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

    Science.gov (United States)

    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

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

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    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.

  4. 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.

  5. 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.

  6. Differential regulation of plastid mRNA stability. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Stern, D.B.

    1993-09-01

    Our goal is to identify cis-acting sequences and transacting factors that function in plastid mRNA maturation, stabilization, and/or decay through an in vitro and in vivo analysis of mRNA:protein interactions. Our previous results emphasized the study of 3{prime}end inverted repeat sequences (IRs) that serve both as mRNA processing elements and stability determinants, and associate with plastid proteins that potentially play enzymatic, structural and/or regulatory roles. We seek to define, by single base and internal deletion mutagenesis, the sequence and structural requirements for protein binding to the 3{prime} IRs of petD and psbA mRNAs; to purify RNA-binding proteins that demonstrate gene- or sequence-specific binding, or that are implicated in RNA stabilization or decay; and to investigate the native form of mRNA in the plastid, by attempting to purify ribonucleoprotein (RNP) particles from organelles. Our view of mRNA decay is that it is regulated by three interactive components: RNA structure, ribonucleases and RNA-binding proteins. We have used mutagenesis to study the role of RNA structure in regulating RNA decay rates, and to identify protein binding and endonuclease recognition sites. We have identified at least three endonuclease activities; one that cleaves psbA RNA; and two whose cleavage patterns with petD 3{prime} IR-RNA has been studied (endoC1 and endoC2). Additionally, we have continued to analyze the properties of the major RNA processing exoribonuclease. We have concentrated our efforts on three RNA-binding proteins. A 100 kd protein with properties suggestive of a mammalian RNP component has been purified. A protein of 55 kd that may also be an endonuclease has been partially purified. We have studied the interaction of a 29 kd protein with the petD stem/loop, and its role in RNA processing. Recently, we have used a novel gel shift/SDS-PAGE technique to identify new RNA-binding proteins.

  7. Rhythmic control of mRNA stability modulates circadian amplitude of mouse Period3 mRNA.

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    Kim, Sung-Hoon; Lee, Kyung-Ha; Kim, Do-Yeon; Kwak, Eunyee; Kim, Seunghwan; Kim, Kyong-Tai

    2015-03-01

    The daily oscillations observed in most living organisms are endogenously generated with a period of 24 h, and the underlying structure of periodic oscillation is an autoregulatory transcription-translation feedback loop. The mechanisms of untranslated region (UTR)-mediated post-transcriptional regulation (e.g., mRNA degradation and internal ribosomal entry site (IRES)-mediated translation) have been suggested to fine-tune the expression of clock genes. Mouse Period3 (mPer3) is one of the paralogs of Period gene and its function is important in peripheral clocks and sleep physiology. mPer3 mRNA displays a circadian oscillation as well as a circadian phase-dependent stability, while the stability regulators still remain unknown. In this study, we identify three proteins - heterogeneous nuclear ribonucleoprotein (hnRNP) K, polypyrimidine tract-binding protein (PTB), and hnRNP D - that bind to mPer3 mRNA 3'-UTR. We show that hnRNP K is a stabilizer that increases the amplitude of circadian mPer3 mRNA oscillation and hnRNP D is a destabilizer that decreases it, while PTB exhibits no effect on mPer3 mRNA expression. Our experiments describe their cytoplasmic roles for the mRNA stability regulation and the circadian amplitude formation. Moreover, our mathematical model suggests a mechanism through which post-transcriptional mRNA stability modulation provides not only the flexibility of oscillation amplitude, but also the robustness of the period and the phase for circadian mPer3 expression. Mouse Period3 (mPer3) is one of well-known clock genes. We identified three 3'-UTR-binding proteins that modulate the mRNA stability, and they influenced to the amplitude of circadian mPer3 mRNA oscillation. Our mathematical model not only showed the relationship between mRNA stability and its oscillation profile but provided the molecular mechanism for the robustness of the period and the phase in circadian oscillation. hnK, heterogeneous nuclear ribonucleoprotein (hnRNP) K; hnD, hn

  8. A MYLK variant regulates asthmatic inflammation via alterations in mRNA secondary structure.

    Science.gov (United States)

    Wang, Ting; Zhou, Tong; Saadat, Laleh; Garcia, Joe G N

    2015-06-01

    Myosin light-chain kinase (MYLK) is a gene known to be significantly associated with severe asthma in African Americans. Here we further examine the molecular function of a single-nucleotide polymorphism (SNP), located in the non-muscle myosin light-chain kinase isoform (nmMLCK), in asthma susceptibility and pathobiology. We identified nmMLCK variant (reference SNP: rs9840993, NM_053025: 721C>T, c.439C>T) with a distinct mRNA secondary structure from the other variants. The nmMLCK variant (721C) secondary structure exhibits increased stability with an elongated half-life in the human endothelial cell, and greater efficiency in protein translation initiation owing to an increased accessibility to translation start site. Finally, nmMLCK expression of 721C- and 721T-containing MYLK transgenes were compared in nmMLCK(-/-) mice and confirmed deleterious effects of nmMLCK expression on asthmatic indices and implicated the augmented influence of MYLK 721C>T (c.439C>T) SNP on asthma severity. The confirmation of the novel mechanism of the regulation of asthmatic inflammation by a MYLK advances knowledge of the genetic basis for asthma disparities, and further suggests the potential of nmMLCK as a therapeutic target. Our study suggests that in addition to altering protein structure and function, non-synonymous SNPs may also lead to phenotypic disparity by altering protein expression. PMID:25271083

  9. Postural Stability is Altered by Blood Shift

    Science.gov (United States)

    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.

  10. 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...

  11. 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.

  12. 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.

  13. 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.

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

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    Karlsson, Oskar; Segerström, Lova; Sjöback, Robert; Nylander, Ingrid; Borén, Mats

    2016-03-01

    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. PMID:27077049

  15. 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.

  16. mRNA Stability and Polysome Loss in Hibernating Arctic Ground Squirrels (Spermophilus parryii)

    OpenAIRE

    Knight, Jason E.; Narus, Erin Nicol; Martin, Sandra L.; JACOBSON, ALLAN; Barnes, Brian M.; Boyer, Bert B.

    2000-01-01

    All small mammalian hibernators periodically rewarm from torpor to high, euthermic body temperatures for brief intervals throughout the hibernating season. The functional significance of these arousal episodes is unknown, but one suggestion is that rewarming may be related to replacement of gene products lost during torpor due to degradation of mRNA. To assess the stability of mRNA as a function of the hibernation state, we examined the poly(A) tail lengths of liver mRNA from arctic ground sq...

  17. Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Lazarus, Kyren A. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122 (Australia); Zhao, Zhe; Knower, Kevin C. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); To, Sarah Q. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia); Chand, Ashwini L. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Clyne, Colin D., E-mail: Colin.clyne@princehenrys.org [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia)

    2013-08-30

    Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.

  18. Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

    International Nuclear Information System (INIS)

    Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E2), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E2, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E2 treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer

  19. Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4.

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    Borchardt, Erin K; Vandoros, Leonidas A; Huang, Michael; Lackey, Patrick E; Marzluff, William F; Asokan, Aravind

    2015-11-01

    The bacterial CRISPR endoribonuclease Csy4 has recently been described as a potential RNA processing tool. Csy4 recognizes substrate RNA through a specific 28-nt hairpin sequence and cleaves at the 3' end of the stem. To further explore applicability in mammalian cells, we introduced this hairpin at various locations in mRNAs derived from reporter transgenes and systematically evaluated the effects of Csy4-mediated processing on transgene expression. Placing the hairpin in the 5' UTR or immediately after the start codon resulted in efficient degradation of target mRNA by Csy4 and knockdown of transgene expression by 20- to 40-fold. When the hairpin was incorporated in the 3' UTR prior to the poly(A) signal, the mRNA was cleaved, but only a modest decrease in transgene expression (∼2.5-fold) was observed. In the absence of a poly(A) tail, Csy4 rescued the target mRNA substrate from degradation, resulting in protein expression, which suggests that the cleaved mRNA was successfully translated. In contrast, neither catalytically inactive (H29A) nor binding-deficient (R115A/R119A) Csy4 mutants were able to exert any of the effects described above. Generation of a similar 3' end by RNase P-mediated cleavage was unable to rescue transgene expression independent of Csy4. These results support the idea that the selective generation of the Csy4/hairpin complex resulting from cleavage of target mRNA might serve as a functional poly(A)/poly(A) binding protein (PABP) surrogate, stabilizing the mRNA and supporting translation. Although the exact mechanism(s) remain to be determined, our studies expand the potential utility of CRISPR nucleases as tools for controlling mRNA stability and translation. PMID:26354771

  20. 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

  1. 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

  2. ZMS regulation of M2 muscarinic receptor mRNA stability requires protein factor

    International Nuclear Information System (INIS)

    Aim The aim of this work is to study the elevation mechanism of ZMS on muscarinic M2 receptor mRNA expression. Methods Actinomycin D was added to cultured CHOm2 cells to stop the de novo synthesis of M2 receptor mRNA and samples were taken at various times to determine the time course of mRNA of M2 receptor with real-time quantitative RT-PCR. Half-life of M2 receptor mRNA and the effect of ZMS on the half-life was obtained from the slope of the exponential curves. Cycloheximide was added at 4 h prior to and 24 h after the addition of ZMS to examine the effect of de novo protein synthesis on the action of ZMS. Results The half-life of m2 mRNA was prolonged by ZMS treatment without cycloheximide (4.75±0.54 h and 2.13 h±0.23 h for ZMS and vehicle treated groups, respectively, P<0.05). When cycloheximide was added to the culture medium 4h prior to the addition of ZMS, the effect of ZMS in prolonging the half-life of m2 mRNA disappeared (3.06 h±0.23 h and 3.00 h±l.20 h for cells with and without ZMS, respectively). However, when the ZMS was added to the medium 24h prior to the addition of cycloheximide, the action of ZMS was not abolished by cycloheximide (half-life was 5.43 h±1.13 h and 2.46 h±0.09 h for cells with and without ZMS, respectively). Conclusion These data suggest that de novo protein synthesis was required for the increase in M2 mRNA stability induced by ZMS. (authors)

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

    Science.gov (United States)

    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

  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. Time Course of Behavioral Alteration and mRNA Levels of Neurotrophic Factor Following Stress Exposure in Mouse.

    Science.gov (United States)

    Hashikawa, Naoya; Ogawa, Takumi; Sakamoto, Yusuke; Ogawa, Mami; Matsuo, Yumi; Zamami, Yoshito; Hashikawa-Hobara, Narumi

    2015-08-01

    Stress is known to affect neurotrophic factor expression, which induces depression-like behavior. However, whether there are time-dependent changes in neurotrophic factor mRNA expression following stress remains unclear. In the present study, we tested whether chronic stress exposure induces long-term changes in depression-related behavior, serum corticosterone, and hippocampal proliferation as well as neurotrophic factor family mRNA levels, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and ciliary neurotrophic factor (CNTF), in the mouse hippocampus. The mRNA level of neurotrophic factors (BDNF, NGF, NT-3, and CNTF) was measured using the real-time PCR. The serum corticosterone level was evaluated by enzyme-linked immunosorbent assay, and, for each subject, the hippocampal proliferation was examined by 5-bromo-2-deoxyuridine immunostaining. Mice exhibited depression-like behavior in the forced-swim test (FST) and decreased BDNF mRNA and hippocampal proliferation in the middle of the stress exposure. After 15 days of stress exposure, we observed increased immobility in the FST, serum corticosterone levels, and BDNF mRNA levels and degenerated hippocampal proliferation, maintained for at least 2 weeks. Anhedonia-like behavior in the sucrose preference test and NGF mRNA levels were decreased following 15 days of stress. NGF mRNA levels were significantly higher 1 week after stress exposure. The current data demonstrate that chronic stress exposure induces prolonged BDNF and NGF mRNA changes and increases corticosterone levels and depression-like behavior in the FST, but does not alter other neurotrophic factors or performance in the sucrose preference test. PMID:25820756

  6. 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

  7. 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

  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. Continuous presence of phorbol ester is required for its IL-1 beta mRNA stabilizing effect.

    Science.gov (United States)

    Siljander, P; Hurme, M

    1993-01-01

    The protein kinase C (PKC) activating phorbol esters are known to prevent the decay of mRNA of several cytokines and proto-oncogenes. To examine whether the phorbol ester signal is continuously required for this stabilizing effect, THP-1 monocytic cells were stimulated either with phorbol 12,13-dibutyrate (PDBu), which can be removed from the cells by washings, or with the more hydrophobic phorbol 12-myristate 13-acetate (PMA). Both of these stimuli induced high levels of interleukin-1 beta (IL-1 beta) mRNA. When the cells were washed at the peak of the IL-1 beta mRNA expression, this mRNA decayed rapidly in the PDBu stimulated cells while in PMA stimulated cells the mRNA levels were not affected. Moreover, this mRNA degradation induced by the removal of PDBu could be inhibited by readdition of the phorbol ester. This restabilization could be prevented by pharmacologic inhibitors of PKC, but not by inhibiting protein or RNA synthesis. Thus these data suggest that the phorbol ester must be continuously present to exert its mRNA stabilizing effect and that its effect is PKC-mediated but does not require active protein or RNA synthesis. PMID:8416817

  10. 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.

  11. 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.

  12. Stability of the gel formed during nuclear glass alteration

    International Nuclear Information System (INIS)

    This thesis is divided in two parts. The first part is dedicated to the study of the protective properties of the gels formed during the alteration of nuclear glass and the second part the stability of these gels after drying and the effect of the sulfate ions are studied. The protective properties of the gel of SON68 glass are assessed at different alteration progress and related to their morphology analyzed by X-ray reflectometry and gas adsorption. Initially a depleted glass layer is obtained, followed by the formation of a gel with an open porosity. Subsequently the gel densifies as the glass alteration rate decreases and finally the pore size increases upon the formation of a dense and thin zone within the film on the glass surface. The increase of the gradient density zone could explain the increase of the protective properties of the gel. A high reaction progress, when the thin and dense zone within the gel is formed, the protective properties are constant. Various simplified glasses are also studied. At high reaction progress there is a dense zone within the gel. The location of this zone depends on the glass composition which control the competition between the species diffusion in solution and their recondensation. The gel morphology, the protective properties, its stability and the glass composition are correlated. The drying induces some modifications of the morphology of the SON68 glass alteration layer (density, thickness and porosity) and of the mechanical properties (cracks, adhesion on glass). For protective gels these modifications are limited and don't raise the protective properties of the gel. The effect of the sulfate ions in a closed system is essentially to complex calcium. For sulfate ions concentrations in geological repository, the stability of the gel is not raised. (author)

  13. Function of plastid mRNA 3' inverted repeats. RNA stabilization and gene-specific protein binding

    International Nuclear Information System (INIS)

    Plastid protein coding regions in plants are generally flanked by 3' inverted repeat (IR) sequences. In a previous work, we have shown that their role may be in RNA stabilization and as a processing signal that establishes the mature mRNA 3' end. In this report we have investigated the stability and protein interaction of chloroplast mRNA 3' IR-RNA sequences in more detail. Progressive deletions into the 3' IR-RNA sequences for the chloroplast cytochrome b6/f subunit IV (petD) mRNA reduce the stability of the RNA, indicating that the potential to form a stem/loop is a minimum requirement for petD 3' IR-RNA stability in vitro. Specific point mutants also destabilize the processed 3' IR-RNA, suggesting an important role for the primary sequence. Gel mobility shift and UV-cross-linking analysis has shown that 3' IR-RNAs of petD and two other chloroplast mRNAs (rbcL and psbA) interact with proteins in vitro. Comparison of the bound petD 3' IR-RNA proteins with proteins that bind to rbcL and psbA reveals that binding of certain proteins is gene-specific. Also, precursor and processed petD 3' IR-RNAs bind different sets of proteins. A single nucleotide transversion (T----A) near the base of the stem eliminates the binding of a 29-kDa protein to the petD 3' IR-RNA precursor. We discuss the possible role of 3' IR-RNA-protein interactions in plastid mRNA 3' end maturation and differential mRNA stability

  14. 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...

  15. 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...

  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 U1 small nuclear ribonucleoprotein particle with altered specificity induces alternative splicing of an adenovirus E1A mRNA precursor.

    OpenAIRE

    Yuo, C Y; Weiner, A.M.

    1989-01-01

    We have altered the specificity of U1 small nuclear RNA by replacing its 5' splice site recognition sequence (nucleotides 3 to 11) with sequences complementary to other regions of either the adenovirus E1A or the rabbit beta-globin mRNA precursor. We then used a HeLa cell transient expression assay to test whether such altered U1 small nuclear ribonucleoprotein particles (snRNPs) could interfere with splicing of the targeted mRNA precursors. The altered U1 snRNPs were able to cause novel spli...

  18. 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....

  19. mRNA stability and the unfolding of gene expression in the long-period yeast metabolic cycle

    Directory of Open Access Journals (Sweden)

    Farina Lorenzo

    2009-02-01

    Full Text Available Abstract Background In yeast, genome-wide periodic patterns associated with energy-metabolic oscillations have been shown recently for both short (approx. 40 min and long (approx. 300 min periods. Results The dynamical regulation due to mRNA stability is found to be an important aspect of the genome-wide coordination of the long-period yeast metabolic cycle. It is shown that for periodic genes, arranged in classes according either to expression profile or to function, the pulses of mRNA abundance have phase and width which are directly proportional to the corresponding turnover rates. Conclusion The cascade of events occurring during the yeast metabolic cycle (and their correlation with mRNA turnover reflects to a large extent the gene expression program observable in other dynamical contexts such as the response to stresses/stimuli.

  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. 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

  2. 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...

  3. 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

  4. 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

  5. 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

  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. 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

    BACKGROUND: The 3' untranslated region (UTR) of p53 mRNA contains two conserved U-rich sequences resembling cytoplasmic polyadenylation elements (CPE). It is not known if these sequences regulate p53 expression by post-transcriptional mechanisms. MATERIALS AND METHODS: Stable p53 3'UTR reporter Ha......-type p53 3'UTR reduced mRNA steady state levels of the reporter gene and point mutations in the CPEs rescued the mRNA steady state levels in the MCF-7 cells, but not in the HaCaT cells. In both cell lines, the CPEs had a significant effect on translation of the reporter and influenced the effect of UV...... 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...

  8. 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...

  9. Stabilization of Dll1 mRNA by Elavl1/HuR in neuroepithelial cells undergoing mitosis

    OpenAIRE

    García-Domínguez, Daniel J.; Morello, Dominique; Cisneros, Elsa; Kontoyiannis, Dimitris L.; Frade, José M

    2011-01-01

    In the vertebrate neuroepithelium, the decision to differentiate is made by neural precursors soon after mitosis, when they are apically located. This process is controlled by lateral inhibitory signals triggered by the Delta/Notch pathway. During mitosis, the capacity of neural precursors to express the neurogenic genes Dll1 and Notch1 is maximal due to mRNA stabilization, but the mechanism controlling this process remains unknown. Here we show that Elav-like (Elavl1)/HuR becomes enriched in...

  10. 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...

  11. 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...

  12. 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...

  13. 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

  14. 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

  15. 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.

  16. Dysregulation of the Axonal Trafficking of Nuclear-encoded Mitochondrial mRNA alters Neuronal Mitochondrial Activity and Mouse Behavior

    OpenAIRE

    Kar, Amar N.; Sun, Ching-Yu; Reichard, Kathryn; Gervasi, Noreen M.; Pickel, James; Nakazawa, Kazu; Gioio, Anthony E.; Kaplan, Barry B.

    2013-01-01

    Local translation of nuclear-encoded mitochondrial mRNAs is essential for mitochondrial activity, yet there is little insight into the role that axonal trafficking of these transcripts play in neuronal function and behavior. Previously, we identified a 38 nucleotide stem-loop structure (zipcode) in the 3′ untranslated region of the Cytochrome C oxidase IV (COXIV) mRNA that directs the transport of a reporter mRNA to the axon of superior cervical ganglion neurons (SCG). Over-expression of a ch...

  17. 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

  18. 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...

  19. 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...

  20. 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...

  1. 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

  2. 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

  3. 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...

  4. 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

  5. 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

  6. 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

  7. Kaposin-B Enhances the PROX1 mRNA Stability during Lymphatic Reprogramming of Vascular Endothelial Cells by Kaposi's Sarcoma Herpes Virus

    Science.gov (United States)

    Yoo, Jaehyuk; Kang, Jinjoo; Lee, Ha Neul; Aguilar, Berenice; Kafka, Darren; Lee, Sunju; Choi, Inho; Lee, Juneyong; Ramu, Swapnika; Haas, Juergen; Koh, Chester J.; Hong, Young-Kwon

    2010-01-01

    Kaposi's sarcoma (KS) is the most common cancer among HIV-positive patients. Histogenetic origin of KS has long been elusive due to a mixed expression of both blood and lymphatic endothelial markers in KS tumor cells. However, we and others discovered that Kaposi's sarcoma herpes virus (KSHV) induces lymphatic reprogramming of blood vascular endothelial cells by upregulating PROX1, which functions as the master regulator for lymphatic endothelial differentiation. Here, we demonstrate that the KSHV latent gene kaposin-B enhances the PROX1 mRNA stability and plays an important role in KSHV-mediated PROX1 upregulation. We found that PROX1 mRNA contains a canonical AU-rich element (ARE) in its 3′-untranslated region that promotes PROX1 mRNA turnover and that kaposin-B stimulates cytoplasmic accumulation of the ARE-binding protein HuR through activation of the p38/MK2 pathway. Moreover, HuR binds to and stabilizes PROX1 mRNA through its ARE and is necessary for KSHV-mediated PROX1 mRNA stabilization. Together, our study demonstrates that kaposin-B plays a key role in PROX1 upregulation during lymphatic reprogramming of blood vascular endothelial cells by KSHV. PMID:20730087

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. Regulation of cyclooxygenase-2 expression by cAMP response element and mRNA stability in a human airway epithelial cell line exposed to zinc

    International Nuclear Information System (INIS)

    Exposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. Cyclooxygenase 2-derived eicosanoids are important modulators of airway inflammation. In this study, we characterized the transcriptional and posttranscriptional events that regulate COX-2 expression in a human bronchial epithelial cell line BEAS-2B exposed to Zn2+. Zn2+ exposure resulted in pronounced increases in COX-2 mRNA and protein expression, which were prevented by pretreatment with the transcription inhibitor actinomycin D, implying the involvement of transcriptional regulation. This was supported by the observation of increased COX-2 promoter activity in Zn2+-treated BEAS-2B cells. Mutation of the cAMP response element (CRE), but not the κB-binding sites in the COX-2 promoter markedly reduced COX-2 promoter activity induced by Zn2+. Inhibition of NFκB activation did not block Zn2+-induced COX-2 expression. Measurement of mRNA stability demonstrated that Zn2+ exposure impaired the degradation of COX-2 mRNA in BEAS-2B cells. This message stabilization effect of Zn2+ exposure was shown to be dependent on the integrity of the 3'-untranslated region found in the COX-2 transcript. Taken together, these data demonstrate that the CRE and mRNA stability regulates COX-2 expression induced in BEAS-2B cells exposed to extracellular Zn2+

  14. 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...

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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 ...

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

    Directory of Open Access Journals (Sweden)

    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.

  2. 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

  3. 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

  4. 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.

  5. 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...

  6. Alterations in hepatic mRNA expression of phase II enzymes and xenobiotic transporters after targeted disruption of hepatocyte nuclear factor 4 alpha.

    Science.gov (United States)

    Lu, Hong; Gonzalez, Frank J; Klaassen, Curtis

    2010-12-01

    Hepatocyte nuclear factor 4 alpha (HNF4a) is a liver-enriched master regulator of liver function. HNF4a is important in regulating hepatic expression of certain cytochrome P450s. The purpose of this study was to use mice lacking HNF4a expression in liver (HNF4a-HNull) to elucidate the role of HNF4a in regulating hepatic expression of phase II enzymes and transporters in mice. Compared with male wild-type mice, HNF4a-HNull male mouse livers had (1) markedly lower messenger RNAs (mRNAs) encoding the uptake transporters sodium taurocholate cotransporting polypeptide, organic anion transporting polypeptide (Oatp) 1a1, Oatp2b1, organic anion transporter 2, sodium phosphate cotransporter type 1, sulfate anion transporter 1, sodium-dependent vitamin C transporter 1, the phase II enzymes Uridine 5'-diphospho (UDP)-glucuronosyltransferase (Ugt) 2a3, Ugt2b1, Ugt3a1, Ugt3a2, sulfotransferase (Sult) 1a1, Sult1b1, Sult5a1, the efflux transporters multidrug resistance-associated protein (Mrp) 6, and multidrug and toxin extrusion 1; (2) moderately lower mRNAs encoding Oatp1b2, organic cation transporter (Oct) 1, Ugt1a5, Ugt1a9, glutathione S-transferase (Gst) m4, Gstm6, and breast cancer resistance protein; but (3) higher mRNAs encoding Oatp1a4, Octn2, Ugt1a1, Sult1e1, Sult2a2, Gsta4, Gstm1-m3, multidrug resistance protein (Mdr) 1a, Mrp3, and Mrp4. Hepatic signaling of nuclear factor E2-related factor 2 and pregnane X receptor appear to be activated in HNF4a-HNull mice. In conclusion, HNF4a deficiency markedly alters hepatic mRNA expression of a large number of phase II enzymes and transporters, probably because of the loss of HNF4a, which is a transactivator and a determinant of gender-specific expression and/or adaptive activation of signaling pathways important in hepatic regulation of these phase II enzymes and transporters. PMID:20935164

  7. Examining the effect of altered redox conditions on deep soil organic matter stability

    Science.gov (United States)

    Gabriel, C.; Kellman, L. M.; Ziegler, S. E.

    2013-12-01

    Since subsoil horizons contribute significantly to terrestrial carbon (C) budgets, understanding the influence of disturbances such as forest harvesting on subsoil C stability is critical. Clearcut harvesting leads to changes in the soil physico-chemical environment, including altering redox conditions arising from changes in soil hydrology that increase soil saturation, soil temperature, and pH. These physico-chemical changes have the potential to alter the adsorption of soil organic matter (SOM) to minerals, particularly at depth where SOM is primarily associated with mineral phases. The objective of this study was to determine the effect of differing redox states (aerobic vs. anaerobic) and temperature upon SOM stability of forested soils representative of the Acadian Forest Region of Eastern North America. Composite soil samples through depth (0-10, 10-20, 20-35, and 35-50 cm) from a mature red spruce forest (110 years) were incubated under optimum (aerobic) or saturated (anaerobic) conditions for 1 or 4 months at two temperatures (5 and 15 C). Following incubation, soil leachate was analyzed for dissolved organic carbon (DOC), and UV-vis absorbance in order to determine soil C losses and its optical character. Specific UV-vis absorbance SUVA (254 nm) and spectral slope ratios were calculated in order to assess the composition of chromophoric dissolved organic matter (CDOM). Preliminary results from the 1 month incubation indicate that under anaerobic conditions, all depths released DOC with a higher SUVA than under aerobic conditions, with the largest change observed in the 0-10 cm depth increment. Soil incubated at 5 C produced leachate with significantly less DOC and with a lower absorbance compared to 15 C under both redox conditions. These results suggest that both temperature and redox state are important in determining the aromaticity of DOC released from soils. Spectral slope ratios revealed that a greater proportion of CDOM of lower molecular weight

  8. 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…

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

    Directory of Open Access Journals (Sweden)

    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.

  10. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  12. 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

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

    Directory of Open Access Journals (Sweden)

    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

  14. 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

  15. The 3′ Untranslated Region of the Rabies Virus Glycoprotein mRNA Specifically Interacts with Cellular PCBP2 Protein and Promotes Transcript Stability

    OpenAIRE

    Palusa, Saiprasad; Ndaluka, Christina; Bowen, Richard A.; Wilusz, Carol J.; Wilusz, Jeffrey

    2012-01-01

    Viral polymerase entry and pausing at intergenic junctions is predicted to lead to a defined polarity in the levels of rhabdovirus gene expression. Interestingly, we observed that the rabies virus glycoprotein mRNA is differentially over-expressed based on this model relative to other transcripts during infection of 293T cells. During infection, the rabies virus glycoprotein mRNA also selectively interacts with the cellular poly(rC)-binding protein 2 (PCBP2), a factor known to influence mRNA ...

