myoglobin would also be up-regulated during HA, this study shows that HA lowers myoglobin expression by 35% and down-regulates iron-related proteins in skeletal muscle, as evidenced...

  15. Combinatorial Control of Transgene Expression by Hypoxia-Responsive Promoter and MicroRNA Regulation for Neural Stem Cell-Based Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Yumei Luo

    2014-01-01

    Full Text Available Owing to their strong migratory capacity, tumor tropism, and tumor inhibitory effect, neural stem cells (NSCs have recently emerged as one of the most attractive gene delivery vectors for cancer therapy. However, further animal studies found that proportional NSC vectors were distributed to nontarget organs after intravenous injection and the nonspecific transgene expression led to significant cytotoxic effects in these organs. Hence, an expression cassette that controls the transgene expression within NSC vectors in a tumor site-specific manner is desired. Considering hypoxia as a hallmark of tumor microenvironment, we have developed a novel NSC vector platform coupling transcriptional targeting with microRNA (miRNA regulation for tumor hypoxia targeting. This combinatorial vector employed a hypoxia-responsive promoter and repeated targeting sequences of an miRNA that is enriched in NSCs but downregulated upon hypoxia induction to control the transgene expression. This resulted in significantly improved hypoxic selectivity over the use of a control vector without miRNA regulation. Thus, incorporating miRNA regulation into a transcriptional targeting vector adds an extra layer of security to prevent off-target transgene expression and should be useful for the development of NSC vectors with high targeting specifcity for cancer therapy.

  16. Stabilization of HIF-2α through redox regulation of mTORC2 activation and initiation of mRNA translation.

    Science.gov (United States)

    Nayak, B K; Feliers, D; Sudarshan, S; Friedrichs, W E; Day, R T; New, D D; Fitzgerald, J P; Eid, A; Denapoli, T; Parekh, D J; Gorin, Y; Block, K

    2013-06-27

    Hypoxia inducible factor-2α (HIF-2α) has a critical role in renal tumorigenesis. HIF-2α is stabilized in von Hippel-Lindau (VHL)-deficient renal cell carcinoma through mechanisms that require ongoing mRNA translation. Mammalian target of rapamycin (mTOR) functions in two distinct complexes: Raptor-associated mTORC1 and Rictor-associated mTORC2. Rictor-associated mTORC2 complex has been linked to maintaining HIF-2α protein in the absence of VHL; however, the mechanisms remain to be elucidated. Although Raptor-associated mTORC1 is a known key upstream regulator of mRNA translation, initiation and elongation, the role of mTORC2 in regulating mRNA translation is not clear. Complex assembly of the mRNA cap protein, eukaryotic translation initiation factor 4 (eIF4)E, with activators (eIF4 gamma (eIF4G)) and inhibitors (eIF4E-binding protein 1 (4E-BP1)) are rate-limiting determinants of mRNA translation. Our laboratory has previously demonstrated that reactive oxygen species, mediated by p22(phox)-based Nox oxidases, are enhanced in VHL-deficient cells and have a role in the activation of Akt on S473, a site phosphorylated by the mTORC2 complex. In this study, we examined the role of Rictor-dependent regulation of HIF-2α through eIF4E-dependent mRNA translation and examined the effects of p22(phox)-based Nox oxidases on TORC2 regulation. We demonstrate for the first time that mTORC2 complex stability and activation is redox sensitive, and further defined a novel role for p22(phox)-based Nox oxidases in eIF4E-dependent mRNA translation through mTORC2. Furthermore, we provide the first evidence that silencing of p22(phox) reduces HIF-2α-dependent gene targeting in vitro and tumor formation in vivo. The clinical relevance of these studies is demonstrated.

  17. Hypoxia-Inducible Hydrogels

    Science.gov (United States)

    Park, Kyung Min; Gerecht, Sharon

    2014-01-01

    Oxygen is vital for the existence of all multicellular organisms, acting as a signaling molecule regulating cellular activities. Specifically, hypoxia, which occurs when the partial pressure of oxygen falls below 5%, plays a pivotal role during development, regeneration, and cancer. Here we report a novel hypoxia-inducible (HI) hydrogel composed of gelatin and ferulic acid that can form hydrogel networks via oxygen consumption in a laccase-mediated reaction. Oxygen levels and gradients within the hydrogels can be accurately controlled and precisely predicted. We demonstrate that HI hydrogels guide vascular morphogenesis in vitro via hypoxia-inducible factors activation of matrix metalloproteinases and promote rapid neovascularization from the host tissue during subcutaneous wound healing. The HI hydrogel is a new class of biomaterials that may prove useful in many applications, ranging from fundamental studies of developmental, regenerative and disease processes through the engineering of healthy and diseased tissue models towards the treatment of hypoxia-regulated disorders. PMID:24909742

  18. MicroRNA-210 regulates mitochondrial free radical response to hypoxia and krebs cycle in cancer cells by targeting iron sulfur cluster protein ISCU.

    Directory of Open Access Journals (Sweden)

    Elena Favaro

    2010-04-01

    Full Text Available Hypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1 and a microRNA, hsa-miR-210 (miR-210 which is associated with a poor prognosis.In human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis.Induction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation.

  19. MicroRNA-210 regulates mitochondrial free radical response to hypoxia and krebs cycle in cancer cells by targeting iron sulfur cluster protein ISCU.

    Science.gov (United States)

    Favaro, Elena; Ramachandran, Anassuya; McCormick, Robert; Gee, Harriet; Blancher, Christine; Crosby, Meredith; Devlin, Cecilia; Blick, Christopher; Buffa, Francesca; Li, Ji-Liang; Vojnovic, Borivoj; Pires das Neves, Ricardo; Glazer, Peter; Iborra, Francisco; Ivan, Mircea; Ragoussis, Jiannis; Harris, Adrian L

    2010-04-26

    Hypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1) and a microRNA, hsa-miR-210 (miR-210) which is associated with a poor prognosis. In human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS) in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis. Induction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation.

  20. ATX-2, the C. elegans ortholog of ataxin 2, functions in translational regulation in the germline.

    Science.gov (United States)

    Ciosk, Rafal; DePalma, Michael; Priess, James R

    2004-10-01

    Human ataxin 2 is a protein of unknown function that is implicated in the neurodegenerative disorder spinocerebellar ataxia type 2. We found that the C. elegans ortholog of ataxin 2, ATX-2, forms a complex with PAB-1, a cytoplasmic polyA-binding protein, and that ATX-2 is required for development of the germline. In the absence of ATX-2, proliferation of stem cells is reduced, and the germline is abnormally masculinized. These defects appear to result from inappropriate translational regulation that normally is mediated by the conserved KH-domain protein GLD-1. We find that MEX-3, a second KH-domain protein, exhibits a novel, ATX-2-dependent role in preventing inappropriate translation in the germline stem cells. Together, our results suggest that ATX-2 functions in translational regulation that is mediated by GLD-1 and MEX-3 proteins.

  1. Roles of Transcriptional and Translational Control Mechanisms in Regulation of Ribosomal Protein Synthesis in Escherichia coli.

    Science.gov (United States)

    Burgos, Hector L; O'Connor, Kevin; Sanchez-Vazquez, Patricia; Gourse, Richard L

    2017-11-01

    Bacterial ribosome biogenesis is tightly regulated to match nutritional conditions and to prevent formation of defective ribosomal particles. In Escherichia coli, most ribosomal protein (r-protein) synthesis is coordinated with rRNA synthesis by a translational feedback mechanism: when r-proteins exceed rRNAs, specific r-proteins bind to their own mRNAs and inhibit expression of the operon. It was recently discovered that the second messenger nucleotide guanosine tetra and pentaphosphate (ppGpp), which directly regulates rRNA promoters, is also capable of regulating many r-protein promoters. To examine the relative contributions of the translational and transcriptional control mechanisms to the regulation of r-protein synthesis, we devised a reporter system that enabled us to genetically separate the cis-acting sequences responsible for the two mechanisms and to quantify their relative contributions to regulation under the same conditions. We show that the synthesis of r-proteins from the S20 and S10 operons is regulated by ppGpp following shifts in nutritional conditions, but most of the effect of ppGpp required the 5' region of the r-protein mRNA containing the target site for translational feedback regulation and not the promoter. These results suggest that most regulation of the S20 and S10 operons by ppGpp following nutritional shifts is indirect and occurs in response to changes in rRNA synthesis. In contrast, we found that the promoters for the S20 operon were regulated during outgrowth, likely in response to increasing nucleoside triphosphate (NTP) levels. Thus, r-protein synthesis is dynamic, with different mechanisms acting at different times.IMPORTANCE Bacterial cells have evolved complex and seemingly redundant strategies to regulate many high-energy-consuming processes. In E. coli, synthesis of ribosomal components is tightly regulated with respect to nutritional conditions by mechanisms that act at both the transcription and translation steps. In this

  2. Regulating NKX3.1 stability and function: Post-translational modifications and structural determinants.

    Science.gov (United States)

    Padmanabhan, Achuth; Rao, Varsha; De Marzo, Angelo M; Bieberich, Charles J

    2016-05-01

    The androgen-regulated homeodomain transcription factor NKX3.1 plays roles in early prostate development and functions as a prostate-specific tumor suppressor. Decreased expression of NKX3.1 protein is common in primary prostate cancer. Discordance between NKX3.1 mRNA and protein levels during prostate carcinogenesis suggested a key role for post-transcriptional modifications in regulating NKX3.1 protein levels in prostate epithelial cells. Subsequent studies revealed NKX3.1 to be modified post-translationally at multiple sites. We reviewed published literature to identify and summarize post-translational modifications and structural elements critical in regulating NKX3.1 stability and levels in prostate epithelial cells. NKX3.1 is modified post-translationally at multiple sites by different protein kinases. These modifications together with several structural determinants were identified to play an important role in NKX3.1 stability and biology. In this review, we provide a comprehensive overview of the known post-translational modifications and structural features that impact NKX3.1. Defining factors that regulate NKX3.1 in prostate epithelial cells will extend our understanding of molecular changes that may contribute to prostate cancer initiation and progression. © 2016 Wiley Periodicals, Inc.

  3. Oxygen sensing and hypoxia signalling pathways in animals: the implications of physiology for cancer.

    Science.gov (United States)

    Ratcliffe, Peter J

    2013-04-15

    Studies of regulation of the haematopoietic growth factor erythropoietin led to the unexpected discovery of a widespread system of direct oxygen sensing that regulates gene expression in animals. The oxygen-sensitive signal is generated by a series of non-haem Fe(II)- and 2-oxoglutarate-dependent dioxygenases that catalyse the post-translational hydroxylation of specific residues in the transcription factor hypoxia-inducible factor (HIF). These hydroxylations promote both oxygen-dependent degradation and oxygen-dependent inactivation of HIF, but are suppressed in hypoxia, leading to the accumulation of HIF and assembly of an active transcriptional complex in hypoxic cells. Hypoxia-inducible factor activates an extensive transcriptional cascade that interfaces with other cell signalling pathways, microRNA networks and RNA-protein translational control systems. The relationship of these cellular signalling pathways to the integrated physiology of oxygen homeostasis and the implication of dysregulating these massive physiological pathways in diseases such as cancer are discussed.

  4. The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through posttranscriptional regulation of the proto-oncogene PIM1 in pancreatic cancer cells.

    Science.gov (United States)

    Blanco, F F; Jimbo, M; Wulfkuhle, J; Gallagher, I; Deng, J; Enyenihi, L; Meisner-Kober, N; Londin, E; Rigoutsos, I; Sawicki, J A; Risbud, M V; Witkiewicz, A K; McCue, P A; Jiang, W; Rui, H; Yeo, C J; Petricoin, E; Winter, J M; Brody, J R

    2016-05-01

    Previously, it has been shown that pancreatic ductal adenocarcinoma (PDA) tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO2) and decreased O2 intracellular perfusion. Chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in an understudied phenomenon known as hypoxia-induced chemoresistance. The hypoxia-inducible, pro-oncogenic, serine-threonine kinase PIM1 (Proviral Integration site for Moloney murine leukemia virus 1) has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. Although its role in therapeutic resistance has been described previously, the molecular mechanism behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present within a 38-base pair region of the PIM1 mRNA 3'-untranslated region mediate a regulatory interaction with the mRNA stability factor HuR (Hu antigen R) in the context of tumor hypoxia. Predominantly expressed in the nucleus in PDA cells, HuR translocates to the cytoplasm in response to hypoxic stress and stabilizes the PIM1 mRNA transcript, resulting in PIM1 protein overexpression. A reverse-phase protein array revealed that HuR-mediated regulation of PIM1 protects cells from hypoxic stress through phosphorylation and inactivation of the apoptotic effector BAD and activation of MEK1/2. Importantly, pharmacological inhibition of HuR by MS-444 inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and markedly enhances PDA cell sensitivity to oxaliplatin and 5-fluorouracil under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has prosurvival properties in PDA cells by enabling them with growth advantages in stressful tumor microenvironment niches. Accordingly, these studies provide evidence that therapeutic disruption of HuR's regulation of PIM1 may be a key strategy in

  5. Regulation of red blood cell deformability is independent of red blood cell-nitric oxide synthase under hypoxia.

    Science.gov (United States)

    Grau, Marijke; Lauten, Alexander; Hoeppener, Steffen; Goebel, Bjoern; Brenig, Julian; Jung, Christian; Bloch, Wilhelm; Suhr, Frank

    2016-09-12

    The aim was to study impacts of mild to severe hypoxia on human red blood cell (RBC)-nitric oxide synthase (NOS)-dependent NO production, protein S-nitrosylation and deformability.Ambient air oxygen concentration of 12 healthy subjects was step-wisely reduced from 20.95% to 16.21%, 12.35%, 10% and back to 20.95%. Additional in vitro experiments involved purging of blood (±sodium nitrite) with gas mixtures corresponding to in vivo intervention.Vital and hypoxia-associated parameters showed physiological adaptation to changing demands. Activation of RBC-NOS decreased with increasing hypoxia. RBC deformability, which is influenced by RBC-NOS activation, decreased under mild hypoxia, but surprisingly increased at severe hypoxia in vivo and in vitro. This was causatively induced by nitrite reduction to NO which increased S-nitrosylation of RBC α- and β-spectrins -a critical step to improve RBC deformability. The addition of sodium nitrite prevented decreases of RBC deformability under hypoxia by sustaining S-nitrosylation of spectrins suggesting compensatory mechanisms of non-RBC-NOS-produced NO.The results first time indicate a direct link between maintenance of RBC deformability under severe hypoxia by non-enzymatic NO production because RBC-NOS activation is reduced. These data improve our understanding of physiological mechanisms supporting adequate blood and, thus, oxygen supply to different tissues under severe hypoxia.

  6. Gene regulation by translational inhibition is determined by Dicer partnering proteins.

    Science.gov (United States)

    Reis, Rodrigo S; Hart-Smith, Gene; Eamens, Andrew L; Wilkins, Marc R; Waterhouse, Peter M

    2015-02-09

    MicroRNAs (miRNAs) are small regulatory RNAs produced by Dicer proteins that regulate gene expression in development and adaptive responses to the environment(1-4). In animals, the degree of base pairing between a miRNA and its target messenger RNA seems to determine whether the regulation occurs through cleavage or translation inhibition(1). In contrast, the selection of regulatory mechanisms is independent of the degree of mismatch between a plant miRNA and its target transcript(5). However, the components and mechanism(s) that determine whether a plant miRNA ultimately regulates its targets by guiding cleavage or translational inhibition are unknown(6). Here we show that the form of regulatory action directed by a plant miRNA is determined by DRB2, a DICER-LIKE1 (DCL1) partnering protein. The dependence of DCL1 on DRB1 for miRNA biogenesis is well characterized(7-9), but we show that it is only required for miRNA-guided transcript cleavage. We found that DRB2 determines miRNA-guided translational inhibition and represses DRB1 expression, thereby allowing the active selection of miRNA regulatory action. Furthermore, our results reveal that the core silencing proteins ARGONAUTE1 (AGO1) and SERRATE (SE) are highly regulated by miRNA-guided translational inhibition. DRB2 has been remarkably conserved throughout plant evolution, raising the possibility that translational repression is the ancient form of miRNA-directed gene regulation in plants, and that Dicer partnering proteins, such as human TRBP, might play a similar role in other eukaryotic systems.

  7. ChIP-seq and in vivo transcriptome analyses of the Aspergillus fumigatus SREBP SrbA reveals a new regulator of the fungal hypoxia response and virulence.

    Directory of Open Access Journals (Sweden)

    Dawoon Chung

    2014-11-01

    Full Text Available The Aspergillus fumigatus sterol regulatory element binding protein (SREBP SrbA belongs to the basic Helix-Loop-Helix (bHLH family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA. How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4-sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveal new insights into SREBPs' complex role in infection site adaptation and fungal virulence.

  8. Regulated Formation of an Amyloid-like Translational Repressor Governs Gametogenesis.

    Science.gov (United States)

    Berchowitz, Luke E; Kabachinski, Greg; Walker, Margaret R; Carlile, Thomas M; Gilbert, Wendy V; Schwartz, Thomas U; Amon, Angelika

    2015-10-08

    Message-specific translational control is required for gametogenesis. In yeast, the RNA-binding protein Rim4 mediates translational repression of numerous mRNAs, including the B-type cyclin CLB3, which is essential for establishing the meiotic chromosome segregation pattern. Here, we show that Rim4 forms amyloid-like aggregates and that it is the amyloid-like form of Rim4 that is the active, translationally repressive form of the protein. Our data further show that Rim4 aggregation is a developmentally regulated process. Starvation induces the conversion of monomeric Rim4 into amyloid-like aggregates, thereby activating the protein to bring about repression of translation. At the onset of meiosis II, Rim4 aggregates are abruptly degraded allowing translation to commence. Although amyloids are best known for their role in the etiology of diseases such as Alzheimer's, Parkinson's, and diabetes by forming toxic protein aggregates, our findings show that cells can utilize amyloid-like protein aggregates to function as central regulators of gametogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. A universal strategy for regulating mRNA translation in prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Cao, Jicong; Arha, Manish; Sudrik, Chaitanya; Mukherjee, Abhirup; Wu, Xia; Kane, Ravi S

    2015-04-30

    We describe a simple strategy to control mRNA translation in both prokaryotic and eukaryotic cells which relies on a unique protein-RNA interaction. Specifically, we used the Pumilio/FBF (PUF) protein to repress translation by binding in between the ribosome binding site (RBS) and the start codon (in Escherichia coli), or by binding to the 5' untranslated region of target mRNAs (in mammalian cells). The design principle is straightforward, the extent of translational repression can be tuned and the regulator is genetically encoded, enabling the construction of artificial signal cascades. We demonstrate that this approach can also be used to regulate polycistronic mRNAs; such regulation has rarely been achieved in previous reports. Since the regulator used in this study is a modular RNA-binding protein, which can be engineered to target different 8-nucleotide RNA sequences, our strategy could be used in the future to target endogenous mRNAs for regulating metabolic flows and signaling pathways in both prokaryotic and eukaryotic cells. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Regulation of endothelin-1 synthesis in human pulmonary arterial smooth muscle cells. Effects of transforming growth factor-beta and hypoxia.

    Science.gov (United States)

    Markewitz, B A; Farrukh, I S; Chen, Y; Li, Y; Michael, J R

    2001-01-01

    Endothelin-1 (ET-1) potently regulates pulmonary vascular tone and promotes vascular smooth muscle cell growth. Clinical and animal studies implicate increased ET-1 production in the pathogenesis of primary and secondary pulmonary hypertension. Although pulmonary arterial smooth muscle cells (PASMCs) synthesize ET-1 under basal conditions, it is unknown whether factors that may be important in pulmonary hypertension, such as transforming growth factor-beta (TGF-beta) or hypoxia, augment ET-1 production by these cells. We determined the effect of TGF-beta and hypoxia on ET-1 release and preproET-1 mRNA from cultured rat and human PASMCs. In the basal state, rat and human PASMCs synthesize, on average (mean+/-S.E.M.), 872+/-114 and 563+/-57 pg ET-1/mg cell protein over 24 h, respectively, a level that causes autocrine and paracrine effects in other tissues. TGF-beta significantly increases the expression of preproET-1 mRNA and ET-1 production by both rat and human PASMCs. Hypoxia for 24 h, however, does not affect ET-1 release from rat or human PASMCs. Cultured rat and human PASMCs are a source of ET-1 production. Enhanced ET-1 release from PASMCs may contribute to the pathophysiology of TGF-beta-induced pulmonary hypertension. ET-1 production by PASMCs is unlikely to contribute to the role of ET-1 in hypoxia-induced pulmonary vasoconstriction.

  11. 47 CFR 74.789 - Broadcast regulations applicable to digital low power television and television translator stations.

    Science.gov (United States)

    2010-10-01

    ... 47 Telecommunication 4 2010-10-01 2010-10-01 false Broadcast regulations applicable to digital low... AND OTHER PROGRAM DISTRIBUTIONAL SERVICES Low Power TV, TV Translator, and TV Booster Stations § 74.789 Broadcast regulations applicable to digital low power television and television translator...

  12. HIF-1α-l-PGDS-PPARγ regulates hypoxia-induced ANP secretion in beating rat atria.

    Science.gov (United States)

    Li, Xiang; Zhang, Ying; Zhang, Bo; Liu, Xia; Hong, Lan; Liu, Li-Ping; Wu, Cheng-Zhe; Cui, Xun

    2018-01-01

    Lipocalin-type prostaglandin D synthase (L-PGDS) and peroxisome proliferator activated receptor γ (PPARγ) play important roles in cardiovascular diseases. Nevertheless, effects of hypoxia-inducible factor 1α (HIF-1α) on L-PGDS and PPARγ protein levels and its role in hypoxia-induced atrial natriuretic peptide (ANP) secretion are unclear. In perfused beating rat atria, we observed that hypoxia significantly increased HIF-1α protein levels and stimulated ANP secretion, while upregulating L-PGDS. Hypoxia-induced ANP secretion was clearly attenuated by HIF-1α antagonist 2-methoxyestradiol, downregulating both HIF-1α and L-PGDS protein levels. It was also attenuated by L-PGDS antagonists, AT-56 and HQL-49, downregulating L-PGDS protein levels. In addition, hypoxia-induced ANP secretion was accompanied by increased PPARγ protein levels and was strongly attenuated by PPARγ antagonist GW9662. Hypoxia-induced increase in atrial PPARγ protein levels were dramatically inhibited by both 2-methoxyestradiol and AT-56. These results indicated that hypoxia promotes ANP secretion, at least in part, by activating HIF-1α-l-PGDS-PPARγ signaling in beating rat atria. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Controlling the Messenger: Regulated Translation of Maternal mRNAs in Xenopus laevis Development.

    Science.gov (United States)

    Sheets, Michael D; Fox, Catherine A; Dowdle, Megan E; Blaser, Susanne Imboden; Chung, Andy; Park, Sookhee

    2017-01-01

    The selective translation of maternal mRNAs encoding cell-fate determinants drives the earliest decisions of embryogenesis that establish the vertebrate body plan. This chapter will discuss studies in Xenopus laevis that provide insights into mechanisms underlying this translational control. Xenopus has been a powerful model organism for many discoveries relevant to the translational control of maternal mRNAs because of the large size of its oocytes and eggs that allow for microinjection of molecules and the relative ease of manipulating the oocyte to egg transition (maturation) and fertilization in culture. Consequently, many key studies have focused on the expression of maternal mRNAs during the oocyte to egg transition (the meiotic cell cycle) and the rapid cell divisions immediately following fertilization. This research has made seminal contributions to our understanding of translational regulatory mechanisms, but while some of the mRNAs under consideration at these stages encode cell-fate determinants, many encode cell cycle regulatory proteins that drive these early cell cycles. In contrast, while maternal mRNAs encoding key developmental (i.e., cell-fate) regulators that function after the first cleavage stages may exploit aspects of these foundational mechanisms, studies reveal that these mRNAs must also rely on distinct and, as of yet, incompletely understood mechanisms. These findings are logical because the functions of such developmental regulatory proteins have requirements distinct from cell cycle regulators, including becoming relevant only after fertilization and then only in specific cells of the embryo. Indeed, key maternal cell-fate determinants must be made available in exquisitely precise amounts (usually low), only at specific times and in specific cells during embryogenesis. To provide an appreciation for the regulation of maternal cell-fate determinant expression, an overview of the maternal phase of Xenopus embryogenesis will be presented

  14. Translational control by the DEAD Box RNA helicase belle regulates ecdysone-triggered transcriptional cascades.

    Directory of Open Access Journals (Sweden)

    Robert J Ihry

    Full Text Available Steroid hormones act, through their respective nuclear receptors, to regulate target gene expression. Despite their critical role in development, physiology, and disease, however, it is still unclear how these systemic cues are refined into tissue-specific responses. We identified a mutation in the evolutionarily conserved DEAD box RNA helicase belle/DDX3 that disrupts a subset of responses to the steroid hormone ecdysone during Drosophila melanogaster metamorphosis. We demonstrate that belle directly regulates translation of E74A, an ets transcription factor and critical component of the ecdysone-induced transcriptional cascade. Although E74A mRNA accumulates to abnormally high levels in belle mutant tissues, no E74A protein is detectable, resulting in misregulation of E74A-dependent ecdysone response genes. The accumulation of E74A mRNA in belle mutant salivary glands is a result of auto-regulation, fulfilling a prediction made by Ashburner nearly 40 years ago. In this model, Ashburner postulates that, in addition to regulating secondary response genes, protein products of primary response genes like E74A also inhibit their own ecdysone-induced transcription. Moreover, although ecdysone-triggered transcription of E74A appears to be ubiquitous during metamorphosis, belle-dependent translation of E74A mRNA is spatially restricted. These results demonstrate that translational control plays a critical, and previously unknown, role in refining transcriptional responses to the steroid hormone ecdysone.

  15. mosR, A Novel Transcriptional Regulator of Hypoxia and Virulence in Mycobacterium tuberculosis

    Science.gov (United States)

    Chronic tuberculosis represents a high-risk burden for one third of the world population. Previous microarray analysis of murine tuberculosis identified a novel transcriptional regulator encoded by rv0348 that could control the establishment of the chronic phase of tuberculosis. Disruption of the ...

  16. Chronic Hypoxia Differentially Up-Regulates Protein Kinase C-Mediated Ovine Uterine Arterial Contraction via Actin Polymerization Signaling in Pregnancy1

    Science.gov (United States)

    Xiao, DaLiao; Huang, Xiaohui; Zhang, Lubo

    2012-01-01

    ABSTRACT Chronic hypoxia (CH) during pregnancy is associated with increased uterine vascular tone. The present study tested the hypothesis that CH up-regulates protein kinase C (PKC)-mediated actin polymerization, resulting in enhanced uterine vascular contraction in pregnancy. Uterine arteries were isolated from nonpregnant (NPUA) and near-term (∼140 days of gestation) pregnant (PUA) sheep that had been maintained at sea level (∼300 m) or exposed to high altitude (3801 m) hypoxia for 110 days. In normoxic animals, the induced contractions by the PKC activator phorbol 12,13-dibutyrate (PDBu) were greater in NPUA than in PUA, which was abrogated by an actin polymerization inhibitor cytochalasin B (Cyto B). In hypoxic animals, PDBu-induced contractions were significantly increased in PUA but not in NPUA, which was inhibited by Cyto B. In contrast, neither pregnancy nor hypoxia affected Cyto B-mediated inhibition of norepinephrine (NE)-induced contractions. Prolonged ex vivo treatment of NPUA with 17beta-estradiol and progesterone significantly attenuated PDBu-induced actin polymerization and contractions, and the hormonal treatment did not alter the inhibitory effect of Cyto B on PDBu- or NE-induced contractions in either normoxic or hypoxic animals. 2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one potentiated PDBu-mediated actin polymerization and enhanced PDBu-induced contractions of PUA in normoxic but not hypoxic animals, which was abrogated by Cyto B. The results suggest that chronic hypoxia during pregnancy causes attenuation of steroid hormone-mediated ERK1/2 signaling and results in increased actin polymerization and uterine vascular tone, linking gestational hypoxia to aberrant uteroplacental circulation. PMID:23136295

  17. Hypoxia inducible factor-2α regulates the development of retinal astrocytic network by maintaining adequate supply of astrocyte progenitors.

    Directory of Open Access Journals (Sweden)

    Li-Juan Duan

    Full Text Available Here we investigate the role of hypoxia inducible factor (HIF-2α in coordinating the development of retinal astrocytic and vascular networks. Three Cre mouse lines were used to disrupt floxed Hif-2α, including Rosa26(CreERT2, Tie2(Cre, and GFAP(Cre. Global Hif-2α disruption by Rosa26(CreERT2 led to reduced astrocytic and vascular development in neonatal retinas, whereas endothelial disruption by Tie2(Cre had no apparent effects. Hif-2α deletion in astrocyte progenitors by GFAP(Cre significantly interfered with the development of astrocytic networks, which failed to reach the retinal periphery and were incapable of supporting vascular development. Perplexingly, the abundance of strongly GFAP(+ mature astrocytes transiently increased at P0 before they began to lag behind the normal controls by P3. Pax2(+ and PDGFRα(+ astrocytic progenitors and immature astrocytes were dramatically diminished at all stages examined. Despite decreased number of astrocyte progenitors, their proliferation index or apoptosis was not altered. The above data can be reconciled by proposing that HIF-2α is required for maintaining the supply of astrocyte progenitors by slowing down their differentiation into non-proliferative mature astrocytes. HIF-2α deficiency in astrocyte progenitors may accelerate their differentiation into astrocytes, a change which greatly interferes with the replenishment of astrocyte progenitors due to insufficient time for proliferation. Rapidly declining progenitor supply may lead to premature cessation of astrocyte development. Given that HIF-2α protein undergoes oxygen dependent degradation, an interesting possibility is that retinal blood vessels may regulate astrocyte differentiation through their oxygen delivery function. While our findings support the consensus that retinal astrocytic template guides vascular development, they also raise the possibility that astrocytic and vascular networks may mutually regulate each other

  18. Hypoxia-inducing factors as master regulators of stemness properties and altered metabolism of cancer- and metastasis-initiating cells

    Science.gov (United States)

    Mimeault, Murielle; Batra, Surinder K

    2013-01-01

    Accumulating lines of experimental evidence have revealed that hypoxia-inducible factors, HIF-1α and HIF-2α, are key regulators of the adaptation of cancer- and metastasis-initiating cells and their differentiated progenies to oxygen and nutrient deprivation during cancer progression under normoxic and hypoxic conditions. Particularly, the sustained stimulation of epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R), stem cell factor (SCF) receptor KIT, transforming growth factor-β receptors (TGF-βRs) and Notch and their downstream signalling elements such as phosphatidylinositol 3′-kinase (PI3K)/Akt/molecular target of rapamycin (mTOR) may lead to an enhanced activity of HIFs. Moreover, the up-regulation of HIFs in cancer cells may also occur in the hypoxic intratumoral regions formed within primary and secondary neoplasms as well as in leukaemic cells and metastatic prostate and breast cancer cells homing in the hypoxic endosteal niche of bone marrow. The activated HIFs may induce the expression of numerous gene products such as induced pluripotency-associated transcription factors (Oct-3/4, Nanog and Sox-2), glycolysis- and epithelial-mesenchymal transition (EMT) programme-associated molecules, including CXC chemokine receptor 4 (CXCR4), snail and twist, microRNAs and angiogenic factors such as vascular endothelial growth factor (VEGF). These gene products in turn can play critical roles for high self-renewal ability, survival, altered energy metabolism, invasion and metastases of cancer cells, angiogenic switch and treatment resistance. Consequently, the targeting of HIF signalling network and altered metabolic pathways represents new promising strategies to eradicate the total mass of cancer cells and improve the efficacy of current therapies against aggressive and metastatic cancers and prevent disease relapse. PMID:23301832

  19. Post-translational Modifications in Regulation of Chloroplast Function: Recent Advances.

    Science.gov (United States)

    Grabsztunowicz, Magda; Koskela, Minna M; Mulo, Paula

    2017-01-01

    Post-translational modifications (PTMs) of proteins enable fast modulation of protein function in response to metabolic and environmental changes. Phosphorylation is known to play a major role in regulating distribution of light energy between the Photosystems (PS) I and II (state transitions) and in PSII repair cycle. In addition, thioredoxin-mediated redox regulation of Calvin cycle enzymes has been shown to determine the efficiency of carbon assimilation. Besides these well characterized modifications, recent methodological progress has enabled identification of numerous other types of PTMs in various plant compartments, including chloroplasts. To date, at least N-terminal and Lys acetylation, Lys methylation, Tyr nitration and S-nitrosylation, glutathionylation, sumoylation and glycosylation of chloroplast proteins have been described. These modifications impact DNA replication, control transcriptional efficiency, regulate translational machinery and affect metabolic activities within the chloroplast. Moreover, light reactions of photosynthesis as well as carbon assimilation are regulated at multiple levels by a number of PTMs. It is likely that future studies will reveal new metabolic pathways to be regulated by PTMs as well as detailed molecular mechanisms of PTM-mediated regulation.

  20. Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation

    Science.gov (United States)

    Chauvier, Adrien; Picard-Jean, Frédéric; Berger-Dancause, Jean-Christophe; Bastet, Laurène; Naghdi, Mohammad Reza; Dubé, Audrey; Turcotte, Pierre; Perreault, Jonathan; Lafontaine, Daniel A.

    2017-01-01

    On the basis of nascent transcript sequencing, it has been postulated but never demonstrated that transcriptional pausing at translation start sites is important for gene regulation. Here we show that the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch contains a regulatory pause site in the translation initiation region that acts as a checkpoint for thiC expression. By biochemically probing nascent transcription complexes halted at defined positions, we find a narrow transcriptional window for metabolite binding, in which the downstream boundary is delimited by the checkpoint. We show that transcription complexes at the regulatory pause site favour the formation of a riboswitch intramolecular lock that strongly prevents TPP binding. In contrast, cotranscriptional metabolite binding increases RNA polymerase pausing and induces Rho-dependent transcription termination at the checkpoint. Early transcriptional pausing may provide a general mechanism, whereby transient transcriptional windows directly coordinate the sensing of environmental cues and bacterial mRNA regulation. PMID:28071751

  1. Post-translational regulation of endothelial nitric oxide synthase in vascular endothelium

    Science.gov (United States)

    Qian, Jin; Fulton, David

    2013-01-01

    Nitric oxide (NO) is a short-lived gaseous signaling molecule. In blood vessels, it is synthesized in a dynamic fashion by endothelial nitric oxide synthase (eNOS) and influences vascular function via two distinct mechanisms, the activation of soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)-dependent signaling and the S-nitrosylation of proteins with reactive thiols (S-nitrosylation). The regulation of eNOS activity and NO bioavailability is critical to maintain blood vessel function. The activity of eNOS and ability to generate NO is regulated at the transcriptional, posttranscriptional, and posttranslational levels. Post-translational modifications acutely impact eNOS activity and dysregulation of these mechanisms compromise eNOS activity and foster the development of cardiovascular diseases (CVDs). This review will intergrate past and current literature on the post-translational modifications of eNOS in both health and disease. PMID:24379783

  2. Post-translational modifications of Hsp90 and their contributions to chaperone regulation

    OpenAIRE

    Mollapour, Mehdi; Neckers, Len

    2011-01-01

    Molecular chaperones, as the name suggests, are involved in folding, maintenance, intracellular transport, and degradation of proteins as well as in facilitating cell signaling. Heat shock protein 90 (Hsp90) is an essential eukaryotic molecular chaperone that carries out these processes in normal and cancer cells. Hsp90 function in vivo is coupled to its ability to hydrolyze ATP and this can be regulated by co-chaperones and post-translational modifications. In this review, we explore the var...

  3. Post-translational Modifications Regulate Class IIa Histone Deacetylase (HDAC) Function in Health and Disease*

    OpenAIRE

    Mathias, Rommel A.; Amanda J Guise; Cristea, Ileana M.

    2015-01-01

    Class IIa histone deacetylases (HDACs4, -5, -7, and -9) modulate the physiology of the human cardiovascular, musculoskeletal, nervous, and immune systems. The regulatory capacity of this family of enzymes stems from their ability to shuttle between nuclear and cytoplasmic compartments in response to signal-driven post-translational modification. Here, we review the current knowledge of modifications that control spatial and temporal histone deacetylase functions by regulating subcellular loca...

  4. Regulation of protein stability of DNA methyltransferase 1 by post-translational modifications

    OpenAIRE

    Scott, Anthony; Song, Jing; Ewing, Rob; Wang, Zhenghe

    2014-01-01

    DNA methylation is an important epigenetic mechanism that ensures correct gene expression and maintains genetic stability. DNA methyltransferase 1 (DNMT1) is the primary enzyme that maintains DNA methylation during replication. Dysregulation of DNMT1 is implicated in a variety of diseases. DNMT1 protein stability is regulated via various post-translational modifications, such as acetylation and ubiquitination, but also through protein–protein interactions. These mechanisms ensure DNMT1 is pro...

  5. Trm9-Catalyzed tRNA Modifications Regulate Global Protein Expression by Codon-Biased Translation.

    Directory of Open Access Journals (Sweden)

    Wenjun Deng

    2015-12-01

    Full Text Available Post-transcriptional modifications of transfer RNAs (tRNAs have long been recognized to play crucial roles in regulating the rate and fidelity of translation. However, the extent to which they determine global protein production remains poorly understood. Here we use quantitative proteomics to show a direct link between wobble uridine 5-methoxycarbonylmethyl (mcm5 and 5-methoxy-carbonyl-methyl-2-thio (mcm5s2 modifications catalyzed by tRNA methyltransferase 9 (Trm9 in tRNAArg(UCU and tRNAGlu(UUC and selective translation of proteins from genes enriched with their cognate codons. Controlling for bias in protein expression and alternations in mRNA expression, we find that loss of Trm9 selectively impairs expression of proteins from genes enriched with AGA and GAA codons under both normal and stress conditions. Moreover, we show that AGA and GAA codons occur with high frequency in clusters along the transcripts, which may play a role in modulating translation. Consistent with these results, proteins subject to enhanced ribosome pausing in yeast lacking mcm5U and mcm5s2U are more likely to be down-regulated and contain a larger number of AGA/GAA clusters. Together, these results suggest that Trm9-catalyzed tRNA modifications play a significant role in regulating protein expression within the cell.

  6. Hypoxia and Fetal Heart Development

    Science.gov (United States)

    Patterson, A.J.; Zhang, L

    2010-01-01

    Fetal hearts show a remarkable ability to develop under hypoxic conditions. The metabolic flexibility of fetal hearts allows sustained development under low oxygen conditions. In fact, hypoxia is critical for proper myocardial formation. Particularly, hypoxia inducible factor 1 (HIF-1) and vascular endothelial growth factor play central roles in hypoxia-dependent signaling in fetal heart formation, impacting embryonic outflow track remodeling and coronary vessel growth. Although HIF is not the only gene involved in adaptation to hypoxia, its role places it as a central figure in orchestrating events needed for adaptation to hypoxic stress. Although “normal” hypoxia (lower oxygen tension in the fetus as compared with the adult) is essential in heart formation, further abnormal hypoxia in utero adversely affects cardiogenesis. Prenatal hypoxia alters myocardial structure and causes a decline in cardiac performance. Not only are the effects of hypoxia apparent during the perinatal period, but prolonged hypoxia in utero also causes fetal programming of abnormality in the heart’s development. The altered expression pattern of cardioprotective genes such as protein kinase c epsilon, heat shock protein 70, and endothelial nitric oxide synthase, likely predispose the developing heart to increased vulnerability to ischemia and reperfusion injury later in life. The events underlying the long-term changes in gene expression are not clear, but likely involve variation in epigenetic regulation. PMID:20712587

  7. Identification of nuclear protein targets for six leukemogenic tyrosine kinases governed by post-translational regulation.

    Directory of Open Access Journals (Sweden)

    Andrew Pierce

    Full Text Available Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases.

  8. Asymmetric distribution of hypoxia-inducible factor α regulates dorsoventral axis establishment in the early sea urchin embryo.

    Science.gov (United States)

    Chang, Wei-Lun; Chang, Yi-Cheng; Lin, Kuan-Ting; Li, Han-Ru; Pai, Chih-Yu; Chen, Jen-Hao; Su, Yi-Hsien

    2017-08-15

    Hypoxia signaling is an ancient pathway by which animals can respond to low oxygen. Malfunction of this pathway disturbs hypoxic acclimation and can result in various diseases, including cancers. The role of hypoxia signaling in early embryogenesis remains unclear. Here, we show that in the blastula of the sea urchin Strongylocentrotus purpuratus , hypoxia-inducible factor α (HIFα), the downstream transcription factor of the hypoxia pathway, is localized and transcriptionally active on the future dorsal side. This asymmetric distribution is attributable to its oxygen-sensing ability. Manipulations of the HIFα level entrained the dorsoventral axis, as the side with the higher level of HIFα tends to develop into the dorsal side. Gene expression analyses revealed that HIFα restricts the expression of nodal to the ventral side and activates several genes encoding transcription factors on the dorsal side. We also observed that intrinsic hypoxic signals in the early embryos formed a gradient, which was disrupted under hypoxic conditions. Our results reveal an unprecedented role of the hypoxia pathway in animal development. © 2017. Published by The Company of Biologists Ltd.

  9. Protein phosphatase 1 (PP1 is a post-translational regulator of the mammalian circadian clock.

    Directory of Open Access Journals (Sweden)

    Isabelle Schmutz

    Full Text Available Circadian clocks coordinate the timing of important biological processes. Interconnected transcriptional and post-translational feedback loops based on a set of clock genes generate and maintain these rhythms with a period of about 24 hours. Many clock proteins undergo circadian cycles of post-translational modifications. Among these modifications, protein phosphorylation plays an important role in regulating activity, stability and intracellular localization of clock components. Several protein kinases were characterized as regulators of the circadian clock. However, the function of protein phosphatases, which balance phosphorylation events, in the mammalian clock mechanism is less well understood. Here, we identify protein phosphatase 1 (PP1 as regulator of period and light-induced resetting of the mammalian circadian clock. Down-regulation of PP1 activity in cells by RNA interference and in vivo by expression of a specific inhibitor in the brain of mice tended to lengthen circadian period. Moreover, reduction of PP1 activity in the brain altered light-mediated clock resetting behavior in mice, enhancing the phase shifts in either direction. At the molecular level, diminished PP1 activity increased nuclear accumulation of the clock component PER2 in neurons. Hence, PP1, may reduce PER2 phosphorylation thereby influencing nuclear localization of this protein. This may at least partially influence period and phase shifting properties of the mammalian circadian clock.

  10. Regulation of mitochondrial biogenesis in erythropoiesis by mTORC1-mediated protein translation.

    Science.gov (United States)

    Liu, Xin; Zhang, Yuannyu; Ni, Min; Cao, Hui; Signer, Robert A J; Li, Dan; Li, Mushan; Gu, Zhimin; Hu, Zeping; Dickerson, Kathryn E; Weinberg, Samuel E; Chandel, Navdeep S; DeBerardinis, Ralph J; Zhou, Feng; Shao, Zhen; Xu, Jian

    2017-06-01

    Advances in genomic profiling present new challenges of explaining how changes in DNA and RNA are translated into proteins linking genotype to phenotype. Here we compare the genome-scale proteomic and transcriptomic changes in human primary haematopoietic stem/progenitor cells and erythroid progenitors, and uncover pathways related to mitochondrial biogenesis enhanced through post-transcriptional regulation. Mitochondrial factors including TFAM and PHB2 are selectively regulated through protein translation during erythroid specification. Depletion of TFAM in erythroid cells alters intracellular metabolism, leading to elevated histone acetylation, deregulated gene expression, and defective mitochondria and erythropoiesis. Mechanistically, mTORC1 signalling is enhanced to promote translation of mitochondria-associated transcripts through TOP-like motifs. Genetic and pharmacological perturbation of mitochondria or mTORC1 specifically impairs erythropoiesis in vitro and in vivo. Our studies support a mechanism for post-transcriptional control of erythroid mitochondria and may have direct relevance to haematologic defects associated with mitochondrial diseases and ageing.

  11. Celecoxib Down-Regulates the Hypoxia-Induced Expression of HIF-1α and VEGF Through the PI3K/AKT Pathway in Retinal Pigment Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Yi-zhou Sun

    2017-12-01

    Full Text Available Background/Aims: The goal of this study was to detect the expression of hypoxia-inducible factor 1α (HIF-1α and vascular endothelial growth factor (VEGF in human retinal pigmented epithelial (RPE cells treated with celecoxib, a selective cyclooxygenase-2 (COX-2 inhibitor, under hypoxic and normoxic conditions and to explore the signaling mechanism involved in regulating the hypoxia-induced expression of HIF-1α and VEGF in RPE cells. Methods: D407 cells were cultured in normoxic or hypoxic conditions, with or without celecoxib or a PI3K inhibitor (LY294002. The anti-proliferative effect of celecoxib was assessed using the MTT assay. RT-PCR, Western blotting and ELISA were performed to detect the levels of PI3K, phosphorylated AKT (p-AKT, HIF-1α, VEGF and COX-2. Results: Celecoxib inhibited the proliferation of RPE cells in a dose-dependent manner. Celecoxib suppressed the expression of VEGF at both the mRNA and protein levels and decreased HIF-1α protein expression. HIF-1α activation was regulated by the PI3K/AKT pathway. The celecoxib-induced down-regulation of HIF-1α and VEGF required the suppression of the hypoxia-induced PI3K/AKT pathway. However, the down-regulation of COX-2 did not occur in cells treated with celecoxib. Conclusions: The antiangiogenic effects of celecoxib in RPE cells under hypoxic conditions resulted from the inhibition of HIF-1α and VEGF expression, which may be partly mediated by a COX-2-independent, PI3K/AKT-dependent pathway.

  12. Folding upon phosphorylation: translational regulation by a disorder-to-order transition.

    Science.gov (United States)

    Metskas, Lauren Ann; Rhoades, Elizabeth

    2015-05-01

    4E binding proteins (4E-BPs) play an important role in the regulation of translation by binding to eukaryotic translation initiation factor 4E (eIF4E) and inhibiting assembly of the eIF4F complex. While phosphorylation of 4E-BPs is known to disrupt their binding to eIF4E, the mechanism by which this occurs has been unclear. In a recent study, Forman-Kay and coworkers demonstrate that this mechanism is primarily structure-based: phosphorylation of 4E-BPs results in a disorder-to-order transition, bringing them from their binding-competent disordered state to a folded state incompatible with eIF4E binding. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Translation Initiation from Conserved Non-AUG Codons Provides Additional Layers of Regulation and Coding Capacity

    Directory of Open Access Journals (Sweden)

    Ivaylo P. Ivanov

    2017-06-01

    Full Text Available Neurospora crassa cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying transcription activators that drive the transcriptional response to amino acid limitation. The cpc-1 mRNA contains two upstream open reading frames (uORFs in its >700-nucleotide (nt 5′ leader, and its expression is controlled at the level of translation in response to amino acid starvation. We used N. crassa cell extracts and obtained data indicating that cpc-1 uORF1 and uORF2 are functionally analogous to GCN4 uORF1 and uORF4, respectively, in controlling translation. We also found that the 5′ region upstream of the main coding sequence of the cpc-1 mRNA extends for more than 700 nucleotides without any in-frame stop codon. For 100 cpc-1 homologs from Pezizomycotina and from selected Basidiomycota, 5′ conserved extensions of the CPC1 reading frame are also observed. Multiple non-AUG near-cognate codons (NCCs in the CPC1 reading frame upstream of uORF2, some deeply conserved, could potentially initiate translation. At least four NCCs initiated translation in vitro. In vivo data were consistent with initiation at NCCs to produce N-terminally extended N. crassa CPC1 isoforms. The pivotal role played by CPC1, combined with its translational regulation by uORFs and NCC utilization, underscores the emerging significance of noncanonical initiation events in controlling gene expression.

  14. Up-regulation of selenoprotein P and HIP/PAP mRNAs in hepatocytes by intermittent hypoxia via down-regulation of miR-203

    Directory of Open Access Journals (Sweden)

    Tomoko Uchiyama

    2017-09-01

    Full Text Available Sleep apnea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH] and is a risk factor for insulin resistance/type 2 diabetes. However, the mechanisms linking IH stress and insulin resistance remain elusive. We exposed human hepatocytes (JHH5, JHH7, and HepG2 to experimental IH or normoxia for 24 h, measured mRNA levels by real-time reverse transcription polymerase chain reaction (RT-PCR, and found that IH significantly increased the mRNA levels of selenoprotein P (SELENOP — a hepatokine — and hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP — one of REG (Regenerating gene family. We next investigated promoter activities of both genes and discovered that they were not increased by IH. On the other hand, a target mRNA search of micro RNA (miRNA revealed that both mRNAs have a potential target sequence for miR-203. The miR-203 level of IH-treated cells was significantly lower than that of normoxia-treated cells. Thus, we introduced miR-203 inhibitor and a non-specific control RNA (miR-203 inhibitor NC into HepG2 cells and measured the mRNA levels of SELENOP and HIP/PAP. The IH-induced expression of SELENOP and HIP/PAP was abolished by the introduction of miR-203 inhibitor but not by miR-203 inhibitor NC. These results demonstrate that IH stress up-regulates the levels of SELENOP in human hepatocytes to accelerate insulin resistance and up-regulates the levels of HIP/PAP mRNAs to proliferate such hepatocytes, via the miR-203 mediated mechanism.

  15. Maternal RNA regulates Aurora C kinase during mouse oocyte maturation in a translation-independent fashion.

    Science.gov (United States)

    Balboula, Ahmed Z; Blengini, Cecilia S; Gentilello, Amanda S; Takahashi, Masashi; Schindler, Karen

    2017-06-01

    During oocyte meiotic maturation, Aurora kinase C (AURKC) is required to accomplish many critical functions including destabilizing erroneous kinetochore-microtubule (K-MT)attachments and regulating bipolar spindle assembly. How localized activity of AURKC is regulated in mammalian oocytes, however, is not fully understood. Female gametes from many species, including mouse, contain stores of maternal transcripts that are required for downstream developmental events. We show here that depletion of maternal RNA in mouse oocytes resulted in impaired meiotic progression, increased incidence of chromosome misalignment and abnormal spindle formation at metaphase I (Met I), and cytokinesis defects. Importantly, depletion of maternal RNA perturbed the localization and activity of AURKC within the chromosomal passenger complex (CPC). These perturbations were not observed when translation was inhibited by cycloheximide (CHX) treatment. These results demonstrate a translation-independent function of maternal RNA to regulate AURKC-CPC function in mouse oocytes. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. HCdc14A is involved in cell cycle regulation of human brain vascular endothelial cells following injury induced by high glucose, free fatty acids and hypoxia.

    Science.gov (United States)

    Su, Jingjing; Zhou, Houguang; Tao, Yinghong; Guo, Zhuangli; Zhang, Shuo; Zhang, Yu; Huang, Yanyan; Tang, Yuping; Hu, Renming; Dong, Qiang

    2015-01-01

    Cell cycle processes play a vital role in vascular endothelial proliferation and dysfunction. Cell division cycle protein 14 (Cdc14) is an important cell cycle regulatory phosphatase. Previous studies in budding yeast demonstrated that Cdc14 could trigger the inactivation of mitotic cyclin-dependent kinases (Cdks), which are required for mitotic exit and cytokinesis. However, the exact function of human Cdc14 (hCdc14) in cell cycle regulation during vascular diseases is yet to be elucidated. There are two HCdc14 homologs: hCdc14A and hCdc14B. In the current study, we investigated the potential role of hCdc14A in high glucose-, free fatty acids (FFAs)-, and hypoxia-induced injury in cultured human brain vascular endothelial cells (HBVECs). Data revealed that high glucose, FFA, and hypoxia down-regulated hCdc14A expression remarkably, and also affected the expression of other cell cycle-related proteins such as cyclin B, cyclin D, cyclin E, and p53. Furthermore, the combined addition of the three stimuli largely blocked cell cycle progression, decreased cell proliferation, and increased apoptosis. We also determined that hCdc14A was localized mainly to centrosomes during interphase and spindles during mitosis using confocal microscopy, and that it could affect the expression of other cycle-related proteins. More importantly, the overexpression of hCdc14A accelerated cell cycle progression, enhanced cell proliferation, and promoted neoplastic transformation, whereas the knockdown of hCdc14A using small interfering RNA produced the opposite effects. Therefore, these findings provide novel evidence that hCdc14A might be involved in cell cycle regulation in cultured HBVECs during high glucose-, FFA-, and hypoxia-induced injury. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. 1,25-Dihydroxycholecalciferol regulates chromogranin-A translatability in bovine parathyroid cells.

