Sample records for altering mrna stability

  1. The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae.

    PubMed

    Huch, Susanne; Müller, Maren; Muppavarapu, Mridula; Gommlich, Jessie; Balagopal, Vidya; Nissan, Tracy

    2016-10-15

    The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization. © 2016. Published by The Company of Biologists Ltd.

  2. The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae

    PubMed Central

    Huch, Susanne; Müller, Maren; Muppavarapu, Mridula; Gommlich, Jessie; Balagopal, Vidya; Nissan, Tracy

    2016-01-01

    ABSTRACT The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization. PMID:27543059

  3. mRNA stability in mammalian cells.

    PubMed Central

    Ross, J

    1995-01-01

    This review concerns how cytoplasmic mRNA half-lives are regulated and how mRNA decay rates influence gene expression. mRNA stability influences gene expression in virtually all organisms, from bacteria to mammals, and the abundance of a particular mRNA can fluctuate manyfold following a change in the mRNA half-life, without any change in transcription. The processes that regulate mRNA half-lives can, in turn, affect how cells grow, differentiate, and respond to their environment. Three major questions are addressed. Which sequences in mRNAs determine their half-lives? Which enzymes degrade mRNAs? Which (trans-acting) factors regulate mRNA stability, and how do they function? The following specific topics are discussed: techniques for measuring eukaryotic mRNA stability and for calculating decay constants, mRNA decay pathways, mRNases, proteins that bind to sequences shared among many mRNAs [like poly(A)- and AU-rich-binding proteins] and proteins that bind to specific mRNAs (like the c-myc coding-region determinant-binding protein), how environmental factors like hormones and growth factors affect mRNA stability, and how translation and mRNA stability are linked. Some perspectives and predictions for future research directions are summarized at the end. PMID:7565413

  4. Connections Underlying Translation and mRNA Stability.

    PubMed

    Radhakrishnan, Aditya; Green, Rachel

    2016-09-11

    Gene expression and regulation in organisms minimally depends on transcription by RNA polymerase and on the stability of the RNA product (for both coding and non-coding RNAs). For coding RNAs, gene expression is further influenced by the amount of translation by the ribosome and by the stability of the protein product. The stabilities of these two classes of RNA, non-coding and coding, vary considerably: tRNAs and rRNAs tend to be long lived while mRNAs tend to be more short lived. Even among mRNAs, however, there is a considerable range in stability (ranging from seconds to hours in bacteria and up to days in metazoans), suggesting a significant role for stability in the regulation of gene expression. Here, we review recent experiments from bacteria, yeast and metazoans indicating that the stability of most mRNAs is broadly impacted by the actions of ribosomes that translate them. Ribosomal recognition of defective mRNAs triggers "mRNA surveillance" pathways that target the mRNA for degradation [Shoemaker and Green (2012) ]. More generally, even the stability of perfectly functional mRNAs appears to be dictated by overall rates of translation by the ribosome [Herrick et al. (1990), Presnyak et al. (2015) ]. Given that mRNAs are synthesized for the purpose of being translated into proteins, it is reassuring that such intimate connections between mRNA and the ribosome can drive biological regulation. In closing, we consider the likelihood that these connections between protein synthesis and mRNA stability are widespread or whether other modes of regulation dominate the mRNA stability landscape in higher organisms. Copyright © 2016. Published by Elsevier Ltd.

  5. An mRNA decapping mutant deficient in P body assembly limits mRNA stabilization in response to osmotic stress.

    PubMed

    Huch, Susanne; Nissan, Tracy

    2017-03-14

    Yeast is exposed to changing environmental conditions and must adapt its genetic program to provide a homeostatic intracellular environment. An important stress for yeast in the wild is high osmolarity. A key response to this stress is increased mRNA stability primarily by the inhibition of deadenylation. We previously demonstrated that mutations in decapping activators (edc3∆ lsm4∆C), which result in defects in P body assembly, can destabilize mRNA under unstressed conditions. We wished to examine whether mRNA would be destabilized in the edc3∆ lsm4∆C mutant as compared to the wild-type in response to osmotic stress, when P bodies are intense and numerous. Our results show that the edc3∆ lsm4∆C mutant limits the mRNA stability in response to osmotic stress, while the magnitude of stabilization was similar as compared to the wild-type. The reduced mRNA stability in the edc3∆ lsm4∆C mutant was correlated with a shorter PGK1 poly(A) tail. Similarly, the MFA2 mRNA was more rapidly deadenylated as well as significantly stabilized in the ccr4∆ deadenylation mutant in the edc3∆ lsm4∆C background. These results suggest a role for these decapping factors in stabilizing mRNA and may implicate P bodies as sites of reduced mRNA degradation.

  6. A General Framework for Interrogation of mRNA Stability Programs Identifies RNA-Binding Proteins that Govern Cancer Transcriptomes.

    PubMed

    Perron, Gabrielle; Jandaghi, Pouria; Solanki, Shraddha; Safisamghabadi, Maryam; Storoz, Cristina; Karimzadeh, Mehran; Papadakis, Andreas I; Arseneault, Madeleine; Scelo, Ghislaine; Banks, Rosamonde E; Tost, Jorg; Lathrop, Mark; Tanguay, Simon; Brazma, Alvis; Huang, Sidong; Brimo, Fadi; Najafabadi, Hamed S; Riazalhosseini, Yasser

    2018-05-08

    Widespread remodeling of the transcriptome is a signature of cancer; however, little is known about the post-transcriptional regulatory factors, including RNA-binding proteins (RBPs) that regulate mRNA stability, and the extent to which RBPs contribute to cancer-associated pathways. Here, by modeling the global change in gene expression based on the effect of sequence-specific RBPs on mRNA stability, we show that RBP-mediated stability programs are recurrently deregulated in cancerous tissues. Particularly, we uncovered several RBPs that contribute to the abnormal transcriptome of renal cell carcinoma (RCC), including PCBP2, ESRP2, and MBNL2. Modulation of these proteins in cancer cell lines alters the expression of pathways that are central to the disease and highlights RBPs as driving master regulators of RCC transcriptome. This study presents a framework for the screening of RBP activities based on computational modeling of mRNA stability programs in cancer and highlights the role of post-transcriptional gene dysregulation in RCC. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Photobiomodulation effects on mRNA levels from genomic and chromosome stabilization genes in injured muscle.

    PubMed

    da Silva Neto Trajano, Larissa Alexsandra; Trajano, Eduardo Tavares Lima; da Silva Sergio, Luiz Philippe; Teixeira, Adilson Fonseca; Mencalha, Andre Luiz; Stumbo, Ana Carolina; de Souza da Fonseca, Adenilson

    2018-04-26

    Muscle injuries are the most prevalent type of injury in sports. A great number of athletes have relapsed in muscle injuries not being treated properly. Photobiomodulation therapy is an inexpensive and safe technique with many benefits in muscle injury treatment. However, little has been explored about the infrared laser effects on DNA and telomeres in muscle injuries. Thus, the aim of this study was to evaluate photobiomodulation effects on mRNA relative levels from genes related to telomere and genomic stabilization in injured muscle. Wistar male rats were randomly divided into six groups: control, laser 25 mW, laser 75 mW, injury, injury laser 25 mW, and injury laser 75 mW. Photobiomodulation was performed with 904 nm, 3 J/cm 2 at 25 or 75 mW. Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly on the tibialis anterior muscle. After euthanasia, skeletal muscle samples were withdrawn and total RNA extracted for evaluation of mRNA levels from genomic (ATM and p53) and chromosome stabilization (TRF1 and TRF2) genes by real-time quantitative polymerization chain reaction. Data show that photobiomodulation reduces the mRNA levels from ATM and p53, as well reduces mRNA levels from TRF1 and TRF2 at 25 and 75 mW in injured skeletal muscle. In conclusion, photobiomodulation alters mRNA relative levels from genes related to genomic and telomere stabilization in injured skeletal muscle.

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

    PubMed

    Fred, Rikard G; Mehrabi, Syrina; Adams, Christopher M; Welsh, Nils

    2016-09-01

    Insulin expression is highly controlled on the posttranscriptional level. The RNA binding proteins (RBPs) responsible for this result are still largely unknown. To identify RBPs that bind to insulin mRNA we performed mass spectrometry analysis on proteins that bound synthetic oligonucloetides mimicing the 5'- and the 3'-untranslated regions (UTRs) of rat and human insulin mRNA in vitro . We observed that the RBPs heterogeneous nuclear ribonucleoprotein (hnRNP) U, polypyrimidine tract binding protein (PTB), hnRNP L and T-cell restricted intracellular antigen 1-related protein (TIA-1-related protein; TIAR) bind to insulin mRNA sequences, and that the in vitro binding affinity of these RBPs changed when INS-1 cells were exposed to glucose, 3-isobutyl-1-methylxanthine (IBMX) or nitric oxide. High glucose exposure resulted in a modest increase in PTB and TIAR binding to an insulin mRNA sequence. The inducer of nitrosative stress DETAnonoate increased markedly hnRNP U and TIAR mRNA binding. An increased PTB to TIAR binding ratio in vitro correlated with higher insulin mRNA levels and insulin biosynthesis rates in INS-1 cells. To further investigate the importance of RNA-binding proteins for insulin mRNA stability, we decreased INS-1 and EndoC-βH1 cell levels of PTB and TIAR by RNAi. In both cell lines, decreased levels of PTB resulted in lowered insulin mRNA levels while decreased levels of TIAR resulted in increased insulin mRNA levels. Thapsigargin-induced stress granule formation was associated with a redistribution of TIAR from the cytosol to stress granules. 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.

  9. Matrin 3 binds and stabilizes mRNA.

    PubMed

    Salton, Maayan; Elkon, Ran; Borodina, Tatiana; Davydov, Aleksey; Yaspo, Marie-Laure; Halperin, Eran; Shiloh, Yosef

    2011-01-01

    Matrin 3 (MATR3) is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM), whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq) identified several small noncoding RNA species. Using microarray analysis to explore MATR3's role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts.

  10. Matrin 3 Binds and Stabilizes mRNA

    PubMed Central

    Salton, Maayan; Elkon, Ran; Borodina, Tatiana; Davydov, Aleksey; Yaspo, Marie-Laure; Halperin, Eran; Shiloh, Yosef

    2011-01-01

    Matrin 3 (MATR3) is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM), whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq) identified several small noncoding RNA species. Using microarray analysis to explore MATR3′s role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts. PMID:21858232

  11. BAG3 directly stabilizes Hexokinase 2 mRNA and promotes aerobic glycolysis in pancreatic cancer cells.

    PubMed

    An, Ming-Xin; Li, Si; Yao, Han-Bing; Li, Chao; Wang, Jia-Mei; Sun, Jia; Li, Xin-Yu; Meng, Xiao-Na; Wang, Hua-Qin

    2017-12-04

    Aerobic glycolysis, a phenomenon known historically as the Warburg effect, is one of the hallmarks of cancer cells. In this study, we characterized the role of BAG3 in aerobic glycolysis of pancreatic ductal adenocarcinoma (PDAC) and its molecular mechanisms. Our data show that aberrant expression of BAG3 significantly contributes to the reprogramming of glucose metabolism in PDAC cells. Mechanistically, BAG3 increased Hexokinase 2 (HK2) expression, the first key enzyme involved in glycolysis, at the posttranscriptional level. BAG3 interacted with HK2 mRNA, and the degree of BAG3 expression altered recruitment of the RNA-binding proteins Roquin and IMP3 to the HK2 mRNA. BAG3 knockdown destabilized HK2 mRNA via promotion of Roquin recruitment, whereas BAG3 overexpression stabilized HK2 mRNA via promotion of IMP3 recruitment. Collectively, our results show that BAG3 promotes reprogramming of glucose metabolism via interaction with HK2 mRNA in PDAC cells, suggesting that BAG3 may be a potential target in the aerobic glycolysis pathway for developing novel anticancer agents. © 2017 An et al.

  12. TP53 and ATM mRNA expression in skin and skeletal muscle after low-level laser exposure.

    PubMed

    Guedes de Almeida, Luciana; Sergio, Luiz Philippe da Silva; de Paoli, Flavia; Mencalha, Andre Luiz; da Fonseca, Adenilson de Souza

    2017-08-01

    Low-level lasers are widespread in regenerative medicine, but the molecular mechanisms involved in their biological effects are not fully understood, particularly those on DNA stability. Therefore, this study aimed to investigate mRNA expression of genes related to DNA genomic stability in skin and skeletal muscle tissue from Wistar rats exposed to low-level red and infrared lasers. For this, TP53 (Tumor Protein 53) and ATM (Ataxia Telangiectasia Mutated gene) mRNA expressions were evaluated by real-time quantitative PCR (RT-qPCR) technique 24 hours after low-level red and infrared laser exposure. Our data showed that relative TP53 mRNA expression was not significantly altered in both tissues exposed to lasers. For ATM, relative mRNA expression in skin tissue was not significantly altered, but in muscle tissue, laser exposure increased relative ATM mRNA expression. Low-level red and infrared laser radiations alter ATM mRNA expression related to DNA stability in skeletal muscle tissue.