  16. 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...

  17. 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

  18. 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.

  19. 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

  20. 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.

  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. 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...

  3. 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...... results suggest a regulatory role for Hfq that specifically facilitates the ompA mRNA degradation in a growth rate-dependent manner....

  4. 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...

  5. 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.

  6. 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

  7. 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)

  8. 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

  9. Biomaterials for mRNA Delivery

    Science.gov (United States)

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

    2015-01-01

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

  10. Long-lasting alteration in mesocorticolimbic structures after repeated social defeat stress in rats: time course of μ-opioid receptor mRNA and FosB/ΔFosB immunoreactivity

    OpenAIRE

    Nikulina, Ella M.; Arrillaga-Romany, Isabel; Miczek, Klaus A.; Hammer, Ronald P

    2008-01-01

    Social defeat stress is a salient stressor that induces neuroadaptive changes in the mesocorticolimbic dopaminergic system. Substantial evidence indicates that μ-opioid receptors (MOR) modulate dopamine transmission in the ventral tegmental area (VTA). FosB/ΔFosB protein accumulation in dopaminergic projections during repeated treatments is thought to be involved in long-term neuroplasticity. In this study we characterize the magnitude and time-course of MOR mRNA expression and FosB/ΔFosB imm...

  11. 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

  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. 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...

  14. Unexpected effects of the alteration of structure and stability of myoglobin and hemoglobin in ammonium-based ionic liquids.

    Science.gov (United States)

    Jha, Indrani; Attri, Pankaj; Venkatesu, Pannuru

    2014-03-28

    The nature of solvent-biomolecule interactions is generally weak and non-specific. The addition of ionic liquids (ILs), which have emerged as a new class of solvents, strengthen the stability of some proteins whereas the same ILs weaken the stability of some other proteins. Although ILs are commonly used for the stabilization of biomolecules, the bimolecular interactions of their stabilization-destabilization is still an active subject of considerable interest and studies on this topic have been limited. To reveal the impact of ILs on the stability of proteins, a series of protic ILs possessing a tetra-alkyl ammonium cation [R4N](+) with a hydroxide [OH](-) anion were synthesized. In this study, we report the structural stability of heme proteins such as myoglobin (Mb) and hemoglobin (Hb) in a series of ammonium-based ILs such as tetramethyl ammonium hydroxide [(CH3)4N](+)[OH](-) (TMAH), tetraethyl ammonium hydroxide [(C2H5)4N](+)[OH](-) (TEAH), tetrapropyl ammonium hydroxide [(C3H7)4N](+)[OH](-) (TPAH) and tetrabutyl ammonium hydroxide [(C4H9)4N](+)[OH](-) (TBAH) by fluorescence and circular dichroism (CD) spectroscopic studies. Our experimental results reveal that less viscous ILs carrying smaller alkyl chain such as TMAH are strong destabilizers of the heme proteins as compared to the ILs carrying bulkier alkyl chains which are more viscous ILs, such as TBAH. Therefore, our results demonstrate that the addition of these ILs to the heme proteins decreases their thermal stability allowing the protein to be in an unfolded state at lower temperatures. Further, we describe the molecular-structural interaction of the heme proteins with the ILs (molecule like a ligand) by the PatchDocking method. PMID:24501743

  15. Levels of myosin heavy chain mRNA transcripts and content of protein isoforms in the slow soleus muscle of 7 month-old rats with altered thyroid status

    Czech Academy of Sciences Publication Activity Database

    Vadászová, Adriana; Hudecová, S.; Križanová, O.; Soukup, Tomáš

    2006-01-01

    Roč. 55, č. 2 (2006), s. 221-225. ISSN 0862-8408 R&D Projects: GA ČR(CZ) GD305/03/H148; GA ČR(CZ) GA304/05/0327 Grant ostatní: VEGA(SK) 2/6078; SAV(SK) APVT-51-027404; NATO(XE) 979876; MYORES(XE) 511978 Institutional research plan: CEZ:AV0Z50110509 Keywords : myosin heavy chain * thyroid hormones status * mRNA transcripts Subject RIV: ED - Physiology Impact factor: 2.093, year: 2006

  16. 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 of an...... is associated with CHOP10 mRNA and regulates its stability. CHOP10 expression is conserved in RNase L(-/-)-MEFs, maintaining preadipocyte state while impairing their terminal differentiation. RNase L(-/-)-MEFs have decreased lipids storage capacity, insulin sensitivity and glucose uptake. Expression...... of ectopic RNase L in RNase L(-/-)-MEFs triggers CHOP10 mRNA instability, allowing increased lipids storage, insulin response and glucose uptake. Similarly, downregulation of CHOP10 mRNA with CHOP10 siRNA in RNase L(-/-)-MEFs improves their differentiation in adipocyte. In vivo, aged RNase L...

  17. 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.

  18. Designing excipient emulsions to increase nutraceutical bioavailability: emulsifier type influences curcumin stability and bioaccessibility by altering gastrointestinal fate.

    Science.gov (United States)

    Zou, Liqiang; Liu, Wei; Liu, Chengmei; Xiao, Hang; McClements, David Julian

    2015-08-01

    The influence of emulsifier type on the ability of excipient emulsions to improve the solubility, stability, and bioaccessibility of powdered curcumin was examined. Oil-in-water emulsions prepared using three different emulsifiers (whey protein isolate, caseinate, or Tween 80) were mixed with curcumin powder and then incubated at either 30 °C (to simulate applications of salad dressings) or 100 °C (to simulate applications of cooking sauces). The transfer of curcumin into the excipient emulsions was appreciably higher for excipient emulsions held at 100 °C than those held at 30 °C, and was appreciably higher for surfactant-stabilized emulsions than protein-stabilized emulsions. For example, the amounts of curcumin transferred into emulsions held at 30 and 100 °C were 66 and 280 μg mL(-1) for Tween 80, but only 17 and 208 μg mL(-1) for caseinate. The total curcumin concentration in the digesta and mixed micelle phases collected after excipient emulsions were exposed to a simulated gastrointestinal tract (mouth, stomach, and small intestine) depended on emulsifier type. The total amount of curcumin within the digesta was higher for protein-stabilized emulsions than surfactant-stabilized ones, which was attributed to the ability of the proteins to protect curcumin from chemical degradation. For example, the digesta contained 204 μg mL(-1) curcumin for caseinate emulsions, but only 111 μg mL(-1) for Tween 80 emulsions. This study shows the potential of designing excipient emulsions to increase the oral bioavailability of curcumin for food and pharmaceutical applications. PMID:26165514

  19. Poly(A)-binding proteins and mRNA localization: who rules the roost?

    Science.gov (United States)

    Gray, Nicola K; Hrabálková, Lenka; Scanlon, Jessica P; Smith, Richard W P

    2015-12-01

    RNA-binding proteins are often multifunctional, interact with a variety of protein partners and display complex localizations within cells. Mammalian cytoplasmic poly(A)-binding proteins (PABPs) are multifunctional RNA-binding proteins that regulate multiple aspects of mRNA translation and stability. Although predominantly diffusely cytoplasmic at steady state, they shuttle through the nucleus and can be localized to a variety of cytoplasmic foci, including those associated with mRNA storage and localized translation. Intriguingly, PABP sub-cellular distribution can alter dramatically in response to cellular stress or viral infection, becoming predominantly nuclear and/or being enriched in induced cytoplasmic foci. However, relatively little is known about the mechanisms that govern this distribution/relocalization and in many cases PABP functions within specific sites remain unclear. Here we discuss the emerging evidence with respect to these questions in mammals. PMID:26614673

  20. 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

  1. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters the mRNA expression of critical genes associated with cholesterol metabolism, bile acid biosynthesis, and bile transport in rat liver: A microarray study

    International Nuclear Information System (INIS)

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent hepatotoxin that exerts its toxicity through binding to the aryl hydrocarbon receptor (AhR) and the subsequent induction or repression of gene transcription. In order to further identify novel genes and pathways that may be associated with TCDD-induced hepatotoxicity, we investigated gene changes in rat liver following exposure to single oral doses of TCDD. Male Sprague-Dawley rats were administered single doses of 0.4 μg/kg bw or 40 μg/kg bw TCDD and killed at 6 h, 24 h, or 7 days, for global analyses of gene expression. In general, low-dose TCDD exposure resulted in greater than 2-fold induction of genes coding for a battery of phase I and phase II metabolizing enzymes including CYP1A1, CYP1A2, NADPH quinone oxidoreductase, UGT1A6/7, and metallothionein 1. However, 0.4 μg/kg bw TCDD also altered the expression of Gadd45a and Cyclin D1, suggesting that even low-dose TCDD exposure can alter the expression of genes indicative of cellular stress or DNA damage and associated with cell cycle control. At the high-dose, widespread changes were observed for genes encoding cellular signaling proteins, cellular adhesion, cytoskeletal and membrane transport proteins as well as transcripts coding for lipid, carbohydrate and nitrogen metabolism. In addition, decreased expression of cytochrome P450 7A1, short heterodimer partner (SHP; gene designation nr0b2), farnesyl X receptor (FXR), Ntcp, and Slc21a5 (oatp2) were observed and confirmed by RT-PCR analyses in independent rat liver samples. Altered expression of these genes implies major deregulation of cholesterol metabolism and bile acid synthesis and transport. We suggest that these early and novel changes have the potential to contribute significantly to TCDD induced hepatotoxicity and hypercholesterolemia

  2. 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...

  3. 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...

  4. Posttranscriptional regulation of sodium-iodide symporter mRNA expression in the rat thyroid gland by acute iodide administration.

    Science.gov (United States)

    Serrano-Nascimento, Caroline; Calil-Silveira, Jamile; Nunes, Maria Tereza

    2010-04-01

    Iodide is an important regulator of thyroid activity. Its excess elicits the Wolff-Chaikoff effect, characterized by an acute suppression of thyroid hormone synthesis, which has been ascribed to serum TSH reduction or TGF-beta increase and production of iodolipids in the thyroid. These alterations take hours/days to occur, contrasting with the promptness of Wolff-Chaikoff effect. We investigated whether acute iodide administration could trigger events that precede those changes, such as reduction of sodium-iodide symporter (NIS) mRNA abundance and adenylation, and if perchlorate treatment could counteract them. Rats subjected or not to methylmercaptoimidazole treatment (0.03%) received NaI (2,000 microg/0.5 ml saline) or saline intraperitoneally and were killed 30 min up to 24 h later. Another set of animals was treated with iodide and perchlorate, in equimolar doses. NIS mRNA content was evaluated by Northern blotting and real-time PCR, and NIS mRNA poly(A) tail length by rapid amplification of cDNA ends-poly(A) test (RACE-PAT). We observed that NIS mRNA abundance and poly(A) tail length were significantly reduced in all periods of iodide treatment. Perchlorate reversed these effects, indicating that iodide was the agent that triggered the modifications observed. Since the poly(A) tail length of mRNAs is directly associated with their stability and translation efficiency, we can assume that the rapid decay of NIS mRNA abundance observed was due to a reduction of its stability, a condition in which its translation could be impaired. Our data show for the first time that iodide regulates NIS mRNA expression at posttranscriptional level, providing a new mechanism by which iodide exerts its autoregulatory effect on thyroid. PMID:20107044

  5. 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

  6. 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...

  7. 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.

  8. 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.

  9. 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

  10. Highly metastatic hepatocellular carcinomas induced in male F344 rats treated with N-nitrosomorpholine in combination with other hepatocarcinogens show a high incidence of p53 gene mutations along with altered mRNA expression of tumor-related genes.

    Science.gov (United States)

    Masui, T; Nakanishi, H; Inada, K; Imai, T; Mizoguchi, Y; Yada, H; Futakuchi, M; Shirai, T; Tatematsu, M

    1997-01-15

    The carcinogenic and metastatic processes are thought to consist of a sequence of steps, and animal models featuring highly metastatic lesions are clearly necessary to allow analysis of the whole process of transformation from preneoplastic changes to high grade metastatic tumors, and to access effectiveness of therapeutic treatments of advanced cancers in vivo. The purpose of the present study was to establish a model and to screen for reported genetic alterations in induced lesions. In the present study, it was confirmed that lung metastasis of hepatocellular carcinomas (HCCs) induced in male F344 rats by N-nitrosomorpholine (NNM), given in the drinking water at a dose of 120 ppm for 24 weeks, was significantly enhanced by additional carcinogenic pretreatments and that a single i.p. injection of 100 mg/kg body weight N-diethylnitrosamine (DEN) alone was sufficient for that purpose. Molecular biological analyses of the induced lesions revealed point mutations in the p53 gene in 60.9% of HCCs, and elevated expression of mRNAs for p53, c-myc, c-fos, TGF-alpha, TGF-beta1, alpha-fetoprotein, GST-P, and GGT, and decreased mRNA expression of EGF and EGFR in HCCs when compared to controls. No obvious association of gene alterations with metastatic potential of primary tumors was found except for an increase in the incidence of p53 mutations. Since the process of metastasis is thought to be sequential and selective, further comparative analysis of metastatic and primary lesions should clarify the mechanisms involved in the multi-step process of metastasis. PMID:9029167

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

    DEFF Research Database (Denmark)

    Glahder, Jacob-Andreas Harald; Norrild, Bodil

    2011-01-01

    Cytoplasmic polyadenylation is a post-transcriptional mechanism regulating mRNA stability and translation. The human p53 3'-untranslated region (3'-UTR) contains two regions similar to cytoplasmic polyadenylation elements (CPEs) just upstream of the poly(A) hexanucleotide. Evaluation of the p53 CPE...... 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...

  12. 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

  13. 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

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

    Science.gov (United States)

    de Bruijn, Wouter J C; Weesepoel, Yannick; Vincken, Jean-Paul; Gruppen, Harry

    2016-03-01

    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 esterification decelerated degradation of all-trans-astaxanthin (RP-UHPLC-PDA), whereas, it had no influence on colour loss over time (spectrophotometry). This difference might be explained by the observation that palmitate esterification influenced the cis-trans equilibrium. Free astaxanthin produced larger amounts of 9-cis isomer whereas monopalmitate esterification resulted in increased 13-cis isomerization. The molar ratios of 9-cis:13-cis after 60min were 1:1.7 (free), 1:4.8 (monopalmitate) and 1:2.6 (dipalmitate). The formation of 9-cis astaxanthin, with its higher molar extinction coefficient than that of all-trans-astaxanthin, might compensate for colour loss induced by conjugated double bond cleavage. As such, it was concluded that spectrophotometry is not an accurate measure of the degradation of the all-trans-astaxanthin molecule. PMID:26471660

  15. 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...

  16. 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...

  17. 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 ...

  18. Main: MRNA3ENDTAH3 [PLACE

    Lifescience Database Archive (English)

    Full Text Available MRNA3ENDTAH3 S000069 17-May-1998 (last modified) kehi Cis element in 3' end region of wheat (T.a ... tion; Also found in histone genes of other plants, yeast , etc; histone H3; mRNA; 3' end formation; meristem ... ; wheat (Triticum aestivum); yeast ; AATGGAAATG ...

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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)

  4. -- mRNA expression in human breast cancer: a meta-analysis

    OpenAIRE

    Gonçalves, Anthony; Finetti, Pascal; Sabatier, Renaud; Gilabert, Marine; Adelaide, José; Borg, Jean-Paul; Chaffanet, Max; Viens, Patrice; Birnbaum, Daniel; Bertucci, François

    2010-01-01

    Although poly(ADP-ribose) polymerase-1 (PARP1) inhibition is a recent promising therapy in breast cancer, PARP1 expression in this disease is not known. Using DNA microarray and array-based comparative genomic hybridization (arrayCGH), we examined mRNA expression and copy number alterations in 326 invasive breast cancer samples and normal breast (NB) samples. A meta-analysis was performed on a large public retrospective gene expression data set ( = 2,485) to analyze correlation between mRNA e...

  5. 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...

  6. Smectite alteration

    International Nuclear Information System (INIS)

    The colloquium was convened to compose a summary of the most recent data and the best scientific appraisal of present knowledge on the process of smectite conversion to illite. A brief account of a consensus view on the probable stability of smectite clays when employed as the buffer material in the Swedish nuclear fuel storage system. It is concluded that if the quantitiy of nuclear waste to be put in each bore hole is carefully limited, temperatures surrounding the cannister will not rise to an acceptable level and the clay buffer material can be relied upon to be effective throughout the life of the repository. (G.B.)

  7. Determination of the Absolute Number of Cytokine mRNA Molecules within Individual Activated Human T Cells

    Science.gov (United States)

    Karr, Laurel J.; Marshall, Gwen; Hockett, Richard D.; Bucy, R. Pat; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    A primary function of activated T cells is the expression and subsequent secretion of cytokines, which orchestrate the differentiation of other lymphocytes, modulate antigen presenting cell activity, and alter vascular endothelium to mediate an immune response. Since many features of immune regulation probably result from modest alterations of endogenous rates of multiple interacting processes, quantitative analysis of the frequency and specific activity of individual T cells is critically important. Using a coordinated set of quantitative methods, the absolute number of molecules of several key cytokine mRNA species in individual T cells has been determined. The frequency of human blood T cells activated in vitro by mitogens and recall protein antigens was determined by intracellular cytokine protein staining, in situ hybridization for cytokine mRNA, and by limiting dilution analysis for cytokine mRNA+ cells. The absolute number of mRNA molecules was simultaneously determined in both homogenates of the entire population of cells and in individual cells obtained by limiting dilution, using a quantitative, competitive RT-PCR assay. The absolute numbers of mRNA molecules in a population of cells divided by the frequency of individual positive cells, yielded essentially the same number of mRNA molecules per cell as direct analysis of individual cells by limiting dilution analysis. Mean numbers of mRNA per positive cell from both mitogen and antigen activated T cells, using these stimulation conditions, were 6000 for IL-2, 6300 for IFN-gamma, and 1600 for IL-4.

  8. 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.

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

    Science.gov (United States)

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

    2016-05-01

    miRNAs play critical roles in heart disease. In addition to differential miRNA expression, miRNA-mediated control is also affected by variable miRNA processing or alternative 3'-end cleavage and polyadenylation (APA) of their mRNA targets. To what extent these phenomena play a role in the heart remains unclear. We sought to explore miRNA processing and mRNA APA in cardiomyocytes, and whether these change during cardiac hypertrophy. Thoracic aortic constriction (TAC) was performed to induce hypertrophy in C57BL/6J mice. RNA extracted from cardiomyocytes of sham-treated, pre-hypertrophic (2days post-TAC), and hypertrophic (7days post-TAC) mice was subjected to small RNA- and poly(A)-test sequencing (PAT-Seq). Differential expression analysis matched expectations; nevertheless we identified ~400 mRNAs and hundreds of noncoding RNA loci as altered with hypertrophy for the first time. Although multiple processing variants were observed for many miRNAs, there was little change in their relative proportions during hypertrophy. PAT-Seq mapped ~48,000 mRNA 3'-ends, identifying novel 3' untranslated regions (3'UTRs) for over 7000 genes. Importantly, hypertrophy was associated with marked changes in APA with a net shift from distal to more proximal mRNA 3'-ends, which is predicted to decrease overall miRNA repression strength. We independently validated several examples of 3'UTR proportion change and showed that alternative 3'UTRs associate with differences in mRNA translation. Our work suggests that APA contributes to altered gene expression with the development of cardiomyocyte hypertrophy and provides a rich resource for a systems-level understanding of miRNA-mediated regulation in physiological and pathological states of the heart. PMID:27032571

  10. 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...

  11. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis

    DEFF Research Database (Denmark)

    Krakauer, Martin; Sorensen, P; Khademi, M; Olsson, T; Sellebjerg, F

    2008-01-01

    volunteers served to confirm initial findings. mRNA was analyzed by real-time reverse transcriptase polymerase chain reaction (PCR). RESULTS: We found elevated expression of interleukin (IL)-23 and IL-10 in untreated MS patients. IFN-beta therapy increased IL-10 and decreased IL-23 expression independently...... of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another...

  12. Chaperone Hsp27 modulates AUF1 proteolysis and AU-rich element-mediated mRNA degradation.

    Science.gov (United States)

    Knapinska, Anna M; Gratacós, Frances M; Krause, Christopher D; Hernandez, Kristina; Jensen, Amber G; Bradley, Jacquelyn J; Wu, Xiangyue; Pestka, Sidney; Brewer, Gary

    2011-04-01

    AUF1 is an AU-rich element (ARE)-binding protein that recruits translation initiation factors, molecular chaperones, and mRNA degradation enzymes to the ARE for mRNA destruction. We recently found chaperone Hsp27 to be an AUF1-associated ARE-binding protein required for tumor necrosis factor alpha (TNF-α) mRNA degradation in monocytes. Hsp27 is a multifunctional protein that participates in ubiquitination of proteins for their degradation by proteasomes. A variety of extracellular stimuli promote Hsp27 phosphorylation on three serine residues--Ser(15), Ser(78), and Ser(82)-by a number of kinases, including the mitogen-activated protein (MAP) pathway kinases p38 and MK2. Activating either kinase stabilizes ARE mRNAs. Likewise, ectopic expression of phosphomimetic mutant forms of Hsp27 stabilizes reporter ARE mRNAs. Here, we continued to examine the contributions of Hsp27 to mRNA degradation. As AUF1 is ubiquitinated and degraded by proteasomes, we addressed the hypothesis that Hsp27 phosphorylation controls AUF1 levels to modulate ARE mRNA degradation. Indeed, selected phosphomimetic mutants of Hsp27 promote proteolysis of AUF1 in a proteasome-dependent fashion and render ARE mRNAs more stable. Our results suggest that the p38 MAP kinase (MAPK)-MK2-Hsp27 signaling axis may target AUF1 destruction by proteasomes, thereby promoting ARE mRNA stabilization. PMID:21245386

  13. Alterations in LMTK2, MSMB and HNF1B gene expression are associated with the development of prostate cancer

    Directory of Open Access Journals (Sweden)

    McCullagh Paul

    2010-06-01

    Full Text Available Abstract Background Genome wide association studies (GWAS have identified several genetic variants that are associated with prostate cancer. Most of these variants, like other GWAS association signals, are located in non-coding regions of potential candidate genes, and thus could act at the level of the mRNA transcript. Methods We measured the expression and isoform usage of seven prostate cancer candidate genes in benign and malignant prostate by real-time PCR, and correlated these factors with cancer status and genotype at the GWAS risk variants. Results We determined that levels of LMTK2 transcripts in prostate adenocarcinomas were only 32% of those in benign tissues (p = 3.2 × 10-7, and that an independent effect of genotype at variant rs6465657 on LMTK2 expression in benign (n = 39 and malignant tissues (n = 21 was also evident (P = 0.002. We also identified that whilst HNF1B(C and MSMB2 comprised the predominant isoforms in benign tissues (90% and 98% of total HNF1B or MSMB expression, HNF1B(B and MSMB1 were predominant in malignant tissue (95% and 96% of total HNF1B or MSMB expression; P = 1.7 × 10-7 and 4 × 10-4 respectively, indicating major shifts in isoform usage. Conclusions Our results indicate that the amount or nature of mRNA transcripts expressed from the LMTK2, HNF1B and MSMB candidate genes is altered in prostate cancer, and provides further evidence for a role for these genes in this disorder. The alterations in isoform usage we detect highlights the potential importance of alternative mRNA processing and moderation of mRNA stability as potentially important disease mechanisms.

  14. Alterations in LMTK2, MSMB and HNF1B gene expression are associated with the development of prostate cancer

    International Nuclear Information System (INIS)

    Genome wide association studies (GWAS) have identified several genetic variants that are associated with prostate cancer. Most of these variants, like other GWAS association signals, are located in non-coding regions of potential candidate genes, and thus could act at the level of the mRNA transcript. We measured the expression and isoform usage of seven prostate cancer candidate genes in benign and malignant prostate by real-time PCR, and correlated these factors with cancer status and genotype at the GWAS risk variants. We determined that levels of LMTK2 transcripts in prostate adenocarcinomas were only 32% of those in benign tissues (p = 3.2 × 10-7), and that an independent effect of genotype at variant rs6465657 on LMTK2 expression in benign (n = 39) and malignant tissues (n = 21) was also evident (P = 0.002). We also identified that whilst HNF1B(C) and MSMB2 comprised the predominant isoforms in benign tissues (90% and 98% of total HNF1B or MSMB expression), HNF1B(B) and MSMB1 were predominant in malignant tissue (95% and 96% of total HNF1B or MSMB expression; P = 1.7 × 10-7 and 4 × 10-4 respectively), indicating major shifts in isoform usage. Our results indicate that the amount or nature of mRNA transcripts expressed from the LMTK2, HNF1B and MSMB candidate genes is altered in prostate cancer, and provides further evidence for a role for these genes in this disorder. The alterations in isoform usage we detect highlights the potential importance of alternative mRNA processing and moderation of mRNA stability as potentially important disease mechanisms

  15. 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

    intermediate expression levels of green fluorescent protein in Escherichia coli. At least in one case, no apparent effect on protein stability was observed, pointing to RNA level effects as the principal reason for the observed expression differences. Targeting a synonymous codon library to the 5' coding......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...

  16. Binding of TDP-43 to the 3'UTR of its cognate mRNA enhances its solubility.

    Science.gov (United States)

    Sun, Yulong; Arslan, Pharhad E; Won, Amy; Yip, Christopher M; Chakrabartty, Avi

    2014-09-23

    TAR DNA binding protein of 43 kDa (TDP-43) has been implicated in the pathogenesis of a broad range of neurodegenerative diseases termed TDP-43 proteinopathies, which encompass a spectrum of diseases ranging from amyotrophic lateral sclerosis to frontotemporal dementia. Pathologically misfolded and aggregated forms of TDP-43 are found in cytoplasmic inclusion bodies of affected neurons in these diseases. The mechanism by which TDP-43 misfolding causes disease is not well-understood. Current hypotheses postulate that the TDP-43 aggregation process plays a major role in pathogenesis. We amplify that hypothesis and suggest that binding of cognate ligands to TDP-43 can stabilize the native functional state of the protein and ameliorate aggregation. We expressed recombinant TDP-43 containing an N-terminal Venus yellow fluorescent protein tag in Escherichia coli and induced its aggregation by altering solvent salt concentrations and examined the extent to which various oligonucleotide molecules affect its aggregation in vitro using aggregation-induced turbidity assays. We show that vYFP-TDP-43 binding to its naturally occurring RNA target that comprises a sequence on the 3'UTR region of its mRNA improves its solubility, suggesting interplay among TDP-43 solubility, oligonucleotide binding, and TDP-43 autoregulation. PMID:25171271

  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. mRNA as gene therapeutic: how to control protein expression.

    Science.gov (United States)

    Tavernier, Geertrui; Andries, Oliwia; Demeester, Jo; Sanders, Niek N; De Smedt, Stefaan C; Rejman, Joanna

    2011-03-30

    For many years, it was generally accepted that mRNA is too unstable to be efficiently used for gene therapy purposes. In the last decade, however, several research groups faced this challenge and not only proved the feasibility of mRNA-mediated transfection with surprising results regarding transfection efficiency and duration of protein expression, but also were able to demonstrate major advantages over the use of pDNA. These advantages will be the first issue discussed in this review, which first of all addresses the notions that mRNA does not need to cross the nuclear barrier to exert its biological activity and in addition lacks CpG motifs, which reduces its immunogenicity. Secondly, it provides insight in the (in)stability of the mRNA molecule, in how mRNA can be modified to increase its half-life and in the necessities of exogenously produced mRNA to be successfully used in transfection protocols. Furthermore, this review gives an in-depth overview of the different techniques and vehicles for intracellular mRNA delivery exploited by us and other groups, comprising electroporation, gene gun injection, lipo- and polyplexes. Finally, it covers recent literature describing specific applications for mRNA based gene delivery, showing that until now most attention has been paid to vaccination strategies. This review offers a comprehensive overview of current knowledge of the major theoretical as well as practical aspects of mRNA-mediated transfection, showing both its possibilities and its pitfalls and should therefore be useful for a diverse scientific audience. PMID:20970469

  20. 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.

  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. 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

    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......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...

