WorldWideScience

Sample records for altering mrna stability

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

    Science.gov (United States)

    Donaldson, Michael E; Saville, Barry J

    2013-07-01

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

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

    Science.gov (United States)

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

    2016-07-01

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

  3. Vibrational force alters mRNA expression in osteoblasts

    Science.gov (United States)

    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.

  4. PTB and TIAR binding to insulin mRNA 3′- and 5′UTRs; implications for insulin biosynthesis and messenger stability

    Directory of Open Access Journals (Sweden)

    Rikard G. Fred

    2016-09-01

    Conclusions: These experiments indicate that alterations in insulin mRNA stability and translation correlate with differential RBP binding. We propose that the balance between PTB on one hand and TIAR on the other participates in the control of insulin mRNA stability and utilization for insulin biosynthesis.

  5. 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.

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. HDAC3 regulates stability of estrogen receptor α mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Oie, Shohei; Matsuzaki, Kazuya; Yokoyama, Wataru; Murayama, Akiko; Yanagisawa, Junn, E-mail: junny@agbi.tsukuba.ac.jp

    2013-03-08

    Highlights: ► HDAC inhibitors decrease the stability of ERα mRNA in MCF-7 cells. ► HDAC3 is involved in maintaining ERα mRNA stability in MCF-7 cells. ► ERα mRNA instability by knockdown of HDAC3 reduces the estrogen-dependent proliferation of ERα-positive MCF-7 cells. ► HDAC3 specific inhibitor will be one of new drugs for ERα-positive breast cancers. -- Abstract: Estrogen receptor alpha (ERα) expression is a risk factor for breast cancer. HDAC inhibitors have been demonstrated to down-regulate ERα expression in ERα-positive breast cancer cell lines, but the molecular mechanisms are poorly understood. Here, we showed that HDAC inhibitors decrease the stability of ERα mRNA, and that knockdown of HDAC3 decreases the stability of ERα mRNA and suppresses estrogen-dependent proliferation of ERα-positive MCF-7 breast cancer cells. In the Oncomine database, expression levels of HDAC3 in ERα-positive tumors are higher than those in ERα-negative tumors, thus suggesting that HDAC3 is necessary for ERα mRNA stability, and is involved in the estrogen-dependent proliferation of ERα-positive tumors.

  9. Major role for mRNA stability in shaping the kinetics of gene induction

    Directory of Open Access Journals (Sweden)

    Zeller Karen I

    2010-04-01

    Full Text Available Abstract Background mRNA levels in cells are determined by the relative rates of RNA production and degradation. Yet, to date, most analyses of gene expression profiles were focused on mechanisms which regulate transcription, while the role of mRNA stability in modulating transcriptional networks was to a large extent overlooked. In particular, kinetic waves in transcriptional responses are usually interpreted as resulting from sequential activation of transcription factors. Results In this study, we examined on a global scale the role of mRNA stability in shaping the kinetics of gene response. Analyzing numerous expression datasets we revealed a striking global anti-correlation between rapidity of induction and mRNA stability, fitting the prediction of a kinetic mathematical model. In contrast, the relationship between kinetics and stability was less significant when gene suppression was analyzed. Frequently, mRNAs that are stable under standard conditions were very rapidly down-regulated following stimulation. Such effect cannot be explained even by a complete shut-off of transcription, and therefore indicates intense modulation of RNA stability. Conclusion Taken together, our results demonstrate the key role of mRNA stability in determining induction kinetics in mammalian transcriptional networks.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. UBE2B mRNA alterations are associated with severe oligozoospermia in infertile men

    Science.gov (United States)

    Yatsenko, Alexander N.; Georgiadis, Andrew P.; Murthy, Lata J.; Lamb, Dolores J.; Matzuk, Martin M.

    2013-01-01

    Oligozoospermia (low sperm count) is a common semen deficiency. However, to date, few genetic defects have been identified to cause this condition. Moreover, even fewer molecular genetic diagnostic tests are available for patients with oligozoospermia in the andrology clinic. Based on animal and gene expression studies of oligozoospermia, several molecular pathways may be disrupted in post-meiotic spermatozoa. One of the disrupted pathways is protein ubiquitination and cell apoptosis. A critical protein involved in this pathway is the ubiquitin-conjugating enzyme 2B, UBE2B. Absence of Ube2b in male mice causes spermatogenic meiotic disruption with increased apoptosis, leading to infertility. To examine the association between messenger RNA defects in UBE2B and severe oligozoospermia (0.1–10 × 106 cells/ml), sequencing of sperm cDNA in 326 oligozoospermic patients and 421 normozoospermic men was performed. mRNA alterations in UBE2B were identified in sperm in 4.6% (15 out of 326) of the oligozoospermic patients, but not found in control men, suggesting strong association between mRNA defects and oligozoospermia (χ2 = 19, P = 0.0001). Identified UBE2B alterations include nine splicing, four missense and two nonsense alterations. The follow-up screen of corresponding DNA regions did not reveal causative DNA mutations, suggesting a post-transcriptional nature of identified defects. None of these variants were reported in the dbSNP database, although other splicing abnormalities with low level of expression were present in 11 out of 421 (2.6%) controls. Our findings suggest that two distinct molecular mechanisms, mRNA editing and splicing processing, are disrupted in oligozoospermia. We speculate that the contribution of post-transcriptional mRNA defects to oligozoospermia could be greater than previously anticipated. PMID:23378580

  12. The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA.

    Science.gov (United States)

    López de Silanes, Isabel; Gorospe, Myriam; Taniguchi, Hiroaki; Abdelmohsen, Kotb; Srikantan, Subramanya; Alaminos, Miguel; Berdasco, María; Urdinguio, Rocío G; Fraga, Mario F; Jacinto, Filipe V; Esteller, Manel

    2009-05-01

    The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3'UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT-qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns.

  13. Gravitational loading of a simulated launch alters mRNA expression in osteoblasts

    Science.gov (United States)

    Fitzgerald, J.; Hughes-Fulford, M.

    1996-01-01

    Serum-deprived mouse osteoblastic cells (MC3T3-E1a) were centrifuged under a regime designed to simulate a space shuttle launch (maximum of 3g). Messenger RNA levels for eight genes involved in bone growth and maintenance were determined using RT-PCR. Following 30 min of centrifugation, mRNA level for early response gene c-fos was significantly increased 89% (P gene osteocalcin was significantly decreased to 44% of control level (P basal mRNA level for TGFbeta3 was detected. In addition, no change in the steady-state synthesis of prostaglandin E2 was detected, possibly due to lack of lipid substrates in serum-deprived cells, suggesting that the increase in c-fos mRNA in response to gravitational loading is a result of mechanical stimulation. These results indicate that a small magnitude mechanical loading, such as that experienced during a shuttle launch, can alter mRNA levels in quiescent osteoblastic cells.

  14. Control of mRNA stability during development of Dictyostelium discoideum.

    Science.gov (United States)

    Mangiarotti, G

    1989-01-01

    A large group of mRNA species (which are mainly pre-spore specific) accumulate only after the formation of multicellular aggregates. They are transcribed at a constant rate from the beginning of development and their accumulation is controlled by a 10-20-fold increase in their stability. This mRNA stabilization is dependent upon multicellularity. When aggregates are dispersed, the mRNAs are destabilized; if cells are allowed to reaggregate, the destabilization is reversed. Destabilization is not due to a selective exclusion of mRNA from polyribosomes, but is a primary control event. It does not require synthesis of new RNA or protein, but it may require an interaction between ribosome and the 5'-end of mRNA molecules.

  15. Codon optimality is a major determinant of mRNA stability.

    Science.gov (United States)

    Presnyak, Vladimir; Alhusaini, Najwa; Chen, Ying-Hsin; Martin, Sophie; Morris, Nathan; Kline, Nicholas; Olson, Sara; Weinberg, David; Baker, Kristian E; Graveley, Brenton R; Coller, Jeff

    2015-03-12

    mRNA degradation represents a critical regulated step in gene expression. Although the major pathways in turnover have been identified, accounting for disparate half-lives has been elusive. We show that codon optimality is one feature that contributes greatly to mRNA stability. Genome-wide RNA decay analysis revealed that stable mRNAs are enriched in codons designated optimal, whereas unstable mRNAs contain predominately non-optimal codons. Substitution of optimal codons with synonymous, non-optimal codons results in dramatic mRNA destabilization, whereas the converse substitution significantly increases stability. Further, we demonstrate that codon optimality impacts ribosome translocation, connecting the processes of translation elongation and decay through codon optimality. Finally, we show that optimal codon content accounts for the similar stabilities observed in mRNAs encoding proteins with coordinated physiological function. This work demonstrates that codon optimization exists as a mechanism to finely tune levels of mRNAs and, ultimately, proteins.

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

    Institute of Scientific and Technical Information of China (English)

    朱长虹; 田红

    2001-01-01

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

  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. Neural Progenitor Cells Promote Axonal Growth and Alter Axonal mRNA Localization in Adult Neurons

    Science.gov (United States)

    Merianda, Tanuja T.; Jin, Ying

    2017-01-01

    Abstract The inhibitory environment of the spinal cord and the intrinsic properties of neurons prevent regeneration of axons following CNS injury. However, both ascending and descending axons of the injured spinal cord have been shown to regenerate into grafts of embryonic neural progenitor cells (NPCs). Previous studies have shown that grafts composed of glial-restricted progenitors (GRPs) and neural-restricted progenitors (NRPs) can provide a permissive microenvironment for axon growth. We have used cocultures of adult rat dorsal root ganglion (DRG) neurons together with NPCs, which have shown significant enhancement of axon growth by embryonic rat GRP and GRPs/NRPs, both in coculture conditions and when DRGs are exposed to conditioned medium from the NPC cultures. This growth-promoting effect of NPC-conditioned medium was also seen in injury-conditioned neurons. DRGs cocultured with GRPs/NRPs showed altered expression of regeneration-associated genes at transcriptional and post-transcriptional levels. We found that levels of GAP-43 mRNA increased in DRG cell bodies and axons. However, hepcidin antimicrobial peptide (HAMP) mRNA decreased in the cell bodies of DRGs cocultured with GRPs/NRPs, which is distinct from the increase in cell body HAMP mRNA levels seen in DRGs after injury conditioning. Endogenous GAP-43 and β-actin mRNAs as well as reporter RNAs carrying axonally localizing 3'UTRs of these transcripts showed significantly increased levels in distal axons in the DRGs cocultured with GRPs/NRPs. These results indicate that axon growth promoted by NPCs is associated not only with enhanced transcription of growth-associated genes but also can increase localization of some mRNAs into growing axons. PMID:28197547

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

    Directory of Open Access Journals (Sweden)

    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

  20. Alterations in lipoxygenase and cyclooxygenase-2 catalytic activity and mRNA expression in prostate carcinoma.

    Science.gov (United States)

    Shappell, S B; Manning, S; Boeglin, W E; Guan, Y F; Roberts, R L; Davis, L; Olson, S J; Jack, G S; Coffey, C S; Wheeler, T M; Breyer, M D; Brash, A R

    2001-01-01

    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 5-HETE 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 COX-2 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 occurs in high

  1. KSRP modulation of GAP-43 mRNA stability restricts axonal outgrowth in embryonic hippocampal neurons.

    Directory of Open Access Journals (Sweden)

    Clark W Bird

    Full Text Available The KH-type splicing regulatory protein (KSRP promotes the decay of AU-rich element (ARE-containing mRNAs. Although KSRP is expressed in the nervous system, very little is known about its role in neurons. In this study, we examined whether KSRP regulates the stability of the ARE-containing GAP-43 mRNA. We found that KSRP destabilizes this mRNA by binding to its ARE, a process that requires the presence of its fourth KH domain (KH4. Furthermore, KSRP competed with the stabilizing factor HuD for binding to these sequences. We also examined the functional consequences of KSRP overexpression and knockdown on the differentiation of primary hippocampal neurons in culture. Overexpression of full length KSRP or KSRP without its nuclear localization signal hindered axonal outgrowth in these cultures, while overexpression of a mutant protein without the KH4 domain that has less affinity for binding to GAP-43's ARE had no effect. In contrast, depletion of KSRP led to a rise in GAP-43 mRNA levels and a dramatic increase in axonal length, both in KSRP shRNA transfected cells and neurons cultured from Ksrp(+/- and Ksrp(-/- embryos. Finally we found that overexpression of GAP-43 rescued the axonal outgrowth deficits seen with KSRP overexpression, but only when cells were transfected with GAP-43 constructs containing 3' UTR sequences targeting the transport of this mRNA to axons. Together, our results suggest that KSRP is an important regulator of mRNA stability and axonal length that works in direct opposition to HuD to regulate the levels of GAP-43 and other ARE-containing neuronal mRNAs.

  2. Codon Usage and 3' UTR Length Determine Maternal mRNA Stability in Zebrafish.

    Science.gov (United States)

    Mishima, Yuichiro; Tomari, Yukihide

    2016-03-17

    The control of mRNA stability plays a central role in regulating gene expression. In metazoans, the earliest stages of development are driven by maternally supplied mRNAs. The degradation of these maternal mRNAs is critical for promoting the maternal-to-zygotic transition of developmental programs, although the underlying mechanisms are poorly understood in vertebrates. Here, we characterized maternal mRNA degradation pathways in zebrafish using a transcriptome analysis and systematic reporter assays. Our data demonstrate that ORFs enriched with uncommon codons promote deadenylation by the CCR4-NOT complex in a translation-dependent manner. This codon-mediated mRNA decay is conditional on the context of the 3' UTR, with long 3' UTRs conferring resistance to deadenylation. These results indicate that the combined effect of codon usage and 3' UTR length determines the stability of maternal mRNAs in zebrafish embryos. Our study thus highlights the codon-mediated mRNA decay as a conserved regulatory mechanism in eukaryotes.

  3. Post-transcriptional regulation of coumarin 7-hydroxylase (P450coh) induction by xenobiotics in mouse liver: mRNA stabilization by pyrazole

    Energy Technology Data Exchange (ETDEWEB)

    Aida, K.; Negishi, M. (NIEHS/NIH, Research Triangle Park, NC (United States))

    1991-03-15

    The induction mechanism by pyrazole or phenobarbital of coumarin 7-hydroxylase was investigated in DBA/2J male mice. The P450coh mRNA in the pyrazole-induced mice was increased gradually to a 20-fold higher level within 48 hr, yet transcription of the P450coh gene was not affected. The half-life of P450coh mRNA, on the other hand, was at least 4-fold longer in the pyrazole-induced DBA2J than in control DBA/2J male mice. The stabilization of P450coh mRNA, therefore, is the primary mechanism for the induction by pyrazole of coumarin 7-hydroxylase. Phenobarbital, on the other hand, regulates the induction translationally or post-translationally. This drug affected neither the P450coh mRNA nor the P450coh gene's transcription levels in the DBA/2J male mice, although Western blots showed a 2- to 3-fold increase of the P450coh protein in the liver microsomes of the drug-treated mice. The results indicate, therefore, that both phenobarbital and pyrazole regulate the P450coh induction post-transcriptional efficiency of P450coh mRNA or alters the degradation rate of P450coh protein, while the latter stabilizes P450coh mRNA.

  4. Posttranscriptional control of Klebsiella pneumoniae nif mRNA stability by the nifL product.

    OpenAIRE

    1986-01-01

    Posttranscriptional control of nif mRNA stability was demonstrated by functional and chemical analyses, using specific probes for four nif transcripts. In the wild type, nif transcripts (except nifLA) were stable during derepression, with half-lives of approximately 30 min. They were dramatically destabilized by O2 or elevated temperature (41 degrees C) and to a lesser extent by NH4+. In contrast, the nifLA message was not particularly stable, and posttranscriptional control was not evident. ...

  5. Stabilization of LCD devices via geometric alteration.

    Science.gov (United States)

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

    2013-02-20

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

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

    Science.gov (United States)

    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 bind mTORC2 via Rictor and/or Sin1 and may signal downstream events promoting apoptosis. Protor-2 dissociates from mTORC2 upon hyperactivation of the kinase and is not required for mTORC2 integrity or activity. We identified Protor-2 in a yeast two-hybrid screen as a TTP interactor using the C-terminal mRNA decay domain of TTP as bait. The interaction of Protor-2 with TTP was also confirmed in vivo in co-immunoprecipitation experiments and Protor-2 was also detected in immunoprecipitates of rictor. Protor-2 was shown to stimulate TTP-mediated mRNA turnover of several TTP-associated mRNAs (TNF-α, GM-CSF, IL-3 and COX-2) in Jurkat cells when overexpressed while the half-lives of transcripts which do not decay via a TTP-mediated mechanism were unaffected. Knockdown of Protor-2 via RNAi inhibited TTP-mediated mRNA turnover of these TTP-associated mRNAs and inhibited association of TTP with cytoplasmic stress granules (SG) or mRNA processing bodies (P-bodies) following induction of the integrated stress response. These results suggest that Protor-2 associates with TTP to accelerate TTP-mediated mRNA turnover and functionally links the control of TTP regulated mRNA stability to mTORC2 activity. PMID:21964062

  7. A dominant nuclear mutation in Chlamydomonas identifies a factor controlling chloroplast mRNA stability by acting on the coding region of the atpA transcript.

    Science.gov (United States)

    Drapier, Dominique; Girard-Bascou, Jacqueline; Stern, David B; Wollman, Francis-André

    2002-09-01

    We have characterized a nuclear mutation, mda1-ncc1, that affects mRNA stability for the atpA gene cluster in the chloroplast of Chlamydomonas. Unlike all nuclear mutations altering chloroplast gene expression described to date, mda1-ncc1 is a dominant mutation that still allows accumulation of detectable amounts of atpA mRNAs. At variance with the subset of these mutations that affect mRNA stability through the 5' UTR of a single chloroplast transcript, the mutated version of MDA1 acts on the coding region of the atpA message. We discuss the action of MDA1 in relation to the unusual pattern of expression of atpA that associates particularly short lived-transcripts with a very high translational efficiency.

  8. Induction of human spermine oxidase SMO(PAOh1) is regulated at the levels of new mRNA synthesis, mRNA stabilization and newly synthesized protein.

    Science.gov (United States)

    Wang, Yanlin; Hacker, Amy; Murray-Stewart, Tracy; Fleischer, Jennifer G; Woster, Patrick M; Casero, Robert A

    2005-03-15

    The oxidation of polyamines induced by antitumour polyamine analogues has been associated with tumour response to specific agents. The human spermine oxidase, SMO(PAOh1), is one enzyme that may play a direct role in the cellular response to the antitumour polyamine analogues. In the present study, the induction of SMO(PAOh1) enzyme activity by CPENSpm [N1-ethyl-N11-(cyclopropyl)methyl-4,8,diazaundecane] is demonstrated to be a result of newly synthesized mRNA and protein. Inhibition of new RNA synthesis by actinomycin D inhibits both the appearance of SMO(PAOh1) mRNA and enzyme activity. Similarly, inhibition of newly synthesized protein with cycloheximide prevents analogue-induced enzyme activity. Half-life determinations indicate that stabilization of SMO(PAOh1) protein does not play a significant role in analogue-induced activity. However, half-life experiments using actinomycin D indicate that CPENSpm treatment not only increases mRNA expression, but also leads to a significant increase in mRNA half-life (17.1 and 8.8 h for CPENSpm-treated cells and control respectively). Using reporter constructs encompassing the SMO(PAOh1) promoter region, a 30-90% increase in transcription is observed after exposure to CPENSpm. The present results are consistent with the hypothesis that analogue-induced expression of SMO(PAOh1) is a result of increased transcription and stabilization of SMO(PAOh1) mRNA, leading to increased protein production and enzyme activity. These data indicate that the major level of control of SMO(PAOh1) expression in response to polyamine analogues exposure is at the level of mRNA.

  9. Human cytomegalovirus induces alteration of (-actin mRNA and microfilaments in human embryo fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    林茂芳; 魏国庆; 黄河; 蔡真

    2004-01-01

    Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time- and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

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

    Science.gov (United States)

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

    2011-01-01

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

  11. The Expression of Antibiotic Resistance Methyltransferase Correlates with mRNA Stability Independently of Ribosome Stalling.

    Science.gov (United States)

    Dzyubak, Ekaterina; Yap, M N

    2016-12-01

    Members of the Erm methyltransferase family modify 23S rRNA of the bacterial ribosome and render cross-resistance to macrolides and multiple distantly related antibiotics. Previous studies have shown that the expression of erm is activated when a macrolide-bound ribosome stalls the translation of the leader peptide preceding the cotranscribed erm Ribosome stalling is thought to destabilize the inhibitory stem-loop mRNA structure and exposes the erm Shine-Dalgarno (SD) sequence for translational initiation. Paradoxically, mutations that abolish ribosome stalling are routinely found in hyper-resistant clinical isolates; however, the significance of the stalling-dead leader sequence is largely unknown. Here, we show that nonsense mutations in the Staphylococcus aureus ErmB leader peptide (ErmBL) lead to high basal and induced expression of downstream ErmB in the absence or presence of macrolide concomitantly with elevated ribosome methylation and resistance. The overexpression of ErmB is associated with the reduced turnover of the ermBL-ermB transcript, and the macrolide appears to mitigate mRNA cleavage at a site immediately downstream of the ermBL SD sequence. The stabilizing effect of antibiotics on mRNA is not limited to ermBL-ermB; cationic antibiotics representing a ribosome-stalling inducer and a noninducer increase the half-life of specific transcripts. These data unveil a new layer of ermB regulation and imply that ErmBL translation or ribosome stalling serves as a "tuner" to suppress aberrant production of ErmB because methylated ribosome may impose a fitness cost on the bacterium as a result of misregulated translation.

  12. 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.

  13. Expression of trkB mRNA is altered in rat hippocampus after experimental brain trauma.

    Science.gov (United States)

    Hicks, R R; Zhang, L; Dhillon, H S; Prasad, M R; Seroogy, K B

    1998-08-31

    Recent investigations have shown that expression of mRNAs for the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) is differentially altered in the hippocampus following traumatic brain injury. In the present study, modulation of neurotrophin receptor expression was examined in the hippocampus in a rat model of traumatic brain injury using in situ hybridization. Messenger RNA for trkB, the high-affinity receptor for BDNF and neurotrophin-4 (NT-4), was increased between 3 and 6 h bilaterally in the dentate gyrus following a lateral fluid-percussion brain injury of moderate severity (2.0-2.1 atm). No time-dependent alterations were observed for trkB mRNA in hippocampal subfields CA1 and CA3. Levels of mRNA for trkC, the high-affinity receptor for NT-3, did not change in any region of the hippocampus. These data demonstrate that lateral fluid-percussion injury modulates expression of trkB mRNA in the hippocampus and support a role for BDNF/trkB signalling mechanisms in secondary events associated with traumatic brain injury.

  14. 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.

  15. 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.

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

    Science.gov (United States)

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

    2016-09-01

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

  17. Effect of the increased stability of the penicillin amidase mRNA on the protein expression levels.

    Science.gov (United States)

    Viegas, Sandra C; Schmidt, Dorothea; Kasche, Volker; Arraiano, Cecília M; Ignatova, Zoya

    2005-09-12

    Several factors at transcriptional, post-transcriptional or post-translational level determine the fate of a target protein and can severely restrict its yield. Here, we focus on the post-transcriptional regulation of the biosynthesis of the periplasmic protein, penicillin amidase (PA). The PA mRNA stability was determined under depleted RNase conditions in strains carrying single or multiple RNase deletions. Single deletion of the endonuclease RNase E yielded, as the highest, a fourfold stabilization of the PA mRNA. This effect, however, was reduced twice at post-translational level. The RNase II, generating secondary exonucleolytic cleavages in the mRNA, although not significantly influencing the PA mRNA decay, led also to an increase of the amount of mature PA. The non-proportional correlation between increased mRNA longevity and amount of active enzyme propose that the rational strategies for yield improvement must be based on a simultaneous tuning of more than one yield restricting factor.

  18. 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......' untranslated region (UTR). We have now identified two major RNA-binding proteins, nucleolin and YB-1, that specifically bind to the JRE. Binding of both proteins is required for IL-2 mRNA stabilization induced by T-cell activation signals and for JNK-induced stabilization in a cell-free system that duplicates...... essential features of regulated mRNA decay. Nucleolin and YB-1 are required for formation of an IL-2 mRNP complex that responds to specific mRNA stabilizing signals....

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

    Science.gov (United States)

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

    2016-04-26

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

  20. Estrogenic environmental contaminants alter the mRNA abundance profiles of genes involved in gonadal differentiation of the American bullfrog

    Science.gov (United States)

    Wolff, Stephanie E.; Veldhoen, Nik; Helbing, Caren C.; Ramirez, Claire A.; Malpas, Janae M.; Propper, Catherine R.

    2015-01-01

    Wildlife and human populations are exposed to anthropogenic mixtures of chemicals in the environment that may adversely influence normal reproductive function and development. We determined the effects of exposure to estrogenic chemicals and wastewater effluent (WWE) on developing gonads of the American bullfrog, Rana (Lithobates) catesbeiana, a species whose widespread distribution make it an ideal model for environmental monitoring for endocrine effects of chemical contaminants. Premetamorphic bullfrog tadpoles were exposed to treatment vehicle, 17β-estradiol (E2; 10−9 M) or 4-tert-octylphenol (OP; 10−9 M, 10−8 M, and 10−7 M). Additionally, gonadal differentiation was evaluated in bullfrog tadpoles from a WWE-containing site versus those from a reference location receiving no WWE. In both studies, phenotypic sex, steroidogenic factor-1 (nr5a1), and aromatase (cyp19a1) mRNA levels using quantitative real-time PCR were determined. Exposure to E2 or OP did not alter sex ratios. In controls, both nr5a1 and cyp19a1 transcript levels exhibited sexual dimorphism, with males demonstrating higher levels of nr5a1 and females greater abundance of cyp19a1. However, E2 exposure increased cyp19a1 mRNA abundance in testes and decreased levels in ovaries, eliminating the sexual dimorphism observed in controls. E2-exposed males exhibited increased nr5a1 transcript levels in the testes compared to controls, while females demonstrated no E2 effect. OP treatment had no effect on female cyp19a1 mRNA abundance, but exposure to 10−7 M OP increased testicular transcript levels. Treatment with 10−9 and 10−8 M OP, but not 10−7 M, resulted in decreased abundance of nr5a1 transcript in both ovaries and testes. Animals from the field had sexually dimorphic gonadal levels of cyp19a1, but both sexes from the WWE site exhibited elevated cyp19a1 transcript abundance compared to the reference location. Individual chemical compounds and anthropogenic wastewater effluent dispersed

  1. Estrogenic environmental contaminants alter the mRNA abundance profiles of genes involved in gonadal differentiation of the American bullfrog.

    Science.gov (United States)

    Wolff, Stephanie E; Veldhoen, Nik; Helbing, Caren C; Ramirez, Claire A; Malpas, Janae M; Propper, Catherine R

    2015-07-15

    Wildlife and human populations are exposed to anthropogenic mixtures of chemicals in the environment that may adversely influence normal reproductive function and development. We determined the effects of exposure to estrogenic chemicals and wastewater effluent (WWE) on developing gonads of the American bullfrog, Rana (Lithobates) catesbeiana, a species whose widespread distribution make it an ideal model for environmental monitoring of endocrine effects of chemical contaminants. Premetamorphic bullfrog tadpoles were exposed to treatment vehicle, 17β-estradiol (E2; 10(-9)M) or 4-tert-octylphenol (OP; 10(-9)M, 10(-8)M, and 10(-7)M). Additionally, gonadal differentiation was evaluated in bullfrog tadpoles from a WWE-containing site versus those from a reference location receiving no WWE. In both studies, phenotypic sex, steroidogenic factor-1 (nr5a1), and aromatase (cyp19a1) mRNA levels using quantitative real-time PCR were determined. Exposure to E2 or OP did not alter sex ratios. In controls, both nr5a1 and cyp19a1 transcript levels exhibited sexual dimorphism, with males demonstrating higher levels of nr5a1 and females greater abundance of cyp19a1. However, E2 exposure increased cyp19a1 mRNA abundance in testes and decreased levels in ovaries, eliminating the sexual dimorphism observed in controls. E2-exposed males exhibited increased nr5a1 transcript levels in the testes compared to controls, while females demonstrated no E2 effect. OP treatment had no effect on female cyp19a1 mRNA abundance, but exposure to 10(-7)M OP increased testicular transcript levels. Treatment with 10(-9) and 10(-8)M OP, but not 10(-7)M, resulted in decreased abundance of nr5a1 transcript in both ovaries and testes. Animals from the field had sexually dimorphic gonadal levels of cyp19a1, but both sexes from the WWE site exhibited elevated cyp19a1 transcript abundance compared to the reference location. Individual chemical compounds and anthropogenic wastewater effluent dispersed within

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

    Science.gov (United States)

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

    2014-01-01

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

  3. The tRNA methyltransferase NSun2 stabilizes p16INK4 mRNA by methylating the 3′-untranslated region of p16

    OpenAIRE

    Zhang, Xiaotian; Liu, Zhenyun; Yi, Jie; Tang, Hao; Xing, Junyue; Yu, Minqwei; Tong, Tanjun; Shang, Yongfeng; Gorospe, Myriam; Wang, Wengong

    2012-01-01

    The impact of methylation of the 3′-untranslated region (UTR) of a messenger RNA (mRNA) remains largely unknown. Here we show that NSun2, a transfer RNA methyltransferase, inhibits the turnover of p16INK4 mRNA. Knockdown of NSun2 reduces p16 expression by shortening the half-life of the p16 mRNA, while overexpression of NSun2 stabilizes the p16 mRNA. In vitro methylation assays show that NSun2 methylates the p16 3′UTR at A988. Knockdown of NSun2 reduces the stability of the EGFP-p16 chimeric ...

  4. Triptolide inhibits COX-2 expression by regulating mRNA stability in TNF-{alpha}-treated A549 cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Lixin; Zhang, Shuang; Jiang, Zhenzhou; Huang, Xin; Wang, Tao; Huang, Xiao; Li, Han [Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing 210009 (China); Zhang, Luyong, E-mail: lyzhang@cpu.edu.cn [Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing 210009 (China)

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Triptolide inhibited COX-2 expression and the half-life of COX-2 mRNA is decreased. Black-Right-Pointing-Pointer The HuR protein shuttling from nucleus to cytoplasm is inhibited by triptolide. Black-Right-Pointing-Pointer Triptolide inhibited 3 Prime -UTR fluorescence reporter gene activity. Black-Right-Pointing-Pointer COX-2 mRNA binding to HuR is decreased by triptolide in pull-down experiments. -- Abstract: Cyclooxygenase-2 (COX-2) over-expression is frequently associated with human non-small-cell lung cancer (NSCLC) and involved in tumor proliferation, invasion, angiogenesis and resistance to apoptosis. In the present study, the effects of triptolide on COX-2 expression in A549 cells were investigated and triptolide was found to inhibit TNF-{alpha}-induced COX-2 expression. In our further studies, it was found that triptolide decreased the half-life of COX-2 mRNA dramatically and that it inhibited 3 Prime -untranslated region (3 Prime -UTR) fluorescence reporter gene activity. Meanwhile, triptolide inhibited the HuR shuttling from nucleus to cytoplasm. After triptolide treatment, decreased COX-2 mRNA in pull-down experiments with anti-HuR antibodies was observed, indicating that the decreased cytoplasmic HuR is responsible for the decreased COX-2 mRNA. Taken together, our results provided evidence for the first time that triptolide inhibited COX-2 expression by COX-2 mRNA stability modulation and post-transcriptional regulation. These results provide a novel mechanism of action for triptolide which may be important in the treatment of lung cancer.

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

    Directory of Open Access Journals (Sweden)

    Jia-Shiuan Tsai

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

  6. Posttranscriptional regulation of GAP-43 gene expression in PC12 cells through protein kinase C-dependent stabilization of the mRNA.

    Science.gov (United States)

    Perrone-Bizzozero, N I; Cansino, V V; Kohn, D T

    1993-03-01

    We have previously shown that nerve growth factor (NGF) selectively stabilizes the GAP-43 mRNA in PC12 cells. To study the cellular mechanisms for this post-transcriptional control and to determine the contribution of mRNA stability to GAP-43 gene expression, we examined the effects of several agents that affect PC12 cell differentiation on the level of induction and rate of degradation of the GAP-43 mRNA. The NGF-mediated increase in GAP-43 mRNA levels and neurite outgrowth was mimicked by the phorbol ester TPA, but not by dibutyryl cAMP or the calcium ionophore A12783. Downregulation of protein kinase C (PKC) by high doses of phorbol esters or selective PKC inhibitors prevented the induction of this mRNA by NGF, suggesting that NGF and TPA act through a common PKC-dependent pathway. In mRNA decay studies, phorbol esters caused a selective 6-fold increase in the half-life of the GAP-43 mRNA, which accounts for most of the induction of this mRNA by TPA. The phorbol ester-induced stabilization of GAP-43 mRNA was blocked by the protein kinase inhibitor polymyxin B and was partially inhibited by dexamethasone, an agent that blocks GAP-43 expression and neuronal differentiation in PC12 cells. In contrast, the rates of degradation and the levels of the GAP-43 mRNA in control and TPA-treated cells were not affected by cycloheximide treatment. Thus, changes in GAP-43 mRNA turnover do not appear to require continuous protein synthesis. In conclusion, these data suggest that PKC activity regulates the levels of the GAP-43 mRNA in PC12 cells through a novel, translation-independent mRNA stabilization mechanism.

  7. 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

  8. 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

  9. The manganese superoxide dismutase Ala16Val dimorphism modulates both mitochondrial import and mRNA stability.

    Science.gov (United States)

    Sutton, Angela; Imbert, Audrey; Igoudjil, Anissa; Descatoire, Véronique; Cazanave, Sophie; Pessayre, Dominique; Degoul, Françoise

    2005-05-01

    A genetic dimorphism incorporates either alanine (Ala) or valine (Val) in the mitochondrial targeting sequence (MTS) of manganese superoxide dismutase (MnSOD). The Ala-MTS confers a 40% higher MnSOD activity than the Val-MTS after import into isolated mitochondria in vitro. The present study aimed to characterize functional consequences in whole cells. HuH7 human hepatoma cells were transfected with vectors encoding for the human Ala- or Val-MnSOD variants fused to a Myc-His-tag. The Ala-variant resulted in four-fold higher levels of the mature exogenous protein and MnSOD activity than the Val-variant. Studies with a proteasome inhibitor indicated that precursor proteins are either imported into the mitochondria or degraded by the proteasome. Despite identical levels 8 h after transfection, mRNA levels at 36 h were two-fold higher for the Ala-encoding mRNA than the Val-mRNA. Decreasing the mitochondrial membrane potential decreased both MnSOD mitochondrial import and its mRNA levels. Much larger differences in the activity of the human Val- and Ala-MnSOD variants are observed in whole cells rather than after import experiments performed in vitro. First, the slowly imported Val-MnSOD is degraded by the proteasome in cells. Second, the slower mitochondrial import of the Val-variant may be associated with decreased mRNA stability, possibly due to impaired cotranslational import.

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

    Directory of Open Access Journals (Sweden)

    Akinori Takahashi

    2015-12-01

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

  11. The circadian deadenylase Nocturnin is necessary for stabilization of the iNOS mRNA in mice.

    Directory of Open Access Journals (Sweden)

    Shuang Niu

    Full Text Available Nocturnin is a member of the CCR4 deadenylase family, and its expression is under circadian control with peak levels at night. Because it can remove poly(A tails from mRNAs, it is presumed to play a role in post-transcriptional control of circadian gene expression, but its target mRNAs are not known. Here we demonstrate that Nocturnin expression is acutely induced by the endotoxin lipopolysaccharide (LPS. Mouse embryo fibroblasts (MEFs lacking Nocturnin exhibit normal patterns of acute induction of TNFα and iNOS mRNAs during the first three hours following LPS treatment, but by 24 hours, while TNFα mRNA levels are indistinguishable from WT cells, iNOS message is significantly reduced 20-fold. Accordingly, analysis of the stability of the mRNAs showed that loss of Nocturnin causes a significant decrease in the half-life of the iNOS mRNA (t(1/2 = 3.3 hours in Nocturnin knockout MEFs vs. 12.4 hours in wild type MEFs, while having no effect on the TNFα message. Furthermore, mice lacking Nocturnin lose the normal nighttime peak of hepatic iNOS mRNA, and have improved survival following LPS injection. These data suggest that Nocturnin has a novel stabilizing activity that plays an important role in the circadian response to inflammatory signals.

  12. 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...

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

    Science.gov (United States)

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

    2011-09-30

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

  14. Ivermectin induces P-glycoprotein expression and function through mRNA stabilization in murine hepatocyte cell line.

    Science.gov (United States)

    Ménez, Cécile; Mselli-Lakhal, Laïla; Foucaud-Vignault, Magali; Balaguer, Patrick; Alvinerie, Michel; Lespine, Anne

    2012-01-15

    Ivermectin is widely used in human and veterinary medicine for the control of helminth infections. Ivermectin is known to interact with P-glycoprotein (P-gp/MDR1), being a good substrate and a potent inhibitor, however, the influence of ivermectin on the expression of the transporter has not been investigated. Expression of P-glycoprotein was investigated in cultured mouse hepatocytes acutely exposed to ivermectin. The two P-glycoprotein murine isoforms, Mdr1a and Mdr1b, mRNA levels were assessed by real-time RT-PCR. Ivermectin induced a clear time- and concentration-dependent up-regulation of Mdr1a and Mdr1b mRNA levels (as early as a 12-h exposure and up to 2.5-fold at 10μM). Moreover, ivermectin-treated cells displayed enhanced cellular efflux of the P-glycoprotein substrate calcein that was inhibited by the P-glycoprotein blocker valspodar, providing evidence that the ivermectin-induced P-glycoprotein was functional. The mechanisms underlying these effects were investigated. Ivermectin-mediated Mdr1 mRNA induction was independent of the two nuclear receptors CAR and PXR, which are known to be involved in drug transporters regulation. Moreover, by using reporter cell lines that detects specific ligand-activated transcription factors, we showed that ivermectin did not displayed CAR, PXR or AhR ligand activities. However, studies with actinomycin D revealed that the half-life of Mdr1a and Mdr1b mRNA were significantly prolonged by two-fold in ivermectin-treated cells suggesting a post-transcriptional mode of ivermectin regulation. This study demonstrates for the first time that ivermectin induces P-glycoprotein overexpression through post-transcriptional mRNA stabilization, thus offering insight into the mechanism of reduced therapeutic efficacy and development of ivermectin-resistant parasites.

  15. HIV-1 frameshift efficiency is primarily determined by the stability of base pairs positioned at the mRNA entrance channel of the ribosome.

    Science.gov (United States)

    Mouzakis, Kathryn D; Lang, Andrew L; Vander Meulen, Kirk A; Easterday, Preston D; Butcher, Samuel E

    2013-02-01

    The human immunodeficiency virus (HIV) requires a programmed -1 ribosomal frameshift for Pol gene expression. The HIV frameshift site consists of a heptanucleotide slippery sequence (UUUUUUA) followed by a spacer region and a downstream RNA stem-loop structure. Here we investigate the role of the RNA structure in promoting the -1 frameshift. The stem-loop was systematically altered to decouple the contributions of local and overall thermodynamic stability towards frameshift efficiency. No correlation between overall stability and frameshift efficiency is observed. In contrast, there is a strong correlation between frameshift efficiency and the local thermodynamic stability of the first 3-4 bp in the stem-loop, which are predicted to reside at the opening of the mRNA entrance channel when the ribosome is paused at the slippery site. Insertion or deletions in the spacer region appear to correspondingly change the identity of the base pairs encountered 8 nt downstream of the slippery site. Finally, the role of the surrounding genomic secondary structure was investigated and found to have a modest impact on frameshift efficiency, consistent with the hypothesis that the genomic secondary structure attenuates frameshifting by affecting the overall rate of translation.

  16. Role of highly conserved pyrimidine-rich sequences in the 3' untranslated region of the GAP-43 mRNA in mRNA stability and RNA-protein interactions.

    Science.gov (United States)

    Kohn, D T; Tsai, K C; Cansino, V V; Neve, R L; Perrone-Bizzozero, N I

    1996-03-01

    We have shown previously that the mRNA for the growth-associated protein GAP-43 is selectively stabilized during neuronal differentiation. In this study, we explored the role of its highly conserved 3' untranslated region (3'UTR) in mRNA stability and RNA-protein interactions. The 3'UTRs of the rat and chicken GAP-43 mRNAs show 78% sequence identity, which is equivalent to the conservation of their coding regions. In rat PC12 cells stably transfected with the full-length rat or chicken GAP-43 cDNAs, the transgene mRNAs decayed with same half-life of about 3 h. The GAP-43 3'UTR also caused the rabbit beta-globin mRNA to decay with a half-life of 4 h, indicating that the major determinants for GAP-43 mRNA stability are localized in its highly conserved 3'UTR. Three brain cytosolic RNA-binding proteins (molecular mass 40, 65 and 95 kDa) were found to interact with both the rat and chicken GAP-43 mRNAs. These RNA-protein interactions were specific and involved pyrimidine-rich sequences in the 3'UTR. Like the GAP-43 mRNA, the activity of these proteins was enriched in brain and increased during development. We propose that highly conserved pyrimidine-rich sequences in the 3'UTR of this mRNA regulate GAP-43 gene expression via interactions with specific RNA-binding proteins.

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

    Science.gov (United States)

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

    2007-01-15

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

  18. Rice Stripe Virus Infection Alters mRNA Levels of Sphingolipid-Metabolizing Enzymes and Sphingolipids Content in Laodelphax striatellus

    Science.gov (United States)

    Li, Fei-Qiang; Bai, Yue-Liang; Shi, Xiao-Xiao; Zhu, Mu-Fei; Zhang, Min-Jing; Mao, Cun-Gui; Zhu, Zeng-Rong

    2017-01-01

    Sphingolipids and their metabolites have been implicated in viral infection and replication in mammal cells but how their metabolizing enzymes in the host are regulated by viruses remains largely unknown. Here we report the identification of 12 sphingolipid genes and their regulation by Rice stripe virus in the small brown planthopper (Laodelphax striatellus Fallén), a serious pest of rice throughout eastern Asia. According to protein sequence similarity, we identified 12 sphingolipid enzyme genes in L. striatellus. By comparing their mRNA levels in viruliferous versus nonviruliferous L. striatellus at different life stages by qPCR, we found that RSV infection upregulated six genes (LsCGT1, LsNAGA1, LsSGPP, LsSMPD4, LsSMS, and LsSPT) in most stages of L. striatellus. Especially, four genes (LsCGT1, LsSMPD2, LsNAGA1, and LsSMS) and another three genes (LsNAGA1, LsSGPP, and LsSMS) were significantly upregulated in viruliferous third-instar and fourth-instar nymphs, respectively. HPLC-MS/MS results showed that RSV infection increased the levels of various ceramides, such as Cer18:0, Cer20:0, and Cer22:0 species, in third and fourth instar L. striatellus nymphs. Together, these results demonstrate that RSV infection alters the transcript levels of various sphingolipid enzymes and the contents of sphingolipids in L. striatellus, indicating that sphingolipids may be important for RSV infection or replication in L. striatellus. PMID:28130458

  19. Nickel ions selectively inhibit lipopolysaccharide-induced interleukin-6 production by decreasing its mRNA stability.

