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Sample records for altering mrna stability

  1. Cloning of the altered mRNA stability (ams) gene of Escherichia coli K-12.

    OpenAIRE

    Claverie-Martin, F; Diaz-Torres, M R; Yancey, S D; Kushner, S R

    1989-01-01

    A temperature-sensitive mutation in the ams gene of Escherichia coli causes an increase in the chemical half-life of pulse-labeled RNA at the nonpermissive temperature. Using lambda clones containing DNA fragments from the 23- to 24-min region on the E. coli chromosome, we have isolated a 5.8-kilobase DNA fragment which, when present in a low-copy-number plasmid, complements the conditional lethality and increased mRNA stability associated with the ams-1 mutation. The approximate initiation s...

  2. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis.

    Science.gov (United States)

    Donaldson, Michael E; Saville, Barry J

    2013-07-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense-antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis.

  3. Estrogen-dependent activation of the avian very low density apolipoprotein II and vitellogenin genes. Transient alterations in mRNA polyadenylation and stability early during induction.

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    Cochrane, A W; Deeley, R G

    1988-10-01

    Administration of estrogen to egg-laying vertebrates activates unscheduled, hepatic expression of major, egg-yolk protein genes in immature animals and mature males. Two avian yolk protein genes, encoding very low density apolipoprotein II (apoVLDLII) and vitellogenin II, are dormant prior to stimulation with estrogen, but within three days their cognate mRNAs accumulate to become two of the most abundant species in the liver. Accumulation of these mRNAs has been attributed to both induction of transcription and selective, estrogen-dependent mRNA stabilization. We have detected alterations in the size of apoVLDLII mRNA that occur during the first 24 hours that are attributable to a shift in the extent of polyadenylation as steady-state is approached. In vitro transcription assays indicate that primary activation of both genes takes place relatively slowly and that maximal rates of mRNA accumulation occur when the apoVLDLII and vitellogenin II genes are expressed at only 30% and 10% of their fully induced levels, respectively. Transcription data combined with the structural alteration of apoVLDLII mRNA suggest that stability of the two mRNAs may change as steady-state is approached. We have assessed the compatibility of this suggestion with earlier estimates of the kinetics of accumulation of both mRNAs by developing a generally useful algorithm that predicts approach to steady-state kinetics under conditions where both the rate of synthesis and mRNA stability change throughout the accumulation phase of the response. The results predict that the stability of both mRNAs decreases by at least two- to threefold during the approach to steady-state and that, although an additional destabilization of apoVLDLII mRNA may occur following withdrawal of estrogen, the steady-state stability of vitellogenin mRNA is not significantly decreased upon removal of hormone. PMID:3210227

  4. Low-level laser irradiation alters mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts

    Science.gov (United States)

    Trajano, L. A. S. N.; Sergio, L. P. S.; Silva, C. L.; Carvalho, L.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

    2016-07-01

    Low-level lasers are used for the treatment of diseases in soft and bone tissues, but few data are available regarding their effects on genomic stability. In this study, we investigated mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts exposed to low-level infrared laser. C2C12 myoblast cultures in different fetal bovine serum concentrations were exposed to low-level infrared laser (10, 35 and 70 J cm-2), and collected for the evaluation of DNA repair gene expression. Laser exposure increased gene expression related to base excision repair (8-oxoguanine DNA glycosylase and apurinic/apyrimidinic endonuclease 1), nucleotide excision repair (excision repair cross-complementation group 1 and xeroderma pigmentosum C protein) and genomic stabilization (ATM serine/threonine kinase and tumor protein p53) in normal and low fetal bovine serum concentrations. Results suggest that genomic stability could be part of a biostimulation effect of low-level laser therapy in injured muscles.

  5. Low-level laser irradiation alters mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts

    Science.gov (United States)

    Trajano, L. A. S. N.; Sergio, L. P. S.; Silva, C. L.; Carvalho, L.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

    2016-07-01

    Low-level lasers are used for the treatment of diseases in soft and bone tissues, but few data are available regarding their effects on genomic stability. In this study, we investigated mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts exposed to low-level infrared laser. C2C12 myoblast cultures in different fetal bovine serum concentrations were exposed to low-level infrared laser (10, 35 and 70 J cm‑2), and collected for the evaluation of DNA repair gene expression. Laser exposure increased gene expression related to base excision repair (8-oxoguanine DNA glycosylase and apurinic/apyrimidinic endonuclease 1), nucleotide excision repair (excision repair cross-complementation group 1 and xeroderma pigmentosum C protein) and genomic stabilization (ATM serine/threonine kinase and tumor protein p53) in normal and low fetal bovine serum concentrations. Results suggest that genomic stability could be part of a biostimulation effect of low-level laser therapy in injured muscles.

  6. Control of gene expression during T cell activation: alternate regulation of mRNA transcription and mRNA stability

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    Gorospe Myriam

    2005-05-01

    Full Text Available Abstract Background Microarray technology has become highly valuable for identifying complex global changes in gene expression patterns. The effective correlation of observed changes in gene expression with shared transcription regulatory elements remains difficult to demonstrate convincingly. One reason for this difficulty may result from the intricate convergence of both transcriptional and mRNA turnover events which, together, directly influence steady-state mRNA levels. Results In order to investigate the relative contribution of gene transcription and changes in mRNA stability regulation to standard analyses of gene expression, we used two distinct microarray methods which individually measure nuclear gene transcription and changes in polyA mRNA gene expression. Gene expression profiles were obtained from both polyA mRNA (whole-cell and nuclear run-on (newly transcribed RNA across a time course of one hour following the activation of human Jurkat T cells with PMA plus ionomycin. Comparative analysis revealed that regulation of mRNA stability may account for as much as 50% of all measurements of changes in polyA mRNA in this system, as inferred by the absence of any corresponding regulation of nuclear gene transcription activity for these groups of genes. Genes which displayed dramatic elevations in both mRNA and nuclear run-on RNA were shown to be inhibited by Actinomycin D (ActD pre-treatment of cells while large numbers of genes regulated only through altered mRNA turnover (both up and down were ActD-resistant. Consistent patterns across the time course were observed for both transcribed and stability-regulated genes. Conclusion We propose that regulation of mRNA stability contributes significantly to the observed changes in gene expression in response to external stimuli, as measured by high throughput systems.

  7. Vibrational force alters mRNA expression in osteoblasts

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

    1997-01-01

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

  8. Postural Stability is Altered by Blood Shift

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    Marais, M.; Denise, P.; Guincetre, J. Y.; Normand, H.

    2008-06-01

    Non-vestibular influences as shift in blood volume changed perception of body posture. Then, factors affecting blood shift may alter postural control. The purpose of our study was to investigate the effects of leg venous contention on postural stability. Twelve subjects were studied on a balance plate for 5 minutes with the eyes closed, in 3 conditions: with no leg venous contention or grade 1 and 3 support stockings. Standard deviation of x and y position was calculated before and after the closure of the eyes. Strong venous contention altered postural stability, after the eyes were closed, during the first 10 s of standing. As support stockings prevent blood shift induced by upright posture, this result is in line with the hypothesis that blood shifts influence the perception of body orientation and postural control among others factors as vision, vestibular inputs... This strong venous contention could induce an increase of fall.

  9. Glucocorticoids enhance stability of human growth hormone mRNA.

    OpenAIRE

    Paek, I; Axel, R.

    1987-01-01

    We have studied the control of expression of the human growth hormone (hGH) gene introduced into the chromosomes of mouse fibroblasts. Cell lines transformed with the hGH gene expressed low levels of intact hGH mRNA and secreted hGH protein into the medium. Although the level of expression of hGH mRNA was low, the gene remained responsive to induction by glucocorticoid hormones. To localize the sequences responsible for induction and to determine the mechanism by which these cis-acting sequen...

  10. Altering Emulsion Stability with Heterogeneous Surface Wettability

    Science.gov (United States)

    Meng, Qiang; Zhang, Yali; Li, Jiang; Lammertink, Rob G. H.; Chen, Haosheng; Tsai, Peichun Amy

    2016-06-01

    Emulsions–liquid droplets dispersed in another immiscible liquid–are widely used in a broad spectrum of applications, including food, personal care, agrochemical, and pharmaceutical products. Emulsions are also commonly present in natural crude oil, hampering the production and quality of petroleum fuels. The stability of emulsions plays a crucial role in their applications, but controlling the stability without external driving forces has been proven to be difficult. Here we show how heterogeneous surface wettability can alter the stability and dynamics of oil-in-water emulsions, generated by a co-flow microfluidic device. We designed a useful methodology that can modify a micro-capillary of desired heterogeneous wettability (e.g., alternating hydrophilic and hydrophobic regions) without changing the hydraulic diameter. We subsequently investigated the effects of flow rates and heterogeneous wettability on the emulsion morphology and motion. The experimental data revealed a universal critical timescale of advective emulsions, above which the microfluidic emulsions remain stable and intact, whereas below they become adhesive or inverse. A simple theoretical model based on a force balance can be used to explain this critical transition of emulsion dynamics, depending on the droplet size and the Capillary number–the ratio of viscous to surface effects. These results give insight into how to control the stability and dynamics of emulsions in microfluidics with flow velocity and different wettability.

  11. Genetic effect of CysLTR2 polymorphisms on its mRNA synthesis and stabilization

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    Chung Il

    2009-10-01

    Full Text Available Abstract Background We previously demonstrated that single nucleotide polymorphism (SNP and haplotypes were associated with aspirin hypersensitivity in asthmatics. We investigated the genetic effects of the SNPs and haplotypes on the expression of the CysLTR2 gene. Methods We measured CysLTR2 protein and mRNA expression in EB virus-infected B cell lines from asthmatics having ht1+/+ and ht2+/+. A gel retardation assay was used to identify nuclear protein binding to the c.-819 promoter site. The function of promoter and 3'-UTR were assessed using pGL3 luciferase and pEGFP reporter system, respectively. Results We found that the expression of CysLTR2 protein was higher in B cell lines of asthmatics having ht2+/+ than in those having ht1+/+. PMA/ionomycin induced higher mRNA expression of CysLTR2 in B cell lines from ht2+/+ asthmatics than those from ht1+/+ asthmatics. A nuclear protein from the B cell lines showed stronger DNA binding affinity with a probe containing c.-819T than one containing c.-819G. The luciferase activity of the c.-819T type of CysLTR2 promoter was higher than that of the c.-819G type. EGFP expression was higher in the EGFP-c.2078T 3'-UTR fusion construct than in the c.2078C construct. Conclusion The sequence variants of CysLTR2 may affect its transcription and the stability of its mRNA, resulting in altered expression of CysLTR2 protein, which in turn causes some asthmatics to be susceptible to aspirin hypersensitivity.

  12. qPCR based mRNA quality score show intact mRNA after heat stabilization

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    Oskar Karlsson

    2016-03-01

    Full Text Available Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method. DNA extraction from heat stabilized brain samples was also examined. The snap frozen samples had RIN values about 1 unit higher than heat stabilized samples for the direct QiaZol extraction but equal with stabilized samples using urea pre-solubilization. qPCR based RNA quality measurement showed no difference in quality between snap frozen and heat inactivated samples. The probable explanation for this discrepancy is that the RIN value is an indirect measure based on rRNA, while the qPCR score is based on actual measurement of mRNA quality. The DNA yield from heat stabilized brain tissue samples was significantly increased, compared to the snap frozen tissue, without any effects on purity or quality. Hence, heat stabilization of tissues opens up the possibility for a two step preservation protocol, where proteins and their modifications can be preserved in the first heat based step, while in a second step, using standard RNA preservation strategies, mRNA be preserved. This collection strategy will enable biobanking of samples where the ultimate analysis is not determined without loss of sample quality.

  13. Heat shock response in yeast involves changes in both transcription rates and mRNA stabilities.

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    Laia Castells-Roca

    Full Text Available We have analyzed the heat stress response in the yeast Saccharomyces cerevisiae by determining mRNA levels and transcription rates for the whole transcriptome after a shift from 25 °C to 37 °C. Using an established mathematical algorithm, theoretical mRNA decay rates have also been calculated from the experimental data. We have verified the mathematical predictions for selected genes by determining their mRNA decay rates at different times during heat stress response using the regulatable tetO promoter. This study indicates that the yeast response to heat shock is not only due to changes in transcription rates, but also to changes in the mRNA stabilities. mRNA stability is affected in 62% of the yeast genes and it is particularly important in shaping the mRNA profile of the genes belonging to the environmental stress response. In most cases, changes in transcription rates and mRNA stabilities are homodirectional for both parameters, although some interesting cases of antagonist behavior are found. The statistical analysis of gene targets and sequence motifs within the clusters of genes with similar behaviors shows that both transcriptional and post-transcriptional regulons apparently contribute to the general heat stress response by means of transcriptional factors and RNA binding proteins.

  14. Stability of Human Follicle-Stimulating Hormone Receptor mRNA in Stably Transfected Cells

    Institute of Scientific and Technical Information of China (English)

    朱长虹; 田红

    2001-01-01

    In order to assess the impact of mRNA degradation on steady state levels of follicle-stimulating hormone receptor (FSHR) mRNA and on regulation of FSHR gene expression, the stability and half-life of FSHR mRNA were determined in transfected cells expressing recombinant FSHR. Time-dependent changes in FSHR mRNA content were determined by nuclease protection-solution hybridization assay (NPA) or by qualitative reverse transcription-competitive polymerase chain reaction (RT-PCR) in cultured hFSHR-YI cells, cell lines stably transfected with a human FSHR cDNA. FSHR mRNA content remained constant during 8 h control incubations of hFSHR-Y1 cells (NPA, 2.9±0.3 μg/mg RNA; RT-PCR, 2.7±0.3 μg/mg RNA). Actinomycin D (ActD, 5 μg/ml) inhibited mRNA synthesis, as assessed by incorporation of [3 H]uridine into total RNA, by 90 % within 1 h in hFSHR-Y1 cells. No effect of ActD on cellular morphology or viability was observed. ActD caused a time-dependent decrease in FSHR mRNA content in hFSHR-Y1 cell lines with a lag time of 1 h. There were no significant differences in the rate of FSHR mRNA degradation between the two methods of mRNA quantification. The half-life of hFSHR mRNA was 3.6±0.2 h by NPA and 3.1±0.1 h by RT-PCR. The results indicated that degradation of mRNA was an important process in maintenance of steady state expression of the FSHR gene in cells stably expressing recombinant receptor.

  15. Alterations in Lipoxygenase and Cyclooxygenase-2 Catalytic Activity and mRNA Expression in Prostate Carcinoma

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    Scott B. Shappell

    2001-01-01

    Full Text Available Recent studies in prostate tissues and especially cell lines have suggested roles for arachidonic acid (AA metabolizing enzymes in prostate adenocarcinoma (Pca development or progression. The goal of this study was to more fully characterize lipoxygenase (LOX and cyclooxygenase-2 (COX-2 gene expression and AA metabolism in benign and malignant prostate using snap-frozen tissues obtained intraoperatively and mRNA analyses and enzyme assays. Formation of 15-hydroxyeicosatetraenoic acid (15-HETE was detected in 23/29 benign samples and 15-LOX-2 mRNA was detected in 21/25 benign samples. In pairs of pure benign and Pca from the same patients, 15-HETE production and 15-LOX-2 mRNA were reduced in Pca versus benign in 9/14 (P=.04 and 14/17 (P=.002, respectively. Under the same conditions, neither 5HETE nor 12-HETE formation was detectable in 29 benign and 24 tumor samples; with a more sensitive assay, traces were detected in some samples, but there was no clear association with tumor tissue. COX-2 mRNA was detected by nuclease protection assay in 7/16 benign samples and 5/16 tumors. In benign and tumor pairs from 10 patients, COX-2 was higher in tumor versus benign in only 2, with similar results by in situ hybridization. Paraffin immunoperoxidase for COX2 was performed in whole mount sections from 87 additional radical prostatectomy specimens, with strong expression in ejaculatory duct as a positive control and corroboration with in situ hybridization. No immunostaining was detected in benign prostate or tumor in 45% of cases. Greater immunostaining in tumor versus benign was present in only 17% of cases, and correlated with high tumor grade (Gleason score 8 and 9 vs. 5 to 7. In conclusion, reduced 15-LOX-2 expression and 15-HETE formation is the most characteristic alteration of AA metabolism in Pca. Increased 12-HETE and 5-HETE formation in Pca were not discernible. Increased COX-2 expression is not a typical abnormality in Pca in general, but

  16. Altered organization of GABAA receptor mRNA expression in the depressed suicide brain

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    Michael O Poulter

    2010-03-01

    Full Text Available Inter-relationships ordinarily exist between mRNA expression of GABA-A subunits in the frontopolar cortex (FPC of individuals that had died suddenly from causes other than suicide. However, these correlations were largely absent in persons that had died by suicide. In the present investigation, these findings were extended by examining GABA-A receptor expression patterns (of controls and depressed individuals that died by suicide in the orbital frontal cortex (OFC, hippocampus, amygdala. locus coeruleus (LC,and paraventricular nucleus (PVN, all of which have been implicated in either depression, anxiety or stress responsivity. Results Using QPCR analysis, we found that in controls the inter-relations between GABA-A subunits varied across brain regions, being high in the hippocampus and amygdala, intermediate in the LC, and low in the OFC and PVN. The GABA-A subunit inter-relations were markedly different in persons that died by suicide, being reduced in hippocampus and amygdala, stable in the LC, but more coordinated in the OFC and to some extent in the PVN. Conclusions It seems that altered brain region-specific inhibitory signaling, stemming from altered GABA-A subunit coordination, are associated with depression/suicide. Although, it is unknown whether GABA-A subunit re-organization was specifically tied to depression, suicide, or the accompanying distress, these data show that the co-ordinate expression of this transcriptome does vary depending on brain region and is plastic.

  17. Enhanced stability of tristetraprolin mRNA protects mice against immune-mediated inflammatory pathologies.

    Science.gov (United States)

    Patial, Sonika; Curtis, Alan D; Lai, Wi S; Stumpo, Deborah J; Hill, Georgette D; Flake, Gordon P; Mannie, Mark D; Blackshear, Perry J

    2016-02-16

    Tristetraprolin (TTP) is an inducible, tandem zinc-finger mRNA binding protein that binds to adenylate-uridylate-rich elements (AREs) in the 3'-untranslated regions (3'UTRs) of specific mRNAs, such as that encoding TNF, and increases their rates of deadenylation and turnover. Stabilization of Tnf mRNA and other cytokine transcripts in TTP-deficient mice results in the development of a profound, chronic inflammatory syndrome characterized by polyarticular arthritis, dermatitis, myeloid hyperplasia, and autoimmunity. To address the hypothesis that increasing endogenous levels of TTP in an intact animal might be beneficial in the treatment of inflammatory diseases, we generated a mouse model (TTPΔARE) in which a 136-base instability motif in the 3'UTR of TTP mRNA was deleted in the endogenous genetic locus. These mice appeared normal, but cultured fibroblasts and macrophages derived from them exhibited increased stability of the otherwise highly labile TTP mRNA. This resulted in increased TTP protein expression in LPS-stimulated macrophages and increased levels of TTP protein in mouse tissues. TTPΔARE mice were protected from collagen antibody-induced arthritis, exhibited significantly reduced inflammation in imiquimod-induced dermatitis, and were resistant to induction of experimental autoimmune encephalomyelitis, presumably by dampening the excessive production of proinflammatory mediators in all cases. These data suggest that increased systemic levels of TTP, secondary to increased stability of its mRNA throughout the body, can be protective against inflammatory disease in certain models and might be viewed as an attractive therapeutic target for the treatment of human inflammatory diseases. PMID:26831084

  18. Influenza A viruses suppress cyclooxygenase-2 expression by affecting its mRNA stability

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    Dudek, Sabine Eva; Nitzsche, Katja; Ludwig, Stephan; Ehrhardt, Christina

    2016-01-01

    Infection with influenza A viruses (IAV) provokes activation of cellular defence mechanisms contributing to the innate immune and inflammatory response. In this process the cyclooxygenase-2 (COX-2) plays an important role in the induction of prostaglandin-dependent inflammation. While it has been reported that COX-2 is induced upon IAV infection, in the present study we observed a down-regulation at later stages of infection suggesting a tight regulation of COX-2 by IAV. Our data indicate the pattern-recognition receptor RIG-I as mediator of the initial IAV-induced COX-2 synthesis. Nonetheless, during on-going IAV replication substantial suppression of COX-2 mRNA and protein synthesis could be detected, accompanied by a decrease in mRNA half-life. Interestingly, COX-2 mRNA stability was not only imbalanced by IAV replication but also by stimulation of cells with viral RNA. Our results reveal tristetraprolin (TTP), which is known to bind COX-2 mRNA and promote its rapid degradation, as regulator of COX-2 expression in IAV infection. During IAV replication and viral RNA accumulation TTP mRNA synthesis was induced, resulting in reduced COX-2 levels. Accordingly, the down-regulation of TTP resulted in increased COX-2 protein expression after IAV infection. These findings indicate a novel IAV-regulated cellular mechanism, contributing to the repression of host defence and therefore facilitating viral replication. PMID:27265729

  19. Wnt signaling stimulates transcriptional outcome of the hedgehog pathway by stabilizing GLI1 mRNA

    OpenAIRE

    Noubissi, Felicite K.; Goswami, Srikanta; Sanek, Nicholas A.; Kawakami, Kazuyuki; Minamoto, Toshinari; Moser, Amy; Grinblat, Yevgenya; Spiegelman, Vladimir S.

    2009-01-01

    Wnt and Hedgehog signaling pathways play central roles in embryogenesis, stem cell maintenance, and tumorigenesis. However, the mechanisms by which these two pathways interact are not well understood. Here, we identified a novel mechanism by which Wnt signaling pathway stimulates the transcriptional output of Hedgehog signaling. Wnt/β-catenin signaling induces expression of an RNA-binding protein, CRD-BP, which in turn binds and stabilizes GLI1 mRNA, causing an elevation of GLI1 expression an...

  20. Wnt signaling stimulates transcriptional outcome of the Hedgehog pathway by stabilizing GLI1 mRNA

    OpenAIRE

    Noubissi, Felicite K.; Goswami, Srikanta; Sanek, Nicholas A.; Kawakami, Kazuyuki; Minamoto, Toshinari; Moser, Amy; Grinblat, Yevgenya; Spiegelman, Vladimir S.

    2009-01-01

    Wnt and Hedgehog signaling pathways play central roles in embryogenesis, stem cell maintenance, and tumorigenesis. However, mechanisms by which these two pathways interact are not well-understood. Here, we identified a novel mechanism by which Wnt signaling pathway stimulates the transcriptional output of Hedgehog signaling. Wnt/β-catenin signaling induces expression of an RNA-binding protein, CRD-BP, which, in turn, binds and stabilizes GLI1 mRNA, causing an elevation of GLI1 expression and ...

  1. Cocaine treatment alters oxytocin receptor binding but not mRNA production in postpartum rat dams☆

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    Jarrett, T.M.; McMurray, M.S.; Walker, C.H.; Johns, J.M.

    2011-01-01

    Gestational cocaine treatment in rat dams results in decreased oxytocin (OT) levels, up-regulated oxytocin receptor (OTR) binding density and decreased receptor affinity in the whole amygdala, all concomitant with a significant increase in maternal aggression on postpartum day six. Rat dams with no gestational drug treatment that received an infusion of an OT antagonist directly into the central nucleus of the amygdala (CeA) exhibited similarly high levels of maternal aggression towards intruders. Additionally, studies indicate that decreased OT release from the hypothalamic division of the paraventricular nucleus (PVN) is coincident with heightened maternal aggression in rats. Thus, it appears that cocaine-induced alterations in OT system dynamics (levels, receptors, production, and/or release) may mediate heightened maternal aggression following cocaine treatment, but the exact mechanisms through which cocaine impacts the OT system have not yet been determined. Based on previous studies, we hypothesized that two likely mechanisms of cocaine’s action would be, increased OTR binding specifically in the CeA, and decreased OT mRNA production in the PVN. Autoradiography and in situ hybridization assays were performed on targeted nuclei in brain regions of rat dams on postpartum day six, following gestational treatment twice daily with cocaine (15 mg/kg) or normal saline (1 ml/kg). We now report cocaine-induced reductions in OTR binding density in the ventromedial hypothalamus (VMH) and bed nucleus of the stria terminalis (BNST), but not the CeA. There was no significant change in OT mRNA production in the PVN following cocaine treatment. PMID:16677710

  2. Stabilization of LCD devices via geometric alteration.

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    Jeon, Il; Yoon, MinSung; Lee, Je-Hoon

    2013-02-20

    Glass bending in LCD displays is an inherent problem that has challenged many engineers. As a solution to this problem, we propose a methodology that can tackle the root of the phenomenon in terms of linear elastic beam theory. Using this hypothesis, we devised a background theory and a solution. In this paper, we present a glass panel to which geometrical changes, such as furrow, groove, and curb have been applied. These geometrical changes are applied to the nonactive area of the glass panel. To confirm the validity of our approach, we conducted simulation tests as well as hands-on experiments to observe the thermo-mechanical behavior of the device under various conditions. The simulation results using the Ansys simulator show that the proposed technique can reduce the deformation level of panel bending by 40%. In the experiment using a bare cell with polarizer films attached and with performing the high temperature reliability test, the deformation level of panel bending is reduced by half compared to the reference glass panel without any geometric alteration.

  3. Sex differences in alcohol consumption and alterations in nucleus accumbens endocannabinoid mRNA in alcohol-dependent rats.

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    Henricks, Angela M; Berger, Anthony L; Lugo, Janelle M; Baxter-Potter, Lydia N; Bieniasz, Kennedy V; Craft, Rebecca M; McLaughlin, Ryan J

    2016-10-29

    Chronic intermittent alcohol (CIA) exposure produces altered motivational states characterized by anxiety and escalated alcohol consumption during withdrawal. The endocannabinoid (ECB) system contributes to these symptoms, and sex differences in alcohol dependence, as well as bidirectional interactions between ECBs and gonadal hormones have been documented. Thus, we evaluated sex differences in alcohol consumption, anxiety-like behavior, and ECB mRNA expression in the nucleus accumbens (NAc) of alcohol-dependent rats during acute withdrawal. Male rats exposed to six weeks of CIA showed escalated alcohol consumption during acute withdrawal and reductions in NAc N-acyl phosphatidylethanolamine phospholipase D (NAPEPLD), DAG lipase alpha (DAGLα), and monoacylglycerol lipase (MAGL) mRNA. Intact alcohol-dependent female rats also escalated their consumption, but notably, this effect was also present in non-dependent females. No differences in NAc ECB mRNA were observed between CIA- and air-exposed females during acute withdrawal. However, when these data were analyzed according to estrous stage, significant differences in NAPEPLD and MAGL mRNA expression emerged in the NAc of air-exposed control rats, which were absent in alcohol-dependent females. We subsequently measured alcohol consumption and NAc ECB mRNA in ovariectomized (OVX) females with or without estradiol (E2) replacement during withdrawal. Neither E2 nor CIA altered alcohol consumption in OVX females. However, E2 reduced both DAGLα and MAGL mRNA, suggesting that E2 may influence the biosynthesis and degradation of 2-arachidonoylglycerol (2-AG) in the NAc. Collectively, these studies indicate sexual dimorphism in alcohol consumption in non-dependent rats and suggest that E2-mediated alterations in NAc ECB mRNA expression during withdrawal may be a mechanism by which sex differences in alcohol dependence emerge. PMID:27578612

  4. Herpes simplex virus virion stimulatory protein mRNA leader contains sequence elements which increase both virus-induced transcription and mRNA stability.

    Science.gov (United States)

    Blair, E D; Blair, C C; Wagner, E K

    1987-08-01

    To investigate the role of 5' noncoding leader sequence of herpes simplex virus type 1 (HSV-1) mRNA in infected cells, the promoter for the 65,000-dalton virion stimulatory protein (VSP), a beta-gamma polypeptide, was introduced into plasmids bearing the chloramphenicol acetyltransferase (cat) gene together with various lengths of adjacent viral leader sequences. Plasmids containing longer lengths of leader sequence gave rise to significantly higher levels of CAT enzyme in transfected cells superinfected with HSV-1. RNase T2 protection assays of CAT mRNA showed that transcription was initiated from an authentic viral cap site in all VSP-CAT constructs and that CAT mRNA levels corresponded to CAT enzyme levels. Use of cis-linked simian virus 40 enhancer sequences demonstrated that the effect was virus specific. Constructs containing 12 and 48 base pairs of the VSP mRNA leader gave HSV infection-induced CAT activities intermediate between those of the leaderless construct and the VSP-(+77)-CAT construct. Actinomycin D chase experiments demonstrated that the longest leader sequences increased hybrid CAT mRNA stability at least twofold in infected cells. Cotransfection experiments with a cosmid bearing four virus-specified transcription factors (ICP4, ICP0, ICP27, and VSP-65K) showed that sequences from -3 to +77, with respect to the viral mRNA cap site, also contained signals responsive to transcriptional activation. PMID:3037112

  5. Estrogen does not regulate CD154 mRNA stability in systemic lupus erythematosus T cells.

    Science.gov (United States)

    Li, X; Rider, V; Kimler, B F; Abdou, N I

    2006-01-01

    Previous studies in our laboratory showed a dose-dependent and hormone-specific increase in CD154 expression in T cells from females with systemic lupus erythematosus (SLE). This present study investigates if the estrogen-dependent increase in CD154 expression is due to stabilization of the messenger RNA. T cells from female SLE patients and controls were cultured for 18 h in serum-free medium without and with estradiol 17-beta (10(-7) M). T cells were either unstimulated (resting) or were activated by further culture on anti-CD3 coated plates. Actinomycin D (25 microg/mL) was added to parallel cultures to inhibit new messenger RNA synthesis. CD154 messenger RNA stability was assessed by reverse-transcription polymerase chain amplification. Resting SLE (n = 10, P = 0.88) and normal (n = 7, P = 0.65) T cells showed no significant differences in message stability in response to estradiol. CD154 messenger RNA was also not significantly stabilized in activated SLE (n = 10, P = 0.15) or activated normal (n = 6, P = 0.077) T cells in response to estradiol. These findings indicate that the estrogen-dependent increase in CD154 in SLE T cells is not due to stability of the mRNA. These data are consistent with the postulate that estradiol stimulates CD154 transcription in SLE T cells. PMID:17211990

  6. Alteration of Na,K-ATPase subunit mRNA and protein levels in hypertrophied rat heart.

    Science.gov (United States)

    Charlemagne, D; Orlowski, J; Oliviero, P; Rannou, F; Sainte Beuve, C; Swynghedauw, B; Lane, L K

    1994-01-14

    To determine if an altered expression of the Na,K-ATPase alpha isoform genes is responsible for an observed increase in cardiac glycoside sensitivity in compensatory hypertrophy, we performed Northern and slot blot analyses of RNA and specific immunological detection of Na,K-ATPase isoforms in rat hearts from normal and pressure overload-treated animals induced by abdominal aortic constriction. During the early phase of hypertrophy, the only alteration is a decrease in the alpha 2 mRNA isoform. In the compensated hypertrophied heart, the levels of the predominant alpha 1 isoform (mRNA and protein) and the beta 1 subunit mRNA are unchanged. In contrast, the alpha 2 isoform (mRNA and protein) is decreased by 35% and up to 61-64% in mild ( 55%) hypertrophy, respectively. The alpha 3 isoform (mRNA and protein), which is extremely low in adult heart, is increased up to 2-fold during hypertrophy but accounts for only approximately equal to 5% of the total alpha isoform mRNA. These findings demonstrate that, in cardiac hypertrophy, the three alpha isoforms of the Na,K-ATPase are independently regulated and that regulation occurs at a pretranslational level. The pattern of expression in hypertrophied adult heart is similar to that of the neonatal heart where the inverse regulation between the alpha 2 and alpha 3 ouabain high affinity isoforms has been reported. This suggests that distinct regulatory mechanisms controlling Na,K-ATPase isoform expression may, at least in part, be involved in the sensitivity to cardiac glycosides. PMID:8288620

  7. Suberoylanilide hydroxamic acid (SAHA) inhibits EGF-induced cell transformation via reduction of cyclin D1 mRNA stability

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jingjie [State Key Laboratory of Natural and Biomimetic Drugs, Department of Pharmacology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Rd, Haidian District, Beijing 100191 (China); Nelson Institute of Environmental Medicine, New York University School of Medicine, 57 Old Forge Rd, Tuxedo, NY 10987 (United States); Ouyang, Weiming; Li, Jingxia; Zhang, Dongyun; Yu, Yonghui; Wang, York [Nelson Institute of Environmental Medicine, New York University School of Medicine, 57 Old Forge Rd, Tuxedo, NY 10987 (United States); Li, Xuejun, E-mail: xjli@bjmu.edu.cn [State Key Laboratory of Natural and Biomimetic Drugs, Department of Pharmacology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Rd, Haidian District, Beijing 100191 (China); Huang, Chuanshu, E-mail: chuanshu.huang@nyumc.org [Nelson Institute of Environmental Medicine, New York University School of Medicine, 57 Old Forge Rd, Tuxedo, NY 10987 (United States)

    2012-09-01

    Suberoylanilide hydroxamic acid (SAHA) inhibiting cancer cell growth has been associated with its downregulation of cyclin D1 protein expression at transcription level or translation level. Here, we have demonstrated that SAHA inhibited EGF-induced Cl41 cell transformation via the decrease of cyclin D1 mRNA stability and induction of G0/G1 growth arrest. We found that SAHA treatment resulted in the dramatic inhibition of EGF-induced cell transformation, cyclin D1 protein expression and induction of G0/G1 growth arrest. Further studies showed that SAHA downregulation of cyclin D1 was only observed with endogenous cyclin D1, but not with reconstitutionally expressed cyclin D1 in the same cells, excluding the possibility of SAHA regulating cyclin D1 at level of protein degradation. Moreover, SAHA inhibited EGF-induced cyclin d1 mRNA level, whereas it did not show any inhibitory effect on cyclin D1 promoter-driven luciferase reporter activity under the same experimental conditions, suggesting that SAHA may decrease cyclin D1 mRNA stability. This notion was supported by the results that treatment of cells with SAHA decreased the half-life of cyclin D1 mRNA from 6.95 h to 2.57 h. Consistent with downregulation of cyclin D1 mRNA stability, SAHA treatment also attenuated HuR expression, which has been well-characterized as a positive regulator of cyclin D1 mRNA stability. Thus, our study identifies a novel mechanism responsible for SAHA inhibiting cell transformation via decreasing cyclin D1 mRNA stability and induction of G0/G1 growth arrest in Cl41 cells. -- Highlights: ► SAHA inhibits cell transformation in Cl41 cells. ► SAHA suppresses Cyclin D1 protein expression. ► SAHA decreases cyclin D1 mRNA stability.

  8. Herpes Simplex Virus 1 Infection Alters the mRNA Translation Processing in L-02 Cells

    Institute of Scientific and Technical Information of China (English)

    Min HONG; Yan-chun CHE; Gui-zhen TANG; Wei CUN; Xue-mei ZHANG; Long-ding LIU; Qi-han LI

    2008-01-01

    HSV-1 infection-mediated regulation of mRNA translation in host cells is a systematic and complicated process. Investigation of the details of this mechanism will facilitate understanding of biological variations in the viral replication process and host cells. In this study, a comparative proteomics technology platform was applied by two-dimension electrophoresis of HSV-1 infected normal human L-02 cell and control cell lysates. The observed protein spots were analyzed qualitatively and quantitatively by the PDQuest software package. A number of the different observed protein spots closely associated with cellular protein synthesis were identified by matrix-assisted laser-desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The expression levels of the RPLP1 protein, which is required for mRNA translation, and KHSRP protein, which is involved in rapid decay of mRNA, were up-regulated, whereas the expression level of RNP H2, which is involved in positive regulation on the mRNA splicing process, was down-regulated. All of these results suggest that HSV-1 infection can influence cellular protein synthesis via modulation of cellular regulatory proteins involved in RNA splicing, translation and decay, resulting in optimisation of viral protein synthesis when cellular protein synthesis is shut off. Although there is need for further investigations regarding the detailed mechanisms of cellular protein control, our studies provide new insight into the targeting of varied virus signaling pathways involved in host cellular protein synthesis.

  9. Cyclic AMP and AKAP-mediated targeting of protein kinase A regulates lactate dehydrogenase subunit A mRNA stability.

    Science.gov (United States)

    Jungmann, Richard A; Kiryukhina, Olga

    2005-07-01

    Expression of the lactate dehydrogenase A subunit (ldh-A) gene is controlled through transcriptional as well as post-transcriptional mechanisms. Both mechanisms involve activation of protein kinase A (PKA) into its subunits and subsequent phosphorylation and activation of several key regulatory factors. In rat C6 glioma cells, post-transcriptional gene regulation occurs through PKA-mediated stabilization of LDH-A mRNA and subsequent increase of intracellular LDH-A mRNA levels. Previous studies have demonstrated a cAMP-stabilizing region (CSR) located in the LDH-A 3'-untranslated region which, in combination with several phosphorylated CSR-binding proteins (CSR-BP), regulates the PKA-mediated stabilization of LDH-A mRNA. However, the mechanistic details of interaction of CSR with proteins as they pertain to mRNA stabilization by PKA are so far largely unknown. In this study we tested the hypothesis that ribosomal protein extracts (RSW) from glioma cells contain PKA regulatory (RII) and catalytic (C) subunits that, in combination with a protein kinase A anchoring protein (AKAP 95) and CSR-BPs participate in forming CSR-protein complexes that are responsible for mRNA stability regulation. To demonstrate the importance of CSR-protein complex formation, the PKA subunits and AKAP 95 were removed from the RSW by immunoprecipitation, and the antigen-deleted RSW were subjected to CSR binding analysis using gel mobility shift and UV cross-linking. It was shown that AKAP 95 as well as RII formed a direct linkage with CSR during CSR-protein complex formation. In contrast, the catalytic subunit formed part of the CSR-protein complex but did not bind to CSR directly in a covalent linkage. To determine whether formation of CSR complexes that included C, RII, and AKAP 95 constituted a functional event and was necessary for mRNA stabilization, cell-free decay reactions were carried out with RSW extracts, and the kinetics of decay of LDH-A mRNA was determined. Depletion of PKA

  10. Stage structure alters how complexity affects stability of ecological networks

    Science.gov (United States)

    Rudolf, V.H.W.; Lafferty, Kevin D.

    2011-01-01

    Resolving how complexity affects stability of natural communities is of key importance for predicting the consequences of biodiversity loss. Central to previous stability analysis has been the assumption that the resources of a consumer are substitutable. However, during their development, most species change diets; for instance, adults often use different resources than larvae or juveniles. Here, we show that such ontogenetic niche shifts are common in real ecological networks and that consideration of these shifts can alter which species are predicted to be at risk of extinction. Furthermore, niche shifts reduce and can even reverse the otherwise stabilizing effect of complexity. This pattern arises because species with several specialized life stages appear to be generalists at the species level but act as sequential specialists that are hypersensitive to resource loss. These results suggest that natural communities are more vulnerable to biodiversity loss than indicated by previous analyses.

  11. Alterations of organ histopathology and metallolhionein mRNA expression in silver barb, Puntius gonionotus during subchronic cadmium exposure

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Common silver barb, Puntius gonionotus exposed to the nominal concentration of 0.06 mg/L Cd for 60 d, were assessed for histopathological alterations (gills, liver and kidney), metal accumulation, and metallothionein (MT) mRNA expression. Fish exhibited pathological symptoms such as hypertrophy and hyperplasia of primary and secondary gill lamellae, vacuolization in hepatocytes, and prominent tubular and glomerular damage in the kidney. In addition, kidney accumulated the highest content of cadmium, more than gills and liver. Expression of MT mRNA was increased in both liver and kidney of treated fish. Hepatic MT levels remained high after fish were removed to Cd-free water. In contrast, MT expression in kidney was peaked after 28 d of treatment and drastically dropped when fish were removed to Cd-free water. The high concentrations of Cd in hepatic tissues indicated an accumulation site or permanent damage on this tissue.

  12. Cadmium chloride alters mRNA levels of angiogenesis related genes in primary human endometrial endothelial cells grown in vitro.

    Science.gov (United States)

    Helmestam, Malin; Stavreus-Evers, Anneli; Olovsson, Matts

    2010-11-01

    Cadmium, is known to cause adverse reproductive effects, and classified as an endocrine disrupting chemical (EDC). Human endometrial endothelial cells (HEEC) have a key role in the regulation of endometrial angiogenesis. These cells are known to express estrogen receptors, a feature that makes them potential targets for EDCs such as cadmium. We have designed a co-culture system, in which HEEC were grown in the same cell culture medium as endometrial stromal cells but in separate, communicating chambers. With quantitative PCR, we investigated changes in mRNA expression of genes associated with angiogenesis, sex steroids and endothelial cell specific functions. We found that cadmium altered the mRNA expression of the two important angiogenic molecules VEGF-A and PLGF. Cadmium might thus affect endometrial angiogenesis and as a consequence cause endometrial dysfunction with an increased risk for fertility problems. PMID:20580663

  13. The Escherichia coli antiterminator protein BglG stabilizes the 5' region of the bgl mRNA

    Indian Academy of Sciences (India)

    Abhilasha Gulati; S Mahadevan

    2001-06-01

    The -glucoside utilization (bgl) genes of Escherichia coli are positively regulated by the product of the bglG gene, which functions as an antiterminator by binding to specific sequences present within the bgl mRNA. BglG is inactivated by phosphorylation in the absence of -glucosides by BglF, the bgl-specific component of the phosphotransferase system (PTS). Here, we present evidence for an additional function for BglG, namely the stabilization of the 5— end of the bgl mRNA. Half-life measurements of the promoter-proximal region of the bgl mRNA indicate a five fold enhancement of stability in the presence of active (unphosphorylated) BglG. This enhancement is lost when the binding of BglG to mRNA is prevented by deletion of the binding site. Interestingly, stabilization by BglG does not extend to downstream sequences. The enhanced stability of the upstream sequences suggest that BglG remains bound to its target on the mRNA even after the downstream sequences have been degraded. Implications of these observations for the mechanism of positive regulation of the operon by BglG are discussed.

  14. Wastewater treatment plant effluent alters pituitary gland gonadotropin mRNA levels in juvenile coho salmon (Oncorhynchus kisutch).

    Science.gov (United States)

    Harding, Louisa B; Schultz, Irvin R; da Silva, Denis A M; Ylitalo, Gina M; Ragsdale, Dave; Harris, Stephanie I; Bailey, Stephanie; Pepich, Barry V; Swanson, Penny

    2016-09-01

    It is well known that endocrine disrupting compounds (EDCs) present in wastewater treatment plant (WWTP) effluents interfere with reproduction in fish, including altered gonad development and induction of vitellogenin (Vtg), a female-specific egg yolk protein precursor produced in the liver. As a result, studies have focused on the effects of EDC exposure on the gonad and liver. However, impacts of environmental EDC exposure at higher levels of the hypothalamic-pituitary-gonad axis are less well understood. The pituitary gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are involved in all aspects of gonad development and are subject to feedback from gonadal steroids making them a likely target of endocrine disruption. In this study, the effects of WWTP effluent exposure on pituitary gonadotropin mRNA expression were investigated to assess the utility of Lh beta-subunit (lhb) as a biomarker of estrogen exposure in juvenile coho salmon (Oncorhynchus kisutch). First, a controlled 72-h exposure to 17α-ethynylestradiol (EE2) and 17β-trenbolone (TREN) was performed to evaluate the response of juvenile coho salmon to EDC exposure. Second, juvenile coho salmon were exposed to 0, 20 or 100% effluent from eight WWTPs from the Puget Sound, WA region for 72h. Juvenile coho salmon exposed to 2 and 10ng EE2L(-1) had 17-fold and 215-fold higher lhb mRNA levels relative to control fish. Hepatic vtg mRNA levels were dramatically increased 6670-fold, but only in response to 10ng EE2L(-1) and Fsh beta-subunit (fshb) mRNA levels were not altered by any of the treatments. In the WWTP effluent exposures, lhb mRNA levels were significantly elevated in fish exposed to five of the WWTP effluents. In contrast, transcript levels of vtg were not affected by any of the WWTP effluent exposures. Mean levels of natural and synthetic estrogens in fish bile were consistent with pituitary lhb expression, suggesting that the observed lhb induction may be due to

  15. Nucleolin and YB-1 are required for JNK-mediated interleukin-2 mRNA stabilization during T-cell activation

    DEFF Research Database (Denmark)

    Chen, C Y; Gherzi, R; Andersen, Jens S.;

    2000-01-01

    Regulated mRNA turnover is a highly important process, but its mechanism is poorly understood. Using interleukin-2 (IL-2) mRNA as a model, we described a role for the JNK-signaling pathway in stabilization of IL-2 mRNA during T-cell activation, acting via a JNK response element (JRE) in the 5...

  16. Transient responses via regulation of mRNA stability as an immuno-logical strategy for countering infectious diseases.

    Science.gov (United States)

    Nakagawa, Junichi

    2008-12-01

    Posttranscriptional regulation of gene expression plays a pivotal role as a fast control system for T-cells and B-cells operating in the defense reactions against rapidly growing infectious agents. The framework of this machinery involves cis-acting elements in the mRNAs of relevant cytokines and trans-acting factors interacting with these elements. The cis- and trans-acting factors enforce rapid mRNA decay with other proteins such as nucleases in the decay machinery. The most prominent cis-element contains A + U- rich sequence (ARE), and is located in the 3'-untranslated region of the target mRNAs. Some ARE-binding proteins promote the rapid decay, and others protect the mRNA from degradation. The 5'-end of nascent mRNA undergoes capping which protects the 5'-end together with the cap-binding protein, and the 3' end is protected with poly (A) tail and associating poly (A) binding protein. Unlike in classical drawing of linear structure of mRNA, the end structures interact with each other through a common platform composed of translation initiation factors, revealing the cross-talk of the 5'-end cap structure and 3'-end poly (A) tail on the translational machinery. The rapid degradation and stabilization of mRNA is triggered by a cellular signaling cascade through phosphorylation of associating protein factors in response to environmental stimuli, and a large nucleolytic complex for specific decay reaction called exosome is formed with the 3'-UTR of mRNA through interaction with the ARE-binding proteins. Possible therapeutic agents modifying stability of ARE-containing mRNA are being screened in order to treat immunological disorders. PMID:19075798

  17. A new function of glucocorticoid receptor: regulation of mRNA stability

    OpenAIRE

    Park, Ok Hyun; Do, Eunjin; Kim, Yoon Ki

    2015-01-01

    It has long been thought that glucocorticoid receptor (GR) functions as a DNA-binding transcription factor in response to its ligand (a glucocorticoid) and thus regulates various cellular and physiological processes. It is also known that GR can bind not only to DNA but also to mRNA; this observation points to the possible role of GR in mRNA metabolism. Recent data revealed a molecular mechanism by which binding of GR to target mRNA elicits rapid mRNA degradation. GR binds to specific RNA seq...

  18. Conserved CPEs in the p53 3' untranslated region influence mRNA stability and protein synthesis

    DEFF Research Database (Denmark)

    Rosenstierne, Maiken W; Vinther, Jeppe; Mittler, Gerhard;

    2008-01-01

    CaT skin and MCF-7 breast cancer cell lines were established. Quantitative PCR and an enzymatic assay were used to quantify the reporter mRNA and protein levels, respectively. Proteins binding to the CPEs were identified by RNA-immunoprecipitation (IP) and quantitative mass spectroscopy. RESULTS: The wild...... irradiation. Several proteins (including GAPDH, heterogeneous nuclear ribonucleoprotein (hnRNP) D and A/B) were identified from the MCF-7 cytoplasmic extracts that bound specifically to the CPEs. CONCLUSION: Two conserved CPEs in the p53 3'UTR regulate stability and translation of a reporter mRNA in non...

  19. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli

    Science.gov (United States)

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J.; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation. PMID:27112822

  20. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

    Science.gov (United States)

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation. PMID:27112822

  1. The p53 target Wig-1 regulates p53 mRNA stability through an AU-rich element

    DEFF Research Database (Denmark)

    Vilborg, Anna; Glahder, Jacob-Andreas Harald; Wilhelm, Margareta T;

    2009-01-01

    The p53 target gene Wig-1 encodes a double-stranded-RNA-binding zinc finger protein. We show here that Wig-1 binds to p53 mRNA and stabilizes it through an AU-rich element (ARE) in the 3' UTR of the p53 mRNA. This effect is mirrored by enhanced p53 protein levels in both unstressed cells and cells...... exposed to p53-activating stress agents. Thus, the p53 target Wig-1 is a previously undescribed ARE-regulating protein that acts as a positive feedback regulator of p53, with implications both for the steady-state levels of p53 and for the p53 stress response. Our data reveal a previously undescribed link...... between the tumor suppressor p53 and posttranscriptional gene regulation via AREs in mRNA....

  2. Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos

    International Nuclear Information System (INIS)

    This work represents the first guide for using steric-block antisense oligos as tools for effective and targeted modification of RNA splicing. Comparison of several steric-block oligo types shows the properties of Morpholinos provide significant advantages over other potential splice-blocking oligos. The procedures and complications of designing effective splice-blocking Morpholino oligos are described. The design process requires complete pre-mRNA sequence for defining suitable targets, which usually generate specific predictable messengers. To validate the targeting procedure, the level and nature of transcript alteration is characterized by RT-PCR analysis of splice modification in a β-globin splice model system. An oligo-walking study reveals that while U1 and U2 small nuclear RiboNucleoProtein (snRNP) binding sites are the most effective targets for blocking splicing, inclusion of these sites is not required to achieve effective splice modifications. The most effective targeting strategy employs simultaneously blocking snRNP binding sites and splice-junctions. The work presented here continues to be the basis for most of the successful Morpholino oligos designed for the worldwide research community to block RNA splicing

  3. Assessment of mRNA and microRNA Stabilization in Peripheral Human Blood for Multicenter Studies and Biobanks

    OpenAIRE

    Beate Pesch; Heinz Otten; Michaela Kreuzer; Maria Gomolka; Dirk Taeger; Oleksandr Bryk; Sandra Zilch- Schöneweis; Peter Rozynek; Martin Lehnert; Swaantje Casjens; Daniel Gilbert Weber; Georg Johnen; Thomas Brüning

    2010-01-01

    In this study we evaluate the suitability of two methods of RNA conservation in blood samples, PAXgene and RNAlater, in combination with variable shipping conditions for their application in multicenter studies and biobanking. RNA yield, integrity, and purity as well as levels of selected mRNA and microRNA species were analyzed in peripheral human blood samples stabilized by PAXgene or RNAlater and shipped on dry ice or at ambient temperatures from the study centers to the central analysis la...

  4. Glucocorticoid regulation of human pulmonary surfactant protein-B (SP-B) mRNA stability is independent of activated glucocorticoid receptor.

    Science.gov (United States)

    Tillis, Ceá C; Huang, Helen W; Bi, Weizhen; Pan, Su; Bruce, Shirley R; Alcorn, Joseph L

    2011-06-01

    Adequate expression of surfactant protein-B (SP-B) is critical in the function of pulmonary surfactant to reduce alveolar surface tension. Expression of SP-B mRNA is restricted to specific lung-airway epithelial cells, and human SP-B mRNA stability is increased in the presence of the synthetic glucocorticoid dexamethasone (DEX). Although the mechanism of SP-B mRNA stabilization by DEX is unknown, studies suggest involvement of the glucocorticoid receptor (GR). We developed a dual-cistronic plasmid-based expression assay in which steady-state levels of SP-B mRNA, determined by Northern analysis, reproducibly reflect changes in SP-B mRNA stability. Using this assay, we found that steady-state levels of SP-B mRNA increased greater than twofold in transfected human-airway epithelial cells (A549) incubated with DEX (10(-7) M). DEX-mediated changes in SP-B mRNA levels required the presence of the SP-B mRNA 3'-untranslated region but did not require ongoing protein synthesis. The effect of DEX on SP-B mRNA levels was dose dependent, with maximal effect at 10(-7) M. DEX increased levels of SP-B mRNA in cells lacking GR, and the presence of the GR antagonist RU486 did not interfere with the effect of DEX. Surprisingly, other steroid hormones (progesterone, estradiol, and vitamin D; 10(-7) M) significantly increased SP-B mRNA levels, suggesting a common pathway of steroid hormone action on SP-B mRNA stability. These results indicate that the effect of DEX to increase SP-B mRNA stability is independent of activated GR and suggests that the mechanism is mediated by posttranscriptional or nongenomic effects of glucocorticoids. PMID:21398497

  5. Cadmium Activates Multiple Signaling Pathways That Coordinately Stimulate Akt Activity to Enhance c-Myc mRNA Stability.

    Directory of Open Access Journals (Sweden)

    Jia-Shiuan Tsai

    Full Text Available Cadmium is a known environmental carcinogen. Exposure of Cd leads to the activation of several proto-oncogenes in cells. We investigated here the mechanism of c-Myc expression in hepatic cells under Cd treatment. The c-Myc protein and mRNA levels increased in dose- and time-dependent manners in HepG2 cells with Cd treatment. This increase was due to an increase in c-Myc mRNA stability. To explore the mechanism involved in enhancing the mRNA stability, several cellular signaling factors that evoked by Cd treatment were analyzed. PI3K, p38, ERK and JNK were activated by Cd. However, ERK did not participate in the Cd-induced c-Myc expression. Further analysis revealed that mTORC2 was a downstream factor of p38. PI3K, JNK and mTORC2 coordinately activated Akt. Akt was phosphorylated at Thr450 in the untreated cells. Cd treatment led to additional phosphorylation at Thr308 and Ser473. Blocking any of the three signaling factors resulted in the reduction of phosphorylation level at all three Akt sites. The activated Akt phosphorylated Foxo1 and allowed the modified protein to translocate into the cytoplasm. We conclude that Cd-induced accumulation of c-Myc requires the activation of several signaling pathways. The signals act coordinately for Akt activation and drive the Foxo1 from the nucleus to the cytoplasm. Reduction of Foxo1 in the nucleus reduces the transcription of its target genes that may affect c-Myc mRNA stability, resulting in a higher accumulation of the c-Myc proteins.

  6. Sequences of a hairpin structure in the 3'-untranslated region mediate regulation of human pulmonary surfactant protein B mRNA stability.

    Science.gov (United States)

    Huang, Helen W; Payne, David E; Bi, Weizhen; Pan, Su; Bruce, Shirley R; Alcorn, Joseph L

    2012-05-15

    The ability of pulmonary surfactant to reduce alveolar surface tension requires adequate expression of surfactant protein B (SP-B). Dexamethasone (DEX, 10(-7) M) increases human SP-B mRNA stability by a mechanism that requires a 126-nt-long segment (the 7.6S region) of the 3'-untranslated region (3'-UTR). The objective of this study was to identify sequences in the 7.6S region that mediate regulation of SP-B mRNA stability. The 7.6S region was found to be sufficient for DEX-mediated stabilization of mRNA. Sequential substitution mutagenesis of the 7.6S region indicates that a 90-nt region is required for DEX-mediated stabilization and maintenance of intrinsic stability. In this region, one 30-nt-long element (002), predicted to form a stem-loop structure, is sufficient for DEX-mediated stabilization of mRNA and intrinsic mRNA stability. Cytosolic proteins specifically bind element 002, and binding activity is unaffected whether proteins are isolated from cells incubated in the absence or presence of DEX. While loop sequences of element 002 have no role in regulation of SP-B mRNA stability, the proximal stem sequences are required for DEX-mediated stabilization and specific binding of proteins. Mutation of the sequences that comprise the proximal or distal arm of the stem negates the destabilizing activity of element 002 on intrinsic SP-B mRNA stability. These results indicate that cytosolic proteins bind a single hairpin structure that mediates intrinsic and hormonal regulation of SP-B mRNA stability via mechanisms that involve sequences of the stems of the hairpin structure. PMID:22367784

  7. Invaded grassland communities have altered stability-maintenance mechanisms but equal stability compared to native communities.

    Science.gov (United States)

    Wilsey, Brian J; Daneshgar, Pedram P; Hofmockel, Kirsten; Polley, H Wayne

    2014-01-01

    Theory predicts that stability should increase with diversity via several mechanisms. We tested predictions in a 5-year experiment that compared low-diversity exotic to high-diversity native plant mixtures under two irrigation treatments. The study included both wet and dry years. Variation in biomass across years (CV) was 50% lower in mixtures than monocultures of both native and exotic species. Growth among species was more asynchronous and overyielding values were greater during and after a drought in native than exotic mixtures. Mean-variance slopes indicated strong portfolio effects in both community types, but the intercept was higher for exotics than for natives, suggesting that exotics were inherently more variable than native species. However, this failed to result in higher CV's in exotic communities because species that heavily dominated plots tended to have lower than expected variance. Results indicate that diversity-stability mechanisms are altered in invaded systems compared to native ones they replaced. PMID:24325664

  8. Invaded grassland communities have altered stability-maintenance mechanisms but equal stability compared to native communities.

    Science.gov (United States)

    Wilsey, Brian J; Daneshgar, Pedram P; Hofmockel, Kirsten; Polley, H Wayne

    2014-01-01

    Theory predicts that stability should increase with diversity via several mechanisms. We tested predictions in a 5-year experiment that compared low-diversity exotic to high-diversity native plant mixtures under two irrigation treatments. The study included both wet and dry years. Variation in biomass across years (CV) was 50% lower in mixtures than monocultures of both native and exotic species. Growth among species was more asynchronous and overyielding values were greater during and after a drought in native than exotic mixtures. Mean-variance slopes indicated strong portfolio effects in both community types, but the intercept was higher for exotics than for natives, suggesting that exotics were inherently more variable than native species. However, this failed to result in higher CV's in exotic communities because species that heavily dominated plots tended to have lower than expected variance. Results indicate that diversity-stability mechanisms are altered in invaded systems compared to native ones they replaced.

  9. Altered mRNA editing and expression of ionotropic glutamate receptors after kainic acid exposure in cyclooxygenase-2 deficient mice.

    Directory of Open Access Journals (Sweden)

    Luca Caracciolo

    Full Text Available Kainic acid (KA binds to the AMPA/KA receptors and induces seizures that result in inflammation, oxidative damage and neuronal death. We previously showed that cyclooxygenase-2 deficient (COX-2(-/- mice are more vulnerable to KA-induced excitotoxicity. Here, we investigated whether the increased susceptibility of COX-2(-/- mice to KA is associated with altered mRNA expression and editing of glutamate receptors. The expression of AMPA GluR2, GluR3 and KA GluR6 was increased in vehicle-injected COX-2(-/- mice compared to wild type (WT mice in hippocampus and cortex, whereas gene expression of NMDA receptors was decreased. KA treatment decreased the expression of AMPA, KA and NMDA receptors in the hippocampus, with a significant effect in COX-2(-/- mice. Furthermore, we analyzed RNA editing levels and found that the level of GluR3 R/G editing site was selectively increased in the hippocampus and decreased in the cortex in COX-2(-/- compared with WT mice. After KA, GluR4 R/G editing site, flip form, was increased in the hippocampus of COX-2(-/- mice. Treatment of WT mice with the COX-2 inhibitor celecoxib for two weeks decreased the expression of AMPA/KA and NMDAR subunits after KA, as observed in COX-2(-/- mice. After KA exposure, COX-2(-/- mice showed increased mRNA expression of markers of inflammation and oxidative stress, such as cytokines (TNF-α, IL-1β and IL-6, inducible nitric oxide synthase (iNOS, microglia (CD11b and astrocyte (GFAP. Thus, COX-2 gene deletion can exacerbate the inflammatory response to KA. We suggest that COX-2 plays a role in attenuating glutamate excitotoxicity by modulating RNA editing of AMPA/KA and mRNA expression of all ionotropic glutamate receptor subunits and, in turn, neuronal excitability. These changes may contribute to the increased vulnerability of COX-2(-/- mice to KA. The overstimulation of glutamate receptors as a consequence of COX-2 gene deletion suggests a functional coupling between COX-2 and the

  10. Patterns of dioxin-altered mRNA expression in livers of dioxin-sensitive versus dioxin-resistant rats

    Energy Technology Data Exchange (ETDEWEB)

    Franc, Monique A. [University of Toronto, Department of Pharmacology and Toxicology, Medical Sciences Building, Toronto, ON (Canada); Johnson and Johnson Pharmaceutical Research and Development, Department of Pharmacogenomics, 1000 Route 202 South, P.O. Box 300, Raritan, NJ (United States); Moffat, Ivy D.; Boutros, Paul C.; Okey, Allan B. [University of Toronto, Department of Pharmacology and Toxicology, Medical Sciences Building, Toronto, ON (Canada); Tuomisto, Jouni T.; Tuomisto, Jouko [National Public Health Institute, Department of Environmental Health, Centre for Environmental Health Risk Analysis, Kuopio (Finland); Pohjanvirta, Raimo [University of Helsinki, Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, Helsinki (Finland)

    2008-11-15

    Dioxins exert their major toxicologic effects by binding to the aryl hydrocarbon receptor (AHR) and altering gene transcription. Numerous dioxin-responsive genes previously were identified both by conventional biochemical and molecular techniques and by recent mRNA expression microarray studies. However, of the large set of dioxin-responsive genes the specific genes whose dysregulation leads to death remain unknown. To identify specific genes that may be involved in dioxin lethality we compared changes in liver mRNA levels following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in three strains/lines of dioxin-sensitive rats with changes in three dioxin-resistant rat strains/lines. The three dioxin-resistant strains/lines all harbor a large deletion in the transactivation domain of the aryl hydrocarbon receptor (AHR). Despite this deletion, many genes exhibited a ''Type-I'' response - that is, their responses were similar in dioxin-sensitive and dioxin-resistant rats. Several genes that previously were well established as being dioxin-responsive or under AHR regulation emerged as Type-I responses (e.g. CYP1A1, CYP1A2, CYP1B1 and Gsta3). In contrast, a relatively small number of genes exhibited a Type-II response - defined as a difference in responsiveness between dioxin-sensitive and dioxin-resistant rat strains. Type-II genes include: malic enzyme 1, ubiquitin C, cathepsin L, S-adenosylhomocysteine hydrolase and ferritin light chain 1. In silico searches revealed that AH response elements are conserved in the 5'-flanking regions of several genes that respond to TCDD in both the Type-I and Type-II categories. The vast majority of changes in mRNA levels in response to 100 {mu}g/kg TCDD were strain-specific; over 75% of the dioxin-responsive clones were affected in only one of the six strains/lines. Selected genes were assessed by quantitative RT-PCR in dose-response and time-course experiments and responses of some genes were

  11. Post-transcriptional Stabilization of Ucp1 mRNA Protects Mice from Diet-Induced Obesity

    Directory of Open Access Journals (Sweden)

    Akinori Takahashi

    2015-12-01

    Full Text Available Uncoupling protein 1 (Ucp1 contributes to thermogenesis, and its expression is regulated at the transcriptional level. Here, we show that Ucp1 expression is also regulated post-transcriptionally. In inguinal white adipose tissue (iWAT of mice fed a high-fat diet (HFD, Ucp1 level decreases concomitantly with increases in Cnot7 and its interacting partner Tob. HFD-fed mice lacking Cnot7 and Tob express elevated levels of Ucp1 mRNA in iWAT and are resistant to diet-induced obesity. Ucp1 mRNA has an elongated poly(A tail and persists in iWAT of Cnot7−/− and/or Tob−/− mice on a HFD. Ucp1 3′-UTR-containing mRNA is more stable in cells expressing mutant Tob that is unable to bind Cnot7 than in WT Tob-expressing cells. Tob interacts with BRF1, which binds to an AU-rich element in the Ucp1 3′-UTR. BRF1 knockdown partially restores the stability of Ucp1 3′-UTR-containing mRNA. Thus, the Cnot7-Tob-BRF1 axis inhibits Ucp1 expression and contributes to obesity.

  12. Sex-specific alterations in mRNA level of key lipid metabolism enzymes in skeletal muscle of overweight and obese subjects following endurance exercise

    OpenAIRE

    Smith, Ira J.; Huffman, Kim M.; Durheim, Michael T.; Duscha, Brian D.; Kraus, William E.

    2008-01-01

    Endurance exercise (EE) leads to beneficial alterations in skeletal muscle lipid metabolism in overweight and obese individuals; however, the mechanisms of these improvements are poorly understood. The primary goal of the current investigation was to test the hypothesis that long-term EE training (6 mo) leads to alterations in the mRNA abundance of key lipid metabolism enzymes in skeletal muscle of overweight and obese middle-aged women and men. A secondary aim of this study was to investigat...

  13. Palmitate alters the rhythmic expression of molecular clock genes and orexigenic neuropeptide Y mRNA levels within immortalized, hypothalamic neurons.

    Science.gov (United States)

    Fick, Laura J; Fick, Gordon H; Belsham, Denise D

    2011-09-30

    The control of energy homeostasis within the hypothalamus is under the regulated control of homeostatic hormones, nutrients and the expression of neuropeptides that alter feeding behavior. Elevated levels of palmitate, a predominant saturated fatty acid in diet and fatty acid biosynthesis, alter cellular function. For instance, a key mechanism involved in the development of insulin resistance is lipotoxicity, through increased circulating saturated fatty acids. Although many studies have begun to determine the underlying mechanisms of lipotoxicity in peripheral tissues, little is known about the effects of excess lipids in the brain. To determine these mechanisms we used an immortalized, clonal, hypothalamic cell line, mHypoE-44, to demonstrate that palmitate directly alters the expression of molecular clock components, by increasing Bmal1 and Clock, or by decreasing Per2, and Rev-erbα, their mRNA levels and altering their rhythmic period within individual neurons. We found that these neurons endogenously express the orexigenic neuropeptides NPY and AgRP, thus we determined that palmitate administration alters the mRNA expression of these neuropeptides as well. Palmitate treatment causes a significant increase in NPY mRNA levels and significantly alters the phase of rhythmic expression. We explored the link between AMPK and the expression of neuropeptide Y using the AMPK inhibitor compound C and the AMP analog AICAR. AMPK inhibition decreased NPY mRNA. AICAR also elevated basal NPY, but prevented the palmitate-mediated increase in NPY mRNA levels. We postulate that this palmitate-mediated increase in NPY and AgRP synthesis may initiate a detrimental positive feedback loop leading to increased energy consumption.

  14. mRNA expression patterns for GH, PRL, SL, IGF-I and IGF-II during altered feeding status in rabbitfish, Siganus guttatus.

    Science.gov (United States)

    Ayson, Felix G; de Jesus-Ayson, Evelyn Grace T; Takemura, Akihiro

    2007-01-15

    Feeding time is a major synchronizer of many physiological rhythms in many organisms. Alteration in the nutritional status, specifically fasting, also affects the secretion rhythms of growth hormone (GH) and insulin-like growth factor-I (IGF-I). In this study, we investigated whether the expression patterns for the mRNAs of GH, prolactin (PRL) and somatolactin (SL) in the pituitary gland, and insulin-like growth factor I and II (IGF-I and IGF-II) in the liver of juvenile rabbitfish (Siganus guttatus) follow a rhythm according to feeding time and whether these hormone rhythms changes with starvation. Hormone mRNA levels were determined by real time PCR. The daily expression pattern for the mRNAs of GH, PRL and SL was not altered whether food was given in the morning (10:00 h) or in the afternoon (15:00 h). The daily GH mRNA expression pattern, however, was affected when food was not available for 3 days. In contrast, the daily expression pattern for IGF-I mRNA reaches its peak at roughly 5-6h after feeding. This pattern, however, was not observed with IGF-II mRNA. During 15-day starvation, GH mRNA levels in starved fish were significantly higher than the control fish starting on the 9th day of starvation until day 15. The levels returned to normal after re-feeding. In contrast to GH, PRL mRNA levels in starved fish were significantly lower than the control group starting on the 6th day of starvation until 3 days after re-feeding. SL mRNA levels were not significantly different between the control and starved group at anytime during the experiment. Both IGF-I and IGF-II mRNA levels in starved group were significantly higher than the control fish on the 3rd and 6th day of starvation. mRNA levels of both IGF-I and II in the starved fish decreased starting on the 9th day of starvation. While IGF-I mRNA levels in the starved group continued to decrease as starvation progressed, IGF-II mRNA levels were not significantly different from the control during the rest of the

  15. Glucocorticoid regulation of human pulmonary surfactant protein-B (SP-B) mRNA stability is independent of activated glucocorticoid receptor

    OpenAIRE

    Tillis, Ceá C.; Huang, Helen W.; Bi, Weizhen; Pan, Su; Bruce, Shirley R.; Alcorn, Joseph L.

    2011-01-01

    Adequate expression of surfactant protein-B (SP-B) is critical in the function of pulmonary surfactant to reduce alveolar surface tension. Expression of SP-B mRNA is restricted to specific lung-airway epithelial cells, and human SP-B mRNA stability is increased in the presence of the synthetic glucocorticoid dexamethasone (DEX). Although the mechanism of SP-B mRNA stabilization by DEX is unknown, studies suggest involvement of the glucocorticoid receptor (GR). We developed a dual-cistronic pl...

  16. Protor-2 interacts with tristetraprolin to regulate mRNA stability during stress

    OpenAIRE

    Holmes, Brent; Artinian, Nicholas; Anderson, Lauren; Martin, Jheralyn; Masri, Janine; Cloninger, Cheri; Bernath, Andrew; Bashir, Tariq; Benavides-Serrato, Angelica; Gera, Joseph

    2011-01-01

    The A/U-rich RNA binding protein tristetraprolin (TTP) is an mRNA destabilizing factor which plays a role in the regulated turnover of many transcripts encoding proteins involved in immune function and cell growth control. TTP also plays a role in stress-induced destabilization of mRNAs. Here we report the interaction of TTP with a component of the mTORC2 kinase, Protor-2 (PRR5-L, protein Q6MZQ0/FLJ14213/CAE45978). Protor-2 is structurally similar to human PRR5 and has been demonstrated to bi...

  17. Regulation of mRNA stability via BRF1 and other AU-binding proteins

    OpenAIRE

    Schmidlin-Stalder, Martin

    2005-01-01

    Steady state levels of mRNAs are determined by the rate of synthesis and degradation. A well-known cis-element conferring instability to mRNA is the so-called AU-rich element (ARE), which is present in the 3’ untranslated region (3’UTR) of many cytokines, chemokines, growth factors or proto-oncogenes. The ARE is recognized by a variety of ARE-binding proteins (AUBPs), which decide about the fate of the RNA. Multiple signaling cascades regulate the activity of the AUBPs. Butyrate response fact...

  18. Decrease in transient receptor potential melastatin 6 mRNA stability caused by rapamycin in renal tubular epithelial cells.

    Science.gov (United States)

    Ikari, Akira; Sanada, Ayumi; Sawada, Hayato; Okude, Chiaki; Tonegawa, Chie; Sugatani, Junko

    2011-06-01

    Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), is used in treatments for transplantation and cancer. Rapamycin causes hypomagnesemia, although precisely how has not been examined. Here, we investigated the effect of rapamycin on the expression of transient receptor potential melastatin 6 (TRPM6), a Mg2+ channel. Rapamycin and LY-294002, an inhibitor of phosphatidilinositol-3 kinase (PI3K) located upstream of mTOR, inhibited epidermal growth factor (EGF)-induced expression of the TRPM6 protein without affecting TRPM7 expression in rat renal NRK-52E epithelial cells. Both rapamycin and LY-294002 decreased EGF-induced Mg2+ influx. U0126, a MEK inhibitor, inhibited EGF-induced increases in c-Fos, p-ERK, and TRPM6 levels. In contrast, neither rapamycin nor LY-294002 inhibited EGF-induced increases in p-ERK and c-Fos levels. EGF increased p-Akt level, an effect inhibited by LY-294002 and 1L-6-hydroxymethyl-chiro-inositol2-[(R)-2-O-methyl-3-O-octadecylcarbonate] (Akt inhibitor). Akt inhibitor decreased TRPM6 level similar to rapamycin and LY-294002. These results suggest that a PI3K/Akt/mTOR pathway is involved in the regulation of TRPM6 expression. Rapamycin inhibited the EGF-induced increase in TRPM6 mRNA but did not inhibit human TRPM6 promoter activity. In the presence of actinomycin D, a transcriptional inhibitor, rapamycin accelerated the decrease in TRPM6 mRNA. Rapamycin decreased the expression and activity of a luciferase linked with the 3'-untranslated region of human TRPM6 mRNA. These results suggest that TRPM6 expression is up-regulated by a PI3K/Akt/mTOR pathway and rapamycin reduces TRPM6 mRNA stability, resulting in a decrease in the reabsorption of Mg2+. PMID:21073857

  19. Stabilization of Oncostatin-M mRNA by Binding of Nucleolin to a GC-Rich Element in Its 3'UTR.

    Science.gov (United States)

    Saha, Sucharita; Chakraborty, Alina; Bandyopadhyay, Sumita Sengupta

    2016-04-01

    Oncostatin-M (OSM) is a patho-physiologically important pleiotropic, multifunctional cytokine. OSM mRNA sequence analysis revealed that its 3'UTR contains three highly conserved GC-rich cis-elements (GCREs) whose role in mRNA stability is unidentified. In the present study, the functional role of the proximal GC-rich region of osm 3'-UTR (GCRE-1) in post-transcriptional regulation of osm expression in U937 cells was assessed by transfecting construct containing GCRE-1 at 3'-end of a fairly stable reporter gene followed by analysis of the expression of the reporter. GCRE-1 showed mRNA destabilizing activity; however, upon PMA treatment the reporter message containing GCRE-1 was stabilized. This stabilization is owing to a time-dependent progressive binding of trans-factors (at least five proteins) to GCRE-1 post-PMA treatment. Nucleolin was identified as one of the proteins in the RNP complex of GCRE-1 with PMA-treated U937 cytosolic extracts by oligo-dT affinity chromatography of poly-adenylated GCRE-1. Immuno-blot revealed time-dependent enhancement of nucleolin in the cytoplasm which in turn directly binds GCRE-1. RNA co-immunoprecipitation confirmed the GCRE-1-nucleolin interaction in vivo. To elucidate the functional role of nucleolin in stabilization of osm mRNA, nucleolin was overexpressed in U937 cells and found to stabilize the intrinsic osm mRNA, where co-transfection with the reporter containing GCRE-1 confirms the role of GCRE-1 in stabilization of the reporter mRNA. Thus, in conclusion, the results asserted that PMA treatment in U937 cells leads to cytoplasmic translocation of nucleolin that directly binds GCRE-1, one of the major GC-rich instability elements, thereby stabilizing the osm mRNA. PMID:26399567

  20. ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK-RSK pathway.