    Science.gov (United States)

    Mouland, A J; Hendy, G N

    1992-11-01

    Our previous studies indicated that regulation of bovine parathyroid chromogranin-A (CgA) by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] may occur at the level of CgA turnover or mRNA translation. In the present study, immunoprecipitation of extracts of bovine parathyroid cells that had been pulse chased with [35S]methionine revealed that 1,25-(OH)2D3 had no effect on the disappearance time of intracellular CgA. Therefore, we examined the effect of 1,25-(OH)2D3 on the polyribosome profile of CgA mRNA analyzed by sucrose density gradients. In the presence of 1,25-(OH)2D3, there was a dose-dependent recruitment of CgA mRNA into the denser polyribosomal fractions by 24 h. To determine whether this increased ribosome loading represents increased or decreased efficiency of mRNA translation, ribosome transit time experiments were conducted. The average ribosome transit time and the specific PTH ribosome transit time were not altered by 1,25-(OH)2D3. However, that for CgA was doubled in the presence of 1,25-(OH)2D3. Thus, parathyroid CgA synthesis is regulated by the vitamin D sterol at the level of peptide chain elongation. These studies, therefore, explain the lack of quantitative correspondence between 1,25-(OH)2D3-induced CgA gene transcription and CgA protein levels by revealing a previously unsuspected level of regulation of mRNA translation in the parathyroid cell.

  18. Regulation of lactate dehydrogenase activity by ascorbic acid in organs of rats with hypoxia of closed space

    Directory of Open Access Journals (Sweden)

    Олександр Костянтинович Будняк

    2015-07-01

    Full Text Available The research of the effects of different doses of ascorbic acid (AA on activity of lactate dehydrogenase (LDG in the organs of white nonlinear rats with hypoxia of closed space (HCS has been conducted. Injections of АА to rats for 30 minutes of HCS reduced the LDG activity in all organs of hypoxic animals

  19. A vigilant, hypoxia-regulated heme oxygenase-1 gene vector in the heart limits cardiac injury after ischemia-reperfusion in vivo.

    Science.gov (United States)

    Tang, Yao Liang; Qian, Keping; Zhang, Y Clare; Shen, Leping; Phillips, M Ian

    2005-12-01

    The effect of a cardiac specific, hypoxia-regulated, human heme oxygenase-1 (hHO-1) vector to provide cardioprotection from ischemia-reperfusion injury was assessed. When myocardial ischemia and reperfusion is asymptomatic, the damaging effects are cumulative and patients miss timely treatment. A gene therapy approach that expresses therapeutic genes only when ischemia is experienced is a desirable strategy. We have developed a cardiac-specific, hypoxia-regulated gene therapy "vigilant vector'' system that amplifies cardioprotective gene expression. Vigilant hHO-1 plasmids, LacZ plasmids, or saline (n = 40 per group) were injected into mouse heart 2 days in advance of ischemia-reperfusion injury. Animals were exposed to 60 minutes of ischemia followed by 24 hours of reperfusion. For that term (24 hours) effects, the protein levels of HO-1, inflammatory responses, apoptosis, and infarct size were determined. For long-term (3 week) effects, the left ventricular remodeling and recovery of cardiac function were assessed. Ischemia-reperfusion resulted in a timely overexpression of HO-1 protein. Infarct size at 24 hours after ischemia-reperfusion was significantly reduced in the HO-1-treated animals compared with the LacZ-treated group or saline-treated group (P < .001). The reduction of infarct size was accompanied by a decrease in lipid peroxidant activity, inflammatory cell infiltration, and proapoptotic protein level in ischemia-reperfusion-injured myocardium. The long-term study demonstrated that timely, hypoxia-induced HO-1 overexpression is beneficial in conserving cardiac function and attenuating left ventricle remodelling. The vigilant HO-1 vector provides a protective therapy in the heart for reducing cellular damage during ischemia-reperfusion injury and preserving heart function.

  20. Involvement of Arabidopsis RACK1 in Protein Translation and Its Regulation by Abscisic Acid

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Jianjun [University of British Columbia, Vancouver; Wang, Shucai [University of British Columbia, Vancouver; Valerius, Oliver [Universitaet Goettingen, Goettingen, Germany; Hall, Hardy [University of British Columbia, Vancouver; Zeng, Qingning [University of British Columbia, Vancouver; Li, Jian-Feng [Harvard University; Weston, David [ORNL; Ellis, Brian [University of British Columbia, Vancouver; Chen, Jay [ORNL

    2011-01-01

    Earlier studies have shown that RACK1 functions as a negative regulator of ABA responses in Arabidopsis, but the molecular mechanism of the action of RACK1 in these processes remains elusive. Global gene expression profiling revealed that approximately 40% of the genes affected by ABA treatment were affected in a similar manner by the rack1 mutation, supporting the view that RACK1 is an important regulator of ABA responses. On the other hand, co-expression analysis revealed that >80% of the genes co-expressed with RACK1 encode ribosome proteins, implying a close relationship between RACK1 s function and the ribosome complex. These results implied that the regulatory role for RACK1 in ABA responses may be partially due to its putative function in protein translation, which is one of the major cellular processes that mammalian and yeast RACK1 is involved in. Consistently, all three Arabidopsis RACK1 homologous genes, namely RACK1A, RACK1B and RACK1C, complemented the growth defects of the S. cerevisiae cpc2/rack1 mutant. In addition, RACK1 physically interacts with Arabidopsis Eukaryotic Initiation Factor 6 (eIF6), whose mammalian homologue is a key regulator of 80S ribosome assembly. Moreover, rack1 mutants displayed hypersensitivity to anisomycin, an inhibitor of protein translation, and displayed characteristics of impaired 80S functional ribosome assembly and 60S ribosomal subunit biogenesis in a ribosome profiling assay. Gene expression analysis revealed that ABA inhibits the expression of both RACK1 and eIF6. Taken together, these results suggest that RACK1 may be required for normal production of 60S and 80S ribosomes and that its action in these processes may be regulated by ABA.

  1. Translational co-regulation of a ligand and inhibitor by a conserved RNA element

    DEFF Research Database (Denmark)

    Zaucker, Andreas; Nagorska, Agnieszka; Kumari, Pooja

    2018-01-01

    In many organisms, transcriptional and post-transcriptional regulation of components of pathways or processes has been reported. However, to date, there are few reports of translational co-regulation of multiple components of a developmental signaling pathway. Here, we show that an RNA element...... which we previously identified as a dorsal localization element (DLE) in the 3'UTR of zebrafish nodal-related1/squint (ndr1/sqt) ligand mRNA, is shared by the related ligand nodal-related2/cyclops (ndr2/cyc) and the nodal inhibitors, lefty1 (lft1) and lefty2 mRNAs. We investigated the activity...... of the DLEs through functional assays in live zebrafish embryos. The lft1 DLE localizes fluorescently labeled RNA similarly to the ndr1/sqt DLE. Similar to the ndr1/sqt 3'UTR, the lft1 and lft2 3'UTRs are bound by the RNA-binding protein (RBP) and translational repressor, Y-box binding protein 1 (Ybx1...

  2. Oxygen-dependent regulation of aquaporin-3 expression

    Directory of Open Access Journals (Sweden)

    Hoogewijs D

    2016-04-01

    Full Text Available David Hoogewijs,1,2 Melanie Vogler,3 Eveline Zwenger,3 Sabine Krull,3 Anke Zieseniss3 1Institute of Physiology, University of Duisburg-Essen, Essen, Germany; 2Institute of Physiology, University of Zürich, Zürich, Switzerland; 3Institute of Cardiovascular Physiology, University Medical Center Göttingen, University of Göttingen, Göttingen, GermanyAbstract: The purpose of this study was to investigate whether aquaporin-3 (AQP3 expression is altered in hypoxia and whether hypoxia-inducible transcription factor (HIF-1 regulates the hypoxic expression. AQP3 mRNA expression was studied in L929 fibrosarcoma cells and in several tissues derived from mice that were subjected to hypoxia. Computational analysis of the AQP3 promoter revealed conserved HIF binding sites within close proximity to the translational start site, and chromatin immunoprecipitation assays confirmed binding of HIF-1 to the endogenous hypoxia response elements. Furthermore, hypoxia resulted in increased expression of AQP3 mRNA in L929 fibrosarcoma cells. Consistently, shRNA-mediated knockdown of HIF-1 greatly reduced the hypoxic induction of AQP3. In addition, mRNA analysis of organs from mice exposed to inspiratory hypoxia demonstrated pronounced hypoxia-inducible expression of AQP3 in the kidney. Overall, our findings suggest that AQP3 expression can be regulated at the transcriptional level and that AQP3 represents a novel HIF-1 target gene. Keywords: transcriptional regulation, oxygen, hypoxia-inducible factor, hypoxia response element

  3. Role of HIF-1 on phosphofructokinase and fructose 1, 6-bisphosphatase expression during hypoxia in the white shrimp Litopenaeus vannamei.

    Science.gov (United States)

    Cota-Ruiz, Keni; Leyva-Carrillo, Lilia; Peregrino-Uriarte, Alma B; Valenzuela-Soto, Elisa M; Gollas-Galván, Teresa; Gómez-Jiménez, Silvia; Hernández, Jesús; Yepiz-Plascencia, Gloria

    2016-08-01

    HIF-1 is a transcription factor that controls a widespread range of genes in metazoan organisms in response to hypoxia and is composed of α and β subunits. In shrimp, phosphofructokinase (PFK) and fructose bisphosphatase (FBP) are up-regulated in hypoxia. We hypothesized that HIF-1 is involved in the regulation of PFK and FBP genes in shrimp hepatopancreas under hypoxia. Long double stranded RNA (dsRNA) intramuscular injection was utilized to silence simultaneously both HIF-1 subunits, and then, we measured the relative expression of PFK and FBP, as well as their corresponding enzymatic activities in hypoxic shrimp hepatopancreas. The results indicated that HIF-1 participates in the up-regulation of PFK transcripts under short-term hypoxia since the induction caused by hypoxia (~1.6 and ~4.2-fold after 3 and 48h, respectively) is significantly reduced in the dsRNA animals treated. Moreover, PFK activity was significantly ~2.8-fold augmented after 3h in hypoxia alongside to an ~1.9-fold increment in lactate. However, when animals were dsRNA treated, both were significantly reduced. On the other hand, FBP transcripts were ~5.3-fold up-regulated in long-term hypoxic conditions (48h). HIF-1 is involved in this process since FBP transcripts were not induced by hypoxia when HIF-1 was silenced. Conversely, the FBP activity was not affected by hypoxia, which suggests its possible regulation at post-translational level. Taken together, these results position HIF-1 as a prime transcription factor in coordinating glucose metabolism through the PFK and FBP genes among others, in shrimp under low oxygen environments. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. The CDK4/6 inhibitor palbociclib synergizes with irinotecan to promote colorectal cancer cell death under hypoxia.

    Science.gov (United States)

    Zhang, Jun; Zhou, Lanlan; Zhao, Shuai; Dicker, David T; El-Deiry, Wafik S

    2017-06-18

    Hypoxia is an inherent impediment to cancer therapy. Palbociclib, a highly selective inhibitor for CDK4/6, has been tested in numerous clinical trials and has been approved by the FDA. We previously reported that CDK inhibitors can destabilize HIF1α regardless of the presence of hypoxia and can sensitize tumor cells to TRAIL through dual blockade of CDK1 and GSK-3β. To translate this knowledge into a cancer therapeutic strategy, we investigated the therapeutic effects and molecular mechanisms of CDK inhibition against colon cancer cells under normoxia and hypoxia. We found that palbociclib sensitizes colon cancer cells to hypoxia-induced apoptotic resistance via deregulation of HIF-1α accumulation. In addition to inhibition of cell proliferation, we observed that palbociclib promotes colon cancer cell death regardless of the presence of hypoxia at a comparatively high concentration via regulating ERK/GSK-3β signaling and GSK-3β expression. Furthermore, palbociclib synergized with irinotecan in a variety of colon cancer cell lines with various molecular subtypes via deregulating irinotecan-induced Rb phosphorylation and reducing HIF-1α accumulation under normoxia or hypoxia. Collectively, our findings provide a novel combination therapy strategy against hypoxic colon cancer cells that may be further translated in the clinic.

  5. Post-translational modifications of hormone-responsive transcription factors: the next level of regulation.

    Science.gov (United States)

    Hill, Kristine

    2015-08-01

    Plants exhibit a high level of developmental plasticity and growth is responsive to multiple developmental and environmental cues. Hormones are small endogenous signalling molecules which are fundamental to this phenotypic plasticity. Post-translational modifications of proteins are a central feature of the signal transduction pathways that regulate gene transcription in response to hormones. Modifications that affect the function of transcriptional regulators may also serve as a mechanism to incorporate multiple signals, mediate cross-talk, and modulate specific responses. This review discusses recent research that suggests hormone-responsive transcription factors are subject to multiple modifications which imply an additional level of regulation conferred by enzymes that mediate specific modifications, such as phosphorylation, ubiquitination, SUMOylation, and S-nitrosylation. These modifications can affect protein stability, sub-cellular localization, interactions with co-repressors and activators, and DNA binding. The focus here is on direct cross-talk involving transcription factors downstream of auxin, brassinosteroid, and gibberellin signalling. However, many of the concepts discussed are more broadly relevant to questions of how plants can modify their growth by regulating subsets of genes in response to multiple cues. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  6. The coupled and uncoupled mechanisms by which trans-acting factors regulate mRNA stability and translation.

    Science.gov (United States)

    Fukao, Akira; Fujiwara, Toshinobu

    2017-04-01

    In mammals, spatiotemporal control of protein synthesis plays a key role in the post-transcriptional regulation of gene expression during cell proliferation, development and differentiation and RNA-binding proteins (RBPs) and microRNAs (miRNAs) are required for this phenomenon. RBPs and miRNAs control the levels of mRNA protein products by regulating mRNA stability and translation. Recent studies have shown that RBPs and miRNAs simultaneously regulate mRNA stability and translation, and that the differential functions of RBPs and miRNAs are dependent on their interaction partners. Here, we summarize the coupled- and uncoupled mechanisms by which trans-acting factors regulate mRNA stability and translation. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  7. Human TM9SF4 Is a New Gene Down-Regulated by Hypoxia and Involved in Cell Adhesion of Leukemic Cells.

    Directory of Open Access Journals (Sweden)

    Rosa Paolillo

    Full Text Available The transmembrane 9 superfamily protein member 4, TM9SF4, belongs to the TM9SF family of proteins highly conserved through evolution. TM9SF4 homologs, previously identified in many different species, were mainly involved in cellular adhesion, innate immunity and phagocytosis. In human, the function and biological significance of TM9SF4 are currently under investigation. However, TM9SF4 was found overexpressed in human metastatic melanoma and in a small subset of acute myeloid leukemia (AMLs and myelodysplastic syndromes, consistent with an oncogenic function of this gene.In this study, we first analyzed the expression and regulation of TM9SF4 in normal and leukemic cells and identified TM9SF4 as a gene highly expressed in human quiescent CD34+ hematopoietic progenitor cells (HPCs, regulated during monocytic and granulocytic differentiation of HPCs, both lineages giving rise to mature myeloid cells involved in adhesion, phagocytosis and immunity. Then, we found that TM9SF4 is markedly overexpressed in leukemic cells and in AMLs, particularly in M2, M3 and M4 AMLs (i.e., in AMLs characterized by the presence of a more or less differentiated granulocytic progeny, as compared to normal CD34+ HPCs. Proliferation and differentiation of HPCs occurs in hypoxia, a physiological condition in bone marrow, but also a crucial component of cancer microenvironment. Here, we investigated the impact of hypoxia on TM9SF4 expression in leukemic cells and identified TM9SF4 as a direct target of HIF-1α, downregulated in these cells by hypoxia. Then, we found that the hypoxia-mediated downregulation of TM9SF4 expression is associated with a decrease of cell adhesion of leukemic cells to fibronectin, thus demonstrating that human TM9SF4 is a new molecule involved in leukemic cell adhesion.Altogether, our study reports for the first time the expression of TM9SF4 at the level of normal and leukemic hematopoietic cells and its marked expression at the level of AMLs

  8. Cerebral hypoxia

    Science.gov (United States)

    ... before, during, or soon after birth such as cerebral palsy Stroke Very low blood pressure Brain cells are very sensitive to a lack of ... of the eye to light Exams and Tests Cerebral hypoxia can usually be diagnosed based on the person's medical history and a physical exam. Tests are done to ...

  9. Hypoxia-increased RAGE expression regulates chemotaxis and pro-inflammatory cytokines release through nuclear translocation of NF-κ B and HIF1α in THP-1 cells.

    Science.gov (United States)

    Bai, Wei; Zhou, Jing; Zhou, Na; Liu, Qin; Cui, Jian; Zou, Wei; Zhang, Wei

    2018-01-15

    The potential role of hypoxia in mediating the receptor for advanced glycation end products (RAGE) expression deserves to be confirmed. And the role of RAGE in hypoxia-induced chemotaxis and inflammation is still unclear. In present study, THP-1 cells were pretreated with siRNA to block HIF1α, NF-κ B, or RAGE, followed by exposed to hypoxia (combined with H 2 O 2 or SNP), and then RAGE expression, nuclear translocation of HIF1α and NF-κ B, release of TNF-α and IL-1β, as well as expression of MCP-1 and CCR2 were measured. The results revealed that RAGE mRNA and protein in THP-1 cells were significantly increased after exposed into hypoxia atmosphere, especially into the solution containing SNP or H 2 O 2 . Moreover, SNP or H 2 O 2 exposure could further amplify hypoxia-induced nuclear translocation of HIF-1α and NF-κ B. Knockdown HIF-1α or NF-κ B by siRNAs could reduce hypoxia- and oxidative stress-induced RAGE hyper-expression. And pretreatment THP-1 cells with RAGE siRNA or NF-κ B siRNA could reduce hypoxia- and oxidative stress-induced expression of MCP-1 and CCR2, and release of TNF-α and IL-1β. Thus, hypoxia not only increases RAGE expression in THP-1 cells by promoting nuclear translocation of NF-κ B and HIF1α, but also regulates chemotaxis and pro-inflammatory cytokines release, which may be partially mediated through upregulation of RAGE expression. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Stress granules: sites of mRNA triage that regulate mRNA stability and translatability.

    Science.gov (United States)

    Kedersha, N; Anderson, P

    2002-11-01

    Mammalian stress granules (SGs) are cytoplasmic domains into which mRNAs are sorted dynamically in response to phosphorylation of eukaryotic initiation factor (eIF) 2alpha, a key regulatory step in translational initiation. The activation of one or more of the eIF2alpha kinases leads to SG assembly by decreasing the levels of eIF2-GTP-tRNA(Met), the ternary complex that is normally required for loading the initiator methionine onto the 48 S preinitiation complex to begin translation. This stress-induced scarcity of eIF2-GTP-tRNA(Met) allows the RNA-binding proteins TIA-1 (T-cell internal antigen-1) and TIAR (TIA-1-related protein) to bind the 48 S complex in lieu of the ternary complex, thereby promoting polysome disassembly and the concurrent routing of the mRNA into a SG. The actual formation of SGs occurs upon auto-aggregation of the prion-like C-termini of TIA-1 proteins; this aggregation is reversed in vivo by overexpression of the heat-shock protein (HSP) chaperone HSP70. Remarkably, HSP70 mRNA is excluded from SGs and is preferentially translated during stress, indicating that the RNA composition of the SG is selective. Moreover, the effects of HSP70 on TIA aggregation suggest a feedback loop whereby HSP70 synthesis is auto-regulated. Proteins that promote mRNA stability [e.g. HuR (Hu protein R)] and destabilize mRNA [i.e. tristetraprolin (TTP)] are also recruited to SGs, suggesting that SGs effect a process of mRNA triage, by promoting polysome disassembly and routing mRNAs to cytoplasmic domains enriched for HuR and TTP. This model reveals connections between the eIF2alpha kinase system, mRNA stability and cellular chaperone levels.

  11. Concordant regulation of translation and mRNA abundance for hundreds of targets of a human microRNA.

    Directory of Open Access Journals (Sweden)

    David G Hendrickson

    2009-11-01

    Full Text Available MicroRNAs (miRNAs regulate gene expression posttranscriptionally by interfering with a target mRNA's translation, stability, or both. We sought to dissect the respective contributions of translational inhibition and mRNA decay to microRNA regulation. We identified direct targets of a specific miRNA, miR-124, by virtue of their association with Argonaute proteins, core components of miRNA effector complexes, in response to miR-124 transfection in human tissue culture cells. In parallel, we assessed mRNA levels and obtained translation profiles using a novel global approach to analyze polysomes separated on sucrose gradients. Analysis of translation profiles for approximately 8,000 genes in these proliferative human cells revealed that basic features of translation are similar to those previously observed in rapidly growing Saccharomyces cerevisiae. For approximately 600 mRNAs specifically recruited to Argonaute proteins by miR-124, we found reductions in both the mRNA abundance and inferred translation rate spanning a large dynamic range. The changes in mRNA levels of these miR-124 targets were larger than the changes in translation, with average decreases of 35% and 12%, respectively. Further, there was no identifiable subgroup of mRNA targets for which the translational response was dominant. Both ribosome occupancy (the fraction of a given gene's transcripts associated with ribosomes and ribosome density (the average number of ribosomes bound per unit length of coding sequence were selectively reduced for hundreds of miR-124 targets by the presence of miR-124. Changes in protein abundance inferred from the observed changes in mRNA abundance and translation profiles closely matched changes directly determined by Western analysis for 11 of 12 proteins, suggesting that our assays captured most of miR-124-mediated regulation. These results suggest that miRNAs inhibit translation initiation or stimulate ribosome drop-off preferentially near the

  12. Nutritional Control of DNA Replication Initiation through the Proteolysis and Regulated Translation of DnaA.

    Directory of Open Access Journals (Sweden)

    David J Leslie

    2015-07-01

    Full Text Available Bacteria can arrest their own growth and proliferation upon nutrient depletion and under various stressful conditions to ensure their survival. However, the molecular mechanisms responsible for suppressing growth and arresting the cell cycle under such conditions remain incompletely understood. Here, we identify post-transcriptional mechanisms that help enforce a cell-cycle arrest in Caulobacter crescentus following nutrient limitation and during entry into stationary phase by limiting the accumulation of DnaA, the conserved replication initiator protein. DnaA is rapidly degraded by the Lon protease following nutrient limitation. However, the rate of DnaA degradation is not significantly altered by changes in nutrient availability. Instead, we demonstrate that decreased nutrient availability downregulates dnaA translation by a mechanism involving the 5' untranslated leader region of the dnaA transcript; Lon-dependent proteolysis of DnaA then outpaces synthesis, leading to the elimination of DnaA and the arrest of DNA replication. Our results demonstrate how regulated translation and constitutive degradation provide cells a means of precisely and rapidly modulating the concentration of key regulatory proteins in response to environmental inputs.

  13. Regulation of multispanning membrane protein topology via post-translational annealing.

    Science.gov (United States)

    Van Lehn, Reid C; Zhang, Bin; Miller, Thomas F

    2015-09-26

    The canonical mechanism for multispanning membrane protein topogenesis suggests that protein topology is established during cotranslational membrane integration. However, this mechanism is inconsistent with the behavior of EmrE, a dual-topology protein for which the mutation of positively charged loop residues, even close to the C-terminus, leads to dramatic shifts in its topology. We use coarse-grained simulations to investigate the Sec-facilitated membrane integration of EmrE and its mutants on realistic biological timescales. This work reveals a mechanism for regulating membrane-protein topogenesis, in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies. The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins. The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis.

  14. CSPGs inhibit axon branching by impairing mitochondria-dependent regulation of actin dynamics and axonal translation.

    Science.gov (United States)

    Sainath, Rajiv; Ketschek, Andrea; Grandi, Leah; Gallo, Gianluca

    2017-04-01

    Chondroitin sulfate proteoglycans (CSPGs) inhibit the formation of axon collateral branches. The regulation of the axonal cytoskeleton and mitochondria are important components of the mechanism of branching. Actin-dependent axonal plasticity, reflected in the dynamics of axonal actin patches and filopodia, is greatest along segments of the axon populated by mitochondria. It is reported that CSPGs partially depolarize the membrane potential of axonal mitochondria, which impairs the dynamics of the axonal actin cytoskeleton and decreases the formation and duration of axonal filopodia, the first steps in the mechanism of branching. The effects of CSPGs on actin cytoskeletal dynamics are specific to axon segments populated by mitochondria. In contrast, CSPGs do not affect the microtubule content of axons, or the localization of microtubules into axonal filopodia, a required step in the mechanism of branch formation. It is also reported that CSPGs decrease the mitochondria-dependent axonal translation of cortactin, an actin associated protein involved in branching. Finally, the inhibitory effects of CSPGs on axon branching, actin cytoskeletal dynamics and the axonal translation of cortactin are reversed by culturing neurons with acetyl-l-carnitine, which promotes mitochondrial respiration. Collectively these data indicate that CSPGs impair mitochondrial function in axons, an effect which contributes to the inhibition of axon branching. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419-437, 2017. © 2016 Wiley Periodicals, Inc.

  15. Cellular DDX3 regulates Japanese encephalitis virus replication by interacting with viral un-translated regions.

    Science.gov (United States)

    Li, Chen; Ge, Ling-ling; Li, Peng-peng; Wang, Yue; Dai, Juan-juan; Sun, Ming-xia; Huang, Li; Shen, Zhi-qiang; Hu, Xiao-chun; Ishag, Hassan; Mao, Xiang

    2014-01-20

    Japanese encephalitis virus is one of the most common causes for epidemic viral encephalitis in humans and animals. Herein we demonstrated that cellular helicase DDX3 is involved in JEV replication. DDX3 knockdown inhibits JEV replication. The helicase activity of DDX3 is crucial for JEV replication. GST-pulldown and co-immunoprecipitation experiments demonstrated that DDX3 could interact with JEV non-structural proteins 3 and 5. Co-immunoprecipitation and confocal microscopy analysis confirmed that DDX3 interacts and colocalizes with these viral proteins and viral RNA during the infection. We determined that DDX3 binds to JEV 5' and 3' un-translated regions. We used a JEV-replicon system to demonstrate that DDX3 positively regulates viral RNA translation, which might affect viral RNA replication at the late stage of virus infection. Collectively, we identified that DDX3 is necessary for JEV infection, suggesting that DDX3 might be a novel target to design new antiviral agents against JEV or other flavivirus infections. © 2013 Published by Elsevier Inc.

  16. Developmentally Regulated Post-translational Modification of Nucleoplasmin Controls Histone Sequestration and Deposition

    Directory of Open Access Journals (Sweden)

    Takashi Onikubo

    2015-03-01

    Full Text Available Nucleoplasmin (Npm is an abundant histone chaperone in vertebrate oocytes and embryos. During embryogenesis, regulation of Npm histone binding is critical for its function in storing and releasing maternal histones to establish and maintain the zygotic epigenome. Here, we demonstrate that Xenopus laevis Npm post-translational modifications (PTMs specific to the oocyte and egg promote either histone deposition or sequestration, respectively. Mass spectrometry and Npm phosphomimetic mutations used in chromatin assembly assays identified hyperphosphorylation on the N-terminal tail as a critical regulator for sequestration. C-terminal tail phosphorylation and PRMT5-catalyzed arginine methylation enhance nucleosome assembly by promoting histone interaction with the second acidic tract of Npm. Electron microscopy reconstructions of Npm and TTLL4 activity toward the C-terminal tail demonstrate that oocyte- and egg-specific PTMs cause Npm conformational changes. Our results reveal that PTMs regulate Npm chaperoning activity by modulating Npm conformation and Npm-histone interaction, leading to histone sequestration in the egg.

  17. Post-translational regulation of SHORT VEGETATIVE PHASE as a major mechanism for thermoregulation of flowering.

    Science.gov (United States)

    Hwan Lee, Jeong; Sook Chung, Kyung; Kim, Soon-Kap; Ahn, Ji Hoon

    2014-01-01

    In contrast to our extensive knowledge of vernalization, we know relatively little about the regulation of ambient temperature-responsive flowering. Recent reports revealed that FLOWERING LOCUS M (FLM) and SHORT VEGETATIVE PHASE (SVP) regulate high ambient temperature-responsive flowering through two different mechanisms: degradation of SVP protein and formation of a non-functional SVP-FLM-δ complex. To investigate further the mechanism of thermoregulation of flowering, we performed real-time quantitative polymerase chain reaction (RT-qPCR) and in vitro pull-down assays. We found that FLM-β and FLM-δ transcripts show similar absolute levels at different temperatures. Also, His-SVP protein bound to the GST-FLM-β or -δ proteins with similar binding intensities. These results suggest that functional SVP-FLM-β and non-functional SVP-FLM-δ complexes form similarly at warmer temperatures, thus indicating that post-translational regulation of SVP functions as a major mechanism for thermoregulation in flowering.

  18. Evidence against Stable Protein S-Nitrosylation as a Widespread Mechanism of Post-translational Regulation.

    Science.gov (United States)

    Wolhuter, Kathryn; Whitwell, Harry J; Switzer, Christopher H; Burgoyne, Joseph R; Timms, John F; Eaton, Philip

    2018-02-01

    S-nitrosation, commonly referred to as S-nitrosylation, is widely regarded as a ubiquitous, stable post-translational modification that directly regulates many proteins. Such a widespread role would appear to be incompatible with the inherent lability of the S-nitroso bond, especially its propensity to rapidly react with thiols to generate disulfide bonds. As anticipated, we observed robust and widespread protein S-nitrosation after exposing cells to nitrosocysteine or lipopolysaccharide. Proteins detected using the ascorbate-dependent biotin switch method are typically interpreted to be directly regulated by S-nitrosation. However, these S-nitrosated proteins are shown to predominantly comprise transient intermediates leading to disulfide bond formation. These disulfides are likely to be the dominant end effectors resulting from elevations in nitrosating cellular nitric oxide species. We propose that S-nitrosation primarily serves as a transient intermediate leading to disulfide formation. Overall, we conclude that the current widely held perception that stable S-nitrosation directly regulates the function of many proteins is significantly incorrect. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. Transcriptional and post-translational regulation of Arc in synaptic plasticity.

    Science.gov (United States)

    Carmichael, Ruth E; Henley, Jeremy M

    2017-09-07

    One of the most interesting features of Arc-dependent synaptic plasticity is how multiple types of synaptic activity can converge to alter Arc transcription and then diverge to induce different plasticity outcomes, ranging from AMPA receptor internalisation that promotes long-term depression (LTD), to actin stabilisation that promotes long-term potentiation (LTP). This diversity suggests that there must be numerous levels of control to ensure the temporal profile, abundance, localisation and function of Arc are appropriately regulated to effect learning and memory in the correct contexts. The activity-dependent transcription and post-translational modification of Arc are crucial regulators of synaptic plasticity, fine-tuning the function of this key protein depending on the specific situation. The extensive cross-talk between signalling pathways and the numerous routes of Arc regulation provide a complex interplay of processes in which Arc-mediated plasticity can be broadly induced, but specifically tailored to synaptic activity. Here we provide an overview what is currently known about these processes and potential future directions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Autophagy Regulates the Post-Translational Cleavage of BCL-2 and Promotes Neuronal Survival

    Directory of Open Access Journals (Sweden)

    Laura Lossi

    2010-01-01

    Full Text Available B-cell lymphoma 2 protein (BCL-2 is one of the more widely investigated anti-apoptotic protein in mammals, and its levels are critical for protecting from programmed cell death. We report here that the cellular content of BCL-2 is regulated at post-translational level along the autophagy/lysosome pathways in organotypic cultures of post-natal mouse cerebellar cortex. Specifically this mechanism appears to be effective in the cerebellar granule cells (CGCs that are known to undergo massive programmed cell death (apoptosis during post-natal maturation. By the use of specific agonists/antagonist of calcium channels at the endoplasmic reticulum it was possible to understand the pivotal role of calcium release from intracellular stores in CGC neuroprotection. The more general significance of these findings is supported by a very recent study Niemann-Pick transgenic mice.

  1. Regulation of the Regulators: Post-Translational Modifications, Subcellular, and Spatiotemporal Distribution of Plant 14-3-3 Proteins.

    Science.gov (United States)

    Wilson, Rashaun S; Swatek, Kirby N; Thelen, Jay J

    2016-01-01

    14-3-3 proteins bind to and modulate the activity of phosphorylated proteins that regulate a variety of metabolic processes in eukaryotes. Multiple 14-3-3 isoforms are expressed in most organisms and display redundancy in both sequence and function. Plants contain the largest number of 14-3-3 isoforms. For example, Arabidopsis thaliana contains thirteen 14-3-3 genes, each of which is expressed. Interest in the plant 14-3-3 field has swelled over the past decade, largely due to the vast number of possibilities for 14-3-3 metabolic regulation. As the field progresses, it is essential to understand these proteins' activities at both the spatiotemporal and subcellular levels. This review summarizes current knowledge of 14-3-3 proteins in plants, including 14-3-3 interactions, regulatory functions, isoform specificity, and post-translational modifications. We begin with a historical overview and structural analysis of 14-3-3 proteins, which describes the basic principles of 14-3-3 function, and then discuss interactions and regulatory effects of plant 14-3-3 proteins in specific tissues and subcellular compartments. We conclude with a summary of 14-3-3 phosphorylation and current knowledge of the functional effects of this modification in plants.

  2. Translational independence between overlapping genes for a restriction endonuclease and its transcriptional regulator

    Directory of Open Access Journals (Sweden)

    Blumenthal Robert M

    2010-11-01

    Full Text Available Abstract Background Most type II restriction-modification (RM systems have two independent enzymes that act on the same DNA sequence: a modification methyltransferase that protects target sites, and a restriction endonuclease that cleaves unmethylated target sites. When RM genes enter a new cell, methylation must occur before restriction activity appears, or the host's chromosome is digested. Transcriptional mechanisms that delay endonuclease expression have been identified in some RM systems. A substantial subset of those systems is controlled by a family of small transcription activators called C proteins. In the PvuII system, C.PvuII activates transcription of its own gene, along with that of the downstream endonuclease gene. This regulation results in very low R.PvuII mRNA levels early after gene entry, followed by rapid increase due to positive feedback. However, given the lethal consequences of premature REase accumulation, transcriptional control alone might be insufficient. In C-controlled RM systems, there is a ± 20 nt overlap between the C termination codon and the R (endonuclease initiation codon, suggesting possible translational coupling, and in many cases predicted RNA hairpins could occlude the ribosome binding site for the endonuclease gene. Results Expression levels of lacZ translational fusions to pvuIIR or pvuIIC were determined, with the native pvuII promoter having been replaced by one not controlled by C.PvuII. In-frame pvuIIC insertions did not substantially decrease either pvuIIC-lacZ or pvuIIR-lacZ expression (with or without C.PvuII provided in trans. In contrast, a frameshift mutation in pvuIIC decreased expression markedly in both fusions, but mRNA measurements indicated that this decrease could be explained by transcriptional polarity. Expression of pvuIIR-lacZ was unaffected when the pvuIIC stop codon was moved 21 nt downstream from its WT location, or 25 or 40 bp upstream of the pvuIIR initiation codon. Disrupting

  3. Translational independence between overlapping genes for a restriction endonuclease and its transcriptional regulator.

    Science.gov (United States)

    Kaw, Meenakshi K; Blumenthal, Robert M

    2010-11-19

    Most type II restriction-modification (RM) systems have two independent enzymes that act on the same DNA sequence: a modification methyltransferase that protects target sites, and a restriction endonuclease that cleaves unmethylated target sites. When RM genes enter a new cell, methylation must occur before restriction activity appears, or the host's chromosome is digested. Transcriptional mechanisms that delay endonuclease expression have been identified in some RM systems. A substantial subset of those systems is controlled by a family of small transcription activators called C proteins. In the PvuII system, C.PvuII activates transcription of its own gene, along with that of the downstream endonuclease gene. This regulation results in very low R.PvuII mRNA levels early after gene entry, followed by rapid increase due to positive feedback. However, given the lethal consequences of premature REase accumulation, transcriptional control alone might be insufficient. In C-controlled RM systems, there is a ± 20 nt overlap between the C termination codon and the R (endonuclease) initiation codon, suggesting possible translational coupling, and in many cases predicted RNA hairpins could occlude the ribosome binding site for the endonuclease gene. Expression levels of lacZ translational fusions to pvuIIR or pvuIIC were determined, with the native pvuII promoter having been replaced by one not controlled by C.PvuII. In-frame pvuIIC insertions did not substantially decrease either pvuIIC-lacZ or pvuIIR-lacZ expression (with or without C.PvuII provided in trans). In contrast, a frameshift mutation in pvuIIC decreased expression markedly in both fusions, but mRNA measurements indicated that this decrease could be explained by transcriptional polarity. Expression of pvuIIR-lacZ was unaffected when the pvuIIC stop codon was moved 21 nt downstream from its WT location, or 25 or 40 bp upstream of the pvuIIR initiation codon. Disrupting the putative hairpins had no significant

  4. Down-regulation of RE-1 silencing transcription factor (REST) in advanced prostate cancer by hypoxia-induced miR-106b~25.

    Science.gov (United States)

    Liang, Hongzi; Studach, Leo; Hullinger, Ronald L; Xie, Jun; Andrisani, Ourania M

    2014-01-15

    Clinically aggressive prostate cancer (PCa) is linked to androgen resistance, metastasis, and expression of neuroendocrine markers. To understand mechanism(s) of neuroendocrine differentiation (NED) of PCa epithelia, we compared neuronal differentiation occurring during embryogenesis, in primary cultures of neural crest (NC) cells, and NED in PCa cell lines (LNCaP and PC3). We demonstrate, hypoxia promotes neuronal and neuroendocrine differentiation of NC cells and PCa cells, respectively, by inducing the miR-106 b~25 cluster. In turn, miR-106b~25 comprised of miR-106b, miR-93 and miR-25, down-regulates the transcriptional repressor REST, which represses neuron-specific protein-coding and miRNA genes. In prostate tumors of high Gleason score (≥ 8), an inverse trend was observed between REST and miR-106b~25 induction. Employing miRNA PCR arrays, we identified miRNAs up-regulated by hypoxia in LNCaP cells and REST-knockdown in NC cells. Significantly, a subset of miRNAs (miR-9, miR-25, miR-30d and miR302b) is up-regulated in high Gleason score (≥ 8) PCa, suggesting a mechanism by which NED contributes to PCa malignancy. We propose that loss of REST and induction of this set of microRNAs can serve as potential novel clinical markers of advanced PCa. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  5. The splicing factor SF2/ASF regulates translation initiation by enhancing phosphorylation of 4E-BP1.

    Science.gov (United States)

    Michlewski, Gracjan; Sanford, Jeremy R; Cáceres, Javier F

    2008-04-25

    The SR protein SF2/ASF has been initially characterized as a splicing factor but has also been shown to mediate postsplicing activities such as mRNA export and translation. Here we demonstrate that SF2/ASF promotes translation initiation of bound mRNAs and that this activity requires the presence of the cytoplasmic cap-binding protein eIF4E. SF2/ASF promotes translation initiation by suppressing the activity of 4E-BP, a competitive inhibitor of cap-dependent translation. This activity is mediated by interactions of SF2/ASF with both mTOR and the phosphatase PP2A, two key regulators of 4E-BP phosphorylation. These findings suggest the model whereby SF2/ASF functions as an adaptor protein to recruit the signaling molecules responsible for regulation of cap-dependent translation of specific mRNAs. Taken together, these data suggest a novel mechanism for the activation of translation initiation of a subset of mRNAs bound by the shuttling protein SF2/ASF.

  6. Self-Regulation and Mechanisms of Action in Psychotherapy: A Theory-Based Translational Perspective

    Science.gov (United States)

    Strauman, Timothy J.; Goetz, Elena L.; Detloff, Allison M.; MacDuffie, Katherine E.; Zaunmüller, Luisa; Lutz, Wolfgang

    2012-01-01

    Psychotherapy is a complex, multi-layered process with the potential to bring about changes at multiple levels of functioning, from the neurobiology of the brain to the individual’s role in the social world. Although studies of the mechanisms by which psychotherapy leads to change continue to appear, there remains much to be learned about how psychological interventions work. To guide explorations of how and for whom particular treatment approaches lead to change, researchers can rely on theory to identify potential loci for change and on translational research methods to integrate basic behavioral science and neuroscience with clinical science. In this article, we describe research linking individual differences in the self-regulation of personal goal pursuit with the etiology and treatment of mood disorders. The research draws upon regulatory focus theory as a model of self-regulation and on microintervention designs – controlled laboratory investigations of a specific therapeutic technique – to generate and test hypotheses about how psychological interventions can help to reverse maladaptive self-regulatory processes. PMID:23072383

  7. Post-translational regulation via Clp protease is critical for survival of Mycobacterium tuberculosis.

    Science.gov (United States)

    Raju, Ravikiran M; Jedrychowski, Mark P; Wei, Jun-Rong; Pinkham, Jessica T; Park, Annie S; O'Brien, Kathryn; Rehren, German; Schnappinger, Dirk; Gygi, Steven P; Rubin, Eric J

    2014-03-01

    Unlike most bacterial species, Mycobacterium tuberculosis depends on the Clp proteolysis system for survival even in in vitro conditions. We hypothesized that Clp is required for the physiologic turnover of mycobacterial proteins whose accumulation is deleterious to bacterial growth and survival. To identify cellular substrates, we employed quantitative proteomics and transcriptomics to identify the set of proteins that accumulated upon the loss of functional Clp protease. Among the set of potential Clp substrates uncovered, we were able to unambiguously identify WhiB1, an essential transcriptional repressor capable of auto-repression, as a substrate of the mycobacterial Clp protease. Dysregulation of WhiB1 turnover had a toxic effect that was not rescued by repression of whiB1 transcription. Thus, under normal growth conditions, Clp protease is the predominant regulatory check on the levels of potentially toxic cellular proteins. Our findings add to the growing evidence of how post-translational regulation plays a critical role in the regulation of bacterial physiology.

  8. Translational regulation of GPx-1 and GPx-4 by the mTOR pathway.

    Directory of Open Access Journals (Sweden)

    Emily N Reinke

    Full Text Available Glutathione peroxidase activity was previously determined to be elevated in lymphocytes obtained from patients treated with the Bcr-Abl kinase inhibitor imatinib mesylate. In order to expand upon this observation, the established chronic myelogenous leukemia cell lines KU812 and MEG-01 were treated with imatinib and the effect on several anti-oxidant proteins was determined. The levels of GPx-1 were significantly increased following treatment with imatinib. This increase was not due to altered steady-state mRNA levels, and appeared to be dependent on the expression of Bcr-Abl, as no increases were observed following imatinib treatment of cells that did not express the fusion protein. The nutrient-sensing signaling protein, mammalian target of rapamycin (mTOR, can be activated by Bcr-Abl and its activity regulates the translation of many different proteins. Treatment of those same cells used in the imatinib studies with rapamycin, an inhibitor of mTOR, resulted in elevated GPx-1 and GPx-4 protein levels independent of Bcr-Abl expression. These proteins all belong to the selenoprotein family of peptides that contain the UGA-encoded amino acid selenocysteine. Collectively, these data provide evidence of a novel means of regulating anti-oxidants of the selenoprotein family via the mTOR pathway.

  9. PPARG Post-translational Modifications Regulate Bone Formation and Bone Resorption

    Directory of Open Access Journals (Sweden)

    L.A. Stechschulte

    2016-08-01

    Full Text Available The peroxisome proliferator-activated receptor gamma (PPARγ regulates osteoblast and osteoclast differentiation, and is the molecular target of thiazolidinediones (TZDs, insulin sensitizers that enhance glucose utilization and adipocyte differentiation. However, clinical use of TZDs has been limited by side effects including a higher risk of fractures and bone loss. Here we demonstrate that the same post-translational modifications at S112 and S273, which influence PPARγ pro-adipocytic and insulin sensitizing activities, also determine PPARγ osteoblastic (pS112 and osteoclastic (pS273 activities. Treatment of either hyperglycemic or normoglycemic animals with SR10171, an inverse agonist that blocks pS273 but not pS112, increased trabecular and cortical bone while normalizing metabolic parameters. Additionally, SR10171 treatment modulated osteocyte, osteoblast, and osteoclast activities, and decreased marrow adiposity. These data demonstrate that regulation of bone mass and energy metabolism shares similar mechanisms suggesting that one pharmacologic agent could be developed to treat both diabetes and metabolic bone disease.

  10. Decoding sORF translation – from small proteins to gene regulation

    OpenAIRE

    Cabrera-Quio, Luis Enrique; Herberg, Sarah; Pauli, Andrea

    2016-01-01

    Translation is best known as the fundamental mechanism by which the ribosome converts a sequence of nucleotides into a string of amino acids. Extensive research over many years has elucidated the key principles of translation, and the majority of translated regions were thought to be known. The recent discovery of wide-spread translation outside of annotated protein-coding open reading frames (ORFs) came therefore as a surprise, raising the intriguing possibility that these newly discovered t...

  11. A Study of Translation Students' Self-Regulation and Metacognitive Awareness in Association with their Gender and Educational Level

    Directory of Open Access Journals (Sweden)

    Mona Hashempour

    2015-07-01

    Full Text Available The present study sought to investigate the probable link between Iranian English translation studies students’ metacognitive awareness, self-regulation, and gender. Furthermore, the role of educational level of translation students in metacognitive awareness and self-regulation was explored. For the purpose of the study, a sample of 230 M.A and B.A senior English translation students comprised the participants of the research. They were asked to complete two questionnaires of Metacognitive Awareness Inventory (MAI and Self-regulation Trait (SRT.The Metacognitive Awareness Inventory (MAI was developed by Schraw and Dennison (1994 and consists of 52 statements. It measures two components of metacognition: metacognitive knowledge and metacognitive regulation. Metacognitive knowledge comprises three subscales: Declarative knowledge, Procedural knowledge, and Conditional knowledge. Metacognitive regulation consists of five subscales: Planning, Information management, Monitoring, Debugging, and Evaluation. The self-regulation trait (SRT questionnaire was designed by O'Neil and Herl (1998. It was developed based on Zimmerman's self –regulation model. It consists of 32 Likert-scale questions. The scale seeks to measure metacognition and motivation dimensions.  Each dimension comprises two sub-scales. Meta-cognition covers the constructs of planning and self-monitoring, and motivation contains effort and self-efficacy. Independent samples t-tests were run to investigate the role of gender and educational level in the level of translation students’ metacognitive awareness and self-regulation. The results of t-test demonstrated that there are not any differences between male and female translation students regarding metacognitive awareness and self-regulation. It was also found that there is a negative significant impact of educational level on total metacognitive awareness, and some components of metacognitive awareness: declarative knowledge

  12. Local translation and retrograde axonal transport of CREB regulates IL-6-induced nociceptive plasticity.

    Science.gov (United States)

    Melemedjian, Ohannes K; Tillu, Dipti V; Moy, Jamie K; Asiedu, Marina N; Mandell, Edward K; Ghosh, Sourav; Dussor, Gregory; Price, Theodore J

    2014-07-04

    Transcriptional regulation of genes by cyclic AMP response element binding protein (CREB) is essential for the maintenance of long-term memory. Moreover, retrograde axonal trafficking of CREB in response to nerve growth factor (NGF) is critical for the survival of developing primary sensory neurons. We have previously demonstrated that hindpaw injection of interleukin-6 (IL-6) induces mechanical hypersensitivity and hyperalgesic priming that is prevented by the local injection of protein synthesis inhibitors. However, proteins that are locally synthesized that might lead to this effect have not been identified. We hypothesized that retrograde axonal trafficking of nascently synthesized CREB might link local, activity-dependent translation to nociceptive plasticity. To test this hypothesis, we determined if IL-6 enhances the expression of CREB and if it subsequently undergoes retrograde axonal transport. IL-6 treatment of sensory neurons in vitro caused an increase in CREB protein and in vivo treatment evoked an increase in CREB in the sciatic nerve consistent with retrograde transport. Importantly, co-injection of IL-6 with the methionine analogue azido-homoalanine (AHA), to assess nascently synthesized proteins, revealed an increase in CREB containing AHA in the sciatic nerve 2 hrs post injection, indicating retrograde transport of nascently synthesized CREB. Behaviorally, blockade of retrograde transport by disruption of microtubules or inhibition of dynein or intrathecal injection of cAMP response element (CRE) consensus sequence DNA oligonucleotides, which act as decoys for CREB DNA binding, prevented the development of IL-6-induced mechanical hypersensitivity and hyperalgesic priming. Consistent with previous studies in inflammatory models, intraplantar IL-6 enhanced the expression of BDNF in dorsal root ganglion (DRG). This effect was blocked by inhibition of retrograde axonal transport and by intrathecal CRE oligonucleotides. Collectively, these findings

  13. PKC Regulates α1-Adrenoceptor-Mediated Contractions and Baseline Ca2+ Sensitivity in the Uterine Arteries of Nonpregnant and Pregnant Sheep Acclimatized to High Altitude Hypoxia

    Science.gov (United States)

    Huang, Xiaohui; Longo, Lawrence D.; Zhang, Lubo

    2010-01-01

    Abstract Xiao, Daliao, Xiaohui Huang, Lawrence D. Longo, and Lubo Zhang. PKC regulates α1-adrenoceptor-mediated contractions and baseline Ca2+ sensitivity in the uterine arteries of nonpregnant and pregnant sheep acclimatized to high alttude hypoxia. High Alt. Med. Biol. 11:153–162, 2010.—Chronic hypoxia has a profound effect on uterine artery adaptation to pregnancy. The present studies tested the hypothesis that pregnant kinase C (PKC) differentially regulates α1-adrenoceptor-mediated contractions and Ca2+ sensitivity in the uterine arteries of nonpregnant and pregnant sheep acclimatized to high altitude hypoxia. Uterine arteries were isolated from nonpregnant (NPUA) and near-term pregnant (PUA) ewes maintained at high altitude (3801 m, Pao2 ∼60 torr) for 110 days. Phorbol 12,13-dibutyrate (PDBu) decreased phenylephrine-induced contractions in PUA but not in NPUA, which was partly inhibited by the PKC inhibitor GF109203X. Additionally, GF109203X shifted the concentration–response curve of phenylephrine-induced contractions to the right in PUA. In β-escin-permeabilized arteries, Ca2+-induced increases in 20-kDa myosin light chain phosphorylation (MLC20-P) were similar in NPUA and PUA. However, Ca2+ produced a concentration-dependent increase in the ratio of tension to MLC20-P in PUA, as compared with NPUA. PKC inhibition decreased Ca2+-induced contractions in both NPUA and PUA. PDBu induced contractions of PUA in the absence of changes in MLC20-P, which was not affected by PD098059. There was a significant increase in the basal activity of PKCɛ in PUA, but not in NPUA, in hypoxic sheep, as compared with normoxic animals. The results demonstrate that the inhibitory effect of PKC on α1-adrenoceptor-mediated contractions of uterine arteries is preserved in pregnant sheep at high altitude. However, the PKC-mediated thin-filament regulatory pathway is upregulated, resulting in increased baseline Ca2+ sensitivity in the uterine artery during pregnancy at

  14. Ectodermal-neural cortex 1 down-regulates Nrf2 at the translational level.

    Directory of Open Access Journals (Sweden)

    Xiao-Jun Wang

    Full Text Available The transcription factor Nrf2 is the master regulator of a cellular defense mechanism against environmental insults. The Nrf2-mediated antioxidant response is accomplished by the transcription of a battery of genes that encode phase II detoxifying enzymes, xenobiotic transporters, and antioxidants. Coordinated expression of these genes is critical in protecting cells from toxic and carcinogenic insults and in maintaining cellular redox homeostasis. Activation of the Nrf2 pathway is primarily controlled by Kelch-like ECH-associated protein 1 (Keap1, which is a molecular switch that turns on or off the Nrf2 signaling pathway according to intracellular redox conditions. Here we report our finding of a novel Nrf2 suppressor ectodermal-neural cortex 1 (ENC1, which is a BTB-Kelch protein and belongs to the same family as Keap1. Transient expression of ENC1 reduced steady-state levels of Nrf2 and its downstream gene expression. Although ENC1 interacted with Keap1 indirectly, the ENC1-mediated down-regulation of Nrf2 was independent of Keap1. The negative effect of ENC1 on Nrf2 was not due to a change in the stability of Nrf2 because neither proteasomal nor lysosomal inhibitors had any effects. Overexpression of ENC1 did not result in a change in the level of Nrf2 mRNA, rather, it caused a decrease in the rate of Nrf2 protein synthesis. These results demonstrate that ENC1 functions as a negative regulator of Nrf2 through suppressing Nrf2 protein translation, which adds another level of complexity in controlling the Nrf2 signaling pathway.

  15. Balancing spatially regulated β-actin translation and dynamin-mediated endocytosis is required to assemble functional epithelial monolayers.