    Directory of Open Access Journals (Sweden)

    Sanki Asakawa

    Full Text Available Nickel (Ni ions easily elute from many alloys and elicit inflammation and allergies. Previous studies have shown that infections due to the implantation of medical devices cause inflammation and enhance the elution of Ni ions (Ni²⁺. However, cross-talk between infection- and Ni²⁺-induced signaling pathways has not yet been elucidated in detail. In the present study, we investigated the effects of Ni2+ on the lipopolysaccharide (LPS-induced production of cytokines in a LPS-induced air pouch-type inflammation model in BALB/c mice and the murine macrophage cell line RAW264. We demonstrated that Ni²⁺ inhibited the LPS-induced production of interleukin (IL-6, but not that of tumor necrosis factor (TNF-α both in vivo and in vitro. This inhibitory effect was also observed with cobalt ion (Co²⁺, but not with chloride ion (Cl⁻, zinc ion (Zn²⁺, or palladium ion (Pd²⁺, and was highly selective to the production of IL-6. Ni²⁺ did not inhibit the activation of ERK1/2, p38 MAPK, or JNK. Although Ni²⁺ decreased IL-6 mRNA levels, it failed to inhibit the LPS-induced activation of the IL-6 promoter. An experiment using actinomycin D, a transcription inhibitor, revealed that Ni²⁺ decreased the stability of IL-6 mRNA. Moreover, Ni²⁺ inhibited the LPS-induced expression of Arid5a, but not regnase-1. These results demonstrated that Ni²⁺ may have selectively inhibited the LPS-induced production of IL-6 by decreasing the Arid5a-dependent stabilization of IL-6 mRNA.

  20. Global change alters the stability of food webs

    NARCIS (Netherlands)

    Emmerson, M.; Bezemer, T.M.; Hunter, M.D.; Jones, T.H.

    2005-01-01

    Recent research has generally shown that a small change in the number of species in a food web can have consequences both for community structure and ecosystem processes. However `change¿ is not limited to just the number of species in a community, but might include an alteration to such properties

  1. Global change alters the stability of food webs

    NARCIS (Netherlands)

    Emmerson, M.; Bezemer, T.M.; Hunter, M.D.; Jones, T.H.

    2005-01-01

    Recent research has generally shown that a small change in the number of species in a food web can have consequences both for community structure and ecosystem processes. However 'change' is not limited to just the number of species in a community, but might include an alteration to such properties

  2. 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.

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

    Science.gov (United States)

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

    2014-09-01

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

  4. miR-346 controls release of TNF-α protein and stability of its mRNA in rheumatoid arthritis via tristetraprolin stabilization.

    Directory of Open Access Journals (Sweden)

    Noha Semaan

    Full Text Available TNF-α is a major cytokine implicated in rheumatoid arthritis. Its expression is regulated both at the transcriptional and posttranscriptional levels and recent data demonstrated that miRNAs are implicated in TNF-α response in macrophages. LPS-activated FLS isolated from RA patients express TNF-α mRNA but not the mature protein. This prompted us to look for miRNAs which could be implicated in this anti-inflammatory effect. Using a microarray, we found two miRNAs, miR-125b and miR-939 predicted to target the 3'-UTR of TNF-α mRNA, to be up-regulated in RA FLS in response to LPS, but their repression did not restore mature TNF-α expression in FLS. We showed previously that miR-346, which is upregulated in LPS-activated FLS, inhibited Btk expression that stabilized TNF-α mRNA. Blocking miR-346 reestablished TNF-α expression in activated FLS. Interestingly, transfection of miR-346 in LPS-activated THP-1 cells inhibited TNF-α secretion. We also demonstrated that TTP, a RNA binding protein which inhibited TNF-α synthesis, is overexpressed in activated FLS and that inhibition of miR-346 decreases its expression. Conversely, transfection of miR-346 in LPS-activated THP-1 cells increased TTP mRNA expression and inhibited TNF-α release. These results indicate that miR-346 controls TNF-α synthesis by regulating TTP expression.

  5. Human cytomegalovirus induces alteration of β-actin mRNA and microfilaments in human embryo fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    林茂芳; 魏国庆; 黄河; 蔡真

    2004-01-01

    Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

  6. Transition State Charge Stabilization and Acid-Base Catalysis of mRNA Cleavage by the Endoribonuclease RelE.

    Science.gov (United States)

    Dunican, Brian F; Hiller, David A; Strobel, Scott A

    2015-12-01

    The bacterial toxin RelE is a ribosome-dependent endoribonuclease. It is part of a type II toxin-antitoxin system that contributes to antibiotic resistance and biofilm formation. During amino acid starvation, RelE cleaves mRNA in the ribosomal A-site, globally inhibiting protein translation. RelE is structurally similar to microbial RNases that employ general acid-base catalysis to facilitate RNA cleavage. The RelE active site is atypical for acid-base catalysis, in that it is enriched with positively charged residues and lacks the prototypical histidine-glutamate catalytic pair, making the mechanism of mRNA cleavage unclear. In this study, we use a single-turnover kinetic analysis to measure the effect of pH and phosphorothioate substitution on the rate constant for cleavage of mRNA by wild-type RelE and seven active-site mutants. Mutation and thio effects indicate a major role for stabilization of increased negative change in the transition state by arginine 61. The wild-type RelE cleavage rate constant is pH-independent, but the reaction catalyzed by many of the mutants is strongly dependent on pH, suggestive of general acid-base catalysis. pH-rate curves indicate that wild-type RelE operates with the pK(a) of at least one catalytic residue significantly downshifted by the local environment. Mutation of any single active-site residue is sufficient to disrupt this microenvironment and revert the shifted pK(a) back above neutrality. pH-rate curves are consistent with K54 functioning as a general base and R81 as a general acid. The capacity of RelE to effect a large pK(a) shift and facilitate a common catalytic mechanism by uncommon means furthers our understanding of other atypical enzymatic active sites.

  7. Glucocorticoid receptor 1B and 1C mRNA transcript alterations in schizophrenia and bipolar disorder, and their possible regulation by GR gene variants.

    Science.gov (United States)

    Sinclair, Duncan; Fullerton, Janice M; Webster, Maree J; Shannon Weickert, Cynthia

    2012-01-01

    Abnormal patterns of HPA axis activation, under basal conditions and in response to stress, are found in individuals with schizophrenia and bipolar disorder. Altered glucocorticoid receptor (GR) mRNA and protein expression in the dorsolateral prefrontal cortex (DLPFC) in psychiatric illness have also been reported, but the cause of these abnormalities is not known. We quantified expression of GR mRNA transcript variants which employ different 5' promoters, in 35 schizophrenia cases, 31 bipolar disorder cases and 34 controls. We also explored whether sequence variation within the NR3C1 (GR) gene is related to GR mRNA variant expression. Total GR mRNA was decreased in the DLPFC in schizophrenia cases relative to controls (15.1%, pbipolar disorder cases (8.9%, pschizophrenia cases relative to controls (20.2%, pschizophrenia and bipolar disorder cases relative to controls (16.1% and 17.2% respectively, both pgene expression changes in psychotic illness, and highlight the potential importance of sequence variation within the NR3C1 gene in modulating GR mRNA expression in the DLPFC.

  8. Increased polyamines alter chromatin and stabilize autoantigens in autoimmune diseases

    Directory of Open Access Journals (Sweden)

    Wesley H. Brooks

    2013-04-01

    Full Text Available Polyamines are small cations with unique combinations of charge and length that give them many putative interactions in cells. Polyamines are essential since they are involved in replication, transcription, translation, and stabilization of macro-molecular complexes. However, polyamine synthesis competes with cellular methylation for S-adenosylmethionine, the methyl donor. Also, polyamine degradation can generate reactive molecules like acrolein. Therefore, polyamine levels are tightly controlled. This control may be compromised in autoimmune diseases since elevated polyamine levels are seen in autoimmune diseases. Here a hypothesis is presented explaining how polyamines can stabilize autoantigens. In addition, the hypothesis explains how polyamines can inappropriately activate enzymes involved in NETosis, a process in which chromatin is modified and extruded from cells as extracellular traps that bind pathogens during an immune response. This polyamine-induced enzymatic activity can lead to an increase in NETosis resulting in release of autoantigenic material and tissue damage.

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

    Directory of Open Access Journals (Sweden)

    Joshua D Doyle

    2015-03-01

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

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

    Science.gov (United States)

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

    2015-11-01

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

  11. The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA

    OpenAIRE

    Lopez de Silanes, I.; Gorospe, M.; Taniguchi, H; Abdelmohsen, K; Srikantan, S.; Alaminos, M.; Berdasco, M.; Urdinguio, R. G.; Fraga, M. F.; Jacinto, F. V.; Esteller, M.

    2009-01-01

    The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3???UTR pr...

  12. Stability of maternal mRNA in Xenopus embryos: role of transcription and translation.

    Science.gov (United States)

    Duval, C; Bouvet, P; Omilli, F; Roghi, C; Dorel, C; LeGuellec, R; Paris, J; Osborne, H B

    1990-08-01

    The first 12 cell divisions of Xenopus laevis embryos do not require gene transcription. This means that the regulation of gene expression during this period is controlled at post transcriptional levels and makes Xenopus early development a potentially interesting biological system with which to study the mechanisms involved. We describe here the stability characteristics of several maternal Xenopus mRNAs which are deadenylated soon after fertilisation (J. Paris and M. Philippe, Dev. Biol., in press). We show that these mRNAs were only degraded in the embryo after the midblastula transition (MBT), when gene transcription was initiated. The kinetics with which the deadenylated maternal mRNAs decreased in the post-MBT embryos showed sequence specificity. The degradation of these mRNAs after the MBT was inhibited by cycloheximide but was not affected by dactinomycin. Therefore, the destabilization of these mRNAs does not appear to be initiated by new embryonic gene transcripts. Sequence comparisons of the 3' untranslated region of these mRNAs identified several motifs which may be involved in the posttranscriptional control of these gene products.

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

    Science.gov (United States)

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

    2015-09-01

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

  14. Glucocorticoid receptor 1B and 1C mRNA transcript alterations in schizophrenia and bipolar disorder, and their possible regulation by GR gene variants.

    Directory of Open Access Journals (Sweden)

    Duncan Sinclair

    Full Text Available Abnormal patterns of HPA axis activation, under basal conditions and in response to stress, are found in individuals with schizophrenia and bipolar disorder. Altered glucocorticoid receptor (GR mRNA and protein expression in the dorsolateral prefrontal cortex (DLPFC in psychiatric illness have also been reported, but the cause of these abnormalities is not known. We quantified expression of GR mRNA transcript variants which employ different 5' promoters, in 35 schizophrenia cases, 31 bipolar disorder cases and 34 controls. We also explored whether sequence variation within the NR3C1 (GR gene is related to GR mRNA variant expression. Total GR mRNA was decreased in the DLPFC in schizophrenia cases relative to controls (15.1%, p<0.0005 and also relative to bipolar disorder cases (8.9%, p<0.05. GR-1B mRNA was decreased in schizophrenia cases relative to controls (20.2%, p<0.05, while GR-1C mRNA was decreased in both schizophrenia and bipolar disorder cases relative to controls (16.1% and 17.2% respectively, both p<0.005. A dose-dependent effect of rs10052957 genotype on GR-1B mRNA expression was observed, where CC homozygotes displayed 18.4% lower expression than TC heterozygotes (p<0.05, and 31.8% lower expression than TT homozygotes (p<0.005. Similarly, a relationship between rs6190 (R23K genotype and GR-1C expression was seen, with 24.8% lower expression in GG homozygotes than GA heterozygotes (p<0.01. We also observed an effect of rs41423247 (Bcl1 SNP on expression of 67 kDa GRα isoform, the most abundant GRα isoform in the DLPFC. These findings suggest possible roles for the GR-1B and GR-1C promoter regions in mediating GR gene expression changes in psychotic illness, and highlight the potential importance of sequence variation within the NR3C1 gene in modulating GR mRNA expression in the DLPFC.

  15. 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

  16. Two tandem RNase III cleavage sites determine betT mRNA stability in response to osmotic stress in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Minji Sim

    Full Text Available While identifying genes regulated by ribonuclease III (RNase III in Escherichia coli, we observed that steady-state levels of betT mRNA, which encodes a transporter mediating the influx of choline, are dependent on cellular concentrations of RNase III. In the present study, we also observed that steady-state levels of betT mRNA are dependent on RNase III activity upon exposure to osmotic stress, indicating the presence of cis-acting elements controlled by RNase III in betT mRNA. Primer extension analyses of betT mRNA revealed two tandem RNase III cleavage sites in its stem-loop region, which were biochemically confirmed via in vitro cleavage assays. Analyses of cleavage sites suggested the stochastic selection of cleavage sites by RNase III, and mutational analyses indicated that RNase III cleavage at either site individually is insufficient for efficient betT mRNA degradation. In addition, both the half-life and abundance of betT mRNA were significantly increased in association with decreased RNase III activity under hyper-osmotic stress conditions. Our findings demonstrate that betT mRNA stability is controlled by RNase III at the post-transcriptional level under conditions of osmotic stress.

  17. Disodium Cromoglycate, A Mast-Cell Stabilizer, Alters Postradiation Regional Cerebral Blood Flow in Primates

    Science.gov (United States)

    1986-01-01

    ranD RISEARCH INSTITUTI SCIENTIFIC REPORTTC SR86-14 ELECTE JUL0186_ _ ~D DISODIUM CROMOGLYCATE , A MAST-CELL STABILIZER, ALTERS POSTRADIATION...were given the mast-cell stabilizers disodium cromoglycate (DSCC) or BRL 22321 (Beecham Phar- maceuticals, Research Division) before exposure to 100 Gy...flow could be mitigated by the pre- radiation administration of either disodium cromoglycate (DSCG) (Fisons Corporation, ledford, Mass.) or BRL 22321

  18. 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.

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

    Science.gov (United States)

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

    2011-08-01

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

  20. Salp15 binding to DC-SIGN inhibits cytokine expression by impairing both nucleosome remodeling and mRNA stabilization.

    Directory of Open Access Journals (Sweden)

    Joppe W R Hovius

    2008-02-01

    Full Text Available Ixodes ticks are major vectors for human pathogens, such as Borrelia burgdorferi, the causative agent of Lyme disease. Tick saliva contains immunosuppressive molecules that facilitate tick feeding and B. burgdorferi infection. We here demonstrate, to our knowledge for the first time, that the Ixodes scapularis salivary protein Salp15 inhibits adaptive immune responses by suppressing human dendritic cell (DC functions. Salp15 inhibits both Toll-like receptor- and B. burgdorferi-induced production of pro-inflammatory cytokines by DCs and DC-induced T cell activation. Salp15 interacts with DC-SIGN on DCs, which results in activation of the serine/threonine kinase Raf-1. Strikingly, Raf-1 activation by Salp15 leads to mitogen-activated protein kinase kinase (MEK-dependent decrease of IL-6 and TNF-alpha mRNA stability and impaired nucleosome remodeling at the IL-12p35 promoter. These data demonstrate that Salp15 binding to DC-SIGN triggers a novel Raf-1/MEK-dependent signaling pathway acting at both cytokine transcriptional and post-transcriptional level to modulate Toll-like receptor-induced DC activation, which might be instrumental to tick feeding and B. burgdorferi infection, and an important factor in the pathogenesis of Lyme disease. Insight into the molecular mechanism of immunosuppression by tick salivary proteins might provide innovative strategies to combat Lyme disease and could lead to the development of novel anti-inflammatory or immunosuppressive agents.

  1. 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

  2. Impact of hydrothermal alteration on lava dome stability: a numerical modelling approach

    Science.gov (United States)

    Detienne, Marie; Delmelle, Pierre

    2016-04-01

    Lava domes are a common feature of many volcanoes worldwide. They represent a serious volcanic hazard as they are prone to repeated collapses, generating devastating debris avalanches and pyroclastic flows. While it has long been known that hydrothermal alteration degrades rock properties and weakens rock mass cohesion and strength, there is still little quantitative information allowing the description of this effect and its consequences for assessing the stability of a volcanic rock mass such as a lava dome. In this study, we use the finite difference numerical model FLAC 3D to investigate the impact of hydrothermal alteration on the stability of a volcanic dome lying on a flat surface. Different hydrothermal alteration distributions were tested to encompass the variability observed in natural lava domes. Rock shear strength parameters (minimum, maximum and mean cohesion "c" and friction angle "φ" values) representative of various degrees of hydrothermal rock alteration were used in the simulations. The model predicts that reduction of the basement rock's shear strength decreases the factor of safety significantly. A similar result is found by increasing the vertical and horizontal extension of hydrothermal alteration in the basement rocks. In addition, pervasive hydrothermal alteration within the lava dome is predicted to exert a strong negative influence on the factor of safety. Through reduction of rock porosity and permeability, hydrothermal alteration may also affect pore fluid pressure within a lava dome. The results of new FLAC 3D runs which simulate the effect of hydrothermal alteration-induced pore pressure changes on lava dome stability will be presented.

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

    Science.gov (United States)

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

    2012-12-01

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

  4. Shrub invasion decreases diversity and alters community stability in northern Chihuahuan Desert plant communities.

    Directory of Open Access Journals (Sweden)

    Selene Báez

    Full Text Available BACKGROUND: Global climate change is rapidly altering species range distributions and interactions within communities. As ranges expand, invading species change interactions in communities which may reduce stability, a mechanism known to affect biodiversity. In aridland ecosystems worldwide, the range of native shrubs is expanding as they invade and replace native grassland vegetation with significant consequences for biodiversity and ecosystem functioning. METHODOLOGY: We used two long-term data sets to determine the effects of shrub encroachment by Larrea tridentata on subdominant community composition and stability in formerly native perennial grassland dominated by Bouteloua eriopoda in New Mexico, USA. PRINCIPAL FINDINGS: Our results indicated that Larrea invasion decreased species richness during the last 100 years. We also found that over shorter temporal scales species-poor subdominant communities in areas invaded by Larrea were less stable (more variable in time compared to species rich communities in grass-dominated vegetation. Compositional stability increased as cover of Bouteloua increased and decreased as cover of Larrea increased. SIGNIFICANCE: Changes in community stability due to altered interspecific interactions may be one mechanism by which biodiversity declines in grasslands following shrub invasion. As global warming increases, shrub encroachment into native grasslands worldwide will continue to alter species interactions and community stability both of which may lead to a decline in biodiversity.

  5. Galectin-3 is a non-classic RNA binding protein that stabilizes the mucin MUC4 mRNA in the cytoplasm of cancer cells

    Science.gov (United States)

    Coppin, Lucie; Vincent, Audrey; Frénois, Frédéric; Duchêne, Belinda; Lahdaoui, Fatima; Stechly, Laurence; Renaud, Florence; Villenet, Céline; Seuningen, Isabelle Van; Leteurtre, Emmanuelle; Dion, Johann; Grandjean, Cyrille; Poirier, Françoise; Figeac, Martin; Delacour, Delphine; Porchet, Nicole; Pigny, Pascal

    2017-01-01

    Pancreatic cancer cells express high levels of MUC1, MUC4 and MUC16 mRNAs that encode membrane-bound mucins. These mRNAs share unusual features such as a long half-life. However, it remains unknown how mucin mRNA stability is regulated. Galectin-3 (Gal-3) is an endogenous lectin playing important biological functions in epithelial cells. Gal-3 is encoded by LGALS3 which is up-regulated in pancreatic cancer. Despite the absence of a RNA-recognition motif, Gal-3 interacts indirectly with pre-mRNAs in the nucleus and promotes constitutive splicing. However a broader role of Gal-3 in mRNA fate is unexplored. We report herein that Gal-3 increases MUC4 mRNA stability through an intermediate, hnRNP-L which binds to a conserved CA repeat element in the 3′UTR in a Gal-3 dependent manner and also controls Muc4 mRNA levels in epithelial tissues of Gal3−/− mice. Gal-3 interacts with hnRNP-L in the cytoplasm, especially during cell mitosis, but only partly associates with protein markers of P-Bodies or Stress Granules. By RNA-IP plus RNA-seq analysis and imaging, we demonstrate that Gal-3 binds to mature spliced MUC4 mRNA in the perinuclear region, probably in hnRNP-L-containing RNA granules. Our findings highlight a new role for Gal-3 as a non-classic RNA-binding protein that regulates MUC4 mRNA post-transcriptionally. PMID:28262838

  6. 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.

  7. HIV-1 Gag Blocks Selenite-Induced Stress Granule Assembly by Altering the mRNA Cap-Binding Complex

    Directory of Open Access Journals (Sweden)

    Alessandro Cinti

    2016-03-01

    Full Text Available Stress granules (SGs are dynamic accumulations of stalled preinitiation complexes and translational machinery that assemble under stressful conditions. Sodium selenite (Se induces the assembly of noncanonical type II SGs that differ in morphology, composition, and mechanism of assembly from canonical SGs. Se inhibits translation initiation by altering the cap-binding activity of eukaryotic translation initiation factor 4E (eIF4E-binding protein 1 (4EBP1. In this work, we show that human immunodeficiency virus type 1 (HIV-1 Gag is able to block the assembly of type II noncanonical SGs to facilitate continued Gag protein synthesis. We demonstrate that expression of Gag reduces the amount of hypophosphorylated 4EBP1 associated with the 5′ cap potentially through an interaction with its target, eIF4E. These results suggest that the assembly of SGs is an important host antiviral defense that HIV-1 has evolved for inhibition through several distinct mechanisms.

  8. Altered biometal homeostasis is associated with CLN6 mRNA loss in mouse neuronal ceroid lipofuscinosis

    Directory of Open Access Journals (Sweden)

    Katja M. Kanninen

    2013-05-01

    Neuronal ceroid lipofuscinoses, the most common fatal childhood neurodegenerative illnesses, share many features with more prevalent neurodegenerative diseases. Neuronal ceroid lipofuscinoses are caused by mutations in CLN genes. CLN6 encodes a transmembrane endoplasmic reticulum protein with no known function. We characterized the behavioural phenotype of spontaneous mutant mice modeling CLN6 disease, and demonstrate progressive motor and visual decline and reduced lifespan in these mice, consistent with symptoms observed in neuronal ceroid lipofuscinosis patients. Alterations to biometal homeostasis are known to play a critical role in pathology in Alzheimer's, Parkinson's, Huntington's and motor neuron diseases. We have previously shown accumulation of the biometals, zinc, copper, manganese and cobalt, in CLN6 Merino and South Hampshire sheep at the age of symptom onset. Here we determine the physiological and disease-associated expression of CLN6, demonstrating regional CLN6 transcript loss, and concurrent accumulation of the same biometals in the CNS and the heart of presymptomatic CLN6 mice. Furthermore, increased expression of the ER/Golgi-localized cation transporter protein, Zip7, was detected in cerebellar Purkinje cells and whole brain fractions. Purkinje cells not only control motor function, an early symptomatic change in the CLN6 mice, but also display prominent neuropathological changes in mouse models and patients with different forms of neuronal ceroid lipofuscinoses. Whole brain fractionation analysis revealed biometal accumulation in fractions expressing markers for ER, Golgi, endosomes and lysosomes of CLN6 brains. These data are consistent with a link between CLN6 expression and biometal homeostasis in CLN6 disease, and provide further support for altered cation transporter regulation as a key factor in neurodegeneration.

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

    Science.gov (United States)

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

    1998-06-01

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

  10. Influence of mandibular fixation method on stability of the maxillary occlusal plane after occlusal plane alteration.

    Science.gov (United States)

    Yosano, Akira; Katakura, Akira; Takaki, Takashi; Shibahara, Takahiko

    2009-05-01

    In this study, we investigated how method of mandibular fixation influenced longterm postoperative stability of the maxilla in Class III cases. In particular, we investigated change in the maxillary occlusal plane after Occlusal Plane Alteration. Therefore, we focused on change in the palatal plane to evaluate stability of the maxillary occlusal plane, as the position of the palatal plane affects the maxillary occlusal plane. This study included 16 patients diagnosed with mandibular protrusion. Alteration of the occlusal plane was achieved by clockwise rotation of the maxilla by Le Fort I osteotomy and mandibular setback was performed by bilateral sagittal split ramus osteotomy. We analyzed and examined lateral cephalometric radiographs taken at 1 month, 3 months, 6 months, and 1 year after surgery. Stability achieved by two methods of mandibular fixation was compared. In one group of patients (group S) titanium screws were used, and in the other group (group P) titanium-locking mini-plates were used. No significant displacement was recognized in group S, whereas an approximately 0.7mm upward vertical displacement was recognized in the anterior nasal spine in group P. As a result, not only the angle of the palatal plane and S-N plane, but also occlusal plane angle in group P showed a greater decrease than that in group S. The results suggest that fixing the mandible with screws yielded greater stability of the maxilla and maxillary occlusal plane than fixing the mandible with titanium plates.

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

    Science.gov (United States)

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

    2014-12-03

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

  12. 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.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    CaT skin and MCF-7 breast cancer cell lines were established. Quantitative PCR and an enzymatic assay were used to quantify the reporter mRNA and protein levels, respectively. Proteins binding to the CPEs were identified by RNA-immunoprecipitation (IP) and quantitative mass spectroscopy. RESULTS: The wild-type...

  14. Conjugation of D-glucosamine to bovine trypsin increases thermal stability and alters functional properties.

    Science.gov (United States)

    Gizurarson, Jóhann Grétar Kröyer; Filippusson, Hörður

    2015-01-01

    D-Glucosamine was conjugated to bovine trypsin by carbodiimide chemistry, involving a water-soluble carbodiimide and a succinimide ester, with the latter being to increase the yield of the conjugation. Mass spectrometric data suggested that several glycoforms were formed, with around 12 D-glucosamine moieties coupled to each trypsin molecule on average. The moieties were probably coupled to eight carboxyl groups (of glutamyl and aspartyl residues) and to four tyrosyl residues on the surface of the enzyme. The glycated trypsin possessed increased thermal stability. When compared with its unmodified counterpart, T50% was increased by 7 °C, thermal inactivation of the first step was increased 34%, and long-term stability assay revealed 71-times higher residual activity at 25 °C (without stabilizing Ca(2+) ions in aqueous buffer) after 67 days. Furthermore, resistance against autolysis was increased almost two-fold. Altered functional properties of the glycated trypsin were also observed. The glycated trypsin was found to become increasingly basophilic, and was found to be slightly structurally altered. This was indicated by 1.2 times higher catalytic efficiency (k(cat)/K(m)) than unmodified trypsin against the substrate N-α-benzoyl-L-arginine-p-nitroanilide. Circular dichroism spectropolarimetry suggested a minor change in spatial arrangement of α-helix/helices, resulting in an increased affinity of the glycated trypsin for this small synthetic substrate.

  15. Detrimental ELAVL-1/HuR-dependent GSK3β mRNA stabilization impairs resolution in acute respiratory distress syndrome

    Science.gov (United States)

    Hoffman, Olivia; Burns, Nana; Vadász, István; Eltzschig, Holger K.; Edwards, Michael G.

    2017-01-01

    A hallmark of acute respiratory distress syndrome (ARDS) is accumulation of protein-rich edema in the distal airspaces and its removal is critical for patient survival. Previous studies have shown a detrimental role of Glycogen Synthase Kinase (GSK) 3β during ARDS via inhibition of alveolar epithelial protein transport. We hypothesized that post-transcriptional regulation of GSK3β could play a functional role in ARDS resolution. To address this hypothesis, we performed an in silico analysis to identify regulatory genes whose expression correlation to GSK3β messenger RNA utilizing two lung cancer cell line array datasets. Among potential regulatory partners of GSK3β, these studies identified the RNA-binding protein ELAVL-1/HuR (Embryonic Lethal, Abnormal Vision, Drosophila-Like) as a central component in a likely GSK3β signaling network. ELAVL-1/HuR is a RNA-binding protein that selectively binds to AU-rich elements of mRNA and enhances its stability thereby increasing target gene expression. Subsequent studies with siRNA suppression of ELAVL-1/HuR demonstrated deceased GSK3β mRNA and protein expression and improved clearance of FITC-albumin in A549 cells. Conversely, stabilization of ELAVL-1/HuR with the proteasome inhibitor MG-132 resulted in induction of GSK3β at mRNA and protein level and attenuated FITC-albumin clearance. Utilizing ventilator-induced lung injury or intra-tracheal installation of hydrochloric acid to induce ARDS in mice, we observed increased mRNA and protein expression of ELAVL-1/HuR and GSK3β. Together, our findings indicate a previously unknown interaction between GSK3β and ELAV-1 during ARDS, and suggest the inhibition of the ELAV-1- GSK3β pathways as a novel ARDS treatment approach. PMID:28196122

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

    Science.gov (United States)

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

    2015-01-01

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

  17. Altered Fibroblast Growth Factor Receptor 4 Stability Promotes Prostate Cancer Progression

    Directory of Open Access Journals (Sweden)

    Jianghua Wang

    2008-08-01

    Full Text Available Fibroblast growth factor receptor 4 (FGFR-4 is expressed at significant levels in almost all human prostate cancers, and expression of its ligands is ubiquitous. A common polymorphism of FGFR-4 in which arginine (Arg388 replaces glycine (Gly388 at amino acid 388 is associated with progression in human prostate cancer. We show that the FGFR-4 Arg388 polymorphism, which is present in most prostate cancer patients, results in increased receptor stability and sustained receptor activation. In patients bearing the FGFR-4 Gly388 variant, expression of Huntingtin-interacting protein 1 (HIP1, which occurs in more than half of human prostate cancers, also results in FGFR-4 stabilization. This is associated with enhanced proliferation and anchorage-independent growth in vitro. Our findings indicate that increased receptor stability and sustained FGFR-4 signaling occur in most human prostate cancers due to either the presence of a common genetic polymorphism or the expression of a protein that stabilizes FGFR-4. Both of these alterations are associated with clinical progression in patients with prostate cancer. Thus, FGFR-4 signaling and receptor turnover are important potential therapeutic targets in prostate cancer.

  18. Isorhapontigenin (ISO) inhibited cell transformation by inducing G0/G1 phase arrest via increasing MKP-1 mRNA Stability.

    Science.gov (United States)

    Gao, Guangxun; Chen, Liang; Li, Jingxia; Zhang, Dongyun; Fang, Yong; Huang, Haishan; Chen, Xiequn; Huang, Chuanshu

    2014-05-15

    The cancer chemopreventive property of Chinese herb new isolate isorhapontigenin (ISO) and mechanisms underlying its activity have never been explored. Here we demonstrated that ISO treatment with various concentrations for 3 weeks could dramatically inhibit TPA/EGF-induced cell transformation of Cl41 cells in Soft Agar assay, whereas co-incubation of cells with ISO at the same concentrations could elicit G0/G1 cell-cycle arrest without redundant cytotoxic effects on non-transformed cells. Further studies showed that ISO treatment resulted in cyclin D1 downregulation in dose- and time-dependent manner. Our results indicated that ISO regulated cyclin D1 at transcription level via targeting JNK/C-Jun/AP-1 activation. Moreover, we found that ISO-inhibited JNK/C-Jun/AP-1 activation was mediated by both upregulation of MKP-1 expression through increasing its mRNA stability and deactivating MKK7. Most importantly, MKP-1 knockdown could attenuate ISO-mediated suppression of JNK/C-Jun activation and cyclin D1 expression, as well as G0/G1 cell cycle arrest and cell transformation inhibition, while ectopic expression of FLAG-cyclin D1 T286A mutant also reversed ISO-induced G0/G1 cell-cycle arrest and inhibition of cell transformation. Our results demonstrated that ISO is a promising chemopreventive agent via upregulating mkp-1 mRNA stability, which is distinct from its cancer therapeutic effect with downregulation of XIAP and cyclin D1 expression.

  19. HuD promotes BDNF expression in brain neurons via selective stabilization of the BDNF long 3'UTR mRNA.

    Directory of Open Access Journals (Sweden)

    Megan Allen

    Full Text Available Complex regulation of brain-derived neurotrophic factor (BDNF governs its intricate functions in brain development and neuronal plasticity. Besides tight transcriptional control from multiple distinct promoters, alternative 3'end processing of the BDNF transcripts generates either a long or a short 3'untranslated region (3'UTR. Previous reports indicate that distinct RNA sequence in the BDNF 3'UTRs differentially regulates BDNF production in the brain to accommodate neuronal activity changes, conceivably through differential interactions with undefined trans-acting factors that regulate stability and translation of these BDNF mRNA isoforms. In this study, we report that the neuronal RNA-binding protein (RBP HuD interacts with a highly conserved AU-rich element (ARE specifically located in the BDNF long 3'UTR. Such interaction is necessary and sufficient for selective stabilization of mRNAs that contain the BDNF long 3'UTR in vitro and in vivo. Moreover, in a HuD transgenic mouse model, the BDNF long 3'UTR mRNA is increased in the hippocampal dentate granule cells (DGCs, leading to elevated expression of BDNF protein that is transported and stored in the mossy fiber (MF terminals. Our results identify HuD as the first trans-acting factor that enhances BDNF expression specifically through the long 3'UTR and a novel mechanism that regulates BDNF protein production in selected neuronal populations by HuD abundance.

  20. Bmx regulates LPS-induced IL-6 and VEGF production via mRNA stability in rheumatoid synovial fibroblasts.

    Science.gov (United States)

    Palmer, Christine D; Mutch, Brenda E; Page, Theresa H; Horwood, Nicole J; Foxwell, Brian M J

    2008-06-13

    Discordant cytokine production is characteristic of chronic inflammatory conditions like rheumatoid arthritis (RA), and anti-cytokine therapeutics are becoming routinely used to treat RA in the clinic. Fibroblasts from rheumatoid synovium have been shown to contribute to cytokine production in inflamed joints; likewise these cells also produce cytokines in response to inflammatory mediators signalling through Toll like receptors (TLRs). Tyrosine kinase activity is essential to LPS-induced cytokine production, and we have previously implicated a role for the Tec kinase, Bmx, in inflammatory cytokine production. Here we show that Bmx kinase activity in RASF is increased following LPS stimulation and that Bmx is involved in the regulation of LPS-induced IL-6 and VEGF production via mRNA stabilisation. This is an important insight into the regulation of VEGF, which is involved in a wide range of different pathologies, and may lead to more effective design of novel anti-inflammatory/angiogenic therapeutics for conditions such as RA.

  1. GABA-A and NMDA receptor subunit mRNA expression is altered in the caudate but not the putamen of the postmortem brains of alcoholics.

    Science.gov (United States)

    Bhandage, Amol K; Jin, Zhe; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R; Birnir, Bryndis

    2014-01-01

    Chronic consumption of alcohol by humans has been shown to lead to impairment of executive and cognitive functions. Here, we have studied the mRNA expression of ion channel receptors for glutamate and GABA in the dorsal striatum of post-mortem brains from alcoholics (n = 29) and normal controls (n = 29), with the focus on the caudate nucleus that is associated with the frontal cortex executive functions and automatic thinking and on the putamen area that is linked to motor cortices and automatic movements. The results obtained by qPCR assay revealed significant changes in the expression of specific excitatory ionotropic glutamate and inhibitory GABA-A receptor subunit genes in the caudate but not the putamen. Thus, in the caudate we found reduced levels of mRNAs encoding the GluN2A glutamate receptor and the δ, ε, and ρ2 GABA-A receptor subunits, and increased levels of the mRNAs encoding GluD1, GluD2, and GABA-A γ1 subunits in the alcoholics as compared to controls. Interestingly in the controls, 11 glutamate and 5 GABA-A receptor genes were more prominently expressed in the caudate than the putamen (fold-increase varied from 1.24 to 2.91). Differences in gene expression patterns between the striatal regions may underlie differences in associated behavioral outputs. Our results suggest an altered balance between caudate-mediated voluntarily controlled and automatic behaviors in alcoholics, including diminished executive control on goal-directed alcohol-seeking behavior.

  2. Maternal consumption of organic trace minerals alters calf systemic and neutrophil mRNA and microRNA indicators of inflammation and oxidative stress.

    Science.gov (United States)

    Jacometo, Carolina B; Osorio, Johan S; Socha, Michael; Corrêa, Marcio N; Piccioli-Cappelli, Fiorenzo; Trevisi, Erminio; Loor, Juan J

    2015-11-01

    Organic trace mineral (ORG) supplementation to dairy cows in substitution of sulfate (INO) sources has been associated with improvement in immune function during stressful states such as the peripartal period. However, the effect of supplemental ORG during pregnancy on the neonatal calf is unknown. Therefore, our aim was to investigate the effects of ORG supplementation during late pregnancy on the immune system and growth of the neonatal calf. Of specific interest was the evaluation of inflammation-related microRNA (miRNA) and target gene expression in blood neutrophils as indicators of possible nutritional programming. Forty multiparous cows were supplemented for 30d prepartum with 40 mg/kg of Zn, 20 mg/kg of Mn, 5 mg/kg of Cu, and 1mg/kg of Co from either organic (ORG) or sulfate (INO) sources (total diet contained supplemental 75 mg/kg of Zn, 65 mg/kg of Mn, 11 mg/kg of Cu, and 1 mg/kg of Co, and additional Zn, Mn, and Co provided by sulfates), and a subset of calves (n=8/treatment) was used for blood immunometabolic marker and polymorphonuclear leukocyte (PMNL) gene and miRNA expression analyses. Samples were collected at birth (before colostrum feeding), 1d (24 h after colostrum intake), and 7 and 21d of age. Data were analyzed as a factorial design with the PROC MIXED procedure of SAS. No differences were detected in BW, but maternal ORG tended to increase calf withers height. Calves from INO-fed cows had greater concentrations of blood glucose, GOT, paraoxonase, myeloperoxidase, and reactive oxygen metabolites. Antioxidant capacity also was greater in INO calves. The PMNL expression of toll-like receptor pathway genes indicated a pro-inflammatory state in INO calves, with greater expression of the inflammatory mediators MYD88, IRAK1, TRAF6, NFKB, and NFKBIA. The lower expression of miR-155 and miR-125b in ORG calves indicated the potential for maternal organic trace minerals in regulating the PMNL inflammatory response at least via alterations in mRNA and

  3. Let-7b/c enhance the stability of a tissue-specific mRNA during mammalian organogenesis as part of a feedback loop involving KSRP.

    Science.gov (United States)

    Repetto, Emanuela; Briata, Paola; Kuziner, Nathalie; Harfe, Brian D; McManus, Michael T; Gherzi, Roberto; Rosenfeld, Michael G; Trabucchi, Michele

    2012-01-01

    Gene silencing mediated by either microRNAs (miRNAs) or Adenylate/uridylate-rich elements Mediated mRNA Degradation (AMD) is a powerful way to post-transcriptionally modulate gene expression. We and others have reported that the RNA-binding protein KSRP favors the biogenesis of select miRNAs (including let-7 family) and activates AMD promoting the decay of inherently labile mRNAs. Different layers of interplay between miRNA- and AMD-mediated gene silencing have been proposed in cultured cells, but the relationship between the two pathways in living organisms is still elusive. We conditionally deleted Dicer in mouse pituitary from embryonic day (E) 9.5 through Cre-mediated recombination. In situ hybridization, immunohistochemistry, and quantitative reverse transcriptase-PCR revealed that Dicer is essential for pituitary morphogenesis and correct expression of hormones. Strikingly, αGSU (alpha glycoprotein subunit, common to three pituitary hormones) was absent in Dicer-deleted pituitaries. αGSU mRNA is unstable and its half-life increases during pituitary development. A transcriptome-wide analysis of microdissected E12.5 pituitaries revealed a significant increment of KSRP expression in conditional Dicer-deleted mice. We found that KSRP directly binds to αGSU mRNA, promoting its rapid decay; and, during pituitary development, αGSU expression displays an inverse temporal relationship to KSRP. Further, let-7b/c downregulated KSRP expression, promoting the degradation of its mRNA by directly binding to the 3'UTR. Therefore, we propose a model in which let-7b/c and KSRP operate within a negative feedback loop. Starting from E12.5, KSRP induces the maturation of let-7b/c that, in turn, post-transcriptionally downregulates the expression of KSRP itself. This event leads to stabilization of αGSU mRNA, which ultimately enhances the steady-state expression levels. We have identified a post-transcriptional regulatory network active during mouse pituitary development in

  4. Let-7b/c enhance the stability of a tissue-specific mRNA during mammalian organogenesis as part of a feedback loop involving KSRP.

    Directory of Open Access Journals (Sweden)

    Emanuela Repetto

    Full Text Available Gene silencing mediated by either microRNAs (miRNAs or Adenylate/uridylate-rich elements Mediated mRNA Degradation (AMD is a powerful way to post-transcriptionally modulate gene expression. We and others have reported that the RNA-binding protein KSRP favors the biogenesis of select miRNAs (including let-7 family and activates AMD promoting the decay of inherently labile mRNAs. Different layers of interplay between miRNA- and AMD-mediated gene silencing have been proposed in cultured cells, but the relationship between the two pathways in living organisms is still elusive. We conditionally deleted Dicer in mouse pituitary from embryonic day (E 9.5 through Cre-mediated recombination. In situ hybridization, immunohistochemistry, and quantitative reverse transcriptase-PCR revealed that Dicer is essential for pituitary morphogenesis and correct expression of hormones. Strikingly, αGSU (alpha glycoprotein subunit, common to three pituitary hormones was absent in Dicer-deleted pituitaries. αGSU mRNA is unstable and its half-life increases during pituitary development. A transcriptome-wide analysis of microdissected E12.5 pituitaries revealed a significant increment of KSRP expression in conditional Dicer-deleted mice. We found that KSRP directly binds to αGSU mRNA, promoting its rapid decay; and, during pituitary development, αGSU expression displays an inverse temporal relationship to KSRP. Further, let-7b/c downregulated KSRP expression, promoting the degradation of its mRNA by directly binding to the 3'UTR. Therefore, we propose a model in which let-7b/c and KSRP operate within a negative feedback loop. Starting from E12.5, KSRP induces the maturation of let-7b/c that, in turn, post-transcriptionally downregulates the expression of KSRP itself. This event leads to stabilization of αGSU mRNA, which ultimately enhances the steady-state expression levels. We have identified a post-transcriptional regulatory network active during mouse pituitary

  5. 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

  6. The phosphorylation of protein S6 modulates the interaction of the 40 S ribosomal subunit with the 5'-untranslated region of a dictyostelium pre-spore-specific mRNA and controls its stability.

    Science.gov (United States)

    Chiaberge, S; Cassarino, E; Mangiarotti, G

    1998-10-16

    AC914 mRNA, a pre-spore-specific mRNA that accumulates only in the post-aggregation stage of development, is transcribed constitutively as shown by nuclear run-off experiments and by fusing its promoter to the luciferase reporter gene. The same mRNA disappears quickly from disaggregated cells. If the 5'-untranslated region (5'UTR) of the constitutively expressed Actin 15 mRNA is substituted for the 5'UTR of AC914 mRNA, this can no longer be destabilized and accumulates both in growing and disaggregated cells. If the 5'UTR of AC914 mRNA is substituted for the 5'UTR of Actin 15 mRNA, the latter accumulates only in aggregated cells. Pactamycin, but not other inhibitors of protein synthesis, prevents AC914 mRNA from being destabilized in disaggregated cells, suggesting a role of 40 S subunits in the destabilization. This has been confirmed by using an in vitro system in which the in vivo stability of different mRNAs is reproduced. A protein kinase A-dependent phosphorylation of ribosomal protein S6 determines whether 40 S subunits are capable or not of destabilizing AC914 mRNA in the in vitro system.

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

    Directory of Open Access Journals (Sweden)

    Feng Cheng

    2017-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

  10. 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.