    Science.gov (United States)

    Adachi, Shungo; Homoto, Masae; Tanaka, Rikou; Hioki, Yusaku; Murakami, Hiroshi; Suga, Hiroaki; Matsumoto, Masaki; Nakayama, Keiichi I; Hatta, Tomohisa; Iemura, Shun-ichiro; Natsume, Tohru

    2014-09-01

    Low-density lipoprotein receptor (LDLR) mRNA is unstable, but is stabilized upon extracellular signal-regulated kinase (ERK) activation, possibly through the binding of certain proteins to the LDLR mRNA 3'-untranslated region (UTR), although the detailed mechanism underlying this stability control is unclear. Here, using a proteomic approach, we show that proteins ZFP36L1 and ZFP36L2 specifically bind to the 3'-UTR of LDLR mRNA and recruit the CCR4-NOT-deadenylase complex, resulting in mRNA destabilization. We also show that the C-terminal regions of ZFP36L1 and ZFP36L2 are directly phosphorylated by p90 ribosomal S6 kinase, a kinase downstream of ERK, resulting in dissociation of the CCR4-NOT-deadenylase complex and stabilization of LDLR mRNA. We further demonstrate that targeted disruption of the interaction between LDLR mRNA and ZFP36L1 and ZFP36L2 using antisense oligonucleotides results in upregulation of LDLR mRNA and protein. These results indicate that ZFP36L1 and ZFP36L2 regulate LDLR protein levels downstream of ERK. Our results also show the usefulness of our method for identifying critical regulators of specific RNAs and the potency of antisense oligonucleotide-based therapeutics.

  1. Signs of Selection in Synonymous Sites of the Mitochondrial Cytochrome b Gene of Baikal Oilfish (Comephoridae by mRNA Secondary Structure Alterations

    Directory of Open Access Journals (Sweden)

    Veronika I. Teterina

    2015-01-01

    Full Text Available Studies over the past decade have shown a significant role of synonymous mutations in posttranscriptional regulation of gene expression, which is particularly associated with messenger RNA (mRNA secondary structure alterations. Most studies focused on prokaryote genomes and the nuclear genomes of eukaryotes while little is known about the regulation of mitochondrial DNA (mtDNA gene expression. This paper reveals signs of selection in synonymous sites of the mitochondrial cytochrome b gene (Cytb of Baikal oilfish or golomyankas (Comephoridae directed towards altering the secondary structure of the mRNA and probably altering the character of mtDNA gene expression. Our findings are based on comparisons of intraspecific genetic variation patterns of small golomyanka (Comephorus dybowski and two genetic groups of big golomyanka (Comephorus dybowskii. Two approaches were used: (i analysis of the distribution of synonymous mutations between weak-AT (W and strong-GC (S nucleotides within species and groups in accordance with mutation directions from central to peripheral haplotypes and (ii approaches based on the predicted mRNA secondary structure.

  2. Diminished reovirus capsid stability alters disease pathogenesis and littermate transmission.

    Directory of Open Access Journals (Sweden)

    Joshua D Doyle

    2015-03-01

    Full Text Available Reovirus is a nonenveloped mammalian virus that provides a useful model system for studies of viral infections in the young. Following internalization into host cells, the outermost capsid of reovirus virions is removed by endosomal cathepsin proteases. Determinants of capsid disassembly kinetics reside in the viral σ3 protein. However, the contribution of capsid stability to reovirus-induced disease is unknown. In this study, we found that mice inoculated intramuscularly with a serotype 3 reovirus containing σ3-Y354H, a mutation that reduces viral capsid stability, succumbed at a higher rate than those infected with wild-type virus. At early times after inoculation, σ3-Y354H virus reached higher titers than wild-type virus at several sites within the host. Animals inoculated perorally with a serotype 1 reassortant reovirus containing σ3-Y354H developed exaggerated myocarditis accompanied by elaboration of pro-inflammatory cytokines. Surprisingly, unchallenged littermates of mice infected with σ3-Y354H virus displayed higher titers in the intestine, heart, and brain than littermates of mice inoculated with wild-type virus. Together, these findings suggest that diminished capsid stability enhances reovirus replication, dissemination, lethality, and host-to-host spread, establishing a new virulence determinant for nonenveloped viruses.

  3. Altered levels of laminin receptor mRNA in various human carcinoma cells that have different abilities to bind laminin

    DEFF Research Database (Denmark)

    Wewer, U M; Liotta, L A; Jaye, M;

    1986-01-01

    isolated after screening a human endothelial lambda gt11 cDNA library with a monoclonal antibody directed against a domain of the laminin receptor involved in ligand binding. Definitive identification of the cDNA clones was based on comparison of cDNA sequence with the amino acid sequence of a cyanogen...... bromide-generated octapeptide of purified placental laminin receptor. The laminin receptor mRNA is approximately 1700 bases long. The level of laminin receptor mRNA in a variety of human carcinoma-derived cell lines correlated with the number of laminin receptors on the cell surfaces of those cells...

  4. Stabilizing and Managing Patients with Altered Mental Status and Delirium.

    Science.gov (United States)

    Odiari, Ebelechukwu A; Sekhon, Navdeep; Han, Jin Y; David, Elizabeth H

    2015-11-01

    Present in all patient populations, altered mental status (AMS) is a common, but nonspecific emergency department (ED) presentation that can signify serious underlying pathology. Delirium is a more defined mental status change caused by another medical condition that carries a high morbidity and mortality if missed. However, ED physicians miss the condition in more than 50% of cases. The ED physician should maintain a high index of suspicion for delirium, because if missed in the ED, delirium is more likely to be missed on the floors as well. Management of delirium is directed toward treating the underlying course.

  5. Acute heat stress up-regulates neuropeptide Y precursor mRNA expression and alters brain and plasma concentrations of free amino acids in chicks.

    Science.gov (United States)

    Ito, Kentaro; Bahry, Mohammad A; Hui, Yang; Furuse, Mitsuhiro; Chowdhury, Vishwajit S

    2015-09-01

    Heat stress causes an increase in body temperature and reduced food intake in chickens. Several neuropeptides and amino acids play a vital role in the regulation of food intake. However, the responses of neuropeptides and amino acids to heat-stress-induced food-intake regulation are poorly understood. In the current study, the hypothalamic mRNA expression of some neuropeptides related to food intake and the content of free amino acids in the brain and plasma was examined in 14-day-old chicks exposed to a high ambient temperature (HT; 40±1 °C for 2 or 5 h) or to a control thermoneutral temperature (CT; 30±1 °C). HT significantly increased rectal temperature and plasma corticosterone level and suppressed food intake. HT also increased the expression of neuropeptide Y (NPY) and agouti-signaling protein (ASIP) precursor mRNA, while no change was observed in pro-opiomelanocortin, cholecystokinin, ghrelin, or corticotropin-releasing hormone precursor mRNA. It was further found that the diencephalic content of free amino acids - namely, tryptophan, leucine, isoleucine, valine and serine - was significantly higher in HT chicks with some alterations in their plasma amino acids in comparison with CT chicks. The induction of NPY and ASIP expression and the alteration of some free amino acids during HT suggest that these changes can be the results or causes the suppression of food intake.

  6. Sequences of a hairpin structure in the 3′-untranslated region mediate regulation of human pulmonary surfactant protein B mRNA stability

    OpenAIRE

    Huang, Helen W.; Payne, David E.; Bi, Weizhen; Pan, Su; Bruce, Shirley R.; Alcorn, Joseph L.

    2012-01-01

    The ability of pulmonary surfactant to reduce alveolar surface tension requires adequate expression of surfactant protein B (SP-B). Dexamethasone (DEX, 10−7 M) increases human SP-B mRNA stability by a mechanism that requires a 126-nt-long segment (the 7.6S region) of the 3′-untranslated region (3′-UTR). The objective of this study was to identify sequences in the 7.6S region that mediate regulation of SP-B mRNA stability. The 7.6S region was found to be sufficient for DEX-mediated stabilizati...

  7. Nutritional Control of mRNA Stability Is Mediated by a Conserved AU-rich Element That Binds the Cytoplasmic Shuttling Protein HuR*

    OpenAIRE

    Yaman, Ibrahim; Fernandez, James; Sarkar, Bedabrata; Schneider, Robert J.; Snider, Martin D.; Nagy, Laura E.; Hatzoglou, Maria

    2002-01-01

    The cationic amino acid transporter, Cat-1, is a high affinity transporter of the essential amino acids, arginine and lysine. Expression of the cat-1 gene increases during nutritional stress as part of the adaptive response to starvation. Amino acid limitation induces coordinate increases in stability and translation of the cat-1 mRNA, at a time when global protein synthesis decreases. It is shown here that increased cat-1 mRNA stability requires an 11 nucleotide AU-rich element within the di...

  8. Comparative transcriptomic analysis reveals the oncogenic fusion protein PAX3-FOXO1 globally alters mRNA and miRNA to enhance myoblast invasion.

    Science.gov (United States)

    Loupe, J M; Miller, P J; Bonner, B P; Maggi, E C; Vijayaraghavan, J; Crabtree, J S; Taylor, C M; Zabaleta, J; Hollenbach, A D

    2016-01-01

    Rhabdomyosarcoma, one of the most common childhood sarcomas, is comprised of two main subtypes, embryonal and alveolar (ARMS). ARMS, the more aggressive subtype, is primarily characterized by the t(2;13)(p35;p14) chromosomal translocation, which fuses two transcription factors, PAX3 and FOXO1 to generate the oncogenic fusion protein PAX3-FOXO1. Patients with PAX3-FOXO1-postitive tumors have a poor prognosis, in part due to the enhanced local invasive capacity of these cells, which leads to the increased metastatic potential for this tumor. Despite this knowledge, little is known about the role that the oncogenic fusion protein has in this increased invasive potential. In this report we use large-scale comparative transcriptomic analyses in physiologically relevant primary myoblasts to demonstrate that the presence of PAX3-FOXO1 is sufficient to alter the expression of 70 mRNA and 27 miRNA in a manner predicted to promote cellular invasion. In contrast the expression of PAX3 alters 60 mRNA and 23 miRNA in a manner predicted to inhibit invasion. We demonstrate that these alterations in mRNA and miRNA translate into changes in the invasive potential of primary myoblasts with PAX3-FOXO1 increasing invasion nearly 2-fold while PAX3 decreases invasion nearly 4-fold. Taken together, these results allow us to build off of previous reports and develop a more expansive molecular model by which the presence of PAX3-FOXO1 alters global gene regulatory networks to enhance the local invasiveness of cells. Further, the global nature of our observed changes highlights the fact that instead of focusing on a single-gene target, we must develop multi-faceted treatment regimens targeting multiple genes of a single oncogenic phenotype or multiple genes that target different oncogenic phenotypes for tumor progression. PMID:27454080

  9. Herpes simplex virus virion stimulatory protein mRNA leader contains sequence elements which increase both virus-induced transcription and mRNA stability.

    OpenAIRE

    Blair, E D; Blair, C C; Wagner, E K

    1987-01-01

    To investigate the role of 5' noncoding leader sequence of herpes simplex virus type 1 (HSV-1) mRNA in infected cells, the promoter for the 65,000-dalton virion stimulatory protein (VSP), a beta-gamma polypeptide, was introduced into plasmids bearing the chloramphenicol acetyltransferase (cat) gene together with various lengths of adjacent viral leader sequences. Plasmids containing longer lengths of leader sequence gave rise to significantly higher levels of CAT enzyme in transfected cells s...

  10. Involvement of the 5'-leader sequence in coupling the stability of a human H3 histone mRNA with DNA replication

    International Nuclear Information System (INIS)

    Two lines of evidence derived from fusion gene constructs indicate that sequences residing in the 5'-nontranslated region of a cell cycle-dependent human H3 histone mRNA are involved in the selective destabilization that occurs when DNA synthesis is terminated. The experimental approach was to construct chimeric genes in which fragments of the mRNA coding regions of the H3 histone gene were fused with fragments of genes not expressed in a cell cycle-dependent manner. After transfection in HeLa S3 cells with the recombinant plasmids, levels of fusion mRNAs were determined by S1 nuclease analysis prior to and following DNA synthesis inhibition. When the first 20 nucleotides of an H3 histone mRNA leader were replaced with 89 nucleotides of the leader from a Drosophila heat-shock (hsp70) mRNA, the fusion transcript remained stable during inhibition of DNA synthesis, in contrast to the rapid destabilization of the endogenous histone mRNA in these cells. In a reciprocal experiment, a histone-globin fusion gene was constructed that produced a transcript with the initial 20 nucleotides of the H3 histone mRNA substituted for the human β-globin mRNA leader. In HeLa cells treated with inhibitors of DNA synthesis and/or protein synthesis, cellular levels of this histone-globin fusion mRNA appeared to be regulated in a manner similar to endogenous histone mRNA levels. These results suggest that the first 20 nucleotides of the leader are sufficient to couple histone mRNA stability with DNA replication

  11. Hypoxia increases rate of transcription and stability of tyrosine hydroxylase mRNA in pheochromocytoma (PC12) cells.

    Science.gov (United States)

    Czyzyk-Krzeska, M F; Furnari, B A; Lawson, E E; Millhorn, D E

    1994-01-01

    Reduced arterial oxygen tension (i.e. hypoxia) is a powerful physiological stimulus that induces synthesis and release of dopamine from O2-sensitive (type I) cells in the mammalian carotid bodies. We reported recently that hypoxia stimulates gene expression for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis in type I cells of the carotid body. Efforts to identify the mechanisms regulating TH gene expression in O2-sensitive cells during hypoxia have been hampered by the lack of an appropriate model cell culture system. Here we report that TH gene expression in the rat pheochromocytoma cell line (PC12) is regulated during hypoxia in a manner similar to that measured in carotid body type I cells. PC12 cells might therefore be useful as an experimental model for identifying the molecular mechanisms that regulate TH gene expression during hypoxia. Nuclear runoff assays revealed that transcription of the wild type TH gene was enhanced during exposures to hypoxia lasting 12 h. Chloramphenicol acetyltransferase assays with constructs that contained different fragments of TH promoter revealed that the regulatory sequences that mediate the hypoxia-induced increase in transcription are located between bases -272 and +27 of the TH gene. Findings from experiments in which transcription was inhibited either with actinomycin D or 5,6-dichloro-1-D-ribofuranosylbenzimidazole, as well as pulse-chase experiments using 4-thiouridine showed that the half-life of TH mRNA was substantially increased during hypoxia. Thus, in the present paper we show that TH gene expression in PC12 cells during hypoxia is regulated by increases in both the rate of TH gene transcription and TH mRNA stability. PMID:7903970

  12. Differential regulation of mRNA stability controls the transient expression of genes encoding Drosophila antimicrobial peptide with distinct immune response characteristics.

    Science.gov (United States)

    Wei, Youheng; Xiao, Qianghai; Zhang, Ting; Mou, Zongchun; You, Jia; Ma, Wei-Jun

    2009-10-01

    The tight regulation of transiently expressed antimicrobial peptides (AMPs) with a distinct antimicrobial spectrum and different expression kinetics contributes greatly to the properly regulated immune response for resistance to pathogens and for the maintenance of mutualistic microbiota in Drosophila. The important role of differential regulation of AMP expression at the posttranscriptional level needs to be elucidated. It was observed that the highly expressed Cecropin A1 (CecA1) mRNA encoding a broad antimicrobial spectrum AMP against both bacteria and fungi decayed more quickly than did the moderately expressed Diptericin mRNA encoding AMP against Gram negative bacteria. The mRNA stability of AMPs is differentially regulated and is attributed to the specific interaction between cis-acting ARE in 3'-UTR of AMP mRNA and the RNA destabilizing protein transactor Tis11 as shown in co-immunoprecipitation of the Tis11 RNP complex with CecA1 mRNA but not other AMP mRNA. The p38MAPK was further demonstrated to play a crucial role in stabilizing ARE-bearing mRNAs by inhibiting Tis11-mediated degradation in LPS induced AMP expression. This evidence suggests an evolutionarily conserved and functionally important molecular basis for and effective approach to exact control of AMP gene expression. These mechanisms thereby orchestrate a well balanced and dynamic antimicrobial spectrum of innate immunity to resist infection and maintain resident microbiota properly. PMID:19726583

  13. Tris(2-butoxyethyl)phosphate and triethyl phosphate alter embryonic development, hepatic mRNA expression, thyroid hormone levels, and circulating bile acid concentrations in chicken embryos

    Energy Technology Data Exchange (ETDEWEB)

    Egloff, Caroline [National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3 (Canada); Crump, Doug, E-mail: doug.crump@ec.gc.ca [National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3 (Canada); Porter, Emily; Williams, Kim L.; Letcher, Robert J.; Gauthier, Lewis T. [National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3 (Canada); Kennedy, Sean W. [National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3 (Canada); Department of Biology, University of Ottawa, Ottawa, ON K1N 6N5 (Canada)

    2014-09-15

    The organophosphate flame retardants tris(2-butoxyethyl) phosphate (TBOEP) and triethyl phosphate (TEP) are used in a wide range of applications to suppress or delay the ignition and spread of fire. Both compounds have been detected in the environment and TBOEP was recently measured in free-living avian species. In this study, TBOEP and TEP were injected into the air cell of chicken embryos at concentrations ranging from 0 to 45,400 ng/g and 0 to 241,500 ng/g egg, respectively. Pipping success, development, hepatic mRNA expression of 9 target genes, thyroid hormone levels, and circulating bile acid concentrations were determined. Exposure to the highest doses of TBOEP and TEP resulted in negligible detection of the parent compounds in embryonic contents at pipping indicating their complete metabolic degradation. TBOEP exposure had limited effects on chicken embryos, with the exception of hepatic CYP3A37 mRNA induction. TEP exposure decreased pipping success to 68%, altered growth, increased liver somatic index (LSI) and plasma bile acids, and modulated genes associated with xenobiotic and lipid metabolism and the thyroid hormone pathway. Plasma thyroxine levels were decreased at all TEP doses, including an environmentally-relevant concentration (8 ng/g), and gallbladder hypotrophy was evident at ≥ 43,200 ng/g. Tarsus length and circulating thyroxine concentration emerged as potential phenotypic anchors for the modulation of transthyretin mRNA. The increase in plasma bile acids and LSI, gallbladder hypotrophy, and discoloration of liver tissue represented potential phenotypic outcomes associated with modulation of hepatic genes involved with xenobiotic and lipid metabolism. - Highlights: • TBOEP is not embryolethal to chicken embryos. • TEP affected embryonic viability, morphometric endpoints, and thyroid hormone levels. • TEP altered mRNA levels of xenobiotic and lipid metabolism genes. • TEP increased plasma bile acids and caused gallbladder hypotrophy

  14. Tris(2-butoxyethyl)phosphate and triethyl phosphate alter embryonic development, hepatic mRNA expression, thyroid hormone levels, and circulating bile acid concentrations in chicken embryos

    International Nuclear Information System (INIS)

    The organophosphate flame retardants tris(2-butoxyethyl) phosphate (TBOEP) and triethyl phosphate (TEP) are used in a wide range of applications to suppress or delay the ignition and spread of fire. Both compounds have been detected in the environment and TBOEP was recently measured in free-living avian species. In this study, TBOEP and TEP were injected into the air cell of chicken embryos at concentrations ranging from 0 to 45,400 ng/g and 0 to 241,500 ng/g egg, respectively. Pipping success, development, hepatic mRNA expression of 9 target genes, thyroid hormone levels, and circulating bile acid concentrations were determined. Exposure to the highest doses of TBOEP and TEP resulted in negligible detection of the parent compounds in embryonic contents at pipping indicating their complete metabolic degradation. TBOEP exposure had limited effects on chicken embryos, with the exception of hepatic CYP3A37 mRNA induction. TEP exposure decreased pipping success to 68%, altered growth, increased liver somatic index (LSI) and plasma bile acids, and modulated genes associated with xenobiotic and lipid metabolism and the thyroid hormone pathway. Plasma thyroxine levels were decreased at all TEP doses, including an environmentally-relevant concentration (8 ng/g), and gallbladder hypotrophy was evident at ≥ 43,200 ng/g. Tarsus length and circulating thyroxine concentration emerged as potential phenotypic anchors for the modulation of transthyretin mRNA. The increase in plasma bile acids and LSI, gallbladder hypotrophy, and discoloration of liver tissue represented potential phenotypic outcomes associated with modulation of hepatic genes involved with xenobiotic and lipid metabolism. - Highlights: • TBOEP is not embryolethal to chicken embryos. • TEP affected embryonic viability, morphometric endpoints, and thyroid hormone levels. • TEP altered mRNA levels of xenobiotic and lipid metabolism genes. • TEP increased plasma bile acids and caused gallbladder hypotrophy

  15. Lethrinas nebulosus fish as a biomarker for petroleum hydrocarbons pollution in Red Sea : Alterations in antioxidants mRNA expression

    OpenAIRE

    Afifi, Mohamed; Ali, Haytham A.; Saber, Taghred M.; El-Murr, Abd elhakeem

    2016-01-01

    Total Petroleum Hydrocarbons (TPHs) are environmental contaminants that are released into the marine water via oil spills and industrial activities. The mRNA expression profile of some antioxidant genes in livers, gills, skin and muscles of Lethrinas nebulosus was used as biomarker of TPHs pollution in six areas at Jeddah and Yanbu coasts in Kingdom of Saudi Arabia (KSA). TPHs were determined in Red Sea water and sediments collected from the studied areas. Ten fish of similar sizes were colle...

  16. miR-143 decreases COX-2 mRNA stability and expression in pancreatic cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Pham, Hung [Department of Surgery, UCLA Center of Excellence in Pancreatic Diseases, UCLA David Geffen School of Medicine, University of California – Los Angeles, Los Angeles, CA 90095 (United States); Department of Medicine, Veterans Affair Greater Los Angeles Healthcare System, Los Angeles, CA 90073 (United States); Ekaterina Rodriguez, C.; Donald, Graham W.; Hertzer, Kathleen M.; Jung, Xiaoman S.; Chang, Hui-Hua; Moro, Aune; Reber, Howard A.; Hines, O. Joe [Department of Surgery, UCLA Center of Excellence in Pancreatic Diseases, UCLA David Geffen School of Medicine, University of California – Los Angeles, Los Angeles, CA 90095 (United States); Eibl, Guido, E-mail: geibl@mednet.ucla.edu [Department of Surgery, UCLA Center of Excellence in Pancreatic Diseases, UCLA David Geffen School of Medicine, University of California – Los Angeles, Los Angeles, CA 90095 (United States)

    2013-09-13

    Highlights: •Pancreatic cancer cells express low miR-143 levels and elevated p-MEK, p-MAPK and RREB1. •MEK inhibitors U0126 and PD98059 increase miR-143 expression. •miR-143 decreases COX-2 mRNA stability and expression and PGE{sub 2}. •miR-143 decreases p-p38MAPK, p-MEK, p-MAPK and RREB1 expression. -- Abstract: Small non-coding RNAs, microRNAs (miRNA), inhibit the translation or accelerate the degradation of message RNA (mRNA) by targeting the 3′-untranslated region (3′-UTR) in regulating growth and survival through gene suppression. Deregulated miRNA expression contributes to disease progression in several cancers types, including pancreatic cancers (PaCa). PaCa tissues and cells exhibit decreased miRNA, elevated cyclooxygenase (COX)-2 and increased prostaglandin E{sub 2} (PGE{sub 2}) resulting in increased cancer growth and metastases. Human PaCa cell lines were used to demonstrate that restoration of miRNA-143 (miR-143) regulates COX-2 and inhibits cell proliferation. miR-143 were detected at fold levels of 0.41 ± 0.06 in AsPC-1, 0.20 ± 0.05 in Capan-2 and 0.10 ± 0.02 in MIA PaCa-2. miR-143 was not detected in BxPC-3, HPAF-II and Panc-1 which correlated with elevated mitogen-activated kinase (MAPK) and MAPK kinase (MEK) activation. Treatment with 10 μM of MEK inhibitor U0126 or PD98059 increased miR-143, respectively, by 187 ± 18 and 152 ± 26-fold in BxPC-3 and 182 ± 7 and 136 ± 9-fold in HPAF-II. miR-143 transfection diminished COX-2 mRNA stability at 60 min by 2.6 ± 0.3-fold in BxPC-3 and 2.5 ± 0.2-fold in HPAF-II. COX-2 expression and cellular proliferation in BxPC-3 and HPAF-II inversely correlated with increasing miR-143. PGE{sub 2} levels decreased by 39.3 ± 5.0% in BxPC-3 and 48.0 ± 3.0% in HPAF-II transfected with miR-143. Restoration of miR-143 in PaCa cells suppressed of COX-2, PGE{sub 2}, cellular proliferation and MEK/MAPK activation, implicating this pathway in regulating miR-143 expression.

  17. miR-143 decreases COX-2 mRNA stability and expression in pancreatic cancer cells

    International Nuclear Information System (INIS)

    Highlights: •Pancreatic cancer cells express low miR-143 levels and elevated p-MEK, p-MAPK and RREB1. •MEK inhibitors U0126 and PD98059 increase miR-143 expression. •miR-143 decreases COX-2 mRNA stability and expression and PGE2. •miR-143 decreases p-p38MAPK, p-MEK, p-MAPK and RREB1 expression. -- Abstract: Small non-coding RNAs, microRNAs (miRNA), inhibit the translation or accelerate the degradation of message RNA (mRNA) by targeting the 3′-untranslated region (3′-UTR) in regulating growth and survival through gene suppression. Deregulated miRNA expression contributes to disease progression in several cancers types, including pancreatic cancers (PaCa). PaCa tissues and cells exhibit decreased miRNA, elevated cyclooxygenase (COX)-2 and increased prostaglandin E2 (PGE2) resulting in increased cancer growth and metastases. Human PaCa cell lines were used to demonstrate that restoration of miRNA-143 (miR-143) regulates COX-2 and inhibits cell proliferation. miR-143 were detected at fold levels of 0.41 ± 0.06 in AsPC-1, 0.20 ± 0.05 in Capan-2 and 0.10 ± 0.02 in MIA PaCa-2. miR-143 was not detected in BxPC-3, HPAF-II and Panc-1 which correlated with elevated mitogen-activated kinase (MAPK) and MAPK kinase (MEK) activation. Treatment with 10 μM of MEK inhibitor U0126 or PD98059 increased miR-143, respectively, by 187 ± 18 and 152 ± 26-fold in BxPC-3 and 182 ± 7 and 136 ± 9-fold in HPAF-II. miR-143 transfection diminished COX-2 mRNA stability at 60 min by 2.6 ± 0.3-fold in BxPC-3 and 2.5 ± 0.2-fold in HPAF-II. COX-2 expression and cellular proliferation in BxPC-3 and HPAF-II inversely correlated with increasing miR-143. PGE2 levels decreased by 39.3 ± 5.0% in BxPC-3 and 48.0 ± 3.0% in HPAF-II transfected with miR-143. Restoration of miR-143 in PaCa cells suppressed of COX-2, PGE2, cellular proliferation and MEK/MAPK activation, implicating this pathway in regulating miR-143 expression

  18. WEREWOLF, a regulator of root hair pattern formation, controls flowering time through the regulation of FT mRNA stability.

    Science.gov (United States)

    Seo, Eunjoo; Yu, Jihyeon; Ryu, Kook Hui; Lee, Myeong Min; Lee, Ilha

    2011-08-01

    A key floral activator, FT, integrates stimuli from long-day, vernalization, and autonomous pathways and triggers flowering by directly regulating floral meristem identity genes in Arabidopsis (Arabidopsis thaliana). Since a small amount of FT transcript is sufficient for flowering, the FT level is strictly regulated by diverse genes. In this study, we show that WEREWOLF (WER), a MYB transcription factor regulating root hair pattern, is another regulator of FT. The mutant wer flowers late in long days but normal in short days and shows a weak sensitivity to vernalization, which indicates that WER controls flowering time through the photoperiod pathway. The expression and double mutant analyses showed that WER modulates FT transcript level independent of CONSTANS and FLOWERING LOCUS C. The histological analysis of WER shows that it is expressed in the epidermis of leaves, where FT is not expressed. Consistently, WER regulates not the transcription but the stability of FT mRNA. Our results reveal a novel regulatory mechanism of FT that is non cell autonomous.

  19. Identification of sequences within the murine granulocyte-macrophage colony-stimulating factor mRNA 3'-untranslated region that mediate mRNA stabilization induced by mitogen treatment of EL-4 thymoma cells.

    Science.gov (United States)

    Iwai, Y; Bickel, M; Pluznik, D H; Cohen, R B

    1991-09-25

    Phorbol esters (TPA) and concanavalin A (ConA) are known to induce granulocyte-macrophage colony-stimulating factor (GM-CSF) production in murine thymoma EL-4 cells by mRNA stabilization. The role of the 3'-untranslated region (3'-UTR) in GM-CSF mRNA stabilization induced by TPA and ConA in EL-4 cells was examined by transfection studies using chloramphenicol acetyltransferase (CAT) constructions. The GM-CSF 3'-UTR contains a 63-nucleotide region at its 3' end with repeating ATTTA motifs which is responsible for mRNA degradation in a variety of cell types (Shaw, G., and Kamen, R. (1986) Cell 46, 659-666). We produced constructs containing most of the GM-CSF 3'-UTR (303 nucleotides, pRSV-CATgm) or the 3'-terminal AT-rich region (116 nucleotides, pRSV-CATau) and measured CAT enzyme activity and CAT mRNA after transient transfection into EL-4 and NIH 3T3 cells. Low levels of CAT activity were seen in both cells with either plasmid compared with levels of CAT activity obtained with pRSV-CAT. TPA treatment caused an approximately 10-fold increase in CAT activity and mRNA in EL-4 cells transfected with pRSV-CATgm. No increases were seen in EL-4 cells transfected with pRSV-CATau or pRSV-CAT. No response to TPA was detected in transfected NIH 3T3 cells, indicating that the response to TPA is relatively cell-specific. There was no increase in CAT activity after ConA treatment in EL-4 or NIH 3T3 cells transfected with any of the constructs suggesting that the GM-CSF 3'-UTR lacks elements that can respond alone to ConA. Nuclear run-on and actinomycin D chase experiments in EL-4 cells showed that TPA induces CAT activity via mRNA stabilization. By linker-substitution mutagenesis we show that TPA inducibility depends on a 60-nucleotide region of the 3'-UTR whose 5' end is located 160 nucleotides upstream of the 5' end of the AU-rich region. PMID:1917935

  20. 5, 8, 11, 14-eicosatetraynoic acid suppresses CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1 mRNA stability

    Directory of Open Access Journals (Sweden)

    Lee Jee

    2012-02-01

    Full Text Available Abstract Background The peroxisome proliferator-activated receptor (PPAR-α activator, 5,8,11,14-eicosatetraynoic acid (ETYA, is an arachidonic acid analog. It is reported to inhibit up-regulation of pro-inflammatory genes; however, its underlying mechanism of action is largely unknown. In the present study, we focused on the inhibitory action of ETYA on the expression of the chemokine, CCL2/MCP-1, which plays a key role in the initiation and progression of inflammation. Methods To determine the effect of ETYA, primary cultured rat astrocytes and microglia were stimulated with IFN-γ in the presence of ETYA and then, expression of CCL2/MCP-1 and MAPK phosphatase (MKP-1 were determined using RT-PCR and ELISA. MKP-1 mRNA stability was evaluated by treating actinomycin D. The effect of MKP-1 and human antigen R (HuR was analyzed by using specific siRNA transfection system. The localization of HuR was analyzed by immunocytochemistry and subcellular fractionation experiment. Results We found that ETYA suppressed CCL2/MCP-1 transcription and secretion of CCL2/MCP-1 protein through up-regulation of MKP-1mRNA levels, resulting in suppression of c-Jun N-terminal kinase (JNK phosphorylation and activator protein 1 (AP1 activity in IFN-γ-stimulated brain glial cells. Moreover, these effects of ETYA were independent of PPAR-α. Experiments using actinomycin D revealed that the ETYA-induced increase in MKP-1 mRNA levels reflected an increase in transcript stability. Knockdown experiments using small interfering RNA demonstrated that this increase in MKP-1 mRNA stability depended on HuR, an RNA-binding protein known to promote enhanced mRNA stability. Furthermore, ETYA-induced, HuR-mediated mRNA stabilization resulted from HuR-MKP-1 nucleocytoplasmic translocation, which served to protect MKP-1 mRNA from the mRNA degradation machinery. Conclusion ETYA induces MKP-1 through HuR at the post-transcriptional level in a receptor-independent manner. The mechanism

  1. Altered mRNA expression of hepatic lipogenic enzyme and PPARalpha in rats fed dietary levan from Zymomonas mobilis.

    Science.gov (United States)

    Kang, Soon Ah; Hong, Kyunghee; Jang, Ki-Hyo; Kim, Yun-Young; Choue, Ryowon; Lim, Yoongho

    2006-06-01

    Levan or high molecular beta-2,6-linked fructose polymer is produced extracellularly from sucrose-based substrates by bacterial levansucrase. In the present study, to investigate the effect of levan feeding on serum leptin, hepatic lipogenic enzyme and peroxisome proliferation-activated receptor (PPAR) alpha expression in high-fat diet-induced obese rats, 4-week-old Sprague-Dawley male rats were fed high-fat diet (beef tallow, 40% of calories as fat), and, 6 weeks later, the rats were fed 0%, 1%, 5% or 10% levan-supplemented diets for 4 weeks. Serum leptin and insulin level were dose dependently reduced in levan-supplemented diet-fed rats. The mRNA expressions of hepatic fatty acid synthase and acetyl CoA carboxylase, which are the key enzymes in fatty acid synthesis, were down-regulated by dietary levan. However, dietary levan did not affect the gene expression of hepatic malic enzyme, phosphatidate phosphohydrolase and HMG CoA reductase. Also, the lipogenic enzyme gene expression in the white adipose tissue (WAT) was not affected by the diet treatments. However, hepatic PPARalpha mRNA expression was dose dependently up-regulated by dietary levan, whereas PPARgamma in the WAT was not changed. The results suggest that the in vivo hypolipidemic effect of dietary levan, including anti-obesity and lipid-lowering, may result from the inhibition of lipogenesis and stimulation of lipolysis, accompanied with regulation of hepatic lipogenic enzyme and PPARalpha gene expression. PMID:16214330

  2. CIL-102 induces matrix metalloproteinase-2 (MMP-2)/MMP-9 down-regulation via simultaneous suppression of genetic transcription and mRNA stability.

    Science.gov (United States)

    Liu, Wen-Hsin; Chen, Yeh-Long; Chang, Long-Sen

    2012-12-01

    This study explores the CIL-102 suppression mechanism on matrix metalloproteinase-2 (MMP-2) and MMP-9 expression in human leukemia K562 cells. CIL-102 attenuated K562 cell invasion with decreased MMP-2/MMP-9 protein expression and mRNA levels. Moreover, CIL-102 reduced luciferase activity of MMP-2/MMP-9 promoter constructs and MMP-2/MMP-9 mRNA stability. CIL-102 treatment induced JNK and p38 MAPK activation but reduced the phospho-ERK level. Transfection of constitutively active MEK1 restored MMP-2 and MMP-9 promoter activity in CIL-102-treated cells, while suppression of p38 MAPK/JNK activation abolished CIL-102-induced MMP-2/MMP-9 mRNA decay. CIL-102-induced p38 MAPK/JNK activation led to protein phosphatase 2A-mediated tristetraprolin (TTP) down-regulation. The reduction in TTP-KH-type splicing regulatory protein (KSRP) complexes formation promoted KSRP-mediated MMP-2/MMP-9 mRNA decay in CIL-102-treated K562 cells. Moreover, CIL-102 reduced invasion and MMP-2/MMP-9 expression in breast and liver cancer cells. Taken together, our data indicate that CIL-102 induces MMP-2/MMP-2 down-regulation via simultaneous suppression of genetic transcription and mRNA stability, and suggest a potential utility for CIL-102 in reducing MMP-2/MMP-9-mediated cancer progression.

  3. Translation and stability of an Escherichia coli beta-galactosidase mRNA expressed under the control of pyruvate kinase sequences in Saccharomyces cerevisiae.

    OpenAIRE

    Purvis, I J; Loughlin, L; Bettany, A J; Brown, A. J.

    1987-01-01

    Plasmids were assembled in which the coding region of the pyruvate kinase (PYK) gene of Saccharomyces cerevisiae was replaced by that of the B-galactosidase (LacZ) gene from Escherichia coli. Analysis of the resultant, chimaeric transcripts from low copy number, centromeric plasmids indicated that this substitution caused a dramatic reduction in the steady-state level of the messenger RNA (mRNA). This fluctuation cannot be wholly accounted for by the 2-fold decrease in mRNA stability observed...

  4. p85α promotes nucleolin transcription and subsequently enhances EGFR mRNA stability and EGF-induced malignant cellular transformation.

    Science.gov (United States)

    Xie, Qipeng; Guo, Xirui; Gu, Jiayan; Zhang, Liping; Jin, Honglei; Huang, Haishan; Li, Jingxia; Huang, Chuanshu

    2016-03-29

    p85α is a regulatory subunit of phosphatidylinositol 3-kinase (PI3K) that is a key lipid enzyme for generating phosphatidylinositol 3, 4, 5-trisphosphate, and subsequently activates signaling that ultimately regulates cell cycle progression, cell growth, cytoskeletal changes, and cell migration. In addition to form a complex with the p110 catalytic subunit, p85α also exists as a monomeric form due to that there is a greater abundance of p85α than p110 in many cell types. Our previous studies have demonstrated that monomeric p85α exerts a pro-apoptotic role in UV response through induction of TNF-α gene expression in PI3K-independent manner. In current studies, we identified a novel biological function of p85α as a positive regulator of epidermal growth factor receptor (EGFR) expression and cell malignant transformation via nucleolin-dependent mechanism. Our results showed that p85α was crucial for EGFR and nucleolin expression and subsequently resulted in an increase of malignant cellular transformation by using both specific knockdown and deletion of p85α in its normal expressed cells. Mechanistic studies revealed that p85α upregulated EGFR protein expression mainly through stabilizing its mRNA, whereas nucleolin (NCL) was able to bind to egfr mRNA and increase its mRNA stability. Consistently, overexpression of NCL in p85α-/- cells restored EGFR mRNA stabilization, protein expression and cell malignant transformation. Moreover, we discovered that p85α upregulated NCL gene transcription via enhancing C-Jun activation. Collectively, our studies demonstrate a novel function of p85α as a positive regulator of EGFR mRNA stability and cell malignant transformation, providing a significant insight into the understanding of biomedical nature of p85α protein in mammalian cells and further supporting that p85α might be a potential target for cancer prevention and therapy. PMID:26918608

  5. p85α promotes nucleolin transcription and subsequently enhances EGFR mRNA stability and EGF-induced malignant cellular transformation

    Science.gov (United States)

    Gu, Jiayan; Zhang, Liping; Jin, Honglei; Huang, Haishan; Li, Jingxia; Huang, Chuanshu

    2016-01-01

    p85α is a regulatory subunit of phosphatidylinositol 3-kinase (PI3K) that is a key lipid enzyme for generating phosphatidylinositol 3, 4, 5-trisphosphate, and subsequently activates signaling that ultimately regulates cell cycle progression, cell growth, cytoskeletal changes, and cell migration. In addition to form a complex with the p110 catalytic subunit, p85α also exists as a monomeric form due to that there is a greater abundance of p85α than p110 in many cell types. Our previous studies have demonstrated that monomeric p85α exerts a pro-apoptotic role in UV response through induction of TNF-α gene expression in PI3K-independent manner. In current studies, we identified a novel biological function of p85α as a positive regulator of epidermal growth factor receptor (EGFR) expression and cell malignant transformation via nucleolin-dependent mechanism. Our results showed that p85α was crucial for EGFR and nucleolin expression and subsequently resulted in an increase of malignant cellular transformation by using both specific knockdown and deletion of p85α in its normal expressed cells. Mechanistic studies revealed that p85α upregulated EGFR protein expression mainly through stabilizing its mRNA, whereas nucleolin (NCL) was able to bind to egfr mRNA and increase its mRNA stability. Consistently, overexpression of NCL in p85α−/− cells restored EGFR mRNA stabilization, protein expression and cell malignant transformation. Moreover, we discovered that p85α upregulated NCL gene transcription via enhancing C-Jun activation. Collectively, our studies demonstrate a novel function of p85α as a positive regulator of EGFR mRNA stability and cell malignant transformation, providing a significant insight into the understanding of biomedical nature of p85α protein in mammalian cells and further supporting that p85α might be a potential target for cancer prevention and therapy. PMID:26918608

  6. Berberine Decreased Inducible Nitric Oxide Synthase mRNA Stability through Negative Regulation of Human Antigen R in Lipopolysaccharide-Induced Macrophages.

    Science.gov (United States)

    Shin, Ji-Sun; Choi, Hye-Eun; Seo, SeungHwan; Choi, Jung-Hye; Baek, Nam-In; Lee, Kyung-Tae

    2016-07-01

    Berberine, a major isoquinoline alkaloid found in medicinal herbs, has been reported to possess anti-inflammatory effects; however, the underlying mechanisms responsible for its actions are poorly understood. In the present study, we investigated the inhibitory effects of berberine and the molecular mechanisms involved in lipopolysaccharide (LPS)-treated RAW 264.7 and THP-1 macrophages and its effects in LPS-induced septic shock in mice. In both macrophage cell types, berberine inhibited the LPS-induced nitric oxide (NO) production and inducible NO synthase (iNOS) protein expression, but it had no effect on iNOS mRNA transcription. Suppression of LPS-induced iNOS protein expression by berberine occurred via a human antigen R (HuR)-mediated reduction of iNOS mRNA stability. Molecular data revealed that the suppression on the LPS-induced HuR binding to iNOS mRNA by berberine was accompanied by a reduction in nucleocytoplasmic HuR shuttling. Pretreatment with berberine reduced LPS-induced iNOS protein expression and the cytoplasmic translocation of HuR in liver tissues and increased the survival rate of mice with LPS-induced endotoxemia. These results show that the suppression of iNOS protein expression by berberine under LPS-induced inflammatory conditions is associated with a reduction in iNOS mRNA stability resulting from inhibition of the cytoplasmic translocation of HuR. PMID:27189969

  7. Effects of two common polymorphisms in the 3' untranslated regions of estrogen receptor β on mRNA stability and translatability

    Directory of Open Access Journals (Sweden)

    Gustafsson Jan-Åke

    2009-09-01

    Full Text Available Abstract Background The present study represents the first attempt to functionally characterize two common single nucleotide polymorphisms (SNPs in the 3'untranslated regions (3'UTRs of estrogen receptor β (ERβ, focusing on the differences between alleles with regard to mRNA stability and translatability. These two ERβ SNPs have been investigated for association with disease in a large number of reports. Results Here we examined allelic expression in breast tumor samples from heterozygous individuals. A significant difference in mRNA levels of the two alleles was observed for one of the SNPs. A cell model system was employed to further investigate potential molecular effects of the two SNPs. We used a modified plasmid, containing the ERβ promoter and ERβ 3'UTRs which include the different alleles of investigated SNPs. Quantitative Real-Time PCR was used to determine mRNA levels after inhibition of transcription by actinomycin D, and a luciferase assay was used to determine protein levels. The obtained results suggested that there was no difference in mRNA stability or translatability between the alleles of investigated SNPs. Conclusion Our results indicate that observed associations between ERβ 3'UTR SNPs and disease susceptibility are due to linkage disequilibrium with another gene variant, rather than the variant itself being the susceptibility factor.

  8. Genomic convergence analysis of schizophrenia: mRNA sequencing reveals altered synaptic vesicular transport in post-mortem cerebellum.

    Directory of Open Access Journals (Sweden)

    Joann Mudge

    Full Text Available Schizophrenia (SCZ is a common, disabling mental illness with high heritability but complex, poorly understood genetic etiology. As the first phase of a genomic convergence analysis of SCZ, we generated 16.7 billion nucleotides of short read, shotgun sequences of cDNA from post-mortem cerebellar cortices of 14 patients and six, matched controls. A rigorous analysis pipeline was developed for analysis of digital gene expression studies. Sequences aligned to approximately 33,200 transcripts in each sample, with average coverage of 450 reads per gene. Following adjustments for confounding clinical, sample and experimental sources of variation, 215 genes differed significantly in expression between cases and controls. Golgi apparatus, vesicular transport, membrane association, Zinc binding and regulation of transcription were over-represented among differentially expressed genes. Twenty three genes with altered expression and involvement in presynaptic vesicular transport, Golgi function and GABAergic neurotransmission define a unifying molecular hypothesis for dysfunction in cerebellar cortex in SCZ.

  9. Reduced stability and bi-allelic, coequal expression of profilaggrin mRNA in keratinocytes cultured from subjects with ichthyosis vulgaris.

    Science.gov (United States)

    Nirunsuksiri, W; Zhang, S H; Fleckman, P

    1998-06-01

    Ichthyosis vulgaris (IV) is an inherited scaling skin disorder in which expression of profilaggrin is reduced. Previous studies have indicated that the reduction is caused by defective post-transcriptional control of gene expression. Here we present evidence that profilaggrin mRNA in keratinocytes cultured from subjects with IV is intrinsically unstable and has a shorter half-life compared with that in normal cells. When IV-affected keratinocytes were treated with the protein synthesis inhibitor cycloheximide, the steady-state level of profilaggrin mRNA was increased due to stabilization of the transcript. In addition, the number of filaggrin repeats within the profilaggrin gene was studied. The number of filaggrin repeats (10-12) in individuals with IV did not differ from that of unaffected subjects. Expression of the gene was bi-allelic and coequal in both control and affected individuals. Our results suggest a model in which a labile ribonuclease and a stabilizing factor may modulate the profilaggrin mRNA steady-state level in normal cells, whereas the stabilizing factor may be absent or functionally inactive in IV-affected keratinocytes.

  10. Post-transcriptional Stabilization of Ucp1 mRNA Protects Mice from Diet-Induced Obesity

    OpenAIRE

    Akinori Takahashi; Shungo Adachi; Masahiro Morita; Miho Tokumasu; Tohru Natsume; Toru Suzuki; Tadashi Yamamoto

    2015-01-01

    Uncoupling protein 1 (Ucp1) contributes to thermogenesis, and its expression is regulated at the transcriptional level. Here, we show that Ucp1 expression is also regulated post-transcriptionally. In inguinal white adipose tissue (iWAT) of mice fed a high-fat diet (HFD), Ucp1 level decreases concomitantly with increases in Cnot7 and its interacting partner Tob. HFD-fed mice lacking Cnot7 and Tob express elevated levels of Ucp1 mRNA in iWAT and are resistant to diet-induced obesity. Ucp1 mRNA ...

  11. Influence of Picual olive ripening on virgin olive oil alteration and stability during potato frying.

    Science.gov (United States)

    Olivero-David, Raul; Mena, Carmen; Pérez-Jimenez, M Angeles; Sastre, Blanca; Bastida, Sara; Márquez-Ruiz, Gloria; Sánchez-Muniz, Francisco J

    2014-12-01

    Ripening modifies oil attributes and composition. However, the influence of olive ripening on virgin olive oil (VOO) thermal oxidative stability on food-frying has not been studied yet. Oils from Picual olives of low (VOO1), medium (VOO2), and high (VOO3) ripeness were obtained, and their thermal oxidative stability during 40 potato-fryings was tested. Unused VOO1 showed higher antioxidant content and oxidative stability than VOO2 and VOO3. Polar compounds (PC), oligomers, and altered fatty acid methyl esters (polar-FAME) increased, whereas linoleic acid, polyphenols, and tocopherols decreased in the three VOOs through frying. The alteration was lower in VOO1, followed by VOO2 (0.105, 0.117, and 0.042 g/100 g oil less of PC, oligomers and polar-FAME per frying, respectively, in VOO1 than in VOO3). In conclusion, VOO obtained from low-ripeness Picual olives should be preferred when frying fresh-potatoes due to its higher thermal and oxidative stability, permitting a higher number of potato-frying uses.

  12. Sex Specific Estrogen Receptor beta (ERβ) mRNA Expression in the Rat Hypothalamus and Amygdala is Altered by Neonatal Bisphenol A (BPA) Exposure

    Science.gov (United States)

    Cao, Jinyan; Joyner, Linwood; Mickens, Jillian A.; Leyrer, Stephanie M; Patisaul, Heather B

    2014-01-01

    Perinatal life is a critical window for sexually dimorphic brain organization, and profoundly influenced by steroid hormones. Exposure to endocrine disrupting compounds (EDCs) may disrupt this process, resulting in compromised reproductive physiology and behavior. To test the hypothesis that neonatal BPA exposure can alter sex specific postnatal ERβ expression in brain regions fundamental to sociosexual behavior we mapped ERβ mRNA levels in the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), paraventricular nucleus (PVN), anterior portion of the medial amygdaloid nucleus (MeA), super optic nucleus (SON), suprachiasmic nucleus (SCN) and lateral habenula (LHb) across postnatal days (PNDs) 0 to 19. Next, rat pups of both sexes were subcutaneously injected over the first three days of life with 10 μg estradiol benzoate (EB), 50 μg/kg BPA (LBPA), or 50 mg/kg BPA (HBPA) and ERβ levels quantified in each region of interest (ROI) on PNDs 4 and 10. EB exposure decreased ERβ signal in most female ROIs, and in the male PVN. In the BNSTp, ERβ expression decreased in LBPA males and HBPA females on PND 10, thereby reversing the sex difference in expression. In the PVN, ERβ mRNA levels were elevated in LBPA females, also resulting in a reversal of sexually dimorphic expression. In the MeA, BPA decreased ERβ expression on PND 4. Collectively, these data demonstrate that region and sex specific ERβ expression is vulnerable to neonatal BPA exposure in regions of the developing rat brain critical to sociosexual behavior. PMID:24352099

  13. Carrying shopping bags does not alter static postural stability and gait parameters in healthy older females.

    Science.gov (United States)

    Bampouras, Theodoros M; Dewhurst, Susan

    2016-05-01

    Food shopping is an important aspect of maintaining independence and social interaction in older age. Carriage of shopping bags alters the body's weight distribution which, depending on load distribution, could potentially increase instability during standing and walking. The study examined the effect of carrying UK style shopping bags on static postural stability and gait in healthy older and young females. Nine older (71.0±6.0 years) and 10 young (26.7±5.2 years) females were assessed in five conditions carrying no bags, one 1.5kg bag in each hand, one 3kg bag in each hand, one 1.5kg bag in preferred hand, one 3kg bag in preferred hand. Antero-posterior and medio-lateral displacement, and 95% ellipse area from a 30s quiet standing were used for postural stability assessment. Stride length and its coefficient of variation, total double support time, step asymmetry and gait stability ratio were calculated from 1min treadmill walking at self-selected speed for gait assessment. Carrying shopping bags did not negatively affect postural stability or gait variables, in either group. Further, in older individuals, a decrease in sway velocity was found when holding bags during the postural stability assessment (psocial and as a physical activity. PMID:27131182

  14. CNOT3 contributes to early B cell development by controlling Igh rearrangement and p53 mRNA stability

    Science.gov (United States)

    Inoue, Takeshi; Morita, Masahiro; Hijikata, Atsushi; Fukuda-Yuzawa, Yoko; Adachi, Shungo; Isono, Kyoichi; Ikawa, Tomokatsu; Kawamoto, Hiroshi; Koseki, Haruhiko; Natsume, Tohru; Fukao, Taro; Ohara, Osamu; Yamamoto, Tadashi

    2015-01-01

    The CCR4–NOT deadenylase complex plays crucial roles in mRNA decay and translational repression induced by poly(A) tail shortening. Although the in vitro activities of each component of this complex have been well characterized, its in vivo role in immune cells remains unclear. Here we show that mice lacking the CNOT3 subunit of this complex, specifically in B cells, have a developmental block at the pro- to pre–B cell transition. CNOT3 regulated generation of germline transcripts in the VH region of the immunoglobulin heavy chain (Igh) locus, compaction of the locus, and subsequent Igh gene rearrangement and destabilized tumor suppressor p53 mRNA. The developmental defect in the absence of CNOT3 could be partially rescued by ablation of p53 or introduction of a pre-rearranged Igh transgene. Thus, our data suggest that the CCR4–NOT complex regulates B cell differentiation by controlling Igh rearrangement and destabilizing p53 mRNA. PMID:26238124

  15. Identification of Cytoplasmic Capping Targets Reveals a Role for Cap Homeostasis in Translation and mRNA Stability

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    Chandrama Mukherjee

    2012-09-01

    Full Text Available The notion that decapping leads irreversibly to messenger RNA (mRNA decay was contradicted by the identification of capped transcripts missing portions of their 5′ ends and a cytoplasmic complex that can restore the cap on uncapped mRNAs. In this study, we used accumulation of uncapped transcripts in cells inhibited for cytoplasmic capping to identify the targets of this pathway. Inhibition of cytoplasmic capping results in the destabilization of some transcripts and the redistribution of others from polysomes to nontranslating messenger ribonucleoproteins, where they accumulate in an uncapped state. Only a portion of the mRNA transcriptome is affected by cytoplasmic capping, and its targets encode proteins involved in nucleotide binding, RNA and protein localization, and the mitotic cell cycle. The 3′ untranslated regions of recapping targets are enriched for AU-rich elements and microRNA binding sites, both of which function in cap-dependent mRNA silencing. These findings identify a cyclical process of decapping and recapping that we term cap homeostasis.

  16. Probing the transcription mechanisms of reovirus cores with molecules that alter RNA duplex stability.

    Science.gov (United States)

    Demidenko, Alexander A; Nibert, Max L

    2009-06-01

    The mammalian reovirus (MRV) genome comprises 10 double-stranded RNA (dsRNA) segments, packaged along with transcriptase complexes inside each core particle. Effects of four small molecules on transcription by MRV cores were studied for this report, chosen for their known capacities to alter RNA duplex stability. Spermidine and spermine, which enhance duplex stability, inhibited transcription, whereas dimethyl sulfoxide and trimethylglycine, which attenuate duplex stability, stimulated transcription. Different mechanisms were identified for inhibition or activation by these molecules. With spermidine, one round of transcription occurred normally, but subsequent rounds were inhibited. Thus, inhibition occurred at the transition between the end of elongation in one round and initiation in the next round of transcription. Dimethyl sulfoxide or trimethylglycine, on the other hand, had no effect on transcription by a constitutively active fraction of cores in each preparation but activated transcription in another fraction that was otherwise silent for the production of elongated transcripts. Activation of this other fraction occurred at the transition between transcript initiation and elongation, i.e., at promoter escape. These results suggest that the relative stability of RNA duplexes is most important for certain steps in the particle-associated transcription cycles of dsRNA viruses and that small molecules are useful tools for probing these and probably other steps. PMID:19297468

  17. Analysis of miRNA and mRNA expression profiles highlights alterations in ionizing radiation response of human lymphocytes under modeled microgravity.

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    Cristina Girardi

    Full Text Available BACKGROUND: Ionizing radiation (IR can be extremely harmful for human cells since an improper DNA-damage response (DDR to IR can contribute to carcinogenesis initiation. Perturbations in DDR pathway can originate from alteration in the functionality of the microRNA-mediated gene regulation, being microRNAs (miRNAs small noncoding RNA that act as post-transcriptional regulators of gene expression. In this study we gained insight into the role of miRNAs in the regulation of DDR to IR under microgravity, a condition of weightlessness experienced by astronauts during space missions, which could have a synergistic action on cells, increasing the risk of radiation exposure. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed miRNA expression profile of human peripheral blood lymphocytes (PBL incubated for 4 and 24 h in normal gravity (1 g and in modeled microgravity (MMG during the repair time after irradiation with 0.2 and 2Gy of γ-rays. Our results show that MMG alters miRNA expression signature of irradiated PBL by decreasing the number of radio-responsive miRNAs. Moreover, let-7i*, miR-7, miR-7-1*, miR-27a, miR-144, miR-200a, miR-598, miR-650 are deregulated by the combined action of radiation and MMG. Integrated analyses of miRNA and mRNA expression profiles, carried out on PBL of the same donors, identified significant miRNA-mRNA anti-correlations of DDR pathway. Gene Ontology analysis reports that the biological category of "Response to DNA damage" is enriched when PBL are incubated in 1 g but not in MMG. Moreover, some anti-correlated genes of p53-pathway show a different expression level between 1 g and MMG. Functional validation assays using luciferase reporter constructs confirmed miRNA-mRNA interactions derived from target prediction analyses. CONCLUSIONS/SIGNIFICANCE: On the whole, by integrating the transcriptome and microRNome, we provide evidence that modeled microgravity can affects the DNA-damage response to IR in human PBL.

  18. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli

    OpenAIRE

    Thomas Esquerré; Marie Bouvier; Catherine Turlan; Carpousis, Agamemnon J.; Laurence Girbal; Muriel Cocaign-Bousquet

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype...

  19. Adenosine receptor antagonists alter the stability of human epileptic GABAA receptors

    Science.gov (United States)

    Roseti, Cristina; Martinello, Katiuscia; Fucile, Sergio; Piccari, Vanessa; Mascia, Addolorata; Di Gennaro, Giancarlo; Quarato, Pier Paolo; Manfredi, Mario; Esposito, Vincenzo; Cantore, Gianpaolo; Arcella, Antonella; Simonato, Michele; Fredholm, Bertil B.; Limatola, Cristina; Miledi, Ricardo; Eusebi, Fabrizio

    2008-01-01

    We examined how the endogenous anticonvulsant adenosine might influence γ-aminobutyric acid type A (GABAA) receptor stability and which adenosine receptors (ARs) were involved. Upon repetitive activation (GABA 500 μM), GABAA receptors, microtransplanted into Xenopus oocytes from neurosurgically resected epileptic human nervous tissues, exhibited an obvious GABAA-current (IGABA) run-down, which was consistently and significantly reduced by treatment with the nonselective adenosine receptor antagonist CGS15943 (100 nM) or with adenosine deaminase (ADA) (1 units/ml), that inactivates adenosine. It was also found that selective antagonists of A2B (MRS1706, 10 nM) or A3 (MRS1334, 30 nM) receptors reduced IGABA run-down, whereas treatment with the specific A1 receptor antagonist DPCPX (10 nM) was ineffective. The selective A2A receptor antagonist SCH58261 (10 nM) reduced or potentiated IGABA run-down in ≈40% and ≈20% of tested oocytes, respectively. The ADA-resistant, AR agonist 2-chloroadenosine (2-CA) (10 μM) potentiated IGABA run-down but only in ≈20% of tested oocytes. CGS15943 administration again decreased IGABA run-down in patch-clamped neurons from either human or rat neocortex slices. IGABA run-down in pyramidal neurons was equivalent in A1 receptor-deficient and wt neurons but much larger in neurons from A2A receptor-deficient mice, indicating that, in mouse cortex, GABAA-receptor stability is tonically influenced by A2A but not by A1 receptors. IGABA run-down from wt mice was not affected by 2-CA, suggesting maximal ARs activity by endogenous adenosine. Our findings strongly suggest that cortical A2–A3 receptors alter the stability of GABAA receptors, which could offer therapeutic opportunities. PMID:18809912

  20. PCBP2 enhances the antiviral activity of IFN-α against HCV by stabilizing the mRNA of STAT1 and STAT2.

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    Zhongshuai Xin

    Full Text Available Interferon-α (IFN-α is a natural choice for the treatment of hepatitis C, but half of the chronically infected individuals do not achieve sustained clearance of hepatitis C virus (HCV during treatment with IFN-α alone. The virus can impair IFN-α signaling and cellular factors that have an effect on the viral life cycles. We found that the protein PCBP2 is down-regulated in HCV-replicon containing cells (R1b. However, the effects and mechanisms of PCBP2 on HCV are unclear. To determine the effect of PCBP2 on HCV, overexpression and knockdown of PCBP2 were performed in R1b cells. Interestingly, we found that PCBP2 can facilitate the antiviral activity of IFN-α against HCV, although the RNA level of HCV was unaffected by either the overexpression or absence of PCBP2 in R1b cells. RIP-qRT-PCR and RNA half-life further revealed that PCBP2 stabilizes the mRNA of STAT1 and STAT2 through binding the 3'Untranslated Region (UTR of these two molecules, which are pivotal for the IFN-α anti-HCV effect. RNA pull-down assay confirmed that there were binding sites located in the C-rich tracts in the 3'UTR of their mRNAs. Stabilization of mRNA by PCBP2 leads to the increased protein expression of STAT1 and STAT2 and a consistent increase of phosphorylated STAT1 and STAT2. These effects, in turn, enhance the antiviral effect of IFN-α. These findings indicate that PCBP2 may play an important role in the IFN-α response against HCV and may benefit the HCV clinical therapy.

  1. mRNA and Protein Levels for GABA[subscript A][alpha]4, [alpha]5, [beta]1 and GABA[subscript B]R1 Receptors are Altered in Brains from Subjects with Autism

    Science.gov (United States)

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rooney, Robert J.; Patel, Diven H.; Thuras, Paul D.

    2010-01-01

    We have shown altered expression of gamma-aminobutyric acid A (GABA[subscript A]) and gamma-aminobutyric acid B (GABA[subscript B]) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3…

  2. Regulation of the stability of poly(I)xpoly(C)-induced human fibroblast interferon mRNA: selective inactivation of interferon mRNA and lack of involvement of 2',5'-oligo(A) synthetase activation during the shutoff of interferon production.

    Science.gov (United States)

    Sehgal, P B; Gupta, S L

    1980-06-01

    The inactivation of interferon mRNA during the shutoff phase of interferon production in poly(I)xpoly(C)-induced human fibroblast cultures is selective. We have determined that the shutoff of interferon production, which takes place from 3 to 8 hr after the beginning of induction, is not associated with an appreciable declined in the rate of bulk cellular protein synthesis or of cellular protein secretion. While the amount of translatable interferon mRNA declined markedly during the shutoff phase, the level of translatable bulk cellular mRNA and the stability of [3H]uridine-labeled mRNA were unaffected. Superinduction with actinomycin D selectively stabilized interferon mRNA with no apparent effect on the stability of bulk cellular mRNA. Furthermore, an activation of the 2',5'-oligo(A) synthetase/endonuclease system does not appear to be involved in the shutoff phenomenon. Uninduced FS-4 cells contained a low basal level of 2'5'-oligo(A) synthetase activity, which was unchanged in poly(I)xpoly(C)-induced cells during the shutoff phase. Treatment of FS-4 cells with interferon for 16-18 hr prior to induction increased the enzyme activity by approximately 200-fold. However, this did not inhibit interferon production after induction with poly(I)xpoly(C) alone or after superinduction with cycloheximide or actinomycin D or both. Furthermore, the rates of decay of interferon production were comparable in cells with either a basal or an increased level of 2',5'-oligo(A) synthetase. Thus a 200-fold increase in 2',5'-oligo(A) synthetase level did not affect either the stability of interferon mRNA or the efficacy of interferon superinduction by metabolic inhibitors.

  3. Jasplakinolide, an actin stabilizing agent, alters anaphase chromosome movements in crane-fly spermatocytes.

    Science.gov (United States)

    Xie, Lele; Forer, Arthur

    2008-11-01

    We added jasplakinolide to anaphase crane-fly spermatocytes and determined its effects on chromosome movement. Previous work showed that the actin depolymerizing agents cytochalasin D or latrunculin B blocked or slowed chromosome movements. We studied the effects of jasplakinolide, a compound that stabilizes actin filaments. Jasplakinolide had the same effect on movements of each half- bivalent in a separating pair of half-bivalents, but different half-bivalent pairs in the same cell often responded differently, even when the concentrations of jasplakinolide varied by a factor of two. Jasplakinolide had no effect on about 20% of the pairs, but otherwise caused movements to slow, or to stop, or, rarely, to accelerate. When cells were kept in jasplakinolide, stopped pairs eventually resumed movement; slowed pairs did not change their speeds. Confocal microscopy indicated that neither the distributions of spindle actin filaments nor the distributions of spindle microtubules were altered by the jasplakinolide. It is possible that jasplakinolide binds to spindle actin and blocks critical binding sites, but we suggest that jasplakinolide affects anaphase chromosome movement by preventing actin-filament depolymerization that is necessary for anaphase to proceed. Overall, our data indicate that actin is involved in one of the redundant mechanisms cells use to move chromosomes. PMID:18688844

  4. T cell LFA-1 engagement induces HuR-dependent cytokine mRNA stabilization through a Vav-1, Rac1/2, p38MAPK and MKK3 signaling cascade.

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    Vinod S Ramgolam

    Full Text Available BACKGROUND: Engagement of the β2 integrin, lymphocyte function-associated antigen-1 (LFA-1, results in stabilization of T cell mRNA transcripts containing AU-rich elements (AREs by inducing rapid nuclear-to-cytosolic translocation of the RNA-stabilizing protein, HuR. However, little is known regarding integrin-induced signaling cascades that affect mRNA catabolism. This study examines the role of the GTPases, Rac 1 and Rac 2, and their downstream effectors, in the LFA-1-induced effects on mRNA. METHODOLOGY/PRINCIPAL FINDINGS: Engagement of LFA-1 to its ligand, ICAM-1, in human peripheral T cells resulted in rapid activation of Rac1 and Rac2. siRNA-mediated knockdown of either Rac1 or Rac2 prevented LFA-1-stimulated stabilization of the labile transcripts encoding IFN-γ and TNF-α, and integrin mediated IFN-γ mRNA stabilization was absent in T cells obtained from Rac2 gene-deleted mice. LFA-1 engagement-induced translocation of HuR and stabilization of TNF- α mRNA was lost in Jurkat cells deficient in the Rac guanine nucleotide exchange factor Vav-1 (J.Vav1. The transfection of J.Vav1 cells with constitutively active Rac1 or Rac2 stabilized a labile β-globin reporter mRNA, in a HuR-dependent manner. Furthermore, LFA-1-mediated mRNA stabilization and HuR translocation in mouse splenic T cells was dependent on the phosphorylation of the mitogen-activated protein kinase kinase, MKK3, and its target MAP kinase p38MAPK, and lost in T cells obtained from MKK3 gene-deleted mice. CONCLUSIONS/SIGNIFICANCE: Collectively, these results demonstrate that LFA-1-induced stabilization of ARE-containing mRNAs in T cells is dependent on HuR, and occurs through the Vav-1, Rac1/2, MKK3 and p38MAPK signaling cascade. This pathway constitutes a molecular switch that enhances immune and pro-inflammatory gene expression in T cells undergoing adhesion at sites of activation and effector function.

  5. Postural stability is altered by the stimulation of pain but not warm receptors in humans

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    Corbeil Philippe

    2003-10-01

    Full Text Available Abstract Background It is now recognized that large diameter myelinated afferents provide the primary source of lower limb proprioceptive information for maintaining an upright standing position. Small diameter afferents transmitting noxious stimuli, however, can also influence motor behaviors. Despite the possible influence of pain on motor behaviors, the effects of pain on the postural control system have not been well documented. Methods Two cutaneous heat stimulations (experiment 1: non-noxious 40 degrees C; experiment 2: noxious 45 degrees C were applied bilaterally on the calves of the subject with two thermal grills to stimulate A delta and C warm receptors and nociceptors in order to examine their effects on postural stability. The non-noxious stimulation induced a gentle sensation of warmth and the noxious stimulation induced a perception of heat pain (visual analogue scores of 0 and 46 mm, respectively. For both experiments, ten healthy young adults were tested with and without heat stimulations of the lower limbs while standing upright on a force platform with eyes open, eyes closed and eyes closed with tendon co-vibration of tibialis anterior and triceps surae muscles. The center of pressure displacements were analyzed to examine how both stimulations affected the regulation of quiet standing and if the effects were exacerbated when vision was removed or ankle proprioception perturbed. Results The stimulation of the warm receptors (40 degrees C did not induce any postural deterioration. With pain (45 degrees C, subjects showed a significant increase in standard deviation, range and mean velocity of postural oscillations as well as standard deviation of the center of pressure velocity. The effects of heat pain were exacerbated when subjects had both their eyes closed and ankle tendons vibrated (increased standard deviation of the center of pressure velocity and mean velocity of the center of pressure. Conclusions A non

  6. Genome-wide expression profiling of in vivo-derived bloodstream parasite stages and dynamic analysis of mRNA alterations during synchronous differentiation in Trypanosoma brucei

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    Ghazal Peter

    2009-09-01

    Full Text Available Abstract Background Trypanosomes undergo extensive developmental changes during their complex life cycle. Crucial among these is the transition between slender and stumpy bloodstream forms and, thereafter, the differentiation from stumpy to tsetse-midgut procyclic forms. These developmental events are highly regulated, temporally reproducible and accompanied by expression changes mediated almost exclusively at the post-transcriptional level. Results In this study we have examined, by whole-genome microarray analysis, the mRNA abundance of genes in slender and stumpy forms of T.brucei AnTat1.1 cells, and also during their synchronous differentiation to procyclic forms. In total, five biological replicates representing the differentiation of matched parasite populations derived from five individual mouse infections were assayed, with RNAs being derived at key biological time points during the time course of their synchronous differentiation to procyclic forms. Importantly, the biological context of these mRNA profiles was established by assaying the coincident cellular events in each population (surface antigen exchange, morphological restructuring, cell cycle re-entry, thereby linking the observed gene expression changes to the well-established framework of trypanosome differentiation. Conclusion Using stringent statistical analysis and validation of the derived profiles against experimentally-predicted gene expression and phenotypic changes, we have established the profile of regulated gene expression during these important life-cycle transitions. The highly synchronous nature of differentiation between stumpy and procyclic forms also means that these studies of mRNA profiles are directly relevant to the changes in mRNA abundance within individual cells during this well-characterised developmental transition.

  7. Tumor-suppressor NFκB2 p100 interacts with ERK2 and stabilizes PTEN mRNA via inhibition of miR-494.

    Science.gov (United States)

    Wang, Y; Xu, J; Gao, G; Li, J; Huang, H; Jin, H; Zhu, J; Che, X; Huang, C

    2016-08-01

    Emerging evidence from The Cancer Genome Atlas has revealed that nuclear factor κB2 (nfκb2) gene encoding p100 is genetically deleted or mutated in human cancers, implicating NFκB2 as a potential tumor suppressor. However, the molecular mechanism underlying the antitumorigenic action of p100 remains poorly understood. Here we report that p100 inhibits cancer cell anchorage-independent growth, a hallmark of cellular malignancy, by stabilizing the tumor-suppressor phosphatase and tensin homolog (PTEN) mRNA via a mechanism that is independent of p100's inhibitory role in NFκB activation. We further demonstrate that the regulatory effect of p100 on PTEN expression is mediated by its downregulation of miR-494 as a result of the inactivation of extracellular signal-regulated kinase 2 (ERK2), in turn leading to inhibition of c-Jun/activator protein-1-dependent transcriptional activity. Furthermore, we identify that p100 specifically interacts with non-phosphorylated ERK2 and prevents ERK2 phosphorylation and nuclear translocation. Moreover, the death domain at C-terminal of p100 is identified as being crucial and sufficient for its interaction with ERK2. Taken together, our findings provide novel mechanistic insights into the understanding of the tumor-suppressive role for NFκB2 p100. PMID:26686085

  8. Runx3 Expression Inhibits Proliferation and Distinctly Alters mRNA Expression of Bax in AGS and A549 Cancer Cells

    Science.gov (United States)

    Torshabi, Maryam; Faramarzi, Mohammad Ali; Tabatabaei Yazdi, Mojtaba; Ostad, Seyyed Naser; Gharemani, Mohammad Hosein

    2011-01-01

    Runx3, a member of Runt-related transcription factor (Runx) proteins with tumor suppressor effect, is a tissue–restricted and cancer related transcription factor that regulate cell proliferation and growth, as well as differentiation. In the present study, exogenous Run3 was transiently expressed in AGS (human gastric adenocarcinoma), with undetectable Runx3 protein and in A549 (human lung carcinoma) with low levels of endogenous Runx3 protein. The GFP tagged Runx3 was transfected into AGS and A549 cells using fugene6 and PolyFect and Runx3 expression was confirmed by fluorescent microscopy and RT-PCR. The effect of Runx3 transfection on cell proliferation was determined by MTT assay and the results were confirmed by the trypan blue dye exclusion method. The effect of Runx3 expression on mRNA expression of BCL2-associated X protein (Bax) was evaluated using RT-PCR. In AGS and A549 cells, Runx3 expression inhibited cell proliferation (p < 0.01). The growth inhibition was less in A549 cells. We show that Runx3 expression increases Bax mRNA expression in AGS cells when compared with control (p < 0.05), but no significant differences in mRNA expression was observed in both examined cells. Runx3 expression has antiproliferative effect in AGS cell perhaps via increase in expression of Bax. The effect of Runx3 on A549 cells’ viability which has endogenous level of Runx3 is not related to Bax. These findings implicate a complex regulation by Runx3 in inhibition of cell proliferation utilizing Bax. PMID:24250365

  9. Environmental changes drive the temporal stability of semi-arid natural grasslands through altering species asynchrony

    NARCIS (Netherlands)

    Xu, Z.; Ren, H.; Li, M.H.; Ruijven, van J.; Han, X.; Wan, S.; Li, H.; Yu, Q.; Jiang, Y.; Jiang, L.

    2015-01-01

    1.Stability is an important property of ecological systems, many of which are experiencing increasing levels of anthropogenic environmental changes. However, how these environmental changes influence ecosystem stability remains poorly understood. 2.We conducted an 8-year field experiment in a semi-a

  10. Leukotriene B(4) BLT receptor signaling regulates the level and stability of cyclooxygenase-2 (COX-2) mRNA through restricted activation of Ras/Raf/ERK/p42 AUF1 pathway.