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

    PubMed Central

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

    1989-01-01

    A temperature-sensitive mutation in the ams gene of Escherichia coli causes an increase in the chemical half-life of pulse-labeled RNA at the nonpermissive temperature. Using lambda clones containing DNA fragments from the 23- to 24-min region on the E. coli chromosome, we have isolated a 5.8-kilobase DNA fragment which, when present in a low-copy-number plasmid, complements the conditional lethality and increased mRNA stability associated with the ams-1 mutation. The approximate initiation site and the direction of transcription of the ams gene were determined from the size of truncated polypeptides produced by Tn1000 insertions and Bal 31 deletions. Overexpression of the ams locus by using a T7 RNA polymerase-promoter system permitted the identification of an ams-encoded polypeptide of 110 kilodaltons. Images PMID:2477358

  14. Impairment of FOS mRNA stabilization following translation arrest in granulocytes from myelodysplastic syndrome patients.

    PubMed

    Feng, Xiaomin; Shikama, Yayoi; Shichishima, Tsutomu; Noji, Hideyoshi; Ikeda, Kazuhiko; Ogawa, Kazuei; Kimura, Hideo; Takeishi, Yasuchika; Kimura, Junko

    2013-01-01

    Although quantitative and qualitative granulocyte defects have been described in myelodysplastic syndromes (MDS), the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. We recently found that FOS mRNA elevation under translation-inhibiting stimuli was significantly smaller in granulocytes from MDS patients than in healthy individuals. The aim of this study is to clarify the cause of the impaired FOS induction in MDS. We first examined the mechanisms of FOS mRNA elevation using granulocytes from healthy donors cultured with the translation inhibitor emetine. Emetine increased both transcription and mRNA stability of FOS. p38 MAPK inhibition abolished the emetine-induced increase of FOS transcription but did not affect FOS mRNA stabilization. The binding of an AU-rich element (ARE)-binding protein HuR to FOS mRNA containing an ARE in 3'UTR was increased by emetine, and the knockdown of HuR reduced the FOS mRNA stabilizing effect of emetine. We next compared the emetine-induced transcription and mRNA stabilization of FOS between MDS patients and healthy controls. Increased rates of FOS transcription by emetine were similar in MDS and controls. In the absence of emetine, FOS mRNA decayed to nearly 17% of initial levels in 45 min in both groups. In the presence of emetine, however, 76.7±19.8% of FOS mRNA remained after 45 min in healthy controls, versus 37.9±25.5% in MDS (P<0.01). To our knowledge, this is the first report demonstrating attenuation of stress-induced FOS mRNA stabilization in MDS granulocytes.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Cup regulates oskar mRNA stability during oogenesis.

    PubMed

    Broyer, Risa M; Monfort, Elena; Wilhelm, James E

    2017-01-01

    The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development. One of the best characterized regulators of maternal transcripts is Cup - a broadly conserved component of the maternal RNP complex that in Drosophila acts as a translational repressor of the localized message oskar. In this study, we have found that loss of cup disrupts the localization of both the oskar mRNA and its associated proteins to the posterior pole of the developing oocyte. This defect is not due to a failure to specify the oocyte or to disruption of RNP transport. Rather, the localization defects are due to a drop in oskar mRNA levels in cup mutant egg chambers. Thus, in addition to its role in regulating oskar mRNA translation, Cup also plays a critical role in controlling the stability of the oskar transcript. This suggests that Cup is ideally positioned to coordinate the translational control function of the maternal RNP complex with its role in storing maternal transcripts in a stable form. Published by Elsevier Inc.

  17. Interrelations between translation and general mRNA degradation in yeast.

    PubMed

    Huch, Susanne; Nissan, Tracy

    2014-01-01

    Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system. © 2014 The Authors. WIREs RNA published by John Wiley & Sons, Ltd.

  18. Heat Shock Response in Yeast Involves Changes in Both Transcription Rates and mRNA Stabilities

    PubMed Central

    Castells-Roca, Laia; García-Martínez, José; Moreno, Joaquín; Herrero, Enrique; Bellí, Gemma; Pérez-Ortín, José E.

    2011-01-01

    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. PMID:21364882

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

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

    Lazarus, Kyren A.; Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122; Zhao, Zhe

    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 highermore » 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.« less

  20. Interrelations between translation and general mRNA degradation in yeast

    PubMed Central

    Huch, Susanne; Nissan, Tracy

    2014-01-01

    Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system. How to cite this article: WIREs RNA 2014, 5:747–763. doi: 10.1002/wrna.1244 PMID:24944158

  1. A small RNA activates CFA synthase by isoform-specific mRNA stabilization

    PubMed Central

    Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

    2013-01-01

    Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5′ end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5′ untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability. PMID:24141880

  2. A small RNA activates CFA synthase by isoform-specific mRNA stabilization.

    PubMed

    Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

    2013-11-13

    Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5' end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5' untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.

  3. Reversible methylation of m6Am in the 5′ cap controls mRNA stability

    PubMed Central

    Mauer, Jan; Luo, Xiaobing; Blanjoie, Alexandre; Jiao, Xinfu; Grozhik, Anya V.; Patil, Deepak P.; Linder, Bastian; Pickering, Brian F.; Vasseur, Jean-Jacques; Chen, Qiuying; Gross, Steven S.; Elemento, Olivier; Debart, Françoise; Kiledjian, Megerditch; Jaffrey, Samie R.

    2017-01-01

    Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5′ end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2′-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5′ cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability. PMID:28002401

  4. Overexpression of nucleolin in chronic lymphocytic leukemia cells induces stabilization of bcl2 mRNA

    PubMed Central

    Otake, Yoko; Soundararajan, Sridharan; Sengupta, Tapas K.; Kio, Ebenezer A.; Smith, James C.; Pineda-Roman, Mauricio; Stuart, Robert K.; Spicer, Eleanor K.

    2007-01-01

    B-cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal B cells that are resistant to apoptosis as a result of bcl2 oncogene overexpression. Studies were done to determine the mechanism for the up-regulation of bcl-2 protein observed in CD19+ CLL cells compared with CD19+ B cells from healthy volunteers. The 11-fold higher level of bcl-2 protein in CLL cells was positively correlated with a 26-fold elevation in the cytosolic level of nucleolin, a bcl2 mRNA–stabilizing protein. Measurements of the bcl2 heterogeneous nuclear/bcl2 mRNA (hnRNA)/mRNA ratios and the rates of bcl2 mRNA decay in cell extracts indicated that the 3-fold higher steady-state level of bcl2 mRNA in CLL cells was the result of increased bcl2 mRNA stability. Nucleolin was present throughout the nucleus and cytoplasm of CLL cells, whereas in normal B cells nucleolin was only detected in the nucleus. The addition of recombinant human nucleolin to extracts of normal B cells markedly slowed the rate of bcl2 mRNA decay. SiRNA knockdown of nucleolin in MCF-7 cells resulted in decreased levels of bcl2 mRNA and protein but no change in β-actin. These results indicate that bcl-2 overexpression in CLL cells is related to stabilization of bcl2 mRNA by nucleolin. PMID:17179226

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

    NASA Technical Reports Server (NTRS)

    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 < 0.05). The c-fos induction was transient and returned to control levels after 3 h. The mRNA level for the mineralization marker gene osteocalcin was significantly decreased to 44% of control level (P < 0.005) 3 h after centrifugation. No changes in mRNA levels were detected for c-myc, TGFbeta1, TGFbeta2, cyclophilin A, or actin. No 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.

  6. Double-Stranded RNA-Binding Protein Regulates Vascular Endothelial Growth Factor mRNA Stability, Translation, and Breast Cancer Angiogenesis▿

    PubMed Central

    Vumbaca, Frank; Phoenix, Kathryn N.; Rodriguez-Pinto, Daniel; Han, David K.; Claffey, Kevin P.

    2008-01-01

    Vascular endothelial growth factor (VEGF) is a key angiogenic factor expressed under restricted nutrient and oxygen conditions in most solid tumors. The expression of VEGF under hypoxic conditions requires transcription through activated hypoxia-inducible factor 1 (HIF-1), increased mRNA stability, and facilitated translation. This study identified double-stranded RNA-binding protein 76/NF90 (DRBP76/NF90), a specific isoform of the DRBP family, as a VEGF mRNA-binding protein which plays a key role in VEGF mRNA stability and protein synthesis under hypoxia. The DRBP76/NF90 protein binds to a human VEGF 3′ untranslated mRNA stability element. RNA interference targeting the DRBP76/NF90 isoform limited hypoxia-inducible VEGF mRNA and protein expression with no change in HIF-1-dependent transcriptional activity. Stable repression of DRBP76/NF90 in MDA-MB-435 breast cancer cells demonstrated reduced polysome-associated VEGF mRNA levels under hypoxic conditions and reduced mRNA stability. Transient overexpression of the DRBP76/NF90 protein increased both VEGF mRNA and protein levels synthesized under normoxic and hypoxic conditions. Cells with stable repression of the DRBP76/NF90 isoform showed reduced tumorigenic and angiogenic potential in an orthotopic breast tumor model. These data demonstrate that the DRBP76/NF90 isoform facilitates VEGF expression by promoting VEGF mRNA loading onto polysomes and translation under hypoxic conditions, thus promoting breast cancer growth and angiogenesis in vivo. PMID:18039850

  7. Vibrational force alters mRNA expression in osteoblasts

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

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

  8. Aging alters mRNA expression of amyloid transporter genes at the blood-brain barrier.

    PubMed

    Osgood, Doreen; Miller, Miles C; Messier, Arthur A; Gonzalez, Liliana; Silverberg, Gerald D

    2017-09-01

    Decreased clearance of potentially toxic metabolites, due to aging changes, likely plays a significant role in the accumulation of amyloid-beta (Aβ) peptides and other macromolecules in the brain of the elderly and in the patients with Alzheimer's disease (AD). Aging is the single most important risk factor for AD development. Aβ transport receptor proteins expressed at the blood-brain barrier are significantly altered with age: the efflux transporters lipoprotein receptor-related protein 1 and P-glycoprotein are reduced, whereas the influx transporter receptor for advanced glycation end products is increased. These receptors play an important role in maintaining brain biochemical homeostasis. We now report that, in a rat model of aging, gene transcription is altered in aging, as measured by Aβ receptor gene messenger RNA (mRNA) at 3, 6, 9, 12, 15, 20, 30, and 36 months. Gene mRNA expression from isolated cerebral microvessels was measured by quantitative polymerase chain reaction. Lipoprotein receptor-related protein 1 and P-glycoprotein mRNA were significantly reduced in aging, and receptor for advanced glycation end products was increased, in parallel with the changes seen in receptor protein expression. Transcriptional changes appear to play a role in aging alterations in blood-brain barrier receptor expression and Aβ accumulation. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Stabilization of Clostridium perfringens collagenase mRNA by VR-RNA-dependent cleavage in 5' leader sequence.

    PubMed

    Obana, Nozomu; Shirahama, Yu; Abe, Kimihiro; Nakamura, Kouji

    2010-09-01

    The small RNA (sRNA), VR-RNA that is directly regulated by the VirR/VirS two-component system, regulates many genes including toxin genes such as collagenase (colA) and phospholipase C (plc) in Clostridium perfringens. Although the VR-RNA 3' region is sufficient to regulate the colA and plc genes, the molecular mechanism of toxin gene regulation by VR-RNA remains unclear. Here, we found that colA mRNA is cleaved at position -79 and -78 from the A of the first codon (ATG) in the presence of VR-RNA. The processed transcripts were stable compared with longer intact transcripts. On the other hand, colA mRNA was labile in a VR-RNA-deficient strain, and processed transcripts were undetectable. The stability and processing of colA mRNA were restored by transformation of the 3' region of VR-RNA-expression vector. The 3' region of VR-RNA and colA mRNA had significant complementation and interacted in vitro. These results show that VR-RNA base pairs with colA mRNA and induces cleavage in the 5' untranslated region (UTR) of colA mRNA, which leads to the stabilization of colA mRNA and the activation of colA expression. © 2010 Blackwell Publishing Ltd.