  11. Altered mRNA Levels of Glucocorticoid Receptor, Mineralocorticoid Receptor, and Co-Chaperones (FKBP5 and PTGES3) in the Middle Frontal Gyrus of Autism Spectrum Disorder Subjects.

    Science.gov (United States)

    Patel, Neil; Crider, Amanda; Pandya, Chirayu D; Ahmed, Anthony O; Pillai, Anilkumar

    2016-05-01

    Although stress has been implicated in the pathophysiology of autistic spectrum disorder (ASD), it is not known whether glucocorticoid receptor (GR) levels are altered in the brain of subjects with ASD. The messenger RNA (mRNA) levels of GR isoforms (GRα, GRβ, GRγ, and GRP), mineralocorticoid receptor (MR), GR co-chaperones (FKBP5, PTGES3, and BAG1), and inflammatory cytokines (IL-6, IL-1β, and IFN-γ) were examined in the postmortem middle frontal gyrus tissues of 13 ASD and 13 age-matched controls by qRT-PCR. The protein levels were examined by Western blotting. We found significant decreases in GRα (64%), GRγ (48%), GRP (20%) and MR (46%) mRNA levels in ASD subjects as compared to controls. However, significant increases in FKBP5 (42%) and PTGES3 (35%) mRNA levels were observed in ASD subjects. There were no differences in the mRNA levels of GRβ and BAG1 in ASD subjects as compared to controls. MR mRNA was found to be negatively correlated with the diagnostic score for abnormality of development. On the protein level, significant reductions in GR and MR, but no change in FKBP5 and PTGES3 were found in ASD subjects as compared to controls. Moreover, we observed significant increases in IL-1β and IFN-γ mRNA levels in ASD subjects, and these cytokines were negatively associated with GR levels. Our data, for the first time, reports dysregulation of GR, MR, FKBP5, and PTGES3 in ASD and suggest a possible role of inflammation in altered GR function in ASD.

  12. The RNA-binding protein HuD is required for GAP-43 mRNA stability, GAP-43 gene expression, and PKC-dependent neurite outgrowth in PC12 cells.

    Science.gov (United States)

    Mobarak, C D; Anderson, K D; Morin, M; Beckel-Mitchener, A; Rogers, S L; Furneaux, H; King, P; Perrone-Bizzozero, N I

    2000-09-01

    The RNA-binding protein HuD binds to a regulatory element in the 3' untranslated region (3' UTR) of the GAP-43 mRNA. To investigate the functional significance of this interaction, we generated PC12 cell lines in which HuD levels were controlled by transfection with either antisense (pDuH) or sense (pcHuD) constructs. pDuH-transfected cells contained reduced amounts of GAP-43 protein and mRNA, and these levels remained low even after nerve growth factor (NGF) stimulation, a treatment that is normally associated with protein kinase C (PKC)-dependent stabilization of the GAP-43 mRNA and neuronal differentiation. Analysis of GAP-43 mRNA stability demonstrated that the mRNA had a shorter half-life in these cells. In agreement with their deficient GAP-43 expression, pDuH cells failed to grow neurites in the presence of NGF or phorbol esters. These cells, however, exhibited normal neurite outgrowth when exposed to dibutyryl-cAMP, an agent that induces outgrowth independently from GAP-43. We observed opposite effects in pcHuD-transfected cells. The GAP-43 mRNA was stabilized in these cells, leading to an increase in the levels of the GAP-43 mRNA and protein. pcHuD cells were also found to grow short spontaneous neurites, a process that required the presence of GAP-43. In conclusion, our results suggest that HuD plays a critical role in PKC-mediated neurite outgrowth in PC12 cells and that this protein does so primarily by promoting the stabilization of the GAP-43 mRNA.

  13. 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…

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

    Science.gov (United States)

    Sehgal, P B; Gupta, S L

    1980-06-01

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

  15. 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

  16. Comparison of stress-induced and LPS-induced depressive-like behaviors and the alterations of central proinflammatory cytokines mRNA in rats.

    Science.gov (United States)

    Guan, Xi-Ting; Lin, Wen-Juan; Tang, Ming-Ming

    2015-09-01

    Although proinflammatory cytokine changes in depression have been studied widely, few investigations have searched for specific and common changes in cytokines. In the present study, two animal models of depression were compared: a chronic stress model using forced swim stress and an immune activation model using repeated central lipopolysaccharide (LPS) infusion. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 mRNA were examined in the brain regions of the prefrontal cortex, amygdala, and hippocampus using real-time polymerase chain reaction (RT-PCR). It was found that both chronic swim stress and repeated central LPS infusion induced depressive-like behaviors, including decreased body weight, reduced saccharin preference, and increased immobility time or shortened latency of immobility in the tail suspension test. Central TNF-α mRNA expression was elevated in both models and central IL-6 mRNA expression was unchanged in both models. Central IL-1β mRNA expression was increased only in the chronic immune activation model. The findings from this study suggest that TNF-α may be a common risk factor for inflammation in depressive disorders.

  17. RNA-binding protein hnRNPLL regulates mRNA splicing and stability during B-cell to plasma-cell differentiation.

    Science.gov (United States)

    Chang, Xing; Li, Bin; Rao, Anjana

    2015-04-14

    Posttranscriptional regulation is a major mechanism to rewire transcriptomes during differentiation. Heterogeneous nuclear RNA-binding protein LL (hnRNPLL) is specifically induced in terminally differentiated lymphocytes, including effector T cells and plasma cells. To study the molecular functions of hnRNPLL at a genome-wide level, we identified hnRNPLL RNA targets and binding sites in plasma cells through integrated Photoactivatable-Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP) and RNA sequencing. hnRNPLL preferentially recognizes CA dinucleotide-containing sequences in introns and 3' untranslated regions (UTRs), promotes exon inclusion or exclusion in a context-dependent manner, and stabilizes mRNA when associated with 3' UTRs. During differentiation of primary B cells to plasma cells, hnRNPLL mediates a genome-wide switch of RNA processing, resulting in loss of B-cell lymphoma 6 (Bcl6) expression and increased Ig production--both hallmarks of plasma-cell maturation. Our data identify previously unknown functions of hnRNPLL in B-cell to plasma-cell differentiation and demonstrate that the RNA-binding protein hnRNPLL has a critical role in tuning transcriptomes of terminally differentiating B lymphocytes.

  18. Short-chain fatty acid-supplemented total parenteral nutrition alters intestinal structure, glucose transporter 2 (GLUT2) mRNA and protein, and proglucagon mRNA abundance in normal rats.

    Science.gov (United States)

    Tappenden, K A; Drozdowski, L A; Thomson, A B; McBurney, M I

    1998-07-01

    Intestinal adaptation is a complex physiologic process that is not completely understood. Intravenous short-chain fatty acids (SCFAs) enhance intestinal adaptation after 80% enterectomy in rats. The purpose of this study was to examine rapid responses to SCFA-supplemented total parenteral nutrition (TPN) in the normal small intestine. After jugular catheterization, 31 Sprague-Dawley rats (weighing 258 +/- 3 g) were randomly assigned to receive standard TPN or an isoenergetic, isonitrogenous TPN solution supplemented with SCFAs (TPN+SCFA). Intestinal samples were obtained after 24 or 72 h of nutrient infusion. TPN+SCFA for 24 h increased (P SCFA for 72 h increased (P SCFA infusion and returned to levels seen with control TPN by 72 h. Glucose transporter 2 (GLUT2) mRNA was significantly higher (P SCFA groups at both time points when compared with control TPN groups. Ileal GLUT2 protein abundance in the 72-h TPN+SCFA group was significantly higher (P SCFA than after 24 h, whereas the 24- and 72-h TPN groups did not differ. In summary, SCFAs led to rapid changes in ileal proglucagon and glucose transporter expression in rats receiving TPN and provide insights into therapeutic management of individuals with short bowel syndrome or intestinal malabsorption syndromes.

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

    Directory of Open Access Journals (Sweden)

    Ghazal Peter

    2009-09-01

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

  20. Early-life infection leads to altered BDNF and IL-1beta mRNA expression in rat hippocampus following learning in adulthood.

    Science.gov (United States)

    Bilbo, Staci D; Barrientos, Ruth M; Eads, Andrea S; Northcutt, Alexis; Watkins, Linda R; Rudy, Jerry W; Maier, Steven F

    2008-05-01

    Neonatal bacterial infection in rats leads to profound hippocampal-dependent memory impairments following a peripheral immune challenge in adulthood. Here, we determined whether neonatal infection plus an immune challenge in adult rats is associated with impaired induction of brain-derived neurotrophic factor (BDNF) within the hippocampus (CA1, CA3, and dentate gyrus) following fear conditioning. BDNF is well characterized for its critical role in learning and memory. Rats injected on postnatal day 4 with PBS (vehicle) or Escherichia coli received as adults either no conditioning or a single 2min trial of fear conditioning. Half of the rats in the conditioned group then received a peripheral injection of 25mug/kg lipopolysaccharide (LPS) and all were sacrificed 1 or 4h later. Basal (unconditioned) BDNF mRNA did not differ between groups. However, following conditioning, neonatal infection with E. coli led to decreased BDNF mRNA induction in all regions compared to PBS-treated rats. This decrease in E. coli-treated rats was accompanied by a large increase in IL-1beta mRNA in CA1. Taken together, these data indicate that early infection strongly influences the induction of IL-1beta and BDNF within distinct regions of the hippocampus, which likely contribute to observed memory impairments in adulthood.

  1. Alterations in trace element levels and mRNA expression of Hsps and inflammatory cytokines in livers of duck exposed to molybdenum or/and cadmium.

    Science.gov (United States)

    Cao, Huabin; Gao, Feiyan; Xia, Bing; Zhang, Mengmeng; Liao, Yilin; Yang, Zhi; Hu, Guoliang; Zhang, Caiying

    2016-03-01

    To evaluate the effects of dietary Molybdenum (Mo) or/and Cadmium (Cd) on trace elements and the mRNA expression levels of heat shock proteins (Hsps) and inflammatory cytokines in duck livers. 240 healthy 11-day-old ducks were randomly divided into six groups with 40 ducks in each group, which were treated with Mo or/and Cd at different doses on the basal diet for 120 days. On days 30, 60, 90 and 120, 10 birds in each group were randomly selected and euthanized and then the livers were collected to determine the contents of Mo, Cd, copper (Cu), iron (Fe), zine (Zn), Selenium (Se) and the mRNA expression levels of Hsps, inflammatory cytokines. In addition, liver tissues at 120 days were subjected to histopathological analysis with the optical microscope. The results showed that the mRNA expression of Hsp60, Hsp70, Hsp90, tumor necrosis factor-α (TNF-α), nuclear factor-kappa B (NF-κB), and cyclooxygenase-2 (COX-2) were significantly (Pcytokines may play a role in the resistance of liver toxicity induced by Mo and Cd.

  2. The effects of 5'-capping, 3'-polyadenylation and leader composition upon the translation and stability of mRNA in a cell-free extract derived from the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Gerstel, B; Tuite, M F; McCarthy, J E

    1992-08-01

    A new modular expression system was developed to direct the in vitro synthesis of defined transcripts that were used as templates for translation in yeast cell-free extracts. The system was used to examine the influence of 5'-capping, 3'-polyadenylation and leader sequence upon the translation and stability of the synthetic Tn9 cat (chloramphenicol acetyl transferase), yeast PGK (phosphoglycerate kinase) and yeast HSP26 (heat-shock protein 26) mRNAs. The addition of a methylated cap (m7Gppp) or of a poly(A) tail enhanced translation and stabilized the mRNA. The dependence of translation upon capping was reduced in the presence of the HSP26 leader sequence. This may indicate the existence of a translational mechanism that enhances cap-independent translation. The enhancement of the translation and stability of mRNA was relatively insensitive to changes in the position of the poly(A) tail relative to the reading frame.

  3. Sodium fluoride affects zebrafish behaviour and alters mRNA expressions of biomarker genes in the brain: Role of Nrf2/Keap1.

    Science.gov (United States)

    Mukhopadhyay, Debdip; Priya, Pooja; Chattopadhyay, Ansuman

    2015-09-01

    Sodium fluoride (NaF), used as pesticides and for industrial purposes are deposited in the water bodies and therefore affects its biota. Zebrafish exposed to NaF in laboratory condition showed hyperactivity and frequent surfacing activity, somersaulting and vertical swimming pattern as compared to the control group. Reactive oxygen species level was elevated and glutathione level was depleted along with increased malondialdehyde content in the brain. Levels of glutathione-s-transferase (GST), catalase (CAT) and superoxide dismutase were also elevated in the treatment groups. Expression of mRNA of nuclear factor erythroid 2 related factor 2 (Nrf2) and its inhibitor Kelch-like ECH-associated protein 1 (Keap1) during stress condition were observed along with Gst, Cat, NADPH: quinone oxidoreductase 1(Nqo1) and p38. Except Keap1, all other genes exhibited elevated expression. Nrf2/Keap1 proteins had similar expression pattern as their corresponding mRNA. The findings in this study might help to understand the molecular mechanism of fluoride induced neurotoxicity in fish.

  4. Alterations in potassium channel gene expression in atria of patients with persistent and paroxysmal atrial fibrillation : Differential regulation of protein and mRNA levels for K+ channels

    NARCIS (Netherlands)

    Brundel, BJJM; Van Gelder, IC; Henning, RH; Tuinenburg, AE; Wietses, M; Grandjean, JG; Wilde, AAM; Van Gilst, WH; Crijns, HJGM

    2001-01-01

    OBJECTIVES Our purpose was to determine whether patients with persistent atrial fibrillation (AF) and patients with paroxysmal AF show alterations in potassium channel expression. BACKGROUND Persistent AF is associated with a sustained shortening of the atrial action potential duration and atrial re

  5. Detecting altered postural control after cerebral concussion in athletes with normal postural stability

    OpenAIRE

    Cavanaugh, J; Guskiewicz, K.; Giuliani, C.; Marshall, S.; Mercer, V; Stergiou, N.

    2005-01-01

    Objective: To determine if approximate entropy (ApEn), a regularity statistic from non-linear dynamics, could detect changes in postural control during quiet standing in athletes with normal postural stability after cerebral concussion.

  6. Splice variants of the human zinc transporter ZnT5 (SLC30A5) are differentially localized and regulated by zinc through transcription and mRNA stability.

    Science.gov (United States)

    Jackson, Kelly A; Helston, Rachel M; McKay, Jill A; O'Neill, Elaine D; Mathers, John C; Ford, Dianne

    2007-04-06

    Maintenance of cellular zinc homeostasis includes regulating the expression of cell membrane zinc transporters. Knowledge about the mechanisms underlying changes in mammalian zinc transporter mRNA abundance is poor. We demonstrated that when expressed in Chinese hamster ovary cells as N-terminal fusions to green fluorescent protein, two splice variants of ZnT5 adopt different subcellular locations (either in the Golgi apparatus or throughout the cell, including at the plasma membrane) indicating discrete roles in cellular zinc homeostasis. We demonstrated, using a beta-galactosidase reporter gene, that both splice variants were expressed from a promoter region that was transcriptionally repressed by increased extracellular zinc (150 microM compared with 3 mum; approximately 40%) and by extracellular zinc depletion, using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine ( approximately 20%). We mapped the zinc-responsive element to the region -154 to +50, relative to the predicted start of transcription, and showed that a consensus metal response element sequence (-410 to -404) was not responsible for these effects. Changes in ZnT5 mRNA abundance in Caco-2 cells at different zinc concentrations were in parallel to the changes in promoter activity ( approximately 40% reduction at 150 microM zinc) but in the presence of actinomycin D, to prevent transcription, we observed a marked stabilization (1.7-2-fold accumulation over 24 h) of ZnT5 mRNA. We conclude that effects of zinc on ZnT5 transcription and mRNA stability act in opposition to balance mRNA abundance for cellular zinc homeostasis. To our knowledge, this is the first report that zinc affects the stability of a transcript with a direct role in cellular zinc homeostasis.

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

    Science.gov (United States)

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

    2010-07-30

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

  8. Alteration in NMDA receptor subunit mRNA expression in vulnerable and resistant regions of in vitro ischemic rat hippocampal slices.

    Science.gov (United States)

    Small, D L; Poulter, M O; Buchan, A M; Morley, P

    1997-08-29

    Brain insults, including cerebral ischemia, can alter glutamate receptor subunit expression in vulnerable neurons. Understanding these post-ischemic changes in glutamate receptors could enhance our ability to identify specific, novel neuroprotective compounds. Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to quantify the altered expression of the N-methyl-D-aspartate (NMDA) NR2A, NR2B and NR2C subunits relative to one another in rat hippocampal slices in resistant and vulnerable regions following in vitro oxygen-glucose deprivation. Ninety minutes after re-oxygenation and return to 10 mM glucose, there was a significant increase in the expression of NR2C relative to NR2B and NR2A in the slice as a whole, as well as in the selectively vulnerable CA1 region and the resistant CA3 and dentate gyrus regions.

  9. The Mef2 Transcription Network Is Disrupted in Myotonic Dystrophy Heart Tissue, Dramatically Altering miRNA and mRNA Expression

    Directory of Open Access Journals (Sweden)

    Auinash Kalsotra

    2014-01-01

    Full Text Available Cardiac dysfunction is the second leading cause of death in myotonic dystrophy type 1 (DM1, primarily because of arrhythmias and cardiac conduction defects. A screen of more than 500 microRNAs (miRNAs in a DM1 mouse model identified 54 miRNAs that were differentially expressed in heart. More than 80% exhibited downregulation toward the embryonic expression pattern and showed a DM1-specific response. A total of 20 of 22 miRNAs tested were also significantly downregulated in human DM1 heart tissue. We demonstrate that many of these miRNAs are direct MEF2 transcriptional targets, including miRNAs for which depletion is associated with arrhythmias or fibrosis. MEF2 protein is significantly reduced in both DM1 and mouse model heart samples, and exogenous MEF2C restores normal levels of MEF2 target miRNAs and mRNAs in a DM1 cardiac cell culture model. We conclude that loss of MEF2 in DM1 heart causes pathogenic features through aberrant expression of both miRNA and mRNA targets.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    Delabastita, Tijs; Desloovere, Kaat; Meyns, Pieter

    2016-01-01

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

  12. 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.

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

    Science.gov (United States)

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

    2007-03-01

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

  14. 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

  15. An alteration in ATG16L1 stability in Crohn disease.

    Science.gov (United States)

    Lassen, Kara G; Xavier, Ramnik J

    2014-10-01

    Individuals who harbor a common coding polymorphism (Thr300Ala) within a structurally unclassified region of ATG16L1 are at increased risk for the development of Crohn disease. Recently, we reported on the generation and characterization of knockin mice carrying the ATG16L1 T300A variant. We demonstrate that multiple cell types from T300A knock-in mice exhibit reduced selective autophagy, and we mechanistically link this phenotype with an increased susceptibility of ATG16L1 T300A to CASP3- and CASP7-mediated cleavage. These findings demonstrate how a single polymorphism can result in cell type- and pathway-specific disruptions of selective autophagy and alterations in the inflammatory milieu that can contribute to disease.

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

    DEFF Research Database (Denmark)

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

    1998-01-01

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

  17. The Alzheimer's Disease-Associated R47H Variant of TREM2 Has an Altered Glycosylation Pattern and Protein Stability

    Science.gov (United States)

    Park, Ji-Seon; Ji, In Jung; Kim, Dong-Hou; An, Hyun Joo; Yoon, Seung-Yong

    2017-01-01

    The R47H coding variant of the triggering receptor expressed on myeloid cells-2 (TREM2) increases the risk of Alzheimer's disease (AD) similar to apolipoprotein E4. TREM2 R47H has recently been shown to have impaired binding to damage-associated lipid or apolipoprotein ligands. However, it is not known how this R47H variant affects the biochemical characteristics of TREM2 and alters the pathogenesis of AD. We previously reported that TREM2-R47H has a slightly different glycosylation pattern from wild-type. A more detailed characterization in our present study confirms that TREM2 R47H has an altered glycosylation pattern and reduced stability. TREM2 R47H shows different glycosylation profiles from analysis using monensin or kifunensine treatment which were confirmed by mass spectrometry. The solubility of TREM2 R47H and its cleaved products such as intracellular domain (ICD) is also decreased, increasing its proteasomal and lysosomal degradation. The different biochemical characteristics of TREM2 R47H, including glycosylation, solubility and processing, may offer insights into a future therapeutic strategy for AD. PMID:28149270

  18. 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.

  19. A study of the alterability of terminal residues stabilized by a mineral-binder-based solidification/stabilization process; Etude de l`alterabilite de residus ultimes stabilises par un procede de solidification, stabilisation a base de liants mineraux

    Energy Technology Data Exchange (ETDEWEB)

    Humez, N.; Prost, R. [Institut National de Recherches Agronomiques (INRA), 78 - Versailles (France). Unite de Science du Sol; Piketty, L.; Schnuriger, B.; Benchara, A.; Pichat, P. [Centre de Solidification, Stabilisation et Stockage, Sarp Industries, 78 - Limay (France)

    1997-12-31

    A new laboratory approach has been developed for the study of the long term behaviour of stabilized terminal residues, and is based on the experimental method for the study of rock alteration. The method consists in examining the migration of elements that are present in the solution contained in the pores, and studying the structural modifications which result from the dissolution of certain constituents, the transformation and the neo-formation of other ones in the material. The method allows for the evaluation, in a short time, of the alterability and perenniality of stabilized terminal wastes. Results show that the long term alterability of solidified/stabilized samples that have been processed using the ECOFIX method, is low

  20. Identification of the cis-elements mediating the autogenous control of ribosomal protein L2 mRNA stability in yeast.

    Science.gov (United States)

    Presutti, C; Villa, T; Hall, D; Pertica, C; Bozzoni, I

    1995-08-15

    The ribosomal protein L2 (rpL2) of Saccharomyces cerevisiae regulates the accumulation of its own mRNA by a feedback mechanism. An RNA sequence is responsible for this control, initially characterized as a 360 nucleotide-long region, localized at the 5' end of the transcript. This region, fused to an unrelated coding sequence, is able to down-regulate the accumulation of the chimeric transcript when increased levels of rpL2 are induced in the cell. The target regulatory region also responds to regulation when inserted inside an intron, demonstrating that the control process can take place inside the nucleus. Deletion analysis from the 5' and 3' borders have restricted the responsive region to approximately 200 nt. The insertion of a poly-G cassette downstream of the regulatory region allowed the identification of truncated 3' cut-off poly(A)+ RNA molecules. The parallel identification of cut-off molecules containing the 5' portion of the transcript allowed us to deduce that the truncated products originate by endonucleolytic cleavage. Altogether, these results are consistent with a mechanism by which the presence of excess amounts of rpL2 in the cell triggers its own mRNA to a degradative pathway; this involves an initial endonucleolytic cleavage that is followed by exonucleolytic trimming. Such a regulatory mechanism shows interesting analogies with the translational regulation of r-proteins in Escherichia coli.

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

    Science.gov (United States)

    Li, Xinrong

    2016-04-01

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

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

    NARCIS (Netherlands)

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

    2006-01-01

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

  3. RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production.

    Science.gov (United States)

    Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

    2015-03-01

    We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis.

  4. The use of Molecular Beacons to Directly Measure Bacterial mRNA Abundances and Transcript Degradation

    Science.gov (United States)

    Kuechenmeister, Lisa J.; Anderson, Kelsi L.; Morrison, John M.; Dunman, Paul M.

    2009-01-01

    The regulation of mRNA turnover is a dynamic means by which bacteria regulate gene expression. Although current methodologies allow characterization of the stability of individual transcripts, procedures designed to measure alterations in transcript abundance/turnover on a high throughput scale are lacking. In the current report, we describe the development of a rapid and simplified molecular beacon-based procedure to directly measure the mRNA abundances and mRNA degradation properties of well-characterized Staphylococcus aureus pathogenicity factors. This method does not require any PCR-based amplification, can monitor the abundances of multiple transcripts within a single RNA sample, and was successfully implemented into a high throughput screen of transposon mutant library members to detect isolates with altered mRNA turnover properties. It is expected that the described methodology will provide great utility in characterizing components of bacterial RNA degradation processes and can be used to directly measure the mRNA levels of virtually any bacterial transcript. PMID:18992285

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

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

    Science.gov (United States)

    Berghauzen-Maciejewska, Klemencja; Wardas, Jadwiga; Kosmowska, Barbara; Głowacka, Urszula; Kuter, Katarzyna; Ossowska, Krystyna

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Klemencja Berghauzen-Maciejewska

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

  8. 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.

  9. 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)

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

    Science.gov (United States)

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

    2012-03-01

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

  11. The alteration of mRNA expression of SOD and GPX genes, and proteins in tomato (Lycopersicon esculentum Mill) under stress of NaCl and/or ZnO nanoparticles.

    Science.gov (United States)

    Alharby, Hesham F; Metwali, Ehab M R; Fuller, Michael P; Aldhebiani, Amal Y

    2016-11-01

    Five cultivars of tomato having different levels of salt stress tolerance were exposed to different treatments of NaCl (0, 3 and 6 g L(-1)) and ZnO-NPs (0, 15 and 30 mg L(-1)). Treatments with NaCl at both 3 and 6 g L(-1) suppressed the mRNA levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX) genes in all cultivars while plants treated with ZnO-NPs in the presence of NaCl, showed increments in the mRNA expression levels. This indicated that ZnO-NPs had a positive response on plant metabolism under salt stress. Superior expression levels of mRNA were observed in the salt tolerant cultivars, Sandpoint and Edkawy while the lowest level was detected in the salt sensitive cultivar, Anna Aasa. SDS-PAGE showed clear differences in patterns of protein expression among the cultivars. A negative protein marker for salt sensitivity and ZnO-NPs was detected in cv. Anna Aasa at a molecular weight of 19.162 kDa, while the tolerant cultivar Edkawy had two positive markers at molecular weights of 74.991 and 79.735 kDa.

  12. An Imprinted Cross-Linked Enzyme Aggregate (iCLEA of Sucrose Phosphorylase: Combining Improved Stability with Altered Specificity

    Directory of Open Access Journals (Sweden)

    Tom Desmet

    2012-09-01

    Full Text Available The industrial use of sucrose phosphorylase (SP, an interesting biocatalyst for the selective transfer of α-glucosyl residues to various acceptor molecules, has been hampered by a lack of long-term stability and low activity towards alternative substrates. We have recently shown that the stability of the SP from Bifidobacterium adolescentis can be significantly improved by the formation of a cross-linked enzyme aggregate (CLEA. In this work, it is shown that the transglucosylation activity of such a CLEA can also be improved by molecular imprinting with a suitable substrate. To obtain proof of concept, SP was imprinted with α-glucosyl glycerol and subsequently cross-linked with glutaraldehyde. As a consequence, the enzyme’s specific activity towards glycerol as acceptor substrate was increased two-fold while simultaneously providing an exceptional stability at 60 °C. This procedure can be performed in an aqueous environment and gives rise to a new enzyme formulation called iCLEA.

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

    Science.gov (United States)

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

    1997-01-01

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

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

    Science.gov (United States)

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

    1997-03-18

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

  15. Administration of flutamide alters sperm ultrastructure, sperm plasma membrane integrity and its stability, and sperm mitochondrial oxidative capability in the boar: in vivo and in vitro approach.

    Science.gov (United States)

    Lydka, M; Piasecka, M; Gaczarzewicz, D; Koziorowski, M; Bilinska, B

    2012-08-01

    Our previous work has shown that an anti-androgen flutamide administered pre- and post-natally induced adverse effects on the epididymal morphology and function of adult boars. The present investigation is aimed to understand the effect of flutamide and its metabolite on changes in sperm plasma membrane integrity and its stability, changes in mitochondrial oxidative capability and frequency of abnormal sperm. In vivo effects of flutamide (50 mg/kg b.w.) on sperm ultrastructure were examined by electron microscopic observations. In vitro effects of 5, 50 and 100 μg/ml hydroxyflutamide, administered for 2 and 24 h, on sperm plasma membrane integrity were measured by LIVE/DEAD Sperm Vitality kit, while those on sperm membrane stability and mitochondrial oxidoreductive activity were investigated using Merocyanine 540 and NADH tests, respectively. The incidence of abnormal spermatozoa increased significantly (p boars compared with controls. In an in vitro approach, low dose of hydroxyflutamide in 2-h incubations appeared less effective in altering the sperm plasma membrane integrity and its stability than two higher doses used (p sperm membrane destabilization and mitochondrial oxidoreductive activity was strengthened after 24 h of hydroxyflutamide administration (p sperm parameters with regard to oxidative capability of mitochondria, plasma membrane changes and sperm ultrastructure provides novel data on the boar sperm sensitivity to anti-androgen action. Results indicate high sensitivity of boar spermatozoa to androgen withdrawal.

  16. Gallium nitrate regulates rat osteoblast expression of osteocalcin protein and mRNA levels.

    Science.gov (United States)

    Guidon, P T; Salvatori, R; Bockman, R S

    1993-01-01

    Gallium nitrate, a group IIIa metal salt, has been found to be clinically effective for the treatment of accelerated bone resorption in cancer-related hypercalcemia and Paget's disease. Here we report the effects of gallium nitrate on osteocalcin mRNA and protein levels on the rat osteoblast-like cell line ROS 17/2.8. Gallium nitrate reduced both constitutive and vitamin D3-stimulated osteocalcin protein levels in culture medium by one-half and osteocalcin mRNA levels to one-third to one-tenth of control. Gallium nitrate also inhibited vitamin D3 stimulation of osteocalcin and osteopontin mRNA levels but did not affect constitutive osteopontin mRNA levels. Among several different metals examined, gallium was unique in its ability to reduce osteocalcin mRNA levels without decreasing levels of other mRNAs synthesized by ROS 17/2.8 cells. The effects of gallium nitrate on osteocalcin mRNA and protein synthesis mimic those seen when ROS 17/2.8 cells are exposed to transforming growth factor beta 1 (TGF beta 1); however, TGF-beta 1 was not detected in gallium nitrate-treated ROS 17/2.8 cell media. Use of the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that gallium nitrate did not alter the stability of osteocalcin mRNA. Transient transfection assays using the rat osteocalcin promoter linked to the bacterial reporter gene chloramphenicol acetyltransferase indicated that gallium nitrate blocked reporter gene expression stimulated by the osteocalcin promoter. This is the first reported effect of gallium nitrate on isolated osteoblast cells.

  17. Massive bowel resection upregulates the intestinal mRNA expression levels of cellular retinol-binding protein II and apolipoprotein A-IV and alters the intestinal vitamin A status in rats.

    Science.gov (United States)

    Hebiguchi, Taku; Mezaki, Yoshihiro; Morii, Mayako; Watanabe, Ryo; Yoshikawa, Kiwamu; Miura, Mitsutaka; Imai, Katsuyuki; Senoo, Haruki; Yoshino, Hiroaki

    2015-03-01

    Short bowel (SB) syndrome causes the malabsorption of various nutrients. Among these, vitamin A is important for a number of physiological activities. Vitamin A is absorbed by epithelial cells of the small intestine and is discharged into the lymphatic vessels as a component of chylomicrons and is delivered to the liver. In the present study, we used a rat model of SB syndrome in order to assess its effects on the expression of genes associated with the absorption, transport and metabolism of vitamin A. In the rats with SB, the intestinal mRNA expression levels of cellular retinol-binding protein II (CRBP II, gene symbol Rbp2) and apolipoprotein A-IV (gene symbol Apoa4) were higher than those in the sham-operated rats, as shown by RT-qPCR. Immunohistochemical analysis revealed that absorptive epithelial cells stained positive for both CRBP II and lecithin retinol acyltransferase, which are both required for the effective esterification of vitamin A. In the rats with SB, the retinol content in the ileum and the retinyl ester content in the jejunum were lower than those in the sham-operated rats, as shown by quantitative analysis of retinol and retinyl esters by high performance liquid chromatography. These results suggest that the elevated mRNA expression levels of Rbp2 and Apoa4 in the rats with SB contribute to the effective esterification and transport of vitamin A.

  18. 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.

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

    Science.gov (United States)

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

    2016-02-01

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

  20. Switching the centromeres on and off: epigenetic chromatin alterations provide plasticity in centromere activity stabilizing aberrant dicentric chromosomes.

    Science.gov (United States)

    Sato, Hiroshi; Saitoh, Shigeaki

    2013-12-01

    The kinetochore, which forms on a specific chromosomal locus called the centromere, mediates interactions between the chromosome and the spindle during mitosis and meiosis. Abnormal chromosome rearrangements and/or neocentromere formation can cause the presence of multiple centromeres on a single chromosome, which results in chromosome breakage or cell cycle arrest. Analyses of artificial dicentric chromosomes suggested that the activity of the centromere is regulated epigenetically; on some stably maintained dicentric chromosomes, one of the centromeres no longer functions as a platform for kinetochore formation, although the DNA sequence remains intact. Such epigenetic centromere inactivation occurs in cells of various eukaryotes harbouring 'regional centromeres', such as those of maize, fission yeast and humans, suggesting that the position of the active centromere is determined by epigenetic markers on a chromosome rather than the nucleotide sequence. Our recent findings in fission yeast revealed that epigenetic centromere inactivation consists of two steps: disassembly of the kinetochore initiates inactivation and subsequent heterochromatinization prevents revival of the inactivated centromere. Kinetochore disassembly followed by heterochromatinization is also observed in normal senescent human cells. Thus epigenetic centromere inactivation may not only stabilize abnormally generated dicentric chromosomes, but also be part of an intrinsic mechanism regulating cell proliferation.

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

    OpenAIRE

    2010-01-01

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

  2. Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: a new gene-silencing approach.

    Science.gov (United States)

    Marsollier, Anne-Charlotte; Ciszewski, Lukasz; Mariot, Virginie; Popplewell, Linda; Voit, Thomas; Dickson, George; Dumonceaux, Julie

    2016-04-15

    Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy.

  3. Occupational health hazards of trichloroethylene among workers in relation to altered mRNA expression of cell cycle regulating genes (p53, p21, bax and bcl-2 and PPARA

    Directory of Open Access Journals (Sweden)

    Meenu Varshney

    2015-01-01

    Full Text Available Trichloroethylene (TCE is widely used as a metal degreaser in industrial processes. The present study reports on the effects of TCE exposure on workers employed in the lock industries. To ensure exposure of the workers to TCE, its toxic metabolites, trichloroacetic acid (TCA, dichloroacetic acid (DCA and trichloroethanol (TCEOH were detected in the plasma of the subjects through solid phase microextraction-gas chromatography-electron capture detection. TCA, DCA and TCEOH were detected in the range of 0.004–2.494 μg/mL, 0.01–3.612 μg/mL and 0.002–0.617 μg/mL, respectively. Quantitative reverse transcription polymerase chain reaction analysis revealed up-regulated expression of p53 (2.4-fold; p < 0.05, p21 (2-fold; p < 0.01, bax (2.9-fold; p < 0.01 mRNAs and down-regulated expression of bcl-2 (67%; p < 0.05 mRNAs, indicating DNA damaging potential of these metabolites. No effects were observed on the levels of p16 and c-myc mRNAs. Further, as TCA and DCA, the ligand of peroxisome proliferator activated receptor alpha (PPARA, are involved in the process of hepatocarcinogenesis in rodents, we examined expression of PPARA mRNA and let-7c miRNA in the workers. No statistically significant differences in expression of PPARA mRNA and let-7c miRNA in patients were observed as compared to values in controls. Dehydroepiandosterone sulfate (DHEAS is a reported endogenous ligand of PPARA so its competitive role was also studied. We observed decreased levels of DHEAS hormone in the subjects. Hence, its involvement in mediation of the observed changes in the levels of various mRNAs analyzed in this study appears unlikely.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  5. 肝癌肝动脉化疗栓塞术后外周血AFP mRNA变化及意义%THE ALTERATION OF AFP mRNA CONCENTRATION IN PERIPHERAL BLOOD WITH HEPATOCELLULAR CARCINOMA DURING TRANSHEPATIC ARTERAL CHEMOTHERAPY AND EMBOLIZATION

    Institute of Scientific and Technical Information of China (English)

    刘迎娣; 梁敏; 孙晓华; 汪鸿志; 程留芳

    2001-01-01

    To find the rule of alterations in AFP mRNA concentration in peripheral blood in patients with hepatocellular carcinoma(HCC) before and after the treatment with transhepatic arterial chemotherapy and embolization(TAE), 15 cases of the pateints with HCC were included in this survey. The AFP mRNA concentration in peripheral blood of the patients were investigated using compatitive RT-PCR technique before and 1,2,3,4, 5,6,7, 14, 28 days after TAE. The results showed that AFP mRNA concentration on the third day after TAE was the highest. It declined to the level before treatment during one week.It suggested that AFP mRNA concentration changed with a rule, and following the rule, preventive measure to distant metastasis could be instituted in clinical work.%通过对肝癌肝动脉化疗栓塞术(TAE)治疗前后外周血AFP mRNA定量检测,探讨治疗前后外周血AFP mRNA定量变化规律,并分析其临床意义。采用分子生物学反转录竞争PCR技术,定量检测了15例原发性肝细胞癌患者TAE治疗前及术后第1、2、3、4、5、6、7、14、28日外周血AFP mRNA水平。结果显示TAE前后外周血AFP mRNA有不同程度的变化,其中术后第3日呈最高水平,1周后基本恢复至术前水平,与术前比较,术后4周1例升高,8例基本相同,6例下降。提示TAE治疗后第3日外周血AFP mRNA水平最高,临床可给予积极治疗,预防肝外转移的发生。

  6. Long-Term Effects of Prenatal Exposure to Undernutrition on Cannabinoid Receptor-Related Behaviors: Sex and Tissue-Specific Alterations in the mRNA Expression of Cannabinoid Receptors and Lipid Metabolic Regulators

    Science.gov (United States)

    Ramírez-López, María T.; Arco, Rocío; Decara, Juan; Vázquez, Mariam; Rivera, Patricia; Blanco, Rosario Noemi; Alén, Francisco; Gómez de Heras, Raquel; Suárez, Juan; Rodríguez de Fonseca, Fernando

    2016-01-01

    Maternal malnutrition causes long-lasting alterations in feeding behavior and energy homeostasis in offspring. It is still unknown whether both, the endocannabinoid (eCB) machinery and the lipid metabolism are implicated in long-term adaptive responses to fetal reprogramming caused by maternal undernutrition. We investigated the long-term effects of maternal exposure to a 20% standard diet restriction during preconceptional and gestational periods on the metabolically-relevant tissues hypothalamus, liver, and perirenal fat (PAT) of male and female offspring at adulthood. The adult male offspring from calorie-restricted dams (RC males) exhibited a differential response to the CB1 antagonist AM251 in a chocolate preference test as well as increased body weight, perirenal adiposity, and plasma levels of triglycerides, LDL, VLDL, bilirubin, and leptin. The gene expression of the cannabinoid receptors Cnr1 and Cnr2 was increased in RC male hypothalamus, but a down-expression of most eCBs-metabolizing enzymes (Faah, Daglα, Daglβ, Mgll) and several key regulators of fatty-acid β-oxidation (Cpt1b, Acox1), mitochondrial respiration (Cox4i1), and lipid flux (Pparγ) was found in their PAT. The female offspring from calorie-restricted dams exhibited higher plasma levels of LDL and glucose as well as a reduction in chocolate and caloric intake at post-weaning periods in the feeding tests. Their liver showed a decreased gene expression of Cnr1, Pparα, Pparγ, the eCBs-degrading enzymes Faah and Mgll, the de novo lipogenic enzymes Acaca and Fasn, and the liver-specific cholesterol biosynthesis regulators Insig1 and Hmgcr. Our results suggest that the long-lasting adaptive responses to maternal caloric restriction affected cannabinoid-regulated mechanisms involved in feeding behavior, adipose β-oxidation, and hepatic lipid and cholesterol biosynthesis in a sex-dependent manner. PMID:28082878

  7. Gene regulation by mRNA editing

    Energy Technology Data Exchange (ETDEWEB)

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

    1997-02-01

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

  8. 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

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

    NARCIS (Netherlands)

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

    2016-01-01

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

  10. 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.

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

    Institute of Scientific and Technical Information of China (English)

    YUAN; Chong; (

    2001-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jerome E Lee

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

  13. Short communication: An evaluation of the effectiveness of Lactobacillus buchneri 40788 to alter fermentation and improve the aerobic stability of corn silage in farm silos.

    Science.gov (United States)

    Mari, L J; Schmidt, R J; Nussio, L G; Hallada, C M; Kung, L

    2009-03-01

    The objective of this study was to determine if the effects of inoculation with Lactobacillus buchneri 40788 were detectable when applied to whole-plant corn stored in farm silos. Corn silage was randomly sampled from farms in Wisconsin, Minnesota, and Pennsylvania, and was untreated (n = 15) or treated with an inoculant (n = 16) containing L. buchneri 40788 alone or this organism combined with Pediococcus pentosaceus during May and June 2007. Corn silage that was removed from the silo face during the morning feeding was sampled, vacuum-packed, and heat sealed in polyethylene bags and shipped immediately to the University of Delaware for analyses. Silage samples were analyzed for dry matter (DM), nutrient composition, fermentation end-products, aerobic stability, and microbial populations. The population of L. buchneri in silages was determined using a real-time quantitative PCR method. Aerobic stability was measured as the time after exposure to air that it took for a 2 degrees C increase above an ambient temperature. The DM and concentrations of lactic and acetic acids were 35.6 and 34.5, 4.17 and 4.85, and 2.24 and 2.41%, respectively, for untreated and inoculated silages and were not different between treatments. The concentration of 1,2-propanediol was greater in inoculated silages (1.26 vs. 0.29%). Numbers of lactic acid bacteria determined on selective agar were not different between treatments. However, the numbers of L. buchneri based on measurements using real-time quantitative PCR analysis were greater and averaged 6.46 log cfu-equivalents/g compared with 4.89 log cfu-equivalent for inoculated silages. There were fewer yeasts and aerobic stability was greater in inoculated silages (4.75 log cfu/g and 74 h of stability) than in untreated silages (5.55 log cfu/g and 46 h of stability). This study supports the effectiveness of L. buchneri 40788 on dairy farms.

  14. Optimal Down Regulation of mRNA Translation

    Science.gov (United States)

    Zarai, Yoram; Margaliot, Michael; Tuller, Tamir

    2017-01-01

    Down regulation of mRNA translation is an important problem in various bio-medical domains ranging from developing effective medicines for tumors and for viral diseases to developing attenuated virus strains that can be used for vaccination. Here, we study the problem of down regulation of mRNA translation using a mathematical model called the ribosome flow model (RFM). In the RFM, the mRNA molecule is modeled as a chain of n sites. The flow of ribosomes between consecutive sites is regulated by n + 1 transition rates. Given a set of feasible transition rates, that models the outcome of all possible mutations, we consider the problem of maximally down regulating protein production by altering the rates within this set of feasible rates. Under certain conditions on the feasible set, we show that an optimal solution can be determined efficiently. We also rigorously analyze two special cases of the down regulation optimization problem. Our results suggest that one must focus on the position along the mRNA molecule where the transition rate has the strongest effect on the protein production rate. However, this rate is not necessarily the slowest transition rate along the mRNA molecule. We discuss some of the biological implications of these results. PMID:28120903

  15. Optimal Down Regulation of mRNA Translation

    Science.gov (United States)

    Zarai, Yoram; Margaliot, Michael; Tuller, Tamir

    2017-01-01

    Down regulation of mRNA translation is an important problem in various bio-medical domains ranging from developing effective medicines for tumors and for viral diseases to developing attenuated virus strains that can be used for vaccination. Here, we study the problem of down regulation of mRNA translation using a mathematical model called the ribosome flow model (RFM). In the RFM, the mRNA molecule is modeled as a chain of n sites. The flow of ribosomes between consecutive sites is regulated by n + 1 transition rates. Given a set of feasible transition rates, that models the outcome of all possible mutations, we consider the problem of maximally down regulating protein production by altering the rates within this set of feasible rates. Under certain conditions on the feasible set, we show that an optimal solution can be determined efficiently. We also rigorously analyze two special cases of the down regulation optimization problem. Our results suggest that one must focus on the position along the mRNA molecule where the transition rate has the strongest effect on the protein production rate. However, this rate is not necessarily the slowest transition rate along the mRNA molecule. We discuss some of the biological implications of these results.