    Science.gov (United States)

    Cruz, Lissette A; Vedula, Pavan; Gutierrez, Natasha; Shah, Neel; Rodriguez, Steven; Ayee, Brian; Davis, Justin; Rodriguez, Alexis J

    2015-12-01

    Regulating adherens junction complex assembly/disassembly is critical to maintaining epithelial homeostasis in healthy epithelial tissues. Consequently, adherens junction structure and function is often perturbed in clinically advanced tumors of epithelial origin. Some of the most studied factors driving adherens junction complex perturbation in epithelial cancers are transcriptional and epigenetic down-regulation of E-cadherin expression. However, numerous reports demonstrate that post-translational regulatory mechanisms such as endocytosis also regulate early phases of epithelial-mesenchymal transition and metastatic progression. In already assembled healthy epithelia, E-cadherin endocytosis recycles cadherin-catenin complexes to regulate the number of mature adherens junctions found at cell-cell contact sites. However, following de novo epithelial cell-cell contact, endocytosis negatively regulates adherens junction assembly by removing E-cadherin from the cell surface. By contrast, following de novo epithelial cell-cell contact, spatially localized β-actin translation drives cytoskeletal remodeling and consequently E-cadherin clustering at cell-cell contact sites and therefore positively regulates adherens junction assembly. In this report we demonstrate that dynamin-mediated endocytosis and β-actin translation-dependent cadherin-catenin complex anchoring oppose each other following epithelial cell-cell contact. Consequently, the final extent of adherens junction assembly depends on which of these processes is dominant following epithelial cell-cell contact. We expressed β-actin transcripts impaired in their ability to properly localize monomer synthesis (Δ3'UTR) in MDCK cells to perturb actin filament remodeling and anchoring, and demonstrate the resulting defect in adherens junction structure and function is rescued by inhibiting dynamin mediated endocytosis. Therefore, we demonstrate balancing spatially regulated β-actin translation and dynamin

  16. Conserved regions of the DMD 3' UTR regulate translation and mRNA abundance in cultured myotubes.

    Science.gov (United States)

    Larsen, C Aaron; Howard, Michael T

    2014-08-01

    Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease, is caused by mutations in the DMD gene, which encodes for the protein dystrophin. Its regulation is of therapeutic interest as even small changes in expression of functional dystrophin can significantly impact the severity of DMD. While tissue-specific distribution and transcriptional regulation of several DMD mRNA isoforms has been well characterized, the post-transcriptional regulation of dystrophin synthesis is not well understood. Here, we utilize qRTPCR and a quantitative dual-luciferase reporter assay to examine the effects of isoform specific DMD 5' UTRs and the highly conserved DMD 3' UTR on mRNA abundance and translational control of gene expression in C2C12 cells. The 5' UTRs were shown to initiate translation with low efficiency in both myoblasts and myotubes. Whereas, two large highly conserved elements in the 3' UTR, which overlap the previously described Lemaire A and D regions, increase mRNA levels and enhance translation upon differentiation of myoblasts into myotubes. The results presented here implicate an important role for DMD UTRs in dystrophin expression and delineate the cis-acting elements required for the myotube-specific regulation of steady-state mRNA levels and translational enhancer activity found in the DMD 3' UTR. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Conserved regions of the DMD 3’ UTR regulate translation and mRNA abundance in cultured myotubes

    Science.gov (United States)

    Larsen, C. Aaron; Howard, Michael T.

    2014-01-01

    Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease, is caused by mutations in the DMD gene, which encodes for the protein dystrophin. Its regulation is of therapeutic interest as even small changes in expression of functional dystrophin can significantly impact the severity of DMD. While tissue-specific distribution and transcriptional regulation of several DMD mRNA isoforms has been well characterized, the post-transcriptional regulation of dystrophin synthesis is not well understood. Here, we utilize qRTPCR and a quantitative dual-luciferase reporter assay to examine the effects of isoform specific DMD 5’ UTRs and the highly conserved DMD 3’ UTR on mRNA abundance and translational control of gene expression in C2C12 cells. The 5’ UTRs were shown to initiate translation with low efficiency in both myoblasts and myotubes. Whereas, two large highly conserved elements in the 3’ UTR, which overlap the previously described Lemaire A and D regions, increase mRNA levels and enhance translation upon differentiation of myoblasts into myotubes. The results presented here implicate an important role for DMD UTRs in dystrophin expression and delineate the cis-acting elements required for the myotube-specific regulation of steady-state mRNA levels and translational enhancer activity found in the DMD 3’ UTR. PMID:24928536

  18. Molecular cloning, characterization and expression of two tryptophan hydroxylase (TPH-1 and TPH-2) genes in the hypothalamus of Atlantic croaker: down-regulation after chronic exposure to hypoxia.

    Science.gov (United States)

    Rahman, M S; Thomas, P

    2009-01-23

    Recently we discovered that hypoxia causes marked impairment of reproductive neuroendocrine function in Atlantic croaker, a marine teleost, which is due to a decline in hypothalamic serotonergic activity. As a first step in understanding the molecular responses of the hypothalamic serotonergic system to hypoxia, we cloned and characterized the genes for the enzymes regulating the rate-limiting step in serotonin biosynthesis, tryptophan hydroxylase (TPH-1 and TPH-2) in the croaker brain. The full-length croaker TPH-1 and TPH-2 cDNAs contain open reading frames encoding proteins with 479 and 487 amino acids, respectively, which are highly homologous to the TPH-1 (76-93%) and TPH-2 (64-92%) proteins of other vertebrates. Croaker TPH-1 and TPH-2 mRNA expression was detected throughout the brain but was greatest in the hypothalamic region. Both Northern blot analysis and real-time PCR showed that TPH-1 (transcript size approximately 2.1 kb) and TPH-2 ( approximately 1.9 kb) mRNA levels were significantly decreased in the hypothalami of croaker exposed for 2 weeks to hypoxic conditions compared with those in fish exposed to normoxic conditions. Immunohistochemistry of hypothalamic neurons with TPH antibodies showed reduced expression of TPHs in hypoxia-exposed fish compared with normoxic fish. Western blot analysis confirmed that hypoxia caused a marked decline in hypothalamic TPH protein levels, which was associated with decreases in hypothalamic TPH enzyme activity and 5-hydroxytryptophan levels. These results suggest that TPH is a major site of hypoxia-induced down-regulation of serotonergic function in croaker brains. Moreover, they provide the first evidence that hypoxia decreases the expression of TPH transcripts in vertebrate brains.

  19. miR-140-5p regulates hypoxia-mediated human pulmonary artery smooth muscle cell proliferation, apoptosis and differentiation by targeting Dnmt1 and promoting SOD2 expression

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanwei; Xu, Jing, E-mail: xujingdoc@163.com

    2016-04-22

    miR-140-5p is down-regulated in patients with pulmonary arterial hypertension (PAH) and experimental models of PAH, and inhibits hypoxia-mediated pulmonary artery smooth muscle cell (PASMC) proliferation in vitro. Delivery of synthetic miR-140-5p prevents and treats established, experimental PAH. DNA methyltransferase 1 (Dnmt1) is up-regulated in PAH associated human PASMCs (HPASMCs), which promotes the development of PAH by hypermethylation of CpG islands within the promoter for superoxide dismutase 2 (SOD2) and down-regulating SOD2 expression. We searched for miR-140-5p targets using TargetScan, PicTar and MiRanda tools, and found that Dnmt1 is a potential target of miR-140-5p. Based on these findings, we speculated that miR-140-5p might target Dnmt1 and regulate SOD2 expression to regulate hypoxia-mediated HPASMC proliferation, apoptosis and differentiation. We detected the expression of miR-140-5p, Dnmt1 and SOD2 by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot assays, respectively, and found down-regulation of miR-140-5p and SOD2 and up-regulation of Dnmt1 exist in PAH tissues and hypoxia-mediated HPASMCs. Cell proliferation, apoptosis and differentiation detection showed that miR-140-5p inhibits proliferation and promotes apoptosis and differentiation of HPASMCs in hypoxia, while the effect of Dnmt1 on hypoxia-mediated HPASMCs is reversed. Luciferase assay confirmed that miR-140-5p targets Dnmt1 directly. An inverse correlation is also found between miR-140-5p and Dnmt1 in HPASMCs. In addition, we further investigated whether miR-140-5p and Dnmt1 regulate HPASMC proliferation, apoptosis and differentiation by regulating SOD2 expression, and the results confirmed our speculation. Taken together, these results indicated that miR-140-5p at least partly targets Dnmt1 and regulates SOD2 expression to inhibit proliferation and promote apoptosis and differentiation of HPASMCs in hypoxia. - Highlights: • miR-140-5p and SOD2 are down-regulated

  20. Regulation of DNA damage tolerance in mammalian cells by post-translational modifications of PCNA.

    Science.gov (United States)

    Kanao, Rie; Masutani, Chikahide

    2017-10-01

    DNA damage tolerance pathways, which include translesion DNA synthesis (TLS) and template switching, are crucial for prevention of DNA replication arrest and maintenance of genomic stability. However, these pathways utilize error-prone DNA polymerases or template exchange between sister DNA strands, and consequently have the potential to induce mutations or chromosomal rearrangements. Post-translational modifications of proliferating cell nuclear antigen (PCNA) play important roles in controlling these pathways. For example, TLS is mediated by mono-ubiquitination of PCNA at lysine 164, for which RAD6-RAD18 is the primary E2-E3 complex. Elaborate protein-protein interactions between mono-ubiquitinated PCNA and Y-family DNA polymerases constitute the core of the TLS regulatory system, and enhancers of PCNA mono-ubiquitination and de-ubiquitinating enzymes finely regulate TLS and suppress TLS-mediated mutagenesis. The template switching pathway is promoted by K63-linked poly-ubiquitination of PCNA at lysine 164. Poly-ubiquitination is achieved by a coupled reaction mediated by two sets of E2-E3 complexes, RAD6-RAD18 and MMS2-UBC13-HTLF/SHPRH. In addition to these mono- and poly-ubiquitinations, simultaneous mono-ubiquitinations on multiple units of the PCNA homotrimeric ring promote an unidentified damage tolerance mechanism that remains to be fully characterized. Furthermore, SUMOylation of PCNA in mammalian cells can negatively regulate recombination. Other modifications, including ISGylation, acetylation, methylation, or phosphorylation, may also play roles in DNA damage tolerance and control of genomic stability. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Neuron-wide RNA transport combines with netrin-mediated local translation to spatially regulate the synaptic proteome.

    Science.gov (United States)

    Kim, Sangmok; Martin, Kelsey C

    2015-01-08

    The persistence of experience-dependent changes in brain connectivity requires RNA localization and protein synthesis. Previous studies have demonstrated a role for local translation in altering the structure and function of synapses during synapse formation and experience-dependent synaptic plasticity. In this study, we ask whether in addition to promoting local translation, local stimulation also triggers directed trafficking of RNAs from nucleus to stimulated synapses. Imaging of RNA localization and translation in cultured Aplysia sensory-motor neurons revealed that RNAs were delivered throughout the arbor of the sensory neuron, but that translation was enriched only at sites of synaptic contact and/or synaptic stimulation. Investigation of the mechanisms that trigger local translation revealed a role for calcium-dependent retrograde netrin-1/DCC receptor signaling. Spatially restricting gene expression by regulating local translation rather than by directing the delivery of mRNAs from nucleus to stimulated synapses maximizes the readiness of the entire neuronal arbor to respond to local cues.

  2. Protein redox chemistry: post-translational cysteine modifications that regulate signal transduction and drug pharmacology

    Directory of Open Access Journals (Sweden)

    Revati eWani

    2014-10-01

    Full Text Available The perception of reactive oxygen species (ROS has evolved over the past decade from agents of cellular damage to secondary messengers which modify signaling proteins in physiology and the disease state (e.g. cancer. New protein targets of specific oxidation are rapidly being identified. One emerging class of redox modification occurs to the thiol side chain of cysteine residues which can produce multiple chemically-distinct alterations to the protein (e.g. sulfenic/sulfinic/sulfonic acid, disulfides. These post-translational modifications (PTM are shown to affect the protein structure and function. Because redox-sensitive proteins can traffic between subcellular compartments that have different redox environments, cysteine oxidation enables a spatio-temporal control to signaling. Understanding ramifications of these oxidative modifications to the functions of signaling proteins is crucial for understanding cellular regulation as well as for informed-drug discovery process. The effects of EGFR oxidation of Cys797 on inhibitor pharmacology are presented to illustrate the principle. Taken together, cysteine redox PTM can impact both cell biology and drug pharmacology.

  3. CLUH regulates mitochondrial metabolism by controlling translation and decay of target mRNAs.

    Science.gov (United States)

    Schatton, Désirée; Pla-Martin, David; Marx, Marie-Charlotte; Hansen, Henriette; Mourier, Arnaud; Nemazanyy, Ivan; Pessia, Alberto; Zentis, Peter; Corona, Teresa; Kondylis, Vangelis; Barth, Esther; Schauss, Astrid C; Velagapudi, Vidya; Rugarli, Elena I

    2017-03-06

    Mitochondria are essential organelles that host crucial metabolic pathways and produce adenosine triphosphate. The mitochondrial proteome is heterogeneous among tissues and can dynamically change in response to different metabolic conditions. Although the transcriptional programs that govern mitochondrial biogenesis and respiratory function are well known, posttranscriptional regulatory mechanisms remain unclear. In this study, we show that the cytosolic RNA-binding protein clustered mitochondria homologue (CLUH) regulates the expression of a mitochondrial protein network supporting key metabolic programs required under nutrient deprivation. CLUH exerts its function by controlling the stability and translation of target messenger RNAs. In the absence of Cluh, mitochondria are severely depleted of crucial enzymes involved in catabolic energy-converting pathways. CLUH preserves oxidative mitochondrial function and glucose homeostasis, thus preventing death at the fetal-neonatal transition. In the adult liver, CLUH ensures maximal respiration capacity and the metabolic response to starvation. Our results shed new light on the posttranscriptional mechanisms controlling the expression of mitochondrial proteins and suggest novel strategies to tailor mitochondrial function to physiological and pathological conditions. © 2017 Schatton et al.

  4. Internal ribosome entry site-mediated translational regulation of ATF4 splice variant in mammalian unfolded protein response.

    Science.gov (United States)

    Chan, Ching-Ping; Kok, Kin-Hang; Tang, Hei-Man Vincent; Wong, Chi-Ming; Jin, Dong-Yan

    2013-10-01

    Activating transcription factor 4 (ATF4) is a master regulator of genes involved in unfolded protein response (UPR) and its translation is regulated through reinitiation at upstream open reading frames. Here, we demonstrate internal ribosome entry site (IRES)-mediated translation of an alternatively spliced variant of human ATF4. This variant that contains four upstream open reading frames in the 5' leader region was expressed in leukocytes and other tissues. mRNA and protein expression of this variant was activated in the UPR. Its translation was neither inhibited by steric hindrance nor affected by eIF4G1 inactivation, indicating a cap-independent and IRES-dependent mechanism not mediated by ribosome scanning-reinitiation. The IRES activity mapped to a highly structured region that partially overlaps with the third and fourth open reading frames was unlikely attributed to cryptic promoter or splicing, but was activated by PERK-induced eIF2α phosphorylation. Taken together, our findings reveal a new mechanism for translational regulation of ATF4 in mammalian UPR. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Toll-like Receptor 3 Regulates Angiogenesis and Apoptosis in Prostate Cancer Cell Lines through Hypoxia-Inducible Factor 1α

    Directory of Open Access Journals (Sweden)

    Alessio Paone

    2010-07-01

    Full Text Available Toll-like receptors (TLRs recognize microbial/viral-derived components that trigger innate immune response and conflicting data implicate TLR agonists in cancer, either as protumor or antitumor agents. We previously demonstrated that TLR3 activation mediated by its agonist poly(I:C induces antitumor signaling, leading to apoptosis of prostate cancer cells LNCaP and PC3 with much more efficiency in the former than in the second more aggressive line. The transcription factor hypoxia-induciblefactor 1 (HIF-1regulates several cellular processes, includingapoptosis, in response to hypoxia and to other stimuli also in normoxic conditions. Here we describe a novel protumor machinery triggered by TLR3 activation in PC3 cells consisting of increased expression of the specific 1.3 isoform of HIF-1α and nuclear accumulation of HIF-1 complex in normoxia, resulting in reduced apoptosis and in secretion of functional vascular endothelial growth factor (VEGF. Moreover, we report that, in the less aggressive LNCaP cells, TLR3 activation fails to induce nuclear accumulation of HIF-1α. However, the transfection of 1.3 isoform of hif-1α in LNCaP cells allows poly(I:CI-induced HIF-1 activation, resulting in apoptosis protection and VEGF secretion. Altogether, our findings demonstrate that differences in the basal level of HIF-1α expression in different prostate cancer cell lines underlie their differential response to TLR3 activation, suggesting a correlation between different stages of malignancy, hypoxic gene expression, and beneficial responsiveness to TLR agonists.

  6. PKC regulates alpha(1)-adrenoceptor-mediated contractions and baseline Ca(2+) sensitivity in the uterine arteries of nonpregnant and pregnant sheep acclimatized to high altitude hypoxia.

    Science.gov (United States)

    Xiao, Daliao; Huang, Xiaohui; Longo, Lawrence D; Zhang, Lubo

    2010-01-01

    Chronic hypoxia has a profound effect on uterine artery adaptation to pregnancy. The present studies tested the hypothesis that pregnant kinase C (PKC) differentially regulates alpha(1)-adrenoceptor-mediated contractions and Ca(2+) sensitivity in the uterine arteries of nonpregnant and pregnant sheep acclimatized to high altitude hypoxia. Uterine arteries were isolated from nonpregnant (NPUA) and near-term pregnant (PUA) ewes maintained at high altitude (3801 m, Pao(2) approximately 60 torr) for 110 days. Phorbol 12,13-dibutyrate (PDBu) decreased phenylephrine-induced contractions in PUA but not in NPUA, which was partly inhibited by the PKC inhibitor GF109203X. Additionally, GF109203X shifted the concentration-response curve of phenylephrine-induced contractions to the right in PUA. In beta-escin-permeabilized arteries, Ca(2+)-induced increases in 20-kDa myosin light chain phosphorylation (MLC(20)-P) were similar in NPUA and PUA. However, Ca(2+) produced a concentration-dependent increase in the ratio of tension to MLC(20)-P in PUA, as compared with NPUA. PKC inhibition decreased Ca(2+)-induced contractions in both NPUA and PUA. PDBu induced contractions of PUA in the absence of changes in MLC(20)-P, which was not affected by PD098059. There was a significant increase in the basal activity of PKCvarepsilon in PUA, but not in NPUA, in hypoxic sheep, as compared with normoxic animals. The results demonstrate that the inhibitory effect of PKC on alpha(1)-adrenoceptor-mediated contractions of uterine arteries is preserved in pregnant sheep at high altitude. However, the PKC-mediated thin-filament regulatory pathway is upregulated, resulting in increased baseline Ca(2+) sensitivity in the uterine artery during pregnancy at high altitude.

  7. 14-3-3 binding and phosphorylation of neuroglobin during hypoxia modulate six-to-five heme pocket coordination and rate of nitrite reduction to nitric oxide.

    Science.gov (United States)

    Jayaraman, Thottala; Tejero, Jesús; Chen, Bill B; Blood, Arlin B; Frizzell, Sheila; Shapiro, Calli; Tiso, Mauro; Hood, Brian L; Wang, Xunde; Zhao, Xuejun; Conrads, Thomas P; Mallampalli, Rama K; Gladwin, Mark T

    2011-12-09

    Neuroglobin protects neurons from hypoxia in vitro and in vivo; however, the underlying mechanisms for this effect remain poorly understood. Most of the neuroglobin is present in a hexacoordinate state with proximal and distal histidines in the heme pocket directly bound to the heme iron. At equilibrium, the concentration of the five-coordinate neuroglobin remains very low (0.1-5%). Recent studies have shown that post-translational redox regulation of neuroglobin surface thiol disulfide formation increases the open probability of the heme pocket and allows nitrite binding and reaction to form NO. We hypothesized that the equilibrium between the six- and five-coordinate states and secondary reactions with nitrite to form NO could be regulated by other hypoxia-dependent post-translational modification(s). Protein sequence models identified candidate sites for both 14-3-3 binding and phosphorylation. In both in vitro experiments and human SH-SY5Y neuronal cells exposed to hypoxia and glucose deprivation, we observed that 1) neuroglobin phosphorylation and protein-protein interactions with 14-3-3 increase during hypoxic and metabolic stress; 2) neuroglobin binding to 14-3-3 stabilizes and increases the half-life of phosphorylation; and 3) phosphorylation increases the open probability of the heme pocket, which increases ligand binding (CO and nitrite) and accelerates the rate of anaerobic nitrite reduction to form NO. These data reveal a series of hypoxia-dependent post-translational modifications to neuroglobin that regulate the six-to-five heme pocket equilibrium and heme access to ligands. Hypoxia-regulated reactions of nitrite and neuroglobin may contribute to the cellular adaptation to hypoxia.

  8. Cross-talk between constitutive androstane receptor and hypoxia-inducible factor in the regulation of gene expression.

    Science.gov (United States)

    Shizu, Ryota; Shindo, Sawako; Yoshida, Takemi; Numazawa, Satoshi

    2013-05-23

    Hypoxia inducible factor (HIF) and 5'-AMP-activated protein kinase are often activated under similar physiological conditions. Constitutive androstane receptor (CAR) translocates into the nucleus in accordance with 5'-AMP-activated protein kinase and thus confers transactivation. The aim of the present study was to investigate a possible link between CAR and HIFα. Phenobarbital (PB), a typical CAR activator, increased the gene expression of HIF-target genes in the livers of mice, including erythropoietin, heme oxygenase-1 and vascular endothelial growth factor-a. PB induced an accumulation of nuclear HIF-1α and an increase in the HIF-responsive element-mediated transactivation in HepG2 cells. Cobalt chloride, a typical HIF activator, induced the gene expression of CAR-target genes, including cyp2b9 and cyp2b10, an accumulation of nuclear CAR and an increase in the PB-responsive enhancer module-mediated transactivation in the mouse liver. Immunoprecipitation-immunoblot and chromatin immunoprecipitation analyses suggest that CAR binds to the PB-responsive enhancer module with HIF-1α in the liver of untreated mice and that the complex dissociates upon PB treatment. Taken together these results suggest that CAR and HIF-α interact and reciprocally modulate the functions of each other. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  9. Translational co-regulation of a ligand and inhibitor by a conserved RNA element

    DEFF Research Database (Denmark)

    Zaucker, Andreas; Nagorska, Agnieszka; Kumari, Pooja

    2017-01-01

    ), whereas deletions in the DLE abolish binding to Ybx1. Analysis of zebrafish ybx1 mutants shows that Ybx1 represses lefty1 translation in embryos. CRISPR/Cas9-mediated inactivation of human YBX1 also results in human NODAL translational de-repression, suggesting broader conservation of the DLE RNA element...

  10. PRMT1-Mediated Translation Regulation Is a Crucial Vulnerability of Cancer.

    Science.gov (United States)

    Hsu, Jessie Hao-Ru; Hubbell-Engler, Benjamin; Adelmant, Guillaume; Huang, Jialiang; Joyce, Cailin E; Vazquez, Francisca; Weir, Barbara A; Montgomery, Philip; Tsherniak, Aviad; Giacomelli, Andrew O; Perry, Jennifer A; Trowbridge, Jennifer; Fujiwara, Yuko; Cowley, Glenn S; Xie, Huafeng; Kim, Woojin; Novina, Carl D; Hahn, William C; Marto, Jarrod A; Orkin, Stuart H

    2017-09-01

    Through an shRNA screen, we identified the protein arginine methyltransferase Prmt1 as a vulnerable intervention point in murine p53/Rb-null osteosarcomas, the human counterpart of which lacks effective therapeutic options. Depletion of Prmt1 in p53-deficient cells impaired tumor initiation and maintenance in vitro and in vivo Mechanistic studies reveal that translation-associated pathways were enriched for Prmt1 downstream targets, implicating Prmt1 in translation control. In particular, loss of Prmt1 led to a decrease in arginine methylation of the translation initiation complex, thereby disrupting its assembly and inhibiting translation. p53/Rb-null cells were sensitive to p53-induced translation stress, and analysis of human cancer cell line data from Project Achilles further revealed that Prmt1 and translation-associated pathways converged on the same functional networks. We propose that targeted therapy against Prmt1 and its associated translation-related pathways offer a mechanistic rationale for treatment of osteosarcomas and other cancers that exhibit dependencies on translation stress response. Cancer Res; 77(17); 4613-25. ©2017 AACR. ©2017 American Association for Cancer Research.

  11. Hypoxia in Tumor Angiogenesis and Metastasis: Evaluation of VEGF and MMP Over-expression and Down-Regulation of HIF-1alpha with RNAi in Hypoxic Tumor Cells

    Science.gov (United States)

    Shah, Shruti

    Background: As tumor mass grows beyond a few millimeters in diameter, the angiogenic "switch" is turned on leading to recruitment of blood vessels from surrounding artery and veins. However, the tumor mass is poorly perfused and there are pockets of hypoxia or lower oxygen concentrations relative to normal tissue. Hypoxia-inducing factor-1a (HIF-1a), a transcription factor, is activated when the oxygen concentration is low. Upon activation of HIF-1a, a number of other genes also turn on that allows the tumor to become more aggressive and resistant to therapy. Purpose: The main objectives of this study were to evaluate the effect of hypoxia-induced HIF-1a followed by over-expression of angiogenic and metastatic markers in tumor cells and down-regulation of HIF-1a using nanoparticle-delivered RNA interference therapy. Methods: Human ovarian (SKOV3) and breast (MDA-MB-231) adenocarcinoma cells were incubated under normoxic and hypoxic conditions. Following hypoxia treatment of the cells, HIF-1α, vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP-2), and MMP-9 expression was analyzed qualitatively and quantitatively. For intracellular delivery of HIF-1a gene silencing small interfering RNA (siRNA), type B gelatin nanoparticles were fabricated using the solvent displacement method and the surface was modified with poly(ethylene glycol) (PEG, Mol. wt. 2kDa). Cellular uptake and distribution of the nanoparticles was observed with Cy3-siRNA loaded, FITC-conjugated gelatin nanoparticles. Cytotoxicity of the nanoparticle formulations was evaluated in both the cell lines. siRNA was transfected in the gelatin nanoparticles under hypoxic conditions. Total cellular protein and RNA were extracted for analysis of HIF1a, VEGF, MMP-2 and MMP-9 expression. Results: MDA-MB-231 and SKOV3 cells show increased expression of HIF1a under hypoxic conditions compared to baseline levels at normoxic conditions. ELISA and western blots of VEGF, MMP-2 and MMP-9 appear to

  12. Myofibrillogenesis regulator-1 attenuated hypoxia/reoxygenation-induced apoptosis by inhibiting the PERK/Nrf2 pathway in neonatal rat cardiomyocytes.

    Science.gov (United States)

    Tao, Tian-Qi; Wang, Xiao-Reng; Liu, Mi; Xu, Fei-Fei; Liu, Xiu-Hua

    2015-03-01

    The purpose of this study was to investigate the role of myofibrillogenesis regulator-1 (MR-1) in cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R), through protein kinase R-like ER kinase (PERK)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. To address this aim, an H/R model of neonatal rat cardiomyocytes was used. MR-1 was overexpressed using an adenoviral vector system and knocked down using MR-1 specific siRNA. Apoptosis was assessed by using Annexin V/PI double staining, terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling assay, and the Bcl-2/Bax ratio. Western blotting was used to detect the protein levels of MR-1, glucose-regulated protein 78 (GRP78), total and phosphorylated PERK, Nrf2, activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), Bcl-2 and Bax. Immunofluorescence staining was used to assess the subcellular location of Nrf2. We found that H/R induced significant apoptosis in neonatal rat cardiomyocytes. MR-1 overexpression attenuated H/R-induced apoptosis, decreased GRP78 (P apoptosis, increased expression of GRP78 and CHOP (P apoptosis through inhibition of the PERK/Nrf2 pathway.

  13. Paradoxical regulation of hypoxia inducible factor-1α (HIF-1α by histone deacetylase inhibitor in diffuse large B-cell lymphoma.

    Directory of Open Access Journals (Sweden)

    Savita Bhalla

    Full Text Available Hypoxia inducible factor (HIF is important in cancer, as it regulates various oncogenic genes as well as genes involved in cell survival, proliferation, and migration. Elevated HIF-1 protein promotes a more aggressive tumor phenotype, and greater HIF-1 expression has been demonstrated to correlate with poorer prognosis, increased risk of metastasis and increased mortality. Recent reports suggest that HIF-1 activates autophagy, a lysosomal degradation pathway which may promote tumor cell survival. We show here that HIF-1α expression is constitutively active in multiple diffuse large B cell lymphoma (DLBCL cell lines under normoxia and it is regulated by the PI3K/AKT pathway. PCI-24781, a pan histone deacetylase inhibitor (HDACI, enhanced accumulation of HIF-1α and induced autophagy initially, while extended incubation with the drug resulted in inhibition of HIF-1α. We tested the hypothesis that PCI-24781- induced autophagy is mediated by HIF-1α and that inhibition of HIF-1α in these cells results in attenuation of autophagy and decreased survival. We also provide evidence that autophagy serves as a survival pathway in DLBCL cells treated with PCI-24781 which suggests that the use of autophagy inhibitors such as chloroquine or 3-methyl adenine in combination with PCI-24781 may enhance apoptosis in lymphoma cells.

  14. Parallel Regulation of von Hippel-Lindau Disease by pVHL-Mediated Degradation of B-Myb and Hypoxia-Inducible Factor α

    Science.gov (United States)

    Uematsu, Keiji; Byrne, Stuart D.; Hirano, Mie; Joo-Okumura, Akiko; Nishikimi, Akihiko; Shuin, Taro; Fukui, Yoshinori; Nakatsukasa, Kunio

    2016-01-01

    pVHL, the protein product of the von Hippel-Lindau (VHL) tumor suppressor gene, is a ubiquitin ligase that targets hypoxia-inducible factor α (HIF-α) for proteasomal degradation. Although HIF-α activation is necessary for VHL disease pathogenesis, constitutive activation of HIF-α alone did not induce renal clear cell carcinomas and pheochromocytomas in mice, suggesting the involvement of an HIF-α-independent pathway in VHL pathogenesis. Here, we show that the transcription factor B-Myb is a pVHL substrate that is degraded via the ubiquitin-proteasome pathway and that vascular endothelial growth factor (VEGF)- and/or platelet-derived growth factor (PDGF)-dependent tyrosine 15 phosphorylation of B-Myb prevents its degradation. Mice injected with B-Myb knockdown 786-O cells developed dramatically larger tumors than those bearing control cell tumors. Microarray screening of B-Myb-regulated genes showed that the expression of HIF-α-dependent genes was not affected by B-Myb knockdown, indicating that B-Myb prevents HIF-α-dependent tumorigenesis through an HIF-α-independent pathway. These data indicate that the regulation of B-Myb by pVHL plays a critical role in VHL disease. PMID:27090638

  15. Identifying intrinsic and extrinsic determinants that regulate internal initiation of translation mediated by the FMR1 5' leader

    Directory of Open Access Journals (Sweden)

    Timmerman Stephanie

    2008-10-01

    Full Text Available Abstract Background Regulating synthesis of the Fragile X gene (FMR1 product, FMRP alters neural plasticity potentially through its role in the microRNA pathway. Cap-dependent translation of the FMR1 mRNA, a process requiring ribosomal scanning through the 5' leader, is likely impeded by the extensive secondary structure generated by the high guanosine/cytosine nucleotide content including the CGG triplet nucleotide repeats in the 5' leader. An alternative mechanism to initiate translation – internal initiation often utilizes secondary structure to recruit the translational machinery. Consequently, studies were undertaken to confirm and extend a previous observation that the FMR1 5' leader contains an internal ribosomal entry site (IRES. Results Cellular transfection of a dicistronic DNA construct containing the FMR1 5' leader inserted into the intercistronic region yielded significant translation of the second cistron, but the FMR1 5' leader was also found to contain a cryptic promoter possibly confounding interpretation of these results. However, transfection of dicistronic and monocistronic RNA ex vivo or in vitro confirmed that the FMR1 5' leader contains an IRES. Moreover, inhibiting cap-dependent translation ex vivo did not affect the expression level of endogenous FMRP indicating a role for IRES-dependent translation of FMR1 mRNA. Analysis of the FMR1 5' leader revealed that the CGG repeats and the 5' end of the leader were vital for internal initiation. Functionally, exposure to potassium chloride or intracellular acidification and addition of polyinosinic:polycytidylic acid as mimics of neural activity and double stranded RNA, respectively, differentially affected FMR1 IRES activity. Conclusion Our results indicate that multiple stimuli influence IRES-dependent translation of the FMR1 mRNA and suggest a functional role for the CGG nucleotide repeats.

  16. Ecosystem impacts of hypoxia: thresholds of hypoxia and pathways to recovery

    Energy Technology Data Exchange (ETDEWEB)

    Steckbauer, A; Duarte, C M; Vaquer-Sunyer, R [Department of Global Change Research, IMEDEA (CSIC-UIB), Institut Mediterrani d' Estudis Avancats, C/Miquel Marques 21, 07190 Esporles (Mallorca), Islas Baleares (Spain); Carstensen, J [Department of Marine Ecology, National Environmental Research Institute, Aarhus University, PO Box 358, DK-4000 Roskilde (Denmark); Conley, D J, E-mail: asteckbauer@imedea.uib-csic.es [Department of Earth and Ecosystem Sciences, Lund University, SE-223 62 Lund (Sweden)

    2011-04-15

    Coastal hypoxia is increasing in the global coastal zone, where it is recognized as a major threat to biota. Managerial efforts to prevent hypoxia and achieve recovery of ecosystems already affected by hypoxia are largely based on nutrient reduction plans. However, these managerial efforts need to be informed by predictions on the thresholds of hypoxia (i.e. the oxygen levels required to conserve biodiversity) as well as the timescales for the recovery of ecosystems already affected by hypoxia. The thresholds for hypoxia in coastal ecosystems are higher than previously thought and are not static, but regulated by local and global processes, being particularly sensitive to warming. The examination of recovery processes in a number of coastal areas managed for reducing nutrient inputs and, thus, hypoxia (Northern Adriatic; Black Sea; Baltic Sea; Delaware Bay; and Danish Coastal Areas) reveals that recovery timescales following the return to normal oxygen conditions are much longer than those of loss following the onset of hypoxia, and typically involve decadal timescales. The extended lag time for ecosystem recovery from hypoxia results in non-linear pathways of recovery due to hysteresis and the shift in baselines, affecting the oxygen thresholds for hypoxia through time.

  17. Eukaryotic initiation factor 3 (eIF3) and 5’ mRNA leader sequences as agents of translational regulation in Arabidopsis. Final report

    Energy Technology Data Exchange (ETDEWEB)

    von Arnim, Albrecht G. [Univ. of Tennessee, Knoxville, TN (United States)

    2015-02-04

    Protein synthesis, or translation, consumes a sizable fraction of the cell’s energy budget, estimated at 5% and up to 50% in differentiated and growing cells, respectively. Plants also invest significant energy and biomass to construct and maintain the translation apparatus. Translation is regulated by a variety of external stimuli. Compared to transcriptional control, attributes of translational control include reduced sensitivity to stochastic fluctuation, a finer gauge of control, and more rapid responsiveness to environmental stimuli. Yet, our murky understanding of translational control allows few generalizations. Consequently, translational regulation is underutilized in the context of transgene regulation, although synthetic biologists are now beginning to appropriate RNA-level gene regulation into their regulatory circuits. We also know little about how translational control contributes to the diversity of plant form and function. This project explored how an emerging regulatory mRNA sequence element, upstream open reading frames (uORFs), is integrated with the general translation initiation machinery to permit translational regulation on specific mRNAs.

  18. Hypoxia-induced miR-15a promotes mesenchymal ablation and adaptation to hypoxia during lung development in chicken.

    Directory of Open Access Journals (Sweden)

    Rui Hao

    Full Text Available The lungs undergo changes that are adaptive for high elevation in certain animal species. In chickens, animals bred at high elevations (e.g., Tibet chickens are better able to hatch and survive under high-altitude conditions. In addition, lowland chicken breeds undergo physiological effects and suffer greater mortality when they are exposed to hypoxic conditions during embryonic development. Although these physiological effects have been noted, the mechanisms that are responsible for hypoxia-induced changes in lung development and function are not known. Here we have examined the role of a particular microRNA (miRNA in the regulation of lung development under hypoxic conditions. When chicks were incubated in low oxygen (hypoxia, miR-15a was significantly increased in embryonic lung tissue. The expression level of miR-15a in hypoxic Tibet chicken embryos increased and remained relatively high at embryonic day (E16-20, whereas in normal chickens, expression increased and peaked at E19-20, at which time the cross-current gas exchange system (CCGS is developing. Bcl-2 was a translationally repressed target of miR-15a in these chickens. miR-16, a cluster and family member of miR-15a, was detected but did not participate in the posttranscriptional regulation of bcl-2. Around E19, the hypoxia-induced decrease in Bcl-2 protein resulted in apoptosis in the mesenchyme around the migrating tubes, which led to an expansion and migration of the tubes that would become the air capillary network and the CCGS. Thus, interfering with miR-15a expression in lung tissue may be a novel therapeutic strategy for hypoxia insults and altitude adaptation.

  19. Hypoxia-Induced miR-15a Promotes Mesenchymal Ablation and Adaptation to Hypoxia during Lung Development in Chicken

    Science.gov (United States)

    Hao, Rui; Hu, Xiaoxiang; Wu, Changxin; Li, Ning

    2014-01-01

    The lungs undergo changes that are adaptive for high elevation in certain animal species. In chickens, animals bred at high elevations (e.g., Tibet chickens) are better able to hatch and survive under high-altitude conditions. In addition, lowland chicken breeds undergo physiological effects and suffer greater mortality when they are exposed to hypoxic conditions during embryonic development. Although these physiological effects have been noted, the mechanisms that are responsible for hypoxia-induced changes in lung development and function are not known. Here we have examined the role of a particular microRNA (miRNA) in the regulation of lung development under hypoxic conditions. When chicks were incubated in low oxygen (hypoxia), miR-15a was significantly increased in embryonic lung tissue. The expression level of miR-15a in hypoxic Tibet chicken embryos increased and remained relatively high at embryonic day (E)16–20, whereas in normal chickens, expression increased and peaked at E19–20, at which time the cross-current gas exchange system (CCGS) is developing. Bcl-2 was a translationally repressed target of miR-15a in these chickens. miR-16, a cluster and family member of miR-15a, was detected but did not participate in the posttranscriptional regulation of bcl-2. Around E19, the hypoxia-induced decrease in Bcl-2 protein resulted in apoptosis in the mesenchyme around the migrating tubes, which led to an expansion and migration of the tubes that would become the air capillary network and the CCGS. Thus, interfering with miR-15a expression in lung tissue may be a novel therapeutic strategy for hypoxia insults and altitude adaptation. PMID:24887070

  20. Regulation of microtubule motors by tubulin isotypes and post-translational modifications

    OpenAIRE

    Vale, Ronald; M. SIRAJUDDIN; Rice, LM; Vale, RD

    2014-01-01

    The 'tubulin-code' hypothesis proposes that different tubulin genes or post-translational modifications (PTMs), which mainly confer variation in the carboxy-terminal tail (CTT), result in unique interactions with microtubule-associated proteins for specifi

  1. PRMT1-Mediated Translation Regulation is a Crucial Vulnerability of Cancer | Office of Cancer Genomics

    Science.gov (United States)

    Through an shRNA screen, we identified the protein arginine methyltransferase Prmt1 as a vulnerable intervention point in murine p53/Rb-null osteosarcomas, the human counterpart of which lacks effective therapeutic options. Depletion of Prmt1 in p53-deficient cells impaired tumor initiation and maintenance in vitro and in vivo Mechanistic studies reveal that translation-associated pathways were enriched for Prmt1 downstream targets, implicating Prmt1 in translation control.

  2. G-quadruplexes regulate Epstein-Barr virus-encoded nuclear antigen 1 mRNA translation.

    Science.gov (United States)

    Murat, Pierre; Zhong, Jie; Lekieffre, Lea; Cowieson, Nathan P; Clancy, Jennifer L; Preiss, Thomas; Balasubramanian, Shankar; Khanna, Rajiv; Tellam, Judy

    2014-05-01

    Viruses that establish latent infections have evolved unique mechanisms to avoid host immune recognition. Maintenance proteins of these viruses regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The mechanisms governing this finely tuned regulation of viral latency are unknown. Here we show that mRNAs encoding gammaherpesviral maintenance proteins contain within their open reading frames clusters of unusual structural elements, G-quadruplexes, which are responsible for the cis-acting regulation of viral mRNA translation. By studying the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) mRNA, we demonstrate that destabilization of G-quadruplexes using antisense oligonucleotides increases EBNA1 mRNA translation. In contrast, pretreatment with a G-quadruplex-stabilizing small molecule, pyridostatin, decreases EBNA1 synthesis, highlighting the importance of G-quadruplexes within virally encoded transcripts as unique regulatory signals for translational control and immune evasion. Furthermore, these findings suggest alternative therapeutic strategies focused on targeting RNA structure within viral ORFs.

  3. Coastal hypoxia responses to remediation

    Science.gov (United States)

    Kemp, W. M.; Testa, J. M.; Conley, D. J.; Gilbert, D.; Hagy, J. D.

    2009-07-01

    The incidence and intensity of hypoxic waters in coastal aquatic ecosystems has been expanding in recent decades coincident with eutrophication of the coastal zone. Because of the negative effects hypoxia has on many organisms, extensive efforts have been made to reduce the size and duration of hypoxia in many coastal waters. Although it has been broadly assumed that reductions in nutrient loading rates would reverse eutrophication and consequently, hypoxia, recent analyses of historical data from European and North American coastal systems suggest little evidence for simple linear response trajectories. We review existing data, analyses, and models that relate variations in the extent and intensity of hypoxia to changes in loading rates for inorganic nutrients and labile organic matter. We also assess existing knowledge of physical and ecological factors regulating oxygen in coastal marine waters and examine a broad range of examples where hypoxia responses to reductions in nutrient (or organic matter) inputs have been documented. Of the 22 systems identified where concurrent time series of loading and O2 were available, half displayed relatively clear and direct recoveries following remediation. We explored in detail 5 well-studied systems that have exhibited complex, non-linear responses to loading, including apparent "regime shifts." A summary of these analyses suggests that O2 conditions improved rapidly and linearly in systems where remediation focused on organic inputs from sewage plants, which were the primary drivers of hypoxia. In larger more open systems where diffuse nutrient loads are more important in fueling O2 depletion and where climatic influences are pronounced, responses to remediation tend to follow non-linear trends that may include hysteresis and time-lags. Improved understanding of hypoxia remediation requires that future studies use comparative approaches and consider multiple regulating factors including: (1) the dominant temporal scales

  4. Nitric oxide signaling in hypoxia.

    Science.gov (United States)

    Ho, J J David; Man, H S Jeffrey; Marsden, Philip A

    2012-03-01

    Endothelial-derived nitric oxide (NO) is classically viewed as a regulator of vasomotor tone. NO plays an important role in regulating O(2) delivery through paracrine control of vasomotor tone locally and cardiovascular and respiratory responses centrally. Very soon after the cloning and functional characterization of the endothelial nitric oxide synthase (eNOS), studies on the interaction between O(2) and NO made the paradoxical finding that hypoxia led to decreases in eNOS expression and function. Why would decreases in O(2) content in tissues elicit a loss of a potent endothelial-derived vasodilator? We now know that restricting our view of NO as a regulator of vasomotor tone or blood pressure limited deeper levels of mechanistic insight. Exciting new studies indicate that functional interactions between NO and O(2) exhibit profound complexity and are relevant to diseases states, especially those associated with hypoxia in tissues. NOS isoforms catalytically require O(2). Hypoxia regulates steady-state expression of the mRNA and protein abundance of the NOS enzymes. Animals genetically deficient in NOS isoforms have perturbations in their ability to adapt to changes in O(2) supply or demand. Most interestingly, the intracellular pathways for O(2) sensing that evolved to ensure an appropriate balance of O(2) delivery and utilization intersect with NO signaling networks. Recent studies demonstrate that hypoxia-inducible factor (HIF) stabilization and transcriptional activity is achieved through two parallel pathways: (1) a decrease in O(2)-dependent prolyl hydroxylation of HIF and (2) S-nitrosylation of HIF pathway components. Recent findings support a role for S-nitrosothiols as hypoxia-mimetics in certain biological and/or disease settings, such as living at high altitude, exposure to small molecules that can bind NO, or anemia.

  5. A novel thiol compound, N-acetylcysteine amide, attenuates allergic airway disease by regulating activation of NF-kappaB and hypoxia-inducible factor-1alpha.

    Science.gov (United States)

    Lee, Kyung Sun; Kim, So Ri; Park, Hee Sun; Park, Seoung Ju; Min, Kyung Hoon; Lee, Ka Young; Choe, Yeong Hun; Hong, Sang Hyun; Han, Hyo Jin; Lee, Young Rae; Kim, Jong Suk; Atlas, Daphne; Lee, Yong Chul

    2007-12-31

    Reactive oxygen species (ROS) play an important role in the pathogenesis of airway inflammation and hyperresponsiveness. Recent studies have demonstrated that antioxidants are able to reduce airway inflammation and hyperreactivity in animal models of allergic airway disease. A newly developed antioxidant, small molecular weight thiol compound, N-acetylcysteine amide (AD4) has been shown to increase cellular levels of glutathione and to attenuate oxidative stress related disorders such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis. However, the effects of AD4 on allergic airway disease such as asthma are unknown. We used ovalbumin (OVA)-inhaled mice to evaluate the role of AD4 in allergic airway disease. In this study with OVA-inhaled mice, the increased ROS generation, the increased levels of Th2 cytokines and VEGF, the increased vascular permeability, the increased mucus production, and the increased airway resistance in the lungs were significantly reduced by the administration of AD4. We also found that the administration of AD4 decreased the increases of the NF-kappaB and hypoxia-inducible factor-1alpha (HIF-1alpha) levels in nuclear protein extracts of lung tissues after OVA inhalation. These results suggest that AD4 attenuates airway inflammation and hyperresponsiveness by regulating activation of NF-kappaB and HIF-1alpha as well as reducing ROS generation in allergic airway disease.

  6. Hypoxia: From Placental Development to Fetal Programming.

    Science.gov (United States)

    Fajersztajn, Lais; Veras, Mariana Matera

    2017-10-16

    Hypoxia may influence normal and different pathological processes. Low oxygenation activates a variety of responses, many of them regulated by hypoxia-inducible factor 1 complex, which is mostly involved in cellular control of O 2 consumption and delivery, inhibition of growth and development, and promotion of anaerobic metabolism. Hypoxia plays a significant physiological role in fetal development; it is involved in different embryonic processes, for example, placentation, angiogenesis, and hematopoiesis. More recently, fetal hypoxia has been associated directly or indirectly with fetal programming of heart, brain, and kidney function and metabolism in adulthood. In this review, the role of hypoxia in fetal development, placentation, and fetal programming is summarized. Hypoxia is a basic mechanism involved in different pregnancy disorders and fetal health developmental complications. Although there are scientific data showing that hypoxia mediates changes in the growth trajectory of the fetus, modulates gene expression by epigenetic mechanisms, and determines the health status later in adulthood, more mechanistic studies are needed. Furthermore, if we consider that intrauterine hypoxia is not a rare event, and can be a consequence of unavoidable exposures to air pollution, nutritional deficiencies, obesity, and other very common conditions (drug addiction and stress), the health of future generations may be damaged and the incidence of some diseases will markedly increase as a consequence of disturbed fetal programming. Birth Defects Research 109:1377-1385, 2017.© 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  7. Successful induction of sclerostin in human-derived fibroblasts by 4 transcription factors and its regulation by parathyroid hormone, hypoxia, and prostaglandin E2.

    Science.gov (United States)

    Fujiwara, Makoto; Kubota, Takuo; Wang, Wei; Ohata, Yasuhisa; Miura, Kohji; Kitaoka, Taichi; Okuzaki, Daisuke; Namba, Noriyuki; Michigami, Toshimi; Kitabatake, Yasuji; Ozono, Keiichi

    2016-04-01

    Sclerostin, coded by SOST, is a secretory protein that is specifically expressed in osteocytes and suppresses osteogenesis by inhibiting WNT signaling. The regulatory mechanism underlying SOST expression remains unclear mainly due to the absence of an adequate human cell model. Thus, we herein attempted to establish a cell model of human dermal fibroblasts in order to investigate the functions of sclerostin. We selected 20 candidate transcription factors (TFs) that induce SOST expression by analyzing gene expression patterns in the human sarcoma cell line, SaOS-2, between differentiation and maintenance cultures using microarrays. An effective set of TFs to induce SOST expression was sought by their viral transduction into fibroblasts, and a combination of four TFs: ATF3, KLF4, PAX4, and SP7, was identified as the most effective inducer of SOST expression. Quantitative PCR demonstrated that the expression levels of SOST in fibroblasts treated with the 4 TFs were 199- and 1439-fold higher than those of the control after 1-week and 4-week cultures, respectively. The level of sclerostin in the conditioned medium, as determined by ELISA, was 21.2pmol/l 4weeks after the transduction of the 4 TFs. Interestingly, the production of Dickkopf1 (DKK1), another secreted inhibitor of WNT signaling, was also increased by transduction of these 4 TFs. Parathyroid hormone (PTH) significantly suppressed the induced SOST by 38% and sclerostin by 82% that of the vehicle. Hypoxia increased the induced SOST by 62% that of normoxia. Furthermore, prostaglandin E2 (PGE2) increased SOST expression levels to 16-fold those of the vehicle. In conclusion, the efficient induction of SOST expression and sclerostin production was achieved in human dermal fibroblasts by the transduction of ATF3, KLF4, PAX4, and SP7, and the induced SOST and sclerostin were regulated by PTH, hypoxia, and PGE2. This model may contribute to elucidating the regulatory mechanisms underlying SOST expression and advancing

  8. Hypoxia-Inducible Lipid Droplet-Associated Is Not a Direct Physiological Regulator of Lipolysis in Adipose Tissue

    DEFF Research Database (Denmark)

    Dijk, Wieneke; Mattijssen, Frits; de la Rosa Rodriguez, Montserrat

    2017-01-01

    , where its levels are induced by peroxisome proliferator-activated receptor γ and β-adrenergic stimulation. In contrast to the reported inhibition of lipolysis by HILPDA in hepatocytes, our data do not support an important direct role of HILPDA in the regulation of lipolysis in adipocytes in vivo...

  9. Post-translational modifications in regulation of pathogen surveillance and signaling in plants: The inside- (and perturbations from) outside story.

    Science.gov (United States)

    Bhattacharjee, Saikat; Noor, Jewel Jameeta; Gohain, Bornali; Gulabani, Hitika; Dnyaneshwar, Ingole Kishor; Singla, Ankit

    2015-07-01

    In its lifetime a plant is exposed to pathogens of diverse types. Although methods of surveillance are broadly pathogen-individualized, immune signaling ultimately connect to common core networks maintained by key protein hubs. Defense elicitations modulate these hubs to re-allocate energy from central metabolic pathway into processes that execute immunity. Because unregulated defenses severely decrease growth and productivity of the host, signaling regulators within the networks function to achieve cellular equilibrium once the threat is minimized. Protein modifications by post-translational processes regulate the molecular switches and crosstalks between interconnected pathways spatially and temporally. Covalent modification of host targets connected to hubs are strategies used by most virulent effectors and result in re-routing signals to suppress host defenses. Resistance is a result of activation of specialized classes of receptors that short-circuit effector activities by co-localizing via post-translational modifications (PTMs) with effector targets. Despite advancement in proteome methodologies, our understanding of how PTMs regulate plant defenses remains elusive. This review presents protein-modifications as forefront regulators of plant innate immunity. © 2015 International Union of Biochemistry and Molecular Biology.

  10. SRSF3 represses the expression of PDCD4 protein by coordinated regulation of alternative splicing, export and translation

    Energy Technology Data Exchange (ETDEWEB)

    Park, Seung Kuk; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

    2016-02-05

    Gene expression is regulated at multiple steps, such as transcription, splicing, export, degradation and translation. Considering diverse roles of SR proteins, we determined whether the tumor-related splicing factor SRSF3 regulates the expression of the tumor-suppressor protein, PDCD4, at multiple steps. As we have reported previously, knockdown of SRSF3 increased the PDCD4 protein level in SW480 colon cancer cells. More interestingly, here we showed that the alternative splicing and the nuclear export of minor isoforms of pdcd4 mRNA were repressed by SRSF3, but the translation step was unaffected. In contrast, only the translation step of the major isoform of pdcd4 mRNA was repressed by SRSF3. Therefore, overexpression of SRSF3 might be relevant to the repression of all isoforms of PDCD4 protein levels in most types of cancer cell. We propose that SRSF3 could act as a coordinator of the expression of PDCD4 protein via two mechanisms on two alternatively spliced mRNA isoforms.