  10. HuR (Elavl1) and HuB (Elavl2) Stabilize Matrix Metalloproteinase-9 mRNA During Seizure-Induced Mmp-9 Expression in Neurons

    PubMed Central

    Zybura-Broda, Katarzyna; Wolder-Gontarek, Malgorzata; Ambrozek-Latecka, Magdalena; Choros, Artur; Bogusz, Agnieszka; Wilemska-Dziaduszycka, Joanna; Rylski, Marcin

    2018-01-01

    Matrix metalloproteinase-9 (Mmp-9) is involved in different general and cell-type–specific processes, both in neuronal and non-neuronal cells. Moreover, it is implicated in an induction or progression of various human disorders, including diseases of the central nervous system. Mechanisms regulating activity-driven Mmp-9 expression in neurons are still not fully understood. Here, we show that stabilization of Mmp-9 mRNA is one of the factors responsible for the neuronal activity-evoked upregulation of Mmp-9 mRNA expression in hippocampal neurons. Furthermore, we demonstrate that the molecular mechanism related to this stabilization is dependent on the neuronal seizure-triggered transiently increased binding of the mRNA stability-inducing protein, HuR, to ARE1 and ARE4 motifs of the 3′UTR for Mmp-9 mRNA as well as the stably augmented association of another mRNA-stabilizing protein, HuB, to the ARE1 element of the 3′UTR. Intriguingly, we demonstrate further that both HuR and HuB are crucial for an incidence of Mmp-9 mRNA stabilization after neuronal activation. This study identifies Mmp-9 mRNA as the first HuB target regulated by mRNA stabilization in neurons. Moreover, these results are the first to describe an existence of HuR-dependent mRNA stabilization in neurons of the brain. PMID:29686606

  11. Altered profile of mRNA expression in atrioventricular node of streptozotocin-induced diabetic rats

    PubMed Central

    Howarth, Frank Christopher; Parekh, Khatija; Jayaprakash, Petrilla; Inbaraj, Edward Samuel; Oz, Murat; Dobrzynski, Halina; Adrian, Thomas Edward

    2017-01-01

    Prolonged action potential duration, reduced action potential firing rate, upstroke velocity and rate of diastolic depolarization have been demonstrated in atrioventricular node (AVN) cells from streptozotocin (STZ)-induced diabetic rats. To further clarify the molecular basis of these electrical disturbances, the mRNA profiles encoding a variety of proteins associated with the generation and conduction of electrical activity in the AVN, were evaluated in the STZ-induced diabetic rat heart. Expression of mRNA was measured in AVN biopsies using reverse transcription-quantitative polymerase chain reaction techniques. Notable differences in mRNA expression included upregulation of genes encoding membrane and intracellular Ca2+ transport, including solute carrier family 8 member A1, transient receptor potential channel 1, ryanodine receptor 2/3, hyperpolarization-activated cyclic-nucleotide 2 and 3, calcium channel voltage-dependent, β2 subunit and sodium channels 3a, 4a, 7a and 3b. In addition to this, potassium channels potassium voltage-gated channel subfamily A member 4, potassium channel calcium activated intermediate/small conductance subfamily N α member 2, potassium voltage-gated channel subfamily J members 3, 5, and 11, potassium channel subfamily K members 1, 2, 3 and natriuretic peptide B (BNP) were upregulated in AVN of STZ heart, compared with controls. Alterations in gene expression were associated with upregulation of various proteins including the inwardly rectifying, potassium channel Kir3.4, NCX1 and BNP. The present study demonstrated notable differences in the profile of mRNA encoding proteins associated with the generation, conduction and regulation of electrical signals in the AVN of the STZ-induced diabetic rat heart. These data will provide a basis for a substantial range of future studies to investigate whether variations in mRNA translate into alterations in electrophysiological function. PMID:28731153

  12. Tristetraprolin regulation of interleukin 23 mRNA stability prevents a spontaneous inflammatory disease

    PubMed Central

    Molle, Céline; Zhang, Tong; Ysebrant de Lendonck, Laure; Gueydan, Cyril; Andrianne, Mathieu; Sherer, Félicie; Van Simaeys, Gaetan; Blackshear, Perry J.; Leo, Oberdan

    2013-01-01

    Interleukin (IL) 12 and IL23 are two related heterodimeric cytokines produced by antigen-presenting cells. The balance between these two cytokines plays a crucial role in the control of Th1/Th17 responses and autoimmune inflammation. Most studies focused on their transcriptional regulation. Herein, we explored the role of the adenine and uridine–rich element (ARE)–binding protein tristetraprolin (TTP) in influencing mRNA stability of IL12p35, IL12/23p40, and IL23p19 subunits. LPS-stimulated bone marrow–derived dendritic cells (BMDCs) from TTP−/− mice produced normal levels of IL12/23p40. Production of IL12p70 was modestly increased in these conditions. In contrast, we observed a strong impact of TTP on IL23 production and IL23p19 mRNA stability through several AREs in the 3′ untranslated region. TTP−/− mice spontaneously develop an inflammatory syndrome characterized by cachexia, myeloid hyperplasia, dermatitis, and erosive arthritis. We observed IL23p19 expression within skin lesions associated with exacerbated IL17A and IL22 production by infiltrating γδ T cells and draining lymph node CD4 T cells. We demonstrate that the clinical and immunological parameters associated with TTP deficiency were completely dependent on the IL23–IL17A axis. We conclude that tight control of IL23 mRNA stability by TTP is critical to avoid severe inflammation. PMID:23940256

  13. Tristetraprolin regulation of interleukin 23 mRNA stability prevents a spontaneous inflammatory disease.

    PubMed

    Molle, Céline; Zhang, Tong; Ysebrant de Lendonck, Laure; Gueydan, Cyril; Andrianne, Mathieu; Sherer, Félicie; Van Simaeys, Gaetan; Blackshear, Perry J; Leo, Oberdan; Goriely, Stanislas

    2013-08-26

    Interleukin (IL) 12 and IL23 are two related heterodimeric cytokines produced by antigen-presenting cells. The balance between these two cytokines plays a crucial role in the control of Th1/Th17 responses and autoimmune inflammation. Most studies focused on their transcriptional regulation. Herein, we explored the role of the adenine and uridine-rich element (ARE)-binding protein tristetraprolin (TTP) in influencing mRNA stability of IL12p35, IL12/23p40, and IL23p19 subunits. LPS-stimulated bone marrow-derived dendritic cells (BMDCs) from TTP(-/-) mice produced normal levels of IL12/23p40. Production of IL12p70 was modestly increased in these conditions. In contrast, we observed a strong impact of TTP on IL23 production and IL23p19 mRNA stability through several AREs in the 3' untranslated region. TTP(-/-) mice spontaneously develop an inflammatory syndrome characterized by cachexia, myeloid hyperplasia, dermatitis, and erosive arthritis. We observed IL23p19 expression within skin lesions associated with exacerbated IL17A and IL22 production by infiltrating γδ T cells and draining lymph node CD4 T cells. We demonstrate that the clinical and immunological parameters associated with TTP deficiency were completely dependent on the IL23-IL17A axis. We conclude that tight control of IL23 mRNA stability by TTP is critical to avoid severe inflammation.

  14. KSRP Modulation of GAP-43 mRNA Stability Restricts Axonal Outgrowth in Embryonic Hippocampal Neurons

    PubMed Central

    Bird, Clark W.; Gardiner, Amy S.; Bolognani, Federico; Tanner, Daniel C.; Chen, Ching-Yi; Lin, Wei-Jye; Yoo, Soonmoon; Twiss, Jeffery L.; Perrone- Bizzozero, Nora

    2013-01-01

    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. PMID:24244461

  15. Minimal alteration in the ratio of circulatory fetal DNA to fetal corticotropin-releasing hormone mRNA level in preeclampsia.

    PubMed

    Zhong, Xiao Yan; Holzgreve, Wolfgang; Gebhardt, Stefan; Hillermann, Renate; Tofa, Kashefa Carelse; Gupta, Anurag Kumar; Huppertz, Berthold; Hahn, Sinuhe

    2006-01-01

    We have recently observed that fetal DNA and fetal corticotropin-releasing hormone (CRH) mRNA are associated with in vitro generated syncytiotrophoblast-derived microparticles, and that the ratio of fetal DNA to mRNA (CRH) varied according to whether the particles were derived by predominantly apoptotic, apo-necrotic or necrotic pathways. Hence, we examined whether these ratios varied in maternal plasma samples taken from normotensive and preeclamptic pregnancies in vivo. Maternal plasma samples were collected from 18 cases with preeclampsia and 29 normotensive term controls. Circulatory fetal CRH mRNA and DNA levels were quantified by real-time PCR and RT-PCR. Circulatory fetal mRNA and fetal DNA levels were significantly elevated in the preeclampsia study group when compared to normotensive controls. Alterations in the fetal mRNA to DNA ratio between the study and control groups were minimal, even when stratified into early (<34 weeks of gestation) and late (>34 weeks of gestation) onset preeclampsia. Our data suggest that although circulatory fetal DNA and mRNA levels are significantly elevated in preeclampsia, the ratios in maternal plasma are not dramatically altered. Copyright 2006 S. Karger AG, Basel.

  16. Neuronal ELAV proteins enhance mRNA stability by a PKCα-dependent pathway

    PubMed Central

    Pascale, Alessia; Amadio, Marialaura; Scapagnini, Giovanni; Lanni, Cristina; Racchi, Marco; Provenzani, Alessandro; Govoni, Stefano; Alkon, Daniel L.; Quattrone, Alessandro

    2005-01-01

    More than 1 in 20 human genes bear in the mRNA 3′ UTR a specific motif called the adenine- and uridine-rich element (ARE), which posttranscriptionally determines its expression in response to cell environmental signals. ELAV (embryonic lethal abnormal vision) proteins are the only known ARE-binding factors that are able to stabilize the bound mRNAs, thereby positively controlling gene expression. Here, we show that in human neuroblastoma SH-SY5Y cells, neuron-specific ELAV (nELAV) proteins (HuB, HuC, and HuD) are up-regulated and redistributed by 15 min of treatment with the activators of PKC phorbol esters and bryostatin-1. PKC stimulation also induces nELAV proteins to colocalize with the translocated PKCα isozyme preferentially on the cytoskeleton, with a concomitant increase of nELAV threonine phosphorylation. The same treatment promotes stabilization of growth-associated protein 43 (GAP-43) mRNA, a well known nELAV target, and induces an early increase in GAP-43 protein concentration, again only in the cytoskeletal cell fraction. Genetic or pharmacological inactivation of PKCα abolishes nELAV protein cytoskeletal up-regulation, GAP-43 mRNA stabilization, and GAP-43 protein increase, demonstrating the primary role of this specific PKC isozyme in the cascade of nELAV recruitment. Finally, in vivo PKC activation is associated with an up-regulation of nELAV proteins in the hippocampal rat brain. These findings suggest a model for gene expression regulation by nELAV proteins through a PKCα-dependent pathway that is relevant for the cellular programs in which ARE-mediated control plays a pivotal role. PMID:16099831

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

    PubMed

    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. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    PubMed Central

    2004-01-01

    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. PMID:15496143

  19. Structural and Functional Analysis of an mRNP Complex That Mediates the High Stability of Human β-Globin mRNA

    PubMed Central

    Yu, Jia; Russell, J. Eric

    2001-01-01

    Human globins are encoded by mRNAs exhibiting high stabilities in transcriptionally silenced erythrocyte progenitors. Unlike α-globin mRNA, whose stability is enhanced by assembly of a specific messenger RNP (mRNP) α complex on its 3′ untranslated region (UTR), neither the structure(s) nor the mechanism(s) that effects the high-level stability of human β-globin mRNA has been identified. The present work describes an mRNP complex assembling on the 3′ UTR of the β-globin mRNA that exhibits many of the properties of the stability-enhancing α complex. The β-globin mRNP complex is shown to contain one or more factors homologous to αCP, a 39-kDa RNA-binding protein that is integral to α-complex assembly. Sequence analysis implicates a specific 14-nucleotide pyrimidine-rich track within its 3′ UTR as the site of β-globin mRNP assembly. The importance of this track to mRNA stability is subsequently verified in vivo using mice expressing human β-globin transgenes that contain informative mutations in this region. In combination, the in vitro and in vivo analyses indicate that the high stabilities of the α- and β-globin mRNAs are maintained through related mRNP complexes that may share a common regulatory pathway. PMID:11486027

  20. Stabilization and cytoskeletal-association of LDL receptor mRNA are mediated by distinct domains in its 3' untranslated region.

    PubMed

    Wilson, G M; Vasa, M Z; Deeley, R G

    1998-05-01

    The mRNA encoding the human low density lipoprotein (LDL) receptor is transiently stabilized after phorbol ester treatment of HepG2 cells and has been shown to associate with components of the cytoskeleton in this cell line (G. M. Wilson, E. A. Roberts, and R. G. Deeley, J. Lipid Res. 1997. 38: 437-446). Using an episomal expression system, fragments of the 3' untranslated region (3'UTR) of LDL receptor mRNA were transcribed in fusion with the coding region of beta-globin mRNA in HepG2 cells. Analyses of the decay kinetics of these beta-globin-LDL receptor fusion mRNA deletion mutants showed that sequences in the proximal 3'UTR of LDL receptor mRNA including several AU-rich elements (AREs) were sufficient to confer short constitutive mRNA half-life in the heterologous system. Stabilization of LDL receptor mRNA in the presence of PMA required sequences in the distal 3'UTR, at or near three Alu-like repetitive elements. Furthermore, the 3'UTR of LDL receptor mRNA conferred cytoskeletal association on the otherwise unassociated beta-globin mRNA, by a mechanism involving at least two distinct RNA elements. Comparisons of decay kinetics and subcellular localization of endogenous LDL receptor mRNA and beta-globin-LDL receptor mRNA fusions in HepG2 cells have demonstrated that several cis-acting elements in the receptor 3'UTR contribute to post-transcriptional regulation of receptor expression, and provide further support for involvement of the cytoskeleton in the regulation of LDL receptor mRNA turnover.

  1. Modulation of Ergot Alkaloids in a Grass-Endophyte Symbiosis by Alteration of mRNA Concentrations of an Ergot Alkaloid Synthesis Gene.