  5. A splice mutation and mRNA decay of EXT2 provoke hereditary multiple exostoses.

    Directory of Open Access Journals (Sweden)

    Chen Tian

    Full Text Available BACKGROUND: Hereditary multiple exostoses (HME is an autosomal dominant disease. The classical paradigm of mutation screening seeks to relate alterations in the exostosin glycosyltransferase genes, EXT1 and EXT2, which are responsible for over 70% of HME cases. However, the pathological significance of the majority of these mutations is often unclear. METHODS: In a Chinese family with HME, EXT1 and EXT2 genes were screened by direct sequencing. The consequence of a detected mutant was predicted by in silico analysis and confirmed by mRNA analysis. The EXT1 and EXT2 mRNA and protein levels and the HS patterns in the HME patients were compared with those in healthy controls. RESULTS: A heterozygous transition (c.743+1G>A in the EXT2 gene, which co-segregated with the HME phenotype in this family, was identified. The G residue at position +1 in intron 4 of EXT2 was predicted to be a 5' donor splice site. The mRNA analysis revealed an alternative transcript with a cryptic splice site 5 bp downstream of the wild-type site, which harbored a premature stop codon. However, the predicted truncated protein was not detected by western blot analysis. Decay of the mutant mRNA was shown by clone sequencing and quantification analysis. The corresponding downregulation of the EXT2 mRNA will contribute to the abnormal EXT1/EXT2 ratio and HS pattern that were detected in the patients with HME. CONCLUSION: The heterozygous mutation c.743+1G>A in the EXT2 gene causes HME as a result of abnormal splicing, mRNA decay, and the resulting haploinsufficiency of EXT2.

  6. Differential gene regulation under altered gravity conditions in follicular thyroid cancer cells: relationship between the extracellular matrix and the cytoskeleton.

    Science.gov (United States)

    Ulbrich, Claudia; Pietsch, Jessica; Grosse, Jirka; Wehland, Markus; Schulz, Herbert; Saar, Katrin; Hübner, Norbert; Hauslage, Jens; Hemmersbach, Ruth; Braun, Markus; van Loon, Jack; Vagt, Nicole; Egli, Marcel; Richter, Peter; Einspanier, Ralf; Sharbati, Soroush; Baltz, Theo; Infanger, Manfred; Ma, Xiao; Grimm, Daniela

    2011-01-01

    Extracellular matrix proteins, adhesion molecules, and cytoskeletal proteins form a dynamic network interacting with signalling molecules as an adaptive response to altered gravity. An important issue is the exact differentiation between real microgravity responses of the cells or cellular reactions to hypergravity and/or vibrations. To determine the effects of real microgravity on human cells, we used four DLR parabolic flight campaigns and focused on the effects of short-term microgravity (22 s), hypergravity (1.8 g), and vibrations on ML-1 thyroid cancer cells. No signs of apoptosis or necrosis were detectable. Gene array analysis revealed 2,430 significantly changed transcripts. After 22 s microgravity, the F-actin and cytokeratin cytoskeleton was altered, and ACTB and KRT80 mRNAs were significantly upregulated after the first and thirty-first parabolas. The COL4A5 mRNA was downregulated under microgravity, whereas OPN and FN were significantly upregulated. Hypergravity and vibrations did not change ACTB, KRT-80 or COL4A5 mRNA. MTSS1 and LIMA1 mRNAs were downregulated/slightly upregulated under microgravity, upregulated in hypergravity and unchanged by vibrations. These data indicate that the graviresponse of ML-1 cells occurred very early, within the first few seconds. Downregulated MTSS1 and upregulated LIMA1 may be an adaptive mechanism of human cells for stabilizing the cytoskeleton under microgravity conditions. PMID:21865726

  7. Nudt3 is an mRNA decapping enzyme that modulates cell migration.

    Science.gov (United States)

    Grudzien-Nogalska, Ewa; Jiao, Xinfu; Song, Man-Gen; Hart, Ronald P; Kiledjian, Megerditch

    2016-05-01

    Removal of the 5'-end 7-methylguanosine cap structure is a critical step in the highly regulated process of mRNA decay. The Nudix hydrolase, Dcp2, was identified as a first decapping enzyme and subsequently shown to preferentially modulate stability of only a subset of mRNAs. This observation led to the hypothesis that mammalian cells possess multiple decapping enzymes that may function in distinct pathways. Here we report Nudt3 is a Nudix protein that possesses mRNA decapping activity in cells and is a modulator of MCF-7 breast cancer cell migration. Reduction of Nudt3 protein levels in MCF-7 cells promotes increased cell migration and corresponding enhanced filopodia extensions. Importantly, this phenotype was reversed by complementation with wild type, but not catalytically inactive Nudt3 protein indicating Nudt3 decapping activity normally functions to control cell migration. Genome-wide analysis of Nudt3 compromised cells identified elevated levels of transcripts involved in cell motility including integrin β6, lipocalin-2, and fibronectin. The observed increase in mRNA abundance was dependent on Nudt3 decapping activity where integrin β6 and lipocalin-2 were modulated directly through mRNA stability, while fibronectin was indirectly controlled. Moreover, increased cell migration observed in Nudt3 knockdown cells was mediated through the extracellular integrin β6 and fibronectin protein nexus. We conclude that Nudt3 is an mRNA decapping enzyme that orchestrates expression of a subset of mRNAs to modulate cell migration and further substantiates the existence of multiple decapping enzymes functioning in distinct cellular pathways in mammals. PMID:26932476

  8. PABPN1-Dependent mRNA Processing Induces Muscle Wasting

    Science.gov (United States)

    Raz, Yotam; van Putten, Maaike; Paniagua-Soriano, Guillem; Krom, Yvonne D.; Florea, Bogdan I.; Raz, Vered

    2016-01-01

    Poly(A) Binding Protein Nuclear 1 (PABPN1) is a multifunctional regulator of mRNA processing, and its expression levels specifically decline in aging muscles. An expansion mutation in PABPN1 is the genetic cause of oculopharyngeal muscle dystrophy (OPMD), a late onset and rare myopathy. Moreover, reduced PABPN1 expression correlates with symptom manifestation in OPMD. PABPN1 regulates alternative polyadenylation site (PAS) utilization. However, the impact of PAS utilization on cell and tissue function is poorly understood. We hypothesized that altered PABPN1 expression levels is an underlying cause of muscle wasting. To test this, we stably down-regulated PABPN1 in mouse tibialis anterior (TA) muscles by localized injection of adeno-associated viruses expressing shRNA to PABPN1 (shPab). We found that a mild reduction in PABPN1 levels causes muscle pathology including myofiber atrophy, thickening of extracellular matrix and myofiber-type transition. Moreover, reduced PABPN1 levels caused a consistent decline in distal PAS utilization in the 3’-UTR of a subset of OPMD-dysregulated genes. This alternative PAS utilization led to up-regulation of Atrogin-1, a key muscle atrophy regulator, but down regulation of proteasomal genes. Additionally reduced PABPN1 levels caused a reduction in proteasomal activity, and transition in MyHC isotope expression pattern in myofibers. We suggest that PABPN1-mediated alternative PAS utilization plays a central role in aging-associated muscle wasting. PMID:27152426

  9. PABPN1-Dependent mRNA Processing Induces Muscle Wasting.

    Science.gov (United States)

    Riaz, Muhammad; Raz, Yotam; van Putten, Maaike; Paniagua-Soriano, Guillem; Krom, Yvonne D; Florea, Bogdan I; Raz, Vered

    2016-05-01

    Poly(A) Binding Protein Nuclear 1 (PABPN1) is a multifunctional regulator of mRNA processing, and its expression levels specifically decline in aging muscles. An expansion mutation in PABPN1 is the genetic cause of oculopharyngeal muscle dystrophy (OPMD), a late onset and rare myopathy. Moreover, reduced PABPN1 expression correlates with symptom manifestation in OPMD. PABPN1 regulates alternative polyadenylation site (PAS) utilization. However, the impact of PAS utilization on cell and tissue function is poorly understood. We hypothesized that altered PABPN1 expression levels is an underlying cause of muscle wasting. To test this, we stably down-regulated PABPN1 in mouse tibialis anterior (TA) muscles by localized injection of adeno-associated viruses expressing shRNA to PABPN1 (shPab). We found that a mild reduction in PABPN1 levels causes muscle pathology including myofiber atrophy, thickening of extracellular matrix and myofiber-type transition. Moreover, reduced PABPN1 levels caused a consistent decline in distal PAS utilization in the 3'-UTR of a subset of OPMD-dysregulated genes. This alternative PAS utilization led to up-regulation of Atrogin-1, a key muscle atrophy regulator, but down regulation of proteasomal genes. Additionally reduced PABPN1 levels caused a reduction in proteasomal activity, and transition in MyHC isotope expression pattern in myofibers. We suggest that PABPN1-mediated alternative PAS utilization plays a central role in aging-associated muscle wasting. PMID:27152426

  10. PABPN1-Dependent mRNA Processing Induces Muscle Wasting.

    Directory of Open Access Journals (Sweden)

    Muhammad Riaz

    2016-05-01

    Full Text Available Poly(A Binding Protein Nuclear 1 (PABPN1 is a multifunctional regulator of mRNA processing, and its expression levels specifically decline in aging muscles. An expansion mutation in PABPN1 is the genetic cause of oculopharyngeal muscle dystrophy (OPMD, a late onset and rare myopathy. Moreover, reduced PABPN1 expression correlates with symptom manifestation in OPMD. PABPN1 regulates alternative polyadenylation site (PAS utilization. However, the impact of PAS utilization on cell and tissue function is poorly understood. We hypothesized that altered PABPN1 expression levels is an underlying cause of muscle wasting. To test this, we stably down-regulated PABPN1 in mouse tibialis anterior (TA muscles by localized injection of adeno-associated viruses expressing shRNA to PABPN1 (shPab. We found that a mild reduction in PABPN1 levels causes muscle pathology including myofiber atrophy, thickening of extracellular matrix and myofiber-type transition. Moreover, reduced PABPN1 levels caused a consistent decline in distal PAS utilization in the 3'-UTR of a subset of OPMD-dysregulated genes. This alternative PAS utilization led to up-regulation of Atrogin-1, a key muscle atrophy regulator, but down regulation of proteasomal genes. Additionally reduced PABPN1 levels caused a reduction in proteasomal activity, and transition in MyHC isotope expression pattern in myofibers. We suggest that PABPN1-mediated alternative PAS utilization plays a central role in aging-associated muscle wasting.

  11. Quantitative studies of mRNA recruitment to the eukaryotic ribosome.

    Science.gov (United States)

    Fraser, Christopher S

    2015-07-01

    The process of peptide bond synthesis by ribosomes is conserved between species, but the initiation step differs greatly between the three kingdoms of life. This is illustrated by the evolution of roughly an order of magnitude more initiation factor mass found in humans compared with bacteria. Eukaryotic initiation of translation is comprised of a number of sub-steps: (i) recruitment of an mRNA and initiator methionyl-tRNA to the 40S ribosomal subunit; (ii) migration of the 40S subunit along the 5' UTR to locate the initiation codon; and (iii) recruitment of the 60S subunit to form the 80S initiation complex. Although the mechanism and regulation of initiation has been studied for decades, many aspects of the pathway remain unclear. In this review, I will focus discussion on what is known about the mechanism of mRNA selection and its recruitment to the 40S subunit. I will summarize how the 43S preinitiation complex (PIC) is formed and stabilized by interactions between its components. I will discuss what is known about the mechanism of mRNA selection by the eukaryotic initiation factor 4F (eIF4F) complex and how the selected mRNA is recruited to the 43S PIC. The regulation of this process by secondary structure located in the 5' UTR of an mRNA will also be discussed. Finally, I present a possible kinetic model with which to explain the process of mRNA selection and recruitment to the eukaryotic ribosome. PMID:25742741

  12. 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

  13. 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.

  14. Effects of simulated microgravity on microRNA and mRNA expression profile of rat soleus

    Science.gov (United States)

    Xu, Hongjie; Wu, Feng; Cao, Hongqing; Kan, Guanghan; Zhang, Hongyu; Yeung, Ella W.; Shang, Peng; Dai, Zhongquan; Li, Yinghui

    2015-02-01

    Spaceflight induces muscle atrophy but mechanism is not well understood. Here, we quantified microRNAs (miRNAs) and mRNA shifts of rat soleus in response to microgravity. MiRNAs and mRNA microarray of soleus after tail suspension (TS) for 7 and 14 days were performed followed by target gene and function annotation analysis and qRT-PCR. Relative muscle mass lost by 37.0% in TS-7 but less than 10% in the following three weeks. TS altered 23 miRNAs and 1313 mRNAs with at least 2-fold. QRT-PCR confirmed some of these changes. MiR-214, miR-486-5p and miR-221 continuously decreased. MiR-674 and Let-7e decreased only in TS-7, while miR-320b and miR-187 decreased only in TS-14. But there was no alteration of miR-320 and miR-206 in both time point. For mRNA detection, actn3 (5.1-fold and 13.8-fold) and myh4 (38-fold and 51.6-fold) increased abundantly and a3galt2 decreased. Predicted targeted genes (whyz, ywhaz and SFRP2) of altered miRNAs decreased. GO terms and cellular pathway of these alteration showed enrichment in regulation of muscle metabolism. Integration analysis of the miRNA and mRNA expression profiles confirmed that eleven genes were differently regulated by four miRNAs. This is the first study that showed expression pattern and synergistical regulation of miRNA and mRNA in rat soleus of TS for up to 14 days.

  15. 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.

  16. 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

  17. 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.

  18. Contributions of transcription and mRNA decay to gene expression dynamics of fission yeast in response to oxidative stress

    Science.gov (United States)

    Marguerat, Samuel; Lawler, Katherine; Brazma, Alvis; Bähler, Jürg

    2014-01-01

    The cooperation of transcriptional and post-transcriptional levels of control to shape gene regulation is only partially understood. Here we show that a combination of two simple and non-invasive genomic techniques, coupled with kinetic mathematical modeling, affords insight into the intricate dynamics of RNA regulation in response to oxidative stress in the fission yeast Schizosaccharomyces pombe. This study reveals a dominant role of transcriptional regulation in response to stress, but also points to the first minutes after stress induction as a critical time when the coordinated control of mRNA turnover can support the control of transcription for rapid gene regulation. In addition, we uncover specialized gene expression strategies associated with distinct functional gene groups, such as simultaneous transcriptional repression and mRNA destabilization for genes encoding ribosomal proteins, delayed mRNA destabilization with varying contribution of transcription for ribosome biogenesis genes, dominant roles of mRNA stabilization for genes functioning in protein degradation, and adjustment of both transcription and mRNA turnover during the adaptation to stress. We also show that genes regulated independently of the bZIP transcription factor Atf1p are predominantly controlled by mRNA turnover, and identify putative cis-regulatory sequences that are associated with different gene expression strategies during the stress response. This study highlights the intricate and multi-faceted interplay between transcription and RNA turnover during the dynamic regulatory response to stress. PMID:25007214

  19. 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.

  20. 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.

  1. 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 ...

  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. Stability Scores: Measuring Coalitional Stability

    OpenAIRE

    Feldman, Michal; Meir, Reshef; Tennenholtz, Moshe

    2011-01-01

    We introduce a measure for the level of stability against coalitional deviations, called \\emph{stability scores}, which generalizes widely used notions of stability in non-cooperative games. We use the proposed measure to compare various Nash equilibria in congestion games, and to quantify the effect of game parameters on coalitional stability. For our main results, we apply stability scores to analyze and compare the Generalized Second Price (GSP) and Vickrey-Clarke-Groves (VCG) ad auctions....

  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. Ideal Stabilization

    CERN Document Server

    Nesterenko, Mikhail

    2009-01-01

    We define and explore the concept of ideal stabilization. The program is ideally stabilizing if its every state is legitimate. Ideal stabilization allows the specification designer to prescribe with arbitrary degree of precision not only the fault-free program behavior but also its recovery operation. Specifications may or may not mention all possible states. We identify approaches to designing ideal stabilization to both kinds of specifications. For the first kind, we state the necessary condition for an ideally stabilizing solution. On the basis of this condition we prove that there is no ideally stabilizing solution to the leader election problem. We illustrate the utility of the concept by providing examples of well-known programs and proving them ideally stabilizing. Specifically, we prove ideal stabilization of the conflict manager, the alternator, the propagation of information with feedback and the alternating bit protocol.

  7. 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.

  8. Successful personalized chemotherapy for metastatic gastric cancer based on quantitative BRCA1 mRNA expression level: A case report

    Science.gov (United States)

    HUANG, YING; WU, PUYUAN; LIU, BAORUI; DU, JUAN

    2016-01-01

    Personalized chemotherapy is based on the specific genetic profile of individual patients and is replacing the traditional ‘one size fits all’ medicine. Breast cancer 1 (BRCA1) plays a central role in the chemotherapy-induced DNA damage response. It has been repeatedly demonstrated that BRCA1 mRNA levels were negatively associated with cisplatin sensitivity, but positively associated with docetaxel sensitivity in patients with gastric cancer in experimental and clinical studies. This feature leads to customized chemotherapy based on the BRCA1 mRNA expression level and results in a high efficacy of treatment. The present study describes the case of a 77-year-old patient with metastatic gastric cancer who was treated with personalized chemotherapy based on quantitative BRCA1 mRNA expression level. This study and the available literature data suggest that the expression level of BRCA1 mRNA is dynamic to BRCA1-based chemotherapy. More importantly, de novo assessment of BRCA1 status is a preferable option for ciscisplatin- or docetaxel-resistant patients, since the expression levels of BRCA1 mRNA in certain patients may alter significantly following treatment. Therefore, BRCA1 expression should be assessed for predicting differential chemosensitivity and tailoring chemotherapy in gastric cancer. PMID:27313763

  9. 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.

  10. 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...

  11. 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...

  12. 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 ...

  13. 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.

  14. 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

  15. Effects of DNA replication on mRNA noise.

    Science.gov (United States)

    Peterson, Joseph R; Cole, John A; Fei, Jingyi; Ha, Taekjip; Luthey-Schulten, Zaida A

    2015-12-29

    There are several sources of fluctuations in gene expression. Here we study the effects of time-dependent DNA replication, itself a tightly controlled process, on noise in mRNA levels. Stochastic simulations of constitutive and regulated gene expression are used to analyze the time-averaged mean and variation in each case. The simulations demonstrate that to capture mRNA distributions correctly, chromosome replication must be realistically modeled. Slow relaxation of mRNA from the low copy number steady state before gene replication to the high steady state after replication is set by the transcript's half-life and contributes significantly to the shape of the mRNA distribution. Consequently both the intrinsic kinetics and the gene location play an important role in accounting for the mRNA average and variance. Exact analytic expressions for moments of the mRNA distributions that depend on the DNA copy number, gene location, cell doubling time, and the rates of transcription and degradation are derived for the case of constitutive expression and subsequently extended to provide approximate corrections for regulated expression and RNA polymerase variability. Comparisons of the simulated models and analytical expressions to experimentally measured mRNA distributions show that they better capture the physics of the system than previous theories. PMID:26669443

  16. 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

  17. 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

  18. Visualization of mRNA translation in living cells

    OpenAIRE

    RODRIGUEZ, ALEXIS J.; Shenoy, Shailesh M; Singer, Robert H.; Condeelis, John

    2006-01-01

    The role of mRNA localization is presumably to effect cell asymmetry by synthesizing proteins in specific cellular compartments. However, protein synthesis has never been directly demonstrated at the sites of mRNA localization. To address this, we developed a live cell method for imaging translation of β-actin mRNA. Constructs coding for β-actin, containing tetracysteine motifs, were transfected into C2C12 cells, and sites of nascent polypeptide chains were detected using the biarsenial dyes ...

  19. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis: interferon-beta treatment increases IL-10 mRNA expression while reducing IL-23 mRNA expression

    DEFF Research Database (Denmark)

    Krakauer, M.; Sorensen, P.; Khademi, M.; Olsson, T.; Sellebjerg, F.

    2008-01-01

    volunteers served to confirm initial findings. mRNA was analyzed by real-time reverse transcriptase polymerase chain reaction (PCR). RESULTS: We found elevated expression of interleukin (IL)-23 and IL-10 in untreated MS patients. IFN-beta therapy increased IL-10 and decreased IL-23 expression independently...... of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another...

  20. 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)

  1. Measurement of mRNA concentration and mRNA half-life as a function of hormonal treatment

    International Nuclear Information System (INIS)

    The paper discusses the use of molecular hybridization techniques to measure mRNA concentrations in cells and tissues that are particularly useful to the analysis of small amounts of tissue. Two methods are presented for the quantitation of casein mRNA levels: (1) cDNA-excess liquid hybridization; and (2) the dot blot assay. The methods are discussed with some specific examples on the hormonal regulation of casein mRNA in the mammary gland explants. Experimental details are given for the isolation of RNA, DNA labelling techniques, RNA labelling techniques, and molecular hybridization techniques

  2. 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)/...

  3. mRNA redistribution during permanent focal cerebral ischemia.

    Science.gov (United States)

    Lewis, Monique K; Jamison, Jill T; Dunbar, Joseph C; DeGracia, Donald J

    2013-12-01

    Translation arrest occurs in neurons following focal cerebral ischemia and is irreversible in penumbral neurons destined to die. Following global cerebral ischemia, mRNA is sequestered away from 40S ribosomal subunits as mRNA granules, precluding translation. Here, we investigated mRNA granule formation using fluorescence in situ histochemistry out to 8 h permanent focal cerebral ischemia using middle cerebral artery occlusion in Long Evans rats with and without diabetes. Neuronal mRNA granules colocalized with PABP, HuR, and NeuN, but not 40S or 60S ribosomal subunits, or organelle markers. The volume of brain with mRNA granule-containing neurons decreased exponentially with ischemia duration, and was zero after 8 h permanent focal cerebral ischemia or any duration of ischemia in diabetic rats. These results show that neuronal mRNA granule response has a limited range of insult intensity over which it is expressed. Identifying the limits of effective neuronal stress response to ischemia will be important for developing effective stroke therapies. PMID:24323415

  4. 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.

  5. Sequence analysis of bone morphogenetic protein receptor type II mRNA from ascitic and nonascitic commercial broilers.

    Science.gov (United States)

    Cisar, C R; Balog, J M; Anthony, N B; Donoghue, A M

    2003-10-01

    Ascites syndrome, also known as pulmonary hypertension syndrome (PHS), is a common metabolic disorder in rapidly growing meat-type chickens. Environmental factors, such as cold, altitude, and diet, play significant roles in development of the disease, but there is also an important genetic component to PHS susceptibility. The human disease familial primary pulmonary hypertension (FPPH) is similar to PHS in broilers both genetically and physiologically. Several recent studies have shown that mutations in the bone morphogenetic protein receptor type II (BMPR2) gene are a cause of FPPH in humans. To determine whether mutations in the chicken BMPR2 gene play a similar role in PHS susceptibility, BMPR-II mRNA from ascitic and nonascitic commercial broilers were sequenced and compared with the published Leghorn chicken BMPR-II mRNA sequence. Fourteen single nucleotide polymorphisms (SNP) were identified in the commercial broiler BMPR-II mRNA. No mutations unique to ascites-susceptible broilers were present in the coding, 5' untranslated or 3' untranslated regions of BMPR-II mRNA. The twelve SNP present within the coding region of BMPR-II mRNA were synonymous substitutions and did not alter the BMPR-II protein sequence. In addition, analysis of BMPR2 gene expression by reverse transcriptase-PCR indicated that there were no differences in BMPR-II mRNA levels in ascitic and nonascitic birds. Therefore, it appears unlikely that mutations in the BMPR2 gene were responsible for susceptibility to PHS in these commercial broilers. PMID:14601724

  6. 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.

  7. 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

  8. 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...

  9. 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

  10. Dynamic proteomics emphasizes the importance of selective mRNA translation and protein turnover during Arabidopsis seed germination.

    Science.gov (United States)

    Galland, Marc; Huguet, Romain; Arc, Erwann; Cueff, Gwendal; Job, Dominique; Rajjou, Loïc

    2014-01-01

    During seed germination, the transition from a quiescent metabolic state in a dry mature seed to a proliferative metabolic state in a vigorous seedling is crucial for plant propagation as well as for optimizing crop yield. This work provides a detailed description of the dynamics of protein synthesis during the time course of germination, demonstrating that mRNA translation is both sequential and selective during this process. The complete inhibition of the germination process in the presence of the translation inhibitor cycloheximide established that mRNA translation is critical for Arabidopsis seed germination. However, the dynamics of protein turnover and the selectivity of protein synthesis (mRNA translation) during Arabidopsis seed germination have not been addressed yet. Based on our detailed knowledge of the Arabidopsis seed proteome, we have deepened our understanding of seed mRNA translation during germination by combining two-dimensional gel-based proteomics with dynamic radiolabeled proteomics using a radiolabeled amino acid precursor, namely [(35)S]-methionine, in order to highlight de novo protein synthesis, stability, and turnover. Our data confirm that during early imbibition, the Arabidopsis translatome keeps reflecting an embryonic maturation program until a certain developmental checkpoint. Furthermore, by dividing the seed germination time lapse into discrete time windows, we highlight precise and specific patterns of protein synthesis. These data refine and deepen our knowledge of the three classical phases of seed germination based on seed water uptake during imbibition and reveal that selective mRNA translation is a key feature of seed germination. Beyond the quantitative control of translational activity, both the selectivity of mRNA translation and protein turnover appear as specific regulatory systems, critical for timing the molecular events leading to successful germination and seedling establishment. PMID:24198433

  11. 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?

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. Current stabilizer

    International Nuclear Information System (INIS)

    A current stabilizer for supplying magnetic lens of β-monochromator at the electron energy up to 1 MeV is described. Stabilization method with use of reference high-stabilized source of direct voltage with switching-in loading (monochromator coil) to circuit of negative feedback of direct-current amplifier with high gain is chosen for stabilization of direct current. The range of current regulation is 0.5 A at available power up to 15 W. Current instability during 10 hour continuous work does not exceed 10-4% that makes it possible to provide instability of electron energy at the monochromator exit using 90Sr+90Y β-nucleide of not more than 10 -4% and number of electrons 2.5x10-4% respectively

  17. 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 the...

  18. Ionizing radiation and bacterial challenge alter splenic cytokine gene expression

    International Nuclear Information System (INIS)

    Irradiation increases susceptibility to bacterial infection. Exogenous proinflammatory cytokines can alter the response of mice to γradiation, but the role of endogenous inflammatory cytokines after bacterial infection in irradiated animals is not known. Gene expression of hematopoietic (GM-CSF) and proinflammatory (IL-1β, IL-6 and TNF-α) cytokines were examined in spleens of B6D2F1/J female mice after irradiation alone (1.0- and 7.0-Gy), and after irradiation followed by Klebsiella pneumoniae s.c. challenge 4 days postirradiation by using the reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot hybridization. At 4, 8, and 24 h after bacterial challenge in 7.0-Gy-irradiated mice, GM-CSF mRNA increased (p<0.05). TNF-α mRNA in irradiated mice were slightly decreased, whereas after bacterial challenge, TNF-α mRNA elevated at 30 h in 7.0-Gy-irradiated mice; at 4, and 8 h in 1.0-Gy-irradiated mice, and at 1 h in sham-irradiated mice (p<0.05). IL-6 mRNA displayed a biphasic response in 7.0-Gy-irradiated mice, and, after bacterial challenge, in both irradiated mice (1.0- and 7.0-Gy) and sham-irradiated mice. IL-1β mRNA remained at or below normal for 8 h and increased at 24 h after bacterial challenge on day 4 in 7.0-Gy-irradiated mice. These results indicate that sublethal gamma radiation alters the patterns of the hematopoietic and proinflammatory cytokine responses to bacterial challenge in vivo. Consequently, treatment protocols may need to take into account changes in cytokine gene responses to resolve infection after irradiation. (author)

  19. Humoral immunity to human breast cancer: antigen definition and quantitative analysis of mRNA expression.

    Science.gov (United States)

    Scanlan, M J; Gout, I; Gordon, C M; Williamson, B; Stockert, E; Gure, A O; Jäger, D; Chen, Y T; Mackay, A; O'Hare, M J; Old, L J

    2001-03-30

    The ability of the immune system to recognize structurally altered, amplified or aberrantly expressed proteins can be used to identify molecules of etiologic relevance to cancer and to define targets for cancer immunotherapy. In the current study, ninety-four distinct antigens reactive with serum IgG from breast cancer patients were identified by immunoscreening breast cancer-derived cDNA expression libraries (SEREX). A serological profile was generated for each antigen on the basis of reactivity with allogeneic sera from normal individuals and cancer patients, and mRNA expression profiles for coding sequences were assembled based upon the tissue distribution of expressed sequence tags, Northern blots and real-time RT-PCR. Forty antigens reacted exclusively with sera from cancer patients. These included well-characterized tumor antigens, e.g. MAGE-3, MAGE-6, NY-ESO-1, Her2neu and p53, as well as newly-defined breast cancer antigens, e.g. kinesin 2, TATA element modulatory factor 1, tumor protein D52 and MAGE D, and novel gene products, e.g. NY-BR-62, NY-BR-75, NY-BR-85, and NY-BR-96. With regard to expression profiles, two of the novel gene products, NY-BR-62 and NY-BR-85, were characterized by a high level of testicular mRNA expression, and were overexpressed in 60% and 90% of breast cancers, respectively. In addition, mRNA encoding tumor protein D52 was overexpressed in 60% of breast cancer specimens, while transcripts encoding SNT-1 signal adaptor protein were downregulated in 70% of these cases. This study adds to the growing list of breast cancer antigens defined by SEREX and to the ultimate objective of identifying the complete repertoire of immunogenic gene products in human cancer (the cancer immunome). PMID:12747765

  20. 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...... mRNA 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...