  11. Sodium arsenite down-regulates the expression of X-linked inhibitor of apoptosis protein via translational and post-translational mechanisms in hepatocellular carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Hong; Hao, Yuqing; Wang, Lijing; Jia, Dongwei [Gene Research Center, Shanghai Medical College, Fudan University, Shanghai 200032 (China); Ruan, Yuanyuan, E-mail: yuanyuanruan@fudan.edu.cn [Gene Research Center, Shanghai Medical College, Fudan University, Shanghai 200032 (China); Gu, Jianxin [Gene Research Center, Shanghai Medical College, Fudan University, Shanghai 200032 (China)

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Sodium arsenite down-regulates the protein expression level of XIAP in HCC. Black-Right-Pointing-Pointer Sodium arsenite inhibits the de novo XIAP synthesis and its IRES activity. Black-Right-Pointing-Pointer Sodium arsenite decreases XIAP stability and promotes its proteasomal degradation. Black-Right-Pointing-Pointer Overexpression of XIAP attenuates the pro-apoptotic effect of sodium arsenite. -- Abstract: X-linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitors of apoptosis protein (IAP) family, and has been reported to exhibit elevated expression levels in hepatocellular carcinoma (HCC) and promote cell survival, metastasis and tumor recurrence. Targeting XIAP has proven effective for the inhibition of cancer cell proliferation and restoration of cancer cell chemosensitivity. Arsenic (or sodium arsenite) is a potent anti-tumor agent used to treat patients with acute promyelocytic leukemia (APL). Additionally, arsenic induces cell growth inhibition, cell cycle arrest and apoptosis in human HCC cells. In this study, we identified XIAP as a target for sodium arsenite-induced cytotoxicity in HCC. The exposure of HCC cell lines to sodium arsenite resulted in inhibition of XIAP expression in both a dose- and time-dependent manner. Sodium arsenite blocked the de novo XIAP synthesis and the activity of its internal ribosome entry site (IRES) element. Moreover, treatment with sodium arsenite decreased the protein stability of XIAP and induced its ubiquitin-proteasomal degradation. Overexpression of XIAP attenuated the pro-apoptotic effect of sodium arsenite in HCC. Taken together, our data demonstrate that sodium arsenite suppresses XIAP expression via translational and post-translational mechanisms in HCC.

  12. Mechanisms of Neuroprotection from Hypoxia-Ischemia (HI) Brain Injury by Up-regulation of Cytoglobin (CYGB) in a Neonatal Rat Model*

    Science.gov (United States)

    Tian, Shu-Feng; Yang, Han-Hua; Xiao, Dan-Ping; Huang, Yue-Jun; He, Gu-Yu; Ma, Hai-Ran; Xia, Fang; Shi, Xue-Chuan

    2013-01-01

    This study was designed to investigate the expression profile of CYGB, its potential neuroprotective function, and underlying molecular mechanisms using a model of neonatal hypoxia-ischemia (HI) brain injury. Cygb mRNA and protein expression were evaluated within the first 36 h after the HI model was induced using RT-PCR and Western blotting. Cygb mRNA expression was increased at 18 h in a time-dependent manner, and its level of protein expression increased progressively in 24 h. To verify the neuroprotective effect of CYGB, a gene transfection technique was employed. Cygb cDNA and shRNA delivery adenovirus systems were established (Cygb-cDNA-ADV and Cygb-shRNA-ADV, respectively) and injected into the brains of 3-day-old rats 4 days before they were induced with HI treatment. Rats from different groups were euthanized 24 h post-HI, and brain samples were harvested. 2,3,5-Triphenyltetrazolium chloride, TUNEL, and Nissl staining indicated that an up-regulation of CYGB resulted in reduced acute brain injury. The superoxide dismutase level was found to be dependent on expression of CYGB. The Morris water maze test in 28-day-old rats demonstrated that CYGB expression was associated with improvement of long term cognitive impairment. Studies also demonstrated that CYGB can up-regulate mRNA and protein levels of VEGF and increase both the density and diameter of the microvessels but inhibits activation of caspase-2 and -3. Thus, this is the first in vivo study focusing on the neuroprotective role of CYGB. The reduction of neonatal HI injury by CYGB may be due in part to antioxidant and antiapoptotic mechanisms and by promoting angiogenesis. PMID:23585565

  13. Polyamine-regulated translation of spermidine/spermine-N1-acetyltransferase.

    Science.gov (United States)

    Perez-Leal, Oscar; Barrero, Carlos A; Clarkson, Allen B; Casero, Robert A; Merali, Salim

    2012-04-01

    Rapid synthesis of the polyamine catabolic enzyme spermidine/spermine-N(1)-acetyltransferase (SSAT) in response to increased polyamines is an important polyamine homeostatic mechanism. Indirect evidence has suggested that there is an important control mechanism involving the release of a translational repressor protein that allows the immediate initiation of SSAT protein synthesis without RNA transcription, maturation, or translocation. To identify a repressor protein, we used a mass spectroscopy-based RNA-protein interaction system and found six proteins that bind to the coding region of SSAT mRNA. Individual small interfering RNA (siRNA) experiments showed that nucleolin knockdown enhances SSAT translation. Nucleolin exists in several isoforms, and we report that the isoform that binds to SSAT mRNA undergoes autocatalysis in the presence of polyamines, a result suggesting that there is a negative feedback system that helps control the cellular content of polyamines. Preliminary molecular interaction data show that a nucleolin isoform binds to a 5' stem-loop of the coding region of SSAT mRNA. The glycine/arginine-rich C terminus of nucleolin is required for binding, and the four RNA recognition motif domains are included in the isoform that blocks SSAT translation. Understanding SSAT translational control mechanisms has the potential for the development of therapeutic strategies against cancer and obesity.

  14. Investigating the Regulation and Potential Role of Nonhypoxic Hypoxia Inducible Factor 1 (HIF 1) in Aromatase Inhibitor Resistant Breast Cancer

    Science.gov (United States)

    2015-12-01

    regulation 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSONUSAMRMC a. REPORT...significantly decrease the number of mammospheres nor their morphology as compared to untreated cells. Thus, PX-478 did not significantly alter the cancer...expression with drug resistance in different cancer cell types, including chronic myeloid leukemia cells (Zhao et al. 2010), gastric cancer cells (Liu et

  15. Leucine Differentially Regulates Gene-Specific Translation in Mouse Skeletal Muscle.

    Science.gov (United States)

    Drummond, Micah J; Reidy, Paul T; Baird, Lisa M; Dalley, Brian K; Howard, Michael T

    2017-09-01

    Background: Amino acids, especially leucine, are particularly effective in promoting protein synthesis. Leucine is known to increase the rate of protein synthesis in skeletal muscle through the mechanistic target of rapamycin complex 1-dependent, as well as -independent, signaling pathways. However, the overall translation program is poorly defined, and it is unknown how the activation of these pathways differentially controls the translation of specific mRNAs.Objective: Ribosome profiling and RNA sequencing were used to precisely define the translational program activated by an acute oral dose of leucine.Methods: Adult male C57BL/6 mice were deprived of food overnight before the delivery of an acute dose of l-leucine (9.4 mg) (n = 6) or vehicle (n = 5) and tissues collected 30 min later. Ribosome footprints and total RNA were isolated and subjected to deep sequencing. Changes in gene-specific mRNA abundance and ribosome occupancy were determined between the leucine-treated and control groups by aligning sequence reads to Reference Sequence database mRNAs and applying statistical features of the Bioconductor package edgeR.Results: Our data revealed mRNA features that confer translational control of skeletal muscle mRNAs in response to an acute dose of leucine. The subset of skeletal muscle mRNAs that are activated consists largely of terminal oligopyrimidine mRNAs (false discovery rate: translation had 5' untranslated regions with increased length. Only the small nuclear RNAs, which are required for ribosome biogenesis, were significantly altered in RNA abundance. The inferred functional translational program activated by dietary leucine includes increased protein synthesis capacity and energy metabolism, upregulation of sarcomere-binding proteins, modulation of circadian rhythm, and suppression of select immune components.Conclusions: These results clarify the translation program acutely stimulated by leucine in mouse skeletal muscle and establish new

  16. Hypoxia tolerance, nitric oxide, and nitrite

    DEFF Research Database (Denmark)

    Fago, Angela; Jensen, Frank Bo

    2015-01-01

    survival resides in concerted physiological responses, including strong metabolic depression, protection against oxidative damage and – in air breathing animals - redistribution of blood flow. Each of these responses is known to be tightly regulated by nitric oxide (NO) and during hypoxia by its metabolite...... of NO and nitrite signaling in the adaptive response to hypoxia in vertebrate animals.......Among vertebrates able to tolerate periods of oxygen deprivation, the painted and red-eared slider turtles (Chrysemys picta and Trachemys scripta) and the crucian carp (Carassius carassius) are the most extreme and can survive even months of total lack of oxygen during winter. The key to hypoxia...

  17. Intra-axonal synthesis of eukaryotic translation initiation factors regulates local protein synthesis and axon growth in rat sympathetic neurons.

    Science.gov (United States)

    Kar, Amar N; MacGibeny, Margaret A; Gervasi, Noreen M; Gioio, Anthony E; Kaplan, Barry B

    2013-04-24

    Axonal protein synthesis is a complex process involving selective mRNA localization and translational regulation. In this study, using in situ hybridization and metabolic labeling, we show that the mRNAs encoding eukaryotic translation initiation factors eIF2B2 and eIF4G2 are present in the axons of rat sympathetic neurons and are locally translated. We also report that a noncoding microRNA, miR16, modulates the axonal expression of eIF2B2 and eIF4G2. Transfection of axons with precursor miR16 and anti-miR16 showed that local miR16 levels modulated axonal eIF2B2 and eIF4G2 mRNA and protein levels, as well as axon outgrowth. siRNA-mediated knock-down of axonal eIF2B2 and eIF4G2 mRNA also resulted in a significant decrease in axonal eIF2B2 and eIF4G2 protein. Moreover, results of metabolic labeling studies showed that downregulation of axonal eIF2B2 and eIF4G2 expression also inhibited local protein synthesis and axon growth. Together, these data provide evidence that miR16 mediates axonal growth, at least in part, by regulating the local protein synthesis of eukaryotic translation initiation factors eIF2B2 and eIF4G2 in the axon.

  18. Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation.

    Science.gov (United States)

    Zhang, Hua; Song, Lei; Cong, Haolong; Tien, Po

    2015-10-01

    Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5' untranslated region (5'UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation. The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially

  19. Cysteine-rich 61-connective tissue growth factor-nephroblastoma-overexpressed 5 (CCN5)/Wnt-1-induced signaling protein-2 (WISP-2) regulates microRNA-10b via hypoxia-inducible factor-1α-TWIST signaling networks in human breast cancer cells.

    Science.gov (United States)

    Haque, Inamul; Banerjee, Snigdha; Mehta, Smita; De, Archana; Majumder, Monami; Mayo, Matthew S; Kambhampati, Suman; Campbell, Donald R; Banerjee, Sushanta K

    2011-12-16

    MicroRNAs (miRNAs) are naturally occurring single-stranded RNA molecules that post-transcriptionally regulate the expression of target mRNA transcripts. Many of these target mRNA transcripts are involved in regulating processes commonly altered during tumorigenesis and metastatic growth. These include cell proliferation, differentiation, apoptosis, migration, and invasion. Among the several miRNAs, miRNA-10b (miR-10b) expression is increased in metastatic breast cancer cells and positively regulates cell migration and invasion through the suppression of the homeobox D10 (HOXD10) tumor suppressor signaling pathway. In breast metastatic cells, miR-10b expression is enhanced by a transcription factor TWIST1. We find that miR-10b expression in breast cancer cells can be suppressed by CCN5, and this CCN5 effect is mediated through the inhibition of TWIST1 expression. Moreover, CCN5-induced inhibition of TWIST1 expression is mediated through the translational inhibition/modification of hypoxia-inducible factor-1α via impeding JNK signaling pathway. Collectively, these studies suggest a novel regulatory pathway exists through which CCN5 exerts its anti-invasive function. On the basis of these findings, it is plausible that reactivation of CCN5 in miR-10b-positive invasive/metastatic breast cancers alone or in combination with current therapeutic regimens could provide a unique, alternative strategy to existing breast cancer therapy.

  20. Conceptualising Multilevel Regulation in the EU: A Legal Translation of Multilevel Governance?

    NARCIS (Netherlands)

    Chowdhury, Nupur; Wessel, Ramses A.

    2012-01-01

    How should we conceive of regulation in the European context? This paper attempts to answer this by developing multilevel regulation as a theoretical concept. The basic aim of the paper is to explore the difference and convergence between regulation and governance and develop multilevel governance

  1. Effects of prolonged exposure to hypobaric hypoxia on oxidative stress, inflammation and gluco-insular regulation: the not-so-sweet price for good regulation.

    Directory of Open Access Journals (Sweden)

    Mario Siervo

    Full Text Available The mechanisms by which low oxygen availability are associated with the development of insulin resistance remain obscure. We thus investigated the relationship between such gluco-insular derangements in response to sustained (hypobaric hypoxemia, and changes in biomarkers of oxidative stress, inflammation and counter-regulatory hormone responses.After baseline testing in London (75 m, 24 subjects ascended from Kathmandu (1,300 m to Everest Base Camp (EBC;5,300 m over 13 days. Of these, 14 ascended higher, with 8 reaching the summit (8,848 m. Assessments were conducted at baseline, during ascent to EBC, and 1, 6 and 8 week(s thereafter. Changes in body weight and indices of gluco-insular control were measured (glucose, insulin, C-Peptide, homeostasis model assessment of insulin resistance [HOMA-IR] along with biomarkers of oxidative stress (4-hydroxy-2-nonenal-HNE, inflammation (Interleukin-6 [IL-6] and counter-regulatory hormones (glucagon, adrenalin, noradrenalin. In addition, peripheral oxygen saturation (SpO2 and venous blood lactate concentrations were determined.SpO2 fell significantly from 98.0% at sea level to 82.0% on arrival at 5,300 m. Whilst glucose levels remained stable, insulin and C-Peptide concentrations increased by >200% during the last 2 weeks. Increases in fasting insulin, HOMA-IR and glucagon correlated with increases in markers of oxidative stress (4-HNE and inflammation (IL-6. Lactate levels progressively increased during ascent and remained significantly elevated until week 8. Subjects lost on average 7.3 kg in body weight.Sustained hypoxemia is associated with insulin resistance, whose magnitude correlates with the degree of oxidative stress and inflammation. The role of 4-HNE and IL-6 as key players in modifying the association between sustained hypoxia and insulin resistance merits further investigation.

  2. A Gene Expressed during Sexual and Asexual Sporulation in Phytophthora infestans is a Member of the Puf Family of Translational Regulators

    DEFF Research Database (Denmark)

    Cvitanich, Cristina; Judelson, Howard S.

    2003-01-01

    of the Puf family of translational regulators. The protein showed up to 51% amino acid identity to other Puf proteins within its 353-amino-acid RNA-binding domain. Little similarity extended beyond this region, as noted for other members of the family. Expression of M90 was measured by using RNA blots....... Potential roles for a translational regulator during both sexual development and asexual sporulation are discussed....

  3. Up-Regulation of Antioxidant Proteins in the Plasma Proteome during Saturation Diving: Unique Coincidence under Hypobaric Hypoxia.

    Directory of Open Access Journals (Sweden)

    Hideharu Domoto

    Full Text Available Saturation diving (SD is one of the safest techniques for tolerating hyperbaric conditions for long durations. However, the changes in the human plasma protein profile that occur during SD are unknown. To identify differential protein expression during or after SD, 65 blood samples from 15 healthy Japanese men trained in SD were analyzed by two-dimensional fluorescence difference gel electrophoresis. The expression of two proteins, one 32.4 kDa with an isoelectric point (pI of 5.8 and the other 44.8 kDa with pI 4.0, were elevated during SD to 60, 100, and 200 meters sea water (msw. The expression of these proteins returned to pre-diving level when the SD training was completed. The two proteins were identified using in-gel digestion and mass spectrometric analysis; the 32.4 kDa protein was transthyretin and the 44.8 kDa protein was alpha-1-acid glycoprotein 1. Oxidation was detected at methionine 13 of transthyretin and at methionine 129 of alpha-1-acid glycoprotein 1 by tandem mass spectrometry. Moreover, haptoglobin was up-regulated during the decompression phase of 200 msw. These plasma proteins up-regulated during SD have a common function as anti-oxidants. This suggests that by coordinating their biological effects, these proteins activate a defense mechanism to counteract the effects of hyperbaric-hyperoxic conditions during SD.

  4. Regulation of hypoxia-inducible factor-1α (HIF-1α expression by interleukin-1β (IL-1 β, insulin-like growth factors I (IGF-I and II (IGF-II in human osteoarthritic chondrocytes

    Directory of Open Access Journals (Sweden)

    Angelica Rossi Sartori-Cintra

    2012-01-01

    Full Text Available OBJECTIVE: Hypoxia-inducible factor 1 alpha regulates genes related to cellular survival under hypoxia. This factor is present in osteroarthritic chondrocytes, and cytokines, such as interleukin-1 beta, participate in the pathogenesis of osteoarthritis, thereby increasing the activities of proteolytic enzymes, such as matrix metalloproteinases, and accelerating cartilage destruction. We hypothesize that Hypoxia Inducible Factor-1 alpha (HIF-1α can regulate cytokines (catabolic action and/or growth factors (anabolic action in osteoarthritis. The purpose of this study was to investigate the modulation of HIF-1α in human osteoarthritic chondrocytes by interleukin-1 beta (IL-1β and insulin-like growth factors I (IGF-I and II (IGF-II and to determine the involvement of the phosphatidylinositol-3kinase (PI-3K pathway in this process. METHODS: Human osteroarthritic chondrocytes were stimulated with IL-1β, IGF-I and IGF-II and LY294002, a specific inhibitor of PI-3K. Nuclear protein levels and gene expression were analyzed by western blot and quantitative reverse transcription-polymerase chain reaction analyses, respectively. RESULTS: HIF-1α expression was upregulated by IL-1β at the protein level but not at the gene level. IGF-I treatment resulted in increases in both the protein and mRNA levels of HIF-1α , whereas IGF-II had no effect on its expression. However, all of these stimuli exploited the PI-3K pathway. CONCLUSION: IL-1β upregulated the levels of HIF-1α protein post-transcriptionally, whereas IGF-I increased HIF-1α at the transcript level. In contrast, IGF-II did not affect the protein or gene expression levels of HIF-1α . Furthermore, all of the tested stimuli exploited the PI-3K pathway to some degree. Based on these findings, we are able to suggest that Hypoxia inducible Factor-1 exhibits protective activity in chondrocytes during osteoarthritis.

  5. Post-translational regulation of macrophage migration inhibitory factor: Basis for functional fine-tuning

    Directory of Open Access Journals (Sweden)

    Lisa Schindler

    2018-05-01

    Full Text Available Macrophage migration inhibitory factor (MIF is a chemokine-like protein and an important mediator in the inflammatory response. Unlike most other pro-inflammatory cytokines, a number of cell types constitutively express MIF and secretion occurs from preformed stores. MIF is an evolutionarily conserved protein that shows a remarkable functional diversity, including specific binding to surface CD74 and chemokine receptors and the presence of two intrinsic tautomerase and oxidoreductase activities. Several studies have shown that MIF is subject to post-translational modification, particularly redox-dependent modification of the catalytic proline and cysteine residues. In this review, we summarize and discuss MIF post-translational modifications and their effects on the biological properties of this protein. We propose that the redox-sensitive residues in MIF will be modified at sites of inflammation and that this will add further depth to the functional diversity of this intriguing cytokine.

  6. A Myeloid Hypoxia-inducible Factor 1α-Krüppel-like Factor 2 Pathway Regulates Gram-positive Endotoxin-mediated Sepsis*

    Science.gov (United States)

    Mahabeleshwar, Ganapati H.; Qureshi, Muhammad Awais; Takami, Yoichi; Sharma, Nikunj; Lingrel, Jerry B.; Jain, Mukesh K.

    2012-01-01

    Although Gram-positive infections account for the majority of cases of sepsis, the molecular mechanisms underlying their effects remains poorly understood. We investigated how cell wall components of Gram-positive bacteria contribute to the development of sepsis. Experimental observations derived from cultured primary macrophages and the cell line indicate that Gram-positive bacterial endotoxins induce hypoxia-inducible factor 1α (HIF-1α) mRNA and protein expression. Inoculation of live or heat-inactivated Gram-positive bacteria with macrophages induced HIF-1 transcriptional activity in macrophages. Concordant with these results, myeloid deficiency of HIF-1α attenuated Gram-positive bacterial endotoxin-induced cellular motility and proinflammatory gene expression in macrophages. Conversely, Gram-positive bacteria and their endotoxins reduced expression of the myeloid anti-inflammatory transcription factor Krüppel-like transcription factor 2 (KLF2). Sustained expression of KLF2 reduced and deficiency of KLF2 enhanced Gram-positive endotoxins induced HIF-1α mRNA and protein expression in macrophages. More importantly, KLF2 attenuated Gram-positive endotoxins induced cellular motility and proinflammatory gene expression in myeloid cells. Consistent with these results, mice deficient in myeloid HIF-1α were protected from Gram-positive endotoxin-induced sepsis mortality and clinical symptomatology. By contrast, myeloid KLF2-deficient mice were susceptible to Gram-positive sepsis induced mortality and clinical symptoms. Collectively, these observations identify HIF-1α and KLF2 as critical regulators of Gram-positive endotoxin-mediated sepsis. PMID:22110137

  7. Crystal Structure of the CTP1L Endolysin Reveals How Its Activity Is Regulated by a Secondary Translation Product.

    Science.gov (United States)

    Dunne, Matthew; Leicht, Stefan; Krichel, Boris; Mertens, Haydyn D T; Thompson, Andrew; Krijgsveld, Jeroen; Svergun, Dmitri I; Gómez-Torres, Natalia; Garde, Sonia; Uetrecht, Charlotte; Narbad, Arjan; Mayer, Melinda J; Meijers, Rob

    2016-03-04

    Bacteriophages produce endolysins, which lyse the bacterial host cell to release newly produced virions. The timing of lysis is regulated and is thought to involve the activation of a molecular switch. We present a crystal structure of the activated endolysin CTP1L that targets Clostridium tyrobutyricum, consisting of a complex between the full-length protein and an N-terminally truncated C-terminal cell wall binding domain (CBD). The truncated CBD is produced through an internal translation start site within the endolysin gene. Mutants affecting the internal translation site change the oligomeric state of the endolysin and reduce lytic activity. The activity can be modulated by reconstitution of the full-length endolysin-CBD complex with free CBD. The same oligomerization mechanism applies to the CD27L endolysin that targets Clostridium difficile and the CS74L endolysin that targets Clostridium sporogenes. When the CTP1L endolysin gene is introduced into the commensal bacterium Lactococcus lactis, the truncated CBD is also produced, showing that the alternative start codon can be used in other bacterial species. The identification of a translational switch affecting oligomerization presented here has implications for the design of effective endolysins for the treatment of bacterial infections. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Crystal Structure of the CTP1L Endolysin Reveals How Its Activity Is Regulated by a Secondary Translation Product*

    Science.gov (United States)

    Dunne, Matthew; Leicht, Stefan; Krichel, Boris; Thompson, Andrew; Gómez-Torres, Natalia; Garde, Sonia; Narbad, Arjan; Mayer, Melinda J.

    2016-01-01

    Bacteriophages produce endolysins, which lyse the bacterial host cell to release newly produced virions. The timing of lysis is regulated and is thought to involve the activation of a molecular switch. We present a crystal structure of the activated endolysin CTP1L that targets Clostridium tyrobutyricum, consisting of a complex between the full-length protein and an N-terminally truncated C-terminal cell wall binding domain (CBD). The truncated CBD is produced through an internal translation start site within the endolysin gene. Mutants affecting the internal translation site change the oligomeric state of the endolysin and reduce lytic activity. The activity can be modulated by reconstitution of the full-length endolysin-CBD complex with free CBD. The same oligomerization mechanism applies to the CD27L endolysin that targets Clostridium difficile and the CS74L endolysin that targets Clostridium sporogenes. When the CTP1L endolysin gene is introduced into the commensal bacterium Lactococcus lactis, the truncated CBD is also produced, showing that the alternative start codon can be used in other bacterial species. The identification of a translational switch affecting oligomerization presented here has implications for the design of effective endolysins for the treatment of bacterial infections. PMID:26683375

  9. Translational regulation of viral secretory proteins by the 5' coding regions and a viral RNA-binding protein.

    Science.gov (United States)

    Nordholm, Johan; Petitou, Jeanne; Östbye, Henrik; da Silva, Diogo V; Dou, Dan; Wang, Hao; Daniels, Robert

    2017-08-07

    A primary function of 5' regions in many secretory protein mRNAs is to encode an endoplasmic reticulum (ER) targeting sequence. In this study, we show how the regions coding for the ER-targeting sequences of the influenza glycoproteins NA and HA also function as translational regulatory elements that are controlled by the viral RNA-binding protein (RBP) NS1. The translational increase depends on the nucleotide composition and 5' positioning of the ER-targeting sequence coding regions and is facilitated by the RNA-binding domain of NS1, which can associate with ER membranes. Inserting the ER-targeting sequence coding region of NA into different 5' UTRs confirmed that NS1 can promote the translation of secretory protein mRNAs based on the nucleotides within this region rather than the resulting amino acids. By analyzing human protein mRNA sequences, we found evidence that this mechanism of using 5' coding regions and particular RBPs to achieve gene-specific regulation may extend to human-secreted proteins. © 2017 Nordholm et al.

  10. Regulation and role of post-translational modifications of enhancer of zeste homologue 2 in cancer development.

    Science.gov (United States)

    Lu, Haiqi; Li, Guangliang; Zhou, Chenyi; Jin, Wei; Qian, Xiaoling; Wang, Zhuo; Pan, Hongming; Jin, Hongchuan; Wang, Xian

    2016-01-01

    Post-translational modifications (PTMs) are critical molecular events which alter protein conformation after their synthesis and diversity protein properties by modulating their stability, localization, interacting partners or the activity of their substrates, consequently exerting pivotal roles in regulating the functions of many important eukaryotic proteins. It has been well acknowledged that PTMs are of great importance in a broad range of biological processes such as gene regulation, cell proliferation, differentiation and apoptosis, tissue development, diseases, tumor progression and drug resistance. As the core and contributing catalytic subunit of Polycomb repressive complex 2(PRC2), Enhancer of zeste homolog 2 (EZH2) is a master epigenetic regulator, often serving as a highly conserved histone methyltransferase (HMTase) to induce histone H3 lysine 27 trimethylation (H3K27me3) and repress gene transcription and expression. Dysregulated EZH2 expression is frequently associated with cancer development and poor prognosis in a wide variety of cancers. Considered its essential role in carcinogenesis, EZH2 is a potential candidate for cancer targeted therapy. Remarkably, mounting evidence highlights that EZH2 expression, activity and stability can be regulated by PTMs including phosphorylation, acetylation, ubiquitination, sumoylation and GlcNAcylation aside from its well-validated modifications in transcriptional and post-transcriptional levels. However, the precise regulatory mechanisms underlying EZH2 PTMs and whether other types of PTMs orchestrate in EZH2 remain largely unclear. In this review, we summarize current advances in the understanding of EZH2 regulation by PTMs and their associated biological functions during tumorigenesis.

  11. The Mycobacterium tuberculosis Clp gene regulator is required for in vitro reactivation from hypoxia-induced dormancy.

    Science.gov (United States)

    McGillivray, Amanda; Golden, Nadia A; Kaushal, Deepak

    2015-01-23

    Mycobacterium tuberculosis (Mtb) is the leading cause of death from an infectious disease worldwide and is the causative agent of tuberculosis (Chao, M. C., and Rubin, E. J. (2010) Annu. Rev. Microbiol. 64, 293-311). Throughout infection, Mtb encounters a variety of host pressures. Thus, responding to these host stresses via the induction of multiple regulatory networks is needed for survival within the host. The Clp protease gene regulator, Rv2745c (clgR), is induced in response to environmental stress conditions, implicating its potential role in Mtb pathogenesis. Transcriptional activation of genes downstream of Rv2745c occurs in a condition-dependent manner. Our isogenic Mtb:ΔRv2745c mutant expresses a significantly different phenotype upon reaeration conditions. Transcriptional analysis revealed differential gene expression profiles relative to wild-type Mtb. Rv2745c is strongly induced in response to hypoxic and reaeration conditions, implicating a role of Rv2745c in vivo during both establishment of infection and reactivation. We found dysregulation of downstream genes within both the σ(H)/σ(E) regulon as well as the dosR regulon in the isogenic mutant, Mtb:ΔRv2745c. Upon hypoxic and reaeration conditions, Clp protease induction occurred within wild-type Mtb, indicating that activation of clgR, which subsequently leads to Clp protease induction, is crucial for degradation of misfolded proteins and ultimately survival of Mtb upon specific stress conditions. Our data indicate the diverse response of Rv2745c, σ(H) and σ(E) in response to a variety of stress conditions. Activation of Rv2745c in response to various stress conditions leads to differential activation of downstream genes, indicating the diverse role of Rv2745c and its importance for Mtb survival in vivo. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Multifaceted regulation of translational readthrough by RNA replication elements in a tombusvirus.

    Directory of Open Access Journals (Sweden)

    Peter A Cimino

    2011-12-01

    Full Text Available Translational readthrough of stop codons by ribosomes is a recoding event used by a variety of viruses, including plus-strand RNA tombusviruses. Translation of the viral RNA-dependent RNA polymerase (RdRp in tombusviruses is mediated using this strategy and we have investigated this process using a variety of in vitro and in vivo approaches. Our results indicate that readthrough generating the RdRp requires a novel long-range RNA-RNA interaction, spanning a distance of ∼3.5 kb, which occurs between a large RNA stem-loop located 3'-proximal to the stop codon and an RNA replication structure termed RIV at the 3'-end of the viral genome. Interestingly, this long-distance RNA-RNA interaction is modulated by mutually-exclusive RNA structures in RIV that represent a type of RNA switch. Moreover, a different long-range RNA-RNA interaction that was previously shown to be necessary for viral RNA replicase assembly was also required for efficient readthrough production of the RdRp. Accordingly, multiple replication-associated RNA elements are involved in modulating the readthrough event in tombusviruses and we propose an integrated mechanistic model to describe how this regulatory network could be advantageous by (i providing a quality control system for culling truncated viral genomes at an early stage in the replication process, (ii mediating cis-preferential replication of viral genomes, and (iii coordinating translational readthrough of the RdRp with viral genome replication. Based on comparative sequence analysis and experimental data, basic elements of this regulatory model extend to other members of Tombusviridae, as well as to viruses outside of this family.

  13. S-acylation dependent post-translational cross-talk regulates large conductance calcium- and voltage- activated potassium (BK channels

    Directory of Open Access Journals (Sweden)

    Michael J Shipston

    2014-08-01

    Full Text Available Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK channels are important determinants of their (pathophysiological function. Indeed, BK channel dysfunction is associated with major human disorders ranging from epilepsy to hypertension and obesity. S-acylation (S-palmitoylation represents a major reversible, post-translational modification controlling the properties and function of many proteins including ion channels. Recent evidence reveals that both pore-forming and regulatory subunits of BK channels are S-acylated and control channel trafficking and regulation by AGC-family protein kinases. The pore-forming α-subunit is S-acylated at two distinct sites within the N- and C-terminus, each site being regulated by different palmitoyl acyl transferases (zDHHCs and acyl thioesterases. (APTs. S-acylation of the N-terminus controls channel trafficking and surface expression whereas S-acylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. S-acylation of the regulatory β4-subunit controls ER exit and surface expression of BK channels but does not affect ion channel kinetics at the plasma membrane. Furthermore, a significant number of previously identified BK-channel interacting proteins have been shown, or are predicted to be, S-acylated. Thus, the BK channel multi-molecular signalling complex may be dynamically regulated by this fundamental post-translational modification and thus S-acylation likely represents an important determinant of BK channel physiology in health and disease.

  14. Regulation of the cardiac Na+ channel NaV1.5 by post-translational modifications.

    Science.gov (United States)

    Marionneau, Céline; Abriel, Hugues

    2015-05-01

    The cardiac voltage-gated Na(+) channel, Na(V)1.5, is responsible for the upstroke of the action potential in cardiomyocytes and for efficient propagation of the electrical impulse in the myocardium. Even subtle alterations of Na(V)1.5 function, as caused by mutations in its gene SCN5A, may lead to many different arrhythmic phenotypes in carrier patients. In addition, acquired malfunctions of Na(V)1.5 that are secondary to cardiac disorders such as heart failure and cardiomyopathies, may also play significant roles in arrhythmogenesis. While it is clear that the regulation of Na(V)1.5 protein expression and function tightly depends on genetic mechanisms, recent studies have demonstrated that Na(V)1.5 is the target of various post-translational modifications that are pivotal not only in physiological conditions, but also in disease. In this review, we examine the recent literature demonstrating glycosylation, phosphorylation by Protein Kinases A and C, Ca(2+)/Calmodulin-dependent protein Kinase II, Phosphatidylinositol 3-Kinase, Serum- and Glucocorticoid-inducible Kinases, Fyn and Adenosine Monophosphate-activated Protein Kinase, methylation, acetylation, redox modifications, and ubiquitylation of Na(V)1.5. Modern and sensitive mass spectrometry approaches, applied directly to channel proteins that were purified from native cardiac tissues, have enabled the determination of the precise location of post-translational modification sites, thus providing essential information for understanding the mechanistic details of these regulations. The current challenge is first, to understand the roles of these modifications on the expression and the function of Na(V)1.5, and second, to further identify other chemical modifications. It is postulated that the diversity of phenotypes observed with Na(V)1.5-dependent disorders may partially arise from the complex post-translational modifications of channel protein components. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Melatonin as a Signaling Molecule for Metabolism Regulation in Response to Hypoxia in the Crab Neohelice granulata

    Directory of Open Access Journals (Sweden)

    Fábio Everton Maciel

    2014-12-01

    Full Text Available Melatonin has been identified in a variety of crustacean species, but its function is not as well understood as in vertebrates. The present study investigates whether melatonin has an effect on crustacean hyperglycemic hormone (CHH gene expression, oxygen consumption (VO2 and circulating glucose and lactate levels, in response to different dissolved-oxygen concentrations, in the crab Neohelice granulata, as well as whether these possible effects are eyestalk- or receptor-dependent. Melatonin decreased CHH expression in crabs exposed for 45 min to 6 (2, 200 or 20,000 pmol·crab−1 or 2 mgO2·L−1 (200 pmol·crab−1. Since luzindole (200 nmol·crab−1 did not significantly (p > 0.05 alter the melatonin effect, its action does not seem to be mediated by vertebrate-typical MT1 and MT2 receptors. Melatonin (200 pmol·crab−1 increased the levels of glucose and lactate in crabs exposed to 6 mgO2·L−1, and luzindole (200 nmol·crab−1 decreased this effect, indicating that melatonin receptors are involved in hyperglycemia and lactemia. Melatonin showed no effect on VO2. Interestingly, in vitro incubation of eyestalk ganglia for 45 min at 0.7 mgO2·L−1 significantly (p < 0.05 increased melatonin production in this organ. In addition, injections of melatonin significantly increased the levels of circulating melatonin in crabs exposed for 45 min to 6 (200 or 20,000 pmol·crab−1, 2 (200 and 20,000 pmol·crab−1 and 0.7 (200 or 20,000 pmol·crab−1 mgO2·L−1. Therefore, melatonin seems to have an effect on the metabolism of N. granulata. This molecule inhibited the gene expression of CHH and caused an eyestalk- and receptor-dependent hyperglycemia, which suggests that melatonin may have a signaling role in metabolic regulation in this crab.

  16. Translator awareness Translator awareness

    Directory of Open Access Journals (Sweden)

    Wolfram Wilss

    2008-04-01

    Full Text Available If we want to encompass adequately the wide-ranging field of human translation, it is necessary to include in translation studies (TS the concept of translator awareness (or translator consciousness, for that matter. However, this is more easily said than done, because this concept does not easily lend itself to definition, let alone to measurement, e. g., by investigating translator behaviour. To put it bluntly: Translator awareness is a fuzzy concept. Like many obviously difficult-to-define concepts, with which dialogue in TS is burdened, translator awareness lacks an articulated theory within which different forms of translator behaviour can be convincingly related to, or distinguished from, one another. Hence, TS has so far not tackled, at least not systematically, the issue of translator awareness. If we want to encompass adequately the wide-ranging field of human translation, it is necessary to include in translation studies (TS the concept of translator awareness (or translator consciousness, for that matter. However, this is more easily said than done, because this concept does not easily lend itself to definition, let alone to measurement, e. g., by investigating translator behaviour. To put it bluntly: Translator awareness is a fuzzy concept. Like many obviously difficult-to-define concepts, with which dialogue in TS is burdened, translator awareness lacks an articulated theory within which different forms of translator behaviour can be convincingly related to, or distinguished from, one another. Hence, TS has so far not tackled, at least not systematically, the issue of translator awareness.

  17. Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor

    Science.gov (United States)

    Amsalem, Ayelet R.; Marom, Barak; Shapira, Keren E.; Hirschhorn, Tal; Preisler, Livia; Paarmann, Pia; Knaus, Petra; Henis, Yoav I.; Ehrlich, Marcelo

    2016-01-01

    The expression and function of transforming growth factor-β superfamily receptors are regulated by multiple molecular mechanisms. The type II BMP receptor (BMPRII) is expressed as two alternatively spliced forms, a long and a short form (BMPRII-LF and –SF, respectively), which differ by an ∼500 amino acid C-terminal extension, unique among TGF-β superfamily receptors. Whereas this extension was proposed to modulate BMPRII signaling output, its contribution to the regulation of receptor expression was not addressed. To map regulatory determinants of BMPRII expression, we compared synthesis, degradation, distribution, and endocytic trafficking of BMPRII isoforms and mutants. We identified translational regulation of BMPRII expression and the contribution of a 3’ terminal coding sequence to this process. BMPRII-LF and -SF differed also in their steady-state levels, kinetics of degradation, intracellular distribution, and internalization rates. A single dileucine signal in the C-terminal extension of BMPRII-LF accounted for its faster clathrin-mediated endocytosis relative to BMPRII-SF, accompanied by mildly faster degradation. Higher expression of BMPRII-SF at the plasma membrane resulted in enhanced activation of Smad signaling, stressing the potential importance of the multilayered regulation of BMPRII expression at the plasma membrane. PMID:26739752

  18. Translation initiation factor 5A in Picrorhiza is up-regulated during leaf senescence and in response to abscisic acid.

    Science.gov (United States)

    Parkash, Jai; Vaidya, Tanmay; Kirti, Shruti; Dutt, Som

    2014-05-25

    Translation initiation, the first step of protein synthesis process is the principal regulatory step controlling translation and involves a pool of translation initiation factors. In plants, from recent studies it is becoming evident that these translation initiation factors impact various aspects of plant growth and development in addition to their role in protein synthesis. Eukaryotic translation initiation factor eIF5A is one such factor which functions in start site selection for the eIF2-GTP-tRNAi ternary complex within the ribosomal-bound preinitiation complex and also stabilizes the binding of GDP to eIF2. In the present study we have cloned and analysed a gene (eIF5a) encoding eIF5A from Picrorhiza (Picrorhiza kurrooa Royle ex Benth.) a medicinal plant of the western Himalayan region. The full length eIF5a cDNA consisted of 838 bp with an open reading frame of 480 bp, 88 bp 5' untranslated region and 270 bp 3' untranslated region. The deduced eIF5A protein contained 159 amino acids with a molecular weight of 17.359 kDa and an isoelectric point of 5.59. Secondary structure analysis revealed eIF5A having 24.53% α-helices, 8.81% β-turns, 23.27% extended strands and 43.40% random coils. pk-eIF5a transcript was found to be expressing during the active growth phase as well as during leaf senescence stage, however, highest expression was observed during leaf senescence stage. Further, its expression was up-regulated in response to exogenous application of abscisic acid. Both high intensity as well as low intensity light decreased the expression of pk-eIF5a. The findings suggest eIF5a to be an important candidate to develop genetic engineering based strategies for delaying leaf senescence. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Do pyrimidine nucleotides regulate translatability of globin mRNA as purine nucleotides do?

    Science.gov (United States)

    Rubin, H N; Almendarez, E; Halim, M N

    1988-01-01

    1. When rabbit globin mRNA was incubated with rabbit reticulocyte lysate in the presence of various concentrations of nucleotides, globin synthesis was inhibited or stimulated dependent on dose. 2. Pyrimidine nucleotides inhibited protein synthesis at 0.3 mM, whereas 2 mM of purine nucleotides were required to cause similar inhibition. 3. Adenosine mono- and diphosphate inhibited globin synthesis at a concentration of only 1 mM; however, the sequence is AMP greater than ADP greater than ATP. 4. Translation arrest by these nucleotides was instantaneous. 5. These results suggest that these nucleotides may provide a structural component for maintaining the integrity, the conformation of mRNA or of the messenger ribonucleoprotein (mRNP).

  20. Migraine induced by hypoxia

    DEFF Research Database (Denmark)

    Arngrim, Nanna; Schytz, Henrik Winther; Britze, Josefine

    2016-01-01

    Migraine with aura is prevalent in high-altitude populations suggesting an association between migraine aura and hypoxia. We investigated whether experimental hypoxia triggers migraine and aura attacks in patients suffering from migraine with aura. We also investigated the metabolic and vascular...... response to hypoxia. In a randomized double-blind crossover study design, 15 migraine with aura patients were exposed to 180 min of normobaric hypoxia (capillary oxygen saturation 70-75%) or sham on two separate days and 14 healthy controls were exposed to hypoxia. Glutamate and lactate concentrations...... in the visual cortex were measured by proton magnetic resonance spectroscopy. The circumference of cranial arteries was measured by 3 T high-resolution magnetic resonance angiography. Hypoxia induced migraine-like attacks in eight patients compared to one patient after sham (P = 0.039), aura in three...

  1. Post-translational Regulation of Neuronal Nitric Oxide Synthase: Implications for sympatho-excitatory states

    Science.gov (United States)

    Sharma, Neeru M; Patel, Kaushik P

    2017-01-01

    Introduction Nitric oxide (NO) synthesized via neuronal nitric oxide synthase (nNOS) plays a significant role in regulation/modulation of autonomic control of circulation. Various pathological states are associated with diminished nNOS expression and blunted autonomic effects of NO in the central nervous system (CNS) including heart failure, hypertension, diabetes mellitus, chronic renal failure etc. Therefore, elucidation of the molecular mechanism/s involved in dysregulation of nNOS is essential to understand the pathogenesis of increased sympathoexcitation in these diseased states. Areas Covered nNOS is a highly regulated enzyme, being regulated at transcriptional and posttranslational levels via protein-protein interactions and modifications viz. phosphorylation, ubiquitination, and sumoylation. The enzyme activity of nNOS also depends on the optimal concentration of substrate, cofactors and association with regulatory proteins. This review focuses on the posttranslational regulation of nNOS in the context of normal and diseased states within the CNS. Expert Opinion Gaining insight into the mechanism/s involved in the regulation of nNOS would provide novel strategies for manipulating nNOS directed therapeutic modalities in the future, including catalytically active dimer stabilization and protein-protein interactions with intracellular protein effectors. Ultimately, this is expected to provide tools to improve autonomic dysregulation in various diseases such as heart failure, hypertension, and diabetes. PMID:27885874

  2. Self-regulation of sleep, emotion, and weight during adolescence: implications for translational research and practice.

    Science.gov (United States)

    Rofey, Dana L; McMakin, Dana L; Shaw, Daniel; Dahl, Ronald E

    2013-06-01

    Self-regulation-the ability to manage motivations, emotions, physiological sensations, and behavior to meet internal and external demands of the environment-is critical to health and development. Adolescence represents a dynamic period of change in both the demand and capacity for self-regulation. As teens mature and become more autonomous, they are confronted with decisions in determining where they spend their time, what they eat, when they go to bed, and how they prioritize and pursue various social, academic, and recreational goals. We highlight opportunities to improve self-regulatory capacities and related health outcomes during this important developmental window. In particular, we focus on emotion regulation, sleep regulation, and weight regulation as three separate but synergistic self-regulatory systems that may provide unique opportunities for intervention to optimize health outcomes. To this end, we begin by describing developmental changes that occur in emotion, sleep and weight regulatory systems during the transitional period of adolescence, as well as how these changes can lead to profound and enduring health consequences. Next, we describe emerging evidence that indicates complex and synergistic interactions among these regulatory systems during adolescence. Last, we end with possible prevention and intervention efforts that capitalize on the interactions among these three regulatory domains. © 2013 Wiley Periodicals, Inc.

  3. Hypoxia-induced irreversible up-regulation of type I collagen and transforming growth factor-beta1 in human peritoneal fibroblasts.

    Science.gov (United States)

    Saed, Ghassan M; Diamond, Michael P

    2002-07-01

    To determine whether restoration of normoxia after a hypoxic insult returns the molecular expression of type I collagen and TGF-beta1 to baseline levels. Prospective experimental study. University medical center. Primary cultures of fibroblasts established from peritoneal tissues of five patients. Hypoxia treatment of the primary cultured fibroblasts. Cultured human peritoneal fibroblasts (HPF) were maintained under hypoxic conditions (2% oxygen) for 24 hours and then transferred into normal culture conditions (normoxia) for another 24 hours. Total cellular RNA from cells was collected and subjected to multiplex reverse transcription polymerase chain reaction to quantitate type I collagen and transforming growth factor (TGF)-beta1 mRNA levels in response to these treatments. Hypoxia treatment resulted in 30% and 50% increases in type I collagen and TGF-beta1 expression, respectively. Restoration of normoxia after hypoxia treatment failed to restore type I collagen and TGF-beta1 expression to their baseline levels. These data support the hypothesis that hypoxia induces irreversible molecular changes in peritoneal fibroblasts that produce a phenotype that increases extracellular matrix expression and thereby would promote adhesion development. Thus once a phenotype consistent with increased adhesion development is manifested, restoration of oxygen supply does not reverse the stimulation of HPF type I collagen and TGF-beta1 expression. This observation may in part explain the clinical observation that adhesion reformation is more difficult to prevent than de novo adhesion formation.

  4. Abolition of reflex bradycardia by cardiac vagotomy has no effect on the regulation of oxygen uptake by Atlantic cod in progressive hypoxia

    DEFF Research Database (Denmark)

    McKenzie, David; Skov, Peter Vilhelm; Taylor, E.W.T.

    2009-01-01

    difference in their mean critical PO2 (P-crit) values, which were 7.40 +/- 0.81 kPa and 8.73 +/- 0.71 kPa, respectively (n=9). Both groups exhibited significant bradycardia during progressive hypoxia, although denervated fish always had higher mean f(H). The incipient threshold for bradycardia coincided...

  5. MicroRNA, SND1, and alterations in translational regulation in colon carcinogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Tsuchiya, Naoto [Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Nakagama, Hitoshi, E-mail: hnakagam@ncc.go.jp [Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Early Oncogenesis Research Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)

    2010-11-10

    Post-transcriptional regulation of gene expression by microRNA (miRNA) has recently attracted major interest in relation to its involvement in cancer development. miRNA is a member of small non-coding RNA, consists of 22-24 nucleotides and regulates expression of target mRNA species in a post-transcriptional manner by being incorporated with RNA-induced silencing complex (RISC). Staphylococcal nuclease homology domain containing 1 (SND1), a component of RISC, is frequently up-regulated in human colon cancers and also chemically induced colon cancers in animals. We here showed that SDN1 is involved in miRNA-mediated gene suppression and overexpression of SND1 in colon cancer cells causes down-regulation of APC without altering APC mRNA levels. As for the miRNA expression profile in human colon cancer, miR-34a was among the list of down-regulated miRNA. Expression of miR-34a is tightly regulated by p53, and ectopic expression of miR-34a in colon cancer cells causes remarkable reduction of cell proliferation and induces senescence-like phenotypes. MiR-34a also participates in the positive feedback loop of the p53 tumor suppressor network. This circuitry mechanism for p53 activation is of interest in understanding the tumor suppressive function of miR-34a in colon carcinogenesis. miRNA should also be considered as novel anti-cancer agents in tumor suppressive therapeutic applications.

  6. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Itoigawa, Yoshiaki [Tohoku University School of Medicine, Sendai (Japan); Juntendo University School of Medicine, Tokyo (Japan); Kishimoto, Koshi N., E-mail: kishimoto@med.tohoku.ac.jp [Tohoku University School of Medicine, Sendai (Japan); Okuno, Hiroshi; Sano, Hirotaka [Tohoku University School of Medicine, Sendai (Japan); Kaneko, Kazuo [Juntendo University School of Medicine, Tokyo (Japan); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan)

    2010-09-03

    Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  7. Hypoxia regulates FGFR3 expression via HIF-1α and miR-100 and contributes to cell survival in non-muscle invasive bladder cancer.

    Science.gov (United States)

    Blick, C; Ramachandran, A; Wigfield, S; McCormick, R; Jubb, A; Buffa, F M; Turley, H; Knowles, M A; Cranston, D; Catto, J; Harris, A L

    2013-07-09

    Non-muscle invasive (NMI) bladder cancer is characterised by increased expression and activating mutations of FGFR3. We have previously investigated the role of microRNAs in bladder cancer and have shown that FGFR3 is a target of miR-100. In this study, we investigated the effects of hypoxia on miR-100 and FGFR3 expression, and the link between miR-100 and FGFR3 in hypoxia. Bladder cancer cell lines were exposed to normoxic or hypoxic conditions and examined for the expression of FGFR3 by quantitative PCR (qPCR) and western blotting, and miR-100 by qPCR. The effect of FGFR3 and miR-100 on cell viability in two-dimensional (2-D) and three-dimensional (3-D) was examined by transfecting siRNA or mimic-100, respectively. In NMI bladder cancer cell lines, FGFR3 expression was induced by hypoxia in a transcriptional and HIF-1α-dependent manner. Increased FGFR3 was also in part dependent on miR-100 levels, which decreased in hypoxia. Knockdown of FGFR3 led to a decrease in phosphorylation of the downstream kinases mitogen-activated protein kinase (MAPK) and protein kinase B (PKB), which was more pronounced under hypoxic conditions. Furthermore, transfection of mimic-100 also decreased phosphorylation of MAPK and PKB. Finally, knocking down FGFR3 profoundly decreased 2-D and 3-D cell growth, whereas introduction of mimic-100 decreased 3-D growth of cells. Hypoxia, in part via suppression of miR-100, induces FGFR3 expression in bladder cancer, both of which have an important role in maintaining cell viability under conditions of stress.

  8. Regulation of Human Macrophage M1–M2 Polarization Balance by Hypoxia and the Triggering Receptor Expressed on Myeloid Cells-1

    Directory of Open Access Journals (Sweden)

    Federica Raggi

    2017-09-01

    Full Text Available Macrophages (Mf are a heterogeneous population of tissue-resident professional phagocytes and a major component of the leukocyte infiltrate at sites of inflammation, infection, and tumor growth. They can undergo diverse forms of activation in response to environmental factors, polarizing into specialized functional subsets. A common hallmark of the pathologic environment is represented by hypoxia. The impact of hypoxia on human Mf polarization has not been fully established. The objective of this study was to elucidate the effects of a hypoxic environment reflecting that occurring in vivo in diseased tissues on the ability of human Mf to polarize into classically activated (proinflammatory M1 and alternatively activated (anti-inflammatory M2 subsets. We present data showing that hypoxia hinders Mf polarization toward the M1 phenotype by decreasing the expression of T cell costimulatory molecules and chemokine homing receptors and the production of proinflammatory, Th1-priming cytokines typical of classical activation, while promoting their acquisition of phenotypic and secretory features of alternative activation. Furthermore, we identify the triggering receptor expressed on myeloid cells (TREM-1, a member of the Ig-like immunoregulatory receptor family, as a hypoxia-inducible gene in Mf and demonstrate that its engagement by an agonist Ab reverses the M2-polarizing effect of hypoxia imparting a M1-skewed phenotype to Mf. Finally, we provide evidence that Mf infiltrating the inflamed hypoxic joints of children affected by oligoarticular juvenile idiopatic arthritis express high surface levels of TREM-1 associated with predominant M1 polarization and suggest the potential of this molecule in driving M1 proinflammatory reprogramming in the hypoxic synovial environment.

  9. The regulation of protein synthesis and translation factors by CD3 and CD28 in human primary T lymphocytes

    Directory of Open Access Journals (Sweden)

    Proud Christopher G

    2002-05-01

    Full Text Available Abstract Background Activation of human resting T lymphocytes results in an immediate increase in protein synthesis. The increase in protein synthesis after 16–24 h has been linked to the increased protein levels of translation initiation factors. However, the regulation of protein synthesis during the early onset of T cell activation has not been studied in great detail. We studied the regulation of protein synthesis after 1 h of activation using αCD3 antibody to stimulate the T cell receptor and αCD28 antibody to provide the co-stimulus. Results Activation of the T cells with both antibodies led to a sustained increase in the rate of protein synthesis. The activities and/or phosphorylation states of several translation factors were studied during the first hour of stimulation with αCD3 and αCD28 to explore the mechanism underlying the activation of protein synthesis. The initial increase in protein synthesis was accompanied by activation of the guanine nucleotide exchange factor, eukaryotic initiation factor (eIF 2B, and of p70 S6 kinase and by dephosphorylation of eukaryotic elongation factor (eEF 2. Similar signal transduction pathways, as assessed using signal transduction inhibitors, are involved in the regulation of protein synthesis, eIF2B activity and p70 S6 kinase activity. A new finding was that the p38 MAPK α/β pathway was involved in the regulation of overall protein synthesis in primary T cells. Unexpectedly, no changes were detected in the phosphorylation state of the cap-binding protein eIF4E and the eIF4E-binding protein 4E-BP1, or the formation of the cap-binding complex eIF4F. Conclusions Both eIF2B and p70 S6 kinase play important roles in the regulation of protein synthesis during the early onset of T cell activation.

  10. Autogenous translational regulation of the Borna disease virus negative control factor X from polycistronic mRNA using host RNA helicases.