    Science.gov (United States)

    Zhai, Beibei; Yang, Huiqing; Mancini, Arturo; He, QingWen; Antoniou, John; Di Battista, John A

    2010-07-30

    Recent studies suggest that active resolution of the inflammatory response in animal models of arthritis may involve leukotriene B(4) (LTB(4))-dependent stimulation of "intermediate" prostaglandin production, which in turn favors the synthesis of "downstream" anti-inflammatory and pro-resolving lipoxins, resolvins, and protectins. We explored a putative mechanism involving LTB(4)-dependent control of cyclooxygenase-2 (COX-2) expression, the rate-limiting step in inflammatory prostaglandin biosynthesis. Indeed, LTB(4) potently up-regulated/stabilized interleukin-1beta-induced COX-2 mRNA and protein expression under conditions of COX-2 inhibitor-dependent blockade of PGE(2) release in human synovial fibroblasts (EC(50) = 16.5 + or - 1.7 nm for mRNA; 19 + or - 2.4 nm for protein, n = 4). The latter response was pertussis toxin-sensitive, and semi-quantitative reverse transcription-PCR confirmed the quantitative predominance of the BLT2 receptor. Transfection experiments, using human COX-2 promoter plasmids and chimeric luciferase-COX-2 mRNA 3'-untranslated region (3'-UTR) reporter constructs, revealed that LTB(4) exerted its stabilizing effect at the post-transcriptional level through a 116-bp adenylate/uridylate-rich sequence in the proximal region of the COX-2 3'-UTR. Using luciferase-COX-2 mRNA 3'-UTR reporter constructs and Ras/c-Raf expression and mutant constructs, we showed that the Ras/c-Raf/MEK1/2/ERK1/2 signaling pathway mediated LTB(4)-dependent COX-2 mRNA stabilization. Knockdown experiments with specific short hairpin RNAs confirmed that LTB(4) stabilization of COX-2 mRNA was apparently mediated through the RNA-binding protein, p42 AUF1. The nuclear export of p42 AUF1 was driven by c-Raf/MEK1/2/ERK1/2 signaling and sensitive to leptomycin B treatment, suggesting a CRM1-dependent mechanism. We conclude that LTB(4) may support the resolution phase of the inflammatory response by stabilizing COX-2, ensuring a reservoir of ambient pro-resolution lipid

  11. Site-specific binding of a PPR protein defines and stabilizes 5′ and 3′ mRNA termini in chloroplasts

    OpenAIRE

    Pfalz, Jeannette; Bayraktar, Omer Ali; Prikryl, Jana; Barkan, Alice

    2009-01-01

    Chloroplast mRNA populations are characterized by overlapping transcripts derived by processing from polycistronic precursors. The mechanisms and functional significance of these processing events are poorly understood. We describe a pentatricopeptide repeat (PPR) protein, PPR10, whose binding defines mRNA segments derived from two transcription units in maize chloroplasts. PPR10 interacts in vivo and in vitro with two intergenic RNA regions of similar sequence. The processed 5′ and 3′ RNA te...

  12. Phosphate Stability in Diagenetic Fluids Constrains the Acidic Alteration Model for Lower Mt. Sharp Sedimentary Rocks in Gale Crater, Mars

    Science.gov (United States)

    Berger, J. A.; Schmidt, M. E.; Izawa, M. R. M.; Gellert, R.; Ming, D. W.; Rampe, E. B.; VanBommel, S. J.; McAdam, A. C.

    2016-01-01

    The Mars rover Curiosity has encountered silica-enriched bedrock (as strata and as veins and associated halos of alteration) in the largely basaltic Murray Fm. of Mt. Sharp in Gale Crater. Alpha Particle X-ray Spectrometer (APXS) investigations of the Murray Fm. revealed decreasing Mg, Ca, Mn, Fe, and Al, and higher S, as silica increased (Fig. 1). A positive correlation between SiO2 and TiO2 (up to 74.4 and 1.7 wt %, respectively) suggests that these two insoluble elements were retained while acidic fluids leached more soluble elements. Other evidence also supports a silica-retaining, acidic alteration model for the Murray Fm., including low trace element abundances consistent with leaching, and the presence of opaline silica and jarosite determined by CheMin. Phosphate stability is a key component of this model because PO4 3- is typically soluble in acidic water and is likely a mobile ion in diagenetic fluids (pH less than 5). However, the Murray rocks are not leached of P; they have variable P2O5 (Fig. 1) ranging from average Mars (0.9 wt%) up to the highest values in Gale Crater (2.5 wt%). Here we evaluate APXS measurements of Murray Fm. bedrock and veins with respect to phosphate stability in acidic fluids as a test of the acidic alteration model for the Lower Mt. Sharp rocks.

  13. Altered mRNA Splicing, Chondrocyte Gene Expression and Abnormal Skeletal Development due to SF3B4 Mutations in Rodriguez Acrofacial Dysostosis

    Science.gov (United States)

    Nevarez, Lisette; Pogue, Robert; Krakow, Deborah; Cohn, Daniel H.

    2016-01-01

    The acrofacial dysostoses (AFD) are a genetically heterogeneous group of inherited disorders with craniofacial and limb abnormalities. Rodriguez syndrome is a severe, usually perinatal lethal AFD, characterized by severe retrognathia, oligodactyly and lower limb abnormalities. Rodriguez syndrome has been proposed to be a severe form of Nager syndrome, a non-lethal AFD that results from mutations in SF3B4, a component of the U2 small nuclear ribonucleoprotein particle (U2 snRNP). Furthermore, a case with a phenotype intermediate between Rodriguez and Nager syndromes has been shown to have an SF3B4 mutation. We identified heterozygosity for SF3B4 mutations in Rodriguez syndrome, confirming that the phenotype is a dominant disorder that is allelic with Nager syndrome. The mutations led to reduced SF3B4 synthesis and defects in mRNA splicing, primarily exon skipping. The mutations also led to reduced expression in growth plate chondrocytes of target genes, including the DLX5, DLX6, SOX9, and SOX6 transcription factor genes, which are known to be important for skeletal development. These data provide mechanistic insight toward understanding how SF3B4 mutations lead to the skeletal abnormalities observed in the acrofacial dysostoses. PMID:27622494

  14. Adult hippocampal neurogenesis and mRNA expression are altered by perinatal arsenic exposure in mice and restored by brief exposure to enrichment.

    Directory of Open Access Journals (Sweden)

    Christina R Tyler

    Full Text Available Arsenic is a common and pervasive environmental contaminant found in drinking water in varying concentrations depending on region. Exposure to arsenic induces behavioral and cognitive deficits in both human populations and in rodent models. The Environmental Protection Agency (EPA standard for the allotment of arsenic in drinking water is in the parts-per-billion range, yet our lab has shown that 50 ppb arsenic exposure during development can have far-reaching consequences into adulthood, including deficits in learning and memory, which have been linked to altered adult neurogenesis. Given that the morphological impact of developmental arsenic exposure on the hippocampus is unknown, we sought to evaluate proliferation and differentiation of adult neural progenitor cells in the dentate gyrus after 50 ppb arsenic exposure throughout the perinatal period of development in mice (equivalent to all three trimesters in humans using a BrdU pulse-chase assay. Proliferation of the neural progenitor population was decreased by 13% in arsenic-exposed mice, but was not significant. However, the number of differentiated cells was significantly decreased by 41% in arsenic-exposed mice compared to controls. Brief, daily exposure to environmental enrichment significantly increased proliferation and differentiation in both control and arsenic-exposed animals. Expression levels of 31% of neurogenesis-related genes including those involved in Alzheimer's disease, apoptosis, axonogenesis, growth, Notch signaling, and transcription factors were altered after arsenic exposure and restored after enrichment. Using a concentration previously considered safe by the EPA, perinatal arsenic exposure altered hippocampal morphology and gene expression, but did not inhibit the cellular neurogenic response to enrichment. It is possible that behavioral deficits observed during adulthood in animals exposed to arsenic during development derive from the lack of differentiated neural

  15. Chronic Exposure to Arsenic in Drinking Water Causes Alterations in Locomotor Activity and Decreases Striatal mRNA for the D2 Dopamine Receptor in CD1 Male Mice

    Directory of Open Access Journals (Sweden)

    Claudia Leticia Moreno Ávila

    2016-01-01

    Full Text Available Arsenic exposure has been associated with sensory, motor, memory, and learning alterations in humans and alterations in locomotor activity, behavioral tasks, and neurotransmitters systems in rodents. In this study, CD1 mice were exposed to 0.5 or 5.0 mg As/L of drinking water for 6 months. Locomotor activity, aggression, interspecific behavior and physical appearance, monoamines levels, and expression of the messenger for dopamine receptors D1 and D2 were assessed. Arsenic exposure produced hypoactivity at six months and other behaviors such as rearing and on-wall rearing and barbering showed both increases and decreases. No alterations on aggressive behavior or monoamines levels in striatum or frontal cortex were observed. A significant decrease in the expression of mRNA for D2 receptors was found in striatum of mice exposed to 5.0 mg As/L. This study provides evidence for the use of dopamine receptor D2 as potential target of arsenic toxicity in the dopaminergic system.

  16. Chronic Exposure to Arsenic in Drinking Water Causes Alterations in Locomotor Activity and Decreases Striatal mRNA for the D2 Dopamine Receptor in CD1 Male Mice.

    Science.gov (United States)

    Moreno Ávila, Claudia Leticia; Limón-Pacheco, Jorge H; Giordano, Magda; Rodríguez, Verónica M

    2016-01-01

    Arsenic exposure has been associated with sensory, motor, memory, and learning alterations in humans and alterations in locomotor activity, behavioral tasks, and neurotransmitters systems in rodents. In this study, CD1 mice were exposed to 0.5 or 5.0 mg As/L of drinking water for 6 months. Locomotor activity, aggression, interspecific behavior and physical appearance, monoamines levels, and expression of the messenger for dopamine receptors D1 and D2 were assessed. Arsenic exposure produced hypoactivity at six months and other behaviors such as rearing and on-wall rearing and barbering showed both increases and decreases. No alterations on aggressive behavior or monoamines levels in striatum or frontal cortex were observed. A significant decrease in the expression of mRNA for D2 receptors was found in striatum of mice exposed to 5.0 mg As/L. This study provides evidence for the use of dopamine receptor D2 as potential target of arsenic toxicity in the dopaminergic system. PMID:27375740

  17. Chronic Exposure to Arsenic in Drinking Water Causes Alterations in Locomotor Activity and Decreases Striatal mRNA for the D2 Dopamine Receptor in CD1 Male Mice

    Science.gov (United States)

    Moreno Ávila, Claudia Leticia

    2016-01-01

    Arsenic exposure has been associated with sensory, motor, memory, and learning alterations in humans and alterations in locomotor activity, behavioral tasks, and neurotransmitters systems in rodents. In this study, CD1 mice were exposed to 0.5 or 5.0 mg As/L of drinking water for 6 months. Locomotor activity, aggression, interspecific behavior and physical appearance, monoamines levels, and expression of the messenger for dopamine receptors D1 and D2 were assessed. Arsenic exposure produced hypoactivity at six months and other behaviors such as rearing and on-wall rearing and barbering showed both increases and decreases. No alterations on aggressive behavior or monoamines levels in striatum or frontal cortex were observed. A significant decrease in the expression of mRNA for D2 receptors was found in striatum of mice exposed to 5.0 mg As/L. This study provides evidence for the use of dopamine receptor D2 as potential target of arsenic toxicity in the dopaminergic system. PMID:27375740

  18. IGF-1 Has Plaque-Stabilizing Effects in Atherosclerosis by Altering Vascular Smooth Muscle Cell Phenotype

    OpenAIRE

    von der Thüsen, Jan H; Borensztajn, Keren S.; Moimas, Silvia; van Heiningen, Sandra; Teeling, Peter; Van Berkel, Theo J. C.; Biessen, Erik A. L.

    2011-01-01

    Insulin-like growth factor-1 (IGF-1) signaling is important for the maintenance of plaque stability in atherosclerosis due to its effects on vascular smooth muscle cell (vSMC) phenotype. To investigate this hypothesis, we studied the effects of the highly inflammatory milieu of the atherosclerotic plaque on IGF-1 signaling and stability-related phenotypic parameters of murine vSMCs in vitro, and the effects of IGF-1 supplementation on plaque phenotype in an atherosclerotic mouse model. M1-pol...

  19. A member of the cathelicidin family of antimicrobial peptides is produced in the upper airway of the chinchilla and its mRNA expression is altered by common viral and bacterial co-pathogens of otitis media.

    Science.gov (United States)

    McGillivary, Glen; Ray, William C; Bevins, Charles L; Munson, Robert S; Bakaletz, Lauren O

    2007-03-01

    Cationic antimicrobial peptides (AMPs), a component of the innate immune system, play a major role in defense of mucosal surfaces against a wide spectrum of microorganisms such as viral and bacterial co-pathogens of the polymicrobial disease otitis media (OM). To further understand the role of AMPs in OM, we cloned a cDNA encoding a cathelicidin homolog (cCRAMP) from upper respiratory tract (URT) mucosae of the chinchilla, the predominant host used to model experimental OM. Recombinant cCRAMP exhibited alpha-helical secondary structure and killed the three main bacterial pathogens of OM. In situ hybridization showed cCRAMP mRNA production in epithelium of the chinchilla Eustachian tube and RT-PCR was used to amplify cCRAMP mRNA from several other tissues of the chinchilla URT. Quantitative RT-PCR analysis of chinchilla middle ear epithelial cells (CMEEs) incubated with either viral (influenza A virus, adenovirus, or RSV) or bacterial (nontypeable H. influenzae, M. catarrhalis, or S. pneumoniae) pathogens associated with OM demonstrated distinct microbe-specific patterns of altered expression. Collectively, these data showed that viruses and bacteria modulate AMP messages in the URT, which likely contributes to the disease course of OM.

  20. Restricted Arm Swing Affects Gait Stability and Increased Walking Speed Alters Trunk Movements in Children with Cerebral Palsy

    Science.gov (United States)

    Delabastita, Tijs; Desloovere, Kaat; Meyns, Pieter

    2016-01-01

    Observational research suggests that in children with cerebral palsy, the altered arm swing is linked to instability during walking. Therefore, the current study investigates whether children with cerebral palsy use their arms more than typically developing children, to enhance gait stability. Evidence also suggests an influence of walking speed on gait stability. Moreover, previous research highlighted a link between walking speed and arm swing. Hence, the experiment aimed to explore differences between typically developing children and children with cerebral palsy taking into account the combined influence of restricting arm swing and increasing walking speed on gait stability. Spatiotemporal gait characteristics, trunk movement parameters and margins of stability were obtained using three dimensional gait analysis to assess gait stability of 26 children with cerebral palsy and 24 typically developing children. Four walking conditions were evaluated: (i) free arm swing and preferred walking speed; (ii) restricted arm swing and preferred walking speed; (iii) free arm swing and high walking speed; and (iv) restricted arm swing and high walking speed. Double support time and trunk acceleration variability increased more when arm swing was restricted in children with bilateral cerebral palsy compared to typically developing children and children with unilateral cerebral palsy. Trunk sway velocity increased more when walking speed was increased in children with unilateral cerebral palsy compared to children with bilateral cerebral palsy and typically developing children and in children with bilateral cerebral palsy compared to typically developing children. Trunk sway velocity increased more when both arm swing was restricted and walking speed was increased in children with bilateral cerebral palsy compared to typically developing children. It is proposed that facilitating arm swing during gait rehabilitation can improve gait stability and decrease trunk movements in

  1. The katG mRNA of Mycobacterium tuberculosis and Mycobacterium smegmatis is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation

    Directory of Open Access Journals (Sweden)

    Magnoni Francesca

    2008-04-01

    Full Text Available Abstract Background In Mycobacterium tuberculosis and in Mycobacterium smegmatis the furA-katG loci, encoding the FurA regulatory protein and the KatG catalase-peroxidase, are highly conserved. In M. tuberculosis furA-katG constitute a single operon, whereas in M. smegmatis a single mRNA covering both genes could not be found. In both species, specific 5' ends have been identified: the first one, located upstream of the furA gene, corresponds to transcription initiation from the furA promoter; the second one is the katG mRNA 5' end, located in the terminal part of furA. Results In this work we demonstrate by in vitro transcription and by RNA polymerase Chromatin immunoprecipitation that no promoter is present in the M. smegmatis region covering the latter 5' end, suggesting that it is produced by specific processing of longer transcripts. Several DNA fragments of M. tuberculosis and M. smegmatis were inserted in a plasmid between the sigA promoter and the lacZ reporter gene, and expression of the reporter gene was measured. A polypurine sequence, located four bp upstream of the katG translation start codon, increased beta-galactosidase activity and stabilized the lacZ transcript. Mutagenesis of this sequence led to destabilization of the mRNA. Analysis of constructs, in which the polypurine sequence of M. smegmatis was followed by an increasing number of katG codons, demonstrated that mRNA stability requires translation of at least 20 amino acids. In order to define the requirements for the 5' processing of the katG transcript, we created several mutations in this region and analyzed the 5' ends of the transcripts: the distance from the polypurine sequence does not seem to influence the processing, neither the sequence around the cutting point. Only mutations which create a double stranded region around the processing site prevented RNA processing. Conclusion This is the first reported case in mycobacteria, in which both a polypurine sequence

  2. Host factor I, Hfq, binds to Escherichia coli ompA mRNA in a growth rate-dependent fashion and regulates its stability

    DEFF Research Database (Denmark)

    Vytvytska, O; Jakobsen, J S; Balcunaite, G;

    1998-01-01

    ompA was purified and identified as Hfq, a host factor initially recognized for its function in phage Qbeta replication. The ompA RNA-binding activity parallels the amount of Hfq, which is elevated in bacteria cultured at slow growth rate, a condition leading to facilitated degradation of the ompA m...... suggest a regulatory role for Hfq that specifically facilitates the ompA mRNA degradation in a growth rate-dependent manner....

  3. Chlorine ions but not sodium ions alter genome stability of Arabidopsis thaliana.

    Science.gov (United States)

    Boyko, Alex; Golubov, Andrey; Bilichak, Andriy; Kovalchuk, Igor

    2010-06-01

    Various environmental stresses influence plant genome stability. Most of these stresses, such as ionizing radiation, heavy metals and organic chemicals, represent potent DNA-damaging agents. Here, we show that exposure to NaCl, the stress that is not thought to cause direct DNA damage, results in an increase in the level of strand breaks and homologous recombination rates (RRs) in Arabidopsis thaliana plants. The effect of salt stress on the RR was found to be primarily associated with Cl(-) ions, since exposure of plants to Na(2)SO(4) did not increase the RR, whereas exposure to MgCl(2) resulted in an increase. Changes in the number of strand breaks and in the RR were also paralleled by transcriptional activation of AtRad51 and down-regulation of AtKu70. The progeny of exposed plants exhibited higher RRs, higher expression of AtRad51, lower expression of AtKu70, higher tolerance to salt and methyl methane sulfate (MMS) stresses, as well as a higher increase in RR upon further exposure to stress. Our experiments showed that NaCl is a genotoxic stress that leads to somatic and transgenerational changes in recombination rates, and these changes are primarily triggered by exposure to Cl(-) ions. PMID:20385609

  4. Disodium cromoglycate, a mast-cell stabilizer, alters postradiation regional cerebral blood flow in primates

    Energy Technology Data Exchange (ETDEWEB)

    Cockerham, L.G.; Doyle, T.F.; Pautler, E.L.; Hampton, J.D.

    1986-01-01

    Early transient incapacitation (ETI) is the complete cessation of performance during the first 30 min after radiation exposure, and performance decrement (PD) is a reduction in performance at the same time. Supralethal doses of radiation have been shown to produce a marked decrease in regional cerebral blood flow in primates concurrent with systemic hypotension and a dramatic release of mast-cell histamine. In an attempt to elucidate mechanisms underlying the radiation-induced ETI/PD phenomena and the postradiation decrease in cerebral blood flow, primates were given the mast-cell stabilizers disodium cromoglycate (DSCG) or BRL 22321 before exposure to 100 Gy whole-body gamma radiation. Hypothalamic and cortical blood flows were measured by hydrogen clearance, before and after radiation exposure. Systemic blood pressures were determined simultaneously. The data indicated that DSCG was successful in diminishing postradiation decrease in cerebral blood flow. Irradiated animals pretreated with DSCG, showed only a 10% decrease in hypothalamic blood flow 60 min postradiation, while untreated, irradiated animals showed a 57% decrease. The cortical blood flow of DSCG treated, irradiated animals showed a triphasic response, with a decrease of 38% at 10 min postradiation, then a rise to 1% below baseline at 20 min, followed by a fall to 42% below baseline by 50 min postradiation. In contrast, the untreated, irradiated animals showed a steady decrease in cortical blood flow to 79% below baseline by 50 min postradiation. There was no significant difference in blood-pressure response between the treated and untreated, irradiated animals. Systemic blood pressure showed a 60% decrease at 10 min postradiation, falling to a 71% decrease by 60 min.

  5. Novel mRNA Targets for Tristetraprolin (TTP) Identified by Global Analysis of Stabilized Transcripts in TTP-Deficient Fibroblasts▿ †

    OpenAIRE

    Lai, Wi S.; Parker, Joel S; Grissom, Sherry F.; Stumpo, Deborah J.; Blackshear, Perry J.

    2006-01-01

    Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that was identified through its rapid induction by mitogens in fibroblasts. Studies of TTP-deficient mice and cells derived from them showed that TTP could bind to certain AU-rich elements in mRNAs, leading to increases in the rates of mRNA deadenylation and destruction. Known physiological target mRNAs for TTP include tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and interleukin-2β. Here we used micro...

  6. AU-rich elements and alternative splicing in the beta-catenin 3'UTR can influence the human beta-catenin mRNA stability.

    NARCIS (Netherlands)

    Thiele, A.; Nagamine, Y.; Hauschildt, S.; Clevers, J.C.

    2006-01-01

    Beta-catenin, the central player of the Wnt signaling cascade, is a well-known oncogene. The regulation of beta-catenin protein stability has been studied extensively while other mechanisms that control cellular levels of beta-catenin have hardly been addressed. In this study, we show that there are

  7. Biological soil crust as a bio-mediator alters hydrological processes in stabilized dune system of the Tengger Desert, China

    Science.gov (United States)

    Li, Xinrong

    2016-04-01

    Biological soil crust (BSC) is a vital component in the stabilized sand dunes with a living cover up to more than 70% of the total, which has been considered as a bio-mediator that directly influences and regulates the sand dune ecosystem processes. However, its influences on soil hydrological processes have been long neglected in Chinese deserts. In this study, BSCs of different successional stages were chose to test their influence on the hydrological processes of stabilized dune, where the groundwater deep exceeds 30m, further to explore why occur the sand-binding vegetation replacement between shrubs and herbs. Our long-term observation (60 years) shows that cyanobacteria crust has been colonized and developed after 3 years since the sand-binding vegetation has been established and dune fixation using planted xerophytic shrubs and made sand barrier (straw-checkerboard) on shifting dune surface, lichen and moss crust occurred after 20 years, and the cover of moss dominated crust could reach 70 % after 50 years. The colonization and development of BSC altered the initial soil water balance of revegetated areas by influencing rainfall infiltration, soil evaporation and dew water entrapment. The results show that BSC obviously reduced the infiltration that occurred during most rainfall events (80%), when rainfall was greater than 5 mm or less than 20 mm. The presence of BSC reduced evaporation of topsoil after small rainfall (facilitated topsoil evaporation when rainfall reached 10 mm. The amount of dew entrapment increases with the succession of BSC. Moreover, the effect of the later successional BSC to dew entrapment, rainfall infiltration and evaporation was more obvious than the early successional BSC on stabilized dunes. In general, BSC reduced the amount of rainfall water that reached deeper soil (0.4-3m), which is where the roots of shrubs are primarily distributed. These changes in the soil moisture pattern induced shifting of sand-binding vegetation from

  8. Biological soil crust as a bio-mediator alters hydrological processes in stabilized dune system of the Tengger Desert, China

    Science.gov (United States)

    Li, Xinrong

    2016-04-01

    Biological soil crust (BSC) is a vital component in the stabilized sand dunes with a living cover up to more than 70% of the total, which has been considered as a bio-mediator that directly influences and regulates the sand dune ecosystem processes. However, its influences on soil hydrological processes have been long neglected in Chinese deserts. In this study, BSCs of different successional stages were chose to test their influence on the hydrological processes of stabilized dune, where the groundwater deep exceeds 30m, further to explore why occur the sand-binding vegetation replacement between shrubs and herbs. Our long-term observation (60 years) shows that cyanobacteria crust has been colonized and developed after 3 years since the sand-binding vegetation has been established and dune fixation using planted xerophytic shrubs and made sand barrier (straw-checkerboard) on shifting dune surface, lichen and moss crust occurred after 20 years, and the cover of moss dominated crust could reach 70 % after 50 years. The colonization and development of BSC altered the initial soil water balance of revegetated areas by influencing rainfall infiltration, soil evaporation and dew water entrapment. The results show that BSC obviously reduced the infiltration that occurred during most rainfall events (80%), when rainfall was greater than 5 mm or less than 20 mm. The presence of BSC reduced evaporation of topsoil after small rainfall (BSC. Moreover, the effect of the later successional BSC to dew entrapment, rainfall infiltration and evaporation was more obvious than the early successional BSC on stabilized dunes. In general, BSC reduced the amount of rainfall water that reached deeper soil (0.4-3m), which is where the roots of shrubs are primarily distributed. These changes in the soil moisture pattern induced shifting of sand-binding vegetation from initial planted xerophytic shrub communities with higher coverage (35%) to complex communities dominated by shallow

  9. The functional half-life of an mRNA depends on the ribosome spacing in an early coding region

    DEFF Research Database (Denmark)

    Pedersen, Margit; Nissen, Søren; Mitarai, Namiko;

    2011-01-01

    . Here, we characterize a determinant of the functional stability of an mRNA, which is located in the early coding region. Using literature values for the mRNA half-lives of variant lacZ mRNAs in Escherichia coli, we modeled how the ribosome spacing is affected by the translation rate of the individual...... of slowly translated codons before codon 20 or after codon 45 should shorten or prolong, respectively, the functional mRNA half-life by altering the ribosome density in the important region. These predictions were tested on eight new lacZ variants, and their experimentally determined mRNA half-lives all...

  10. Alterations of BDNF and trkB mRNA expression in the 6-hydroxydopamine-induced model of preclinical stages of Parkinson's disease: an influence of chronic pramipexole in rats.

    Directory of Open Access Journals (Sweden)

    Klemencja Berghauzen-Maciejewska

    Full Text Available Our recent study has indicated that a moderate lesion of the mesostriatal and mesolimbic pathways in rats, modelling preclinical stages of Parkinson's disease, induces a depressive-like behaviour which is reversed by chronic treatment with pramipexole. The purpose of the present study was to examine the role of brain derived neurotrophic factor (BDNF signalling in the aforementioned model of depression. Therefore, we investigated the influence of 6-hydoxydopamine (6-OHDA administration into the ventral region of the caudate-putamen on mRNA levels of BDNF and tropomyosin-related kinase B (trkB receptor. The BDNF and trkB mRNA levels were determined in the nigrostriatal and limbic structures by in situ hybridization 2 weeks after the operation. Pramipexole (1 mg/kg sc twice a day and imipramine (10 mg/kg ip once a day were injected for 2 weeks. The lesion lowered the BDNF and trkB mRNA levels in the hippocampus [CA1, CA3 and dentate gyrus (DG] and amygdala (basolateral/lateral as well as the BDNF mRNA content in the habenula (medial/lateral. The lesion did not influence BDNF and trkB expression in the caudate-putamen, substantia nigra, nucleus accumbens (shell and core and ventral tegmental area (VTA. Chronic imipramine reversed the lesion-induced decreases in BDNF mRNA in the DG. Chronic pramipexole increased BDNF mRNA, but decreased trkB mRNA in the VTA in lesioned rats. Furthermore, it reduced BDNF and trkB mRNA expression in the shell and core of the nucleus accumbens, BDNF mRNA in the amygdala and trkB mRNA in the caudate-putamen in these animals. The present study indicates that both the 6-OHDA-induced dopaminergic lesion and chronic pramipexole influence BDNF signalling in limbic structures, which may be related to their pro-depressive and antidepressant activity in rats, respectively.

  11. A study on the pathogenesis of the radiation pneumonitis. Alterations in pulmonary mRNA encoding adhesion molecules ICAM-1, VCAM-1, and P-selectin following thoracic irradiation in mice

    Energy Technology Data Exchange (ETDEWEB)

    Tsujino, Kayoko; Kodama, Akihisa; Kono, Michio [Kobe Univ. (Japan). School of Medicine

    1997-12-01

    To investigate the role of the adhesion molecules in the pathogenesis of the radiation pneumonitis, we quantified the mRNA expression of the adhesion molecules in the lung by Northern blot method following whole thorax irradiation to C57BL/6J mice. After irradiation of 12 Gy to the whole thorax, there were increase of mRNA for ICAM-1 by 42% at 4 hours (p<0.05), 76% at 24 hours (p<0.01) and 51% at 48 hours (p<0.05) compared with controls. And it returned to control level at 1 week. No significant change was observed thereafter until 8 weeks. The expression of VCAM-1 mRNA were also increased by 49% (p<0.01) at 12 hours and were still increased by 25% at 1 week. P-selectin mRNA as transiently increased by 59% at 12 hours. We examined the relationship between the ICAM-1 induction and the radiation dose, and found that ICAM-1 expression was increased by 3 Gy of irradiation and it was increased in radiation dose dependent manner up to 24 Gy. These early inductions of mRNA for ICAM-1, VCAM-1 and P-selectin in mice lungs following thoracic irradiation were transient but significant, and they were one of the most immediate change reported in vivo. It is suggested that these adhesion molecules are possibly related to the pathogenesis of the radiation pneumonitis. (author)

  12. Stabilization

    Directory of Open Access Journals (Sweden)

    Muhammad H. Al-Malack

    2016-07-01

    Full Text Available Fuel oil flyash (FFA produced in power and water desalination plants firing crude oils in the Kingdom of Saudi Arabia is being disposed in landfills, which increases the burden on the environment, therefore, FFA utilization must be encouraged. In the current research, the effect of adding FFA on the engineering properties of two indigenous soils, namely sand and marl, was investigated. FFA was added at concentrations of 5%, 10% and 15% to both soils with and without the addition of Portland cement. Mixtures of the stabilized soils were thoroughly evaluated using compaction, California Bearing Ratio (CBR, unconfined compressive strength (USC and durability tests. Results of these tests indicated that stabilized sand mixtures could not attain the ACI strength requirements. However, marl was found to satisfy the ACI strength requirement when only 5% of FFA was added together with 5% of cement. When the FFA was increased to 10% and 15%, the mixture’s strength was found to decrease to values below the ACI requirements. Results of the Toxicity Characteristics Leaching Procedure (TCLP, which was performed on samples that passed the ACI requirements, indicated that FFA must be cautiously used in soil stabilization.

  13. The functional half-life of an mRNA depends on the ribosome spacing in an early coding region.

    Science.gov (United States)

    Pedersen, Margit; Nissen, Søren; Mitarai, Namiko; Lo Svenningsen, Sine; Sneppen, Kim; Pedersen, Steen

    2011-03-18

    Bacterial mRNAs are translated by closely spaced ribosomes and degraded from the 5'-end, with half-lives of around 2 min at 37 °C in most cases. Ribosome-free or "naked" mRNA is known to be readily degraded, but the initial event that inactivates the mRNA functionally has not been fully described. Here, we characterize a determinant of the functional stability of an mRNA, which is located in the early coding region. Using literature values for the mRNA half-lives of variant lacZ mRNAs in Escherichia coli, we modeled how the ribosome spacing is affected by the translation rate of the individual codons. When comparing the ribosome spacing at various segments of the mRNA to its functional half-life, we found a clear correlation between the functional mRNA half-life and the ribosome spacing in the mRNA region approximately between codon 20 and codon 45. From this finding, we predicted that inserts of slowly translated codons before codon 20 or after codon 45 should shorten or prolong, respectively, the functional mRNA half-life by altering the ribosome density in the important region. These predictions were tested on eight new lacZ variants, and their experimentally determined mRNA half-lives all supported the model. We thus suggest that translation-rate-mediated differences in the spacing between ribosomes in this early coding region is a parameter that determines the mRNAs functional half-life. We present a model that is in accordance with many earlier observations and that allows a prediction of the functional half-life of a given mRNA sequence. PMID:21255584

  14. Stabilization of garnet in metamorphosed altered turbidites near the St. Eugene lead-zinc deposit, southeastern British Columbia: Equilibrium and kinetic controls

    Science.gov (United States)

    Pattison, David R. M.; Seitz, JennaLee D.

    2012-03-01

    The St. Eugene lead-zinc deposit, near Moyie, southeastern British Columbia, is a Mesoproterozoic vein deposit hosted by metaturbidites of the 1.5-1.4 Ga Belt-Purcell Supergroup. The regional metamorphic grade of the rocks is biotite zone, with the age of metamorphism being Mesoproterozoic (ca. 1.35 Ga). The vein system is enveloped by a metamorphosed alteration zone of increasing intensity as the vein is approached. Thin argillaceous tops of turbidite beds away from the vein are garnet-free, whereas those in the inner alteration zone are garnet-bearing. Compared to rocks away from the vein, those near the vein are enriched in Fe, Mn, Pb and Zn, with proportional reduction in other compositional parameters. Thermodynamic modeling of three rocks across the alteration gradient predicts increasing stabilization of garnet with increasing degree of alteration. Predicted and observed modes of garnet in the samples are in close agreement. In the most altered rock, the garnet-in line is displaced down-temperature by ~ 100 °C relative to the least altered rock. Approximately 2/3 of the garnet stabilization is accounted for by increase in Mn content and the rest by increase in Fe/(Fe + Mg). Kinetic factors played a role in the development of the mineral assemblages, including metastable persistence of zoisite, and disequilibrium (overstepped) initial growth of garnet. Estimates of peak pressure-temperature conditions from mineral assemblage constraints and from compositional isopleths are complicated by the kinetic effects but yield similar results: 490-510 °C and 3.6-4.0 kbar. The pressure-temperature estimates imply an average linear geothermal gradient of ~ 35 °C/km, broadly consistent with burial metamorphism in the Belt-Purcell extensional basin. However, the estimated pressure, equivalent to a depth of 13-15 km, is greater than the estimated ~ 8 km (~ 2.2 kbar) of stratigraphic overburden at the time of metamorphism. The results of this study support the idea that

  15. Regulation of LPS-induced mRNA expression of pro-inflammatory cytokines via alteration of NF-κB activity in mouse peritoneal macrophages exposed to fluoride.

    Science.gov (United States)

    Tian, Yuhu; Huo, Meijun; Li, Guangsheng; Li, Yanyan; Wang, Jundong

    2016-10-01

    F toxicity to immune system, especially to macrophage, has been studied a lot recently. Nuclear factor-kappa B (NF-κB), as a transcription factor, plays a central role in immune and inflammatory responses via the regulation of downstream gene expression. Recent studies indicated that fluoride effect on inflammatory cytokine secretion, however, the molecular mechanism was less understood. In our study, peritoneal macrophages (PMs) were divided several groups and were administrated sodium fluoride (NaF, 50, 100, 200, 400, 800 μM) and/or lipopolysaccharide (LPS, 30 ng/mg). The mRNA expression of p65, inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) in macrophages exposed to fluoride was determined by quantitative real-time RT-PCR respectively. The translocation of NF-κB from cytoplasm to nucleus, which in a way reflects NF-κB activity, was demonstrated by Immunofluorescence and ELISA. Our results showed that fluoride had a dose-dependent effect on NF-κB activity, which coincided with LPS-induced mRNA expression of its downstream genes, iNOS and IL-1β. Fluoride alone causes no effect on gene expression. However, the mRNA expression of TNF-α showed non-NF-κB-dependent manner. Therefore, we come to the conclusion that fluoride can regulate LPS-induced mRNA expression of iNOS and IL-1β via NF-κB pathway in mouse peritoneal macrophages. PMID:27421105

  16. Triosephosphate Isomerase I170V Alters Catalytic Site, Enhances Stability and Induces Pathology in a Drosophila Model of TPI Deficiency

    Science.gov (United States)

    Roland, Bartholomew P.; Amrich, Christopher G.; Kammerer, Charles J.; Stuchul, Kimberly A.; Larsen, Samantha B.; Rode, Sascha; Aslam, Anoshé A.; Heroux, Annie; Wetzel, Ronald; VanDemark, Andrew P.; Palladino, Michael J.

    2014-01-01

    Triosephosphate isomerase (TPI) is a glycolytic enzyme which homodimerizes for full catalytic activity. Mutations of the TPI gene elicit a disease known as TPI Deficiency, a glycolytic enzymopathy noted for its unique severity of neurological symptoms. Evidence suggests that TPI Deficiency pathogenesis may be due to conformational changes of the protein, likely affecting dimerization and protein stability. In this report, we genetically and physically characterize a human disease-associated TPI mutation caused by an I170V substitution. Human TPII170V elicits behavioral abnormalities in Drosophila. An examination of hTPII170V enzyme kinetics revealed this substitution reduced catalytic turnover, while assessments of thermal stability demonstrated an increase in enzyme stability. The crystal structure of the homodimeric I170V mutant reveals changes in the geometry of critical residues within the catalytic pocket. Collectively these data reveal new observations of the structural and kinetic determinants of TPI deficiency pathology, providing new insights into disease pathogenesis. PMID:25463631

  17. Chronic Exposure to Arsenic in Drinking Water Causes Alterations in Locomotor Activity and Decreases Striatal mRNA for the D2 Dopamine Receptor in CD1 Male Mice

    OpenAIRE

    Moreno Ávila, Claudia Leticia; Limón-Pacheco, Jorge H.; Giordano., Magda; Rodríguez, Verónica M.

    2016-01-01

    Arsenic exposure has been associated with sensory, motor, memory, and learning alterations in humans and alterations in locomotor activity, behavioral tasks, and neurotransmitters systems in rodents. In this study, CD1 mice were exposed to 0.5 or 5.0 mg As/L of drinking water for 6 months. Locomotor activity, aggression, interspecific behavior and physical appearance, monoamines levels, and expression of the messenger for dopamine receptors D1 and D2 were assessed. Arsenic exposure produced h...

  18. Hyperresponsive febrile reactions to interleukin (IL) 1α and IL-1β, and altered brain cytokine mRNA and serum cytokine levels, in IL-1β-deficient mice

    Science.gov (United States)

    Alheim, Katarina; Chai, Zhen; Fantuzzi, Giamila; Hasanvan, Homa; Malinowsky, David; Di Santo, Elena; Ghezzi, Pietro; Dinarello, Charles A.; Bartfai, Tamas

    1997-01-01

    IL-1β is an endogenous pyrogen that is induced during systemic lipopolysaccharide (LPS)- or IL-1-induced fever. We have examined the fever and cytokine responses following i.p. injection of IL-1 agonists, IL-1α and IL-1β, and compared these with response to LPS (i.p.) in wild-type and IL-1β-deficient mice. The IL-1β deficient mice appear to have elevated body temperature but exhibit a normal circadian temperature cycle. Exogenously injected IL-1β, IL-1α, or LPS induced hyperresponsive fevers in the IL-1β-deficient mice. We also observed phenotypic differences between wild-type and IL-1β-deficient mice in hypothalamic basal mRNA levels for IL-1α and IL-6, but not for IL-1β-converting enzyme or IL-1 receptor type I or type II. The IL-1α mRNA levels were down-regulated, whereas the IL-6 mRNA levels were up-regulated in the hypothalamus of IL-1β-deficient mice as compared with wild-type mice. The IL-1β-deficient mice also responded to LPS challenge with significantly higher serum corticosterone and with lower serum tumor necrosis factor type α levels than the wild-type mice. The data suggest that, in the redundant cascade of proinflammatory cytokines, IL-1β plays an important but not obligatory role in fever induction by LPS or IL-1α, as well as in the induction of serum tumor necrosis factor type α and corticosterone responses either by LPS or by IL-1α or IL-1β. PMID:9122256

  19. Hyperresponsive febrile reactions to interleukin (IL) 1alpha and IL-1beta, and altered brain cytokine mRNA and serum cytokine levels, in IL-1beta-deficient mice.

    Science.gov (United States)

    Alheim, K; Chai, Z; Fantuzzi, G; Hasanvan, H; Malinowsky, D; Di Santo, E; Ghezzi, P; Dinarello, C A; Bartfai, T

    1997-03-18

    IL-1beta is an endogenous pyrogen that is induced during systemic lipopolysaccharide (LPS)- or IL-1-induced fever. We have examined the fever and cytokine responses following i.p. injection of IL-1 agonists, IL-1alpha and IL-1beta, and compared these with response to LPS (i.p.) in wild-type and IL-1beta-deficient mice. The IL-1beta deficient mice appear to have elevated body temperature but exhibit a normal circadian temperature cycle. Exogenously injected IL-1beta, IL-1alpha, or LPS induced hyperresponsive fevers in the IL-1beta-deficient mice. We also observed phenotypic differences between wild-type and IL-1beta-deficient mice in hypothalamic basal mRNA levels for IL-1alpha and IL-6, but not for IL-1beta-converting enzyme or IL-1 receptor type I or type II. The IL-1alpha mRNA levels were down-regulated, whereas the IL-6 mRNA levels were up-regulated in the hypothalamus of IL-1beta-deficient mice as compared with wild-type mice. The IL-1beta-deficient mice also responded to LPS challenge with significantly higher serum corticosterone and with lower serum tumor necrosis factor type alpha levels than the wild-type mice. The data suggest that, in the redundant cascade of proinflammatory cytokines, IL-1beta plays an important but not obligatory role in fever induction by LPS or IL-1alpha, as well as in the induction of serum tumor necrosis factor type alpha and corticosterone responses either by LPS or by IL-1alpha or IL-1beta.

  20. Messenger RNA (mRNA) nanoparticle tumour vaccination

    Science.gov (United States)

    Phua, Kyle K. L.; Nair, Smita K.; Leong, Kam W.

    2014-06-01

    Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

  1. The Role of RNA Binding Proteins in Insulin Messenger Stability and Translation

    OpenAIRE

    Fred, Rikard G.

    2010-01-01

    Although the reason for insufficient release of insulin in diabetes mellitus may vary depending on the type and stage of the disease, it is of vital importance that an amplified insulin biosynthesis can meet the increased need during periods of hyperglycemia. The insulin mRNA is highly abundant in beta cells and changes in insulin mRNA levels are, at least in part, controlled by altered rates of mRNA degradation. Since the mechanisms behind the control of insulin messenger stability and trans...

  2. The role of altered proximal femoral geometry in impaired pelvis stability and hip control during CP gait: A simulation study.

    Science.gov (United States)

    Bosmans, Lode; Jansen, Karen; Wesseling, Mariska; Molenaers, Guy; Scheys, Lennart; Jonkers, Ilse

    2016-02-01

    Children with cerebral palsy (CP) often present aberrant hip geometry, more specifically increased femoral anteversion and neck-shaft angle. Furthermore, altered gait patterns are present within this population. This study analyzed the effect of aberrant femoral geometry, as present in subjects with CP, on the ability of muscles to control hip and knee joint kinematics. Given the specific gait deficits observed during crouch gait, increased ability to abduct, externally rotate the hip and extend the knee and hip were denoted as beneficial effects. We ran dynamic simulations of CP and normal gait using two musculoskeletal models, one reflecting normal femoral geometry and one reflecting proximal femoral deformities. The results show that the combination of aberrant bone geometry and CP-specific gait characteristics beneficially increased the ability of gluteus medius and maximus to extend the hip and knee. In contrast, the potentials of the hamstrings to extend the hip decreased whereas the potentials to flex the knee increased. These changes closely followed the observed changes in the muscle moment arm lengths. In conclusion, this study emphasizes the concomitant effect of the presence of proximal femoral deformity and CP gait characteristics on the muscle control of hip and knee joint kinematics during single stance. Not accounting for subject-specific geometry will affect the calculated muscles' potential during gait. Therefore, the use of generic models to assess muscle function in the presence of femoral deformity and CP gait should be treated with caution.

  3. Maternal dietary vitamin D carry-over alters offspring growth, skeletal mineralisation and tissue mRNA expressions of genes related to vitamin D, calcium and phosphorus homoeostasis in swine.

    Science.gov (United States)

    Amundson, Laura A; Hernandez, Laura L; Laporta, Jimena; Crenshaw, Thomas D

    2016-09-01

    Maternal dietary vitamin D carry-over effects were assessed in young pigs to characterise skeletal abnormalities in a diet-induced model of kyphosis. Bone abnormalities were previously induced and bone mineral density (BMD) reduced in offspring from sows fed diets with inadequate vitamin D3. In a nested design, pigs from sows (n 23) fed diets with 0 (-D), 8·125 (+D) or 43·750 (++D) µg D3/kg from breeding through lactation were weaned and, within litter, fed nursery diets arranged as a 2×2 factorial design with 0 (-D) or 7·0 (+D) µg D3/kg, each with 95 % (95P) or 120 % (120P) of P requirements. Selected pigs were euthanised before colostrum consumption at birth (0 weeks, n 23), weaning (3 weeks, n 22) and after a growth period (8 weeks, n 185) for BMD, bone mechanical tests and tissue mRNA analysis. Pigs produced by +D or ++D sows had increased gain at 3 weeks (Pdiets depended on maternal diets (Pdiet interaction (Phumans and animals about maternal dietary influence on offspring skeletal health. PMID:27480125

  4. Regulation of ABCG2 expression at the 3' untranslated region of its mRNA through modulation of transcript stability and protein translation by a putative microRNA in the S1 colon cancer cell line

    DEFF Research Database (Denmark)

    To, Kenneth K W; Zhan, Zhirong; Litman, Thomas;

    2008-01-01

    ABCG2 is recognized as an important efflux transporter in clinical pharmacology and is potentially important in resistance to chemotherapeutic drugs. To identify epigenetic mechanisms regulating ABCG2 mRNA expression at its 3' untranslated region (3'UTR), we performed 3' rapid amplification of cD...

  5. Induction of fetal hemoglobin through enhanced translation efficiency of γ-globin mRNA

    OpenAIRE

    Hahn, Cynthia K.; Lowrey, Christopher H.

    2014-01-01

    HbF induction by salubrinal is not mediated through changes in globin mRNA stability, mRNA cellular localization, or HbA levels.Translation efficiency of γ-globin mRNA is increased during stress recovery following salubrinal-enhanced eIF2α phosphorylation.

  6. Gene regulation by mRNA editing

    Energy Technology Data Exchange (ETDEWEB)

    Ashkenas, J. [Univ. of Washington, Seattle, WA (United States)

    1997-02-01

    The commonly cited figure of 10{sup 5} genes in the human genome represents a tremendous underestimate of our capacity to generate distinct gene products with unique functions. Our cells possess an impressive collection of tools for altering the products of a single gene to create a variety of proteins. The different gene products may have related but distinct functions, allowing cells of different types or at different developmental stages to fine-tune their patterns of gene expression. These tools may act in the cytoplasm, as when proteins undergo post-translational modifications, or in the nucleus, in the processing of pre-mRNA. Two forms of intranuclear fine-tuning are well established and widely studied: alternative splicing of pre-mRNAs and alternative polyadenylation site selection. In recent years it has become clear that cells possess yet another tool to create RNA sequence diversity, mRNA editing. The term {open_quotes}editing{close_quotes} is applied to posttranscriptional modifications of a purine or pyrimidine, which alter an mRNA sequence as it is read, for example, by ribosomes. Covalent changes to the structure of nucleotide bases are well known to occur on tRNA and rRNA molecules, but such changes in mRNA sequence are novel in that they have the capacity to change specific protein sequences. 43 refs., 1 fig.

  7. Synthetic mRNA with Superior Properties that Mimics the Intracellular Fates of Natural Histone mRNA.

    Science.gov (United States)

    Su, Wei; Slevin, Michael K; Marzluff, William F; Rhoads, Robert E

    2016-01-01

    Since DNA and histone levels must be closely balanced for cell survival, histone expressions are highly regulated. The regulation of replication-dependent histone expression is mainly achieved at the mRNA level, as the mRNAs are rapidly removed when DNA replication is inhibited during S-phase. Histone mRNA degradation initiates with addition of multiple uridines (oligouridylation) following the 3' stem-loop (SL) catalyzed by terminal uridyltransferase (TUTase). Previous studies showed that histone mRNA degradation occurs through both 5' → 3' and 3' → 5' processes, but the relative contributions are difficult to dissect due to lack of established protocols. The translational efficiency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by structural modifications at the both 5' and 3' termini. In this chapter, we present methods of utilizing modified cap dinucleotide analogs to block 5' → 3' degradation of a reporter mRNA containing canonical histone mRNA 3' SL and monitoring how oligouridylation and 3' → 5' degradation occur. Protocols are presented for synthesis of reporter mRNA containing the histone 3' SL and modified cap analogs, monitoring mRNA stability and unidirectional degradation either from 5' or 3' termini, and detection of oligo(U) tracts from degradation products by either traditional or deep sequencing. PMID:27236794

  8. Overexpression of CsrA (BB0184) Alters the Morphology and Antigen Profiles of Borrelia burgdorferi▿

    OpenAIRE

    Sanjuan, Eva; Maria D Esteve-Gassent; Maruskova, Mahulena; Seshu, J.

    2009-01-01

    Borrelia burgdorferi, the agent of Lyme disease, alters its gene expression in response to highly disparate environmental signals encountered in its hosts. Among the relatively few regulators of adaptive gene expression present in the borrelial genome is an open reading frame (ORF), BB0184, annotated as CsrA (carbon storage regulator A). CsrA, in several bacterial species, has been characterized as a small RNA binding protein that functions as a global regulator affecting mRNA stability or le...

  9. The staphylococcal accessory regulator, SarA, is an RNA-binding protein that modulates the mRNA turnover properties of late-exponential and stationary phase Staphylococcus aureus cells

    Directory of Open Access Journals (Sweden)

    John M Morrison

    2012-03-01

    Full Text Available The modulation of mRNA turnover is gaining recognition as a mechanism by which Staphylococcus aureus regulates gene expression, but the factors that orchestrate alterations in transcript degradation are poorly understood. In that regard, we previously found that 138 mRNA species, including the virulence factors protein A (spa and collagen binding protein (cna, are stabilized in a sarA-dependent manner during exponential phase growth, suggesting that SarA protein may directly or indirectly effect the RNA turnover properties of these transcripts. Herein, we expanded our characterization of the effects of sarA on mRNA turnover during late exponential and stationary phases of growth. Results revealed that the locus affects the RNA degradation properties of cells during both growth phases. Further, using gel mobility shift assays and RIP-ChIP, it was found that SarA protein is capable of binding mRNA species that it stabilizes both in vitro and within bacterial cells. Taken together, these results suggest that SarA post-transcriptionally regulates S. aureus gene expression in a manner that involves binding to and consequently altering the mRNA turnover properties of target transcripts.

  10. Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation.

    Directory of Open Access Journals (Sweden)

    Adam D Cohen

    Full Text Available In vivo GITR ligation has previously been shown to augment T-cell-mediated anti-tumor immunity, yet the underlying mechanisms of this activity, particularly its in vivo effects on CD4+ foxp3+ regulatory T cells (Tregs, have not been fully elucidated. In order to translate this immunotherapeutic approach to the clinic it is important gain better understanding of its mechanism(s of action. Utilizing the agonist anti-GITR monoclonal antibody DTA-1, we found that in vivo GITR ligation modulates regulatory T cells (Tregs directly during induction of melanoma tumor immunity. As a monotherapy, DTA-1 induced regression of small established B16 melanoma tumors. Although DTA-1 did not alter systemic Treg frequencies nor abrogate the intrinsic suppressive activity of Tregs within the tumor-draining lymph node, intra-tumor Treg accumulation was significantly impaired. This resulted in a greater Teff:Treg ratio and enhanced tumor-specific CD8+ T-cell activity. The decreased intra-tumor Treg accumulation was due both to impaired infiltration, coupled with DTA-1-induced loss of foxp3 expression in intra-tumor Tregs. Histological analysis of B16 tumors grown in Foxp3-GFP mice showed that the majority of GFP+ cells had lost Foxp3 expression. These "unstable" Tregs were absent in IgG-treated tumors and in DTA-1 treated TDLN, demonstrating a tumor-specific effect. Impairment of Treg infiltration was lost if Tregs were GITR(-/-, and the protective effects of DTA-1 were reduced in reconstituted RAG1(-/- mice if either the Treg or Teff subset were GITR-negative and absent if both were negative. Our results demonstrate that DTA-1 modulates both Teffs and Tregs during effective tumor treatment. The data suggest that DTA-1 prevents intra-tumor Treg accumulation by altering their stability, and as a result of the loss of foxp3 expression, may modify their intra-tumor suppressive capacity. These findings provide further support for the continued development of agonist

  11. Fatty acids attached to all-trans-astaxanthin alter its cis-trans equilibrium, and consequently its stability, upon light-accelerated autoxidation

    NARCIS (Netherlands)

    Bruijn, De Wouter J.C.; Weesepoel, Y.; Vincken, J.P.; Gruppen, H.

    2016-01-01

    Fatty acid esterification, common in naturally occurring astaxanthin, has been suggested to influence both colour stability and degradation of all-trans-astaxanthin. Therefore, astaxanthin stability was studied as influenced by monoesterification and diesterification with palmitate. Increased est

  12. SNP detection in mRNA in living cells using allele specific FRET probes.

    Directory of Open Access Journals (Sweden)

    Liya Dahan

    Full Text Available Live mRNA detection allows real time monitoring of specific transcripts and genetic alterations. The main challenge of live genetic detection is overcoming the high background generated by unbound probes and reaching high level of specificity with minimal off target effects. The use of Fluorescence Resonance Energy Transfer (FRET probes allows differentiation between bound and unbound probes thus decreasing background. Probe specificity can be optimized by adjusting the length and through use of chemical modifications that alter binding affinity. Herein, we report the use of two oligonucleotide FRET probe system to detect a single nucleotide polymorphism (SNP in murine Hras mRNA, which is associated with malignant transformations. The FRET oligonucleotides were modified with phosphorothioate (PS bonds, 2'OMe RNA and LNA residues to enhance nuclease stability and improve SNP discrimination. Our results show that a point mutation in Hras can be detected in endogenous RNA of living cells. As determined by an Acceptor Photobleaching method, FRET levels were higher in cells transfected with perfect match FRET probes whereas a single mismatch showed decreased FRET signal. This approach promotes in vivo molecular imaging methods and could further be applied in cancer diagnosis and theranostic strategies.

  13. Single amino-acid substitution in the N-terminal arm altered the tetramer stability of rat muscle lactate dehydrogenase A

    Institute of Scientific and Technical Information of China (English)

    YUAN; Chong; (

    2001-01-01

    [1]Price, N. C., Assembly of multi-subunit structures, in Mechanisms of Protein Folding (ed. Pain, R. H.), New York: Oxford University Press, 1994, 160-193.[2]Casal, J. I., Ahern, T. J., Davenport, R. C. et al., Subunit interface of triosephosphate isomerase: Site-directed mutagenesis and characterization of the altered enzyme, Biochemistry, 1987, 26: 1258-1264.[3]Chakerian, A. E., Matthews, K. S., Characterization of mutations in oligomerization domain of lac repressor protein, J. Biol. Chem., 1991, 266: 22206-22214.[4]Mandelman, D., Schwarz, F. P., Li, H. Y. et al., The role of quaternary interactions on the stability and activity of ascorbate peroxidase, Protein Sci., 1998, 7: 2089-2098.[5]Thomas, M. C., Ballantine, S. P., Bethell, S. S. et al., Single amino acid substitutions disrupt tetramer formation in the dihydroneopterin aldolase enzyme of Pneumocystis carinii, Biochemistry, 1998, 37: 11629-11636.[6]Holbrook, J. J., Liljas, A., Steindel, S. J. et al., Lactate dehydrogenase, in The Enzymes (ed. Boyer, P. D.), Vol. 11, 3rd ed., New York: Academic Press, 1975, 191-292.[7]Zettlmeissl, G., Rudolph, R., Jaenicke, R., Reconstitution of lactic dehydrogenase after acid dissociation, Eur. J. Biochem., 1981, 121: 169-175.[8]Zettlmeissl, G., Rudolph, R., Jaenicke, R., Rate-determining folding and association reactions on the reconstitution pathway of porcine skeletal muscle lactic dehydrogenase after denaturation by guanidine hydrochloride, Biochemistry, 1982, 21: 3946-3950.[9]Hermann, R., Jaenicke, R., Rudolph, R., Analysis of the reconstitution of oligomeric enzymes by cross-linking with glutaraldehyde: Kinetics of reassociation of lactic dehydrogenase, Biochemistry, 1981, 20: 5195-5201.[10]Jaenicke, R., Folding and association of protein, Prog. Biophys. Mol. Biol., 1987, 49: 117-237.[11]Opitz, U., Rudolph, R., Jaenicke, R. et al., Proteolytic dimeric of porcine muscle lactate dehydrogenase: Characterization, folding, and

  14. Alternative splicing of dystrophin mRNA complicates carrier determination: report of a DMD family.

    OpenAIRE

    Lenk, U; Demuth, S.; Kräft, U; Hanke, R; Speer, A

    1993-01-01

    Carrier determination is important for genetic counselling in DMD/BMD families. The detection of altered PCR amplified dystrophin mRNA fragments owing to deletions, insertions, or point mutations has increased the possibilities of carrier determination. However, problems may occur because of alternative splicing events. Here we present a family with a DMD patient characterised by a deletion of exons 45 to 54. At the mRNA level we detected a corresponding altered fragment which served for carr...

  15. GBR 12909 administration as an animal model of bipolar mania: time course of behavioral, brain oxidative alterations and effect of mood stabilizing drugs.

    Science.gov (United States)

    Queiroz, Ana Isabelle G; de Araújo, Maíra Moraes; da Silva Araújo, Tatiane; de Souza, Greicy Coelho; Cavalcante, Lígia Menezes; de Jesus Souza Machado, Michel; de Lucena, David Freitas; Quevedo, João; Macêdo, Danielle

    2015-10-01

    Polymorphisms in the human dopamine transporter (DAT) are associated with bipolar endophenotype. Based on this, the acute inhibition of DAT using GBR12909 causes behavioral alterations that are prevented by valproate (VAL), being related to a mania-like model. Herein our first aim was to analyze behavioral and brain oxidative alterations during a 24 h period post-GBR12909 to better characterize this model. Our second aim was to determine the preventive effects of lithium (Li) or VAL 2 h post-GBR12909. For this, adult male mice received GBR12909 or saline being evaluated at 2, 4, 8, 12 or 24 h post-administration. Hyperlocomotion, levels of reduced glutathione (GSH) and lipid peroxidation in brain areas were assessed at all these time-points. GBR12909 caused hyperlocomotion at 2 and 24 h. Rearing behavior increased only at 2 h. GSH levels decreased in the hippocampus and striatum at the time points of 2, 4, 8 and 12 h. Increased lipid peroxidation was detected at the time-points of 2 and 12 h in all brain areas studied. At the time-point of 2 h post-GBR12909 Li prevented the hyperlocomotion and rearing alterations, while VAL prevented only rearing alterations. Both drugs prevented pro-oxidative changes. In conclusion, we observed that the main behavioral and oxidative alterations took place at the time-period of 2 h post-GBR12909, what points to this time-period as the best for the assessment of alterations in this model. Furthermore, the present study expands the predictive validity of the model by the determination of the preventive effects of Li. PMID:26073232

  16. Systematic analysis of cis-elements in unstable mRNAs demonstrates that CUGBP1 is a key regulator of mRNA decay in muscle cells.

    Directory of Open Access Journals (Sweden)

    Jerome E Lee

    Full Text Available BACKGROUND: Dramatic changes in gene expression occur in response to extracellular stimuli and during differentiation. Although transcriptional effects are important, alterations in mRNA decay also play a major role in achieving rapid and massive changes in mRNA abundance. Moreover, just as transcription factor activity varies between different cell types, the factors influencing mRNA decay are also cell-type specific. PRINCIPAL FINDINGS: We have established the rates of decay for over 7000 transcripts expressed in mouse C2C12 myoblasts. We found that GU-rich (GRE and AU-rich (ARE elements are over-represented in the 3'UTRs of short-lived mRNAs and that these mRNAs tend to encode factors involved in cell cycle and transcription regulation. Stabilizing elements were also identified. By comparing mRNA decay rates in C2C12 cells with those previously measured for pluripotent and differentiating embryonic stem (ES cells, we identified several groups of transcripts that exhibit cell-type specific decay rates. Further, whereas in C2C12 cells the impact of GREs on mRNA decay appears to be greater than that of AREs, AREs are more significant in ES cells, supporting the idea that cis elements make a cell-specific contribution to mRNA stability. GREs are recognized by CUGBP1, an RNA-binding protein and instability factor whose function is affected in several neuromuscular diseases. We therefore utilized RNA immunoprecipitation followed by microarray (RIP-Chip to identify CUGBP1-associated transcripts. These mRNAs also showed dramatic enrichment of GREs in their 3'UTRs and encode proteins linked with cell cycle, and intracellular transport. Interestingly several CUGBP1 substrate mRNAs, including those encoding the myogenic transcription factors Myod1 and Myog, are also bound by the stabilizing factor HuR in C2C12 cells. Finally, we show that several CUGBP1-associated mRNAs containing 3'UTR GREs, including Myod1, are stabilized in cells depleted of CUGBP1

  17. Ethylmalonic encephalopathy ETHE1 R163W/R163Q mutations alter protein stability and redox properties of the iron centre.

    Science.gov (United States)

    Henriques, Bárbara J; Lucas, Tânia G; Rodrigues, João V; Frederiksen, Jane H; Teixeira, Miguel S; Tiranti, Valeria; Bross, Peter; Gomes, Cláudio M

    2014-01-01

    ETHE1 is an iron-containing protein from the metallo β-lactamase family involved in the mitochondrial sulfide oxidation pathway. Mutations in ETHE1 causing loss of function result in sulfide toxicity and in the rare fatal disease Ethylmalonic Encephalopathy (EE). Frequently mutations resulting in depletion of ETHE1 in patient cells are due to severe structural and folding defects. However, some ETHE1 mutations yield nearly normal protein levels and in these cases disease mechanism was suspected to lie in compromised catalytic activity. To address this issue and to elicit how ETHE1 dysfunction results in EE, we have investigated two such pathological mutations, ETHE1-p.Arg163Gln and p.Arg163Trp. In addition, we report a number of benchmark properties of wild type human ETHE1, including for the first time the redox properties of the mononuclear iron centre. We show that loss of function in these variants results from a combination of decreased protein stability and activity. Although structural assessment revealed that the protein fold is not perturbed by mutations, both variants have decreased thermal stabilities and higher proteolytic susceptibilities. ETHE1 wild type and variants bind 1 ± 0.2 mol iron/protein and no zinc; however, the variants exhibited only ≈ 10% of wild-type catalytically activity. Analysis of the redox properties of ETHE1 mononuclear iron centre revealed that the variants have lowered reduction potentials with respect to that of the wild type. This illustrates how point mutation-induced loss of function may arise via very discrete subtle conformational effects on the protein fold and active site chemistry, without extensive disruption of the protein structure or protein-cofactor association. PMID:25198162

  18. Ethylmalonic encephalopathy ETHE1 R163W/R163Q mutations alter protein stability and redox properties of the iron centre.

    Directory of Open Access Journals (Sweden)

    Bárbara J Henriques

    Full Text Available ETHE1 is an iron-containing protein from the metallo β-lactamase family involved in the mitochondrial sulfide oxidation pathway. Mutations in ETHE1 causing loss of function result in sulfide toxicity and in the rare fatal disease Ethylmalonic Encephalopathy (EE. Frequently mutations resulting in depletion of ETHE1 in patient cells are due to severe structural and folding defects. However, some ETHE1 mutations yield nearly normal protein levels and in these cases disease mechanism was suspected to lie in compromised catalytic activity. To address this issue and to elicit how ETHE1 dysfunction results in EE, we have investigated two such pathological mutations, ETHE1-p.Arg163Gln and p.Arg163Trp. In addition, we report a number of benchmark properties of wild type human ETHE1, including for the first time the redox properties of the mononuclear iron centre. We show that loss of function in these variants results from a combination of decreased protein stability and activity. Although structural assessment revealed that the protein fold is not perturbed by mutations, both variants have decreased thermal stabilities and higher proteolytic susceptibilities. ETHE1 wild type and variants bind 1 ± 0.2 mol iron/protein and no zinc; however, the variants exhibited only ≈ 10% of wild-type catalytically activity. Analysis of the redox properties of ETHE1 mononuclear iron centre revealed that the variants have lowered reduction potentials with respect to that of the wild type. This illustrates how point mutation-induced loss of function may arise via very discrete subtle conformational effects on the protein fold and active site chemistry, without extensive disruption of the protein structure or protein-cofactor association.

  19. Identification of a competitive translation determinant in the 3' untranslated region of alfalfa mosaic virus coat protein mRNA.

    Science.gov (United States)

    Hann, L E; Webb, A C; Cai, J M; Gehrke, L

    1997-01-01

    We report that the competitive translational activity of alfalfa mosaic virus coat protein mRNA (CP RNA), a nonadenylated mRNA, is determined in part by the 3' untranslated region (UTR). Competitive translation was characterized both in vitro, with cotranslation assays, and in vivo, with microinjected Xenopus laevis oocytes. In wheat germ extracts, coat protein synthesis was constant when a fixed amount of full-length CP RNA was cotranslated with increasing concentrations of competitor globin mRNA. However, translation of CP RNA lacking the 3' UTR decreased significantly under competitive conditions. RNA stabilities were equivalent. In X. laevis oocytes, which are translationally saturated and are an inherently competitive translational environment, full-length CP RNA assembled into large polysomes and coat protein synthesis was readily detectable. Alternatively, CP RNA lacking the 3' UTR sedimented as small polysomes, and little coat protein was detected. Again, RNA stabilities were equivalent. Site-directed mutagenesis was used to localize RNA sequences or structures required for competitive translation. Since the CP RNA 3' UTR has an unusually large number of AUG nucleotide triplets, two AUG-containing sites were altered in full-length RNA prior to oocyte injections. Nucleotide substitutions at the sequence GAUG, 20 nucleotides downstream of the coat protein termination codon, specifically reduced full-length CP RNA translation, while similar substitutions at the next AUG triplet had little effect on translation. The competitive influence of the 3' UTR could be explained by RNA-protein interactions that affect translation initiation or by ribosome reinitiation at downstream AUG codons, which would increase the number of ribosomes committed to coat protein synthesis. PMID:9121448

  20. Regulation of the mRNA half-life in breast cancer.

    Science.gov (United States)

    Griseri, Paola; Pagès, Gilles

    2014-08-10

    The control of the half-life of mRNA plays a central role in normal development and in disease progression. Several pathological conditions, such as breast cancer, correlate with deregulation of the half-life of mRNA encoding growth factors, oncogenes, cell cycle regulators and inflammatory cytokines that participate in cancer. Substantial stability means that a mRNA will be available for translation for a longer time, resulting in high levels of protein gene products, which may lead to prolonged responses that subsequently result in over-production of cellular mediators that participate in cancer. The stability of these mRNA is regulated at the 3'UTR level by different mechanisms involving mRNA binding proteins, micro-RNA, long non-coding RNA and alternative polyadenylation. All these events are tightly inter-connected to each other and lead to steady state levels of target mRNAs. Compelling evidence also suggests that both mRNA binding proteins and regulatory RNAs which participate to mRNA half-life regulation may be useful prognostic markers in breast cancers, pointing to a potential therapeutic approach to treatment of patients with these tumors. In this review, we summarize the main mechanisms involved in the regulation of mRNA decay and discuss the possibility of its implication in breast cancer aggressiveness and the efficacy of targeted therapy. PMID:25114848

  1. Single amino-acid substitution in the N-terminal arm altered the tetramer stability of rat muscle lactate dehydrogenase A

    Institute of Scientific and Technical Information of China (English)

    袁翀; 胡红雨; 许根俊

    2001-01-01

    Lactate dehydrogenase A (LDHA) is a well-characterized tetrameric enzyme. Its N-terminal arm, comprised of an (-helix and a (-strand, was suggested to be essential for subunit interactions. To examine the critical amino acid residues in the N-terminus involved in the subunit association, two single-point mutants, Leu3Pro (L3P) and Ile8Glu (I8E), have been constructed. We compared the stability of WT-LDHA (WT) and its variants by unfolding experiments. For WT, a dimeric but inactive intermediate was observed by size-exclusion chromatography at 0.6-0.8 mol/L GdmCl. Leu3Pro exists in an active tetrameric structure in aqueous solution as WT does, but it dissociates into dimers under lower concentration of GdmCl (0.2 mol/L). In aqueous solution, the Ile8Glu variant exists predominantly in the dimeric form with increased KM and decreased kcat as compared with those of WT and L3P. However, the activity of Ile8Glu increases significantly in the presence of sodium sulfate. In conclusion, two mutants are less stable than WT in oligomer structure. Results also support the fact that some residues in the N-terminal arm, especially the Leu8 in the (-structure, contribute the important binding energies to the dimerization of dimers, which might affect the assembly of the enzyme as well as the catalytic function.

  2. Splicing of juvenile and adult tau mRNA variants is regulated by thyroid hormone.

    OpenAIRE

    Aniello, F; Couchie, D; Bridoux, A M; Gripois, D.; Nunez, J.

    1991-01-01

    The effect of thyroid hormone on the expression of tau transcripts was studied during postnatal brain development. The level of tau mRNA was only slightly changed postnatally in the cerebral hemispheres of hypothyroid rats, whereas the level of tau mRNA in the cerebellum was maintained at a higher level than in the euthyroid controls. As shown by in situ hybridization studies, such an alteration in tau mRNA expression can be ascribed to an effect of thyroid hormone on the rate of migration of...

  3. c-jun N-terminal kinase is involved in AUUUA-mediated interleukin-3 mRNA turnover in mast cells.

    OpenAIRE

    Ming, X F; Kaiser, M.; Moroni, C

    1998-01-01

    Whereas signalling pathways involved in transcriptional control have been studied extensively, the pathways regulating mRNA turnover remain poorly understood. We are interested in the role of mRNA stability in cell activation and oncogenesis using PB-3c mast cells as a model system. In these cells the short-lived interleukin-3 (IL-3) mRNA is stabilized by ionomycin treatment and following oncogenesis. To identify the signalling pathways involved in these mechanisms, we analysed the effect of ...

  4. Investigating the role of TTP in mRNA decay and pre-mRNA processing

    OpenAIRE

    Reznik, Boris

    2012-01-01

    The AU-rich element (ARE) is a cis-encoded determinant within mRNA 3' untranslated regions (UTRs) that contributes to mRNA translation and stability in the cell. Tristetraprolin (TTP) is an RNA binding protein that specifically binds to mRNAs containing AREs and activates their rapid decay. TTP is rapidly activated following external stimulus and modulates the gene expression program of the responding cell. To better understand TTP- mediated mRNA decay activity, I identified the RNA binding p...

  5. Systems perspectives on mRNA processing

    Institute of Scientific and Technical Information of China (English)

    Adrienne E McKee; Pamela A Silver

    2007-01-01

    The application of genomic technologies to the study of mRNA processing is increasingly conducted in metazoan organisms in order to understand the complex events that occur during and after transcription. Large-scale systems analyses of mRNA-protein interactions and mRNA dynamics have revealed specificity in mRNA transcription, splicing, transport, translation, and turnover, and have begun to make connections between the different layers of mRNA processing. Here, we review global studies of post-transcriptional processes and discuss the challenges facing our understanding of mRNA regulation in metazoan organisms. In parallel, we examine genome-scale investigations that have expanded our knowledge of RNA-binding proteins and the networks of mRNAs that they regulate.

  6. Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences.

    Science.gov (United States)

    Rhoads, Robert E

    2016-01-01

    Recent advances have made it possible to synthesize mRNA in vitro that is relatively stable when introduced into mammalian cells, has a diminished ability to activate the innate immune response against exogenous (virus-like) RNA, and can be efficiently translated into protein. Synthetic methods have also been developed to produce mRNA with unique investigational properties such as photo-cross-linking, fluorescence emission, and attachment of ligands through click chemistry. Synthetic mRNA has been proven effective in numerous applications beneficial for human health such as immunizing patients against cancer and infections diseases, alleviating diseases by restoring deficient proteins, converting somatic cells to pluripotent stem cells to use in regenerative medicine therapies, and engineering the genome by making specific alterations in DNA. This introductory chapter provides background information relevant to the following 20 chapters of this volume that present protocols for these applications of synthetic mRNA. PMID:27236789

  7. Impact of STAT/SOCS mRNA Expression Levels after Major Injury

    Directory of Open Access Journals (Sweden)

    M. Brumann

    2014-01-01

    Full Text Available Background. Fulminant changes in cytokine receptor signalling might provoke severe pathological alterations after multiple trauma. The aim of this study was to evaluate the posttraumatic imbalance of the innate immune system with a special focus on the STAT/SOCS family. Methods. 20 polytraumatized patients were included. Blood samples were drawn 0 h–72 h after trauma; mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3 were quantified by qPCR. Results. IL-10 mRNA expression increased significantly in the early posttraumatic period. STAT 3 mRNA expressions showed a significant maximum at 6 h after trauma. SOCS 1 levels significantly decreased 6 h–72 h after trauma. SOCS 3 levels were significantly higher in nonsurvivors 6 h after trauma. Conclusion. We present a serial, sequential investigation in human neutrophil granulocytes of major trauma patients evaluating mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3. Posttraumatically, immune disorder was accompanied by a significant increase of IL-10 and STAT 3 mRNA expression, whereas SOCS 1 mRNA levels decreased after injury. We could demonstrate that death after trauma was associated with higher SOCS 3 mRNA levels already at 6 h after trauma. To support our results, further investigations have to evaluate protein levels of STAT/SOCS family in terms of posttraumatic immune imbalance.