  1. Stability of the aryl hydrocarbon receptor and its regulated genes in the low activity variant of Hepa-1 cell line.

    Science.gov (United States)

    Humphrey-Johnson, Andria; Abukalam, Rawia; Eltom, Sakina E

    2015-03-01

    We examined the expression kinetics of some of the aryl hydrocarbon receptor (AhR)-regulated genes in LA1 variant cells compared to wild type (WT) Hepa-1 mouse hepatoma cell lines, and we investigated the stability of AhR protein as a key step in the function of this receptor. Treatment of both cell types with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in increased CYP1A1 and CYP1B1 mRNA with a subsequent down regulation of AhR. We show here that co-treatment with transcription inhibitor actinomycin D (ActD) has reversed the TCDD-induced depletion of AhR protein in WT. However, the proteolytic degradation of AhR in absence of TCDD was significantly higher in LA1 cells than in WT, and ActD treatment reduced this loss. Induction of CYP1A1 and CYP1B1 mRNA by TCDD in WT cells each exhibited bursts of activity in the initial hour which were about 3-fold greater than in LAI cells. The induced mRNA levels in LA1 exhibited a slow and sustained increase approximating the WT levels by 20h. The induction of two other AhR-regulated genes also showed comparable turnover differences between the two types of cell. Thus, altered regulation of the AhR responsive genes in LA1 may result from a difference in AhR stability. PMID:25637755

  2. 3'-UTR-located inverted Alu repeats facilitate mRNA translational repression and stress granule accumulation.

    Science.gov (United States)

    Fitzpatrick, Terry; Huang, Sui

    2012-07-01

    Alu repeats within human genes may potentially alter gene expression. Here, we show that 3'-UTR-located inverted Alu repeats significantly reduce expression of an AcGFP reporter gene. Mutational analysis demonstrates that the secondary structure, but not the primary nucleotide sequence, of the inverted Alu repeats is critical for repression. The expression levels and nucleocytoplasmic distribution of reporter mRNAs with or without 3'-UTR inverted Alu repeats are similar; suggesting that reporter gene repression is not due to changes in mRNA levels or mRNA nuclear sequestration. Instead, reporter gene mRNAs harboring 3'-UTR inverted Alu repeats accumulate in cytoplasmic stress granules. These findings may suggest a novel mechanism whereby 3'-UTR-located inverted Alu repeats regulate human gene expression through sequestration of mRNAs within stress granules. PMID:22688648

  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. 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

  5. Recruitment of Nanos to hunchback mRNA by Pumilio

    OpenAIRE

    Sonoda, Junichiro; Wharton, Robin P.

    1999-01-01

    Translational regulation of hunchback (hb) mRNA is essential for posterior patterning of the Drosophila embryo. This regulation is mediated by sequences in the 3′-untranslated region of hb mRNA (the Nanos response elements or NREs), as well as two trans-acting factors—Nanos and Pumilio. Pumilio recognizes the NREs via a conserved binding motif. The mechanism of Nanos action has not been clear. In this report we use protein–protein and protein–RNA interaction assays in yeast and in vitro to sh...

  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. Multiple scenarios of bentonite alteration

    International Nuclear Information System (INIS)

    Performance assessment for TRU waste repositories has shown that soluble and poorly sorbing nuclides such as I-129 and C-14 dominate the dose. These nuclides are expected to migrate with groundwater flow, hence hydraulic conditions and their evolution with time in the repository are key issues for repository safety. Cementitious material will be used for waste packaging, backfilling and structural material in a TRU waste repository. Bentonite is also expected to be used for some TRU wastes to provide the function of a hydraulic barrier in the disposal system. There is concern that the coexistence of cementitious material and bentonite cause the alteration of smectite due to interaction with hyperalkaline leachates and consequent deleterious perturbation of the function of bentonite as a hydraulic barrier. Many research studies have been performed to identify possible mechanisms of cement-bentonite interaction. However, uncertainties still exist in our understanding of the precise chemical scheme of bentonite alteration in highly alkaline conditions, especially the space and time variation of secondary mineral occurrences. In order to reflect this uncertainty, multiple scenarios of bentonite alteration were developed based on the possible mineralogical changes derived from knowledge of both experiments and observation of natural systems. It was focused that the mineral reaction involving hyperalkaline fluids would thermodynamically depend on the variable chemical condition in bentonite buffer and that kinetics would be important as well as thermodynamic stability in controlling their occurrence, i.e., the kinetic controls may operate to remain metastable minerals over the long term. The mineralogical consequences of the interaction between clays and alkaline fluids are summarized as follows. Clay → C-S-H gel and other solids which can rapidly precipitate. Clay and gel → illite. Clay and gel → metastable zeolite. Clay and gel → metastable zeolite → stable

  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. 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 ...

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. Mucin 1 and cytokines mRNA in endometrium of dairy cows with postpartum uterine disease or repeat breeding.

    Science.gov (United States)

    Kasimanickam, R; Kasimanickam, V; Kastelic, J P

    2014-04-15

    Mucin (MUC) 1 is an inducible innate immune effector, an important component of defense against bacterial invasion, and is linked with infertility in humans. The objectives were to evaluate messenger RNA (mRNA) expression of MUC1 and cytokine genes in the endometrium of cows with various postpartum uterine inflammatory conditions or with a history of repeat breeding. Endometrial samples were collected from lactating dairy cows diagnosed with metritis (n = 4), endometritis (n = 4), subclinical endometritis (n = 4), or no uterine pathology (normal; n = 4). In addition, endometrial samples were collected from repeat breeder cows with (n = 4) or without (n = 4) subclinical endometritis, and unaffected cows (n = 4). Quantitative polymerase chain reaction was used to determine mRNA abundances of MUC1, Toll-like receptor (TLR) 4, interleukin (IL) 1β, IL6, IL8, tumor necrosis factor (TNF) α, insulin-like growth factor (IGF) 1, and IGF-binding protein (BP) 2. The mRNA expressions were significantly greater for cows with metritis and clinical endometritis compared with cows with no uterine inflammation, except for IL6. However, mRNA expressions for these target genes were not different for cows with subclinical endometritis, compared with cows without uterine inflammation, except for IL1β and TNFα mRNA (P cows with subclinical endometritis compared with normal cows. However, in repeat breeder cows without subclinical endometritis, only expressions of MUC1, IGF1, and IGF BP2 were greater compared with normal cows (P cows. Perhaps, these altered gene expressions contribute to endometrial insufficiency and consequently pregnancy wastage. PMID:24576715

  15. 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.

  16. Recruitment of Nanos to hunchback mRNA by Pumilio

    Science.gov (United States)

    Sonoda, Junichiro; Wharton, Robin P.

    1999-01-01

    Translational regulation of hunchback (hb) mRNA is essential for posterior patterning of the Drosophila embryo. This regulation is mediated by sequences in the 3′-untranslated region of hb mRNA (the Nanos response elements or NREs), as well as two trans-acting factors—Nanos and Pumilio. Pumilio recognizes the NREs via a conserved binding motif. The mechanism of Nanos action has not been clear. In this report we use protein–protein and protein–RNA interaction assays in yeast and in vitro to show that Nanos forms a ternary complex with the RNA-binding domain of Pumilio and the NRE. Mutant forms of the NRE, Nos, and Pum that do not regulate hb mRNA normally in embryos do not assemble normally into a ternary complex. In particular, recruitment of Nos is dependent on bases in the center of the NRE, on the carboxy-terminal Cys/His domain of Nos, and on residues in the eighth repeat of the Pum RNA-binding domain. These residues differ in a closely related human protein that also binds to the NRE but cannot recruit Drosophila Nos. Taken together, these findings suggest models for how Nos and Pum collaboratively target hb mRNA. More generally, they suggest that Pum-like proteins from other species may also act by recruiting cofactors to regulate translation. PMID:10541556

  17. 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

  18. Regulation of mRNA Trafficking by Nuclear Pore Complexes

    Directory of Open Access Journals (Sweden)

    Amandine Bonnet

    2014-09-01

    Full Text Available Over the last two decades, multiple studies have explored the mechanisms governing mRNA export out of the nucleus, a crucial step in eukaryotic gene expression. During transcription and processing, mRNAs are assembled into messenger ribonucleoparticles (mRNPs. mRNPs are then exported through nuclear pore complexes (NPCs, which are large multiprotein assemblies made of several copies of a limited number of nucleoporins. A considerable effort has been put into the dissection of mRNA export through NPCs at both cellular and molecular levels, revealing the conserved contributions of a subset of nucleoporins in this process, from yeast to vertebrates. Several reports have also demonstrated the ability of NPCs to sort out properly-processed mRNPs for entry into the nuclear export pathway. Importantly, changes in mRNA export have been associated with post-translational modifications of nucleoporins or changes in NPC composition, depending on cell cycle progression, development or exposure to stress. How NPC modifications also impact on cellular mRNA export in disease situations, notably upon viral infection, is discussed.

  19. Sites of amyloid SAA mRNA production

    International Nuclear Information System (INIS)

    To investigate possible extrahepatic sites of SAA production, male BALB/c mice were given a single 0.5 ml injection of either 10% casein or lipopolysaccharide (LPS; 100 mg/ml). Twenty hours after injection, RNA was extracted from liver, kidney, adrenal, testis, brain, spleen, skeletal muscle, heart, lung and small intestine. Northern blots of total RNA were hybridized with nick-translated 32P-labeled cDNA probes (length approximately 150 base pairs) corresponding to an homologous region of the three known SAA genes. Both casein and LPS elevated the mRNA in liver to about 200-fold above control levels; mRNA was elevated in adrenals from O to approximately 2% of liver. mRNA in some other tissues responded only to LPS injection: levels in kidney reached 15% of liver; pituitary, testis and brain reached 0.02 to 0.5% of liver; no apoSAA mRNA was detected in heart, skeletal muscle, lung, spleen or small intestine. Thus, some organs other than liver appear to have operational genes for apoSAA. The expression of apoSAA genes in different tissues is shared with other apoproteins; it remains to be seen whether all three or only selected genes are transcribed and translated in different tissues

  20. 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

  1. 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.

  2. 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

  3. 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

  4. Altered adipocyte structure and function in nutritionally programmed microswine offspring.

    Science.gov (United States)

    DuPriest, E A; Kupfer, P; Lin, B; Sekiguchi, K; Morgan, T K; Saunders, K E; Chatkupt, T T; Denisenko, O N; Purnell, J Q; Bagby, S P

    2012-06-01

    Adipose tissue (AT) dysfunction links obesity of any cause with cardiometabolic disease, but whether early-life nutritional deficiency can program adipocyte dysfunction independently of obesity is untested. In 3-5-month-old juvenile microswine offspring exposed to isocaloric perinatal maternal protein restriction (MPR) and exhibiting accelerated prepubertal fat accrual without obesity, we assessed markers of acquired obesity: adiponectin and tumor necrosis factor (TNF)-α messenger ribonucleic acid (mRNA) levels and adipocyte size in intra-abdominal (ABD-AT) and subcutaneous (SC-AT) adipose tissues. Plasma cortisol, leptin and insulin levels were measured in fetal, neonatal and juvenile offspring. In juvenile low-protein offspring (LPO), adipocyte size in ABD-AT was reduced 22% (P = 0.011 v. controls), whereas adipocyte size in SC-AT was increased in female LPO (P = 0.05) and normal in male LPO; yet, adiponectin mRNA in LPO was low in both sexes and in both depots (P programming of adipocyte size and gene expression and subtly altered glucose homeostasis. Reduced adiponectin mRNA and adipokine dysregulation in juvenile LPO following accelerated growth occurred independently of obesity, adipocyte hypertrophy or inflammatory markers; thus, perinatal MPR and/or growth acceleration can alter adipocyte structure and disturb adipokine homeostasis in metabolically adverse patterns predictive of enhanced disease risk. PMID:25102010

  5. 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...

  6. 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...

  7. 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.

  8. 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.

  9. 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.

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

    International Nuclear Information System (INIS)

    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

  11. 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.

  12. Colorectal cancer cell lines made resistant to SN38-and Oxaliplatin: Roles of altered ion transporter function in resistance?

    DEFF Research Database (Denmark)

    Sandra, Christensen; Jensen, Niels Frank; Stoeckel, Johanne Danmark; Belling, Kirstine C.; Romer, Maria Unni; Gupta, Ramneek; Brunner, Nils; Pedersen, Stine Helene Falsig; Stenvang, Jan

    2013-01-01

    resistance in HCT-116, HT-29 and LoVo cells. Microarray analysis and qPCR validation showed that mRNA expression of glutamate transporters SLC1A1 and SLC1A3 were markedly altered in resistant cells. Remarkably, mRNA levels of SLC1A3 were increased by ~40-and ~2500-fold in SN38-and Oxp-resistant HT29 cells...

  13. 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.

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

    International Nuclear Information System (INIS)

    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

  15. 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

  16. Frequency stability

    Czech Academy of Sciences Publication Activity Database

    Kroupa, Věnceslav František

    Bratislava: Department of Radio and Electronics, FEI STU, 2006 - (Píš, P.; Krajčušková, Z.), I.1-I.9 ISBN 80-227-2388-6. [ Radio elektronika 2006. Conference proceedings. Bratislava (SK), 25.04.2006-26.04.2006] R&D Projects: GA ČR(CZ) GA102/05/0852 Institutional research plan: CEZ:AV0Z20670512 Keywords : noise * frequency stability * frequency control Subject RIV: JW - Navigation, Links, Detection ; Counter-Measures

  17. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

    Holm, Christian; Nyvold, C.G.; Paludan, Søren Riis; Thomsen, A.R.; Hokland, Marianne

    2006-01-01

    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...

  18. Collagen mRNA levels changes during colorectal cancer carcinogenesis

    DEFF Research Database (Denmark)

    Skovbjerg, Hanne; Anthonsen, Dorit; Lothe, Inger M B;

    2009-01-01

    BACKGROUND: Invasive growth of epithelial cancers is a complex multi-step process which involves dissolution of the basement membrane. Type IV collagen is a major component in most basement membranes. Type VII collagen is related to anchoring fibrils and is found primarily in the basement membrane...... zone of stratified epithelia. Immunohistochemical studies have previously reported changes in steady-state levels of different alpha(IV) chains in several epithelial cancer types. In the present study we aimed to quantitatively determine the mRNA levels of type IV collagen (alpha1/alpha 4/alpha 6) and...... type VII collagen (alpha1) during colorectal cancer carcinogenesis. METHODS: Using quantitative RT-PCR, we have determined the mRNA levels for alpha1(IV), alpha 4(IV), alpha 6(IV), and alpha1(VII) in colorectal cancer tissue (n = 33), adenomas (n = 29) and in normal tissue from the same individuals. In...

  19. 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 ...

  20. 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

  1. Heat-shock-induced refolding entails rapid degradation of bsrG toxin mRNA by RNases Y and J1.

    Science.gov (United States)

    Jahn, Natalie; Brantl, Sabine

    2016-02-01

    Gene regulation accomplished by alternative folding of an mRNA is a widely used mechanism. Classical examples are the various transcriptional attenuation mechanisms that employ, for example, leader peptide translation, or binding of a modified protein, an uncharged tRNA or an antisense RNA to the 5' untranslated region of an mRNA. With the discovery of transcriptional and translational riboswitches, it became clear that small metabolites or even metal ions can also alter RNA secondary structures and, hence, gene expression. In addition, biophysical factors like temperature can affect RNA folding, as exemplified by RNA thermometers. We have investigated in detail the type I toxin-antitoxin system bsrG/SR4 from Bacillus subtilis. The antitoxin SR4 is a cis-encoded regulatory RNA that neutralizes BsrG toxin action. SR4 prevents toxin expression by promoting degradation of the toxin mRNA and inhibiting its translation. In addition, upon temperature shock the amount of toxin mRNA decreases significantly. Here, we demonstrate that heat shock induces a refolding in the central region of the toxin mRNA that makes it more accessible to degradation by RNases Y and J1. Furthermore, we show that BsrG might play a role at the onset of stationary phase, when the antitoxin SR4 can no longer prevent toxin synthesis. PMID:26802042

  2. mRNA and microRNA analysis reveals modulation of biochemical pathways related to addiction in the ventral tegmental area of methamphetamine self-administering rats

    OpenAIRE

    Bosch, P J; Benton, M C; Macartney-Coxson, D; Kivell, B M

    2015-01-01

    Background Methamphetamine is a highly addictive central nervous system stimulant with increasing levels of abuse worldwide. Alterations to mRNA and miRNA expression within the mesolimbic system can affect addiction-like behaviors and thus play a role in the development of drug addiction. While many studies have investigated the effects of high-dose methamphetamine, and identified neurotoxic effects, few have looked at the role that persistent changes in gene regulation play following methamp...

  3. 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

  4. 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

  5. Hormonal change and cytokine mRNA expression in peripheral blood mononuclear cells during the development of canine autoimmune thyroiditis.

    Science.gov (United States)

    Choi, E-W; Shin, I-S; Bhang, D-H; Lee, D-H; Bae, B-K; Kang, M-S; Kim, D-Y; Hwang, C-Y; Lee, C-W; Youn, H-Y

    2006-10-01

    To elucidate the hormonal change and alteration in cytokine expression in peripheral blood mononuclear cells (PBMC) during the early stage of autoimmune thyroiditis, we have developed a canine model of this disease, in which normal dogs were immunized with bovine thyroglobulin (Tg) and/or canine thyroid extract. Serum samples were collected weekly, anti-canine Tg antibody was measured by enzyme-linked immunosorbent assay (ELISA) and thyroid stimulating hormone (TSH) and total T4 levels by radioimmunoassay. We also assayed T lymphocyte proliferation in response to Tg, as well as measuring cytokine mRNA by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). All six dogs immunized with bovine Tg had both canine Tg autoantibody and anti-T4 antibody. When the sample from the highest TgAA titre time-point was compared with baseline the expression of mRNA encoding the Th1-type cytokine such as interferon (IFN)-gamma, interleukin (IL)-18 and IL-15 was increased during the development of autoimmune thyroiditis. Expression of the Th2-type cytokine, IL-6 showed minimal change and IL-4 expression was not detected in any of the samples. Expression of the T suppressive cytokine, IL-10 and transforming growth factor (TGF)-beta was increased in the presence of antigen stimulation. These findings suggest that, although autoimmune thyroiditis is an organ-specific autoimmune disease, systemic cytokine mRNA expression is also changed. PMID:16968404

  6. 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

  7. 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 pro...... nucleotide features of 16S-like ribosomal RNA sequences and related to mechanisms for maintaining the correct reading frame by the ribosome. (C) 1996 Wiley-Liss, Inc....

  8. 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...

  9. Regulation of mRNA translation influences hypoxia tolerance

    International Nuclear Information System (INIS)

    Hypoxia is a heterogenous but common characteristic of human tumours and poor oxygenation is associated with poor prognosis. We believe that the presence of viable hypoxic tumor cells reflects in part an adaptation and tolerance of these cells to oxygen deficiency. Since oxidative phosphorylation is compromized during hypoxia, adaptation may involve both the upregulation of glycolysis as well as downregulation of energy consumption. mRNA translation is one of the most energy costly cellular processes, and we and others have shown that global mRNA translation is rapidly inhibited during hypoxia. However, some mRNAs, including those coding for HIF-1 α and VEGF, remain efficiently translated during hypoxia. Clearly, the mechanisms responsible for the overall inhibition of translation during hypoxia does not compromize the translation of certain hypoxia-induced mRNA species. We therefore hypothesize that the inhibition of mRNA translation serves to promote hypoxia tolerance in two ways: i) through conservation of energy and ii) through differential gene expression involved in hypoxia adaptation. We have recently identified two pathways that are responsible for the global inhibition of translation during hypoxia. The phosphorylation of the eukaryotic initiation factor eIF2 α by the ER resident kinase PERK results in down-regulation of protein synthesis shortly after the onset of hypoxia. In addition, the initiation complex eIF4F is disrupted during long lasting hypoxic conditions. The identification of the molecular pathways responsible for the inhibition of overall translation during hypoxia has rendered it possible to investigate their importance for hypoxia tolerance. We have found that mouse embryo fibroblasts that are knockout for PERK and therefore not able to inhibit protein synthesis efficiently during oxygen deficiency are significantly less tolerant to hypoxia than their wildtype counterparts. We are currently also investigating the functional significance

  10. On the dimensionality of ecological stability.

    Science.gov (United States)

    Donohue, Ian; Petchey, Owen L; Montoya, José M; Jackson, Andrew L; McNally, Luke; Viana, Mafalda; Healy, Kevin; Lurgi, Miguel; O'Connor, Nessa E; Emmerson, Mark C

    2013-04-01

    Ecological stability is touted as a complex and multifaceted concept, including components such as variability, resistance, resilience, persistence and robustness. Even though a complete appreciation of the effects of perturbations on ecosystems requires the simultaneous measurement of these multiple components of stability, most ecological research has focused on one or a few of those components analysed in isolation. Here, we present a new view of ecological stability that recognises explicitly the non-independence of components of stability. This provides an approach for simplifying the concept of stability. We illustrate the concept and approach using results from a field experiment, and show that the effective dimensionality of ecological stability is considerably lower than if the various components of stability were unrelated. However, strong perturbations can modify, and even decouple, relationships among individual components of stability. Thus, perturbations not only increase the dimensionality of stability but they can also alter the relationships among components of stability in different ways. Studies that focus on single forms of stability in isolation therefore risk underestimating significantly the potential of perturbations to destabilise ecosystems. In contrast, application of the multidimensional stability framework that we propose gives a far richer understanding of how communities respond to perturbations. PMID:23419041

  11. 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

  12. 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 ...

  13. 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.

  14. Calcyon mRNA expression in the frontal-striatal circuitry and its relationship to vesicular processes and ADHD

    Directory of Open Access Journals (Sweden)

    Castellanos F Xavier

    2007-07-01

    Full Text Available Abstract Background Calcyon is a single transmembrane protein predominantly expressed in the brain. Very recently, calcyon has been implicated in clathrin mediated endocytosis, a critical component of synaptic plasticity. At the genetic level, preliminary evidence supports an association between attention-deficit/hyperactivity disorder (ADHD and polymorphisms in the calcyon gene. As little is known about the potential role of calcyon in ADHD, animal models may provide important insights into this issue. Methods We examined calcyon mRNA expression in the frontal-striatal circuitry of three-, five-, and ten-week-old Spontaneously Hypertensive Rats (SHR, the most commonly used animal model of ADHD, and Wistar-Kyoto (WKY; the strain from which SHR were derived. As a complement, we performed a co-expression network analysis using a database of mRNA gene expression profiles of multiple brain regions in order to explore potential functional links of calcyon to other genes. Results In all age groups, SHR expressed significantly more calcyon mRNA in the medial prefrontal and orbital frontal cortices than WKY rats. In contrast, in the motor cortex, dorsal striatum and nucleus accumbens, calcyon mRNA expression was only significantly elevated in SHR in younger animals. In both strains, calcyon mRNA levels decreased significantly with age in all regions studied. In the co-expression network analysis, we found a cluster of genes (many of them poorly studied so far strongly connected to calcyon, which may help elucidate its role in the brain. The pair-wise relations of calcyon with other genes support its involvement in clathrin mediated endocytosis and, potentially, some other membrane/vesicular processes. Interestingly, no link was found between calcyon and the dopamine D1 receptor, which was previously shown to interact with the C-terminal of calcyon. Conclusion The results indicate an alteration in calcyon expression within the frontal-striatal circuitry

  15. 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

  16. APOBEC3A is implicated in a novel class of G-to-A mRNA editing in WT1 transcripts.

    Science.gov (United States)

    Niavarani, Ahmadreza; Currie, Erin; Reyal, Yasmin; Anjos-Afonso, Fernando; Horswell, Stuart; Griessinger, Emmanuel; Luis Sardina, Jose; Bonnet, Dominique

    2015-01-01

    Classic deamination mRNA changes, including cytidine to uridine (C-to-U) and adenosine to inosine (A-to-I), are important exceptions to the central dogma and lead to significant alterations in gene transcripts and products. Although there are a few reports of non-classic mRNA alterations, as yet there is no molecular explanation for these alternative changes. Wilms Tumor 1 (WT1) mutations and variants are implicated in several diseases, including Wilms tumor and acute myeloid leukemia (AML). We observed two alternative G-to-A changes, namely c.1303G>A and c.1586G>A in cDNA clones and found them to be recurrent in a series of 21 umbilical cord blood mononuclear cell (CBMC) samples studied. Two less conserved U-to-C changes were also observed. These alternative changes were found to be significantly higher in non-progenitor as compared to progenitor CBMCs, while they were found to be absent in a series of AML samples studied, indicating they are targeted, cell type-specific mRNA editing modifications. Since APOBEC/ADAR family members are implicated in RNA/DNA editing, we screened them by RNA-interference (RNAi) for WT1-mRNA changes and observed near complete reversal of WT1 c.1303G>A alteration upon APOBEC3A (A3A) knockdown. The role of A3A in mediating this change was confirmed by A3A overexpression in Fujioka cells, which led to a significant increase in WT1 c.1303G>A mRNA editing. Non-progenitor CBMCs showed correspondingly higher levels of A3A-mRNA and protein as compared to the progenitor ones. To our knowledge, this is the first report of mRNA modifying activity for an APOBEC3 protein and implicates A3A in a novel G-to-A form of editing. These findings open the way to further investigations into the mechanisms of other potential mRNA changes, which will help to redefine the RNA editing paradigm in both health and disease. PMID:25807502

  17. 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.