    Directory of Open Access Journals (Sweden)

    Yohei Watanabe

    2009-11-01

    Full Text Available Borna disease virus (BDV is a nonsegmented, negative-strand RNA virus that employs several unique strategies for gene expression. The shortest transcript of BDV, X/P mRNA, encodes at least three open reading frames (ORFs: upstream ORF (uORF, X, and P in the 5' to 3' direction. The X is a negative regulator of viral polymerase activity, while the P phosphoprotein is a necessary cofactor of the polymerase complex, suggesting that the translation of X is controlled rigorously, depending on viral replication. However, the translation mechanism used by the X/P polycistronic mRNA has not been determined in detail. Here we demonstrate that the X/P mRNA autogenously regulates the translation of X via interaction with host factors. Transient transfection of cDNA clones corresponding to the X/P mRNA revealed that the X ORF is translated predominantly by uORF-termination-coupled reinitiation, the efficiency of which is upregulated by expression of P. We found that P may enhance ribosomal reinitiation at the X ORF by inhibition of the interaction of the DEAD-box RNA helicase DDX21 with the 5' untranslated region of X/P mRNA, via interference with its phosphorylation. Our results not only demonstrate a unique translational control of viral regulatory protein, but also elucidate a previously unknown mechanism of regulation of polycistronic mRNA translation using RNA helicases.

  11. Antioxidant Systems are Regulated by Nitric Oxide-Mediated Post-translational Modifications (NO-PTMs)

    Science.gov (United States)

    Begara-Morales, Juan C.; Sánchez-Calvo, Beatriz; Chaki, Mounira; Valderrama, Raquel; Mata-Pérez, Capilla; Padilla, María N.; Corpas, Francisco J.; Barroso, Juan B.

    2016-01-01

    Nitric oxide (NO) is a biological messenger that orchestrates a plethora of plant functions, mainly through post-translational modifications (PTMs) such as S-nitrosylation or tyrosine nitration. In plants, hundreds of proteins have been identified as potential targets of these NO-PTMs under physiological and stress conditions indicating the relevance of NO in plant-signaling mechanisms. Among these NO protein targets, there are different antioxidant enzymes involved in the control of reactive oxygen species (ROS), such as H2O2, which is also a signal molecule. This highlights the close relationship between ROS/NO signaling pathways. The major plant antioxidant enzymes, including catalase, superoxide dismutases (SODs) peroxiredoxins (Prx) and all the enzymatic components of the ascorbate-glutathione (Asa-GSH) cycle, have been shown to be modulated to different degrees by NO-PTMs. This mini-review will update the recent knowledge concerning the interaction of NO with these antioxidant enzymes, with a special focus on the components of the Asa-GSH cycle and their physiological relevance. PMID:26909095

  12. High throughput selection of novel plant growth regulators: Assessing the translatability of small bioactive molecules from Arabidopsis to crops.

    Science.gov (United States)

    Rodriguez-Furlán, Cecilia; Miranda, Giovanna; Reggiardo, Martín; Hicks, Glenn R; Norambuena, Lorena

    2016-04-01

    Plant growth regulators (PGRs) have become an integral part of agricultural and horticultural practices. Accordingly, there is an increased demand for new and cost-effective products. Nevertheless, the market is limited by insufficient innovation. In this context chemical genomics has gained increasing attention as a powerful approach addressing specific traits. Here is described the successful implementation of a highly specific, sensitive and efficient high throughput screening approach using Arabidopsis as a model. Using a combination of techniques, 10,000 diverse compounds were screened and evaluated for several important plant growth traits including root and leaf growth. The phenotype-based selection allowed the compilation of a collection of putative Arabidopsis growth regulators with a broad range of activities and specificities. A subset was selected for evaluating their bioactivity in agronomically valuable plants. Their validation as growth regulators in commercial species such as tomato, lettuce, carrot, maize and turfgrasses reinforced the success of the screening in Arabidopsis and indicated that small molecules activity can be efficiently translated to commercial species. Therefore, the chemical genomics approach in Arabidopsis is a promising field that can be incorporated in PGR discovery programs and has a great potential to develop new products that can be efficiently used in crops. Copyright © 2016. Published by Elsevier Ireland Ltd.

  13. Selective vulnerability in brain hypoxia

    DEFF Research Database (Denmark)

    Cervos-Navarro, J.; Diemer, Nils Henrik

    1991-01-01

    Neuropathology, selective vulnerability, brain hypoxia, vascular factors, excitotoxicity, ion homeostasis......Neuropathology, selective vulnerability, brain hypoxia, vascular factors, excitotoxicity, ion homeostasis...

  14. Overexpression of Hypoxia-Inducible Factor-1α Exacerbates Endothelial Barrier Dysfunction Induced by Hypoxia

    Directory of Open Access Journals (Sweden)

    Pei Wang

    2013-09-01

    Full Text Available Background/Aims: The mechanisms involved in endothelial barrier dysfunction induced by hypoxia are incompletely understood. There is debate about the role of hypoxia-inducible factor-1α (HIF-1α in endothelial barrier disruption. The aim of this study was to investigate the effect of genetic overexpression of HIF-1α on barrier function and the underlying mechanisms in hypoxic endothelial cells. Methods: The plasmid pcDNA3.1/V5-His-HIF-1α was stably transfected into human endothelial cells. The cells were exposed to normoxia or hypoxia. The mRNA and protein expressions of HIF-1α were detected by RT-PCR and Western blot respectively. The barrier function was assessed by measuring the transendothelial electrical resistance (TER. The Western blot analysis was used to determine the protein expression of glucose transporter-1 (GLUT-1, zonular occludens-1 (ZO-1, occludin, and myosin light chain kinase (MLCK in endothelial cells. The mRNA expression of proinflammatory cytokines was detected by qRT-PCR. Results: Genetic overexpression of HIF-1α significantly increased the mRNA and protein expression of HIF-1α in endothelial cells. The overexpression of HIF-1α enhanced the hypoxia-induced increase of HIF-1α and GLUT-1 protein expression. HIF-1α overexpression not only exacerbated hypoxia-induced endothelial barrier dysfunction but also augmented hypoxia-induced up-regulation of MLCK protein expression. HIF-1α overexpression also enhanced IL-1β, IL-6 and TNF-α mRNA expression. Conclusion: We provide evidence that genetic overexpression of HIF-1α aggravates the hypoxia-induced endothelial barrier dysfunction via enhancing the up-regulation of MLCK protein expression caused by hypoxia, suggesting a potential role for HIF-1α in the pathogenesis of endothelial barrier dysfunction in hypoxia.

  15. Translation of myelin basic protein mRNA in oligodendrocytes is regulated by integrin activation and hnRNP-K

    DEFF Research Database (Denmark)

    Laursen, Lisbeth Schmidt; Chan, Colin W; ffrench-Constant, Charles

    2011-01-01

    of protein synthesis represents one mechanism used to control the different requirements for myelin sheath at each axo–glia interaction. Prior work has established that β1-integrins are involved in the axoglial interactions that initiate myelination. Here, we show that integrin activation regulates...... translation of a key sheath protein, myelin basic protein (MBP), by reversing the inhibitory effect of the mRNA 3′UTR. During oligodendrocyte differentiation and myelination α6β1-integrin interacts with hnRNP-K, an mRNA-binding protein, which binds to MBP mRNA and translocates from the nucleus to the myelin...... sheath. Furthermore, knockdown of hnRNP-K inhibits MBP protein synthesis during myelination. Together, these results identify a novel pathway by which axoglial adhesion molecules coordinate MBP synthesis with myelin sheath formation...

  16. Notch-mediated post-translational control of Ngn3 protein stability regulates pancreatic patterning and cell fate commitment

    DEFF Research Database (Denmark)

    Qu, Xiaoling; Afelik, Solomon; Jensen, Jan Nygaard

    2013-01-01

    of ducts. On one hand, Ngn3 cell-intrinsically activates endocrine target genes; on the other, Ngn3 cell-extrinsically promotes lateral signaling via the Dll1>Notch>Hes1 pathway which substantially limits its ability to sustain endocrine formation. Prior to endocrine commitment, the Ngn3-mediated...... activation of the Notch>Hes1 pathway impacts formation of the trunk domain in the pancreas causing multipotent progenitors to lose acinar, while gaining endocrine and ductal, competence. The subsequent selection of fate from such bipotential progenitors is then governed by lateral inhibition, where Notch>Hes...... protein stabilization in the normal mouse pancreas explants. We conclude that the mutually exclusive expression pattern of Ngn3/Hes1 proteins in the mammalian pancreas is partially controlled through Notch-mediated post-translational regulation and we demonstrate that the formation of insulin...

  17. Environmental stress-mediated changes in transcriptional and translational regulation of protein synthesis in crop plants

    Energy Technology Data Exchange (ETDEWEB)

    Key, J.L.; Nagao, R.T.

    1991-06-01

    The major research activities accomplished during the renewal period focused on defining regulatory mechanisms operative in the heat shock (HS) response and assessing the mechanism of HS-induced thermotolerance in Arabidopsis. HS gene regulation was studied by transcriptional run-off assays, and self regulation of heat shock protein (hsp) levels and synthesis was studied through use of amino acid analogs and protein synthesis inhibitors. Also studied were subcellular localization of hsps during HS and HS recovery, characterization of membrane bound hsps and their effects on membrane proton transport, and the influence of over- or underexpression of HS mRNAs and hsps plant phenotype using transgenic plants. In addition, hsp70 and hsp83 cDNAs/genes are being characterized and their expression patterns will be evaluated. 10 refs. (MHB)

  18. Hypoxia, Hypoxia-inducible Transcription Factors, and Renal Cancer.

    Science.gov (United States)

    Schödel, Johannes; Grampp, Steffen; Maher, Eamonn R; Moch, Holger; Ratcliffe, Peter J; Russo, Paul; Mole, David R

    2016-04-01

    Renal cancer is a common urologic malignancy, and therapeutic options for metastatic disease are limited. Most clear cell renal cell carcinomas (ccRCC) are associated with loss of von Hippel-Lindau tumor suppressor (pVHL) function and deregulation of hypoxia pathways. This review summarizes recent evidence from genetic and biological studies showing that hypoxia and hypoxia-related pathways play critical roles in the development and progress of renal cancer. We used a systematic search for articles using the keywords hypoxia, HIF, renal cancer, and VHL. Identification of the tumor suppressor pVHL has allowed the characterization of important ccRCC-associated pathways. pVHL targets α-subunits of hypoxia-inducible transcription factors (HIF) for proteasomal degradation. The two main HIF-α isoforms have opposing effects on RCC biology, possibly through distinct interactions with additional oncogenes. Furthermore, HIF-1α activity is commonly diminished by chromosomal deletion in ccRCCs, and increased HIF-1 activity reduces tumor burden in xenograft tumor models. Conversely, polymorphisms at the HIF-2α gene locus predispose to the development of ccRCCs, and HIF-2α promotes tumor growth. Genetic studies have revealed a prominent role for chromatin-modifying enzyme genes in ccRCC, and these may further modulate specific aspects of the HIF response. This suggests that, rather than global activation of HIF, specific components of the response are important in promoting kidney cancer. Some of these processes are already targets for current therapeutic strategies, and further dissection of this pathway might yield novel methods of treating RCC. In contrast to many tumor types, HIF-1α and HIF-2α have opposing effects in ccRCC biology, with HIF-1α acting as a tumor suppressor and HIF-2α acting as an oncogene. The overall effect of VHL inactivation will depend on fine-tuning of the HIF response. High levels of hypoxia-inducible transcription factors (HIF) are

  19. Hypoxia and brain development

    NARCIS (Netherlands)

    Nyakas, Csaba; Buwalda, Bauke; Luiten, P.

    Hypoxia threatens brain function during the entire life-span starting from early fetal age up to senescence. This review compares the short-term, long-term and life-spanning effects of fetal chronic hypoxia and neonatal anoxia on several behavioural paradigms including novelty-induced spontaneous

  20. USP2-45 Is a Circadian Clock Output Effector Regulating Calcium Absorption at the Post-Translational Level.

    Directory of Open Access Journals (Sweden)

    Daniel Pouly

    Full Text Available The mammalian circadian clock influences most aspects of physiology and behavior through the transcriptional control of a wide variety of genes, mostly in a tissue-specific manner. About 20 clock-controlled genes (CCGs oscillate in virtually all mammalian tissues and are generally considered as core clock components. One of them is Ubiquitin-Specific Protease 2 (Usp2, whose status remains controversial, as it may be a cogwheel regulating the stability or activity of core cogwheels or an output effector. We report here that Usp2 is a clock output effector related to bodily Ca2+ homeostasis, a feature that is conserved across evolution. Drosophila with a whole-body knockdown of the orthologue of Usp2, CG14619 (dUsp2-kd, predominantly die during pupation but are rescued by dietary Ca2+ supplementation. Usp2-KO mice show hyperabsorption of dietary Ca2+ in small intestine, likely due to strong overexpression of the membrane scaffold protein NHERF4, a regulator of the Ca2+ channel TRPV6 mediating dietary Ca2+ uptake. In this tissue, USP2-45 is found in membrane fractions and negatively regulates NHERF4 protein abundance in a rhythmic manner at the protein level. In clock mutant animals (Cry1/Cry2-dKO, rhythmic USP2-45 expression is lost, as well as the one of NHERF4, confirming the inverse relationship between USP2-45 and NHERF4 protein levels. Finally, USP2-45 interacts in vitro with NHERF4 and endogenous Clathrin Heavy Chain. Taken together these data prompt us to define USP2-45 as the first clock output effector acting at the post-translational level at cell membranes and possibly regulating membrane permeability of Ca2+.

  1. Expression of lactate dehydrogenase A and B genes in different tissues of rats adapted to chronic hypobaric hypoxia.

    Science.gov (United States)

    Rossignol, Fabrice; Solares, Magali; Balanza, Elfride; Coudert, Jean; Clottes, Eric

    2003-05-01

    Lactate dehydrogenase (LDH) is a tetramer made up of two different subunits A and B. In cellular models, severe hypoxia increases LDH A gene expression whereas LDH B gene does not exhibit any regulation. The aim of our work was to characterise LDH expression in different tissues of rats bred at high altitude. For this purpose, we chose a Sprague-Dawley rat strain adapted to chronic hypoxia in La Paz (3700 m), Bolivia. Two normoxic control groups were bred at low altitude in Clermont-Ferrand (350 m), France, one group was ad libitum with free access to food and water as was the hypoxic one, and the second normoxic group was nourished with the food intakes measured for the animals from La Paz. We measured total LDH specific activity, isoform distribution and LDH A and B mRNA amounts in three skeletal muscles (soleus, extensor digitorum longus (EDL), plantaris), heart and brain. Our study demonstrates that, unlike what has been shown in cellular models under severe hypoxia, LDH A gene is not systematically up-regulated in tissues of rats living at high altitude. Furthermore, chronic hypoxia limits LDH B gene transcription or its mRNA stability in both soleus and EDL. These regulations occur at various molecular levels like gene transcription, mRNA stabilisation or translation and protein stability, depending on the tissue studied, and are partly attributed to caloric restriction provoked by high altitude. These data provide insight into LDH gene expression underlying the diverse and complex tissue-specific response to chronic hypoxia. Copyright 2003 Wiley-Liss, Inc.

  2. Role of hypoxia and hypoxia inducible factor in physiological and pathological conditions

    Directory of Open Access Journals (Sweden)

    Mozhgan Jahani

    2017-11-01

    Full Text Available Introduction: Organisms are exposed to oxygen deprivation (Hypoxia in various physiological and pathological conditions. There are different conserve evolutionary responses to counterview with this stress that primary transcriptional response to stress related to hypoxia is interceded by hypoxia-inducible factor (HIF-1 in mammals. This factor can regulate different genes that have essential roles in adaptation to this condition. In this review, the role of this factor in physiological and pathological conditions under hypoxic condition has been evaluated after examining structural features and regulation characteristics of HIF-1. Methods: First, articles related to the keywords of hypoxia and HIF-1 (from 1991-2016 were searched from valid databases such as Springer Link, Google Scholar, PubMed and Science direct. Then, the articles correlated with hypoxia, HIF-1 and their roles in physiological and pathological conditions (120 articles were searched and just 64 articles were selected for this study. Result: According to studies, there are different genes in cells and organs that can be regulated by HIF-1. Activation of genes expression by this protein occurs through its linkage to cis-acting of 50 base pair hypoxia response element (HRE region located in their promotor and enhancer. Depending on circumstances, activation of these genes can be beneficial or harmful. Conclusion: Activation of different genes in hypoxia by HIF-1 has different effects on physiological and pathological conditions. Therefore, HIF-1, as a hypoxia-inducible factor in hypoxic conditions, plays an essential role in the adaptation of cells and organs to changes related to the presence of oxygen.

  3. Investigating the Regulation and Potential Role of Nonhypoxic Hypoxia-Inducible Factor 1 (HIF-1) in Aromatase Inhibitor Resistant Breast Cancer

    Science.gov (United States)

    2013-10-01

    obese and overweight patients with ER+ breast cancer to neoadjuvant aromatase inhibitor therapy. My role in this clinical trial is to analyze HIF-1 and...with drug resistance in different cancer cell types, including chronic myeloid leukemia cells (Zhao et al. Oncogene. 2010), gastric cancer cells (Liu...Hypoxia- Inducible Factor 1 (HIF-1) in Aromatase Inhibitor Resistant Breast Cancer PRINCIPAL INVESTIGATOR: Armina Kazi CONTRACTING

  4. Expression of MUC17 is regulated by HIF1α-mediated hypoxic responses and requires a methylation-free hypoxia responsible element in pancreatic cancer.

    Directory of Open Access Journals (Sweden)

    Sho Kitamoto

    Full Text Available MUC17 is a type 1 membrane-bound glycoprotein that is mainly expressed in the digestive tract. Recent studies have demonstrated that the aberrant overexpression of MUC17 is correlated with the malignant potential of pancreatic ductal adenocarcinomas (PDACs; however, the exact regulatory mechanism of MUC17 expression has yet to be identified. Here, we provide the first report of the MUC17 regulatory mechanism under hypoxia, an essential feature of the tumor microenvironment and a driving force of cancer progression. Our data revealed that MUC17 was significantly induced by hypoxic stimulation through a hypoxia-inducible factor 1α (HIF1α-dependent pathway in some pancreatic cancer cells (e.g., AsPC1, whereas other pancreatic cancer cells (e.g., BxPC3 exhibited little response to hypoxia. Interestingly, these low-responsive cells have highly methylated CpG motifs within the hypoxia responsive element (HRE, 5'-RCGTG-3', a binding site for HIF1α. Thus, we investigated the demethylation effects of CpG at HRE on the hypoxic induction of MUC17. Treatment of low-responsive cells with 5-aza-2'-deoxycytidine followed by additional hypoxic incubation resulted in the restoration of hypoxic MUC17 induction. Furthermore, DNA methylation of HRE in pancreatic tissues from patients with PDACs showed higher hypomethylation status as compared to those from non-cancerous tissues, and hypomethylation was also correlated with MUC17 mRNA expression. Taken together, these findings suggested that the HIF1α-mediated hypoxic signal pathway contributes to MUC17 expression, and DNA methylation of HRE could be a determinant of the hypoxic inducibility of MUC17 in pancreatic cancer cells.

  5. Reciprocal Regulation of Hypoxia-Inducible Factor 2α and GLI1 Expression Associated With the Radioresistance of Renal Cell Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Jiancheng [Department of Urology, First Affiliated Hospital of Medical School, Xi' an Jiaotong University, Xi' an (China); Department of Urology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas (United States); Wu, Kaijie [Department of Urology, First Affiliated Hospital of Medical School, Xi' an Jiaotong University, Xi' an (China); Gao, Dexuan [Department of Urology, Shandong Provincial Hospital affiliated with Shandong University, Ji' nan (China); Zhu, Guodong; Wu, Dapeng; Wang, Xinyang; Chen, Yule; Du, Yuefeng; Song, Wenbin; Ma, Zhenkun [Department of Urology, First Affiliated Hospital of Medical School, Xi' an Jiaotong University, Xi' an (China); Authement, Craig; Saha, Debabrata [Department of Urology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas (United States); Hsieh, Jer-Tsong, E-mail: jt.hsieh@utsouthwestern.edu [Department of Urology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas (United States); He, Dalin, E-mail: dalinhe@yahoo.com [Department of Urology, First Affiliated Hospital of Medical School, Xi' an Jiaotong University, Xi' an (China)

    2014-11-15

    Purpose: Renal cell carcinoma (RCC) is often considered a radioresistant tumor, but the molecular mechanism underlying its radioresistance is poorly understood. This study explored the roles of hypoxia-inducible factor 2α (HIF2α) and sonic hedgehog (SHH)-GLI1 signaling in mediating the radioresistance of RCC cells and to unveil the interaction between these 2 signaling pathways. Methods and Materials: The activities of SHH-GLI1 signaling pathway under normoxia and hypoxia in RCC cells were examined by real-time polymerase chain reaction, Western blot, and luciferase reporter assay. The expression of HIF2α and GLI1 in RCC patients was examined by immunohistochemistry, and their correlation was analyzed. Furthermore, RCC cells were treated with HIF2α-specific shRNA (sh-HIF2α), GLI1 inhibitor GANT61, or a combination to determine the effect of ionizing radiation (IR) on RCC cells based on clonogenic assay and double-strand break repair assay. Results: RCC cells exhibited elevated SHH-GLI1 activities under hypoxia, which was mediated by HIF2α. Hypoxia induced GLI1 activation through SMO-independent pathways that could be ablated by PI3K inhibitor or MEK inhibitor. Remarkably, the SHH-GLI1 pathway also upregulated HIF2α expression in normoxia. Apparently, there was a positive correlation between HIF2α and GLI1 expression in RCC patients. The combination of sh-HIF2α and GLI1 inhibitor significantly sensitized RCC cells to IR. Conclusions: Cross-talk between the HIF2α and SHH-GLI1 pathways was demonstrated in RCC. Cotargeting these 2 pathways, significantly sensitizing RCC cells to IR, provides a novel strategy for RCC treatment.

  6. Post-translational regulation of P2X receptor channels: modulation by phospholipids

    Directory of Open Access Journals (Sweden)

    Louis-Philippe eBernier

    2013-11-01

    Full Text Available P2X receptor channels mediate fast excitatory signaling by ATP and play major roles in sensory transduction, neuro-immune communication and inflammatory response. P2X receptors constitute a gene family of calcium-permeable ATP-gated cation channels therefore the regulation of P2X signaling is critical for both membrane potential and intracellular calcium homeostasis. Phosphoinositides (PIPn are anionic signaling phospholipids that act as functional regulators of many types of ion channels. Direct PIPn binding was demonstrated for several ligand- or voltage-gated ion channels, however no generic motif emerged to accurately predict lipid-protein binding sites. This review presents what is currently known about the modulation of the different P2X subtypes by phospholipids and about critical determinants underlying their sensitivity to PIPn levels in the plasma membrane.All functional mammalian P2X subtypes tested, with the notable exception of P2X5, have been shown to be positively modulated by PIPn, i.e. homomeric P2X1, P2X2, P2X3, P2X4, and P2X7, as well as heteromeric P2X1/5 and P2X2/3 receptors. Based on various results reported on the aforementioned subtypes including mutagenesis of the prototypical PIPn-sensitive P2X4 and PIPn-insensitive P2X5 receptor subtypes, an increasing amount of functional, biochemical and structural evidence converges on the modulatory role of a short polybasic domain located in the proximal C-terminus of P2X subunits. This linear motif, semi-conserved in the P2X family, seems necessary and sufficient for encoding direct modulation of ATP-gated channels by PIPn. Furthermore, the physiological impact of the regulation of ionotropic purinergic responses by phospholipids on pain pathways was recently revealed in the context of native crosstalks between phospholipase C-linked metabotropic receptors and P2X receptor channels in DRG sensory neurons and microglia.

  7. Possible involvement of caspase-6 and -7 but not caspase-3 in the regulation of hypoxia-induced apoptosis in tube-forming endothelial cells.

    Science.gov (United States)

    Eguchi, Ryoji; Toné, Shigenobu; Suzuki, Akio; Fujimori, Yoshihiro; Nakano, Takashi; Kaji, Kazuhiko; Ohta, Toshiro

    2009-01-15

    We recently reported that a broad-spectrum caspase inhibitor zVAD-fmk failed, while p38 inhibitor SB203580 succeeded, to prevent chromatin condensation and nuclear fragmentation induced by hypoxia in tube-forming HUVECs. In this study, we investigated the reasons for zVAD-fmk's inability to inhibit these morphological changes at the molecular level. The inhibitor effectively inhibited DNA ladder formation and activation of caspase-3 and -6, but it surprisingly failed to inhibit caspase-7 activation. On the other hand, SB203580 successfully inhibited all of these molecular events. When zLEHD-fmk, which specifically inhibits initiator caspase-9 upstream of caspase-3, was used, it inhibited caspase-3 activation but failed to inhibit caspase-6 and -7 activation. It also failed to inhibit hypoxia-induced chromatin condensation, nuclear fragmentation and DNA ladder formation. Taken together, our results indicate that, during hypoxia, caspase-7 is responsible for chromatin condensation and nuclear fragmentation while caspase-6 is responsible for DNA ladder formation.

  8. Opposing regulation of the late phase TNF response by mTORC1-IL-10 signaling and hypoxia in human macrophages

    Science.gov (United States)

    Huynh, Linda; Kusnadi, Anthony; Park, Sung Ho; Murata, Koichi; Park-Min, Kyung-Hyun; Ivashkiv, Lionel B.

    2016-01-01

    Tumor necrosis factor (TNF) is best known for inducing a rapid but transient NF-κB-mediated inflammatory response. We investigated later phases of TNF signaling, after the initial transient induction of inflammatory genes has subsided, in primary human macrophages. TNF signaling induced expression of late response genes, including inhibitors of NF-κB and TLR signaling, with delayed and sustained kinetics 6–24 hr after TNF stimulation. A subset of late phase genes was expressed in rheumatoid arthritis synovial macrophages, confirming their expression under chronic inflammatory conditions in vivo. Expression of a subset of late phase genes was mediated by autocrine IL-10, which activated STAT3 with delayed kinetics. Hypoxia, which occurs at sites of infection or inflammation where TNF is expressed, suppressed this IL-10-STAT3 autocrine loop and expression of late phase genes. TNF-induced expression of IL-10 and downstream genes was also dependent on signaling by mTORC1, which senses the metabolic state of cells and is modulated by hypoxia. These results reveal an mTORC1-dependent IL-10-mediated late phase response to TNF by primary human macrophages, and identify suppression of IL-10 responses as a new mechanism by which hypoxia can promote inflammation. Thus, hypoxic and metabolic pathways may modulate TNF responses during chronic inflammation. PMID:27558590

  9. Neuronal exosomal miRNA-dependent translational regulation of astroglial glutamate transporter GLT1.

    Science.gov (United States)

    Morel, Lydie; Regan, Melissa; Higashimori, Haruki; Ng, Seng Kah; Esau, Christine; Vidensky, Svetlana; Rothstein, Jeffrey; Yang, Yongjie

    2013-03-08

    Perisynaptic astrocytes express important glutamate transporters, especially excitatory amino acid transporter 2 (EAAT2, rodent analog GLT1) to regulate extracellular glutamate levels and modulate synaptic activation. In this study, we investigated an exciting new pathway, the exosome-mediated transfer of microRNA (in particular, miR-124a), in neuron-to-astrocyte signaling. Exosomes isolated from neuron-conditioned medium contain abundant microRNAs and small RNAs. These exosomes can be directly internalized into astrocytes and increase astrocyte miR-124a and GLT1 protein levels. Direct miR-124a transfection also significantly and selectively increases protein (but not mRNA) expression levels of GLT1 in cultured astrocytes. Consistent with our in vitro findings, intrastriatal injection of specific antisense against miR-124a into adult mice dramatically reduces GLT1 protein expression and glutamate uptake levels in striatum without reducing GLT1 mRNA levels. MiR-124a-mediated regulation of GLT1 expression appears to be indirect and is not mediated by its suppression of the putative GLT1 inhibitory ligand ephrinA3. Moreover, miR-124a is selectively reduced in the spinal cord tissue of end-stage SOD1 G93A mice, the mouse model of ALS. Subsequent exogenous delivery of miR-124a in vivo through stereotaxic injection significantly prevents further pathological loss of GLT1 proteins, as determined by GLT1 immunoreactivity in SOD1 G93A mice. Together, our study characterized a new neuron-to-astrocyte communication pathway and identified miRNAs that modulate GLT1 protein expression in astrocytes in vitro and in vivo.

  10. Regulation of non-homologous end joining via post-translational modifications of components of the ligation step.

    Science.gov (United States)

    Durdíková, Kristína; Chovanec, Miroslav

    2017-08-01

    DNA double-strand breaks are the most serious type of DNA damage and non-homologous end joining (NHEJ) is an important pathway for their repair. In Saccharomyces cerevisiae, three complexes mediate the canonical NHEJ pathway, Ku (Ku70/Ku80), MRX (Mre11/Rad50/Xrs2) and DNA ligase IV (Dnl4/Lif1). Mammalian NHEJ is more complex, primarily as a consequence of the fact that more factors are involved in the process, and also because higher chromatin organization and more complex regulatory networks exist in mammals. In addition, a stronger interconnection between the NHEJ and DNA damage response (DDR) pathways seems to occur in mammals compared to yeast. DDR employs multiple post-translational modifications (PTMs) of the target proteins and mutual crosstalk among them to ensure highly efficient down-stream effects. Checkpoint-mediated phosphorylation is the best understood PTM that regulates DDR, although recently SUMOylation has also been shown to be involved. Both phosphorylation and SUMOylation affect components of NHEJ. In this review, we discuss a role of these two PTMs in regulation of NHEJ via targeting the components of the ligation step.

  11. Regulation of glyoxysomal enzymes during germination of cucumber. Temporal changes in translatable mRNAs for isocitrate lyase and malate synthase.

    Science.gov (United States)

    Weir, E M; Riezman, H; Grienenberger, J M; Becker, W M; Leaver, C J

    1980-12-01

    The relative levels of translatable messenger RNA for isocitrate lyase and malate synthase were determined in the dry seed and for the first seven days of development of cucumber cotyledons. After extraction and quantification of total and poly(A)-rich RNA each day, the RNA fractions were translated in an optimized wheat germ system and the specific polypeptides were immunoprecipitated quantitatively. The radiolabeled isocitrate lyase and malate synthase polypeptides were then fractionated on dodecylsulphate/polyacrylamide gels, visualized by exposure to X-ray film and quantified densitometrically. The relative levels of translatable messenger RNA for these enzymes rise and fall with a developmental program similar to the enzyme activities, but preceding the latter by about one day. This implies that the rise in enzyme activity is dependent upon a prior postgerminative increase in translatable messenger RNA for the enzymes. These studies also suggest that messenger RNA levels may be regulated, at least in part, by light.

  12. Trans-splicing and operons in metazoans: translational control in maternally regulated development and recovery from growth arrest.

    Science.gov (United States)

    Danks, Gemma B; Raasholm, Martina; Campsteijn, Coen; Long, Abby M; Manak, J Robert; Lenhard, Boris; Thompson, Eric M

    2015-03-01

    Polycistronic mRNAs transcribed from operons are resolved via the trans-splicing of a spliced-leader (SL) RNA. Trans-splicing also occurs at monocistronic transcripts. The phlyogenetically sporadic appearance of trans-splicing and operons has made the driving force(s) for their evolution in metazoans unclear. Previous work has proposed that germline expression drives operon organization in Caenorhabditis elegans, and a recent hypothesis proposes that operons provide an evolutionary advantage via the conservation of transcriptional machinery during recovery from growth arrested states. Using a modified cap analysis of gene expression protocol we mapped sites of SL trans-splicing genome-wide in the marine chordate Oikopleura dioica. Tiled microarrays revealed the expression dynamics of trans-spliced genes across development and during recovery from growth arrest. Operons did not facilitate recovery from growth arrest in O. dioica. Instead, we found that trans-spliced transcripts were predominantly maternal. We then analyzed data from C. elegans and Ciona intestinalis and found that an enrichment of trans-splicing and operon gene expression in maternal mRNA is shared between all three species, suggesting that this may be a driving force for operon evolution in metazoans. Furthermore, we found that the majority of known terminal oligopyrimidine (TOP) mRNAs are trans-spliced in O. dioica and that the SL contains a TOP-like motif. This suggests that the SL in O. dioica confers nutrient-dependent translational control to trans-spliced mRNAs via the TOR-signaling pathway. We hypothesize that SL-trans-splicing provides an evolutionary advantage in species that depend on translational control for regulating early embryogenesis, growth and oocyte production in response to nutrient levels. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Genetic Heterogeneity of Breast Cancer Metastasis May Be Related to miR-21 Regulation of TIMP-3 in Translation

    Directory of Open Access Journals (Sweden)

    Jianyi Li

    2013-01-01

    Full Text Available Purpose. MicroRNAs are noncoding RNA molecules that posttranscriptionally regulated expression of target gene and implicate the progress of cancer proliferation, differentiation, and apoptosis. The aim of this study is to determine whether microRNA-21 (miR-21, a specific microRNA implicated in multiple aspects of carcinogenesis, promoted breast cancer metastasis by regulating the tissue inhibitor of metalloproteinase 3 (TIMP-3 gene. Methods. miR-21 of serum and tissue from 40 patients (30 patients with breast cancer were detected by real-time quantitative reverse transcriptase polymerase chain reaction (RT-qPCR. TIMP-3 of tissue from the patient was tested by real-time RT-qPCR. Protein expression of TIMP-3 was evaluated by western blotting. Correlation analysis was performed between miR-21 and TIMP-3. Results. Of the 40 samples from tissue and serum analyzed, the miR-21 expression was significantly higher in high invasion metastasis group (HIMG that in low invasion metastasis group (LIMG; the latter was higher than that in normal group (NG. Additionally, the TIMP-3 expression was significantly lower in HIMG than in LIMG; the latter was lower than that in NG. There was significantly inverse correlation between miR-21 and TIMP-3 extracted from tissue. Conclusion. Our data suggest that miR-21 could promote metastasis in breast cancer via the regulation of TIMP3 translation, and there was consistency between miR-21 of serum and miR-21 in tissue.

  14. Post-translational regulation of endothelial nitric oxide synthase (eNOS) by estrogens in the rat vagina.

    Science.gov (United States)

    Musicki, Biljana; Liu, Tongyun; Strong, Travis D; Lagoda, Gwen A; Bivalacqua, Trinity J; Burnett, Arthur L

    2010-05-01

    Estrogens control vaginal blood flow during female sexual arousal mostly through nitric oxide (NO). Although vascular effects of estrogens are attributed to an increase in endothelial NO production, the mechanisms of endothelial NO synthase (eNOS) regulation by estrogens in the vagina are largely unknown. Our hypothesis was that estrogens regulate eNOS post-translationally in the vagina, providing a mechanism to affect NO bioavailability without changes in eNOS protein expression. We measured eNOS phosphorylation and eNOS interaction with caveolin-1 and heat shock protein 90 (HSP90) in the distal and proximal vagina of female rats at diestrus, 7 days after ovariectomy and 2 days after replacement of ovariectomized rats with estradiol-17beta (15 microg). Molecular mechanisms of eNOS regulation by estrogen in the rat vagina. We localized phospho-eNOS (Ser-1177) immunohistochemically to the endothelium lining blood vessels and vaginal sinusoids. Estrogen withdrawal decreased phosphorylation of eNOS on its positive regulatory site (Ser-1177) and increased eNOS binding to its negative regulator caveolin-1 (without affecting eNOS/HSP90 interaction), and they were both normalized by estradiol replacement. Protein expressions of phosphorylated Akt (protein kinase B) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) were not affected by estrogen status, suggesting that the effect of estrogens on eNOS (Ser-1177) phosphorylation was not mediated by activated AKT or ERK1/2. eNOS phosphorylation on its negative regulatory site (Ser-114) was increased in the vagina by estrogen withdrawal and normalized by estradiol replacement, implying that the maintenance of low phosphorylation of eNOS on this site by estradiol may limit eNOS interaction with caveolin-1 and preserve the enzyme's activity. Total eNOS, inducible NOS, caveolin-1, and HSP90 protein expressions were not affected by ovariectomy or estradiol replacement in the distal or proximal vagina. These results

  15. Post-Translational Derepression of Invertase Activity in Source Leaves via Down-Regulation of Invertase Inhibitor Expression Is Part of the Plant Defense Response

    OpenAIRE

    Bonfig, Katharina B.; Gabler, Andrea; Simon, Uwe K.; Luschin-Ebengreuth, Nora; Hatz, Martina; Berger, Susanne; Muhammad, Naseem; Zeier, Jürgen; Alok K Sinha; Roitsch, Thomas

    2010-01-01

    There is increasing evidence that pathogens do not only elicit direct defense responses, but also cause pronounced changes in primary carbohydrate metabolism. Cell-wall-bound invertases belong to the key regulators of carbohydrate partitioning and source-sink relations. Whereas studies have focused so far only on the transcriptional induction of invertase genes in response to pathogen infection, the role of post-translational regulation of invertase activity has been neglected and was the foc...

  16. DMPD: Translational mini-review series on Toll-like receptors: networks regulated byToll-like receptors mediate innate and adaptive immunity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available e receptors: networks regulated byToll-like receptors mediate innate and adaptive...ed byToll-like receptors mediate innate and adaptive immunity. Authors Parker LC, Prince LR, Sabroe I. Publi...d byToll-like receptors mediate innate and adaptive immunity. Parker LC, Prince LR, Sabroe I. Clin Exp Immun...17223959 Translational mini-review series on Toll-like receptors: networks regulate

  17. Translational control and differential RNA decay are key elements regulating postsegregational expression of the killer protein encoded by the parB locus of plasmid R1

    DEFF Research Database (Denmark)

    Gerdes, K; Helin, K; Christensen, O W

    1988-01-01

    The parB locus of plasmid R1, which mediates plasmid stability via postsegregational killing of plasmid-free cells, encodes two genes, hok and sok. The hok gene product is a potent cell-killing protein. The hok gene is regulated at the translational level by the sok gene-encoded repressor, a small...

  18. The Crc global regulator binds to an unpaired A-rich motif at the Pseudomonas putida alkS mRNA coding sequence and inhibits translation initiation.

    Science.gov (United States)

    Moreno, Renata; Marzi, Stefano; Romby, Pascale; Rojo, Fernando

    2009-12-01

    Crc is a key global translational regulator in Pseudomonads that orchestrates the hierarchy of induction of several catabolic pathways for amino acids, sugars, hydrocarbons or aromatic compounds. In the presence of amino acids, which are preferred carbon sources, Crc inhibits translation of the Pseudomonas putida alkS and benR mRNAs, which code for transcriptional regulators of genes required to assimilate alkanes (hydrocarbons) and benzoate (an aromatic compound), respectively. Crc binds to the 5'-end of these mRNAs, but the sequence and/or structure recognized, and the way in which it inhibits translation, were unknown. We have determined the secondary structure of the alkS mRNA 5'-end through its sensitivity to several ribonucleases and chemical reagents. Footprinting and band-shift assays using variant alkS mRNAs have shown that Crc specifically binds to a short unpaired A-rich sequence located adjacent to the alkS AUG start codon. This interaction is stable enough to prevent formation of the translational initiation complex. A similar Crc-binding site was localized at benR mRNA, upstream of the Shine-Dalgarno sequence. This allowed predicting binding sites at other Crc-regulated genes, deriving a consensus sequence that will help to validate new Crc targets and to discriminate between direct and indirect effects of this regulator.

  19. Hypoxia and hypoxia mimetics decrease aquaporin 5 (AQP5) expression through both hypoxia inducible factor-1α and proteasome-mediated pathways.

    Science.gov (United States)

    Kawedia, Jitesh D; Yang, Fan; Sartor, Maureen A; Gozal, David; Czyzyk-Krzeska, Maria; Menon, Anil G

    2013-01-01

    The alveolar epithelium plays a central role in gas exchange and fluid transport, and is therefore critical for normal lung function. Since the bulk of water flux across this epithelium depends on the membrane water channel Aquaporin 5 (AQP5), we asked whether hypoxia had any effect on AQP5 expression. We show that hypoxia causes a significant (70%) decrease in AQP5 expression in the lungs of mice exposed to hypoxia. Hypoxia and the hypoxia mimetic, cobalt, also caused similar decreases in AQP5 mRNA and protein expression in the mouse lung epithelial cell line MLE-12. The action of hypoxia and cobalt on AQP5 transcription was demonstrated by directly quantifying heternonuclear RNA by real-time PCR. Dominant negative mutants of Hypoxia Inducible Factor (HIF-1α) and HIF-1α siRNA blocked the action of cobalt, showing that HIF-1α is a key component in this mechanism. The proteasome inhibitors, lactacystin or proteasome inhibitor-III completely abolished the effect of hypoxia and cobalt both at the protein and mRNA level indicating that the proteasome pathway is probably involved not only for the stability of HIF-1α protein, but for the stability of unidentified transcription factors that regulate AQP5 transcription. These studies reveal a potentially important physiological mechanism linking hypoxic stress and membrane water channels.

  20. Inflammatory conditions induce IRES-dependent translation of cyp24a1.

    Directory of Open Access Journals (Sweden)

    Daniela Rübsamen

    Full Text Available Rapid alterations in protein expression are commonly regulated by adjusting translation. In addition to cap-dependent translation, which is e.g. induced by pro-proliferative signaling via the mammalian target of rapamycin (mTOR-kinase, alternative modes of translation, such as internal ribosome entry site (IRES-dependent translation, are often enhanced under stress conditions, even if cap-dependent translation is attenuated. Common stress stimuli comprise nutrient deprivation, hypoxia, but also inflammatory signals supplied by infiltrating immune cells. Yet, the impact of inflammatory microenvironments on translation in tumor cells still remains largely elusive. In the present study, we aimed at identifying translationally deregulated targets in tumor cells under inflammatory conditions. Using polysome profiling and microarray analysis, we identified cyp24a1 (1,25-dihydroxyvitamin D3 24-hydroxylase to be translationally upregulated in breast tumor cells co-cultured with conditioned medium of activated monocyte-derived macrophages (CM. Using bicistronic reporter assays, we identified and validated an IRES within the 5' untranslated region (5'UTR of cyp24a1, which enhances translation of cyp24a1 upon CM treatment. Furthermore, IRES-dependent translation of cyp24a1 by CM was sensitive to phosphatidyl-inositol-3-kinase (PI3K inhibition, while constitutive activation of Akt sufficed to induce its IRES activity. Our data provide evidence that cyp24a1 expression is translationally regulated via an IRES element, which is responsive to an inflammatory environment. Considering the negative feedback impact of cyp24a1 on the vitamin D responses, the identification of a novel, translational mechanism of cyp24a1 regulation might open new possibilities to overcome the current limitations of vitamin D as tumor therapeutic option.

  1. Interleukin-17 (IL-17) and IL-1 activate translation of overlapping sets of mRNAs, including that of the negative regulator of inflammation, MCPIP1.

    Science.gov (United States)

    Dhamija, Sonam; Winzen, Reinhard; Doerrie, Anneke; Behrens, Gesine; Kuehne, Nancy; Schauerte, Celina; Neumann, Elena; Dittrich-Breiholz, Oliver; Kracht, Michael; Holtmann, Helmut

    2013-06-28

    Changes in gene expression during inflammation are in part caused by post-transcriptional mechanisms. A transcriptome-wide screen for changes in ribosome occupancy indicated that the inflammatory cytokine IL-17 activates translation of a group of mRNAs that overlaps partially with those affected similarly by IL-1. Included are mRNAs of IκBζ and of MCPIP1, important regulators of the quality and course of immune and inflammatory responses. Evidence for increased ribosome association of these mRNAs was also obtained in LPS-activated RAW264.7 macrophages and human peripheral blood mononuclear cells. Like IL-1, IL-17 activated translation of IκBζ mRNA by counteracting the function of a translational silencing element in its 3'-UTR defined previously. Translational silencing of MCPIP1 mRNA in unstimulated cells resulted from the combined suppressive activities of its 5'-UTR, which contains upstream open reading frames, and of its 3'-UTR, which silences independently of the 5'-UTR. Only the silencing function of the 3'-UTR was counteracted by IL-17 as well as by IL-1. Translational silencing by the 3'-UTR was dependent on a putative stem-loop-forming region previously associated with rapid degradation of the mRNA. The results suggest that translational control exerted by IL-1 and IL-17 plays an important role in the coordination of an inflammatory reaction.

  2. Translation Theory 'Translated'

    DEFF Research Database (Denmark)

    Wæraas, Arild; Nielsen, Jeppe

    2016-01-01

    common theoretical approaches to translation within the organization and management discipline: actor-network theory, knowledge-based theory, and Scandinavian institutionalism. Although each of these approaches already has borne much fruit in research, the literature is diverse and somewhat fragmented......, but also overlapping. We discuss the ways in which the three versions of translation theory may be combined and enrich each other so as to inform future research, thereby offering a more complete understanding of translation in and across organizational settings....

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-01-01

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

  4. Drosophila NAT1, a homolog of the vertebrate translational regulator NAT1/DAP5/p97, is required for embryonic germband extension and metamorphosis.

    Science.gov (United States)

    Yoshikane, Nami; Nakamura, Nao; Ueda, Ryu; Ueno, Naoto; Yamanaka, Shinya; Nakamura, Makoto

    2007-09-01

    Translational regulation has been to shown to play major roles in the patterning of the early Drosophila embryo. The eIF4G family member NAT1/p97/DAP5 has been identified as a novel translational repressor. To genetically dissect the in vivo function of this unconventional eIF4G-related translational regulator, Drosophila NAT1 (dNAT1) mutants were isolated using a reverse-genetics approach. Four transposon insertion mutants and a deletion mutant affecting the dNAT1 locus were analyzed. Genetic complementation tests and germline rescue using a 12 kb dNAT1 genomic DNA fragment revealed these to be loss-of-function mutants. One P-element insertion line, dNAT1(GS1.), shows severe embryonic lethality and abnormal germband extension. Abnormalities at metamorphosis were also found, including defective head eversion and salivary gland degeneration in the hypomorphic allele dNAT(ex1). A phenotypic analysis of dNAT1 mutants suggests that dNAT protein plays a specific rather than general role in translational regulation.

  5. Mechanism of hypoxia-induced NFκB

    Science.gov (United States)

    Melvin, Andrew; Mudie, Sharon

    2011-01-01

    The cellular response to hypoxia relies on the activation of a specific transcriptional program. Although, most of the attention is focused on the transcription factor HIF, other transcription factors are also activated in hypoxia. We have recently described the mechanism for hypoxia induced NFκB. We have demonstrated the crucial dependency on the IKK complex as well as in the upstream IKK kinase TAK1. TAK1 and IKK activation is dependent upon the calcium calmodulin kinase, CaMK2 and requires Ubc13 as the E2 ubiquitin conjugation enzyme. We report a role for XIAP as the possible E3-ubiquitin ligase for this system. Interestingly, hypoxia induced IKK mediated phosphorylation of IκBα, does not lead to degradation. Hypoxia prevents IκBα de-sumoylation of Sumo-2/3 chains on critical lysine residues, normally required for K-48 linked polyubiquitination. Our results define a novel pathway regulating NFκB activation. PMID:21325892

  6. Hypoxia causes transgenerational impairments in reproduction of fish

    Science.gov (United States)

    Wang, Simon Yuan; Lau, Karen; Lai, Keng-Po; Zhang, Jiang-Wen; Tse, Anna Chung-Kwan; Li, Jing-Woei; Tong, Yin; Chan, Ting-Fung; Wong, Chris Kong-Chu; Chiu, Jill Man-Ying; Au, Doris Wai-Ting; Wong, Alice Sze-Tsai; Kong, Richard Yuen-Chong; Wu, Rudolf Shiu-Sun

    2016-01-01

    Hypoxia is amongst the most widespread and pressing problems in aquatic environments. Here we demonstrate that fish (Oryzias melastigma) exposed to hypoxia show reproductive impairments (retarded gonad development, decrease in sperm count and sperm motility) in F1 and F2 generations despite these progenies (and their germ cells) having never been exposed to hypoxia. We further show that the observed transgenerational reproductive impairments are associated with a differential methylation pattern of specific genes in sperm of both F0 and F2 coupled with relevant transcriptomic and proteomic alterations, which may impair spermatogenesis. The discovered transgenerational and epigenetic effects suggest that hypoxia might pose a dramatic and long-lasting threat to the sustainability of fish populations. Because the genes regulating spermatogenesis and epigenetic modifications are highly conserved among vertebrates, these results may also shed light on the potential transgenerational effects of hypoxia on other vertebrates, including humans. PMID:27373813

  7. Role of 3'UTRs in the translation of mRNAs regulated by oncogenic eIF4E--a computational inference.

    Directory of Open Access Journals (Sweden)

    Arti N Santhanam

    Full Text Available Eukaryotic cap-dependent mRNA translation is mediated by the initiation factor eIF4E, which binds mRNAs and stimulates efficient translation initiation. eIF4E is often overexpressed in human cancers. To elucidate the molecular signature of eIF4E target mRNAs, we analyzed sequence and structural properties of two independently derived polyribosome recruited mRNA datasets. These datasets originate from studies of mRNAs that are actively being translated in response to cells over-expressing eIF4E or cells with an activated oncogenic AKT: eIF4E signaling pathway, respectively. Comparison of eIF4E target mRNAs to mRNAs insensitive to eIF4E-regulation has revealed surprising features in mRNA secondary structure, length and microRNA-binding properties. Fold-changes (the relative change in recruitment of an mRNA to actively translating polyribosomal complexes in response to eIF4E overexpression or AKT upregulation are positively correlated with mRNA G+C content and negatively correlated with total and 3'UTR length of the mRNAs. A machine learning approach for predicting the fold change was created. Interesting tendencies of secondary structure stability are found near the start codon and at the beginning of the 3'UTR region. Highly upregulated mRNAs show negative selection (site avoidance for binding sites of several microRNAs. These results are consistent with the emerging model of regulation of mRNA translation through a dynamic balance between translation initiation at the 5'UTR and microRNA binding at the 3'UTR.

  8. Role of 3'UTRs in the translation of mRNAs regulated by oncogenic eIF4E--a computational inference.

    Science.gov (United States)

    Santhanam, Arti N; Bindewald, Eckart; Rajasekhar, Vinagolu K; Larsson, Ola; Sonenberg, Nahum; Colburn, Nancy H; Shapiro, Bruce A

    2009-01-01

    Eukaryotic cap-dependent mRNA translation is mediated by the initiation factor eIF4E, which binds mRNAs and stimulates efficient translation initiation. eIF4E is often overexpressed in human cancers. To elucidate the molecular signature of eIF4E target mRNAs, we analyzed sequence and structural properties of two independently derived polyribosome recruited mRNA datasets. These datasets originate from studies of mRNAs that are actively being translated in response to cells over-expressing eIF4E or cells with an activated oncogenic AKT: eIF4E signaling pathway, respectively. Comparison of eIF4E target mRNAs to mRNAs insensitive to eIF4E-regulation has revealed surprising features in mRNA secondary structure, length and microRNA-binding properties. Fold-changes (the relative change in recruitment of an mRNA to actively translating polyribosomal complexes in response to eIF4E overexpression or AKT upregulation) are positively correlated with mRNA G+C content and negatively correlated with total and 3'UTR length of the mRNAs. A machine learning approach for predicting the fold change was created. Interesting tendencies of secondary structure stability are found near the start codon and at the beginning of the 3'UTR region. Highly upregulated mRNAs show negative selection (site avoidance) for binding sites of several microRNAs. These results are consistent with the emerging model of regulation of mRNA translation through a dynamic balance between translation initiation at the 5'UTR and microRNA binding at the 3'UTR.

  9. Cardiac atria are the primary source of ANP release in hypoxia-adapted rats.

    Science.gov (United States)

    Casserly, Brian; Pietras, Linda; Schuyler, Joy; Wang, Richard; Hill, Nicholas S; Klinger, James R

    2010-09-11

    atrial natriuretic peptide (ANP) is released from the heart in response to hypoxia and helps mitigate the development of pulmonary hypertension. However, the mechanism of hypoxia-induced ANP release is not clear. The cardiac atria are the primary source of ANP secretion under normal conditions, but right ventricular ANP expression is markedly up-regulated during adaptation to hypoxia. We sought to better understand mechanisms of cardiac ANP release during adaptation to hypoxia. we measured hypoxia-induced ANP release from isolated perfused rat hearts obtained from normoxia and hypoxia-adapted rats before and after removal of the atria. in both normoxia- and hypoxia-adapted hearts, ANP levels in the perfusate increased within 15 min of hypoxia. Hypoxia-induced ANP release was greater from hypoxia-adapted than normoxia-adapted hearts. Baseline and hypoxia-induced ANP release were considerably greater with the atria intact (213±29 to 454±62 and 281±26 to 618±87 pg/ml for normoxia- and hypoxia-adapted hearts respectively, PANP release was reduced over 80% by removing the atria in both normoxia- and in hypoxia-adapted hearts. Acute hypoxia caused a transient increase in lactate release and reductions in pH and left ventricular generated force, but no differences in pH or left ventricular generated force were seen between normoxia- and hypoxia-adapted rats. we conclude that the right ventricle is not a major source of cardiac ANP release in normoxia- or hypoxia-adapted rats. 2010 Elsevier Inc. All rights reserved.