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    Bárbara J Henriques

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

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

    Science.gov (United States)

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

    2014-01-01

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

  19. Diurnal expression of Dnmt3b mRNA in mouse liver is regulated by feeding and hepatic clockwork.

    Science.gov (United States)

    Maekawa, Fumihiko; Shimba, Shigeki; Takumi, Shota; Sano, Tomoharu; Suzuki, Takehiro; Bao, Jinhua; Ohwada, Mika; Ehara, Tatsuya; Ogawa, Yoshihiro; Nohara, Keiko

    2012-09-01

    DNA methyltransferase 3B (DNMT3B) is critically involved in de novo DNA methylation and genomic stability, while the regulatory mechanism in liver is largely unknown. We previously reported that diurnal variation occurs in the mRNA expression of Dnmt3b in adult mouse liver. The aim of this study was to determine the mechanism underlying the diurnal expression pattern. The highest level and the lowest level of Dnmt3b mRNA expression were confirmed to occur at dawn and in the afternoon, respectively, and the expression pattern of Dnmt3b closely coincided with that of Bmal1. Since the diurnal pattern of Dnmt3b mRNA expression developed at weaning and scheduled feeding to separate the feeding cycle from the light/dark cycle led to a phase-shift in the expression, it could be assumed that feeding plays a critical role as an entrainment signal. In liver-specific Bmal1 knockout (L-Bmal1 KO) mice, L-Bmal1 deficiency resulted in significantly higher levels of Dnmt3b at all measured time points, and the time when the expression was the lowest in wild-type mice was shifted to earlier. Investigation of global DNA methylation revealed a temporal decrease of 5-methyl-cytosine percentage in the genome of wild-type mice in late afternoon. By contrast, no such decrease in 5-methyl-cytosine percentage was detected in L-Bmal1 KO mice, suggesting that altered Dnmt3b expression affects the DNA methylation state. Taken together, the results suggest that the feeding and hepatic clockwork generated by the clock genes, including Bmal1, regulate the diurnal variation in Dnmt3b mRNA expression and the consequent dynamic changes in global DNA methylation.

  20. Target and specificity of a nuclear gene product that participates in mRNA 3'-end formation in Chlamydomonas chloroplasts.

    Science.gov (United States)

    Levy, H; Kindle, K L; Stern, D B

    1999-12-10

    Chloroplast mRNA maturation is catalyzed by nucleus-encoded processing enzymes. We previously described a recessive nuclear mutation (crp3) that affects 3'-end formation of several chloroplast mRNAs in Chlamydomonas reinhardtii (Levy, H., Kindle, K. L., and Stern, D. B. (1997) Plant Cell 9, 825-836). In the crp3 background, atpB mRNA lacking a 3'-inverted repeat normally required for stability accumulates as a discrete transcript. The mutation also affects the atpA gene cluster; polycistronic mRNAs with psbI or cemA 3'-ends accumulate to a lower level in the crp3 background. Here, we demonstrate that the crp3 mutation also alters 3'-end formation of psbI mRNA and cemA-containing mRNAs. A novel 3'-end is formed in monocistronic psbI transcripts, and this is the only terminus observed when the psbI 3'-untranslated region is fused to an aadA reporter gene. Accumulation of mRNAs with 3'-ends between cemA and atpH, which is immediately downstream, was reduced. However, this sequence was not recognized as a 3'-end formation element in chimeric genes. The crp3 mutation was able to confer stability to three different atpB 3'-stem-loop-disrupting mutations that lack sequence similarity, but are located at a similar distance from the translation termination codon. We propose that the wild-type CRP3 gene product is part of the general 3' --> 5' processing machinery.

  1. 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.) histone H3 mRNA; 3' end formation; Also found in histone genes of other plants, yeast, etc; histone H3; mRNA

  2. Ressourcenorientierte Diagnostik im Alter

    OpenAIRE

    Forstmeier, Simon; Maercker, Andreas

    2008-01-01

    Trotz der im Alter zunehmenden körperlichen, kognitiven und sozialen Verlusten bleibt das subjektive Wohlbefinden relativ stabil. Dies weist auf die vielen Ressourcen älterer Menschen hin. Dieser Artikel stellt für die klinische Ressourcendiagnostik relevante Verfahren vor und erläutert die zugrunde liegenden Konzepte. Berücksichtigt werden Aktivitäten und Erlebnisse als Ressourcen, emotionale Ressourcen (positiver Affekt, Lebenszufriedenheit, Selbstwerterleben, Lebensqualität), motivationale...

  3. Altering Emulsion Stability with Heterogeneous Surface Wettability

    NARCIS (Netherlands)

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

    2016-01-01

    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

  4. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function.

    Directory of Open Access Journals (Sweden)

    Cristian Del Campo

    2015-10-01

    Full Text Available Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation.

  5. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function.

    Science.gov (United States)

    Del Campo, Cristian; Bartholomäus, Alexander; Fedyunin, Ivan; Ignatova, Zoya

    2015-10-01

    Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation.

  6. Alteration of μ-opioid Receptor mRNA Expression in PAG and Hippocampus of Rat Following the Immunosuppression Induced by Intrathecal Injection of Morphine%鞘内注射吗啡抑制大鼠免疫功能时PAG和海马μ-阿片受体mRNA表达的变化

    Institute of Scientific and Technical Information of China (English)

    孙涛; 杜莅娜; 姜建伟; 吴根诚; 曹小定

    2001-01-01

    Objective To observe the activity of μ-opioid receptor mRNA inperiaqueductal gray(PAG) and hippocampus of rat brains following the immunosuppression induced by intrathecal injection (ith) of morphine. Methods Tested by in situ hybridization histochemistry technique. Results Ith morphine enhanced the μ-opioid receptor mRNA expression in PAG and hippocampus of rats. Conclusions There is a potential relationship between μ-opioid receptors in PAG and hippocampus and the immunosuppression induced by intrathecal injection of morphine ,and further studies are needed to elucidate the mechanism.%目的 观察鞘内注射吗啡抑制大鼠免疫功能时中脑导水管周围灰质(PAG)和海马μ-阿片受体mRNA表达的变化。方法 原位杂交组织化学法。结果 鞘内注射吗啡可促进大鼠PAG和海马μ-阿片受体mRNA表达增加。结论 中枢PAG和海马μ-阿片受体可能参与鞘内注射吗啡引起的免疫功能的抑制,但具体机制尚有待于进一步研究。

  7. Reference: MRNA3ENDTAH3 [PLACE

    Lifescience Database Archive (English)

    Full Text Available MRNA3ENDTAH3 Ohtsubo N, Iwabuchi M The conserved 3'-flanking sequence, AATGGAAATG, ...of the wheat histone H3 gene is necessary for the accurate 3'-end formation of mRNA. Nucleic Acids Res 22:1052-1058 (1994) PubMed: 8152910; ...

  8. 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.

  9. 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.

  10. METHOD FOR STABILIZING KLYSTRONS

    Science.gov (United States)

    Magnuson, D.W.; Smith, D.F.

    1959-04-14

    High-frequency oscillators for the generation of microwaves, particularly a system for stabilizing frequency-modulated klystron oscillators of the reflex type, are described. The system takos advantage of the fact that a change in oscillator frequency will alter the normal phase displacement between the cavity and its modulator, creating an error voltage which is utilized to regulate the frequency of the oscillator and stabilize it.

  11. Self-amplifying mRNA vaccines.

    Science.gov (United States)

    Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

    2015-01-01

    This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization.

  12. Extraction of mRNA from coagulated horse blood and analysis of inflammation-related cytokine responses to coagulation

    DEFF Research Database (Denmark)

    Bovbjerg, Kirsten Katrine Lindegaard; Heegaard, Peter M. H.; Skovgaard, Kerstin

    2010-01-01

    .5-9.2), comparable with that purified by standard methods from stabilized blood. Cytokine mRNA expression was assessed by reverse transcribed quantitative real time PCR and it was found that 24-hour clotting led to a significant increase in the concentrations of mRNA of the pro-inflammatory cytokines interleukin-1β......Coagulated blood is a rich source of mRNA that allows the study of the regulation of expression of cytokine and other genes. However, while several methods are available for isolation of RNA from whole blood and tissues, protocols for purification of mRNA from clotted blood are not generally...... available. Here, a protocol for RNA extraction from highly clotted blood was optimized and the regulation of a number of cytokine genes compared to stabilized blood was studied. Whole blood samples from 10 clinically healthy horses were incubated for 24 hours at 37°C and RNA was extracted from...

  13. 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.

  14. Tuning protein expression using synonymous codon libraries targeted to the 5' mRNA coding region

    DEFF Research Database (Denmark)

    Goltermann, Lise; Borch Jensen, Martin; Bentin, Thomas

    2011-01-01

    In bacteria, the 5' mRNA coding region plays an important role in determining translation output. Here, we report synthetic sequences that when placed in the 5'-mRNA coding region, leading to recombinant proteins containing short N-terminal extensions, virtually abolish, enhance or produce...... 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...... and hence is important to recombinant and, most certainly, endogenous gene expression....

  15. 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.

  16. 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 (2 days post-TAC), and hypertrophic (7 days post-TAC) mice was subjected to small RNA- and poly(A)-test sequencing (PAT-Seq). Differential expression analysis matched expectations; nevertheless we identified ~400 mRNAs and hundreds of noncoding RNA loci as altered with hypertrophy for the first time. Although multiple processing variants were observed for many miRNAs, there was little change in their relative proportions during hypertrophy. PAT-Seq mapped ~48,000 mRNA 3'-ends, identifying novel 3' untranslated regions (3'UTRs) for over 7000 genes. Importantly, hypertrophy was associated with marked changes in APA with a net shift from distal to more proximal mRNA 3'-ends, which is predicted to decrease overall miRNA repression strength. We independently validated several examples of 3'UTR proportion change and showed that alternative 3'UTRs associate with differences in mRNA translation. Our work suggests that APA contributes to altered gene expression with the development of cardiomyocyte hypertrophy and provides a rich resource for a systems-level understanding of miRNA-mediated regulation in physiological and pathological states of the heart.

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

    Science.gov (United States)

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

    2008-05-01

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

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

    Science.gov (United States)

    Kalynchuk, Lisa E; Meaney, Michael J

    2003-12-01

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

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

    DEFF Research Database (Denmark)

    Krakauer, Martin; Sorensen, P; Khademi, M

    2008-01-01

    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 target of IFN...

  20. Enhanced alpha 1(I) mRNA expression in frozen shoulder and dupuytren tissue.

    Science.gov (United States)

    Kilian, Olaf; Pfeil, U; Wenisch, S; Heiss, C; Kraus, R; Schnettler, R

    2007-12-14

    The purpose of this study has been to investigate collagen I and III synthesis during the fibrosing stage of frozen shoulder and Dupuytren samples in comparison to normal capsule tissue. - By using the quantitative PCR significantly increased levels of alpha 1(I) mRNA transcription in samples of frozen shoulder (p = 0.016) and Duypuytren (p = 0.041) could be demonstrated, whereas alpha 2(I) and alpha 1(III) chains have shown the same mRNA levels as in normal capsule tissue. - Despite an enhancement of alpha 1(I) mRNA transcription in frozen shoulder and Dupuytren samples the intracellular precursor procollagen I and extracellular mature collagen I was detected immunohistochemically in reduced levels. - The structural alteration of collagen I assembly might be caused by disturbed post-translation from the polypeptide chains into the triple helices procollagen I though alpha 1(I) mRNA transcription was significantly increased and alpha 2(I) mRNA transcription was in normal range. Fibroblasts might release high quantities of free alpha 1(I) polypeptide chains or (alpha 1(I)) 3 homotrimer into the extracellular space during the fibrosing stage of frozen shoulder and Dupuytren disease. - In all samples neither differences of alpha 1(III) mRNA transcription nor differences of immunohistochemical staining intensity of collagen III could be seen. This might result from apoptosis of myofibroblasts in the final phase of the fibrosing processes. - The stimulating effect of insulin-like growth factor type I (IGF-I) to induce fibrosis in connective tissue such as scarlet is known. In all patients suffering from frozen shoulder and Dupuytren disease the serum IGF-I level was in a normal range and the IGF-I receptor - (IGFR-I) mRNA transcription in the samples was also in the same level compared with normal capsule tissue.

  1. Changes in the expression of estrogen receptor mRNA in the utero-vaginal junction containing sperm storage tubules in laying hens after repeated artificial insemination.

    Science.gov (United States)

    Das, Shubash Chandra; Nagasaka, Naohiro; Yoshimura, Yukinori

    2006-03-01

    The objective was to determine whether expression of estrogen receptor (ER) mRNA in the utero-vaginal junction (UVJ) of laying hens was altered after repeated artificial insemination (AI). Semi-quantitative RT-PCR was used to determine the expression of mRNA of the two types of receptor, ERalpha and ERbeta. Only ERalpha mRNA was expressed in all segments of the oviducts of both virgin and artificially inseminated birds, whereas ERbeta mRNA was expressed in ovarian follicles but not in the oviduct. The expression of ERalpha mRNA in the UVJ was significantly decreased after repeated AI, whereas that in the uterus was not significantly different between virgin and inseminated birds. Since estrogen may be involved in maintaining the sperm storage function of sperm storage tubules, the decreased expression of ERalpha mRNA in the UVJ after repeated AI may contribute to reduced fertility in these birds.

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

    Directory of Open Access Journals (Sweden)

    Johannes H Urban

    2008-03-01

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

  3. 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.

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

    DEFF Research Database (Denmark)

    Glahder, Jacob-Andreas Harald; Norrild, Bodil

    2011-01-01

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

  5. CDKN2A (p16) mRNA decreased expression is a marker of poor prognosis in malignant high-grade glioma.

    Science.gov (United States)

    Sibin, M K; Bhat, Dhananjaya I; Narasingarao, K V L; Lavanya, Ch; Chetan, G K

    2015-09-01

    Human high-grade glioma is heterogeneous in nature based on pathological and genetic profiling. Various tumour suppressor gene alterations are considered as prognostic markers in high-grade glioma. Gene expression of CDKN2A (p16) is used in various cancers as a prognostic biomarker along with methylation and deletion status of this gene. Expression levels of p16 mRNA were not studied as a biomarker in gliomas before. In this study, we have performed mRNA quantification analysis on 48 high-grade glioma tissues and checked for a possible prognostic role. The decreased expression of p16 mRNA in majority of the tumour tissues (57.1 %) was observed when compared to control tissues (P = 0.02). mRNA expression level was correlated with clinical variables also. p16 deletion status and BMI1 mRNA expression were also considered for comparison. p16 mRNA was negatively correlated with the BMI1 mRNA (P = p16 deletion. p16 mRNA expression, midline shift in MRI and tumour type were able to predict patient survival in overall survival (OS) and progression-free survival (PFS). p16 mRNA could independently predict prognosis of OS (P = 0.0146) and PFS (P = 0.0305) in multivariate analysis. We have shown that p16 mRNA expression can act as an independent prognostic biomarker in high-grade glioma.

  6. AGEs and Glucose Levels Modulate Type I and III Procollagen mRNA Synthesis in Dermal Fibroblasts Cells Culture

    Directory of Open Access Journals (Sweden)

    Serban Iren Andreea

    2008-01-01

    Full Text Available In the dermis, fibroblasts play an important role in the turnover of the dermal extracellular matrix. Collagen I and III, the most important dermal proteins of the extracellular matrix, are progressively altered during ageing and diabetes. For mimicking diabetic conditions, the cultured human dermal fibroblasts were incubated with increasing amounts of AGE-modified BSA and D-glucose for 24 hours. The expression of procollagen α2(I and procollagen α1(III mRNA was analyzed by quantitative real-time PCR. Our data revealed that the treatment of fibroblasts with AGE-modified BSA upregulated the expression of procollagen α2(I and procollagen α1(III mRNA in a dose-dependent manner. High glucose levels mildly induced a profibrogenic pattern, increasing the procollagen α2(I mRNA expression whereas there was a downregulation tendency of procollagen α1(III mRNA.

  7. Comparing efficiency of micro-RNA and mRNA biomarker liberation with microbubble-enhanced ultrasound exposure.

    Science.gov (United States)

    Forbrich, Alex; Paproski, Robert; Hitt, Mary; Zemp, Roger

    2014-09-01

    Blood biomarkers are potentially powerful diagnostic tools that are limited clinically by low concentrations, the inability to determine biomarker origin and unknown patient baseline. Recently, ultrasound has been shown to liberate proteins and large mRNA biomarkers, overcoming many of these limitations. We have since demonstrated that adding lipid-stabilized microbubbles elevates mRNA concentration an order of magnitude compared with ultrasound without microbubbles, in vitro. Unfortunately the large size of some mRNA molecules may limit efficiency of release and hinder efficacy as an ultrasound-liberated biomarker. We hypothesize that smaller molecules will be released more efficiently with ultrasound than larger molecules. Although investigation of large libraries of biomarkers should be performed to fully validate this hypothesis, we focus on a small subset of mRNA and micro-RNAs. Specifically, we focus on miR-21 (22 base pairs [bp]), which is upregulated in certain forms of cancer, compared with previously investigated mammaglobin mRNA (502 bp). We also report release of micro-RNA miR-155 (22 bp) and housekeeping rRNA S18 (1869 bp). More than 10 million additional miR-21 copies per 100,000 cells are released with ultrasound-microbubble exposure. The low- molecular-weight miR-21 proved to be liberated 50 times more efficiently than high-molecular-weight mammaglobin mRNA, releasing orders of magnitude more miR-21 than mammaglobin mRNA under comparable conditions.

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

    Science.gov (United States)

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

    1992-05-25

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

  9. 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.

  10. BDNF and trkB mRNA expression in the rat hippocampus following entorhinal cortex lesions.

    Science.gov (United States)

    Lapchak, P A; Araujo, D M; Hefti, F

    1993-02-01

    Quantitative in situ hybridization was used to determine whether the prevalence or topographical distribution of brain-derived neurotrophic factor (BDNF) or tyrosine receptor kinase (trk) B mRNA is altered in the hippocampal formation following lesions of excitatory afferents from the entorhinal cortex which provides an external source of innervation for the hippocampal formation. BDNF mRNA levels were not altered in the hippocampal formation up to 10 days following entorhinal cortex lesions (ECLs). The levels of mRNA coding for all known forms of trkB receptors also remained unchanged. The prevalence of the synaptic plasticity marker SNAP-25 mRNA was increased in the CA2 and CA3 pyramidal cell layers and the dentate gyrus by 6 days following ECLs and remained elevated at 10 days following ECLs. Our findings indicate that hippocampal neuron sprouting which occurs in response to ECLs is not the result of changes in the expression of the BDNF or trkB mRNA.

  11. Anabolic androgenic steroid nandrolone decanoate reduces hypothalamic proopiomelanocortin mRNA levels.

    Science.gov (United States)

    Lindblom, Jonas; Kindlundh, Anna M S; Nyberg, Fred; Bergström, Lena; Wikberg, Jarl E S

    2003-10-03

    Supratherapeutical doses of anabolic androgenic steroids (AASs) have dramatic effects on metabolism in humans, and also inhibit feeding and reduce the rate of body weight gain in rats. In order to test the hypothesis that the AAS metabolic syndrome is accompanied by alterations in the central melanocortin system, we evaluated body weight, food intake and hypothalamic agouti-related protein (AgRP) and proopiomelanocortin (POMC) mRNA levels following administration of different doses of the anabolic androgenic steroid nandrolone decanoate. In order to distinguish changes induced by the steroid treatment per se from those resulting from the reduced food intake and growth rate, we also compared the effect of nandrolone decanoate on AgRP and POMC mRNA expression with both normally fed, and food restricted control groups. We here report that administration of nandrolone specifically reduces arcuate nucleus POMC mRNA levels while not affecting the expression level of AgRP. The effect on POMC expression was not observed in the food restricted controls, excluding the possibility that the observed effect was a mere response to the reduced food intake and body weight. These results raise the possibility that some of the metabolic and behavioural consequences of AAS abuse may be the result of alterations in the melanocortin system.

  12. The influence of eccentric exercise on mRNA expression of skeletal muscle regulators.

    Science.gov (United States)

    Jensky, Nicole E; Sims, Jennifer K; Rice, Judd C; Dreyer, Hans C; Schroeder, E Todd

    2007-11-01

    To evaluate change in myostatin, follistatin, MyoD and SGT mRNA gene expression using eccentric exercise to study mechanisms of skeletal muscle hypertrophy. Young (28+/-5 years) and older (68+/-6 years) men participated in a bout of maximal single-leg eccentric knee extension on an isokinetic dynamometer at 60 degrees /s: six sets, 12-16 maximal eccentric repetitions. Muscle biopsies of the vastus lateralis were obtained from the dominant leg before exercise and 24 h after exercise. Paired t tests were used to compare change (pre versus post-exercise) for normalized gene expression in all variables. Independent t tests were performed to test group differences (young vs. older). A probability level of Peccentric exercise. Similarly, we did not observe significant change in myostatin (-3.83+/-8.8; P=0.23), follistatin (-2.66+/-5.2; P=0.17), MyoD (-0.13+/-3.1; P=0.90), or SGT (-1.6+/-3.5; P=0.19) mRNA expression in older subjects. Furthermore, the non-significant changes in mRNA expression were not different between young and older subjects, P>0.23 for all variables. Our data suggests that a single bout of maximal eccentric exercise does not alter myostatin, follistatin, MyoD or SGT mRNA gene expression in young or older subjects.

  13. Recent innovations in mRNA vaccines.

    Science.gov (United States)

    Ulmer, Jeffrey B; Geall, Andrew J

    2016-08-01

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

  14. 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.

  15. Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region

    Energy Technology Data Exchange (ETDEWEB)

    Pelletier, J.; Kaplan, G.; Racaniello, V.R.; Sonenberg, N.

    1988-03-01

    Poliovirus polysomal RNA is naturally uncapped, and as such, its translation must bypass any 5' cap-dependent ribosome recognition event. To elucidate the manner by which poliovirus mRNA is translated, the authors determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. They found striking differences in translatability among the altered mRNAs when assayed in mock-infected and poliovirus-infected HeLa cell extracts. The results identify a functional cis-acting element within the 5' noncoding region of the poliovirus mRNA which enables it to translate in a cap-independent fashion. The major determinant of this element maps between nucleotides 320 and 631 of the 5' end of the poliovirus mRNA. They also show that this region (320 to 631), when fused to a heterologous mRNA, can function in cis to render the mRNA cap independent in translation.

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

    Science.gov (United States)

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

    2014-03-28

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

  17. Methods to analyze microRNA-mediated control of mRNA translation.

    Science.gov (United States)

    Clancy, Jennifer L; Nousch, Marco; Humphreys, David T; Westman, Belinda J; Beilharz, Traude H; Preiss, Thomas

    2007-01-01

    MicroRNAs (miRs) are an important class of gene regulators that affect a wide range of biological processes. Despite the early recognition of miRs as translational regulators and intense interest in studying this phenomenon, it has so far not been possible to derive a consensus model for the underlying molecular mechanism(s). The potential of miRs to act in a combinatorial manner and to also promote mRNA decay creates conceptual and technical challenges in their study. Here, we discuss critical parameters in design and analysis of experiments used to study miR function including creation of synthetic miR and mRNA partners for assay of translational inhibition using luciferase reporters; measurement of mRNA stability after miR action; defining poly(A) tail length in miR target mRNA; determining the distribution of miRs and their target mRNAs in polysome profiles; and visualization of P-body components. We describe protocols for each of these procedures.

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

    Science.gov (United States)

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

    2014-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Emma Abernathy

    2014-01-01

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

  20. Global analysis of mRNA decay intermediates in Saccharomyces cerevisiae.

    Science.gov (United States)

    Harigaya, Yuriko; Parker, Roy

    2012-07-17

    The general pathways of eukaryotic mRNA decay occur via deadenylation followed by 3' to 5' degradation or decapping, although some endonuclease sites have been identified in metazoan mRNAs. To determine the role of endonucleases in mRNA degradation in Saccharomyces cerevisiae, we mapped 5' monophosphate ends on mRNAs in wild-type and dcp2 xrn1 yeast cells, wherein mRNA endonuclease cleavage products are stabilized. This led to three important observations. First, only few mRNAs that undergo low-level endonucleolytic cleavage were observed, suggesting that endonucleases are not a major contributor to yeast mRNA decay. Second, independent of known decapping enzymes, we observed low levels of 5' monophosphates on some mRNAs, suggesting that an unknown mechanism can generate 5' exposed ends, although for all substrates tested, Dcp2 was the primary decapping enzyme. Finally, we identified debranched lariat intermediates from intron-containing genes, demonstrating a significant discard pathway for mRNAs during the second step of pre-mRNA splicing, which is a potential step to regulate gene expression.

  1. 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.

  2. [Altered states of consciousness].

    Science.gov (United States)

    Gora, E P

    2005-01-01

    The review of modern ideas concerning the altered states of consciousness is presented in this article. Various methods of entry into the altered states of consciousness are looked over. It is shown that the altered states of consciousness are insufficiently known, but important aspects of human being existence. The role of investigation of the altered states of consciousness for the creation of integrative scientific conception base is discussed.

  3. 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

    that mRNA expression of PTGDS and AKR1C3 is deregulated in rapid-cycling disorder patients in a euthymic or current affective state compared with healthy control subjects, and that expression alters with affective states. METHODS: PTGDS and AKR1C3 mRNA expression in peripheral blood mononuclear cells...... was measured in 37 rapid-cycling bipolar disorder patients and 40 age- and gender-matched healthy control subjects using reverse transcription quantitative real-time polymerase chain reaction. Repeated measurements of PTGDS and AKR1C3 mRNA expression were obtained in various affective states during 6-12 months...

  4. Trans-acting factors governing acetylcholinesterase mRNA metabolism in neurons

    Directory of Open Access Journals (Sweden)

    Lucas M. Bronicki

    2012-03-01

    Full Text Available The most characterized function of acetylcholinesterase (AChE is to terminate cholinergic signaling at neuron-neuron and neuro-muscular synapses. In addition, AChE is causally or casually implicated in neuronal development, stress-response, cognition and neurodegenerative diseases. Given the importance of AChE, many studies have focused on identifying the molecular mechanisms that govern its expression. Despite these efforts, post-transcriptional control of AChE mRNA expression is still relatively unclear. Here, we review the trans-acting factors and cis-acting elements that are known to control AChE pre-mRNA splicing, mature mRNA stability and translation. Moreover, since the Hu/ELAV family of RNA-binding proteins (RBPs have emerged in recent years as ‘master’ post-transcriptional regulators, we discuss the possibility that predominantly neuronal ELAVs (nELAVs play multiple roles in regulating splicing, stability, localization and translation of AChE mRNA.

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

    Science.gov (United States)

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

    2000-01-01

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

  6. Low-level lasers and mRNA levels of reference genes used in Escherichia coli

    Science.gov (United States)

    Teixeira, A. F.; Machado, Y. L. R. C.; Fonseca, A. S.; Mencalha, A. L.

    2016-11-01

    Low-level lasers are widely used for the treatment of diseases and antimicrobial photodynamic therapy. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is widely used to evaluate mRNA levels and output data from a target gene are commonly relative to a reference mRNA that cannot vary according to treatment. In this study, the level of reference genes from Escherichia coli exposed to red or infrared lasers at different fluences was evaluated. E. coli AB1157 cultures were exposed to red (660 nm) and infrared (808 nm) lasers, incubated (20 min, 37 °C), the total RNA was extracted, and cDNA synthesis was performed to evaluate mRNA levels from arcA, gyrA and rpoA genes by RT-qPCR. Melting curves and agarose gel electrophoresis were carried out to evaluate specific amplification. Data were analyzed by geNorm, NormFinder and BestKeeper. The melting curve and agarose gel electrophoresis showed specific amplification. Although mRNA levels from arcA, gyrA or rpoA genes presented no significant variations trough a traditional statistical analysis, Excel-based tools revealed that these reference genes are not suitable for E. coli cultures exposed to lasers. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from arcA, gyrA and rpoA in E. coli cells.

  7. 3'-UTR-dependent regulation of mRNA turnover is critical for differential distribution patterns of cyclic gene mRNAs.

    Science.gov (United States)

    Nitanda, Yasuhide; Matsui, Takaaki; Matta, Tatsuro; Higami, Aya; Kohno, Kenji; Nakahata, Yasukazu; Bessho, Yasumasa

    2014-01-01

    Somite segmentation, a prominent periodic event in the development of vertebrates, is instructed by cyclic expression of several genes, including Hes7 and Lunatic fringe (Lfng). Transcriptional regulation accounts for the cyclic expression. In addition, because the expression patterns vary in a cycle, rapid turnover of mRNAs should be involved in the cyclic expression, although its contribution remains unclear. Here, we demonstrate that 3'-UTR-dependent rapid turnover of Lfng and Hes7 plays a critical role in their dynamic expression patterns. The regions active in the transcription of Lfng and Hes7 are wholly overlapped in the posterior presomitic mesoderm (PSM) of the mouse embryo. However, their distribution patterns are slightly different; Hes7 mRNA shows a broader distribution pattern than Lfng mRNA in the posterior PSM. Lfng mRNA is less stable than Hes7 mRNA, where their 3'-UTRs are responsible for the different stability. Using transgenic mice expressing Venus under the control of the Hes7 promoter, which leads to cyclic transcription in the PSM, we reveal that the Lfng 3'-UTR provides the narrow distribution pattern of Lfng mRNA, whereas the Hes7 3'-UTR contributes the relatively broad distribution pattern of Hes7 mRNA. Thus, we conclude that 3'-UTR-dependent mRNA stability accounts for the differential distribution patterns of Lfng and Hes7 mRNA. Our findings suggest that 3'-UTR-dependent regulation of mRNA turnover plays a crucial role in the diverse patterns of mRNA distribution during development.

  8. Database for mRNA Half-Life of 19 977 Genes Obtained by DNA Microarray Analysis of Pluripotent and Differentiating Mouse Embryonic Stem Cells

    Science.gov (United States)

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

    2009-01-01

    Degradation of mRNA is one of the key processes that control the steady-state level of gene expression. However, the rate of mRNA decay for the majority of genes is not known. We successfully obtained the rate of mRNA decay for 19 977 non-redundant genes by microarray analysis of RNA samples obtained from mouse embryonic stem (ES) cells. Median estimated half-life was 7.1 h and only <100 genes, including Prdm1, Myc, Gadd45 g, Foxa2, Hes5 and Trib1, showed half-life less than 1 h. In general, mRNA species with short half-life were enriched among genes with regulatory functions (transcription factors), whereas mRNA species with long half-life were enriched among genes related to metabolism and structure (extracellular matrix, cytoskeleton). The stability of mRNAs correlated more significantly with the structural features of genes than the function of genes: mRNA stability showed the most significant positive correlation with the number of exon junctions per open reading frame length, and negative correlation with the presence of PUF-binding motifs and AU-rich elements in 3′-untranslated region (UTR) and CpG di-nucleotides in the 5′-UTR. The mRNA decay rates presented in this report are the largest data set for mammals and the first for ES cells. PMID:19001483

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

    Science.gov (United States)

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

    2009-02-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-05-01

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

  11. Stability Functions

    CERN Document Server

    Burns, Daniel; Wang, Zuoqin

    2008-01-01

    In this article we discuss the role of stability functions in geometric invariant theory and apply stability function techniques to problems in toric geometry. In particular we show how one can use these techniques to recover results of Burns-Guillemin-Uribe and Shiffman-Tate-Zelditch on asymptotic properties of sections of holomorphic line bundles over toric varieties.

  12. The effect of hypothermia on the expression of neurotrophin mRNA in the hippocampus following transient cerebral ischemia in the rat.

    Science.gov (United States)

    Boris-Möller, F; Kamme, F; Wieloch, T

    1998-12-10

    The expression of the mRNAs of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3) and the neurotrophin receptor, TrkB, was studied in the rat hippocampus by in situ hybridization following normothermic (37 degreesC) and protective hypothermic (33 degreesC) transient cerebral ischemia of 15 min duration. In the resistant dentate gyrus, normothermic ischemia transiently induced NGF mRNA at around 8 h of recovery, while the NT3 mRNA levels were depressed over at least a 24-h recovery period. The levels of BDNF and TrkB were transiently and markedly elevated with a maximal expression at 24 h of recovery. Intraischemic hypothermia reduced the induction of NGF mRNA, while the increase of BDNF mRNA expression occurred earlier during recovery, and the post-ischemic NT3 mRNA depression was not affected. Also, the expression of TrkB mRNA was enhanced, and occurred concomitantly with the elevation of BDNF mRNA. In contrast, there were no changes in neurotrophin and TrkB mRNA in the CA3 and CA1 regions. The expression of BDNF mRNA at 24 h after normothermic ischemia, was attenuated by intraischemic hypothermia. We conclude that, the expressions of NGF, BDNF, NT3 or TrkB mRNA in ischemia-sensitive hippocampal subregions are not increased by protective hypothermia. In contrast, hypothermia induces neurotrophin mRNA alterations in the ischemia-resistant dentate gyrus that may convey protection to sensitive regions.

  13. Intestinal PTGS2 mRNA levels, PTGS2 gene polymorphisms, and colorectal carcinogenesis.

    Directory of Open Access Journals (Sweden)

    Lotte K Vogel

    Full Text Available BACKGROUND & AIMS: Inflammation is a major risk factor for development of colorectal cancer (CRC. Prostaglandin synthase cyclooxygenase-2 (COX-2 encoded by the PTGS2 gene is the rate limiting enzyme in prostaglandin synthesis and therefore plays a distinct role as regulator of inflammation. METHODS: PTGS2 mRNA levels were determined in intestinal tissues from 85 intestinal adenoma cases, 115 CRC cases, and 17 healthy controls. The functional PTGS2 polymorphisms A-1195G (rs689466, G-765C (rs20417, T8473C (rs5275 were assessed in 200 CRC cases, 991 adenoma cases and 399 controls from the Norwegian KAM cohort. RESULTS: PTGS2 mRNA levels were higher in mild/moderate adenoma tissue compared to morphologically normal tissue from the same individual (P<0.0001 and (P<0.035 and compared to mucosa from healthy individuals (P<0.0039 and (P<0.0027, respectively. In CRC patients, PTGS2 mRNA levels were 8-9 times higher both in morphologically normal tissue and in cancer tissue, compared to healthy individuals (P<0.0001. PTGS2 A-1195G variant allele carriers were at reduced risk of CRC (odds ratio (OR = 0.52, 95% confidence interval (95% CI: 0.28-0.99, P = 0.047. Homozygous carriers of the haplotype encompassing the A-1195G and G-765C wild type alleles and the T8473C variant allele (PTGS2 AGC were at increased risk of CRC as compared to homozygous carriers of the PTGS2 AGT (A-1195G, G-765C, T8473C haplotype (OR = 5.37, 95% CI: 1.40-20.5, P = 0.014. No association between the investigated polymorphisms and PTGS2 mRNA levels could be detected. CONCLUSION: High intestinal PTGS2 mRNA level is an early event in colorectal cancer development as it occurs already in mild/moderate dysplasia. PTGS2 polymorphisms that have been associated with altered PTGS2 mRNA levels/COX-2 activity in some studies, although not the present study, were associated with colorectal cancer risk. Thus, both PTGS2 polymorphisms and PTGS2 mRNA levels may provide information

  14. Geochemical modelling of the weathering zone of the 'Mina Fe' U deposit (Spain): A natural analogue for nuclear spent fuel alteration and stability processes in radwaste disposal

    Energy Technology Data Exchange (ETDEWEB)

    Arcos, D. [AMPHOS XXI Consulting S.L., Passeig de Rubi, 29-31, 08197 Valldoreix, Barcelona (Spain)], E-mail: david.arcos@amphos21.com; Perez del Villar, L. [CIEMAT, Dpto.de Medio Ambiente, Avda, Complutense 22, 28040 Madrid (Spain); Bruno, J.; Domenech, C. [AMPHOS XXI Consulting S.L., Passeig de Rubi, 29-31, 08197 Valldoreix, Barcelona (Spain)

    2008-04-15

    The 'Mina Fe' U deposit (Salamanca, Spain) has been studied in the context of Enresa's programme for U-mine sites restoration and also as a natural analogue for processes in high-level nuclear waste (HLNW) geological disposal. The investigations encompassed an array of geoscience disciplines, such as structural geology, mineralogy, hydrogeology and elemental and isotopic geochemistry and hydrogeochemistry of the site. Based on the obtained results, a conceptual mineralogical and geochemical model was performed integrating the main geochemical processes occurring at the site: the interaction between oxidised and slightly acidic water with pyrite, pitchblende, calcite and dolomite, as essential minerals of the U fracture-filling mineralisation, and hydroxyapatite from the host rock, as the main source of P. This conceptual model has been tested in a systematic numerical model, which includes the main kinetic (pyrite and pitchblende dissolution) and equilibrium processes (carbonate mineral dissolution, and goethite, schoepite and autunite secondary precipitation). The results obtained from the reactive-transport model satisfactorily agree with the conceptual model previously established. The assumption of the precipitation of coffinite as a secondary mineral in the system cannot be correctly evaluated due to the lack of hydrochemical data from the reducing zone of the site and valid thermodynamic and kinetic data for this hydrated U(IV)-silicate. This precipitation can also be hampered by the probable existence of dissolved U(IV)-organic matter and/or uranyl carbonate complexes, which are thermodynamically stable under the alkaline and reducing conditions that prevail in the reducing zone of the system. Finally, the intense downwards oxic and acidic alteration in the upper part of the system is of no relevance for the performance assessment of a HLNW disposal. However, the acidic and oxidised conditions are quickly buffered to neutral-alkaline and

  15. ACTH Action on Messenger RNA Stability Mechanisms

    Science.gov (United States)

    Desroches-Castan, Agnès; Feige, Jean-Jacques; Cherradi, Nadia

    2017-01-01

    The regulation of mRNA stability has emerged as a critical control step in dynamic gene expression. This process occurs in response to modifications of the cellular environment, including hormonal variations, and regulates the expression of subsets of proteins whose levels need to be rapidly adjusted. Modulation of messenger RNA stability is usually mediated by stabilizing or destabilizing RNA-binding proteins (RNA-BP) that bind to the 3′-untranslated region regulatory motifs, such as AU-rich elements (AREs). Destabilizing ARE-binding proteins enhance the decay of their target transcripts by recruiting the mRNA decay machineries. Failure of such mechanisms, in particular misexpression of RNA-BP, has been linked to several human diseases. In the adrenal cortex, the expression and activity of mRNA stability regulatory proteins are still understudied. However, ACTH- or cAMP-elicited changes in the expression/phosphorylation status of the major mRNA-destabilizing protein TIS11b/BRF1 or in the subcellular localization of the stabilizing protein Human antigen R have been reported. They suggest that this level of regulation of gene expression is also important in endocrinology.

  16. Integrated microRNA and mRNA Signature Associated with the Transition from the Locally Confined to the Metastasized Clear Cell Renal Cell Carcinoma Exemplified by miR-146-5p.

    Directory of Open Access Journals (Sweden)

    Zofia Wotschofsky

    Full Text Available MicroRNAs (miRNAs regulate gene expression by interfering translation or stability of target transcripts. This interplay between miRNA and their mRNA has been proposed as an important process in cancer development and progression. We have investigated molecular networks impacted by predicted mRNA targets of differentially expressed miRNAs in patients with clear cell renal cell carcinoma (ccRCC diagnosed with or without metastasis.miRNA and mRNA microarray expression profiles derived from primary ccRCC from patients with (16 samples or without diagnosed metastasis (22 samples were used to identify anti-correlated miRNA-mRNA interaction in ccRCC. For this purpose, Ingenuity pathway analysis microRNA Target Filter, which enables prioritization of experimentally validated and predicted mRNA targets was used. By applying an expression pairing tool, the analysis was focused on targets exhibiting altered expression in our analysis, finding miRNAs and their target genes with opposite or same expression. The resulting identified interactions were revalidated by RT-qPCR in another cohort of ccRCC patients. A selection of the predicted miRNA-mRNA interactions was tested by functional analyses using miRNA knockdown and overexpression experiments in renal cancer cell lines.Among the significantly differentially expressed miRNAs, we have identified three miRNAs (miR-146a-5p, miR-128a-3p, and miR-17-5p that were upregulated in primary tumors from patients without metastasis and downregulated in primary tumors from patients with metastasis. We have further identified mRNA targets, which expression were inversely correlated to these 3 miRNAs, and have been previously experimentally demonstrated in cancer setting in humans. Specifically, we showed that CXCL8/IL8, UHRF1, MCM10, and CDKN3 were downregulated and targeted by miR-146a-5p. The interaction between miR-146a-5p and their targets CXCL8 and UHRF1 was validated in cell culture experiments.We identified novel

  17. A poly(A) binding protein-specific sequence motif: MRTENGKSKGFGFVC binding to mRNA poly(A) and polynucleotides and its role on mRNA translation.

    Science.gov (United States)

    Rubin, H N; Halim, M N; Leavis, P C

    1994-06-01

    A consensus sequence (GKSKGFGFV) was recognized in all the sequenced poly(A) binding proteins. We synthesized a 15-amino acid peptide (corresponding to 354-368 in the yeast poly(A) binding protein) which includes the consensus sequence to test its binding affinity to different nucleotides, polynucleotides and mRNA with or without a poly(A) tail. Biochemical and biophysical studies revealed that the 15-amino acid peptide has a strong binding affinity to poly(A) alone or poly(A) attached at the 3' end of mRNA. Circular dichroism spectroscopy demonstrated that the secondary structure of the 15-mer is consistent with that expected based on the structure of the native RNP domain. Furthermore, among the various mononucleotides performed in the present studies, ATP was preferentially found to bind to the 15-mer. To further examine the biological significance of the binding of the 15-mer to the poly(A) tail of mRNA, in vitro translation of the mRNA poly(A)+ in the presence of the 15-mer drastically increased globin synthesis by almost 2-fold, while translation of the deadenylated mRNA in the presence of the 15-mer almost did not alter the rate of incorporation of radiolabeled leucine into globin.

  18. 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.

  19. Ensuring Stability

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

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

  20. mRNA pseudoknot structures can act as ribosomal roadblocks

    DEFF Research Database (Denmark)

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

    2012-01-01

    Several viruses utilize programmed ribosomal frameshifting mediated by mRNA pseudoknots in combination with a slippery sequence to produce a well defined stochiometric ratio of the upstream encoded to the downstream-encoded protein. A correlation between the mechanical strength of mRNA pseudoknot...