  18. Montmorillonite stability - experimental results

    International Nuclear Information System (INIS)

    Document available in extended abstract form only. Several repository concepts for spent nuclear fuel comprise a bentonite buffer which encloses the fuel containers. The function of this bentonite buffer is critically dependent on the properties of the swelling mineral in the bentonite, which typically is montmorillonite in commercial high quality bentonites. The physical and chemical properties of the montmorillonite in bentonites have consequently been studied in various types of analyses in several countries, and the results have been the basis for the design of the bentonite buffer with respect to size, degree of compaction etc (SKB TR-10-47). Significant changes in the montmorillonite structure with time may change the properties of the bentonite buffer and jeopardize the isolating function. In nature, illite is the most common alteration product from montmorillonite, and a 50% alteration reduces, for instance, the swelling pressure by one order of magnitude. Fortunately, the process is very slow at typical repository temperatures, and also in the perspective of repository life-time no significant alteration is expected according to the available kinetic models. The models are, however, based on laboratory batch experiments performed at temperatures, which by necessity are much higher than what is planned for in a repository. Alternatively, the models are based on natural montmorillonite-to-illite alteration in e.g. deep sediments. In both cases there are divergences from repository conditions which limit the arguments for the bentonite buffer stability. The mechanism by which the montmorillonite-to-illite reaction takes place is not fully understood, but the overall process may be represented by the following schematic reaction: Na+ montmorillonite + K+ + Al3+ → illite + SiO2 + Na+. Aluminum may be supplied from the bentonite itself, but potassium has to be transported to the buffer from an external source in order to give a significant alteration in a

  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. Alteration of podocyte phenotype in the urine of women with preeclampsia

    Science.gov (United States)

    Zhai, Tianyue; Furuta, Itsuko; Akaishi, Rina; Ishikawa, Satoshi; Morikawa, Mamoru; Yamada, Takahiro; Koyama, Takahiro; Minakami, Hisanori

    2016-01-01

    Podocyte injury has been suggested to induce phenotypic alteration of glomerular podocytes and accelerate the detachment of podocytes from the glomeruli resulting in podocyturia. However, it is not clear whether podocyte phenotypic alteration occurs in the urine of women with preeclampsia (PE). Seventy-seven and 116 pelleted urine samples from 38 and 18 women at various stages of normal and PE pregnancies, respectively underwent quantitative analysis of podocyte-specific or associated protein mRNA expression, including podocin, nephrin, and synaptopodin using RT-PCR. Significant proteinuria in pregnancy (SPIP) is defined as protein:creatinine ratio (P/Cr, mg/mg) ≥0.27 in the urine supernatant. All three urine-pellet mRNAs expression levels were significantly positively correlated with P/Cr levels, suggesting that podocyturia increased with proteinuria. The podocin:nephrin mRNA ratio (PNR) and synaptopodin:nephrin mRNA ratio (SNR) increased significantly with increasing P/Cr, while the podocin:synaptopodin mRNA ratio (PSR) did not change significantly according to P/Cr, resulting in significantly higher PNR and SNR, but not PSR levels, in urine from PE women with than without SPIP. The PNR, SNR, and PSR in urine from PE women before onset of SPIP were comparable to those from controls. Thus, nephrin mRNA expression was reduced in the podocytes recovered from PE women. PMID:27052160

  2. 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

  3. Chromatoid Body Protein TDRD6 Supports Long 3’ UTR Triggered Nonsense Mediated mRNA Decay

    Science.gov (United States)

    Fanourgakis, Grigorios; Akpinar, Müge; Dahl, Andreas; Jessberger, Rolf

    2016-01-01

    Chromatoid bodies (CBs) are spermiogenesis-specific organelles of largely unknown function. CBs harbor various RNA species, RNA-associated proteins and proteins of the tudor domain family like TDRD6, which is required for a proper CB architecture. Proteome analysis of purified CBs revealed components of the nonsense-mediated mRNA decay (NMD) machinery including UPF1. TDRD6 is essential for UPF1 localization to CBs, for UPF1-UPF2 and UPF1-MVH interactions. Upon removal of TDRD6, the association of several mRNAs with UPF1 and UPF2 is disturbed, and the long 3’ UTR-stimulated but not the downstream exon-exon junction triggered pathway of NMD is impaired. Reduced association of the long 3’ UTR mRNAs with UPF1 and UPF2 correlates with increased stability and enhanced translational activity. Thus, we identified TDRD6 within CBs as required for mRNA degradation, specifically the extended 3’ UTR-triggered NMD pathway, and provide evidence for the requirement of NMD in spermiogenesis. This function depends on TDRD6-promoted assembly of mRNA and decay enzymes in CBs. PMID:27149095

  4. Mitochondrial Polyadenylation Is a One-Step Process Required for mRNA Integrity and tRNA Maturation

    Science.gov (United States)

    Calvo-Garrido, Javier; Maffezzini, Camilla; Felser, Andrea; Wibom, Rolf; Wedell, Anna; Wredenberg, Anna

    2016-01-01

    Polyadenylation has well characterised roles in RNA turnover and translation in a variety of biological systems. While polyadenylation on mitochondrial transcripts has been suggested to be a two-step process required to complete translational stop codons, its involvement in mitochondrial RNA turnover is less well understood. We studied knockdown and knockout models of the mitochondrial poly(A) polymerase (MTPAP) in Drosophila melanogaster and demonstrate that polyadenylation of mitochondrial mRNAs is exclusively performed by MTPAP. Further, our results show that mitochondrial polyadenylation does not regulate mRNA stability but protects the 3' terminal integrity, and that despite a lack of functioning 3' ends, these trimmed transcripts are translated, suggesting that polyadenylation is not required for mitochondrial translation. Additionally, loss of MTPAP leads to reduced steady-state levels and disturbed maturation of tRNACys, indicating that polyadenylation in mitochondria might be important for the stability and maturation of specific tRNAs. PMID:27176048

  5. 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.

  6. 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

  7. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

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

    2006-01-01

    Interleukin-21 is a cytokine with profound impact on the proliferation and differentiation of activated leukocytes of both the innate and adaptive immune system. In experiments in vitro, antigen activation induces IL-21 production in CD4+ T cells. Where, when, and how the proliferative 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...

  8. 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

  9. 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

  10. 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.

  11. 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...

  12. Interaction between the poly(A)-binding protein Pab1 and the eukaryotic release factor eRF3 regulates translation termination but not mRNA decay in Saccharomyces cerevisiae.

    Science.gov (United States)

    Roque, Sylvain; Cerciat, Marie; Gaugué, Isabelle; Mora, Liliana; Floch, Aurélie G; de Zamaroczy, Miklos; Heurgué-Hamard, Valérie; Kervestin, Stephanie

    2015-01-01

    Eukaryotic release factor 3 (eRF3) is implicated in translation termination and also interacts with the poly(A)-binding protein (PABP, Pab1 in yeast), a major player in mRNA metabolism. Despite conservation of this interaction, its precise function remains elusive. First, we showed experimentally that yeast eRF3 does not contain any obvious consensus PAM2 (PABP-interacting motif 2). Thus, in yeast this association is different from the well described interaction between the metazoan factors. To gain insight into the exact function of this interaction, we then analyzed the phenotypes resulting from deleting the respective binding domains. Deletion of the Pab1 interaction domain on eRF3 did not affect general mRNA stability or nonsense-mediated mRNA decay (NMD) pathway and induced a decrease in translational readthrough. Furthermore, combined deletions of the respective interacting domains on eRF3 and on Pab1 were viable, did not affect Pab1 function in mRNA stability and harbored an antisuppression phenotype. Our results show that in Saccharomyces cerevisiae the role of the Pab1 C-terminal domain in mRNA stability is independent of eRF3 and the association of these two factors negatively regulates translation termination. PMID:25411355

  13. 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.

  14. 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

  15. Adiponectin mRNA Expression in the Cat (Felis domesticus

    Directory of Open Access Journals (Sweden)

    Angela L. Lusby

    2010-01-01

    Full Text Available Problem statement: Adiponectin is a hormone expressed from adipose tissue in people, rodents and dogs. Adiponectin has anti-inflammatory action with beneficial effects on cardiovascular health and insulin sensitivity. With increasing fat mass, adiponectin concentrations paradoxically decrease. Adiponectin’s role in metabolism and diabetes mellitus is of interest in feline medicine because cats are susceptible to developing type II diabetes with weight gain. This study determined relative amounts of adiponectin mRNA expression from various body tissues and organs in domestic cats. Approach: Two intact male cats and one intact female cat were evaluated post-mortem. All cats were estimated to be young adults and had lean body conditions. Tissues samples from inguinal subcutaneous adipose, visceral mesenteric adipose, liver, skeletal muscle, cardiac muscle, aorta, stomach fundus, duodenum, pancreas, thyroid gland, adrenal gland (cortex and medulla and renal cortex were collected and frozen. Following RNA extraction, adiponectin mRNA expression of each tissue was detected using Reverse Transcriptase (RT real-time (Q PCR. Results: Visceral adipose tissue had the highest level of expression, averaging 12% higher than subcutaneous adipose. All other tissues had negligible levels of expression compared to adipose samples. Conclusion: This study provided a valuable step for adiponectin research in cats by determining which tissues express this hormone. Cats differ from human beings by expressing higher levels of adiponectin in visceral compared to subcutaneous fat. The metabolic impact of this expression pattern is not known and provides a basis for future research.

  16. 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.

  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. Changes in pro-opiomelanocortin and pre-proenkephalin mRNA levels in the ovine brain during pregnancy, parturition and lactation and in response to oestrogen and progesterone.

    Science.gov (United States)

    Broad, K D; Kendrick, K M; Sirinathsinghji, D J; Keverne, E B

    1993-12-01

    In the female sheep opioids act centrally to influence both oxytocin release and maternal behaviour. We have used in situ hybridization and histochemistry to investigate the changes in mRNA expression of the two opioid precursor genes, pro-opiomelanocortin (POMC) and pre-proenkephalin (PPE), in discrete hypothalamic nuclei as a function of pregnancy, parturition and lactation and following treatment with oestrogen and progesterone. Quantitative in situ hybridization histochemistry demonstrated that POMC mRNA expression in the arcuate nucleus (ARC) decreased at parturition and increased during lactation compared to late pregnant and ovariectomized animals. Oestradiol and progesterone treatments increased POMC mRNA expression compared to ovariectomized controls. Pre-proenkephalin mRNA expression was quantified in three discrete hypothalamic nuclei, the ventromedial nucleus (VMN), the paraventricular nucleus (PVN) and the suprachiasmatic nucleus (SCN). In the VMN, PPE mRNA expression increased during lactation compared to late pregnancy and parturition. Expression levels during late pregnancy and parturition were decreased compared to ovariectomized animals. Oestradiol increased, and progesterone decreased, PPE mRNA levels compared to ovariectomized controls. Combined progesterone followed by oestrogen treatment produced significant increases in PPE mRNA expression. In the PVN, PPE expression increased at parturition compared to late pregnant, lactating and ovariectomized animals. Expression levels in late pregnant animals were decreased compared to lactating or ovariectomized ones. However, sex steroid treatment produced no changes in PPE expression in the PVN. No changes were observed in PPE mRNA expression in the SCN in response to any of the experimental conditions. This data shows that both POMC and PPE mRNA levels are altered in the sheep brain during pregnancy, parturition and lactation and in response to sex steroids, although the direction of the changes is

  19. STAMP alters the growth of transformed and ovarian cancer cells

    International Nuclear Information System (INIS)

    Steroid receptors play major roles in the development, differentiation, and homeostasis of normal and malignant tissue. STAMP is a novel coregulator that not only enhances the ability of p160 coactivator family members TIF2 and SRC-1 to increase gene induction by many of the classical steroid receptors but also modulates the potency (or EC50) of agonists and the partial agonist activity of antisteroids. These modulatory activities of STAMP are not limited to gene induction but are also observed for receptor-mediated gene repression. However, a physiological role for STAMP remains unclear. The growth rate of HEK293 cells stably transfected with STAMP plasmid and overexpressing STAMP protein is found to be decreased. We therefore asked whether different STAMP levels might also contribute to the abnormal growth rates of cancer cells. Panels of different stage human cancers were screened for altered levels of STAMP mRNA. Those cancers with the greatest apparent changes in STAMP mRNA were pursued in cultured cancer cell lines. Higher levels of STAMP are shown to have the physiologically relevant function of reducing the growth of HEK293 cells but, unexpectedly, in a steroid-independent manner. STAMP expression was examined in eight human cancer panels. More extensive studies of ovarian cancers suggested the presence of higher levels of STAMP mRNA. Lowering STAMP mRNA levels with siRNAs alters the proliferation of several ovarian cancer tissue culture lines in a cell line-specific manner. This cell line-specific effect of STAMP is not unique and is also seen for the conventional effects of STAMP on glucocorticoid receptor-regulated gene transactivation. This study indicates that a physiological function of STAMP in several settings is to modify cell growth rates in a manner that can be independent of steroid hormones. Studies with eleven tissue culture cell lines of ovarian cancer revealed a cell line-dependent effect of reduced STAMP mRNA on cell growth rates. This cell

  20. Incore stabilizer

    International Nuclear Information System (INIS)

    The present invention provides an incore stabilizer for preventing collision of neutron guide tubes with each other or with other equipments in a BWR-type reactor even if oscillations in one identical phase such as earthquakes are exerted. Namely, a first connection member connected to a reactor core shroud is inserted between a tuning fork-like branched second connecting members connected to a neutron instrumentation guide tube. A pin is pierced to the second connecting member from above through a pin hole, to connect both of the connection members by a nut disposed below. In this case, double eccentric sleeves are fitted to the first connection member and it is inserted to the second connection members, and they are connected by using pins and nuts. With such a constitution, even if the pin hole of the second connection member is eccentrically deviated, the eccentricity can easily be absorbed by rotating the double eccentric sleeves. As a result, pins can be attached easily. In addition, abrasion resistance of the inner surface of the pin hole is enhanced by using a material of high abrasion resistance for the eccentric sleeves. (I.S.)

  1. 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

  2. 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

  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. 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

  5. Imaging oncogene MRNA for early diagnosis of cancer

    International Nuclear Information System (INIS)

    In 2005 breast cancer attacked approximately 210 000 and took the lives of 40 000 women in the United States of America. Standard screening with breast self-examination and mammography, recommended to minimize breast cancer morbidity, miss 10-20% (up to 40% in young women) of breast cancer. Moreover, if an abnormality is found, an invasive diagnostic procedure is required to determine if the breast contains hyperplasia, atypical, or cancer. Approximately 80% of invasive procedures detect a benign pathology. Breast cancer cells express a gene product, cell surface receptor VPAC1, so named because the endogenous growth hormones vasoctive intestinal peptide and pituitary adenylate cylcase activating peptide bind to VPAC1 receptors with high affinity. VPAC1 receptors are overexpressed on 100% of human breast cancer cells. Cyclin D1 is a key regulator of the cell cycle and overexpressed in 50-80% of breast cells, whereas it is low or absent in normal breast tissues. The human breast cancer cell line MCF7 displays elevated levels of CCND1 mRNA, encoding cyclin D1, and an elevated level of IGF1R mRNA, encoding insulin-like growth factor 1 receptor. The authors hypothesize that 99mTc or 64Cu labelled VIP analogues, or a peptide nucleic acid (PNA) chimera specific for IGFI receptor and CCND1 mRNA, will permit the early imaging of breast cancer by planar, SPECT or PET methods. The authors synthesized, characterized and administered i.v. 99mTc-AcGly-D (Ala)-Gly-Gly-aminobutanoyl-VIP (TP3654), 64Cu diaminodithiol-aminobutanoyl-VIP (TP3982), 99mTc-AcGly-D(Ala)- Gly-Gly-PNA-D(Cys-ser-lys-Cys) chimera (WT4185) and 64Cu-DOTA-PNA-D(cys-serlys- cys) WT4348. A 12mer, CTGGTGTTCCAT nucleic acid sequence served as the PNA and 3 or 4 mer mismatched PNAs as negative controls. Using 99mTc-TP3654, the authors have successfully imaged human breast cancers not detectable by current modalities. In athymic, nude mice bearing MCF-7 human breast cancer xenorgraphs, 64Cu-TP3982 tumour uptake was

  6. 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.

  7. 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

  8. 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. PMID:27068463

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

    Directory of Open Access Journals (Sweden)

    Dmitry Zabezhinsky

    2016-04-01

    Full Text Available 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.

  10. 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.

  11. Chronic toxicity of pesticides to the mRNA expression levels of metallothioneins and cytochrome P450 1A genes in rainbow trout.

    Science.gov (United States)

    Ceyhun, Saltuk Bugrahan; Aksakal, Ercüment; Kirim, Birsen; Atabeyoglu, Kübra; Erdogan, Orhan

    2012-03-01

    The hazardous effects of pesticides on various metabolic pathways are a great problem for environmental health and should be well determined. In the present study, the authors treated rainbow trout with 0.6 μg/L deltamethrin for 28 days and 1.6 mg/L 2,2-dichlorovinyl dimethyl phosphate for 21 days. After this time period, the authors observed alterations in mRNA expression levels of MT-A, MT-B and CYP-1A. Chronic exposure to low levels of pesticides may have a more significant effect on fish populations than acute poisoning. While both pesticides caused a significant increase on mRNA levels of MT-A and CYP-1A, MT-B mRNA levels were increased significantly only upon deltamethin administration. The significant increase in mRNA levels of the corresponding genes may be considered as a defence mechanism in addition to the antioxidants against oxidative stress, as well as a detoxification mechanism against adverse effects of pesticides. PMID:21665904

  12. Transport of Β-globin mRNA from nuclei of friend erythroleukemia cells - Reversible redox-dependent inhibition of mRNA transport

    International Nuclear Information System (INIS)

    An in vitro assay system for routine analysis of the release of Β-globin mRNA from isolated nuclei of Friend erythroleukemia cells is described. By direct blotting of the released material, hybridization and densitometric scanning of the autoradiographs, the rate of mRNA release was quantified. The degradation of mRNA was prevented by addition of the placental ribonuclease inhibitor RNasin to the incubation medium. The oxidizing sulfhydryl reagent o-iodosobenzoate, in contrast to the non-oxidizing reagent iodoacetate, inhibited Β-globin mRNA transport from the isolated nuclei. The inhibition could be reversed by the reducing agent dithiothreitol indicating that mRNA transport is affected by thiol-disulfide conversions. This observation might be useful for further elucidation of the basic mechanism of RNA transport

  13. 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, S. G.;

    2011-01-01

    women). Expressions of androgen receptor (AR) mRNA levels in granulosa cells, and of androstenedione and testosterone in follicular fluid, were correlated to the expression of the FSH receptor (FSHR), LH receptor (LHR), CYP19 and anti-Mullerian Hormone-receptor II (AMHRII) mRNA in the granulosa cells...... and to the follicular fluid concentrations of AMH, inhibin-B, progesterone and estradiol. AR mRNA expression in granulosa cells and the follicular fluid content of androgens both showed a highly significant positive association with the expression of FSHR mRNA in granulosa cells. AR mRNA expression...... between the follicular fluid levels of androgen and FSHR expression. This suggests that follicular sensitivity towards FSH stimulation may be augmented by stimulation of androgens via the AR....

  14. Identification of proteins specifically interacting with YB-1 mRNA 3' UTR and the effect of hnRNP Q on YB-1 mRNA translation.

    Science.gov (United States)

    Lyabin, D N; Nigmatullina, L F; Doronin, A N; Eliseeva, I A; Ovchinnikov, L P

    2013-06-01

    In this study, proteins specifically interacting with the 3' untranslated region (UTR) of mRNA of the multifunctional Y-box-binding protein 1 (YB-1) were identified. One of these, hnRNP Q, was shown to specifically interact with the regulatory element (RE) in YB-1 mRNA 3' UTR and to inhibit translation of this mRNA. Its binding to the RE was accompanied by displacement from this element of the poly(A)-binding protein (PABP), a positive regulator of YB-1 mRNA translation, and by enhanced binding of the negative YB-1 mRNA translation regulator - YB-1 itself. PMID:23980891

  15. 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.

  16. 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 ...

  17. Translational Influence on Messenger Stability

    DEFF Research Database (Denmark)

    Eriksen, Mette

    composition of the coding sequence of a gene. This thesis focussed on illuminating the impact of ribosome binding sites on the functional messenger half-life using a range of ribosome binding sites with altered binding strength. Furthermore, the additive in uence of the transcription terminator, Rho, on...... pre-termination to be a global phenomena in gene regulation. The influence of codon usage in the early coding region on messenger stability was examined, in order to establish how fast or slow the ribosome has to decode the sequence for it to protect the messenger from degradation. The experiments...

  18. 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

  19. 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

  20. IMP3 RNP safe houses prevent miRNA-directed HMGA2 mRNA decay in cancer and development

    DEFF Research Database (Denmark)

    Jønson, Lars; Christiansen, Jan; Hansen, Thomas van Overeem;

    2014-01-01

    The IMP3 RNA-binding protein is associated with metastasis and poor outcome in human cancer. Using solid cancer transcriptome data, we found that IMP3 correlates with HMGA2 mRNA expression. Cytoplasmic IMP3 granules contain HMGA2, and IMP3 dose-dependently increases HMGA2 mRNA. HMGA2 is regulated...... by let-7, and let-7 antagomiRs make HMGA2 refractory to IMP3. Removal of let-7 target sites eliminates IMP3-dependent stabilization, and IMP3-containing bodies are depleted of Ago1-4 and miRNAs. The relationship between Hmga2 mRNA and IMPs also exists in the developing limb bud, where IMP1-deficient...... embryos show dose-dependent Hmga2 mRNA downregulation. Finally, IMP3 ribonucleoproteins (RNPs) contain other let-7 target mRNAs, including LIN28B, and a global gene set enrichment analysis demonstrates that miRNA-regulated transcripts in general are upregulated following IMP3 induction. We conclude that...

  1. 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

    BACKGROUND: Disturbances related to the arachidonic acid cascade and prostaglandin metabolism may be involved in the pathophysiology of bipolar disorder, as supported by a recent genome-wide association study meta-analysis; however, evidence from clinical studies on a transcriptional level...... disorder. The sample size was limited; replication of the findings in larger, independent samples is warranted to further explore the role of the arachidonic acid cascade and prostaglandin metabolism as a potential therapeutic target in bipolar disorder....... 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...

  2. 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.

  3. 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.

  4. 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.

  5. Study on in vivo imaging of 99Tcm-hTERT mRNA as antisense molecular probe in breast cancer tumor-bearing nude mice

    International Nuclear Information System (INIS)

    Objective: Antisense imaging is one of the important modalities in the domain of molecular nuclear medicine. The purpose of this study was to design and synthesize an antisense oligonucleotide (ASON) molecular probe targeting human telomerase reverse transcriptase (hTERT) mRNA, and to validate the potential application value using animal model experimental study in early diagnosis of the tumor. Methods: Antisense and sense molecular probes targeting hTERT mRNA were radiolabeled with 99Tcm through bifunctional chelator N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (S-Acetyl NHS-MAG3). The BALB/c nu/nu nude mice were inoculated with MCF-7 mammary tumor cells in the right upper limbs. 99Tcm-hTERT mRNA ASON and 99Tcm-hTERT mRNA sense oligonucleotide (SON) with or without mediated by liposome was injected intravenously in mammary tumor-bearing BALB/c nude mice, respectively. Imaging it, vivo was performed periodically. All data were analyzed by the statistic software of SPSS 12.0. Results: The in vitro study showed that the labeling efficiencies of 99Tcm-hTERT mRNA ASON reached (76 ± 5)%, with radiochemical purity greater than 96% and specific activity of 1850 kBq/μg. The stability of 99Tcm-hTERT mRNA ASON in room temperature and serum incubation after 24 h was still above 93%. The in vivo study showed that tumor uptake of 99Tcm-hTERT mRNA ASON was high from 4 to 8 h after injection. On the contrary, there was little 99Tcm-hTERT mRNA SON accumulated in tumor within 8 h. The radioactivity ratio of tumor-to-nontumor (T/NT) of antisense probe group with or' without liposome mediation was 8.02 ± 0.03 and 7.55 ± 0.12, respectively (t=-1.99, P>0.05), and that of sense probe group with or without liposome mediation was 1.23 ± 0.06 and 1.33 ± 0.15, respectively (t=0.42, P>0.05). However, there was significant difference between antisense and sense probe groups with or without liposome mediation (t= 26.30, 28.71, both P99Tcm could be used as a

  6. 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...

  7. 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...

  8. Dimethyl sulphoxide and haemin induce ferrochelatase mRNA by different mechanisms in murine erythroleukaemia cells.

    Science.gov (United States)

    Fukuda, Y; Fujita, H; Taketani, S; Sassa, S

    1993-03-01

    The level of mRNA encoding ferrochelatase (FeC), the terminal enzyme of the haem biosynthetic pathway, was examined in murine erythroleukaemia (MEL) cells when they were induced to undergo erythroid cell differentiation by treatment with dimethyl sulphoxide (DMSO), or haemin. FeC mRNA increased within 12 h after DMSO or haemin treatment of MEL cells, and its level continued to increase for 48 h. Treatment of cells with succinylacetone (SA), a potent inhibitor of haem synthesis, suppressed a DMSO-mediated increase in FeC mRNA, and haemin treatment reversed a SA-mediated decrease in FeC mRNA. Nuclear runoff analyses showed that, while DMSO increased the rate of transcription of FeC mRNA, haemin did not. These results indicate that the induction of FeC mRNA by DMSO is largely transcriptional, while that by haemin is post-transcriptional. PMID:8485055

  9. 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

  10. 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.

  11. 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...

  12. 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...

  13. 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

  14. IER5 gene's mRNA expression after irradiation

    International Nuclear Information System (INIS)

    Objective: To explore the effect of irradiation on IER5 gene expression. Methods: Two kinds of cells (AHH-1 and HeLa) and the BALB/c-nu mice inoculated with tumor cells were exposed to 60Co γ- rays and analyzed by real-time PCR. The above-mentioned irradiated objects were firstly divided into groups by different doses and post-radiation time, then mRNA were extracted and reverse-transcripted to DNA before real-time PCR test. Results: Under the same condition, AHH-1 was more sensitive to radiation than HeLa. The dose level corresponding to the expression peak of AHH-1 was less than that of HeLa. For AHH-1 cells, the response to 2 Gy irradiation was earlier than that to 10 Gy. But there was not remarkable difference for HeLa response between 2 and 10 Gy, and the top transcriptional levels for both cells nearly simultaneously appeared at 2 h after irradiation. In addition, the IER5 gene of human liver tumor was more sensitive than that of lung cancer and brain tumor. Conclusions: IER5 might be a candidate biomarker of radiation injury, and had the potential value in radiation-therapy for liver tumor. (authors)

  15. Export of mRNA from microinjected nuclei of Xenopus laevis oocytes

    OpenAIRE

    1992-01-01

    Export of mRNA from the nucleus to the cytoplasm was studied in mature Xenopus laevis oocytes. In vitro transcribed, capped 32P-labeled mRNA was microinjected into nuclei, and its appearance in the cytoplasm measured by counting radioactivity or by RNA extraction and gel electrophoresis. Both for a 5.0-kb transferrin receptor mRNA and a 2.0- kb 4F2 antigen heavy chain mRNA we found saturable transport with an apparent Km of 3.6 x 10(8) molecules per oocyte nucleus. Under non- saturating condi...

  16. 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...

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Trapped particle stability for the kinetic stabilizer

    International Nuclear Information System (INIS)

    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.

  2. 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

  3. Temporal Stability of the Human Skin Microbiome.

    Science.gov (United States)

    Oh, Julia; Byrd, Allyson L; Park, Morgan; Kong, Heidi H; Segre, Julia A

    2016-05-01

    Biogeography and individuality shape the structural and functional composition of the human skin microbiome. To explore these factors' contribution to skin microbial community stability, we generated metagenomic sequence data from longitudinal samples collected over months and years. Analyzing these samples using a multi-kingdom, reference-based approach, we found that despite the skin's exposure to the external environment, its bacterial, fungal, and viral communities were largely stable over time. Site, individuality, and phylogeny were all determinants of stability. Foot sites exhibited the most variability; individuals differed in stability; and transience was a particular characteristic of eukaryotic viruses, which showed little site-specificity in colonization. Strain and single-nucleotide variant-level analysis showed that individuals maintain, rather than reacquire, prevalent microbes from the environment. Longitudinal stability of skin microbial communities generates hypotheses about colonization resistance and empowers clinical studies exploring alterations observed in disease states. PMID:27153496

  4. 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.

  5. 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...

  6. 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

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

    Science.gov (United States)

    Trivedi, Malav S; Deth, Richard C

    2012-01-01

    Homeostatic synaptic scaling in response to neuronal stimulus or activation, and due to changes in cellular niche, is an important phenomenon for memory consolidation, retrieval, and other similar cognitive functions (Turrigiano and Nelson, 2004). Neurological disorders and cognitive disabilities in autism, Rett syndrome, schizophrenia, dementia, etc., are strongly correlated to alterations in protein expression (both synaptic and cytoplasmic; Cajigas et al., 2010). 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 (Cajigas et al., 2010). Thus a regulatory switch, which controls 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 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 and RNA binding proteins 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 anti-oxidant levels (Lertratanangkoon et al., 1997), the redox status of

  8. 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

  9. 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.

  10. 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.

  11. Chromatic alteration on marble surfaces analysed by molecular biology tools

    OpenAIRE

    Franco Palla; Elena Tartamella

    2007-01-01

    The patina represents a superficial natural alteration of the constituting matter of the work of art. It emerges from the natural and usual stabilization process that the materials of the surface undergo because of the interaction with outdoor agents characterizing the surrounding environment. Besides, it is not linked to an obvious phenomenon of degradation that can be noticed through the change in the original colour of the matter. This is what we intend when we talk about biological patina...

  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. 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.

  14. 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.