  10. Global Gene Expression Profiling in Three Tumor Cell Lines Subjected to Experimental Cycling and Chronic Hypoxia

    Science.gov (United States)

    Olbryt, Magdalena; Habryka, Anna; Student, Sebastian; Jarząb, Michał; Tyszkiewicz, Tomasz; Lisowska, Katarzyna Marta

    2014-01-01

    Hypoxia is one of the most important features of the tumor microenvironment, exerting an adverse effect on tumor aggressiveness and patient prognosis. Two types of hypoxia may occur within the tumor mass, chronic (prolonged) and cycling (transient, intermittent) hypoxia. Cycling hypoxia has been shown to induce aggressive tumor cell phenotype and radioresistance more significantly than chronic hypoxia, though little is known about the molecular mechanisms underlying this phenomenon. The aim of this study was to delineate the molecular response to both types of hypoxia induced experimentally in tumor cells, with a focus on cycling hypoxia. We analyzed in vitro gene expression profile in three human cancer cell lines (melanoma, ovarian cancer, and prostate cancer) exposed to experimental chronic or transient hypoxia conditions. As expected, the cell-type specific variability in response to hypoxia was significant. However, the expression of 240 probe sets was altered in all 3 cell lines. We found that gene expression profiles induced by both types of hypoxia were qualitatively similar and strongly depend on the cell type. Cycling hypoxia altered the expression of fewer genes than chronic hypoxia (6,132 vs. 8,635 probe sets, FDR adjusted pcycling hypoxia than by prolonged hypoxia, such as IL8, PLAU, and epidermal growth factor (EGF) pathway-related genes (AREG, HBEGF, and EPHA2). These transcripts were, in most cases, validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Our results indicate that experimental cycling hypoxia exerts similar, although less intense effects, on the examined cancer cell lines than its chronic counterpart. Nonetheless, we identified genes and molecular pathways that seem to be preferentially regulated by cyclic hypoxia. PMID:25122487

  11. Cardiovascular disease in obstructive sleep apnoea syndrome: the role of intermittent hypoxia and inflammation.

    LENUS (Irish Health Repository)

    Garvey, J F

    2012-02-01

    There is increasing evidence that intermittent hypoxia plays a role in the development of cardiovascular risk in obstructive sleep apnoea syndrome (OSAS) through the activation of inflammatory pathways. The development of translational models of intermittent hypoxia has allowed investigation of its role in the activation of inflammatory mechanisms and promotion of cardiovascular disease in OSAS. There are noticeable differences in the response to intermittent hypoxia between body tissues but the hypoxia-sensitive transcription factors hypoxia-inducible factor-1 and nuclear factor-kappaB appear to play a key role in mediating the inflammatory and cardiovascular consequences of OSAS. Expanding our understanding of these pathways, the cross-talk between them and the activation of inflammatory mechanisms by intermittent hypoxia in OSAS will provide new avenues of therapeutic opportunity for the disease.

  12. Understanding Translation

    DEFF Research Database (Denmark)

    Schjoldager, Anne Gram; Gottlieb, Henrik; Klitgård, Ida

    Understanding Translation is designed as a textbook for courses on the theory and practice of translation in general and of particular types of translation - such as interpreting, screen translation and literary translation. The aim of the book is to help you gain an in-depth understanding of the...... - translators, language teachers, translation users and literary, TV and film critics, for instance. Discussions focus on translation between Danish and English....

  13. Regulation of Ribosomal Protein Operons rplM-rpsI, rpmB-rpmG, and rplU-rpmA at the Transcriptional and Translational Levels.

    Science.gov (United States)

    Aseev, Leonid V; Koledinskaya, Ludmila S; Boni, Irina V

    2016-09-15

    It is widely assumed that in the best-characterized model bacterium Escherichia coli, transcription units encoding ribosomal proteins (r-proteins) and regulation of their expression have been already well defined. However, transcription start sites for several E. coli r-protein operons have been established only very recently, so that information concerning the regulation of these operons at the transcriptional or posttranscriptional level is still missing. This paper describes for the first time the in vivo regulation of three r-protein operons, rplM-rpsI, rpmB-rpmG, and rplU-rpmA The results demonstrate that transcription of all three operons is subject to ppGpp/DksA-dependent negative stringent control under amino acid starvation, in parallel with the rRNA operons. By using single-copy translational fusions with the chromosomal lacZ gene, we show here that at the translation level only one of these operons, rplM-rpsI, is regulated by the mechanism of autogenous repression involving the 5' untranslated region (UTR) of the operon mRNA, while rpmB-rpmG and rplU-rpmA are not subject to this type of regulation. This may imply that translational feedback control is not a general rule for modulating the expression of E. coli r-protein operons. Finally, we report that L13, a primary protein in 50S ribosomal subunit assembly, serves as a repressor of rplM-rpsI expression in vivo, acting at a target within the rplM translation initiation region. Thus, L13 represents a novel example of regulatory r-proteins in bacteria. It is important to obtain a deeper understanding of the regulatory mechanisms responsible for coordinated and balanced synthesis of ribosomal components. In this paper, we highlight the major role of a stringent response in regulating transcription of three previously unexplored r-protein operons, and we show that only one of them is subject to feedback regulation at the translational level. Improved knowledge of the regulatory pathways controlling ribosome

  14. Role of Hypoxia-inducible factor-1 and its target genes in human lung adenocarcinoma cells after photon- versus carbon ion irradiation; Expression HIF-1-abhaengiger Gene in humanen Lungenadenokarzinom (A549)-Zellen und deren Regulation nach Photonen- und Schwerionenbestrahlung

    Energy Technology Data Exchange (ETDEWEB)

    Bill, Verena Maria

    2013-11-26

    Exposed to hypoxia tumor cells are notably resistant to photon irradiation. The hypoxiainducible transcription factor 1α (HIF-1α) seems to play a fundamental role in this resistance, while its role after heavy-ion beam remains unknown. The intention of this study was to determine how A549-cells (non-small-cell lung carcinoma) react in different oxygenation states after irradiation with photons or heavy ions, particularly in regards to their expression of HIF-1 target genes. Resistance of hypoxic A549 cells after photon irradiation was documented by cellular and clonogenic survival. In contrast, cellular survival after heavy-ion irradiation in hypoxic cells was not elevated to normoxic cells. Among the oxygen dependent regulation of HIF-1 target genes, gene expression analyses showed an increased expression of GLUT-1, LDH-A, PDK-1 and VEGF after photon irradiation but not after heavy-ion irradiation after 48 hours in normoxic cells. As expected, CDKN1A as inhibitor of cell cycle progression showed higher expression after both radiation forms; interestingly CDKN1A was also in an oxygen dependent manner lightly upregulated. In western blot analyses we demonstrated a significant increase of HIF-1 and GLUT-1 caused by hypoxia, but only a tendency of increased protein level in hypoxia after photon irradiation and no changes after heavy-ion irradiation. Significantly higher protein level of secreted VEGF-A could be measured 72 hours after photon irradiation in normoxic cells by ELISA analyses. Controversially discussed, I could not detect an association between HIF-1 and SCF or Trx-1 in A549-cells in this study. Whereas Trx-1-expression was neither influenced by changed oxygen partial pressure nor irradiation, I could show increased SCF mRNA by quantitative Real Time-PCR and secreted protein level by ELISA after photon irradiation independent of oxygen state. In summary, this study showed that HIF-1 and its target genes (GLUT-1, LDHA; PDK, VEGF) and also SCF was

  15. Post-translational regulation of miRNA pathway components, AGO1 and HYL1, in plants

    DEFF Research Database (Denmark)

    Cho, Seok Keun; Ryu, Moon Young; Shah, Pratik

    2016-01-01

    Post-translational modifications (PTMs) of proteins are essential to increase the functional diversity of the proteome. By adding chemical groups to proteins, or degrading entire proteins by phosphorylation, glycosylation, ubiquitination, neddylation, acetylation, lipidation, and proteolysis...

  16. DUBs, new members in the hypoxia signaling clUb

    Directory of Open Access Journals (Sweden)

    Amelie S Schober

    2016-03-01

    Full Text Available Cellular protein homeostasis is tightly regulated by ubiquitination. Responsible for target protein ubiquitination is a class of enzymes, the so-called ubiquitin (Ub E3-ligases. They are opposed to a second class of enzymes, called DeUBiquitinating enzymes (DUBs, which can remove polyubiquitin chains from their specific target proteins. The coaction of the two sets of enzymes allow the cell to adapt its overall protein content and the abundance of particular proteins to a variety of cellular and environmental stresses, including hypoxia. In recent years, DUBs have been highlighted to play major roles in many diseases, including cancer, both as tumor suppressors and oncogenes. Therefore, DUBs are emerging as promising targets for cancer-cell specific treatment. Here, we will review the current understanding of DUBs implicated in the control of hypoxia-inducible factor (HIF, the regulation of DUBs by hypoxia and the use of DUB-specific drugs to target tumor hypoxia signaling.

  17. Activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) during hypoxia in cerebral cortical nuclei of guinea pig fetus at term: role of nitric oxide.

    Science.gov (United States)

    Maulik, Dev; Ashraf, Qazi M; Mishra, Om P; Delivoria-Papadopoulos, Maria

    2008-07-04

    Previously we have shown that cerebral tissue hypoxia results in generation of nitric oxide (NO) free radicals as well as increased expression of mitogen-activated protein kinase like extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK). The present study tested the hypothesis that administration of l-nitro-l-arginine methyl ester (L-NAME), a NOS inhibitor, prior to hypoxia prevents the hypoxia-induced activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) and in the cerebral cortex of the term guinea pig fetus. To test this hypothesis normoxic (Nx, n=6), hypoxic (Hx, n=7) and hypoxic pretreated with l-NAME (Hx+L-NAME, n=6) guinea pig fetuses at 60 days gestation were studied to determine the phosphorylated p38, ERK and JNK. Hypoxia was induced by exposing pregnant guinea pigs to FiO2 of 0.07 for 1h. l-NAME (30mg/kg i.p.) was administered to pregnant mothers 60min prior to hypoxia. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Neuronal nuclei were isolated, purified and proteins separated using 12% SDS-PAGE, and then probed with specific phosphorylated ERK, JNK and p38 antibodies. Protein bands were detected by enhanced chemiluminescence, analyzed by imaging densitometry and expressed as absorbance (ODxmm2). The relative level of p-p38 was 51.41+/-9.80 (Nx), 173.67+/-3.63 (Hx), 58.56+/-3.40 (Hx+L-NAME), phypoxia decreased the relative level of phosphorylated p38, ERK and JNK at term gestation. Since a NOS inhibitor prevented the hypoxia-induced phosphorylation of p38, ERK and JNK, we conclude that the hypoxia-induced activation of p38, ERK and JNK in the cerebral cortical nuclei of guinea pig fetus at term is NO-mediated. We speculate that NO-mediated modification of cysteine residue leading to inhibition of MAP kinase phosphatases results in increased activation of p38, ERK and JNK

  18. Identification of hypoxia-induced genes in human SGBS adipocytes by microarray analysis.

    Directory of Open Access Journals (Sweden)

    Kathrin Geiger

    Full Text Available Hypoxia in adipose tissue is suggested to be involved in the development of a chronic mild inflammation, which in obesity can further lead to insulin resistance. The effect of hypoxia on gene expression in adipocytes appears to play a central role in this inflammatory response observed in obesity. However, the global impact of hypoxia on transcriptional changes in human adipocytes is unclear. Therefore, we compared gene expression profiles of human Simpson-Golabi-Behmel syndrome (SGBS adipocytes under normoxic or hypoxic conditions to detect hypoxia-responsive genes in adipocytes by using whole human genome microarrays. Microarray analysis showed more than 500 significantly differentially regulated mRNAs after incubation of the cells under low oxygen levels. To gain further insight into the biological processes, hypoxia-regulated genes after 16 hours of hypoxia were classified according to their function. We identified an enrichment of genes involved in important biological processes such as glycolysis, response to hypoxia, regulation of cellular component movement, response to nutrient levels, regulation of cell migration, and transcription regulator activity. Real-time PCR confirmed eight genes to be consistently upregulated in response to 3, 6 and 16 hours of hypoxia. For adipocytes the hypoxia-induced regulation of these genes is shown here for the first time. Moreover in six of these eight genes we identified HIF response elements in the proximal promoters, specific for the HIF transcription factor family members HIF1A and HIF2A. In the present study, we demonstrated that hypoxia has an extensive effect on gene expression of SGBS adipocytes. In addition, the identified hypoxia-regulated genes are likely involved in the regulation of obesity, the incidence of type 2 diabetes, and the metabolic syndrome.

  19. Comparative and Experimental Studies on the Genes Altered by Chronic Hypoxia in Human Brain Microendothelial Cells

    Directory of Open Access Journals (Sweden)

    Eugenia Mata-Greenwood

    2017-05-01

    Full Text Available Background : Hypoxia inducible factor 1 alpha (HIF1A is a master regulator of acute hypoxia; however, with chronic hypoxia, HIF1A levels return to the normoxic levels. Importantly, the genes that are involved in the cell survival and viability under chronic hypoxia are not known. Therefore, we tested the hypothesis that chronic hypoxia leads to the upregulation of a core group of genes with associated changes in the promoter DNA methylation that mediates the cell survival under hypoxia.Results : We examined the effect of chronic hypoxia (3 days; 0.5% oxygen on human brain micro endothelial cells (HBMEC viability and apoptosis. Hypoxia caused a significant reduction in cell viability and an increase in apoptosis. Next, we examined chronic hypoxia associated changes in transcriptome and genome-wide promoter methylation. The data obtained was compared with 16 other microarray studies on chronic hypoxia. Nine genes were altered in response to chronic hypoxia in all 17 studies. Interestingly, HIF1A was not altered with chronic hypoxia in any of the studies. Furthermore, we compared our data to three other studies that identified HIF-responsive genes by various approaches. Only two genes were found to be HIF dependent. We silenced each of these 9 genes using CRISPR/Cas9 system. Downregulation of EGLN3 significantly increased the cell death under chronic hypoxia, whereas downregulation of ERO1L, ENO2, adrenomedullin, and spag4 reduced the cell death under hypoxia.Conclusions : We provide a core group of genes that regulates cellular acclimatization under chronic hypoxic stress, and most of them are HIF independent.

  20. PKCδ Regulates Translation Initiation through PKR and eIF2α in Response to Retinoic Acid in Acute Myeloid Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Bulent Ozpolat

    2012-01-01

    Full Text Available Translation initiation and activity of eukaryotic initiation factor-alpha (eIF2α, the rate-limiting step of translation initiation, is often overactivated in malignant cells. Here, we investigated the regulation and role of eIF2α in acute promyelocytic (APL and acute myeloid leukemia (AML cells in response to all-trans retinoic acid (ATRA and arsenic trioxide (ATO, the front-line therapies in APL. ATRA and ATO induce Ser-51 phosphorylation (inactivation of eIF2α, through the induction of protein kinase C delta (PKCδ and PKR, but not other eIF2α kinases, such as GCN2 and PERK in APL (NB4 and AML cells (HL60, U937, and THP-1. Inhibition of eIF2α reduced the expression of cellular proteins that are involved in apoptosis (DAP5/p97, cell cycle (p21Waf1/Cip1, differentiation (TG2 and induced those regulating proliferation (c-myc and survival (p70S6K. PI3K/Akt/mTOR pathway is involved in regulation of eIF2α through PKCδ/PKR axis. PKCδ and p-eIF2α protein expression levels revealed a significant association between the reduced levels of PKCδ (P=0.0378 and peIF2 (P=0.0041 and relapses in AML patients (n=47. In conclusion, our study provides the first evidence that PKCδ regulates/inhibits eIF2α through induction of PKR in AML cells and reveals a novel signaling mechanism regulating translation initiation.

  1. Understanding Translation

    DEFF Research Database (Denmark)

    Schjoldager, Anne Gram; Gottlieb, Henrik; Klitgård, Ida

    Understanding Translation is designed as a textbook for courses on the theory and practice of translation in general and of particular types of translation - such as interpreting, screen translation and literary translation. The aim of the book is to help you gain an in-depth understanding...... of the phenomenon of translation and to provide you with a conceptual framework for the analysis of various aspects of professional translation. Intended readers are students of translation and languages, but the book will also be relevant for others who are interested in the theory and practice of translation...

  2. The utilization of selenocysteine-tRNA([Ser]Sec) isoforms is regulated in part at the level of translation in vitro.

    Science.gov (United States)

    Carlson, Bradley A; Gupta, Nirupama; Pinkerton, Mark H; Hatfield, Dolph L; Copeland, Paul R

    2017-01-01

    The tRNA for the 21st proteinogenic amino acid, selenocysteine, exists in mammalian cells as 2 isoforms differing by a single 2'-O-methylribosyl moiety at position 34 (Um34). These isoforms contain either 5-methoxycarbonylmethyluridine (mcm(5)U) or 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm(5)Um) at position 34. The accumulation of the mcm(5)Um isoform is tightly correlated with the expression of nonessential "stress response" selenoproteins such as glutathione peroxidase 1 (GPX1). The expression of essential selenoproteins, such as thioredoxin reductase 1 (TXNRD1), is not affected by changes in Sec-tRNA([Ser]Sec) isoform accumulation. In this work we used purified mcm(5)U and mcm(5)Um Sec-tRNA([Ser]Sec) isoforms to analyze possible differences in binding to the selenocysteine-specific elongation factor, EEFSEC, and the translation of GPX1 and TXNRD1in vitro. Our results indicate that no major distinction between mcm(5)U and mcm(5)Um isoforms is made by the translation machinery, but a small consistent increase in GPX1 translation is associated with the mcm(5)Um isoform. These results implicate fundamental differences in translation efficiency in playing a role in regulating selenoprotein expression as a function of isoform accumulation.

  3. Hypoxia and Angiogenesis in Endometrioid Endometrial Carcinogenesis

    Directory of Open Access Journals (Sweden)

    Nicole Horrée

    2007-01-01

    Full Text Available Background: Hypoxia-inducible factor 1α (HIF-1α plays an essential role in the adaptive response of cells to hypoxia, triggering biologic events associated with aggressive tumor behavior. Methods: Expression of HIF-1α and proteins in the HIF-1α pathway (Glut-1, CAIX, VEGF in paraffin-embedded specimens of normal (n = 17, premalignant (n = 17 and endometrioid endometrial carcinoma (n = 39 was explored by immunohistochemistry, in relation to microvessel density (MVD. Results: HIF-1α overexpression was absent in inactive endometrium but present in hyperplasia (61% and carcinoma (87%, with increasing expression in a perinecrotic fashion pointing to underlying hypoxia. No membranous expression of Glut-1 and CAIX was noticed in inactive endometrium, in contrast with expression in hyperplasia (Glut-1 0%, CAIX 61%, only focal and diffuse and carcinoma (Glut-1 94.6%, CAIX 92%, both mostly perinecrotically. Diffuse HIF-1α was accompanied by activation of downstream targets. VEGF was significantly higher expressed in hyperplasias and carcinomas compared to inactive endometrium. MVD was higher in hyperplasias and carcinomas than in normal endometrium (p < 0.001. Conclusion: HIF-1α and its downstream genes are increasingly expressed from normal through premalignant to endometrioid adenocarcinoma of the endometrium, paralleled by activation of its downstream genes and increased angiogenesis. This underlines the potential importance of hypoxia and its key regulator HIF-1α in endometrial carcinogenesis.

  4. The Translation Factor eIF6 Is a Notch-Dependent Regulator of Cell Migration and Invasion

    OpenAIRE

    Dario Benelli; Samantha Cialfi; Michela Pinzaglia; Claudio Talora; Paola Londei

    2012-01-01

    A growing body of evidence indicates that protein factors controlling translation play an important role in tumorigenesis. The protein known as eIF6 is a ribosome anti-association factor that has been implicated in translational initiation and in ribosome synthesis. Over-expression of eIF6 is observed in many natural tumours, and causes developmental and differentiation defects in certain animal models. Here we show that the transcription of the gene encoding eIF6 is modulated by the receptor...

  5. Upstream open reading frames of the barley Mla13 powdery mildew resistance gene function co-operatively to down-regulate translation.

    Science.gov (United States)

    Halterman, Dennis A; Wise, Roger P

    2006-05-01

    SUMMARY To protect themselves against pathogen infection, plants have evolved multifarious defence mechanisms that include the production of antimicrobial compounds and hypersensitive cell death. This defence includes the regulation of expression of resistance proteins, which function to detect pathogen effectors and control the response. Barley Mla is one of several Ml genes that confer specific resistance to the powdery mildew fungus, Blumeria graminis f. sp. hordei. The Mla13 allele contains a long (523 nt) transcript leader region with three upstream open reading frames (uORFs) and two alternatively spliced introns. The lengths of the Mla13 uORFs, which encode up to 70 amino acid peptides, are directly influenced by the splicing of these introns. We have used in vivo and in vitro translation assays to show that Mla13 uORFs function co-operatively to down-regulate translation of the parent cistron up to 13-fold. However, resistance conferred by constitutively transcribed Mla appears to be unaffected by removal of these uORFs, signifying that a potential increase in MLA13 protein is innocuous to barley cells. In addition, cells that possess a predicted increase in MLA13 translation via over-expression and uORF mutation retain their requirement for RAR1, suggesting that this increase is not sufficient to overcome the concentration threshold required for activation of RAR1-independent resistance.

  6. Factors regulated by interferon gamma and hypoxia-inducible factor 1A contribute to responses that protect mice from Coccidioides immitis infection

    Science.gov (United States)

    2012-01-01

    Background Coccidioidomycosis results from airborne infections caused by either Coccidioides immitis or C. posadasii. Both are pathogenic fungi that live in desert soil in the New World and can infect normal hosts, but most infections are self-limited. Disseminated infections occur in approximately 5% of cases and may prove fatal. Mouse models of the disease have identified strains that are resistant (e.g. DBA/2) or susceptible (e.g. C57BL/6) to these pathogens. However, the genetic and immunological basis for this difference has not been fully characterized. Results Microarray technology was used to identify genes that were differentially expressed in lung tissue between resistant DBA/2 and sensitive C57BL/6 mice after infection with C. immitis. Differentially expressed genes were mapped onto biological pathways, gene ontologies, and protein interaction networks, which revealed that innate immune responses mediated by Type II interferon (i.e., IFNG) and the signal transducer and activator of transcription 1 (STAT1) contribute to the resistant phenotype. In addition, upregulation of hypoxia inducible factor 1A (HIF1A), possibly as part of a larger inflammatory response mediated by tumor necrosis factor alpha (TNFA), may also contribute to resistance. Microarray gene expression was confirmed by real-time quantitative PCR for a subset of 12 genes, which revealed that IFNG HIF1A and TNFA, among others, were significantly differentially expressed between the two strains at day 14 post-infection. Conclusion These results confirm the finding that DBA/2 mice express more Type II interferon and interferon stimulated genes than genetically susceptible strains and suggest that differential expression of HIF1A may also play a role in protection. PMID:23006927

  7. Factors regulated by interferon gamma and hypoxia-inducible factor 1A contribute to responses that protect mice from Coccidioides immitis infection

    Directory of Open Access Journals (Sweden)

    Woelk Christopher H

    2012-09-01

    Full Text Available Abstract Background Coccidioidomycosis results from airborne infections caused by either Coccidioides immitis or C. posadasii. Both are pathogenic fungi that live in desert soil in the New World and can infect normal hosts, but most infections are self-limited. Disseminated infections occur in approximately 5% of cases and may prove fatal. Mouse models of the disease have identified strains that are resistant (e.g. DBA/2 or susceptible (e.g. C57BL/6 to these pathogens. However, the genetic and immunological basis for this difference has not been fully characterized. Results Microarray technology was used to identify genes that were differentially expressed in lung tissue between resistant DBA/2 and sensitive C57BL/6 mice after infection with C. immitis. Differentially expressed genes were mapped onto biological pathways, gene ontologies, and protein interaction networks, which revealed that innate immune responses mediated by Type II interferon (i.e., IFNG and the signal transducer and activator of transcription 1 (STAT1 contribute to the resistant phenotype. In addition, upregulation of hypoxia inducible factor 1A (HIF1A, possibly as part of a larger inflammatory response mediated by tumor necrosis factor alpha (TNFA, may also contribute to resistance. Microarray gene expression was confirmed by real-time quantitative PCR for a subset of 12 genes, which revealed that IFNG HIF1A and TNFA, among others, were significantly differentially expressed between the two strains at day 14 post-infection. Conclusion These results confirm the finding that DBA/2 mice express more Type II interferon and interferon stimulated genes than genetically susceptible strains and suggest that differential expression of HIF1A may also play a role in protection.

  8. Factors regulated by interferon gamma and hypoxia-inducible factor 1A contribute to responses that protect mice from Coccidioides immitis infection.

    Science.gov (United States)

    Woelk, Christopher H; Zhang, Jin X; Walls, Lorraine; Viriyakosol, Suganya; Singhania, Akul; Kirkland, Theo N; Fierer, Joshua

    2012-09-24

    Coccidioidomycosis results from airborne infections caused by either Coccidioides immitis or C. posadasii. Both are pathogenic fungi that live in desert soil in the New World and can infect normal hosts, but most infections are self-limited. Disseminated infections occur in approximately 5% of cases and may prove fatal. Mouse models of the disease have identified strains that are resistant (e.g. DBA/2) or susceptible (e.g. C57BL/6) to these pathogens. However, the genetic and immunological basis for this difference has not been fully characterized. Microarray technology was used to identify genes that were differentially expressed in lung tissue between resistant DBA/2 and sensitive C57BL/6 mice after infection with C. immitis. Differentially expressed genes were mapped onto biological pathways, gene ontologies, and protein interaction networks, which revealed that innate immune responses mediated by Type II interferon (i.e., IFNG) and the signal transducer and activator of transcription 1 (STAT1) contribute to the resistant phenotype. In addition, upregulation of hypoxia inducible factor 1A (HIF1A), possibly as part of a larger inflammatory response mediated by tumor necrosis factor alpha (TNFA), may also contribute to resistance. Microarray gene expression was confirmed by real-time quantitative PCR for a subset of 12 genes, which revealed that IFNG HIF1A and TNFA, among others, were significantly differentially expressed between the two strains at day 14 post-infection. These results confirm the finding that DBA/2 mice express more Type II interferon and interferon stimulated genes than genetically susceptible strains and suggest that differential expression of HIF1A may also play a role in protection.

  9. Understanding Translation

    DEFF Research Database (Denmark)

    Schjoldager, Anne Gram; Gottlieb, Henrik; Klitgård, Ida

    Understanding Translation is designed as a textbook for courses on the theory and practice of translation in general and of particular types of translation - such as interpreting, screen translation and literary translation. The aim of the book is to help you gain an in-depth understanding...... of the phenomenon of translation and to provide you with a conceptual framework for the analysis of various aspects of professional translation. Intended readers are students of translation and languages, but the book will also be relevant for others who are interested in the theory and practice of translation...... - translators, language teachers, translation users and literary, TV and film critics, for instance. Discussions focus on translation between Danish and English....

  10. Cyclin-dependent kinase five mediates activation of lung xanthine oxidoreductase in response to hypoxia.

    Directory of Open Access Journals (Sweden)

    Bo S Kim

    Full Text Available Xanthine oxidoreductase (XOR is involved in oxidative metabolism of purines and is a source of reactive oxygen species (ROS. As such, XOR has been implicated in oxidant-mediated injury in multiple cardiopulmonary diseases. XOR enzyme activity is regulated, in part, via a phosphorylation-dependent, post-translational mechanism, although the kinase(s responsible for such hyperactivation are unknown.Using an in silico approach, we identified a cyclin-dependent kinase 5 (CDK5 consensus motif adjacent to the XOR flavin adenine dinucleotide (FAD binding domain. CDK5 is a proline-directed serine/threonine kinase historically linked to neural development and injury. We tested the hypothesis that CDK5 and its activators are mediators of hypoxia-induced hyperactivation of XOR in pulmonary microvascular endothelial cells (EC and the intact murine lung. Using complementary molecular and pharmacologic approaches, we demonstrated that hypoxia significantly increased CDK5 activity in EC. This was coincident with increased expression of the CDK5 activators, cyclin-dependent kinase 5 activator 1 (CDK5r1 or p35/p25, and decreased expression of the CDK5 inhibitory peptide, p10. Expression of p35/p25 was necessary for XOR hyperactivation. Further, CDK5 physically associated with XOR and was necessary and sufficient for XOR phosphorylation and hyperactivation both in vitro and in vivo. XOR hyperactivation required the target threonine (T222 within the CDK5-consensus motif.These results indicate that p35/CDK5-mediated phosphorylation of T222 is required for hypoxia-induced XOR hyperactivation in the lung. Recognizing the contribution of XOR to oxidative injury in cardiopulmonary disease, these observations identify p35/CDK5 as novel regulators of XOR and potential modifiers of ROS-mediated injury.

  11. Altered expression of hypoxia-inducible factor-1α (HIF-1α and its regulatory genes in gastric cancer tissues.

    Directory of Open Access Journals (Sweden)

    Jihan Wang

    Full Text Available Tissue hypoxia induces reprogramming of cell metabolism and may result in normal cell transformation and cancer progression. Hypoxia-inducible factor 1-alpha (HIF-1α, the key transcription factor, plays an important role in gastric cancer development and progression. This study aimed to investigate the underlying regulatory signaling pathway in gastric cancer using gastric cancer tissue specimens. The integration of gene expression profile and transcriptional regulatory element database (TRED was pursued to identify HIF-1α ↔ NFκB1 → BRCA1 → STAT3 ← STAT1 gene pathways and their regulated genes. The data showed that there were 82 differentially expressed genes that could be regulated by these five transcription factors in gastric cancer tissues and these genes formed 95 regulation modes, among which seven genes (MMP1, TIMP1, TLR2, FCGR3A, IRF1, FAS, and TFF3 were hub molecules that are regulated at least by two of these five transcription factors simultaneously and were associated with hypoxia, inflammation, and immune disorder. Real-Time PCR and western blot showed increasing of HIF-1α in mRNA and protein levels as well as TIMP1, TFF3 in mRNA levels in gastric cancer tissues. The data are the first study to demonstrate HIF-1α-regulated transcription factors and their corresponding network genes in gastric cancer. Further study with a larger sample size and more functional experiments is needed to confirm these data and then translate into clinical biomarker discovery and treatment strategy for gastric cancer.

  12. Sympathetic Vasoconstrictor Responsiveness of the Leg Vasculature During Experimental Endotoxemia and Hypoxia in Humans

    DEFF Research Database (Denmark)

    Brassard, Patrice; Zaar, Morten; Thaning, Pia

    2016-01-01

    OBJECTIVE: Sympathetic vasoconstriction regulates peripheral circulation and controls blood pressure, but sepsis is associated with hypotension. We evaluated whether apparent loss of sympathetic vasoconstrictor responsiveness relates to distended smooth muscles or to endotoxemia and/or hypoxia...... was calculated during 1) adenosine infusion (vasodilator control), 2) hypoxia (FIO2 = 10%), 3) endotoxemia, and 4) endotoxemia + hypoxia. Leg sympathetic vasoconstrictor responsiveness (reduction in leg vascular conductance) was evaluated by femoral artery tyramine infusion. MEASUREMENTS AND MAIN RESULTS...

  13. Machine Translation

    Institute of Scientific and Technical Information of China (English)

    张严心

    2015-01-01

    As a kind of ancillary translation tool, Machine Translation has been paid increasing attention to and received different kinds of study by a great deal of researchers and scholars for a long time. To know the definition of Machine Translation and to analyse its benefits and problems are significant for translators in order to make good use of Machine Translation, and helpful to develop and consummate Machine Translation Systems in the future.

  14. A preclinical model for noninvasive imaging of hypoxia-induced gene expression; comparison with an exogenous marker of tumor hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Wen Bixiu; Burgman, Paul; Zanzonico, Pat; O' Donoghue, Joseph; Li, Gloria C.; Ling, C. Clifton [Memorial Sloan-Kettering Cancer Center, Department of Medical Physics, New York (United States); Cai Shangde; Finn, Ron [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York (United States); Serganova, Inna [Memorial Sloan-Kettering Cancer Center, Department of Neurology, New York (United States); Blasberg, Ronald; Gelovani, Juri [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York (United States); Memorial Sloan-Kettering Cancer Center, Department of Neurology, New York (United States)

    2004-11-01

    Hypoxia is associated with tumor aggressiveness and is an important cause of resistance to radiation therapy and chemotherapy. Assays of tumor hypoxia could provide selection tools for hypoxia-modifying treatments. The purpose of this study was to develop and characterize a rodent tumor model with a reporter gene construct that would be transactivated by the hypoxia-inducible molecular switch, i.e., the upregulation of HIF-1. The reporter gene construct is the herpes simplex virus 1-thymidine kinase (HSV1-tk) fused with the enhanced green fluorescent protein (eGFP) under the regulation of an artificial hypoxia-responsive enhancer/promoter. In this model, tumor hypoxia would up-regulate HIF-1, and through the hypoxia-responsive promoter transactivate the HSV1-tkeGFPfusion gene. The expression of this reporter gene can be assessed with the {sup 124}I-labeled reporter substrate 2'-fluoro-2'-deoxy-1-{beta}-d-arabinofuranosyl-5-iodouracil ({sup 124}I-FIAU), which is phosphorylated by the HSV1-tk enzyme and trapped in the hypoxic cells. Animal positron emission tomography (microPET) and phosphor plate imaging (PPI) were used in this study to visualize the trapped {sup 124}I-FIAU, providing a distribution of the hypoxia-induced molecular events. The distribution of {sup 124}I-FIAU was also compared with that of an exogenous hypoxic cell marker, {sup 18}F-fluoromisonidazole (FMISO). Our results showed that {sup 124}I-FIAU microPET imaging of the hypoxia-induced reporter gene expression is feasible, and that the intratumoral distributions of {sup 124}I-FIAU and {sup 18}F-FMISO are similar. In tumor sections, detailed radioactivity distributions were obtained with PPI which also showed similarity between {sup 124}I-FIAU and {sup 18}F-FMISO. This reporter system is sufficiently sensitive to detect hypoxia-induced transcriptional activation by noninvasive imaging and might provide a valuable tool in studying tumor hypoxia and in validating existing and future

  15. Hypoxia inhibits colonic ion transport via activation of AMP kinase.

    LENUS (Irish Health Repository)

    Collins, Danielle

    2012-02-01

    BACKGROUND AND AIMS: Mucosal hypoxia is a common endpoint for many pathological processes including ischemic colitis, colonic obstruction and anastomotic failure. Previous studies suggest that hypoxia modulates colonic mucosal function through inhibition of chloride secretion. However, the molecular mechanisms underlying this observation are poorly understood. AMP-activated protein kinase (AMPK) is a metabolic energy regulator found in a wide variety of cells and has been linked to cystic fibrosis transmembrane conductance regulator (CFTR) mediated chloride secretion in several different tissues. We hypothesized that AMPK mediates many of the acute effects of hypoxia on human and rat colonic electrolyte transport. METHODS: The fluorescent chloride indicator dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide was used to measure changes in intracellular chloride concentrations in isolated single rat colonic crypts. Ussing chamber experiments in human colonic mucosa were conducted to evaluate net epithelial ion transport. RESULTS: This study demonstrates that acute hypoxia inhibits electrogenic chloride secretion via AMPK mediated inhibition of CFTR. Pre-treatment of tissues with the AMPK inhibitor 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyyrazolo [1,5-a] pyrimidine (compound C) in part reversed the effects of acute hypoxia on chloride secretion. CONCLUSION: We therefore suggest that AMPK is a key component of the adaptive cellular response to mucosal hypoxia in the colon. Furthermore, AMPK may represent a potential therapeutic target in diseased states or in prevention of ischemic intestinal injury.

  16. Ethics of translational medical research.

    Science.gov (United States)

    Mandal, Jharna; Ponnambath, Dinoop Korol; Parija, Subhash Chandra

    2017-01-01

    Translational research is the science of application of basic research knowledge to develop novel methods of improving public health outcome. This review briefly discusses the various phases involved and the hurdles faced in the translational pathway, how translational science originated, and the infrastructures consigned for translational research. Translational research also faces numerous diverse ethical issues similar to those observed in biomedical research. The various types of ethical issues faced whereas conducting translational research are discussed briefly in this review to help researchers identify and prevent unethical practices while conducting or regulating translational research.

  17. Translatress, Translator, Translation

    OpenAIRE

    Margala, Miriam

    2009-01-01

    I still manage to surprise a few scholars from other fields when they hear that there is such a thing as research of gender issues within the field of translation studies. It may seem as such a narrow niche – but only deceivingly so. It is language, linguistics, pragmatics, culture, history, literature, anthropology, gender metaphorics, communication, interpreting, cultural politics, social studies and politics, psychology and I can go on and on. History seems to be a very appropriate startin...

  18. Intermittent hypoxia and neurorehabilitation

    Science.gov (United States)

    Gonzalez-Rothi, Elisa J.; Lee, Kun-Ze; Dale, Erica A.; Reier, Paul J.; Mitchell, Gordon S.

    2015-01-01

    In recent years, it has become clear that brief, repeated presentations of hypoxia [i.e., acute intermittent hypoxia (AIH)] can boost the efficacy of more traditional therapeutic strategies in certain cases of neurologic dysfunction. This hypothesis derives from a series of studies in animal models and human subjects performed over the past 35 yr. In 1980, Millhorn et al. (Millhorn DE, Eldridge FL, Waldrop TG. Respir Physiol 41: 87-103, 1980) showed that electrical stimulation of carotid chemoafferent neurons produced a persistent, serotonin-dependent increase in phrenic motor output that outlasts the stimulus for more than 90 min (i.e., a “respiratory memory”). AIH elicits similar phrenic “long-term facilitation” (LTF) by a mechanism that requires cervical spinal serotonin receptor activation and de novo protein synthesis. From 2003 to present, a series of studies demonstrated that AIH can induce neuroplasticity in the injured spinal cord, causing functional recovery of breathing capacity after cervical spinal injury. Subsequently, it was demonstrated that repeated AIH (rAIH) can induce recovery of limb function, and the functional benefits of rAIH are greatest when paired with task-specific training. Since uncontrolled and/or prolonged intermittent hypoxia can elicit pathophysiology, a challenge of intermittent hypoxia research is to ensure that therapeutic protocols are well below the threshold for pathogenesis. This is possible since many low dose rAIH protocols have induced functional benefits without evidence of pathology. We propose that carefully controlled rAIH is a safe and noninvasive modality that can be paired with other neurorehabilitative strategies including traditional activity-based physical therapy or cell-based therapies such as intraspinal transplantation of neural progenitors. PMID:25997947

  19. Quantifying circulating hypoxia-induced RNA transcripts in maternal blood to determine in utero fetal hypoxic status.

    Science.gov (United States)

    Whitehead, Clare; Teh, Wan Tinn; Walker, Susan P; Leung, Cheryl; Mendis, Sonali; Larmour, Luke; Tong, Stephen

    2013-12-09

    Hypoxia in utero can lead to stillbirth and severe perinatal injury. While current prenatal tests can identify fetuses that are hypoxic, none can determine the severity of hypoxia/acidemia. We hypothesized a hypoxic/acidemic fetus would up-regulate and release hypoxia-induced mRNA from the fetoplacental unit into the maternal circulation, where they can be sampled and quantified. Furthermore, we hypothesized the abundance of hypoxia induced mRNA in the maternal circulation would correlate with severity of fetal hypoxia/acidemia in utero. We therefore examined whether abundance of hypoxia-induced mRNA in the maternal circulation correlates with the degree of fetal hypoxia in utero. We performed a prospective study of two cohorts: 1) longitudinal study of pregnant women undergoing an induction of labor (labor induces acute fetal hypoxia) and 2) pregnancies complicated by severe preterm growth restriction (chronic fetal hypoxia). For each cohort, we correlated hypoxia induced mRNA in the maternal blood with degree of fetal hypoxia during its final moments in utero, evidenced by umbilical artery pH or lactate levels obtained at birth. Gestational tissues and maternal bloods were sampled and mRNAs quantified by microarray and RT-PCR. Hypoxia-induced mRNAs in maternal blood rose across labor, an event that induces acute fetal hypoxia. They exhibited a precipitous increase across the second stage of labor, a particularly hypoxic event. Importantly, a hypoxia gene score (sum of the relative expression of four hypoxia-induced genes) strongly correlated with fetal acidemia at birth. Hypoxia-induced mRNAs were also increased in the blood of women carrying severely growth restricted preterm fetuses, a condition of chronic fetal hypoxia. The hypoxia gene score correlated with the severity of ultrasound Doppler velocimetry abnormalities in fetal vessels. Importantly, the hypoxia gene score (derived from mRNA abundance in maternal blood) was significantly correlated with the

  20. Characterization of CgHIFα-Like, a Novel bHLH-PAS Transcription Factor Family Member, and Its Role under Hypoxia Stress in the Pacific Oyster Crassostrea gigas.

    Science.gov (United States)

    Wang, Ting; Meng, Jie; Li, Li; Zhang, Guofan

    2016-01-01

    Hypoxia-inducible factor (HIF), a critical member of the basic-helix-loop-helix (bHLH)-containing Per-Arnt-Sim (PAS) protein family, is a master transcription factor involved in maintaining oxygen homeostasis. In the present study, we isolated and characterized a novel bHLH-PAS family member, CgHIFα-like gene, from the Pacific oyster Crassostrea gigas, and determined its importance during hypoxia stress. The 3020-bp CgHIFα-like cDNA encoded a protein of 888 amino acids. The predicted CgHIFα-like amino acid sequence was conserved in the N-terminal bHLH, PAS, and PAC domains (but not in the C-terminal domain) and was most closely related to the HIF family in the bHLH-PAS protein phylogenic tree. Similar to the mammalian HIF-1α, CgHIFα-like could be expressed as four mRNA isoforms containing alternative 5'-untranslated regions and different translation initiation codons. At the mRNA level, these isoforms were expressed in a tissue-specific manner and showed increased transcription to varying degrees under hypoxic conditions. Additionally, the western blot analysis demonstrated that CgHIFα-like was induced by hypoxia. Electrophoretic mobility shift assay indicated that CgHIFα-like could bind to the hypoxia responsive element (HRE), whereas dual-luciferase reporter analysis demonstrated that CgHIFα-like could transactivate the reporter gene containing the HREs. In addition to CgHIFα-like, we identified CgARNT from the C. gigas, analyzed its expression pattern, and confirmed its interaction with CgHIFα-like using a yeast two-hybrid assay. In conclusion, this is the first report on the cloning and characterization of a novel hypoxia transcription factor in mollusks, which could accumulate under hypoxia and regulate hypoxia related gene expression by binding to HRE and dimerizing with CgARNT. As only one member of HIF has been identified in invertebrates to date, our results provide new insights into the unique mechanisms of hypoxia tolerance in mollusks.

  1. Characterization of CgHIFα-Like, a Novel bHLH-PAS Transcription Factor Family Member, and Its Role under Hypoxia Stress in the Pacific Oyster Crassostrea gigas.

    Directory of Open Access Journals (Sweden)

    Ting Wang

    Full Text Available Hypoxia-inducible factor (HIF, a critical member of the basic-helix-loop-helix (bHLH-containing Per-Arnt-Sim (PAS protein family, is a master transcription factor involved in maintaining oxygen homeostasis. In the present study, we isolated and characterized a novel bHLH-PAS family member, CgHIFα-like gene, from the Pacific oyster Crassostrea gigas, and determined its importance during hypoxia stress. The 3020-bp CgHIFα-like cDNA encoded a protein of 888 amino acids. The predicted CgHIFα-like amino acid sequence was conserved in the N-terminal bHLH, PAS, and PAC domains (but not in the C-terminal domain and was most closely related to the HIF family in the bHLH-PAS protein phylogenic tree. Similar to the mammalian HIF-1α, CgHIFα-like could be expressed as four mRNA isoforms containing alternative 5'-untranslated regions and different translation initiation codons. At the mRNA level, these isoforms were expressed in a tissue-specific manner and showed increased transcription to varying degrees under hypoxic conditions. Additionally, the western blot analysis demonstrated that CgHIFα-like was induced by hypoxia. Electrophoretic mobility shift assay indicated that CgHIFα-like could bind to the hypoxia responsive element (HRE, whereas dual-luciferase reporter analysis demonstrated that CgHIFα-like could transactivate the reporter gene containing the HREs. In addition to CgHIFα-like, we identified CgARNT from the C. gigas, analyzed its expression pattern, and confirmed its interaction with CgHIFα-like using a yeast two-hybrid assay. In conclusion, this is the first report on the cloning and characterization of a novel hypoxia transcription factor in mollusks, which could accumulate under hypoxia and regulate hypoxia related gene expression by binding to HRE and dimerizing with CgARNT. As only one member of HIF has been identified in invertebrates to date, our results provide new insights into the unique mechanisms of hypoxia tolerance in

  2. Function and ribosomal localization of aIF6, a translational regulator shared by archaea and eukarya.

    Science.gov (United States)

    Benelli, Dario; Marzi, Stefano; Mancone, Carmine; Alonzi, Tonino; la Teana, Anna; Londei, Paola

    2009-01-01

    The translation factor IF6 is shared by the Archaea and the Eukarya, but is not found in Bacteria. The properties of eukaryal IF6 (eIF6) have been extensively studied, but remain somewhat elusive. eIF6 behaves as a ribosome-anti-association factor and is involved in miRNA-mediated gene silencing; however, it also seems to participate in ribosome synthesis and export. Here we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homologue (aIF6). We find that aIF6 binds specifically to the 50S ribosomal subunits, hindering the formation of 70S ribosomes and strongly inhibiting translation. aIF6 is uniformly expressed along the cell cycle, but it is upregulated following both cold- and heat shock. The aIF6 ribosomal binding site lies in the middle of the 30-S interacting surface of the 50S subunit, including a number of critical RNA and protein determinants involved in subunit association. The data suggest that the IF6 protein evolved in the archaeal-eukaryal lineage to modulate translational efficiency under unfavourable environmental conditions, perhaps acquiring additional functions during eukaryotic evolution.

  3. Hypoxia and Fetal Heart Development

    OpenAIRE

    Patterson, A.J.; Zhang, L

    2010-01-01

    Fetal hearts show a remarkable ability to develop under hypoxic conditions. The metabolic flexibility of fetal hearts allows sustained development under low oxygen conditions. In fact, hypoxia is critical for proper myocardial formation. Particularly, hypoxia inducible factor 1 (HIF-1) and vascular endothelial growth factor play central roles in hypoxia-dependent signaling in fetal heart formation, impacting embryonic outflow track remodeling and coronary vessel growth. Although HIF is not th...

  4. Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation

    Energy Technology Data Exchange (ETDEWEB)

    Velasquez, Celestino; Cheng, Erdong; Shuda, Masahiro; Lee-Oesterreich, Paula J.; Pogge von Strandmann, Lisa; Gritsenko, Marina A.; Jacobs, Jon M.; Moore, Patrick S.; Chang, Yuan

    2016-07-11

    mTOR-directed 4E-BP1 phosphorylation promotes cap-dependent translation and tumorigen-esis. During mitosis, CDK1 substitutes for mTOR and fully phosphorylates 4E-BP1 at canoni-cal as well a non-canonical S83 site resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. While S83 phosphorylation of 4E-BP1 does not affect in vitro cap-dependent translation, nor eIF4G/4E-BP1 cap-binding, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus (MCV) small T (sT) antigen viral oncoprotein. In contrast to inhibitory mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain-of-function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function.

  5. Tumor growth and hypoxia: it's all about location, location and location

    NARCIS (Netherlands)

    Blouw, B.

    2007-01-01

    In response to hypoxia during tumor growth, a number of genetic changes are induced that allow the cancer cells to survive. These changes are orchestrated by the Hypoxia Inducible Factors (HIFs), respectively HIF-1alpha, HIF-2alpha and HIF-3alpha. HIF-1alpha is the main regulator of this response

  6. Post-translational derepression of invertase activity in source leaves via down-regulation of invertase inhibitor expression is part of the plant defense response.

    Science.gov (United States)

    Bonfig, Katharina B; Gabler, Andrea; Simon, Uwe K; Luschin-Ebengreuth, Nora; Hatz, Martina; Berger, Susanne; Muhammad, Naseem; Zeier, Jürgen; Sinha, Alok K; Roitsch, Thomas

    2010-11-01

    There is increasing evidence that pathogens do not only elicit direct defense responses, but also cause pronounced changes in primary carbohydrate metabolism. Cell-wall-bound invertases belong to the key regulators of carbohydrate partitioning and source-sink relations. Whereas studies have focused so far only on the transcriptional induction of invertase genes in response to pathogen infection, the role of post-translational regulation of invertase activity has been neglected and was the focus of the present study. Expression analyses revealed that the high mRNA level of one out of three proteinaceous invertase inhibitors in source leaves of Arabidopsis thaliana is strongly repressed upon infection by a virulent strain of Pseudomonas syringae pv. tomato DC3000. This repression is paralleled by a decrease in invertase inhibitor activity. The physiological role of this regulatory mechanism is revealed by the finding that in situ invertase activity was detectable only upon infection by P. syringae. In contrast, a high invertase activity could be measured in vitro in crude and cell wall extracts prepared from both infected and non-infected leaves. The discrepancy between the in situ and in vitro invertase activity of control leaves and the high in situ invertase activity in infected leaves can be explained by the pathogen-dependent repression of invertase inhibitor expression and a concomitant reduction in invertase inhibitor activity. The functional importance of the release of invertase from post-translational inhibition for the defense response was substantiated by the application of the competitive chemical invertase inhibitor acarbose. Post-translational inhibition of extracellular invertase activity by infiltration of acarbose in leaves was shown to increase the susceptibility to P. syringae. The impact of invertase inhibition on spatial and temporal dynamics of the repression of photosynthesis and promotion of bacterial growth during pathogen infection supports

  7. Radiation-induced mitotic cell death and glioblastoma radioresistance: a new regulating pathway controlled by integrin-linked kinase, hypoxia-inducible factor 1 alpha and survivin in U87 cells.

    Science.gov (United States)

    Lanvin, Olivia; Monferran, Sylvie; Delmas, Caroline; Couderc, Bettina; Toulas, Christine; Cohen-Jonathan-Moyal, Elizabeth

    2013-09-01

    We have previously shown that integrin-linked kinase (ILK) regulates U87 glioblastoma cell radioresistance by modulating the main radiation-induced cell death mechanism in solid tumours, the mitotic cell death. To decipher the biological pathways involved in these mechanisms, we constructed a U87 glioblastoma cell model expressing an inducible shRNA directed against ILK (U87shILK). We then demonstrated that silencing ILK enhanced radiation-induced centrosome overduplication, leading to radiation-induced mitotic cell death. In this model, ionising radiations induce hypoxia-inducible factor 1 alpha (HIF-1α) stabilisation which is inhibited by silencing ILK. Moreover, silencing HIF-1α in U87 cells reduced the surviving fraction after 2 Gy irradiation by increasing cell sensitivity to radiation-induced mitotic cell death and centrosome amplification. Because it is known that HIF-1α controls survivin expression, we then looked at the ILK silencing effect on survivin expression. We show that survivin expression is decreased in U87shILK cells. Furthermore, treating U87 cells with the specific survivin suppressor YM155 significantly increased the percentage of giant multinucleated cells, centrosomal overduplication and thus U87 cell radiosensitivity. In consequence, we decipher here a new pathway of glioma radioresistance via the regulation of radiation-induced centrosome duplication and therefore mitotic cell death by ILK, HIF-1α and survivin. This work identifies new targets in glioblastoma with the intention of radiosensitising these highly radioresistant tumours. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Function and ribosomal localization of aIF6, a translational regulator shared by archaea and eukarya

    OpenAIRE

    Benelli, Dario; Marzi, Stefano; Mancone, Carmine; Alonzi, Tonino; La Teana, Anna; Londei, Paola

    2008-01-01

    The translation factor IF6 is shared by the Archaea and the Eukarya, but is not found in Bacteria. The properties of eukaryal IF6 (eIF6) have been extensively studied, but remain somewhat elusive. eIF6 behaves as a ribosome-anti-association factor and is involved in miRNA-mediated gene silencing; however, it also seems to participate in ribosome synthesis and export. Here we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homologue (aIF6)....

  9. Escherichia coli Quorum-Sensing EDF, A Peptide Generated by Novel Multiple Distinct Mechanisms and Regulated by trans-Translation.