  1. Probing dimensionality beyond the linear sequence of mRNA.

    Science.gov (United States)

    Del Campo, Cristian; Ignatova, Zoya

    2016-05-01

    mRNA is a nexus entity between DNA and translating ribosomes. Recent developments in deep sequencing technologies coupled with structural probing have revealed new insights beyond the classic role of mRNA and place it more centrally as a direct effector of a variety of processes, including translation, cellular localization, and mRNA degradation. Here, we highlight emerging approaches to probe mRNA secondary structure on a global transcriptome-wide level and compare their potential and resolution. Combined approaches deliver a richer and more complex picture. While our understanding on the effect of secondary structure for various cellular processes is quite advanced, the next challenge is to unravel more complex mRNA architectures and tertiary interactions.

  2. TP53 Promoter Methylation in Primary Glioblastoma: Relationship with TP53 mRNA and Protein Expression and Mutation Status

    OpenAIRE

    JESIONEK-KUPNICKA, DOROTA; Szybka, Malgorzata; Malachowska, Beata; Fendler, Wojciech; Potemski, Piotr; Piaskowski, Sylwester; Jaskolski, Dariusz; Papierz, Wielislaw; Skowronski, Wieslaw; Och, Waldemar; Kordek, Radzislaw; ZAWLIK, IZABELA

    2014-01-01

    Reduced expression of TP53 by promoter methylation has been reported in several neoplasms. It remains unclear whether TP53 promoter methylation is associated with reduced transcriptional and protein expression in glioblastoma (GB). The aim of our work was to study the impact of TP53 methylation and mutations on TP53 mRNA level and protein expression in 42 molecularly characterized primary GB tumors. We also evaluate the impact of all molecular alterations on the overall patient survival. The ...

  3. Effect of atrophy and contractions on myogenin mRNA concentration in chick and rat myoblast omega muscle cells

    Science.gov (United States)

    Krebs, J. M.; Denney, R. M.

    1997-01-01

    The skeletal rat myoblast omega (RMo) cell line forms myotubes that exhibit spontaneous contractions under appropriate conditions in culture. We examined if the RMo cells would provide a model for studying atrophy and muscle contraction. To better understand how to obtain contractile cultures, we examined levels of contraction under different growing conditions. The proliferation medium and density of plating affected the subsequent proportion of spontaneously contracting myotubes. Using a ribonuclease protection assay, we found that exponentially growing RMo myoblasts contained no detectable myogenin or herculin mRNA, while differentiating myoblasts contained high levels of myogenin mRNA but no herculin mRNA. There was no increase in myogenin mRNA concentration in either primary chick or RMo myotubes whose contractions were inhibited by depolarizing concentrations of potassium (K+). Thus, altered myogenin mRNA concentrations are not involved in atrophy of chick myotubes. Depolarizing concentrations of potassium inhibited spontaneous contractions in both RMo cultures and primary chick myotube cultures. However, we found that the myosin concentration of 6-d-old contracting RMo cells fed medium plus AraC was 11 +/- 3 micrograms myosin/microgram DNA, not significantly different from 12 +/- 4 micrograms myosin/microgram DNA (n = 3), the myosin concentration of noncontracting RMo cells (treated with 12 mM K+ for 6 d). Resolving how RMo cells maintained their myosin content when contraction is inhibited may be important for understanding atrophy.

  4. Dexamethasone increases growth hormone (GH)-releasing hormone (GRH) receptor mRNA levels in cultured rat anterior pituitary cells.

    Science.gov (United States)

    Tamaki, M; Sato, M; Matsubara, S; Wada, Y; Takahara, J

    1996-06-01

    To examine the effects of glucocorticoid (GC) on growth hormone (GH)-releasing hormone (GRH) receptor gene expression, a highly-sensitive and quantitative reverse-transcribed polymerase chain reaction (RT-PCR) method was used in this study. Rat anterior pituitary cells were isolated and cultured for 4 days. The cultured cells were treated with dexamethasone for 2, 6, and 24 h. GRH receptor mRNA levels were determined by competitive RT-PCR using a recombinant RNA as the competitor. Dexamethasone significantly increased GRH receptor mRNA levels at 5 nM after 6- and 24 h-incubations, and the maximal effect was found at 25 nM. The GC receptor-specific antagonist, RU 38486 completely eliminated the dexamethasone-induced enhancement of GRH receptor mRNA levels. Dexamethasone did not alter the mRNA levels of beta-actin and prolactin at 5 nM for 24 h, whereas GH mRNA levels were significantly increased by the same treatment. The GH response to GRH was significantly enhanced by the 24-h incubation with 5 nM dexamethasone. These findings suggest that GC stimulates GRH receptor gene expression through the ligand-activated GC receptors in the rat somatotrophs. The direct effects of GC on the GRH receptor gene could explain the enhancement of GRH-induced GH secretion.

  5. Total RNA-seq to identify pharmacological effects on specific stages of mRNA synthesis.

    Science.gov (United States)

    Boswell, Sarah A; Snavely, Andrew; Landry, Heather M; Churchman, L Stirling; Gray, Jesse M; Springer, Michael

    2017-03-06

    Pharmacological perturbation is a powerful tool for understanding mRNA synthesis, but identification of the specific steps of this multi-step process that are targeted by small molecules remains challenging. Here we applied strand-specific total RNA sequencing (RNA-seq) to identify and distinguish specific pharmacological effects on transcription and pre-mRNA processing in human cells. We found unexpectedly that the natural product isoginkgetin, previously described as a splicing inhibitor, inhibits transcription elongation. Compared to well-characterized elongation inhibitors that target CDK9, isoginkgetin caused RNA polymerase accumulation within a broader promoter-proximal band, indicating that elongation inhibition by isoginkgetin occurs after release from promoter-proximal pause. RNA-seq distinguished isoginkgetin and CDK9 inhibitors from topoisomerase I inhibition, which alters elongation across gene bodies. We were able to detect these and other specific defects in mRNA synthesis at low sequencing depth using simple metagene-based metrics. These metrics now enable total-RNA-seq-based screening for high-throughput identification of pharmacological effects on individual stages of mRNA synthesis.

  6. 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.

  7. Bidirectional regulation of mRNA translation in mammalian cells by using PUF domains.

    Science.gov (United States)

    Cao, Jicong; Arha, Manish; Sudrik, Chaitanya; Schaffer, David V; Kane, Ravi S

    2014-05-05

    The regulation of gene expression is crucial in diverse areas of biological science, engineering, and medicine. A genetically encoded system based on the RNA binding domain of the Pumilio and FBF (PUF) proteins was developed for the bidirectional regulation (i.e., either upregulation or downregulation) of the translation of a target mRNA. PUF domains serve as designable scaffolds for the recognition of specific RNA elements and the specificity can be easily altered to target any 8-nucleotide RNA sequence. The expression of a reporter could be varied by over 17-fold when using PUF-based activators and repressors. The specificity of the method was established by using wild-type and mutant PUF domains. Furthermore, this method could be used to activate the translation of target mRNA downstream of PUF binding sites in a light-dependent manner. Such specific bidirectional control of mRNA translation could be particularly useful in the fields of synthetic biology, developmental biology, and metabolic engineering.

  8. Targeted mRNA oxidation regulates sunflower seed dormancy alleviation during dry after-ripening.

    Science.gov (United States)

    Bazin, Jérémie; Langlade, Nicolas; Vincourt, Patrick; Arribat, Sandrine; Balzergue, Sandrine; El-Maarouf-Bouteau, Hayat; Bailly, Christophe

    2011-06-01

    After-ripening is the mechanism by which dormant seeds become nondormant during their dry storage after harvest. The absence of free water in mature seeds does not allow detectable metabolism; thus, the processes associated with dormancy release under these conditions are largely unknown. We show here that sunflower (Helianthus annuus) seed alleviation of dormancy during after-ripening is associated with mRNA oxidation and that this oxidation is prevented when seeds are maintained dormant. In vitro approaches demonstrate that mRNA oxidation results in artifacts in cDNA-amplified fragment length polymorphim analysis and alters protein translation. The oxidation of transcripts is not random but selective, and, using microarrays, we identified 24 stored mRNAs that became highly oxidized during after-ripening. Oxidized transcripts mainly correspond to genes involved in responses to stress and in cell signaling. Among them, protein phosphatase 2C PPH1, mitogen-activated protein kinase phosphatase 1, and phenyl ammonia lyase 1 were identified. We propose that targeted mRNA oxidation during dry after-ripening of dormant seeds could be a process that governs cell signaling toward germination in the early steps of seed imbibition.

  9. Stabilizing Niger

    DEFF Research Database (Denmark)

    Hahonou, Eric Komlavi

    international intervention in Niger. Their main objective is to secure their own strategic, economic and political interests by strengthening the Nigerien authorities through direct intervention and capacity building activities. For western states reinforcing state security institutions and stabilizing elite...

  10. 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.

  11. Increased mRNA Levels of TCF7L2 and MYC of the Wnt Pathway in Tg-ArcSwe Mice and Alzheimer's Disease Brain

    Directory of Open Access Journals (Sweden)

    Elin S. Blom

    2011-01-01

    Full Text Available Several components in the Wnt pathway, including β-catenin and glycogen synthase kinase 3 beta, have been implied in AD pathogenesis. Here, mRNA brain levels from five-month-old tg-ArcSwe and nontransgenic mice were compared using Affymetrix microarray analysis. With surprisingly small overall changes, Wnt signaling was the most affected pathway with altered expression of nine genes in tg-ArcSwe mice. When analyzing mRNA levels of these genes in human brain, transcription factor 7-like 2 (TCF7L2 and v-myc myelocytomatosis viral oncogene homolog (MYC, were increased in Alzheimer's disease (AD (P<.05. Furthermore, no clear differences in TCF7L2 and MYC mRNA were found in brains with frontotemporal lobar degeneration, suggesting that altered regulation of these Wnt-related genes could be specific to AD. Finally, mRNA levels of three neurogenesis markers were analyzed. Increased mRNA levels of dihydropyrimidinase-like 3 were observed in AD brain, suggesting that altered Wnt pathway regulation may signify synaptic rearrangement or neurogenesis.

  12. Epigenetic Alterations in Muscular Disorders

    Directory of Open Access Journals (Sweden)

    Chiara Lanzuolo

    2012-01-01

    Full Text Available Epigenetic mechanisms, acting via chromatin organization, fix in time and space different transcriptional programs and contribute to the quality, stability, and heritability of cell-specific transcription programs. In the last years, great advances have been made in our understanding of mechanisms by which this occurs in normal subjects. However, only a small part of the complete picture has been revealed. Abnormal gene expression patterns are often implicated in the development of different diseases, and thus epigenetic studies from patients promise to fill an important lack of knowledge, deciphering aberrant molecular mechanisms at the basis of pathogenesis and diseases progression. The identification of epigenetic modifications that could be used as targets for therapeutic interventions could be particularly timely in the light of pharmacologically reversion of pathological perturbations, avoiding changes in DNA sequences. Here I discuss the available information on epigenetic mechanisms that, altered in neuromuscular disorders, could contribute to the progression of the disease.

  13. Dual-acting riboswitch control of translation initiation and mRNA decay.

    Science.gov (United States)

    Caron, Marie-Pier; Bastet, Laurène; Lussier, Antony; Simoneau-Roy, Maxime; Massé, Eric; Lafontaine, Daniel A

    2012-12-11

    Riboswitches are mRNA regulatory elements that control gene expression by altering their structure in response to specific metabolite binding. In bacteria, riboswitches consist of an aptamer that performs ligand recognition and an expression platform that regulates either transcription termination or translation initiation. Here, we describe a dual-acting riboswitch from Escherichia coli that, in addition to modulating translation initiation, also is directly involved in the control of initial mRNA decay. Upon lysine binding, the lysC riboswitch adopts a conformation that not only inhibits translation initiation but also exposes RNase E cleavage sites located in the riboswitch expression platform. However, in the absence of lysine, the riboswitch folds into an alternative conformation that simultaneously allows translation initiation and sequesters RNase E cleavage sites. Both regulatory activities can be individually inhibited, indicating that translation initiation and mRNA decay can be modulated independently using the same conformational switch. Because RNase E cleavage sites are located in the riboswitch sequence, this riboswitch provides a unique means for the riboswitch to modulate RNase E cleavage activity directly as a function of lysine. This dual inhibition is in contrast to other riboswitches, such as the thiamin pyrophosphate-sensing thiM riboswitch, which triggers mRNA decay only as a consequence of translation inhibition. The riboswitch control of RNase E cleavage activity is an example of a mechanism by which metabolite sensing is used to regulate gene expression of single genes or even large polycistronic mRNAs as a function of environmental changes.

  14. mRNA changes in nucleus accumbens related to methamphetamine addiction in mice

    Science.gov (United States)

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

    2016-11-01

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

  15. Modulation of IGFBP2 mRNA expression in white adipose tissue upon aging and obesity.

    Science.gov (United States)

    Li, Z; Picard, F

    2010-10-01

    The insulin/IGF-1 signaling pathway is a determinant of aging and age-related diseases. IGF-binding protein 2 (IGFBP2) is secreted by white adipocytes and contributes to the prevention of diet-induced obesity and age-related insulin resistance in mice. However, the expression levels of IGFBP2 in insulin resistance disorders have not been evaluated. The present study was aimed at determining IGFBP2 mRNA levels in adipose tissue in conditions of insulin resistance such as aging and obesity. In visceral white adipose tissue (WAT), but not in subcutaneous WAT, IGFBP2 mRNA levels were significantly lower in obese OB/OB, DB/DB and high fat-fed mice compared with those of their respective lean and chow-fed littermates. IGFBP2 mRNA levels were also decreased in visceral WAT of 12 and 24 months old mice compared with those of their 4 months old counterparts. Visceral WAT IGFBP2 expression was significantly associated with IGFBP2 circulating levels in mice, suggesting an important contribution from this tissue. The negative effect of aging on IGFBP2 mRNA levels in visceral WAT was confirmed in obese men. These findings demonstrate that the transcription of the IGFBP2 gene is modulated in a depot-specific fashion in obesity and aging in mice and men. Because IGFBP2 is an adipokine, an altered production from visceral WAT depots could impact on IGF-1 signaling and its downstream targets. This supports the need for further molecular and clinical studies to determine the factors regulating IGFBP2 expression and its relevance to metabolic diseases.

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

    Science.gov (United States)

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

    2014-03-01

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

  17. 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.

  18. Identification of a quality-control mechanism for mRNA 5'-end capping.

    Science.gov (United States)

    Jiao, Xinfu; Xiang, Song; Oh, Chanseok; Martin, Charles E; Tong, Liang; Kiledjian, Megerditch

    2010-09-30

    The 7-methylguanosine cap structure at the 5' end of eukaryotic messenger RNAs is a critical determinant of their stability and translational efficiency. It is generally believed that 5'-end capping is a constitutive process that occurs during mRNA maturation and lacks the need for a quality-control mechanism to ensure its fidelity. We recently reported that the yeast Rai1 protein has pyrophosphohydrolase activity towards mRNAs lacking a 5'-end cap. Here we show that, in vitro as well as in yeast cells, Rai1 possesses a novel decapping endonuclease activity that can also remove the entire cap structure dinucleotide from an mRNA. This activity is targeted preferentially towards mRNAs with unmethylated caps in contrast to the canonical decapping enzyme, Dcp2, which targets mRNAs with a methylated cap. Capped but unmethylated mRNAs generated in yeast cells with a defect in the methyltransferase gene are more stable in a rai1-gene-disrupted background. Moreover, rai1Δ yeast cells with wild-type capping enzymes show significant accumulation of mRNAs with 5'-end capping defects under nutritional stress conditions of glucose starvation or amino acid starvation. These findings provide evidence that 5'-end capping is not a constitutive process that necessarily always proceeds to completion and demonstrates that Rai1 has an essential role in clearing mRNAs with aberrant 5'-end caps. We propose that Rai1 is involved in an as yet uncharacterized quality control process that ensures mRNA 5'-end integrity by an aberrant-cap-mediated mRNA decay mechanism.

  19. 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.

  20. mRNA trafficking and local translation: the Yin and Yang of regulating mRNA localization in neurons

    Institute of Scientific and Technical Information of China (English)

    John R. Sinnamon; Kevin Czaplinski

    2011-01-01

    Localized translation and the requisite trafficking of the mRNA template play significant roles in the nervous system including the establishment of dendrites and axons,axon path-finding,and synaptic plasticity.We provide a brief review on the regulation of localizing mRNA in mammalian neurons through critical posttranslational modifications of the factors involved.These examples highlight the relationship between mRNA trafficking and the translational regulation of trafficked mRNAs and provide insight into how extracellular signals target these events during signal transduction.

  1. 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.

    2008-01-01

    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 target of IFN...

  2. 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...

  3. 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.

  4. Post-transcriptional regulation of the GAP-43 gene by specific sequences in the 3' untranslated region of the mRNA.

    Science.gov (United States)

    Tsai, K C; Cansino, V V; Kohn, D T; Neve, R L; Perrone-Bizzozero, N I

    1997-03-15

    We have shown previously that GAP-43 gene expression during neuronal differentiation is controlled by selective changes in mRNA stability. This process was found to depend on highly conserved sequences in the 3' untranslated region (3' UTR) of the mRNA. To map the sequences in the GAP-43 3' UTR that mediate this post-transcriptional event, we generated specific 3' UTR deletion mutants and chimeras with the beta-globin gene and measured their half-lives in transfected PC12 cells. Our results indicate that there are two distinct instability-conferring elements localized at the 5' and 3' ends of the GAP-43 3' UTR. Of these destabilizing elements, only the one at the 3' end is required for the stabilization of the mRNA in response to treatment with the phorbol ester TPA. This 3' UTR element consists of highly conserved uridine-rich sequences and contains specific recognition sites for two neural-specific GAP-43 mRNA-binding proteins. Analysis of the levels of mRNA and protein derived from various 3' UTR deletion mutants indicated that all mutants were translated effectively and that differences in gene expression in response to TPA were attributable to changes in GAP-43 mRNA stability. In addition, the phorbol ester was found to affect the binding of specific RNA-binding proteins to the 3' UTR of the GAP-43 mRNA. Given that, like the GAP-43 mRNA, its degradation machinery and the GAP-43 mRNA-binding proteins are expressed primarily in neural cells, we propose that these factors may be involved in the post-transcriptional regulation of GAP-43 gene expression during neuronal differentiation.

  5. Mechanism of regulation of bcl-2 mRNA by nucleolin and A+U-rich element-binding factor 1 (AUF1).

    Science.gov (United States)

    Ishimaru, Daniella; Zuraw, Lisa; Ramalingam, Sivakumar; Sengupta, Tapas K; Bandyopadhyay, Sumita; Reuben, Adrian; Fernandes, Daniel J; Spicer, Eleanor K

    2010-08-27

    The antiapoptotic Bcl-2 protein is overexpressed in a variety of cancers, particularly leukemias. In some cell types this is the result of enhanced stability of bcl-2 mRNA, which is controlled by elements in its 3'-untranslated region. Nucleolin is one of the proteins that binds to bcl-2 mRNA, thereby increasing its half-life. Here, we examined the site on the bcl-2 3'-untranslated region that is bound by nucleolin as well as the protein binding domains important for bcl-2 mRNA recognition. RNase footprinting and RNA fragment binding assays demonstrated that nucleolin binds to a 40-nucleotide region at the 5' end of the 136-nucleotide bcl-2 AU-rich element (ARE(bcl-2)). The first two RNA binding domains of nucleolin were sufficient for high affinity binding to ARE(bcl-2). In RNA decay assays, ARE(bcl-2) transcripts were protected from exosomal decay by the addition of nucleolin. AUF1 has been shown to recruit the exosome to mRNAs. When MV-4-11 cell extracts were immunodepleted of AUF1, the rate of decay of ARE(bcl-2) transcripts was reduced, indicating that nucleolin and AUF1 have opposing roles in bcl-2 mRNA turnover. When the function of nucleolin in MV-4-11 cells was impaired by treatment with the nucleolin-targeting aptamer AS1411, association of AUF1 with bcl-2 mRNA was increased. This suggests that the degradation of bcl-2 mRNA induced by AS1411 results from both interference with nucleolin protection of bcl-2 mRNA and recruitment of the exosome by AUF1. Based on our findings, we propose a model that illustrates the opposing roles of nucleolin and AUF1 in regulating bcl-2 mRNA stability.

  6. Estrogen regulates the development of brain-derived neurotrophic factor mRNA and protein in the rat hippocampus.

    Science.gov (United States)

    Solum, Derek T; Handa, Robert J

    2002-04-01

    During development, estrogen has a variety of effects on morphological and electrophysiological properties of hippocampal neurons. Brain-derived neurotrophic factor (BDNF) also plays an important role in the survival and differentiation of neurons during development. We examined the effects of gonadectomy with and without estrogen replacement on the mRNA and protein of BDNF and its receptor, trkB, during early postnatal development of the rat hippocampus. We used immunocytochemistry to demonstrate that estrogen receptor alpha (ERalpha) and BDNF were localized to the same cells within the developing hippocampus. BDNF and ERalpha were colocalized in pyramidal cells of the CA3 subregion and to a lesser extent in CA1. To determine whether BDNF mRNA was regulated by estrogen during development, we gonadectomized male rat pups at postnatal day 0 (P0) and examined mRNA and protein levels from P0 to P25 using real-time reverse transcription-PCR and Western blot analysis. After gonadectomy, BDNF mRNA levels are significantly reduced on P7, but after treatment of gonadectomized animals with estradiol benzoate on P0, levels at all ages were similar to those in intact animals. BDNF mRNA changes after gonadectomy are accompanied by an increase in the levels of BDNF protein, which were reduced by estrogen treatment at P0. We also examined the effect of postnatal estrogen treatment on trkB. There were no significant changes in trkB mRNA or protein in gonadectomized or estrogen-replaced animals. These results suggest that a direct interaction may exist between ERalpha and BDNF to alter hippocampal physiology during development in the rat.

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

    Science.gov (United States)

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

    2005-01-10

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

  8. Duodenal calcium transporter mRNA expression in stressed male rats treated with diazepam, fluoxetine, reboxetine, or venlafaxine.

    Science.gov (United States)

    Charoenphandhu, Narattaphol; Teerapornpuntakit, Jarinthorn; Lapmanee, Sarawut; Krishnamra, Nateetip; Charoenphandhu, Jantarima

    2012-10-01

    Chronic stress has been reported to decrease bone density and intestinal calcium absorption, but its underlying mechanism remains elusive. Since long-term exposure to glucocorticoids, major stress hormones from adrenal gland, is known to downregulate the mRNA expression of intestinal calcium transporter TRPV6, the present study aimed to demonstrate whether decreases in mRNA expressions of duodenal calcium transporter genes were observed in male rats subjected to restraint stress for 4 weeks. The results from quantitative real-time PCR showed that restraint stress significantly downregulated the mRNA expressions of apical calcium channels (TRPV6 and Ca(v)1.3), cytoplasmic calcium-binding protein (calbindin-D(9k)), and basolateral calcium pump (PMCA(1b)), but not the expression of TRPV5 or NCX1. The mRNA expressions of paracellular genes, ZO-1, occludin, and claudin-3, were not altered by restraint stress. Since several antidepressant or anxiolytic drugs effectively alleviate stress-induced depressive and anxiety symptoms, we further hypothesized that these drugs may also enhance calcium transporter gene expression in stressed rats. As expected, 4-week daily administration of 10 mg/kg fluoxetine, 10 mg/kg reboxetine, or 10 mg/kg venlafaxine differentially increased calcium transporter mRNA expression in stressed rats, whereas 2 mg/kg diazepam had no such effect. It could, therefore, be concluded that 4-week restraint stress downregulated some important calcium transporter mRNA expression in the duodenal epithelial cells of male rats, which could be prevented by oral administration of fluoxetine, reboxetine, and venlafaxine. The present findings may be applied to help alleviate the stress-induced bone loss and osteoporosis by restoring intestinal calcium absorption to provide calcium for bone formation.

  9. 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.

  10. 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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  12. 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?

  13. Music and Alterity Processes

    Directory of Open Access Journals (Sweden)

    Josep Martí

    2014-10-01

    Full Text Available The concept of alterity constitutes an important issue in anthropological research and, therefore, in the study of musical practices, as well. Without it, we could hardly understand other kinds of music situated in different spaces and time from the observer. In order to effectively approach these musical practices, we have to develop strategies to help us reduce as much as possible that which distorts the vision of the other. However, beyond the strictly epistemological and methodological issues, the study of music cannot ignore the ethical question related to the manner in which Western thought has understood and treated the other: through a hierarchical and stereotypical type of thinking based on the condition of otherness. Throughout the article, different alterity procedures are presented and discussed, such as synecdochization, exoticization, undervaluation, overvaluation, misunderstanding and exclusion. Taking these different alterity strategies into account may help us to better understand how the musical other is constructed, used and ultimately instrumentalized.

  14. 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.

  15. Determination of the human cardiomyocyte mRNA and miRNA differentiation network by fine-scale profiling.

    Science.gov (United States)

    Babiarz, Joshua E; Ravon, Morgane; Sridhar, Sriram; Ravindran, Palanikumar; Swanson, Brad; Bitter, Hans; Weiser, Thomas; Chiao, Eric; Certa, Ulrich; Kolaja, Kyle L

    2012-07-20

    To gain insight into the molecular regulation of human heart development, a detailed comparison of the mRNA and miRNA transcriptomes across differentiating human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and biopsies from fetal, adult, and hypertensive human hearts was performed. Gene ontology analysis of the mRNA expression levels of the hiPSCs differentiating into cardiomyocytes revealed 3 distinct groups of genes: pluripotent specific, transitional cardiac specification, and mature cardiomyocyte specific. Hierarchical clustering of the mRNA data revealed that the transcriptome of hiPSC cardiomyocytes largely stabilizes 20 days after initiation of differentiation. Nevertheless, analysis of cells continuously cultured for 120 days indicated that the cardiomyocytes continued to mature toward a more adult-like gene expression pattern. Analysis of cardiomyocyte-specific miRNAs (miR-1, miR-133a/b, and miR-208a/b) revealed an miRNA pattern indicative of stem cell to cardiomyocyte specification. A biostatistitical approach integrated the miRNA and mRNA expression profiles revealing a cardiomyocyte differentiation miRNA network and identified putative mRNAs targeted by multiple miRNAs. Together, these data reveal the miRNA network in human heart development and support the notion that overlapping miRNA networks re-enforce transcriptional control during developmental specification.

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

    Directory of Open Access Journals (Sweden)

    Igor Ruiz de los Mozos

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

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

    Science.gov (United States)

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

    2014-06-01

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

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

    Science.gov (United States)

    Ruiz de los Mozos, Igor; Vergara-Irigaray, Marta; Segura, Victor; Villanueva, Maite; Bitarte, Nerea; Saramago, Margarida; Domingues, Susana; Arraiano, Cecilia M; Fechter, Pierre; Romby, Pascale; Valle, Jaione; Solano, Cristina; Lasa, Iñigo; Toledo-Arana, Alejandro

    2013-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  20. Alpha-synuclein mRNA expression in oligodendrocytes in MSA.

    Science.gov (United States)

    Asi, Yasmine T; Simpson, Julie E; Heath, Paul R; Wharton, Stephen B; Lees, Andrew J; Revesz, Tamas; Houlden, Henry; Holton, Janice L

    2014-06-01

    Multiple system atrophy (MSA) is a progressive neurodegenerative disease presenting clinically with parkinsonian, cerebellar, and autonomic features. α-Synuclein (αsyn), encoded by the gene SNCA, is the main constituent of glial cytoplasmic inclusion (GCI) found in oligodendrocytes in MSA, but the methods of its accumulation have not been established. The aim of this study is to investigate alterations in regional and cellular SNCA mRNA expression in MSA as a possible substrate for GCI formation. Quantitative reverse transcription polymerase chain reaction (qPCR) was performed on postmortem brain samples from 15 MSA, 5 IPD, and 5 control cases to investigate regional expression in the frontal and occipital regions, dorsal putamen, pontine base, and cerebellum. For cellular expression analysis, neurons and oligodendrocytes were isolated by laser-capture microdissection from five MSA and five control cases. SNCA mRNA expression was not significantly different between the MSA, IPD and control cases in all regions (multilevel model, P = 0.14). After adjusting for group effect, the highest expression was found in the occipital cortex while the lowest was in the putamen (multilevel model, P MSA oligodendrocytes expressed more SNCA than control oligodendrocytes and expression in MSA neurons was slightly lower than that in controls, however, these results did not reach statistical significance. We have demonstrated regional variations in SNCA expression, which is higher in cortical than subcortical regions. This study is the first to demonstrate SNCA mRNA expression by oligodendrocytes in human postmortem tissue using qPCR and, although not statistically significant, could suggest that this may be increased in MSA compared to controls.

  1. Correlation of mRNA Profiles, miRNA Profiles, and Functional Immune Response in Rainbow Trout (Oncorrhynkus Mykiss) Infected With Viral Hemorrhagic Septicemia Virus (VHSV) and in Fish Vaccinated With a DNA Vaccine Against VHSV

    DEFF Research Database (Denmark)

    Bela-Ong, Dennis; Schyth, Brian Dall; Jørgensen, Hanne

    2011-01-01

    and are incorporated into the RNA-Induced Silencing Complex (RISC), which target specific mRNA sequences, causing either mRNA degradation or translation repression. This results in altered mRNA and protein profiles characteristic of a particular cellular phenotype or physiological state. By targeting immune relevant m...... with the highly pathogenic fish rhabdovirus Viral hemorrhagic septicemia virus (VHSV). This talk will discuss our overall strategy and present preliminary data on the expression of miRNAs and the type I interferon-inducible Mx gene in the liver and the skeletal muscle tissue of fish injected with a DNA vaccine...

  2. 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

  3. 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...

  4. 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.

  5. 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.

  6. Complexity on Acute Myeloid Leukemia mRNA Transcript Variant

    Directory of Open Access Journals (Sweden)

    Carlo Cattani

    2011-01-01

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

  7. 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.

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

    Science.gov (United States)

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

    2015-06-01

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

  9. Occupational toluene exposure induces cytochrome P450 2E1 mRNA expression in peripheral lymphocytes.

    Science.gov (United States)

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

    2006-04-01

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

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

    Science.gov (United States)

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

    2006-01-01

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

  11. Cis-regulatory variants affect CHRNA5 mRNA expression in populations of African and European ancestry.

    Directory of Open Access Journals (Sweden)

    Jen-Chyong Wang

    Full Text Available Variants within the gene cluster encoding α3, α5, and β4 nicotinic receptor subunits are major risk factors for substance dependence. The strongest impact on risk is associated with variation in the CHRNA5 gene, where at least two mechanisms are at work: amino acid variation and altered mRNA expression levels. The risk allele of the non-synonymous variant (rs16969968; D398N primarily occurs on the haplotype containing the low mRNA expression allele. In populations of European ancestry, there are approximately 50 highly correlated variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster and the adjacent PSMA4 gene region that are associated with CHRNA5 mRNA levels. It is not clear which of these variants contribute to the changes in CHRNA5 transcript level. Because populations of African ancestry have reduced linkage disequilibrium among variants spanning this gene cluster, eQTL mapping in subjects of African ancestry could potentially aid in defining the functional variants that affect CHRNA5 mRNA levels. We performed quantitative allele specific gene expression using frontal cortices derived from 49 subjects of African ancestry and 111 subjects of European ancestry. This method measures allele-specific transcript levels in the same individual, which eliminates other biological variation that occurs when comparing expression levels between different samples. This analysis confirmed that substance dependence associated variants have a direct cis-regulatory effect on CHRNA5 transcript levels in human frontal cortices of African and European ancestry and identified 10 highly correlated variants, located in a 9 kb region, that are potential functional variants modifying CHRNA5 mRNA expression levels.

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

    Science.gov (United States)

    Fu, Zidong Donna; Klaassen, Curtis D

    2014-01-01

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

  13. Hypoxia-inducible Factor-1α mRNA Contains an Internal Ribosome Entry Site That Allows Efficient Translation during Normoxia and Hypoxia

    OpenAIRE

    Lang, Kenneth J. D.; Kappel, Andreas; Gregory J. Goodall

    2002-01-01

    HIF-1α is the regulated subunit of the HIF-1 transcription factor, which induces transcription of a number of genes involved in the cellular response to hypoxia. The HIF-1α protein is rapidly degraded in cells supplied with adequate oxygen but is stabilized in hypoxic cells. Using polysome profile analysis, we found that translation of HIF-1α mRNA in NIH3T3 cells is spared the general reduction in translation rate that occurs during hypoxia. To assess whether the 5′UTR of the HIF-1α mRNA cont...

  14. 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.

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

    Science.gov (United States)

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

    2015-02-27

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

  16. 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.

  17. 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.

  18. Stabilizing brokerage.

    Science.gov (United States)

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

    2011-12-27

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

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

    Science.gov (United States)

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

    2013-07-11

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

  20. Bacteria differently regulate mRNA abundance to specifically respond to various stresses.

    Science.gov (United States)

    Bartholomäus, Alexander; Fedyunin, Ivan; Feist, Peter; Sin, Celine; Zhang, Gong; Valleriani, Angelo; Ignatova, Zoya

    2016-03-13

    Environmental stress is detrimental to cell viability and requires an adequate reprogramming of cellular activities to maximize cell survival. We present a global analysis of the response of Escherichia coli to acute heat and osmotic stress. We combine deep sequencing of total mRNA and ribosome-protected fragments to provide a genome-wide map of the stress response at transcriptional and translational levels. For each type of stress, we observe a unique subset of genes that shape the stress-specific response. Upon temperature upshift, mRNAs with reduced folding stability up- and downstream of the start codon, and thus with more accessible initiation regions, are translationally favoured. Conversely, osmotic upshift causes a global reduction of highly translated transcripts with high copy numbers, allowing reallocation of translation resources to not degraded and newly synthesized mRNAs.

  1. 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

    2016-05-27

    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.

  2. Role of mRNA Methylation in Prostate Cancer

    Science.gov (United States)

    2015-02-01

    we carried out high-performance liquid chromatography coupled with triple - quadrupole tandem mass spectrometry (HPLC–QqQ-MS/MS) of mRNA isolated from...time publications. 8 Nothing to Report  Website(s) or other Internet site(s) Nothing to Report  Technologies or techniques Nothing to report

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

    Science.gov (United States)

    Hannibal, Jens; Georg, Birgitte; Fahrenkrug, Jan

    2013-01-01

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

  5. Effect of“Plaque - Stablizing Decoction”on Unstable Plaque Bax mRNA and Bcl -2 mRNA Expression of ApoE Knockout Rats with AS%稳斑汤对ApoE基因敲除小鼠主动脉粥样硬化不稳定斑块形成Bax和Bcl-2mRNA的影响

    Institute of Scientific and Technical Information of China (English)

    程修平; 宫丽鸿

    2015-01-01

    Objective To observe the effect of compound plaque - stablizing decoction on unstable plaque Bax mRNA and Bcl - 2 mRNA expression of ApoE knockout rats with Atherosclerosis. Methods A number of 6 to 8 weeks old ApoE gene - deficient rats were selected to establish AS unstable plaque model, and were randomly divided into the model group,the western medicine statin group and the stable plaque de-coction low,medium and high dose groups,and the control group. They were given normal saline,low dose, medium dose,high dose compound stabilizing plaque decoction,and Atorvastatin for a month. Aortic tissue was observed under optical microscope pathology after HE staining. Detect the expression levels of Bax mRNA and Bcl - 2 mRNA for each group with RT - PCR. Results Bax mRNA expression levels in the model group was significantly higher than the control group(P 0. 05). Compared with western medicine statin group,Bax mRNA expression levels in the stable plaque decoction low and medium dose groups were signifi-cantly higher(P 0. 05). Compared with western medicine statin group,Bax mRNA expression levels in the stable plaque decoction low and medium dose groups were significantly higher(P 0.05)。光镜下观察稳斑汤各剂量组和西药组斑块面积均小于模型组。结论搜风祛痰法复方中药稳斑汤治疗 AS 的机制可能与抑制 Bax mRNA 的表达,促进 Bcl -2 mRNA 表达有关。

  6. Reciprocal regulation of microRNA and mRNA profiles in neuronal development and synapse formation

    Directory of Open Access Journals (Sweden)

    Grant Seth GN

    2009-09-01

    Full Text Available Abstract Background Synapse formation and the development of neural networks are known to be controlled by a coordinated program of mRNA synthesis. microRNAs are now recognized to be important regulators of mRNA translation and stability in a wide variety of organisms. While specific microRNAs are known to be involved in neural development, the extent to which global microRNA and mRNA profiles are coordinately regulated in neural development is unknown. Results We examined mouse primary neuronal cultures, analyzing microRNA and mRNA expression. Three main developmental patterns of microRNA expression were observed: steady-state levels, up-regulated and down-regulated. Co-expressed microRNAs were found to have related target recognition sites and to be encoded in distinct genomic locations. A number of 43 differentially expressed miRNAs were located in five genomic clusters. Their predicted mRNA targets show reciprocal levels of expression. We identified a set of reciprocally expressed microRNAs that target mRNAs encoding postsynaptic density proteins and high-level steady-state microRNAs that target non-neuronal low-level expressed mRNAs. Conclusion We characterized hundreds of miRNAs in neuronal culture development and identified three major modes of miRNA expression. We predict these miRNAs to regulate reciprocally expressed protein coding genes, including many genes involved in synaptogenesis. The identification of miRNAs that target mRNAs during synaptogenesis indicates a new level of regulation of the synapse.

  7. 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.

  8. Cigarette smoke exposure-associated alterations to noncoding RNA

    Directory of Open Access Journals (Sweden)

    Matthew Alan Maccani

    2012-04-01

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

  9. 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.

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

    Science.gov (United States)

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

    2008-07-01

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

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

    Science.gov (United States)

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

    2016-04-01

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

  12. Cyclic AMP stabilizes a class of developmentally regulated Dictyostelium discoideum mRNAs.

    Science.gov (United States)

    Mangiarotti, G; Ceccarelli, A; Lodish, H F

    The stability of mRNA is an important facet of the regulation of protein synthesis. In mammalian cells most mRNAs have long half-lives (5-15 hours) but a substantial fraction are much less stable. There are few examples where the stability of a particular mRNA or class of mRNAs is specifically affected by environmental or developmental stimuli. Certain hormones cause specific stabilization of mRNAs species and preferential mRNA stability is important in the accumulation of globin and myosin mRNAs during the terminal stages of erythropoesis or myogenesis, respectively. Disaggregation of Dictyostelium discoideum aggregates induces the specific destabilization of a large class of developmentally regulated mRNAs; thus, this system is an excellent one in which to determine how such controls are effected. Here we show that addition of cyclic AMP to disaggregated cells specifically prevents the destabilization of these mRNAs.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2009-12-01

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

  15. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

  17. 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

  18. 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

  19. Genetics of the DST-mediated mRNA decay pathway using a transgene-based selection.

    Science.gov (United States)

    Lidder, P; Johnson, M A; Sullivan, M L; Thompson, D M; Pérez-Amador, M A; Howard, C J; Green, P J

    2004-08-01

    mRNA sequences that control abundance, localization and translation initiation have been identified, yet the factors that recognize these sequences are largely unknown. In this report, a transgene-based strategy designed to isolate mutants of Arabidopsis thaliana that fail to recognize these sequences is described. In this strategy, a selectable gene and a screenable marker gene are put under the control of the sequence element being analysed and mutants are selected with altered abundance of the corresponding marker RNAs. The selection of mutants deficient in recognition of the DST (downstream) mRNA degradation signal is used as a test-case to illustrate some of the technical aspects that have facilitated success. Using this strategy, we report the isolation of a new mutant, dst3, deficient in the DST-mediated mRNA decay pathway. The targeted genetic strategy described circumvents certain technical limitations of biochemical approaches. Hence, it provides a means to investigate a variety of other mechanisms responsible for post-transcriptional regulation.

  20. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  1. Membrane-Coupled mRNA Trafficking in Fungi.

    Science.gov (United States)

    Haag, Carl; Steuten, Benedikt; Feldbrügge, Michael

    2015-01-01

    Intracellular logistics are essential for delivery of newly synthesized material during polar growth of fungal hyphae. Proteins and lipids are actively transported throughout the cell by motor-dependent movement of small vesicles or larger units such as endosomes and the endoplasmic reticulum. A remarkably tight link is emerging between active membrane trafficking and mRNA transport, a process that determines the precise subcellular localization of translation products within the cell. Here, we report on recent insights into the mechanism and biological role of these intricate cotransport processes in fungal models such as Saccharomyces cerevisiae, Candida albicans, and Ustilago maydis. In the latter, we focus on the new finding of endosomal mRNA transport and its implications for protein targeting, complex assembly, and septin biology.

  2. 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 ...

  3. 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....... In addition, corresponding tissue was examined from healthy volunteers (n = 20). mRNA levels were normalized to beta-actin. Immunohistochemical analysis of the distributions of type IV and type VII collagens were performed on normal and affected tissues from colorectal cancer patients. RESULTS: The alpha1(IV......). The level of alpha 6(IV) was 5-fold lower in colorectal cancer tissue as compared to healthy individuals (p collagen was visualized by immunohistochemical staining. CONCLUSION: Our results suggest that the down-regulation of alpha 6(IV) mRNA coincides...

  4. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

    Holm, Christian; Nyvold, C.G.; Paludan, Søren Riis;

    2006-01-01

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

  5. 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

    2014-07-03

    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.

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

    DEFF Research Database (Denmark)

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

    1986-01-01

    of the receptor from different carcinoma sources and from normal placental tissue is in the range of 68-72 kDa. Isoelectric focusing and two-dimensional gel electrophoresis indicated that the receptor protein consists of one major polypeptide chain with a pI value of 6.4 +/- 0.2. Laminin receptor cDNA clones were...

  7. Analysis of specific mRNA destabilization during Dictyostelium development.

    Science.gov (United States)

    Mangiarotti, G; Bulfone, S; Giorda, R; Morandini, P; Ceccarelli, A; Hames, B D

    1989-07-01

    A number of specific mRNAs are destabilized upon disaggregation of developing Dictyostelium discoideum cells. Analysis of a family of cloned genes indicates that only prespore-enriched mRNAs are affected; constitutive mRNAs that are expressed throughout development and mRNAs that accumulate preferentially in prestalk cells are stable under these conditions. The decay of sensitive prespore mRNAs can be halted by allowing the cells to reaggregate, indicating that destabilization occurs by the progressive selection of individual molecules rather than on all members of an mRNA subpopulation at the time of disaggregation. Individual molecules of the sensitive mRNA species remain engaged in protein synthesis in the disaggregated cells until selected. Destabilization of sensitive mRNAs is induced by cell dissociation even in the presence of concentrations of nogalamycin that inhibit RNA synthesis. The reported prevention of disaggregation-induced mRNA decay by actinomycin D and daunomycin is therefore probably a secondary effect unrelated to the inhibition of transcription.

  8. mRNA and DNA PCR tests in cutaneous tuberculosis

    Directory of Open Access Journals (Sweden)

    Chandanmal Suthar

    2013-01-01

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

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

    Science.gov (United States)

    Shiflett, Lora A; Read, G Sullivan

    2013-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-04-15

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

  12. 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.

  13. A selection of reference genes and early-warning mRNA biomarkers for environmental monitoring using Mytilus spp. as sentinel species.