  8. The optional long 5'-untranslated region of human ACAT1 mRNAs impairs the production of ACAT1 protein by promoting its mRNA decay

    Institute of Scientific and Technical Information of China (English)

    Xiaonan Zhao; Baoliang Song; Tayuan Chang; Boliang Li; Jia Chen; Lei Lei; Guangjing Hu; Ying Xiong; Jiajia Xu; Qin Li; Xinying Yang; Catherine C.Y.Chang

    2009-01-01

    We have previously reported that human ACAT1 mRNAs produce the 50 kDa protein using the AUG1397-1399 initiation codon,and also a minor 56 kDa isoform using the upstream in-frame GGC1274-1276initiation codon.The GGC1274-1276 codon is located at the optional long 5'-untranslated region(5'-UTR,nt 1-1396)of the mRNAs.The DNA sequences corresponding to this 5'-UTR are located in two different chromosomes,7 and 1.In the current work,we report that the optional long 5'-UTR significantly impairs the production of human ACAT1 protein initiated from the AUG1397-1399 codon,mainly by promoting its mRNA decay.The western blot analyses indicated that the optional long 5'-UTR potently impaired the production of different proteins initiated from the AUG1397-1399codon,meaning that this impairing effect was not influenced by the 3'-UTR or the coding sequence of ACAT1 mRNA.The results of reverse transcription-quantitative polymerase chain reaction demonstrated that this 5'-UTR dramatically reduced the contents of its linked mRNAs.Analyses of the protein to mRNA ratios showed that this 5'-UTR mainly decreased its mRNA stability rather than altering its translational efficiency.We next performed the plasmid transfection experiments and used actinomycin D to inhibit transcription.The results showed that this 5'-UTR promoted its mRNA decay.Additional transfection and nucleofection experiments using RNAs prepared in vitro illustrated that,in both the cytoplasm and the nucleus of cells,the optional long 5'-UTR-linked mRNAs decayed faster than those without the link.Overall,our study brings new insight to the regulation of the human ACAT1 gene expression at the post-transcription level.

  9. Smectite alteration

    International Nuclear Information System (INIS)

    This report contains the proceedings of a second workshop in Washington DC December 8-9, 1983 on the alteration of smectites intended for use as buffer materials in the long-term containment of nuclear wastes. It includes extended summaries of all presentations and a transcript of the detailed scientific discussion. The discussions centered on three main questions: What is the prerequisite for and what is the precise mechanism by which smectite clays may be altered to illite. What are likly sources of potassium with respect to the KBS project. Is it likely that the conversion of smectite to illite will be of importance in the 10 5 to the 10 6 year time frame. The workshop was convened to review considerations and conclusions in connection to these questions and also to broaden the discussion to consider the use of smectite clays as buffer materials for similar applications in different geographical and geological settings. SKBF/KBS technical report 83-03 contains the proceedings from the first workshop on these matters that was held at the State University of New York, Buffalo May 26-27, 1982. (Author)

  10. Kinetin improves IKBKAP mRNA splicing in patients with familial dysautonomia

    OpenAIRE

    Axelrod, Felicia B.; Liebes, Leonard; Gold-von Simson, Gabrielle; Mendoza, Sandra; Mull, James; Leyne, Maire; Norcliffe-Kaufmann, Lucy; Kaufmann, Horacio; Slaugenhaupt, Susan A.

    2011-01-01

    Familial dysautonomia (FD) is caused by an intronic splice mutation in the IKBKAP gene that leads to partial skipping of exon 20 and tissue-specific reduction in I-κ-B kinase complex associated protein/ elongation protein 1 (IKAP/ELP-1) expression. Kinetin (6-furfurylaminopurine) has been shown to improve splicing and increase wild-type IKBKAP mRNA and IKAP protein expression in FD cell lines and carriers. To determine if oral kinetin treatment could alter mRNA splicing in FD subjects and was...

  11. Adaptive and maladaptive expression of the mRNA regulatory protein HuR

    Institute of Scientific and Technical Information of China (English)

    Suman; Govindaraju; Beth; S; Lee

    2013-01-01

    The RNA-binding proteins involved in regulation of mRNA post-transcriptional processing and translation control the fates of thousands of mRNA transcripts and basic cellular processes. The best studied of these, HuR, is well characterized as a mediator of mRNA stability and translation, and more recently, as a factor in nuclear functions such as pre-mRNA splicing. Due to HuR’s role in regulating thousands of mRNA transcripts, including those for other RNA-binding proteins, HuR can act as a master regulator of cell survival and proliferation. HuR itself is subject to multiple post-translationa modifications including regulation of its nucleocytoplasmic distribution. However, the mechanisms that govern HuR levels in the cell have only recently begun to be defined. These mechanisms are critical to cell health, as it has become clear in recent years that aberrant expression of HuR can lead alternately to decreased cell viability or to promotion of pathological proliferation and invasiveness. HuR is expressed as alternate mRNAs that vary in their untranslated regions, leading to differences in transcript stability and translatability. Multiple transcription factors and modulators of mRNA stability that regulate HuR mRNA expression have been identified. In addition, translation of HuR is regulated by numerous microRNAs, several of which have been demonstrated to have anti-tumor properties due to their suppression of HuR expression. This review summarizes the current state of knowledge of the factors that regulate HuR expression, along with the circumstances under which these factors contribute to cancer and inflammation.

  12. Hyperosmolarity regulates SOX9 mRNA posttranscriptionally in human articular chondrocytes

    OpenAIRE

    Tew, Simon R.; Peffers, Mandy J.; McKay, Tristan R; Lowe, Emma T.; Khan, Wasim S; Hardingham, Timothy E.; Clegg, Peter D

    2009-01-01

    The transcription factor SOX9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. We previously showed that activation of p38 MAPK by cycloheximide in human chondrocytes leads to stabilization of SOX9 mRNA (Tew SR and Hardingham TE. J Biol Chem 281: 39471–39479, 2006). In this study we investigated whether regulation of p38 MAPK caused by changes in osmotic pressure could control SOX9 mRNA levels expression by a similar mechanism. Primary...

  13. The transcription factor ERG recruits CCR4-NOT to control mRNA decay and mitotic progression.

    Science.gov (United States)

    Rambout, Xavier; Detiffe, Cécile; Bruyr, Jonathan; Mariavelle, Emeline; Cherkaoui, Majid; Brohée, Sylvain; Demoitié, Pauline; Lebrun, Marielle; Soin, Romuald; Lesage, Bart; Guedri, Katia; Beullens, Monique; Bollen, Mathieu; Farazi, Thalia A; Kettmann, Richard; Struman, Ingrid; Hill, David E; Vidal, Marc; Kruys, Véronique; Simonis, Nicolas; Twizere, Jean-Claude; Dequiedt, Franck

    2016-07-01

    Control of mRNA levels, a fundamental aspect in the regulation of gene expression, is achieved through a balance between mRNA synthesis and decay. E26-related gene (Erg) proteins are canonical transcription factors whose previously described functions are confined to the control of mRNA synthesis. Here, we report that ERG also regulates gene expression by affecting mRNA stability and identify the molecular mechanisms underlying this function in human cells. ERG is recruited to mRNAs via interaction with the RNA-binding protein RBPMS, and it promotes mRNA decay by binding CNOT2, a component of the CCR4-NOT deadenylation complex. Transcriptome-wide mRNA stability analysis revealed that ERG controls the degradation of a subset of mRNAs highly connected to Aurora signaling, whose decay during S phase is necessary for mitotic progression. Our data indicate that control of gene expression by mammalian transcription factors may follow a more complex scheme than previously anticipated, integrating mRNA synthesis and degradation. PMID:27273514

  14. Amygdala kindling increases fear responses and decreases glucocorticoid receptor mRNA expression in hippocampal regions.

    Science.gov (United States)

    Kalynchuk, Lisa E; Meaney, Michael J

    2003-12-01

    Amygdala kindling dramatically increases fearful behavior in rats. Because kindling-induced fear increases in magnitude as rats receive more stimulations, kindling provides an excellent model for studying the nature and neural mechanisms of fear sensitization. In the present experiment, we studied whether the development of kindling-induced fear is related to changes in glucocorticoid receptor (GR) mRNA expression in various brain regions. Rats received 20, 60 or 100 amygdala kindling stimulations or 100 sham stimulations. One day after the final stimulation, their fearful behavior was assessed in an unfamiliar open field. Then, the rats were sacrificed and their brains were processed for in situ hybridization of GR mRNA expression. We found that compared with the sham-stimulated rats, the rats that received 60 or 100 kindling stimulations were significantly more fearful in the open field and also had significantly less GR mRNA expression in the dentate gyrus and CA1 subfield of the hippocampus. Importantly, the changes in fearful behavior were significantly correlated with the changes in GR mRNA expression. These results suggest that alterations in GR mRNA expression in hippocampal regions may play a role in the development of kindling-induced fear.

  15. Acute stress increases neuropsin mRNA expression in the mouse hippocampus through the glucocorticoid pathway.

    Science.gov (United States)

    Harada, Akiko; Shiosaka, Sadao; Ishikawa, Yasuyuki; Komai, Shoji

    2008-05-01

    Stress affects synaptic plasticity and may alter various types of behaviour, including anxiety or memory formation. In the present study, we examined the effects of acute stress (1 h restraint with or without tail-shock) on mRNA levels of a plasticity-related serine protease neuropsin (NP) in the hippocampus using semiquantitative RT-PCR and in situ hybridization. We found that NP mRNA expression was dramatically increased shortly after exposure to the acute restraint tail-shock stress and remained at high level for at least 24 h. The level of NP mRNA would be correlated to the elevated plasma concentration of the glucocorticoid corticosterone (CORT) and to the stress intensity. Application of CORT either onto primary cultured hippocampal neurons (5 nM) or in vivo to adrenalectomized (ADX) mice (10 mg/kg B.W., s.c.) mimicked the effect of stress and significantly elevated NP mRNA. These results suggest that the upregulation of NP mRNA after stress is CORT-dependent and point to a role for neuropsin in stress-induced neuronal plasticity.

  16. Diaphragm Unloading via Controlled Mechanical Ventilation Alters the Gene Expression Profile

    OpenAIRE

    DeRuisseau, Keith C.; Shanely, R Andrew; Akunuri, Nagabhavani; Hamilton, Marc T.; Van Gammeren, Darin; Zergeroglu, A. Murat; McKenzie, Michael; Powers, Scott K.

    2005-01-01

    Rationale: Prolonged controlled mechanical ventilation results in diaphragmatic inactivity and promotes oxidative injury, atrophy, and contractile dysfunction in this important inspiratory muscle. However, the impact of controlled mechanical ventilation on global mRNA alterations in the diaphragm remains unknown.

  17. A ribonucleoprotein complex protects the interleukin-6 mRNA from degradation by distinct herpesviral endonucleases.

    Directory of Open Access Journals (Sweden)

    Mandy Muller

    2015-05-01

    Full Text Available During lytic Kaposi's sarcoma-associated herpesvirus (KSHV infection, the viral endonuclease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA. However, select mRNAs escape SOX-induced cleavage and remain robustly expressed. Prominent among these is interleukin-6 (IL-6, a growth factor important for survival of KSHV infected B cells. IL-6 escape is notable because it contains a sequence within its 3' untranslated region (UTR that can confer protection when transferred to a SOX-targeted mRNA, and thus overrides the endonuclease targeting mechanism. Here, we pursued how this protective RNA element functions to maintain mRNA stability. Using affinity purification and mass spectrometry, we identified a set of proteins that associate specifically with the protective element. Although multiple proteins contributed to the escape mechanism, depletion of nucleolin (NCL most severely impacted protection. NCL was re-localized out of the nucleolus during lytic KSHV infection, and its presence in the cytoplasm was required for protection. After loading onto the IL-6 3' UTR, NCL differentially bound to the translation initiation factor eIF4H. Disrupting this interaction, or depleting eIF4H, reinstated SOX targeting of the RNA, suggesting that interactions between proteins bound to distant regions of the mRNA are important for escape. Finally, we found that the IL-6 3' UTR was also protected against mRNA degradation by the vhs endonuclease encoded by herpes simplex virus, despite the fact that its mechanism of mRNA targeting is distinct from SOX. These findings highlight how a multitude of RNA-protein interactions can impact endonuclease targeting, and identify new features underlying the regulation of the IL-6 mRNA.

  18. A ribonucleoprotein complex protects the interleukin-6 mRNA from degradation by distinct herpesviral endonucleases.

    Science.gov (United States)

    Muller, Mandy; Hutin, Stephanie; Marigold, Oliver; Li, Kathy H; Burlingame, Al; Glaunsinger, Britt A

    2015-05-01

    During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, the viral endonuclease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs escape SOX-induced cleavage and remain robustly expressed. Prominent among these is interleukin-6 (IL-6), a growth factor important for survival of KSHV infected B cells. IL-6 escape is notable because it contains a sequence within its 3' untranslated region (UTR) that can confer protection when transferred to a SOX-targeted mRNA, and thus overrides the endonuclease targeting mechanism. Here, we pursued how this protective RNA element functions to maintain mRNA stability. Using affinity purification and mass spectrometry, we identified a set of proteins that associate specifically with the protective element. Although multiple proteins contributed to the escape mechanism, depletion of nucleolin (NCL) most severely impacted protection. NCL was re-localized out of the nucleolus during lytic KSHV infection, and its presence in the cytoplasm was required for protection. After loading onto the IL-6 3' UTR, NCL differentially bound to the translation initiation factor eIF4H. Disrupting this interaction, or depleting eIF4H, reinstated SOX targeting of the RNA, suggesting that interactions between proteins bound to distant regions of the mRNA are important for escape. Finally, we found that the IL-6 3' UTR was also protected against mRNA degradation by the vhs endonuclease encoded by herpes simplex virus, despite the fact that its mechanism of mRNA targeting is distinct from SOX. These findings highlight how a multitude of RNA-protein interactions can impact endonuclease targeting, and identify new features underlying the regulation of the IL-6 mRNA. PMID:25965334

  19. Two seemingly homologous noncoding RNAs act hierarchically to activate glmS mRNA translation.

    Directory of Open Access Journals (Sweden)

    Johannes H Urban

    2008-03-01

    Full Text Available Small noncoding RNAs (sRNA can function as posttranscriptional activators of gene expression to regulate stress responses and metabolism. We here describe the mechanisms by which two sRNAs, GlmY and GlmZ, activate the Escherichia coli glmS mRNA, coding for an essential enzyme in amino-sugar metabolism. The two sRNAs, although being highly similar in sequence and structure, act in a hierarchical manner. GlmZ, together with the RNA chaperone, Hfq, directly activates glmS mRNA translation by an anti-antisense mechanism. In contrast, GlmY acts upstream of GlmZ and positively regulates glmS by antagonizing GlmZ RNA inactivation. We also report the first example, to our knowledge, of mRNA expression being controlled by the poly(A status of a chromosomally encoded sRNA. We show that in wild-type cells, GlmY RNA is unstable due to 3' end polyadenylation; whereas in an E. coli pcnB mutant defective in RNA polyadenylation, GlmY is stabilized and accumulates, which in turn stabilizes GlmZ and causes GlmS overproduction. Our study reveals hierarchical action of two well-conserved sRNAs in a complex regulatory cascade that controls the glmS mRNA. Similar cascades of noncoding RNA regulators may operate in other organisms.

  20. Involvement of hGLD-2 in cytoplasmic polyadenylation of human p53 mRNA

    DEFF Research Database (Denmark)

    Glahder, Jacob-Andreas Harald; Norrild, Bodil

    2011-01-01

    -like elements was performed by luciferase reporter assays, qPCR, and poly(A) assays. Herein, we report the down regulation of a luciferase reporter fused to the p53 3'-UTR, when human CPE-binding protein 1 (hCPEB1) is overexpressed. This inhibition is partially rescued when hCPEB1fused to hGLD-2 [a human...... cytoplasmic poly(A) polymerase] is overexpressed instead. The stability of a luciferase mRNA containing the p53 3'-UTR downstream, is decreased when hCPEB1 is overexpressed as seen by qPCR. Expression of hGLD-2 restores the mRNA stability. This is due to elongation of the poly(A) tail as seen by a PCR...

  1. Decrease in class pi glutathione transferase mRNA levels by ultraviolet irradiation of cultured rat keratinocytes

    International Nuclear Information System (INIS)

    The effect of ultraviolet (UV) B irradiation on pi class glutathione transferase (GST-P) gene expression was examined in cultured rat keratinocytes. Immunoblotting demonstrated GST-P to be the major GST form in the cells, and it was significantly decreased following irradiation. Northern blot analysis revealed that the mRNA decreased to 10-25% of the initial value 24 h after irradiation at a dose of 40 mJ/cm2. No remarkable changes were observed at earlier time points. Hydrogen peroxide treatment enhanced GST-P mRNA expression, with a 70% increase at 250 μM concentration. Alterations in possible trans-acting factors were examined to clarify the mechanism of repression by UV irradiation. c-Jun mRNA was induced 3.5-fold at 4 h after irradiation, but by 24 h fell to a lower level than that observed initially. c-Fos mRNA was increased 10-fold at 1 h but was completely suppressed at 12 and 24 h. Thus, the changes of c-Jun and c-Fos mRNA differed from that of GST-P mRNA. The level of mRNA for silencer factor-B was decreased to less than 10% at 12 h. UV irradiation of cells transfected with the chloramphenicol acetyltransferase (CAT) reporter gene containing enhancer (GPE I) or silencer regions of the GST-P gene did not suppress CAT activity. Although basal expression of the GST-P gene was mainly dependent on GPE I, altered expression of c-jun, c-fos and other genes coding for factors possibly trans-acting on GPE I did not appear to be responsible for the decreased GST-P mRNA levels. (author)

  2. Post-transcriptional regulation of cytokine mRNA controls the initiation and resolution of inflammation.

    OpenAIRE

    Mino, Takashi; Takeuchi, Osamu

    2013-01-01

    Cytokines are critical mediators of inflammation and host defense. Cytokine production is regulated during transcription and post-transcription. Post-transcriptional regulation modifies mRNA stability and translation, allowing for the rapid and flexible control of gene expression, which is important for coordinating the initiation and resolution of inflammation. We review here a variety of post-transcriptional control mechanisms that regulate inflammation and discuss how these mechanisms are ...

  3. Circadian Rhythm of Surfactant Protein A, B and C mRNA in Rats

    OpenAIRE

    Kim, Chung Mi; Sohn, Jang Won; Yoon, Ho Joo; Shin, Dong Ho; Park, Sung Soo

    2003-01-01

    Background: All organisms have developed an internal timing system capable of reacting to and anticipating environmental stimuli with a program of appropriately timed metabolic, physiologic and behavioral events. The alveolar epithelial type II cell of the mammalian lung synthesizes, stores, and secretes a lipoprotein pulmonary surfactant, which functions to stabilize alveoli at low lung volumes. Methods: The authors investigated the diurnal variation of surfactant protein A, B and C mRNA acc...

  4. Distinct regulation of vasoactive intestinal peptide (VIP) expression at mRNA and peptide levels in human neuroblastoma cells.

    Science.gov (United States)

    Agoston, D V; Colburn, S; Krajniak, K G; Waschek, J A

    1992-05-25

    Neuronal differentiation was induced in cultures of the human neuroblastoma cell line subclone SH-SY5Y by 14-day treatment with dibutyryl cAMP (dBcAMP), retinoic acid, and phorbol 12-myristate 13-acetate (PMA). An approximate 4-fold increase in vasoactive intestinal peptide (VIP) mRNA concentration was observed after differentiation with retinoic acid, whereas no change in VIP mRNA concentration was observed after differentiation with dBcAMP or PMA. A short-term treatment of cells with PMA did however result in a 5-fold transient increase in VIP mRNA; prior differentiation with retinoic acid or dBcAMP diminished this effect. Observed increases in VIP mRNA were in all cases accompanied by increases in VIP immunoreactivity. Remarkably, however, long-term treatment of cells with dBcAMP, which caused no change in mRNA levels, resulted in a six-fold increase in VIP immunoreactivity. Acute (36-h) treatment with carbachol also caused an increase in VIP immunoreactivity (about 2-fold, and blocked by atropine) without an increase in VIP mRNA level. Thus, a quantitative change in gene transcription or mRNA stability appears not to be a prerequisite for increased VIP expression, indicating that regulation can occur at translational or post-translational steps.

  5. Cable Stability

    CERN Document Server

    Bottura, L

    2014-01-01

    Superconductor stability is at the core of the design of any successful cable and magnet application. This chapter reviews the initial understanding of the stability mechanism, and reviews matters of importance for stability such as the nature and magnitude of the perturbation spectrum and the cooling mechanisms. Various stability strategies are studied, providing criteria that depend on the desired design and operating conditions.

  6. Identification of European starling GnRH-I precursor mRNA and its seasonal regulation.

    Science.gov (United States)

    Ubuka, Takayoshi; Cadigan, Penelope A; Wang, Ariel; Liu, Jennifer; Bentley, George E

    2009-07-01

    Songbirds show dynamic seasonal changes in their reproductive activities during the year. Gonadotropin-releasing hormone-I (GnRH-I) is critical for the control of reproduction in vertebrates. The molecular mechanisms controlling reproduction are not well understood in songbirds, largely because the GnRH-I precursor polypeptide gene was unknown until now. Here, we report the complete sequence and seasonal regulation of GnRH-I precursor polypeptide mRNA in a songbird, European starling (Sturnus vulgaris). The translated starling GnRH-I precursor polypeptide contained an amino acid sequence that can be processed into chicken GnRH-I peptide (pEHWSYGLQPG-NH(2)). However, the overall homology of GnRH-I precursor polypeptide (including a 23 amino acid signal peptide, the decapeptide hormone and Gly-Lys-Arg cleavage site followed by 55 amino acid GnRH-associated peptide sequences) between starling and chicken was only 58%. GnRH-I mRNA and GnRH-I peptide were observed to be co-localized in the preoptic area of sexually mature birds using in situ hybridization and immunocytochemistry. GnRH-I mRNA exhibited large variance in photosensitive birds, and converged to a high level in photostimulated birds. Subsequently, GnRH-I mRNA decreased to below detectability in most of the photorefractory birds. Changes were also observed in GnRH-I peptide levels, although changes in GnRH-I peptide were not as marked. Our data indicate that GnRH-I mRNA synthesis commences but is variable in photosensitive birds, stabilizes in photostimulated birds, then ceases when birds become photorefractory. Finer-scale investigation into temporal regulation of GnRH-I precursor polypeptide mRNA will provide insight into its regulation by environmental, social and physiological cues. PMID:19362556

  7. New insights on the role of epigenetic alterations in hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Frau M

    2014-06-01

    Full Text Available Maddalena Frau,1 Claudio F Feo,2 Francesco Feo,1 Rosa M Pascale11Department of Clinical and Experimental Medicine, Division of Experimental Pathology and Oncology, 2Department of Clinical and Experimental Medicine, Division of Surgery. University of Sassari, Sassari, ItalyAbstract: Emerging evidence assigns to epigenetic mechanisms heritable differences in gene function that come into being during cell development or via the effect of environmental factors. Epigenetic deregulation is strongly involved in the development of hepatocellular carcinoma (HCC. It includes changes in methionine metabolism, promoter hypermethylation, or increased proteasomal degradation of oncosuppressors, as well as posttranscriptional deregulation by microRNA or messenger RNA (mRNA binding proteins. Alterations in the methylation of the promoter of methyl adenosyltransferase MAT1A and MAT2A genes in HCC result in decreased S-adenosylmethionine level, global DNA hypomethylation, and deregulation of signal transduction pathways linked to methionine metabolism and methyl adenosyltransferases activity. Changes in S-adenosylmethionine levels may also depend on MAT1A mRNA destabilization associated with MAT2A mRNA stabilization by specific proteins. Decrease in MAT1A expression has also been attributed to miRNA upregulation in HCC. A complex deregulation of miRNAs is also strongly involved in hepatocarcinogenesis, with up-regulation of different miRNAs targeting oncosuppressor genes and down-regulation of miRNAs targeting genes involved in cell-cycle and signal transduction control. Oncosuppressor gene down-regulation in HCC is also induced by promoter hypermethylation or posttranslational deregulation, leading to proteasomal degradation. The role of epigenetic changes in hepatocarcinogenesis has recently suggested new promising therapeutic approaches for HCC on the basis of the administration of methylating agents, inhibition of methyl adenosyltransferases, and restoration

  8. Food deprivation decreases vasopressin mRNA in the supraoptic and paraventricular nuclei of the hypothalamus in rats.

    Directory of Open Access Journals (Sweden)

    Ogasa,Takashi

    1991-08-01

    Full Text Available We examined the effect of food deprivation for three days on hypothalamic arginine vasopressin (AVP mRNA in rats. Simultaneously the effect of water deprivation for the same period was examined as a model of dehydration. Levels of AVP mRNA in the supraoptic nucleus (SON and the paraventricular nucleus (PVN were determined by semiquantitative in situ hybridization histochemistry. Water deprivation increased AVP mRNA in both nuclei as previously reported. In contrast, food deprivation decreased AVP mRNA in these nuclei. The changes in AVP mRNA levels in the PVN were observed in the magnocellular subdivision of the nucleus. Plasma levels of ACTH and corticosterone were greatly increased in both treated groups of rats. Plasma AVP and osmolality levels were significantly elevated in water-deprived rats but not in food-deprived rats. These observations indicated that both food deprivation and water deprivation stimulated the pituitary-adrenal axis and that a reduction in AVP mRNA levels in food-deprived rats was caused by food deprivation but not by glucocorticoid feedback suppression nor by altered plasma osmolality.

  9. Recent innovations in mRNA vaccines.

    Science.gov (United States)

    Ulmer, Jeffrey B; Geall, Andrew J

    2016-08-01

    Nucleic acid-based vaccines are being developed as a means to combine the positive attributes of both live-attenuated and subunit vaccines. Viral vectors and plasmid DNA vaccines have been extensively evaluated in human clinical trials and have been shown to be safe and immunogenic, although none have yet been licensed for human use. Recently, mRNA based vaccines have emerged as an alternative approach. They promise the flexibility of plasmid DNA vaccines, without the need for electroporation, but with enhanced immunogenicity and safety. In addition, they avoid the limitations of anti-vector immunity seen with viral vectors, and can be dosed repeatedly. This review highlights the key papers published over the past few years and summarizes prospects for the near future.

  10. Interaction between thymidylate synthase and its cognate mRNA in zebrafish embryos.

    Directory of Open Access Journals (Sweden)

    Yuyan Zhang

    Full Text Available Thymidylate synthase (TS, which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 microM 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 microM ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

  11. Cell cycle-dependent regulation of Aurora kinase B mRNA by the Microprocessor complex.

    Science.gov (United States)

    Jung, Eunsun; Seong, Youngmo; Seo, Jae Hong; Kwon, Young-Soo; Song, Hoseok

    2014-03-28

    Aurora kinase B regulates the segregation of chromosomes and the spindle checkpoint during mitosis. In this study, we showed that the Microprocessor complex, which is responsible for the processing of the primary transcripts during the generation of microRNAs, destabilizes the mRNA of Aurora kinase B in human cells. The Microprocessor-mediated cleavage kept Aurora kinase B at a low level and prevented premature entrance into mitosis. The cleavage was reduced during mitosis leading to the accumulation of Aurora kinase B mRNA and protein. In addition to Aurora kinase B mRNA, the processing of other primary transcripts of miRNAs were also decreased during mitosis. We found that the cleavage was dependent on an RNA helicase, DDX5, and the association of DDX5 and DDX17 with the Microprocessor was reduced during mitosis. Thus, we propose a novel mechanism by which the Microprocessor complex regulates stability of Aurora kinase B mRNA and cell cycle progression.

  12. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells.

    Science.gov (United States)

    Carlile, Thomas M; Rojas-Duran, Maria F; Zinshteyn, Boris; Shin, Hakyung; Bartoli, Kristen M; Gilbert, Wendy V

    2014-11-01

    Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function--it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1-4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease.

  13. Gammaherpesviral gene expression and virion composition are broadly controlled by accelerated mRNA degradation.

    Directory of Open Access Journals (Sweden)

    Emma Abernathy

    2014-01-01

    Full Text Available Lytic gammaherpesvirus infection restricts host gene expression by promoting widespread degradation of cytoplasmic mRNA through the activity of the viral endonuclease SOX. Though generally assumed to be selective for cellular transcripts, the extent to which SOX impacts viral mRNA stability has remained unknown. We addressed this issue using the model murine gammaherpesvirus MHV68 and, unexpectedly, found that all stages of viral gene expression are controlled through mRNA degradation. Using both comprehensive RNA expression profiling and half-life studies we reveal that the levels of the majority of viral mRNAs but not noncoding RNAs are tempered by MHV68 SOX (muSOX activity. The targeting of viral mRNA by muSOX is functionally significant, as it impacts intracellular viral protein abundance and progeny virion composition. In the absence of muSOX-imposed gene expression control the viral particles display increased cell surface binding and entry as well as enhanced immediate early gene expression. These phenotypes culminate in a viral replication defect in multiple cell types as well as in vivo, highlighting the importance of maintaining the appropriate balance of viral RNA during gammaherpesviral infection. This is the first example of a virus that fails to broadly discriminate between cellular and viral transcripts during host shutoff and instead uses the targeting of viral messages to fine-tune overall gene expression.

  14. Differential expression of melanopsin mRNA and protein in the Brown Norwegian rats

    DEFF Research Database (Denmark)

    Hannibal, Jens; Georg, Birgitte; Fahrenkrug, Jan

    2012-01-01

    on melanopsin expression in the pigmented retina of the Brown Norwegian rat (Rattus norvegicus). The diurnal and circadian expressions were examined in retinal extracts from rats euthanized every 4 h during a 24 h light/dark (LD) and a 24 h dark cycle (DD) using quantitative real-time PCR and Western blotting...... in immunoreactivity in the dendritic processes. In conclusion we found that light and darkness are important for regulation of melanopsin protein expression whereas input from a retinal networks regulates melanopsin mRNA expression. It is likely to speculate that altered level of melanopsin is one way in which....... To study whether light regulates melanopsin expression, rats were sacrificed after being placed in either constant light (LL) or darkness for 3 or 21 d. Flat mount retinas from animals kept during either LL or DD were also examined by immunohistochemistry. Melanopsin mRNA expression displayed a significant...

  15. Pattern of mRNA expression of β-defensins in basal cell carcinoma

    International Nuclear Information System (INIS)

    Although the human β-defensins hBDs today seem to have diverse functional activities in innate antimicrobial immunity, a few reports also indicated an altered expression of these antimicrobial peptides (AMPs) in tissues of cancers such as oral squamous cell carcinoma. The present work was aimed on the study of hBD gene expression in basal cell carcinoma (BCC) which is the most common cancer in humans. Twenty-two non-ulcerated BCCs (12 nodular type, 10 superficial type) have been analysed for the presence of hBD (1–3) mRNA by quantitative real-time RT-PCR. As controls, non-lesional skin specimens of BCC patients as well as samples of healthy subjects were assessed by RT-PCR. hBD-1 levels in healthy controls and non-lesional skin of BCC patients were significantly (P < 0.05) higher than the levels observed in tumour tissue. Moreover, BCCs showed significantly (P < 0.05) increased mRNA expression of hBD-2 as compared to controls. There was no significant (P > 0.05) difference between lesional mRNA levels for hBD-3 and those levels observed in controls. The mRNA expression of hBDs (1–3) found in nodular and superficial BCCs did not significantly (P > 0.05) differ. The gene expression patterns of hBD-1 and hBD-2 are for the first time shown to be significantly altered in non-ulcerated BCCs as compared to intra-individual and inter-individual controls, respectively. The present findings may indicate that beside the antimicrobial activity of AMPs, hBDs may also play a role in the pathogenesis of BCC. However, functional and immunohistological studies investigating hBDs in patients with BCC are needed to confirm our data

  16. Dynein light chain binding to a 3′-untranslated sequence mediates parathyroid hormone mRNA association with microtubules

    Science.gov (United States)

    Epstein, Eyal; Sela-Brown, Alin; Ringel, Israel; Kilav, Rachel; King, Stephen M.; Benashski, Sharon E.; Yisraeli, Joel K.; Silver, Justin; Naveh-Many, Tally

    2000-01-01

    The 3′-untranslated region (UTR) of mRNAs binds proteins that determine mRNA stability and localization. The 3′-UTR of parathyroid hormone (PTH) mRNA specifically binds cytoplasmic proteins. We screened an expression library for proteins that bind the PTH mRNA 3′-UTR, and the sequence of 1 clone was identical to that of the dynein light chain LC8, a component of the dynein complexes that translocate cytoplasmic components along microtubules. Recombinant LC8 binds PTH mRNA 3′-UTR, as shown by RNA electrophoretic mobility shift assay. We showed that PTH mRNA colocalizes with microtubules in the parathyroid gland, as well as with a purified microtubule preparation from calf brain, and that this association was mediated by LC8. To our knowledge, this is the first report of a dynein complex protein binding an mRNA. The dynein complex may be the motor that is responsible for transporting mRNAs to specific locations in the cytoplasm and for the consequent is asymmetric distribution of translated proteins in the cell. PMID:10683380

  17. ErbB3 mRNA leukocyte levels as a biomarker for major depressive disorder

    Directory of Open Access Journals (Sweden)

    Milanesi Elena

    2012-09-01

    Full Text Available Abstract Background In recent years, the identification of peripheral biomarkers that are associated with psychiatric diseases, such as Major Depressive Disorder (MDD, has become relevant because these biomarkers may improve the efficiency of the differential diagnosis process and indicate targets for new antidepressant drugs. Two recent candidate genes, ErbB3 and Fgfr1, are growth factors whose mRNA levels have been found to be altered in the leukocytes of patients that are affected by bipolar disorder in a depressive state. On this basis, the aim of the study was to determine if ErbB3 and Fgfr1 mRNA levels could be a biomarkers of MDD. Methods We measured by Real Time PCR ErbB3 and Fgfr1 mRNA expression levels in leukocytes of MDD patients compared with controls. Successively, to assess whether ErbB3 mRNA levels were influenced by previous antidepressant treatment we stratified our patients sample in two cohorts, comparing drug-naive versus drug-free patients. Moreover, we evaluated the levels of the transcript in MDD patients after 12 weeks of antidepressant treatment, and in prefrontal cortex of rats stressed and treated with an antidepressant drug of the same class. Results These results showed that ErbB3 but not Fgfr1 mRNA levels were reduced in leukocytes of MDD patients compared to healthy subjects. Furthermore, ErbB3 levels were not affected by antidepressant treatment in either human or animal models Conclusions Our data suggest that ErbB3 might be considered as a biomarker for MDD and that its deficit may underlie the pathopsysiology of the disease and is not a consequence of treatment. Moreover the study supports the usefulness of leukocytes as a peripheral system for identifying biomarkers in psychiatric diseases.

  18. Sperm mRNA transcripts are indicators of sub-chronic low dose testicular injury in the Fischer 344 rat.

    Directory of Open Access Journals (Sweden)

    Sara E Pacheco

    Full Text Available Current human reproductive risk assessment methods rely on semen and serum hormone analyses, which are not easily comparable to the histopathological endpoints and mating studies used in animal testing. Because of these limitations, there is a need to develop universal evaluations that reliably reflect male reproductive function. We hypothesized that toxicant-induced testicular injury can be detected in sperm using mRNA transcripts as indicators of insult. To test this, we exposed adult male Fischer 344 rats to low doses of model testicular toxicants and classically characterized the testicular injury while simultaneously evaluating sperm mRNA transcripts from the same animals. Overall, this study aimed to: 1 identify sperm transcripts altered after exposure to the model testicular toxicant, 2,5-hexanedione (HD using microarrays; 2 expand on the HD-induced transcript changes in a comprehensive time course experiment using qRT-PCR arrays; and 3 test these injury indicators after exposure to another model testicular toxicant, carbendazim (CBZ. Microarray analysis of HD-treated adult Fischer 344 rats identified 128 altered sperm mRNA transcripts when compared to control using linear models of microarray analysis (q<0.05. All transcript alterations disappeared after 3 months of post-exposure recovery. In the time course experiment, time-dependent alterations were observed for 12 candidate transcripts selected from the microarray data based upon fold change and biological relevance, and 8 of these transcripts remained significantly altered after the 3-month recovery period (p<0.05. In the last experiment, 8 candidate transcripts changed after exposure to CBZ (p<0.05. The two testicular toxicants produced distinct molecular signatures with only 4 overlapping transcripts between them, each occurring in opposite directions. Overall, these results suggest that sperm mRNA transcripts are indicators of low dose toxicant-induced testicular injury in the rat.

  19. MAPKAP Kinase 2 Blocks Tristetraprolin-directed mRNA Decay by Inhibiting CAF1 Deadenylase Recruitment

    OpenAIRE

    Marchese, Francesco P.; Aubareda, Anna; Tudor, Corina; Saklatvala, Jeremy; Clark, Andrew R; Dean, Jonathan L. E.

    2010-01-01

    Tristetraprolin (TTP) directs its target AU-rich element (ARE)-containing mRNAs for degradation by promoting removal of the poly(A) tail. The p38 MAPK pathway regulates mRNA stability via the downstream kinase MAPK-activated protein kinase 2 (MAPKAP kinase 2 or MK2), which phosphorylates and prevents the mRNA-destabilizing function of TTP. We show that deadenylation of endogenous ARE-containing tumor necrosis factor mRNA is inhibited by p38 MAPK. To investigate whether phosphorylation of TTP ...

  20. Regional induction of c-fos and heat shock protein-72 mRNA following fluid-percussion brain injury in the rat

    Energy Technology Data Exchange (ETDEWEB)

    Raghupathi, R.; Welsh, F.A.; Gennarelli, T.A. [Univ. of Pennsylvania, Philadelphia, PA (United States)] [and others

    1995-05-01

    To evaluate the cellular response to traumatic brain injury, the expression of mRNA for c-fos and the 72-kDa heat shock protein (hsp72) was determined using in situ hybridization following lateral fluid-percussion injury (2.2-2.4 atm) in rat brain. At 2 h after injury, induction of c-fos mRNA was restricted to regions of the cortex surrounding the contusion area. An increase in c-fos mRNA, but not hsp72 mRNA, was observed bilaterally in the CA{sub 3} subfield of the hippocampus and the granule cells of the dentate gyrus and in the thalamus ipsilateral to the impact site. By 6 h, increased expression of c-fos mRNA was observed only in the corpus callosum on the impact side; hsp72 mRNA persisted in the deep cortical layers and upper layers of the subcortical white matter below the site of maximal injury. By 24 h, both c-fos and hsp72 mRNA had returned to control levels in all regions of the brain. These results demonstrate that lateral fluid-percussion brain injury triggers regionally and temporally specific expression of c-fos and hsp72 mRNA, which may be suggestive of differential neurochemical alterations in neurons and glia following experimental brain injury. 33 refs., 3 figs., 1 tab.

  1. Database for mRNA half-life of 19 977 genes obtained by DNA microarray analysis of pluripotent and differentiating mouse embryonic stem cells.

    Science.gov (United States)

    Sharova, Lioudmila V; Sharov, Alexei A; Nedorezov, Timur; Piao, Yulan; Shaik, Nabeebi; Ko, Minoru S H

    2009-02-01

    Degradation of mRNA is one of the key processes that control the steady-state level of gene expression. However, the rate of mRNA decay for the majority of genes is not known. We successfully obtained the rate of mRNA decay for 19 977 non-redundant genes by microarray analysis of RNA samples obtained from mouse embryonic stem (ES) cells. Median estimated half-life was 7.1 h and only genes, including Prdm1, Myc, Gadd45 g, Foxa2, Hes5 and Trib1, showed half-life less than 1 h. In general, mRNA species with short half-life were enriched among genes with regulatory functions (transcription factors), whereas mRNA species with long half-life were enriched among genes related to metabolism and structure (extracellular matrix, cytoskeleton). The stability of mRNAs correlated more significantly with the structural features of genes than the function of genes: mRNA stability showed the most significant positive correlation with the number of exon junctions per open reading frame length, and negative correlation with the presence of PUF-binding motifs and AU-rich elements in 3'-untranslated region (UTR) and CpG di-nucleotides in the 5'-UTR. The mRNA decay rates presented in this report are the largest data set for mammals and the first for ES cells.

  2. Differential regulation of amyloid-β-protein mRNA expression within hippocampal neuronal subpopulations in Alzheimer disease

    International Nuclear Information System (INIS)

    The authors have mapped the neuroanatomical distribution of amyloid-β-protein mRNA within neuronal subpopulations of the hippocampal formation in the cynomolgus monkey (Macaca fascicularis), normal aged human, and patients with Alzheimer disease. Amyloid-β-protein mRNA appears to be expressed in all hippocampal neurons, but at different levels of abundance. In the central nervous system of monkey and normal aged human, image analysis shows that neurons of the dentate gyrus and cornu Ammonis fields contain a 2.5-times-greater hybridization signal than is present in neurons of the subiculum and entorhinal cortex. In contrast, in the Alzheimer disease hippocampal formation, the levels of amyloid-β-protein mRNA in the cornu Ammonis field 3 and parasubiculum are equivalent. These findings suggest that within certain neuronal subpopulations cell type-specific regulation of amyloid-β-protein gene expression may be altered in Alzheimer disease

  3. Functional challenge affects aquaporin mRNA abundance in mouse blastocysts

    DEFF Research Database (Denmark)

    Offenberg, Hanne Kjær; Thomsen, Preben Dybdahl

    2005-01-01

    The aquaporins (AQPs) are a family of channel proteins that facilitate diffusion of water across cell membranes. Three members of the AQP family have been detected in the mouse blastocyst: AQP 3 and 8 are located in the basolateral domain and AQP 9 predominantly in the apical domain of the tropho...... in vivo developed blastocysts. We found that in vitro culture resulted in lower levels of AQP 8, 9, and 11 compared to in vivo development. These experiments show that mouse embryos are capable of regulating AQP mRNA abundances in response to environmental alterations....

  4. Experimental research on water stability alteration features of warm recycled asphalt mixture%温再生沥青混合料水稳定性变化特征试验研究

    Institute of Scientific and Technical Information of China (English)

    李斌; 蔡伟; 刘康; 董建勋

    2014-01-01

    为提高废旧沥青混合料的利用率,以EvothermTM-DAT添加剂作为试验用温拌剂,研究了旧沥青混合料掺量分别为0%,20%,40%和60%下温再生沥青混合料的水稳性能,并对试验结果进行了分析,指出温再生沥青混合料有效实现了废旧沥青混合料的二次利用。%In order to improve wasted asphalt mixture utilizing rate,the paper takes EvothermTM-DAT additive as testing warm mixing agent,stud-ies water stability performance of old asphalt mixture with 0%,20%,40% and 60% mixing amount,analyzes the testing results,and finally points out that:warm mixing recycled asphalt mixture effectively realizes the secondary utilization of waste asphalt mixture.

  5. Seasonal changes in hepatic progesterone receptor mRNA, estrogen receptor mRNA, and vitellogenin mRNA in the painted turtle, Chrysemys picta.

    Science.gov (United States)

    Custodia-Lora, Noemí; Callard, Ian P

    2002-10-01

    Previous studies using the fresh water turtle Chrysemys picta have demonstrated that progesterone (P) inhibits estradiol (E)-induced vitellogenin (vtg) secretion in this species. Further, there is evidence for the differential expression of the two P receptor isoforms (PRA and PRB) in the liver during the turtle seasonal cycle, correlating with hepatic vitellogenesis. In this study we report changes in the hepatic PR mPNA, ER mRNA, and vitellogenin (vtg) mRNA transcripts during the reproductive cycle of the turtle. Fragments of the turtle hepatic PR and ER cDNAs were cloned and sequenced and a previously cloned turtle vtg cDNA were used as probes in Northern blotting. No 3.7-kb PR mRNA, corresponding to the smaller PR transcript, PRA of other species was found, although, a smaller 1.8-kb transcript (putative PRC mRNA) was present. These observations suggest that the turtle as in the chicken and human, the 4.5-kb PR mRNA transcript encodes both PRA and PRB proteins. Only the larger PR mRNA transcript (4.5-kb), was found to vary significantly during the annual cycle, being highest when vitellogenesis was inhibited in winter and summer. Vtg mRNA could not be detected during the summer or winter, was highest during vitellogenesis in the spring, and reappeared during the fall period of vitellogenesis and ovarian recrudescence. ER mRNA followed a similar pattern, being highest during spring and early fall, when vtg synthesis is high. The data suggest that P/PR, as well as E/ER, may be involved in the seasonal regulation of hepatic vitellogenesis in this species. PMID:12392693

  6. Exosome-bound WD repeat protein Monad inhibits breast cancer cell invasion by degrading amphiregulin mRNA.

    Directory of Open Access Journals (Sweden)

    Makio Saeki

    Full Text Available Increased stabilization of mRNA coding for key cancer genes can contribute to invasiveness. This is achieved by down-regulation of exosome cofactors, which bind to 3'-UTR in cancer-related genes. Here, we identified amphiregulin, an EGFR ligand, as a target of WD repeat protein Monad, a component of R2TP/prefoldin-like complex, in MDA-MB-231 breast cancer cells. Monad specifically interacted with both the 3'-UTR of amphiregulin mRNA and the RNA degrading exosome, and enhanced decay of amphiregulin transcripts. Knockdown of Monad increased invasion and this effect was abolished with anti-amphiregulin neutralizing antibody. These results suggest that Monad could prevent amphiregulin-mediated invasion by degrading amphiregulin mRNA.

  7. Hypoxia stimulates binding of a cytoplasmic protein to a pyrimidine-rich sequence in the 3'-untranslated region of rat tyrosine hydroxylase mRNA.

    Science.gov (United States)

    Czyzyk-Krzeska, M F; Dominski, Z; Kole, R; Millhorn, D E

    1994-04-01

    Reduced oxygen tension (hypoxia) induces a 3-fold increase in stability of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell line. To investigate the possibility that RNA-protein interactions are involved in mediating this increase in stability, RNA gel shift assays were performed using different fragments of labeled TH mRNA and the S-100 fraction of PC12 cytoplasmic protein extracts. We identified a sequence within the 3'-untranslated region of TH mRNA that binds cytoplasmic protein. RNase T1 mapping revealed that the protein was bound to a 28 nucleotide long sequence that is located between bases 1551-1579 of TH mRNA. Moreover, protein binding to this fragment was prevented with an antisense oligonucleotide directed against bases 1551-1579 and subsequent RNase H digestion. This fragment of the 3'-untranslated region of TH mRNA is rich in pyrimidine nucleotides, and the binding of cytoplasmic protein to this fragment was reduced by competition with other polypyrimidine sequences including poly(C) but not poly(U) polymers. The binding of the protein to TH mRNA was increased when cytoplasmic proteins were extracted from PC12 cells exposed to hypoxia (5% O2) for 24 h. Electrophoresis of the UV cross-linked RNA-protein complex on SDS-polyacrylamide gel electrophoresis revealed a complex of 74 kDa. The potential role of this protein-TH mRNA interaction in regulation of TH mRNA stability during hypoxia is discussed. PMID:7908289

  8. Global mRNA degradation during lytic gammaherpesvirus infection contributes to establishment of viral latency.

    Directory of Open Access Journals (Sweden)

    Justin M Richner

    2011-07-01

    Full Text Available During a lytic gammaherpesvirus infection, host gene expression is severely restricted by the global degradation and altered 3' end processing of mRNA. This host shutoff phenotype is orchestrated by the viral SOX protein, yet its functional significance to the viral lifecycle has not been elucidated, in part due to the multifunctional nature of SOX. Using an unbiased mutagenesis screen of the murine gammaherpesvirus 68 (MHV68 SOX homolog, we isolated a single amino acid point mutant that is selectively defective in host shutoff activity. Incorporation of this mutation into MHV68 yielded a virus with significantly reduced capacity for mRNA turnover. Unexpectedly, the MHV68 mutant showed little defect during the acute replication phase in the mouse lung. Instead, the virus exhibited attenuation at later stages of in vivo infections suggestive of defects in both trafficking and latency establishment. Specifically, mice intranasally infected with the host shutoff mutant accumulated to lower levels at 10 days post infection in the lymph nodes, failed to develop splenomegaly, and exhibited reduced viral DNA levels and a lower frequency of latently infected splenocytes. Decreased latency establishment was also observed upon infection via the intraperitoneal route. These results highlight for the first time the importance of global mRNA degradation during a gammaherpesvirus infection and link an exclusively lytic phenomenon with downstream latency establishment.

  9. DCP1 forms asymmetric trimers to assemble into active mRNA decapping complexes in metazoa

    OpenAIRE

    Tritschler, Felix; Braun, Joerg E.; Motz, Carina; Igreja, Catia; Haas, Gabrielle; Truffault, Vincent; Izaurralde, Elisa; Weichenrieder, Oliver

    2009-01-01

    DCP1 stimulates the decapping enzyme DCP2, which removes the mRNA 5′ cap structure committing mRNAs to degradation. In multicellular eukaryotes, DCP1-DCP2 interaction is stabilized by additional proteins, including EDC4. However, most information on DCP2 activation stems from studies in S. cerevisiae, which lacks EDC4. Furthermore, DCP1 orthologs from multicellular eukaryotes have a C-terminal extension, absent in fungi. Here, we show that in metazoa, a conserved DCP1 C-terminal domain drives...

  10. Functional characterization of the mammalian mRNA decapping enzyme hDcp2

    OpenAIRE

    PICCIRILLO, CHRISTOPHER; Khanna, Richie; Kiledjian, Megerditch

    2003-01-01

    Regulation of decapping is a critical determinant of mRNA stability. We recently identified hDcp2 as a human decapping enzyme with intrinsic decapping activity. This activity is specific to N7-methylated guanosine containing RNA. The hDcp2 enzyme does not function on the cap structure alone and is not sensitive to competition by cap analog, suggesting that hDcp2 requires the RNA for cap recognition. We now demonstrate that hDcp2 is an RNA-binding protein and its recognition and hydrolysis of ...

  11. Metastasis-suppressor transcript destabilization through TARBP2 binding of mRNA hairpins

    OpenAIRE

    Goodarzi, Hani; Zhang, Steven; Buss, Colin G.; Fish, Lisa; Tavazoie, Saeed; Tavazoie, Sohail F

    2014-01-01

    Aberrant regulation of RNA stability plays an important role in many disease states1,2. Deregulated post-transcriptional modulation, such as that governed by microRNAs targeting linear sequence elements in mRNAs, has been implicated in the progression of many cancer types3-7. A defining feature of RNA is its ability to fold into structures. However, the roles of structural mRNA elements in cancer progression remain unexplored. We performed an unbiased search for post-transcriptional modulator...

  12. Propranolol and verapamil inhibit mRNA expression of RyR2 and SERCA in L-thyroxin-induced rat ventricular hypertrophy

    Institute of Scientific and Technical Information of China (English)

    Xiao-dong WU; De-zai DAI; Qiu-pin ZHANG; Feng GAO

    2004-01-01

    AIM: To study the alteration in the mRNA level of cardiac ryanodine receptor 2 (RyR2) and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) in L-thyroxin-induced hypertrophy. METHODS: L-thyroxin (500 g/kg) daily was injected for 10 d. RT-PCR was used to determine mRNA expression. RESULTS: An increase in the relative amount of RyR2 (111%) and SERCA mRNA (65 %) expression was observed in the hypertrophied rats (RyR2:77± 11; SERCA: 87± 10, n=9) compared with the normal rats (RyR2: 36± 10; SERCA: 53± 10, n=9). Propranolol was effective to inhibit the increase in RyR2 (51±7) and SERCA (63±13) mRNA expression in hypertrophied rats,respectively. Verapamil also reduced RyR2 (62±5) and SERCA (75±8) mRNA expression. CONCLUSION: Both RyR2 and SERCA mRNA level in L-thyroxin-induced cardiac hypertrophy was over-expressed and propranolol or verapamil inhibited the alteration.

  13. Induction of fetal hemoglobin through enhanced translation efficiency of γ-globin mRNA.

    Science.gov (United States)

    Hahn, Cynthia K; Lowrey, Christopher H

    2014-10-23

    Fetal hemoglobin (HbF) induction can ameliorate the clinical severity of sickle cell disease and β-thalassemia. We previously reported that activation of the eukaryotic initiation factor 2α (eIF2α) stress pathway increased HbF through a posttranscriptional mechanism. In this study, we explored the underlying means by which salubrinal, an activator of eIF2α signaling, enhances HbF production in primary human erythroid cells. Initial experiments eliminated changes in globin messenger RNA (mRNA) stability or cellular location and reduction of adult hemoglobin as possible salubrinal mechanisms. We then determined that salubrinal selectively increased the number of actively translating ribosomes on γ-globin mRNA. This enhanced translation efficiency occurred in the recovery phase of the stress response as phosphorylation of eIF2α and global protein synthesis returned toward baseline. These findings highlight γ-globin mRNA translation as a novel mechanism for regulating HbF production and as a pharmacologic target for induction of HbF. PMID:25170120

  14. CNP2 mRNA directs synthesis of both CNP1 and CNP2 polypeptides.

    Science.gov (United States)

    O'Neill, R C; Minuk, J; Cox, M E; Braun, P E; Gravel, M

    1997-10-15

    The ribosome scanning model for translational initiation predicts that eukaryotic mRNAs should, as a rule, be monocistronic. However, cases have recently been described of eukaryotic mRNAs producing more than one protein through alternative translational initiation at several different AUG codons. The present work reports the occurrence of two translational start sites on the mRNA encoding isoform 2 of the myelin marker enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in rat and mouse. We show that the CNP2 mRNA is able to direct synthesis of not only CNP2, but also CNP1 polypeptide. Immunoprecipitation experiments using a polyclonal antibody directed against CNP detect both CNP isoforms in tissues or cell lines expressing only the CNP2 transcript. Thus, the synthesis of CNP1 and CNP2 polypeptides must be encoded by the CNP2 transcript. In vitro translation of synthetic CNP2 mRNA demonstrates that both CNP isoforms are synthesized by initiation at different AUG codons. Furthermore, by introducing mutations to "switch off" translation from the second in-frame AUG codon in the CNP2 cDNA, and transfecting 293T cells with those constructs, we are able to correlate the production of CNP1 and CNP2 with different translational start sites. These results lead us to conclude that the CNP2 mRNA is able to produce both CNP1 and CNP2 polypeptides. This investigation has altered our understanding of the temporal expression of the CNP protein isoforms during development of the central nervous system (CNS). PMID:9373034

  15. Hormonal regulation of vasotocin receptor mRNA in a seasonally breeding songbird.

    Science.gov (United States)

    Grozhik, Anya V; Horoszko, Christopher P; Horton, Brent M; Hu, Yuchen; Voisin, Dene A; Maney, Donna L

    2014-03-01

    Behaviors associated with breeding are seasonally modulated in a variety of species. These changes in behavior are mediated by sex steroids, levels of which likewise vary with season. The effects of androgens on behaviors associated with breeding may in turn be partly mediated by the nonapeptides vasopressin (VP) and oxytocin (OT) in mammals, and vasotocin (VT) in birds. The effects of testosterone (T) on production of these neuropeptides have been well-studied; however, the regulation of VT receptors by T is not well understood. In this study, we investigated steroid-dependent regulation of VT receptor (VTR) mRNA in a seasonally breeding songbird, the white-throated sparrow (Zonotrichia albicollis). We focused on VTR subtypes that have been most strongly implicated in social behavior: V1a and oxytocin-like receptor (OTR). Using in situ hybridization, we show that T-treatment of non-breeding males altered V1a and OTR mRNA expression in several regions associated with seasonal reproductive behaviors. For example, T-treatment increased V1a mRNA expression in the medial preoptic area, bed nucleus of the stria terminalis, and ventromedial hypothalamus. T-treatment also affected both V1a and OTR mRNA expression in nuclei of the song system; some of these effects depended on the presence or absence of a chromosomal rearrangement that affects singing behavior, plasma T, and VT immunolabeling in this species. Overall, our results strengthen evidence that VT helps mediate the behavioral effects of T in songbirds, and suggest that the chromosomal rearrangement in this species may affect the sensitivity of the VT system to seasonal changes in T.

  16. Integrating microRNA and mRNA expression profiles in response to radiation-induced injury in rat lung

    International Nuclear Information System (INIS)

    Exposure to radiation provokes cellular responses, which are likely regulated by gene expression networks. MicroRNAs are small non-coding RNAs, which regulate gene expression by promoting mRNA degradation or inhibiting protein translation. The expression patterns of both mRNA and miRNA during the radiation-induced lung injury (RILI) remain less characterized and the role of miRNAs in the regulation of this process has not been studied. The present study sought to evaluate miRNA and mRNA expression profiles in the rat lung after irradiation. Male Wistar rats were subjected to single dose irradiation with 20 Gy using 6 MV x-rays to the right lung. (A dose rate of 5 Gy/min was applied). Rats were sacrificed at 3, 12 and 26 weeks after irradiation, and morphological changes in the lung were examined by haematoxylin and eosin. The miRNA and mRNA expression profiles were evaluated by microarrays and followed by quantitative RT-PCR analysis. A cDNA microarray analysis found 2183 transcripts being up-regulated and 2917 transcripts down-regulated (P ≤ 0.05, ≥2.0 fold change) in the lung tissues after irradiation. Likewise, a miRNAs microarray analysis indicated 15 miRNA species being up-regulated and 8 down-regulated (P ≤ 0.05). Subsequent bioinformatics anal -yses of the differentially expressed mRNA and miRNAs revealed that alterations in mRNA expression following irradiation were negatively correlated with miRNAs expression. Our results provide evidence indicating that irradiation induces alterations of mRNA and miRNA expression in rat lung and that there is a negative correlation of mRNA and miRNA expression levels after irradiation. These findings significantly advance our understanding of the regulatory mechanisms underlying the pathophysiology of radiation-induced lung injury. In summary, RILI does not develop gradually in a linear process. In fact, different cell types interact via cytokines in a very complex network. Furthermore, this study suggests that

  17. Ensuring Stability

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    "Stable"will be a key word for China’s economy in 2012.That’s the beat set at the annual Central Economic Work Conference held in Beijing on December 12-14,which reviewed this year’s development and mapped out plans for the next year.Policymakers at the conference decided to keep macroeconomic policies stable,seek a stable and relatively fast economic growth,stabilize consumer prices and maintain social stability in 2012.On the basis of stability,the government will transform the development model,deepen reform and improve people’s livelihood.

  18. Against Stabilization

    Directory of Open Access Journals (Sweden)

    Roger Mac Ginty

    2012-11-01

    Full Text Available This is a polemic against the concept and practice of stabilization as practiced by leading states from the global north in peace support interventions. It is not an argument against stability. Instead, it depicts stabilization as an essentially conservative doctrine that runs counter to its stated aims of enhancing local participation and legitimacy. It is an agenda of control that privileges notions of assimilation with international (western standards and mainstreams the military into peace-support operations. As a result, the value of peace is undercut.

  19. Changes in apoptotic microRNA and mRNA expression profiling in Caenorhabditis elegans during the Shenzhou-8 mission

    International Nuclear Information System (INIS)

    Radiation and microgravity exposure have been proven to induce abnormal apoptosis in microRNA (miRNA) and mRNA expression, but whether space conditions, including radiation and microgravity, activate miRNAs to regulate the apoptosis is undetermined. For that purpose, we investigated miRNome and mRNA expression in the ced-1 Caenorhabditis elegans mutant vs the wild-type, both of which underwent spaceflight, spaceflight 1g-centrifuge control and ground control conditions during the Shenzhou-8 mission. Results showed that no morphological changes in the worms were detected, but differential miRNA expression increased from 43 (ground control condition) to 57 and 91 in spaceflight and spaceflight control conditions, respectively. Microgravity altered miRNA expression profiling by decreasing the number and significance of differentially expressed miRNA compared with 1 g incubation during spaceflight. Alterations in the miRNAs were involved in alterations in apoptosis, neurogenesis larval development, ATP metabolism and GTPase-mediated signal transduction. Among these, 17 altered miRNAs potentially involved in apoptosis were screened and showed obviously different expression signatures between space conditions. By integrated analysis of miRNA and mRNA, miR-797 and miR-81 may be involved in apoptosis by targeting the genes ced-10 and both drp-1 and hsp-1, respectively. Compared with ground condition, space conditions regulated apoptosis though a different manner on transcription, by altering expression of seven core apoptotic genes in spaceflight condition, and eight in spaceflight control condition. Results indicate that, miRNA of Caenorhabditis elegans probably regulates apoptotic gene expression in response to space environmental stress, and shows different behavior under microgravity condition compared with 1 g condition in the presence of space radiation. (author)

  20. Regulation of mRNA Translation by Signaling Pathways

    OpenAIRE

    Roux, Philippe P.; Topisirovic, Ivan

    2012-01-01

    mRNA translation is the most energy consuming process in the cell. In addition, it plays a pivotal role in the control of gene expression and is therefore tightly regulated. In response to various extracellular stimuli and intracellular cues, signaling pathways induce quantitative and qualitative changes in mRNA translation by modulating the phosphorylation status and thus the activity of components of the translational machinery. In this work we focus on the phosphoinositide 3-kinase (PI3K)/...

  1. Assembly Properties of Divergent Tubulin Isotypes and Altered Tubulin Polypeptides in Vivo

    Science.gov (United States)

    Gu, Wei

    1990-01-01

    Mbeta1 is one of the closely related (though distinct) gene products termed isotypes encoded by the mouse beta-tubulin multigene family. These isotypes typically share 95%-98% homology at the amino acid level. However, Mbeta 1 is unusual in its relatively high degree of divergence compared to other beta-tubulin isotypes; furthermore, its tissue-restricted pattern of expression (Mbeta1 is only expressed in hematopoietic tissue) led to speculation that this isotype might be specialized for assembly into unique microtubule structures (such as the marginal band in some erythropoietic cell types). To test if this isotype is capable of coassembly into microtubules in cell types other than those in which it is normally expressed, a method was developed for the generation of an anti-Mbeta1 specific antibody. The Mbeta1 tubulin isotype was introduced into tissue culture cells by transfection and its expression and assembly properties were studied in both transiently transfected cells and stable cell lines using the anti -Mbeta1 specific antibody. The successful expression and coassembly of a 'foreign' tubulin isotype into microtubules in tissue culture cells and the generation of an antibody that can specifically recognize this isotype provided an approach to study the properties of altered beta-tubulin polypeptides in vivo. beta-tubulin synthesis in eukaryotic cells is autoregulated by a posttranscriptional mechanism in which the first four amino acids are responsible for determining the stability of beta -tubulin mRNA. To test if the beta -tubulin amino-terminal regulatory domain also contributes to the capacity of the tubulin monomer to polymerize into microtubules, altered sequences encoding Mbeta 1 but containing deletions encompassing amino acids 2-5 were expressed in HeLa cells. Stable cell lines expressing the altered Mbeta1 isotype were also generated. The assembly properties and stability of these altered Mbeta1 tubulin polypeptides were tested using the anti

  2. Cytokine mRNA quantification in histologically normal canine duodenal mucosa by real-time RT-PCR.

    Science.gov (United States)

    Peters, I R; Helps, C R; Calvert, E L; Hall, E J; Day, M J

    2005-01-10

    CD4(+) T helper cells are important for the regulation of immune responses in the intestinal mucosa and they exert their effects through the secretion of pro-inflammatory and immunomodulatory cytokines. Human patients with inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis have alterations in the normal intestinal cytokine profile. These cytokine abnormalities have been shown at both the protein and messenger RNA (mRNA) level. The role that mucosal cytokines play in the pathogenesis of canine IBD has only been investigated using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of gut tissue, as cytokine antisera are not available for this species. Real-time RT-PCR has been recognised to be a more accurate and sensitive method of quantifying mRNA transcripts, so in this study TaqMan real-time RT-PCR assays for the quantification of mRNA encoding IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, IFN-gamma, TNF-alpha and TGF-beta in canine intestinal mucosa were developed. The amount of these templates was quantified in normal canine duodenal mucosa (n = 8). IL-18, TGF-beta and TNF-alpha were found to be the most abundant transcripts, with IL-10 and IFN-gamma present at levels approximately 10-fold less. IL-2, IL-4, IL-5, IL-6 and IL-12 were the least abundant templates, with some RNA samples having no detectable mRNA copies. The methods developed in this study will form the basis of further work investigating the expression of mRNA encoding cytokines in mucosa from dogs with chronic enteropathies. In addition, these real-time PCR assays can also be used for the quantification of canine cytokine mRNA in other diseases.

  3. AT1a Receptor Has Interacted with Angiotensin-converting Enzymes 2 mRNA Expression in Mouse Brainstem

    Institute of Scientific and Technical Information of China (English)

    Zhanyi Lin; Shuguang Lin

    2008-01-01

    Objectives To examine in vivo interactions between angiotensin Ⅱ(Ang Ⅱ) AT1a receptor (AT1aR),angiotensin-converting enzymes (ACE) and ACE2 using small hairpin RNA (shRNA) gene-silencing methods in mice brainstem nucleus ttactus solitarius (NTS).Methods C57BL mice (n=8) were used as animal model.Method of microinjection in the nucleus of NTS was adopted.After ten days,mice were killed and their brain tissue were fixed and sectioned.The expression levels of AT1 aR,ACE and ACE2 mRNA at both sides of NTS were examined by in situ hybridization.Based on compared t-test,the changing for mRNA expression was examined.Results After the expression of AT1aR mRNA was significantly inhibited (61.6%±6.8% ) by AT1aR-shRNA,it was associated with decreases in ACE2 mRNA expression from (1.05±0.12) μCi/mg to (0.74±0.09) μCi/mg (29.0%±14.5%,P<0.01) on the same side of the brainstem.ACE mRNA expression was consistent at both sides (0.50 μCi/mg±0.09 μCi/mg and 0.53 μCi/mg±0.08 μCi/mg),with insignificant difference (P>0.05).Condusions The gene silencing result showed that there were interactions between brainstem AT1aR and ACE2.ACE mRNA expression was not altered by RNA interference treatment at AT1aR.

  4. Final Report [Regulated mRNA Decay in Arabidopsis: A global analysis of differential control by hormones and the circadian clock

    Energy Technology Data Exchange (ETDEWEB)

    Green, Pamela J.

    2010-03-18

    The long-term goal of this research was to better understand the influence of mRNA stability on gene regulation, particularly in response to hormones and the circadian clock. The primary aim of this project was to examine this using DNA microarrays, small RNA analysis and other approaches. We accomplished these objectives, although we were only able to detect small changes in mRNA stability in response to these stimuli. However, the work also contributed to a major breakthrough allowing the identification of small RNAs on a genomic scale in eukaryotes. Moreover, the project prompted us to develop a new way to analyze mRNA decay genome wide. Thus, the research was hugely successful beyond our objectives.

  5. 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

  6. Transient recombinant protein expression in mammalian cells: the role of mRNA level and stability

    OpenAIRE

    Wulhfard, Sarah

    2009-01-01

    Transient gene expression (TGE) is a rapid method for generating recombinant proteins in mammalian cells, but the volumetric productivities for secreted proteins in transiently transfected CHO DG44 cells are typically more than an order of magnitude lower than the yields achieved with recombinant CHO-derived cell lines. The goals of the thesis are to identify the limitations to higher TGE yields in CHO DG44 cells and to find possible solutions to overcome the problems. Initially an attempt wa...

  7. Rrp6p controls mRNA poly(a) tail length and its decoration with poly(a) binding proteins

    DEFF Research Database (Denmark)

    Schmid, Manfred; Poulsen, Mathias Bach; Olszewski, Pawel;

    2012-01-01

    Poly(A) (pA) tail binding proteins (PABPs) control mRNA polyadenylation, stability, and translation. In a purified system, S. cerevisiae PABPs, Pab1p and Nab2p, are individually sufficient to provide normal pA tail length. However, it is unknown how this occurs in more complex environments. Here ...

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

    Energy Technology Data Exchange (ETDEWEB)

    Maquat, Lynne

    2002-12-01

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

  9. PARALLEL STABILIZATION

    Institute of Scientific and Technical Information of China (English)

    J.L.LIONS

    1999-01-01

    A new algorithm for the stabilization of (possibly turbulent, chaotic) distributed systems, governed by linear or non linear systems of equations is presented. The SPA (Stabilization Parallel Algorithm) is based on a systematic parallel decomposition of the problem (related to arbitrarily overlapping decomposition of domains) and on a penalty argument. SPA is presented here for the case of linear parabolic equations: with distrjbuted or boundary control. It extends to practically all linear and non linear evolution equations, as it will be presented in several other publications.

  10. Early-life stress induces persistent alterationsin 5-HT1Areceptor and serotonin transporter mRNA expression in the adultrat brain.

    Directory of Open Access Journals (Sweden)

    Javier A. Bravo

    2014-04-01

    Full Text Available Early-life experience plays a major role in the stress response throughout life. Neonatal maternal separation (MS is an animal model of depression with an altered serotonergic response. We hypothesize that this alteration may be caused by differences in 5-HT1A receptor and serotonin transporter (SERT mRNA expression in brain areas involved in the control of emotions, memory and fear as well as in regions controlling the central serotonergic tone.To test this, Sprague-Dawley rats were subjected to MS for 3h daily during post-natal days 2-12. As control, age matched rats were not separated (NS from their dams. When animals reached adulthood (11-13 weeks brain was extracted and mRNA expression of 5-HT1A receptor in amygdala, hippocampus and dorsal raphé nucleus (DRN and SERT in the DRN was analyzed through in-situ hybridisation.Densitometric analysis revealed that MS increased 5-HT1A receptor mRNA expression in the amygdala, and reduced its expression in the DRN, but no changes were observed in the hippocampus in comparison to NS controls. Also, MS reduced SERT mRNA expression in the DRN when compared to NS rats.These results suggest that early-life stress induces persistent changes in 5-HT1A receptor and SERT mRNA expression in key brain regions involved in the development of stress-related psychiatric disorders. The reduction in SERT mRNA indicates an alteration that is in line with clinical findings such as polymorphic variants in individuals with higher risk of depression. These data may help to understand how early-life stress contributes to the development of mood disorders in adulthood.

  11. Stabilizing Movements

    DEFF Research Database (Denmark)

    Norbäck, Maria; Helin, Jenny; Raviola, Elena

    2014-01-01

    of turbulent times. Departing from a process perspective to organizational change and insights from Bakhtin's notion of ‘double-voicing’, which means that people borrow other people's words in their own talk, two main contributions are offered. First, we show how stability cannot be taken for granted...

  12. Before Stabilization

    DEFF Research Database (Denmark)

    Plesner, Ursula; Horst, Maja

    2013-01-01

    to the literature on the relationship between ICTs and organizing, but with a distinct focus on innovation communication and distributed innovation processes taking place before ICTs are stabilized, issues which cannot be captured by studies of diffusion and adaptation of new ICTs within single organizations....

  13. Macroeconomic stability

    DEFF Research Database (Denmark)

    Jespersen, Jesper

    2004-01-01

    It is demonstrated that full employment and sustainable development not necessarily are conflicting goals. On the other hand macroeconomic stability cannot be obtained without a deliberate labour sharing policy and a shift in the composition of private consumption away from traditional material...

  14. Nebulisation of IVT mRNA Complexes for Intrapulmonary Administration.

    Directory of Open Access Journals (Sweden)

    Sarah M Johler

    Full Text Available During the last years the potential role of in vitro transcribed (IVT mRNA as a vehicle to deliver genetic information has come into focus. IVT mRNA could be used for anti-cancer therapies, vaccination purposes, generation of pluripotent stem cells and also for genome engineering or protein replacement. However, the administration of IVT mRNA into the target organ is still challenging. The lung with its large surface area is not only of interest for delivery of genetic information for treatment of e.g. for cystic fibrosis or alpha-1-antitrypsin deficiency, but also for vaccination purposes. Administration of IVT mRNA to the lung can be performed by direct intratracheal instillation or by aerosol inhalation/nebulisation. The latter approach shows a non-invasive tool, although it is not known, if IVT mRNA is resistant during the process of nebulisation. Therefore, we investigated the transfection efficiency of non-nebulised and nebulised IVT mRNA polyplexes and lipoplexes in human bronchial epithelial cells (16HBE. A slight reduction in transfection efficiency was observed for lipoplexes (Lipofectamine 2000 in the nebulised part compared to the non-nebulised which can be overcome by increasing the amount of Lipofectamine. However, Lipofectamine was more than three times more efficient in transfecting 16HBE than DMRIE and linear PEI performed almost 10 times better than its branched derivative. By contrast, the nebulisation process did not affect the cationic polymer complexes. Furthermore, aerosolisation of IVT mRNA complexes did neither affect the protein duration nor the toxicity of the cationic complexes. Taken together, these data show that aerosolisation of cationic IVT mRNA complexes constitute a potentially powerful means to transfect cells in the lung with the purpose of protein replacement for genetic diseases such as cystic fibrosis or alpha-1-antitrypsin deficiency or for infectious disease vaccines, while bringing along the advantages

  15. The R1441C mutation alters the folding properties of the ROC domain of LRRK2

    OpenAIRE

    Li, Yongchao; Dunn, Laura; Greggio, Elisa; Krumm, Brian; Jackson, Graham S.; Cookson, Mark R; Lewis, Patrick A.; Deng, Junpeng

    2009-01-01

    Abstract LRRK2 is a 250kDa multidomain protein, mutations in which cause familial Parkinson's disease. Previously, we have demonstrated that the R1441C mutation in the ROC domain decreases GTPase activity. Here we show that the R1441C alters the folding properties of the ROC domain, lowering its thermodynamic stability. Similar to small GTPases, binding of different guanosine nucleotides alters the stability of the ROC domain, suggesting that there is an alteration in conformation ...