    Science.gov (United States)

    Kumar, Sathish; Kolodkin-Gal, Ilana; Vesper, Oliver; Alam, Nawsad; Schueler-Furman, Ora; Moll, Isabella; Engelberg-Kulka, Hanna

    2016-01-26

    Eshcerichia coli mazEF is a stress-induced toxin-antitoxin module mediating cell death and requiring a quorum-sensing (QS) extracellular death factor (EDF), the pentapeptide NNWNN. Here we uncovered several distinct molecular mechanisms involved in its generation from the zwf mRNA encoding glucose-6-phosphate dehydrogenase. In particular, we show that, under stress conditions, the endoribonuclease MazF cleaves specific ACA sites, thereby generating a leaderless zwf mRNA which is truncated 30 codons after the EDF-encoding region. Since the nascent ribosome peptide exit tunnel can accommodate up to 40 amino acids, this arrangement allows the localization of the EDF residues inside the tunnel when the ribosome is stalled at the truncation site. Moreover, ribosome stalling activates the trans-translation system, which provides a means for the involvement of ClpPX in EDF generation. Furthermore, the trans-translation is described as a regulatory system that attenuated the generation of EDF, leading to low levels of EDF in the single cell. Therefore, the threshold EDF molecule concentration required is achieved only by the whole population, as expected for QS. Bacteria communicate with one another via quorum-sensing (QS) signal molecules. QS provides a mechanism for bacteria to monitor each other's presence and to modulate gene expression in response to population density. Previously, we added E. coli pentapeptide EDF to this list of QS molecules. We showed that, under stress conditions, the induced MazF, an endoribonuclease cleaving at ACA sites, generates EDF from zwf. Here we studied the mechanism of EDF generation and asked whether it is related to EDF density dependency. We illustrated that, under stress conditions, multiple distinct complex mechanisms are involved in EDF generation. This includes formation of leaderless truncated zwf mRNA by MazF, configuration of a length corresponding to the nascent ribosome peptide exit tunnel, rescue performed by the trans-translation

  10. The translation initiation factor eIF4E regulates the sex-specific expression of the master switch gene Sxl in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Patricia L Graham

    2011-07-01

    Full Text Available In female fruit flies, Sex-lethal (Sxl turns off the X chromosome dosage compensation system by a mechanism involving a combination of alternative splicing and translational repression of the male specific lethal-2 (msl-2 mRNA. A genetic screen identified the translation initiation factor eif4e as a gene that acts together with Sxl to repress expression of the Msl-2 protein. However, eif4e is not required for Sxl mediated repression of msl-2 mRNA translation. Instead, eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs. Like other factors required for Sxl regulation of splicing, eif4e shows maternal-effect female-lethal interactions with Sxl. This female lethality can be enhanced by mutations in other co-factors that promote female-specific splicing and is caused by a failure to properly activate the Sxl-positive autoregulatory feedback loop in early embryos. In this feedback loop Sxl proteins promote their own synthesis by directing the female-specific alternative splicing of Sxl-Pm pre-mRNAs. Analysis of pre-mRNA splicing when eif4e activity is compromised demonstrates that Sxl-dependent female-specific splicing of both Sxl-Pm and msl-2 pre-mRNAs requires eif4e activity. Consistent with a direct involvement in Sxl-dependent alternative splicing, eIF4E is associated with unspliced Sxl-Pm pre-mRNAs and is found in complexes that contain early acting splicing factors--the U1/U2 snRNP protein Sans-fils (Snf, the U1 snRNP protein U1-70k, U2AF38, U2AF50, and the Wilms' Tumor 1 Associated Protein Fl(2d--that have been directly implicated in Sxl splicing regulation.

  11. Differential phosphorylation of translation initiation regulators 4EBP1, S6k1, and Erk 1/2 following inhibition of alcohol metabolism in mouse heart.

    Science.gov (United States)

    Vary, Thomas C; Lang, Charles H

    2008-03-01

    Acute alcohol intoxication leads to an inhibition of protein synthesis in heart that results in part through altered phosphorylation of protein factors controlling mRNA translation initiation. The purpose of the present set of experiments was designed to examine the effects of inhibitors of ethanol metabolism on the phosphorylation of 4E-binding protein (4EBP1) and S6k1(Thr(389)), two factors regulating mRNA translation initiation. Phosphorylation of 4E-BP1, S6k1(Thr(389)), and Erk 1/2 was reduced 2 h following IP injection of alcohol. Pretreatment with 4-methylpyrazole (4-MP), an inhibitor of alcohol dehydrogenase (ADH), did not attenuate the ethanol-induced decrease in phosphorylation of 4EBP1 and S6k1(Thr(389)). In contrast, 4-MP prevented the decrease in Erk 1/2 phosphorylation observed with acute ethanol intoxication. Pretreatment with cyanamide, an inhibitor of aldehyde dehydrogenase, did not attenuate the ethanol-induced decrease in phosphorylation S6k1(Thr(389)), but partially prevented the ethanol-induced lowering of 4EBP1 phosphorylation. The studies indicate that modulation of ethanol metabolism through inhibition of ADH or aldehyde dehydrogenase leads to preferential modulation of the phosphorylation of distinct myocardial signaling systems involved in regulating protein synthesis.

  12. Translational Creativity

    DEFF Research Database (Denmark)

    Nielsen, Sandro

    2010-01-01

    A long-established approach to legal translation focuses on terminological equivalence making translators strictly follow the words of source texts. Recent research suggests that there is room for some creativity allowing translators to deviate from the source texts. However, little attention...... is given to genre conventions in source texts and the ways in which they can best be translated. I propose that translators of statutes with an informative function in expert-to-expert communication may be allowed limited translational creativity when translating specific types of genre convention....... This creativity is a result of translators adopting either a source-language or a target-language oriented strategy and is limited by the pragmatic principle of co-operation. Examples of translation options are provided illustrating the different results in target texts. The use of a target-language oriented...

  13. NITRIC OXIDE INTERFERES WITH HYPOXIA SIGNALING DURING COLONIC INFLAMMATION

    Directory of Open Access Journals (Sweden)

    Cintia Rabelo e Paiva CARIA

    2014-12-01

    Full Text Available Context Intestinal inflammation can induce a local reduction in oxygen levels that triggers an adaptive response centered on the expression of hypoxia-inducible factors (HIFs. Nitric oxide, a well-described inflammatory mediator, may interfere with hypoxia signaling. Objectives We aimed to evaluate the role of nitric oxide in hypoxia signaling during colonic inflammation. Methods Colitis was induced by single (acute or repeated (reactivated colitis trinitrobenzenosulfonic acid administration in rats. In addition, one group of rats with reactivated colitis was also treated with Nw-Nitro-L-arginine methyl ester hydrochloride to block nitric oxide synthase. Colitis was assessed by macroscopic score and myeloperoxidase activity in the colon samples. Hypoxia was determined using the oxygen-dependent probe, pimonidazole. The expression of HIF-1α and HIF-induced factors (vascular endothelial growth factor - VEGF and apelin was assessed using Western blotting. Results The single or repeated administration of trinitrobenzenosulfonic acid to rats induced colitis which was characterized by a high macroscopic score and myeloperoxidase activity. Hypoxia was observed with both protocols. During acute colitis, HIF-1α expression was not increased, but VEGF and apelin were increased. HIF-1α expression was inhibited during reactivated colitis, and VEGF and apelin were not increased. Nw-Nitro-L-arginine methyl ester hydrochloride blockade during reactivated colitis restored HIF-1α, VEGF and apelin expression. Conclusions Nitric oxide could interfere with hypoxia signaling during reactivated colitis inflammation modifying the expression of proteins regulated by HIF-1α.

  14. Nitric oxide interferes with hypoxia signaling during colonic inflammation.

    Science.gov (United States)

    Caria, Cintia Rabelo e Paiva; Moscato, Camila Henrique; Tomé, Renata Bortolin Guerra; Pedrazzoli, José; Ribeiro, Marcelo Lima; Gambero, Alessandra

    2014-01-01

    Intestinal inflammation can induce a local reduction in oxygen levels that triggers an adaptive response centered on the expression of hypoxia-inducible factors (HIFs). Nitric oxide, a well-described inflammatory mediator, may interfere with hypoxia signaling. We aimed to evaluate the role of nitric oxide in hypoxia signaling during colonic inflammation. Colitis was induced by single (acute) or repeated (reactivated colitis) trinitrobenzenosulfonic acid administration in rats. In addition, one group of rats with reactivated colitis was also treated with Nw-Nitro-L-arginine methyl ester hydrochloride to block nitric oxide synthase. Colitis was assessed by macroscopic score and myeloperoxidase activity in the colon samples. Hypoxia was determined using the oxygen-dependent probe, pimonidazole. The expression of HIF-1α and HIF-induced factors (vascular endothelial growth factor - VEGF and apelin) was assessed using Western blotting. The single or repeated administration of trinitrobenzenosulfonic acid to rats induced colitis which was characterized by a high macroscopic score and myeloperoxidase activity. Hypoxia was observed with both protocols. During acute colitis, HIF-1α expression was not increased, but VEGF and apelin were increased. HIF-1α expression was inhibited during reactivated colitis, and VEGF and apelin were not increased. Nw-Nitro-L-arginine methyl ester hydrochloride blockade during reactivated colitis restored HIF-1α, VEGF and apelin expression. Nitric oxide could interfere with hypoxia signaling during reactivated colitis inflammation modifying the expression of proteins regulated by HIF-1α.

  15. Amyloid β production is regulated by β2-adrenergic signaling-mediated post-translational modifications of the ryanodine receptor.

    Science.gov (United States)

    Bussiere, Renaud; Lacampagne, Alain; Reiken, Steven; Liu, Xiaoping; Scheuerman, Valerie; Zalk, Ran; Martin, Cécile; Checler, Frederic; Marks, Andrew R; Chami, Mounia

    2017-06-16

    Alteration of ryanodine receptor (RyR)-mediated calcium (Ca(2+)) signaling has been reported in Alzheimer disease (AD) models. However, the molecular mechanisms underlying altered RyR-mediated intracellular Ca(2+) release in AD remain to be fully elucidated. We report here that RyR2 undergoes post-translational modifications (phosphorylation, oxidation, and nitrosylation) in SH-SY5Y neuroblastoma cells expressing the β-amyloid precursor protein (βAPP) harboring the familial double Swedish mutations (APPswe). RyR2 macromolecular complex remodeling, characterized by depletion of the regulatory protein calstabin2, resulted in increased cytosolic Ca(2+) levels and mitochondrial oxidative stress. We also report a functional interplay between amyloid β (Aβ), β-adrenergic signaling, and altered Ca(2+) signaling via leaky RyR2 channels. Thus, post-translational modifications of RyR occur downstream of Aβ through a β2-adrenergic signaling cascade that activates PKA. RyR2 remodeling in turn enhances βAPP processing. Importantly, pharmacological stabilization of the binding of calstabin2 to RyR2 channels, which prevents Ca(2+) leakage, or blocking the β2-adrenergic signaling cascade reduced βAPP processing and the production of Aβ in APPswe-expressing SH-SY5Y cells. We conclude that targeting RyR-mediated Ca(2+) leakage may be a therapeutic approach to treat AD. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Escherichia coli Quorum-Sensing EDF, A Peptide Generated by Novel Multiple Distinct Mechanisms and Regulated by trans-Translation

    Directory of Open Access Journals (Sweden)

    Sathish Kumar

    2016-01-01

    Full Text Available Eshcerichia coli mazEF is a stress-induced toxin-antitoxin module mediating cell death and requiring a quorum-sensing (QS extracellular death factor (EDF, the pentapeptide NNWNN. Here we uncovered several distinct molecular mechanisms involved in its generation from the zwf mRNA encoding glucose-6-phosphate dehydrogenase. In particular, we show that, under stress conditions, the endoribonuclease MazF cleaves specific ACA sites, thereby generating a leaderless zwf mRNA which is truncated 30 codons after the EDF-encoding region. Since the nascent ribosome peptide exit tunnel can accommodate up to 40 amino acids, this arrangement allows the localization of the EDF residues inside the tunnel when the ribosome is stalled at the truncation site. Moreover, ribosome stalling activates the trans-translation system, which provides a means for the involvement of ClpPX in EDF generation. Furthermore, the trans-translation is described as a regulatory system that attenuated the generation of EDF, leading to low levels of EDF in the single cell. Therefore, the threshold EDF molecule concentration required is achieved only by the whole population, as expected for QS.

  17. High-intensity interval training in hypoxia does not affect muscle HIF responses to acute hypoxia in humans.

    Science.gov (United States)

    De Smet, Stefan; D'Hulst, Gommaar; Poffé, Chiel; Van Thienen, Ruud; Berardi, Emanuele; Hespel, Peter

    2018-02-08

    The myocellular response to hypoxia is primarily regulated by hypoxia-inducible factors (HIFs). HIFs thus conceivably are implicated in muscular adaptation to altitude training. Therefore, we investigated the effect of hypoxic versus normoxic training during a period of prolonged hypoxia ('living high') on muscle HIF activation during acute ischaemia. Ten young male volunteers lived in normobaric hypoxia for 5 weeks (5 days per week, ~ 15.5 h per day, FiO2: 16.4-14.0%). One leg was trained in hypoxia (TRHYP, 12.3% FiO2) whilst the other leg was trained in normoxia (TRNOR, 20.9% FiO2). Training sessions (3 per week) consisted of intermittent unilateral knee extensions at 20-25% of the 1-repetition maximum. Before and after the intervention, a 10-min arterial occlusion and reperfusion of the leg was performed. Muscle oxygenation status was continuously measured by near-infrared spectroscopy. Biopsies were taken from m. vastus lateralis before and at the end of the occlusion. Irrespective of training, occlusion elevated the fraction of HIF-1α expressing myonuclei from ~ 54 to ~ 64% (P Training in both TRNOR and TRHYP raised muscular oxygen extraction rate upon occlusion by ~ 30%, whilst muscle hyperperfusion immediately following the occlusion increased by ~ 25% in either group (P training during 'living high' altered muscle HIF translocation, stabilisation, or transcription in response to acute hypoxia induced by arterial occlusion.

  18. Comparative Analysis of Muscle Hypertrophy Models Reveals Divergent Gene Transcription Profiles and Points to Translational Regulation of Muscle Growth through Increased mTOR Signaling

    Directory of Open Access Journals (Sweden)

    Marcelo G. Pereira

    2017-12-01

    Full Text Available Skeletal muscle mass is a result of the balance between protein breakdown and protein synthesis. It has been shown that multiple conditions of muscle atrophy are characterized by the common regulation of a specific set of genes, termed atrogenes. It is not known whether various models of muscle hypertrophy are similarly regulated by a common transcriptional program. Here, we characterized gene expression changes in three different conditions of muscle growth, examining each condition during acute and chronic phases. Specifically, we compared the transcriptome of Extensor Digitorum Longus (EDL muscles collected (1 during the rapid phase of postnatal growth at 2 and 4 weeks of age, (2 24 h or 3 weeks after constitutive activation of AKT, and (3 24 h or 3 weeks after overload hypertrophy caused by tenotomy of the Tibialis Anterior muscle. We observed an important overlap between significantly regulated genes when comparing each single condition at the two different timepoints. Furthermore, examining the transcriptional changes occurring 24 h after a hypertrophic stimulus, we identify an important role for genes linked to a stress response, despite the absence of muscle damage in the AKT model. However, when we compared all different growth conditions, we did not find a common transcriptional fingerprint. On the other hand, all conditions showed a marked increase in mTORC1 signaling and increased ribosome biogenesis, suggesting that muscle growth is characterized more by translational, than transcriptional regulation.

  19. Nocturnal hypoxia and neuropsychological variables.

    Science.gov (United States)

    Berry, D T; Webb, W B; Block, A J; Bauer, R M; Switzer, D A

    1986-06-01

    Hypoxia is a well known cause of brain dysfunction. Neuropsychological impairments have been observed in normal subjects experiencing hypoxia iatrogenically as well as in patients with chronic lung disease. Recent investigations have demonstrated significant nocturnal hypoxia in subjects with sleep-disordered breathing. In the present study, heavy-snoring males, a group known to experience frequent episodes of sleep-disordered breathing received neuropsychological testing and a night of continuous monitoring of respiratory parameters. Partial correlations, controlling for age, weight, and education, indicated reliable relationships between nocturnal hypoxia and measures of general intelligence, verbal and nonverbal memory, and expressive verbal fluency. It is proposed that heavy-snoring males may potentially serve as a population in which to model the neurobehavioral effects of hypoxia. Further research in subjects with sleep-disordered breathing may help clarify the extent of the possible cognitive deficits as well as point out possible ameliorative treatments.

  20. Intersections between cardiac physiology, emotion regulation and interpersonal warmth in preschoolers: Implications for drug abuse prevention from translational neuroscience.

    Science.gov (United States)

    Clark, Caron A C; Skowron, Elizabeth A; Giuliano, Ryan J; Fisher, Philip A

    2016-06-01

    Early childhood is characterized by dramatic gains in emotion regulation skills that support social adjustment and mental health. Understanding the physiological substrates of healthy emotion regulation may offer new directions for altering trajectories toward initiation and escalation of substance abuse. Here, we describe the intersections between parasympathetic and sympathetic tone, emotion regulation and prosocial behavior in a high-risk sample of preschoolers. Fifty-two 3-6 year old children completed an assessment of attention regulation in response to affective stimuli. Cardiac respiratory sinus arrhythmia, an index of parasympathetic tone, and pre-ejection period, a marker of sympathetic activation, were recorded at rest and while children engaged in social interactions with their mothers and an unfamiliar research assistant. Mothers reported on children's emotional reactivity and prosocial behavior. Controlling for age and psychosocial risk, higher parasympathetic tone predicted better attention regulation in response to angry emotion and higher levels of prosocial behavior, whereas a reciprocal pattern of higher parasympathetic tone and lower sympathetic arousal predicted better attention in response to positive emotion and lower emotional reactivity. Children exposed to fewer risk factors and higher levels of maternal warmth were more able to sustain a high level of parasympathetic tone during interaction episodes. Findings suggest that autonomic measures represent biomarkers for socio-emotional competence in young children. They also point to the importance of early experiences in the establishment of physiological regulation and the promise of family-based intervention to promote healthy emotion regulation and prevent substance dependence in high-risk populations. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  1. The ribB FMN riboswitch from Escherichia coli operates at the transcriptional and translational level and regulates riboflavin biosynthesis.

    Science.gov (United States)

    Pedrolli, Danielle; Langer, Simone; Hobl, Birgit; Schwarz, Julia; Hashimoto, Masayuki; Mack, Matthias

    2015-08-01

    FMN riboswitches are genetic elements that, in many bacteria, control genes responsible for biosynthesis and/or transport of riboflavin (vitamin B2 ). We report that the Escherichia coli ribB FMN riboswitch controls expression of the essential gene ribB coding for the riboflavin biosynthetic enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase (RibB; EC 4.1.99.12). Our data show that the E. coli ribB FMN riboswitch is unusual because it operates at the transcriptional and also at the translational level. Expression of ribB is negatively affected by FMN and by the FMN analog roseoflavin mononucleotide, which is synthesized enzymatically from roseoflavin and ATP. Consequently, in addition to flavoenzymes, the E. coli ribB FMN riboswitch constitutes a target for the antibiotic roseoflavin produced by Streptomyces davawensis. © 2015 FEBS.

  2. Single β-actin mRNA detection in neurons reveals a mechanism for regulating its translatability.

    Science.gov (United States)

    Buxbaum, Adina R; Wu, Bin; Singer, Robert H

    2014-01-24

    The physical manifestation of learning and memory formation in the brain can be expressed by strengthening or weakening of synaptic connections through morphological changes. Local actin remodeling underlies some forms of plasticity and may be facilitated by local β-actin synthesis, but dynamic information is lacking. In this work, we use single-molecule in situ hybridization to demonstrate that dendritic β-actin messenger RNA (mRNA) and ribosomes are in a masked, neuron-specific form. Chemically induced long-term potentiation prompts transient mRNA unmasking, which depends on factors active during synaptic activity. Ribosomes and single β-actin mRNA motility increase after stimulation, indicative of release from complexes. Hence, the single-molecule assays we developed allow for the quantification of activity-induced unmasking and availability for active translation. Further, our work demonstrates that β-actin mRNA and ribosomes are in a masked state that is alleviated by stimulation.

  3. Translating Europe

    Directory of Open Access Journals (Sweden)

    Yves Chevrel

    2007-07-01

    Europe thinks in many languages and Europe is a land of translation. Translation is a means of transmitting culture, a means of making it available to others and an invitation to share. It is a cement which binds Europe together.

  4. Tubulin tail sequences and post-translational modifications regulate closure of mitochondrial voltage-dependent anion channel (VDAC).

    Science.gov (United States)

    Sheldon, Kely L; Gurnev, Philip A; Bezrukov, Sergey M; Sackett, Dan L

    2015-10-30

    It was previously shown that tubulin dimer interaction with the mitochondrial outer membrane protein voltage-dependent anion channel (VDAC) blocks traffic through the channel and reduces oxidative metabolism and that this requires the unstructured anionic C-terminal tail peptides found on both α- and β-tubulin subunits. It was unclear whether the α- and β-tubulin tails contribute equally to VDAC blockade and what effects might be due to sequence variations in these tail peptides or to tubulin post-translational modifications, which mostly occur on the tails. The nature of the contribution of the tubulin body beyond acting as an anchor for the tails had not been clarified either. Here we present peptide-protein chimeras to address these questions. These constructs allow us to easily combine a tail peptide with different proteins or combine different tail peptides with a particular protein. The results show that a single tail grafted to an inert protein is sufficient to produce channel closure similar to that observed with tubulin. We show that the β-tail is more than an order of magnitude more potent than the α-tail and that the lower α-tail activity is largely due to the presence of a terminal tyrosine. Detyrosination activates the α-tail, and activation is reversed by the removal of the glutamic acid penultimate to the tyrosine. Nitration of tyrosine reverses the tyrosine inhibition of binding and even induces prolonged VDAC closures. Our results demonstrate that small changes in sequence or post-translational modification of the unstructured tails of tubulin result in substantial changes in VDAC closure. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. The rice cold-responsive calcium-dependent protein kinase OsCPK17 is regulated by alternative splicing and post-translational modifications.

    Science.gov (United States)

    Cecília Almadanim, M; Gonçalves, Nuno M; Rosa, Margarida T G; Alexandre, Bruno M; Cordeiro, André M; Rodrigues, Mafalda; Saibo, Nelson J M; Soares, Cláudio M; Romão, Célia V; Margarida Oliveira, M; Abreu, Isabel A

    2017-10-31

    Plant calcium-dependent protein kinases (CDPKs) are key proteins implicated in calcium-mediated signaling pathways of a wide range of biological events in the organism. The action of each particular CDPK is strictly regulated by many mechanisms in order to ensure an accurate signal translation and the activation of the adequate response processes. In this work, we investigated the regulation of a CDPK involved in rice cold stress response, OsCPK17, to better understand its mode of action. We identified two new alternative splicing (AS) mRNA forms of OsCPK17 encoding truncated versions of the protein, missing the CDPK activation domain. We analyzed the expression patterns of all AS variants in rice tissues and examined their subcellular localization in onion epidermal cells. The results indicate that the AS of OsCPK17 putatively originates truncated forms of the protein with distinct functions, and different subcellular and tissue distributions. Additionally, we addressed the regulation of OsCPK17 by post-translational modifications in several in vitro experiments. Our analysis indicated that OsCPK17 activity depends on its structural rearrangement induced by calcium binding, and that the protein can be autophosphorylated. The identified phosphorylation sites mostly populate the OsCPK17 N-terminal domain. Exceptions are phosphosites T107 and S136 in the kinase domain and S558 in the C-terminal domain. These phosphosites seem conserved in CDPKs and may reflect a common regulatory mechanism for this protein family. Copyright © 2017. Published by Elsevier B.V.

  6. Structural integration in hypoxia-inducible factors

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Dalei; Potluri, Nalini; Lu, Jingping; Kim, Youngchang; Rastinejad, Fraydoon

    2015-08-20

    The hypoxia-inducible factors (HIFs) coordinate cellular adaptations to low oxygen stress by regulating transcriptional programs in erythropoiesis, angiogenesis and metabolism. These programs promote the growth and progression of many tumours, making HIFs attractive anticancer targets. Transcriptionally active HIFs consist of HIF-alpha and ARNT (also called HIF-1 beta) subunits. Here we describe crystal structures for each of mouse HIF-2 alpha-ARNT and HIF-1 alpha-ARNT heterodimers in states that include bound small molecules and their hypoxia response element. A highly integrated quaternary architecture is shared by HIF-2 alpha-ARNT and HIF-1 alpha-ARNT, wherein ARNT spirals around the outside of each HIF-alpha subunit. Five distinct pockets are observed that permit small-molecule binding, including PAS domain encapsulated sites and an interfacial cavity formed through subunit heterodimerization. The DNA-reading head rotates, extends and cooperates with a distal PAS domain to bind hypoxia response elements. HIF-alpha mutations linked to human cancers map to sensitive sites that establish DNA binding and the stability of PAS domains and pockets.

  7. Hypoxia inhibits hypertrophic differentiation and endochondral ossification in explanted tibiae.

    Directory of Open Access Journals (Sweden)

    Jeroen C H Leijten

    Full Text Available Hypertrophic differentiation of growth plate chondrocytes induces angiogenesis which alleviates hypoxia normally present in cartilage. In the current study, we aim to determine whether alleviation of hypoxia is merely a downstream effect of hypertrophic differentiation as previously described or whether alleviation of hypoxia and consequent changes in oxygen tension mediated signaling events also plays an active role in regulating the hypertrophic differentiation process itself.Fetal mouse tibiae (E17.5 explants were cultured up to 21 days under normoxic or hypoxic conditions (21% and 2.5% oxygen respectively. Tibiae were analyzed on growth kinetics, histology, gene expression and protein secretion.The oxygen level had a strong influence on the development of explanted fetal tibiae. Compared to hypoxia, normoxia increased the length of the tibiae, length of the hypertrophic zone, calcification of the cartilage and mRNA levels of hypertrophic differentiation-related genes e.g. MMP9, MMP13, RUNX2, COL10A1 and ALPL. Compared to normoxia, hypoxia increased the size of the cartilaginous epiphysis, length of the resting zone, calcification of the bone and mRNA levels of hyaline cartilage-related genes e.g. ACAN, COL2A1 and SOX9. Additionally, hypoxia enhanced the mRNA and protein expression of the secreted articular cartilage markers GREM1, FRZB and DKK1, which are able to inhibit hypertrophic differentiation.Collectively our data suggests that oxygen levels play an active role in the regulation of hypertrophic differentiation of hyaline chondrocytes. Normoxia stimulates hypertrophic differentiation evidenced by the expression of hypertrophic differentiation related genes. In contrast, hypoxia suppresses hypertrophic differentiation of chondrocytes, which might be at least partially explained by the induction of GREM1, FRZB and DKK1 expression.

  8. TRANSLATING FEMINISM

    OpenAIRE

    Gajewska, Agnieszka

    2012-01-01

    Translating feminism Pointing to manifold and long-lasting connections between feminism and translation, the article first presents a selection of multilingual writers (Narcyza Żmichowska and Deborah Vogel), translators (Zofia Żeleńska and Kazimiera Iłłakowiczówna) and translation commentators (Joanna Lisek and Karolina Szymaniak) to ponder why the work of early Polish feminists is neglected. It seems that one of the reasons might be the current colonization of Polish femini...

  9. Divergent protein motifs direct elongation factor P-mediated translational regulation in Salmonella enterica and Escherichia coli

    DEFF Research Database (Denmark)

    Hersch, Steven J; Wang, Mengchi; Zou, S Betty

    2013-01-01

    are not misregulated in an EF-P-deficient background, suggesting that the factors that govern EF-P-mediated regulation are complex. Finally, we analyzed the specific region of the PoxB protein that is modulated by EF-P and found that mutation of any residue within a specific GSCGPG sequence eliminates the requirement...

  10. The CDKN2A/p16(INK) (4a) 5'UTR sequence and translational regulation: impact of novel variants predisposing to melanoma.

    Science.gov (United States)

    Andreotti, Virginia; Bisio, Alessandra; Bressac-de Paillerets, Brigitte; Harland, Mark; Cabaret, Odile; Newton-Bishop, Julia; Pastorino, Lorenza; Bruno, William; Bertorelli, Roberto; De Sanctis, Veronica; Provenzani, Alessandro; Menin, Chiara; Fronza, Gilberto; Queirolo, Paola; Spitale, Robert C; Bianchi-Scarrà, Giovanna; Inga, Alberto; Ghiorzo, Paola

    2016-03-01

    Many variants of uncertain functional significance in cancer susceptibility genes lie in regulatory regions, and clarifying their association with disease risk poses significant challenges. We studied 17 germline variants (nine of which were novel) in the CDKN2A 5'UTR with independent approaches, which included mono and bicistronic reporter assays, Western blot of endogenous protein, and allelic representation after polysomal profiling to investigate their impact on CDKN2A mRNA translation regulation. Two of the novel variants (c.-27del23, c.-93-91delAGG) were classified as causal mutations (score ≥3), along with the c.-21C>T, c.-34G>T, and c.-56G>T, which had already been studied by a subset of assays. The novel c.-42T>A as well as the previously described c.-67G>C were classified as potential mutations (score 1 or 2). The remaining variants (c.-14C>T, c.-20A>G, c.-25C>T+c.-180G>A, c.-30G>A, c.-40C>T, c.-45G>A, c.-59C>G, c.-87T>A, c.-252A>T) were classified as neutral (score 0). In conclusion, we found evidence that nearly half of the variants found in this region had a negative impact on CDKN2A mRNA translation, supporting the hypothesis that 5'UTR can act as a cellular Internal Ribosome Entry Site (IRES) to modulate p16(INK) (4a) translation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Machine translation

    Energy Technology Data Exchange (ETDEWEB)

    Nagao, M.

    1982-04-01

    Each language has its own structure. In translating one language into another one, language attributes and grammatical interpretation must be defined in an unambiguous form. In order to parse a sentence, it is necessary to recognize its structure. A so-called context-free grammar can help in this respect for machine translation and machine-aided translation. Problems to be solved in studying machine translation are taken up in the paper, which discusses subjects for semantics and for syntactic analysis and translation software. 14 references.

  12. Transcription and translation products of the cytolysin gene psm-mec on the mobile genetic element SCCmec regulate Staphylococcus aureus virulence.

    Directory of Open Access Journals (Sweden)

    Chikara Kaito

    Full Text Available The F region downstream of the mecI gene in the SCCmec element in hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA contains two bidirectionally overlapping open reading frames (ORFs, the fudoh ORF and the psm-mec ORF. The psm-mec ORF encodes a cytolysin, phenol-soluble modulin (PSM-mec. Transformation of the F region into the Newman strain, which is a methicillin-sensitive S. aureus (MSSA strain, or into the MW2 (USA400 and FRP3757 (USA300 strains, which are community-acquired MRSA (CA-MRSA strains that lack the F region, attenuated their virulence in a mouse systemic infection model. Introducing the F region to these strains suppressed colony-spreading activity and PSMα production, and promoted biofilm formation. By producing mutations into the psm-mec ORF, we revealed that (i both the transcription and translation products of the psm-mec ORF suppressed colony-spreading activity and promoted biofilm formation; and (ii the transcription product of the psm-mec ORF, but not its translation product, decreased PSMα production. These findings suggest that both the psm-mec transcript, acting as a regulatory RNA, and the PSM-mec protein encoded by the gene on the mobile genetic element SCCmec regulate the virulence of Staphylococcus aureus.

  13. Coastal hypoxia and sediment biogeochemistry

    Directory of Open Access Journals (Sweden)

    J. J. Middelburg

    2009-07-01

    Full Text Available The intensity, duration and frequency of coastal hypoxia (oxygen concentration <63 μM are increasing due to human alteration of coastal ecosystems and changes in oceanographic conditions due to global warming. Here we provide a concise review of the consequences of coastal hypoxia for sediment biogeochemistry. Changes in bottom-water oxygen levels have consequences for early diagenetic pathways (more anaerobic at expense of aerobic pathways, the efficiency of re-oxidation of reduced metabolites and the nature, direction and magnitude of sediment-water exchange fluxes. Hypoxia may also lead to more organic matter accumulation and burial and the organic matter eventually buried is also of higher quality, i.e. less degraded. Bottom-water oxygen levels also affect the organisms involved in organic matter processing with the contribution of metazoans decreasing as oxygen levels drop. Hypoxia has a significant effect on benthic animals with the consequences that ecosystem functions related to macrofauna such as bio-irrigation and bioturbation are significantly affected by hypoxia as well. Since many microbes and microbial-mediated biogeochemical processes depend on animal-induced transport processes (e.g. re-oxidation of particulate reduced sulphur and denitrification, there are indirect hypoxia effects on biogeochemistry via the benthos. Severe long-lasting hypoxia and anoxia may result in the accumulation of reduced compounds in sediments and elimination of macrobenthic communities with the consequences that biogeochemical properties during trajectories of decreasing and increasing oxygen may be different (hysteresis with consequences for coastal ecosystem dynamics.

  14. A tRNA-derived fragment competes with mRNA for ribosome binding and regulates translation during stress.

    Science.gov (United States)

    Gebetsberger, Jennifer; Wyss, Leander; Mleczko, Anna M; Reuther, Julia; Polacek, Norbert

    2017-10-03

    Posttranscriptional processing of RNA molecules is a common strategy to enlarge the structural and functional repertoire of RNomes observed in all 3 domains of life. Fragmentation of RNA molecules of basically all functional classes has been reported to yield smaller non-protein coding RNAs (ncRNAs) that typically possess different roles compared with their parental transcripts. Here we show that a valine tRNA-derived fragment (Val-tRF) that is produced under certain stress conditions in the halophilic archaeon Haloferax volcanii is capable of binding to the small ribosomal subunit. As a consequence of Val-tRF binding mRNA is displaced from the initiation complex which results in global translation attenuation in vivo and in vitro. The fact that the archaeal Val-tRF also inhibits eukaryal as well as bacterial protein biosynthesis implies a functionally conserved mode of action. While tRFs and tRNA halves have been amply identified in recent RNA-seq project, Val-tRF described herein represents one of the first functionally characterized tRNA processing products to date.

  15. The Measurement of Reversible Redox Dependent Post-translational Modifications and Their Regulation of Mitochondrial and Skeletal Muscle Function

    Energy Technology Data Exchange (ETDEWEB)

    Kramer, Philip A.; Duan, Jicheng; Qian, Wei-Jun; Marcinek, David J.

    2015-11-25

    Mitochondrial oxidative stress is a common feature of skeletal myopathies across multiple conditions; however, the mechanism by which it contributes to skeletal muscle dysfunction remains controversial. Oxidative damage to proteins, lipids, and DNA has received the most attention, yet an important role for reversible redox post-translational modifications (PTMs) in pathophysiology is emerging. The possibility that these PTMs can exert dynamic control of muscle function implicates them as a mechanism contributing to skeletal muscle dysfunction in chronic disease. Herein, we discuss the significance of thiol-based redox dependent modifications to mitochondrial, myofibrillar and excitation-contraction (EC) coupling proteins with an emphasis on how these changes could alter skeletal muscle performance under chronically stressed conditions. A major barrier to a better mechanistic understanding of the role of reversible redox PTMs in muscle function is the technical challenges associated with accurately measuring the changes of site-specific redox PTMs. Here we will critically review current approaches with an emphasis on sample preparation artifacts, quantitation, and specificity. Despite these challenges, the ability to accurately quantify reversible redox PTMs is critical to understanding the mechanisms by which mitochondrial oxidative stress contributes to skeletal muscle dysfunction in chronic diseases.

  16. A long non-coding RNA, LncMyoD, regulates skeletal muscle differentiation by blocking IMP2-mediated mRNA translation.

    Science.gov (United States)

    Gong, Chenguang; Li, Zhizhong; Ramanujan, Krishnan; Clay, Ieuan; Zhang, Yunyu; Lemire-Brachat, Sophie; Glass, David J

    2015-07-27

    Increasing evidence suggests that long non-coding RNAs (LncRNAs) represent a new class of regulators of stem cells. However, the roles of LncRNAs in stem cell maintenance and myogenesis remain largely unexamined. For this study, hundreds of intergenic LncRNAs were identified that are expressed in myoblasts and regulated during differentiation. One of these LncRNAs, termed LncMyoD, is encoded next to the Myod gene and is directly activated by MyoD during myoblast differentiation. Knockdown of LncMyoD strongly inhibits terminal muscle differentiation, largely due to a failure to exit the cell cycle. LncMyoD directly binds to IGF2-mRNA-binding protein 2 (IMP2) and negatively regulates IMP2-mediated translation of proliferation genes such as N-Ras and c-Myc. While the RNA sequence of LncMyoD is not well conserved between human and mouse, its locus, gene structure, and function are preserved. The MyoD-LncMyoD-IMP2 pathway elucidates a mechanism as to how MyoD blocks proliferation to create a permissive state for differentiation. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Oligogalacturonic acids promote tomato fruit ripening through the regulation of 1-aminocyclopropane-1-carboxylic acid synthesis at the transcriptional and post-translational levels.

    Science.gov (United States)

    Ma, Yingxuan; Zhou, Leilei; Wang, Zhichao; Chen, Jianting; Qu, Guiqin

    2016-01-09

    Oligogalacturonic acids (OGs) are oligomers of alpha-1,4-linked galacturonosyl residues that are released from cell walls by the hydrolysis of polygalacturonic acids upon fruit ripening and under abiotic/biotic stress. OGs may induce ethylene production and fruit ripening, however, the mechanism(s) behind these processes is unknown. Tomato cultivar 'Ailsa Craig' (AC) and mutant Neverripe, ripening inhibitor, non-ripening, and colorless non-ripening fruits were treated with OGs at different stages. Only AC fruits at mature green stage 1 showed an advanced ripening phenomenon, although transient ethylene production was detected in all of the tomato fruits. Ethylene synthesis genes LeACS2 and LeACO1 were rapidly up-regulated, and the phosphorylated LeACS2 protein was detected after OGs treatment. Protein kinase/phosphatase inhibitors significantly affected the ripening process induced by the OGs. As a potential receptor of OGs, LeWAKL2 was also up-regulated in their presence. We demonstrated that OGs promoted tomato fruit ripening by inducing ethylene synthesis through the regulation of LeACS2 at transcriptional and post-translational levels.

  18. Translational regulation of gene expression by an anaerobically induced small non-coding RNA in Escherichia coli

    DEFF Research Database (Denmark)

    Boysen, Anders; Møller-Jensen, Jakob; Kallipolitis, Birgitte H.

    2010-01-01

    Small non-coding RNAs (sRNA) have emerged as important elements of gene regulatory circuits. In enterobacteria such as Escherichia coli and Salmonella many of these sRNAs interact with the Hfq protein, an RNA chaperone similar to mammalian Sm-like proteins and act in the post...... of at least one sRNA regulator. Here, we extend this view by the identification and characterization of a highly conserved, anaerobically induced small sRNA in E. coli, whose expression is strictly dependent on the anaerobic transcriptional fumarate and nitrate reductase regulator (FNR). The sRNA, named Fnr...... that adaptation to anaerobic growth involves the action of a small regulatory RNA....

  19. Regulation of Mammary Stem Cell Quiescence via Post-Translational Modification of DeltaNp63alpha

    Science.gov (United States)

    2012-12-01

    This document is the Annual Summary Report on the training grant awarded to Andrew DeCastro entitled Regulation of Mammary Stem Cell Quiescence via...screen) mediated phosphorylation of deltaNPdelta3 on stem cell behavior and mitotic activity. Task 1 aims to determine the effects of wild-type, phospho...ablative and phospho-mimetic alleles of deltaNP63delta phosphorylation on stem cell behavior in vitro. Thus far, we demonstrate that stem cell enriched

  20. Environmental stress mediated changes in transpirational and translational regulation of protein synthesis in crop plants: Progress report, 1987--1988

    Energy Technology Data Exchange (ETDEWEB)

    Key, Joe L.

    1988-04-01

    These studies have emphasized the characterization of HS cDNA and genomic clones, development and partial characterization of a HS-cassette for expression of various genes under the control of a very strong HS promoter, analyses on the regulation of the HS response and some aspects of thermotolerance using amino acid analogues, including studies of arsenite and cadmium on HS and thermotolerance. 11 refs.

  1. Redox regulation of plant S-nitrosoglutathione reductase activity through post-translational modifications of cysteine residues.

    Science.gov (United States)

    Tichá, Tereza; Lochman, Jan; Činčalová, Lucie; Luhová, Lenka; Petřivalský, Marek

    2017-12-09

    Nitric oxide (NO) is considered as a signalling molecule involved in a variety of important physiological and pathological processes in plant and animal systems. The major pathway of NO reactions in vivo represents S-nitrosation of thiols to form S-nitrosothiols. S-nitrosoglutathione reductase (GSNOR) is the key enzyme in the degradation pathway of S-nitrosoglutathione (GSNO), a low-molecular weight adduct of NO and glutathione. GSNOR indirectly regulates the level of protein S-nitrosothiol in the cells. This study was focused on the dynamic regulation of the activity of plant GSNORs through reversible S-nitrosation and/or oxidative modifications of target cysteine residues. Pre-incubation with NO/NO - donors or hydrogen peroxide resulted in a decreased reductase and dehydrogenase activity of all studied plant GSNORs. Incubation with thiol reducing agent completely reversed inhibitory effects of nitrosative modifications and partially also oxidative inhibition. In biotin-labelled samples, S-nitrosation of plant GSNORs was confirmed after immunodetection and using mass spectrometry S-nitrosation of conserved Cys271 was identified in tomato GSNOR. Negative regulation of constitutive GSNOR activity in vivo by nitrosative or oxidative modifications might present an important mechanism to control GSNO levels, a critical mediator of the downstream signalling effects of NO, as well as for formaldehyde detoxification in dehydrogenase reaction mode. Copyright © 2017. Published by Elsevier Inc.

  2. Microneedle-array patches loaded with hypoxia-sensitive vesicles provide fast glucose-responsive insulin delivery.

    Science.gov (United States)

    Yu, Jicheng; Zhang, Yuqi; Ye, Yanqi; DiSanto, Rocco; Sun, Wujin; Ranson, Davis; Ligler, Frances S; Buse, John B; Gu, Zhen

    2015-07-07

    A glucose-responsive "closed-loop" insulin delivery system mimicking the function of pancreatic cells has tremendous potential to improve quality of life and health in diabetics. Here, we report a novel glucose-responsive insulin delivery device using a painless microneedle-array patch ("smart insulin patch") containing glucose-responsive vesicles (GRVs; with an average diameter of 118 nm), which are loaded with insulin and glucose oxidase (GOx) enzyme. The GRVs are self-assembled from hypoxia-sensitive hyaluronic acid (HS-HA) conjugated with 2-nitroimidazole (NI), a hydrophobic component that can be converted to hydrophilic 2-aminoimidazoles through bioreduction under hypoxic conditions. The local hypoxic microenvironment caused by the enzymatic oxidation of glucose in the hyperglycemic state promotes the reduction of HS-HA, which rapidly triggers the dissociation of vesicles and subsequent release of insulin. The smart insulin patch effectively regulated the blood glucose in a mouse model of chemically induced type 1 diabetes. The described work is the first demonstration, to our knowledge, of a synthetic glucose-responsive device using a hypoxia trigger for regulation of insulin release. The faster responsiveness of this approach holds promise in avoiding hyperglycemia and hypoglycemia if translated for human therapy.

  3. Hypoxia-on-a-chip

    Directory of Open Access Journals (Sweden)

    Busek Mathias

    2016-09-01

    Full Text Available In this work a microfluidic cell cultivation device for perfused hypoxia assays as well as a suitable controlling unit are presented. The device features active components like pumps for fluid actuation and valves for fluid direction as well as an oxygenator element to ensure a sufficient oxygen transfer. It consists of several individually structured layers which can be tailored specifically to the intended purpose. Because of its clearness, its mechanical strength and chemical resistance as well as its well-known biocompatibility polycarbonate was chosen to form the fluidic layers by thermal diffusion bonding. Several oxygen sensing spots are integrated into the device and monitored with fluorescence lifetime detection. Furthermore an oxygen regulator module is implemented into the controlling unit which is able to mix different process gases to achieve a controlled oxygenation. First experiments show that oxygenation/deoxygenation of the system is completed within several minutes when pure nitrogen or air is applied to the oxygenator. Lastly the oxygen input by the pneumatically driven micro pump was quantified by measuring the oxygen content before and after the oxygenator.

  4. Global hypoxia induced impairment in learning and spatial memory is associated with precocious hippocampal aging.

    Science.gov (United States)

    Biswal, Suryanarayan; Sharma, Deepti; Kumar, Kushal; Nag, Tapas Chandra; Barhwal, Kalpana; Hota, Sunil Kumar; Kumar, Bhuvnesh

    2016-09-01

    Both chronological aging and chronic hypoxia stress have been reported to cause degeneration of hippocampal CA3 neurons and spatial memory impairment through independent pathways. However, the possible occurrence of precocious biological aging on exposure to single episode of global hypoxia resulting in impairment of learning and memory remains to be established. The present study thus aimed at bridging this gap in existing literature on hypoxia induced biological aging. Male Sprague Dawley rats were exposed to simulated hypobaric hypoxia (25,000ft) for different durations and were compared with aged rats. Behavioral studies in Morris Water Maze showed decline in learning abilities of both chronologically aged as well as hypoxic rats as evident from increased latency and pathlength to reach target platform. These behavioral changes in rats exposed to global hypoxia were associated with deposition of lipofuscin and ultrastructural changes in the mitochondria of hippocampal neurons that serve as hallmarks of aging. A single episode of chronic hypobaric hypoxia exposure also resulted in the up-regulation of pro-aging protein, S100A9 and down regulation of Tau, SNAP25, APOE and Sod2 in the hippocampus similar to that in aged rats indicating hypoxia induced accelerated aging. The present study therefore provides evidence for role of biological aging of hippocampal neurons in hypoxia induced impairment of learning and memory. Copyright © 2016. Published by Elsevier Inc.

  5. Hypoxia in models of lung cancer

    DEFF Research Database (Denmark)

    Graves, Edward E; Vilalta, Marta; Cecic, Ivana K

    2010-01-01

    PURPOSE: To efficiently translate experimental methods from bench to bedside, it is imperative that laboratory models of cancer mimic human disease as closely as possible. In this study, we sought to compare patterns of hypoxia in several standard and emerging mouse models of lung cancer...... to establish the appropriateness of each for evaluating the role of oxygen in lung cancer progression and therapeutic response. EXPERIMENTAL DESIGN: Subcutaneous and orthotopic human A549 lung carcinomas growing in nude mice as well as spontaneous K-ras or Myc-induced lung tumors grown in situ......H2AX foci in vitro and in vivo. Finally, our findings were compared with oxygen electrode measurements of human lung cancers. RESULTS: Minimal fluoroazomycin arabinoside and pimonidazole accumulation was seen in tumors growing within the lungs, whereas subcutaneous tumors showed substantial trapping...

  6. Systems Level Analysis of Histone H3 Post-translational Modifications (PTMs) Reveals Features of PTM Crosstalk in Chromatin Regulation*

    Science.gov (United States)

    Schwämmle, Veit; Sidoli, Simone; Ruminowicz, Chrystian; Wu, Xudong; Lee, Chung-Fan; Helin, Kristian; Jensen, Ole N.

    2016-01-01

    Histones are abundant chromatin constituents carrying numerous post-translational modifications (PTMs). Such PTMs mediate a variety of biological functions, including recruitment of enzymatic readers, writers and erasers that modulate DNA replication, transcription and repair. Individual histone molecules contain multiple coexisting PTMs, some of which exhibit crosstalk, i.e. coordinated or mutually exclusive activities. Here, we present an integrated experimental and computational systems level molecular characterization of histone PTMs and PTM crosstalk. Using wild type and engineered mouse embryonic stem cells (mESCs) knocked out in components of the Polycomb Repressive Complex 2 (PRC2, Suz12−/−), PRC1 (Ring1A/B−/−) and (Dnmt1/3a/3b−/−) we performed comprehensive PTM analysis of histone H3 tails (50 aa) by utilizing quantitative middle-down proteome analysis by tandem mass spectrometry. We characterized combinatorial PTM features across the four mESC lines and then applied statistical data analysis to predict crosstalk between histone H3 PTMs. We detected an overrepresentation of positive crosstalk (codependent marks) between adjacent mono-methylated and acetylated marks, and negative crosstalk (mutually exclusive marks) among most of the seven characterized di- and tri-methylated lysine residues in the H3 tails. We report novel features of PTM interplay involving hitherto poorly characterized arginine methylation and lysine methylation sites, including H3R2me, H3R8me and H3K37me. Integration of the H3 data with RNAseq data by coabundance clustering analysis of histone PTMs and histone modifying enzymes revealed correlations between PTM and enzyme levels. We conclude that middle-down proteomics is a powerful tool to determine conserved or dynamic interdependencies between histone marks, which paves the way for detailed investigations of the histone code. Histone H3 PTM data is publicly available in the CrossTalkDB repository at http

  7. Systems Level Analysis of Histone H3 Post-translational Modifications (PTMs) Reveals Features of PTM Crosstalk in Chromatin Regulation.

    Science.gov (United States)

    Schwämmle, Veit; Sidoli, Simone; Ruminowicz, Chrystian; Wu, Xudong; Lee, Chung-Fan; Helin, Kristian; Jensen, Ole N

    2016-08-01

    Histones are abundant chromatin constituents carrying numerous post-translational modifications (PTMs). Such PTMs mediate a variety of biological functions, including recruitment of enzymatic readers, writers and erasers that modulate DNA replication, transcription and repair. Individual histone molecules contain multiple coexisting PTMs, some of which exhibit crosstalk, i.e. coordinated or mutually exclusive activities. Here, we present an integrated experimental and computational systems level molecular characterization of histone PTMs and PTM crosstalk. Using wild type and engineered mouse embryonic stem cells (mESCs) knocked out in components of the Polycomb Repressive Complex 2 (PRC2, Suz12(-/-)), PRC1 (Ring1A/B(-/-)) and (Dnmt1/3a/3b(-/-)) we performed comprehensive PTM analysis of histone H3 tails (50 aa) by utilizing quantitative middle-down proteome analysis by tandem mass spectrometry. We characterized combinatorial PTM features across the four mESC lines and then applied statistical data analysis to predict crosstalk between histone H3 PTMs. We detected an overrepresentation of positive crosstalk (codependent marks) between adjacent mono-methylated and acetylated marks, and negative crosstalk (mutually exclusive marks) among most of the seven characterized di- and tri-methylated lysine residues in the H3 tails. We report novel features of PTM interplay involving hitherto poorly characterized arginine methylation and lysine methylation sites, including H3R2me, H3R8me and H3K37me. Integration of the H3 data with RNAseq data by coabundance clustering analysis of histone PTMs and histone modifying enzymes revealed correlations between PTM and enzyme levels. We conclude that middle-down proteomics is a powerful tool to determine conserved or dynamic interdependencies between histone marks, which paves the way for detailed investigations of the histone code. Histone H3 PTM data is publicly available in the CrossTalkDB repository at http

  8. The Interplay of Cofactor Interactions and Post-translational Modifications in the Regulation of the AAA+ ATPase p97

    Science.gov (United States)

    Hänzelmann, Petra; Schindelin, Hermann

    2017-01-01

    The hexameric type II AAA ATPase (ATPase associated with various activities) p97 (also referred to as VCP, Cdc48, and Ter94) is critically involved in a variety of cellular activities including pathways such as DNA replication and repair which both involve chromatin remodeling, and is a key player in various protein quality control pathways mediated by the ubiquitin proteasome system as well as autophagy. Correspondingly, p97 has been linked to various pathophysiological states including cancer, neurodegeneration, and premature aging. p97 encompasses an N-terminal domain, two highly conserved ATPase domains and an unstructured C-terminal tail. This enzyme hydrolyzes ATP and utilizes the resulting energy to extract or disassemble protein targets modified with ubiquitin from stable protein assemblies, chromatin and membranes. p97 participates in highly diverse cellular processes and hence its activity is tightly controlled. This is achieved by multiple regulatory cofactors, which either associate with the N-terminal domain or interact with the extreme C-terminus via distinct binding elements and target p97 to specific cellular pathways, sometimes requiring the simultaneous association with more than one cofactor. Most cofactors are recruited to p97 through conserved binding motifs/domains and assist in substrate recognition or processing by providing additional molecular properties. A tight control of p97 cofactor specificity and diversity as well as the assembly of higher-order p97-cofactor complexes is accomplished by various regulatory mechanisms, which include bipartite binding, binding site competition, changes in oligomeric assemblies, and nucleotide-induced conformational changes. Furthermore, post-translational modifications (PTMs) like acetylation, palmitoylation, phosphorylation, SUMOylation, and ubiquitylation of p97 have been reported which further modulate its diverse molecular activities. In this review, we will describe the molecular basis of p97

  9. Environmental stress-mediated changes in transcriptional and translational regulation of protein synthesis in crop plants. Final report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-12-31

    The research described in this final report focused on the influence of stress agents on protein synthesis in crop plants (primarily soybean). Investigations into the `heat shock` (HS) stress mediated changes in transcriptional and translocational regulation of protein synthesis coupled with studies on anaerobic water deficit and other stress mediated alterations in protein synthesis in plants provided the basis of the research. Understanding of the HS gene expression and function(s) of the HSPs may clarify regulatory mechanisms operative in development. Since the reproductive systems of plants if often very temperature sensitive, it may be that the system could be manipulated to provide greater thermotolerance.