    Science.gov (United States)

    Lacroix, C; Coquillé, V; Guyomarch, J; Auffret, M; Moraga, D

    2014-09-15

    mRNA biomarkers are promising tools for environmental health assessment and reference genes are needed to perform relevant qPCR analyses in tissue samples of sentinel species. In the present study, potential reference genes and mRNA biomarkers were tested in the gills and digestive glands of native and caged mussels (Mytilus spp.) exposed to harbor pollution. Results highlighted the difficulty to find stable reference genes in wild, non-model species and suggested the use of normalization indices instead of single genes as they exhibit a higher stability. Several target genes were found differentially expressed between mussel groups, especially in gills where cyp32, π-gst and CuZn-sod mRNA levels could be biomarker candidates. Multivariate analyses confirmed the ability of mRNA levels to highlight site-effects and suggested the use of several combined markers instead of individual ones. These findings support the use of qPCR technology and mRNA levels as early-warning biomarkers in marine monitoring programs.

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

    Directory of Open Access Journals (Sweden)

    Ahmadreza Niavarani

    Full Text Available 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.

  15. FEATURES OF LOCAL mRNA SYNTHESIS FOR SOME CC- AND CXC-CHEMOKINES AND THEIR RECEPTORS IN ENDOMETRIAL HYPERPLASIA

    Directory of Open Access Journals (Sweden)

    N. V. Kipich

    2011-01-01

    Full Text Available Аbstract.  Endometrial  hyperplasia  (EH  represents  an  excessive  increase  in  thickness  and  volume  of proliferating endometrium accompanied by altered glandular structure. This disorder is higly prevalent among women in their premenopausal period. There exist only scarce data concerning possible role of chemokines and their receptors in EH pathogenesis and clinical course. Hence, the aim of our study was to analyze mRNA expression  of  several  key  chemokines  and  their  receptors  in  endometrial  tissue  samples  from  EH  patients. This work included sixty-three women with disturbed menstrual  cycle  and/or  pathological  changes  of endometrium, as assessed by sonographic studies. The patients were 32 to 61 years old (a mean of 48.4±0.6 years. The levels of mRNA expression were determined by  gene-specific  PCR  in  a  semiquantitative  manner,  whereas promoter genotypes of matrix metalloproteinases (ММР1 -16071G/2G and ММР3 -11715А/6A were identified by means of allele-specific PCR. Results of the study included a significant increase of mRNA for MIP-1α, eotaxin 2, along with decreased amounts of mRNA for CCR-3 (a specific receptor for eotaxins, in polyps developing from hyperplastic endometrium. MIP-1α synthesis fades away with increasing age. An increased level of MIP-1β was shown in prolonged and recurrent disturbances of menstrual cycle, whereas elevation of MIP-1α and CXCR-1 was registered in cases of multiple pregnancies. In threatening abortions, an increase of MIP-1β gene expression was revealed. Hence, the local chemokine system reacts to inflammatory and hemorrhagic complications with increased mRNA expression of certain chemokine genes. Determination of the chemokine mRNA levels, as well as their receptors in patients with endometrial hyperplasia may reflect a general background of this disorder. (Med. Immunol., 2011, vol. 13, N 2-3, pp 189-196

  16. Mixture effects of levonorgestrel and ethinylestradiol: estrogenic biomarkers and hormone receptor mRNA expression during sexual programming.

    Science.gov (United States)

    Säfholm, Moa; Jansson, Erika; Fick, Jerker; Berg, Cecilia

    2015-04-01

    Synthetic progesterone (progestins) and estrogens are widely used pharmaceuticals. Given that their simultaneous unintentional exposure occurs in wildlife and also in human infants, data on mixture effects of combined exposures to these hormones during development is needed. Using the Xenopus (Silurana) tropicalis test system we investigated mixture effects of levonorgestrel (LNG) and ethinylestradiol (EE2) on hormone sensitive endpoints. After larval exposure to LNG (0.1nM), or EE2 (0.1nM) singly, or in combination with LNG (0.01, 0.1, 1.0nM), the gonadal sex ratio was determined histologically and hepatic mRNA levels of genes encoding vitellogenin (vtg beta1) and the estrogen (esr1, esr2), progesterone (ipgr) and androgen (ar) receptors were quantified using quantitative PCR. All EE2-exposed groups showed female-biased sex ratios and increased vtg beta1 mRNA levels compared with the controls. Compared with the EE2-alone group (positive control) there were no significant alterations in vtg beta1 levels or in sex ratios in the co-exposure groups. Exposure to LNG-alone caused an increase in ar mRNA levels in females, but not in males, compared to the controls and the co-exposed groups, indicating that co-exposure to EE2 counteracted the LNG-induced ar levels. No treatment related impacts on the mRNA expression of esr1, esr2, and ipgr in female tadpoles were found, suggesting that these endpoints are insensitive to long-term exposure to estrogen or progestin. Due to the EE2-induced female-biased sex ratios, the mRNA expression data for the low number of males in the EE2-exposed groups were not statistically analyzed. In conclusion, our results suggest that induced vtg expression is a robust biomarker for estrogenic activity in exposure scenarios involving both estrogens and progestins. Developmental exposure to LNG caused an induction of hepatic ar mRNA expression that was antagonized by combined exposure to EE2 and LNG. To our knowledge this is the first study to

  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. Monitoring protein stability in vivo

    Directory of Open Access Journals (Sweden)

    Ignatova Zoya

    2005-08-01

    Full Text Available Abstract Reduced protein stability in vivo is a prerequisite to aggregation. While this is merely a nuisance factor in recombinant protein production, it holds a serious impact for man. This review focuses on specific approaches to selectively determine the solubility and/or stability of a target protein within the complex cellular environment using different detection techniques. Noninvasive techniques mapping folding/misfolding events on a fast time scale can be used to unravel the complexity and dynamics of the protein aggregation process and factors altering protein solubility in vivo. The development of approaches to screen for folding and solubility in vivo should facilitate the identification of potential components that improve protein solubility and/or modulate misfolding and aggregation and may provide a therapeutic benefit.

  19. Monitoring protein stability in vivo.

    Science.gov (United States)

    Ignatova, Zoya

    2005-08-24

    Reduced protein stability in vivo is a prerequisite to aggregation. While this is merely a nuisance factor in recombinant protein production, it holds a serious impact for man. This review focuses on specific approaches to selectively determine the solubility and/or stability of a target protein within the complex cellular environment using different detection techniques. Noninvasive techniques mapping folding/misfolding events on a fast time scale can be used to unravel the complexity and dynamics of the protein aggregation process and factors altering protein solubility in vivo. The development of approaches to screen for folding and solubility in vivo should facilitate the identification of potential components that improve protein solubility and/or modulate misfolding and aggregation and may provide a therapeutic benefit.

  20. Extensive Mitochondrial mRNA Editing and Unusual Mitochondrial Genome Organization in Calcaronean Sponges.

    Science.gov (United States)

    Lavrov, Dennis V; Adamski, Marcin; Chevaldonné, Pierre; Adamska, Maja

    2016-01-11

    One of the unusual features of DNA-containing organelles in general and mitochondria in particular is the frequent occurrence of RNA editing [1]. The term "RNA editing" refers to a variety of mechanistically unrelated biochemical processes that alter RNA sequence during or after transcription [2]. The editing can be insertional, deletional, or substitutional and has been found in all major types of RNAs [3, 4]. Although mitochondrial mRNA editing is widespread in some eukaryotic lineages [5-7], it is rare in animals, with reported cases limited both in their scope and in phylogenetic distribution [8-11] (see also [12]). While analyzing genomic data from calcaronean sponges Sycon ciliatum and Leucosolenia complicata, we were perplexed by the lack of recognizable mitochondrial coding sequences. Comparison of genomic and transcriptomic data from these species revealed the presence of mitochondrial cryptogenes whose transcripts undergo extensive editing. This editing consisted of single or double uridylate (U) insertions in pre-existing short poly(U) tracts. Subsequent analysis revealed the presence of similar editing in Sycon coactum and the loss of editing in Petrobiona massiliana, a hypercalcified calcaronean sponge. In addition, mitochondrial genomes of at least some calcaronean sponges were found to have a highly unusual architecture, with nearly all genes located on individual and likely linear chromosomes. Phylogenetic analysis of mitochondrial coding sequences revealed accelerated rates of sequence evolution in this group. The latter observation presents a challenge for the mutational-hazard hypothesis [13], which posits that mRNA editing should not occur in lineages with an elevated mutation rate.

  1. 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.

  2. RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution

    Directory of Open Access Journals (Sweden)

    Dobric Nada

    2007-04-01

    Full Text Available Abstract Background In protein drug development, in vitro molecular optimization or protein maturation can be used to modify protein properties. One basic approach to protein maturation is the introduction of random DNA mutations into the target gene sequence to produce a library of variants that can be screened for the preferred protein properties. Unfortunately, the capability of this approach has been restricted by deficiencies in the methods currently available for random DNA mutagenesis and library generation. Current DNA based methodologies generally suffer from nucleotide substitution bias that preferentially mutate particular base pairs or show significant bias with respect to transitions or transversions. In this report, we describe a novel RNA-based random mutagenesis strategy that utilizes Qβ replicase to manufacture complex mRNA libraries with a mutational spectrum that is close to the ideal. Results We show that Qβ replicase generates all possible base substitutions with an equivalent preference for mutating A/T or G/C bases and with no significant bias for transitions over transversions. To demonstrate the high diversity that can be sampled from a Qβ replicase-generated mRNA library, the approach was used to evolve the binding affinity of a single domain VNAR shark antibody fragment (12Y-2 against malarial apical membrane antigen-1 (AMA-1 via ribosome display. The binding constant (KD of 12Y-2 was increased by 22-fold following two consecutive but discrete rounds of mutagenesis and selection. The mutagenesis method was also used to alter the substrate specificity of β-lactamase which does not significantly hydrolyse the antibiotic cefotaxime. Two cycles of RNA mutagenesis and selection on increasing concentrations of cefotaxime resulted in mutants with a minimum 10,000-fold increase in resistance, an outcome achieved faster and with fewer overall mutations than in comparable studies using other mutagenesis strategies

  3. Food Fortification Stability Study

    Science.gov (United States)

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

    2017-01-01

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

  4. 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.

  5. Genetic Alterations in Glioma

    Energy Technology Data Exchange (ETDEWEB)

    Bralten, Linda B. C.; French, Pim J., E-mail: p.french@erasmusmc.nl [Department of Neurology, Erasmus University Medical Center, Erasmus University Rotterdam, Dr Molewaterplein 50, 3000 CA, Rotterdam (Netherlands)

    2011-03-07

    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.

  6. Context-dependent dual role of SKI8 homologs in mRNA synthesis and turnover.

    Directory of Open Access Journals (Sweden)

    Eavan Dorcey

    Full Text Available Eukaryotic mRNA transcription and turnover is controlled by an enzymatic machinery that includes RNA polymerase II and the 3' to 5' exosome. The activity of these protein complexes is modulated by additional factors, such as the nuclear RNA polymerase II-associated factor 1 (Paf1c and the cytoplasmic Superkiller (SKI complex, respectively. Their components are conserved across uni- as well as multi-cellular organisms, including yeast, Arabidopsis, and humans. Among them, SKI8 displays multiple facets on top of its cytoplasmic role in the SKI complex. For instance, nuclear yeast ScSKI8 has an additional function in meiotic recombination, whereas nuclear human hSKI8 (unlike ScSKI8 associates with Paf1c. The Arabidopsis SKI8 homolog VERNALIZATION INDEPENDENT 3 (VIP3 has been found in Paf1c as well; however, whether it also has a role in the SKI complex remains obscure so far. We found that transgenic VIP3-GFP, which complements a novel vip3 mutant allele, localizes to both nucleus and cytoplasm. Consistently, biochemical analyses suggest that VIP3-GFP associates with the SKI complex. A role of VIP3 in the turnover of nuclear encoded mRNAs is supported by random-primed RNA sequencing of wild-type and vip3 seedlings, which indicates mRNA stabilization in vip3. Another SKI subunit homolog mutant, ski2, displays a dwarf phenotype similar to vip3. However, unlike vip3, it displays neither early flowering nor flower development phenotypes, suggesting that the latter reflect VIP3's role in Paf1c. Surprisingly then, transgenic ScSKI8 rescued all aspects of the vip3 phenotype, suggesting that the dual role of SKI8 depends on species-specific cellular context.

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

    Science.gov (United States)

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

    2015-09-01

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

  8. Mechanisms of mRNA translation of interferon stimulated genes.

    Science.gov (United States)

    Joshi, Sonali; Kaur, Surinder; Kroczynska, Barbara; Platanias, Leonidas C

    2010-01-01

    Over the last two decades, a lot of research work has been focused on the interferon (IFN)-regulated JAK-STAT pathway and understanding the mechanisms governing the transcription of interferon stimulated genes (ISGs). Evidence suggests that the JAK-STAT pathway alone does not account in its entirety for mediating cellular responses to IFNs. There is emerging evidence that non-Stat pathways play important roles in mediating signals for the generation of IFN-responses. Various studies have underscored the importance of mitogen activated protein kinases (MAPKs), especially p38 and ERK1/2, as well as the PI 3'K/AKT pathway in transmitting signals that are of critical importance for the biological effects of IFNs. Besides regulating the transcription of ISGs in some cases, engagement of these signaling pathways by the IFN-receptor (IFNR) associated complexes also plays an important role in mediating the translation of ISGs. The mechanisms regulating mRNA translation of ISGs is an area of ongoing active research and a lot more efforts will be required to complete our understanding of the various cellular elements involved in this process. In this review we highlight the mechanisms regulating translation of ISGs. We focus on the proteins regulated by the PI 3'K/AKT pathway, their role in mediating mRNA translation of ISGs and the functional consequences of this regulation. In addition, MAPKs are known to regulate the phosphorylation of various eukaryotic initiation factors and we summarize the roles of eIF4B and eIF4E phosphorylations on the translation of ISGs. The emerging roles of microRNAs in mRNA translation of ISGs are also discussed.

  9. 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

  10. mRNA analysis of single living cells

    Directory of Open Access Journals (Sweden)

    Ikai Atsushi

    2003-02-01

    Full Text Available Abstract Analysis of specific gene expression in single living cells may become an important technique for cell biology. So far, no method has been available to detect mRNA in living cells without killing or destroying them. We have developed here a novel method to examine gene expression of living cells using an atomic force microscope (AFM. AFM tip was inserted into living cells to extract mRNAs. The obtained mRNAs were analyzed with RT-PCR, nested PCR, and quantitative PCR. This method enabled us to examine time-dependent gene expression of single living cells without serious damage to the cells.

  11. Control of Klebsiella pneumoniae nif mRNA synthesis.

    OpenAIRE

    1985-01-01

    Four probes, each specific for a single nif transcript, were used for an analysis of the regulation of nif mRNA synthesis. Transcription of the nifLA operon was repressed by NH4+ but not by amino acids, O2, or temperatures above 37 degrees C. The nifA gene product was required for the activation of transcription of the other nif operons but not nifLA. Synthesis of the other nif transcripts was rapidly turned off by the addition of O2, NH4+, serine, or glutamine. These regulatory effects requi...

  12. Transcriptional activators enhance polyadenylation of mRNA precursors

    OpenAIRE

    Nagaike, Takashi; Manley, James L.

    2011-01-01

    3′ processing of mRNA precursors is frequently coupled to transcription by RNA polymerase II (RNAP II). This coupling is well known to involve the C-terminal domain of the RNAP II largest subunit, but a variety of other transcription-associated factors have also been suggested to mediate coupling. Our recent studies have provided direct evidence that transcriptional activators can enhance the efficiency of transcription-coupled 3′ processing. In this point-of-view, we discuss the mechanisms t...

  13. Postnatal morphine administration alters hippocampal development in rats.

    Science.gov (United States)

    Traudt, Christopher M; Tkac, Ivan; Ennis, Kathleen M; Sutton, Leah M; Mammel, Daniel M; Rao, Raghavendra

    2012-01-01

    Morphine is frequently used as an analgesic and sedative in preterm infants. Adult rats exposed to morphine have an altered hippocampal neurochemical profile and decreased neurogenesis in the dentate gyrus of the hippocampus. To evaluate whether neonatal rats are similarly affected, rat pups were injected twice daily with 2 mg/kg morphine or normal saline from postnatal days 3 to 7. On postnatal day 8, the hippocampal neurochemical profile was determined using in vivo (1)H NMR spectroscopy. The mRNA and protein concentrations of specific analytes were measured in hippocampus, and cell division in dentate gyrus was assessed using bromodeoxyuridine. The concentrations of γ-aminobutyric acid (GABA), taurine, and myo-insotol were decreased, whereas concentrations of glutathione, phosphoethanolamine, and choline-containing compounds were increased in morphine-exposed rats relative to control rats. Morphine decreased glutamic acid decarboxylase enzyme levels and myelin basic protein mRNA expression in the hippocampus. Bromodeoxyuridine labeling in the dentate gyrus was decreased by 60-70% in morphine-exposed rats. These results suggest that recurrent morphine administration during brain development alters hippocampal structure.

  14. Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts

    DEFF Research Database (Denmark)

    Dabelsteen, S; Wandall, H H; Grøn, B

    1997-01-01

    controls. KGF mRNA was found in all cultured fibroblasts. Upon addition of 10% FCS to the cell cultures, an increase in KGF mRNA levels was noticed especially after 72 h. Furthermore, RT-PCR analysis of material scraped from the tooth root surface indicated the presence of KGF mRNA even in noncultured...

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

    Science.gov (United States)

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

    1993-01-01

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

  16. Plutonium inventories for stabilization and stabilized materials

    Energy Technology Data Exchange (ETDEWEB)

    Williams, A.K.

    1996-05-01

    The objective of the breakout session was to identify characteristics of materials containing plutonium, the need to stabilize these materials for storage, and plans to accomplish the stabilization activities. All current stabilization activities are driven by the Defense Nuclear Facilities Safety Board Recommendation 94-1 (May 26, 1994) and by the recently completed Plutonium ES&H Vulnerability Assessment (DOE-EH-0415). The Implementation Plan for accomplishing stabilization of plutonium-bearing residues in response to the Recommendation and the Assessment was published by DOE on February 28, 1995. This Implementation Plan (IP) commits to stabilizing problem materials within 3 years, and stabilizing all other materials within 8 years. The IP identifies approximately 20 metric tons of plutonium requiring stabilization and/or repackaging. A further breakdown shows this material to consist of 8.5 metric tons of plutonium metal and alloys, 5.5 metric tons of plutonium as oxide, and 6 metric tons of plutonium as residues. Stabilization of the metal and oxide categories containing greater than 50 weight percent plutonium is covered by DOE Standard {open_quotes}Criteria for Safe Storage of Plutonium Metals and Oxides{close_quotes} December, 1994 (DOE-STD-3013-94). This standard establishes criteria for safe storage of stabilized plutonium metals and oxides for up to 50 years. Each of the DOE sites and contractors with large plutonium inventories has either started or is preparing to start stabilization activities to meet these criteria.

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

    Science.gov (United States)

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

    1988-03-01

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

  18. A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication

    Directory of Open Access Journals (Sweden)

    Stoltzfus C Martin

    2006-02-01

    Full Text Available Abstract Background Inefficient alternative splicing of the human immunodeficiency virus type 1(HIV-1 primary RNA transcript results in greater than half of all viral mRNA remaining unspliced. Regulation of HIV-1 alternative splicing occurs through the presence of suboptimal viral 5' and 3' splice sites (5' and 3'ss, which are positively regulated by exonic splicing enhancers (ESE and negatively regulated by exonic splicing silencers (ESS and intronic splicing silencers (ISS. We previously showed that splicing at HIV-1 3'ss A2 is repressed by ESSV and enhanced by the downstream 5'ss D3 signal. Disruption of ESSV results in increased vpr mRNA accumulation and exon 3 inclusion, decreased accumulation of unspliced viral mRNA, and decreased virus production. Results Here we show that optimization of the 5'ss D2 signal results in increased splicing at the upstream 3'ss A1, increased inclusion of exon 2 into viral mRNA, decreased accumulation of unspliced viral mRNA, and decreased virus production. Virus production from the 5'ss D2 and ESSV mutants was rescued by transient expression of HIV-1 Gag and Pol. We further show that the increased inclusion of either exon 2 or 3 does not significantly affect the stability of viral mRNA but does result in an increase and decrease, respectively, in HIV-1 mRNA levels. The changes in viral mRNA levels directly correlate with changes in tat mRNA levels observed upon increased inclusion of exon 2 or 3. Conclusion These results demonstrate that splicing at HIV-1 3'ss A1 is regulated by the strength of the downstream 5'ss signal and that suboptimal splicing at 3'ss A1 is necessary for virus replication. Furthermore, the replication defective phenotype resulting from increased splicing at 3'ss A1 is similar to the phenotype observed upon increased splicing at 3'ss A2. Further examination of the role of 5'ss D2 and D3 in the alternative splicing of 3'ss A1 and A2, respectively, is necessary to delineate a role for non

  19. Myc Regulation of mRNA Cap Methylation

    Science.gov (United States)

    Cowling, Victoria H.; Cole, Michael D.

    2010-01-01

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

  20. 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.

  1. 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.

  2. Imaging mRNA and protein interactions within neurons

    Science.gov (United States)

    Eliscovich, Carolina; Shenoy, Shailesh M.

    2017-01-01

    RNA–protein interactions are essential for proper gene expression regulation, particularly in neurons with unique spatial constraints. Currently, these interactions are defined biochemically, but a method is needed to evaluate them quantitatively within morphological context. Colocalization of two-color labels using wide-field microscopy is a method to infer these interactions. However, because of chromatic aberrations in the objective lens, this approach lacks the resolution to determine whether two molecules are physically in contact or simply nearby by chance. Here, we developed a robust super registration methodology that corrected the chromatic aberration across the entire image field to within 10 nm, which is capable of determining whether two molecules are physically interacting or simply in proximity by random chance. We applied this approach to image single-molecule FISH in combination with immunofluorescence (smFISH-IF) and determined whether the association between an mRNA and binding protein(s) within a neuron was significant or accidental. We evaluated several mRNA-binding proteins identified from RNA pulldown assays to determine which of these exhibit bona fide interactions. Surprisingly, many known mRNA-binding proteins did not bind the mRNA in situ, indicating that adventitious interactions are significant using existing technology. This method provides an ability to evaluate two-color registration compatible with the scale of molecular interactions. PMID:28223507

  3. Cytokine mRNA quantification by real-time PCR.

    Science.gov (United States)

    Stordeur, Patrick; Poulin, Lionel F; Craciun, Ligia; Zhou, Ling; Schandené, Liliane; de Lavareille, Aurore; Goriely, Stanislas; Goldman, Michel

    2002-01-01

    Real-time PCR represents a new methodology that accurately quantifies nucleic acids. This has been made possible by the use of fluorogenic probes, which are presented in two forms, namely hydrolysis probes (also called TaqMan probes) and hybridisation probes. We decided to apply this methodology to cytokine mRNA quantification and this led us to the development of a protocol that provides an easy way to develop and perform rapidly real-time PCR on a Lightcycler instrument. It was made possible by the use of freely available software that permits a choice of both the hydrolysis probe and the primers. We firstly demonstrated that the reproducibility of the method using hydrolysis probes compares favourably with that obtained with hybridisation probes. We then applied this technique to determine the kinetics of IL-1ra, IL-1beta, IL-5, IL-13, TNF-alpha and IFN-gamma induction upon stimulation of human peripheral blood mononuclear cells (PBMC) by phytohaemagglutinin (PHA). Finally, the method was also used successfully to demonstrate that IFN-alpha induces IL-10 mRNA accumulation in human monocytes.

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  5. Nicotinic stimulation modulates tyrosine hydroxylase mRNA half-life and protein binding to the 3'UTR in a manner that requires transcription.

    Science.gov (United States)

    Roe, David F; Craviso, Gale L; Waymire, Jack C

    2004-01-05

    Tyrosine hydroxylase (TH) expression increases in adrenal chromaffin cells treated with the nicotinic agonist, dimethylphenylpiperazinium (DMPP; 1 microM). We are using this response as a model of the changes in TH level that occur during increased cholinergic neural activity. Here we report a 4-fold increase in TH mRNA half-life in DMPP-treated cells chromaffin cells that is apparent when using a pulse-chase analysis to measure TH mRNA half-life. No increase is apparent using actinomycin D to measure half-life, indicating a requirement for ongoing transcription. Characterization of protein binding to the TH 3'UTR responsible for stabilization using labeled TH 3'UTR probes and electro-mobility shift assays shows the presence of two complexes both of which are increased by DMPP-treatment. The faster migrating complex (FMC) increases 2.5-fold and the slower migrating complex (SMC) increases 1.5-fold. Both changes are prevented by actinomycin D. Characterization of the protein binding to the TH UTR probes indicates SMC is disrupted by polyribonucleotides, poly (A) and poly (U), while binding to FMC is reduced by poly (CU). Separation of UV crosslinked RNA-protein complexes on SDS polyacrylamide gels shows FMC to contain a single protein whereas SMC contains three proteins. Northwesterns yielded similar results. Comparison of DMPP-induced protein binding with the poly C binding protein (PCBP) involved in hypoxia induced rat PC12 TH mRNA stability indicates none of the bovine UTR binding proteins are the PCBP. Thus, nicotinic stimulation produces a transcription-dependent increase in TH mRNA half-life that is mediated by previously unrecognized TH mRNA binding proteins.

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

    Science.gov (United States)

    Kash, J C; Menon, K M

    1999-12-21

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

  7. Temporal Stability of the Human Skin Microbiome.

    Science.gov (United States)

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

    2016-05-05

    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.

  8. Tearing mode stability calculations with pressure flattening

    CERN Document Server

    Ham, C J; Cowley, S C; Hastie, R J; Hender, T C; Liu, Y Q

    2013-01-01

    Calculations of tearing mode stability in tokamaks split conveniently into an external region, where marginally stable ideal MHD is applicable, and a resonant layer around the rational surface where sophisticated kinetic physics is needed. These two regions are coupled by the stability parameter. Pressure and current perturbations localized around the rational surface alter the stability of tearing modes. Equations governing the changes in the external solution and - are derived for arbitrary perturbations in axisymmetric toroidal geometry. The relationship of - with and without pressure flattening is obtained analytically for four pressure flattening functions. Resistive MHD codes do not contain the appropriate layer physics and therefore cannot predict stability directly. They can, however, be used to calculate -. Existing methods (Ham et al. 2012 Plasma Phys. Control. Fusion 54 025009) for extracting - from resistive codes are unsatisfactory when there is a finite pressure gradient at the rational surface ...

  9. Saturated fatty acids induce post-transcriptional regulation of HAMP mRNA via AU-rich element-binding protein, human antigen R (HuR).

    Science.gov (United States)

    Lu, Sizhao; Mott, Justin L; Harrison-Findik, Duygu Dee

    2015-10-02

    Iron is implicated in fatty liver disease pathogenesis. The human hepcidin gene, HAMP, is the master switch of iron metabolism. The aim of this study is to investigate the regulation of HAMP expression by fatty acids in HepG2 cells. For these studies, both saturated fatty acids (palmitic acid (PA) and stearic acid (SA)) and unsaturated fatty acid (oleic acid (OA)) were used. PA and, to a lesser extent, SA, but not OA, up-regulated HAMP mRNA levels, as determined by real-time PCR. To understand whether PA regulates HAMP mRNA at the transcriptional or post-transcriptional level, the transcription inhibitor actinomycin D was employed. PA-mediated induction of HAMP mRNA expression was not blocked by actinomycin D. Furthermore, PA activated HAMP 3'-UTR, but not promoter, activity, as shown by reporter assays. HAMP 3'-UTR harbors a single AU-rich element (ARE). Mutation of this ARE abolished the effect of PA, suggesting the involvement of ARE-binding proteins. The ARE-binding protein human antigen R (HuR) stabilizes mRNA through direct interaction with AREs on 3'-UTR. HuR is regulated by phosphorylation-mediated nucleo-cytoplasmic shuttling. PA activated this process. The binding of HuR to HAMP mRNA was also induced by PA in HepG2 cells. Silencing of HuR by siRNA abolished PA-mediated up-regulation of HAMP mRNA levels. PKC is known to phosphorylate HuR. Staurosporine, a broad-spectrum PKC inhibitor, inhibited both PA-mediated translocation of HuR and induction of HAMP expression. Similarly, rottlerin, a novel class PKC inhibitor, abrogated PA-mediated up-regulation of HAMP expression. In conclusion, lipids mediate post-transcriptional regulation of HAMP throughPKC- and HuR-dependent mechanisms.

  10. Integrated mRNA and micro RNA profiling reveals epigenetic mechanism of differential sensitivity of Jurkat T cells to AgNPs and Ag ions.

    Science.gov (United States)

    Eom, Hyun-Jeong; Chatterjee, Nivedita; Lee, Jeongsoo; Choi, Jinhee

    2014-08-17

    In our previous in vitro study of the toxicity on silver nanoparticles (AgNPs), we observed a dramatically higher sensitivity of Jurkat T cells to AgNPs than to Ag ions, and DNA damage and apoptosis were found to be involved in that toxicity. In this study, to understand underlying mechanism of different sensitivity of Jurket T cells to AgNPs and Ag ions, mRNA microarray and micro RNA microarray were concomitantly conducted on AgNPs and Ag ions exposed Jurkat T cells. Surprisingly only a small number of genes were differentially expressed by exposure to each of the silver (15 altered mRNA by AgNPs exposure, whereas 4 altered mRNA by Ag ions exposure, as determined 1.5-fold change as the cut-off value). miRNA microarray revealed that the expression of 63 miRNAs was altered by AgNPs exposure, whereas that of 32 miRNAs was altered by Ag ions exposure. An integrated analysis of mRNA and miRNA expression revealed that the expression of hsa-miR-219-5p, was negatively correlated with the expression of metallothionein 1F (MT1F) and tribbles homolog 3 (TRIB3), in cells exposed to AgNPs; whereas, the expression of hsa-miR-654-3p was negatively correlated with the expression of mRNA, endonuclease G-like 1 (EDGL1) in cells exposed to Ag ions. Network analysis were further conducted on mRNA-miRNA pairs, which revealed that miR-219-5p-MT1F and -TRIB3 pairs by AgNPs are being involved in various cellular processes, such as, oxidative stress, cell cycle and apoptosis, whereas, miR-654-3p and ENDOGL1 pair by Ag ions generated a much simpler network. The putative target genes of AgNPs-induced miR-504, miR-33 and miR-302 identified by Tarbase 6.0 are also found to be involved in DNA damage and apoptosis. These results collectively suggest that distinct epigenetic regulation may be an underlying mechanism of different sensitivity of Jurkat T cells to AgNPs and Ag ion. Further identification of putative target genes of DE miRNA by AgNPs and Ag ions may provide additional clues for the

  11. Mutations in DCPS and EDC3 in autosomal recessive intellectual disability indicate a crucial role for mRNA decapping in neurodevelopment.

    Science.gov (United States)

    Ahmed, Iltaf; Buchert, Rebecca; Zhou, Mi; Jiao, Xinfu; Mittal, Kirti; Sheikh, Taimoor I; Scheller, Ute; Vasli, Nasim; Rafiq, Muhammad Arshad; Brohi, M Qasim; Mikhailov, Anna; Ayaz, Muhammad; Bhatti, Attya; Sticht, Heinrich; Nasr, Tanveer; Carter, Melissa T; Uebe, Steffen; Reis, André; Ayub, Muhammad; John, Peter; Kiledjian, Megerditch; Vincent, John B; Jamra, Rami Abou

    2015-06-01

    There are two known mRNA degradation pathways, 3' to 5' and 5' to 3'. We identified likely pathogenic variants in two genes involved in these two pathways in individuals with intellectual disability. In a large family with multiple branches, we identified biallelic variants in DCPS in three affected individuals; a splice site variant (c.636+1G>A) that results in an in-frame insertion of 45 nucleotides and a missense variant (c.947C>T; p.Thr316Met). DCPS decaps the cap structure generated by 3' to 5' exonucleolytic degradation of mRNA. In vitro decapping assays showed an ablation of decapping function for both variants in DCPS. In another family, we identified a homozygous mutation (c.161T>C; p.Phe54Ser) in EDC3 in two affected children. EDC3 stimulates DCP2, which decaps mRNAs at the beginning of the 5' to 3' degradation pathway. In vitro decapping assays showed that altered EDC3 is unable to enhance DCP2 decapping at low concentrations and even inhibits DCP2 decapping at high concentration. We show that individuals with biallelic mutations in these genes of seemingly central functions are viable and that these possibly lead to impairment of neurological functions linking mRNA decapping to normal cognition. Our results further affirm an emerging theme linking aberrant mRNA metabolism to neurological defects.

  12. Chronic corticosterone decreases brain-derived neurotrophic factor (BDNF) mRNA and protein in the hippocampus, but not in the frontal cortex, of the rat.

    Science.gov (United States)

    Jacobsen, Jacob P R; Mørk, Arne

    2006-09-19

    This study examined the effects of chronic corticosterone (32 mg/kg/day, s.c., 21 days) on brain-derived neurotrophic factor (BDNF) mRNA and protein in the frontal cortex and hippocampus of the rat. Because evidence suggests that BDNF is an important determinant of the function of the 5-hydroxytryptamine (5-HT) system, we also quantified tissue levels of 5-HT and its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA), to investigate if changes in BDNF mRNA and protein paralleled changes in the 5-HT system. Corticosterone modestly decreased BDNF protein (-16.6%) in whole hippocampus and BDNF mRNA (-19%) in the CA3 area. In contrast, BDNF mRNA and protein in the frontal cortex were unchanged. In both the frontal cortex and hippocampus, tissue levels of 5-HT and 5-HIAA were increased and decreased, respectively. Combined, these data suggests that the effects of corticosterone on the BDNF system are not linked to the effects on the 5-HT systems. However, our findings do suggest that chronic corticosterone impairs hippocampal BDNF function, a finding with potential relevance for the hippocampal atrophy reported in major depression. Additionally, as inferred from the alterations in tissue levels of 5-HT and 5-HIAA, chronic corticosterone may influence the function of the 5-HT system.

  13. Effect of Hyperosmotic Stimulation and Adrenalectomy on Vasopressin mRNA Levels in the Paraventricular and Supraoptic Nuclei of the Hypothalamus:

    Directory of Open Access Journals (Sweden)

    Suemaru,Shuso

    1990-10-01

    Full Text Available The effects of salt loading and adrenalectomy on arginine vasopressin (AVP mRNA levels in the paraventricular nucleus (PVN and the supraoptic nucleus (SON of the hypothalamus were studied by semiquantitative in situ hybridization histochemistry, using a synthetic oligonucleotide probe and a computer-assisted image analysis system. Salt loading (2% NaCl for 7 days produced marked increases in AVP mRNA levels in the magnocellular neurons of the PVN, SON, and accessory nuclei. Adrenalectomy caused an increase in AVP mRNA expression in the magnocellular part of the PVN and the expansion of hybridization signals into its medial parvocellular region, where the cell bodies of corticotropin-releasing hormone (CRH neurons are located. No apparent alteration of AVP mRNA levels was observed in the SON following adrenalectomy. These results indicate that hyperosmotic stimulation and the loss of circulating glucocorticoids had differential effects on AVP gene expression in the PVN and SON, and that the magnocellular PVN and SON neurons responded in different manners to the loss of feedback signals.

  14. 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.

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

    Science.gov (United States)

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

    2012-01-01

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

  16. 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.

  17. 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.

  18. The Circadian Clock Modulates Global Daily Cycles of mRNA Ribosome Loading.

    Science.gov (United States)

    Missra, Anamika; Ernest, Ben; Lohoff, Tim; Jia, Qidong; Satterlee, James; Ke, Kenneth; von Arnim, Albrecht G

    2015-09-01

    Circadian control of gene expression is well characterized at the transcriptional level, but little is known about diel or circadian control of translation. Genome-wide translation state profiling of mRNAs in Arabidopsis thaliana seedlings grown in long day was performed to estimate ribosome loading per mRNA. The experiments revealed extensive translational regulation of key biological processes. Notably, translation of mRNAs for ribosomal proteins and mitochondrial respiration peaked at night. Central clock mRNAs are among those subject to fluctuations in ribosome loading. There was no consistent phase relationship between peak translation states and peak transcript levels. The overlay of distinct transcriptional and translational cycles can be expected to alter the waveform of the protein synthesis rate. Plants that constitutively overexpress the clock gene CCA1 showed phase shifts in peak translation, with a 6-h delay from midnight to dawn or from noon to evening being particularly common. Moreover, cycles of ribosome loading that were detected under continuous light in the wild type collapsed in the CCA1 overexpressor. Finally, at the transcript level, the CCA1-ox strain adopted a global pattern of transcript abundance that was broadly correlated with the light-dark environment. Altogether, these data demonstrate that gene-specific diel cycles of ribosome loading are controlled in part by the circadian clock.

  19. Hypoxia and Human Genome Stability: Downregulation of BRCA2 Expression in Breast Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Daniele Fanale

    2013-01-01

    Full Text Available Previously, it has been reported that hypoxia causes increased mutagenesis and alteration in DNA repair mechanisms. In 2005, an interesting study showed that hypoxia-induced decreases in BRCA1 expression and the consequent suppression of homologous recombination may lead to genetic instability. However, nothing is yet known about the involvement of BRCA2 in hypoxic conditions in breast cancer. Initially, a cell proliferation assay allowed us to hypothesize that hypoxia could negatively regulate the breast cancer cell growth in short term in vitro studies. Subsequently, we analyzed gene expression in breast cancer cell lines exposed to hypoxic condition by microarray analysis. Interestingly, genes involved in DNA damage repair pathways such as mismatch repair, nucleotide excision repair, nonhomologous end-joining and homologous recombination repair were downregulated. In particular, we focused on the BRCA2 downregulation which was confirmed at mRNA and protein level. In addition, breast cancer cells were treated with dimethyloxalylglycine (DMOG, a cell-permeable inhibitor of both proline and asparaginyl hydroxylases able to induce HIF-1α stabilization in normoxia, providing results comparable to those previously described. These findings may provide new insights into the mechanisms underlying genetic instability mediated by hypoxia and BRCA involvement in sporadic breast cancers.

  20. 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.

  1. Selenoprotein P mRNA expression in human hepatic tissues

    Institute of Scientific and Technical Information of China (English)

    Chun-Li Li; Ke-Jun Nan; Tao Tian; Chen-Guang Sui; Yan-Fang Liu

    2007-01-01

    AIM: To investigate the expression of Selenoprotein P mRNA (SePmRNA) in tissues of normal liver, liver cirrhosis and hepatocellular carcinoma (HCC), and its relationship with HCC occurrence and development.METHODS: The expression of SePmRNA in tissues of normal liver, liver cirrhosis and HCC were detected by in situ hybridization using a cDNA probe.RESULTS: The enzyme digesting products of pBluescript-Human Selenoprotein P were evaluated by electrophoresis.The positive expression of SePmRNA was found in the tissues of normal liver,liver cirrhosis and HCC.The expression of SeP mRNA was found in hepatic interstitial substance,especially in endothelial cells and lymphocytes of vasculature.The positive rate of SePmRNA in normal liver tissue was 84.6% (11/13) and the positive signals appeared in the nucleus and cytoplasm,mostly in the nucleolus,and the staining granules were larger in the nucleolus and around the nucleus.The positive rate of SePmRNA in liver cirrhosis tissue was 45.O% (9/20) and the positive signals were mainly in the nucleolus and cytoplasm,being less around the nucleus and inner nucleus than that in normal liver tissue. The positive rate of SePmRNA in HCC tissue was 30.0% (9/30) and the positive signals were in the cytoplasm, but less in the nucleus.CONCLUSION: SePmRNA expression in the tissues of normal liver and HCC is significantly different (84.6% vs 30.0%, P = 0.003), suggesting that SeP might play a role in the occurrence and development of HCC.

  2. Genetic alteration in hepatocellular carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yoo Chul; Kang, Tae Woong; Lee, Jin Oh [Korea Cancer Center Hospital of Korea Atomic Energy Research Institute, Seoul (Korea, Republic of)

    1994-12-01

    Cancer of stomach, colon and liver are a group of the most common cancer in Korea. However, results with current therapeutic modalities are still unsatisfactory. The intensive efforts have been made to understand basic pathogenesis and to find better therapeutic tools for the treatment of this miserable disease. We studied the alteration of tumor suppressor genes and oncogenes in hepatocellular carcinoma in Korea. We found that alteration of Rb gene, APC were 33 %, 13 % respectively. But alterations of oncogenes such as myc, ras and mdm2 were rarely found. Our results suggests that HBV may act as oncogenic role in hepatocarcinogenesis instead of oncogenes. 6 figs, 2 tabs. (Author).

  3. 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.

  4. Base Pairing Interaction between 5′- and 3′-UTRs Controls icaR mRNA Translation in Staphylococcus aureus

    Science.gov (United States)

    Ruiz de los Mozos, Igor; Vergara-Irigaray, Marta; Segura, Victor; Villanueva, Maite; Bitarte, Nerea; Saramago, Margarida; Domingues, Susana; Arraiano, Cecilia M.; Fechter, Pierre; Romby, Pascale; Valle, Jaione; Solano, Cristina; Lasa, Iñigo; Toledo-Arana, Alejandro

    2013-01-01

    The presence of regulatory sequences in the 3′ untranslated region (3′-UTR) of eukaryotic mRNAs controlling RNA stability and translation efficiency is widely recognized. In contrast, the relevance of 3′-UTRs in bacterial mRNA functionality has been disregarded. Here, we report evidences showing that around one-third of the mapped mRNAs of the major human pathogen Staphylococcus aureus carry 3′-UTRs longer than 100-nt and thus, potential regulatory functions. We selected the long 3′-UTR of icaR, which codes for the repressor of the main exopolysaccharidic compound of the S. aureus biofilm matrix, to evaluate the role that 3′-UTRs may play in controlling mRNA expression. We showed that base pairing between the 3′-UTR and the Shine-Dalgarno (SD) region of icaR mRNA interferes with the translation initiation complex and generates a double-stranded substrate for RNase III. Deletion or substitution of the motif (UCCCCUG) within icaR 3′-UTR was sufficient to abolish this interaction and resulted in the accumulation of IcaR repressor and inhibition of biofilm development. Our findings provide a singular example of a new potential post-transcriptional regulatory mechanism to modulate bacterial gene expression through the interaction of a 3′-UTR with the 5′-UTR of the same mRNA. PMID:24367275

  5. Zinc finger protein 407 (ZFP407) regulates insulin-stimulated glucose uptake and glucose transporter 4 (Glut4) mRNA.

    Science.gov (United States)

    Buchner, David A; Charrier, Alyssa; Srinivasan, Ethan; Wang, Li; Paulsen, Michelle T; Ljungman, Mats; Bridges, Dave; Saltiel, Alan R

    2015-03-06

    The glucose transporter GLUT4 facilitates insulin-stimulated glucose uptake in peripheral tissues including adipose, muscle, and heart. GLUT4 function is impaired in obesity and type 2 diabetes leading to hyperglycemia and an increased risk of cardiovascular disease and neuropathy. To better understand the regulation of GLUT4 function, a targeted siRNA screen was performed and led to the discovery that ZFP407 regulates insulin-stimulated glucose uptake in adipocytes. The decrease in insulin-stimulated glucose uptake due to ZFP407 deficiency was attributed to a reduction in GLUT4 mRNA and protein levels. The decrease in GLUT4 was due to both decreased transcription of Glut4 mRNA and decreased efficiency of Glut4 pre-mRNA splicing. Interestingly, ZFP407 coordinately regulated this decrease in transcription with an increase in the stability of Glut4 mRNA, resulting in opposing effects on steady-state Glut4 mRNA levels. More broadly, transcriptome analysis revealed that ZFP407 regulates many peroxisome proliferator-activated receptor (PPAR) γ target genes beyond Glut4. ZFP407 was required for the PPARγ agonist rosiglitazone to increase Glut4 expression, but was not sufficient to increase expression of a PPARγ target gene reporter construct. However, ZFP407 and PPARγ co-overexpression synergistically activated a PPARγ reporter construct beyond the level of PPARγ alone. Thus, ZFP407 may represent a new modulator of the PPARγ signaling pathway.