  15. 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

  16. 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

  17. Financial stability concept

    OpenAIRE

    Nikolay Beketov

    2007-01-01

    This article deals with a relatively new concept of the economics, i.e. financial stability. The essence of the financial stability concept, academic and practical definitions of financial stability, relationships between financial stability and other notions of the theory of finance (financial crisis, financial fragility, financial soundness, etc.) are considered. The author argues convincingly that there is a need for promoting scientific debate about financial stability problems in the Rep...

  18. 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...

  19. 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.

  20. 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

  1. 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.

  2. 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)

  3. PROLONGED FASTING AND CORTISOL REDUCE MYOSTATIN MRNA LEVELS IN TILAPIA LARVAE, SHORT-TERM FASTING ELEVATES

    Science.gov (United States)

    Myostatin negatively regulates muscle growth and development and has recently been characterized in several fishes. We measured fasting myostatin mRNA levels in adult tilapia skeletal muscle and in whole larvae. Although fasting reduced some growth indices in adults, skeletal muscle myostatin mRNA...

  4. 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......: AT IL-18 mRNA content and plasma IL-18 concentration were higher (p < 0.05) in the obese group than in the non-obese group. AT IL-18 mRNA content and plasma IL-18 concentration was positively correlated (p < 0.05) with insulin resistance. While acute exercise did not affect IL-18 mRNA expression at...... the studied time-points, exercise training reduced AT IL-18 mRNA content by 20% in both sexes. DISCUSSION: Because obesity and insulin resistance were associated with elevated AT IL-18 mRNA and plasma IL-18 levels, the training-induced lowering of AT IL-18 mRNA content may contribute to the beneficial...

  5. Disrupted-in-schizophrenia 1 regulates transport of ITPR1 mRNA for synaptic plasticity.

    Science.gov (United States)

    Tsuboi, Daisuke; Kuroda, Keisuke; Tanaka, Motoki; Namba, Takashi; Iizuka, Yukihiko; Taya, Shinichiro; Shinoda, Tomoyasu; Hikita, Takao; Muraoka, Shinsuke; Iizuka, Michiro; Nimura, Ai; Mizoguchi, Akira; Shiina, Nobuyuki; Sokabe, Masahiro; Okano, Hideyuki; Mikoshiba, Katsuhiko; Kaibuchi, Kozo

    2015-05-01

    Disrupted-in-schizophrenia 1 (DISC1) is a susceptibility gene for major psychiatric disorders, including schizophrenia. DISC1 has been implicated in neurodevelopment in relation to scaffolding signal complexes. Here we used proteomic analysis to screen for DISC1 interactors and identified several RNA-binding proteins, such as hematopoietic zinc finger (HZF), that act as components of RNA-transporting granules. HZF participates in the mRNA localization of inositol-1,4,5-trisphosphate receptor type 1 (ITPR1), which plays a key role in synaptic plasticity. DISC1 colocalizes with HZF and ITPR1 mRNA in hippocampal dendrites and directly associates with neuronal mRNAs, including ITPR1 mRNA. The binding potential of DISC1 for ITPR1 mRNA is facilitated by HZF. Studies of Disc1-knockout mice have revealed that DISC1 regulates the dendritic transport of Itpr1 mRNA by directly interacting with its mRNA. The DISC1-mediated mRNA regulation is involved in synaptic plasticity. We show that DISC1 binds ITPR1 mRNA with HZF, thereby regulating its dendritic transport for synaptic plasticity. PMID:25821909

  6. 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

  7. Leveraging Rules of Nonsense-Mediated mRNA Decay for Genome Engineering and Personalized Medicine.

    Science.gov (United States)

    Popp, Maximilian W; Maquat, Lynne E

    2016-06-01

    Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA quality control and regulatory process that plays direct roles in human health and disease. In this Minireview, we discuss how understanding the molecular events that trigger NMD can facilitate strategic targeting of genes via CRISPR/Cas9 technologies and also inform disease diagnostics and treatments. PMID:27259145

  8. Ultraviolet light-induced crosslinking of mRNA to proteins

    International Nuclear Information System (INIS)

    Irradiation of intact or EDTA-dissociated L-cell polyribosomes with 254 nm UV light at doses of 1-2 x 105 ergs/mm2 extensively crosslinks mRNA to proteins. The crosslinked mRNA-protein complexes can be isolated on the basis of buoyant density in urea-containing Cs2S04 gradients that dissociate non-covalent complexes. Crosslinking of mRNA can also be assayed by phenol-chloroform extraction. mRNA recovered from the crosslinked complexes by digestion with proteinase K has the same electrophoretic mobility in polyacrylamide gels as unirradiated mRNa. Therefore, irradiation does not either crosslink RNA molecules to RNA molecules or break phosphodiester bonds. With these methods it has been found that more than 70% of high molecular weight polydisperse mRNA, but only 25-40% of histone mRNA, can be crosslinked to protein. On the basis of buoyant density the histone mRNA-protein complex had a protein content of 26%, whereas the mean protein content of most non-histone mRNA-protein complexes was 65%. It is concluded that most mRNA in polyribosomes is in close contact with proteins, and that histone mRNA can be crosslinked to many fewer proteins than most other mRNAs. (author)

  9. 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

  10. 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

  11. 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.

  12. 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...

  13. 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

  14. 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.

  15. Photothermal Breaking of Emulsions Stabilized with Graphene.

    Science.gov (United States)

    Quinn, Matthew D J; Vu, Khu; Madden, Stephen; Notley, Shannon M

    2016-04-27

    Pristine graphene particles prepared using an aqueous phase exfoliation technique have been used to promote the stabilization of emulsions through adsorption at the oil-water interface. Highly localized phase separation of these ultrastable emulsions could, however, be induced through photothermal heating of the graphene particles at the interface exposed to near-infrared light. The graphene wettability, which is a key determinant in preventing droplet coalescence was altered through the adsorption of nonionic block copolymer surfactants. Varying the aqueous solution conditions influenced the hydration of the hydrophilic component of the surfactant providing a further opportunity to alter the overall particle wettability and, hence, stability of the emulsion. In this way, highly stable-oil-in water emulsions were produced with decane; however, water-in-oil emulsions were formed with toluene as the oil phase. PMID:27054548

  16. 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...

  17. 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...

  18. 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...

  19. 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...

  20. Treatment of neurological disorders by introducing mRNA in vivo using polyplex nanomicelles.

    Science.gov (United States)

    Baba, Miyuki; Itaka, Keiji; Kondo, Kenji; Yamasoba, Tatsuya; Kataoka, Kazunori

    2015-03-10

    Sensory nerve disorders are difficult to cure completely considering poor nerve regeneration capacity and difficulties in accurately targeting neural tissues. Administering mRNA is a promising approach for treating neurological disorders because mRNA can provide proteins and peptides in their native forms for mature non-dividing neural cells, without the need of entering their nuclei. However, direct mRNA administration into neural tissues in vivo has been challenging due to too unstable manner of mRNA and its strong immunogenicity. Thus, using a suitable carrier is essential for effective mRNA administration. For this purpose, we established a novel carrier based on the self-assembly of polyethylene glycol (PEG)-polyamino acid block copolymer, i.e. polyplex nanomicelles. To investigate the feasibility and efficacy of mRNA administration for the treatment of sensory nerve disorders, we used a mouse model of experimentally induced olfactory dysfunction. Intranasal administration of mRNA-loaded nanomicelles provided an efficient and sustained protein expression for nearly two days in nasal tissues, particularly in the lamina propria which contains olfactory nerve fibers, with effectively regulating the immunogenicity of mRNA. Consequently, once-daily intranasal administration of brain-derived neurotrophic factor (BDNF)-expressing mRNA using polyplex nanomicelles remarkably enhanced the neurological recovery of olfactory function along with repairing the olfactory epithelium to a nearly normal architecture. To the best of our knowledge, this is the first study to show the therapeutic potential of introducing exogenous mRNA for the treatment of neurological disorders. These results indicate the feasibility and safety of using mRNA, and provide a novel strategy of mRNA-based therapy. PMID:25599855

  1. 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 tissue (p  0.05), but ERCC2 mRNA expression decreases in skin (p tissue (p tissue exposed to low-intensity lasers depending on wavelengths and fluences used in therapeutic protocols. PMID:26796702

  2. Phosphorylation at intrinsically disordered regions of PAM2 motif-containing proteins modulates their interactions with PABPC1 and influences mRNA fate.

    Science.gov (United States)

    Huang, Kai-Lieh; Chadee, Amanda B; Chen, Chyi-Ying A; Zhang, Yueqiang; Shyu, Ann-Bin

    2013-03-01

    Cytoplasmic poly(A)-binding protein (PABP) C1 recruits different interacting partners to regulate mRNA fate. The majority of PABP-interacting proteins contain a PAM2 motif to mediate their interactions with PABPC1. However, little is known about the regulation of these interactions or the corresponding functional consequences. Through in silico analysis, we found that PAM2 motifs are generally embedded within an extended intrinsic disorder region (IDR) and are located next to cluster(s) of potential serine (Ser) or threonine (Thr) phosphorylation sites within the IDR. We hypothesized that phosphorylation at these Ser/Thr sites regulates the interactions between PAM2-containing proteins and PABPC1. In the present study, we have tested this hypothesis using complementary approaches to increase or decrease phosphorylation. The results indicate that changing the extent of phosphorylation of three PAM2-containing proteins (Tob2, Pan3, and Tnrc6c) alters their ability to interact with PABPC1. Results from experiments using phospho-blocking or phosphomimetic mutants in PAM2-containing proteins further support our hypothesis. Moreover, the phosphomimetic mutations appreciably affected the functions of these proteins in mRNA turnover and gene silencing. Taken together, these results provide a new framework for understanding the roles of intrinsically disordered proteins in the dynamic and signal-dependent control of cytoplasmic mRNA functions. PMID:23340509

  3. 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.

  4. 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

  5. Dynamic expression of tyrosine hydroxylase mRNA and protein in neurons of the striatum and amygdala of mice, and experimental evidence of their multiple embryonic origin.

    Science.gov (United States)

    Bupesh, Munisamy; Vicario, Alba; Abellán, Antonio; Desfilis, Ester; Medina, Loreta

    2014-05-01

    Emotional and motivational dysfunctions observed in Parkinson's disease, schizophrenia, and drug addiction are associated to an alteration of the mesocortical and mesolimbic dopaminergic pathways, which include axons projecting to the prefrontal cortex, the ventral striatum, and the amygdala. Subpopulations of catecholaminergic neurons have been described in the cortex and striatum of several mammals, but the presence of such cells in the adult amygdala is unclear in murine rodents, and in other rodents appears to show variations depending on the species. Moreover, the embryonic origin of telencephalic tyrosine hydroxylase (TH) cells is unknown, which is essential for trying to understand aspects of their evolution, distribution and function. Herein we investigated the expression of TH mRNA and protein in cells of the striatum and amygdala of developing and adult mice, and analyzed the embryonic origin of such cells using in vitro migration assays. Our results showed the presence of TH mRNA and protein expressing cells in the striatum (including nucleus accumbens), central and medial extended amygdala during development, which are persistent in adulthood although they are less numerous, generally show weak mRNA expression, and some appear to lack the protein. Fate mapping analysis showed that these cells include at least two subpopulations with different embryonic origin in either the commissural preoptic area of the subpallium or the supraopto-paraventricular domain of the alar hypothalamus. These data are important for future studies trying to understand the role of catecholamines in modulation of emotion, motivation, and reward. PMID:23479178

  6. Regular endurance training reduces the exercise induced HIF-1alpha and HIF-2alpha mRNA expression in human skeletal muscle in normoxic conditions

    DEFF Research Database (Denmark)

    Lundby, Carsten; Gassmann, Max; Pilegaard, Henriette

    2005-01-01

    single exercise bout, and that this response is blunted with training. We obtained muscle biopsies from a trained (5 days/week during 4 weeks) and untrained leg from the same human subject before, immediately after, and during the recovery from a 3 h two-legged knee extensor exercise bout, where the two...... legs exercised at the same absolute workload. In the untrained leg, the exercise bout induced an increase (P<0.05) in HIF-1alpha fold and HIF-2alpha fold mRNA at 6 h of recovery. In contrast, HIF-1alpha and HIF-2alpha mRNA levels were not altered at any time point in the trained leg. Obviously, HIF-1......alpha and HIF-2alpha mRNA levels are transiently increased in untrained human skeletal muscle in response to an acute exercise bout, but this response is blunted after exercise training. We propose that HIFs expression is upregulated with exercise and that it may be an important transcription factor...

  7. Sodium bicarbonate ingestion augments the increase in PGC-1α mRNA expression during recovery from intense interval exercise in human skeletal muscle.

    Science.gov (United States)

    Percival, Michael E; Martin, Brian J; Gillen, Jenna B; Skelly, Lauren E; MacInnis, Martin J; Green, Alex E; Tarnopolsky, Mark A; Gibala, Martin J

    2015-12-01

    We tested the hypothesis that ingestion of sodium bicarbonate (NaHCO3) prior to an acute session of high-intensity interval training (HIIT) would augment signaling cascades and gene expression linked to mitochondrial biogenesis in human skeletal muscle. On two occasions separated by ∼1 wk, nine men (mean ± SD: age 22 ± 2 yr, weight 78 ± 13 kg, V̇O(2 peak) 48 ± 8 ml·kg(-1)·min(-1)) performed 10 × 60-s cycling efforts at an intensity eliciting ∼90% of maximal heart rate (263 ± 40 W), interspersed with 60 s of recovery. In a double-blind, crossover manner, subjects ingested a total of 0.4 g/kg body weight NaHCO3 before exercise (BICARB) or an equimolar amount of a placebo, sodium chloride (PLAC). Venous blood bicarbonate and pH were elevated at all time points after ingestion (P 0.05). However, the increase in PGC-1α mRNA expression after 3 h of recovery was higher in BICARB vs. PLAC (approximately sevenfold vs. fivefold compared with rest, P HIIT alters the mRNA expression of this key regulatory protein associated with mitochondrial biogenesis. The elevated PGC-1α mRNA response provides a putative mechanism to explain the enhanced mitochondrial adaptation observed after chronic HIIT supplemented with NaHCO3 in rats. PMID:26384407

  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. TRM6/61 connects PKCα with translational control through tRNAi(Met) stabilization: impact on tumorigenesis.

    Science.gov (United States)

    Macari, F; El-Houfi, Y; Boldina, G; Xu, H; Khoury-Hanna, S; Ollier, J; Yazdani, L; Zheng, G; Bièche, I; Legrand, N; Paulet, D; Durrieu, S; Byström, A; Delbecq, S; Lapeyre, B; Bauchet, L; Pannequin, J; Hollande, F; Pan, T; Teichmann, M; Vagner, S; David, A; Choquet, A; Joubert, D

    2016-04-01

    Accumulating evidence suggests that changes of the protein synthesis machinery alter translation of specific mRNAs and participate in malignant transformation. Here we show that protein kinase C α (PKCα) interacts with TRM61, the catalytic subunit of the TRM6/61 tRNA methyltransferase. The TRM6/61 complex is known to methylate the adenosine 58 of the initiator methionine tRNA (tRNAi(Met)), a nuclear post-transcriptional modification associated with the stabilization of this crucial component of the translation-initiation process. Depletion of TRM6/61 reduced proliferation and increased death of C6 glioma cells, effects that can be partially rescued by overexpression of tRNAi(Met). In contrast, elevated TRM6/61 expression regulated the translation of a subset of mRNAs encoding proteins involved in the tumorigenic process and increased the ability of C6 cells to form colonies in soft agar or spheres when grown in suspension. In TRM6/61/tRNAi(Met)-overexpressing cells, PKCα overexpression decreased tRNAi(Met) expression and both colony- and sphere-forming potentials. A concomitant increase in TRM6/TRM61 mRNA and tRNAi(Met) expression with decreased expression of PKCα mRNA was detected in highly aggressive glioblastoma multiforme as compared with Grade II/III glioblastomas, highlighting the clinical relevance of our findings. Altogether, we suggest that PKCα tightly controls TRM6/61 activity to prevent translation deregulation that would favor neoplastic development. PMID:26234676

  10. 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....

  11. 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.

  12. 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

  13. PURE mRNA display for in vitro selection of single-chain antibodies.

    Science.gov (United States)

    Nagumo, Yu; Fujiwara, Kei; Horisawa, Kenichi; Yanagawa, Hiroshi; Doi, Nobuhide

    2016-05-01

    mRNA display is a method to form a covalent linkage between a cell-free synthesized protein (phenotype) and its encoding mRNA (genotype) through puromycin for in vitro selection of proteins. Although a wheat germ cell-free translation system has been previously used in our mRNA display system, a protein synthesis using recombinant elements (PURE) system is a more attractive approach because it contains no endogenous nucleases and proteases and is optimized for folding of antibodies with disulphide bonds. However, when we used the PURE system for mRNA display of single-chain Fv (scFv) antibodies, the formation efficiency of the mRNA-protein conjugates was quite low. To establish an efficient platform for the PURE mRNA display of scFv, we performed affinity selection of a library of scFv antibodies with a C-terminal random sequence and obtained C-terminal sequences that increased the formation of mRNA-protein conjugates. We also identified unexpected common substitution mutations around the start codon of scFv antibodies, which were inferred to destabilize the mRNA secondary structure. This destabilization causes an increase in protein expression and the efficiency of the formation of mRNA-protein conjugates. We believe these improvements should make the PURE mRNA display more efficient for selecting antibodies for diagnostic and therapeutic applications. PMID:26711234

  14. 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

  15. 99mTc-sestamibi to monitor treatment with antisense oligodeoxynucleotide complementary to MRP mRNA in human breast cancer cells

    International Nuclear Information System (INIS)

    Technetium-99m sestamibi (MIBI) has been utilized to evaluate multi-drug resistance (MDR) phenomenon of malignant tumors and to predict chemotherapeutic effects on them. The current investigation examined the possibility of monitoring changes with respect to mRNA expression of multi-drug resistance associated protein (MRP) following antisense oligodeoxynucleotide (AS-ODN) treatment involving 99mTc-MIBI. The human breast cancer MCF-7 cell line and its MDR-induced MCF-7/VP cell line were employed. Cell suspensions of the two cell lines at 1 x 104 cells/ml were inoculated in 24-well plates (0.2 ml/well) and incubated for one day. Antisense (AS) 20-mer phosphorothioate ODN complementary to the coding region of MRP mRNA and its sense (S) ODN were administered at final concentrations up to 25 μM, followed by a 5-day incubation. 99mTc-MIBI solution was added to each well and incubated for 30 min. Cellular 99mTc-MIBI uptake was corrected for protein concentration. MRP mRNA expression levels were analyzed via the reverse transcription polymerase chain reaction (RT-PCR). Cellular uptake of 99mTc-MIBI in MCF-7/VP cells was only 15% of that of MCF-7 cells. Following AS-ODN treatment at 25 μM for five days, 99mTc-MIBI uptake in MCF-7/VP cells increased 2.4-fold in comparison with non-treated control cells. 99mTc-MIBI uptake in MCF-7 cells was unaffected by AS-ODN administration. Sense ODN did not alter uptake in either cell line. RT-PCR confirmed reduction of MRP mRNA in MCF-7/VP cells following AS-ODN treatment. Effects of AS-ODN administration on MRP function can be monitored via assessment of cellular uptake of 99mTc-MIBI. (author)

  16. 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

  17. 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

  18. 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

  19. 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.

  20. Prefrontal cortical-striatal dopamine receptor mRNA expression predicts distinct forms of impulsivity.

    Science.gov (United States)

    Simon, Nicholas W; Beas, Blanca S; Montgomery, Karienn S; Haberman, Rebecca P; Bizon, Jennifer L; Setlow, Barry

    2013-06-01

    Variation in dopamine receptor levels has been associated with different facets of impulsivity. To further delineate the neural substrates underlying impulsive action (inability to withhold a prepotent motor response) and impulsive choice (delay aversion), we characterised rats in the Differential Reinforcement of Low Rates of Responding task and a delay discounting task. We also measured performance on an effort-based discounting task. We then assessed D1 and D2 dopamine receptor mRNA expression in subregions of the prefrontal cortex and nucleus accumbens using in situ hybridisation, and compared these data with behavioral performance. Expression of D1 and D2 receptor mRNA in distinct brain regions was predictive of impulsive action. A dissociation within the nucleus accumbens was observed between subregions and receptor subtypes; higher D1 mRNA expression in the shell predicted greater impulsive action, whereas lower D2 mRNA expression in the core predicted greater impulsive action. We also observed a negative correlation between impulsive action and D2 mRNA expression in the prelimbic cortex. Interestingly, a similar relationship was present between impulsive choice and prelimbic cortex D2 mRNA, despite the fact that behavioral indices of impulsive action and impulsive choice were uncorrelated. Finally, we found that both high D1 mRNA expression in the insular cortex and low D2 mRNA expression in the infralimbic cortex were associated with willingness to exert effort for rewards. Notably, dopamine receptor mRNA in these regions was not associated with either facet of impulsivity. The data presented here provide novel molecular and neuroanatomical distinctions between different forms of impulsivity, as well as effort-based decision-making. PMID:23510331

  1. Prefrontal cortical–striatal dopamine receptor mRNA expression predicts distinct forms of impulsivity

    Science.gov (United States)

    Simon, Nicholas W.; Beas, Blanca S.; Montgomery, Karienn S.; Haberman, Rebecca P.; Bizon, Jennifer L.; Setlow, Barry

    2014-01-01

    Variation in dopamine receptor levels has been associated with different facets of impulsivity. To further delineate the neural substrates underlying impulsive action (inability to withhold a prepotent motor response) and impulsive choice (delay aversion), we characterised rats in the Differential Reinforcement of Low Rates of Responding task and a delay discounting task. We also measured performance on an effort-based discounting task. We then assessed D1 and D2 dopamine receptor mRNA expression in subregions of the prefrontal cortex and nucleus accumbens using in situ hybridisation, and compared these data with behavioral performance. Expression of D1 and D2 receptor mRNA in distinct brain regions was predictive of impulsive action. A dissociation within the nucleus accumbens was observed between subregions and receptor subtypes; higher D1 mRNA expression in the shell predicted greater impulsive action, whereas lower D2 mRNA expression in the core predicted greater impulsive action. We also observed a negative correlation between impulsive action and D2 mRNA expression in the prelimbic cortex. Interestingly, a similar relationship was present between impulsive choice and prelimbic cortex D2 mRNA, despite the fact that behavioral indices of impulsive action and impulsive choice were uncorrelated. Finally, we found that both high D1 mRNA expression in the insular cortex and low D2 mRNA expression in the infralimbic cortex were associated with willingness to exert effort for rewards. Notably, dopamine receptor mRNA in these regions was not associated with either facet of impulsivity. The data presented here provide novel molecular and neuroanatomical distinctions between different forms of impulsivity, as well as effort-based decision-making. PMID:23510331

  2. Effect of lead on globin mRNA in vivo and in vitro

    International Nuclear Information System (INIS)

    Plumbous ion has been shown to be a potent catalyst for the depolymerization of RNA in vitro but the question of whether or not Lead-catalyzed RNA degradation also occurs in vivo has never been addressed. Our experimental design, to answer this question, was to transfuse rabbit reticulocytes into normal rabbits and rabbits that had been injected with different doses of lead acetate. After 24 hours the mRNA was isolated form the reticulocytes of each rabbit by phenol extraction and affinity chromatography on oligo dT cellulose. The amount of mRNA per ml of packed reticulocytes was determined. The integrity of the mRNA was then determined with a cell-free reticulocyte translation system that was dependent on exogenous mRNA. The results showed that there was little difference in the amount of mRNA recovered from control and treated rabbits, but the ability of the mRNA to support globin synthesis was decreased by as much as 86% in the lead-treated rabbits. These data suggest that not only is mRNA attacked by lead in vivo but that the lead attacks the mRNA at just one or at least very few sites. In the in vivo studies, purified rabbit globin mRNA was incubated with lead acetate and the products of this reaction were labelled with 32P using T4 polynucleotide kinase. A labelled fragment of slightly greater mobility than tRNA was isolated by gel electrophoresis. This fragment was digested to the monomers and analyzed by TLC to identify the nucleotide at the 5' end

  3. Stability of symplectic leaves

    OpenAIRE

    Crainic, M.; Fernandes, R.L.

    2008-01-01

    We find computable criteria for stability of symplectic leaves of Poisson manifolds. Using Poisson geometry as an inspiration, we also give a general criterion for stability of leaves of Lie algebroids, including singular ones. This not only extends but also provides a new approach (and proofs) to the classical stability results for foliations and group actions.

  4. 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

  5. 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.

  6. 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…

  7. 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.

  8. Ochratoxin a lowers mRNA levels of genes encoding for key proteins of liver cell metabolism.

    Science.gov (United States)

    Hundhausen, Christoph; Boesch-Saadatmandi, Christine; Matzner, Nicole; Lang, Florian; Blank, Ralf; Wolffram, Siegfried; Blaschek, Wolfgang; Rimbach, Gerald

    2008-01-01

    Ochratoxin A (OTA) is a nephro- and hepatotoxic mycotoxin that frequently contaminates food and feedstuffs. Although recent studies have indicated that OTA modulates renal gene expression, little is known regarding its impact on differential gene expression in the liver. Therefore a microarray study of the HepG2 liver cell transcriptome in response to OTA exposure (0, 0.25, 2.5 micromol/l for 24 h) was performed using Affymetrix GeneChip technology. Selected microarray results were verified by real-time PCR and Western blotting as independent methods. Out of 14,500 genes present on the microarray, 13 and 250 genes were down-regulated by 0.25 and 2.5 micromol/l OTA, respectively. Reduced mRNA levels of calcineurin A beta (PPP3CB), which regulates inflammatory signalling pathways in immune cells, and of the uncoupling protein 2 (UCP2), which has been suggested to control the production of reactive oxygen species (ROS), were observed in response to 0.25 micromol/l OTA. A particularly strong down-regulation due to 2.5 micromol/l OTA was evident for the mRNA levels of insulin-like growth factor binding protein 1 (IGFBP1) and tubulin beta 1 (TUBB1) which have been demonstrated to function as a pro-survival factor in hepatocytes and as an important cytoskeletal component, respectively. In addition, many genes involved in energy and xenobiotic metabolism, including phosphoglycerate kinase 1 (PGK1), stearoyl-Coenzyme A desaturase 1 (SCD), and glutathione S-transferase omega 1 (GSTO1), were down-regulated by OTA. Furthermore, OTA significantly inhibited the capacitative calcium entry into the HepG2 cells, indicating an alteration of calcium homeostasis. Overall, OTA dose-dependently affects multiple genes encoding for key proteins of liver cell metabolism. PMID:19287073

  9. 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.

  10. 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...

  11. 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...

  12. 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.

  13. 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 ...

  14. 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

  15. 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.

  16. 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...

  17. 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...

  18. 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...

  19. 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

  20. 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

  1. 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

  2. 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

  3. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production.

    Science.gov (United States)

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent; Gupta, Ishaan; Steinmetz, Lars M; Jensen, Torben Heick

    2015-07-01

    Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S. 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-dependent process. We conclude that Nab2p is required to protect early mRNA and therefore constitutes a crucial nuclear mRNA biogenesis factor. PMID:26119729

  4. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production

    Directory of Open Access Journals (Sweden)

    Manfred Schmid

    2015-07-01

    Full Text Available Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A tails on mRNA metabolism, the precise roles of poly(A-binding proteins (PABPs in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S. 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-dependent process. We conclude that Nab2p is required to protect early mRNA and therefore constitutes a crucial nuclear mRNA biogenesis factor.

  5. 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

  6. In ovo effects of perfluorohexane sulfonate and perfluorohexanoate on pipping success, development, mRNA expression, and thyroid hormone levels in chicken embryos.