    PubMed

    Mulinti, Prashanthi; Florea, Simona; Schardl, Christopher L; Panaccione, Daniel G

    2016-06-22

    The profile of ergot alkaloids in perennial ryegrass (Lolium perenne) containing the endophytic fungus Epichloë typhina × festucae includes high concentrations of the early pathway metabolites ergotryptamine and chanoclavine-I in addition to the pathway end-product ergovaline. Because these alkaloids differ in activity, we investigated strategies to alter their relative concentrations. An RNAi-based approach reduced the concentration of mRNA from the gene easA, which encodes an enzyme required for a ring closure that separates ergotryptamine and chanoclavine-I from ergovaline. Lower easA mRNA concentrations correlated with lower concentrations of ergovaline and higher concentrations of ergotryptamine and chanoclavine-I. Overexpression of easA led to higher concentrations of ergovaline in leaf blades but not in pseudostems; concentrations of the early pathway metabolites were not altered in overexpression strains. The data indicate that altering the concentration of mRNA from a single gene can change alkaloid flux, but the magnitude of the change was limited and variable.

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

    PubMed

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

    2016-06-06

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

  3. p62/SQSTM1 enhances breast cancer stem-like properties by stabilizing MYC mRNA

    PubMed Central

    Xu, L-Z; Li, S-S; Zhou, W; Kang, Z-J; Zhang, Q-X; Kamran, M; Xu, J; Liang, D-P; Wang, C-L; Hou, Z-J; Wan, X-B; Wang, H-J; Lam, E W-F; Zhao, Z-W; Liu, Q

    2017-01-01

    Aberrant p62 overexpression has been implicated in breast cancer development. Here, we found that p62 expression was elevated in breast cancer stem cells (BCSCs), including CD44+CD24− fractions, mammospheres, ALDH1+ populations and side population cells. Indeed, short-hairpin RNA (shRNA)-mediated knockdown of p62 impaired breast cancer cells from self-renewing under anchorage-independent conditions, whereas ectopic overexpression of p62 enhanced the self-renewal ability of breast cancer cells in vitro. Genetic depletion of p62 robustly inhibited tumor-initiating frequencies, as well as growth rates of BCSC-derived tumor xenografts in immunodeficient mice. Consistently, immunohistochemical analysis of clinical breast tumor tissues showed that high p62 expression levels were linked to poorer clinical outcome. Further gene expression profiling analysis revealed that p62 was positively correlated with MYC expression level, which mediated the function of p62 in promoting breast cancer stem-like properties. MYC mRNA level was reduced upon p62 deletion by siRNA and increased with p62 overexpression in breast cancer cells, suggesting that p62 positively regulated MYC mRNA. Interestingly, p62 did not transactivate MYC promoter. Instead, p62 delayed the degradation of MYC mRNA by repressing the expression of let-7a and let-7b, thus promoting MYC mRNA stabilization at the post-transcriptional level. Consistently, let-7a and let-7b mimics attenuated p62-mediated MYC mRNA stabilization. Together, these findings unveiled a previously unappreciated role of p62 in the regulation of BCSCs, assigning p62 as a promising therapeutic target for breast cancer treatments. PMID:27345399

  4. Nest box exploration may stimulate breeding physiology and alter mRNA expression in the medial preoptic area of female European starlings.

    PubMed

    Spool, Jeremy A; Jay, Melannie D; Riters, Lauren V

    2018-04-25

    Environmental resources are proposed to fine-tune the timing of breeding, yet how they may do so remains unclear. In female European starlings ( Sturnus vulgaris ), nest cavities are limited resources that are necessary for breeding. Females that explore nest cavities, compared to those that do not, readily perform sexually-motivated behaviors. We assigned female starlings to aviaries with 1) no nest boxes, 2) nest boxes, or 3) nest boxes, plants, flowing water, insects and berries to test the hypothesis that environmental resources alter neural systems to stimulate mating behavior. Compared to other females, females that were housed with and explored nest boxes had higher estradiol, higher preproenkephalin (PENK) mRNA, and lower levels of D1 and D2 dopamine receptor mRNA in the medial preoptic area (mPOA), a region in which opioids and dopamine modify female sexual behaviors and sexual motivation. Additionally, in the mPOA, PENK and tyrosine hydroxylase mRNA positively predicted, whereas estrogen receptor beta mRNA negatively predicted nest box exploration. In the ventromedial hypothalamus, a region in which estradiol acts to stimulate sexual behavior, estrogen receptor alpha mRNA was highest in females that had access to but did not explore nest cavities. It is likely that seasonal increases in estradiol modify mRNA in the mPOA to facilitate nest cavity exploration. It is also possible that nest cavity exploration further alters gene expression in the mPOA, functioning to coordinate mating with resource availability. Thus nest cavity exploration may be a form of self-stimulation that alters neural systems to fine-tune sexual behavior. © 2018. Published by The Company of Biologists Ltd.

  5. The mTOR kinase inhibitor rapamycin decreases iNOS mRNA stability in astrocytes

    PubMed Central

    2011-01-01

    NOS mRNA were detected after 48 hours, suggesting that rapamycin can modify iNOS mRNA stability. In this regard, we found that rapamycin significantly reduced the half-life of iNOS mRNA, from 4 h to 50 min when cells were co-incubated with cytokine mixture and 10 nM rapamycin. Similarly, rapamycin induced a significant up-regulation of tristetraprolin (TTP), a protein involved in the regulation of iNOS mRNA stability. Conclusion The present findings show that mTOR controls the rate of iNOS mRNA degradation in astrocytes. Together with the marked anti-inflammatory effects that we previously observed in microglial cells, these data suggest possible beneficial effects of mTOR inhibitors in the treatment of inflammatory-based CNS pathologies. PMID:21208419

  6. The importance of mRNA structure in determining the pathogenicity of synonymous and non-synonymous mutations in haemophilia

    PubMed Central

    Hamasaki-Katagiri, Nobuko; Lin, Brian C.; Simon, Jonathan; Hunt, Ryan C.; Schiller, Tal; Russek-Cohen, Estelle; Komar, Anton A.; Bar, Haim; Kimchi-Sarfaty, Chava

    2016-01-01

    Introduction Mutational analysis is commonly used to support the diagnosis and management of haemophilia. This has allowed for the generation of large mutation databases which provide unparalleled insight into genotype-phenotype relationships. Haemophilia is associated with inversions, deletions, insertions, nonsense and missense mutations. Both synonymous and non-synonymous mutations influence the base pairing of messenger RNA (mRNA), which can alter mRNA structure, cellular half-life and ribosome processivity/elongation. However, the role of mRNA structure in determining the pathogenicity of point mutations in haemophilia has not been evaluated. Aim To evaluate mRNA thermodynamic stability and associated RNA prediction software as a means to distinguish between neutral and disease-associated mutations in haemophilia. Methods Five mRNA structure prediction software programs were used to assess the thermodynamic stability of mRNA fragments carrying neutral vs. disease-associated and synonymous vs. non-synonymous point mutations in F8, F9 and a third X-linked gene, DMD (dystrophin). Results In F8 and DMD, disease-associated mutations tend to occur in more structurally stable mRNA regions, represented by lower MFE (minimum free energy) levels. In comparing multiple software packages for mRNA structure prediction, a 101–151 nucleotide fragment length appears to be a feasible range for structuring future studies. Conclusion mRNA thermodynamic stability is one predictive characteristic, which when combined with other RNA and protein features, may offer significant insight when screening sequencing data for novel disease-associated mutations. Our results also suggest potential utility in evaluating the mRNA thermodynamic stability profile of a gene when determining the viability of interchanging codons for biological and therapeutic applications. PMID:27933712

  7. LAST, a c-Myc-inducible long noncoding RNA, cooperates with CNBP to promote CCND1 mRNA stability in human cells

    PubMed Central

    Li, Jinming

    2017-01-01

    Cyclin D1 is a critical regulator of cell cycle progression and works at the G1 to S-phase transition. Here, we report the isolation and characterization of the novel c-Myc-regulated lncRNA LAST (LncRNA-Assisted Stabilization of Transcripts), which acts as a CCND1 mRNA stabilizer. Mechanistically, LAST was shown to cooperate with CNBP to bind to the 5′UTR of CCND1 mRNA to protect against possible nuclease targeting. In addition, data from CNBP RIP-seq and LAST RNA-seq showed that CCND1 mRNA might not be the only target of LAST and CNBP; three additional mRNAs were shown to be post-transcriptional targets of LAST and CNBP. In a xenograft model, depletion of LAST diminished and ectopic expression of LAST induced tumor formation, which are suggestive of its oncogenic function. We thus report a previously unknown lncRNA involved in the fine-tuned regulation of CCND1 mRNA stability, without which CCND1 exhibits, at most, partial expression. PMID:29199958

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

    PubMed

    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.

  9. Sodium 4-phenylbutyrate downregulates HSC70 expression by facilitating mRNA degradation.

    PubMed

    Rubenstein, R C; Lyons, B M

    2001-07-01

    Intracellular trafficking of the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) is repaired by sodium 4-phenylbutyrate (4PBA) by an undetermined mechanism. 4PBA downregulates protein and mRNA expression of the heat shock cognate protein HSC70 (the constitutively expressed member of the 70-kDa heat shock protein family) by approximately 40-50% and decreases formation of a HSC70-DeltaF508 CFTR complex that may be important in the intracellular degradation of DeltaF508 CFTR. We examined the potential mechanisms by which 4PBA decreases HSC70 mRNA and protein expression. In IB3-1 cells, 1 mM 4PBA did not alter the activity of the Chinese hamster ovary HSC70 promoter or of a human HSC70 promoter fragment in luciferase reporter assays nor did it alter HSC70 mRNA synthesis in nuclear runoff assays. In contrast, preincubation with 4PBA increased the rate of HSC70 mRNA degradation by approximately 40%. The initial rate of 35S-HSC70 protein synthesis in 4PBA-treated IB3-1 cells was reduced by approximately 40%, consistent with the steady-state mRNA level, whereas its rate of degradation was unaltered by 4PBA. 4PBA also reduced the steady-state accumulation of (35)S-HSC70 by approximately 40%. These data suggest that 4PBA decreases the expression of HSC70 mRNA and protein by inducing cellular adaptations that result in the decreased stability of HSC70 mRNA.

  10. Ventilation-induced increases in EGFR ligand mRNA are not altered by intra-amniotic LPS or ureaplasma in preterm lambs.

    PubMed

    Hillman, Noah H; Gisslen, Tate; Polglase, Graeme R; Kallapur, Suhas G; Jobe, Alan H

    2014-01-01

    Chorioamnionitis and mechanical ventilation are associated with bronchopulmonary dysplasia (BPD) in preterm infants. Mechanical ventilation at birth activates both inflammatory and acute phase responses. These responses can be partially modulated by previous exposure to intra-amniotic (IA) LPS or Ureaplasma parvum (UP). Epidermal growth factor receptor (EGFR) ligands participate in lung development, and angiotensin converting enzyme (ACE) 1 and ACE2 contribute to lung inflammation. We asked whether brief mechanical ventilation at birth altered EGFR and ACE pathways and if antenatal exposure to IA LPS or UP could modulate these effects. Ewes were exposed to IA injections of UP, LPS or saline multiple days prior to preterm delivery at 85% gestation. Lambs were either immediately euthanized or mechanically ventilated for 2 to 3 hr. IA UP and LPS cause modest changes in the EGFR ligands amphiregulin (AREG), epiregulin (EREG), heparin binding epidermal growth factor (HB-EGF), and betacellulin (BTC) mRNA expression. Mechanical ventilation greatly increased mRNA expression of AREG, EREG, and HB-EGF, with no additional increases resulting from IA LPS or UP. With ventilation AREG and EREG mRNA localized to cells in terminal airspace. EGFR mRNA also increased with mechanical ventilation. IA UP and LPS decreased ACE1 mRNA and increased ACE2 mRNA, resulting in a 4 fold change in the ACE1/ACE2 ratio. Mechanical ventilation with large tidal volumes increased both ACE1 and ACE2 expression. The alterations seen in ACE with IA exposures and EGFR pathways with mechanical ventilation may contribute to the development of BPD in preterm infants.

  11. Low levels of the AhR in chronic obstructive pulmonary disease (COPD)-derived lung cells increases COX-2 protein by altering mRNA stability

    PubMed Central

    Zago, Michela; Sheridan, Jared A.; Traboulsi, Hussein; Hecht, Emelia; Zhang, Yelu; Guerrina, Necola; Matthews, Jason; Nair, Parameswaran; Eidelman, David H.; Hamid, Qutayba

    2017-01-01

    Heightened inflammation, including expression of COX-2, is associated with chronic obstructive pulmonary disease (COPD) pathogenesis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is reduced in COPD-derived lung fibroblasts. The AhR also suppresses COX-2 in response to cigarette smoke, the main risk factor for COPD, by destabilizing the Cox-2 transcript by mechanisms that may involve the regulation of microRNA (miRNA). Whether reduced AhR expression is responsible for heightened COX-2 in COPD is not known. Here, we investigated the expression of COX-2 as well as the expression of miR-146a, a miRNA known to regulate COX-2 levels, in primary lung fibroblasts derived from non-smokers (Normal) and smokers (At Risk) with and without COPD. To confirm the involvement of the AhR, AhR knock-down via siRNA in Normal lung fibroblasts and MLE-12 cells was employed as were A549-AhRko cells. Basal expression of COX-2 protein was higher in COPD lung fibroblasts compared to Normal or Smoker fibroblasts but there was no difference in Cox-2 mRNA. Knockdown of AhR in lung structural cells increased COX-2 protein by stabilizing the Cox-2 transcript. There was less induction of miR-146a in COPD-derived lung fibroblasts but this was not due to the AhR. Instead, we found that RelB, an NF-κB protein, was required for transcriptional induction of both Cox-2 and miR-146a. Therefore, we conclude that the AhR controls COX-2 protein via mRNA stability by a mechanism independent of miR-146a. Low levels of the AhR may therefore contribute to the heightened inflammation common in COPD patients. PMID:28749959

  12. Low levels of the AhR in chronic obstructive pulmonary disease (COPD)-derived lung cells increases COX-2 protein by altering mRNA stability.