  10. Phosphorylation of xanthine dehydrogenase/oxidase in hypoxia.

    Science.gov (United States)

    Kayyali, U S; Donaldson, C; Huang, H; Abdelnour, R; Hassoun, P M

    2001-04-27

    The enzyme xanthine oxidase (XO) has been implicated in the pathogenesis of several disease processes, such as ischemia-reperfusion injury, because of its ability to generate reactive oxygen species. The expression of XO and its precursor xanthine dehydrogenase (XDH) is regulated at pre- and posttranslational levels by agents such as lipopolysaccharide and hypoxia. Posttranslational modification of the protein, for example through thiol oxidation or proteolysis, has been shown to be important in converting XDH to XO. The possibility of posttranslational modification of XDH/XO through phosphorylation has not been adequately investigated in mammalian cells, and studies have reported conflicting results. The present report demonstrates that XDH/XO is phosphorylated in rat pulmonary microvascular endothelial cells (RPMEC) and that phosphorylation is greatly increased ( approximately 50-fold) in response to acute hypoxia (4 h). XDH/XO phosphorylation appears to be mediated, at least in part, by casein kinase II and p38 kinase as inhibitors of these kinases partially prevent XDH/XO phosphorylation. In addition, the results indicate that p38 kinase, a stress-activated kinase, becomes activated in response to hypoxia (an approximately 4-fold increase after 1 h of exposure of RPMEC to hypoxia) further supporting a role for this kinase in hypoxia-stimulated XDH/XO phosphorylation. Finally, hypoxia-induced XDH/XO phosphorylation is accompanied by a 2-fold increase in XDH/XO activity, which is prevented by inhibitors of phosphorylation. In summary, this study shows that XDH/XO is phosphorylated in hypoxic RPMEC through a mechanism involving p38 kinase and casein kinase II and that phosphorylation is necessary for hypoxia-induced enzymatic activation.

  11. Hypoxia signaling pathways: modulators of oxygen-related organelles

    OpenAIRE

    Schönenberger, Miriam J.; Kovacs, Werner J.

    2015-01-01

    Oxygen (O2) is an essential substrate in cellular metabolism, bioenergetics, and signaling and as such linked to the survival and normal function of all metazoans. Low O2 tension (hypoxia) is a fundamental feature of physiological processes as well as pathophysiological conditions such as cancer and ischemic diseases. Central to the molecular mechanisms underlying O2 homeostasis are the hypoxia-inducible factors-1 and -2 alpha (HIF-1α and EPAS1/HIF-2α) that function as master regulators of th...

  12. Hypoxia and the hypoxic response pathway protect against pore-forming toxins in C. elegans.

    Directory of Open Access Journals (Sweden)

    Audrey Bellier

    2009-12-01

    Full Text Available Pore-forming toxins (PFTs are by far the most abundant bacterial protein toxins and are important for the virulence of many important pathogens. As such, cellular responses to PFTs critically modulate host-pathogen interactions. Although many cellular responses to PFTs have been recorded, little is understood about their relevance to pathological or defensive outcomes. To shed light on this important question, we have turned to the only genetic system for studying PFT-host interactions-Caenorhabditis elegans intoxication by Crystal (Cry protein PFTs. We mutagenized and screened for C. elegans mutants resistant to a Cry PFT and recovered one mutant. Complementation, sequencing, transgenic rescue, and RNA interference data demonstrate that this mutant eliminates a gene normally involved in repression of the hypoxia (low oxygen response pathway. We find that up-regulation of the C. elegans hypoxia pathway via the inactivation of three different genes that normally repress the pathway results in animals resistant to Cry PFTs. Conversely, mutation in the central activator of the hypoxia response, HIF-1, suppresses this resistance and can result in animals defective in PFT defenses. These results extend to a PFT that attacks mammals since up-regulation of the hypoxia pathway confers resistance to Vibrio cholerae cytolysin (VCC, whereas down-regulation confers hypersusceptibility. The hypoxia PFT defense pathway acts cell autonomously to protect the cells directly under attack and is different from other hypoxia pathway stress responses. Two of the downstream effectors of this pathway include the nuclear receptor nhr-57 and the unfolded protein response. In addition, the hypoxia pathway itself is induced by PFT, and low oxygen is protective against PFT intoxication. These results demonstrate that hypoxia and induction of the hypoxia response protect cells against PFTs, and that the cellular environment can be modulated via the hypoxia pathway to

  13. Translating Inclusion

    DEFF Research Database (Denmark)

    Fallov, Mia Arp; Birk, Rasmus

    2018-01-01

    The purpose of this paper is to explore how practices of translation shape particular paths of inclusion for people living in marginalized residential areas in Denmark. Inclusion, we argue, is not an end-state, but rather something which must be constantly performed. Active citizenship, today......, is not merely a question of participation, but of learning to become active in all spheres of life. The paper draws on empirical examples from a multi-sited field work in 6 different sites of local community work in Denmark, to demonstrate how different dimensions of translation are involved in shaping active...... citizenship. We propose the following different dimensions of translation: translating authority, translating language, translating social problems. The paper takes its theoretical point of departure from assemblage urbanism, arguing that cities are heterogeneous assemblages of socio-material interactions...

  14. Hypoxia signaling pathways: modulators of oxygen-related organelles

    Directory of Open Access Journals (Sweden)

    Miriam Johanna Schönenberger

    2015-07-01

    Full Text Available Oxygen (O2 is an essential substrate in cellular metabolism, bioenergetics, and signaling and as such linked to the survival and normal function of all metazoans. Low O2 tension (hypoxia is a fundamental feature of physiological processes as well as pathophysiological conditions such as cancer and ischemic diseases. Central to the molecular mechanisms underlying O2 homeostasis are the hypoxia-inducible factors-1 and -2 alpha (HIF-1a and EPAS1/HIF-2a that function as master regulators of the adaptive response to hypoxia. HIF-induced genes promote characteristic tumor behaviors, including angiogenesis and metabolic reprogramming. The aim of this review is to critically explore current knowledge of how HIF-a signaling regulates the abundance and function of major O2-consuming organelles. Abundant evidence suggests key roles for HIF-1a in the regulation of mitochondrial homeostasis. An essential adaptation to sustained hypoxia is repression of mitochondrial respiration and induction of glycolysis. HIF-1a activates several genes that trigger mitophagy and represses regulators of mitochondrial biogenesis. Several lines of evidence point to a strong relationship between hypoxia, the accumulation of misfolded proteins in the endoplasmic reticulum, and activation of the unfolded protein response. Surprisingly, although peroxisomes depend highly on molecular O2 for their function, there has been no evidence linking HIF signaling to peroxisomes. We discuss our recent findings that establish HIF-2a as a negative regulator of peroxisome abundance and suggest a mechanism by which cells attune peroxisomal function with O2 availability. HIF-2a activation augments peroxisome turnover by pexophagy and thereby changes lipid composition reminiscent of peroxisomal disorders. We discuss potential mechanisms by which HIF-2a might trigger pexophagy and place special emphasis on the potential pathological implications of HIF-2a-mediated pexophagy for human health.

  15. Environmental stress mediated changes in transcriptional and translational regulation of protein synthesis in crop plants. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Kochert, G.; Key, J.L.

    1985-04-01

    This progress report summarizes research accomplished during 1985. This research has focused on: (1) an analysis of the transport of heat shock proteins into chloroplasts and isolation of cDNA clones to those proteins, (2) development of monoclonal antibodies to 15 to 18 kD heat shock proteins, (3) additional analyses of heat shock protein localization focusing on ''heat shock granules'' and ribosome association of 15 to 18 kD heat shock proteins, (4) isolation of cDNA and genomic clones to heat shock proteins, hsp 70 and hsp 84, (5) Cadmium/arsenite-mediated induction of heat shock mRNAs and proteins and (6) some aspects of the regulation of heat shock mRNA levels.

  16. PRC2 regulates RNA polymerase III transcribed non-translated RNA gene transcription through EZH2 and SUZ12 interaction with TFIIIC complex

    Science.gov (United States)

    Liu, Chang; Li, Shuai; Dai, Xiaoyan; Ma, Ji; Wan, Junhu; Jiang, Hao; Wang, Peng; Liu, Zhaoli; Zhang, Hongquan

    2015-01-01

    Polycomb repression complex 2 (PRC2) component EZH2 tri-methylates H3K27 and exerts epigenetic repression on target gene expression. EZH2-mediated epigenetic control of RNA polymerase II (Pol II) transcribed coding gene transcription has been well established. However, little is known about EZH2-mediated epigenetic regulation of RNA polymerase III (Pol III) transcription. Here we present a paradigm that EZH2 is involved in the repression of Pol III transcription via interaction with transcriptional factor complex IIIC (TFIIIC). EZH2 and H3K27me3 co-occupy the promoter of tRNATyr, 5S rRNA and 7SL RNA genes. Depletion of EZH2 or inhibition of EZH2 methyltransferase activity led to upregulation of Pol III target gene transcription. EZH2-mediated repression of Pol III transcribed gene expression requires presence of SUZ12. SUZ12 was able to interact with TFIIIC complex and knockdown of SUZ12 decreased occupancy of EZH2 and H3K27me3 at the promoter of Pol III target genes. Our findings pointed out a previously unidentified role of PRC2 complex in suppressing transcription of Pol III transcribed non-translated RNA genes, putting Pol III on a new layer of epigenetic regulation. PMID:26038315

  17. The Germline Linker Histone dBigH1 and the Translational Regulator Bam Form a Repressor Loop Essential for Male Germ Stem Cell Differentiation.

    Science.gov (United States)

    Carbonell, Albert; Pérez-Montero, Salvador; Climent-Cantó, Paula; Reina, Oscar; Azorín, Fernando

    2017-12-12

    Drosophila spermatogenesis constitutes a paradigmatic system to study maintenance, proliferation, and differentiation of adult stem cell lineages. Each Drosophila testis contains 6-12 germ stem cells (GSCs) that divide asymmetrically to produce gonialblast cells that undergo four transit-amplifying (TA) spermatogonial divisions before entering spermatocyte differentiation. Mechanisms governing these crucial transitions are not fully understood. Here, we report the essential role of the germline linker histone dBigH1 during early spermatogenesis. Our results suggest that dBigH1 is a general silencing factor that represses Bam, a key regulator of spermatogonia proliferation that is silenced in spermatocytes. Reciprocally, Bam represses dBigH1 during TA divisions. This double-repressor mechanism switches dBigH1/Bam expression from off/on in spermatogonia to on/off in spermatocytes, regulating progression into spermatocyte differentiation. dBigH1 is also required for GSC maintenance and differentiation. These results show the critical importance of germline H1s for male GSC lineage differentiation, unveiling a regulatory interaction that couples transcriptional and translational repression. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Post-transcriptional regulation of the Streptomyces coelicolor stress responsive sigma factor, SigH, involves translational control, proteolytic processing, and an anti-sigma factor homolog.

    Science.gov (United States)

    Viollier, Patrick H; Weihofen, Andreas; Folcher, Marc; Thompson, Charles J

    2003-01-24

    The sigH gene encodes a sigma factor whose transcription is controlled by stress regulatory systems and the developmental program in Streptomyces coelicolor. Here, we describe developmentally regulated post-transcriptional control systems for SigH. sigH is expressed as three primary translation products, SigH-sigma(37), SigH-sigma(51), and SigH-sigma(52). In vitro, SigH-sigma(52) was comparable to SigH-sigma(37) in its ability to associate with RNA polymerase core enzyme and specifically initiate transcription in vitro. While SigH-sigma(51/52) were the primary gene products observed throughout early phases of growth, their abundance decreased during later stages in liquid or solid phase cultures while levels of shorter, C-terminally encoded products increased. These included SigH-sigma(37), a product of the downstream translational initiation site, as well as two proteolytic derivatives of SigH-sigma(51/52) (34kDa and 38kDa). Accumulation of SigH-sigma(37) and processing of SigH-sigma(51/52) into these stable 34kDa and 38kDa derivatives correlated with morphological changes on solid medium and physiological maturation in liquid medium. SigH-sigma(51/52) processing did not occur on medium non-permissive for aerial mycelium formation or in one particular developmental mutant (brgA). The proteolytic activity could be detected in vitro using crude extracts of stationary phase cultures, but was absent from exponential phase cultures. prsH, the gene upstream of sigH having sequence similarity to known anti-sigma factors, was able to bind to, and thus presumably inactivate SigH-sigma(52), SigH-sigma(51), and SigH-sigma(37). We have shown elsewhere that prsH was conditionally required for colonial development. Thus, while at least one transcriptional regulator is known to bring about the accumulation of sigH mRNA at different times and different locations in colonies, the post-transcriptional processes described here regulate the activity of different SigH isoforms and

  19. A murine retrovirus co-Opts YB-1, a translational regulator and stress granule-associated protein, to facilitate virus assembly.

    Science.gov (United States)

    Bann, Darrin V; Beyer, Andrea R; Parent, Leslie J

    2014-04-01

    The Gag protein of the murine retrovirus mouse mammary tumor virus (MMTV) orchestrates the assembly of immature virus particles in the cytoplasm which are subsequently transported to the plasma membrane for release from the cell. The morphogenetic pathway of MMTV assembly is similar to that of Saccharomyces cerevisiae retrotransposons Ty1 and Ty3, which assemble virus-like particles (VLPs) in intracytoplasmic ribonucleoprotein (RNP) complexes. Assembly of Ty1 and Ty3 VLPs depends upon cellular mRNA processing factors, prompting us to examine whether MMTV utilizes a similar set of host proteins to facilitate viral capsid assembly. Our data revealed that MMTV Gag colocalized with YB-1, a translational regulator found in stress granules and P bodies, in intracytoplasmic foci. The association of MMTV Gag and YB-1 in cytoplasmic granules was not disrupted by cycloheximide treatment, suggesting that these sites were not typical stress granules. However, the association of MMTV Gag and YB-1 was RNA dependent, and an MMTV RNA reporter construct colocalized with Gag and YB-1 in cytoplasmic RNP complexes. Knockdown of YB-1 resulted in a significant decrease in MMTV particle production, indicating that YB-1 plays a role in MMTV capsid formation. Analysis by live-cell imaging with fluorescence recovery after photobleaching (FRAP) revealed that the population of Gag proteins localized within YB-1 complexes was relatively immobile, suggesting that Gag forms stable complexes in association with YB-1. Together, our data imply that the formation of intracytoplasmic Gag-RNA complexes is facilitated by YB-1, which promotes MMTV virus assembly. Cellular mRNA processing factors regulate the posttranscriptional fates of mRNAs, affecting localization and utilization of mRNAs under normal conditions and in response to stress. RNA viruses such as retroviruses interact with cellular mRNA processing factors that accumulate in ribonucleoprotein complexes known as P bodies and stress granules

  20. Further insights into the mechanism of hypoxia-induced NFκB. [corrected].

    Science.gov (United States)

    Melvin, Andrew; Mudie, Sharon; Rocha, Sonia

    2011-03-15

    The cellular response to hypoxia relies on the activation of a specific transcriptional program. Although, most of the attention is focused on the transcription factor HIF, other transcription factors are also activated in hypoxia. We have recently described the mechanism for hypoxia induced NFκB. We have demonstrated the crucial dependency on the IKK complex as well as in the upstream IKK kinase TAK1. TAK1 and IKK activation is dependent upon the calcium calmodulin kinase, CaMK2 and requires Ubc13 as the E2 ubiquitin conjugation enzyme. We report a role for XIAP as the possible E3-ubiquitin ligase for this system. Interestingly, hypoxia induced IKK mediated phosphorylation of IκBα, does not lead to degradation. Hypoxia prevents IκBα de-sumoylation of Sumo-2/3 chains on critical lysine residues, normally required for K-48 linked polyubiquitination. Our results define a novel pathway regulating NFκB activation.

  1. Hypoxia, Epithelial-Mesenchymal Transition, and TET-Mediated Epigenetic Changes

    Directory of Open Access Journals (Sweden)

    Shih-Han Kao

    2016-02-01

    Full Text Available Tumor hypoxia is a pathophysiologic outcome of disrupted microcirculation with inadequate supply of oxygen, leading to enhanced proliferation, epithelial-mesenchymal transition (EMT, metastasis, and chemo-resistance. Epigenetic changes induced by hypoxia are well documented, and they lead to tumor progression. Recent advances show that DNA demethylation mediated by the Ten-eleven translocation (TET proteins induces major epigenetic changes and controls key steps of cancer development. TET enzymes serve as 5mC (5-methylcytosine-specific dioxygenases and cause DNA demethylation. Hypoxia activates the expression of TET1, which also serves as a co-activator of HIF-1α transcriptional regulation to modulate HIF-1α downstream target genes and promote epithelial-mesenchymal transition. As HIF is a negative prognostic factor for tumor progression, hypoxia-activated prodrugs (HAPs may provide a favorable therapeutic approach to lessen hypoxia-induced malignancy.

  2. Hypobaric intermittent hypoxia attenuates hypoxia-induced depressor response.

    Directory of Open Access Journals (Sweden)

    Fang Cui

    Full Text Available Hypobaric intermittent hypoxia (HIH produces many favorable effects in the cardiovascular system such as anti-hypertensive effect. In this study, we showed that HIH significantly attenuated a depressor response induced by acute hypoxia.Sprague-Dawley rats received HIH in a hypobaric chamber simulating an altitude of 5000 m. The artery blood pressure (ABP, heart rate (HR and renal sympathetic nerve activity (RSNA were recorded in anesthetized control rats and rats received HIH. The baseline ABP, HR and RSNA were not different between HIH and control rats. Acute hypoxia-induced decrease in ABP was significantly attenuated in HIH rat compared with control rats. However, acute hypoxia-induced increases in HR and RSNA were greater in HIH rat than in control rats. After removal of bilateral ascending depressor nerves, acute hypoxia-induced depressor and sympathoexcitatory responses were comparable in control and HIH rats. Furthermore, acute hypoxia-induced depressor and sympathoexcitatory responses did not differ between control and HIH groups after blocking ATP-dependent K(+ channels by glibenclamide. The baroreflex function evaluated by intravenous injection of phenylephrine and sodium nitroprusside was markedly augmented in HIH rats compared with control rats. The pressor and sympathoexcitatory responses evoked by intravenous injection of cyanide potassium were also significantly greater in HIH rats than in control rats.Our findings suggest that HIH suppresses acute hypoxia-induced depressor response through enhancement of baroreflex and chemoreflex function, which involves activation of ATP-dependent K(+ channels. This study provides new information and underlying mechanism on the beneficiary effect of HIH on maintaining cardiovascular homeostasis.

  3. Gene Expression Programs in Response to Hypoxia: Cell Type Specificity and Prognostic Significance in Human Cancers.

    Directory of Open Access Journals (Sweden)

    2006-01-01

    Full Text Available BACKGROUND: Inadequate oxygen (hypoxia triggers a multifaceted cellular response that has important roles in normal physiology and in many human diseases. A transcription factor, hypoxia-inducible factor (HIF, plays a central role in the hypoxia response; its activity is regulated by the oxygen-dependent degradation of the HIF-1alpha protein. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia among different cell types or how this variation might relate to tissue- and cell-specific diseases. METHODS AND FINDINGS: We analyzed the temporal changes in global transcript levels in response to hypoxia in primary renal proximal tubule epithelial cells, breast epithelial cells, smooth muscle cells, and endothelial cells with DNA microarrays. The extent of the transcriptional response to hypoxia was greatest in the renal tubule cells. This heightened response was associated with a uniquely high level of HIF-1alpha RNA in renal cells, and it could be diminished by reducing HIF-1alpha expression via RNA interference. A gene-expression signature of the hypoxia response, derived from our studies of cultured mammary and renal tubular epithelial cells, showed coordinated variation in several human cancers, and was a strong predictor of clinical outcomes in breast and ovarian cancers. In an analysis of a large, published gene-expression dataset from breast cancers, we found that the prognostic information in the hypoxia signature was virtually independent of that provided by the previously reported wound signature and more predictive of outcomes than any of the clinical parameters in current use. CONCLUSIONS: The transcriptional response to hypoxia varies among human cells. Some of this variation is traceable to variation in expression of the HIF1A gene. A gene-expression signature of the cellular response to hypoxia is associated with a significantly poorer prognosis

  4. Gene expression programs in response to hypoxia: cell type specificity and prognostic significance in human cancers.

    Directory of Open Access Journals (Sweden)

    Jen-Tsan Chi

    2006-03-01

    Full Text Available Inadequate oxygen (hypoxia triggers a multifaceted cellular response that has important roles in normal physiology and in many human diseases. A transcription factor, hypoxia-inducible factor (HIF, plays a central role in the hypoxia response; its activity is regulated by the oxygen-dependent degradation of the HIF-1alpha protein. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia among different cell types or how this variation might relate to tissue- and cell-specific diseases.We analyzed the temporal changes in global transcript levels in response to hypoxia in primary renal proximal tubule epithelial cells, breast epithelial cells, smooth muscle cells, and endothelial cells with DNA microarrays. The extent of the transcriptional response to hypoxia was greatest in the renal tubule cells. This heightened response was associated with a uniquely high level of HIF-1alpha RNA in renal cells, and it could be diminished by reducing HIF-1alpha expression via RNA interference. A gene-expression signature of the hypoxia response, derived from our studies of cultured mammary and renal tubular epithelial cells, showed coordinated variation in several human cancers, and was a strong predictor of clinical outcomes in breast and ovarian cancers. In an analysis of a large, published gene-expression dataset from breast cancers, we found that the prognostic information in the hypoxia signature was virtually independent of that provided by the previously reported wound signature and more predictive of outcomes than any of the clinical parameters in current use.The transcriptional response to hypoxia varies among human cells. Some of this variation is traceable to variation in expression of the HIF1A gene. A gene-expression signature of the cellular response to hypoxia is associated with a significantly poorer prognosis in breast and ovarian cancer.

  5. Prader-Willi Critical Region, a Non-Translated, Imprinted Central Regulator of Bone Mass: Possible Role in Skeletal Abnormalities in Prader-Willi Syndrome.

    Directory of Open Access Journals (Sweden)

    Ee-Cheng Khor

    Full Text Available Prader-Willi Syndrome (PWS, a maternally imprinted disorder and leading cause of obesity, is characterised by insatiable appetite, poor muscle development, cognitive impairment, endocrine disturbance, short stature and osteoporosis. A number of causative loci have been located within the imprinted Prader-Willi Critical Region (PWCR, including a set of small non-translated nucleolar RNA's (snoRNA. Recently, micro-deletions in humans identified the snoRNA Snord116 as a critical contributor to the development of PWS exhibiting many of the classical symptoms of PWS. Here we show that loss of the PWCR which includes Snord116 in mice leads to a reduced bone mass phenotype, similar to that observed in humans. Consistent with reduced stature in PWS, PWCR KO mice showed delayed skeletal development, with shorter femurs and vertebrae, reduced bone size and mass in both sexes. The reduction in bone mass in PWCR KO mice was associated with deficiencies in cortical bone volume and cortical mineral apposition rate, with no change in cancellous bone. Importantly, while the length difference was corrected in aged mice, consistent with continued growth in rodents, reduced cortical bone formation was still evident, indicating continued osteoblastic suppression by loss of PWCR expression in skeletally mature mice. Interestingly, deletion of this region included deletion of the exclusively brain expressed Snord116 cluster and resulted in an upregulation in expression of both NPY and POMC mRNA in the arcuate nucleus. Importantly, the selective deletion of the PWCR only in NPY expressing neurons replicated the bone phenotype of PWCR KO mice. Taken together, PWCR deletion in mice, and specifically in NPY neurons, recapitulates the short stature and low BMD and aspects of the hormonal imbalance of PWS individuals. Moreover, it demonstrates for the first time, that a region encoding non-translated RNAs, expressed solely within the brain, can regulate bone mass in health

  6. Prader-Willi Critical Region, a Non-Translated, Imprinted Central Regulator of Bone Mass: Possible Role in Skeletal Abnormalities in Prader-Willi Syndrome.

    Science.gov (United States)

    Khor, Ee-Cheng; Fanshawe, Bruce; Qi, Yue; Zolotukhin, Sergei; Kulkarni, Rishikesh N; Enriquez, Ronaldo F; Purtell, Louise; Lee, Nicola J; Wee, Natalie K; Croucher, Peter I; Campbell, Lesley; Herzog, Herbert; Baldock, Paul A

    2016-01-01

    Prader-Willi Syndrome (PWS), a maternally imprinted disorder and leading cause of obesity, is characterised by insatiable appetite, poor muscle development, cognitive impairment, endocrine disturbance, short stature and osteoporosis. A number of causative loci have been located within the imprinted Prader-Willi Critical Region (PWCR), including a set of small non-translated nucleolar RNA's (snoRNA). Recently, micro-deletions in humans identified the snoRNA Snord116 as a critical contributor to the development of PWS exhibiting many of the classical symptoms of PWS. Here we show that loss of the PWCR which includes Snord116 in mice leads to a reduced bone mass phenotype, similar to that observed in humans. Consistent with reduced stature in PWS, PWCR KO mice showed delayed skeletal development, with shorter femurs and vertebrae, reduced bone size and mass in both sexes. The reduction in bone mass in PWCR KO mice was associated with deficiencies in cortical bone volume and cortical mineral apposition rate, with no change in cancellous bone. Importantly, while the length difference was corrected in aged mice, consistent with continued growth in rodents, reduced cortical bone formation was still evident, indicating continued osteoblastic suppression by loss of PWCR expression in skeletally mature mice. Interestingly, deletion of this region included deletion of the exclusively brain expressed Snord116 cluster and resulted in an upregulation in expression of both NPY and POMC mRNA in the arcuate nucleus. Importantly, the selective deletion of the PWCR only in NPY expressing neurons replicated the bone phenotype of PWCR KO mice. Taken together, PWCR deletion in mice, and specifically in NPY neurons, recapitulates the short stature and low BMD and aspects of the hormonal imbalance of PWS individuals. Moreover, it demonstrates for the first time, that a region encoding non-translated RNAs, expressed solely within the brain, can regulate bone mass in health and disease.

  7. YelA, a putative Dictyostelium translational regulator, acts as antagonist of DIF-1 signaling to control cell-type proportioning.

    Science.gov (United States)

    Yamada, Yoko; Sugden, Chris; Williams, Jeffrey G

    2017-01-01

    DIF-1 (differentiation-inducing factor1) is a polyketide produced by Dictyostelium prespore cells which induces initially uncommitted cells to differentiate as prestalk cells. Exposure of cells to DIF-1 causes transitory hypo-phosphorylation of seven serine residues in YelA, a protein with a region of strong homology to the MIF4G domain of the eukaryotic initiation factor eIF4G. Based upon its domain architecture, which in one important aspect closely resembles that of Death-Associated Protein 5 (DAP5), we predict a role in stimulating internal ribosome entry-driven mRNA translation. The two paradigmatic DIF-1 inducible genes are ecmA (extracellular matrixA) and ecmB. In support of a YelA function in DIF-1 signaling, a YelA null strain showed greatly increased expression of ecmA and ecmB in response to DIF-1. Also, during normal development in the null strain, expression of the two genes is accelerated. This is particularly evident for ecmB, a marker of stalk tube and supporting structure differentiation. Mutants in DIF-1 bio-synthesis or signaling display a rudimentary or no basal disc and, conversely, YelA null mutants produce fruiting bodies with a highly enlarged basal disc that ectopically expresses a stalk tube-specific marker. Thus YelA acts as an antagonist of DIF-1 signaling, with a consequent effect on cell type proportioning and it is predicted to act as a translational regulator.

  8. Translation Competence

    DEFF Research Database (Denmark)

    Vandepitte, Sonia; Mousten, Birthe; Maylath, Bruce

    2014-01-01

    at different stages in the translation processes in the Trans-Atlantic and Pacific Project, a long-term cross-cultural virtual team. It describes the forms of collaborative learning practised in this multilateral international project in technical communication and translator training programmes and explores...

  9. Translator: Expert of ‘What’? Translator Training and the Changing/Changeful Identity of the Translator

    Directory of Open Access Journals (Sweden)

    Senem Öner

    2013-07-01

    Full Text Available The aim of this paper is to trace the contemporary perceptions/definitions of the translator and translation in Turkey where the translation profession is not a legally regulated profession. (Invisibility of the translator and the scope of his/her expertise are widely debated issues among both the scholars/students of translation and the various players in the world of translation. Although translation is no longer being defined as a mere linguistic activity, reductionist views of translation that equate translation to ‘know’ing a foreign language still continues to prevail. What accompanies the said reductionist view is the claim that a translator can not translate ‘all the texts’ in different fields of expertise, at least not as successfully as the ‘real’ experts of the field who also know a foreign language such as engineer-translators or lawyer-translators who know English. However, recent developments concerning legal translation in plurilingual settings evidence a completely different understanding of translation and the identity/expertise of the translator. In such settings, translators work together with the drafters of the legal texts and are responsible for the source legal texts as well as the target ones, broadening the scope of the ‘expertise’ of the translator in an unprecedented way. In the light of the implications of the said transformation of the identity of the legal translator, this paper argues that the identity/definition of the translator/translation needs to be reconsidered and the results should be reflected onto the translator training programs with the aim of helping students of translation (redefine their identity as real ‘experts’ of the translation field and contributing to the transformation of the perception of translation and the translator.

  10. Translator: Expert of ‘What’? Translator Training and the Changing/Changeful Identity of the Translator

    Directory of Open Access Journals (Sweden)

    Senem Öner

    2013-06-01

    Full Text Available The aim of this paper is to trace the contemporary perceptions/definitions of the translator and translation in Turkey where the translation profession is not a legally regulated profession. (Invisibility of the translator and the scope of his/her expertise are widely debated issues among both the scholars/students of translation and the various players in the world of translation. Although translation is no longer being defined as a mere linguistic activity, reductionist views of translation that equate translation to ‘know’ing a foreign language still continues to prevail. What accompanies the said reductionist view is the claim that a translator can not translate ‘all the texts’ in different fields of expertise, at least not as successfully as the ‘real’ experts of the field who also know a foreign language such as engineer-translators or lawyer-translators who know English. However, recent developments concerning legal translation in plurilingual settings evidence a completely different understanding of translation and the identity/expertise of the translator. In such settings, translators work together with the drafters of the legal texts and are responsible for the source legal texts as well as the target ones, broadening the scope of the ‘expertise’ of the translator in an unprecedented way. In the light of the implications of the said transformation of the identity of the legal translator, this paper argues that the identity/definition of the translator/translation needs to be reconsidered and the results should be reflected onto the translator training programs with the aim of helping students of translation (redefine their identity as real ‘experts’ of the translation field and contributing to the transformation of the perception of translation and the translator.

  11. Hypoxia-Related Hormonal Appetite Modulation in Humans during Rest and Exercise: Mini Review

    Directory of Open Access Journals (Sweden)

    Tadej Debevec

    2017-05-01

    Full Text Available Obesity is associated with numerous chronic ailments and represents one of the major health and economic issues in the modernized societies. Accordingly, there is an obvious need for novel treatment approaches. Recently, based on the reports of reduced appetite and subsequent weight loss following high-altitude sojourns, exposure to hypoxia has been proposed as a viable weight-reduction strategy. While altitude-related appetite modulation is complex and not entirely clear, hypoxia-induced alterations in hormonal appetite modulation might be among the key underlying mechanisms. The present paper summarizes the up-to-date research on hypoxia/altitude-induced changes in the gut and adipose tissue derived peptides related to appetite regulation. Orexigenic hormone ghrelin and anorexigenic peptides leptin, glucagon-like peptide-1, peptide YY, and cholecystokinin have to-date been investigated as potential modulators of hypoxia-driven appetite alterations. Current evidence suggests that hypoxia can, especially acutely, lead to decreased appetite, most probably via reduction of acylated ghrelin concentration. Hypoxia-related short and long-term changes in other hormonal markers are more unclear although hypoxia seems to importantly modulate leptin levels, especially following prolonged hypoxic exposures. Limited evidence also suggests that different activity levels during exposures to hypoxia do not additively affect hormonal appetite markers. Although very few studies have been performed in obese/overweight individuals, the available data indicate that hypoxia/altitude exposures do not seem to differentially affect appetite regulation via hormonal pathways in this cohort. Given the lack of experimental data, future well-controlled acute and prolonged studies are warranted to expand our understanding of hypoxia-induced hormonal appetite modulation and its kinetics in health and disease.

  12. Induction of gastrin expression in gastrointestinal cells by hypoxia or cobalt is independent of hypoxia-inducible factor (HIF).

    Science.gov (United States)

    Xiao, Lin; Kovac, Suzana; Chang, Mike; Shulkes, Arthur; Baldwin, Graham S; Patel, Oneel

    2012-07-01

    Gastrin and its precursors have been shown to promote mitogenesis and angiogenesis in gastrointestinal tumors. Hypoxia stimulates tumor growth, but its effect on gastrin gene regulation has not been examined in detail. Here we have investigated the effect of hypoxia on the transcription of the gastrin gene in human gastric cancer (AGS) cells. Gastrin mRNA was measured by real-time PCR, gastrin peptides were measured by RIA, and gastrin promoter activity was measured by dual-luciferase reporter assay. Exposure to a low oxygen concentration (1%) increased gastrin mRNA concentrations in wild-type AGS cells (AGS) and in AGS cells overexpressing the gastrin receptor (AGS-cholecystokinin receptor 2) by 2.1 ± 0.4- and 4.1 ± 0.3-fold (P gastrin promoter activity in AGS cells by 2.4 ± 0.3-fold (P gastrin promoter of the putative binding sites for the transcription factor hypoxia-inducible factor 1 (HIF-1) or knockdown of either the HIF-1α or HIF-1β subunit did not affect gastrin promoter inducibility under hypoxia indicated that the hypoxic activation of the gastrin gene is likely HIF independent. Mutational analysis of previously identified Sp1 regulatory elements in the gastrin promoter also failed to abrogate the induction of promoter activity by hypoxia. The observations that hypoxia up-regulates the gastrin gene in AGS cells by HIF-independent mechanisms, and that this effect is enhanced by the presence of gastrin receptors, provide potential targets for gastrointestinal cancer therapy.

  13. Transcriptional targeting of acute hypoxia in the tumour stroma is a novel and viable strategy for cancer gene therapy.

    Science.gov (United States)

    Ingram, N; Porter, C D

    2005-07-01

    Deregulated tumour growth and neovascularization result in an inadequate tumour blood supply, leading to areas of chronic hypoxia and necrosis. Irregular vascular structure and abnormal tumour physiology also cause erratic blood flow in tumour vessels. We reasoned that tumour stroma, including vascular endothelial cells, would consequently experience transient hypoxia that may allow transcriptional targeting as part of an antivascular gene therapy approach to cancer. To exploit hypoxia for transcriptional regulation, retroviral vectors were generated with modified LTRs: a 6-mer of hypoxia response elements in place of the viral enhancer produced near wild-type levels of expression in hypoxia but was functionally inert in normoxia. In a tumour xenograft model, expression was mainly around areas of necrosis, which were shown to be hypoxic; no expression was detected in tumour stroma. Time-course experiments in vitro demonstrated that expression was transient in response to a hypoxic episode, such that a reporter gene would be insensitive to acute hypoxia in vivo. In contrast, a significant therapeutic effect was seen upon ganciclovir administration with a vector expressing thymidine kinase (TK) in the tumour stroma. Expression of TK was more effective when targeted to acute hypoxia in the stroma compared to chronic hypoxia in the poorly vascularized regions of the tumour cell compartment. The data presented here are evidence that hypoxia in the stromal compartment does occur and that transient hypoxia constitutes a valid therapeutic target.

  14. Intermittent Hypoxia Effect on Osteoclastogenesis Stimulated by Neuroblastoma Cells

    Science.gov (United States)

    Bhaskara, Vasantha Kumar; Mohanam, Indra; Gujrati, Meena; Mohanam, Sanjeeva

    2014-01-01

    Background Neuroblastoma is the most common extracranial pediatric solid tumor. Intermittent hypoxia, which is characterized by cyclic periods of hypoxia and reoxygenation, has been shown to positively modulate tumor development and thereby induce tumor growth, angiogenic processes, and metastasis. Bone is one of the target organs of metastasis in advanced neuroblastoma Neuroblastoma cells produce osteoclast-activating factors that increase bone resorption by the osteoclasts. The present study focuses on how intermittent hypoxia preconditioned SH-SY5Y neuroblastoma cells modulate osteoclastogenesis in RAW 264.7 cells compared with neuroblastoma cells grown at normoxic conditions. Methods We inhibited HIF-1α and HIF-2α in neuroblastoma SH-SY5Y cells by siRNA/shRNA approaches. Protein expression of HIF-1α, HIF-2α and MAPKs were investigated by western blotting. Expression of osteoclastogenic factors were determined by real-time RT-PCR. The influence of intermittent hypoxia and HIF-1α siRNA on migration of neuroblastoma cells and in vitro differentiation of RAW 264.7 cells were assessed. Intratibial injection was performed with SH-SY5Y stable luciferase-expressing cells and in vivo bioluminescence imaging was used in the analysis of tumor growth in bone. Results Upregulation of mRNAs of osteoclastogenic factors VEGF and RANKL was observed in intermittent hypoxia-exposed neuroblastoma cells. Conditioned medium from the intermittent hypoxia-exposed neuroblastoma cells was found to enhance osteoclastogenesis, up-regulate the mRNAs of osteoclast marker genes including TRAP, CaSR and cathepsin K and induce the activation of ERK, JNK, and p38 in RAW 264.7 cells. Intermittent hypoxia-exposed neuroblastoma cells showed an increased migratory pattern compared with the parental cells. A significant increase of tumor volume was found in animals that received the intermittent hypoxia-exposed cells intratibially compared with parental cells. Conclusions Intermittent hypoxic

  15. The Linkage between Breast Cancer, Hypoxia, and Adipose Tissue

    Directory of Open Access Journals (Sweden)

    Linda K. Rausch

    2017-09-01

    Full Text Available ObjectiveThe development of breast cancer cells is linked to hypoxia. The hypoxia-induced factor HIF-1α influences metastasis through neovascularization. Hypoxia seems to decrease the responsiveness to hormonal treatment due to loss of estrogen receptors (ERs. Obesity is discussed to increase hypoxia in adipocytes, which promotes a favorable environment for tumor cells in mammary fat tissue, whereas, tumor cells profit from good oxygen supply and are influenced by its deprivation as target regions within tumors show. This review gives an overview of the current state on research of hypoxia and breast cancer in human adipose tissue.MethodsA systematic literature search was conducted on PubMed (2000–2016 by applying hypoxia and/or adipocytes and breast cancer as keywords. Review articles were excluded as well as languages other than English or German. There was no restriction regarding the study design or type of breast cancer. A total of 35 papers were found. Eight studies were excluded due to missing at least two of the three keywords. One paper was removed due to Russian language, and one was dismissed due to lack of adherence. Seven papers were identified as reviews. After applying exclusion criteria, 18 articles were eligible for inclusion.ResultsTwo articles describe the impairment of mammary epithelial cell polarization through hypoxic preconditioning. A high amount of adipocytes enhances cancer progression due to the increased expression of HIF-1α which causes the loss of ER α protein as stated in four articles. Four articles analyzed that increased activation of HIF’s induces a series of transcriptions resulting in tumor angiogenesis. HIF inhibition, especially when combined with cytotoxic chemotherapy, holds strong potential for tumor suppression as stated in further four articles. In two articles there is evidence of a strong connection between hypoxia, oxidative stress and a poor prognosis for breast cancer via HIF regulated

  16. Hypoxia-induced cytotoxic drug resistance in osteosarcoma is independent of HIF-1Alpha.

    Directory of Open Access Journals (Sweden)

    Jennifer Adamski

    Full Text Available Survival rates from childhood cancer have improved dramatically in the last 40 years, such that over 80% of children are now cured. However in certain subgroups, including metastatic osteosarcoma, survival has remained stubbornly poor, despite dose intensive multi-agent chemotherapy regimens, and new therapeutic approaches are needed. Hypoxia is common in adult solid tumours and is associated with treatment resistance and poorer outcome. Hypoxia induces chemotherapy resistance in paediatric tumours including neuroblastoma, rhabdomyosarcoma and Ewing's sarcoma, in vitro, and this drug resistance is dependent on the oxygen-regulated transcription factor hypoxia inducible factor-1 (HIF-1. In this study the effects of hypoxia on the response of the osteosarcoma cell lines 791T, HOS and U2OS to the clinically relevant cytotoxics cisplatin, doxorubicin and etoposide were evaluated. Significant hypoxia-induced resistance to all three agents was seen in all three cell lines and hypoxia significantly reduced drug-induced apoptosis. Hypoxia also attenuated drug-induced activation of p53 in the p53 wild-type U2OS osteosarcoma cells. Drug resistance was not induced by HIF-1α stabilisation in normoxia by cobalt chloride nor reversed by the suppression of HIF-1α in hypoxia by shRNAi, siRNA, dominant negative HIF or inhibition with the small molecule NSC-134754, strongly suggesting that hypoxia-induced drug resistance in osteosarcoma cells is independent of HIF-1α. Inhibition of the phosphoinositide 3-kinase (PI3K pathway using the inhibitor PI-103 did not reverse hypoxia-induced drug resistance, suggesting the hypoxic activation of Akt in osteosarcoma cells does not play a significant role in hypoxia-induced drug resistance. Targeting hypoxia is an exciting prospect to improve current anti-cancer therapy and combat drug resistance. Significant hypoxia-induced drug resistance in osteosarcoma cells highlights the potential importance of hypoxia as a target

  17. Evolutionary conserved regulation of HIF-1β by NF-κB.

    Directory of Open Access Journals (Sweden)

    Patrick van Uden

    2011-01-01

    Full Text Available Hypoxia Inducible Factor-1 (HIF-1 is essential for mammalian development and is the principal transcription factor activated by low oxygen tensions. HIF-α subunit quantities and their associated activity are regulated in a post-translational manner, through the concerted action of a class of enzymes called Prolyl Hydroxylases (PHDs and Factor Inhibiting HIF (FIH respectively. However, alternative modes of HIF-α regulation such as translation or transcription are under-investigated, and their importance has not been firmly established. Here, we demonstrate that NF-κB regulates the HIF pathway in a significant and evolutionary conserved manner. We demonstrate that NF-κB directly regulates HIF-1β mRNA and protein. In addition, we found that NF-κB-mediated changes in HIF-1β result in modulation of HIF-2α protein. HIF-1β overexpression can rescue HIF-2α protein levels following NF-κB depletion. Significantly, NF-κB regulates HIF-1β (tango and HIF-α (sima levels and activity (Hph/fatiga, ImpL3/ldha in Drosophila, both in normoxia and hypoxia, indicating an evolutionary conserved mode of regulation. These results reveal a novel mechanism of HIF regulation, with impact in the development of novel therapeutic strategies for HIF-related pathologies including ageing, ischemia, and cancer.

  18. Evolutionary conserved regulation of HIF-1β by NF-κB.

    Science.gov (United States)

    van Uden, Patrick; Kenneth, Niall S; Webster, Ryan; Müller, H Arno; Mudie, Sharon; Rocha, Sonia

    2011-01-27

    Hypoxia Inducible Factor-1 (HIF-1) is essential for mammalian development and is the principal transcription factor activated by low oxygen tensions. HIF-α subunit quantities and their associated activity are regulated in a post-translational manner, through the concerted action of a class of enzymes called Prolyl Hydroxylases (PHDs) and Factor Inhibiting HIF (FIH) respectively. However, alternative modes of HIF-α regulation such as translation or transcription are under-investigated, and their importance has not been firmly established. Here, we demonstrate that NF-κB regulates the HIF pathway in a significant and evolutionary conserved manner. We demonstrate that NF-κB directly regulates HIF-1β mRNA and protein. In addition, we found that NF-κB-mediated changes in HIF-1β result in modulation of HIF-2α protein. HIF-1β overexpression can rescue HIF-2α protein levels following NF-κB depletion. Significantly, NF-κB regulates HIF-1β (tango) and HIF-α (sima) levels and activity (Hph/fatiga, ImpL3/ldha) in Drosophila, both in normoxia and hypoxia, indicating an evolutionary conserved mode of regulation. These results reveal a novel mechanism of HIF regulation, with impact in the development of novel therapeutic strategies for HIF-related pathologies including ageing, ischemia, and cancer.

  19. Hypoxia activated EGFR signaling induces epithelial to mesenchymal transition (EMT.

    Directory of Open Access Journals (Sweden)

    Ashish Misra

    Full Text Available Metastasis is a multi-step process which requires the conversion of polarized epithelial cells to mesenchymal cells, Epithelial-Mesenchymal Transition (EMT. EMT is essential during embryonic morphogenesis and has been implicated in the progression of primary tumors towards metastasis. Hypoxia is known to induce EMT; however the molecular mechanism is still poorly understood. Using the A431 epithelial cancer cell line, we show that cells grown under hypoxic conditions migrated faster than cells grown under normal oxygen environment. Cells grown under hypoxia showed reduced adhesion to the extracellular matrix (ECM probably due to reduced number of Vinculin patches. Growth under hypoxic conditions also led to down regulation of E-cadherin and up regulation of vimentin expression. The increased motility of cells grown under hypoxia could be due to redistribution of Rac1 to the plasma membrane as opposed to increased expression of Rac1. EGF (Epidermal Growth Factor is a known inducer of EMT and growth of A431 cells in the absence of oxygen led to increased expression of EGFR (EGF Receptor. Treatment of A431 cells with EGF led to reduced cell adhesion to ECM, increased cell motility and other EMT characteristics. Furthermore, this transition was blocked by the monoclonal antibody Cetuximab. Cetuximab also blocked the hypoxia-induced EMT suggesting that cell growth under hypoxic conditions led to activation of EGFR signaling and induction of EMT phenotype.

  20. Binary translation using peephole translation rules

    Science.gov (United States)

    Bansal, Sorav; Aiken, Alex

    2010-05-04

    An efficient binary translator uses peephole translation rules to directly translate executable code from one instruction set to another. In a preferred embodiment, the translation rules are generated using superoptimization techniques that enable the translator to automatically learn translation rules for translating code from the source to target instruction set architecture.

  1. Lung Oxidative Damage by Hypoxia

    Directory of Open Access Journals (Sweden)

    O. F. Araneda

    2012-01-01

    Full Text Available One of the most important functions of lungs is to maintain an adequate oxygenation in the organism. This organ can be affected by hypoxia facing both physiological and pathological situations. Exposure to this condition favors the increase of reactive oxygen species from mitochondria, as from NADPH oxidase, xanthine oxidase/reductase, and nitric oxide synthase enzymes, as well as establishing an inflammatory process. In lungs, hypoxia also modifies the levels of antioxidant substances causing pulmonary oxidative damage. Imbalance of redox state in lungs induced by hypoxia has been suggested as a participant in the changes observed in lung function in the hypoxic context, such as hypoxic vasoconstriction and pulmonary edema, in addition to vascular remodeling and chronic pulmonary hypertension. In this work, experimental evidence that shows the implied mechanisms in pulmonary redox state by hypoxia is reviewed. Herein, studies of cultures of different lung cells and complete isolated lung and tests conducted in vivo in the different forms of hypoxia, conducted in both animal models and humans, are described.

  2. Physiological determinants of human acute hypoxia tolerance.

    Science.gov (United States)

    2013-11-01

    AbstractIntroduction. We investigated possible physiological determinants of variability in hypoxia tolerance in subjects given a 5-minute normobaric exposure to 25,000 ft equivalent. Physiological tolerance to hypoxia was defined as the magnitude of...

  3. HIF1 Contributes to Hypoxia-Induced Pancreatic Cancer Cells Invasion via Promoting QSOX1 Expression

    Directory of Open Access Journals (Sweden)

    Chen-Ye Shi

    2013-08-01

    Full Text Available Background: Quiescin sulfhydryl oxidase 1 (QSOX1, which oxidizes sulfhydryl groups to form disulfide bonds in proteins, is found to be over-expressed in various pancreatic cancer cell lines and patients. QSOX1 promotes invasion of pancreatic cancer cells by activating MMP-2 and MMP-9. However, its regulatory mechanism remains largely undefined. Methods: Real-time PCR and Western blot were employed to detect the expression of QSOX1 in human pancreatic cancer cell lines under hypoxic condition. Luciferase reporter and ChIP assays were used to assess the regulation of QSOX1 by hypoxia-inducible factor 1 (HIF-1. Small interfering RNA (siRNA was applied to knock down endogenous expression of QSOX1. Matrigel-coated invasion chamber essays were conducted to detect the invasion capacity of QSOX1-depleted cells. Results: Both hypoxia and hypoxia mimicking reagent up-regulated the expression of QSOX1 in human pancreatic cancer cell lines. Knockdown of HIF-1α eliminated hypoxia induced QSOX1 expression. HIF-1α was found directly bound to two hypoxia-response elements (HRE of QSOX1 gene, both of which were required for HIF-1 induced QSOX1 expression. Moreover, QSOX1 silencing blocked hypoxia-induced pancreatic cancer cells invasion. Conclusion: QSOX1 is a direct target of HIF-1 and may contribute to hypoxia-induced pancreatic cancer cells invasion.

  4. Differential effect of hypoxia and acidity on lung cancer cell and fibroblast metabolism.

    Science.gov (United States)

    Giatromanolaki, Alexandra; Liousia, Maria; Arelaki, Stella; Kalamida, Dimitra; Pouliliou, Stamatia; Mitrakas, Achilleas; Tsolou, Avgi; Sivridis, Efthimios; Koukourakis, Michael

    2017-06-01

    This study examined the metabolic response of lung cancer cells and normal lung fibroblasts to hypoxia and acidity. GLUT1 and HXKII mRNA/protein expression was up-regulated under hypoxia in the MRC5 fibroblasts and in the A549 and H1299 lung cancer cell lines, indicating intensified glucose absorption and glycolysis. Under hypoxia, the LDHA mRNA and LDH5 protein levels increased in the cancer cells but not in the fibroblasts. Acidity suppressed the above-mentioned hypoxia effect. PDH-kinase-1 (PDK1 mRNA and protein) and inactive phosphorylated-PDH protein levels were induced under hypoxia in the cancer cells, whereas these were reduced in the MRC5 lung fibroblasts. In human tissue sections, the prevalent expression patterns supported the contrasting metabolic behavior of cancer cells vs. tumor fibroblasts. The monocarboxylate/lactate transporter 1 (MCT1) was up-regulated in all the cell lines under hypoxic conditions, but it was suppressed under acidic conditions. The mitochondrial DNA (mtDNA) content per cell decreased significantly in the A549 cancer cell line under hypoxia, but it increased in the MRC5 fibroblasts. Taking into account these findings, we suggest that, under hypoxia, cancer cells intensify the anaerobic direction in glycolysis, while normal fibroblasts prefer to seek energy by intensifying the aerobic use of the available oxygen.

  5. Revising Translations

    DEFF Research Database (Denmark)

    Rasmussen, Kirsten Wølch; Schjoldager, Anne

    2011-01-01

    survey. Results clearly show that most translation companies regard both unilingual and comparative revisions as essential components of professional quality assurance. Data indicate that revision is rarely fully comparative, as the preferred procedure seems to be a unilingual revision followed by a more...... or less comparative rereading. Though questionnaire data seem to indicate that translation companies use linguistic correctness and presentation as the only revision parameters, interview data reveal that textual and communicative aspects are also considered. Generally speaking, revision is not carried...... out by specialised revisers, but by staff translators, who revise the work of colleagues and freelancers on an ad hoc basis. Corrections are mostly given in a peer-to-peer fashion, though the work of freelancers and inexperienced in-house translators is often revised in an authoritative (nonnegotiable...

  6. Impaired response of mature adipocytes of diabetic mice to hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Seok Jong, E-mail: seok-hong@northwestern.edu; Jin, Da P.; Buck, Donald W.; Galiano, Robert D.; Mustoe, Thomas A., E-mail: tmustoe@nmh.org

    2011-10-01

    Adipose tissue contains various cells such as infiltrated monocytes/macrophages, endothelial cells, preadipocytes, and adipocytes. Adipocytes have an endocrine function by secreting adipokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-{alpha}, leptin, and adiponectin. Dysregulation of adipokines in adipose tissues leads to a chronic low-grade inflammation which could result in atherosclerosis, hypertension, and type 2 diabetes. A sustained inflammatory state, which is characterized by prolonged persistence of macrophages and neutrophils, is found in diabetic wounds. In addition, subcutaneous adipocytes are enormously increased in amount clinically in type 2 diabetes. However, the function of subcutaneous adipocytes, which play an important role in injured tissue subjected to hypoxia, has not been well characterized in vitro due to the difficulty of maintaining mature adipocytes in culture using conventional methods because of their buoyancy. In this study, we established a novel in vitro culture method of mature adipocytes by enclosing them in a hyaluronan (HA) based hydrogel to study their role in response to stress such as hypoxia. BrdU labeling and Ki67 immunostaining experiments showed that hydrogel enclosed mature adipocytes proliferate in vitro. Both mRNA and protein expression analyses for hypoxia regulated genes, such as vascular endothelial growth factor (VEGF) and heme oxygenase 1 (HO1), showed that mature adipocytes of wild type mice respond to hypoxia. In contrast, mature adipocytes of diabetic db/db and TallyHo mice did not efficiently respond to hypoxia. Our studies suggest that mature adipocytes are functionally active cells, and their abnormal function to hypoxia can be one of underlining mechanisms in type 2 diabetes.

  7. Post-translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold-induced sweetening of potato tubers.

    Science.gov (Unit