    Science.gov (United States)

    Cassone, Cristina G; Vongphachan, Viengtha; Chiu, Suzanne; Williams, Kim L; Letcher, Robert J; Pelletier, Eric; Crump, Doug; Kennedy, Sean W

    2012-05-01

    Perfluoroalkyl acids (PFAAs), specifically perfluorinated sulfonates and carboxylates, are synthetic substances known for their chemical stability, resistance to degradation, and potential to biomagnify in food chains. The toxicological and biological effects of PFAAs in avian species are not well characterized, although there is some evidence to suggest that they can impact neurodevelopment and hatching success. Our laboratory recently reported significant effects of perfluorohexane sulfonate (PFHxS) and perfluorohexanoate (PFHxA) on messenger RNA (mRNA) levels of thyroid hormone (TH)-responsive genes in chicken embryonic neuronal cells. In this study, we determined in ovo effects of PFHxS and PFHxA exposure (maximum dose = 38,000 and 9700 ng/g egg, respectively) on embryonic death, developmental endpoints, tissue accumulation, mRNA expression in liver and cerebral cortex, and plasma TH levels. Pipping success was reduced to 63% at the highest dose of PFHxS; no effects were observed for PFHxA. PFHxS exposure (38,000 ng/g) decreased tarsus length and embryo mass. PFHxS and PFHxA accumulated in the three tissue compartments analyzed as follows: yolk sac > liver > cerebral cortex. Type II and type III 5'-deiodinases (D2 and D3) and cytochrome P450 3A37 mRNA levels were induced in liver tissue of chicken embryos exposed to PFHxS, whereas D2, neurogranin (RC3), and octamer motif binding factor 1 mRNA levels were upregulated in cerebral cortex. Plasma TH levels were reduced in a concentration-dependent manner following PFHxS exposure; PFHxA had no effect. This in ovo study successfully validated previous in vitro results concerning the modulation of TH-responsive genes and identified adverse effects associated with TH homeostasis in response to PFHxS treatment. PMID:22302310

  7. 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

  8. 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.

  9. Dietary glycerol for quail: association between productive performance and COX III mRNA expression.

    Science.gov (United States)

    Silva, S C C; Gasparino, E; Batista, E; Tanamati, F; Vesco, A P D; Lala, B; de Oliveira, D P

    2016-01-01

    This study was carry out to evaluate mRNA expression of mitochondrial cytochrome c oxidase III in the Pectoralis superficialis muscle of 28-day-old quails fed diets containing 0, 8, and 12% glycerol. Total RNA was extracted (N = 10) and cDNA was amplified using specifics primers for qRT-PCR. Feed efficiency and feed intake were evaluated. COX III mRNA expression in breast muscle was higher in the group fed with 12% glycerol (0.863 AU); no differences were observed in the expression of this gene between the muscle of animals fed diets without glycerol (0.357 AU) and 8% glycerol (0.415 AU). Quails that showed greater COX III mRNA expression also showed the lowest feed efficiency. These results show that there is a difference in COX III mRNA expression in breast muscle of 28-day-old quail fed diets different concentrations of glycerol. PMID:27323091

  10. 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.

  11. 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.

  12. Nerve growth factor mRNA in brain: localization by in situ hybridization

    International Nuclear Information System (INIS)

    Nerve Growth Factor is a 118 amino acid polypeptide that plays an important role in the differentiation and survival of neurons. The recent discovery that a mRNA that encodes beta Nerve Growth Factor is present in brain suggests that the Nerve Growth Factor gene may not only regulate gene expression of peripheral but also of central neurons. To identify the site(s) of Nerve Growth Factor mRNA production in the brain and to determine which cells express the Nerve Growth Factor gene, the technique of in situ hybridization was employed. A 32P-labeled RNA probe complementary to Nerve Growth Factor mRNA hybridized to cells in the stratum granulosum of the dentate gyrus and the stratum pyramidale of the hippocampus. These observations identify for the first time cellular sites of Nerve Growth Factor gene expression in the central nervous system, and suggest that Nerve Growth Factor mRNA is produced by neurons

  13. 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

  14. 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

  15. Low-power millimeter wave radiations do not alter stress-sensitive gene expression of chaperone proteins.

    Science.gov (United States)

    Zhadobov, M; Sauleau, R; Le Coq, L; Debure, L; Thouroude, D; Michel, D; Le Dréan, Y

    2007-04-01

    This article reports experimental results on the influence of low-power millimeter wave (MMW) radiation at 60 GHz on a set of stress-sensitive gene expression of molecular chaperones, namely clusterin (CLU) and HSP70, in a human brain cell line. Selection of the exposure frequency is determined by its near-future applications for the new broadband civil wireless communication systems including wireless local area networks (WLAN) for domestic and professional uses. Frequencies around 60 GHz are strongly attenuated in the earth's atmosphere and such radiations represent a new environmental factor. An exposure system operating in V-band (50-75 GHz) was developed for cell exposure. U-251 MG glial cell line was sham-exposed or exposed to MMW radiation for different durations (1-33 h) and two different power densities (5.4 microW/cm(2) or 0.54 mW/cm(2)). As gene expression is a multiple-step process, we analyzed chaperone proteins induction at different levels. First, using luciferase reporter gene, we investigated potential effect of MMWs on the activation of transcription factors (TFs) and gene promoter activity. Next, using RT-PCR and Western blot assays, we verified whether MMW exposure could alter RNA accumulation, translation, or protein stability. Experimental data demonstrated the absence of significant modifications in gene transcription, mRNA, and protein amount for the considered stress-sensitive genes for the exposure durations and power densities investigated. The main results of this study suggest that low-power 60 GHz radiation does not modify stress-sensitive gene expression of chaperone proteins. PMID:17080454

  16. 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 ...

  17. 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...

  18. 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.

  19. Improved protocol for the extraction of bacterial mRNA from soils.

    Science.gov (United States)

    Sharma, Shilpi; Mehta, Ravikumar; Gupta, Rashi; Schloter, Michael

    2012-10-01

    An improved protocol for extraction of prokaryotic mRNA from soil samples was developed by modifying the extraction procedure to obtain higher yields of mRNA and to reduce co-extraction of humic acids. The modified protocol was found to be more robust and efficient compared to the original protocol by Griffiths et al. (2000) without compromising with the quality and quantity of RNA. PMID:22841738

  20. Mediator Complex Regulates Alternative mRNA Processing via the Med23 Subunit

    OpenAIRE

    Yan HUANG; Li, Wencheng; Yao, Xiao; Lin, Qi-jiang; Yin, Jing-wen; Liang, Yan; Heiner, Monika; Tian, Bin; HUI, JINGYI; Wang, Gang

    2012-01-01

    Mediator complex is an integrative hub for transcriptional regulation. Here we show that Mediator regulates alternative mRNA processing via its Med23 subunit. Combining tandem affinity purification and mass spectrometry, we identified a number of mRNA processing factors that bind to a soluble recombinant Mediator subunit MED23 but not to several other Mediator components. One of these factors, hnRNP L, specifically interacts with MED23 in vitro and in vivo. Consistently, Mediator partially co...

  1. The human insulin receptor mRNA contains a functional internal ribosome entry segment

    OpenAIRE

    Spriggs, Keith A.; Cobbold, Laura C.; Ridley, Simon H.; Coldwell, Mark; Bottley, Andrew; Bushell, Martin; Willis, Anne E.; Siddle, Kenneth

    2009-01-01

    Regulation of mRNA translation is an important mechanism determining the level of expression of proteins in eukaryotic cells. Translation is most commonly initiated by cap-dependent scanning, but many eukaryotic mRNAs contain internal ribosome entry segments (IRESs), providing an alternative means of initiation capable of independent regulation. Here, we show by using dicistronic luciferase reporter vectors that the 5'-UTR of the mRNA encoding human insulin receptor (hIR) contains a functiona...

  2. 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...

  3. 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...

  4. 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.

  5. 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...

  6. 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...

  7. Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing.

    Science.gov (United States)

    Roundtree, Ian A; He, Chuan

    2016-06-01

    N(6)-Methyladenosine (m(6)A) is emerging as a chemical mark that broadly affects the flow of genetic information in various biological processes in eukaryotes. Recently, Xiao et al. reported that the nuclear m(6)A reader protein YTHDC1 impacts mRNA splicing, providing a transcriptome-wide glance of splicing changes affected by this mRNA methylation reader protein. PMID:27050931

  8. 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...

  9. 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

  10. Altered expression and insulin-induced trafficking of Na+-K+-ATPase in rat skeletal muscle

    DEFF Research Database (Denmark)

    Galuska, Dana; Kotova, Olga; Barres, Romain;

    2009-01-01

    . Skeletal muscle insulin resistance was observed after 12 wk of HFD. Na(+)-K(+)-ATPase alpha(1)-subunit protein expression was increased 1.6-fold (P <0.05), whereas alpha(2)- and beta(1)-subunits and protein expression were decreased twofold (P <0.01) in parallel with decrease in plasma membrane Na......) and alpha(1) mRNA expression were increased after HFD and restored by ET. DNA binding activity of Sp-1, a transcription factor involved in the regulation of alpha(2)- and beta(1)-subunit expression, was decreased after HFD. ET increased phosphorylation of the Na(+)-K(+)-ATPase regulatory protein...... phospholemman. Phospholemman mRNA and protein expression were increased after HFD and restored to control levels after ET. Insulin-stimulated translocation of the alpha(2)-subunit to plasma membrane was impaired by HFD, whereas alpha(1)-subunit translocation remained unchanged. Alterations in sodium pump...

  11. Energy metabolism in developing chicken lymphocytes is altered during the embryonic to posthatch transition.

    Science.gov (United States)

    Rudrappa, Shashidhara G; Humphrey, Brooke D

    2007-02-01

    Adequate energy status in lymphocytes is vital for their development. The ability of developing chicken lymphocytes to acquire and metabolize energy substrates was determined during embryonic days (e) and neonatal days (d) of life when primary-energy substrate metabolism is altered at the whole-animal level. In 3 experiments, bursacytes and thymocytes were isolated on e17, e20, d1, d3, d7, or d14 to analyze markers associated with glucose, glutamine, and lipid metabolism. Bursacyte glucose transporter-3 (Glut-3) mRNA abundance increased from d1 to d14 and hexokinase-1 (HK-1) mRNA abundance was maximum on e20 (Pglutamine metabolism. Understanding the factors that regulate lymphocyte development in neonatal chicks may help promote their adaptive immune responses to pathogens in early life. PMID:17237322

  12. Evolutionary Conservation and Diversification of Puf RNA Binding Proteins and Their mRNA Targets.

    Science.gov (United States)

    Hogan, Gregory J; Brown, Patrick O; Herschlag, Daniel

    2015-11-01

    Reprogramming of a gene's expression pattern by acquisition and loss of sequences recognized by specific regulatory RNA binding proteins may be a major mechanism in the evolution of biological regulatory programs. We identified that RNA targets of Puf3 orthologs have been conserved over 100-500 million years of evolution in five eukaryotic lineages. Focusing on Puf proteins and their targets across 80 fungi, we constructed a parsimonious model for their evolutionary history. This model entails extensive and coordinated changes in the Puf targets as well as changes in the number of Puf genes and alterations of RNA binding specificity including that: 1) Binding of Puf3 to more than 200 RNAs whose protein products are predominantly involved in the production and organization of mitochondrial complexes predates the origin of budding yeasts and filamentous fungi and was maintained for 500 million years, throughout the evolution of budding yeast. 2) In filamentous fungi, remarkably, more than 150 of the ancestral Puf3 targets were gained by Puf4, with one lineage maintaining both Puf3 and Puf4 as regulators and a sister lineage losing Puf3 as a regulator of these RNAs. The decrease in gene expression of these mRNAs upon deletion of Puf4 in filamentous fungi (N. crassa) in contrast to the increase upon Puf3 deletion in budding yeast (S. cerevisiae) suggests that the output of the RNA regulatory network is different with Puf4 in filamentous fungi than with Puf3 in budding yeast. 3) The coregulated Puf4 target set in filamentous fungi expanded to include mitochondrial genes involved in the tricarboxylic acid (TCA) cycle and other nuclear-encoded RNAs with mitochondrial function not bound by Puf3 in budding yeast, observations that provide additional evidence for substantial rewiring of post-transcriptional regulation. 4) Puf3 also expanded and diversified its targets in filamentous fungi, gaining interactions with the mRNAs encoding the mitochondrial electron transport

  13. 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...

  14. Chemosensory alterations and cancer therapies

    International Nuclear Information System (INIS)

    Taste and olfaction provide sensory information and sensory pleasure. Cancer therapies affect both. Chemotherapy has not been shown to produce dramatic losses of taste or smell, but systematic studies on various chemotherapeutic agents and types of cancer are lacking. Radiation therapy does produce clear losses of both taste and smell. Both chemotherapy and radiation therapy alter the pleasure produced by taste and smell through the formation of conditioned aversions. That is, foods consumed in proximity with the nausea of therapy come to be unpleasant. The impact of conditioned aversions can be diminished by providing a scapegoat food just before therapy. Alterations in foods may be beneficial to the cancer patient. Increasing the concentrations of flavor ingredients can compensate for sensory losses, and providing pureed foods that retain the cognitive integrity of a meal can benefit the patient who has chewing or swallowing problems

  15. Targeted Mutagenesis in Plant Cells through Transformation of Sequence-Specific Nuclease mRNA

    Science.gov (United States)

    Stoddard, Thomas J.; Clasen, Benjamin M.; Baltes, Nicholas J.; Demorest, Zachary L.; Voytas, Daniel F.; Zhang, Feng; Luo, Song

    2016-01-01

    Plant genome engineering using sequence-specific nucleases (SSNs) promises to advance basic and applied plant research by enabling precise modification of endogenous genes. Whereas DNA is an effective means for delivering SSNs, DNA can integrate randomly into the plant genome, leading to unintentional gene inactivation. Further, prolonged expression of SSNs from DNA constructs can lead to the accumulation of off-target mutations. Here, we tested a new approach for SSN delivery to plant cells, namely transformation of messenger RNA (mRNA) encoding TAL effector nucleases (TALENs). mRNA delivery of a TALEN pair targeting the Nicotiana benthamiana ALS gene resulted in mutation frequencies of approximately 6% in comparison to DNA delivery, which resulted in mutation frequencies of 70.5%. mRNA delivery resulted in three-fold fewer insertions, and 76% were 10bp. In an effort to increase mutation frequencies using mRNA, we fused several different 5’ and 3’ untranslated regions (UTRs) from Arabidopsis thaliana genes to the TALEN coding sequence. UTRs from an A. thaliana adenine nucleotide α hydrolases-like gene (At1G09740) enhanced mutation frequencies approximately two-fold, relative to a no-UTR control. These results indicate that mRNA can be used as a delivery vehicle for SSNs, and that manipulation of mRNA UTRs can influence efficiencies of genome editing. PMID:27176769

  16. SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export.

    Science.gov (United States)

    Müller-McNicoll, Michaela; Botti, Valentina; de Jesus Domingues, Antonio M; Brandl, Holger; Schwich, Oliver D; Steiner, Michaela C; Curk, Tomaz; Poser, Ina; Zarnack, Kathi; Neugebauer, Karla M

    2016-03-01

    Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends. PMID:26944680

  17. Conserved noncoding sequences are associated with rates of mRNA decay in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Jacob B Spangler

    2013-05-01

    Full Text Available Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs, DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near α duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near α duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both nontranscribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression.

  18. Uridylation and PABP Cooperate to Repair mRNA Deadenylated Ends in Arabidopsis.

    Science.gov (United States)

    Zuber, Hélène; Scheer, Hélène; Ferrier, Emilie; Sement, François Michaël; Mercier, Pierre; Stupfler, Benjamin; Gagliardi, Dominique

    2016-03-22

    Uridylation emerges as a key modification promoting mRNA degradation in eukaryotes. In addition, uridylation by URT1 prevents the accumulation of excessively deadenylated mRNAs in Arabidopsis. Here, we show that the extent of mRNA deadenylation is controlled by URT1. By using TAIL-seq analysis, we demonstrate the prevalence of mRNA uridylation and the existence, at lower frequencies, of mRNA cytidylation and guanylation in Arabidopsis. Both URT1-dependent and URT1-independent types of uridylation co-exist but only URT1-mediated uridylation prevents the accumulation of excessively deadenylated mRNAs. Importantly, uridylation repairs deadenylated extremities to restore the size distribution observed for non-uridylated oligo(A) tails. In vivo and in vitro data indicate that Poly(A) Binding Protein (PABP) binds to uridylated oligo(A) tails and determines the length of U-extensions added by URT1. Taken together, our results uncover a role for uridylation and PABP in repairing mRNA deadenylated ends and reveal that uridylation plays diverse roles in eukaryotic mRNA metabolism. PMID:26972004

  19. A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines.

    Science.gov (United States)

    Toki, Yasumichi; Sasaki, Katsunori; Tanaka, Hiroki; Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro; Torimoto, Yoshihiro; Ohtake, Takaaki; Kohgo, Yutaka

    2016-08-01

    Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. PMID:27264950

  20. Three-Dimensional Mapping of mRNA Export through the Nuclear Pore Complex

    Directory of Open Access Journals (Sweden)

    Steven J. Schnell

    2014-11-01

    Full Text Available The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE. Plenty of nuclear pore complexes (NPCs embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms.

  1. Uridylation and PABP Cooperate to Repair mRNA Deadenylated Ends in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Hélène Zuber

    2016-03-01

    Full Text Available Uridylation emerges as a key modification promoting mRNA degradation in eukaryotes. In addition, uridylation by URT1 prevents the accumulation of excessively deadenylated mRNAs in Arabidopsis. Here, we show that the extent of mRNA deadenylation is controlled by URT1. By using TAIL-seq analysis, we demonstrate the prevalence of mRNA uridylation and the existence, at lower frequencies, of mRNA cytidylation and guanylation in Arabidopsis. Both URT1-dependent and URT1-independent types of uridylation co-exist but only URT1-mediated uridylation prevents the accumulation of excessively deadenylated mRNAs. Importantly, uridylation repairs deadenylated extremities to restore the size distribution observed for non-uridylated oligo(A tails. In vivo and in vitro data indicate that Poly(A Binding Protein (PABP binds to uridylated oligo(A tails and determines the length of U-extensions added by URT1. Taken together, our results uncover a role for uridylation and PABP in repairing mRNA deadenylated ends and reveal that uridylation plays diverse roles in eukaryotic mRNA metabolism.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. Altered integrity and decreased expression of hepatocyte tight junctions in rifampicin-induced cholestasis in mice

    International Nuclear Information System (INIS)

    Rifampicin is a well-known hepatotoxicant, but little is known about the mechanism of rifampicin-induced hepatotoxicity. The aim of this study was to characterize the expression and localization of hepatocyte tight junctions in rifampicin-induced cholestasis in mice. Cholestasis was induced by administration of rifampicin (200 mg/kg) for 7 consecutive days or treatment with a single dose of rifampicin (200 mg/kg) by gastric intubation. The expression of mRNA for hepatic zonula occludens (ZO)-1, ZO-2, ZO-3, occludin and claudin-1 was determined using RT-PCR. Localization of ZO-1 and occludin was detected using immunofluorescence. Results showed that there was an 82-fold increase in the conjugated bilirubin in serum in rifampicin-treated mice. In addition, an 8-fold increase in total bile acid in serum was observed after a seven-day administration of rifampicin. The integrity of hepatocyte ZO-1 and occludin was altered by a seven-day administration of rifampicin. Importantly, the integrity and intensity of hepatocyte tight junctions were altered as early as 30 min after a single dose of rifampicin. The expression of hepatic ZO-1 and ZO-2 mRNA was significantly decreased, beginning as early as 30 min and remaining a lower level 12 h after a single dose of rifampicin. Taken together, these results suggest that the altered integrity and internalization of hepatocyte tight junctions are associated with rifampicin-induced cholestasis.

  8. Prognostic significance of serum ERBB3 and ERBB4 mRNA in lung adenocarcinoma patients.

    Science.gov (United States)

    Masroor, Mirza; Javid, Jamsheed; Mir, Rashid; Y, Prasant; A, Imtiyaz; Z, Mariyam; Mohan, Anant; Ray, P C; Saxena, Alpana

    2016-01-01

    Serum messenger RNA (mRNA) is an emerging prognostic tool for noninvasive malignant disease prognosis, and to study serum mRNA may have importance in the prognosis and detection of disease. This study aimed to evaluate the possible prognostic role of serum ERBB3 and ERBB4 mRNA expressions in lung adenocarcinoma patients. One hundred newly diagnosed lung adenocarcinoma patients and 100 age- and sex-matched healthy controls were included. Expression was analysed by quantitative real-time PCR and overall survival was analysed by Kaplan-Meier analysis. Serum ERBB3 and ERBB4 mRNA expressions was found to be significantly associated with distant metastases and TNM stages. It was observed that patients with distant metastases had 4.8- and 3.4-fold high ERBB3 and ERBB4 expression in contrast to patients without distant metastases, respectively. It was also found that ERBB3 and ERBB4 mRNA expression was 7.7-fold and 6.7-fold high in TNM stage IV compared to TNM stage I, respectively. Significantly, 2.6-fold increased serum ERBB4 mRNA expression was found in patients with pleural effusion compared to patients without pleural effusion (p = 0.005). Lung adenocarcinoma patients with ≤8- and >8-fold increased serum ERBB3 mRNA expression had 10.0 and 5.5 months of overall median survival while serum ERBB4 mRNA with ≤10- and >10-fold increased expression showed 11.4 and 5.0 months overall median survival, respectively. ERBB3 and ERBB4 together also found to be significantly associated with poor overall median survival. Patients with ≤8 + ≤10- and >8 + >10-fold expression showed 11.3 vs 4.8 months of overall median survival, respectively. In conclusion, serum ERBB3 and ERBB4 mRNA expressions may be a prognostic marker and monitoring of serum ERBB3 and ERBB4 mRNA can be one of the predictive factors for metastases and poor overall survival of lung adenocarcinoma patients. PMID:26254096

  9. 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…

  10. Anguillicola crassus infection significantly affects the silvering related modifications in steady state mRNA levels in gas gland tissue of the European eel

    Directory of Open Access Journals (Sweden)

    Bernd ePelster

    2016-05-01

    Full Text Available Using Illumina sequencing, transcriptional changes occurring during silvering in swimbladder tissue of the European eel have been analyzed by comparison of yellow eel and silver eel tissue samples. Functional annotation analysis based on GO terms revealed significant expression changes in a number of genes related to the extracellular matrix, important for the control of gas permeability of the swimbladder, and to ROS (reactive oxygen species defense, important to cope with reactive oxygen species generated under hyperbaric oxygen partial pressures. Focusing on swimbladder tissue metabolism, levels of several mRNA species encoding glucose transport proteins were several-fold higher in silver eels, while enzymes of the glycolytic pathway were not affected. The significantly higher steady state level of a transcript encoding for membrane bound carbonic anhydrase, however, suggested that CO2 production in the pentose phosphate shunt and diffusion of CO2 was of particular importance in silver eel swimbladder. In addition, the mRNA level of a large number of genes related to immune response and to sexual maturation was significantly modified in the silver eel swimbladder. The modification of several processes related to protein metabolism and transport, cell cycle, and apoptosis suggested that these changes in swimbladder metabolism and permeability were achieved by increasing cell turn-over. The impact of an infection of the swimbladder with the nematode Anguillicola crassus has been assessed by comparing these expression changes with expression changes observed between uninfected yellow eel swimbladder tissue and infected silver eel swimbladder tissue. In contrast to uninfected silver eel swimbladder tissue, in infected tissue the mRNA level of several glycolytic enzymes was significantly elevated, and with respect to extracellular matrix, several mucin genes were many-fold higher in their mRNA level. Modification of many immune related genes and

  11. Anguillicola crassus Infection Significantly Affects the Silvering Related Modifications in Steady State mRNA Levels in Gas Gland Tissue of the European Eel.

    Science.gov (United States)

    Pelster, Bernd; Schneebauer, Gabriel; Dirks, Ron P

    2016-01-01

    Using Illumina sequencing, transcriptional changes occurring during silvering in swimbladder tissue of the European eel have been analyzed by comparison of yellow and silver eel tissue samples. Functional annotation analysis based on GO terms revealed significant expression changes in a number of genes related to the extracellular matrix, important for the control of gas permeability of the swimbladder, and to reactive oxygen species (ROS) defense, important to cope with ROS generated under hyperbaric oxygen partial pressures. Focusing on swimbladder tissue metabolism, levels of several mRNA species encoding glucose transport proteins were several-fold higher in silver eels, while enzymes of the glycolytic pathway were not affected. The significantly higher steady state level of a transcript encoding for membrane bound carbonic anhydrase, however, suggested that CO2 production in the pentose phosphate shunt and diffusion of CO2 was of particular importance in silver eel swimbladder. In addition, the mRNA level of a large number of genes related to immune response and to sexual maturation was significantly modified in the silver eel swimbladder. The modification of several processes related to protein metabolism and transport, cell cycle, and apoptosis suggested that these changes in swimbladder metabolism and permeability were achieved by increasing cell turn-over. The impact of an infection of the swimbladder with the nematode Anguillicola crassus has been assessed by comparing these expression changes with expression changes observed between uninfected yellow eel swimbladder tissue and infected silver eel swimbladder tissue. In contrast to uninfected silver eel swimbladder tissue, in infected tissue the mRNA level of several glycolytic enzymes was significantly elevated, and with respect to extracellular matrix, several mucin genes were many-fold higher in their mRNA level. Modification of many immune related genes and of the functional categories "response to

  12. Decreased hepatotoxic bile acid composition and altered synthesis in progressive human nonalcoholic fatty liver disease

    Energy Technology Data Exchange (ETDEWEB)

    Lake, April D. [University of Arizona, Department of Pharmacology and Toxicology, Tucson, AZ 85721 (United States); Novak, Petr [Biology Centre ASCR, Institute of Plant Molecular Biology, Ceske Budejovice 37001 (Czech Republic); Shipkova, Petia; Aranibar, Nelly; Robertson, Donald; Reily, Michael D. [Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Co., Princeton, NJ 08543 (United States); Lu, Zhenqiang [The Arizona Statistical Consulting Laboratory, University of Arizona, Tucson, AZ 85721 (United States); Lehman-McKeeman, Lois D. [Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Co., Princeton, NJ 08543 (United States); Cherrington, Nathan J., E-mail: cherrington@pharmacy.arizona.edu [University of Arizona, Department of Pharmacology and Toxicology, Tucson, AZ 85721 (United States)

    2013-04-15

    Bile acids (BAs) have many physiological roles and exhibit both toxic and protective influences within the liver. Alterations in the BA profile may be the result of disease induced liver injury. Nonalcoholic fatty liver disease (NAFLD) is a prevalent form of chronic liver disease characterized by the pathophysiological progression from simple steatosis to nonalcoholic steatohepatitis (NASH). The hypothesis of this study is that the ‘classical’ (neutral) and ‘alternative’ (acidic) BA synthesis pathways are altered together with hepatic BA composition during progression of human NAFLD. This study employed the use of transcriptomic and metabolomic assays to study the hepatic toxicologic BA profile in progressive human NAFLD. Individual human liver samples diagnosed as normal, steatosis, and NASH were utilized in the assays. The transcriptomic analysis of 70 BA genes revealed an enrichment of downregulated BA metabolism and transcription factor/receptor genes in livers diagnosed as NASH. Increased mRNA expression of BAAT and CYP7B1 was observed in contrast to decreased CYP8B1 expression in NASH samples. The BA metabolomic profile of NASH livers exhibited an increase in taurine together with elevated levels of conjugated BA species, taurocholic acid (TCA) and taurodeoxycholic acid (TDCA). Conversely, cholic acid (CA) and glycodeoxycholic acid (GDCA) were decreased in NASH liver. These findings reveal a potential shift toward the alternative pathway of BA synthesis during NASH, mediated by increased mRNA and protein expression of CYP7B1. Overall, the transcriptomic changes of BA synthesis pathway enzymes together with altered hepatic BA composition signify an attempt by the liver to reduce hepatotoxicity during disease progression to NASH. - Highlights: ► Altered hepatic bile acid composition is observed in progressive NAFLD. ► Bile acid synthesis enzymes are transcriptionally altered in NASH livers. ► Increased levels of taurine and conjugated bile acids

  13. Brain-derived neurotrophic factor signaling is altered in the forebrain of Engrailed-2 knockout mice.

    Science.gov (United States)

    Zunino, G; Messina, A; Sgadò, P; Baj, G; Casarosa, S; Bozzi, Y

    2016-06-01

    Engrailed-2 (En2), a homeodomain transcription factor involved in regionalization and patterning of the midbrain and hindbrain regions has been associated to autism spectrum disorders (ASDs). En2 knockout (En2(-/-)) mice show ASD-like features accompanied by a significant loss of GABAergic subpopulations in the hippocampus and neocortex. Brain-derived neurotrophic factor (BDNF) is a crucial factor for the postnatal development of forebrain GABAergic neurons, and altered GABA signaling has been hypothesized to underlie the symptoms of ASD. Here we sought to determine whether interneuron loss in the En2(-/-) forebrain might be related to altered expression of BDNF and its signaling receptors. We first evaluated the expression of different BDNF mRNA isoforms in the neocortex and hippocampus of wild-type (WT) and En2(-/-) mice. Quantitative RT-PCR showed a marked down-regulation of several splicing variants of BDNF mRNA in the neocortex but not hippocampus of adult En2(-/-) mice, as compared to WT controls. Accordingly, levels of mature BDNF protein were lower in the neocortex but not hippocampus of En2(-/-) mice, as compared to WT. Increased levels of phosphorylated TrkB and decreased levels of p75 receptor were also detected in the neocortex of mutant mice. Accordingly, the expression of low density lipoprotein receptor (LDLR) and RhoA, two genes regulated via p75 was significantly altered in forebrain areas of mutant mice. These data indicate that BDNF signaling alterations might be involved in the anatomical changes observed in the En2(-/-) forebrain and suggest a pathogenic role of altered BDNF signaling in this mouse model of ASD. PMID:26987954

  14. Decreased hepatotoxic bile acid composition and altered synthesis in progressive human nonalcoholic fatty liver disease

    International Nuclear Information System (INIS)

    Bile acids (BAs) have many physiological roles and exhibit both toxic and protective influences within the liver. Alterations in the BA profile may be the result of disease induced liver injury. Nonalcoholic fatty liver disease (NAFLD) is a prevalent form of chronic liver disease characterized by the pathophysiological progression from simple steatosis to nonalcoholic steatohepatitis (NASH). The hypothesis of this study is that the ‘classical’ (neutral) and ‘alternative’ (acidic) BA synthesis pathways are altered together with hepatic BA composition during progression of human NAFLD. This study employed the use of transcriptomic and metabolomic assays to study the hepatic toxicologic BA profile in progressive human NAFLD. Individual human liver samples diagnosed as normal, steatosis, and NASH were utilized in the assays. The transcriptomic analysis of 70 BA genes revealed an enrichment of downregulated BA metabolism and transcription factor/receptor genes in livers diagnosed as NASH. Increased mRNA expression of BAAT and CYP7B1 was observed in contrast to decreased CYP8B1 expression in NASH samples. The BA metabolomic profile of NASH livers exhibited an increase in taurine together with elevated levels of conjugated BA species, taurocholic acid (TCA) and taurodeoxycholic acid (TDCA). Conversely, cholic acid (CA) and glycodeoxycholic acid (GDCA) were decreased in NASH liver. These findings reveal a potential shift toward the alternative pathway of BA synthesis during NASH, mediated by increased mRNA and protein expression of CYP7B1. Overall, the transcriptomic changes of BA synthesis pathway enzymes together with altered hepatic BA composition signify an attempt by the liver to reduce hepatotoxicity during disease progression to NASH. - Highlights: ► Altered hepatic bile acid composition is observed in progressive NAFLD. ► Bile acid synthesis enzymes are transcriptionally altered in NASH livers. ► Increased levels of taurine and conjugated bile acids

  15. 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)

  16. Acute phase cytokines, TAC1, and toll-like receptor4 mRNA expression and health associated with group size in veal calves.