  6. Dissecting non-canonical interactions in frameshift-stimulating mRNA pseudoknots

    Energy Technology Data Exchange (ETDEWEB)

    Cornish, Peter V. [University of Illinois at Urbana-Champaign, Department of Physics (United States); Giedroc, David P. [2128 TAMU, Texas A and M University, Department of Biochemistry and Biophysics (United States); Hennig, Mirko [Scripps Research Institute, MB33, Department of Molecular Biology (United States)], E-mail: hennig@musc.edu

    2006-07-15

    A variety of powerful NMR experiments have been introduced over the last few years that allow for the direct identification of different combinations of donor and acceptor atoms involved in hydrogen bonds in biomolecules. This ability to directly observe tertiary structural hydrogen bonds in solution tremendously facilitates structural studies of nucleic acids. We show here that an adiabatic HNN-COSY pulse scheme permits observation and measurement of J(N,N) couplings for nitrogen sites that are separated by up to 140 ppm in a single experiment at a proton resonance frequency of 500 MHz. Crucial hydrogen bond acceptor sites in nucleic acids, such as cytidine N3 nitrogens, can be unambiguously identified even in the absence of detectable H41 and H42 amino protons using a novel triple-resonance two-dimensional experiment, denoted H5(C5C4)N3. The unambiguous identification of amino nitrogen donor and aromatic nitrogen acceptor sites associated with both major groove as well as minor groove triple base pairs reveal the details of hydrogen bonding networks that stabilize the complex architecture of frameshift-stimulating mRNA pseudoknots. Another key tertiary interaction involving a 2'-OH hydroxyl proton that donates a hydrogen bond to an aromatic nitrogen acceptor in a cis Watson-Crick/sugar edge interaction can also be directly detected using a quantitative J(H,N) {sup 1}H,{sup 15}N-HSQC experiment.

  7. 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.

  8. Complement Activation Alters Platelet Function

    Science.gov (United States)

    2015-12-01

    Award Number: W81XWH-12-1-0523 TITLE: Complement Activation Alters Platelet Function PRINCIPAL INVESTIGATOR: George Tsokos, M.D. CONTRACTING...Activation Alters Platelet Function 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-12-1-0523 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) George Tsokos, M.D...a decreased level of disease. Further studies will expand upon these observations better outlining the function of platelets in the injury associated

  9. Gene expression analysis in sections and tissue microarrays of archival tissues by mRNA in situ hybridization.

    Science.gov (United States)

    Henke, R T; Maitra, A; Paik, S; Wellstein, A

    2005-01-01

    Altered expression of genes in diseased tissues can prognosticate a distinct natural progression of the disease as well as predict sensitivity or resistance to particular therapies. Archival tissues from patients with a known medical history and treatments are an invaluable resource to validate the utility of candidate genes for prognosis and prediction of therapy outcomes. However, stored tissues with associated long-term follow-up information typically are formalin-fixed, paraffin-embedded specimen and this can severely restrict the methods applicable for gene expression analysis. We report here on the utility of tissue microarrays (TMAs) that use valuable tissues sparingly and provide a platform for simultaneous analysis of gene expression in several hundred samples. In particular, we describe a stable method applicable to mRNA expression screening in such archival tissues. TMAs are constructed from sections of small drill cores, taken from tissue blocks of archival tissues and multiple samples can thus be arranged on a single microscope slide. We used mRNA in situ hybridization (ISH) on >500 full sections and >100 TMAs for >10 different cDNAs that yielded >10,000 data points. We provide detailed experimental protocols that can be implemented without major hurdles in a molecular pathology laboratory and discuss quantitative analysis and the advantages and limitations of ISH. We conclude that gene expression analysis in archival tissues by ISH is reliable and particularly useful when no protein detection methods are available for a candidate gene.

  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. Highest trkB mRNA expression in the entorhinal cortex among hippocampal subregions in the adult rat: contrasting pattern with BDNF mRNA expression.

    Science.gov (United States)

    Tokuyama, W; Hashimoto, T; Li, Y X; Okuno, H; Miyashita, Y

    1998-11-20

    Brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, regulate synaptic functions in the hippocampus of the adult rodent. In previous studies, in situ hybridization methods have been used to evaluate regional differences in BDNF and trkB mRNA expression levels in hippocampal subregions. However, these studies have failed to reach consensus regarding the regional differences in the mRNA expression levels. In the present study, we quantitated mRNA expression levels using two different methods, ribonuclease protection assays and a quantitative reverse-transcription polymerase chain reaction technique, in four hippocampal subregions: the entorhinal cortex, dentate gyrus (DG), CA3 and CA1. These two methods yielded the same results. We found that BDNF and trkB mRNA expression levels did not covary in the four subregions. BDNF and full length trkB (trkB FL) mRNA in the entorhinal cortex and the DG show contrasting expression patterns. The expression level of BDNF mRNA was highest in the DG among the hippocampal subregions and low in the entorhinal cortex and the CA1, whereas the trkB FL mRNA expression level was highest in the entorhinal cortex, low in the DG and lowest in the CA3. These results suggest regional differences in BDNF/TrkB signaling for maintenance and modifiability of neuronal connections in the hippocampal formation.

  12. Decreased AMPA GluR2, but not GluR3, mRNA expression in rat amygdala and dorsal hippocampus following morphine-induced behavioural sensitization.

    Science.gov (United States)

    Sepehrizadeh, Zargham; Bahrololoumi Shapourabadi, Mina; Ahmadi, Shamseddin; Hashemi Bozchlou, Saeed; Zarrindast, Mohammad-Reza; Sahebgharani, Mousa

    2008-11-01

    1. Repeated administration of psychostimulants and micro-opioid receptor agonists elicits a progressive enhancement of drug-induced behavioural responses, a phenomenon termed behavioural sensitization. These changes in behaviour may reflect plastic changes requiring regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptor function. 2. In the present study, rats were treated for 7 days with saline or morphine (10 mg/kg). After a washout period of either 24 h or 7 days, locomotion, oral stereotypy and state-dependent memory in a passive avoidance test were measured in the presence or absence of 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt (CNQX; 3 mg/kg), an AMPA receptor antagonist. In order to evaluate the mechanism underlying the behavioural responses, quantitative real-time reverse transcription-polymerase chain reaction was used to evaluate mRNA expression of the AMPA receptor subunits GluR2 and GluR3 in the striatum, prefrontal cortex, hippocampus, hypothalamus and amygdala of animals treated repeatedly with morphine. 3. The results indicate that repeated morphine treatment followed by 7 days (but not 24 h) washout produces behavioural sensitization, as determined by locomotion, oral stereotypy and state-dependent memory. Blockade of AMPA receptors with CNQX on the test day did not alter these behavioural responses. In addition, repeated morphine treatment followed by 7 days (but not 24 h) washout decreased GluR2 mRNA expression in both the amygdala (by 50%) and hippocampus (by 35%). Repeated morphine treatment did not alter GluR3 mRNA expression in any brain area assessed. 4. These data imply that AMPA receptors are involved in the development (but not expression) phase of behavioural sensitization. The decreases in GluR2 mRNA expression in the amygdala and hippocampus may result in the formation of calcium-permeable AMPA receptors, which are believed to play an important role in behavioural sensitization.

  13. 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.

  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. Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

    Science.gov (United States)

    Tong, X; Kono, T; Evans-Molina, C

    2015-06-18

    The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b

  16. 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.

  17. A FQ-RT-PCR method for quantitative determination of IL-2 mRNA and IL-4 mRNA and its application

    Institute of Scientific and Technical Information of China (English)

    QING HUI ZHU; QING LU; GU SHENG ZHANG

    2006-01-01

    The purpose of the study is to establish a fluorescence quantitative reverse transcription polymerase chain response (FQ-RT-PCR) method for the quantitative determination of IL-2 mRNA and IL-4mRNA in Th cells, with which the Th cells status of the patients with gynaecological tumors and chronic renal failure (CRF) can be analyzed. IL-2 cDNA and IL-4 cDNA were prepared, and the plasmid pMD18 carrying IL-2 cDNA or IL-4 cDNA fragment was constructed and cloned as the template for quantitative determination. The primers and probes labelled with 6-carboxy-fluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) were prepared, and the experimental conditions were optimized to set up the FQ-RT-PCR method for quantitative determination of IL-2 mRNA and IL-4 mRNA. Th cells enriched from peripheral blood mononuclear cells (PBMCs) of 20 healthy volunteers (HVs), 16 gynaecological benign (GB) cases, 18 gynaecological malignant (GM) tumor cases and 16 chronic renal failure (CRF) patients were tested for IL-2 mRNA and IL-4 mRNA by FQ-RT-PCR. The house-keeping gene 3-actin was used as the internal control gene of the experiment. The standard curve for log concentration of series of quantitative templates vs threshold cycle (CT) was established by linear regression, and the linear range was 102-107 copies/μl. The imprecision test showed the CV of inter-assay and intra-assay of a high content sample by FQ-RT-PCR were 7.8% and 12.5%, respectively. The CV of inter-assay and intra-assay of a low content sample were 10.8% and 19.5%, respectively. The IL-2 mRNA expressions in Th of the patients with gynaecological malignant tumor (compared with the HVs and the patients with gynaecological benign disease) and in Th of the CRF patients (compared with the HVs) were declined significantly and at the same time the IL-4 mRNA expression increased significantly ( P < 0. 001 ). A simple, sensitive and accurate FQ-RT-PCR method for the quantitative detection of IL-2 mRNA and IL-4 mRNA has

  18. Relative Expression of HIF-1α mRNA in Rat Heart, Brain and Blood During Induced Systemic Hypoxia

    Directory of Open Access Journals (Sweden)

    Syarifah Dewi

    2009-11-01

    Full Text Available Hypoxia is a pathological condition in which the body as a whole or region of the body (tissue or cell deprived of adequate oxygen supply. The transcriptional regulator hypoxia inducible factor-1 (HIF-1 is an essential mediator of O2 homeostasis. Unlike the β sub unit (HIF-1β, the activity of HIF-1α is controlled in an oxygen-dependent manner. It has been reported that the stability and expression of HIF-1α during hypoxia is remarkably higher than those under normoxic conditions.The aim of this study was to analyze the adaptive tissue responses during induced systemic hypoxia by comparation of relative expression of mRNA HIF-1α in rat heart, brain and blood. Twenty-five male Sprague Dawley rats were subjected to systemic hypoxia by placing them in the hypoxic chamber supplied by 8-10% of O2 for 0, 1, 7, 14 and 21 days, respectively. The relative expression level of HIF-1α mRNA in brain, heart and leucocyte cells were analyzed using quantitative RT-PCR assay (Real Time PCR based on Pfaff's formula. This study demonstrates that the increased of relative expression of HIF-1α mRNA during induced systemic hypoxia reached its maximum level at day 7 (in heart or at day 14 (in brain, whereas in leucocyte cells the stimulation of HIF-1α expression was intensively maintained up to 21 days although the expression has reached the remarkably high level. We could conclude that HIF-1α as an oxygen sensing during systemic hypoxia has different capacity and sensitivity in brain, heart and blood tissues, due to the importance of oxygen homeostasis in each tissue.

  19. Analysis of MDM2 and MDM4 single nucleotide polymorphisms, mRNA splicing and protein expression in retinoblastoma.

    Directory of Open Access Journals (Sweden)

    Justina McEvoy

    Full Text Available Retinoblastoma is a childhood cancer of the developing retina that begins in utero and is diagnosed in the first years of life. Biallelic RB1 gene inactivation is the initiating genetic lesion in retinoblastoma. The p53 gene is intact in human retinoblastoma but the pathway is believed to be suppressed by increased expression of MDM4 (MDMX and MDM2. Here we quantify the expression of MDM4 and MDM2 mRNA and protein in human fetal retinae, primary retinoblastomas, retinoblastoma cell lines and several independent orthotopic retinoblastoma xenografts. We found that MDM4 is the major p53 antagonist expressed in retinoblastoma and in the developing human retina. We also discovered that MDM4 protein steady state levels are much higher in retinoblastoma than in human fetal retinae. This increase would not have been predicted based on the mRNA levels. We explored several possible post-transcriptional mechanisms that may contribute to the elevated levels of MDM4 protein. A proportion of MDM4 transcripts are alternatively spliced to produce protein products that are reported to be more stable and oncogenic. We also discovered that a microRNA predicted to target MDM4 (miR191 was downregulated in retinoblastoma relative to human fetal retinae and a subset of samples had somatic mutations that eliminated the miR-191 binding site in the MDM4 mRNA. Taken together, these data suggest that post-transcriptional mechanisms may contribute to stabilization of the MDM4 protein in retinoblastoma.

  20. Iron deficiency alters megakaryopoiesis and platelet phenotype independent of thrombopoietin.

    Science.gov (United States)

    Evstatiev, Rayko; Bukaty, Adam; Jimenez, Kristine; Kulnigg-Dabsch, Stefanie; Surman, Lidia; Schmid, Werner; Eferl, Robert; Lippert, Kathrin; Scheiber-Mojdehkar, Barbara; Kvasnicka, Hans Michael; Khare, Vineeta; Gasche, Christoph

    2014-05-01

    Iron deficiency is a common cause of reactive thrombocytosis, however, the exact pathways have not been revealed. Here we aimed to study the mechanisms behind iron deficiency-induced thrombocytosis. Within few weeks, iron-depleted diet caused iron deficiency in young Sprague-Dawley rats, as reflected by a drop in hemoglobin, mean corpuscular volume, hepatic iron content and hepcidin mRNA in the liver. Thrombocytosis established in parallel. Moreover, platelets produced in iron deficient animals displayed a higher mean platelet volume and increased aggregation. Bone marrow studies revealed subtle alterations that are suggestive of expansion of megakaryocyte progenitors, an increase in megakaryocyte ploidy and accelerated megakaryocyte differentiation. Iron deficiency did not alter the production of hematopoietic growth factors such as thrombopoietin, interleukin 6 or interleukin 11. Megakaryocytic cell lines grown in iron-depleted conditions exhibited reduced proliferation but increased ploidy and cell size. Our data suggest that iron deficiency increases megakaryopoietic differentiation and alters platelet phenotype without changes in megakaryocyte growth factors, specifically TPO. Iron deficiency-induced thrombocytosis may have evolved to maintain or increase the coagulation capacity in conditions with chronic bleeding.

  1. Occlusal effects on longitudinal bone alterations of the temporomandibular joint.

    Science.gov (United States)

    Zhang, J; Jiao, K; Zhang, M; Zhou, T; Liu, X-D; Yu, S-B; Lu, L; Jing, L; Yang, T; Zhang, Y; Chen, D; Wang, M-Q

    2013-03-01

    The pathological changes of subchondral bone during osteoarthritis (OA) development in the temporomandibular joint (TMJ) are poorly understood. In the present study, we investigated the longitudinal alterations of subchondral bone using a rat TMJ-OA model developed in our laboratory. Changes in bone mass were examined by micro-CT, and changes in osteoblast and osteoclast activities were analyzed by real-time PCR, immunohistochemistry, and TRAP staining. Subchondral bone loss was detected from 8 weeks after dental occlusion alteration and reached the maximum at 12 weeks, followed by a repair phase until 32 weeks. Although bone mass increased at late stages, poor mechanical structure and lower bone mineral density (BMD) were found in these rats. The numbers of TRAP-positive cells were increased at 12 weeks, while the numbers of osteocalcin-expressing cells were increased at both 12 and 32 weeks. Levels of mRNA expression of TRAP and cathepsin K were increased at 12 weeks, while levels of ALP and osteocalcin were increased at both 12 and 32 weeks. These findings demonstrated that there is an active bone remodeling in subchondral bone in TMJs in response to alteration in occlusion, although new bone was formed with lower BMD and poor mechanical properties.

  2. Altered Esophageal Mucosal Structure in Patients with Celiac Disease

    Science.gov (United States)

    Pinto-Sánchez, María Inés; Nachman, Fabio D.; Fuxman, Claudia; Iantorno, Guido; Hwang, Hui Jer; Ditaranto, Andrés; Costa, Florencia; Longarini, Gabriela; Wang, Xuan Yu; Huang, Xianxi; Vázquez, Horacio; Moreno, María L.; Niveloni, Sonia; Bercik, Premysl; Smecuol, Edgardo; Mazure, Roberto; Bilder, Claudio; Mauriño, Eduardo C.; Verdu, Elena F.; Bai, Julio C.

    2016-01-01

    Background/Aim. Reflux symptoms (RS) are common in patients with celiac disease (CD), a chronic enteropathy that affects primarily the small intestine. We evaluated mucosal integrity and motility of the lower esophagus as mechanisms contributing to RS generation in patients with CD. Methods. We enrolled newly diagnosed CD patients with and without RS, nonceliac patients with classical reflux disease (GERD), and controls (without RS). Endoscopic biopsies from the distal esophagus were assessed for dilated intercellular space (DIS) by light microscopy and electron microscopy. Tight junction (TJ) mRNA proteins expression for zonula occludens-1 (ZO-1) and claudin-2 and claudin-3 (CLDN-2; CLDN-3) was determined using qRT-PCR. Results. DIS scores were higher in patients with active CD than in controls, but similar to GERD patients. The altered DIS was found even in CD patients without RS and normalized after one year of a gluten-free diet. CD patients with and without RS had lower expression of ZO-1 than controls. The expression of CLDN-2 and CLDN-3 was similar in CD and GERD patients. Conclusions. Our study shows that patients with active CD have altered esophageal mucosal integrity, independently of the presence of RS. The altered expression of ZO-1 may underlie loss of TJ integrity in the esophageal mucosa and may contribute to RS generation. PMID:27446827

  3. Repeated stress increases catalytic TrkB mRNA in rat hippocampus.

    Science.gov (United States)

    Nibuya, M; Takahashi, M; Russell, D S; Duman, R S

    1999-05-28

    Northern blot analysis was utilized to distinguish between catalytic and truncated TrkB mRNA on the basis of transcript size. Repeated (10 days), but not acute, immobilization stress significantly increased levels of catalytic TrkB mRNA, but did not influence expression of truncated TrkB transcripts in rat hippocampus. Exposure to another paradigm, a combination of different, unpredictable stressors, also increased levels of catalytic, but not truncated, TrkB mRNA. In situ hybridization analysis demonstrated that chronic stress up-regulated TrkB mRNA in CA1 and CA3 pyramidal and dentate gyrus granule cells layers of hippocampus. As previously reported, both acute and chronic immobilization stress decreased expression of BDNF mRNA, suggesting that up-regulation of catalytic TrkB mRNA may be a compensatory adaptation to repeated stress.

  4. Schistocephalus solidus infections increase gonadotropins and gonadotropin releasing hormone (GnRH3) mRNA levels in the three-spined stickleback, Gasterosteus aculeatus.

    Science.gov (United States)

    Shao, Yi Ta; Tseng, Yung Che; Trombley, Susanne; Hwang, Pung Pung; Schmitz, Monika; Borg, Bertil

    2012-09-01

    Parasites often impair the reproduction of their hosts, one well known case being the cestode Schistocephalus solidus which is a common parasite in three-spined sticklebacks, Gasterosteus aculeatus. One of the possible ways that this could be exerted is by suppression on the brain-pituitary-gonadal (BPG) axis. In this study, mRNA levels of FSH-β and LH-β and of GnRH2 (cGnRH II) and GnRH3 (sGnRH) were measured via Q-PCR in infected and uninfected fish sampled from the field a few weeks before the onset of breeding. The pituitary mRNA levels of both FSH-β and LH-β were higher in infected males than in uninfected males. Also in females, FSH-β mRNA levels were higher in infected individuals than in others, whereas there was no significant difference found in LH-β expression. Brain mRNA levels of GnRH3 were higher in infected fish than in uninfected fish in both sexes, but no difference was found in GnRH2 mRNA levels. Thus, infection by S. solidus was able to alter the expressions not only of gonadotropins (GtHs), but also of GnRH which has not been observed previously. However, the effects are opposite to what should be expected if the parasite suppressed reproduction via actions on the brain-pituitary level. The gonads are perhaps more likely to be impaired by the parasites in other ways, and changed feedbacks on the BPG axis could then lead to the increases in GtHs and GnRH.

  5. Gonadotropin-induced changes in oviducal mRNA expression levels of sex steroid hormone receptors and activin-related signaling factors in the alligator

    Science.gov (United States)

    Moore, Brandon C.; Forouhar, Sara; Kohno, Satomi; Botteri, Nicole L.; Hamlin, Heather J.; Guillette, Louis J.

    2011-01-01

    Oviducts respond to hormonal cues from ovaries with tissue proliferation and differentiation in preparation of transporting and fostering gametes. These responses produce oviducal microenvironments conducive to reproductive success. Here we investigated changes in circulating plasma sex steroid hormones concentrations and ovarian and oviducal mRNA expression to an in vivo gonadotropin (FSH) challenge in sexually immature, five-month-old alligators. Further, we investigated differences in these observed responses between alligators hatched from eggs collected at a heavily-polluted (Lake Apopka, FL) and minimally-polluted (Lake Woodruff, FL) site. In oviducts, we measured mRNA expression of estrogen, progesterone, and androgen receptors and also beta A and B subunits which homo- or heterodimerize to produce the transforming growth factor activin. In comparison, minimal inhibin alpha subunit mRNA expression suggests that these oviducts produce a primarily activin-dominated signaling milieu. Ovaries responded to a five-day FSH challenge with increased expression of steroidogenic enzyme mRNA which was concomitant with increased circulating sex steroid hormone concentrations. Oviducts in the FSH-challenged Lake Woodruff alligators increased mRNA expression of progesterone and androgen receptors, proliferating cell nuclear antigen, and the activin signaling antagonist follistatin. In contrast, Lake Apopka alligators displayed a diminished increase in ovarian CYP19A1 aromatase expression and no increase in oviducal AR expression, as compared to those observed in Lake Woodruff alligators. These results demonstrate that five-month-old female alligators display an endocrine-responsive ovarian-oviducal axis and environmental pollution exposure may alter these physiological responses. PMID:22154572

  6. Dynamics of translation by single ribosomes through mRNA secondary structures

    OpenAIRE

    Chen, Chunlai; Zhang, Haibo; Broitman, Steven L.; Reiche, Michael; Farrell, Ian; Cooperman, Barry S.; Goldman, Yale E.

    2013-01-01

    During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary (2°) structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we employ single molecule fluorescence resonance energy transfer to determine reaction rates for specific steps within the elongation cycle as the Escherichia coli ribosome encounters stem loop or pseudoknot mRNA 2° structures. Downstream ...

  7. 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.

  8. 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.

  9. Transcriptome-wide mapping reveals widespread dynamic regulated pseudouridylation of ncRNA and mRNA

    Science.gov (United States)

    Schwartz, Schraga; Bernstein, Douglas A.; Mumbach, Maxwell R.; Jovanovic, Marko; Herbst, Rebecca H.; León-Ricardo, Brian X.; Engreitz, Jesse M.; Guttman, Mitchell; Satija, Rahul; Lander, Eric S.; Fink, Gerald; Regev, Aviv

    2014-01-01

    SUMMARY Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of novel sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUSs) uncovers which PUSs modify each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided PUSs. Upon heat shock in yeast, Pus7-mediated pseudouridylation is induced at >200 sites and Pus7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved, but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcritome-wide scope for pseudouridine, and methods to dissect its underlying mechanisms and function. PMID:25219674

  10. Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA.

    Science.gov (United States)

    Schwartz, Schraga; Bernstein, Douglas A; Mumbach, Maxwell R; Jovanovic, Marko; Herbst, Rebecca H; León-Ricardo, Brian X; Engreitz, Jesse M; Guttman, Mitchell; Satija, Rahul; Lander, Eric S; Fink, Gerald; Regev, Aviv

    2014-09-25

    Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function.

  11. Effects of Hypoxanthine Substitution in Peptide Nucleic Acids Targeting KRAS2 Oncogenic mRNA Molecules: Theory and Experiment

    Science.gov (United States)

    Sanders, Jeffrey M.; Wampole, Matthew E.; Chen, Chang-Po; Sethi, Dalip; Singh, Amrita; Dupradeau, François-Yves; Wang, Fan; Gray, Brian D.; Thakur, Mathew L.; Wickstrom, Eric

    2013-01-01

    Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multi-mutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick basepairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA-PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA-PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition. PMID:23972113

  12. Aging of dynamically stabilized microtubules

    CERN Document Server

    Ebbinghaus, M

    2009-01-01

    The microtubule network, an important part of the cytoskeleton, is constantly remodeled by alternating phases of growth and shrinkage of individual filaments. Plus-end tracking proteins (+TIPs) interact with the microtubule and in many cases alter its dynamics. While it is established that the prototypal CLIP-170 enhances microtubule stability by increasing rescues, the plus-end tracking mechanism is still under debate. We present a model for microtubule dynamics in which a rescue factor is dynamically added to the filament while growing. As a consequence, the filament shows aging behavior which should be experimentally accessible and thus allow one to exclude some hypothesized models of the inclusion of rescue factors at the microtubule plus end. Additionally, we show the strong influence of the cell geometry on the quantitative results.

  13. Daily rhythms of plasma melatonin, but not plasma leptin or leptin mRNA, vary between lean, obese and type 2 diabetic men.

    Science.gov (United States)

    Mäntele, Simone; Otway, Daniella T; Middleton, Benita; Bretschneider, Silvia; Wright, John; Robertson, M Denise; Skene, Debra J; Johnston, Jonathan D

    2012-01-01

    Melatonin and leptin exhibit daily rhythms that may contribute towards changes in metabolic physiology. It remains unclear, however, whether this rhythmicity is altered in obesity or type 2 diabetes (T2DM). We tested the hypothesis that 24-hour profiles of melatonin, leptin and leptin mRNA are altered by metabolic status in laboratory conditions. Men between 45-65 years old were recruited into lean, obese-non-diabetic or obese-T2DM groups. Volunteers followed strict sleep-wake and dietary regimes for 1 week before the laboratory study. They were then maintained in controlled light-dark conditions, semi-recumbent posture and fed hourly iso-energetic drinks during wake periods. Hourly blood samples were collected for hormone analysis. Subcutaneous adipose biopsies were collected 6-hourly for gene expression analysis. Although there was no effect of subject group on the timing of dim light melatonin onset (DLMO), nocturnal plasma melatonin concentration was significantly higher in obese-non-diabetic subjects compared to weight-matched T2DM subjects (peffect of subject group on absolute plasma leptin concentration and, when expressed relative to an individual's 24-hour mean, plasma leptin showed significant (peffect of time on leptin mRNA expression. Despite an overall effect (peffects of group on leptin mRNA expression. Altered plasma melatonin rhythms in weight-matched T2DM and non-diabetic individuals supports a possible role of melatonin in T2DM aetiology. However, neither obesity nor T2DM changed 24-hour rhythms of plasma leptin relative to cycle mean, or expression of subcutaneous adipose leptin gene expression, compared with lean subjects.

  14. Correlation of mRNA and protein in complex biological samples.

    Science.gov (United States)

    Maier, Tobias; Güell, Marc; Serrano, Luis

    2009-12-17

    The correlation between mRNA and protein abundances in the cell has been reported to be notoriously poor. Recent technological advances in the quantitative analysis of mRNA and protein species in complex samples allow the detailed analysis of this pathway at the center of biological systems. We give an overview of available methods for the identification and quantification of free and ribosome-bound mRNA, protein abundances and individual protein turnover rates. We review available literature on the correlation of mRNA and protein abundances and discuss biological and technical parameters influencing the correlation of these central biological molecules.

  15. Nuclear imprisonment: viral strategies to arrest host mRNA nuclear export.

    Science.gov (United States)

    Kuss, Sharon K; Mata, Miguel A; Zhang, Liang; Fontoura, Beatriz M A

    2013-07-01

    Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

  16. Regulation of mRNA translation during mitosis.

    Science.gov (United States)

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

    2015-08-25

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

  17. 6-Hydroxydopamine-induced glutathione alteration occurs via glutathione enzyme system in primary cultured astrocytes

    Institute of Scientific and Technical Information of China (English)

    Ji ZHANG; Jun HU; Jian-hua DING; Hong-hong YAO; Gang HU

    2005-01-01

    Aim: To define the role of enzymes involved in glutathione metabolism in 6-hydroxydopamine (6-OHDA)-induced glutathione alteration in primary cultured astrocytes.Methods: Total glutathione (GSx) levels were determined using the modified enzymatic microtiter plate assay.The mRNA levels ofγ-glutamylcysteine synthetase (γGCS), γ-glutamyltransferase (γGT), glutathione peroxidase (GPx), GR (glutathione reductase), and glutathione transferases (GST) were determined using RT-PCR.γGT activity was determined using γGT assay kits.Results: In primary cultured astrocytes, 6-OHDA induced a significant elevation of cellular GSx levels after treatment for 24 h.However, the GSx levels decreased after 24 h and the values were even lower than the value in the control group without 6-OHDA at 48 h.RT-PCR data showed that the mRNA levels of γGCS, the ratelimiting enzyme of γ-L-glutamyl-L-cysteinylglycine (GSH) synthesis, were increased by 6-OHDA after treatment for 24 h and 48 h; the mRNA levels of GPx, GR, and GST did not alter in 6-OHDA-treated astrocytes after treatment for 24 h and 48 h; and 6-OHDA increased the mRNA levels and the activity of γGT after treatment for 48 h,which induced a decrease in GSx levels, despite the up-regulation of γGCS after exposure to 6-OHDA for 48 h.Conclusion: The change in γGCS correlated with the increase in GSH levels induced by 6-OHDA after treatment for 24 h.GSx levels decreased because of increased γGT mRNA levels and γGT activity induced by 6-OHDA after treatment for 48 h.

  18. 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.

  19. Topological stabilization for synchronized dynamics on networks

    Science.gov (United States)

    Cencetti, Giulia; Bagnoli, Franco; Battistelli, Giorgio; Chisci, Luigi; Di Patti, Francesca; Fanelli, Duccio

    2017-01-01

    A general scheme is proposed and tested to control the symmetry breaking instability of a homogeneous solution of a spatially extended multispecies model, defined on a network. The inherent discreteness of the space makes it possible to act on the topology of the inter-nodes contacts to achieve the desired degree of stabilization, without altering the dynamical parameters of the model. Both symmetric and asymmetric couplings are considered. In this latter setting the web of contacts is assumed to be balanced, for the homogeneous equilibrium to exist. The performance of the proposed method are assessed, assuming the Complex Ginzburg-Landau equation as a reference model. In this case, the implemented control allows one to stabilize the synchronous limit cycle, hence time-dependent, uniform solution. A system of coupled real Ginzburg-Landau equations is also investigated to obtain the topological stabilization of a homogeneous and constant fixed point.

  20. Elongation factor-2 phosphorylation in dendrites and the regulation of dendritic mRNA translation in neurons

    Directory of Open Access Journals (Sweden)

    Christopher eHeise

    2014-02-01

    Full Text Available Neuronal activity results in long lasting changes in synaptic structure and function by regulating mRNA translation in dendrites. These activity dependent events yield the synthesis of proteins known to be important for synaptic modifications and diverse forms of synaptic plasticity. Worthy of note, there is accumulating evidence that the eukaryotic Elongation Factor 2 Kinase (eEF2K/eukaryotic Elongation Factor 2 (eEF2 pathway may be strongly involved in this process. Upon activation, eEF2K phosphorylates and thereby inhibits eEF2, resulting in a dramatic reduction of mRNA translation. eEF2K is activated by elevated levels of calcium and binding of Calmodulin (CaM, hence its alternative name calcium/CaM-dependent protein kinase III (CaMKIII. In dendrites, this process depends on glutamate signaling and N-methyl-D-aspartate receptor (NMDAR activation. Interestingly, it has been shown that eEF2K can be activated in dendrites by the metabotropic glutamate receptor (mGluR 1/5 signaling, as well. Therefore, neuronal activity can induce local proteomic changes at the postsynapse by altering eEF2K activity. Well-established targets of eEF2K in dendrites include Brain-derived neurotrophic factor (BDNF, activity-regulated cytoskeletal-associated protein (Arc, the alpha subunit of calcium/CaM-dependent protein kinase II (αCaMKII, and Microtubule-associated protein 1B (MAP1B, all of which have well-known functions in different forms of synaptic plasticity.In this review we will give an overview of the involvement of the eEF2K/eEF2 pathway at dendrites in regulating the translation of dendritic mRNA in the context of altered NMDAR- and neuronal activity, and diverse forms of synaptic plasticity, such as metabotropic glutamate receptor-dependent-long-term depression (mGluR-LTD. For this, we draw on studies carried out both in vitro and in vivo.

  1. Yeast mitochondrial biogenesis: a role for the PUF RNA-binding protein Puf3p in mRNA localization.

    Directory of Open Access Journals (Sweden)

    Yann Saint-Georges

    Full Text Available The asymmetric localization of mRNA plays an important role in coordinating posttranscriptional events in eukaryotic cells. We investigated the peripheral mitochondrial localization of nuclear-encoded mRNAs (MLR in various conditions in which the mRNA binding protein context and the translation efficiency were altered. We identified Puf3p, a Pumilio family RNA-binding protein, as the first trans-acting factor controlling the MLR phenomenon. This allowed the characterization of two classes of genes whose mRNAs are translated to the vicinity of mitochondria. Class I mRNAs (256 genes have a Puf3p binding motif in their 3'UTR region and many of them have their MLR properties deeply affected by PUF3 deletion. Conversely, mutations in the Puf3p binding motif alter the mitochondrial localization of BCS1 mRNA. Class II mRNAs (224 genes have no Puf3p binding site and their asymmetric localization is not affected by the absence of PUF3. In agreement with a co-translational import process, we observed that the presence of puromycin loosens the interactions between most of the MLR-mRNAs and mitochondria. Unexpectedly, cycloheximide, supposed to solidify translational complexes, turned out to destabilize a class of mRNA-mitochondria interactions. Classes I and II mRNAs, which are therefore transported to the mitochondria through different pathways, correlated with different functional modules. Indeed, Class I genes code principally for the assembly factors of respiratory chain complexes and the mitochondrial translation machinery (ribosomes and translation regulators. Class II genes encode proteins of the respiratory chain or proteins involved in metabolic pathways. Thus, MLR, which is intimately linked to translation control, and the activity of mRNA-binding proteins like Puf3p, may provide the conditions for a fine spatiotemporal control of mitochondrial protein import and mitochondrial protein complex assembly. This work therefore provides new openings

  2. Dose-response of Berberine on Hepatic Cytochromes P450 mRNA Expression and Activities in Mice

    Science.gov (United States)

    Guo, Ying; Pope, Chad; Cheng, Xingguo; Zhou, Honghao; Klaassen, Curtis D.

    2011-01-01

    Ethnopharmacological relevance Berberine is an isoquinoline alkaloid isolated from the root and bark of plants such as goldenseal, Berberis, and Chinese goldthread. Berberine-containing crude drugs have been used as an antimicrobial remedy against gastrointestinal infections for thousands of years. It is also widely used in Asian countries for diabetes, hypertension, and hypercholesterolemia therapy. Aim of the Study Potential drug-drug interactions are of concern because of the wide usage of berberine. A few studies have reported interactions between berberine and cytochromes P450 (CYPs) in vitro, but little is known about whether berberine influences CYPs in vivo, especially after repeated administration. In this study, eight-week-old male C57BL/6 mice were given berberine orally (0,10, 30, 100, 300 mg/kg, i.g., daily for 14 days), and the effect of berberine on over 20 major Cyps and related nuclear receptors in mice livers were examined at both the mRNA and enzyme activity levels. Results In general, liver function of mice treated with various doses of berberine had no significant change, and repeated oral administration of the 3 lower doses of berberine for 14 days did not affect the expression of genes examined. However, after the highest dose of berberine (300 mg/kg), Cyp3a11 and Cyp3a25 mRNA decreased 67.6 and 87.4%, respectively, whereas Cyp1a2 mRNA increased 43.2%, and enzyme activities of Cyp3a11 and Cyp2d22 decreased 67.9 and 32.4%, respectively. Cyp2a4, 2b10 and Cyp2c29 were not altered at both mRNA and enzyme activity levels. Conclusions If studies in mice extrapolate to humans, lower doses of berberine appear to present a low risk of producing drug-drug interactions as a result of changed Cyp enzyme activity. However, high doses of berberine may suppress Cyp activities and result in drug-drug interactions. PMID:21920422

  3. 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.

  4. Effects of Cerium Nitrate on Expression of CaM Ⅰ and PMCA Ca2+-ATPase mRNA in Rat Liver

    Institute of Scientific and Technical Information of China (English)

    杨维东; 王艇; 刘洁生; 雷衡毅; 杨燕生

    2001-01-01

    The effect of cerium nitrate on expression of CaM Ⅰ and PMCA1b in rat liver was studied by means of reverse transcription-polymerase chain reaction (RT-PCR). The result shows that neither a high dose (50 mg*kg-1) nor a low dose (1 mg*kg-1) of cerium nitrate induces any alterations of expression of CaM Ⅰ and PMCA 1b mRNA after recurrent intraperitoneal injection of cerium nitrate, which suggests that effect of cerium nitrate on CaM and Ca2+-ATPase might be at posttranscription level.

  5. 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 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.

  6. [Neurocutaneous syndrome with hair alterations].

    Science.gov (United States)

    Camacho-Martínez, F

    1997-09-01

    There are multiple neurocutaneous syndromes that may show hair alterations such as the interglabellar peak or 'widow's peak', which is an alteration of the hair implantation, in addition to the genohypotrichosis, hypertrichosis and hair shaft dysplasias. In this chapter we will focus on the latter. Out of the unspecific hair shaft dysplasias the only ones showing neurological alterations are trichorrhexis invaginata, observed in the syndrome of Netherton. Among the specific dysplasias we would like to point out monilethrix, and very especially the moniliform hair syndrome, the trichorrhexis nodosa, the pili torti and trichotiodystrophy. The latter is actually a group of syndromes which associates a series of diverse symptoms that have in common hair brittleness, fertility problems and physical and mental retardation, and they constitute the basic syndrome know as 'BIDS syndrome.

  7. 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.

  8. BUB1 mRNA is significantly co-expressed with AURKA and AURKB mRNA in advanced-stage ovarian serous carcinoma.

    Science.gov (United States)

    Davidson, Ben; Nymoen, Dag Andre; Elgaaen, Bente Vilming; Staff, Anne Cathrine; Tropé, Claes G; Kærn, Janne; Reich, Reuven; Falkenthal, Thea E Hetland

    2014-06-01

    The objective of this study was to investigate the expression and clinical role of the spindle checkpoint kinase budding uninhibited by benzimidazole 1 (Bub1) in primary and metastatic advanced-stage ovarian serous carcinoma. BUB1 mRNA expression was analyzed in 178 tumors (88 effusions, 38 primary carcinomas, and 52 solid metastases) from 144 patients with advanced-stage disease using quantitative real-time polymerase chain reaction (PCR). Bub1 protein expression by Western blotting was studied in 63 carcinomas (30 effusions and 33 solid lesions). BUB1 mRNA expression at different anatomic sites was studied for association with clinicopathologic parameters, including chemotherapy resistance and survival. BUB1 mRNA was universally expressed in serous carcinomas, irrespective of anatomic site. BUB1 mRNA levels were uniformly low in six ovarian surface epithelium specimens analyzed for comparative purposes. Bub1 protein was expressed in 22/30 effusions and 28/33 solid lesions. BUB1 mRNA expression was significantly higher in chemo-naïve primary carcinomas and solid metastases compared to specimens obtained following neoadjuvant chemotherapy (p cancer. BUB1 mRNA levels are lower following chemotherapy exposure in solid lesions, though its presence is unrelated to clinical behavior including response to chemotherapy and survival. BUB1 is co-expressed with AURKA and AURKB suggesting biological relationship between these spindle cell components.

  9. Glucocorticoid regulates TrkB protein levels via c-Cbl dependent ubiquitination: a decrease in c-Cbl mRNA in the prefrontal cortex of suicide subjects.

    Science.gov (United States)

    Pandya, Chirayu; Kutiyanawalla, Ammar; Turecki, Gustavo; Pillai, Anilkumar

    2014-07-01

    Brain derived neurotrophic factor (BDNF) signaling through its receptor TrkB plays a crucial role in neurodevelopment and plasticity. Stress and glucocorticoids have been shown to alter TrkB signaling in neurons, and defects in TrkB expression have been reported in the prefrontal cortex of suicide subjects. Glucocorticoid treatment has been shown to induce deleterious effects on the neuronal maturation. However, the mechanisms involved in the regulation of TrkB by glucocorticoid during neurodevelopment are not clear. Here we show that acute corticosterone exposure induced posttranslational upregulation of TrkB in primary cortical neurons (days in vitro 4, DIV4), which was blocked by the proteasome inhibitors. Acute corticosterone-induced increase in TrkB protein levels was dependent on glucocorticoid receptor (GR). At the cellular level, ubiquitin E3 ligase c-Cbl mediates TrkB stabilization and corticosterone-induced TrkB levels. Moreover, the tyrosine kinase binding domain in c-Cbl plays a critical role in corticosterone-induced TrkB levels. Chronic treatment of neurons with corticosterone induced significant decreases in both TrkB and c-Cbl protein levels. Acute corticosterone treatment failed to induce any significant change in TrkB and c-Cbl protein levels in mature neurons (DIV 12), where as chronic corticosterone exposure reduced TrkB levels. Under an in vivo condition, chronic corticosterone exposure induced down-regulation of c-Cbl in mouse frontal cortex and hippocampus. Importantly, we demonstrate for the first time a significant decrease in c-Cbl mRNA levels in the prefrontal cortex of suicide subjects indicating the possible role of c-Cbl in the pathophysiology of suicidal behavior. Thus, ubiquitin-proteasome-mediated TrkB regulation may be an important mechanism for improving BDNF signaling and maintaining neuroplasticity in stress-related neuropsychiatric disorders.

  10. 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.

  11. Identifying mRNA, microRNA and protein profiles of melanoma exosomes.

    Directory of Open Access Journals (Sweden)

    Deyi Xiao

    Full Text Available BACKGROUND: Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and in various disease conditions. Tumor exosomes contain intact and functional mRNAs, small RNAs (including miRNAs, and proteins that can alter the cellular environment to favor tumor growth. Molecular profiling may increase our understanding of the role of exosomes in melanoma progression and may lead to discovery of useful biomarkers. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we used mRNA array profiling to identify thousands of exosomal mRNAs associated with melanoma progression and metastasis. Similarly, miRNA array profiling identified specific miRNAs, such as hsa-miR-31, -185, and -34b, involved in melanoma invasion. We also used proteomic analysis and discovered differentially expressed melanoma exosomal proteins, including HAPLN1, GRP78, syntenin-1, annexin A1, and annexin A2. Importantly, normal melanocytes acquired invasion ability through molecules transported in melanoma cell-derived exosomes. CONCLUSIONS/SIGNIFICANCE: Our results indicate that melanoma-derived exosomes have unique gene expression signatures, miRNA and proteomics profiles compared to exosomes from normal melanocytes. To the best of our knowledge, this is the first in-depth screening of the whole transcriptome/miRNome/proteome expression in melanoma exosomes. These results provide a starting point for future more in-depth studies of tumor-derived melanoma exosomes, which will aid our understanding of melanoma biogenesis and new drug-targets that may be translated into clinical applications, or as non-invasive biomarkers for melanoma.