  16. SURVIV for survival analysis of mRNA isoform variation.

    Science.gov (United States)

    Shen, Shihao; Wang, Yuanyuan; Wang, Chengyang; Wu, Ying Nian; Xing, Yi

    2016-01-01

    The rapid accumulation of clinical RNA-seq data sets has provided the opportunity to associate mRNA isoform variations to clinical outcomes. Here we report a statistical method SURVIV (Survival analysis of mRNA Isoform Variation), designed for identifying mRNA isoform variation associated with patient survival time. A unique feature and major strength of SURVIV is that it models the measurement uncertainty of mRNA isoform ratio in RNA-seq data. Simulation studies suggest that SURVIV outperforms the conventional Cox regression survival analysis, especially for data sets with modest sequencing depth. We applied SURVIV to TCGA RNA-seq data of invasive ductal carcinoma as well as five additional cancer types. Alternative splicing-based survival predictors consistently outperform gene expression-based survival predictors, and the integration of clinical, gene expression and alternative splicing profiles leads to the best survival prediction. We anticipate that SURVIV will have broad utilities for analysing diverse types of mRNA isoform variation in large-scale clinical RNA-seq projects. PMID:27279334

  17. Base pairing interaction between 5'- and 3'-UTRs controls icaR mRNA translation in Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Igor Ruiz de los Mozos

    Full Text Available The presence of regulatory sequences in the 3' untranslated region (3'-UTR of eukaryotic mRNAs controlling RNA stability and translation efficiency is widely recognized. In contrast, the relevance of 3'-UTRs in bacterial mRNA functionality has been disregarded. Here, we report evidences showing that around one-third of the mapped mRNAs of the major human pathogen Staphylococcus aureus carry 3'-UTRs longer than 100-nt and thus, potential regulatory functions. We selected the long 3'-UTR of icaR, which codes for the repressor of the main exopolysaccharidic compound of the S. aureus biofilm matrix, to evaluate the role that 3'-UTRs may play in controlling mRNA expression. We showed that base pairing between the 3'-UTR and the Shine-Dalgarno (SD region of icaR mRNA interferes with the translation initiation complex and generates a double-stranded substrate for RNase III. Deletion or substitution of the motif (UCCCCUG within icaR 3'-UTR was sufficient to abolish this interaction and resulted in the accumulation of IcaR repressor and inhibition of biofilm development. Our findings provide a singular example of a new potential post-transcriptional regulatory mechanism to modulate bacterial gene expression through the interaction of a 3'-UTR with the 5'-UTR of the same mRNA.

  18. [Expression of the Drosophila melanogaster limk1 gene 3'-UTRs mRNA in Yeast Saccharomyces cerevisiae].

    Science.gov (United States)

    Rumyantsev, A M; Zakharov, G A; Zhuravlev, A V; Padkina, M V; Savvateeva-Popova, E V; Sambuk, E V

    2014-06-01

    The stability of mRNA and its translation efficacy in higher eukaryotes are influenced by the interaction of 3'-untranscribed regions (3'-UTRs) with microRNAs and RNA-binding proteins. Since Saccharomyces cerevisiae lack microRNAs, it is possible to evaluate the contribution of only 3'-UTRs' and RNA-binding proteins' interaction in post-transcriptional regulation. For this, the post-transcriptional regulation of Drosophila limk1 gene encoding for the key enzyme of actin remodeling was studied in yeast. Analysis of limkl mRNA 3'-UTRs revealed the potential sites of yeast transcriptional termination. Computer remodeling demonstrated the possibility of secondary structure formation in limkl mRNA 3'-UTRs. For an evaluation of the functional activity of Drosophila 3'-UTRs in yeast, the reporter gene PHO5 encoding for yeast acid phosphatase (AP) fused to different variants of Drosophila limk1 mRNA 3'-UTRs (513, 1075, 1554 bp) was used. Assessments of AP activity and RT-PCR demonstrated that Drosophila limkl gene 3'-UTRs were functionally active and recognized in yeast. Therefore, yeast might be used as an appropriate model system for studies of 3'-UTR's role in post-transcriptional regulation.

  19. Attention Alters Perceived Attractiveness.

    Science.gov (United States)

    Störmer, Viola S; Alvarez, George A

    2016-04-01

    Can attention alter the impression of a face? Previous studies showed that attention modulates the appearance of lower-level visual features. For instance, attention can make a simple stimulus appear to have higher contrast than it actually does. We tested whether attention can also alter the perception of a higher-order property-namely, facial attractiveness. We asked participants to judge the relative attractiveness of two faces after summoning their attention to one of the faces using a briefly presented visual cue. Across trials, participants judged the attended face to be more attractive than the same face when it was unattended. This effect was not due to decision or response biases, but rather was due to changes in perceptual processing of the faces. These results show that attention alters perceived facial attractiveness, and broadly demonstrate that attention can influence higher-level perception and may affect people's initial impressions of one another. PMID:26966228

  20. Biofilm formation by Bacillus subtilis requires an endoribonuclease-containing multisubunit complex that controls mRNA levels for the matrix gene repressor SinR.

    Science.gov (United States)

    DeLoughery, Aaron; Dengler, Vanina; Chai, Yunrong; Losick, Richard

    2016-01-01

    Biofilm formation by Bacillus subtilis is largely governed by a circuit in which the response regulator Spo0A turns on the gene for the anti-repressor SinI. SinI, in turn, binds to and inactivates SinR, a dedicated repressor of genes for matrix production. Mutants of the genes ylbF, ymcA and yaaT are blocked in biofilm formation, but the mechanism by which they act has been mysterious. A recent report attributed their role in biofilm formation to stimulating Spo0A activity. However, we detect no measurable effect on the transcription of sinI. Instead, we find that the block in biofilm formation is caused by an increase in the levels of SinR and of its mRNA. Evidence is presented that YlbF, YmcA and YaaT interact with, and control the activity of, RNase Y, which is known to destabilize sinR mRNA. We also show that the processing of another target of RNase Y, cggR-gapA mRNA, similarly depends on YlbF and YmcA. Our work suggests that sinR mRNA stability is an additional posttranscriptional control mechanism governing the switch to multicellularity and raises the possibility that YlbF, YmcA and YaaT broadly regulate mRNA stability as part of an RNase Y-containing, multi-subunit complex.

  1. Transcriptome kinetics is governed by a genome-wide coupling of mRNA production and degradation: a role for RNA Pol II.

    Directory of Open Access Journals (Sweden)

    Ophir Shalem

    2011-09-01

    Full Text Available Transcriptome dynamics is governed by two opposing processes, mRNA production and degradation. Recent studies found that changes in these processes are frequently coordinated and that the relationship between them shapes transcriptome kinetics. Specifically, when transcription changes are counter-acted with changes in mRNA stability, transient fast-relaxing transcriptome kinetics is observed. A possible molecular mechanism underlying such coordinated regulation might lay in two RNA polymerase (Pol II subunits, Rpb4 and Rpb7, which are recruited to mRNAs during transcription and later affect their degradation in the cytoplasm. Here we used a yeast strain carrying a mutant Pol II which poorly recruits these subunits. We show that this mutant strain is impaired in its ability to modulate mRNA stability in response to stress. The normal negative coordinated regulation is lost in the mutant, resulting in abnormal transcriptome profiles both with respect to magnitude and kinetics of responses. These results reveal an important role for Pol II, in regulation of both mRNA synthesis and degradation, and also in coordinating between them. We propose a simple model for production-degradation coupling that accounts for our observations. The model shows how a simple manipulation of the rates of co-transcriptional mRNA imprinting by Pol II may govern genome-wide transcriptome kinetics in response to environmental changes.

  2. Effects of tris(1,3-dichloro-2-propyl) phosphate and triphenyl phosphate on receptor-associated mRNA expression in zebrafish embryos/larvae

    International Nuclear Information System (INIS)

    Highlights: ► TDCPP or TPP exposure caused developmental toxicity. ► Receptor-centered PCR array was developed. ► TDCPP or TPP exposure altered mRNA expression in receptor-centered network. -- Abstract: Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and triphenyl phosphate (TPP) are frequently detected in biota, including fish. However, knowledge of the toxicological and molecular effects of these currently used flame retardants is limited. In the present study, an in vivo screening approach was developed to evaluate effects of TDCPP and TPP on developmental endpoints and receptor-associated expression of mRNA in zebrafish embryos/larvae. Exposure to TDCPP or TPP resulted in significantly smaller rates of hatching and survival, in dose- and time-dependent manners. The median lethal concentration (LC50) was 7.0 mg/L for TDCPP and 29.6 mg/L for TPP at 120 hour post-fertilization (hpf). Real-time PCR revealed alterations in expression of mRNAs involved in aryl hydrocarbon receptors (AhRs)-, peroxisome proliferator-activated receptor alpha (PPARα)-, estrogenic receptors (ERs)-, thyroid hormone receptor alpha (TRα)-, glucocorticoid receptor (GR)-, and mineralocorticoid receptor (MR)-centered gene networks. Exposure to positive control chemicals significantly altered abundances of mRNA in corresponding receptor-centered gene networks, a result that suggests that it is feasible to use zebrafish embryos/larvae to evaluate effects of chemicals on mRNA expression in these gene networks. Exposure to TDCPP altered transcriptional profiles in all six receptor-centered gene networks, thus exerting multiple toxic effects. TPP was easily metabolized and its potency to change expression of mRNA involved in receptor-centered gene networks was weaker than that of TDCPP. The PPARα- and TRα-centered gene networks might be the primary pathways affected by TPP. Taken together, these results demonstrated that TDCPP and TPP could alter mRNA expression of genes involved in the six

  3. Effects of tris(1,3-dichloro-2-propyl) phosphate and triphenyl phosphate on receptor-associated mRNA expression in zebrafish embryos/larvae

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chunsheng, E-mail: liuchunshengidid@126.com [State Key Laboratory of Pollution Control and Resource Reuse and School of the Environment, Nanjing University, Nanjing (China); Wang, Qiangwei [State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 (China); Liang, Kang; Liu, Jingfu [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, Beijing 100085 (China); Zhou, Bingsheng [State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 (China); Zhang, Xiaowei; Liu, Hongling [State Key Laboratory of Pollution Control and Resource Reuse and School of the Environment, Nanjing University, Nanjing (China); Giesy, John P. [Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B3 (Canada); Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B3 (Canada); Zoology Department, Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong (China); Yu, Hongxia, E-mail: yuhx@nju.edu.cn [State Key Laboratory of Pollution Control and Resource Reuse and School of the Environment, Nanjing University, Nanjing (China)

    2013-03-15

    Highlights: ► TDCPP or TPP exposure caused developmental toxicity. ► Receptor-centered PCR array was developed. ► TDCPP or TPP exposure altered mRNA expression in receptor-centered network. -- Abstract: Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and triphenyl phosphate (TPP) are frequently detected in biota, including fish. However, knowledge of the toxicological and molecular effects of these currently used flame retardants is limited. In the present study, an in vivo screening approach was developed to evaluate effects of TDCPP and TPP on developmental endpoints and receptor-associated expression of mRNA in zebrafish embryos/larvae. Exposure to TDCPP or TPP resulted in significantly smaller rates of hatching and survival, in dose- and time-dependent manners. The median lethal concentration (LC{sub 50}) was 7.0 mg/L for TDCPP and 29.6 mg/L for TPP at 120 hour post-fertilization (hpf). Real-time PCR revealed alterations in expression of mRNAs involved in aryl hydrocarbon receptors (AhRs)-, peroxisome proliferator-activated receptor alpha (PPARα)-, estrogenic receptors (ERs)-, thyroid hormone receptor alpha (TRα)-, glucocorticoid receptor (GR)-, and mineralocorticoid receptor (MR)-centered gene networks. Exposure to positive control chemicals significantly altered abundances of mRNA in corresponding receptor-centered gene networks, a result that suggests that it is feasible to use zebrafish embryos/larvae to evaluate effects of chemicals on mRNA expression in these gene networks. Exposure to TDCPP altered transcriptional profiles in all six receptor-centered gene networks, thus exerting multiple toxic effects. TPP was easily metabolized and its potency to change expression of mRNA involved in receptor-centered gene networks was weaker than that of TDCPP. The PPARα- and TRα-centered gene networks might be the primary pathways affected by TPP. Taken together, these results demonstrated that TDCPP and TPP could alter mRNA expression of genes involved in

  4. mRNA pseudoknot structures can act as ribosomal roadblocks

    DEFF Research Database (Denmark)

    Hansen, Jesper Tholstrup; Oddershede, Lene Broeng; Sørensen, Michael Askvad

    2012-01-01

    Several viruses utilize programmed ribosomal frameshifting mediated by mRNA pseudoknots in combination with a slippery sequence to produce a well defined stochiometric ratio of the upstream encoded to the downstream-encoded protein. A correlation between the mechanical strength of mRNA pseudoknots...... and frameshifting efficiency has previously been found; however, the physical mechanism behind frameshifting still remains to be fully understood. In this study, we utilized synthetic sequences predicted to form mRNA pseudoknot-like structures. Surprisingly, the structures predicted to be strongest lead only...... to limited frameshifting. Two-dimensional gel electrophoresis of pulse labelled proteins revealed that a significant fraction of the ribosomes were frameshifted but unable to pass the pseudoknot-like structures. Hence, pseudoknots can act as ribosomal roadblocks, prohibiting a significant fraction...

  5. Protein Structure and the Sequential Structure of mRNA

    DEFF Research Database (Denmark)

    Brunak, Søren; Engelbrecht, Jacob

    1996-01-01

    A direct comparison of experimentally determined protein structures and their corresponding protein coding mRNA sequences has been performed, We examine whether real world data support the hypothesis that clusters of rare codons correlate with the location of structural units in the resulting...... protein, The degeneracy of the genetic code allows for a biased selection of codons which may control the translational rate of the ribosome, and may thus in vivo have a catalyzing effect on the folding of the polypeptide chain, A complete search for GenBank nucleotide sequences coding for structural...... entries in the Brookhaven Protein Data Bank produced 719 protein chains with matching mRNA sequence, amino acid sequence, and secondary structure assignment, By neural network analysis, we found strong signals in mRNA sequence regions surrounding helices and sheets, These signals do not originate from...

  6. mRNA expression of dopamine receptors in peripheral blood lymphocytes of computer game addicts.

    Science.gov (United States)

    Vousooghi, Nasim; Zarei, Seyed Zeinolabedin; Sadat-Shirazi, Mitra-Sadat; Eghbali, Fatemeh; Zarrindast, Mohammad Reza

    2015-10-01

    Excessive playing of computer games like some other behaviors could lead to addiction. Addictive behaviors may induce their reinforcing effects through stimulation of the brain dopaminergic mesolimbic pathway. The status of dopamine receptors in the brain may be parallel to their homologous receptors in peripheral blood lymphocytes (PBLs). Here, we have investigated the mRNA expression of dopamine D3, D4 and D5 receptors in PBLs of computer game addicts (n = 20) in comparison to normal subjects (n = 20), using a real-time PCR method. The results showed that the expression level of D3 and D4 dopamine receptors in computer game addicts were not statistically different from the control group. However, the expression of the mRNA of D5 dopamine receptor was significantly down-regulated in PBLs of computer game addicts and reached 0.42 the amount of the control group. It is concluded that unlike with drug addiction, the expression levels of the D3 and D4 dopamine receptors in computer game addicts are not altered compared to the control group. However, reduced level of the D5 dopamine receptor in computer game addicts may serve as a peripheral marker in studies where the confounding effects of abused drugs are unwanted. PMID:25967984

  7. Epithelial expression of mRNA and protein for IL-6, IL-10 and TNF-α in endobronchial biopsies in horses with recurrent airway obstruction

    Directory of Open Access Journals (Sweden)

    Art Tatiana

    2008-02-01

    Full Text Available Abstract Background The aim of this study was to evaluate the contribution of bronchial epithelium to airway inflammation, with focus on mRNA and protein expression of cytokines of innate immunity IL-6, IL-10 and TNF-α, in horses with Recurrent Airway Obstruction (RAO during exacerbation and in remission. Results Despite marked clinical and physiologic alterations between exacerbation and after remission in the RAO horses no differences were detected in either cytokine mRNA or protein levels. Moreover, the expression of investigated cytokines in RAO horses on pasture did not differ from controls. In comparing real-time PCR analysis to results of immunohistochemistry only IL-10 mRNA and protein levels in RAO horses on pasture were significantly correlated (rs = 0.893, p = 0.007. Curiously, in controls examined on pasture the TNF-α protein level was positively correlated to IL-10 mRNA expression (rs = 0.967, p = 0.007 and negatively correlated to IL-6 mRNA expression (rs = -0.971, p = 0.001. Conclusion Given the complementary relationship of assessing cytokines directly by immunohistochemistry, or indirectly by PCR to mRNA, the lack of significant changes in either mRNA or protein levels of IL-6, IL-10 or TNF-α mRNA in RAO horses in exacerbation suggests that these particular cytokines in bronchial tissue may not play a substantive role in the active inflammation of this disease. To support this contention further studies examining time dependency of expression of IL-6, IL-10 or TNF-α are needed, as is expansion of the range of cytokines to include other key regulators of airway inflammation.

  8. mRNA expression profiling reveals a role of Helicobacter pylorivacuolating toxin in escaping host defense

    Institute of Scientific and Technical Information of China (English)

    Jian-Ping Yuan; Tao Li; Zhen-Hong Li; Gui-Zhen Yang; Bao-Yu Hu; Xiao-Dong Shi; Tie-Liu Shi; Shan-Qing Tong; Xiao-Kui Guo

    2004-01-01

    AIM: To study the immune response of host to Helicobacter pylori VacA.METHODS: The monocyte/macrophage-like U937 cells were infected with Helicobacter pylori vacA-positive strain NCTC 11638 or isogenic vacA-negative mutant. Differentially expressed genes were identified at 2, 6, 10, and 24 h postinfection by cDNA microarray. Differential expressions of some genes were confirmed by Northern blot.RESULTS: More than 100 genes altered their mRNA expression at different time points respectively, many of which were identified to be related to immune evasion.CONCLUSION: VacA is a crucial element for H pylorito escape from host immune defense by means of differentially regulating the expression of some related genes. These genes, previously known or unknown to be involved in the mechanism of immune evasion, deserve further investigation to unearth much more information complicated in the immune response.

  9. Complexity on Acute Myeloid Leukemia mRNA Transcript Variant

    Directory of Open Access Journals (Sweden)

    Carlo Cattani

    2011-01-01

    Full Text Available This paper deals with the sequence analysis of acute myeloid leukemia mRNA. Six transcript variants of mlf1 mRNA, with more than 2000 bps, are analyzed by focusing on the autocorrelation of each distribution. Through the correlation matrix, some patches and similarities are singled out and commented, with respect to similar distributions. The comparison of Kolmogorov fractal dimension will be also given in order to classify the six variants. The existence of a fractal shape, patterns, and symmetries are discussed as well.

  10. The quantification of COMT mRNA in post mortem cerebellum tissue: diagnosis, genotype, methylation and expression

    Directory of Open Access Journals (Sweden)

    Craig Ian W

    2006-02-01

    Full Text Available Abstract Background The COMT gene is located on chromosome 22q11, a region strongly implicated in the aetiology of several psychiatric disorders, in particular schizophrenia. Previous research has suggested that activity and expression of COMT is altered in schizophrenia, and is mediated by one or more polymorphisms within the gene, including the functional Val158Met polymorphism. Method In this study we examined the expression levels of COMT mRNA using quantitative RT-PCR in 60 post mortem cerebellum samples derived from individuals with schizophrenia, bipolar disorder, depression, and no history of psychopathology. Furthermore, we have examined the methylation status of two CpG sites in the promoter region of the gene. Results We found no evidence of altered COMT expression or methylation in any of the psychiatric diagnoses examined. We did, however, find evidence to suggest that genotype is related to COMT gene expression, replicating the findings of two previous studies. Specifically, val158met (rs165688; Val allele rs737865 (G allele and rs165599 (G allele all showed reduced expression (P COMT expression, with females exhibiting significantly greater levels of COMT mRNA. Conclusion The expression of COMT does not appear to be altered in the cerebellum of individuals suffering from schizophrenia, bipolar disorder or depression, but does appear to be influenced by single nucleotide polymorphisms within the gene.

  11. Occupational Toluene Exposure Induces Cytochrome P450 2E1 mRNA Expression in Peripheral Lymphocytes

    Science.gov (United States)

    Mendoza-Cantú, Ania; Castorena-Torres, Fabiola; de León, Mario Bermúdez; Cisneros, Bulmaro; López-Carrillo, Lizbeth; Rojas-García, Aurora E.; Aguilar-Salinas, Alberto; Manno, Maurizio; Albores, Arnulfo

    2006-01-01

    Print workers are exposed to organic solvents, of which the systemic toxicant toluene is a main component. Toluene induces expression of cytochrome P450 2E1 (CYP2E1), an enzyme involved in its own metabolism and that of other protoxicants, including some procarcinogens. Therefore, we investigated the association between toluene exposure and the CYP2E1 response, as assessed by mRNA content in peripheral lymphocytes or the 6-hydroxychlorzoxazone (6OH-CHZ)/chlorzoxazone (CHZ) quotient (known as CHZ metabolic ratio) in plasma, and the role of genotype (5′-flanking region RsaI/PstI polymorphic sites) in 97 male print workers. The geometric mean (GM) of toluene concentration in the air was 52.80 ppm (10–760 ppm); 54% of the study participants were exposed to toluene concentrations that exceeded the maximum permissible exposure level (MPEL). The GM of urinary hippuric acid at the end of a work shift (0.041 g/g creatinine) was elevated relative to that before the shift (0.027 g/g creatinine; p < 0.05). The GM of the CHZ metabolic ratio was 0.33 (0–9.3), with 40% of the subjects having ratios below the GM. However, the average CYP2E1 mRNA level in peripheral lymphocytes was 1.07 (0.30–3.08), and CYP2E1 mRNA levels within subjects correlated with the toluene exposure ratio (environmental toluene concentration:urinary hippuric acid concentration) (p = 0.014). Genotype did not alter the association between the toluene exposure ratio and mRNA content. In summary, with further validation, CYP2E1 mRNA content in peripheral lymphocytes could be a sensitive and noninvasive biomarker for the continuous monitoring of toluene effects in exposed persons. PMID:16581535

  12. Integrated miRNA and mRNA expression profiling in inflamed colon of patients with ulcerative colitis.

    Directory of Open Access Journals (Sweden)

    Jan Van der Goten

    Full Text Available BACKGROUND: Ulcerative colitis (UC is associated with differential colonic expression of genes involved in immune response (e.g. IL8 and barrier integrity (e.g. cadherins. MicroRNAs (miRNAs are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1 if miRNA expression in UC mucosa is altered and (2 if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA. METHODOLOGY: Colonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays. RESULTS: When comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression. CONCLUSION: Differential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression

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

    Science.gov (United States)

    Fu, Zidong Donna; Klaassen, Curtis D

    2014-01-01

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

  14. Notch mRNA expression in Drosophila embryos is negatively regulated at the level of mRNA 3' processing.

    Directory of Open Access Journals (Sweden)

    Andrew K Shepherd

    Full Text Available Notch receptor regulates differentiation of almost all tissues and organs during animal development. Many mechanisms function at the protein level to finely regulate Notch activity. Here we provide evidence for Notch regulation at an earlier step - mRNA 3' processing. Processing at the Notch consensus polyadenylation site appears by default to be suppressed in Drosophila embryos. Interference with this suppression, by a mutation, results in increased levels of polyadenylated Notch mRNA, excess Notch signaling, and severe developmental defects. We propose that Notch mRNA 3' processing is negatively regulated to limit the production of Notch protein and render it a controlling factor in the generation of Notch signaling.

  15. Regulation of ARE-mRNA Stability by Cellular Signaling

    DEFF Research Database (Denmark)

    Damgaard, Christian Kroun; Lykke-Andersen, Jens

    2013-01-01

    but as a response to different cellular cues they can become either stabilized, allowing expression of a given gene, or further destabilized to silence their expression. These tightly regulated mRNAs include many that encode growth factors, proto-oncogenes, cytokines, and cell cycle regulators. Failure to properly......During recent years, it has become clear that regulation of mRNA stability is an important event in the control of gene expression. The stability of a large class of mammalian mRNAs is regulated by AU-rich elements (AREs) located in the mRNA 3′ UTRs. mRNAs with AREs are inherently labile...... regulate their stability can therefore lead to uncontrolled expression of factors associated with cell proliferation and has been implicated in several human cancers. A number of transfactors that recognize AREs and regulate the translation and degradation of ARE-mRNAs have been identified...

  16. Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines

    Directory of Open Access Journals (Sweden)

    Barredo Julio C

    2002-03-01

    Full Text Available Abstract Background We describe an alternative method to determine mRNA half-life (t1/2 based on the Real-Time RT-PCR procedure. This approach was evaluated by using the β-actin gene as a reference molecule for measuring of mRNA stability. Results Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGreen® fluorescent dye with the VersaFluor Fluorometer System. One μg of total RNA was reverse transcribed and used as template for the amplification of a region of the β-actin gene (231 bp. To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, β-actin mRNAs were quantified by Real-Time RT-PCR using the SYBR® Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the β-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t1/2 value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h. Conclusions We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that β-actin mRNA half-life can be affected by the cellular growth rate.

  17. Stem cells. m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation.

    Science.gov (United States)

    Geula, Shay; Moshitch-Moshkovitz, Sharon; Dominissini, Dan; Mansour, Abed AlFatah; Kol, Nitzan; Salmon-Divon, Mali; Hershkovitz, Vera; Peer, Eyal; Mor, Nofar; Manor, Yair S; Ben-Haim, Moshe Shay; Eyal, Eran; Yunger, Sharon; Pinto, Yishay; Jaitin, Diego Adhemar; Viukov, Sergey; Rais, Yoach; Krupalnik, Vladislav; Chomsky, Elad; Zerbib, Mirie; Maza, Itay; Rechavi, Yoav; Massarwa, Rada; Hanna, Suhair; Amit, Ido; Levanon, Erez Y; Amariglio, Ninette; Stern-Ginossar, Noam; Novershtern, Noa; Rechavi, Gideon; Hanna, Jacob H

    2015-02-27

    Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N(6)-methyladenosine (m(6)A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m(6)A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m(6)A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner. PMID:25569111

  18. Stem cells. m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation.

    Science.gov (United States)

    Geula, Shay; Moshitch-Moshkovitz, Sharon; Dominissini, Dan; Mansour, Abed AlFatah; Kol, Nitzan; Salmon-Divon, Mali; Hershkovitz, Vera; Peer, Eyal; Mor, Nofar; Manor, Yair S; Ben-Haim, Moshe Shay; Eyal, Eran; Yunger, Sharon; Pinto, Yishay; Jaitin, Diego Adhemar; Viukov, Sergey; Rais, Yoach; Krupalnik, Vladislav; Chomsky, Elad; Zerbib, Mirie; Maza, Itay; Rechavi, Yoav; Massarwa, Rada; Hanna, Suhair; Amit, Ido; Levanon, Erez Y; Amariglio, Ninette; Stern-Ginossar, Noam; Novershtern, Noa; Rechavi, Gideon; Hanna, Jacob H

    2015-02-27

    Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N(6)-methyladenosine (m(6)A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m(6)A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m(6)A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner.

  19. Quantification of low-expressed mRNA using 5' LNA-containing real-time PCR primers

    International Nuclear Information System (INIS)

    Real-time RT-PCR is the most sensitive and accurate method for mRNA quantification. Using specific recombinant DNA as a template, real-time PCR allows accurate quantification within a 7-log range and increased sensitivity below 10 copies. However, when using RT-PCR to quantify mRNA in biological samples, a stochastic off-targeted amplification can occur. Classical adjustments of assay parameters have minimal effects on such amplification. This undesirable amplification appears mostly to be dependent on specific to non-specific target ratio rather than on the absolute quantity of the specific target. This drawback, which decreases assay reliability, mostly appears when quantifying low-expressed transcript in a whole organ. An original primer design using properties of LNA allows to block off-target amplification. 5'-LNA substitution strengthens 5'-hybridization. Consequently on-target hybridization is stabilized and the probability for the off-target to lead to amplification is decreased

  20. Neonatal hyper- and hypothyroidism alter the myoglobin gene expression program in adulthood

    Energy Technology Data Exchange (ETDEWEB)

    Picoli Souza, K. de [Faculdade de Ciências Biológicas e Ambientais, Universidade Federal da Grande Dourados, Dourados, MS (Brazil); Nunes, M.T. [Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade de São Paulo, São Paulo, SP (Brazil)

    2014-06-24

    Myoglobin acts as an oxygen store and a reactive oxygen species acceptor in muscles. We examined myoglobin mRNA in rat cardiac ventricle and skeletal muscles during the first 42 days of life and the impact of transient neonatal hypo- and hyperthyroidism on the myoglobin gene expression pattern. Cardiac ventricle and skeletal muscles of Wistar rats at 7-42 days of life were quickly removed, and myoglobin mRNA was determined by Northern blot analysis. Rats were treated with propylthiouracil (5-10 mg/100 g) and triiodothyronine (0.5-50 µg/100 g) for 5, 15, or 30 days after birth to induce hypo- and hyperthyroidism and euthanized either just after treatment or at 90 days. During postnatal (P) days 7-28, the ventricle myoglobin mRNA remained unchanged, but it gradually increased in skeletal muscle (12-fold). Triiodothyronine treatment, from days P0-P5, increased the skeletal muscle myoglobin mRNA 1.5- to 4.5-fold; a 2.5-fold increase was observed in ventricle muscle, but only when triiodothyronine treatment was extended to day P15. Conversely, hypothyroidism at P5 markedly decreased (60%) ventricular myoglobin mRNA. Moreover, transient hyperthyroidism in the neonatal period increased ventricle myoglobin mRNA (2-fold), and decreased heart rate (5%), fast muscle myoglobin mRNA (30%) and body weight (20%) in adulthood. Transient hypothyroidism in the neonatal period also permanently decreased fast muscle myoglobin mRNA (30%) and body weight (14%). These results indicated that changes in triiodothyronine supply in the neonatal period alter the myoglobin expression program in ventricle and skeletal muscle, leading to specific physiological repercussions and alterations in other parameters in adulthood.

  1. Neonatal hyper- and hypothyroidism alter the myoglobin gene expression program in adulthood

    Directory of Open Access Journals (Sweden)

    K. de Picoli Souza

    2014-08-01

    Full Text Available Myoglobin acts as an oxygen store and a reactive oxygen species acceptor in muscles. We examined myoglobin mRNA in rat cardiac ventricle and skeletal muscles during the first 42 days of life and the impact of transient neonatal hypo- and hyperthyroidism on the myoglobin gene expression pattern. Cardiac ventricle and skeletal muscles of Wistar rats at 7-42 days of life were quickly removed, and myoglobin mRNA was determined by Northern blot analysis. Rats were treated with propylthiouracil (5-10 mg/100 g and triiodothyronine (0.5-50 µg/100 g for 5, 15, or 30 days after birth to induce hypo- and hyperthyroidism and euthanized either just after treatment or at 90 days. During postnatal (P days 7-28, the ventricle myoglobin mRNA remained unchanged, but it gradually increased in skeletal muscle (12-fold. Triiodothyronine treatment, from days P0-P5, increased the skeletal muscle myoglobin mRNA 1.5- to 4.5-fold; a 2.5-fold increase was observed in ventricle muscle, but only when triiodothyronine treatment was extended to day P15. Conversely, hypothyroidism at P5 markedly decreased (60% ventricular myoglobin mRNA. Moreover, transient hyperthyroidism in the neonatal period increased ventricle myoglobin mRNA (2-fold, and decreased heart rate (5%, fast muscle myoglobin mRNA (30% and body weight (20% in adulthood. Transient hypothyroidism in the neonatal period also permanently decreased fast muscle myoglobin mRNA (30% and body weight (14%. These results indicated that changes in triiodothyronine supply in the neonatal period alter the myoglobin expression program in ventricle and skeletal muscle, leading to specific physiological repercussions and alterations in other parameters in adulthood.

  2. Moderate cyclic tensile strain alters the assembly of cartilage extracellular matrix proteins in vitro.

    Science.gov (United States)

    Bleuel, Judith; Zaucke, Frank; Brüggemann, Gert-Peter; Heilig, Juliane; Wolter, Marie-Louise; Hamann, Nina; Firner, Sara; Niehoff, Anja

    2015-06-01

    Mechanical loading influences the structural and mechanical properties of articular cartilage. The cartilage matrix protein collagen II essentially determines the tensile properties of the tissue and is adapted in response to loading. The collagen II network is stabilized by the collagen II-binding cartilage oligomeric matrix protein (COMP), collagen IX, and matrilin-3. However, the effect of mechanical loading on these extracellular matrix proteins is not yet understood. Therefore, the aim of this study was to investigate if and how chondrocytes assemble the extracellular matrix proteins collagen II, COMP, collagen IX, and matrilin-3 in response to mechanical loading. Primary murine chondrocytes were applied to cyclic tensile strain (6%, 0.5 Hz, 30 min per day at three consecutive days). The localization of collagen II, COMP, collagen IX, and matrilin-3 in loaded and unloaded cells was determined by immunofluorescence staining. The messenger ribo nucleic acid (mRNA) expression levels and synthesis of the proteins were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and western blots. Immunofluorescence staining demonstrated that the pattern of collagen II distribution was altered by loading. In loaded chondrocytes, collagen II containing fibrils appeared thicker and strongly co-stained for COMP and collagen IX, whereas the collagen network from unloaded cells was more diffuse and showed minor costaining. Further, the applied load led to a higher amount of COMP in the matrix, determined by western blot analysis. Our results show that moderate cyclic tensile strain altered the assembly of the extracellular collagen network. However, changes in protein amount were only observed for COMP, but not for collagen II, collagen IX, or matrilin-3. The data suggest that the adaptation to mechanical loading is not always the result of changes in RNA and/or protein expression but might also be the result of changes in matrix assembly and structure.

  3. Influenza virus mRNA trafficking through host nuclear speckles.

    Science.gov (United States)

    Mor, Amir; White, Alexander; Zhang, Ke; Thompson, Matthew; Esparza, Matthew; Muñoz-Moreno, Raquel; Koide, Kazunori; Lynch, Kristen W; García-Sastre, Adolfo; Fontoura, Beatriz M A

    2016-01-01

    Influenza A virus is a human pathogen with a genome composed of eight viral RNA segments that replicate in the nucleus. Two viral mRNAs are alternatively spliced. The unspliced M1 mRNA is translated into the matrix M1 protein, while the ion channel M2 protein is generated after alternative splicing. These proteins are critical mediators of viral trafficking and budding. We show that the influenza virus uses nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies and exports the spliced M2 mRNA from the nucleus. Given that nuclear speckles are storage sites for splicing factors, which leave these sites to splice cellular pre-mRNAs at transcribing genes, we reveal a functional subversion of nuclear speckles to promote viral gene expression. PMID:27572970

  4. MRNA-based skin identification for forensic applications

    NARCIS (Netherlands)

    M. Visser (Mijke); D. Zubakov (Dmitry); K. Ballantyne (Kaye); M.H. Kayser (Manfred)

    2011-01-01

    textabstractAlthough the identification of human skin cells is of important relevance in many forensic cases, there is currently no reliable method available. Here, we present a highly specific and sensitive messenger RNA (mRNA) approach for skin identification, meeting the key requirements in foren

  5. Localization of histidine decarboxylase mRNA in rat brain.

    Science.gov (United States)

    Bayliss, D A; Wang, Y M; Zahnow, C A; Joseph, D R; Millhorn, D E

    1990-08-01

    The recent cloning of a cDNA encoding fetal rat liver histidine decarboxylase (HDC), the synthesizing enzyme for histamine, allows the study of the central histaminergic system at the molecular level. To this end, Northern blot and in situ hybridization analyses were used to determine the regional and cellular distribution of neurons which express HDC mRNA in rat brain. Three hybridizing species which migrate as 1.6-, 2.6-, and 3.5-kb RNA were identified with Northern blots. The major (2.6 kb) and minor (3.5 kb) species, characteristic of HDC mRNA in fetal liver, were expressed at high levels in diencephalon and at just detectable levels in hippocampus, but not in other brain regions. In contrast, the 1.6-kb species was present in all brain regions examined except the olfactory bulb. Cells which contain HDC mRNA were found by in situ hybridization in the hypothalamus; HDC mRNA-containing cells were not detected in other areas, including the hippocampus. Hypothalamic neurons which express HDC mRNA were localized to all aspects of the tuberomammillary nucleus, a result consistent with previous immunohistochemical findings. PMID:19912749

  6. A minimum principle in mRNA editing of Physarum ?

    CERN Document Server

    Frappat, L; Sorba, Paul

    2000-01-01

    mRNA editing in three sequences of Physarum polycephalum is analyzed. Once fixed the edited peptide chain, the nature of the inserted nucleotides and the position of the insertion sites are explained by introducing a minimum principle in the framework of the crystal basis model of the genetic code introduced by the authors.

  7. Differential expression of melanopsin mRNA and protein in Brown Norwegian rats.

    Science.gov (United States)

    Hannibal, Jens; Georg, Birgitte; Fahrenkrug, Jan

    2013-01-01

    retinal networks regulates melanopsin mRNA expression. It is likely to speculate that altered level of melanopsin is one way in which the retina adapts to environmental light and darkness conditions ensuring optimal light sensitivity for the transmission to the brain.

  8. Increased mRNA expression of cytochrome oxidase in dorsal raphe nucleus of depressive suicide victims

    Directory of Open Access Journals (Sweden)

    A Sanchez-Bahillo

    2008-04-01

    Full Text Available A Sanchez-Bahillo1, V Bautista-Hernandez1, Carlos Barcia Gonzalez1, R Bañon2, A Luna2, EC Hirsch3, Maria-Trinidad Herrero11Clinical and Experimental Neuroscience, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED; 2Department of Legal Medicine, Department of Human Anatomy, School of Medicine, University of Murcia, Campus de Espinardo, Murcia 30100, Spain; 3INSERM U679 Hôpital de la Salpêtrière, Boulevard de l’Hôpital, Paris, FranceAbstract: Suicidal behavior is a problem with important social repercussions. Some groups of the population show a higher risk of suicide; for example, depression, alcoholism, psychosis or drug abuse frequently precedes suicidal behavior. However, the relationship between metabolic alterations in the brain and premorbid clinical symptoms of suicide remains uncertain. The serotonergic and noradrenergic systems have frequently been, implicated in suicidal behavior and the amount of serotonin in the brain and CSF of suicide victims has been found to be low compared with normal subjects. However, there are contradictory results regarding the role of noradrenergic neurons in the mediation of suicide attempts, possibly reflecting the heterogeneity of conditions that lead to a common outcome. In the present work we focus on the subgroup of suicide victims that share a common diagnosis of major depression. Based on post-mortem studies analyzing mRNA expression by in situ hybridization, serotonergic neurons from the dorsal raphe nucleus (DRN from depressive suicide victims are seen to over-express cytochrome oxidase mRNA. However, no corresponding changes were found in the expression of tyrosine hydroxylase (TH mRNA in the noradrenergic neurons of the Locus Coeruleus (LC. These results suggest that, despite of the low levels of serotonin described in suicide victims, the activity of DRN neurons could increase in the suicidally depressed, probably due to the over activation of

  9. LARP1 specifically recognizes the 3' terminus of poly(A) mRNA.

    Science.gov (United States)

    Aoki, Kazuma; Adachi, Shungo; Homoto, Masae; Kusano, Hideo; Koike, Katsuyuki; Natsume, Tohru

    2013-07-11

    A poly(A) tail functions in mRNA turnover and in facilitating translation as a ribonucleoprotein complex with poly(A) binding proteins (PABPs). However, factors that associate with the poly(A) tail other than PABPs have not been described. Using proteomics, we identified candidate proteins that interact to the 3' terminus of the poly(A) tail. Among these proteins, we focused on La motif-related protein 1 (LARP1) and found that LARP1 specifically recognizes the 3' termini of normal poly(A) tails. We also reveal that LARP1 stabilizes multiple mRNAs carrying 5' terminal oligopyrimidine tract (5'TOP). Our findings suggest that LARP1 may be involved in the post-transcriptional regulation of gene expression, at least in several 5'TOP mRNAs, through the binding to 3' terminus of the poly(A) tail.

  10. The mRNA expression of hTERT in human breast carcinomas correlates with VEGF expression

    Directory of Open Access Journals (Sweden)

    Kirkpatrick Katharine L

    2004-01-01

    Full Text Available Abstract Background Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal stability leading to cellular immortalisation. hTERT (human telomerase reverse transcriptase is the rate-limiting determinant of telomerase reactivation. Telomerase has been associated with negative prognostic indicators in some studies. The present study aims to detect any correlation between hTERT and the negative prognostic indicators VEGF and PCNA by quantitatively measuring the mRNA expression of these genes in human breast cancer and in adjacent non-cancerous tissue (ANCT. Materials and methods RNA was extracted from 38 breast carcinomas and 40 ANCT. hTERT and VEGF165, VEGF189 and PCNA mRNA expressions were estimated by reverse transcriptase-PCR (RT-PCR and Taqman methodology. Results The level of expression of VEGF-165 and PCNA was significantly higher in carcinoma tissue than ANCT (p = 0.02. The ratio of VEGF165/189 expression was significantly higher in breast carcinoma than ANCT (p = 0.025. hTERT mRNA expression correlated with VEGF-189 mRNA (p = 0.008 and VEGF165 (p = 0.07. Conclusions hTERT mRNA expression is associated with the expression of the VEGF189 and 165 isoforms. This could explain the poorer prognosis reported in breast tumours expressing high levels of hTERT. The relative expression of the VEGF isoforms is significantly different in breast tumour to ANCT, and this may be important in breast carcinogenesis.

  11. Nutritional and growth control of ribosomal protein mRNA and rRNA in Neurospora crassa.

    OpenAIRE

    Cujec, T P; Tyler, B M

    1996-01-01

    The effects of changing growth rates on the levels of 40S pre-rRNA and two r-protein mRNAs were examined to gain insight into the coordinate transcriptional regulation of ribosomal genes in the ascomycete fungus Neurospora crassa. Growth rates were varied either by altering carbon nutritional conditions, or by subjecting the isolates to inositol-limiting conditions. During carbon up- or down-shifts, r-protein mRNA levels were stoichiometrically coordinated. Changes in 40S pre-rRNA levels para...

  12. RNase L controls terminal adipocyte differentiation, lipids storage and insulin sensitivity via CHOP10 mRNA regulation

    DEFF Research Database (Denmark)

    Fabre, Odile Martine Julie; Salehzada, T; Lambert, K;

    2012-01-01

    Adipose tissue structure is altered during obesity, leading to deregulation of whole-body metabolism. Its function depends on its structure, in particular adipocytes number and differentiation stage. To better understand the mechanisms regulating adipogenesis, we have investigated the role...... an expanded adipose tissue, which, however, is unable to correctly store lipids, illustrated by ectopic lipids storage in the liver and in the kidney. These findings highlight RNase L as an essential regulator of adipogenesis via the regulation of CHOP10 mRNA....

  13. LARP6 Meets Collagen mRNA: Specific Regulation of Type I Collagen Expression

    Directory of Open Access Journals (Sweden)

    Yujie Zhang

    2016-03-01

    Full Text Available Type I collagen is the most abundant structural protein in all vertebrates, but its constitutive rate of synthesis is low due to long half-life of the protein (60–70 days. However, several hundred fold increased production of type I collagen is often seen in reparative or reactive fibrosis. The mechanism which is responsible for this dramatic upregulation is complex, including multiple levels of regulation. However, posttranscriptional regulation evidently plays a predominant role. Posttranscriptional regulation comprises processing, transport, stabilization and translation of mRNAs and is executed by RNA binding proteins. There are about 800 RNA binding proteins, but only one, La ribonucleoprotein domain family member 6 (LARP6, is specifically involved in type I collagen regulation. In the 5′untranslated region (5’UTR of mRNAs encoding for type I and type III collagens there is an evolutionally conserved stem-loop (SL structure; this structure is not found in any other mRNA, including any other collagen mRNA. LARP6 binds to the 5′SL in sequence specific manner to regulate stability of collagen mRNAs and their translatability. Here, we will review current understanding of how is LARP6 involved in posttranscriptional regulation of collagen mRNAs. We will also discuss how other proteins recruited by LARP6, including nonmuscle myosin, vimentin, serine threonine kinase receptor associated protein (STRAP, 25 kD FK506 binding protein (FKBP25 and RNA helicase A (RHA, contribute to this process.

  14. Cigarette smoke exposure-associated alterations to noncoding RNA

    Directory of Open Access Journals (Sweden)

    Matthew Alan Maccani

    2012-04-01

    Full Text Available Environmental exposures vary by timing, severity, and frequency and may have a number of deleterious effects throughout the life course. The period of in utero development, for example, is one of the most crucial stages of development during which adverse environmental exposures can both alter the growth and development of the fetus as well as lead to aberrant fetal programming, increasing disease risk. During fetal development and beyond, the plethora of exposures, including nutrients, drugs, stress, and trauma, influence health, development, and survival. Recent research in environmental epigenetics has investigated the roles of environmental exposures in influencing epigenetic modes of gene regulation during pregnancy and at various stages of life. Many relatively common environmental exposures, such as cigarette smoking, alcohol consumption, and drug use, may have consequences for the expression and function of noncoding RNA (ncRNA, important post-transcriptional regulators of gene expression. A number of ncRNA have been discovered, including microRNA (miRNA, Piwi-interacting RNA (piRNA, and long noncoding RNA (long ncRNA. The best-characterized species of ncRNA are miRNA, the mature forms of which are ~22 nucleotides in length and capable of post-transcriptionally regulating target mRNA utilizing mechanisms based largely on the degree of complementarity between miRNA and target mRNA. Because miRNA can still negatively regulate gene expression when imperfectly base-paired with a target mRNA, a single miRNA can have a large number of potential mRNA targets and can regulate many different biological processes critical for health and development. The following review analyzes the current literature detailing links between cigarette smoke exposure and aberrant expression and function of noncoding RNA, assesses how such alterations may have consequences throughout the life course, and proposes future directions for this intriguing field of

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

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Zidong Donna [Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Klaassen, Curtis D., E-mail: cklaasse@kumc.edu [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS 66160 (United States)

    2014-01-01

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

  16. Neonatal phthalate ester exposure induced placental MTs, FATP1 and HFABP mRNA expression in two districts of southeast China.

    Science.gov (United States)

    Li, Bin; Xu, Xijin; Zhu, Yueqin; Cao, Junjun; Zhang, Yuling; Huo, Xia

    2016-01-01

    Plastic production releases phthalate esters (PAEs), which can alter the expression of metallothioneins (MTs), fatty acid transport protein 1 (FATP1) and heart fatty acid binding protein (HFABP). A total of 187 mother-infant pairs were recruited, 127 from Chenghai (high exposed group) and 60 from Haojiang (low exposed group), to investigate the association between neonatal PAE exposure and mRNA expression of placental MTs, FATP1 and HFABP. Umbilical cord blood and placenta samples were collected for measuring five PAE concentrations and detecting mRNA levels of MTs, FATP1 and HFABP. Butylbenzyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DNOP) were significantly higher in the high exposed group compared to the low exposed group. FATP1 and HFABP mRNA in the high exposed group were higher than that in the low exposed group while MT-1A was contrary. Both dimethyl phthalate (DMP) and DEHP were correlated with higher MT and MT-2A expression, while diethyl phthalate (DEP) was also positively correlated with MT-1A and FATP1 expression in female infants. DEHP exposure was negatively correlated with birth weight and gestational age in male infants. These results show that neonatal PAE exposure alters the mRNA expression of placental MTs and FATP1, which are related to fetal growth and development. PMID:26867681

  17. Inducible expression of Pisum sativum xyloglucan fucosyltransferase in the pea root cap meristem, and effects of antisense mRNA expression on root cap cell wall structural integrity.

    Science.gov (United States)

    Wen, Fushi; Celoy, Rhodesia M; Nguyen, Trang; Zeng, Weiqing; Keegstra, Kenneth; Immerzeel, Peter; Pauly, Markus; Hawes, Martha C

    2008-07-01

    Mitosis and cell wall synthesis in the legume root cap meristem can be induced and synchronized by the nondestructive removal of border cells from the cap periphery. Newly synthesized cells can be examined microscopically as they differentiate progressively during cap development, and ultimately detach as a new population of border cells. This system was used to demonstrate that Pisum sativum L. fucosyl transferase (PsFut1) mRNA expression is strongly expressed in root meristematic tissues, and is induced >2-fold during a 5-h period when mitosis in the root cap meristem is increased. Expression of PsFut1 antisense mRNA in pea hairy roots under the control of the CaMV35S promoter, which exhibits meristem localized expression in pea root caps, resulted in a 50-60% reduction in meristem localized endogenous PsFut1 mRNA expression measured using whole mount in situ hybridization. Changes in gross levels of cell wall fucosylated xyloglucan were not detected, but altered surface localization patterns were detected using whole mount immunolocalization with CCRC-M1, an antibody that recognizes fucosylated xyloglucan. Emerging hairy roots expressing antisense PsFut1 mRNA appeared normal macroscopically but scanning electron microscopy of tissues with altered CCRC-M1 localization patterns revealed wrinkled, collapsed cell surfaces. As individual border cells separated from the cap periphery, cell death occurred in correlation with extrusion of cellular contents through breaks in the wall.

  18. Low-intensity red and infrared lasers affect mRNA expression of DNA nucleotide excision repair in skin and muscle tissue.

    Science.gov (United States)

    Sergio, Luiz Philippe S; Campos, Vera Maria A; Vicentini, Solange C; Mencalha, Andre Luiz; de Paoli, Flavia; Fonseca, Adenilson S

    2016-04-01

    Lasers emit light beams with specific characteristics, in which wavelength, frequency, power, fluence, and emission mode properties determine the photophysical, photochemical, and photobiological responses. Low-intensity lasers could induce free radical generation in biological tissues and cause alterations in macromolecules, such as DNA. Thus, the aim of this work was to evaluate excision repair cross-complementing group 1 (ERCC1) and excision repair cross-complementing group 2 (ERCC2) messenger RNA (mRNA) expression in biological tissues exposed to low-intensity lasers. Wistar rat (n = 28, 4 for each group) skin and muscle were exposed to low-intensity red (660 nm) and near-infrared (880 nm) lasers at different fluences (25, 50, and 100 J/cm(2)), and samples of these tissues were withdrawn for RNA extraction, cDNA synthesis, and gene expression evaluation by quantitative polymerase chain reaction. Laser exposure was in continuous wave and power of 100 mW. Data show that ERCC1 and ERCC2 mRNA expressions decrease in skin (p laser, but increase in muscle tissue (p  0.05), but ERCC2 mRNA expression decreases in skin (p laser. Our results show that ERCC1 and ERCC2 mRNA expression is differently altered in skin and muscle tissue exposed to low-intensity lasers depending on wavelengths and fluences used in therapeutic protocols.

  19. Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune; Daugaard, Tina Fuglsang; Holm, Ida E;

    2013-01-01

    The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapa is the most predominant isoform. The Gfapd isoform is expressed in proliferating......RNA localization patterns were dependent on the different 39-exon sequences included in Gfapd and Gfapa mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential...

  20. UIF, a New mRNA export adaptor that works together with REF/ALY, requires FACT for recruitment to mRNA.

    Science.gov (United States)

    Hautbergue, Guillaume M; Hung, Ming-Lung; Walsh, Matthew J; Snijders, Ambrosius P L; Chang, Chung-Te; Jones, Rachel; Ponting, Chris P; Dickman, Mark J; Wilson, Stuart A

    2009-12-01

    Messenger RNA (mRNA) export adaptors play an important role in the transport of mRNA from the nucleus to the cytoplasm. They couple early mRNA processing events such as 5' capping and 3' end formation with loading of the TAP/NXF1 export receptor onto mRNA. The canonical adaptor REF/ALY/Yra1 is recruited to mRNA via UAP56 and subsequently delivers the mRNA to NXF1 [1]. Knockdown of UAP56 [2, 3] and NXF1 [4-7] in higher eukaryotes efficiently blocks mRNA export, whereas knockdown of REF only causes a modest reduction, suggesting the existence of additional adaptors [8-10]. Here we identify a new UAP56-interacting factor, UIF, which functions as an export adaptor, binding NXF1 and delivering mRNA to the nuclear pore. REF and UIF are simultaneously found on the same mRNA molecules, and both proteins are required for efficient export of mRNA. We show that the histone chaperone FACT specifically binds UIF, but not REF, via the SSRP1 subunit, and this interaction is required for recruitment of UIF to mRNA. Together the results indicate that REF and UIF represent key human adaptors for the export of cellular mRNAs via the UAP56-NXF1 pathway.

  1. mRNAs involved in copper homeostasis are regulated by the nonsense-mediated mRNA decay pathway depending on environmental conditions.

    Science.gov (United States)

    Peccarelli, Megan; Scott, Taylor D; Steele, Megan; Kebaara, Bessie W

    2016-01-01

    The nonsense-mediated mRNA decay pathway (NMD) is an mRNA degradation pathway that degrades mRNAs that prematurely terminate translation. These mRNAs include mRNAs with premature termination codons as well as many natural mRNAs. In Saccharomyces cerevisiae a number of features have been shown to target natural mRNAs to NMD. However, the extent to which natural mRNAs from the same functional group are regulated by NMD and how environmental conditions influence this regulation is not known. Here, we examined mRNAs involved in copper homeostasis and are predicted to be sensitive to NMD. We found that the majority of these mRNAs have long 3'-UTRs that could target them for degradation by NMD. Analysis of one of these mRNAs, COX19, found that the long 3'-UTR contributes to regulation of this mRNA by NMD. Furthermore, we examined an additional mRNA, MAC1 under low copper conditions. We found that low copper growth conditions affect NMD sensitivity of the MAC1 mRNA demonstrating that sensitivity to NMD can be altered by environmental conditions. MAC1 is a copper sensitive transcription factor that regulates genes involved with high affinity copper transport. Our results expand our understanding of how NMD regulates mRNAs from the same functional group and how the environment influences this regulation.

  2. The differential expression of alternatively polyadenylated transcripts is a common stress-induced response mechanism that modulates mammalian mRNA expression in a quantitative and qualitative fashion.

    Science.gov (United States)

    Hollerer, Ina; Curk, Tomaz; Haase, Bettina; Benes, Vladimir; Hauer, Christian; Neu-Yilik, Gabriele; Bhuvanagiri, Madhuri; Hentze, Matthias W; Kulozik, Andreas E

    2016-09-01

    Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells. PMID:27407180

  3. Stabilizing brokerage.

    Science.gov (United States)

    Stovel, Katherine; Golub, Benjamin; Milgrom, Eva M Meyersson

    2011-12-27

    A variety of social and economic arrangements exist to facilitate the exchange of goods, services, and information over gaps in social structure. Each of these arrangements bears some relationship to the idea of brokerage, but this brokerage is rarely like the pure and formal economic intermediation seen in some modern markets. Indeed, for reasons illuminated by existing sociological and economic models, brokerage is a fragile relationship. In this paper, we review the causes of instability in brokerage and identify three social mechanisms that can stabilize fragile brokerage relationships: social isolation, broker capture, and organizational grafting. Each of these mechanisms rests on the emergence or existence of supporting institutions. We suggest that organizational grafting may be the most stable and effective resolution to the tensions inherent in brokerage, but it is also the most institutionally demanding.

  4. STABILIZED OSCILLATOR

    Science.gov (United States)

    Jessen, P.L.; Price, H.J.

    1958-03-18

    This patent relates to sine-wave generators and in particular describes a generator with a novel feedback circuit resulting in improved frequency stability. The generator comprises two triodes having a common cathode circuit connected to oscillate at a frequency and amplitude at which the loop galn of the circutt ls unity, and another pair of triodes having a common cathode circuit arranged as a conventional amplifier. A signal is conducted from the osciliator through a frequency selective network to the amplifier and fed back to the osciliator. The unique feature of the feedback circuit is the amplifier operates in the nonlinear portion of its tube characteristics thereby providing a relatively constant feedback voltage to the oscillator irrespective of the amplitude of its input signal.

  5. Role of a redox-based methylation switch in mRNA life cycle ( pre- & post- transcriptional maturation and protein turnover : Implications in neurological disorders

    Directory of Open Access Journals (Sweden)

    MALAV SUCHIN TRIVEDI

    2012-06-01

    Full Text Available Homeostatic synaptic scaling in response to neuronal stimulus or activation, as well as due to changes in cellular niche, is an important phenomenon for memory consolidation, retrieval, and other similar cognitive functions. Neurological disorders and cognitive disabilities in autism, Rett syndrome, schizophrenia, dementia etc., are strongly correlated to alterations in protein expression (both synaptic and cytoplasmic. This correlation suggests that efficient temporal regulation of synaptic protein expression is important for synaptic plasticity. In addition, equilibrium between mRNA processing, protein translation and protein turnover is a critical sensor/trigger for recording synaptic information, normal cognition and behavior. Thus a regulatory switch, controlling the lifespan, maturation and processing of mRNA, might influence cognition and adaptive behavior. Here, we propose a two part novel hypothesis that methylation might act as this suggested coordinating switch to critically regulate mRNA maturation at 1.The pre-transcription level, by regulating precursor-RNA (pre-RNA processing into mRNA, via other non-coding RNAs and their influence on splicing phenomenon, and 2. the post-transcription level by modulating the regulatory functions of ribonucleoproteins (RNP and RNA binding proteins (RNABP in mRNA translation, dendritic translocation as well as protein synthesis and synaptic turnover. DNA methylation changes are well recognized and highly correlated to gene expression levels as well as, learning and memory; however, RNA methylation changes are recently characterized and yet their functional implications are not established. This review article provides some insight on the intriguing consequences of changes in methylation levels on mRNA life-cycle. We also suggest that, since methylation is under the control of glutathione antioxidant levels, the redox status of neurons might be the central regulatory switch for methylation

  6. Hippocampal damage and kainic acid injection induce a rapid increase in mRNA for BDNF and NGF in the rat brain.

    Science.gov (United States)

    Ballarín, M; Ernfors, P; Lindefors, N; Persson, H

    1991-10-01

    In situ hybridization and Northern blots were used to study expression of mRNAs for members of the nerve growth factor family in the rat brain following an excitatory stimulus. One hour after a unilateral needle insertion or saline injection into the dorsal hippocampus, the level of brain-derived neurotrophic factor (BDNF) mRNA increased markedly in granular neurons of the dentate gyrus and in the piriform cortex ipsilateral to the injection. The same treatment also increased the level of NGF mRNA in granular neurons of the ipsilateral dentate gyrus. The rapid increase in BDNF and NGF mRNA after a needle insertion or injection of saline was transient and preceded by an increase in c-fos mRNA in the same brain regions. In contrast to a needle insertion per se or a saline injection, 7 h after a unilateral injection of kainic acid into the dorsal hippocampus, the level of BDNF mRNA was dramatically increased in the ipsilateral hippocampus, as well as in the ipsilateral frontoparietal, piriform and perihinal cortex, the amygdaloid complex, claustrum, and ventromedial hypothalamus. A less pronounced increase was also seen in these brain areas on the contralateral side. Northern blots revealed that the level of BDNF mRNA increased 5- and 40-fold in the contra- and ipsilateral hippocampus, respectively, compared to sham-operated control animals. In contrast to BDNF and NGF, the level of hippocampus-derived neurotrophic factor/neurotrohin-3 (HDNF/NT-3) mRNA was not altered by either needle insertion or injection of saline or kainic acid.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1915733

  7. Adenosine methylation in Arabidopsis mRNA is associated with the 3’ end and reduced levels cause developmental defects

    Directory of Open Access Journals (Sweden)

    Zsuzsanna eBodi

    2012-03-01

    Full Text Available We previously showed that the N6-methyladenosine (m6A mRNA methylase is essential during Arabidopsis thaliana embryonic development. We also demonstrated that this modification is present at varying levels in all mature tissues. However, the requirement for the m6A in the mature plant was not tested. Here we show that a 95% reduction in m6A levels during later growth stages gives rise to plants with altered growth patterns and reduced apical dominance. The flowers of these plants commonly show defects in their floral organ number, size and identity. The global analysis of gene expression from reduced m6A plants show that a significant number of down regulated genes are involved in transport, or targeted transport, and most of the upregulated genes are involved in stress and stimulus response processes. An analysis of m6A distribution in fragmented mRNA suggests that the m6A is predominantly positioned towards the 3’ end of transcripts in a region 100-150 bp before the poly(A tail. In addition to the analysis of the phenotypic changes in the low methylation Arabidopsis plants we will review the latest advances in the field of mRNA internal methylation

  8. Neuron-specific ELAV/Hu proteins suppress HuR mRNA during neuronal differentiation by alternative polyadenylation

    OpenAIRE

    Mansfield, Kyle D.; Keene, Jack D.

    2011-01-01

    The ubiquitously expressed RNA-binding protein HuR increases the stability and translation of mRNAs encoding growth regulatory proteins that promote proliferation in a variety of cell types. However, the three neuron-specific ELAV/Hu proteins, HuB, HuC and HuD, while binding to the same types of mRNAs, are required instead for neuronal differentiation, and it becomes difficult to reconcile these contrary functions when all four Hu proteins are expressed in the same neuron. HuR mRNA exists as ...

  9. Combinatorial Control of mRNA Fates by RNA-Binding Proteins and Non-Coding RNAs

    Directory of Open Access Journals (Sweden)

    Valentina Iadevaia

    2015-09-01

    Full Text Available Post-transcriptional control of gene expression is mediated by RNA-binding proteins (RBPs and small non-coding RNAs (e.g., microRNAs that bind to distinct elements in their mRNA targets. Here, we review recent examples describing the synergistic and/or antagonistic effects mediated by RBPs and miRNAs to determine the localisation, stability and translation of mRNAs in mammalian cells. From these studies, it is becoming increasingly apparent that dynamic rearrangements of RNA-protein complexes could have profound implications in human cancer, in synaptic plasticity, and in cellular differentiation.

  10. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

    Holm, Christian; Nyvold, Charlotte Guldborg; Paludan, Søren Riis;

    2006-01-01

    and activational effects of IL-21 on different leukocytes come into play in vivo in an immune response has so far not been fully investigated. We show here for the first time in vivo, that IL-21 mRNA is produced in the spleen when mice are challenged with herpes simplex virus type 2 (HSV-2) or lymphocytic...... choriomeningitis virus (LCMV). We show in HSV-2 challenged mice that this production takes place in CD4+ T cell fractions and is absent in CD4+ T cell-depleted fractions. We also show that the peak of IL-21 mRNA production in both the HSV-2 and LCMV-challenged mice coincides with the onset of the adaptive immune...... response. Thus, our data suggest a role for IL-21 in the early stages of adaptive immune response against virus infections....

  11. Translation with frameshifting of ribosome along mRNA transcript

    CERN Document Server

    Li, Jingwei

    2015-01-01

    Translation is an important process for prokaryotic and eukaryotic cells to produce necessary proteins for cell growth. Numerious experiments have been performed to explore the translational properties. Diverse models have also been developed to determine the biochemical mechanism of translation. However, to simplify the majority of the existing models, the frameshifting of ribosome along the mRNA transcript is neglected, which actually occurs in real cells and has been extensively experimentally studied. The frameshifting of ribosome evidently influences the efficiency and speed of translation, considering that the peptide chains synthesized by shifted ribosomes will not fold into functional proteins and will degrade rapidly. In this study, a theoretical model is presented to describe the translational process based on the model for totally asymmetric simple exclusion process. In this model, the frameshifting of the ribosome along the mRNA transcript and the attachment/detachment of the ribosome to/from the ...

  12. MRNA-based skin identification for forensic applications

    OpenAIRE

    Visser, Mijke; Zubakov, Dmitry; Ballantyne, Kaye; Kayser, Manfred

    2011-01-01

    Although the identification of human skin cells is of important relevance in many forensic cases, there is currently no reliable method available. Here, we present a highly specific and sensitive messenger RNA (mRNA) approach for skin identification, meeting the key requirements in forensic analyses. We examined 11 candidate genes with skin-specific expression, as ascertained from expression databases and the literature, as well as five candidate reference genes ascertained from previous stud...

  13. mRNA decay during herpes simplex virus (HSV) infections: mutations that affect translation of an mRNA influence the sites at which it is cleaved by the HSV virion host shutoff (Vhs) protein.

    Science.gov (United States)

    Shiflett, Lora A; Read, G Sullivan

    2013-01-01

    During lytic infections, the herpes simplex virus (HSV) virion host shutoff (Vhs) endoribonuclease degrades many host and viral mRNAs. Within infected cells it cuts mRNAs at preferred sites, including some in regions of translation initiation. Vhs binds the translation initiation factors eIF4H, eIF4AI, and eIF4AII, suggesting that its mRNA degradative function is somehow linked to translation. To explore how Vhs is targeted to preferred sites, we examined the in vitro degradation of a target mRNA in rabbit reticulocyte lysates containing in vitro-translated Vhs. Vhs caused rapid degradation of mRNAs beginning with cleavages at sites in the first 250 nucleotides, including a number near the start codon and in the 5' untranslated region. Ligation of the ends to form a circular mRNA inhibited Vhs cleavage at the same sites at which it cuts capped linear molecules. This was not due to an inability to cut any circular RNA, since Vhs cuts circular mRNAs containing an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) at the same sites as linear molecules with the IRES. Cutting linear mRNAs at preferred sites was augmented by the presence of a 5' cap. Moreover, mutations that altered the 5' proximal AUG abolished Vhs cleavage at nearby sites, while mutations that changed sequences surrounding the AUG to improve their match to the Kozak consensus sequence enhanced Vhs cutting near the start codon. The results indicate that mutations in an mRNA that affect its translation affect the sites at which it is cut by Vhs and suggest that Vhs is directed to its preferred cut sites during translation initiation.

  14. Stochastic mRNA synthesis in mammalian cells.

    Science.gov (United States)

    Raj, Arjun; Peskin, Charles S; Tranchina, Daniel; Vargas, Diana Y; Tyagi, Sanjay

    2006-10-01

    Individual cells in genetically homogeneous populations have been found to express different numbers of molecules of specific proteins. We investigated the origins of these variations in mammalian cells by counting individual molecules of mRNA produced from a reporter gene that was stably integrated into the cell's genome. We found that there are massive variations in the number of mRNA molecules present in each cell. These variations occur because mRNAs are synthesized in short but intense bursts of transcription beginning when the gene transitions from an inactive to an active state and ending when they transition back to the inactive state. We show that these transitions are intrinsically random and not due to global, extrinsic factors such as the levels of transcriptional activators. Moreover, the gene activation causes burst-like expression of all genes within a wider genomic locus. We further found that bursts are also exhibited in the synthesis of natural genes. The bursts of mRNA expression can be buffered at the protein level by slow protein degradation rates. A stochastic model of gene activation and inactivation was developed to explain the statistical properties of the bursts. The model showed that increasing the level of transcription factors increases the average size of the bursts rather than their frequency. These results demonstrate that gene expression in mammalian cells is subject to large, intrinsically random fluctuations and raise questions about how cells are able to function in the face of such noise. PMID:17048983

  15. mRNA and DNA PCR tests in cutaneous tuberculosis

    Directory of Open Access Journals (Sweden)

    Chandanmal Suthar

    2013-01-01

    Full Text Available Background: The microbiologic diagnosis of cutaneous tuberculosis is difficult because most lesions harbor only a small number of mycobacteria that cannot usually be detected by staining for the organism or by culture. Nucleic acid amplification tests based on the polymerase chain reaction (PCR are potentially useful in this situation. Aims: To evaluate the utility of mRNA PCR and DNA PCR in the diagnosis of cutaneous tuberculosis. Methods: Biopsies from 28 cases of cutaneous tuberculosis and 19 controls with other diseases were subjected to microbiologic tests including direct smears for mycobacteria, culture and both mRNA PCR and DNA PCR. The laboratory was blinded to the clinical diagnosis. Results: None of the patients or controls showed a positive reaction on mRNA PCR test. Seven of 28 cases and 5 out of 19 controls showed a positive result on DNA PCR test yielding a sensitivity of 25% and a specificity of 73.7%. Conclusion: The results of PCR tests in cutaneous tuberculosis should be interpreted in the light of clinical and histopathological findings.

  16. Sodium arsenite-induced inhibition of cell proliferation is related to inhibition of IL-2 mRNA expression in mouse activated T cells

    Energy Technology Data Exchange (ETDEWEB)

    Conde, Patricia; Acosta-Saavedra, Leonor C.; Calderon-Aranda, Emma S. [Centro de Investigacion y de Estudios Avanzados, CINVESTAV, Seccion Toxicologia, P.O. Box 14-740, Mexico, D.F. (Mexico); Goytia-Acevedo, Raquel C. [Universidad Juarez del Estado de Durango, Facultad de Medicina, Gomez Palacio, Durango (Mexico)

    2007-04-15

    A proposed mechanism for the As-induced inhibition of cell proliferation is the inhibition of IL-2 secretion. However, the effects of arsenite on IL-2 mRNA expression or on the ERK pathway in activated-T cells have not yet been described. We examined the effect of arsenite on IL-2 mRNA expression, cell activation and proliferation in PHA-stimulated murine lymphocytes. Arsenite (1 and 10 {mu}M) decreased IL-2 mRNA expression, IL-2 secretion and cell proliferation. Arsenite (10 {mu}M) strongly inhibited ERK-phosphorylation. However, the partial inhibition (50%) of IL-2 mRNA produced by 1 {mu}M, consistent with the effects on IL-2 secretion and cell proliferation, could not be explained by the inhibition of ERK-phosphorylation, which was not affected at this concentration. The inhibition of IL-2 mRNA expression caused by 1 {mu}M could be associated to effects on pathways located downstream or parallel to ERK. Arsenite also decreased early activation (surface CD69{sup +} expression) in both CD4{sup +} and CD8{sup +}, and decreased total CD8{sup +} count without significantly affecting CD4{sup +}, supporting that the cellular immune response mediated by cytotoxic T cells is an arsenic target. Thus, our results suggest that arsenite decreases IL-2 mRNA levels and T-cell activation and proliferation. However, further studies on the effects of arsenite on IL-2 gene transcription and IL-2 mRNA stability are needed. (orig.)

  17. EXOSC8 mutations alter mRNA metabolism and cause hypomyelination with spinal muscular atrophy and cerebellar hypoplasia

    Science.gov (United States)

    Boczonadi, Veronika; Müller, Juliane S.; Pyle, Angela; Munkley, Jennifer; Dor, Talya; Quartararo, Jade; Ferrero, Ileana; Karcagi, Veronika; Giunta, Michele; Polvikoski, Tuomo; Birchall, Daniel; Princzinger, Agota; Cinnamon, Yuval; Lützkendorf, Susanne; Piko, Henriett; Reza, Mojgan; Florez, Laura; Santibanez-Koref, Mauro; Griffin, Helen; Schuelke, Markus; Elpeleg, Orly; Kalaydjieva, Luba; Lochmüller, Hanns; Elliott, David J.; Chinnery, Patrick F.; Edvardson, Shimon; Horvath, Rita

    2014-01-01

    The exosome is a multi-protein complex, required for the degradation of AU-rich element (ARE) containing messenger RNAs (mRNAs). EXOSC8 is an essential protein of the exosome core, as its depletion causes a severe growth defect in yeast. Here we show that homozygous missense mutations in EXOSC8 cause progressive and lethal neurological disease in 22 infants from three independent pedigrees. Affected individuals have cerebellar and corpus callosum hypoplasia, abnormal myelination of the central nervous system or spinal motor neuron disease. Experimental downregulation of EXOSC8 in human oligodendroglia cells and in zebrafish induce a specific increase in ARE mRNAs encoding myelin proteins, showing that the imbalanced supply of myelin proteins causes the disruption of myelin, and explaining the clinical presentation. These findings show the central role of the exosomal pathway in neurodegenerative disease. PMID:24989451

  18. Prenatal alcohol exposure alters methyl metabolism and programs serotonin transporter and glucocorticoid receptor expression in brain

    Science.gov (United States)

    Ngai, Ying Fai; Sulistyoningrum, Dian C.; O'Neill, Ryan; Innis, Sheila M.; Weinberg, Joanne

    2015-01-01

    Prenatal alcohol exposure (PAE) programs the fetal hypothalamic-pituitary-adrenal (HPA) axis, resulting in HPA dysregulation and hyperresponsiveness to stressors in adulthood. Molecular mechanisms mediating these alterations are not fully understood. Disturbances in one-carbon metabolism, a source of methyl donors for epigenetic processes, contributes to alcoholic liver disease. We assessed whether PAE affects one-carbon metabolism (including Mtr, Mat2a, Mthfr, and Cbs mRNA) and programming of HPA function genes (Nr3c1, Nr3c2, and Slc6a4) in offspring from ethanol-fed (E), pair-fed (PF), and ad libitum-fed control (C) dams. At gestation day 21, plasma total homocysteine and methionine concentrations were higher in E compared with C dams, and E fetuses had higher plasma methionine concentrations and lower whole brain Mtr and Mat2a mRNA compared with C fetuses. In adulthood (55 days), hippocampal Mtr and Cbs mRNA was lower in E compared with C males, whereas Mtr, Mat2a, Mthfr, and Cbs mRNA were higher in E compared with C females. We found lower Nr3c1 mRNA and lower nerve growth factor inducible protein A (NGFI-A) protein in the hippocampus of E compared with PF females, whereas hippocampal Slc6a4 mRNA was higher in E than C males. By contrast, hypothalamic Slc6a4 mRNA was lower in E males and females compared with C offspring. This was accompanied by higher hypothalamic Slc6a4 mean promoter methylation in E compared with PF females. These findings demonstrate that PAE is associated with alterations in one-carbon metabolism and has long-term and region-specific effects on gene expression in the brain. These findings advance our understanding of mechanisms of HPA dysregulation associated with PAE. PMID:26180184

  19. Inhibitory Effects of Atorvastatin on the mRNA Expression of ICAM-1 and VCAM-1 Activated by TNF-α in Cultured Human Umbilical Vein Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    Zhiming Yang; Zhanhai Li; Gaiying Fan; Bin Liang; Ying Ma; Chuanshi Xiao; Yuming Kang

    2007-01-01

    To investigate the effects of atorvastatin on the mRNA expression of intercellular adhesion molecule-1 ( ICAM-1 ) and vascular cell adhesion molecule-1 ( VCAM-1 ) activated by TNF-α in cultured human umbilical vein endothelial cells (HUVEC).Methods and Results Lactic dehydrogenase (LDH) activity in the culture media increased when HUVEC were incubated with TNF-α,suggesting a cytotoxic effect of TNF-α on HUVEC.The mRNA expression of ICAM-1 and VCAM-1 increased in HUVEC incubated with 10μg/L TNF-α and reached peak in HUVEC incubated with 30μg/L TNF-α.The mRNA expression of ICAM-1 and VCAM-1 in HUVEC incubated with 30μg/L TNF-α began to increase at 6 h,reached peak at 48 h,and kept a plateau until 72 h.Atorvastatin dose-dependently inhibited the mRNA expressions of ICAM-1 and VCAM-1 activated by incubating HUVEC with 30μg/L TNF-α for 48 hours.Conclusions Atorvastatin might stabilize plaque and decelerate the process of AS by inhibiting the mRNA expressions of ICAM-1 and VCAM-1.