    PubMed

    Zago, Michela; Sheridan, Jared A; Traboulsi, Hussein; Hecht, Emelia; Zhang, Yelu; Guerrina, Necola; Matthews, Jason; Nair, Parameswaran; Eidelman, David H; Hamid, Qutayba; Baglole, Carolyn J

    2017-01-01

    Heightened inflammation, including expression of COX-2, is associated with chronic obstructive pulmonary disease (COPD) pathogenesis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is reduced in COPD-derived lung fibroblasts. The AhR also suppresses COX-2 in response to cigarette smoke, the main risk factor for COPD, by destabilizing the Cox-2 transcript by mechanisms that may involve the regulation of microRNA (miRNA). Whether reduced AhR expression is responsible for heightened COX-2 in COPD is not known. Here, we investigated the expression of COX-2 as well as the expression of miR-146a, a miRNA known to regulate COX-2 levels, in primary lung fibroblasts derived from non-smokers (Normal) and smokers (At Risk) with and without COPD. To confirm the involvement of the AhR, AhR knock-down via siRNA in Normal lung fibroblasts and MLE-12 cells was employed as were A549-AhRko cells. Basal expression of COX-2 protein was higher in COPD lung fibroblasts compared to Normal or Smoker fibroblasts but there was no difference in Cox-2 mRNA. Knockdown of AhR in lung structural cells increased COX-2 protein by stabilizing the Cox-2 transcript. There was less induction of miR-146a in COPD-derived lung fibroblasts but this was not due to the AhR. Instead, we found that RelB, an NF-κB protein, was required for transcriptional induction of both Cox-2 and miR-146a. Therefore, we conclude that the AhR controls COX-2 protein via mRNA stability by a mechanism independent of miR-146a. Low levels of the AhR may therefore contribute to the heightened inflammation common in COPD patients.

  13. Fragile X syndrome: loss of local mRNA regulation alters synaptic development and function.

    PubMed

    Bassell, Gary J; Warren, Stephen T

    2008-10-23

    Fragile X syndrome is the most common inherited form of cognitive deficiency in humans and perhaps the best-understood single cause of autism. A trinucleotide repeat expansion, inactivating the X-linked FMR1 gene, leads to the absence of the fragile X mental retardation protein. FMRP is a selective RNA-binding protein that regulates the local translation of a subset of mRNAs at synapses in response to activation of Gp1 metabotropic glutamate receptors (mGluRs) and possibly other receptors. In the absence of FMRP, excess and dysregulated mRNA translation leads to altered synaptic function and loss of protein synthesis-dependent plasticity. Recent evidence indicates the role of FMRP in regulated mRNA transport in dendrites. New studies also suggest a possible local function of FMRP in axons that may be important for guidance, synaptic development, and formation of neural circuits. The understanding of FMRP function at synapses has led to rationale therapeutic approaches.

  14. RNA-binding Protein Quaking Stabilizes Sirt2 mRNA during Oligodendroglial Differentiation*

    PubMed Central

    Thangaraj, Merlin P.; Furber, Kendra L.; Gan, Jotham K.; Ji, Shaoping; Sobchishin, Larhonda; Doucette, J. Ronald; Nazarali, Adil J.

    2017-01-01

    Myelination is controlled by timely expression of genes involved in the differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes (OLs). Sirtuin 2 (SIRT2), a NAD+-dependent deacetylase, plays a critical role in OL differentiation by promoting both arborization and downstream expression of myelin-specific genes. However, the mechanisms involved in regulating SIRT2 expression during OL development are largely unknown. The RNA-binding protein quaking (QKI) plays an important role in myelination by post-transcriptionally regulating the expression of several myelin specific genes. In quaking viable (qkv/qkv) mutant mice, SIRT2 protein is severely reduced; however, it is not known whether these genes interact to regulate OL differentiation. Here, we report for the first time that QKI directly binds to Sirt2 mRNA via a common quaking response element (QRE) located in the 3′ untranslated region (UTR) to control SIRT2 expression in OL lineage cells. This interaction is associated with increased stability and longer half-lives of Sirt2.1 and Sirt2.2 transcripts leading to increased accumulation of Sirt2 transcripts. Consistent with this, overexpression of qkI promoted the expression of Sirt2 mRNA and protein. However, overexpression of the nuclear isoform qkI-5 promoted the expression of Sirt2 mRNA, but not SIRT2 protein, and delayed OL differentiation. These results suggest that the balance in the subcellular distribution and temporal expression of QKI isoforms control the availability of Sirt2 mRNA for translation. Collectively, our study demonstrates that QKI directly plays a crucial role in the post-transcriptional regulation and expression of Sirt2 to facilitate OL differentiation. PMID:28188285

  15. The Vibrio parahaemolyticus small RNA RyhB promotes production of the siderophore vibrioferrin by stabilizing the polycistronic mRNA.

    PubMed

    Tanabe, Tomotaka; Funahashi, Tatsuya; Nakao, Hiroshi; Maki, Jun; Yamamoto, Shigeo

    2013-08-01

    High-affinity iron acquisition in Vibrio parahaemolyticus is mediated by the cognate siderophore vibrioferrin. We have previously reported that the vibrioferrin biosynthesis operon (pvsOp) is regulated at the transcriptional level by the iron-responsive repressor Fur (T. Tanabe, T. Funahashi, H. Nakao, S. Miyoshi, S. Shinoda, and S. Yamamoto, J. Bacteriol. 185:6938-6949, 2003). In this study, we identified the Fur-regulated small RNA RyhB and the RNA chaperone Hfq protein as additional regulatory proteins of vibrioferrin biosynthesis. We found that vibrioferrin production was greatly impaired in both the ryhB and hfq deletion mutants, and a TargetRNA search (http://snowwhite.wellesley.edu/targetRNA/index2.html) revealed that the 5'-untranslated region of pvsOp mRNA (pvsOp 5'-UTR) contains a potential base-pairing region required for the formation of the RyhB-pvsOp 5'-UTR duplex. An electrophoresis mobility shift assay indicated that RyhB can directly bind to the pvsOp 5'-UTR with the aid of Hfq. Rifampin chase experiments indicated that the half-life of pvsOp mRNA in the ryhB and hfq mutants was approximately 3-fold shorter than that in the parental strain, suggesting that both RyhB and Hfq are engaged in the stabilization of pvsOp mRNA. Chrome azurol S assays followed by electrophoresis mobility shift assays and rifampin chase experiments carried out for mutant strains indicated that base pairing between RyhB and the pvsOp 5'-UTR results in an increase in the stability of pvsOp mRNA, thereby leading to the promotion of vibrioferrin production. It is unprecedented that RyhB confers increased stability on a polycistronic mRNA involved in siderophore biosynthesis as a direct target.

  16. The Vibrio parahaemolyticus Small RNA RyhB Promotes Production of the Siderophore Vibrioferrin by Stabilizing the Polycistronic mRNA

    PubMed Central

    Funahashi, Tatsuya; Nakao, Hiroshi; Maki, Jun; Yamamoto, Shigeo

    2013-01-01

    High-affinity iron acquisition in Vibrio parahaemolyticus is mediated by the cognate siderophore vibrioferrin. We have previously reported that the vibrioferrin biosynthesis operon (pvsOp) is regulated at the transcriptional level by the iron-responsive repressor Fur (T. Tanabe, T. Funahashi, H. Nakao, S. Miyoshi, S. Shinoda, and S. Yamamoto, J. Bacteriol. 185:6938–6949, 2003). In this study, we identified the Fur-regulated small RNA RyhB and the RNA chaperone Hfq protein as additional regulatory proteins of vibrioferrin biosynthesis. We found that vibrioferrin production was greatly impaired in both the ryhB and hfq deletion mutants, and a TargetRNA search (http://snowwhite.wellesley.edu/targetRNA/index2.html) revealed that the 5′-untranslated region of pvsOp mRNA (pvsOp 5′-UTR) contains a potential base-pairing region required for the formation of the RyhB-pvsOp 5′-UTR duplex. An electrophoresis mobility shift assay indicated that RyhB can directly bind to the pvsOp 5′-UTR with the aid of Hfq. Rifampin chase experiments indicated that the half-life of pvsOp mRNA in the ryhB and hfq mutants was approximately 3-fold shorter than that in the parental strain, suggesting that both RyhB and Hfq are engaged in the stabilization of pvsOp mRNA. Chrome azurol S assays followed by electrophoresis mobility shift assays and rifampin chase experiments carried out for mutant strains indicated that base pairing between RyhB and the pvsOp 5′-UTR results in an increase in the stability of pvsOp mRNA, thereby leading to the promotion of vibrioferrin production. It is unprecedented that RyhB confers increased stability on a polycistronic mRNA involved in siderophore biosynthesis as a direct target. PMID:23772063

  17. Stability of the Osmoregulated Promoter-Derived proP mRNA Is Posttranscriptionally Regulated by RNase III in Escherichia coli

    PubMed Central

    Lim, Boram

    2015-01-01

    ABSTRACT The enzymatic activity of Escherichia coli endo-RNase III determines the stability of a subgroup of mRNA species, including bdm, betT, and proU, whose protein products are associated with the cellular response to osmotic stress. Here, we report that the stability of proP mRNA, which encodes a transporter of osmoprotectants, is controlled by RNase III in response to osmotic stress. We observed that steady-state levels of proP mRNA and ProP protein are inversely correlated with cellular RNase III activity and, in turn, affect the proline uptake capacity of the cell. In vitro and in vivo analyses of proP mRNA revealed RNase III cleavage sites in a stem-loop within the 5′ untranslated region present only in proP mRNA species synthesized from the osmoregulated P1 promoter. Introduction of nucleotide substitutions in the cleavage site identified inhibited the ribonucleolytic activity of RNase III on proP mRNA, increasing the steady-state levels and half-life of the mRNA. In addition, decreased RNase III activity coincided with a significant increase in both the half-life and abundance of proP mRNA under hyperosmotic stress conditions. Analysis of the RNA bound to RNase III via in vivo cross-linking and immunoprecipitation indicated that this phenomenon is related to the decreased RNA binding capacity of RNase III. Our findings suggest the existence of an RNase III-mediated osmoregulatory network that rapidly balances the expression levels of factors associated with the cellular response to osmotic stress in E. coli. IMPORTANCE Our results demonstrate that RNase III activity on proP mRNA degradation is downregulated in Escherichia coli cells under osmotic stress. In addition, we show that the downregulation of RNase III activity is associated with decreased RNA binding capacity of RNase III under hyperosmotic conditions. In particular, our findings demonstrate a link between osmotic stress and RNase III activity, underscoring the growing importance of

  18. 5, 8, 11, 14-eicosatetraynoic acid suppresses CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1 mRNA stability.

    PubMed

    Lee, Jee Hoon; Kim, Hyunmi; Woo, Joo Hong; Joe, Eun-hye; Jou, Ilo

    2012-02-18

    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. 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. 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. ETYA induces MKP-1 through HuR at the post-transcriptional level in a receptor-independent manner. The mechanism revealed here suggests eicosanoids as potential therapeutic

  19. Gad1 mRNA as a reliable indicator of altered GABA release from orexigenic neurons in the hypothalamus

    PubMed Central

    Dicken, Matthew S.; Hughes, Alexander R.; Hentges, Shane T.

    2016-01-01

    The strength of γ-aminobutyric acid (GABA)-mediated inhibitory synaptic input is a principle determinant of neuronal activity. However, because of differences in the number of GABA afferent inputs and the sites of synapses, it is difficult to directly assay for altered GABA transmission between specific cells. The present study tested the hypothesis that the level of mRNA for the GABA synthetic enzyme glutamate decarboxylase (GAD) can provide a reliable proxy for GABA release. This was tested in a mouse hypothalamic circuit important in the regulation of energy balance. Fluorescent in situ hybridization results show that the expression of Gad1 mRNA (encoding the GAD67 enzyme) was increased in hypothalamic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons after an overnight fast, consistent with the ability of GABA from these neurons to stimulate food intake. Optogenetic studies confirmed that the observed increase in Gad1 mRNA correlated with an increase in the probability of GABA release from NPY/AgRP neurons onto downstream proopiomelanocortin neurons. Likewise, there was an increase in the readily releasable pool of GABA in NPY/AgRP neurons. Selective inhibition of GAD activity in NPY/AgRP neurons decreased GABA release, indicating that GAD67 activity, which is largely dictated by expression level, is a key determinant of GABA release. Altogether, it appears that Gad expression may be a reliable proxy of altered GABAergic transmission. Examining changes in Gad mRNA as a proxy for GABA release may be particularly helpful when the downstream targets are not known or when limited tools exist for detecting GABA release at a particular synapse. PMID:26370162

  20. Postural Stability is Altered by Blood Shift

    NASA Astrophysics Data System (ADS)

    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.