    Science.gov (United States)

    Abdelfattah, E M; Karousa, M M; Schutz, M M; Lay, D C; Marchant-Forde, J N; Eicher, S D

    2015-04-15

    Chronic stressors are a major health and well-being issue in animals. Immune status of animals under chronic stress is compromised, thus reducing disease resistance and compromising well-being of the animal. The objective of this study was to determine the influence of group size of veal calves on immune status and leukocyte mRNA expression of acute phase cytokines, toll-like receptor 4 (TLR4) and tachykinin 1 (TAC1) over a five-month finishing period. Holstein bull calves (n=168), 44±3 days of age were assigned to one of three treatments; 2, 4, or 8 calves/pen (pen space allowance of 1.82m(2)/calf). Jugular blood samples were collected at the day of grouping and then monthly for 4 months. The differential leukocyte counts were determined and mRNA was extracted from the leukocytes. Reverse transcription-qPCR was used to measure the gene expression of interleukin-1 (IL-1β), IL-1 receptor antagonist (IL-1Ra), tumor necrosis factor (TNF-α), TLR4, and TAC1 in leukocytes. Health was evaluated before grouping and monthly for 4 months. On the 1st month after grouping, veal calves that were housed in groups of 8 have greater expression of IL-1β mRNA than calves housed in groups of 4 or 2 (treatment×month, P=0.04). Also at 1 month, groups of 8 had greater TAC1 expression (Pveal calves in a group, given the same space during the finishing period did not alter IL-1Ra, TNF-α, and TLR4 mRNA expression. However, housing of calves in groups of 8 was associated with greater expression of IL-1β and TAC1 mRNA in peripheral blood leukocytes, and coughing during the first 2 months after grouping. Therefore, housing of veal calves in larger groups may lead to greater susceptibility to respiratory disease and stress. PMID:25746346

  17. The mRNA expression of pro- and anti-inflammatory cytokines in T regulatory cells in children with type 1 diabetes.

    Directory of Open Access Journals (Sweden)

    Maria Górska

    2010-06-01

    Full Text Available Type 1 diabetes mellitus (T1DM is caused by the autoimmune-mediated destruction of insulin-producing beta cells in the pancreas. T regulatory cells (Tregs represent an active mechanism of suppressing autoreactive T cells that escape central tolerance. The aim of our study was to test the hypothesis that T regulatory cells express pro- and anti-inflammatory cytokines, elements of cytotoxicity and OX40/4-1BB molecules. The examined group consisted of 50 children with T1DM. Fifty two healthy individuals (control group were enrolled into the study. A flow cytometric analysis of T-cell subpopulations was performed using the following markers: anti-CD3, anti-CD4, anti-CD25, anti-CD127, anti-CD134 and anti-CD137. Concurrently with the flow cytometric assessment of Tregs we separated CD4+CD25+CD127dim/- cells for further mRNA analysis. mRNA levels for transcription factor FoxP3, pro- and anti-inflammatory cytokines (interferon gamma, interleukin-2, interleukin-4, interleukin-10, transforming growth factor beta1 and tumor necrosis factor alpha, activatory molecules (OX40, 4-1BB and elements of cytotoxicity (granzyme B, perforin 1 were determined by real-time PCR technique. We found no alterations in the frequency of CD4+CD25highCD127low cells between diabetic and control children. Treg cells expressed mRNA for pro- and anti-inflammatory cytokines. Lower OX40 and higher 4-1BB mRNA but not protein levels in Treg cells in diabetic patients compared to the healthy children were noted. Our observations confirm the presence of mRNA for pro- and anti-inflammatory cytokines in CD4+CD25+CD127dim/- cells in the peripheral blood of children with T1DM. Further studies with the goal of developing new strategies to potentiate Treg function in autoimmune diseases are warranted.

  18. Tilt-tuned etalon locking for tunable laser stabilization.

    Science.gov (United States)

    Gibson, Bradley M; McCall, Benjamin J

    2015-06-15

    Locking to a fringe of a tilt-tuned etalon provides a simple, inexpensive method for stabilizing tunable lasers. Here, we describe the use of such a system to stabilize an external-cavity quantum cascade laser; the locked laser has an Allan deviation of approximately 1 MHz over a one-second integration period, and has a single-scan tuning range of approximately 0.4  cm(-1). The system is robust, with minimal alignment requirements and automated lock acquisition, and can be easily adapted to different wavelength regions or more stringent stability requirements with minor alterations. PMID:26076239

  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. Radiation-induced alterations of fracture healing biomechanics

    International Nuclear Information System (INIS)

    The effects of irradiation on the normal temporal progression of the physical properties of healing fractures were studied in a rat model. Fractures were surgically produced in the femur, stabilized with an intramedullary pin, and irradiated. One group of rats was exposed to 2,500 rads in divided doses over 2 weeks, beginning 3 days after fracture, and compared to a control group with fractures which were not irradiated. Animals were sacrificed at periodic intervals and the bones were tested to failure in torsion. The torque, stiffness, and energy increased and the angle decreased for the nonirradiated specimens in the expected fashion. This progression was deleteriously altered in the irradiated femurs

  1. Molecular cloning and regulation of murine fatty acid synthase mRNA

    International Nuclear Information System (INIS)

    Mouse liver mRNA that was enriched in sequences coding for fatty acid synthase (FAS) by sucrose-density gradient centrifugation was used as a template for cDNA synthesis. Double-stranded cDNA sequences were inserted into pBR322 and λgt10 and cloned. Clones containing putative cDNA sequences for FAS were identified by differential hybridization where 32P-cDNAs, synthesized from sucrose gradient purified liver mRNA from mice starved or starved and refed a fat-free diet, were used as probes. Two of these clones were further studied and found to contain sequences complementary to FAS mRNA by hybrid-selected translation and specific immunoprecipitation. Using these clones as probes, they selected 33 additional clones containing cDNA sequences for FAS. Partial DNA sequence data for these clones were obtained. Northern blot analysis revealed a single mRNA size of 9.3 kb when a cDNA clone with a 3.1 kb insert was used as a probe. This is in contrast to rat liver FAS which showed two mRNAs sizes of 9.2 and 10.0 kb. They also studied FAS mRNA level of 3T3-L1 preadipocytes during differentiation into adipocytes. An approximate 10-fold increase in FAS mRNA content was observed which corresponded with an increased rate of FAS synthesis indicating pretranslational regulation. The FAS cDNA probe was also employed to demonstrate that induction of FAS in the livers of previously starved mice that were fed a fat-free diet was controlled pretranslationally by a parallel modulation of the FAS mRNA concentration

  2. UCP2 mRNA expression is dependent on glucose metabolism in pancreatic islets

    International Nuclear Information System (INIS)

    Highlights: ► UCP2 mRNA levels are decreased in islets of Langerhans from glucokinase deficient mice. ► UCP2 mRNA up-regulation by glucose is dependent on glucokinase. ► Absence of UCP2 increases GSIS of glucokinase heterozygous pancreatic islets. ► This may protect glucokinase deficient mice from hyperglycemic damages. -- Abstract: 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 mimetic, did not prevent the glucose-induced up-regulation of UCP2. Glucose-stimulated insulin secretion was increased in UCP2−/− and GK+/− islets compared with GK+/− islets and UCP2 deficiency improved glucose tolerance of GK+/− mice. Accordingly, UCP2 deficiency increased ATP-levels of GK+/− mice. Thus, the compensatory down-regulation of UCP2 is involved in preserving the insulin secretory capacity of GK mutant mice and might also be implicated in limiting disease progression in MODY2 patients.

  3. The ribosome structure controls and directs mRNA entry, translocation and exit dynamics

    International Nuclear Information System (INIS)

    The protein-synthesizing ribosome undergoes large motions to effect the translocation of tRNAs and mRNA; here, the domain motions of this system are explored with a coarse-grained elastic network model using normal mode analysis. Crystal structures are used to construct various model systems of the 70S complex with/without tRNA, elongation factor Tu and the ribosomal proteins. Computed motions reveal the well-known ratchet-like rotational motion of the large subunits, as well as the head rotation of the small subunit and the high flexibility of the L1 and L7/L12 stalks, even in the absence of ribosomal proteins. This result indicates that these experimentally observed motions during translocation are inherently controlled by the ribosomal shape and only partially dependent upon GTP hydrolysis. Normal mode analysis further reveals the mobility of A- and P-tRNAs to increase in the absence of the E-tRNA. In addition, the dynamics of the E-tRNA is affected by the absence of the ribosomal protein L1. The mRNA in the entrance tunnel interacts directly with helicase proteins S3 and S4, which constrain the mRNA in a clamp-like fashion, as well as with protein S5, which likely orients the mRNA to ensure correct translation. The ribosomal proteins S7, S11 and S18 may also be involved in assuring translation fidelity by constraining the mRNA at the exit site of the channel. The mRNA also interacts with the 16S 3' end forming the Shine–Dalgarno complex at the initiation step; the 3' end may act as a 'hook' to reel in the mRNA to facilitate its exit

  4. Thymidylate synthase protein and p53 mRNA form an in vivo ribonucleoprotein complex.

    Science.gov (United States)

    Chu, E; Copur, S M; Ju, J; Chen, T M; Khleif, S; Voeller, D M; Mizunuma, N; Patel, M; Maley, G F; Maley, F; Allegra, C J

    1999-02-01

    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 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression. PMID:9891091

  5. Prognostic impact of mRNA levels of osteopontin splice variants in soft tissue sarcoma patients

    International Nuclear Information System (INIS)

    It is well known that osteopontin (OPN) plays an important role in tumor progression and that a high OPN expression level in several tumor entities correlates with poor prognosis in cancer patients. However, little is known about the prognostic relevance of the OPN mRNA splice variants. We analyzed the mRNA expression levels of different OPN splice variants in tumor tissue of 124 soft tissue sarcoma (STS) patients. Quantitative real-time PCR (qRT-PCR) was used to analyze the mRNA expression level of three OPN splice variants (OPN-a, -b and -c). The multivariate Cox's proportional hazard regression model revealed that high mRNA expression levels of OPN splice variants are significantly associated with poor prognosis in STS patients (n = 124). Women (n = 68) with high mRNA expression levels of OPN-a and OPN-b have an especially elevated risk of tumor-related death (OPN-a: RR = 3.0, P = 0.01, CI = 1.3-6.8; OPN-b: RR = 3.4, P = 0.01, CI = 1.4-8.2). In particular, we found that high mRNA expression levels of OPN-b and OPN-c correlated with a high risk of tumor-related death in STS patients that received radiotherapy (n = 52; OPN-b: RR = 10.3, P < 0.01, CI = 2.0-53.7; OPN-c: RR = 11.4, P < 0.01, CI = 2.2-59.3). Our study shows that elevated mRNA expression levels of OPN splice variants are negative prognostic and predictive markers for STS patients. Further studies are needed to clarify the impact of the OPN splice variants on prognosis

  6. Chronic neonatal nicotine exposure increases mRNA expression of neurotrophic factors in the postnatal rat hippocampus.

    Science.gov (United States)

    Son, Jong-Hyun; Winzer-Serhan, Ursula H

    2009-06-30

    Nicotine, the psychoactive ingredient in tobacco, can be neuroprotective but the mechanism is unknown. In the adult hippocampus, chronic nicotine can increase expression of growth factors which could contribute to nicotine's neuroprotective effects. During development, nicotine could also increase expression of neurotrophic factors. Therefore, we determined whether chronic neonatal nicotine (CNN) exposure increased mRNA expression levels of brain-derived neurotrophic factor (BDNF), nerve-growth factor (NGF), neurotrophin-3 (NT-3), fibroblast growth factor-2 (FGF-2), and insulin-like growth factor-1 (IGF-1). Nicotine (6 mg/kg/day in milk formula) or milk formula (controls) were delivered in three daily doses via oral gastric intubation to rat pups from postnatal day (P)1 to P8, and then sacrificed. Brains were processed for in situ hybridization using specific (35)S-labeled cRNA probes. At P8, CNN had a significant stimulant treatment effect on the expression of BDNF, FGF-2, NT-3 and IGF-1 [pCNN increased the number of IGF-1-expressing cells in CA1 (18.0%), CA3 (20.9%) and DG (17.7%). Thus, nicotine exposure during early postnatal development differentially up-regulated expression of neurotrophic factor mRNAs in the hippocampus, which could increase neurotrophic tone and alter developmental processes. PMID:19410565

  7. Acid Sulfate Alteration on Mars

    Science.gov (United States)

    Ming, D. W.; Morris, R. V.

    2016-01-01

    A variety of mineralogical and geochemical indicators for aqueous alteration on Mars have been identified by a combination of surface and orbital robotic missions, telescopic observations, characterization of Martian meteorites, and laboratory and terrestrial analog studies. Acid sulfate alteration has been identified at all three landing sites visited by NASA rover missions (Spirit, Opportunity, and Curiosity). Spirit landed in Gusev crater in 2004 and discovered Fe-sulfates and materials that have been extensively leached by acid sulfate solutions. Opportunity landing on the plains of Meridiani Planum also in 2004 where the rover encountered large abundances of jarosite and hematite in sedimentary rocks. Curiosity landed in Gale crater in 2012 and has characterized fluvial, deltaic, and lacustrine sediments. Jarosite and hematite were discovered in some of the lacustrine sediments. The high elemental abundance of sulfur in surface materials is obvious evidence that sulfate has played a major role in aqueous processes at all landing sites on Mars. The sulfate-rich outcrop at Meridiani Planum has an SO3 content of up to 25 wt.%. The interiors of rocks and outcrops on the Columbia Hills within Gusev crater have up to 8 wt.% SO3. Soils at both sites generally have between 5 to 14 wt.% SO3, and several soils in Gusev crater contain around 30 wt.% SO3. After normalization of major element compositions to a SO3-free basis, the bulk compositions of these materials are basaltic, with a few exceptions in Gusev crater and in lacustrine mudstones in Gale crater. These observations suggest that materials encountered by the rovers were derived from basaltic precursors by acid sulfate alteration under nearly isochemical conditions (i.e., minimal leaching). There are several cases, however, where acid sulfate alteration minerals (jarosite and hematite) formed in open hydrologic systems, e.g., in Gale crater lacustrine mudstones. Several hypotheses have been suggested for the

  8. Altered surfactant function and structure in SP-A gene targeted mice.

    OpenAIRE

    Korfhagen, T R; Bruno, M D; Ross, G F; Huelsman, K. M.; Ikegami, M; Jobe, A H; Wert, S E; Stripp, B R; Morris, R E; Glasser, S W; Bachurski, C J; Iwamoto, H S; Whitsett, J A

    1996-01-01

    The surfactant protein A (SP-A) gene was disrupted by homologous recombination in embryonic stem cells that were used to generate homozygous SP-A-deficient mice. SP-A mRNA and protein were not detectable in the lungs of SP-A(-/-) mice, and perinatal survival of SP-A(-/-) mice was not altered compared with wild-type mice. Lung morphology, surfactant proteins B-D, lung tissue, alveolar phospholipid pool sizes and composition, and lung compliance in SP-A(-/-) mice were unaltered. At the highest ...

  9. BVDV alters uterine prostaglandin production during pregnancy recognition in cows.

    Science.gov (United States)

    Cheng, Zhangrui; Abudureyimu, Ayimuguli; Oguejiofor, Chike F; Ellis, Rebekah; Barry, Amy Teresa; Chen, Xing; Anstaett, Olivia L; Brownlie, Joe; Wathes, D Claire

    2016-06-01

    Embryonic mortality in cows is at least in part caused by failure of pregnancy recognition (PR). Evidence has shown that bovine viral diarrhoea virus (BVDV) infection can disrupt pregnancy. Prostaglandins (PG) play important roles in many reproductive processes, such as implantation. The aim of this study was to investigate the effect of BVDV infection on uterine PG production and PR using an in vitro PR model. Bovine uterine endometrial cells isolated from ten BVDV-free cows were cultured and treated with 0 or 100ng/mL interferon-τ (IFNT) in the absence or presence of non-cytopathic BVDV (ncpBVDV). PGF2α and PGE2 concentrations in the spent medium were measured using radioimmunoassays, and in the treated cells expression of the genes associated with PG production and signalling was quantified using qPCR. The results showed that the IFNT challenge significantly stimulated PTGS1 and PTGER3 mRNA expression and PGE2 production; however, these stimulatory effects were neutralised in the presence of ncpBVDV infection. ncpBVDV infection significantly increased PTGS1 and mPGES1 mRNA expression and decreased AKR1B1 expression, leading to increased PGE2 and decreased PGF2α concentrations and an increased PGE2:PGF2α ratio. The other tested genes, including PGR, ESR1, OXTR, PTGS2, PTGER2 and PTGFR, were not significantly altered by IFNT, ncpBVDV or their combination. Our study suggests that BVDV infection may impair PR by (1) inhibiting the effect of IFNT on uterine PG production and (2) inducing an endocrine switch of PG production from PGF2α to PGE2 to decrease uterine immunity, thereby predisposing the animals to uterine disease. PMID:26952097

  10. Tissue cholesterol content alterations in streptozotocin-induced diabetic rats

    Institute of Scientific and Technical Information of China (English)

    Xin-ting WANG; Jia LI; Li LIU; Nan HU; Shi JIN; Can LIU; Dan MEI; Xiao-dong LIU

    2012-01-01

    Aim:Diabetes is associated with elevated serum total cholesterol level and disrupted lipoprotein subfractions.The aim of this study was to examine alterations in the tissue cholesterol contents closely related to diabetic complications.Methods:Intraperitoneal injection of streptozotocin was used to induce type 1 diabetes in adult male Sprague-Dawley rats.On d 35 after the injection,liver,heart,intestine,kidney,pancreas,cerebral cortex and hippocampus were isolated from the rats.The content of total and free cholesterol in the tissues was determined using HPLC.The ATP-binding cassette protein A1 (ABCA1) protein and ApoE mRNA were measured using Western blot and QT-PCR analyses,respectively.Results:In diabetic rats,the level of free cholesterol was significantly decreased in the peripheral tissues,but significantly elevated in hippocampus,as compared with those in the control rats.Diabetic rats showed a trend of decreasing the total cholesterol level in the peripheral tissues,but significant change was only found in kidney and liver.In diabetic rats,the level of the ABCA1 protein was significantly increased in the peripheral tissues and cerebral cortex; the expression of ApoE mRNA was slightly decreased in hippocampus and cerebral cortex,but the change had no statistical significance.Conclusion:Type 1 diabetes decreases the free cholesterol content in the peripheral tissues and increases the free cholesterol content in hippocampus.The decreased free cholesterol level in the peripheral tissues may be partly due to the increased expression of the ABCA1 protein.

  11. 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).

  12. Homological stabilizer codes

    International Nuclear Information System (INIS)

    In this paper we define homological stabilizer codes on qubits which encompass codes such as Kitaev’s toric code and the topological color codes. These codes are defined solely by the graphs they reside on. This feature allows us to use properties of topological graph theory to determine the graphs which are suitable as homological stabilizer codes. We then show that all toric codes are equivalent to homological stabilizer codes on 4-valent graphs. We show that the topological color codes and toric codes correspond to two distinct classes of graphs. We define the notion of label set equivalencies and show that under a small set of constraints the only homological stabilizer codes without local logical operators are equivalent to Kitaev’s toric code or to the topological color codes. - Highlights: ► We show that Kitaev’s toric codes are equivalent to homological stabilizer codes on 4-valent graphs. ► We show that toric codes and color codes correspond to homological stabilizer codes on distinct graphs. ► We find and classify all 2D homological stabilizer codes. ► We find optimal codes among the homological stabilizer codes.

  13. 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

  14. 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.

  15. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Massa, Christopher B.; Scott, Pamela; Abramova, Elena; Gardner, Carol; Laskin, Debra L.; Gow, Andrew J., E-mail: Gow@rci.rutgers.edu

    2014-07-01

    Acute Cl{sub 2} 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 Cl{sub 2} 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 Cl{sub 2} 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. Cl{sub 2} exposure had no effect on NOS2 mRNA or iNOS protein expression, nor on BAL NO{sub 3}{sup −} or NO{sub 2}{sup −}. 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. Cl{sub 2} 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 Cl{sub 2} inhalation. - Highlights: • Effect of 60 ppm*hr Cl{sub 2} gas on lung inflammation and mechanical function examined. • Pulmonary inflammation is transient and minor.

  17. Characterization of the ptr5+ gene involved in nuclear mRNA export in fission yeast

    International Nuclear Information System (INIS)

    Highlights: ► We cloned the ptr5+ gene involved in nuclear mRNA export in fission yeast. ► The ptr5+ gene was found to encode nucleoporin 85 (Nup85). ► Seh1p and Mlo3p are multi-copy suppressors for the ptr5 mutation. ► Ptr5p/Nup85p functions in nuclear mRNA export through the mRNA export factor Rae1p. ► Ptr5p/Nup85p interacts genetically with pre-mRNA splicing factors. -- Abstract: To analyze the mechanisms of mRNA export from the nucleus to the cytoplasm, we have isolated eleven mutants, ptr [poly(A)+ RNA transport] 1 to 11, which accumulate poly(A)+ RNA in the nucleus at a nonpermissive temperature in Schizosaccharomyces pombe. Of those, the ptr5–1 mutant shows dots- or a ring-like accumulation of poly(A)+ RNA at the nuclear periphery after shifting to the nonpermissive temperature. We cloned the ptr5+ gene and found that it encodes a component of the nuclear pore complex (NPC), nucleoporin 85 (Nup85). The ptr5–1 mutant shows no defects in protein transport, suggesting the specific involvement of Ptr5p/Nup85p in nuclear mRNA export in S. pombe. We identified Seh1p, a nucleoporin interacting with Nup85p, an mRNA-binding protein Mlo3p, and Sac3p, a component of the TREX-2 complex involved in coupling of nuclear mRNA export with transcription, as multi-copy suppressors for the ptr5–1 mutation. In addition, we found that the ptr5–1 mutation is synthetically lethal with a mutation of the mRNA export factor Rae1p, and that the double mutant exaggerates defective nuclear mRNA export, suggesting that Ptr5p/Nup85p is involved in nuclear mRNA export through Rae1p. Interestingly, the ptr5–1 mutation also showed synthetic effects with several prp pre-mRNA splicing mutations, suggesting a functional linkage between the NPCs and the splicing apparatus in the yeast nucleus.

  18. Correlated alteration effects in CM carbonaceous chondrites

    Science.gov (United States)

    Browning, Lauren B.; McSween, Harry Y., Jr.; Zolensky, Michael E.

    1996-07-01

    Three parameters are proposed to determine the relative extent of alteration in CM chondrites. The mineralogic alteration index monitors the relative progress of coupled substitutions in the progressive alteration of cronstedtite to Mg-serpentine and increases with increasing alteration. To calculate values of this index, an algorithm has been developed to estimate the average matrix phyllosilicate composition in individual CM chondrites. The second parameter is the volume percent of isolated matrix silicates, which decreases with progressive alteration due to mineral hydration. Finally, the volume percent of chondrule alteration monitors the extent of chondrule phyllosilicate production and increases as alteration proceeds. These parameters define the first CM alteration scale that relies on multiple indicators of progressive alteration. The following relative order of increasing alteration is established by this model: Murchison ≤ Bells Cochabamba and Boriskino experienced is less precisely constrained, although both fall near the middle of this sequence. A comparison between the mineralogic alteration index and literature values for the whole-rock chemistry of CM chondrites reveals several correlations. A positive, nearly linear correlation between bulk H content and progressive CM alteration suggests an approximately constant production rate of new phyllosilicates relative to the mineralogical transition from cronstedtite to Mg-serpentine. The abundance of trapped planetary 36Ar decreases systematically in progressively altered CM chondrites, suggesting the wholesale destruction of primary noble gas carrier phase (s) by aqueous reactions. Because low temperature fluid-rock reactions are generally associated with large isotopic mass fractionation factors, we also compared our model predictions with δ18O values for bulk CM samples. Although some of these data are poorly resolved, the order of increasing δ18O values approximates the order of increasing

  19. TWO STAGE FRAMEWORK FOR ALTERED FINGERPRINT MATCHING

    OpenAIRE

    T. R. Anoop; M.G. Mini

    2015-01-01

    Fingerprint alteration is the process of masking one’s identity from personal identification systems especially in boarder control security systems. Failure of matching the altered fingerprint of the criminals against the watch list of fingerprints can help them to break the security system. This fact leads to the need of a method for altered fingerprint matching. This paper presents a two stage method for altered fingerprint matching. In first stage, approximated global ridge orientation fie...

  20. Genetic alterations and epigenetic changes in hepatocarcinogenesis

    Directory of Open Access Journals (Sweden)

    Luz Stella Hoyos Giraldo

    2007-02-01

    -family: QuorumITCbyBT-Book">Dietary factors have a role in the modification of epigenetic changes. Altered expression of DNMT mRNA and DNA hypermethylation of TSGs, has been observed in HCC. The mechanism of the interaction between chemical carcinogens and changes of methylation is still unclear and need much more research. Risk factors for hepatocarcinogenesis and their genetic and epigenetic reactions remains poorly understood.