  20. PAB-1, a Caenorhabditis elegans poly(A-binding protein, regulates mRNA metabolism in germline by interacting with CGH-1 and CAR-1.

    Directory of Open Access Journals (Sweden)

    Sunhee Ko

    Full Text Available Poly(A-binding proteins are highly conserved among eukaryotes and regulate stability of mRNA and translation. Among C. elegans homologues, pab-1 mutants showed defects in germline mitotic proliferation. Unlike pab-1 mutants, pab-1 RNAi at every larval stage caused arrest of germline development at the following stage, indicating that pab-1 is required for the entire postembryonic germline development. This idea is supported by the observations that the mRNA level of pab-1 increased throughout postembryonic development and its protein expression was germline-enriched. PAB-1 localized to P granules and the cytoplasm in the germline. PAB-1 colocalized with CGH-1 and CAR-1 and affected their localization, suggesting that PAB-1 is a component of processing (P-bodies that interacts with them. The mRNA and protein levels of representative germline genes, rec-8, GLP-1, rme-2, and msp-152, were decreased after pab-1 RNAi. Although the mRNA level of msp-152 was increased in cgh-1 mutant, it was also significantly reduced by pab-1 RNAi. Our results suggest that PAB-1 positively regulates the mRNA levels of germline genes, which is likely facilitated by the interaction of PAB-1 with other P-body components, CGH-1 and CAR-1.

  1. N (6)-Methyladenosine (m(6)A) Methylation in mRNA with A Dynamic and Reversible Epigenetic Modification.

    Science.gov (United States)

    Wu, Ruifan; Jiang, Denghu; Wang, Yizhen; Wang, Xinxia

    2016-07-01

    N (6)-methyladenosine (m(6)A) is the most abundant and reversible internal modification ubiquitously occurring in eukaryotic mRNA, albeit the significant biological roles of m(6)A methylation have remained largely unclear. The well-known DNA and histone methylations play crucial roles in epigenetic modification of biologic processes in eukaryotes. Analogously, the dynamic and reversible m(6)A RNA modification, which is installed by methyltransferase (METTL3, METTL14, and WTAP), reversed by demethylases (FTO, ALKBH5) and mediated by m(6)A-binding proteins (YTHDF1-3, YTHDC1), may also have a profound impact on gene expression regulation. Recent discoveries of the distributions, functions, and mechanisms of m(6)A modification suggest that this methylation functionally modulates the eukaryotic transcriptome to influence mRNA transcription, splicing, nuclear export, localization, translation, and stability. This reversible mRNA methylation shed light on a new dimension of post-transcriptional regulation of gene expression at the RNA level. m(6)A methylation also plays significant and broad roles in various physiological processes, such as development, fertility, carcinogenesis, stemness, early mortality, meiosis and circadian cycle, and links to obesity, cancer, and other human diseases. This review mainly describes the current knowledge of m(6)A and perspectives on future investigations. PMID:27179969

  2. Synaptic adaptations by alcohol and drugs of abuse: changes in microRNA expression and mRNA regulation

    Directory of Open Access Journals (Sweden)

    Dana eMost

    2014-12-01

    Full Text Available Local translation of mRNAs is a mechanism by which cells can rapidly remodel synaptic structure and function. There is ample evidence for a role of synaptic translation in the neuroadaptations resulting from chronic drug use and abuse. Persistent and coordinated changes of many mRNAs, globally and locally, may have a causal role in complex disorders such as addiction. In this review we examine the evidence that translational regulation by microRNAs drives synaptic remodeling and mRNA expression, which may regulate the transition from recreational to compulsive drug use.MicroRNAs are small, non-coding RNAs that control the translation of mRNAs in the cell and within spatially restricted sites such as the synapse. MicroRNAs typically repress the translation of mRNAs into protein by binding to the 3’UTR of their targets. As ‘master regulators’ of many mRNAs, changes in microRNAs could account for the systemic alterations in mRNA and protein expression observed with drug abuse and dependence. Recent studies indicate that manipulation of microRNAs affects addiction-related behaviors such as the rewarding properties of cocaine, cocaine-seeking behavior and self-administration rates of alcohol. There is limited evidence, however, regarding how synaptic microRNAs control local mRNA translation during chronic drug exposure and how this contributes to the development of dependence.Here, we discuss research supporting microRNA regulation of local mRNA translation and how drugs of abuse may target this process. The ability of synaptic microRNAs to rapidly regulate mRNAs provides a discrete, localized system that could potentially be used as diagnostic and treatment tools for alcohol and other addiction disorders.

  3. Intronless β-Globin Reporter: A Tool for Studying Nuclear RNA Stability Elements.

    Science.gov (United States)

    Brown, Jessica A; Steitz, Joan A

    2016-01-01

    The intronless β-globin reporter, whose mRNA is intrinsically unstable due to the lack of introns, is a useful tool to study RNA stability elements in a heterologous transcript. Insertion of a stability element leads to the accumulation of intronless β-globin mRNA that can be visualized by conventional Northern blot analyses. In this chapter, we explain how to perform the β-globin reporter assay using the ENE (expression and nuclear retention element), a triple-helix-forming RNA stability element that protects reporter mRNA from 3'- 5' decay. A list of considerations is included for the use of ENEs as a tool to stabilize other RNAs. In this chapter, we provide a brief description of how to insert an ENE sequence into the 3'-untranslated region of an intronless β-globin reporter plasmid using basic cloning technology. Then, we provide a detailed protocol for quantitative measurements of steady-state levels of β-globin mRNA. This entails the transient transfection of mammalian cells with β-globin reporter plasmids, isolation of total cellular RNA, and detection of reporter mRNA via Northern blot. This methodology can be applied for the study of any nuclear RNA stability element using the intronless β-globin reporter. PMID:27236793

  4. LMKB/MARF1 localizes to mRNA processing bodies, interacts with Ge-1, and regulates IFI44L gene expression.

    Directory of Open Access Journals (Sweden)

    Donald B Bloch

    Full Text Available The mRNA processing body (P-body is a cellular structure that regulates the stability of cytoplasmic mRNA. MARF1 is a murine oocyte RNA-binding protein that is associated with maintenance of mRNA homeostasis and genomic stability. In this study, autoantibodies were used to identify Limkain B (LMKB, the human orthologue of MARF1, as a P-body component. Indirect immunofluorescence demonstrated that Ge-1 (a central component of the mammalian core-decapping complex co-localized with LMKB in P-bodies. Two-hybrid and co-immunoprecipitation assays were used to demonstrate interaction between Ge-1 and LMKB. The C-terminal 120 amino acids of LMKB mediated interaction with Ge-1 and the N-terminal 1094 amino acids of Ge-1 were required for interaction with LMKB. LMKB is the first protein identified to date that interacts with this portion of Ge-1. LMKB was expressed in human B and T lymphocyte cell lines; depletion of LMKB increased expression of IFI44L, a gene that has been implicated in the cellular response to Type I interferons. The interaction between LMKB/MARF1, a protein that contains RNA-binding domains, and Ge-1, which interacts with core-decapping proteins, suggests that LMKB has a role in the regulation of mRNA stability. LMKB appears to have different functions in different cell types: maintenance of genomic stability in developing oocytes and possible dampening of the inflammatory response in B and T cells.

  5. Relative workload determines exercise-induced increases in PGC-1alpha mRNA

    DEFF Research Database (Denmark)

    Nordsborg, Nikolai Baastrup; Lundby, Carsten; Leick, Lotte;

    2010-01-01

    after exercise at 85% of VO2 peak. Likewise, PDK4 and HKII mRNA expression were only increased (PHIF2alpha mRNA only increased (P... trial. No change in HIF1alpha, PFK, CS, LDH-A or LDH-B mRNA expression was detected after any of the exercise trials. CONCLUSION:: The relative intensity of brief intermittent exercise is of major importance for the exercise induced increase of several mRNA's, including PGC-1alpha....

  6. Downregulation of the microtubule associated protein tau impairs process outgrowth and myelin basic protein mRNA transport in oligodendrocytes.

    Science.gov (United States)

    Seiberlich, Veronika; Bauer, Nina G; Schwarz, Lisa; Ffrench-Constant, Charles; Goldbaum, Olaf; Richter-Landsberg, Christiane

    2015-09-01

    Oligodendrocytes, the myelin forming cells of the CNS, are characterized by their numerous membranous extensions, which enwrap neuronal axons and form myelin sheaths. During differentiation oligodendrocytes pass different morphological stages, downregulate the expression of the proteoglycan NG2, and acquire major myelin specific proteins, such as myelin basic proteins (MBP) and proteolipid protein. MBP mRNA is transported in RNA granules along the microtubules (MTs) to the periphery and translated locally. MTs participate in the elaboration and stabilization of the myelin forming extensions and are essential for cellular sorting processes. Their dynamic properties are regulated by microtubule associated proteins (MAPs). The MAP tau is present in oligodendrocytes and involved in the regulation and stabilization of the MT network. To further elucidate the functional significance of tau in oligodendrocytes, we have downregulated tau by siRNA technology and studied the effects on cell differentiation and neuron-glia contact formation. The data show that tau knockdown impairs process outgrowth and leads to a decrease in MBP expression. Furthermore, MBP mRNA transport to distant cellular extensions is impaired and cells remain in the NG2 stage. In myelinating cocultures with dorsal root ganglion neurons, oligodendrocyte precursor cells after tau miR RNA lentiviral knockdown develop into NG2 positive cells with very long and thin processes, contacting axons loosely, but fail to form internodes. This demonstrates that tau is important for MBP mRNA transport and involved in process formation. The disturbance of the balance of tau leads to abnormalities in oligodendrocyte differentiation, neuron-glia contact formation and the early myelination process.

  7. Thyroid hormone may regulate mRNA abundance in liver by acting on microRNAs.

    Directory of Open Access Journals (Sweden)

    Hongyan Dong

    Full Text Available MicroRNAs (miRNAs are extensively involved in diverse biological processes. However, very little is known about the role of miRNAs in mediating the action of thyroid hormones (TH. Appropriate TH levels are known to be critically important for development, differentiation and maintenance of metabolic balance in mammals. We induced transient hypothyroidism in juvenile mice by short-term exposure to methimazole and perchlorate from post natal day (PND 12 to 15. The expression of miRNAs in the liver was analyzed using Taqman Low Density Arrays (containing up to 600 rodent miRNAs. We found the expression of 40 miRNAs was significantly altered in the livers of hypothyroid mice compared to euthyroid controls. Among the miRNAs, miRs-1, 206, 133a and 133b exhibited a massive increase in expression (50- to 500-fold. The regulation of TH on the expression of miRs-1, 206, 133a and 133b was confirmed in various mouse models including: chronic hypothyroid, short-term hyperthyroid and short-term hypothyroid followed by TH supplementation. TH regulation of these miRNAs was also confirmed in mouse hepatocyte AML 12 cells. The expression of precursors of miRs-1, 206, 133a and 133b were examined in AML 12 cells and shown to decrease after TH treatment, only pre-mir-206 and pre-mir-133b reached statistical significance. To identify the targets of these miRNAs, DNA microarrays were used to examine hepatic mRNA levels in the short-term hypothyroid mouse model relative to controls. We found transcripts from 92 known genes were significantly altered in these hypothyroid mice. Web-based target predication software (TargetScan and Microcosm identified 14 of these transcripts as targets of miRs-1, 206, 133a and 133b. The vast majority of these mRNA targets were significantly down-regulated in hypothyroid mice, corresponding with the up-regulation of miRs-1, 206, 133a and 133b in hypothyroid mouse liver. To further investigate target genes, miR-206 was over-expressed in

  8. Thyroid Hormone May Regulate mRNA Abundance in Liver by Acting on MicroRNAs

    Science.gov (United States)

    Dong, Hongyan; Paquette, Martin; Williams, Andrew; Zoeller, R. Thomas; Wade, Mike; Yauk, Carole

    2010-01-01

    MicroRNAs (miRNAs) are extensively involved in diverse biological processes. However, very little is known about the role of miRNAs in mediating the action of thyroid hormones (TH). Appropriate TH levels are known to be critically important for development, differentiation and maintenance of metabolic balance in mammals. We induced transient hypothyroidism in juvenile mice by short-term exposure to methimazole and perchlorate from post natal day (PND) 12 to 15. The expression of miRNAs in the liver was analyzed using Taqman Low Density Arrays (containing up to 600 rodent miRNAs). We found the expression of 40 miRNAs was significantly altered in the livers of hypothyroid mice compared to euthyroid controls. Among the miRNAs, miRs-1, 206, 133a and 133b exhibited a massive increase in expression (50- to 500-fold). The regulation of TH on the expression of miRs-1, 206, 133a and 133b was confirmed in various mouse models including: chronic hypothyroid, short-term hyperthyroid and short-term hypothyroid followed by TH supplementation. TH regulation of these miRNAs was also confirmed in mouse hepatocyte AML 12 cells. The expression of precursors of miRs-1, 206, 133a and 133b were examined in AML 12 cells and shown to decrease after TH treatment, only pre-mir-206 and pre-mir-133b reached statistical significance. To identify the targets of these miRNAs, DNA microarrays were used to examine hepatic mRNA levels in the short-term hypothyroid mouse model relative to controls. We found transcripts from 92 known genes were significantly altered in these hypothyroid mice. Web-based target predication software (TargetScan and Microcosm) identified 14 of these transcripts as targets of miRs-1, 206, 133a and 133b. The vast majority of these mRNA targets were significantly down-regulated in hypothyroid mice, corresponding with the up-regulation of miRs-1, 206, 133a and 133b in hypothyroid mouse liver. To further investigate target genes, miR-206 was over-expressed in AML 12 cells. TH

  9. Altered fingerprints: analysis and detection.

    Science.gov (United States)

    Yoon, Soweon; Feng, Jianjiang; Jain, Anil K

    2012-03-01

    The widespread deployment of Automated Fingerprint Identification Systems (AFIS) in law enforcement and border control applications has heightened the need for ensuring that these systems are not compromised. While several issues related to fingerprint system security have been investigated, including the use of fake fingerprints for masquerading identity, the problem of fingerprint alteration or obfuscation has received very little attention. Fingerprint obfuscation refers to the deliberate alteration of the fingerprint pattern by an individual for the purpose of masking his identity. Several cases of fingerprint obfuscation have been reported in the press. Fingerprint image quality assessment software (e.g., NFIQ) cannot always detect altered fingerprints since the implicit image quality due to alteration may not change significantly. The main contributions of this paper are: 1) compiling case studies of incidents where individuals were found to have altered their fingerprints for circumventing AFIS, 2) investigating the impact of fingerprint alteration on the accuracy of a commercial fingerprint matcher, 3) classifying the alterations into three major categories and suggesting possible countermeasures, 4) developing a technique to automatically detect altered fingerprints based on analyzing orientation field and minutiae distribution, and 5) evaluating the proposed technique and the NFIQ algorithm on a large database of altered fingerprints provided by a law enforcement agency. Experimental results show the feasibility of the proposed approach in detecting altered fingerprints and highlight the need to further pursue this problem. PMID:21808092

  10. Cocaine differentially regulates activator protein-1 mRNA levels and DNA-binding complexes in the rat striatum and cerebellum.

    Science.gov (United States)

    Couceyro, P; Pollock, K M; Drews, K; Douglass, J

    1994-10-01

    Cocaine is a psychomotor stimulant that exerts many of its behavioral and physiological effects through alteration of catecholamine reuptake systems. One early cellular response to cocaine administration is a brain region-specific alteration in the transcriptional pattern of immediate early genes belonging to the Fos/Jun family of nucleotide sequence-specific [activator protein-1 (AP-1)] DNA-binding proteins. The work described here compares cocaine-induced transcriptional regulation of immediate early gene mRNA levels, as well as AP-1 DNA-binding activity, within the striatum and cerebellum. In the striatum, acute cocaine administration increases cellular levels of c-fos and jun-B mRNA, whereas transcriptional effects in the cerebellum are limited to c-fos mRNA. After chronic cocaine treatment a desensitization of c-fos mRNA induction is observed in the striatum, with sensitization of the same transcriptional effect occurring in the cerebellum. Pharmacological studies further reveal that the dopamine D1, dopamine D2, gamma-aminobutyric acid type B, and N-methyl-D-aspartate receptor systems mediate the effects of cocaine on cerebellar neurons, whereas striatal effects are modulated through D1 and N-methyl-D-aspartate receptors. Gel retention analysis using antibodies to the various Fos and Jun proteins was used to characterize cocaine-dependent alterations in the composition of striatal and cerebellar AP-1 DNA-binding complexes. In striatum, cocaine increases the relative levels of c-Fos, Fos-B, Jun-B, and Jun-D proteins that bind the AP-1 DNA sequence element, whereas in the cerebellum only c-Fos and Jun-D binding activities are increased. These data suggest two possible neuroanatomical sites where tolerance and sensitization to cocaine can be examined at the genomic level. PMID:7969045

  11. Body Fluid Identification Using mRNA Profiling.

    Science.gov (United States)

    Roeder, Amy D; Haas, Cordula

    2016-01-01

    RNA analysis is a valuable tool for the identification of the forensically relevant body fluids, saliva, blood, menstrual blood, cervicovaginal fluid, and semen. Multiple human mRNA and bacterial RNA markers have been identified for each of these body fluids. RNA and DNA can be coextracted from the same portion of a sample and RNA markers for different body fluids can be multiplexed in a single PCR, thereby maximizing the number of analyses that can be performed with limited sample material. PMID:27259728

  12. [Age-dependent changes in mRNA transport (nucleus-cytoplasm)].

    Science.gov (United States)

    Müller, W E; Agutter, P S; Prochnow, D J; Fasold, H; Sève, A P; Tsiapalis, C M; Schröder, H C

    1993-01-01

    Transport of mRNA from nucleus to cytoplasm is an ATP-dependent process which occurs strictly vectorially. Because the mRNA is structurally bound during transport, mRNA transport is a "solid-state" process consisting of i) mRNA release from the nuclear matrix, ii) mRNA translocation through the nuclear pore, and iii) cytoskeletal binding. We identified and purified the following components involved in the translocation step: i) the nuclear envelope (NE) nucleoside triphosphatase (NTPase) which is stimulated by the 3'poly(A) tail of mRNA, ii) the poly(A)-recognizing mRNA carrier, iii) the NE protein kinase, and iv) the NE phosphatase. In addition, we found that an RNA helicase activity is present in NE, which also may be involved in RNA transport. Our results show that, besides poly(A), also double-stranded RNA structures may modulate RNA export. The amount of mRNA released from nuclei markedly decreases with age. Evidence is presented that this age-dependent change is caused by an impairment of polyadenylation of mRNA, hnRNA processing, release of mRNA from nuclear matrix, and translocations of mRNA from nuclear to cytoplasmic compartment (decrease in activities of NE NTPase, protein kinase, and phosphatase; decrease in poly(A)-binding affinity of mRNA carrier).

  13. RNase III cleavage of Escherichia coli beta-galactosidase and tryptophan operon mRNA.

    OpenAIRE

    Shen, V; Imamoto, F; Schlessinger, D

    1982-01-01

    Purified RNase III of Escherichia coli cleaved the initial 479-nucleotide sequence of lac operon mRNA at four specific sites and also gave limited cleavage of trp operon mRNA. This action explains the inactivation of mRNA coding capacity by RNase III in vitro.

  14. Differential regulation of host mRNA translation during obligate pathogen-plant interactions

    Science.gov (United States)

    Virus infection reprograms the plant messenger RNA (mRNA) transcriptome by activating or interfering with a variety of signaling pathways, but the effects on host mRNA translation have not been explored on a genome-wide scale. To address this issue, Arabidopsis thaliana mRNA transcripts were quantif...

  15. Transcript-Specific Decapping and Regulated Stability by the Human Dcp2 Decapping Protein▿ †

    OpenAIRE

    Li,You; Song, Man-Gen; Kiledjian, Megerditch

    2007-01-01

    mRNA decapping is a critical step in the control of mRNA stability and gene expression and is carried out by the Dcp2 decapping enzyme. Dcp2 is an RNA binding protein that must bind RNA in order to recognize the cap for hydrolysis. We demonstrate that human Dcp2 (hDcp2) preferentially binds to a subset of mRNAs and identify sequences at the 5′ terminus of the mRNA encoding Rrp41, a core subunit component of the RNA exosome, as a specific hDcp2 substrate. A 60-nucleotide element at the 5′ end ...

  16. Functional dissection of nuclear envelope mRNA translocation system: effects of phorbol ester and a monoclonal antibody recognizing cytoskeletal structures.

    Science.gov (United States)

    Schröder, H C; Diehl-Seifert, B; Rottmann, M; Messer, R; Bryson, B A; Agutter, P S; Müller, W E

    1988-03-01

    Unidirectional transport of poly(A)-containing mRNA [poly(A)+ mRNA] through the nuclear envelope pore complex is thought to be an energy (ATP or GTP)-dependent process which involves a nuclear envelope nucleoside triphosphatase (NTPase). In the intact envelope, this enzyme is regulatable by poly(A) binding and by poly(A)-dependent phosphorylation/dephosphorylation of other components of the mRNA translocation system, which are as yet unidentified. Monoclonal antibodies (mAbs) were elicited against the poly(A) binding nuclear envelope fraction isolated from rat liver. The mAbs were screened for their modulatory effects on mRNA transport in vitro. One stable clone decreased the efflux of rapidly labeled RNA and of one specific mRNA (ovalbumin) from isolated nuclei. It increased the binding of poly(A) to the envelope and increased the maximal catalytic rate of the NTPase, but it did not alter the apparent Km of the enzyme or the extent of its stimulation by poly(A). The nuclear envelope-associated protein kinase that down-regulates the NTPase was inhibited by the antibody, while other protein kinases were not affected. Because both the NTPase and mRNA efflux were inhibited by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, the sensitive kinase is probably protein kinase C. Protein kinase C was found to be associated with the isolated nuclear envelope. The antibody reacted with both a Mr 83,000 and a Mr 65,000 nuclear envelope polypeptide from rat liver and other tissues. By immunofluorescence microscopy in CV-1 cells, the antibody localized to the nuclear envelope and, in addition, to cytoplasmic filaments which show some superposition with the microfilament network.

  17. Decreased GATA5 mRNA expression associates with CpG island methylation and shortened recurrence-free survival in clear cell renal cell carcinoma

    International Nuclear Information System (INIS)

    GATA-5, a zinc-finger transcription factor and member of the GATA family proteins 1–6, is known to be involved in cellular differentiation. We recently found that tumor-specific hypermethylation of the GATA5 CpG island (CGI) occurs in renal cell carcinoma (RCC) and is associated with an adverse clinical outcome. In this study, we investigated whether epigenetic GATA5 alterations may result in changes in GATA5 mRNA expression levels and correlate with the observed prognostic impact of epigenetic changes in GATA5 in RCC. Quantitative real-time reverse-transcribed polymerase chain reaction was applied to measure relative GATA5 mRNA expression levels in 135 kidney tissue samples, including 77 clear cell RCC (ccRCC) tissues and 58 paired adjacent normal renal tissue samples. Relative GATA5 expression levels were determined using the ΔΔCt method and detection of three endogenous control genes then compared to previously measured values of relative methylation. The mean relative GATA5 mRNA expression level exhibited an approximately 31-fold reduction in tumor specimens compared with corresponding normal tissues (p < 0.001, paired t-test). Decreased GATA5 mRNA expression was inversely correlated with increased GATA5 CGI methylation (p < 0.001) and was associated with shortened recurrence-free survival in ccRCC patients (p = 0.023, hazard ratio = 0.25). GATA5 mRNA expression is decreased in ccRCC, likely due to gene silencing by methylation of the GATA5 CGI. Moreover, reduced GATA5 mRNA levels were associated with a poor clinical outcome, indicating a possible role of GATA5 for the development of aggressive ccRCC phenotypes

  18. Genetic Alterations in Glioma

    International Nuclear Information System (INIS)

    Gliomas are the most common type of primary brain tumor and have a dismal prognosis. Understanding the genetic alterations that drive glioma formation and progression may help improve patient prognosis by identification of novel treatment targets. Recently, two major studies have performed in-depth mutation analysis of glioblastomas (the most common and aggressive subtype of glioma). This systematic approach revealed three major pathways that are affected in glioblastomas: The receptor tyrosine kinase signaling pathway, the TP53 pathway and the pRB pathway. Apart from frequent mutations in the IDH1/2 gene, much less is known about the causal genetic changes of grade II and III (anaplastic) gliomas. Exceptions include TP53 mutations and fusion genes involving the BRAF gene in astrocytic and pilocytic glioma subtypes, respectively. In this review, we provide an update on all common events involved in the initiation and/or progression across the different subtypes of glioma and provide future directions for research into the genetic changes

  19. Music alters visual perception.

    Directory of Open Access Journals (Sweden)

    Jacob Jolij

    Full Text Available BACKGROUND: Visual perception is not a passive process: in order to efficiently process visual input, the brain actively uses previous knowledge (e.g., memory and expectations about what the world should look like. However, perception is not only influenced by previous knowledge. Especially the perception of emotional stimuli is influenced by the emotional state of the observer. In other words, how we perceive the world does not only depend on what we know of the world, but also by how we feel. In this study, we further investigated the relation between mood and perception. METHODS AND FINDINGS: We let observers do a difficult stimulus detection task, in which they had to detect schematic happy and sad faces embedded in noise. Mood was manipulated by means of music. We found that observers were more accurate in detecting faces congruent with their mood, corroborating earlier research. However, in trials in which no actual face was presented, observers made a significant number of false alarms. The content of these false alarms, or illusory percepts, was strongly influenced by the observers' mood. CONCLUSIONS: As illusory percepts are believed to reflect the content of internal representations that are employed by the brain during top-down processing of visual input, we conclude that top-down modulation of visual processing is not purely predictive in nature: mood, in this case manipulated by music, may also directly alter the way we perceive the world.

  20. Cytoskeleton-Dependent Transport as a Potential Target for Interfering with Post-transcriptional HuR mRNA Regulons.

    Science.gov (United States)

    Eberhardt, Wolfgang; Badawi, Amel; Biyanee, Abhiruchi; Pfeilschifter, Josef

    2016-01-01

    The ubiquitous mRNA binding protein human antigen R (HuR), a member of the embryonal lethal abnormal vision protein family has a critical impact on the post-transcriptional control of AU-rich element bearing mRNA regulons implied in inflammation, senescence, and carcinogenesis. HuR in addition to mRNA stability can affect many other aspects of mRNA processing including splicing, polyadenylation, translation, modulation of miRNA repression, and intracellular mRNA trafficking. Since many of the identified HuR mRNA targets ("HuR mRNA regulons") encode tumor-related proteins, HuR is not only discussed as an useful biomarker but also as promising therapeutic target for treatment of various human cancers. HuR which is most abundantly localized in the nucleus is translocated to the cytoplasm which is fundamental for most of the described HuR functions on target mRNAs. Accordingly, an elevation in cytoplasmic HuR was found in many tumors and correlated with a high grade of malignancy and a poor prognosis of patients. Therefore, direct interference with the intracellular trafficking of HuR offers an attractive approach to intervene with pathologically deregulated HuR functions. Data from several laboratories implicate that the integrity of the cytoskeleton is critical for HuR-mediated intracellular mRNA localization and translation. This review will particularly focus on drugs which have proven a direct inhibitory effect on HuR translocation. Based on the results from those studies, we will also discuss on the principle value of targeting cytoskeleton-dependent transport of HuR by natural or synthetic inhibitors as a potential therapeutic avenue for interfering with dysregulated post-transcriptional HuR mRNA regulons and related tumor cell functions. In spite of that, interfering with cytoplasmic HuR transport could highlight a so far underestimated action of microtubule inhibitors clinically used for cancer chemotherapy. PMID:27582706

  1. Optimization of antisense drug design againstconservative local motif in simulant secondary structures of HER-2 mRNA and QSAR analysis

    Institute of Scientific and Technical Information of China (English)

    YANGShuan-Ping; SONGSan-Tai; TANGZhong-Ming; SONGHai-Feng

    2003-01-01

    AIM: To study the role of mRNA secondary structure stability in antisense drug design and obtain better antisensecandidates against neu/HER-2/erbB-2 mRNA than previous report. METHODS: Program RNAstructure wasutilized to simulate the secondary structures of HER-2 mRNA. Then 21 antisense phosphorothioateoligodeoxynucleotides (S-ODN) targeting different parts of secondary structural motif were designed. HA4 wasset as positive control. Mean 50 % inhibitory effects (IC50) of S-ODN on proliferations of SK-BR-3 breast cancercells were evaluated. The expression of target mRNA was detected by RT-PCR. The multiple regression andquantitative structure-activity relationship (QSAR) analysis was preformed by SPSS software. RESULTS: Oneoptimal and two suboptimal secondary structures of target mRNA were obtained. Nine out of 11 S-ODN againstcompletely conservative local motif (LM) (conservative among all simulant secondary structures) got lower orsimilar IC50 values compared with HA4. On the other hand, 2 out of 3 S-ODN against relatively conservative LM(conservative between any two simulant secondary structures) got lower or similar IC50 values compared withHA4. Only 2 out of 5 S-ODN targeting variable LM (variable among different predicted secondary structures) hadacceptable activities. Average IC50 of S-ODN against completely conservative LM was significantly lower than thatof S-ODN against diverse LM. QSAR analysis suggested that stability, base number of bulge loops, and target freeenergies △G°T were statistically significant. In the multiple regression, R was 0.967, P=0.005. CONCLUSION:Antisense drug design against conservative LM was helpful for improving the positive rate. Several S-ODN candidates better than positive control were screened.

  2. Sequence-specific binding of a hormonally regulated mRNA binding protein to cytidine-rich sequences in the lutropin receptor open reading frame.

    Science.gov (United States)

    Kash, J C; Menon, K M

    1999-12-21

    In previous studies, a lutropin receptor mRNA binding protein implicated in the hormonal regulation of lutropin receptor mRNA stability was identified. This protein, termed LRBP-1, was shown by RNA gel electrophoretic mobility shift assay to specifically interact with lutropin receptor RNA sequences. The present studies have examined the specificity of lutropin receptor mRNA recognition by LRBP-1 and mapped the contact site by RNA footprinting and by site-directed mutagenesis. LRBP-1 was partially purified by cation-exchange chromatography, and the mRNA binding properties of the partially purified LRBP-1 were examined by RNA gel electrophoretic mobility shift assay and hydroxyl-radical RNA footprinting. These data showed that the LRBP-1 binding site is located between nucleotides 203 and 220 of the receptor open reading frame, and consists of the bipartite polypyrimidine sequence 5'-UCUC-X(7)-UCUCCCU-3'. Competition RNA gel electrophoretic mobility shift assays demonstrated that homoribopolymers of poly(rC) were effective RNA binding competitors, while poly(rA), poly(rG), and poly(rU) showed no effect. Mutagenesis of the cytidine residues contained within the LRBP-1 binding site demonstrated that all the cytidines in the bipartite sequence contribute to LRBP-1 binding specificity. Additionally, RNA gel electrophoretic mobility supershift analysis showed that LRBP-1 was not recognized by antibodies against two well-characterized poly(rC) RNA binding proteins, alphaCP-1 and alphaCP-2, implicated in the regulation of RNA stability of alpha-globin and tyrosine hydroxylase mRNAs. In summary, we show that partially purified LRBP-1 binds to a polypyrimidine sequence within nucleotides 203 and 220 of lutropin receptor mRNA with a high degree of specificity which is indicative of its role in posttranscriptional control of lutropin receptor expression.

  3. Prolonged exposure of chromaffin cells to nitric oxide down-regulates the activity of soluble guanylyl cyclase and corresponding mRNA and protein levels

    Science.gov (United States)

    Ferrero, Rut; Torres, Magdalena

    2002-01-01

    Background Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one α and one β subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model. Results Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and β1 subunit protein levels. Both sGC activity and β1 subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases β1 subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in β1 subunits. Conclusions These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing β1 subunit stability. PMID:12350235

  4. Heterogeneity of zein mRNA and protein in maize.

    Science.gov (United States)

    Park, W D; Lewis, E D; Rubenstein, I

    1980-01-01

    Zein, the prolamine fraction of maize, is localized in the endosperm in membrane-bound structures called protein bodies, which have polyribosomes on their surfaces. These polysomes or the mRNA fraction isolated from them will direct the synthesis of zein-like proteins in vitro. The in vitro products consist primarily of two molecular weight classes but show considerable charge heterogeneity when analyzed by isoelectric focusing. Although the molecular weight classes are very similar for different inbred lines, the isoelectric focusing patterns differ.Results given here suggest that the extensive charge heterogeneity of zein proteins does not result from the presence of a large number of totally distinct mRNAs. Zein proteins synthesized in vitro fall into several families related by sequence homologies in their mRNAs. In Illinois High Protein (IHP) the major zein mRNAs can be classified into three families based on their binding to cloned complimentary DNA copies of IHP zein mRNA. Each of three other lines we have studied (W22, Oh43, and W64A) has zein mRNAs that are related to those of IHP. Among these four lines the molecular weights of the members of a given family are generally similar, but the number of members in a family and their isoelectric points differ. PMID:16661153

  5. Myc Regulation of mRNA Cap Methylation

    Science.gov (United States)

    Cowling, Victoria H.; Cole, Michael D.

    2010-01-01

    The c-myc proto-oncogene regulates the expression of 15% to 20% of all genes, depending on the cell type, and the regulation is usually modest (1.5- to 2.0-fold). The authors discovered that in addition to regulating mRNA abundance, c-Myc regulates the formation of the 7-methylguanosine cap on many mRNAs, including transcriptional target genes and others not transcriptionally activated. Because the 7-methylguanosine cap is required for effective translation, enhanced methyl cap formation leads to increased protein production from Myc-responsive genes that exceeds the transcriptional induction. Increased cap methylation is linked to Myc-dependent enhanced activity of 2 critical kinases, TFIIH and p-TEFb, which phosphorylate the RNA polymerase II carboxy-terminal domain (CTD). Phosphorylation of the CTD recruits RNGTT and RNMT, the enzymes involved in mRNA capping, to the nascent transcript. Evidence is accumulating that enhanced cap methylation makes a significant contribution to Myc-dependent gene regulation and protein production. PMID:21170289

  6. Tracking single mRNA molecules in live cells

    Science.gov (United States)

    Moon, Hyungseok C.; Lee, Byung Hun; Lim, Kiseong; Son, Jae Seok; Song, Minho S.; Park, Hye Yoon

    2016-06-01

    mRNAs inside cells interact with numerous RNA-binding proteins, microRNAs, and ribosomes that together compose a highly heterogeneous population of messenger ribonucleoprotein (mRNP) particles. Perhaps one of the best ways to investigate the complex regulation of mRNA is to observe individual molecules. Single molecule imaging allows the collection of quantitative and statistical data on subpopulations and transient states that are otherwise obscured by ensemble averaging. In addition, single particle tracking reveals the sequence of events that occur in the formation and remodeling of mRNPs in real time. Here, we review the current state-of-the-art techniques in tagging, delivery, and imaging to track single mRNAs in live cells. We also discuss how these techniques are applied to extract dynamic information on the transcription, transport, localization, and translation of mRNAs. These studies demonstrate how single molecule tracking is transforming the understanding of mRNA regulation in live cells.

  7. mRNA quality control pathways in Saccharomyces cerevisiae

    Indian Academy of Sciences (India)

    Satarupa Das; Biswadip Das

    2013-09-01

    Efficient production of translation-competent mRNAs involves processing and modification events both in the nucleus and cytoplasm which require a number of complex machineries at both co-transcriptional and post-transcriptional levels. Mutations in the genomic sequence sometimes result in the formation of mutant non-functional defective messages. In addition, the enormous amounts of complexities involved in the biogenesis of mRNPs in the nucleus very often leads to the formation of aberrant and faulty messages along with their functional counterpart. Subsequent translation of these mutant and defective populations of messenger RNAs could possibly result in the unfaithful transmission of genetic information and thus is considered a threat to the survival of the cell. To prevent this possibility, mRNA quality control systems have evolved both in the nucleus and cytoplasm in eukaryotes to scrutinize various stages of mRNP biogenesis and translation. In this review, we will focus on the physiological role of some of these mRNA quality control systems in the simplest model eukaryote Saccharomyces cerevisiae.

  8. Specific miRNA Stabilization by Gld2-Catalyzed Monoadenylation

    Directory of Open Access Journals (Sweden)

    Andrea D’Ambrogio

    2012-12-01

    Full Text Available MicroRNAs (miRNAs are small, noncoding RNAs that inhibit translation and promote mRNA decay. The levels of mature miRNAs are the result of different rates of transcription, processing, and turnover. The noncanonical polymerase Gld2 has been implicated in the stabilization of miR-122, possibly through catalyzing 3′ monoadenylation; however, there is little evidence that this relationship is one of cause and effect. Here, we biochemically characterize Gld2’s involvement in miRNA monoadenylation and its effect on miRNA stability. We find that Gld2 directly monoadenylates and stabilizes specific miRNA populations in human fibroblasts and that sensitivity to monoadenylation-induced stability depends on nucleotides in the miRNA 3′ end. These results establish a mechanism of miRNA stability and resulting posttranscriptional gene regulation.

  9. Food Fortification Stability Study

    Science.gov (United States)

    Sirmons, T.; Cooper, M.; Douglas, G.

    2017-01-01

    NASA has established the goal of traveling beyond low-Earth orbit and extending manned exploration to Mars. The length of proposed Mars missions and the lack of resupply missions increases the importance of nutritional content in the food system, which will need a five-year shelf life. The purpose of this research is to assess the stability of vitamin supplementation in traditionally processed spaceflight foods. It is expected that commercially available fortification nutrients will remain stable through a long duration exploration mission at sufficient levels if compatible formulation, processing, and storage temperatures are achieved. Five vitamins (vitamin E, vitamin K, pantothenic acid, folic acid, and thiamin) were blended into a vitamin premix (DSM, Freeport, TX) such that the vitamin concentration per serving equaled 25% of the recommended daily intake after two years of ambient storage. Four freeze-dried foods (Scrambled Eggs, Italian Vegetables, Potatoes Au Gratin, Noodles and Chicken) and four thermo-stabilized foods (Curry Sauce with Vegetables, Chicken Noodle Soup, Grilled Pork Chop, Rice with Butter) were produced, with and without the vitamin premix, to assess the impact of the added fortification on color and taste and to determine the stability of supplemental vitamins in spaceflight foods. The addition of fortification to spaceflight foods did not greatly alter the organoleptic properties of most products. In most cases, overall acceptability scores remained above 6.0 (minimum acceptable score) following six months and one year of low-temperature storage. Likewise, the color of fortified products appears to be preserved over one year of storage. The only exceptions were Grilled pork Chop and Chicken Noodle Soup whose individual components appeareddegrade rapidly over one year of storage. Finally, most vitamins appeared to be stable during long-term storage. The only exception was thiamin, which degraded rapidly during the first year of storage at

  10. Acute chlorine gas exposure produces transient inflammation and a progressive alteration in surfactant composition with accompanying mechanical dysfunction

    International Nuclear Information System (INIS)

    Acute Cl2 exposure following industrial accidents or military/terrorist activity causes pulmonary injury and severe acute respiratory distress. Prior studies suggest that antioxidant depletion is important in producing dysfunction, however a pathophysiologic mechanism has not been elucidated. We propose that acute Cl2 inhalation leads to oxidative modification of lung lining fluid, producing surfactant inactivation, inflammation and mechanical respiratory dysfunction at the organ level. C57BL/6J mice underwent whole-body exposure to an effective 60 ppm-hour Cl2 dose, and were euthanized 3, 24 and 48 h later. Whereas pulmonary architecture and endothelial barrier function were preserved, transient neutrophilia, peaking at 24 h, was noted. Increased expression of ARG1, CCL2, RETLNA, IL-1b, and PTGS2 genes was observed in bronchoalveolar lavage (BAL) cells with peak change in all genes at 24 h. Cl2 exposure had no effect on NOS2 mRNA or iNOS protein expression, nor on BAL NO3− or NO2−. Expression of the alternative macrophage activation markers, Relm-α and mannose receptor was increased in alveolar macrophages and pulmonary epithelium. Capillary surfactometry demonstrated impaired surfactant function, and altered BAL phospholipid and surfactant protein content following exposure. Organ level respiratory function was assessed by forced oscillation technique at 5 end expiratory pressures. Cl2 exposure had no significant effect on either airway or tissue resistance. Pulmonary elastance was elevated with time following exposure and demonstrated PEEP refractory derecruitment at 48 h, despite waning inflammation. These data support a role for surfactant inactivation as a physiologic mechanism underlying respiratory dysfunction following Cl2 inhalation. - Highlights: • Effect of 60 ppm*hr Cl2 gas on lung inflammation and mechanical function examined. • Pulmonary inflammation is transient and minor. • Alterations in surfactant homeostasis and pulmonary mechanics

  11. Microarray-based mRNA expression profiling of leukemia cells treated with the flavonoid, casticin.

    Science.gov (United States)

    Righeschi, Chiara; Eichhorn, Tolga; Karioti, Anastasia; Bilia, Anna Rita; Efferth, Thomas

    2012-01-01

    Natural polyphenols play an important role in tumor inhibition. We used a doxorubicin-sensitive acute T-lymphoblastic leukemia cell line (CCRF-CEM) and its multidrug-resistant subline (CEM/ADR5000) to evaluate the activity of 15 plant polyphenols isolated in our laboratory (hypericin and pseudohypericin, verbascoside, ellagic acid, casticin, kaempferol-3-O-(2'',6''-di-E-p-coumaroyl)-glucopyranoside, kaempferol-3-O-(3,4-diacetyl-2,6-di-E-p-coumaroyl) -glucopyranoside, tiliroside, salvianolic acid B, oleuropein, rosmarinic acid, bergenin) or of others from commercial sources (curcumin, epigallocatechin-3-gallate, silymarin). Casticin was the most potent compound (IC50 values of 0.28 ± 0.02 μM in CCRF-CEM and 0.44 ± 0.17 μM in CEM/ADR5000 cells. The IC50 values of the other compounds tested ranged from 1.52 μM to 164.1 μM. A microarray-based mRNA expression profiling of CCRF-CEM cells treated with casticin was performed in order to identify genes with altered expression following casticin treatment. Networks related to NF-κB, p38MAPK, histones H3 and H4, and follicle stimulating hormone were identified.

  12. Comparison of mRNA Splicing Assay Protocols across Multiple Laboratories

    DEFF Research Database (Denmark)

    Whiley, Phillip J; de la Hoya, Miguel; Thomassen, Mads;

    2014-01-01

    and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501......Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data...... in turn relies on appropriate assay design, interpretation, and reporting.METHODS: We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities...

  13. An Essential Role for COPI in mRNA Localization to Mitochondria and Mitochondrial Function.

    Science.gov (United States)

    Zabezhinsky, Dmitry; Slobodin, Boris; Rapaport, Doron; Gerst, Jeffrey E

    2016-04-19

    Nuclear-encoded mRNAs encoding mitochondrial proteins (mMPs) can localize directly to the mitochondrial surface, yet how mMPs target mitochondria and whether RNA targeting contributes to protein import into mitochondria and cellular metabolism are unknown. Here, we show that the COPI vesicle coat complex is necessary for mMP localization to mitochondria and mitochondrial function. COPI inactivation leads to reduced mMP binding to COPI itself, resulting in the dissociation of mMPs from mitochondria, a reduction in mitochondrial membrane potential, a decrease in protein import in vivo and in vitro, and severe deficiencies in mitochondrial respiration. Using a model mMP (OXA1), we observed that COPI inactivation (or mutation of the potential COPI-interaction site) led to altered mRNA localization and impaired cellular respiration. Overall, COPI-mediated mMP targeting is critical for mitochondrial protein import and function, and transcript delivery to the mitochondria or endoplasmic reticulum is regulated by cis-acting RNA sequences and trans-acting proteins.

  14. Defence transcriptome profiling of Zingiber zerumbet (L.) Smith by mRNA differential display

    Indian Academy of Sciences (India)

    P G Kavitha; George Thomas

    2008-03-01

    Soft rot is a serious disease in ginger (Zingiber officinale Roscoe), imposing a considerable economic loss annually in all ginger-producing countries. In this study, mRNA differential display was employed to identify genes whose expression was altered in a soft rot-resistant accession of Zingiber zerumbet (L.) Smith, a wild relative of ginger, in response to Pythium aphanidermatum (Edson) Fitzp., which is the principal causative agent of soft-rot disease in ginger. Analysis using 68 primer combinations identified 70 differentially expressed transcript-derived fragments (TDFs), of which 34 TDFs were selected for further analysis following reverse northern screening. Cloning and sequence characterization of the 34 TDFs yielded a total of 54 distinct clones. Functional categorization of these clones revealed seven categories, of which the defence/stress/signalling group was the largest, with clones homologous to genes known to be actively involved in various pathogenesis-related functions in other plant species. The significance of these genes in relation to the resistance response in Z. zerumbet is discussed. This study has provided a pool of candidate genes for detailed molecular dissection of the defence mechanisms in Z. zerumbet and for accessing wild genetic resources for the transgenic improvement of ginger.

  15. Tissue-specific alterations in expression and function of P-glycoprotein in streptozotocininduced diabetic rats

    Institute of Scientific and Technical Information of China (English)

    Lu-lu ZHANG; Guang-ji WANG; Lin XIE; Liang LU; Shi JIN; Xin-yue JING; Dan YAO; Nan HU; Li LIU; Ru DUAN; Xiao-dong LIU

    2011-01-01

    Aim: To investigate the changes of expression and function of P-glycoprotein (P-GP) in cerebral cortex, hippocampus, liver, intestinal mucosa and kidney of streptozocin-induced diabetic rats.Methods: Diabetic rats were prepared via a single dose of streptozocin (65 mg/kg, ip). Abcb1/P-GP mRNA and protein expression levels in tissues were evaluated using quantitative real time polymerase chain reaction (QRT-PCR) analysis and Western blot, respectively.P-GP function was investigated via measuring tissue-to-plasma concentration ratios and body fluid excretion percentages of rhodamine 123.Results: In 5- and 8-week diabetic rats, Abcb1a mRNA levels were significantly decreased in cerebral cortices and intestinal mucosa,but dramatically increased in hippocampus and kidney. In liver, the level was increased in 5-week diabetic rats, and decreased in 8-week diabetic rats. Abcb1b mRNA levels were increased in cerebral cortex, hippocampus and kidney, but reduced in liver and intestinal mucosa in the diabetic rats. Western blot results were in accordance with the alterations of Abcb1a mRNA levels in most tissues examined. P-GP activity was markedly decreased in most tissues of diabetic rats, except kidney tissues.Conclusion: Alterations in the expression and function of Abcb1/P-GP under diabetic conditions are tissue specific, Abcb1 specific and diabetic duration-dependent.

  16. Plutonium inventories for stabilization and stabilized materials

    Energy Technology Data Exchange (ETDEWEB)

    Williams, A.K.

    1996-05-01

    The objective of the breakout session was to identify characteristics of materials containing plutonium, the need to stabilize these materials for storage, and plans to accomplish the stabilization activities. All current stabilization activities are driven by the Defense Nuclear Facilities Safety Board Recommendation 94-1 (May 26, 1994) and by the recently completed Plutonium ES&H Vulnerability Assessment (DOE-EH-0415). The Implementation Plan for accomplishing stabilization of plutonium-bearing residues in response to the Recommendation and the Assessment was published by DOE on February 28, 1995. This Implementation Plan (IP) commits to stabilizing problem materials within 3 years, and stabilizing all other materials within 8 years. The IP identifies approximately 20 metric tons of plutonium requiring stabilization and/or repackaging. A further breakdown shows this material to consist of 8.5 metric tons of plutonium metal and alloys, 5.5 metric tons of plutonium as oxide, and 6 metric tons of plutonium as residues. Stabilization of the metal and oxide categories containing greater than 50 weight percent plutonium is covered by DOE Standard {open_quotes}Criteria for Safe Storage of Plutonium Metals and Oxides{close_quotes} December, 1994 (DOE-STD-3013-94). This standard establishes criteria for safe storage of stabilized plutonium metals and oxides for up to 50 years. Each of the DOE sites and contractors with large plutonium inventories has either started or is preparing to start stabilization activities to meet these criteria.

  17. Trapped particle stability for the kinetic stabilizer

    Science.gov (United States)

    Berk, H. L.; Pratt, J.

    2011-08-01

    A kinetically stabilized axially symmetric tandem mirror (KSTM) uses the momentum flux of low-energy, unconfined particles that sample only the outer end-regions of the mirror plugs, where large favourable field-line curvature exists. The window of operation is determined for achieving magnetohydrodynamic (MHD) stability with tolerable energy drain from the kinetic stabilizer. Then MHD stable systems are analysed for stability of the trapped particle mode. This mode is characterized by the detachment of the central-cell plasma from the kinetic-stabilizer region without inducing field-line bending. Stability of the trapped particle mode is sensitive to the electron connection between the stabilizer and the end plug. It is found that the stability condition for the trapped particle mode is more constraining than the stability condition for the MHD mode, and it is challenging to satisfy the required power constraint. Furthermore, a severe power drain may arise from the necessary connection of low-energy electrons in the kinetic stabilizer to the central region.

  18. EGF mRNA Expression in Pig Ovary

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Epidermal growth factor(EGF)is known to enhance oocyte maturation,embryo development and implantation in many species. To study the rules of EGF in pig ovary,the level of EGF mRNA activity was measured by RT-PCR technique. A strong EGF transcripts band was detected in the follicles and oocytes. The expression of EGF is strongest in the small follicle or oocyte from small follicle. A EGF transcripts band could be detectable in the granulosa cell. The expression of EGF in the granulosa cell was lower than that in the oocyte. Also,the expression of EGF in the granulosa cell from the small follicle is stronger than in another.These results suggest EGF has important roles in the pig follicular development by autocrine/paracrine fashion.

  19. Probing the Transcription Mechanisms of Reovirus Cores with Molecules That Alter RNA Duplex Stability▿

    OpenAIRE

    Demidenko, Alexander A.; Nibert, Max L.

    2009-01-01

    The mammalian reovirus (MRV) genome comprises 10 double-stranded RNA (dsRNA) segments, packaged along with transcriptase complexes inside each core particle. Effects of four small molecules on transcription by MRV cores were studied for this report, chosen for their known capacities to alter RNA duplex stability. Spermidine and spermine, which enhance duplex stability, inhibited transcription, whereas dimethyl sulfoxide and trimethylglycine, which attenuate duplex stability, stimulated transc...

  20. Selenoprotein P mRNA expression in human hepatic tissues

    Institute of Scientific and Technical Information of China (English)

    Chun-Li Li; Ke-Jun Nan; Tao Tian; Chen-Guang Sui; Yan-Fang Liu

    2007-01-01

    AIM: To investigate the expression of Selenoprotein P mRNA (SePmRNA) in tissues of normal liver, liver cirrhosis and hepatocellular carcinoma (HCC), and its relationship with HCC occurrence and development.METHODS: The expression of SePmRNA in tissues of normal liver, liver cirrhosis and HCC were detected by in situ hybridization using a cDNA probe.RESULTS: The enzyme digesting products of pBluescript-Human Selenoprotein P were evaluated by electrophoresis.The positive expression of SePmRNA was found in the tissues of normal liver,liver cirrhosis and HCC.The expression of SeP mRNA was found in hepatic interstitial substance,especially in endothelial cells and lymphocytes of vasculature.The positive rate of SePmRNA in normal liver tissue was 84.6% (11/13) and the positive signals appeared in the nucleus and cytoplasm,mostly in the nucleolus,and the staining granules were larger in the nucleolus and around the nucleus.The positive rate of SePmRNA in liver cirrhosis tissue was 45.O% (9/20) and the positive signals were mainly in the nucleolus and cytoplasm,being less around the nucleus and inner nucleus than that in normal liver tissue. The positive rate of SePmRNA in HCC tissue was 30.0% (9/30) and the positive signals were in the cytoplasm, but less in the nucleus.CONCLUSION: SePmRNA expression in the tissues of normal liver and HCC is significantly different (84.6% vs 30.0%, P = 0.003), suggesting that SeP might play a role in the occurrence and development of HCC.

  1. Altered vesicular glutamate transporter expression in human temporal lobe epilepsy with hippocampal sclerosis

    OpenAIRE

    Van Liefferinge, J.; Jensen, C.J.; Albertini, G.; Bentea, E.; Demuyser, T.; Merckx, E.; Aronica, E.; Smolders, I; Massie, A.

    2015-01-01

    Vesicular glutamate transporters (VGLUTs) are responsible for loading glutamate into synaptic vesicles. Altered VGLUT protein expression has been suggested to affect quantal size and glutamate release under both physiological and pathological conditions. In this study, we investigated mRNA and protein expression levels of the three VGLUT subtypes in hippocampal tissue of patients suffering from temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS), International League Against Epilepsy...

  2. Tearing mode stability calculations with pressure flattening

    CERN Document Server

    Ham, C J; Cowley, S C; Hastie, R J; Hender, T C; Liu, Y Q

    2013-01-01

    Calculations of tearing mode stability in tokamaks split conveniently into an external region, where marginally stable ideal MHD is applicable, and a resonant layer around the rational surface where sophisticated kinetic physics is needed. These two regions are coupled by the stability parameter. Pressure and current perturbations localized around the rational surface alter the stability of tearing modes. Equations governing the changes in the external solution and - are derived for arbitrary perturbations in axisymmetric toroidal geometry. The relationship of - with and without pressure flattening is obtained analytically for four pressure flattening functions. Resistive MHD codes do not contain the appropriate layer physics and therefore cannot predict stability directly. They can, however, be used to calculate -. Existing methods (Ham et al. 2012 Plasma Phys. Control. Fusion 54 025009) for extracting - from resistive codes are unsatisfactory when there is a finite pressure gradient at the rational surface ...

  3. Transforming Growth Factor β1 (TGF-β1) Activates Hepcidin mRNA Expression in Hepatocytes.

    Science.gov (United States)

    Chen, Simeng; Feng, Teng; Vujić Spasić, Maja; Altamura, Sandro; Breitkopf-Heinlein, Katja; Altenöder, Jutta; Weiss, Thomas S; Dooley, Steven; Muckenthaler, Martina U

    2016-06-17

    The hepatic hormone hepcidin is the master regulator of systemic iron homeostasis. Its expression level is adjusted to alterations in iron levels, inflammatory cues, and iron requirements for erythropoiesis. Bone morphogenetic protein 6 (BMP6) contributes to the iron-dependent control of hepcidin. In addition, TGF-β1 may stimulate hepcidin mRNA expression in murine hepatocytes and human leukocytes. However, receptors and downstream signaling proteins involved in TGF-β1-induced hepcidin expression are still unclear. Here we show that TGF-β1 treatment of mouse and human hepatocytes, as well as ectopic expression of TGF-β1 in mice, increases hepcidin mRNA levels. The hepcidin response to TGF-β1 depends on functional TGF-β1 type I receptor (ALK5) and TGF-β1 type II receptor (TβRII) and is mediated by a noncanonical mechanism that involves Smad1/5/8 phosphorylation. Interestingly, increasing availability of canonical Smad2/3 decreases TGF-β1-induced hepcidin regulation, whereas the BMP6-hepcidin signal was enhanced, indicating a signaling component stoichiometry-dependent cross-talk between the two pathways. Although ALK2/3-dependent hepcidin activation by BMP6 can be modulated by each of the three hemochromatosis-associated proteins: HJV (hemojuvelin), HFE (hemochromatosis protein), and TfR2 (transferrin receptor 2), these proteins do not control the ALK5-mediated hepcidin response to TGF-β1. TGF-β1 mRNA levels are increased in mouse models of iron overload, indicating that TGF-β1 may contribute to hepcidin synthesis under these conditions. In conclusion, these data demonstrate that a complex regulatory network involving TGF-β1 and BMP6 may control the sensing of systemic and/or hepatic iron levels. PMID:27129231

  4. Transforming Growth Factor β1 (TGF-β1) Activates Hepcidin mRNA Expression in Hepatocytes.

    Science.gov (United States)

    Chen, Simeng; Feng, Teng; Vujić Spasić, Maja; Altamura, Sandro; Breitkopf-Heinlein, Katja; Altenöder, Jutta; Weiss, Thomas S; Dooley, Steven; Muckenthaler, Martina U

    2016-06-17

    The hepatic hormone hepcidin is the master regulator of systemic iron homeostasis. Its expression level is adjusted to alterations in iron levels, inflammatory cues, and iron requirements for erythropoiesis. Bone morphogenetic protein 6 (BMP6) contributes to the iron-dependent control of hepcidin. In addition, TGF-β1 may stimulate hepcidin mRNA expression in murine hepatocytes and human leukocytes. However, receptors and downstream signaling proteins involved in TGF-β1-induced hepcidin expression are still unclear. Here we show that TGF-β1 treatment of mouse and human hepatocytes, as well as ectopic expression of TGF-β1 in mice, increases hepcidin mRNA levels. The hepcidin response to TGF-β1 depends on functional TGF-β1 type I receptor (ALK5) and TGF-β1 type II receptor (TβRII) and is mediated by a noncanonical mechanism that involves Smad1/5/8 phosphorylation. Interestingly, increasing availability of canonical Smad2/3 decreases TGF-β1-induced hepcidin regulation, whereas the BMP6-hepcidin signal was enhanced, indicating a signaling component stoichiometry-dependent cross-talk between the two pathways. Although ALK2/3-dependent hepcidin activation by BMP6 can be modulated by each of the three hemochromatosis-associated proteins: HJV (hemojuvelin), HFE (hemochromatosis protein), and TfR2 (transferrin receptor 2), these proteins do not control the ALK5-mediated hepcidin response to TGF-β1. TGF-β1 mRNA levels are increased in mouse models of iron overload, indicating that TGF-β1 may contribute to hepcidin synthesis under these conditions. In conclusion, these data demonstrate that a complex regulatory network involving TGF-β1 and BMP6 may control the sensing of systemic and/or hepatic iron levels.

  5. mRNA expression of diacylglycerol kinase isoforms in insulin-sensitive tissues: effects of obesity and insulin resistance.

    Science.gov (United States)

    Mannerås-Holm, Louise; Kirchner, Henriette; Björnholm, Marie; Chibalin, Alexander V; Zierath, Juleen R

    2015-04-01

    Diacylglycerol kinase (DGK) isoforms regulate signal transduction and lipid metabolism. DGKδ deficiency leads to hyperglycemia, peripheral insulin resistance, and metabolic inflexibility. Thus, dysregulation of other DGK isoforms may play a role in metabolic dysfunction. We investigated DGK isoform mRNA expression in extensor digitorum longus (EDL) and soleus muscle, liver as well as subcutaneous and epididymal adipose tissue in C57BL/6J mice and obese and insulin-resistant ob/ob mice. All DGK isoforms, except for DGKκ, were detectable, although with varying mRNA expression. Liver DGK expression was generally lowest, with several isoforms undetectable. In soleus muscle, subcutaneous and epididymal adipose tissue, DGKδ was the most abundant isoform. In EDL muscle, DGKα and DGKζ were the most abundant isoforms. In liver, DGKζ was the most abundant isoform. Comparing obese insulin-resistant ob/ob mice to lean C57BL/6J mice, DGKβ, DGKι, and DGKθ were increased and DGKε expression was decreased in EDL muscle, while DGKβ, DGKη and DGKθ were decreased and DGKδ and DGKι were increased in soleus muscle. In liver, DGKδ and DGKζ expression was increased in ob/ob mice. DGKη was increased in subcutaneous fat, while DGKζ was increased and DGKβ, DGKδ, DGKη and DGKε were decreased in epididymal fat from ob/ob mice. In both adipose tissue depots, DGKα and DGKγ were decreased and DGKι was increased in ob/ob mice. In conclusion, DGK mRNA expression is altered in an isoform- and tissue-dependent manner in obese insulin-resistant ob/ob mice. DGK isoforms likely have divergent functional roles in distinct tissues, which may contribute to metabolic dysfunction. PMID:25847921

  6. Dissecting non-canonical interactions in frameshift-stimulating mRNA pseudoknots

    Energy Technology Data Exchange (ETDEWEB)

    Cornish, Peter V. [University of Illinois at Urbana-Champaign, Department of Physics (United States); Giedroc, David P. [2128 TAMU, Texas A and M University, Department of Biochemistry and Biophysics (United States); Hennig, Mirko [Scripps Research Institute, MB33, Department of Molecular Biology (United States)], E-mail: hennig@musc.edu

    2006-07-15

    A variety of powerful NMR experiments have been introduced over the last few years that allow for the direct identification of different combinations of donor and acceptor atoms involved in hydrogen bonds in biomolecules. This ability to directly observe tertiary structural hydrogen bonds in solution tremendously facilitates structural studies of nucleic acids. We show here that an adiabatic HNN-COSY pulse scheme permits observation and measurement of J(N,N) couplings for nitrogen sites that are separated by up to 140 ppm in a single experiment at a proton resonance frequency of 500 MHz. Crucial hydrogen bond acceptor sites in nucleic acids, such as cytidine N3 nitrogens, can be unambiguously identified even in the absence of detectable H41 and H42 amino protons using a novel triple-resonance two-dimensional experiment, denoted H5(C5C4)N3. The unambiguous identification of amino nitrogen donor and aromatic nitrogen acceptor sites associated with both major groove as well as minor groove triple base pairs reveal the details of hydrogen bonding networks that stabilize the complex architecture of frameshift-stimulating mRNA pseudoknots. Another key tertiary interaction involving a 2'-OH hydroxyl proton that donates a hydrogen bond to an aromatic nitrogen acceptor in a cis Watson-Crick/sugar edge interaction can also be directly detected using a quantitative J(H,N) {sup 1}H,{sup 15}N-HSQC experiment.

  7. Hypoxia and Human Genome Stability: Downregulation of BRCA2 Expression in Breast Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Daniele Fanale

    2013-01-01

    Full Text Available Previously, it has been reported that hypoxia causes increased mutagenesis and alteration in DNA repair mechanisms. In 2005, an interesting study showed that hypoxia-induced decreases in BRCA1 expression and the consequent suppression of homologous recombination may lead to genetic instability. However, nothing is yet known about the involvement of BRCA2 in hypoxic conditions in breast cancer. Initially, a cell proliferation assay allowed us to hypothesize that hypoxia could negatively regulate the breast cancer cell growth in short term in vitro studies. Subsequently, we analyzed gene expression in breast cancer cell lines exposed to hypoxic condition by microarray analysis. Interestingly, genes involved in DNA damage repair pathways such as mismatch repair, nucleotide excision repair, nonhomologous end-joining and homologous recombination repair were downregulated. In particular, we focused on the BRCA2 downregulation which was confirmed at mRNA and protein level. In addition, breast cancer cells were treated with dimethyloxalylglycine (DMOG, a cell-permeable inhibitor of both proline and asparaginyl hydroxylases able to induce HIF-1α stabilization in normoxia, providing results comparable to those previously described. These findings may provide new insights into the mechanisms underlying genetic instability mediated by hypoxia and BRCA involvement in sporadic breast cancers.

  8. Trapped Particle Stability for the Kinetic Stabilizer

    CERN Document Server

    Berk, H L

    2011-01-01

    A kinetically stabilized axially symmetric tandem mirror (KSTM) uses the momentum flux of low-energy, unconfined particles that sample only the outer end-regions of the mirror plugs, where large favorable field-line curvature exists. The window of operation is determined for achieving MHD stability with tolerable energy drain from the kinetic stabilizer. Then MHD stable systems are analyzed for stability of the trapped particle mode. This mode is characterized by the detachment of the central-cell plasma from the kinetic stabilizer region without inducing field-line bending. Stability of the trapped particle mode is sensitive to the electron connection between the stabilizer and the end plug. It is found that the stability condition for the trapped particle mode is more constraining than the stability condition for the MHD mode, and it is challenging to satisfy the required power constraint. Furthermore a severe power drain may arise from the necessary connection of low-energy electrons in the kinetic stabili...

  9. Zebrafish usp39 mutation leads to rb1 mRNA splicing defect and pituitary lineage expansion.

    Directory of Open Access Journals (Sweden)

    Yesenia Ríos

    Full Text Available Loss of retinoblastoma (Rb tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39 mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130, and cdkn1a (p21 expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.

  10. Gene expression analysis in sections and tissue microarrays of archival tissues by mRNA in situ hybridization.

    Science.gov (United States)

    Henke, R T; Maitra, A; Paik, S; Wellstein, A

    2005-01-01

    Altered expression of genes in diseased tissues can prognosticate a distinct natural progression of the disease as well as predict sensitivity or resistance to particular therapies. Archival tissues from patients with a known medical history and treatments are an invaluable resource to validate the utility of candidate genes for prognosis and prediction of therapy outcomes. However, stored tissues with associated long-term follow-up information typically are formalin-fixed, paraffin-embedded specimen and this can severely restrict the methods applicable for gene expression analysis. We report here on the utility of tissue microarrays (TMAs) that use valuable tissues sparingly and provide a platform for simultaneous analysis of gene expression in several hundred samples. In particular, we describe a stable method applicable to mRNA expression screening in such archival tissues. TMAs are constructed from sections of small drill cores, taken from tissue blocks of archival tissues and multiple samples can thus be arranged on a single microscope slide. We used mRNA in situ hybridization (ISH) on >500 full sections and >100 TMAs for >10 different cDNAs that yielded >10,000 data points. We provide detailed experimental protocols that can be implemented without major hurdles in a molecular pathology laboratory and discuss quantitative analysis and the advantages and limitations of ISH. We conclude that gene expression analysis in archival tissues by ISH is reliable and particularly useful when no protein detection methods are available for a candidate gene.

  11. How the Magnitude of Prey Genetic Variation Alters Predator-Prey Eco-Evolutionary Dynamics.

    Science.gov (United States)

    Cortez, Michael H

    2016-09-01

    Evolution can alter the stability and dynamics of ecological communities; for example, prey evolution can drive cyclic dynamics in predator-prey systems that are not possible in the absence of evolution. However, it is unclear how the magnitude of additive genetic variation in the evolving species mediates those effects. In this study, I explore how the magnitude of prey additive genetic variation determines what effects prey evolution has on the dynamics and stability of predator-prey systems. I use linear stability analysis to decompose the stability of a general eco-evolutionary predator-prey model into components representing the stabilities of the ecological and evolutionary subsystems as well as the interactions between those subsystems. My results show that with low genetic variation, the cyclic dynamics and stability of the system are determined by the ecological subsystem. With increased genetic variation, disruptive selection always destabilizes stable communities, stabilizing selection can stabilize or destabilize communities, and prey evolution can alter predator-prey phase lags. Stability changes occur approximately when the magnitude of genetic variation balances the (in)stabilities of the ecological and evolutionary subsystems. I discuss the connections between my stability results and prior results from the theory of adaptive dynamics. PMID:27501090

  12. Epigenetic alterations in gastric carcinogenesis

    Institute of Scientific and Technical Information of China (English)

    In-Seon CHOI; Tsung-Teh WU

    2005-01-01

    Gastric cancer is believed to result in part from the accumulation of multiple genetic alterations leading to oncogene overexpression and tumor suppressor loss. Epigenetic alterations as a distinct and crucial mechanism to silence a variety of methylated tissue-specific and imprinted genes, have been extensively studied in gastric carcinoma and play important roles in gastric carcinogenesis. This review will briefly discuss the basic aspects of DNA methylation and CpG island methylation, in particular the epigenetic alterations of certain critical genes implicated in gastric carcinogenesis and its relevance of clinical implications.

  13. Stability criteria for unsupervised temporal association networks.

    Science.gov (United States)

    Wallis, Guy

    2005-03-01

    A biologically realizable, unsupervised learning rule is described for the online extraction of object features, suitable for solving a range of object recognition tasks. Alterations to the basic learning rule are proposed which allow the rule to better suit the parameters of a given input space. One negative consequence of such modifications is the potential for learning instability. The criteria for such instability are modeled using digital filtering techniques and predicted regions of stability and instability tested. The result is a family of learning rules which can be tailored to the specific environment, improving both convergence times and accuracy over the standard learning rule, while simultaneously insuring learning stability. PMID:15787138

  14. Developed and evaluated a multiplex mRNA profiling system for body fluid identification in Chinese Han population.

    Science.gov (United States)

    Song, Feng; Luo, Haibo; Hou, Yiping

    2015-10-01

    In forensic casework, identification the cellular origin from a biological sample is crucial to the case investigation and reconstruction in crime scene. DNA/RNA co-extraction for STR typing and human body fluids identification has been proposed as an efficient and comprehensive assay for forensic analysis. Several cell-specific messenger RNA (mRNA) markers for identification of the body fluids have been proposed by previous studies. In this study, a novel multiplex mRNA profiling system included 19 markers was developed and performed by reverse transcription endpoint polymerase chain reaction (RT-PCR). The multiplex combined 3 housekeeping gene markers and 16 cell-specific markers that have been used to identify five types of human body fluids: peripheral blood, semen, saliva, vaginal secretions and menstrual blood. The specificity, sensitivity, stability and detectability of the mixture were explored in our study. Majority of the cell-specific mRNA markers showed high specificity, although cross-reactivity was observed sporadically. Specific profiling for per body fluid was obtained. Moreover, the interpretation guidelines for inference of body fluid types were performed according to the A. Lindenbergh et al. The scoring guidelines can be applied to any RNA multiplex, which was based on six different scoring categories (observed, observed and fits, sporadically observed and fits, not observed, sporadically observed, not reliable, and non-specific due to high input). The simultaneous extraction of DNA showed positive full or partial profiling results of all samples. It demonstrated that the approach of combined STR-profiling and RNA profiling was suitable and reliable to detect the donor and origin of human body fluids in Chinese Han population. PMID:26311108

  15. Food Fortification Stability Study

    Science.gov (United States)

    Sirmons, T.; Cooper, M.; Douglas, G.

    2016-01-01

    NASA has established the goal of traveling beyond low-Earth orbit and extending manned exploration to Mars. The length of proposed Mars missions and the lack of resupply missions increases the importance of nutritional content in the food system, which will need a five year shelf life. The purpose of this research is to assess the stability of vitamin supplementation in traditionally processed spaceflight foods. It is expected that commercially available fortification nutrients will remain stable through a long duration exploration mission at sufficient levels if compatible formulation, processing, and storage temperatures are achieved. Five vitamins (vitamin E, vitamin K, pantothenic acid, folic acid, and thiamin) were blended into a vitamin premix (DSM, Freeport, TX) such that the vitamin concentration per serving equaled 25% of the recommended daily intake after two years of ambient storage. Four freeze-dried foods (Scrambled Eggs, Italian Vegetables, Potatoes Au Gratin, Noodles and Chicken) and four thermostabilized foods (Curry Sauce with Vegetables, Chicken Noodle Soup, Grilled Pork Chop, Rice with Butter) were produced, with and without the vitamin premix, to assess the impact of the added fortification on color and taste and to determine the stability of supplemental vitamins in spaceflight foods. The addition of fortification to spaceflight foods did not greatly alter the organoleptic properties of most products. In most cases, overall acceptability scores remained above 6.0 (minimum acceptable score) following six months and one year of low-temperature storage. Likewise, the color of fortified products appears to be preserved over one year of storage. The only exception was Grilled pork Chop and Chicken Noodle Soup whose individual components appear to degrade rapidly over one year of storage. Finally, most vitamins appear to be stable during long-term storage. The only exception was thiamin, which degraded rapidly during the first year of storage at 35

  16. Pulmonary alterations in Behcet's disease

    International Nuclear Information System (INIS)

    Purpose: This study aims to demonstrate pulmonary alterations (PA) in patients with Behcet's disease by using CT. Materials and methods: CTs of 50 patients with Behcet's disease and 20 others in a control group have been evaluated retrospectively for PA (septal, reticular, nodular, atelectatic opacities). Results: Eight out of 50 patients (16%) with Behcet's disease showed PA. Three out of 20 (15%) in the control group showed PA. No differences were observed between Behcet's disease patients and the control group regarding pulmonary alterations (p = 0.917). No differences were observed in the disease duration, ages and sex in either group in those with and without PA. Conclusion: Pulmonary alterations can be seen in patients with Behcet's disease, but these alterations are not significant.

  17. Workshop on Feedback Stabilization of MHD Stabilities

    Energy Technology Data Exchange (ETDEWEB)

    McGuire, K.; Kugel, H. [Princeton Univ., NJ (United States). Plasma Physics Lab.; La Haye, R. [General Atomics, San Diego, CA (United States); Mauel, M. [Columbia Univ., New York, NY (United States). Dept. of Applied Physics; Nevins, W. [Lawrence Livermore National Lab., CA (United States); Prager, S. [Wisconsin Univ., Madison, WI (United States). Dept. of Physics

    1996-12-31

    The feedback stabilization of MHD instabilities is an area of research that is critical for improving the performance and economic attractiveness of magnetic confinement devices. A Workshop dedicated to feedback stabilization of MHD instabilities was held from December 11-13, 1996 at the Princeton Plasma Physics Laboratory, Princeton NJ, USA. The resulting presentations, conclusions, and recommendations are summarized.

  18. Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS Gene

    Directory of Open Access Journals (Sweden)

    Yoko Nakajima

    2016-01-01

    Full Text Available Dihydropyrimidinase (DHP deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.

  19. Musashi regulates the temporal order of mRNA translation during Xenopus oocyte maturation

    OpenAIRE

    Charlesworth, Amanda; Wilczynska, Anna; Thampi, Prajitha; Cox, Linda L.; MacNicol, Angus M.

    2006-01-01

    A strict temporal order of maternal mRNA translation is essential for meiotic cell cycle progression in oocytes of the frog Xenopus laevis. The molecular mechanisms controlling the ordered pattern of mRNA translational activation have not been elucidated. We report a novel role for the neural stem cell regulatory protein, Musashi, in controlling the translational activation of the mRNA encoding the Mos proto-oncogene during meiotic cell cycle progression. We demonstrate that Musashi interacts...