  1. Low-level lasers alter mRNA levels from traditional reference genes used in breast cancer cells

    NASA Astrophysics Data System (ADS)

    Teixeira, A. F.; Canuto, K. S.; Rodrigues, J. A.; Fonseca, A. S.; Mencalha, A. L.

    2017-07-01

    Cancer is among the leading causes of mortality worldwide, increasing the importance of treatment development. Low-level lasers are used in several diseases, but some concerns remains on cancers. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a technique used to understand cellular behavior through quantification of mRNA levels. Output data from target genes are commonly relative to a reference that cannot vary according to treatment. This study evaluated reference genes levels from MDA-MB-231 cells exposed to red or infrared lasers at different fluences. Cultures were exposed to red and infrared lasers, incubated (4 h, 37 °C), total RNA was extracted and cDNA synthesis was performed to evaluate mRNA levels from ACTB, GUSB and TRFC genes by RT-qPCR. Specific amplification was verified by melting curves and agarose gel electrophoresis. RefFinder enabled data analysis by geNorm, NormFinder and BestKeeper. Specific amplifications were obtained and, although mRNA levels from ACTB, GUSB or TRFC genes presented no significant variation through traditional statistical analysis, Excel-based tools revealed that the use of these reference genes are dependent of laser characteristics. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from ACTB, GUSB and TRFC in MDA-MB-231 cells.

  2. IMP3 Stabilization of WNT5B mRNA Facilitates TAZ Activation in Breast Cancer.

    PubMed

    Samanta, Sanjoy; Guru, Santosh; Elaimy, Ameer L; Amante, John J; Ou, Jianhong; Yu, Jun; Zhu, Lihua J; Mercurio, Arthur M

    2018-05-29

    Insulin-like growth factor-2 mRNA-binding protein 3 (IMP3) is an oncofetal protein associated with many aggressive cancers and implicated in the function of breast cancer stem cells (CSCs). The mechanisms involved, however, are poorly understood. We observed that IMP3 facilitates the activation of TAZ, a transcriptional co-activator of Hippo signaling that is necessary for the function of breast CSCs. The mechanism by which IMP3 activates TAZ involves both mRNA stability and transcriptional regulation. IMP3 stabilizes the mRNA of an alternative WNT ligand (WNT5B) indirectly by repressing miR145-5p, which targets WNT5B, resulting in TAZ activation by alternative WNT signaling. IMP3 also facilitates the transcription of SLUG, which is necessary for TAZ nuclear localization and activation, by a mechanism that is also mediated by WNT5B. These results demonstrate that TAZ can be regulated by an mRNA-binding protein and that this regulation involves the integration of Hippo and alternative WNT-signaling pathways. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  3. v-Src oncogene product increases sphingosine kinase 1 expression through mRNA stabilization: alteration of AU-rich element-binding proteins.

    PubMed

    Sobue, S; Murakami, M; Banno, Y; Ito, H; Kimura, A; Gao, S; Furuhata, A; Takagi, A; Kojima, T; Suzuki, M; Nozawa, Y; Murate, T

    2008-10-09

    Sphingosine kinase 1 (SPHK1) is overexpressed in solid tumors and leukemia. However, the mechanism of SPHK1 overexpression by oncogenes has not been defined. We found that v-Src-transformed NIH3T3 cells showed a high SPHK1 mRNA, SPHK1 protein and SPHK enzyme activity. siRNA of SPHK1 inhibited the growth of v-Src-NIH3T3, suggesting the involvement of SPHK1 in v-Src-induced oncogenesis. v-Src-NIH3T3 showed activations of protein kinase C-alpha, signal transducers and activators of transcription 3 and c-Jun NH(2)-terminal kinase. Their inhibition suppressed SPHK1 expression in v-Src-NIH3T3, whereas their overexpression increased SPHK1 mRNA in NIH3T3. Unexpectedly, the nuclear run-on assay and the promoter analysis using 5'-promoter region of mouse SPHK1 did not show any significant difference between mock- and v-Src-NIH3T3. Furthermore, the half-life of SPHK1 mRNA in mock-NIH3T3 was nearly 15 min, whereas that of v-Src-NIH3T3 was much longer. Examination of two AU-rich region-binding proteins, AUF1 and HuR, that regulate mRNA decay reciprocally, showed decreased total AUF1 protein associated with increased tyrosine-phosphorylated form and increased serine-phosphorylated HuR protein in v-Src-NIH3T3. Modulation of AUF1 and HuR by their overexpression or siRNA revealed that SPHK1 mRNA in v-Src- and mock-NIH3T3 was regulated reciprocally by these factors. Our results showed, for the first time, a novel mechanism of v-Src-induced SPHK1 overexpression.

  4. Gad1 mRNA as a reliable indicator of altered GABA release from orexigenic neurons in the hypothalamus.

    PubMed

    Dicken, Matthew S; Hughes, Alexander R; Hentges, Shane T

    2015-11-01

    The strength of γ-aminobutyric acid (GABA)-mediated inhibitory synaptic input is a principle determinant of neuronal activity. However, because of differences in the number of GABA afferent inputs and the sites of synapses, it is difficult to directly assay for altered GABA transmission between specific cells. The present study tested the hypothesis that the level of mRNA for the GABA synthetic enzyme glutamate decarboxylase (GAD) can provide a reliable proxy for GABA release. This was tested in a mouse hypothalamic circuit important in the regulation of energy balance. Fluorescent in situ hybridization results show that the expression of Gad1 mRNA (encoding the GAD67 enzyme) was increased in hypothalamic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons after an overnight fast, consistent with the ability of GABA from these neurons to stimulate food intake. Optogenetic studies confirmed that the observed increase in Gad1 mRNA correlated with an increase in the probability of GABA release from NPY/AgRP neurons onto downstream proopiomelanocortin neurons. Likewise, there was an increase in the readily releasable pool of GABA in NPY/AgRP neurons. Selective inhibition of GAD activity in NPY/AgRP neurons decreased GABA release, indicating that GAD67 activity, which is largely dictated by expression level, is a key determinant of GABA release. Altogether, it appears that Gad expression may be a reliable proxy of altered GABAergic transmission. Examining changes in Gad mRNA as a proxy for GABA release may be particularly helpful when the downstream targets are not known or when limited tools exist for detecting GABA release at a particular synapse. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  5. Pulsed low-level infrared laser alters mRNA levels from muscle repair genes dependent on power output in Wistar rats

    NASA Astrophysics Data System (ADS)

    Trajano, L. A. S. N.; Trajano, E. T. L.; Thomé, A. M. C.; Sergio, L. P. S.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

    2017-10-01

    Satellite cells are present in skeletal muscle functioning in the repair and regeneration of muscle injury. Activation of these cells depends on the expression of myogenic factor 5 (Myf5), myogenic determination factor 1(MyoD), myogenic regulatory factor 4 (MRF4), myogenin (MyoG), paired box transcription factors 3 (Pax3), and 7 (Pax7). Low-level laser irradiation accelerates the repair of muscle injuries. However, data from the expression of myogenic factors have been controversial. Furthermore, the effects of different laser beam powers on the repair of muscle injuries have been not evaluated. The aim of this study was to evaluate the effects of low-level infrared laser at different powers and in pulsed emission mode on the expression of myogenic regulatory factors and on Pax3 and Pax7 in injured skeletal muscle from Wistar rats. Animals that underwent cryoinjury were divided into three groups: injury, injury laser 25 Mw, and injury laser 75 mW. Low-level infrared laser irradiation (904 nm, 3 J cm-2, 5 kHz) was carried out at 25 and 75 mW. After euthanasia, skeletal muscle samples were withdrawn and the total RNA was extracted for the evaluation of mRNA levels from the MyoD, MyoG, MRF4, Myf5, Pax3, and Pax7 gene. Pax 7 mRNA levels did not alter, but Pax3 mRNA levels increased in the injured and laser-irradiated group at 25 mW. MyoD, MyoG, and MYf5 mRNA levels increased in the injured and laser-irradiated animals at both powers, and MRF4 mRNA levels decreased in the injured and laser-irradiated group at 75 mW. In conclusion, exposure to pulsed low-level infrared laser, by power-dependent effect, could accelerate the muscle repair process altering mRNA levels from paired box transcription factors and myogenic regulatory factors.

  6. Double-stranded RNA-dependent protein kinase (pkr) is essential for thermotolerance, accumulation of HSP70, and stabilization of ARE-containing HSP70 mRNA during stress.

    PubMed

    Zhao, Meijuan; Tang, Dan; Lechpammer, Stanislav; Hoffman, Alexander; Asea, Alexzander; Stevenson, Mary Ann; Calderwood, Stuart K

    2002-11-15

    We have investigated the role of the double-stranded RNA-dependent protein kinase gene (pkr) in the regulation of the heat shock response. We show that the pkr gene is essential for efficient activation of the heat shock response and that pkr disruption profoundly inhibits heat shock protein 70 (HSP70) synthesis and blocks the development of thermotolerance. Despite these profound effects, pkr disruption did not markedly affect the activation of heat shock factor 1 by heat and did not reduce the rate of transcription of the HSP70 gene after heat shock. However, despite the lack of effect of pkr disruption on HSP70 gene transcription, we found a significant decrease in the expression of HSP70 mRNA in pkr-/- cells after heat shock. Kinetic studies of mRNA turnover suggested a block in the thermal stabilization of HSP70 mRNA in pkr-/- cells. As the thermal stabilization of HSP70 mRNA is thought to involve cis-acting A+U rich (ARE) elements in the 3'-untranslated region (UTR), we examined a potential role for pkr in this process. We found that a reporter beta-galactosidase mRNA destabilized by introduction of a functional ARE into the 3'-UTR became stabilized by heat but only in cells containing an intact pkr gene. Our studies suggest therefore that pkr plays a significant role in the stabilization of mRNA species containing ARE destruction sequences in the 3'-UTR and through this mechanism, contributes to the regulation of the heat shock response and other processes.

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

    PubMed

    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

  8. Tumor protein D52 expression is post-transcriptionally regulated by T-cell intercellular antigen (TIA) 1 and TIA-related protein via mRNA stability.

    PubMed

    Motohashi, Hiromi; Mukudai, Yoshiki; Ito, Chihiro; Kato, Kosuke; Shimane, Toshikazu; Kondo, Seiji; Shirota, Tatsuo

    2017-05-04

    Although tumor protein D52 (TPD52) family proteins were first identified nearly 20 years ago, their molecular regulatory mechanisms remain unclear. Therefore, we investigated the post-transcriptional regulation of TPD52 family genes. An RNA immunoprecipitation (RIP) assay showed the potential binding ability of TPD52 family mRNAs to several RNA-binding proteins, and an RNA degradation assay revealed that TPD52 is subject to more prominent post-transcriptional regulation than are TPD53 and TPD54. We subsequently focused on the 3'-untranslated region (3'-UTR) of TPD52 as a cis -acting element in post-transcriptional gene regulation. Several deletion mutants of the 3'-UTR of TPD52 mRNA were constructed and ligated to the 3'-end of a reporter green fluorescence protein gene. An RNA degradation assay revealed that a minimal cis -acting region, located in the 78-280 region of the 5'-proximal region of the 3'-UTR, stabilized the reporter mRNA. Biotin pull-down and RIP assays revealed specific binding of the region to T-cell intracellular antigen 1 (TIA-1) and TIA-1-related protein (TIAR). Knockdown of TIA-1/TIAR decreased not only the expression, but also the stability of TPD52 mRNA; it also decreased the expression and stability of the reporter gene ligated to the 3'-end of the 78-280 fragment. Stimulation of transforming growth factor-β and epidermal growth factor decreased the binding ability of these factors, resulting in decreased mRNA stability. These results indicate that the 78-280 fragment and TIA-1/TIAR concordantly contribute to mRNA stability as a cis -acting element and trans -acting factor(s), respectively. Thus, we here report the specific interactions between these elements in the post-transcriptional regulation of the TPD52 gene. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

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

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

    Sun, Lixin; Zhang, Shuang; Jiang, Zhenzhou

    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.more » 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.« less

  10. Biomaterials for mRNA Delivery

    PubMed Central

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

    2015-01-01

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

  11. Altered PIWI-LIKE 1 and PIWI-LIKE 2 mRNA expression in ejaculated spermatozoa of men with impaired sperm characteristics.