  12. 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.

  13. Gallium nitrate increases type I collagen and fibronectin mRNA and collagen protein levels in bone and fibroblast cells.

    Science.gov (United States)

    Bockman, R S; Guidon, P T; Pan, L C; Salvatori, R; Kawaguchi, A

    1993-08-01

    Gallium is a Group IIIa transitional element with therapeutic efficacy in the treatment of metabolic bone disorders. Previously described antiresorptive effects of gallium on osteoclasts are not sufficient to account for the full range of effects of gallium on bone structure and metabolism. We have recently shown that gallium nitrate inhibits osteocalcin gene expression and the synthesis of osteocalcin protein, an osteoblast-specific bone matrix protein that is thought to serve as a signal to trigger osteoclastic resorption. Here we present evidence for an additional mechanism by which gallium may function to augment bone mass by altering matrix protein synthesis by osteoblastic and fibroblastic cells. Rat calvarial explants exposed to gallium nitrate for 48 h showed increased incorporation of 3H-proline into hydroxyproline and collagenase digestible protein. In addition, gallium treatment increased steady-state mRNA levels for fibronectin and type I procollagen chains in primary rat calvarial osteoblast-enriched cultures, the ROS 17/2.8 osteoblastic osteosarcoma line, and nontransformed human dermal fibroblasts. These findings suggest that the exposure of mesenchymally-derived cells to gallium results in an altered pattern of matrix protein synthesis that would favor increased bone formation.

  14. 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…

  15. Impaired expression of glycogen synthase mRNA in skeletal muscle of NIDDM patients

    DEFF Research Database (Denmark)

    Vestergaard, H; Bjørbaek, C; Andersen, P H

    1991-01-01

    with NIDDM were accompanied by a 39% reduction (P less than 0.02) in the steady state level of GS mRNA per microgram DNA of muscle. In both diabetic and control subjects, the mRNA expression of GS was unaffected after euglycemic-hyperinsulinemic clamp for 4 h. With single-stranded conformation polymorphism...

  16. Distribution of the mRNA for protein phosphatase T in rat brain

    NARCIS (Netherlands)

    Becker, W; Buttini, M; Limonta, S; Boddeke, H; Joost, HG

    1996-01-01

    We have recently cloned a novel protein serine/threonine phosphatase (PPT) from rat mRNA which is predominantly expressed in the brain (Becker et al., J. Biol. Chem., 269 (1994) 22586-22592). In the present study, the regional distribution of PPT mRNA in the brain of adult rats was characterized by

  17. Applying the breaks on gene expression - mRNA deadenylation by Pop2p

    DEFF Research Database (Denmark)

    Andersen, Kasper Røjkjær; Jonstrup, Anette Thyssen; Van, Lan Bich;

    When driving a car, control of the brakes is just as important as control of the accelerator pedal. Likewise, in gene expression, regulation of mRNA degradation is as important as regulation of its synthesis (Mühlemann, 2005). The rate-determining step of mRNA decay in eukaryotes seems to be the ...

  18. Differential targeting of VDAC3 mRNA isoforms influences mitochondria morphology.

    Science.gov (United States)

    Michaud, Morgane; Ubrig, Elodie; Filleur, Sophie; Erhardt, Mathieu; Ephritikhine, Geneviève; Maréchal-Drouard, Laurence; Duchêne, Anne-Marie

    2014-06-17

    Intracellular targeting of mRNAs has recently emerged as a prevalent mechanism to control protein localization. For mitochondria, a cotranslational model of protein import is now proposed in parallel to the conventional posttranslational model, and mitochondrial targeting of mRNAs has been demonstrated in various organisms. Voltage-dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane and the major transport pathway for numerous metabolites. Four nucleus-encoded VDACs have been identified in Arabidopsis thaliana. Alternative cleavage and polyadenylation generate two VDAC3 mRNA isoforms differing by their 3' UTR. By using quantitative RT-PCR and in vivo mRNA visualization approaches, the two mRNA variants were shown differentially associated with mitochondria. The longest mRNA presents a 3' extension named alternative UTR (aUTR) that is necessary and sufficient to target VDAC3 mRNA to the mitochondrial surface. Moreover, aUTR is sufficient for the mitochondrial targeting of a reporter transcript, and can be used as a tool to target an unrelated mRNA to the mitochondrial surface. Finally, VDAC3-aUTR mRNA variant impacts mitochondria morphology and size, demonstrating the role of mRNA targeting in mitochondria biogenesis.

  19. Neurotrophin-3 mRNA expression in rat intrafusal muscle fibres after denervation and reinnervation

    NARCIS (Netherlands)

    Copray, JCVM; Brouwer, N

    1997-01-01

    We have studied the regulation of the expression of neurotrophin-3 (NT-3) mRNA in neonatal and adult rat muscle spindles after denervation and after denervation followed by reinnervation. Denervation of the intrafusal fibres did not result in an upregulation of the NT-3 mRNA expression but decreased

  20. Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons

    NARCIS (Netherlands)

    Mochida, Sumiko; Poulain, Bernard; Eisel, Ulrich; Binz, Thomas; Kurazono, Hisao; Niemann, Heiner; Tauc, Ladislav; Bullock, Theodore H.

    1990-01-01

    Injection of exogenous mRNA purified from various tissue preparations into cellular translation systems such as Xenopus oocytes has allowed expression of complex proteins (e.g., receptors for neurotransmitters). No evidence for expression of injected exogenous mRNA, however, has been reported in ter

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    trial. No change in HIF1alpha, PFK, CS, LDH-A or LDH-B mRNA expression was detected after any of the exercise trials. CONCLUSION:: The relative intensity of brief intermittent exercise is of major importance for the exercise induced increase of several mRNA's, including PGC-1alpha....

  2. Modelling glass alteration in an altered argillaceous environment

    Science.gov (United States)

    Bildstein, O.; Trotignon, L.; Pozo, C.; Jullien, M.

    2007-05-01

    The long term behaviour of materials such as glass, steel and clay has been investigated in the context of deep geological disposal of radioactive wastes. The interactions between vitrified wastes, canister corrosion products (CPs) and clay are studied using a modified version of the reaction-transport code Crunch, especially looking at pH changes and possible cementation at the interface with the clayey materials. These perturbations may indeed affect the lifetime of glass matrix in deep repositories, e.g., high pH enhances the rate of glass alteration. This work focuses on the argillite of Bure. The calculations were performed at 323 K with a glass alteration rate switching from a high initial rate to a residual rate according to the sorption capacity of CPs. The time at which this sorption capacity is saturated is crucial to the system in terms of wastes package lifetime. The results show that the glass alteration imposes a high pH value at the interface with CPs and clay: up to a value of 9.2, compared to 7.3 which is the initial pH value in the argillite. Experimental data show that the rate of glass alteration is much higher in such pH conditions. For a R7T7-type glass, the rate is about five times higher at pH 9 than at pH 7. This pH perturbation migrates through the clayey domain as a result of the migration of mobile elements such as boron and sodium, and despite the existence of strong pH buffers in the argillite. The cementation of porosity at the interface between glass and clay is predicted by the model due to the massive precipitation of iron corrosion products and glass alteration products. At this point of the evolution of the system, the pH starts to decrease and the alteration rate of the glass could be significantly reduced. This porosity clogging effect is difficult to confirm by experiments especially since existing data on short term experiments tend to show a pervasive precipitation of silica in the domain instead of a localized precipitation

  3. 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…

  4. Triphenylphosphine Stabilized Silver Carboxylates

    Institute of Scientific and Technical Information of China (English)

    Jian Lin HAN; Ying Zhong SHEN; Yi PAN

    2005-01-01

    A series of novel triphenylphosphine stabilized silver carboxylates, potential precursors for CVD growth of ultrafast interconnection link in microelectronic devices, have been prepared and characterized.

  5. Relationship Between Heart Damages and HSPs mRNA in Persistent Heat Stressed Broilers

    Institute of Scientific and Technical Information of China (English)

    SUN Pei-ming; LIU Yu-tian; ZHAO Yong-gang; BAO En-dong; WANG Zhi-liang

    2007-01-01

    The relationship between myocardial cell damages and HSPs mRNA transcription in heat stressed broilers was studied using a spectrophotometer, the histopathological technique, and fluorescence quantitative reverse transcription PCR (FQ RT-PCR). The results showed that the activities of creatine kinase (CK) and glutamic-pyruvic transaninase (GPT) were induction during the persistent heat stress. The major lesions of the myocardial fibers were granular degeneration and necrosis. The transcription of constitutive or cognate heat shock protein 70 (HSC70) mRNA was changeable. The transcription of heat shock protein 70 (HSP70) mRNA was increased obviously in the course of persistent heat stress. The results showed that the change of HSC70 mRNA transcription was contrary to the activity of CK, and the level of HSC70 mRNA transcription must be used as a symbol of the myocardial cell damages in the course of persistent heat stress.

  6. Challenges and advances towards the rational design of mRNA vaccines.

    Science.gov (United States)

    Pollard, Charlotte; De Koker, Stefaan; Saelens, Xavier; Vanham, Guido; Grooten, Johan

    2013-12-01

    In recent years, mRNA vaccines have emerged as a safe and potent approach for the induction of cellular immune responses. Whereas initial studies were limited to the ex vivo loading of dendritic cells (DCs) with antigen-encoding mRNA, recent progress has led to the development of improved mRNA vaccines that enable direct in vivo targeting of DCs. Although preclinical studies demonstrated their potency in inducing antitumor immunity, several bottlenecks hinder the broader application of mRNA vaccines. In this review, we discuss the challenges associated with mRNA-based vaccination strategies, the technological advances that have been made to overcome these limitations, and the hurdles that remain to be tackled for the development of an optimal mRNA vaccine.

  7. Glucocorticoids modulate BDNF mRNA expression in the rat hippocampus after traumatic brain injury.

    Science.gov (United States)

    Grundy, P L; Patel, N; Harbuz, M S; Lightman, S L; Sharples, P M

    2000-10-20

    Brain-derived neurotrophic factor (BDNF) expression in rat hippocampus is increased after experimental traumatic brain injury (TBI) and may be neuroprotective. Glucocorticoids are important regulators of brain neurotrophin levels and are often prescribed following TBI. The effect of adrenalectomy (ADX) on the expression of BDNF mRNA in the hippocampus after TBI has not been investigated to date. We used fluid percussion injury (FPI) and in situ hybridization to evaluate the expression of BDNF mRNA in the hippocampus 4 h after TBI in adrenal-intact or adrenalectomized rats (with or without corticosterone replacement). FPI and ADX independently increased expression of BDNF mRNA. In animals undergoing FPI, prior ADX caused further elevation of BDNF mRNA and this upregulation was prevented by corticosterone replacement in ADX rats. These findings suggest that glucocorticoids are involved in the modulation of the BDNF mRNA response to TBI.

  8. Glucocorticoids modulate the NGF mRNA response in the rat hippocampus after traumatic brain injury.

    Science.gov (United States)

    Grundy, P L; Patel, N; Harbuz, M S; Lightman, S L; Sharples, P M

    2001-02-23

    Nerve growth factor (NGF) expression in the rat hippocampus is increased after experimental traumatic brain injury (TBI) and is neuroprotective. Glucocorticoids are regulators of brain neurotrophin levels and are often prescribed following TBI. The effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on the expression of NGF mRNA in the hippocampus after TBI has not been investigated to date. We used fluid percussion injury and in situ hybridisation to evaluate the expression of NGF mRNA in the hippocampus 4 h after TBI in adrenal-intact or adrenalectomised rats (with or without CORT replacement). TBI increased expression of NGF mRNA in sham-ADX rats, but not in ADX rats. Furthermore, CORT replacement in ADX rats restored the increase in NGF mRNA induced by TBI. These findings suggest that glucocorticoids have an important role in the induction of hippocampal NGF mRNA after TBI.

  9. Simultaneous isolation of mRNA and native protein from minute samples of cells

    DEFF Research Database (Denmark)

    Petersen, Tonny Studsgaard; Andersen, Claus Yding

    2014-01-01

    proteins from samples containing small numbers of cells. Our approach utilizes oligodeoxythymidylate [oligo(dT)25]-coated paramagnetic beads in an optimized reaction buffer to isolate mRNA comparable in quantity and quality to mRNA isolated with existing methods, while maintaining the proteins...... in their native state for traditional protein assays. We validated the procedure using neonatal rat ovaries and small numbers of human granulosa cells, demonstrating the extraction of mRNA suitable for gene expression analysis with simultaneous isolation of native proteins suitable for downstream characterization......Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native...

  10. 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.

  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. Quantification of mRNA Levels Using Real-Time Polymerase Chain Reaction (PCR).

    Science.gov (United States)

    Li, Yiyi; Wang, Kai; Chen, Longhua; Zhu, Xiaoxia; Zhou, Jie

    2016-01-01

    Real-time quantitative reverse transcription PCR technique has advanced greatly over the past 20 years. Messenger RNA (mRNA) levels in cells or tissues can be quantified by this approach. It is well known that changes in mRNA expression in disease, and correlation of mRNA expression profiles with clinical parameters, serve as clinically relevant biomarkers. Hence, accurate determination of the mRNA levels is critically important in describing the biological, pathological, and clinical roles of genes in health and disease. This chapter describes a real-time PCR approach to detect and quantify mRNA expression levels, which can be used for both laboratorial and clinical studies in breast cancer research.

  13. 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

    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 effects...... of regular physical activity with improved insulin sensitivity.......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...

  14. Ontogeny of pituitary growth hormone and growth hormone mRNA in the chicken.

    Science.gov (United States)

    McCann-Levorse, L M; Radecki, S V; Donoghue, D J; Malamed, S; Foster, D N; Scanes, C G

    1993-01-01

    The changes in pituitary growth hormone (GH) mRNA levels have been determined by Northern blot analysis and laser densitometry during embryonic development and posthatch growth of white Leghorn cockerels. Pituitary GH mRNA levels were observed to progressively increase between 18 days of embryonic development to a maximum at 4 weeks of age (posthatch). Subsequently, pituitary GH mRNA levels declined between 4 and 8 weeks of age, and between 12 weeks of age and adulthood. Pituitary GH contents showed increases during embryonic development and posthatch growth that paralleled the rise in GH mRNA. The decline in pituitary GH mRNA levels between 4 weeks of age and adulthood occurs when GH secretion has been observed previously to decline.

  15. mRNA secondary structures fold sequentially but exchange rapidly in vivo.

    Directory of Open Access Journals (Sweden)

    Elisabeth M Mahen

    2010-02-01

    Full Text Available RNAs adopt defined structures to perform biological activities, and conformational transitions among alternative structures are critical to virtually all RNA-mediated processes ranging from metabolite-activation of bacterial riboswitches to pre-mRNA splicing and viral replication in eukaryotes. Mechanistic analysis of an RNA folding reaction in a biological context is challenging because many steps usually intervene between assembly of a functional RNA structure and execution of a biological function. We developed a system to probe mechanisms of secondary structure folding and exchange directly in vivo using self-cleavage to monitor competition between mutually exclusive structures that promote or inhibit ribozyme assembly. In previous work, upstream structures were more effective than downstream structures in blocking ribozyme assembly during transcription in vitro, consistent with a sequential folding mechanism. However, upstream and downstream structures blocked ribozyme assembly equally well in vivo, suggesting that intracellular folding outcomes reflect thermodynamic equilibration or that annealing of contiguous sequences is favored kinetically. We have extended these studies to learn when, if ever, thermodynamic stability becomes an impediment to rapid equilibration among alternative RNA structures in vivo. We find that a narrow thermodynamic threshold determines whether kinetics or thermodynamics govern RNA folding outcomes in vivo. mRNA secondary structures fold sequentially in vivo, but exchange between adjacent secondary structures is much faster in vivo than it is in vitro. Previous work showed that simple base-paired RNA helices dissociate at similar rates in vivo and in vitro so exchange between adjacent structures must occur through a different mechanism, one that likely involves facilitation of branch migration by proteins associated with nascent transcripts.

  16. Effect of AMP on mRNA binding by yeast NAD+-specific isocitrate dehydrogenase.

    Science.gov (United States)

    Anderson, Sondra L; Schirf, Virgil; McAlister-Henn, L

    2002-06-04

    Yeast mitochondrial NAD+-specific isocitrate dehydrogenase (IDH) has previously been shown to bind specifically to 5'-untranslated regions of yeast mitochondrial mRNAs, and transcripts containing these regions have been found to allosterically inhibit activity of the enzyme. This inhibition is relieved by AMP, an allosteric activator of this regulatory enzyme of the tricarboxylic acid cycle. We further investigated these enzyme/ligand interactions to determine if binding of RNA and AMP by IDH is competitive or independent. Gel mobility shift experiments indicated no effect of AMP on formation of an IDH/RNA complex. Similarly, sedimentation velocity ultracentrifugation experiments used to analyze interactions in solution indicated that AMP alone had little effect on the formation or stability of an RNA/IDH complex. However, when these sedimentation experiments were conducted in the presence of isocitrate, which has been shown to be essential for binding of AMP by IDH, the proportion of RNA sedimenting in a complex with IDH was significantly reduced by AMP. These results suggest that AMP can affect the binding of RNA by IDH but that this effect is apparent only in the presence of substrate. They also suggest that the catalytic activity of IDH in vivo may be subject to complex allosteric control determined by relative mitochondrial concentrations of mRNA, isocitrate, and AMP. We also found evidence for binding of 5'-untranslated regions of mitochondrial mRNAs by yeast mitochondrial NADP+-specific isocitrate dehydrogenase (IDP1) but not by the corresponding cytosolic isozyme (IDP2). However, this appears to be a nonspecific interaction since no evidence was obtained for any effect on the catalytic activity of IDP1.

  17. Down-regulation of flavonoid 3'-hydroxylase gene expression by virus-induced gene silencing in soybean reveals the presence of a threshold mRNA level associated with pigmentation in pubescence.

    Science.gov (United States)

    Nagamatsu, Atsushi; Masuta, Chikara; Matsuura, Hideyuki; Kitamura, Keisuke; Abe, Jun; Kanazawa, Akira

    2009-01-01

    Changes in flavonoid content are often manifested as altered pigmentation in plant tissues. Two loci have been identified as controlling pigmentation in soybean pubescence. Of these, the T locus appears to encode flavonoid 3'-hydroxylase (F3'H) protein: the T and t alleles are associated with tawny and gray colors, respectively, in pubescence. We previously down-regulated F3'H gene expression by virus-induced gene silencing (VIGS) in soybean. Despite this successful VIGS, the tawny pubescence pigmentation proved to be unchanged in greenhouse-grown plants. We hypothesized that the reduced mRNA level of the F3'H gene resulting from VIGS remained high enough to induce pigmentation. To verify this hypothesis, in the present study, we performed F3'H VIGS on plants grown under controlled conditions, in which the steady-state mRNA level of the F3'H gene was reduced to approximately 5% of that of greenhouse-grown plants. This VIGS treatment resulted in the loss of tawny pigmentation in pubescence, suggesting that the sf3'h1 gene is involved in the control of pigmentation in pubescence. We detected a marked decrease in target mRNA, an accumulation of short interfering RNAs (siRNAs), and a decrease in quercetin content relative to kaempferol in leaf tissues, indicating that sequence-specific mRNA degradation of the F3'H gene was induced. These results suggest that leaf tissues have a threshold mRNA level of the F3'H gene, which is associated with the occurrence of tawny pigmentation in pubescence. The estimated threshold mRNA level for pigmentation in pubescence was approximately 3% of the steady-state mRNA level of the F3'H gene in greenhouse-grown plants.

  18. Starch supplementation modulates amylase enzymatic properties and amylase B mRNA level in the digestive gland of the Pacific oyster Crassostrea gigas.

    Science.gov (United States)

    Huvet, A; Jeffroy, F; Daniel, J Y; Quéré, C; Le Souchu, P; Van Wormhoudt, A; Boudry, P; Moal, J; Samain, J F

    2012-09-01

    In the oyster Crassostrea gigas consumption-related traits, amylase properties and growth were found to be linked through genotypes that differed for polymorphism in the two amylase genes AMYA and AMYB. Modulation of AMYA mRNA level had already been observed in response to food availability, whereas the functional role of AMYB was still unknown. To improve knowledge about the regulation of amylase expression in C. gigas and the respective roles of the two genes, we made an assay of amylase expression at mRNA and enzymatic levels in the digestive gland of oysters that had received dietary supplements of starch. After 18 days, a significant increase of translatable mRNA for AMYB was observed, with a correlated increase in Michaelis-Menten constant Km values and a decrease in total amylase activity. This modulation is the first evidence of observable functioning of AMYB in digestive processes. Amylase B is suggested to display a higher Km than amylase A, offering a means of adapting to high substrate concentrations. The highest starch supplement level (10 mgL(-1)) induced alteration in oyster physiology. The 1 mgL(-1) treatment should be tested as a practical food supplement that could lead to growth benefits for oysters.

  19. Age determines the magnitudes of angiotensin II-induced contractions, mRNA, and protein expression of angiotensin type 1 receptors in rat carotid arteries.

    Science.gov (United States)

    Vamos, Zoltan; Cseplo, Peter; Ivic, Ivan; Matics, Robert; Hamar, Janos; Koller, Akos

    2014-05-01

    In this study, we hypothesized that aging alters angiotensin II (Ang II)-induced vasomotor responses and expression of vascular mRNA and protein angiotensin type 1 receptor (AT1R). Thus, carotid arteries were isolated from the following age groups of rats: 8 days, 2-9 months, 12-20 months, and 20-30 months, and their vasomotor responses were measured in a myograph after repeated administrations of Ang II. Vascular relative AT1R mRNA level was determined by quantitative reverse-transcriptase polymerase chain reaction and the AT1R protein density was measured by Western blot. Contractions to the first administration of Ang II increased from 8 days to 6 months and then they decreased to 30 months. In general, second administration of Ang II elicited reduced contractions, but they also increased from 8 days until 2 months and then they decreased to 30 months. Similarly the AT1R mRNA level increased from 8 days to 12 months and then decreased to 30 months. Similarly the AT1R protein density increased from 8 days until 16 months and then they decreased to 30 months. The pattern of these changes correlated with functional vasomotor data. We conclude that aging (newborn to senescence) has substantial effects on Ang II-induced vasomotor responses and AT1R signaling suggesting the importance of genetic programs.

  20. Chronic stress induces upregulation of brain-derived neurotrophic factor (BDNF) mRNA and integrin alpha5 expression in the rat pineal gland.

    Science.gov (United States)

    Dagnino-Subiabre, Alexies; Zepeda-Carreño, Rodrigo; Díaz-Véliz, Gabriela; Mora, Sergio; Aboitiz, Francisco

    2006-05-01

    Chronic stress affects brain areas involved in learning and emotional responses. These alterations have been related with the development of cognitive deficits in major depression. Moreover, stress induces deleterious actions on the epithalamic pineal organ, a gland involved in a wide range of physiological functions. The aim of this study was to investigate whether the stress effects on the pineal gland are related with changes in the expression of neurotrophic factors and cell adhesion molecules. Using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, we analyzed the effect of chronic immobilization stress on the BDNF mRNA and integrin alpha5 expression in the rat pineal gland. We found that BDNF is produced in situ in the pineal gland. Chronic immobilization stress induced upregulation of BDNF mRNA and integrin alpha5 expression in the rat pineal gland but did not produce changes in beta-actin mRNA or in GAPDH expression. Stressed animals also evidenced an increase in anxiety-like behavior and acute gastric lesions. These results suggest that BDNF and integrin alpha5 may have a counteracting effect to the deleterious actions of immobilization stress on functionally stimulated pinealocytes. Furthermore, this study proposes that the pineal gland may be a target of glucocorticoid damage during stress.

  1. Altered expression of adipose differentiation-related protein gene in placental tissue of pre-eclampsia

    Institute of Scientific and Technical Information of China (English)

    ZHANG Chun-li; YAO Yuan-qing; LI Dong-hong; ZHANG Wei

    2006-01-01

    Objective: To investigate the altered expression of lipid metabolism-related gene adipose differentiation-related protein (ADRP) in pre-eclampsia. Methods: Semi-quantitative RT-PCR and Western blotting were used to validate the altered expression of ADRP gene between pre-eclamptic placentas (preeclampsia group) and normotensive placentas (control group) respectively. In situ hybridization (ISH)was used to localize ADRP mRNA in pre-eclamptic placentas. Results: There was a significant difference in the levels of placental ADRP mRNA between pre-eclampsia group and control group (1.98± 0. 50 vs 1. 09±0. 20, P<0.01). Western blotting showed that placentas both in pre-eclampsia group and control group expressed the special ADRP band at 48. 1 kD. The relative levels of ADRP protein in pre-eclampsia group were significantly higher than those of control group (0. 40 ±0. 19 vs 0. 19 ±0. 09, P< 0. 01).ADRP mRNA was diffusely distributed in pre-eclamptic placentas. Their positive staining existed in cytoplasm of trophoblast. Conclusion: Abnormal expression of ADRP gene in pre-eclamptic placenta may be associated with the pathogenesis of pre-eclampsia.

  2. Mucin dynamics in the chick small intestine are altered by starvation.

    Science.gov (United States)

    Smirnov, Asya; Sklan, David; Uni, Zehava

    2004-04-01

    The absorptive surface of the small intestine is covered by a layer of mucus secreted by goblet cells. The secreted mucins and thickness of the adherent layer influence nutrient digestion and absorption processes as well as the functionality of the mucosa. In this study, methods for the analysis of mucin synthesis and dynamics in the chick small intestine are described. A fragment of chicken mucin cDNA was isolated and characterized; this fraction had 60% homology to human mucin MUC-5AC. The thickness of the mucus adherent layer and the relative amounts of mucin glycoprotein and mRNA were also examined in the small intestines of control and starved chicks. Relative amounts of intestinal mucin mRNA and protein increased in the duodenum and jejunum of starved chicks, and mucus adherent layer thickness decreased throughout the small intestine. In starved chicks, higher mRNA expression and protein concentrations with lower amounts of adherent mucus may be related to a higher rate of degradation of the mucus layer, a lower rate of mucus secretion, or an altered rate of mucin turnover. It thus appears that starvation alters mucus dynamics in the small intestine, and this may affect intestinal digestive function and defense.

  3. 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.

  4. Hemorrhage and resuscitation induce alterations in cytokine expression and the development of acute lung injury.

    Science.gov (United States)

    Shenkar, R; Coulson, W F; Abraham, E

    1994-03-01

    Acute pulmonary injury occurs frequently following hemorrhage and injury. In order to better examine the sequence of events leading to lung injury in this setting, we investigated lung histology as well as in vivo mRNA levels for cytokines with proinflammatory and immunoregulatory properties (IL-1 beta, IL-6, IL-10, TNF-alpha, TGF-beta, IFN-gamma) over the 3 days following hemorrhage and resuscitation. Significant increases in mRNA levels for IL-1 beta, IL-6, IL-10, and IFN-gamma, but not TNF-alpha, were present among intraparenchymal pulmonary mononuclear cells obtained 1 and 3 days after hemorrhage. Among alveolar macrophages, TNF-alpha and IL-1 beta mRNA levels were increased 3 days after hemorrhage. Few changes in cytokine mRNA levels, with the exception of TNF-alpha at 3 days after hemorrhage, were present among peripheral blood mononuclear cells. Histologic examination of lungs from hemorrhaged animals showed no alterations 1 day after hemorrhage, but neutrophil and mononuclear cell infiltrates, edema, intra-alveolar hemorrhage, and fibrin generation were present 3 days after hemorrhage. These results suggest that hemorrhage-induced enhancement of proinflammatory cytokine gene transcription may be an important mechanism contributing to the frequent development of acute lung injury following blood loss and injury.

  5. Chronic unpredictive mild stress leads to altered hepatic metabolic profile and gene expression.

    Science.gov (United States)

    Jia, Hong-Mei; Li, Qi; Zhou, Chao; Yu, Meng; Yang, Yong; Zhang, Hong-Wu; Ding, Gang; Shang, Hai; Zou, Zhong-Mei

    2016-03-23

    Depression is a complex disease characterized by a series of pathological changes. Research on depression is mainly focused on the changes in brain, but not on liver. Therefore, we initially explored the metabolic profiles of hepatic extracts from rats treated with chronic unpredictive mild stress (CUMS) by UPLC-Q-TOF/MS. Using multivariate statistical analysis, a total of 26 altered metabolites distinguishing CUMS-induced depression from normal control were identified. Using two-stage receiver operating characteristic (ROC) analysis, 18 metabolites were recognized as potential biomarkers related to CUMS-induced depression via 12 metabolic pathways. Subsequently, we detected the mRNA expressions levels of apoptosis-associated genes such as Bax and Bcl-2 and four key enzymes including Pla2g15, Pnpla6, Baat and Gad1 involved in phospholipid and primary bile acid biosynthesis in liver tissues of CUMS rats by real-time qRT-PCR assay. The expression levels of Bax, Bcl-2, Pla2g15, Pnpla6 and Gad1 mRNA were 1.43,1.68, 1.74, 1.67 and 1.42-fold higher, and those of Baat, Bax/Bcl-2 ratio mRNA were 0.83, 0.85-fold lower in CUMS rats compared with normal control. Results of liver-targeted metabonomics and mRNA expression demonstrated that CUMS-induced depression leads to variations in hepatic metabolic profile and gene expression, and ultimately results in liver injury.

  6. Suppression of serotonin hyperinnervation does not alter the dysregulatory influences of dopamine depletion on striatal neuropeptide gene expression in rodent neonates.

    Science.gov (United States)

    Basura, G J; Walker, P D

    1999-10-15

    Sixty days following neonatal dopamine depletion (>98%) with 6-hydroxydopamine, preprotachykinin and preprodynorphin mRNA levels were significantly reduced (67 and 78% of vehicle controls, respectively) in the anterior striatum as determined by in situ hybridization while preproenkephalin mRNA expression was elevated (133% of vehicle controls). Suppression of the serotonin hyperinnervation phenomenon in the dopamine-depleted rat with 5,7-dihydroxytryptamine yielded no significant alterations in reduced striatal preprotachykinin (66%) or preprodynorphin (64%) mRNA levels, while preproenkephalin mRNA expression remained significantly elevated (140%). These data suggest that striatal serotonin hyperinnervation does not contribute to the development of dysregulated striatal neuropeptide transmission in either direct or indirect striatal output pathways following neonatal dopamine depletion.

  7. In vivo intra-luteal implants of prostaglandin (PG) E1 or E2 (PGE1, PGE2) prevent luteolysis in cows. II: mRNA for PGF2α, EP1, EP2, EP3 (A-D), EP3A, EP3B, EP3C, EP3D, and EP4 prostanoid receptors in luteal tissue.

    Science.gov (United States)

    Weems, Yoshie S; Bridges, Phillip J; Jeoung, Myoungkun; Arreguin-Arevalo, J Alejandro; Nett, Torrance M; Vann, Rhonda C; Ford, Stephen P; Lewis, Andrew W; Neuendorff, Don A; Welsh, Thomas H; Randel, Ronald D; Weems, Charles W

    2012-01-01

    Previously, it was reported that chronic intra-uterine infusion of PGE(1) or PGE(2) every 4h inhibited luteolysis in ewes by altering luteal mRNA for luteinizing hormone (LH) receptors and unoccupied and occupied luteal LH receptors. However, estradiol-17β or PGE(2) given intra-uterine every 8h did not inhibit luteolysis in cows, but infusion of estradiol+PGE(2) inhibited luteolysis. In contrast, intra-luteal implants containing PGE(1) or PGE(2) in Angus or Brahman cows also inhibited the decline in circulating progesterone, mRNA for LH receptors, and loss of unoccupied and occupied receptors for LH to prevent luteolysis. The objective of this experiment was to determine how intra-luteal implants of PGE(1) or PGE(2) alter mRNA for prostanoid receptors and how this could influence luteolysis in Brahman or Angus cows. On day-13 Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE(1), or PGE(2) and corpora lutea were retrieved on day-19. Corpora lutea slices were analyzed for mRNA for prostanoid receptors (FP, EP1, EP2, EP3 (A-D), EP3A, EP3B, EP3C, EP3D, and EP4) by RT-PCR. Day-13 Angus cow luteal tissue served as pre-luteolytic controls. mRNA for FP receptors decreased in day-19 Vehicle controls compared to day-13 Vehicle controls regardless of breed. PGE(1) and PGE(2) up-regulated FP gene expression on day-19 compared to day-19 Vehicle controls regardless of breed. EP1 mRNA was not altered by any treatment. PGE(1) and PGE(2) down-regulated EP2 and EP4 mRNA compared to day-19 Vehicle controls regardless of breed. PGE(1) or PGE(2) up-regulated mRNA EP3B receptor subtype compared to day-19 Vehicle control cows regardless of breed. The similarities in relative gene expression profiles induced by PGE(1) and PGE(2) support their agonistic effects. We conclude that both PGE(1) and PGE(2) may prevent luteolysis by altering expression of mRNA for prostanoid

  8. Ornithopter flight stabilization

    Science.gov (United States)

    Dietl, John M.; Garcia, Ephrahim

    2007-04-01

    The quasi-steady aerodynamics model and the vehicle dynamics model of ornithopter flight are explained, and numerical methods are described to capture limit cycle behavior in ornithopter flight. The Floquet method is used to determine stability in forward flight, and a linear discrete-time state-space model is developed. This is used to calculate stabilizing and disturbance-rejecting controllers.

  9. Conformational stability of calreticulin

    DEFF Research Database (Denmark)

    Jørgensen, C.S.; Trandum, C.; Larsen, N.

    2005-01-01

    The conformational stability of calreticulin was investigated. Apparent unfolding temperatures (T-m) increased from 31 degrees C at pH 5 to 51 degrees C at pH 9, but electrophoretic analysis revealed that calreticulin oligomerized instead of unfolding. Structural analyses showed that the single C......-terminal a-helix was of major importance to the conformational stability of calreticulin....

  10. Conformational stability of calreticulin

    DEFF Research Database (Denmark)

    Jørgensen, Charlotte S; Trandum, Christa; Larsen, Nanna Brink

    2005-01-01

    The conformational stability of calreticulin was investigated. Apparent unfolding temperatures (Tm) increased from 31 degrees C at pH 5 to 51 degrees C at pH 9, but electrophoretic analysis revealed that calreticulin oligomerized instead of unfolding. Structural analyses showed that the single C......-terminal alpha-helix was of major importance to the conformational stability of calreticulin....

  11. Visual attention and stability

    NARCIS (Netherlands)

    Mathot, Sebastiaan; Theeuwes, Jan

    2011-01-01

    In the present review, we address the relationship between attention and visual stability. Even though with each eye, head and body movement the retinal image changes dramatically, we perceive the world as stable and are able to perform visually guided actions. However, visual stability is not as co

  12. Basic principles of stability.

    Science.gov (United States)

    Egan, William; Schofield, Timothy

    2009-11-01

    An understanding of the principles of degradation, as well as the statistical tools for measuring product stability, is essential to management of product quality. Key to this is management of vaccine potency. Vaccine shelf life is best managed through determination of a minimum potency release requirement, which helps assure adequate potency throughout expiry. Use of statistical tools such a least squares regression analysis should be employed to model potency decay. The use of such tools provides incentive to properly design vaccine stability studies, while holding stability measurements to specification presents a disincentive for collecting valuable data. The laws of kinetics such as Arrhenius behavior help practitioners design effective accelerated stability programs, which can be utilized to manage stability after a process change. Design of stability studies should be carefully considered, with an eye to minimizing the variability of the stability parameter. In the case of measuring the degradation rate, testing at the beginning and the end of the study improves the precision of this estimate. Additional design considerations such as bracketing and matrixing improve the efficiency of stability evaluation of vaccines.

  13. Glucose-induced binding of the polypyrimidine tract-binding protein (PTB) to the 3'-untranslated region of the insulin mRNA (ins-PRS) is inhibited by rapamycin.

    Science.gov (United States)

    Tillmar, Linda; Welsh, Nils

    2004-05-01

    Despite considerable knowledge on the regulation of insulin gene transcription, little is known about the post-transcriptional control mechanisms of this gene. We have recently reported glucose- and hypoxia-regulated binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich sequence of the 3'-untranslated insulin mRNA (ins-PRS), an event which may control insulin mRNA stability. The present aim was to probe for the signaling pathways that control this binding activity. Rat islets were exposed to pharmacological inhibitors against several molecules, previously shown to be involved in glucose signaling. The inhibitors used were; LY 294002 (PI3 kinase), Rp-cAMP triatylamine (the cAMP-dependent protein kinase PKA), bisindolylmaleimide I hydrochloride (PKC), PD 098059 (ERK1/ERK2), SB 203580 (p38/SAPK2a), rapamycin (mTOR) and okadaic acid (PP1/2A). PTB-binding activity to the ins-PRS was then analyzed by elecrophoretic mobility shift assay (EMSA). The glucose-induced PTB-binding was only inhibited by the mTOR inhibitor rapamycin. Rapamycin also reduced glucose-induced insulin mRNA expression. Thus, our results suggest an involvement of mTOR in glucose-induced PTB/ins-PRS binding and insulin mRNA stability.

  14. Dose-dependent effects of morphine exposure on mRNA and microRNA (miR) expression in hippocampus of stressed neonatal mice.

    Science.gov (United States)

    McAdams, Ryan M; McPherson, Ronald J; Beyer, Richard P; Bammler, Theo K; Farin, Frederico M; Juul, Sandra E

    2015-01-01

    Morphine is used to sedate critically ill infants to treat painful or stressful conditions associated with intensive care. Whether neonatal morphine exposure affects microRNA (miR) expression and thereby alters mRNA regulation is unknown. We tested the hypothesis that repeated morphine treatment in stress-exposed neonatal mice alters hippocampal mRNA and miR expression. C57BL/6 male mice were treated from postnatal day (P) 5 to P9 with morphine sulfate at 2 or 5 mg/kg ip twice daily and then exposed to stress consisting of hypoxia (100% N2 1 min and 100% O2 5 min) followed by 2h maternal separation. Control mice were untreated and dam-reared. mRNA and miR expression profiling was performed on hippocampal tissues at P9. Overall, 2 and 5 mg/kg morphine treatment altered expression of a total of 150 transcripts (>1.5 fold change, Pmorphine affected 63 mRNAs exclusively. The most upregulated mRNAs were fidgetin, arginine vasopressin, and resistin-like alpha, and the most down-regulated were defensin beta 11, aquaporin 1, calmodulin-like 4, chloride intracellular channel 6, and claudin 2. Gene Set Enrichment Analysis revealed that morphine treatment affected pathways related to cell cycle, membrane function, signaling, metabolism, cell death, transcriptional regulation, and immune response. Morphine decreased expression of miR-204-5p, miR-455-3p, miR-448-5p, and miR-574-3p. Nine morphine-responsive mRNAs that are involved in neurodevelopment, neurotransmission, and inflammation are predicted targets of the aforementioned differentially expressed miRs. These data establish that morphine produces dose-dependent changes in both hippocampal mRNA and miR expression in stressed neonatal mice. If permanent, morphine-mediated neuroepigenetic effects may affect long-term hippocampal function, and this provides a mechanism for the neonatal morphine-related impairment of adult learning.

  15. Dose-dependent effects of morphine exposure on mRNA and microRNA (miR expression in hippocampus of stressed neonatal mice.

    Directory of Open Access Journals (Sweden)

    Ryan M McAdams

    Full Text Available Morphine is used to sedate critically ill infants to treat painful or stressful conditions associated with intensive care. Whether neonatal morphine exposure affects microRNA (miR expression and thereby alters mRNA regulation is unknown. We tested the hypothesis that repeated morphine treatment in stress-exposed neonatal mice alters hippocampal mRNA and miR expression. C57BL/6 male mice were treated from postnatal day (P 5 to P9 with morphine sulfate at 2 or 5 mg/kg ip twice daily and then exposed to stress consisting of hypoxia (100% N2 1 min and 100% O2 5 min followed by 2h maternal separation. Control mice were untreated and dam-reared. mRNA and miR expression profiling was performed on hippocampal tissues at P9. Overall, 2 and 5 mg/kg morphine treatment altered expression of a total of 150 transcripts (>1.5 fold change, P<0.05 from which 100 unique mRNAs were recognized (21 genes were up- and 79 genes were down-regulated, and 5 mg/kg morphine affected 63 mRNAs exclusively. The most upregulated mRNAs were fidgetin, arginine vasopressin, and resistin-like alpha, and the most down-regulated were defensin beta 11, aquaporin 1, calmodulin-like 4, chloride intracellular channel 6, and claudin 2. Gene Set Enrichment Analysis revealed that morphine treatment affected pathways related to cell cycle, membrane function, signaling, metabolism, cell death, transcriptional regulation, and immune response. Morphine decreased expression of miR-204-5p, miR-455-3p, miR-448-5p, and miR-574-3p. Nine morphine-responsive mRNAs that are involved in neurodevelopment, neurotransmission, and inflammation are predicted targets of the aforementioned differentially expressed miRs. These data establish that morphine produces dose-dependent changes in both hippocampal mRNA and miR expression in stressed neonatal mice. If permanent, morphine-mediated neuroepigenetic effects may affect long-term hippocampal function, and this provides a mechanism for the neonatal morphine

  16. Reduced transcript stabilization restricts TNF-alpha expression in RAW264.7 macrophages infected with pathogenic mycobacteria: evidence for an involvement of lipomannan.

    Science.gov (United States)

    Basler, Tina; Holtmann, Helmut; Abel, Jens; Eckstein, Torsten; Baumer, Wolfgang; Valentin-Weigand, Peter; Goethe, Ralph

    2010-01-01

    Despite the critical role that TNF-alpha plays in the containment of mycobacterial infection, the mechanisms involved in regulation of its expression by mycobacteria are poorly defined. We addressed this question by studying MAP, which causes a chronic enteritis in ruminants and is linked to human Crohn's disease. We found that in MAP infected macrophages, TNF-alpha gene expression was substantially lower than in macrophages infected with nonpathogenic MS or stimulated with LPS. TNF-alpha transcriptional one could not fully explain the differential TNF-alpha mRNA expression, suggesting that there must be a substantial contribution by post-transcriptional mechanisms.Accordingly, we found reduced TNF-alpha mRNA stability in MAP-infected macrophages. Further comparison of MAP- and MS-infected macrophages revealed that lower TNF-alpha mRNA stability combined with lower mRNA and protein expression in MAP-infected macrophages correlated with lower p38 MAPK phosphorylation. These findings were independent of viability of MAP and MS. We demonstrate that the major mycobacterial cell-wall lipoglycan LM of MAP and MS induced TNF-alpha mRNA transcription,but only the MS-LM induced p38 MAPK-dependent transcript stabilization. Overall, our data suggest that pathogenic mycobacteria cause weak p38 and TNF-alpha mRNA stabilization as a result of their structural cell-wall components such as LM and thereby, restrict TNF-alpha expression in macrophages.

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

    Science.gov (United States)

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

    2016-1