  20. Targeted mRNA Profiling of Transfected Breast Cancer Gene in a Living Cell

    OpenAIRE

    Nawarathna, D.; Chang, R; Nelson, E.; Wickramasinghe, H. Kumar

    2010-01-01

    Selective mRNA profiling of transfected breast cancer gene expression in a living cell is demonstrated. Atomic Force Microscope (AFM) probe tips are structurally modified to create a dielectrophoretic force that attracts mRNA molecules within the cell nucleus. The tip end is chemically modified to hybridize only to the target mRNA from a pool of molecules within the nucleus. We successfully combined this scheme with standard assay techniques to develop an assay technology that can be used for...

  1. Anemia induces accumulation of erythropoietin mRNA in the kidney and liver.

    OpenAIRE

    Bondurant, M C; Koury, M J

    1986-01-01

    Regulation of the production of erythropoietin occurs in the kidney and liver largely through control of accumulation of erythropoietin mRNA. Erythropoietin mRNA was first detected in kidneys at 1.5 h postanemia and reached a plateau value at least 200-fold above the control value by 4 to 8 h. A 20-base sequence immediately upstream from the reported erythropoietin mRNA initiation site is complementary to a hypervariable sequence in 18S rRNA.

  2. Nuclear mRNA degradation pathway(s are implicated in Xist regulation and X chromosome inactivation.

    Directory of Open Access Journals (Sweden)

    Constance Ciaudo

    2006-06-01

    Full Text Available A critical step in X-chromosome inactivation (XCI, which results in the dosage compensation of X-linked gene expression in mammals, is the coating of the presumptive inactive X chromosome by the large noncoding Xist RNA, which then leads to the recruitment of other factors essential for the heterochromatinisation of the inactive X and its transcriptional silencing. In an approach aimed at identifying genes implicated in the X-inactivation process by comparative transcriptional profiling of female and male mouse gastrula, we identified the Eif1 gene involved in translation initiation and RNA degradation. We show here that female embryonic stem cell lines, silenced by RNA interference for the Eif1 gene, are unable to form Xist RNA domains upon differentiation and fail to undergo X-inactivation. To probe further an effect involving RNA degradation pathways, the inhibition by RNA interference of Rent1, a factor essential for nonsense-mediated decay and Exosc10, a specific nuclear component of the exosome, was analysed and shown to similarly impair Xist upregulation and XCI. In Eif1-, Rent1-, and Exosc10-interfered clones, Xist spliced form(s are strongly downregulated, while the levels of unspliced form(s of Xist and the stability of Xist RNA remain comparable to that of the control cell lines. Our data suggests a role for mRNA nuclear degradation pathways in the critical regulation of spliced Xist mRNA levels and the onset of the X-inactivation process.

  3. Transcriptome-wide mapping reveals widespread dynamic regulated pseudouridylation of ncRNA and mRNA

    Science.gov (United States)

    Schwartz, Schraga; Bernstein, Douglas A.; Mumbach, Maxwell R.; Jovanovic, Marko; Herbst, Rebecca H.; León-Ricardo, Brian X.; Engreitz, Jesse M.; Guttman, Mitchell; Satija, Rahul; Lander, Eric S.; Fink, Gerald; Regev, Aviv

    2014-01-01

    SUMMARY Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of novel sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUSs) uncovers which PUSs modify each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided PUSs. Upon heat shock in yeast, Pus7-mediated pseudouridylation is induced at >200 sites and Pus7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved, but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcritome-wide scope for pseudouridine, and methods to dissect its underlying mechanisms and function. PMID:25219674

  4. Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA.

    Science.gov (United States)

    Schwartz, Schraga; Bernstein, Douglas A; Mumbach, Maxwell R; Jovanovic, Marko; Herbst, Rebecca H; León-Ricardo, Brian X; Engreitz, Jesse M; Guttman, Mitchell; Satija, Rahul; Lander, Eric S; Fink, Gerald; Regev, Aviv

    2014-09-25

    Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function.

  5. Effects of Hypoxanthine Substitution in Peptide Nucleic Acids Targeting KRAS2 Oncogenic mRNA Molecules: Theory and Experiment

    Science.gov (United States)

    Sanders, Jeffrey M.; Wampole, Matthew E.; Chen, Chang-Po; Sethi, Dalip; Singh, Amrita; Dupradeau, François-Yves; Wang, Fan; Gray, Brian D.; Thakur, Mathew L.; Wickstrom, Eric

    2013-01-01

    Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multi-mutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick basepairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA-PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA-PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition. PMID:23972113

  6. Hypothalamic-pituitary thyroid axis alterations in female mice with deletion of the neuromedin B receptor gene.

    Science.gov (United States)

    Oliveira, Karen J; Paula, Gabriela S M; Império, Guinever E; Bressane, Nina O; Magalhães, Carolina M A; Miranda-Alves, Leandro; Ortiga-Carvalho, Tania M; Pazos-Moura, Carmen C

    2014-11-01

    Neuromedin B, a peptide highly expressed at the pituitary, has been shown to act as autocrine/paracrine inhibitor of thyrotropin (TSH) release. Here we studied the thyroid axis of adult female mice lacking neuromedin B receptor (NBR-KO), compared to wild type (WT) littermates. They exhibited slight increase in serum TSH (18%), with normal pituitary expression of mRNA coding for α-glycoprotein subunit (Cga), but reduced TSH β-subunit mRNA (Tshb, 41%), lower intra-pituitary TSH content (24%) and increased thyroid hormone transporter MCT-8 (Slc16a2, 44%) and thyroid hormone receptor β mRNA expression (Thrb, 39%). NBR-KO mice exhibited normal thyroxine (T4) and reduced triiodothyronine (T3) (30%), with no alterations in the intra-thyroidal content of T4 and T3 or thyroid morphological changes. Hypothalamic thyrotropin-releasing hormone (TRH) mRNA (Trh) was increased (68%), concomitant with a reduction in type 2 deiodinase mRNA (Dio2, 30%) and no changes in MCT-8 and thyroid hormone receptor mRNA expression. NBR-KO mice exhibited a 56% higher increase in serum TSH in response to an acute single intraperitoneal injection of TRH concomitant with a non-significant increase in pituitary TRH receptor (Trhr) mRNA at basal state. The phenotype of female NBR-KO mice at the hypothalamus-pituitary axis revealed alterations in pituitary and hypothalamic gene expression, associated with reduced serum T3, and higher TSH response to TRH, with apparently normal thyroid morphology and hormonal production. Thus, results confirm that neuromedin B pathways are importantly involved in secretory pathways of TSH and revealed its participation in the in vivo regulation of gene expression of TSH β-subunit and pituitary MCT8 and Thrb and hypothalamic TRH and type 2 deiodinase. PMID:25454367

  7. Altered Esophageal Mucosal Structure in Patients with Celiac Disease

    Science.gov (United States)

    Pinto-Sánchez, María Inés; Nachman, Fabio D.; Fuxman, Claudia; Iantorno, Guido; Hwang, Hui Jer; Ditaranto, Andrés; Costa, Florencia; Longarini, Gabriela; Wang, Xuan Yu; Huang, Xianxi; Vázquez, Horacio; Moreno, María L.; Niveloni, Sonia; Bercik, Premysl; Smecuol, Edgardo; Mazure, Roberto; Bilder, Claudio; Mauriño, Eduardo C.; Verdu, Elena F.; Bai, Julio C.

    2016-01-01

    Background/Aim. Reflux symptoms (RS) are common in patients with celiac disease (CD), a chronic enteropathy that affects primarily the small intestine. We evaluated mucosal integrity and motility of the lower esophagus as mechanisms contributing to RS generation in patients with CD. Methods. We enrolled newly diagnosed CD patients with and without RS, nonceliac patients with classical reflux disease (GERD), and controls (without RS). Endoscopic biopsies from the distal esophagus were assessed for dilated intercellular space (DIS) by light microscopy and electron microscopy. Tight junction (TJ) mRNA proteins expression for zonula occludens-1 (ZO-1) and claudin-2 and claudin-3 (CLDN-2; CLDN-3) was determined using qRT-PCR. Results. DIS scores were higher in patients with active CD than in controls, but similar to GERD patients. The altered DIS was found even in CD patients without RS and normalized after one year of a gluten-free diet. CD patients with and without RS had lower expression of ZO-1 than controls. The expression of CLDN-2 and CLDN-3 was similar in CD and GERD patients. Conclusions. Our study shows that patients with active CD have altered esophageal mucosal integrity, independently of the presence of RS. The altered expression of ZO-1 may underlie loss of TJ integrity in the esophageal mucosa and may contribute to RS generation. PMID:27446827

  8. Correlation of mRNA and protein in complex biological samples.

    Science.gov (United States)

    Maier, Tobias; Güell, Marc; Serrano, Luis

    2009-12-17

    The correlation between mRNA and protein abundances in the cell has been reported to be notoriously poor. Recent technological advances in the quantitative analysis of mRNA and protein species in complex samples allow the detailed analysis of this pathway at the center of biological systems. We give an overview of available methods for the identification and quantification of free and ribosome-bound mRNA, protein abundances and individual protein turnover rates. We review available literature on the correlation of mRNA and protein abundances and discuss biological and technical parameters influencing the correlation of these central biological molecules.

  9. Nuclear imprisonment: viral strategies to arrest host mRNA nuclear export.

    Science.gov (United States)

    Kuss, Sharon K; Mata, Miguel A; Zhang, Liang; Fontoura, Beatriz M A

    2013-07-01

    Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

  10. Functional cloning of BRF1, a regulator of ARE-dependent mRNA turnover

    OpenAIRE

    Stoecklin, Georg; Colombi, Marco; Raineri, Ines; Leuenberger, Sabrina; Mallaun, Michel; Schmidlin, Martin; Gross, Brigitte; Lu, Min; Kitamura, Toshio; Moroni, Christoph

    2002-01-01

    To identify regulators of AU-rich element (ARE)-dependent mRNA turnover we have followed a genetic approach using a mutagenized cell line (slowC) that fails to degrade cytokine mRNA. Accordingly, a GFP reporter construct whose mRNA is under control of the ARE from interleukin-3 gives an increased fluorescence signal in slowC. Here we describe rescue of slowC by a retroviral cDNA library. Flow cytometry allowed us to isolate revertants with reconstituted rapid mRNA decay. The cDNA was identifi...

  11. STRUCTURAL ALTERATIONS OF SKELETAL MUSCLE IN COPD

    Directory of Open Access Journals (Sweden)

    Sunita eMathur

    2014-03-01

    Full Text Available Background: Chronic obstructive pulmonary disease (COPD is a respiratory disease associated with a systemic inflammatory response. Peripheral muscle dysfunction has been well characterized in individuals with COPD and results from a complex interaction between systemic and local factors. Objective: In this narrative review, we will describe muscle wasting in people with COPD, the associated structural changes, muscle regenerative capacity and possible mechanisms for muscle wasting. We will also discuss how structural changes relate to impaired muscle function and mobility in people with COPD. Key Observations: Approximately 30-40% of individuals with COPD experience muscle mass depletion. Furthermore, muscle atrophy is a predictor of physical function and mortality in this population. Associated structural changes include a decreased proportion and size of type-I fibers, reduced oxidative capacity and mitochondrial density mainly in the quadriceps. Observations related to impaired muscle regenerative capacity in individuals with COPD include a lower proportion of central nuclei in the presence or absence of muscle atrophy and decreased maximal telomere length, which has been correlated with reduced muscle cross-sectional area. Potential mechanisms for muscle wasting in COPD may include excessive production of reactive oxygen species, altered amino acid metabolism and lower expression of peroxisome proliferator-activated receptors-gamma-coactivator 1-alpha mRNA. Despite a moderate relationship between muscle atrophy and function, impairments in oxidative metabolism only seems weakly related to muscle function. Conclusion: This review article demonstrates the cellular modifications in the peripheral muscle of people with COPD and describes the evidence of its relationship to muscle function. Future research will focus on rehabilitation strategies to improve muscle wasting and maximize function.

  12. QUANTIFICATION OF P4HA2 mRNA OF FIBROBLASTS WITH SYBR GREEN BASED RT-PCR FOR CORRECTING CMV INACTIVATION EFFICIENCY IN DONOR BLOOD

    Institute of Scientific and Technical Information of China (English)

    FANG Feng-qin; ZHANG Yue; LU Ping; ZHANG Li; JI Yu-hua

    2009-01-01

    Objective To quantify proline 4-hydroxylase, alpha polypeptide Ⅱ (P4HA2) mRNA of human embryo lung fibroblast (HELF) with SYBR green based reversed transcript PCR (RT-PCR) for correcting cytomegalovirus (CMV) inactivation or clearance efficiency in donor blood.Methods A pair of specific primers of exon 12a of P4HA2 was designed, and the related PCR-reaction system and condition were optimized. Then the recombinant plasmid containing the target fragment was constructed for making standard curve with SYBR green based real-time RT-PCR. Finally, the sensitivity, reproducibility, and specificity of this method were fully estimated.Results The sensitivity of the method was 1.5E+04 copies/mL of P4HA2 mRNA, corresponding to 103 fibroblasts. In addition, existence of 8.67E+06 leukocytes could not interfere with the accurate quantification of HELF in the large dynamic range. The intra-assay variability and inter-assay variability both varied in different concentrations, being higher in low concentrations and lower in high concentrations. But all of them were below 13.76% in variation, which showed acceptable stability of this method.Conclusion SYBR green and specific primer based real-time RT-PCR show up a good quality for quantifying HELF P4HA2 mRNA with good specificity, stability, and high sensitivity. Approximate 10 copies of P4HA2 mRNA per cell in average can be detected by the method. Therefore, this method can be used to deduct fibroblast-associated CMV for correcting CMV inactivation efficiency in leukocytes.

  13. Regulation of mRNA translation during mitosis.

    Science.gov (United States)

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

    2015-01-01

    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.

  14. mRNA related to insulin family in human placenta

    International Nuclear Information System (INIS)

    The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A+) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A+) RNA templates. Five hundred transformants were initially screened by colony hybridization using a 32P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II

  15. The role of the CNOT1 subunit of the CCR4-NOT complex in mRNA deadenylation and cell viability

    Institute of Scientific and Technical Information of China (English)

    Kentaro Ito; Akinori Takahashi; Masahiro Morita; Toru Suzuki; Tadashi Yamamoto

    2011-01-01

    The human CCR4-NOT deadenylase complex consists of at least nine enzymatic and non-enzymatic subunits.Accumulating evidence suggests that the non-enzymatic subunits are involved in the regulation of mRNA deadenylation,although their precise roles remain to be established.In this study,we addressed the function of the CNOT1 subunit by depleting its expression in HeLa cells.Flow cytometric analysis revealed that the sub G1 fraction was increased in CNOT1-depleted cells.Virtually,the same level of the sub G1 fraction was seen when cells were treated with a mixture of siRNAs targeted against all enzymatic subunits,suggesting that CNOT1 depletion induces apoptosis by destroying the CCR4-NOT-associated deadenylase activity.Further analysis revealed that CNOT1 depletion leads to a reduction in the amount of other CCR4-NOT subunits.Importantly,the specific activity of the CNOT6L immunoprecipitates-associated deadenylase from CNOT1-depleted cells was less than that from control cells.The formation of P-bodies,where mRNA decay is reported to take place,was largely suppressed in CNOT1-depleted cells.Therefore,CNOT1 has an important role in exhibiting enzymatic activity of the CCR4-NOT complex,and thus is critical in control of mRNA deadenylation and mRNA decay.We further showed that CNOT1 depletion enhanced CHOP mRNA levels and activated caspase-4,which is associated with endoplasmic reticulum ER stress-induced apoptosie.Taken together,CNOT1 depletion structurally and functionally deteriorates the CCR4-NOT complex and induces stabilization of mRNAs,which results in the increment of translation causing ER stress-mediated apoptosie.We conclude that CNOT1 contributes to cell viability by securing the activity of the CCR4-NOT deadenylase.

  16. Androgen regulation of corticotropin-releasing hormone receptor 2 (CRHR2) mRNA expression and receptor binding in the rat brain

    Science.gov (United States)

    Weiser, Michael J.; Goel, Nirupa; Sandau, Ursula S.; Bale, Tracy L.; Handa, Robert J.

    2008-01-01

    vivo studies, DHT significantly increased CRHR2 mRNA expression in hippocampal neurons (p<.02) compared to vehicle treated controls. Flutamide treatment prevented the effect of DHT on CRHR2 mRNA indicating that DHT’s effect on CRHR2 expression is AR-mediated. Thus, the CRHR2 gene appears to be a target for regulation by AR and these data suggest a potential mechanism by which androgen may alter mood and anxiety-related behaviors. PMID:18706413

  17. 6-Hydroxydopamine-induced glutathione alteration occurs via glutathione enzyme system in primary cultured astrocytes

    Institute of Scientific and Technical Information of China (English)

    Ji ZHANG; Jun HU; Jian-hua DING; Hong-hong YAO; Gang HU

    2005-01-01

    Aim: To define the role of enzymes involved in glutathione metabolism in 6-hydroxydopamine (6-OHDA)-induced glutathione alteration in primary cultured astrocytes.Methods: Total glutathione (GSx) levels were determined using the modified enzymatic microtiter plate assay.The mRNA levels ofγ-glutamylcysteine synthetase (γGCS), γ-glutamyltransferase (γGT), glutathione peroxidase (GPx), GR (glutathione reductase), and glutathione transferases (GST) were determined using RT-PCR.γGT activity was determined using γGT assay kits.Results: In primary cultured astrocytes, 6-OHDA induced a significant elevation of cellular GSx levels after treatment for 24 h.However, the GSx levels decreased after 24 h and the values were even lower than the value in the control group without 6-OHDA at 48 h.RT-PCR data showed that the mRNA levels of γGCS, the ratelimiting enzyme of γ-L-glutamyl-L-cysteinylglycine (GSH) synthesis, were increased by 6-OHDA after treatment for 24 h and 48 h; the mRNA levels of GPx, GR, and GST did not alter in 6-OHDA-treated astrocytes after treatment for 24 h and 48 h; and 6-OHDA increased the mRNA levels and the activity of γGT after treatment for 48 h,which induced a decrease in GSx levels, despite the up-regulation of γGCS after exposure to 6-OHDA for 48 h.Conclusion: The change in γGCS correlated with the increase in GSH levels induced by 6-OHDA after treatment for 24 h.GSx levels decreased because of increased γGT mRNA levels and γGT activity induced by 6-OHDA after treatment for 48 h.

  18. In vitro Splicing of Influenza Viral NS1 mRNA and NS1-β -globin Chimeras: Possible Mechanisms for the Control of Viral mRNA Splicing

    Science.gov (United States)

    Plotch, Stephen J.; Krug, Robert M.

    1986-08-01

    In influenza virus-infected cells, the splicing of the viral NS1 mRNA catalyzed by host nuclear enzymes is controlled so that the steady-state amount of the spliced NS2 mRNA is only 5-10% of that of the unspliced NS1 mRNA. Here we examine the splicing of NS1 mRNA in vitro, using nuclear extracts from HeLa cells. We show that in addition to its consensus 5' and 3' splice sites, NS1 mRNA has an intron branch-point adenosine residue that was functional in lariat formation. Nonetheless, this RNA was not detectably spliced in vitro under conditions in which a human β -globin precursor was efficiently spliced. Using chimeric RNA precursors containing both NS1 and β -globin sequences, we show that the NS1 5' splice site was effectively utilized by the β -globin branch-point sequence and 3' splice site to form a spliced RNA, whereas the NS1 3' splice site did not function in detectable splicing in vitro, even in the presence of the β -globin branch-point sequence or in the presence of both the branch-point sequence and 5' exon and splice site from β -globin With the chimeric precursors that were not detectably spliced, as with NS1 mRNA itself, a low level of a lariat structure containing only intron and not 3' exon sequences was formed. The inability of the consensus 3' splice site of NS1 mRNA to function effectively in in vitro splicing suggests that this site is structurally inaccessible to components of the splicing machinery. Based on these results, we propose two mechanisms whereby NS1 mRNA splicing in infected cells is controlled via the accessibility of its 3' splice site.

  19. Interrogation of brain miRNA and mRNA expression profiles reveals a molecular regulatory network that is perturbed by mutant huntingtin

    OpenAIRE

    Jin, Jing; Cheng, Yong; Zhang, Yongqing; Wood, William; Peng, Qi; Hutchison, Emmette; Mattson, Mark P; Becker, Kevin G.; Duan, Wenzhen

    2012-01-01

    Emerging evidence indicates that microRNAs (miRNAs) may play an important role in the pathogenesis of Huntington’s disease (HD). To identify the individual miRNAs that are altered in HD and may therefore regulate a gene network underlying mutant huntingtin-induced neuronal dysfunction in HD, we performed miRNA array analysis combined with mRNA profiling in the cerebral cortex from N171-82Q HD mice. Expression profiles of miRNAs as well as mRNAs in HD mouse cerebral cortex were analyzed and co...

  20. Inhibition of glutathione biosynthesis alters compartmental redox status and the thiol proteome in organogenesis-stage rat conceptuses.

    Science.gov (United States)

    Harris, Craig; Shuster, Daniel Z; Roman Gomez, Rosaicela; Sant, Karilyn E; Reed, Matthew S; Pohl, Jan; Hansen, Jason M

    2013-10-01

    oxidation was seen in the BSO-treated AF compartment after 6 h. Biotinylated iodoacetamide (BIAM) labeling of proteins revealed the significant thiol oxidation of many EMB proteins following BSO treatment. Quantitative changes in the thiol proteome, associated with developmentally relevant pathways, were detected using isotope coded affinity tag (ICAT) labeling and mass spectroscopy. Adaptive pathways were selectively enriched with increased concentrations of proteins involved in mRNA processing (splicesome) and mRNA stabilization (glycolysis, GAPDH), as well as protein synthesis (aminoacyl-tRNA) and protein folding (antigen processing, Hsp70, protein disulfide isomerase). These results show the ability of chemical and environmental modulators to selectively alter compartmental intracellular and extracellular GSH and Cys concentrations and change their corresponding E(h) within the intact viable conceptus. The altered E(h) were also of sufficient magnitude to alter the redox proteome and change relative protein concentrations, suggesting that the mechanistic links through which environmental factors inform and regulate developmental signaling pathways may be discovered using systems developmental biology techniques. PMID:23736079

  1. Identification of reference housekeeping-genes for mRNA expression studies in patients with type 1 diabetes.

    Science.gov (United States)

    Kar, Parmita; Chawla, Himika; Saha, Soma; Tandon, Nikhil; Goswami, Ravinder

    2016-06-01

    Selection of appropriate housekeeping-genes as reference is important in mRNA expression-related experiments. It is more important in diabetes since hyperglycemia per se can influence expression of housekeeping-genes. RNA expression of Glyceraldehyde-3-phosphate-dehydrogenase, β-actin and 18S-ribosomal-RNA, Hypoxanthine-phosphoribosyl-transferase (HPRT), Tyrosine-3-monooxygenase/tryptophan (YHWAZ), β2-microglobin (β2M), TATA-binding-protein (TBP), and Ubiquitin C and cytochrome1 (CYC1) assessed in circulating-lymphocytes-(PBMC) of patients with type-1-diabetes and healthy controls. The stability ('M' value housekeeping-genes required for normalization in qRT-PCR were determined by 'ge-norm software.' Vitamin-D-receptor (VDR) was used as a target gene. All the nine genes tested had sufficient 'M' value in diabetes and healthy controls. However, housekeeping-genes indicated a relatively higher stability of expression in healthy controls in comparison to diabetes. Use of single housekeeping-genes brought gross variation in the calculation of VDR-mRNA copies. The ge-norm software suggested geometric mean of five housekeeping-genes for ideal normalization in diabetes (CYC1, β-actin, YHWAZ, HPRT, and β2M) and only three in controls (CYC1, β-actin, and TBP). HbA1c did not correlate with expression of any of the nine housekeeping-genes. Thus, geometric mean of CYC1, β-actin, YHWAZ, HPRT, and β2M needs to be used for ideal normalization of mRNA in type-1-diabetes. Similar studies are required in other population. PMID:27160934

  2. Developmentally Regulated Expression of HDNF/NT-3 mRNA in Rat Spinal Cord Motoneurons and Expression of BDNF mRNA in Embryonic Dorsal Root Ganglion.

    Science.gov (United States)

    Ernfors, Patrik; Persson, Håkan

    1991-01-01

    Northern blot analysis was used to demonstrate high levels of hippocampus-derived neurotrophic factor/neurotrophin-3 (HDNF/NT-3) mRNA in the embryonic day (E) 13 - 14 and 15 - 16 spinal cord. The level decreased at E18 - 19 and remained the same until postnatal day (P) 1, after which it decreased further to a level below the detection limit in the adult. In situ hybridization revealed that the NT-3 mRNA detected in the developing spinal cord was derived from motoneurons and the decrease seen at E18 - 19 was caused by a reduction in the number of motoneurons expressing NT-3 mRNA. The distribution of NT-3 mRNA-expressing cells in the E15 spinal cord was very similar to the distribution of cells expressing choline acetyltransferase or nerve growth factor receptor (NGFR) mRNA. Moreover, a striking similarity between the developmentally regulated expression of NT-3 and NGFR mRNA was noted in spinal cord motoneurons. A subpopulation of all neurons in the dorsal root ganglia expressed brain-derived neurotrophic factor (BDNF) mRNA from E13, the earliest time examined, to adulthood. These results are consistent with a trophic role of NT-3 for proprioceptive sensory neurons innervating the ventral horn, and imply a local action of BDNF for developing sensory neurons within the dorsal root ganglia. PMID:12106253

  3. Effect of Exercise on the Expression of Adiponectin mRNA and GLUT4 mRNA in Type 2 Diabetic Rats

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To investigate the impact of exercise on the expression of adiponectin and GLUT4 mR NA in type 2 diabetic rats, type 2 diabetic rat model was made. The diabetic rats were treated with swimming training for 8 weeks. The expression of adiponectin mRNA in perirenal fat and GLUT4mRNA in skeletal muscles were assessed by reverse transcription polymerase chain reaction (RT PCR) and the levels of blood glucose, serum insulin, and blood lipid were measured. Our results showed that the expression of adiponectin mRNA and GLUT4 mRNA in diabetic model group was decreased by 45 % (P<0.01), 43 % (P<0.01) respectively. The gene expression of adiponectin and GLUT4 was increased significantly in swimming group (P<0.05 and P<0.01, respectively).Compared with the model group, fasting insulin, TG, TC and FFA were decreased significantly in the training group (P<0.05 or P<0.01) as compared with model group. It is concluded that exercise can promote the expression of adiponectin mRNA and GLUT4 mRNA in type 2 diabetic rats,which may be one of the mechanisms responsible for the amelioration of insulin resistance in the rats.

  4. Stability of Dolos Slopes

    DEFF Research Database (Denmark)

    Brorsen, Michael; Burcharth, Hans F.; Larsen, Torben

    The stability of dolos armour blocks against wave attack has been investigated in wave model studies.......The stability of dolos armour blocks against wave attack has been investigated in wave model studies....

  5. K stability and stability of chiral ring

    CERN Document Server

    Collins, Tristan C; Yau, Shing-Tung

    2016-01-01

    We define a notion of stability for chiral ring of four dimensional N=1 theory by introducing test chiral rings and generalized a maximization. We conjecture that a chiral ring is the chiral ring of a superconformal field theory if and only if it is stable. We then study N=1 field theory derived from D3 branes probing a three-fold singularity X, and show that the K stability which implies the existence of Ricci-flat conic metric on X is equivalent to the stability of chiral ring of the corresponding field theory.

  6. Stability of parallel flows

    CERN Document Server

    Betchov, R

    2012-01-01

    Stability of Parallel Flows provides information pertinent to hydrodynamical stability. This book explores the stability problems that occur in various fields, including electronics, mechanics, oceanography, administration, economics, as well as naval and aeronautical engineering. Organized into two parts encompassing 10 chapters, this book starts with an overview of the general equations of a two-dimensional incompressible flow. This text then explores the stability of a laminar boundary layer and presents the equation of the inviscid approximation. Other chapters present the general equation

  7. Preparation of 99Tcm labeled survivin mRNA antisense PNA and gene imaging in nude mice bearing lung carcinoma A549 xenografts

    International Nuclear Information System (INIS)

    Objective: To prepare the 99Tcm-survivin mRNA antisense peptide nucleic acid (PNA)and investigate its value as a gene imaging agent in tumor bearing mice and early diagnosis in tumor. Methods: Survivin mRNA antisense PNA and mismatch PNA were synthesized. Four amino acids (Gly- (D)Ala-Gly-Gly) and Aba (4-aminobutyric acid) were linked to the 5' end of PNA. Gly- (D)Ala-Gly-Gly served as a chelating moiety for strong chelation of 99Tcm and Aba acted as a spacer to minimize the steric hindrance. PNAs were labeled with 99Tcm by the ligand-exchange method. The labeling efficiency and radiochemical purity were measured by HPLC and ITLC methods. There were five BALB/c nude mice bearing human lung carcinoma (A549) in each of antisense PNA and mismatch PNA groups. Gene imaging of 99Tcm-survivin mRNA antisense and mismatch PNAs were performed at 1, 2 and 4 h post the injection, respectively, and the T/NT ratio was measured by the method of ROI. The statistical comparisons of average values were performed with the two-group t-test for independent sample by SPSS 13.0. Results: The product kept stable in vitro. The labeling efficiency of 99Tcm-survivin mRNA antisense PNA was (95.48 ±1.92)% and more than 85% after the incubation for 24 h in serum. The radiochemical purity was >95%. The labeling efficiency of mismatch PNA was similar to the antisense PNA. 99Tcm-survivin mRNA antisense PNA was especially uptaken by tumor lesion, and its accumulation reached the top at 4 h post the injection. T/NT ratios at 1, 2, and 4 h were 2.70 ± 0.28, 3.44 ± 0.35,4.21 ± 0.63, respectively. In the comparison, the T/NT ratio of 99Tcm-survivin mRNA mismatch PNA at 4 h (3.12 ±0.50) was significantly lower (t=2.918, P=0.019). Conclusions: 99Tcm-survivin mRNA antisense PNA has high labeling efficiency,good stability and no need of purification. Its characteristic of especial uptake by tumor lesion provides the potential value in early diagnosis of tumor. (authors)

  8. Disease-associated mutations that alter the RNA structural ensemble.

    Directory of Open Access Journals (Sweden)

    Matthew Halvorsen

    2010-08-01

    Full Text Available Genome-wide association studies (GWAS often identify disease-associated mutations in intergenic and non-coding regions of the genome. Given the high percentage of the human genome that is transcribed, we postulate that for some observed associations the disease phenotype is caused by a structural rearrangement in a regulatory region of the RNA transcript. To identify such mutations, we have performed a genome-wide analysis of all known disease-associated Single Nucleotide Polymorphisms (SNPs from the Human Gene Mutation Database (HGMD that map to the untranslated regions (UTRs of a gene. Rather than using minimum free energy approaches (e.g. mFold, we use a partition function calculation that takes into consideration the ensemble of possible RNA conformations for a given sequence. We identified in the human genome disease-associated SNPs that significantly alter the global conformation of the UTR to which they map. For six disease-states (Hyperferritinemia Cataract Syndrome, beta-Thalassemia, Cartilage-Hair Hypoplasia, Retinoblastoma, Chronic Obstructive Pulmonary Disease (COPD, and Hypertension, we identified multiple SNPs in UTRs that alter the mRNA structural ensemble of the associated genes. Using a Boltzmann sampling procedure for sub-optimal RNA structures, we are able to characterize and visualize the nature of the conformational changes induced by the disease-associated mutations in the structural ensemble. We observe in several cases (specifically the 5' UTRs of FTL and RB1 SNP-induced conformational changes analogous to those observed in bacterial regulatory Riboswitches when specific ligands bind. We propose that the UTR and SNP combinations we identify constitute a "RiboSNitch," that is a regulatory RNA in which a specific SNP has a structural consequence that results in a disease phenotype. Our SNPfold algorithm can help identify RiboSNitches by leveraging GWAS data and an analysis of the mRNA structural ensemble.

  9. Glucocorticoid Regulation of Human Pulmonary Surfactant Protein-B mRNA Stability Involves the 3′-Untranslated Region

    OpenAIRE

    Huang, Helen W.; Bi, Weizhen; Jenkins, Gaye N.; Alcorn, Joseph L.

    2007-01-01

    Expression of pulmonary surfactant, a complex mixture of lipids and proteins that acts to reduce alveolar surface tension, is developmentally regulated and restricted to lung alveolar type II cells. The hydrophobic protein surfactant protein-B (SP-B) is essential in surfactant function, and insufficient levels of SP-B result in severe respiratory dysfunction. Glucocorticoids accelerate fetal lung maturity and surfactant synthesis both experimentally and clinically. Glucocorticoids act transcr...

  10. Elevated Intracellular Calcium Increases Ferritin H Expression Through an NFAT-Independent Posttranscriptional Mechanism Involving mRNA Stabilization

    OpenAIRE

    MacKenzie, Elizabeth L.; Tsuji, Yoshiaki

    2008-01-01

    An increase in intracellular Ca2+ is one of the initiating events in T cell activation. A calcium-mediated signaling cascade in T cells involves activation of calcineurin and the dephosphorylation and translocation of Nuclear Factor of Activated T-cells (NFAT), resulting in the transcriptional activation of target genes such as IL-2. In the present study, we found that increased intracellular calcium leads to induction of the antioxidant protein ferritin H. We previously reported that the fer...

  11. Elevating calcium in Th2 cells activates multiple pathways to induce IL-4 transcription and mRNA stabilization

    OpenAIRE

    Guo, Liying; Joseph F. Urban; Zhu, Jinfang; Paul, William E.

    2008-01-01

    PMA and ionomycin cause T cell cytokine production. We report that ionomycin alone induces IL-4 and IFNγ, but not IL-2, from in vivo and in vitro generated murine Th2 and Th1 cells. Ionomycin-induced cytokine production requires nuclear factor of activated T cells (NFAT), p38; and calmodulin-dependent kinase IV (CaMKIV). Ionomycin induces p38 phosphorylation through a calcium-dependent, cyclosporine A-inhibitable pathway. “Knocking-down” apoptosis signal-regulating kinase 1 (ASK1) inhibits io...

  12. Formation of mRNA 3' termini: stability and dissociation of a complex involving the AAUAAA sequence.

    OpenAIRE

    Zarkower, D; Wickens, M

    1987-01-01

    Formation of the 3' termini of mRNAs in animal cells involves endonucleolytic cleavage of a pre-mRNA, followed by polyadenylation of the newly formed end. Here we demonstrate that, during cleavage in vitro, the highly conserved AAUAAA sequence of the pre-mRNA forms a complex with a factor present in a crude nuclear extract. This complex is required for cleavage and polyadenylation. It normally is transient, but is very stable on cleaved RNA to which a single terminal cordycepin residue has be...

  13. Neurotrophin-3 mRNA expression in rat intrafusal muscle fibres after denervation and reinnervation

    NARCIS (Netherlands)

    Copray, JCVM; Brouwer, N

    1997-01-01

    We have studied the regulation of the expression of neurotrophin-3 (NT-3) mRNA in neonatal and adult rat muscle spindles after denervation and after denervation followed by reinnervation. Denervation of the intrafusal fibres did not result in an upregulation of the NT-3 mRNA expression but decreased

  14. Adipose tissue interleukin-18 mRNA and plasma interleukin-18: effect of obesity and exercise

    DEFF Research Database (Denmark)

    Leick, Lotte; Lindegaard, Birgitte; Stensvold, Dorthe;

    2007-01-01

    OBJECTIVES: Obesity and a physically inactive lifestyle are associated with increased risk of developing insulin resistance. The hypothesis that obesity is associated with increased adipose tissue (AT) interleukin (IL)-18 mRNA expression and that AT IL-18 mRNA expression is related to insulin...

  15. CUGBP1 and MBNL1 preferentially bind to 3 ' UTRs and facilitate mRNA decay

    DEFF Research Database (Denmark)

    Masuda, A.; Andersen, H. S.; Doktor, T. K.;

    2012-01-01

    - and MBNL1-bound 3' UTRs demonstrated that both factors mediate accelerated mRNA decay and temporal profiles of expression arrays supported this. Role of CUGBP1 on accelerated mRNA decay has been previously reported, but the similar function of MBNL1 has not been reported to date. It is well established...

  16. Distribution of the mRNA for protein phosphatase T in rat brain

    NARCIS (Netherlands)

    Becker, W; Buttini, M; Limonta, S; Boddeke, H; Joost, HG

    1996-01-01

    We have recently cloned a novel protein serine/threonine phosphatase (PPT) from rat mRNA which is predominantly expressed in the brain (Becker et al., J. Biol. Chem., 269 (1994) 22586-22592). In the present study, the regional distribution of PPT mRNA in the brain of adult rats was characterized by

  17. In situ localization of chalcone synthase mRNA in pea root nodule development.

    NARCIS (Netherlands)

    Yang, W.C.; Canter Cremers, H.C.J.; Hogendijk, P.; Katinakis, P.; Wijffelman, C.A.; Franssen, H.J.; Kammen, van A.; Bisseling, T.

    1992-01-01

    In this paper studies on the role of flavonoids in pea root nodule development are reported. Flavonoid synthesis was followed by localizing chalcone synthase (CHS) mRNA in infected pea roots and in root nodules. In a nodule primordium, CHS mRNA is present in all cells of the primordium. Therefore it

  18. Interactions between mRNA export commitment, 3'-end quality control, and nuclear degradation

    DEFF Research Database (Denmark)

    Libri, Domenico; Dower, Ken; Boulay, Jocelyne;

    2002-01-01

    Several aspects of eukaryotic mRNA processing are linked to transcription. In Saccharomyces cerevisiae, overexpression of the mRNA export factor Sub2p suppresses the growth defect of hpr1 null cells, yet the protein Hpr1p and the associated THO protein complex are implicated in transcriptional el...

  19. Stability of cosmological deflagration fronts

    CERN Document Server

    Megevand, Ariel

    2013-01-01

    In a cosmological first-order phase transition, bubbles of the stable phase nucleate and expand in the supercooled metastable phase. In many cases, the growth of bubbles reaches a stationary state, with bubble walls propagating as detonations or deflagrations. However, these hydrodynamical solutions may be unstable under corrugation of the interface. Such instability may drastically alter some of the cosmological consequences of the phase transition. Here, we study the hydrodynamical stability of deflagration fronts. We improve upon previous studies by making a more careful and detailed analysis. In particular, we take into account the fact that the equation of motion for the phase interface depends separately on the temperature and fluid velocity on each side of the wall. Fluid variables on each side of the wall are similar for weakly first-order phase transitions, but differ significantly for stronger phase transitions. As a consequence, we find that for large enough supercooling, any subsonic wall velocity...

  20. Feedback stabilization initiative

    International Nuclear Information System (INIS)

    Much progress has been made in attaining high confinement regimes in magnetic confinement devices. These operating modes tend to be transient, however, due to the onset of MHD instabilities, and their stabilization is critical for improved performance at steady state. This report describes the Feedback Stabilization Initiative (FSI), a broad-based, multi-institutional effort to develop and implement methods for raising the achievable plasma betas through active MHD feedback stabilization. A key element in this proposed effort is the Feedback Stabilization Experiment (FSX), a medium-sized, national facility that would be specifically dedicated to demonstrating beta improvement in reactor relevant plasmas by using a variety of MHD feedback stabilization schemes

  1. Expression of hippocampal adrenergic receptor mRNA in a rat model of depression

    Institute of Scientific and Technical Information of China (English)

    Jianbin Zhang; Lingling Wang; Xinjun Wang; Jingfeng Jiang; Xiaoren Xiang; Tianjun Wang

    2011-01-01

    Adrenergic receptor dysfunction is suggested as a potential cause of hippocampal vulnerability to stress-related pathology. We examined mRNA expression of adrenergic receptor (AR) subtypes α1-AR, α2-AR, and β1-AR in hippocampal subregions (CA1, CA3, dentate gyrus) using in situ hybridization in a depression model induced by chronic unpredictable mild stress and social isolation. α1-AR mRNA expression was significantly increased in the CA3 and dentate gyrus, β1-AR mRNA was significantly increased in the CA1, and α2-AR mRNA remained unchanged in all regions of depression rats compared with controls. Thus, different AR subtypes exhibit a differing pattern of mRNA expression in various hippocampal subregions following depression.

  2. The potential role of IGF-I receptor mRNA in rats with diabetic retinopathy

    Institute of Scientific and Technical Information of China (English)

    匡洪宇; 邹伟; 刘丹; 史榕荇; 程丽华; 殷慧清; 刘晓民

    2003-01-01

    Objective To evaluate the potential role of insulin-like growth factor-1 receptor mRNA(IGF-IR mRNA) in the onset and development of retinopathy in diabetic rats.Methods A diabetic model was duplicated in Wistar rats. The early changes in the retina were examined using light and transmission electron microscopy. Expression of IGF-IR mRNA was analyzed using in situ hybridization.Results Weak expression of IGF-IR mRNA(5%) was found in retinas of normal rats, but was significantly increased (15% and 18%) in the retinas of diabetic rats after 3 and 6 months of diabetes (P<0.01). In situ hybridization and morphological study demonstrated that there was a positive correlation between IGF-IR mRNA expression and retinal changes at various stages.Conclusion Increased IGF-IR mRNA might play an important role in the onset and development of diabetic retinopathy.

  3. Quantification of mRNA Levels Using Real-Time Polymerase Chain Reaction (PCR).

    Science.gov (United States)

    Li, Yiyi; Wang, Kai; Chen, Longhua; Zhu, Xiaoxia; Zhou, Jie

    2016-01-01

    Real-time quantitative reverse transcription PCR technique has advanced greatly over the past 20 years. Messenger RNA (mRNA) levels in cells or tissues can be quantified by this approach. It is well known that changes in mRNA expression in disease, and correlation of mRNA expression profiles with clinical parameters, serve as clinically relevant biomarkers. Hence, accurate determination of the mRNA levels is critically important in describing the biological, pathological, and clinical roles of genes in health and disease. This chapter describes a real-time PCR approach to detect and quantify mRNA expression levels, which can be used for both laboratorial and clinical studies in breast cancer research.

  4. Glucocorticoids modulate BDNF mRNA expression in the rat hippocampus after traumatic brain injury.

    Science.gov (United States)

    Grundy, P L; Patel, N; Harbuz, M S; Lightman, S L; Sharples, P M

    2000-10-20

    Brain-derived neurotrophic factor (BDNF) expression in rat hippocampus is increased after experimental traumatic brain injury (TBI) and may be neuroprotective. Glucocorticoids are important regulators of brain neurotrophin levels and are often prescribed following TBI. The effect of adrenalectomy (ADX) on the expression of BDNF mRNA in the hippocampus after TBI has not been investigated to date. We used fluid percussion injury (FPI) and in situ hybridization to evaluate the expression of BDNF mRNA in the hippocampus 4 h after TBI in adrenal-intact or adrenalectomized rats (with or without corticosterone replacement). FPI and ADX independently increased expression of BDNF mRNA. In animals undergoing FPI, prior ADX caused further elevation of BDNF mRNA and this upregulation was prevented by corticosterone replacement in ADX rats. These findings suggest that glucocorticoids are involved in the modulation of the BDNF mRNA response to TBI.

  5. Glucocorticoids modulate the NGF mRNA response in the rat hippocampus after traumatic brain injury.

    Science.gov (United States)

    Grundy, P L; Patel, N; Harbuz, M S; Lightman, S L; Sharples, P M

    2001-02-23

    Nerve growth factor (NGF) expression in the rat hippocampus is increased after experimental traumatic brain injury (TBI) and is neuroprotective. Glucocorticoids are regulators of brain neurotrophin levels and are often prescribed following TBI. The effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on the expression of NGF mRNA in the hippocampus after TBI has not been investigated to date. We used fluid percussion injury and in situ hybridisation to evaluate the expression of NGF mRNA in the hippocampus 4 h after TBI in adrenal-intact or adrenalectomised rats (with or without CORT replacement). TBI increased expression of NGF mRNA in sham-ADX rats, but not in ADX rats. Furthermore, CORT replacement in ADX rats restored the increase in NGF mRNA induced by TBI. These findings suggest that glucocorticoids have an important role in the induction of hippocampal NGF mRNA after TBI.

  6. Relationship Between Heart Damages and HSPs mRNA in Persistent Heat Stressed Broilers

    Institute of Scientific and Technical Information of China (English)

    SUN Pei-ming; LIU Yu-tian; ZHAO Yong-gang; BAO En-dong; WANG Zhi-liang

    2007-01-01

    The relationship between myocardial cell damages and HSPs mRNA transcription in heat stressed broilers was studied using a spectrophotometer, the histopathological technique, and fluorescence quantitative reverse transcription PCR (FQ RT-PCR). The results showed that the activities of creatine kinase (CK) and glutamic-pyruvic transaninase (GPT) were induction during the persistent heat stress. The major lesions of the myocardial fibers were granular degeneration and necrosis. The transcription of constitutive or cognate heat shock protein 70 (HSC70) mRNA was changeable. The transcription of heat shock protein 70 (HSP70) mRNA was increased obviously in the course of persistent heat stress. The results showed that the change of HSC70 mRNA transcription was contrary to the activity of CK, and the level of HSC70 mRNA transcription must be used as a symbol of the myocardial cell damages in the course of persistent heat stress.

  7. UCP2 mRNA expression is dependent on glucose metabolism in pancreatic islets

    DEFF Research Database (Denmark)

    Dalgaard, Louise Torp

    2012-01-01

    Uncoupling Protein 2 (UCP2) is expressed in the pancreatic β-cell, where it partially uncouples the mitochondrial proton gradient, decreasing both ATP-production and glucose-stimulated insulin secretion (GSIS). Increased glucose levels up-regulate UCP2 mRNA and protein levels, but the mechanism for...... UCP2 up-regulation in response to increased glucose is unknown. The aim was to examine the effects of glucokinase (GK) deficiency on UCP2 mRNA levels and to characterize the interaction between UCP2 and GK with regard to glucose-stimulated insulin secretion in pancreatic islets. UCP2 mRNA expression...... was reduced in GK+/- islets and GK heterozygosity prevented glucose-induced up-regulation of islet UCP2 mRNA. In contrast to UCP2 protein function UCP2 mRNA regulation was not dependent on superoxide generation, but rather on products of glucose metabolism, because MnTBAP, a superoxide dismutase...

  8. Genetic diagnostic test of hepatocellular carcinoma by telomerase catalytic subunit mRNA.

    Science.gov (United States)

    Wada, E; Hisatomi, H; Moritoyo, T; Kanamaru, T; Hikiji, K

    1998-01-01

    This study investigated the relationship between telomerase activity and telomere length and between telomerase reverse transcriptase (hTERT) mRNA and telomere length. Both cancerous and non-cancerous tissues were studied in individuals with hepatic carcinoma. In this study, the telomere length in HCC livers had a wide range, no clear significant correlation was found between hTERT mRNA and telomere length. Telomerase activity was more strongly correlated with hTERT mRNA than with telomere length. The correlation between hTERT mRNA and telomerase activity shown here indicates that hTERT mRNA has potential for cancer diagnosis. PMID:9769378

  9. Starch supplementation modulates amylase enzymatic properties and amylase B mRNA level in the digestive gland of the Pacific oyster Crassostrea gigas.

    Science.gov (United States)

    Huvet, A; Jeffroy, F; Daniel, J Y; Quéré, C; Le Souchu, P; Van Wormhoudt, A; Boudry, P; Moal, J; Samain, J F

    2012-09-01

    In the oyster Crassostrea gigas consumption-related traits, amylase properties and growth were found to be linked through genotypes that differed for polymorphism in the two amylase genes AMYA and AMYB. Modulation of AMYA mRNA level had already been observed in response to food availability, whereas the functional role of AMYB was still unknown. To improve knowledge about the regulation of amylase expression in C. gigas and the respective roles of the two genes, we made an assay of amylase expression at mRNA and enzymatic levels in the digestive gland of oysters that had received dietary supplements of starch. After 18 days, a significant increase of translatable mRNA for AMYB was observed, with a correlated increase in Michaelis-Menten constant Km values and a decrease in total amylase activity. This modulation is the first evidence of observable functioning of AMYB in digestive processes. Amylase B is suggested to display a higher Km than amylase A, offering a means of adapting to high substrate concentrations. The highest starch supplement level (10 mgL(-1)) induced alteration in oyster physiology. The 1 mgL(-1) treatment should be tested as a practical food supplement that could lead to growth benefits for oysters.

  10. The 5' leader of the mRNA encoding the mouse neurotrophin receptor TrkB contains two internal ribosomal entry sites that are differentially regulated.

    Directory of Open Access Journals (Sweden)

    Stephanie L Timmerman

    Full Text Available A single internal ribosomal entry site (IRES in conjunction with IRES transactivating factors (ITAFs is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5' leaders (1428 nt and 448 nt, both of which include the common 3' exon (Ex2, 344 nt. Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5' leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1. Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5' leader are differentially regulated, in part by PTB1.

  11. The 5' leader of the mRNA encoding the mouse neurotrophin receptor TrkB contains two internal ribosomal entry sites that are differentially regulated.

    Science.gov (United States)

    Timmerman, Stephanie L; Pfingsten, Jennifer S; Kieft, Jeffrey S; Krushel, Les A

    2008-01-01

    A single internal ribosomal entry site (IRES) in conjunction with IRES transactivating factors (ITAFs) is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5' leaders (1428 nt and 448 nt), both of which include the common 3' exon (Ex2, 344 nt). Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5' leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a) also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1). Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5' leader are differentially regulated, in part by PTB1. PMID:18779873

  12. Reduced mRNA expression of PTGDS in peripheral blood mononuclear cells of rapid-cycling bipolar disorder patients compared with healthy control subjects

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Peijs, Lone; Kessing, Lars Vedel;

    2015-01-01

    is lacking. Two enzymes in the arachidonic acid cascade are the prostaglandin D synthase (PTGDS), which catalyzes the conversion of prostaglandin H2 to prostaglandin D2 (PGD2), and the aldo-keto reductase family 1 member C3 (AKR1C3), which catalyzes the reduction of PGD2. We aimed to test the hypothesis...... that mRNA expression of PTGDS and AKR1C3 is deregulated in rapid-cycling disorder patients in a euthymic or current affective state compared with healthy control subjects, and that expression alters with affective states. METHODS: PTGDS and AKR1C3 mRNA expression in peripheral blood mononuclear cells...... was measured in 37 rapid-cycling bipolar disorder patients and 40 age- and gender-matched healthy control subjects using reverse transcription quantitative real-time polymerase chain reaction. Repeated measurements of PTGDS and AKR1C3 mRNA expression were obtained in various affective states during 6-12 months...

  13. Estrogen alters proenkephalin RNAs in the paraventricular nucleus of the hypothalamus following stress.

    Science.gov (United States)

    Yukhananov, R Y; Handa, R J

    1997-08-01

    Gonadal steroids modulate activity of the hypothalamo-pituitary-adrenal axis (HPA) following stress, but the regulatory pathways of this modulation are unknown. A possible site of action is the synthesis of CRH and/or enkephalin in cells of the paraventricular nucleus of the hypothalamus (PVN). To investigate this possibility, we utilized two stressors, i.p. hypertonic saline injection (HSI) or exposure to novel environment, and examined the response of CRH or c-fos mRNAs and proenkephalin (PPE) mRNA and heteronuclear RNA (hnRNA, primary transcript). Male rats were gonadectomized and treated with estrogen or dihydrotestosterone propionate (DHTP) for 2 weeks. In situ hybridization revealed that novelty or HSI elevated levels of PPE hnRNA and c-fos mRNA in the PVN. Estrogen attenuated the elevation of PPE hnRNA in the PVN following HSI, and enhanced the c-fos mRNA response to novelty. In contrast, DHTP did not affect PPE hnRNA, but inhibited the c-fos mRNA response to novelty. These data indicate that in male rats estrogen receptor but not androgen receptor may modulate the endocrine stress response by altering PPE transcription in the PVN and that this effect depends on the type of stressor. PMID:9295199

  14. Early repeated maternal separation induces alterations of hippocampus reelin expression in rats

    Indian Academy of Sciences (India)

    Jianlong Zhang; Lina Qin; Hu Zhao

    2013-03-01

    The long-term effects of repeated maternal separation (MS) during early postnatal life on reelin expression in the hippocampus of developing rats were investigated in the present study. MS was carried out by separating Wistar rat pups singly from their mothers for 3 h a day during postnatal days (PND) 2–14. Reelin mRNA and protein levels in the hippocampus were determined using qRT-PCR and Western blotting, at PND 22, PND 60 and PND 90. MS resulted in the loss of body weight in the developing rats, and reelin mRNA and protein levels in the hippocampus generally were down-regulated over the developing period, but the reelin mRNA and protein levels in the hippocampus of 90-day-old male rats were up-regulated. These findings suggest that the long-term effects of MS on the expression levels of hippocampal reelin mRNA and protein depends on the age at which the stressed rats’ brains were collected; reelin had important implications for the maternal-neonate interaction needed for normal brain development. In conclusion, repeated MS occurring during early postnatal life may cause the alterations of hippocampal reelin expression with the increasing age of developing rats.

  15. Art as Alterity in Education

    Science.gov (United States)

    Zhao, Guoping

    2014-01-01

    In education, art has often been perceived as entertainment and decoration and is the first subject to go when there are budget cuts or test-score pressures. Drawing on Emmanuel Lévinas's idea of the primacy of radical alterity that breaks the totality of our being, enables self-transformation and ethics, and ensures community as a totality…

  16. Altered expression of adipose differentiation-related protein gene in placental tissue of pre-eclampsia

    Institute of Scientific and Technical Information of China (English)

    ZHANG Chun-li; YAO Yuan-qing; LI Dong-hong; ZHANG Wei

    2006-01-01

    Objective: To investigate the altered expression of lipid metabolism-related gene adipose differentiation-related protein (ADRP) in pre-eclampsia. Methods: Semi-quantitative RT-PCR and Western blotting were used to validate the altered expression of ADRP gene between pre-eclamptic placentas (preeclampsia group) and normotensive placentas (control group) respectively. In situ hybridization (ISH)was used to localize ADRP mRNA in pre-eclamptic placentas. Results: There was a significant difference in the levels of placental ADRP mRNA between pre-eclampsia group and control group (1.98± 0. 50 vs 1. 09±0. 20, P<0.01). Western blotting showed that placentas both in pre-eclampsia group and control group expressed the special ADRP band at 48. 1 kD. The relative levels of ADRP protein in pre-eclampsia group were significantly higher than those of control group (0. 40 ±0. 19 vs 0. 19 ±0. 09, P< 0. 01).ADRP mRNA was diffusely distributed in pre-eclamptic placentas. Their positive staining existed in cytoplasm of trophoblast. Conclusion: Abnormal expression of ADRP gene in pre-eclamptic placenta may be associated with the pathogenesis of pre-eclampsia.

  17. Stability Testing of Herbal Drugs: Challenges, Regulatory Compliance and Perspectives.

    Science.gov (United States)

    Bansal, Gulshan; Suthar, Nancy; Kaur, Jasmeen; Jain, Astha

    2016-07-01

    Stability testing is an important component of herbal drugs and products (HDPs) development process. Drugs regulatory agencies across the globe have recommended guidelines for the conduct of stability studies on HDPs, which require that stability data should be included in the product registration dossier. From the scientific viewpoint, numerous chemical constituents in an herbal drug are liable to varied chemical reactions under the influence of different conditions during its shelf life. These reactions can lead to altered chemical composition of HDP and consequently altered therapeutic profile. Many reports on stability testing of HDPs have appeared in literature since the last 10 years. A review of these reports reveals that there is wide variability in temperature (-80 to 100 °C), humidity (0-100%) and duration (a few hours-36 months) for stability assessment of HDPs. Of these, only 1% studies are conducted in compliance with the regulatory guidelines for stability testing. The present review is aimed at compiling all stability testing reports, understanding key challenges in stability testing of HDPs and suggesting possible solutions for these. The key challenges are classified as chemical complexity and biochemical composition variability in raw material, selection of marker(s) and influences of enzymes. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27073177

  18. Cardiac calcium release channel (ryanodine receptor) in control and cardiomyopathic human hearts: mRNA and protein contents are differentially regulated.

    Science.gov (United States)

    Sainte Beuve, C; Allen, P D; Dambrin, G; Rannou, F; Marty, I; Trouvé, P; Bors, V; Pavie, A; Gandgjbakch, I; Charlemagne, D

    1997-04-01

    Abnormal intracellular calcium handling in cardiomyopathic human hearts has been associated with an impaired function of the sarcoplasmic reticulum, but previous reports on the gene expression of the ryanodine receptors (Ry2) are contradictory. We measured the mRNA levels, the protein levels and the number of high affinity [3H]ryanodine binding sites in the left ventricle of non-failing (n = 9) and failing human hearts [idiopathic dilated (IDCM n = 16), ischemic (ICM n = 7) or mixed (MCM n = 8) cardiomyopathies]. Ry2 mRNA levels were significantly reduced in IDCM (-30%) and unchanged in MCM and ICM and Ry2 protein levels were similar. In contrast, we observed a two-fold increase in the number of high affinity Ry2 (B(max) = 0.43 +/- 0.11 v 0.22 +/- 0.13 pmol/mg protein, respectively; P<0.01) and an unchanged K(d). Furthermore, levels of myosin heavy chain mRNA and protein per g of tissue were similar in failing and non-failing hearts, suggesting that the observed differences in Ry2 are not caused by the increase in fibrosis in failing heart. Therefore, the dissociation between the two-fold increase in the number of high affinity ryanodine receptors observed in all failing hearts and the slightly decreased mRNA level or unchanged protein level suggests that the ryanodine binding properties are affected in failing myocardium and that such modifications rather than a change in gene expression alter the channel activity and could contribute to abnormalities in intracellular Ca2+ handling. PMID:9160875

  19. An evaluation of the effects of acute and chronic L-tyrosine administration on BDNF levels and BDNF mRNA expression in the rat brain.

    Science.gov (United States)

    Ferreira, Gabriela K; Scaini, Giselli; Jeremias, Isabela C; Carvalho-Silva, Milena; Gonçalves, Cinara L; Pereira, Talita C B; Oliveira, Giovanna M T; Kist, Luiza W; Bogo, Maurício R; Schuck, Patrícia F; Ferreira, Gustavo C; Streck, Emilio L

    2014-04-01

    Tyrosinemia type II, which is also known as Richner-Hanhart syndrome, is an inborn error of metabolism that is due to a block in the transamination reaction that converts tyrosine to p-hydroxyphenylpyruvate. Because the mechanisms of neurological dysfunction in hypertyrosinemic patients are poorly known and the symptoms of these patients are related to the central nervous system, the present study evaluated brain-derived neurotrophic factor (BDNF) levels and bdnf mRNA expression in young rats and during growth. In our acute protocol, Wistar rats (10 and 30 days old) were killed 1 h after a single intraperitoneal L-tyrosine injection (500 mg/kg) or saline. Chronic administration consisted of L-tyrosine (500 mg/kg) or saline injections 12 h apart for 24 days in Wistar rats (7 days old), and the rats were killed 12 h after the last injection. The brains were rapidly removed, and we evaluated the BDNF levels and bdnf mRNA expression. The present results showed that the acute administration of L-tyrosine decreased both BDNF and bdnf mRNA levels in the striatum of 10-day-old rats. In the 30-day-old rats, we observed decreased BDNF levels without modifications in bdnf transcript level in the hippocampus and striatum. Chronic administration of L-tyrosine increased the BDNF levels in the striatum of rats during their growth, whereas bdnf mRNA expression was not altered. We hypothesize that oxidative stress can interact with the BDNF system to modulate synaptic plasticity and cognitive function. The present results enhance our knowledge of the pathophysiology of hypertyrosinemia.

  20. The relative expression levels of insulin-like growth factor 1 and myostatin mRNA in the asynchronous development of skeletal muscle in ducks during early development.

    Science.gov (United States)

    Hu, Yan; Liu, Hongxiang; Shan, Yanju; Ji, Gaige; Xu, Wenjuan; Shu, Jingting; Li, Huifang

    2015-08-10

    Genetic selection is a powerful tool for modifying poultry muscle yield. Insulin-like growth factor I (IGF-I) and myostatin (MSTN) are important regulators of muscle growth, especially in the myogenesis stage. This study compared the developmental pattern of the pectoralis major (PM) and lateral gastrocnemius (LM) muscles, mRNA expression characterization of IGF-I and MSTN-A and their correlation between 14 days in ovo and 1 week post-hatch in two Chinese local duck breeds. During early development, the growth of duck PM and LM followed an asynchronous pattern. Variations in PM growth rate observed with development followed the relative variations of MSTN and IGF-I expression; however, the same behavior was not observed in LM. Moreover, the profile of IGF-I expression in duck skeletal muscles indicated that genetic selection for high meat-yield poultry has altered the temporal expression of IGF-I and affected cellular characteristics and mass by hatch in a PM-specific manner. The MSTN-A expression profile showed synchronization with the growth of skeletal muscle and peaks of myofiber proliferation. The expression patterns of IGF-I and MSTN suggest that duck pectoralis fibers are prioritized for proliferation in embryogenesis. The IGF-1/MSTN-A mRNA ratios in PM and LM presented very similar trends in the changes of myofiber characteristics, and differences in the IGF-1/MSTN-A mRNA ratio in PM between the two breeds corresponded to the timing of differences in PM mass between the varieties. Our results support the hypothesis that relative levels of IGF-I and MSTN mRNA may participate in ordering muscle growth rates with selected development.

  1. Effect of ration size on fillet fatty acid composition, phospholipid allostasis and mRNA expression patterns of lipid regulatory genes in gilthead sea bream (Sparus aurata).

    Science.gov (United States)

    Benedito-Palos, Laura; Calduch-Giner, Josep A; Ballester-Lozano, Gabriel F; Pérez-Sánchez, Jaume

    2013-04-14

    The effect of ration size on muscle fatty acid (FA) composition and mRNA expression levels of key regulatory enzymes of lipid and lipoprotein metabolism have been addressed in juveniles of gilthead sea bream fed a practical diet over the course of an 11-week trial. The experimental setup included three feeding levels: (i) full ration until visual satiety, (ii) 70 % of satiation and (iii) 70 % of satiation with the last 2 weeks at the maintenance ration. Feed restriction reduced lipid content of whole body by 30 % and that of fillet by 50 %. In this scenario, the FA composition of fillet TAG was not altered by ration size, whereas that of phospholipids was largely modified with a higher retention of arachidonic acid and DHA. The mRNA transcript levels of lysophosphatidylcholine acyltransferases, phosphatidylethanolamine N-methyltransferase and FA desaturase 2 were not regulated by ration size in the present experimental model. In contrast, mRNA levels of stearoyl-CoA desaturases were markedly down-regulated by feed restriction. An opposite trend was found for a muscle-specific lipoprotein lipase, which is exclusive of fish lineage. Several upstream regulatory transcriptions were also assessed, although nutritionally mediated changes in mRNA transcripts were almost reduced to PPARα and β, which might act in a counter-regulatory way on lipolysis and lipogenic pathways. This gene expression pattern contributes to the construction of a panel of biomarkers to direct marine fish production towards muscle lean phenotypes with increased retentions of long-chain PUFA.

  2. Effect of ration size on fillet fatty acid composition, phospholipid allostasis and mRNA expression patterns of lipid regulatory genes in gilthead sea bream (Sparus aurata).

    Science.gov (United States)

    Benedito-Palos, Laura; Calduch-Giner, Josep A; Ballester-Lozano, Gabriel F; Pérez-Sánchez, Jaume

    2013-04-14

    The effect of ration size on muscle fatty acid (FA) composition and mRNA expression levels of key regulatory enzymes of lipid and lipoprotein metabolism have been addressed in juveniles of gilthead sea bream fed a practical diet over the course of an 11-week trial. The experimental setup included three feeding levels: (i) full ration until visual satiety, (ii) 70 % of satiation and (iii) 70 % of satiation with the last 2 weeks at the maintenance ration. Feed restriction reduced lipid content of whole body by 30 % and that of fillet by 50 %. In this scenario, the FA composition of fillet TAG was not altered by ration size, whereas that of phospholipids was largely modified with a higher retention of arachidonic acid and DHA. The mRNA transcript levels of lysophosphatidylcholine acyltransferases, phosphatidylethanolamine N-methyltransferase and FA desaturase 2 were not regulated by ration size in the present experimental model. In contrast, mRNA levels of stearoyl-CoA desaturases were markedly down-regulated by feed restriction. An opposite trend was found for a muscle-specific lipoprotein lipase, which is exclusive of fish lineage. Several upstream regulatory transcriptions were also assessed, although nutritionally mediated changes in mRNA transcripts were almost reduced to PPARα and β, which might act in a counter-regulatory way on lipolysis and lipogenic pathways. This gene expression pattern contributes to the construction of a panel of biomarkers to direct marine fish production towards muscle lean phenotypes with increased retentions of long-chain PUFA. PMID:22856503

  3. Nonsense mutations in the human β-globin gene affect mRNA metabolism

    International Nuclear Information System (INIS)

    A number of premature translation termination mutations (nonsense mutations) have been described in the human α- and β-globin genes. Studies on mRNA isolated from patients with β0-thalassemia have shown that for both the β-17 and the β-39 mutations less than normal levels of β-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human β-globin mRNA). In vitro studies using the cloned β-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. The authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human β-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation

  4. Evaluation of mRNA Localization Using Double Barrel Scanning Ion Conductance Microscopy.

    Science.gov (United States)

    Nashimoto, Yuji; Takahashi, Yasufumi; Zhou, Yuanshu; Ito, Hidenori; Ida, Hiroki; Ino, Kosuke; Matsue, Tomokazu; Shiku, Hitoshi

    2016-07-26

    Information regarding spatial mRNA localization in single cells is necessary for a better understanding of cellular functions in tissues. Here, we report a method for evaluating localization of mRNA in single cells using double-barrel scanning ion conductance microscopy (SICM). Two barrels in a nanopipette were filled with aqueous and organic electrolyte solutions and used for SICM and as an electrochemical syringe, respectively. We confirmed that the organic phase barrel could be used to collect cytosol from living cells, which is a minute but sufficient amount to assess cellular status using qPCR analysis. The water phase barrel could be used for SICM to image topography with subcellular resolution, which could be used to determine positions for analyzing mRNA expression. This system was able to evaluate mRNA localization in single cells. After puncturing the cellular membrane in a minimally invasive manner, using SICM imaging as a guide, we collected a small amount cytosol from different positions within a single cell and showed that mRNA expression depends on cellular position. In this study, we show that SICM imaging can be utilized for the analysis of mRNA localization in single cells. In addition, we fully automated the pipet movement in the XYZ-directions during the puncturing processes, making it applicable as a high-throughput system for collecting cytosol and analyzing mRNA localization. PMID:27399804

  5. Negative regulation of neuromedin U mRNA expression in the rat pars tuberalis by melatonin.

    Science.gov (United States)

    Aizawa, Sayaka; Sakata, Ichiro; Nagasaka, Mai; Higaki, Yuriko; Sakai, Takafumi

    2013-01-01

    The pars tuberalis (PT) is part of the anterior pituitary gland surrounding the median eminence as a thin cell layer. The characteristics of PT differ from those of the pars distalis (PD), such as cell composition and gene expression, suggesting that the PT has a unique physiological function compared to the PD. Because the PT highly expresses melatonin receptor type 1, it is considered a mediator of seasonal and/or circadian signals of melatonin. Expression of neuromedin U (NMU) that is known to regulate energy balance has been previously reported in the rat PT; however, the regulatory mechanism of NMU mRNA expression and secretion in the PT are still obscure. In this study, we examined both the diurnal change of NMU mRNA expression in the rat PT and the effects of melatonin on NMU in vivo. In situ hybridization and quantitative PCR analysis of laser microdissected PT samples revealed that NMU mRNA expression in the PT has diurnal variation that is high during the light phase and low during the dark phase. Furthermore, melatonin administration significantly suppressed NMU mRNA expression in the PT in vivo. On the other hand, 48 h fasting did not have an effect on PT-NMU mRNA expression, and the diurnal change of NMU mRNA expression was maintained. We also found the highest expression of neuromedin U receptor type 2 (NMUR2) mRNA in the third ventricle ependymal cell layer, followed by the arcuate nucleus and the spinal cord. These results suggest that NMU mRNA expression in the PT is downregulated by melatonin during the dark phase and shows diurnal change. Considering that NMU mRNA in the PT showed the highest expression level in the brain, PT-NMU may act on NMUR2 in the brain, especially in the third ventricle ependymal cell layer, with a circadian rhythm. PMID:23843987

  6. Negative regulation of neuromedin U mRNA expression in the rat pars tuberalis by melatonin.

    Directory of Open Access Journals (Sweden)

    Sayaka Aizawa

    Full Text Available The pars tuberalis (PT is part of the anterior pituitary gland surrounding the median eminence as a thin cell layer. The characteristics of PT differ from those of the pars distalis (PD, such as cell composition and gene expression, suggesting that the PT has a unique physiological function compared to the PD. Because the PT highly expresses melatonin receptor type 1, it is considered a mediator of seasonal and/or circadian signals of melatonin. Expression of neuromedin U (NMU that is known to regulate energy balance has been previously reported in the rat PT; however, the regulatory mechanism of NMU mRNA expression and secretion in the PT are still obscure. In this study, we examined both the diurnal change of NMU mRNA expression in the rat PT and the effects of melatonin on NMU in vivo. In situ hybridization and quantitative PCR analysis of laser microdissected PT samples revealed that NMU mRNA expression in the PT has diurnal variation that is high during the light phase and low during the dark phase. Furthermore, melatonin administration significantly suppressed NMU mRNA expression in the PT in vivo. On the other hand, 48 h fasting did not have an effect on PT-NMU mRNA expression, and the diurnal change of NMU mRNA expression was maintained. We also found the highest expression of neuromedin U receptor type 2 (NMUR2 mRNA in the third ventricle ependymal cell layer, followed by the arcuate nucleus and the spinal cord. These results suggest that NMU mRNA expression in the PT is downregulated by melatonin during the dark phase and shows diurnal change. Considering that NMU mRNA in the PT showed the highest expression level in the brain, PT-NMU may act on NMUR2 in the brain, especially in the third ventricle ependymal cell layer, with a circadian rhythm.

  7. COX-2 mRNA expression in esophageal squamous cell carcinoma (ESCC) and effect by NSAID.

    Science.gov (United States)

    Liu, X; Li, P; Zhang, S-T; You, H; Jia, J-D; Yu, Z-L

    2008-01-01

    To investigate cyclooxygenase-2 (COX-2) mRNA expression in human esophageal squamous cell carcinoma and the effect of a non-steroidal anti-inflammatory drug (NSAID) on it, in order to explore the mechanism of COX-2 in esophageal squamous cell carcinoma (ESCC) carcinogenesis and the ability of NSAID to prevent or treat ESCC. Frozen specimens of human ESCC and adjacent normal esophageal squamous epithelium pairs (n = 22) were examined for COX-2 mRNA expression by reverse-transcription polymerase chain reaction (RT-PCR). After incubation with aspirin (a non-selective COX inhibitor) or Nimesulide (a selective COX-2 inhibitor), the proliferation status of two human esophageal squamous cancer cell lines, EC-9706 and EC-109, was quantified by 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide assay. The expression of COX-2 mRNA in these cells was detected by RT-PCR. COX-2 mRNA was expressed in 12 of 22 (54.5%) ESCC tissue samples, but it was undetectable in all the specimens of adjacent normal esophageal squamous epithelium COX-2 mRNA expression. Both aspirin (5-20 mmol/L) and Nimesulide (0.1-0.8 mmol/L) inhibited EC-9706 cell line proliferation and suppressed its COX-2 mRNA expression dose-dependently. However, only aspirin (5-20 mmol/L) could inhibit proliferation in the EC-109 cell line and suppress COX-2 mRNA expression. Nimesulide (0.1-0.8 mmol/L) could neither inhibit EC-109 cell growth nor suppress COX-2 mRNA expression. COX-2 mRNA expression is a frequent phenomenon in human ESCC tissue samples and plays an important role in the carcinogenesis of ESCC. NSAID may be useful in the chemoprevention and therapy of human ESCC and its effects are likely to be mediated by modulating COX-2 activity.

  8. Internet Addiction: Stability and Change

    Science.gov (United States)

    Huang, Chiungjung

    2010-01-01

    This longitudinal study examined five indices of stability and change in Internet addiction: structural stability, mean-level stability, differential stability, individual-level stability, and ipsative stability. The study sample was 351 undergraduate students from end of freshman year to end of junior year. Convergent findings revealed stability…

  9. DEPENDENCE OF YKL-40 mRNA TISSUE LEVELS ON KRAS MUTATION STATUS IN COLORECTAL CANCER - PRELIMINARY RESULTS

    Directory of Open Access Journals (Sweden)

    Yana Feodorova

    2015-08-01

    Full Text Available Colorectal cancer (CRC is one of the most deadly cancers worldwide. Despite the introduction of targeted molecular therapies in the last 10 years, overall survival has not increased substantially. CRC progression is accompanied by numerous genetic and epigenetic alterations and dysregulation of several signaling pathways, among which activation of Wnt and inactivation of TGF-β signaling. The molecular heterogeneity of CRC, however, hinders the molecular subtyping of CRC and thus the identification of common biomarkers for this pathology. The only three well established biomarkers for advanced-colorectal-cancer drug treatment are negative biomarkers. These are mutations in the genes KRAS, NRAS and BRAF which determine resistance to therapy with anti-EGFR antibodies. YKL-40 is a chitin-binding glycoprotein that has been shown to play a role in extracellular tissue remodeling, angiogenesis, cell migration and inflammation. Increased serum levels of this protein have been detected in patients with CRC but the role of YKL-40 in this neoplastic disease has not been studied extensively and the precise function of YKL-40 in CRC progression is not known. In the present study we determined the KRAS mutation status and measured the mRNA levels of YKL-40 of 24 patients with sporadic CRC. In addition, we assessed the association between these two parameters by statistical analysis. We are the first to show that in CRC YKL-40 mRNA levels are dependent on the presence of KRAS mutations, being prominently elevated in the wild type background. Our results indicate the potential role of YKL-40 as a target molecule for CRC therapy.