    PubMed

    Giebler, Maria; Greither, Thomas; Müller, Lisa; Mösinger, Carina; Behre, Hermann M

    2018-01-01

    In about half the cases of involuntary childlessness, a male infertility factor is involved. The PIWI-LIKE genes, a subclade of the Argonaute protein family, are involved in RNA silencing and transposon control in the germline. Knockout of murine Piwi-like 1 and 2 homologs results in complete infertility in males. The aim of this study was to analyze whether the mRNA expression of human PIWI-LIKE 1-4 genes is altered in ejaculated spermatozoa of men with impaired sperm characteristics. Ninety male participants were included in the study, among which 47 were with normozoospermia, 36 with impaired semen characteristics according to the World Health Organization (WHO) manual, 5 th edition, and 7 with azoospermia serving as negative control for the PIWI-LIKE 1-4 mRNA expression in somatic cells in the ejaculate. PIWI-LIKE 1-4 mRNA expression in the ejaculated spermatozoa of the participants was measured by quantitative real-time PCR. In nonazoospermic men, PIWI-LIKE 1-4 mRNA was measurable in ejaculated spermatozoa in different proportions. PIWI-LIKE 1 (100.0%) and PIWI-LIKE 2 (49.4%) were more frequently expressed than PIWI-LIKE 3 (9.6%) and PIWI-LIKE 4 (15.7%). Furthermore, a decreased PIWI-LIKE 2 mRNA expression showed a significant correlation with a decreased sperm count (P = 0.022) and an increased PIWI-LIKE 1 mRNA expression with a decreased progressive motility (P = 0.048). PIWI-LIKE 1 and PIWI-LIKE 2 mRNA expression exhibited a significant association with impaired sperm characteristics and may be a useful candidate for the evaluation of the impact of PIWI-LIKE 1-4 mRNA expression on male infertility.

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

    PubMed

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

    2017-03-06

    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.

  13. Emphysema induced by elastase alters the mRNA relative levels from DNA repair genes in acute lung injury in response to sepsis induced by lipopolysaccharide administration in Wistar rats.

    PubMed

    Sergio, Luiz Philippe S; Lucinda, Leda M F; Reboredo, Maycon M; de Paoli, Flavia; Fonseca, Lídia M C; Pinheiro, Bruno V; Mencalha, Andre L; Fonseca, Adenilson S

    2018-03-01

    Purpose/Aim of the study: Patients suffering from chronic obstructive pulmonary disease (COPD) in association with acute respiratory distress syndrome (ARDS) present oxidative stress in lung cells, with production of free radicals and DNA lesions in pulmonary and adjacent cells. Once the DNA molecule is damaged, a set of enzymatic mechanisms are trigged to preserve genetic code integrity and cellular homeostasis. These enzymatic mechanisms include the base and the nucleotide excision repair pathways, as well as telomere regulation. Thus, the aim of this work was to evaluate the mRNA levels from APEX1, ERCC2, TP53, and TRF2 genes in lung tissue from Wistar rats affected by acute lung injury in response to sepsis and emphysema. Adult male Wistar rats were randomized into 4 groups (n = 6, for each group): control, emphysema, sepsis, and emphysema with sepsis. Pulmonary emphysema was induced by intratracheal instillation of elastase (12 IU/animal) and sepsis induced by intraperitoneal Escherichia coli lipopolysaccharide (LPS) injection (10 mg/kg). Lungs were removed, and samples were withdrawn for histological analysis and total RNA extraction, cDNA synthesis, and mRNA level evaluation by real time quantitative polymerase chain reaction. Data show acute lung injury by LPS and emphysema by elastase and that APEX1, ERCC2, TP53, and TRF2 mRNA levels are increased significantly (p < 0.01) in emphysema with sepsis group. Our results suggest that alteration in mRNA levels from DNA repair and genomic stability could be part of cell response to acute lung injury in response to emphysema and sepsis.

  14. Manganese exposure alters extracellular GABA, GABA receptor and transporter protein and mRNA levels in the developing rat brain

    PubMed Central

    Anderson, Joel G.; Fordahl, Steve C.; Cooney, Paula T.; Weaver, Tara L.; Colyer, Christa L.; Erikson, Keith M.

    2011-01-01

    Unlike other essential trace elements (e.g., zinc and iron) it is the toxicity of manganese (Mn) that is more common in human populations than its deficiency. Data suggest alterations in dopamine biology may drive the effects associated with Mn neurotoxicity, though recently γ-aminobutyric acid (GABA) has been implicated. In addition, iron deficiency (ID), a common nutritional problem, may cause disturbances in neurochemistry by facilitating accumulation of Mn in the brain. Previous data from our lab have shown decreased brain tissue levels of GABA as well as decreased 3H-GABA uptake in synaptosomes as a result of Mn exposure and ID. These results indicate a possible increase in the concentration of extracellular GABA due to alterations in expression of GABA transport and receptor proteins. In this study weanling-male Sprague-Dawley rats were randomly placed into one of four dietary treatment groups: control (CN; 35 mg Fe/kg diet), iron-deficient (ID; 6 mg Fe/kg diet), CN with Mn supplementation (via the drinking water; 1 g Mn/L) (CNMn), and ID with Mn supplementation (IDMn). Using in vivo microdialysis, an increase in extracellular GABA concentrations in the striatum was observed in response to Mn exposure and ID although correlational analysis reveals that extracellular GABA is related more to extracellular iron levels and not Mn. A diverse effect of Mn exposure and ID was observed in the regions examined via Western blot and RT-PCR analysis, with effects on mRNA and protein expression of GAT-1, GABAA, and GABAB differing between and within the regions examined. For example, Mn exposure reduced GAT-1 protein expression by approximately 50% in the substantia nigra, while increasing mRNA expression approximately four-fold, while in the caudate putamen mRNA expression was decreased with no effect on protein expression. These data suggest that Mn exposure results in an increase in extracellular GABA concentrations via altered expression of transport and receptor

  15. Dietary fatty acids regulate hepatic low density lipoprotein (LDL) transport by altering LDL receptor protein and mRNA levels.

    PubMed Central

    Horton, J D; Cuthbert, J A; Spady, D K

    1993-01-01

    The concentration of LDL in plasma is strongly influenced by the amount and the type of lipid in the diet. Recent studies in the hamster have shown that dietary fatty acids differentially affect circulating LDL levels primarily by altering receptor-dependent LDL uptake in the liver. To investigate the mechanistic basis of this effect, rates of receptor-dependent LDL transport in the liver were correlated with LDL receptor protein and mRNA levels in hamsters fed safflower oil or coconut oil and varying amounts of cholesterol. Hepatic LDL receptor activity was significantly lower in animals fed coconut oil than in animals fed safflower oil at all levels of cholesterol intake (26, 53, and 61% lower at cholesterol intakes of 0, 0.06, and 0.12%, respectively). These fatty acid-induced changes in hepatic LDL receptor activity were accompanied by parallel changes in hepatic LDL receptor protein and mRNA levels, suggesting that dietary fatty acids regulate the LDL receptor pathway largely at the mRNA level. Images PMID:8349814

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

    PubMed Central

    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

  17. c-fms mRNA is regulated posttranscriptionally by 1,25(OH)2D3 in HL-60 cells.

    PubMed

    Biskobing, D M; Fan, D; Rubin, J

    1997-09-01

    Macrophage colony-stimulating factor (MCSF) is required for normal osteoclast and macrophage development. The receptor for MCSF (c-fms) is expressed on the pluripotent precursor and mature osteoclasts and macrophages. We have previously shown in myelomonocytic HL-60 cells that phorbol myristate acetate (PMA) upregulates c-fms mRNA expression. This induction of c-fms is inhibited by 1,25(OH)2D3. The major regulatory control of c-fms mRNA levels by PMA has been identified as posttranscriptional. However, a role of transcript elongation in controlling levels of c-fms mRNA has also been suggested. To better understand the 1,25(OH)2D3 regulation of c-fms mRNA expression we studied nuclear run on, mRNA stability, and transcript elongation in HL-60 cells treated with 10 ng/ml phorbol myristate acetate, 10 nM 1,25(OH)2D3 alone or combined. We demonstrated by nuclear run on that c-fms was constitutively transcribed in 1,25(OH)2D3 as well as control and PMA-treated cells. Transcript elongation was evaluated by RT-PCR for exon 2 or exon 3. Both exons were minimally expressed in control and 1,25(OH)2D3-treated cells, and increased in PMA-treated cells; this increased expression was inhibited by the addition of 1,25(OH)2D3. These results fail to show differential transcript elongation. Measurement of mRNA stability demonstrated decreased mRNA half-life to 5 hours in cells treated with PMA and 1,25(OH)2D3 compared with a half-life of 8 hours in cells treated with PMA alone. Our findings demonstrate that c-fms is regulated by 1,25(OH)2D3 at the posttranscriptional level by changes in mRNA stability. This gives the cell the ability to respond to local signals with rapid changes in c-fms levels altering the ability of the cell to respond to MCSF.

  18. Phosphorylation alters Bim-mediated Mcl-1 stabilization and priming.

    PubMed

    Conage-Pough, Jason E; Boise, Lawrence H

    2018-05-18

    Mcl-1 is a highly labile protein, subject to extensive post-translational regulation. This distinguishes Mcl-1 from other antiapoptotic proteins and necessitates further study to better understand how interactions with proapoptotic Bcl-2 proteins affect its regulation. One such protein, Bim, is known to stabilize Mcl-1, and Bim phosphorylation has been associated with increased Mcl-1 binding. Consequently, we investigated the potential impact of Bim phosphorylation on Mcl-1 stability. We found that Bim stabilizes and primes Mcl-1 in RPCI-WM1 cells and is constitutively phosphorylated. Additionally, introduction of several phospho-mimetic and unphosphosphorylateable Bim mutations resulted in altered Mcl-1 stability and distinct Bim binding to antiapoptotic proteins. These findings suggest Bim phosphorylation not only regulates Mcl-1 stability but also is a potential mechanism for enforcing Mcl-1 dependence. © 2018 Federation of European Biochemical Societies.

  19. The Arabidopsis polyamine transporter LHR1/PUT3 modulates heat responsive gene expression by enhancing mRNA stability.

    PubMed

    Shen, Yun; Ruan, Qingxia; Chai, Haoxi; Yuan, Yongze; Yang, Wannian; Chen, Junping; Xin, Zhanguo; Shi, Huazhong

    2016-12-01

    Polyamines involve in gene regulation by interacting with and modulating the functions of various anionic macromolecules such as DNA, RNA and proteins. In this study, we identified an important function of the polyamine transporter LHR1 (LOWER EXPRESSION OF HEAT RESPONSIVE GENE1) in heat-inducible gene expression in Arabidopsis thaliana. The lhr1 mutant was isolated through a forward genetic screening for altered expression of the luciferase reporter gene driven by the promoter from the heat-inducible gene AtHSP18.2. The lhr1 mutant showed reduced induction of the luciferase gene in response to heat stress and was more sensitive to high temperature than the wild type. Map-based cloning identified that the LHR1 gene encodes the polyamine transporter PUT3 (POLYAMINE UPTAKE TRANSPORTER 3) localized in the plasma membrane. The LHR1/PUT3 is required for the uptake of extracellular polyamines and plays an important role in stabilizing the mRNAs of several crucial heat stress responsive genes under high temperature. Genome-wide gene expression analysis using RNA-seq identified an array of differentially expressed genes, among which the transcript levels of some of the heat shock protein genes significantly reduced in response to prolonged heat stress in the lhr1 mutant. Our findings revealed an important heat stress response and tolerance mechanism involving polyamine influx which modulates mRNA stability of heat-inducible genes under heat stress conditions. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  20. Regulation of cytoplasmic mRNA decay

    PubMed Central

    Schoenberg, Daniel R.; Maquat, Lynne E.

    2012-01-01

    Discoveries made over the past 20 years highlight the importance of mRNA decay as a means to modulate gene expression and thereby protein production. Up until recently, studies focused largely on identifying cis-acting sequences that serve as mRNA stability or instability elements, the proteins that bind these elements, how the process of translation influences mRNA decay, and the ribonucleases that catalyze decay. Now, current studies have begun to elucidate how the decay process is regulated. This review examines our current understanding of how mammalian-cell mRNA decay is controlled by different signaling pathways and lays out a framework for future research. PMID:22392217

  1. Tipping the balance of RNA stability by 3' editing of the transcriptome.

    PubMed

    Chung, Christina Z; Seidl, Lauren E; Mann, Mitchell R; Heinemann, Ilka U

    2017-11-01

    The regulation of active microRNAs (miRNAs) and maturation of messenger RNAs (mRNAs) that are competent for translation is a crucial point in the control of all cellular processes, with established roles in development and differentiation. Terminal nucleotidyltransferases (TNTases) are potent regulators of RNA metabolism. TNTases promote the addition of single or multiple nucleotides to an RNA transcript that can rapidly alter transcript stability. The well-known polyadenylation promotes transcript stability while the newly discovered but ubiquitious 3'-end polyuridylation marks RNA for degradation. Monoadenylation and uridylation are essential control mechanisms balancing mRNA and miRNA homeostasis. This review discusses the multiple functions of non-canonical TNTases, focusing on their substrate range, biological functions, and evolution. TNTases directly control mRNA and miRNA levels, with diverse roles in transcriptome stabilization, maturation, silencing, or degradation. We will summarize the current state of knowledge on non-canonical nucleotidyltransferases and their function in regulating miRNA and mRNA metabolism. We will review the discovery of uridylation as an RNA degradation pathway and discuss the evolution of nucleotidyltransferases along with their use in RNA labeling and future applications as therapeutic targets. The biochemically and evolutionarily highly related adenylyl- and uridylyltransferases play antagonizing roles in the cell. In general, RNA adenylation promotes stability, while uridylation marks RNA for degradation. Uridylyltransferases evolved from adenylyltransferases in multiple independent evolutionary events by the insertion of a histidine residue into the active site, altering nucleotide, but not RNA specificity. Understanding the mechanisms regulating RNA stability in the cell and controlling the transcriptome is essential for efforts aiming to influence cellular fate. Selectively enhancing or reducing RNA stability allows for

  2. The Mitochondrion-Targeted PENTATRICOPEPTIDE REPEAT78 Protein Is Required for nad5 Mature mRNA Stability and Seed Development in Maize.