  10. An exploration of heat tolerance in mice utilizing mRNA and microRNA expression analysis.

    Directory of Open Access Journals (Sweden)

    Aminul Islam

    Full Text Available BACKGROUND: Individuals who rapidly develop hyperthermia during heat exposure (heat-intolerant are vulnerable to heat associated illness and injury. We recently reported that heat intolerant mice exhibit complex alterations in stress proteins in response to heat exposure. In the present study, we further explored the role of genes and molecular networks associated with heat tolerance in mice. METHODOLOGY: Heat-induced physiological and biochemical changes were assessed to determine heat tolerance levels in mice. We performed RNA and microRNA expression profiling on mouse gastrocnemius muscle tissue samples to determine novel biological pathways associated with heat tolerance. PRINCIPAL FINDINGS: Mice (n = 18 were assigned to heat-tolerant (TOL and heat-intolerant (INT groups based on peak core temperatures during heat exposures. This was followed by biochemical assessments (Hsp40, Hsp72, Hsp90 and Hsf1 protein levels. Microarray analysis identified a total of 3,081 mRNA transcripts that were significantly misregulated in INT compared to TOL mice (p<0.05. Among them, Hspa1a, Dnajb1 and Hspb7 were differentially expressed by more than two-fold under these conditions. Furthermore, we identified 61 distinct microRNA (miRNA sequences significantly associated with TOL compared to INT mice; eight miRNAs corresponded to target sites in seven genes identified as being associated with heat tolerance pathways (Hspa1a, Dnajb1, Dnajb4, Dnajb6, Hspa2, Hspb3 and Hspb7. CONCLUSIONS: The combination of mRNA and miRNA data from the skeletal muscle of adult mice following heat stress provides new insights into the pathophysiology of thermoregulatory disturbances of heat intolerance.

  11. Safety of Herbal Medicinal Products: Echinacea and Selected Alkylamides Do Not Induce CYP3A4 mRNA Expression

    Directory of Open Access Journals (Sweden)

    Maryam Modarai

    2011-01-01

    Full Text Available A major safety concern with the use of herbal medicinal products (HMP is their interactions with conventional medicines, which are often mediated via the cytochrome P450 (CYP system. Echinacea is a widely used over-the-counter HMP, with proven immunomodulatory properties. Its increasing use makes research into its safety an urgent concern. Previously, we showed that Echinacea extracts and its alkylamides (thought to be important for Echinacea's immunomodulatory activity mildly inhibit the enzymatic activity of the main drug metabolising CYP isoforms, but to this date, there is insufficient work on its ability to alter CYP expression levels. We now report for the first time the effect of a commercial Echinacea extract (Echinaforce and four Echinacea alkylamides on the transcription of the major drug metabolizing enzyme CYP3A4. HepG2 cells were exposed for 96 h to clinically relevant concentrations of Echinaforce (22, 11.6 and 1.16 μg mL−1 or the alkylamides (1.62 and 44 nM. CYP3A4 mRNA levels were quantified using real-time reverse transcription polymerase chain reaction (RT-PCR. Neither Echinaforce nor the alkylamides produced any significant changes in the steady-state CYP3A4 mRNA levels, under these conditions. In contrast, treatment with 50 μM rifampicin resulted in a 3.8-fold up-regulation over the vehicle control. We conclude that Echinaforce is unlikely to affect CYP3A4 transcriptional levels, even at concentrations which can inhibit the enzymatic activity of CYP3A4. Overall, our data provides further evidence for the lack of interactions between Echinacea and conventional drugs.

  12. Severe insulin resistance alters metabolism in mesenchymal progenitor cells.

    Science.gov (United States)

    Balhara, Bharti; Burkart, Alison; Topcu, Vehap; Lee, Youn-Kyoung; Cowan, Chad; Kahn, C Ronald; Patti, Mary-Elizabeth

    2015-06-01

    Donohue syndrome (DS) is characterized by severe insulin resistance due to mutations in the insulin receptor (INSR) gene. To identify molecular defects contributing to metabolic dysregulation in DS in the undifferentiated state, we generated mesenchymal progenitor cells (MPCs) from induced pluripotent stem cells derived from a 4-week-old female with DS and a healthy newborn male (control). INSR mRNA and protein were significantly reduced in DS MPC (for β-subunit, 64% and 89% reduction, respectively, P consumption in both the basal state (87% higher, P =.09) and in response to the uncoupler carbonyl cyanide-p-triflouromethoxyphenylhydrazone (2-fold increase, P =.06). Although mitochondrial DNA or mass did not differ, oxidative phosphorylation protein complexes III and V were increased in DS (by 37% and 6%, respectively; P < .05). Extracellular acidification also tended to increase in DS (91% increase, P = .07), with parallel significant increases in lactate secretion (34% higher at 4 h, P < .05). In summary, DS MPC maintain signaling downstream of the INSR, suggesting that IGF-1R signaling may partly compensate for INSR mutations. However, alterations in receptor expression and pathway-specific defects in insulin signaling, even in undifferentiated cells, can alter cellular oxidative metabolism, potentially via transcriptional mechanisms. PMID:25811318

  13. Endoplasmic reticulum-directed recombinant mRNA displays subcellular localization equal to endogenous mRNA during transient expression in CHO cells

    DEFF Research Database (Denmark)

    Beuchert Kallehauge, Thomas; Kol, Stefan; Andersen, Mikael Rørdam;

    2016-01-01

    When expressing pharmaceutical recombinant proteins in mammalian cells, the protein is commonly directed through the secretory pathway, in a signal peptide-dependent manner, to acquire specific post-translational modifications and to facilitate secretion into the culture medium. One key premise...... for this is the direction of the mRNA encoding the recombinant protein to the surface of the endoplasmic reticulum (ER) for subsequent protein translocation into the secretory pathway. To evaluate the efficiency of this process in Chinese hamster ovary (CHO) cells, the subcellular localization of recombinant mRNA encoding...

  14. In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA

    DEFF Research Database (Denmark)

    Nielsen, M. E.; Rasmussen, I. A.; Kristensen, Stine Gry;

    2011-01-01

    RNA analysis (24 women). Expression of Androgen Receptor (AR) mRNA levels in granulosa cells, and of androstenedione and testosterone in FF, were correlated to the expression of FSH receptor (FSHR), LH receptor (LHR), CYP19 and anti-Müllerian Hormone-receptor2 (AMHR2) mRNA in the granulosa cells and to the FF...... with the expression of AMHR2, but did not correlate with any of the hormones in the FF. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the FF levels of androgen and FSHR expression...

  15. An Experimental Study on the Expression of Heme Oxygenase-2 mRNA in Hirschsprung's Disease

    Institute of Scientific and Technical Information of China (English)

    朱珉; 魏明发; 刘芳

    2002-01-01

    Summary: In order to investigate the relationship between the expression of heme oxygenase-2 (HO-2) mRNA and the pathogenesis of Hirschsprung's disease (HD), total ribonucleic acid (RNA) was extracted in the aganglionic and ganglionic segments of colon respectively from 15 cases of HD. The single-stranded cDNA of HO-2 was synthesized and further amplified by reverse transcription-poly merase chain reaction (RT-PCR). The expression of HO-2 mRNA was normal in ganglionic seg ments, but absent in aganglionic segments. It is concluded that the absence of HO-2 mRNA expres sion may be an important mechanism responsible for HD.

  16. Translationally Repressed mRNA Transiently Cycles through Stress Granules during Stress

    OpenAIRE

    Mollet, Stephanie; Cougot, Nicolas; Wilczynska, Ania; Dautry, François; Kress, Michel; Bertrand, Edouard; Weil, Dominique

    2008-01-01

    In mammals, repression of translation during stress is associated with the assembly of stress granules in the cytoplasm, which contain a fraction of arrested mRNA and have been proposed to play a role in their storage. Because physical contacts are seen with GW bodies, which contain the mRNA degradation machinery, stress granules could also target arrested mRNA to degradation. Here we show that contacts between stress granules and GW bodies appear during stress-granule assembly and not after ...

  17. The topological configuration and conformational analysis of mRNA in translation

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The theoretical model construction of mRNA hairpin structure and single-stranded structure as well as the simulation studies on RNA structure determined by the X-ray crystal diffraction and nuclear magnetic resonance revealed that in translation, after mRNA being unfolded into single-stranded structure, its topological configuration was closely correlative with the original hairpin structure. The conformational features of single-stranded mRNA appeared as helical regions alternating with curly regions to different extents, which might exert the influence on the folding of nascent polypeptide by various regulating effects including different translational rates.

  18. Mutation of genes controlling mRNA metabolism and protein synthesis predisposes to neurodevelopmental disorders.

    Science.gov (United States)

    Sartor, Francesca; Anderson, Jihan; McCaig, Colin; Miedzybrodzka, Zosia; Müller, Berndt

    2015-12-01

    Brain development is a tightly controlled process that depends upon differentiation and function of neurons to allow for the formation of functional neural networks. Mutation of genes encoding structural proteins is well recognized as causal for neurodevelopmental disorders (NDDs). Recent studies have shown that aberrant gene expression can also lead to disorders of neural development. Here we summarize recent evidence implicating in the aetiology of NDDs mutation of factors acting at the level of mRNA splicing, mRNA nuclear export, translation and mRNA degradation. This highlights the importance of these fundamental processes for human health and affords new strategies and targets for therapeutic intervention.

  19. Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

    OpenAIRE

    Chu, Edward; Copur, Sitki M.; Ju, Jingfang; Chen, Tian-men; Khleif, Samir; Voeller, Donna M.; Mizunuma, Nobuyuki; Patel, Mahendra; Maley, Gladys F.; Maley, Frank; Allegra, Carmen J.

    1999-01-01

    A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRN...

  20. mRNA turnover rate limits siRNA and microRNA efficacy

    OpenAIRE

    Larsson, Erik; Sander, Chris; Marks, Debora

    2010-01-01

    What determines how strongly an mRNA responds to a microRNA or an siRNA? We know that properties of the sequence match between the small RNA and the mRNA are crucial. However, large-scale validations of siRNA efficacies have shown that certain transcripts remain recalcitrant to perturbation even after repeated redesign of the siRNA (Krueger et al, 2007). Weak response to RNAi may thus be an inherent property of the mRNA, but the underlying factors have proven difficult to uncover. siRNAs indu...

  1. Nonsense mutations in the human beta-globin gene affect mRNA metabolism.

    OpenAIRE

    Baserga, S J; Benz, E J

    1988-01-01

    A number of premature translation termination mutations (nonsense mutations) have been described in the human alpha- and beta-globin genes. Studies on mRNA isolated from patients with beta zero-thalassemia have shown that for both the beta-17 and the beta-39 mutations less than normal levels of beta-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human beta-globin mRNA.) In vitro studies usi...

  2. Transcript Abundance Explains mRNA Mobility Data in Arabidopsis thaliana.

    Science.gov (United States)

    Calderwood, Alexander; Kopriva, Stanislav; Morris, Richard J

    2016-03-01

    Recently, a large population of mRNA was shown to be able to travel between plant organs via sieve elements as a putative long-distance signaling molecule. However, a mechanistic basis by which transcripts are selected for transport has not yet been identified. Here, we show that experimental mRNA mobility data in Arabidopsis can be explained by transcript abundance and half-life. This suggests that the majority of identified mobile transcripts can be accounted for by non-sequence-specific movement of mRNA from companion cells into sieve elements. PMID:26952566

  3. Increased thymidylate synthase mRNA concentration in blood leukocytes following an experimental stressor

    DEFF Research Database (Denmark)

    Ehrnrooth, Eva; Zacharia, Robert; Svendsen, Gunner;

    2002-01-01

    to a computerized mental stressor; (2) relaxation, and (3) control. Measurements included TS mRNA levels, total leukocyte number, leukocyte subtypes, and serum cortisol before (baseline), immediately after, and 1 h after each experimental condition. RESULTS: While no significant differences were found between...... experimental conditions at baseline in cortisol (p = 0.9) or TS mRNA levels (p = 0.1), significantly higher TS mRNA expression was found immediately after stress compared to pretreatment levels (p cortisol levels indicated an effect of the experimental stressor, with higher cortisol levels...... seen immediately after stress as compared to both relaxation (p cortisol and percentage of lymphocytes and significantly greater decreases...

  4. Tuning protein expression using synonymous codon libraries targeted to the 5' mRNA coding region

    DEFF Research Database (Denmark)

    Goltermann, Lise; Borch Jensen, Martin; Bentin, Thomas

    2011-01-01

    In bacteria, the 5' mRNA coding region plays an important role in determining translation output. Here, we report synthetic sequences that when placed in the 5'-mRNA coding region, leading to recombinant proteins containing short N-terminal extensions, virtually abolish, enhance or produce...... sequence allowed tuning of protein expression over ~300-fold with preservation of amino acid identity. This approach is simple and should be generally applicable in bacteria. The data support that features in the 5' mRNA coding region near the AUG start codon are key in determining translation output...

  5. Mechanisms of RNAi: mRNA cleavage fragments may indicate stalled RISC

    OpenAIRE

    Holen, Torgeir

    2005-01-01

    The molecular mechanism of RNA interference (RNAi) is under intense investigation. We previously demonstrated the existence of inactive siRNAs and also of mRNA cleavage in vivo in human cells. Here it is shown that some siRNAs with low activity leave mRNA cleavage fragments while an siRNA with higher activity does not. The pattern is consistent with both short-term (4-24 hours) and long-term (1-4 days) time-series. Analysis of the putative 3′ mRNA cleavage product showed high GC content immed...

  6. [Factors that alter taste perception].

    Science.gov (United States)

    Maffeis, E R; Silva-Netto, C R

    1990-01-01

    Dysfunction of taste perception is a significant problem for many individuals. Taste anomalies may affect health not only by directly affecting liquid and solid food intake, but also by creating a state of depression due to the loss of an important source of pleasure. Many factors alter taste perception, such as lesions of the oral mucosa, cigarette smoking, radiation, chemotherapy, renal disease, hepatitis, leprosy, hormones, nutrition, use of dentures, medications, and aging. Gum or ice chewing may temporarily help loss of taste. Patients should be encouraged to chew their food thoroughly, alternating the sides of the mouth, or alternating different foods. Unfortunately, in many cases there is no cure for this alteration, and patience is then the only possibility.

  7. Altered states: psychedelics and anesthetics.

    Science.gov (United States)

    Icaza, Eduardo E; Mashour, George A

    2013-12-01

    The psychedelic experience has been reported since antiquity, but there is relatively little known about the underlying neural mechanisms. A recent neuroimaging study on psilocybin revealed a pattern of decreased cerebral blood flow and functional disconnections that is surprisingly similar to that caused by various anesthetics. In this article, the authors review historical examples of psychedelic experiences induced by general anesthetics and then contrast the mechanisms by which these two drug classes generate altered states of consciousness. PMID:24061599

  8. Cholesteryl Ester Transfer Protein (CETP polymorphisms affect mRNA splicing, HDL levels, and sex-dependent cardiovascular risk.

    Directory of Open Access Journals (Sweden)

    Audrey C Papp

    Full Text Available Polymorphisms in and around the Cholesteryl Ester Transfer Protein (CETP gene have been associated with HDL levels, risk for coronary artery disease (CAD, and response to therapy. The mechanism of action of these polymorphisms has yet to be defined. We used mRNA allelic expression and splice isoform measurements in human liver tissues to identify the genetic variants affecting CETP levels. Allelic CETP mRNA expression ratios in 56 human livers were strongly associated with several variants 2.5-7 kb upstream of the transcription start site (e.g., rs247616 p = 6.4 × 10(-5, allele frequency 33%. In addition, a common alternatively spliced CETP isoform lacking exon 9 (Δ9, has been shown to prevent CETP secretion in a dominant-negative manner. The Δ 9 expression ranged from 10 to 48% of total CETP mRNA in 94 livers. Increased formation of this isoform was exclusively associated with an exon 9 polymorphism rs5883-C>T (p = 6.8 × 10(-10 and intron 8 polymorphism rs9930761-T>C (5.6 × 10(-8 (in high linkage disequilibrium with allele frequencies 6-7%. rs9930761 changes a key splicing branch point nucleotide in intron 8, while rs5883 alters an exonic splicing enhancer sequence in exon 9.The effect of these polymorphisms was evaluated in two clinical studies. In the Whitehall II study of 4745 subjects, both rs247616 and rs5883T/rs9930761C were independently associated with increased HDL-C levels in males with similar effect size (rs247616 p = 9.6 × 10(-28 and rs5883 p = 8.6 × 10(-10, adjusted for rs247616. In an independent multiethnic US cohort of hypertensive subjects with CAD (INVEST-GENE, rs5883T/rs9930761C alone were significantly associated with increased incidence of MI, stroke, and all-cause mortality in males (rs5883: OR 2.36 (CI 1.29-4.30, p = 0.005, n = 866. These variants did not reach significance in females in either study. Similar to earlier results linking low CETP activity with poor outcomes in males, our results suggest genetic, sex

  9. Cholesteryl Ester Transfer Protein (CETP) polymorphisms affect mRNA splicing, HDL levels, and sex-dependent cardiovascular risk.

    Science.gov (United States)

    Papp, Audrey C; Pinsonneault, Julia K; Wang, Danxin; Newman, Leslie C; Gong, Yan; Johnson, Julie A; Pepine, Carl J; Kumari, Meena; Hingorani, Aroon D; Talmud, Philippa J; Shah, Sonia; Humphries, Steve E; Sadee, Wolfgang

    2012-01-01

    Polymorphisms in and around the Cholesteryl Ester Transfer Protein (CETP) gene have been associated with HDL levels, risk for coronary artery disease (CAD), and response to therapy. The mechanism of action of these polymorphisms has yet to be defined. We used mRNA allelic expression and splice isoform measurements in human liver tissues to identify the genetic variants affecting CETP levels. Allelic CETP mRNA expression ratios in 56 human livers were strongly associated with several variants 2.5-7 kb upstream of the transcription start site (e.g., rs247616 p = 6.4 × 10(-5), allele frequency 33%). In addition, a common alternatively spliced CETP isoform lacking exon 9 (Δ9), has been shown to prevent CETP secretion in a dominant-negative manner. The Δ 9 expression ranged from 10 to 48% of total CETP mRNA in 94 livers. Increased formation of this isoform was exclusively associated with an exon 9 polymorphism rs5883-C>T (p = 6.8 × 10(-10)) and intron 8 polymorphism rs9930761-T>C (5.6 × 10(-8)) (in high linkage disequilibrium with allele frequencies 6-7%). rs9930761 changes a key splicing branch point nucleotide in intron 8, while rs5883 alters an exonic splicing enhancer sequence in exon 9.The effect of these polymorphisms was evaluated in two clinical studies. In the Whitehall II study of 4745 subjects, both rs247616 and rs5883T/rs9930761C were independently associated with increased HDL-C levels in males with similar effect size (rs247616 p = 9.6 × 10(-28) and rs5883 p = 8.6 × 10(-10), adjusted for rs247616). In an independent multiethnic US cohort of hypertensive subjects with CAD (INVEST-GENE), rs5883T/rs9930761C alone were significantly associated with increased incidence of MI, stroke, and all-cause mortality in males (rs5883: OR 2.36 (CI 1.29-4.30), p = 0.005, n = 866). These variants did not reach significance in females in either study. Similar to earlier results linking low CETP activity with poor outcomes in males, our results suggest genetic, sex

  10. Depletion of three combined THOC5 mRNA export protein target genes synergistically induces human hepatocellular carcinoma cell death.

    Science.gov (United States)

    Saran, S; Tran, D D H; Ewald, F; Koch, A; Hoffmann, A; Koch, M; Nashan, B; Tamura, T

    2016-07-21

    Hepatocellular carcinoma (HCC) is a frequent form of cancer with a poor prognosis and with limited possibilities of medical intervention. It has been shown that over 100 putative driver genes are associated with multiple recurrently altered pathways in HCC, suggesting that multiple pathways will need to be inhibited for any therapeutic method. mRNA processing is regulated by a complex RNA-protein network that is essential for the maintenance of homeostasis. THOC5, a member of mRNA export complex, has a role in less than 1% of mRNA processing, and is required for cell growth and differentiation, but not for cell survival in normal fibroblasts, hepatocytes and macrophages. In this report, we show that 50% depletion of THOC5 in human HCC cell lines Huh7 and HepG2 induced apoptosis. Transcriptome analysis using THOC5-depleted cells revealed that 396 genes, such as transmembrane BAX inhibitor motif containing 4 (TMBIM4), transmembrane emp24-like trafficking protein 10 (Tmed10) and D-tyrosyl-tRNA deacylase 2 (Dtd2) genes were downregulated in both cell lines. The depletion of one of these THOC5 target genes in Huh7 or HepG2 did not significantly induce cell death, suggesting that these may be fine tuners for HCC cell survival. However, the depletion of a combination of these genes synergistically increased the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive HCC. It must be noted that the depletion of these genes did not induce cell death in the hepatocyte cell line, THLE-2 cells. THOC5 expression was enhanced in 78% of cytological differentiation grading G2 and G3 tumor in primary HCC. Furthermore, the expression of a putative glycoprotein, Tmed10, is correlated to THOC5 expression level in primary HCCs, suggesting that this protein may be a novel biomarker for HCC. These data imply that the suppression of the multiple THOC5 target genes may represent a novel strategy for HCC therapy. PMID:26549021

  11. Oral alterations among chemical dependents

    Directory of Open Access Journals (Sweden)

    Estela Vanessa COLODEL

    2009-03-01

    Full Text Available Introduction: It has been daily observed a significant increase ofchemical dependent individuals, as well as the lack of depth on thisissue in the dentistry area. Nevertheless many times the dentalclinicians are the first professionals to diagnose possible alterations,which appear due to the consumption of tobacco, alcohol and other illicit drugs. Objectives: To make a literature review of oral alterations and to identify them on a specific group of persons, which are addicted to different types of drugs. Material and methods: The clinical history of the selected individuals was added to the answers of a questionnaire,comprising the data of the present research. Results: Besides other minor soft tissue alterations, a high prevalence of caries and periodontal diseases were found in the studied population. Conclusion: It was concluded that the role of the dental clinician is very important to the health rehabilitation of drug addicts, individuals with physical and mental disorders that need specific oral care, which sometimes is neglected.

  12. Buccal alterations in diabetes mellitus

    Directory of Open Access Journals (Sweden)

    Negrato Carlos

    2010-01-01

    Full Text Available Abstract Long standing hyperglycaemia besides damaging the kidneys, eyes, nerves, blood vessels, heart, can also impair the function of the salivary glands leading to a reduction in the salivary flow. When salivary flow decreases, as a consequence of an acute hyperglycaemia, many buccal or oral alterations can occur such as: a increased concentration of mucin and glucose; b impaired production and/or action of many antimicrobial factors; c absence of a metalloprotein called gustin, that contains zinc and is responsible for the constant maturation of taste papillae; d bad taste; e oral candidiasis f increased cells exfoliation after contact, because of poor lubrication; g increased proliferation of pathogenic microorganisms; h coated tongue; i halitosis; and many others may occur as a consequence of chronic hyperglycaemia: a tongue alterations, generally a burning mouth; b periodontal disease; c white spots due to demineralization in the teeth; d caries; e delayed healing of wounds; f greater tendency to infections; g lichen planus; h mucosa ulcerations. Buccal alterations found in diabetic patients, although not specific of this disease, have its incidence and progression increased when an inadequate glycaemic control is present.

  13. Severe acute respiratory syndrome coronavirus nsp1 facilitates efficient propagation in cells through a specific translational shutoff of host mRNA.

    Science.gov (United States)

    Tanaka, Tomohisa; Kamitani, Wataru; DeDiego, Marta L; Enjuanes, Luis; Matsuura, Yoshiharu

    2012-10-01

    Severe acute respiratory syndrome (SARS) coronavirus (SCoV) is an enveloped virus containing a single-stranded, positive-sense RNA genome. Nine mRNAs carrying a set of common 5' and 3' untranslated regions (UTR) are synthesized from the incoming viral genomic RNA in cells infected with SCoV. A nonstructural SCoV nsp1 protein causes a severe translational shutoff by binding to the 40S ribosomal subunits. The nsp1-40S ribosome complex further induces an endonucleolytic cleavage near the 5'UTR of host mRNA. However, the mechanism by which SCoV viral proteins are efficiently produced in infected cells in which host protein synthesis is impaired by nsp1 is unknown. In this study, we investigated the role of the viral UTRs in evasion of the nsp1-mediated shutoff. Luciferase activities were significantly suppressed in cells expressing nsp1 together with the mRNA carrying a luciferase gene, while nsp1 failed to suppress luciferase activities of the mRNA flanked by the 5'UTR of SCoV. An RNA-protein binding assay and RNA decay assay revealed that nsp1 bound to stem-loop 1 (SL1) in the 5'UTR of SCoV RNA and that the specific interaction with nsp1 stabilized the mRNA carrying SL1. Furthermore, experiments using an SCoV replicon system showed that the specific interaction enhanced the SCoV replication. The specific interaction of nsp1 with SL1 is an important strategy to facilitate efficient viral gene expression in infected cells, in which nsp1 suppresses host gene expression. Our data indicate a novel mechanism of viral gene expression control by nsp1 and give new insight into understanding the pathogenesis of SARS.

  14. CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage.

    Science.gov (United States)

    Yakhnin, Alexander V; Baker, Carol S; Vakulskas, Christopher A; Yakhnin, Helen; Berezin, Igor; Romeo, Tony; Babitzke, Paul

    2013-02-01

    Csr is a conserved global regulatory system that controls expression of several hundred Escherichia coli genes. CsrA protein represses translation of numerous genes by binding to mRNA and inhibiting ribosome access. CsrA also activates gene expression, although an activation mechanism has not been reported. CsrA activates flhDC expression, encoding the master regulator of flagellum biosynthesis and chemotaxis, by stabilizing the mRNA. Computer modelling, gel mobility shift and footprint analyses identified two CsrA binding sites extending from positions 1-12 (BS1) and 44-55 (BS2) of the 198 nt flhDC leader transcript. flhD'-'lacZ expression was reduced by mutations in csrA and/or the CsrA binding sites. The position of BS1 suggested that bound CsrA might inhibit 5' end-dependent RNase E cleavage of flhDC mRNA. Consistent with this hypothesis, CsrA protected flhDC leader RNA from RNase E cleavage in vitro and protection depended on BS1 and BS2. Primer extension studies identified flhDC decay intermediates in vivo that correspond to in vitro RNase E cleavage sites. Deletion of these RNase E cleavage sites resulted in increased flhD'-'lacZ expression. Data from mRNA decay studies and quantitative primer extension assays support a model in which bound CsrA activates flhDC expression by inhibiting the 5' end-dependent RNase E cleavage pathway. PMID:23305111

  15. Inhibition of GLI1 Expression by Targeting the CRD-BP-GLI1 mRNA Interaction Using a Specific Oligonucleotide.

    Science.gov (United States)

    Mehmood, Kashif; Akhtar, Daud; Mackedenski, Sebastian; Wang, Chuyi; Lee, Chow H

    2016-06-01

    The stabilization of glioma-associated oncogene 1 (GLI1) mRNA by coding region determinant binding protein (CRD-BP) through the Wnt/β-catenin signaling pathway is implicated in the proliferation of colorectal cancer and basal cell carcinoma. Here, we set out to characterize the physical interaction between CRD-BP and GLI1 mRNA so as to find inhibitors for such interaction. Studies using CRD-BP variants with a point mutation in the GXXG motif at each KH domain showed that KH1 and KH2 domain are critical for the binding of GLI1 RNA. The smallest region of GLI1 RNA binding to CRD-BP was mapped to nucleotides (nts) 320-380. A 37-nt S1 RNA sense oligonucleotide, containing two distinct stem-loops present in nts 320-380 of GLI1 RNA, was found to be effective in blocking CRD-BP-GLI1 RNA interaction. Studies using various competitor RNAs with modifications to S1 RNA oligonucleotide further displayed that both the sequences and the structure of the two stem-loops are important for CRD-BP-GLI1 RNA binding. The role of the two-stem-loop motif in influencing CRD-BP-RNA interaction was further investigated in cells. The 2'-O-methyl derivative of the S1 RNA oligonucleotide significantly decreased GLI1, c-myc, and CD44 mRNA levels, in a panel of colon and breast cancer cells. The results from this study demonstrate the potential importance of the two-stem-loop motif as a target region for the inhibition of the CRD-BP-GLI1 RNA interaction and Hedgehog signaling pathway. Such results pave the way for the development of novel inhibitors that act by destabilizing the CRD-BP-GLI1 mRNA interaction. PMID:27036131

  16. Infant formula alters surfactant protein A (SP-A) and SP-B expression in pulmonary epithelial cells.

    Science.gov (United States)

    Chen, Maurice G; Atkins, Constance L; Bruce, Shirley R; Khan, Amir M; Liu, Yuying; Alcorn, Joseph L

    2011-09-01

    Surfactant proteins A (SP-A) and SP-B are critical in the ability of pulmonary surfactant to reduce alveolar surface tension and provide innate immunity. Aspiration of infant milk formula can lead to lung dysfunction, but direct effects of aspirated formula on surfactant protein expression in pulmonary cells have not been described. The hypothesis that infant formula alters surfactant protein homeostasis was tested in vitro by assessing surfactant protein gene expression in cultured pulmonary epithelial cell lines expressing SP-A and SP-B that were transiently exposed (6 hr) to infant formula. Steady-state levels of SP-A protein and mRNA and SP-B mRNA in human bronchiolar (NCI-H441) and mouse alveolar (MLE15) epithelial cells were reduced in a dose-dependent manner 18 hr after exposure to infant formula. SP-A mRNA levels remained reduced 42 hr after exposure, but SP-B mRNA levels increased 10-fold. Neither soy formula nor non-fat dry milk affected steady-state SP-A and SP-B mRNA levels; suggesting a role of a component of infant formula derived from cow milk. These results indicate that infant formula has a direct, dose-dependent effect to reduce surfactant protein gene expression. Ultimately, milk aspiration may potentially result in a reduced capacity of the lung to defend against environmental insults. PMID:21520433

  17. Leishmania donovani infection induces anemia in hamsters by differentially altering erythropoiesis in bone marrow and spleen.

    Directory of Open Access Journals (Sweden)

    William P Lafuse

    Full Text Available Leishmania donovani is a parasite that causes visceral leishmaniasis by infecting and replicating in macrophages of the bone marrow, spleen, and liver. Severe anemia and leucopenia is associated with the disease. Although immune defense mechanisms against the parasite have been studied, we have a limited understanding of how L. donovani alters hematopoiesis. In this study, we used Syrian golden hamsters to investigate effects of L. donovani infection on erythropoiesis. Infection resulted in severe anemia and leucopenia by 8 weeks post-infection. Anemia was associated with increased levels of serum erythropoietin, which indicates the hamsters respond to the anemia by producing erythropoietin. We found that infection also increased numbers of BFU-E and CFU-E progenitor populations in the spleen and bone marrow and differentially altered erythroid gene expression in these organs. In the bone marrow, the mRNA expression of erythroid differentiation genes (α-globin, β-globin, ALAS2 were inhibited by 50%, but mRNA levels of erythroid receptor (c-kit, EpoR and transcription factors (GATA1, GATA2, FOG1 were not affected by the infection. This suggests that infection has a negative effect on differentiation of erythroblasts. In the spleen, erythroid gene expression was enhanced by infection, indicating that the anemia activates a stress erythropoiesis response in the spleen. Analysis of cytokine mRNA levels in spleen and bone marrow found that IFN-γ mRNA is highly increased by L. donovani infection. Expression of the IFN-γ inducible cytokine, TNF-related apoptosis-inducing ligand (TRAIL, was also up-regulated. Since TRAIL induces erythroblasts apoptosis, apoptosis of bone marrow erythroblasts from infected hamsters was examined by flow cytometry. Percentage of erythroblasts that were apoptotic was significantly increased by L. donovani infection. Together, our results suggest that L. donovani infection inhibits erythropoiesis in the bone marrow by

  18. Conformational stability of calreticulin

    DEFF Research Database (Denmark)

    Jørgensen, Charlotte S; Trandum, Christa; Larsen, Nanna Brink;

    2005-01-01

    The conformational stability of calreticulin was investigated. Apparent unfolding temperatures (Tm) increased from 31 degrees C at pH 5 to 51 degrees C at pH 9, but electrophoretic analysis revealed that calreticulin oligomerized instead of unfolding. Structural analyses showed that the single C......-terminal alpha-helix was of major importance to the conformational stability of calreticulin....

  19. Temperature stabilized phase detector

    Science.gov (United States)

    Lo, Y.

    1981-01-01

    The construction, tests, and performance of a temperature stabilized phase detector are discussed. It has a frequency stability of 5 parts in 10 to the 16th power at 100 MHz, with a temperature step of 20 C (15 to 35 C).

  20. Conformational stability of calreticulin

    DEFF Research Database (Denmark)

    Jørgensen, C.S.; Trandum, C.; Larsen, N.;

    2005-01-01

    The conformational stability of calreticulin was investigated. Apparent unfolding temperatures (T-m) increased from 31 degrees C at pH 5 to 51 degrees C at pH 9, but electrophoretic analysis revealed that calreticulin oligomerized instead of unfolding. Structural analyses showed that the single C......-terminal a-helix was of major importance to the conformational stability of calreticulin....

  1. Radion Cosmology and Stabilization

    CERN Document Server

    Chakraborty, Sumanta

    2014-01-01

    We have solved the Einstein equation in 5D spacetime for Randall Sundrum Brane world model with time dependent radion field. We have shown how inflation could compactify the extra dimension such that it become unobservable today. We also propose a mechanism for stabilizing the time dependent radion field following Goldberger Wise mechanism and have obtained the stabilized value.

  2. Stability: general considerations

    International Nuclear Information System (INIS)

    This chapter is concerned with determining the stability properties of ideal MHD equilibria. The problem of stability can be stated qualitatively as follows. The existence of an MHD equilibrium state implies a situation where the sum of the forces acting on the plasma is zero. If the plasma is perturbed from this state, the resulting perturbed forces either restore the plasma to its original equilibrium (stability) or cause a further enhancement of the initial disturbance (instability). Chapter 8 begins with a discussion of a mathematical definition of stability particularly applicable to ideal MHD. Next, a short derivation is presented of the dispersion relation for waves in an infinite homogeneous MHD plasma. These waves play an important role in providing intuition into the dynamical behavior of an MHD plasma. Following this is a rather extensive discussion of the formulation of the general linearized stability problem, starting with the equations of motion and culminating with the development of the Energy Principle, a powerful method for testing stability. Having established the Energy Principle, it is then possible to address the role of plasma compressibility on ideal MHD stability in a general manner. This demonstrates the relationship between the ideal and collisionless MHD stability boundaries. Next, a rather subtle point is investigated concerning whether the region outside the main plasma core is more accurately described by a vacuum or by a relatively cold, but still perfectly conducting, force-free plasma. Finally, a brief discussion is presented of the general classification of ideal MHD instabilities

  3. Stability of charged membranes

    OpenAIRE

    Bensimon, D; David, F.; Leibler, S.; Pumir, A.

    1990-01-01

    The electrostatic contribution to the bending elastic modulus of charged phospholipid bilayers in an ionic solution is computed. It is found to be the same for conducting and non-conducting membranes and is always stabilizing. This stability for free membranes is shown to be a simple consequence of the vanishing of the physical surface tension.

  4. Alteration of biological samples in speciation analysis of metalloproteins.

    Science.gov (United States)

    Wolf, Christian; Wenda, Nadine; Richter, Andrea; Kyriakopoulos, Antonios

    2007-10-01

    For investigations of metalloproteins by speciation analysis, the integrity of the protein-metal complexes before and during separation is crucial. Knowledge about potential alterations of the samples is thus essential to avoid misinterpretations of the analytical results. Chromatographic element profiles of different cytosolic samples from animal tissues were measured repeatedly to estimate the sample stability. The dependence of the signals on the dwell time of the sample in an autosampling device at 4 degrees C for a period of 10 h was observed. Alterations in the element content of different metal-containing fractions were quantified by means of recovery values. Some metalloprotein fractions (e.g. approximately 27-kDa arsenic, approximately 27-kDa iron and different zinc fractions) were stable or only minor alterations were observed and for their investigation an autosampling device is therefore suitable. However, most of the other metalloprotein fractions, especially nickel-containing proteins, showed major alterations: these samples should therefore be analysed immediately after preparation or directly after thawing.

  5. UPF1, a conserved nonsense-mediated mRNA decay factor, regulates cyst wall protein transcripts in Giardia lamblia.

    Directory of Open Access Journals (Sweden)

    Yi-Hsiu Chen

    Full Text Available The Giardia lamblia cyst wall is required for survival outside the host and infection. Three cyst wall protein (cwp genes identified to date are highly up-regulated during encystation. However, little is known of the molecular mechanisms governing their gene regulation. Messenger RNAs containing premature stop codons are rapidly degraded by a nonsense-mediated mRNA decay (NMD system to avoid production of non-functional proteins. In addition to RNA surveillance, NMD also regulates thousands of naturally occurring transcripts through a variety of mechanisms. It is interesting to know the NMD pathway in the primitive eukaryotes. Previously, we have found that the giardial homologue of a conserved NMD factor, UPF1, may be functionally conserved and involved in NMD and in preventing nonsense suppression. In this study, we tested the hypothesis that NMD factors can regulate some naturally occurring transcripts in G. lamblia. We found that overexpression of UPF1 resulted in a significant decrease of the levels of CWP1 and cyst formation and of the endogenous cwp1-3, and myb2 mRNA levels and stability. This indicates that NMD could contribute to the regulation of the cwp1-3 and myb2 transcripts, which are key to G. lamblia differentiation into cyst. Interestingly, we also found that UPF1 may be involved in regulation of eight other endogenous genes, including up-regulation of the translation elongation factor gene, whose product increases translation which is required for NMD. Our results indicate that NMD factor could contribute to the regulation of not only nonsense containing mRNAs, but also mRNAs of the key encystation-induced genes and other endogenous genes in the early-diverging eukaryote, G. lamblia.

  6. The change in thyroid hormone signaling by altered training intensity in male rat skeletal muscle.

    Science.gov (United States)

    Lesmana, Ronny; Iwasaki, Toshiharu; Iizuka, Yuki; Amano, Izuki; Shimokawa, Noriaki; Koibuchi, Noriyuki

    2016-08-31

    Aerobic (sub lactate threshold; sub-LT) exercise training facilitates oxidative phosphorylation and glycolysis of skeletal muscle. Thyroid hormone (TH) also facilitates such metabolic events. Thus, we studied whether TH signaling pathway is activated by treadmill training. Male adult rats received 30 min/day treadmill training with different exercise intensity for 12 days. Then plasma lactate and thyrotropin (TSH) levels were measured. By lactate levels, rats were divided into stationary control (SC, 0 m/min), sub-LT (15 m/min) and supra lactate threshold (supra-LT; 25 m/min) training groups. Immediately after the last training, the soleus muscles were dissected out to measure TH receptor (TR) mRNA and protein expressions. Other rats received intraperitoneal injection of T3, 24 h after the last training and sacrificed 6 h after the injection to measure TH target gene expression. TSH level was suppressed in both sub-LT and supra-LT groups during the exercise. TRβ1 mRNA and protein levels were increased in sub-LT group. Sensitivity to T3 was altered in several TH-target genes by training. Particularly, induction of Na(+)/K(+)-ATPase β1 expression by T3 was significantly augmented in sub-LT group. These results indicate that sub-LT training alters TH signaling at least in part by increasing TRβ1 expression. Such TH signaling alteration may contribute metabolic adaptation in skeletal muscle during physical training.

  7. Correlation of genomic and expression alterations of AS3 with esophageal squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Yu Zhang; Xiaoping Huang; Jun Qi; Cai Yan; Xin Xu; Yaling Han; Mingrong Wang

    2008-01-01

    Androgen-induced proliferation shutoff gene AS3, also known as APRIN, is a growth inhibitory gene that is in itially implicated inprostate cancer. This gene is required for androgen-dependent growth arrest and is a primary target for 1,25(OH)2D3 and androgens. Alle-lic loss at AS3 locus has been linked to a variety of cancers. However, the correlation of genomic and expression alterations of AS3 with esophageal squamous cell carcinoma (ESCC) is not well established. In this study, the genomic and expression alterations of AS3 in ESCC and their clinical significance are evaluated. Loss of beterozygosity (LOH) analysis using an AS3 intragenic mierosatellite marker D13S171 revealed 72% allelic loss at AS3 locus in ESCC, which is significantly correlated with higher pathological grade (P=0.042).RT-PCR examination showed that AS3 mRNA obviously decreased in 44% tumors and its down-regulation was correlated with the sex of patients (P=0.03). Furthermore, the correlation between genomic and expression alterations of AS3 gene was analyzed in 18 ESCC specimens, which indicated that the consistency between allelic loss and decreased mRNA expression of AS3 was relatively poor. The results of this study indicate that the aberrant expression of AS3 may be involved in the tumorigenesis of esophagus and is responsible for the male predominance of ESCC.

  8. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

    Directory of Open Access Journals (Sweden)

    Beatriz M. A. Fontoura

    2013-07-01

    Full Text Available Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

  9. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    International Nuclear Information System (INIS)

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures

  10. Expression of D2 dopamine receptor mRNA in the arterial chemoreceptor afferent pathway.

    Science.gov (United States)

    Czyzyk-Krzeska, M F; Lawson, E E; Millhorn, D E

    1992-11-01

    Dopamine is a major neurotransmitter in the arterial chemoreceptor pathway. In the present study we wished to determine if messenger RNAs for dopamine D1 and D2 receptor are expressed in carotid body (type I cells), in sensory neurons of the petrosal ganglion which innervate the carotid body and in sympathetic neurons of the superior cervical ganglion. We failed to detect D1 receptor mRNA in any of these tissues. However, we found that D2 receptor mRNA was expressed by dopaminergic carotid body type I cells. D2 receptor mRNA was also found in petrosal ganglion neurons that innervated the carotid sinus and carotid body. In addition, a large number of sympathetic postganglionic neurons in the superior cervical ganglion expressed D2 receptor mRNA. PMID:1362730

  11. Visual detection of Akt mRNA in living cell using gold nanoparticle beacon

    Science.gov (United States)

    Ma, Yi; Tian, Caiping; Li, Siwen; Wang, Zhaohui; Gu, Yueqing

    2014-09-01

    PI3K-Akt signaling pathway plays the key role in cell apoptosis and survival, and the components of PI3K /Akt signaling pathway are often abnormally expressed in human tumors. Therefore, determination of the Akt (protein kinase B, PKB) messenger ribonucleic acid (mRNA) expression is significantly important in understanding the mechanism of tumor progression. In this study, we designed a special hairpin deoxyribonucleic acid (DNA) functionalized with gold nanoparticles and fluorescein isothiocyanate(FITC) as a beacon for detecting human Akt mRNA. Spectrofluorometer was used to detect the fluorescence quenching and recovery of the beacons, and laser confocal scanning microscopy was adopted to image Akt mRNA in cells. The results showed that this beacon could sensitively and quantitatively measure the Akt mRNA in living cells . This strategy is potentially useful for the cellular imaging of RNA or protein expression in living cells.

  12. G–quadruplex RNA structure as a signal for neurite mRNA targeting

    OpenAIRE

    Subramanian, Murugan; Rage, Florence; Tabet, Ricardos; Flatter, Eric; Mandel, Jean-Louis; Moine, Hervé

    2011-01-01

    Guanine-quadruplex structures in the 3'-UTR of neuronal mRNAs are shown to function as a neurite mRNA localization signal in mouse cortical neurons in a metabotrobic glutamate receptor-responsive manner.

  13. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M. (Cleveland Clinic Foundation, OH (USA))

    1990-08-01

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.

  14. Mapping interactions between mRNA export factors in living cells.

    Directory of Open Access Journals (Sweden)

    I-Fang Teng

    Full Text Available The TREX complex couples nuclear mRNA processing events with subsequent export to the cytoplasm. TREX also acts as a binding platform for the mRNA export receptor Nxf1. The sites of mRNA transcription and processing within the nucleus have been studied extensively. However, little is known about where TREX assembly takes place and where Nxf1 is recruited to TREX to form the export competent mRNP. Here we have used sensitized emission Förster resonance energy transfer (FRET and fluorescence lifetime imaging (FLIM-FRET, to produce a spatial map in living cells of the sites for the interaction of two TREX subunits, Alyref and Chtop, with Nxf1. Prominent assembly sites for export factors are found in the vicinity of nuclear speckles in regions known to be involved in transcription, splicing and exon junction complex formation highlighting the close coupling of mRNA export with mRNP biogenesis.

  15. Stress-induced alterations in 5-HT1A receptor transcriptional modulators NUDR and Freud-1.

    Science.gov (United States)

    Szewczyk, Bernadeta; Kotarska, Katarzyna; Daigle, Mireille; Misztak, Paulina; Sowa-Kucma, Magdalena; Rafalo, Anna; Curzytek, Katarzyna; Kubera, Marta; Basta-Kaim, Agnieszka; Nowak, Gabriel; Albert, Paul R

    2014-11-01

    The effect of stress on the mRNA and protein level of the 5-HT1A receptor and two of its key transcriptional modulators, NUDR and Freud-1, was examined in the prefrontal cortex (PFC) and hippocampus (Hp) using rodent models: olfactory bulbectomy (OB) and prenatal stress (PS) in male and female rats; chronic mild stress in male rats (CMS) and pregnancy stress. In PFC, CMS induced the most widespread changes, with significant reduction in both mRNA and protein levels of NUDR, 5-HT1A receptor and in Freud-1 mRNA; while in Hp 5-HT1A receptor and Freud-1 protein levels were also decreased. In male, but not female OB rats PFC Freud-1 and 5-HT1A receptor protein levels were reduced, while in Hp 5-HT1A receptor, Freud-1 and NUDR mRNA's but not protein were reduced. In PS rats PFC 5-HT1A receptor protein was reduced more in females than males; while in Hp Freud-1 protein was increased in females. In pregnancy stress, PFC NUDR, Freud-1 and 5-HT1A protein receptor levels were reduced, and in HP 5-HT1A receptor protein levels were also reduced; in HP only NUDR and Freud-1 mRNA levels were reduced. Overall, CMS and stress during pregnancy produced the most salient changes in 5-HT1A receptor and transcription factor expression, suggesting a primary role for altered transcription factor expression in chronic regulation of 5-HT1A receptor expression. By contrast, OB (in males) and PS (in females) produced gender-specific reductions in PFC 5-HT1A receptor protein levels, suggesting a role for post-transcriptional regulation. These and previous data suggest that chronic stress might be a key regulator of NUDR/Freud-1 gene expression.

  16. OX40 mRNA in peripheral blood as a biomarker of acute renal allograft rejection

    Institute of Scientific and Technical Information of China (English)

    WANG Yu-liang; FU Ying-xin; ZHU Zhi-jun; WANG Hui; SHEN Zhong-yang

    2012-01-01

    Background Acute rejection remains an important cause of renal allograft dysfunction and the need for accurate diagnosis is essential to successfully treat transplant recipients.The purpose of this study was to determine the costimulatory molecules OX40 and OX40L messenger RNA (mRNA) levels in peripheral blood mononuclear cells (PBMCs) to predict acute renal transplant rejection.Methods The whole blood samples from 20 recipients with biopsy-confirmed acute rejection (rejection group),20 recipients with stable graft function and normal biopsy results (stable group) after kidney transplantation,and 20 healthy volunteers (control group) were collected.The mRNA levels of OX40 and OX40L were analyzed with TaqMan real-time reverse transcriptase polymerase chain reaction (RT-PCR).The association of OX40 and OX40L mRNA levels with disease severity was investigated.Results There was no significant difference of OX40,OX40L mRNA levels in PBMCs between the stable group and control group (P>0.05).The levels of OX40 and OX40L mRNA were significantly higher in the rejection group than in the control group (P<0.01 and P<0.05,respectively).Non-significantly higher OX40L mRNA and significantly higher OX40 mRNA in PBMCs were observed in subjects in the rejection group compared with the stable group (P >0.05 and P <0.01,respectively).Receiver operating characteristic (ROC) curve analysis demonstrated that OX40 mRNA levels could discriminate recipients who subsequently suffered acute allograft rejection (area under the curve,0.908).OX40 and OX40L mRNA levels did not significantly correlate with serum creatinine levels in the rejection group (P >0.05).Levels of OX40 mRNA after anti-rejection therapy were lower than those at the time of protocol biopsy in the rejection group (P<0.05).Conclusion Our data suggest that measurement of OX40 mRNA levels after transplant might offer a noninvasive means for recognizing recipients at risk of acute renal allograft rejection.

  17. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production

    DEFF Research Database (Denmark)

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent;

    2015-01-01

    . cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation...

  18. Mammalian peptidylglycine alpha-amidating monooxygenase mRNA expression can be modulated by the La autoantigen

    DEFF Research Database (Denmark)

    Brenet, Fabienne; Dussault, Nadège; Borch, Jonas;

    2005-01-01

    interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3' UTR of PAM m...

  19. Altered DNA methylation in leukocytes with trisomy 21.

    Directory of Open Access Journals (Sweden)

    Kristi Kerkel

    2010-11-01

    Full Text Available The primary abnormality in Down syndrome (DS, trisomy 21, is well known; but how this chromosomal gain produces the complex DS phenotype, including immune system defects, is not well understood. We profiled DNA methylation in total peripheral blood leukocytes (PBL and T-lymphocytes from adults with DS and normal controls and found gene-specific abnormalities of CpG methylation in DS, with many of the differentially methylated genes having known or predicted roles in lymphocyte development and function. Validation of the microarray data by bisulfite sequencing and methylation-sensitive Pyrosequencing (MS-Pyroseq confirmed strong differences in methylation (p<0.0001 for each of 8 genes tested: TMEM131, TCF7, CD3Z/CD247, SH3BP2, EIF4E, PLD6, SUMO3, and CPT1B, in DS versus control PBL. In addition, we validated differential methylation of NOD2/CARD15 by bisulfite sequencing in DS versus control T-cells. The differentially methylated genes were found on various autosomes, with no enrichment on chromosome 21. Differences in methylation were generally stable in a given individual, remained significant after adjusting for age, and were not due to altered cell counts. Some but not all of the differentially methylated genes showed different mean mRNA expression in DS versus control PBL; and the altered expression of 5 of these genes, TMEM131, TCF7, CD3Z, NOD2, and NPDC1, was recapitulated by exposing normal lymphocytes to the demethylating drug 5-aza-2'deoxycytidine (5aza-dC plus mitogens. We conclude that altered gene-specific DNA methylation is a recurrent and functionally relevant downstream response to trisomy 21 in human cells.

  20. Plasminogen mRNA induction in the mouse brain after kainate excitation: codistribution with plasminogen activator inhibitor-2 (PAI-2) mRNA.

    Science.gov (United States)

    Sharon, Ronit; Abramovitz, Rene; Miskin, Ruth

    2002-08-15

    Plasminogen (Plg), which can be converted to the active protease plasmin by plasminogen activators, has been previously implicated in brain plasticity and in toxicity inflicted in hippocampal pyramidal neurons by kainate. Here we have localized Plg. mRNA through in situ hybridization in brain cryosections derived from normal adult mice or after kainate injection (i.p.). The results indicated that Plg mRNA was undetectable in the normal brain, but after kainate injection it was induced in neuronal cells in multiple, but specific areas, including layers II-III of the neocortex; the olfactory bulb, anterior olfactory nucleus, and the piriform cortex; the caudate/putamen and accumbens nucleus shell; throughout the amygdaloid complex; and in the CAI/CA3 subfields of the hippocampus. Interestingly, this distribution pattern coincided with what we have recently described for the plasminogen activator inhibitor-2 (PAI-2) mRNA, however differing from that of the plasminogen activator inhibitor-1 (PAI-1) mRNA, as also shown here. These results suggest that enhanced Plg gene expression could be involved in events associated with olfactory, striatal, and limbic structures. Furthermore, because PAI-2 is thought to intracellularly counteract cytotoxic events, our results raise the possibility that PAI-2 can act in the brain as an intracellular neuroprotector against potential plasmin-mediated toxicity.

  1. Fip1 regulates mRNA alternative polyadenylation to promote stem cell self-renewal

    OpenAIRE

    Lackford, Brad; Yao, Chengguo; Charles, Georgette M.; Weng, Lingjie; Zheng, Xiaofeng; Choi, Eun-A; Xie, Xiaohui; Wan, Ji; Xing, Yi; Freudenberg, Johannes M.; Yang, Pengyi; Jothi, Raja; Hu, Guang; Shi, Yongsheng

    2014-01-01

    mRNA alternative polyadenylation (APA) plays a critical role in post-transcriptional gene control and is highly regulated during development and disease. However, the regulatory mechanisms and functional consequences of APA remain poorly understood. Here, we show that an mRNA 3′ processing factor, Fip1, is essential for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Fip1 promotes stem cell maintenance, in part, by activating the ESC-specific APA profiles to ensure the ...

  2. The polyadenylation code: a unified model for the regulation of mRNA alternative polyadenylation*

    OpenAIRE

    Davis, Ryan; Shi, Yongsheng

    2014-01-01

    The majority of eukaryotic genes produce multiple mRNA isoforms with distinct 3′ ends through a process called mRNA alternative polyadenylation (APA). Recent studies have demonstrated that APA is dynamically regulated during development and in response to environmental stimuli. A number of mechanisms have been described for APA regulation. In this review, we attempt to integrate all the known mechanisms into a unified model. This model not only explains most of previous results, but also prov...

  3. Maternally inherited npm2 mRNA is crucial for egg developmental competence in zebrafish.

    Science.gov (United States)

    Bouleau, Aurélien; Desvignes, Thomas; Traverso, Juan Martin; Nguyen, Thaovi; Chesnel, Franck; Fauvel, Christian; Bobe, Julien

    2014-08-01

    The molecular mechanisms underlying and determining egg developmental competence remain poorly understood in vertebrates. Nucleoplasmin (Npm2) is one of the few known maternal effect genes in mammals, but this maternal effect has never been demonstrated in nonmammalian species. A link between developmental competence and the abundance of npm2 maternal mRNA in the egg was previously established using a teleost fish model for egg quality. The importance of maternal npm2 mRNA for egg developmental competence remains unknown in any vertebrate species. In the present study, we aimed to characterize the contribution of npm2 maternal mRNA to early developmental success in zebrafish using a knockdown strategy. We report here the oocyte-specific expression of npm2 and maternal inheritance of npm2 mRNA in zebrafish eggs. The knockdown of the protein translated from this maternal mRNA results in developmental arrest before the onset of epiboly and subsequent embryonic death, a phenotype also observed in embryos lacking zygotic transcription. Npm2 knockdown also results in impaired transcription of the first-wave zygotic genes. Our results show that npm2 is also a maternal effect gene in a nonmammalian vertebrate species and that maternally inherited npm2 mRNA is crucial for egg developmental competence. We also show that de novo protein synthesis from npm2 maternal mRNA is critical for developmental success beyond the blastula stage and required for zygotic genome activation. Finally, our results suggest that npm2 maternal mRNA is an important molecular factor of egg quality in fish and possibly in all vertebrates.

  4. Expression of preproenkephalin mRNA by cultured astrocytes and neurons.

    OpenAIRE

    Vilijn, M H; Vaysse, P J; Zukin, R S; Kessler, J A

    1988-01-01

    Expression of preproenkephalin mRNA by developing glia and neurons was examined in cultures of embryonic and neonatal rat brain. Cultured glia from specific regions of embryonic day 17 and neonatal day 1 rat brain were identified as astrocytes on the basis of both morphology and expression of immunoreactivity for glial fibrillary acidic protein. The level of preproenkephalin mRNA in cultured neonatal hypothalamic astrocytes was comparable to levels present in cultured embryonic striatal and h...

  5. Phosphorylation site analysis of the anti-inflammatory and mRNA destabilizing protein tristetraprolin

    OpenAIRE

    Cao, Heping; Deterding, Leesa J.; Blackshear, Perry J.

    2007-01-01

    Tristetraprolin (TTP) is a member of the CCCH zinc finger proteins and is an anti-inflammatory protein. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. TTP binds to mRNA AU-rich elements with high affinity for UUAUUUAUU nucleotides and causes destabilization of those mRNA molecules. TTP is phosphorylated extensively in vivo and is a substrate for multiple protein kinases in vitro. A number of approaches have been use...

  6. Expression of hepcidin mRNA is uniformly suppressed in hepatocellular carcinoma

    OpenAIRE

    Tomosugi Naohisa; Sawada Tokihiko; Kijima Hiroaki; Kubota Keiichi

    2008-01-01

    Abstract Background The present study evaluated the expression of hepcidin mRNA in hepatocellular carcinoma (HCC). Methods Samples of cancerous and non-cancerous liver tissue were taken from 40 patients with HCC who underwent hepatectomy. Expression of hepcidin mRNA was evaluated by real-time PCR, and compared in tumors differing in their degree of differentiation, number of tumors, and vessel invasion. Correlations between hepcidin expression and the interval until HCC recurrence, and the se...

  7. Upstream AUGs in embryonic proinsulin mRNA control its low translation level

    OpenAIRE

    Hernández-Sánchez, Catalina; Mansilla, Alicia; De La Rosa, Enrique J; Pollerberg, G. Elisabeth; Martínez-Salas, Encarna; Pablo, Flora de

    2003-01-01

    Proinsulin is expressed prior to development of the pancreas and promotes cell survival. Here we study the mechanism affecting the translation efficiency of a specific embryonic proinsulin mRNA. This transcript shares the coding region with the pancreatic form, but presents a 32 nt extended leader region. Translation of proinsulin is markedly reduced by the presence of two upstream AUGs within the 5′ extension of the embryonic mRNA. This attenuation is lost when the two upstream AUGs are muta...

  8. Modified Foxp3 mRNA protects against asthma through an IL-10 dependent mechanism

    OpenAIRE

    Mays, Lauren E.; Ammon-Treiber, Susanne; Mothes, Benedikt; Alkhaled, Mohammed; Rottenberger, Jennifer; Müller-Hermelink, Eva Sophie; Grimm, Melanie; Mezger, Markus; Beer-Hammer, Sandra; von Stebut, Esther; Rieber, Nikolaus; Nürnberg, Bernd; Schwab, Matthias; Handgretinger, Rupert; Idzko, Marco

    2013-01-01

    Chemically modified mRNA is capable of inducing therapeutic levels of protein expression while circumventing the threat of genomic integration often associated with viral vectors. We utilized this novel therapeutic tool to express the regulatory T cell transcription factor, FOXP3, in a time- and site-specific fashion in murine lung, in order to prevent allergic asthma in vivo. We show that modified Foxp3 mRNA rebalanced pulmonary T helper cell responses and protected from allergen-induced tis...

  9. A comparative quantitative assessment of axonal and dendritic mRNA transport in maturing hippocampal neurons.

    Directory of Open Access Journals (Sweden)

    Gunja K Pathak

    Full Text Available Translation of mRNA in axons and dendrites enables a rapid supply of proteins to specific sites of localization within the neuron. Distinct mRNA-containing cargoes, including granules and mitochondrial mRNA, are transported within neuronal projections. The distributions of these cargoes appear to change during neuronal development, but details on the dynamics of mRNA transport during these transitions remain to be elucidated. For this study, we have developed imaging and image processing methods to quantify several transport parameters that can define the dynamics of RNA transport and localization. Using these methods, we characterized the transport of mitochondrial and non-mitochondrial mRNA in differentiated axons and dendrites of cultured hippocampal neurons varying in developmental maturity. Our results suggest differences in the transport profiles of mitochondrial and non-mitochondrial mRNA, and differences in transport parameters at different time points, and between axons and dendrites. Furthermore, within the non-mitochondrial mRNA pool, we observed two distinct populations that differed in their fluorescence intensity and velocity. The net axonal velocity of the brighter pool was highest at day 7 (0.002±0.001 µm/s, mean ± SEM, raising the possibility of a presynaptic requirement for mRNA during early stages of synapse formation. In contrast, the net dendritic velocity of the brighter pool increased steadily as neurons matured, with a significant difference between day 12 (0.0013±0.0006 µm/s and day 4 (-0.003±0.001 µm/s suggesting a postsynaptic role for mRNAs in more mature neurons. The dim population showed similar trends, though velocities were two orders of magnitude higher than of the bright particles. This study provides a baseline for further studies on mRNA transport, and has important implications for the regulation of neuronal plasticity during neuronal development and in response to neuronal injury.

  10. mRNA levels of TLR4 and TLR5 are independent of H pylori

    Institute of Scientific and Technical Information of China (English)

    Elvira Garza-González; Virgilio Bocanegra-García; Francisco Javier Bosques-Padilla; Juan Pablo Flores-Gutiérrez; Francisco Moreno; Guillermo Ignacio Perez-Perez

    2008-01-01

    AIM:To determine if the presence Hpylori or its virulence affect toll-like receptor 4 (TLR4) and TLR5 mRNA expression levels.METHODS:For the in vivo assays,gastric biopsies were obtained from 40 patients and H pylori status was determined.For the in vitro assays,human gastric adenocarcinoma mucosal cells (AGS) were cultured in the presence or absence of twelve selected H pylori strains.H pylori strains isolated from culture-positive patients and selected strains were genotyped for cagA and vacA.The cDNA was obtained from mRNA extracted from biopsies and from infected AGS cells.TLR4 and TLR5 mRNA levels were examined by real-time PCR.RESULTS:The presence of Hpylori did not affect the mRNA levels of TLR4 or TLR5 in gastric biopsies.The mRNA levels of both receptors were not influenced by the vacA status (P>0.05 for both receptors) and there were no differences in TLR4 or TLR5 mRNA levels among the different clinical presentations/histological findings (P>0.05).In the in vitro assay,the mRNA levels of TLR4 or TLR5 in AGS cells were not influenced by the vacAsl status or the clinical condition associated with the strains (P>0.05 for both TLR4 and TLR5).CONCLUSION:The results of this study show that the mRNA levels of TLR4 and TLR5 in gastric cells,both in vivo and in vitro,are independent of H pylori colonization and suggest that vacA may not be a significant player in the first step of innate immune recognition mediated by TLR4 or TLR5.

  11. mRNA levels of TLR4 and TLR5 are independent of H pylori

    Science.gov (United States)

    Garza-González, Elvira; Bocanegra-García, Virgilio; Bosques-Padilla, Francisco Javier; Flores-Gutiérrez, Juan Pablo; Moreno, Francisco; Perez-Perez, Guillermo Ignacio

    2008-01-01

    AIM: To determine if the presence H pylori or its virulence affect toll-like receptor 4 (TLR4) and TLR5 mRNA expression levels. METHODS: For the in vivo assays, gastric biopsies were obtained from 40 patients and H pylori status was determined. For the in vitro assays, human gastric adenocarcinoma mucosal cells (AGS) were cultured in the presence or absence of twelve selected H pylori strains. H pylori strains isolated from culture-positive patients and selected strains were genotyped for cagA and vacA. The cDNA was obtained from mRNA extracted from biopsies and from infected AGS cells. TLR4 and TLR5 mRNA levels were examined by real-time PCR. RESULTS: The presence of H pylori did not affect the mRNA levels of TLR4 or TLR5 in gastric biopsies. The mRNA levels of both receptors were not influenced by the vacA status (P > 0.05 for both receptors) and there were no differences in TLR4 or TLR5 mRNA levels among the different clinical presentations/histological findings (P > 0.05). In the in vitro assay, the mRNA levels of TLR4 or TLR5 in AGS cells were not influenced by the vacAs1 status or the clinical condition associated with the strains (P > 0.05 for both TLR4 and TLR5). CONCLUSION: The results of this study show that the mRNA levels of TLR4 and TLR5 in gastric cells, both in vivo and in vitro, are independent of H pylori colonization and suggest that vacA may not be a significant player in the first step of innate immune recognition mediated by TLR4 or TLR5. PMID:18785283

  12. Changes in mRNA for metabotropic glutamate receptors after transient cerebral ischaemia

    DEFF Research Database (Denmark)

    Rosdahl, D; Seitzberg, D A; Christensen, Thomas;

    1994-01-01

    Using a rat 4-vessel occlusion model of cerebral ischaemia we studied the changes in the mRNA level for the metabotropic receptor subtypes mGluR1 alpha, mGluR1 beta, mGluR2, mGluR3, mGluR4, and mGluR5 by means of in situ hybridization with oligonucleotides. After 24 hours of reperfusion the mRNA ...

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

    Science.gov (United States)

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

    2015-11-01

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

  14. Expression of local renin and angiotensinogen mRNA in cirrhotic portal hypertensive patient

    Institute of Scientific and Technical Information of China (English)

    Li Zhang; Zhen Yang; Bao-Min Shi; Da-Peng Li; Chong-Yun Fang; Fa-Zu Qiu

    2003-01-01

    AIM: To investigate the expression of local renin and angiotensinogen mRNA in cirrhotic portal hypertensive patients.METHODS: The expression of local renin and angiotensinogen mRNA in the liver, splenic artery and vein of PH patients was detected by RT-PCR analysis.RESULTS: Expression of local renin mRNA in the liver of control group was (0.19±0.11), significantly lower than that in splenic artery(0.45±0.17)or splenic vein(0.39±0.12)respectively, (P<0.05). Expression of local angiotensinogen mRNA in the liver was (0.64±0.21), significantly higher than that in splenic artery(0.41±0.15) or in splenic vein (0.35±0.18)respectively, (P<0.05). Expression of local renin mRNA in the liver, splenic artery and vein of PH group was (0.78±0.28),(0.86±0.35) and (0.81±0.22) respectively, significantly higher than that in the control group, (P<0.05). Expression of local angiotensinogen mRNA in the liver, splenic artery and vein of PH group was (0.96±0.25), (0.83±0.18) and (0.79±0.23)respectively, significantly higher than that in the control group,(P<0.05). There was no significant difference between the liver, splenic artery and vein in the expression of local renin or local angiotensinogen mRNA in PH group, (P<0.05).CONCLUSION: In normal subjects the expression of local renin and angiotensinogen mRNA was organ specific, but with increase of the expression of LRAS, the organ-specificity became lost in cirrhotic patients. LRAS may contribute to increased resistance of portal vein with liver and formation of splanchnic vasculopathy.

  15. Methylation Inactivation Mechanism of Parkin Gene mRNA Expression in Nasopharyngeal Carcinoma

    Institute of Scientific and Technical Information of China (English)

    Ni Haifeng; Jiang Bo; Zhou Zhen; Li Yong; Huang Guangwu

    2014-01-01

    Objective:To investigate the methylation inactivation and the clinical signiifcance of Parkin gene mRNA expression in nasopharyngeal carcinoma (NPC). Methods: The methylation-speciifc PCR (MSP) and semi-quantitative reverse transcription PCR (RT-PCR) were used to detect methylation and the mRNA expression level of Parkin gene in 54 cases of NPC tissues and 16 cases of normal nasopharyngeal epithelial (NNE) tissues.The mRNA expression of Parkin gene in two NPC cell lines (CNE1 and CNE2) were detected before and after treatment with the methyltransferase inhibitor 5-aza-2-deoxycytidine so as to analyze the effects of Parkin gene methylation and demethylation on Parkin gene mRNA expression and the relationship between Parkin gene mRNA expression and clinical factors. Results: The methylation frequency of Parkin gene in human NPC tissues was 62.96% (34/54), but didn’t happen in any of 16 cases of NNE tissues. The mRNA expression level was (0.3430±0.4947) in 54 cases of NPC tissues and (1.0052±0.4911) in NNE tissues, showing that the mRNA expression level of NPC tissues was significantly down-regulated (P0.05), but was closely related to lymph node metastasis (P<0.05). Conclusion:Parkin gene mRNA expression, serving as a cancer suppressor gene in the occurrence and development of NPC, is inactivated and regulated by methylation, which also has a negative correlation with lymph node metastasis and could be considered as the judgment of predictive index of clinical prognosis of NPC.

  16. Stability of cocaine impurity profiles during 12 months of storage

    DEFF Research Database (Denmark)

    Nielsen, Louise Stride; Villesen, Palle; Lindholst, Christian

    2016-01-01

    During the lifetime of a cocaine batch from production end to consumption, several alterations may occur, leading to possible changes in the original impurity profile. Such profile changes may eventually result in erroneous forensic evaluations. In the present study, the stability of both...

  17. Alterations of ultraviolet irradiated DNA

    International Nuclear Information System (INIS)

    Thymine dimers production has been studied in several DNA-3H irradiated at various wave lenght of U.V. Light. The influence of dimers on the hydrodynamic and optic properties, thermal structural stability and transformant capacity of DNA have been studied too. At last the recognition and excision of dimers by the DNA-UV-Endonuclease and DNA-Polimerase-I was also studied. (author)

  18. Effect of α-Hederin on IL-2 and IL-17 mRNA and miRNA-133a Levels in Lungs of Ovalbumin-Sensitized Male Rats.

    Science.gov (United States)

    Ebrahimi, Hadi; Fallahi, Maryam; Khamaneh, Amir Mahdi; Ebrahimi Saadatlou, Mohammad Ali; Saadat, Saeideh; Keyhanmanesh, Rana

    2016-03-01

    α-hederin, a saponin that is a major constituent of English Ivy (Hedera helix) is effective in the treatment of asthma. In the present study, the effect of α-hederin on lung tissue pathology and the levels of the inflammatory mediators; IL-2 mRNA, IL-17 mRNA, and MicroRNAs (miRNA)-133a was evaluated in a rat ovalbumin (OVA)-sensitized model of asthma. Rats were divided randomly into control (C), OVA-sensitized (S), OVA-sensitized pretreated with the antioxidant, thymoquinone (3 mg/kg, S + TQ) or OVA-sensitized pretreated with α-hederin (0.02 mg/kg, S + AH) groups. Levels of IL-2 and IL-17 mRNA were higher in the OVA-sensitized group than controls while the level of miRNA-133a gene expression was lower. IL-2 mRNA and miRNA-133a gene expression in the S + TQ group was higher than in the control and OVA-sensitized groups while the level of IL-17 mRNA in the S + TQ group was lower than in the OVA-sensitized group. Pretreatment with α-hederin decreased IL-17 mRNA levels and increased miRNA-133a gene expression compared with OVA-sensitized animals. All pathological changes in pretreated groups were lower than the OVA-sensitized group. These results showed a beneficial effect of α-hederin in OVA-sensitized rats, suggesting that α-hederin affects the IL-2 and IL-17 secretion pathways, altering miRNA-133a expression. PMID:26865286

  19. Characterization of 4-HNE modified L-FABP reveals alterations in structural and functional dynamics.

    Directory of Open Access Journals (Sweden)

    Rebecca L Smathers

    Full Text Available 4-Hydroxynonenal (4-HNE is a reactive α,β-unsaturated aldehyde produced during oxidative stress and subsequent lipid peroxidation of polyunsaturated fatty acids. The reactivity of 4-HNE towards DNA and nucleophilic amino acids has been well established. In this report, using proteomic approaches, liver fatty acid-binding protein (L-FABP is identified as a target for modification by 4-HNE. This lipid binding protein mediates the uptake and trafficking of hydrophobic ligands throughout cellular compartments. Ethanol caused a significant decrease in L-FABP protein (P<0.001 and mRNA (P<0.05, as well as increased poly-ubiquitinated L-FABP (P<0.001. Sites of 4-HNE adduction on mouse recombinant L-FABP were mapped using MALDI-TOF/TOF mass spectrometry on apo (Lys57 and Cys69 and holo (Lys6, Lys31, His43, Lys46, Lys57 and Cys69 L-FABP. The impact of 4-HNE adduction was found to occur in a concentration-dependent manner; affinity for the fluorescent ligand, anilinonaphthalene-8-sulfonic acid, was reduced from 0.347 µM to Kd(1 = 0.395 µM and Kd(2 = 34.20 µM. Saturation analyses revealed that capacity for ligand is reduced by approximately 50% when adducted by 4-HNE. Thermal stability curves of apo L-FABP was also found to be significantly affected by 4-HNE adduction (ΔTm = 5.44°C, P<0.01. Computational-based molecular modeling simulations of adducted protein revealed minor conformational changes in global protein structure of apo and holo L-FABP while more apparent differences were observed within the internal binding pocket, revealing reduced area and structural integrity. New solvent accessible portals on the periphery of the protein were observed following 4-HNE modification in both the apo and holo state, suggesting an adaptive response to carbonylation. The results from this study detail the dynamic process associated with L-FABP modification by 4-HNE and provide insight as to how alterations in structural integrity and ligand

  20. Drosophila glutamate receptor mRNA expression and mRNP particles.

    Science.gov (United States)

    Ganesan, Subhashree; Karr, Julie E; Featherstone, David E

    2011-01-01

    The processes controlling glutamate receptor expression early in synaptogenesis are poorly understood. Here, we examine glutamate receptor (GluR) subunit mRNA expression and localization in Drosophila embryonic/larval neuromuscular junctions (NMJs). We show that postsynaptic GluR subunit gene expression is triggered by contact from the presynaptic nerve, approximately halfway through embryogenesis. After contact, GluRIIA and GluRIIB mRNA abundance rises quickly approximately 20-fold, then falls within a few hours back to very low levels. Protein abundance, however, gradually increases throughout development. At the same time that mRNA levels decrease following their initial spike, GluRIIA, GluRIIB, and GluRIIC subunit mRNA aggregates become visible in the cytoplasm of postsynaptic muscle cells. These mRNA aggregates do not colocalize with eIF4E, but nevertheless presumably represent mRNP particles of unknown function. Multiplex FISH shows that different GluR subunit mRNAs are found in different mRNPs. GluRIIC mRNPs are most common, followed by GluRIIA and then GluRIIB mRNPs. GluR mRNP density is not increased near NMJs, for any subunit; if anything, GluR mRNP density is highest away from NMJs and near nuclei. These results reveal some of the earliest events in postsynaptic development and provide a foundation for future studies of GluR mRNA biology.