    PubMed

    Zhang, Ya-Feng; Suzuki, Masaharu; Sun, Feng; Tan, Bao-Cai

    2017-10-09

    Pentatricopepetide repeat (PPR) proteins are a large family of RNA-binding proteins involved in RNA metabolism in plant organelles. Although many PPR proteins have been functionally studied, few of them are identified with a function in mitochondrial RNA stability. By using a reverse genetic approach, we characterized the role of the mitochondrion-targeted PPR78 protein in nad5 mature mRNA stability and maize (Zea mays) seed development. Loss of PPR78 function leads to a dramatic reduction in the steady-state level of mitochondrial nad5 mature mRNA, blocks the assembly of complex I in the electron transport chain, and causes an arrest in embryogenesis and endosperm development. Characterization of a second strong allele confirms the function of PPR78 in nad5 mRNA accumulation and maize seed development. The generation of mature nad5 requires the assembly of three distinct precursor RNAs via trans-splicing reactions, and the accumulation of nad5T1 precursor is reduced in the ppr78 mutants. However, it is the instability of mature nad5 rather than nad5T1 causing loss of the full-length nad5 transcript, and degradation of nad5 losing both translation start and stop codons is enriched in the mutant. Our data imply the assembly of mature nad5 mRNA precedes the protection of PPR78. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  3. The stargazin-related protein γ7 interacts with the mRNA binding protein hnRNP A2 and regulates the stability of specific mRNAs including CaV2.2

    PubMed Central

    Ferron, Laurent; Davies, Anthony; Page, Karen M.; Cox, David J.; Leroy, Jerôme; Waithe, Dominic; Butcher, Adrian J.; Sellaturay, Priya; Bolsover, Steven; Pratt, Wendy S.; Moss, Fraser J.; Dolphin, Annette C.

    2009-01-01

    The role(s) of the novel stargazin-like γ-subunit proteins remain controversial. We have shown previously that the neuron-specific γ7 suppresses the expression of certain calcium channels, particularly CaV2.2, and is therefore unlikely to operate as a calcium channel subunit. We now show that the effect of γ7 on CaV2.2 expression is via an increase in the degradation rate of CaV2.2 mRNA, and hence a reduction of CaV2.2 protein level. Furthermore, exogenous expression of γ7 in PC12 cells also decreased the endogenous CaV2.2 mRNA level. Conversely, knockdown of endogenous γ7 with short-hairpin RNAs produced a reciprocal enhancement of CaV2.2 mRNA stability and an increase in endogenous calcium currents in PC12 cells. Moreover, both endogenous and expressed γ7 are present on intracellular membranes, rather than the plasma membrane. The cytoplasmic C-terminus of γ7 is essential for all its effects, and we show that γ7 binds directly via its C-terminus to a ribonucleoprotein (hnRNP A2), which also binds to a motif in CaV2.2 mRNA, and is associated with native CaV2.2 mRNA in PC12 cells. The expression of hnRNP A2 enhances CaV2.2 IBa and this enhancement is prevented by a concentration of γ7 that alone has no effect on IBa. The effect of γ7 is selective for certain mRNAs as it had no effect on α2δ-2 mRNA stability, but it decreased the mRNA stability for the potassium-chloride co-transporter, KCC1, which contains a similar hnRNP A2 binding motif to that in CaV2.2 mRNA. Our results indicate that γ7 plays a role in stabilizing CaV2.2 mRNA. PMID:18923037

  4. Stress- and Rho-activated ZO-1–associated nucleic acid binding protein binding to p21 mRNA mediates stabilization, translation, and cell survival

    PubMed Central

    Nie, Mei; Balda, Maria S.; Matter, Karl

    2012-01-01

    A central component of the cellular stress response is p21WAF1/CIP1, which regulates cell proliferation, survival, and differentiation. Inflammation and cell stress often up-regulate p21 posttranscriptionally by regulatory mechanisms that are poorly understood. ZO-1–associated nucleic acid binding protein (ZONAB)/DbpA is a Y-box transcription factor that is regulated by components of intercellular junctions that are affected by cytokines and tissue damage. We therefore asked whether ZONAB activation is part of the cellular stress response. Here, we demonstrate that ZONAB promotes cell survival in response to proinflammatory, hyperosmotic, and cytotoxic stress and that stress-induced ZONAB activation involves the Rho regulator GEF-H1. Unexpectedly, stress-induced ZONAB activation does not stimulate ZONAB’s activity as a transcription factor but leads to the posttranscriptional up-regulation of p21 protein and mRNA. Up-regulation is mediated by ZONAB binding to specific sites in the 3′-untranslated region of the p21 mRNA, resulting in mRNA stabilization and enhanced translation. Binding of ZONAB to mRNA is activated by GEF-H1 via Rho stimulation and also mediates Ras-induced p21 expression. We thus identify a unique type of stress and Rho signaling activated pathway that drives mRNA stabilization and translation and links the cellular stress response to p21 expression and cell survival. PMID:22711822

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

    PubMed

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

    2015-12-01

    RNA-binding proteins are often multifunctional, interact with a variety of protein partners and display complex localizations within cells. Mammalian cytoplasmic poly(A)-binding proteins (PABPs) are multifunctional RNA-binding proteins that regulate multiple aspects of mRNA translation and stability. Although predominantly diffusely cytoplasmic at steady state, they shuttle through the nucleus and can be localized to a variety of cytoplasmic foci, including those associated with mRNA storage and localized translation. Intriguingly, PABP sub-cellular distribution can alter dramatically in response to cellular stress or viral infection, becoming predominantly nuclear and/or being enriched in induced cytoplasmic foci. However, relatively little is known about the mechanisms that govern this distribution/relocalization and in many cases PABP functions within specific sites remain unclear. Here we discuss the emerging evidence with respect to these questions in mammals. © 2015 Authors; published by Portland Press Limited.

  6. Nitric Oxide Increases the Decay of Matrix Metalloproteinase 9 mRNA by Inhibiting the Expression of mRNA-Stabilizing Factor HuR

    PubMed Central

    Akool, El-Sayed; Kleinert, Hartmut; Hamada, Farid M. A.; Abdelwahab, Mohamed H.; Förstermann, Ulrich; Pfeilschifter, Josef; Eberhardt, Wolfgang

    2003-01-01

    Dysregulation of extracellular matrix turnover is an important feature of many inflammatory processes. Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin-1 beta. We demonstrate that NO does strongly destabilize MMP-9 mRNA, since different luciferase reporter gene constructs containing the MMP-9 3′ untranslated region (UTR) displayed significant reduced luciferase activity in response to the presence of NO. Moreover, by use of an in vitro degradation assay we found that the cytoplasmic fractions of NO-treated cells contained a higher capacity to degrade MMP-9 transcripts than those obtained from control cells. An RNA electrophoretic mobility shift assay demonstrated that three of four putative AU-rich elements present in the 3′ UTR of MMP-9 were constitutively occupied by the mRNA-stabilizing factor HuR and that the RNA binding was strongly attenuated by the presence of NO. The addition of recombinant glutathione transferase-HuR prevented the rapid decay of MMP-9 mRNA, whereas the addition of a neutralizing anti-HuR antibody caused an acceleration of MMP-9 mRNA degradation. Furthermore, the expression of HuR mRNA and protein was significantly reduced by exogenously and endogenously produced NO. These inhibitory effects were mimicked by the cGMP analog 8-bromo-cGMP and reversed by LY-83583, an inhibitor of soluble guanylyl cyclase. These results demonstrate that NO acts in a cGMP-dependent mechanism to inhibit the expression level of HuR, thereby reducing the stability of MMP-9 mRNA. PMID:12832476

  7. Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries

    PubMed Central

    Toro-Ascuy, Daniela; Rojas-Araya, Bárbara; Valiente-Echeverría, Fernando; Soto-Rifo, Ricardo

    2016-01-01

    The human immunodeficiency virus type-1 (HIV-1) unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs) containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1), Staufen double-stranded RNA binding protein 1/2 (STAU1/2), or components of miRNA-induced silencing complex (miRISC) and processing bodies (PBs). More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A), allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries. PMID:27886048

  8. RNActive® Technology: Generation and Testing of Stable and Immunogenic mRNA Vaccines.

    PubMed

    Rauch, Susanne; Lutz, Johannes; Kowalczyk, Aleksandra; Schlake, Thomas; Heidenreich, Regina

    2017-01-01

    Developing effective mRNA vaccines poses certain challenges concerning mRNA stability and ability to induce sufficient immune stimulation and requires a specific panel of techniques for production and testing. Here, we describe the production of stabilized mRNA with enhanced immunogenicity, generated using conventional nucleotides only, by introducing changes to the mRNA sequence and by complexation with the nucleotide-binding peptide protamine (RNActive® technology). Methods described here include the synthesis, purification, and protamine complexation of mRNA vaccines as well as a comprehensive panel of in vitro and in vivo methods for evaluation of vaccine quality and immunogenicity.

  9. Profiling of m6A RNA modifications identified an age-associated regulation of AGO2 mRNA stability.

    PubMed

    Min, Kyung-Won; Zealy, Richard W; Davila, Sylvia; Fomin, Mikhail; Cummings, James C; Makowsky, Daniel; Mcdowell, Catherine H; Thigpen, Haley; Hafner, Markus; Kwon, Sang-Ho; Georgescu, Constantin; Wren, Jonathan D; Yoon, Je-Hyun

    2018-06-01

    Gene expression is dynamically regulated in a variety of mammalian physiologies. During mammalian aging, there are changes that occur in protein expression that are highly controlled by the regulatory steps in transcription, post-transcription, and post-translation. Although there are global profiles of human transcripts during the aging processes available, the mechanism(s) by which transcripts are differentially expressed between young and old cohorts remains unclear. Here, we report on N6-methyladenosine (m6A) RNA modification profiles of human peripheral blood mononuclear cells (PBMCs) from young and old cohorts. An m6A RNA profile identified a decrease in overall RNA methylation during the aging process as well as the predominant modification on proteincoding mRNAs. The m6A-modified transcripts tend to be more highly expressed than nonmodified ones. Among the many methylated mRNAs, those of DROSHA and AGO2 were heavily methylated in young PBMCs which coincided with a decreased steady-state level of AGO2 mRNA in the old PBMC cohort. Similarly, downregulation of AGO2 in proliferating human diploid fibroblasts (HDFs) also correlated with a decrease in AGO2 mRNA modifications and steady-state levels. In addition, the overexpression of RNA methyltransferases stabilized AGO2 mRNA but not DROSHA and DICER1 mRNA in HDFs. Moreover, the abundance of miRNAs also changed in the young and old PBMCs which are possibly due to a correlation with AGO2 expression as observed in AGO2-depleted HDFs. Taken together, we uncovered the role of mRNA methylation on the abundance of AGO2 mRNA resulting in the repression of miRNA expression during the process of human aging. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  10. Messenger RNA (mRNA) nanoparticle tumour vaccination

    NASA Astrophysics Data System (ADS)

    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.

  11. Altering Emulsion Stability with Heterogeneous Surface Wettability

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

  12. Neurotrophin-3 mRNA a putative target of miR21 following status epilepticus.

    PubMed

    Risbud, Rashmi M; Lee, Carolyn; Porter, Brenda E

    2011-11-18

    Status epilepticus induces a cascade of protein expression changes contributing to the subsequent development of epilepsy. By identifying the cascade of molecular changes that contribute to the development of epilepsy we hope to be able to design therapeutics for preventing epilepsy. MicroRNAs influence gene expression by altering mRNA stability and/or translation and have been implicated in the pathology of multiple diseases. MiR21 and its co-transcript miR21, microRNAs produced from either the 5' or 3' ends of the same precursor RNA strand, are increased in the hippocampus following status epilepticus. We have identified a miR21 binding site, in the 3' UTR of neurotrophin-3 that inhibits translation. Neurotrophin-3 mRNA levels decrease in the hippocampus following SE concurrent with the increase in miR21. MiR21 levels in cultured hippocampal neurons inversely correlate with neurotrophin-3 mRNA levels. Treatment of hippocampal neuronal cultures with excess K(+)Cl(-), a depolarizing agent mimicking the episode of status epilepticus, also results in an increase in miR21 and a decrease in neurotrophin-3 mRNA. MiR21 is a candidate for regulating neurotrophin-3 signaling in the hippocampus following status epilepticus. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Base substitutions at scissile bond sites are sufficient to alter RNA-binding and cleavage activity of RNase III.

    PubMed

    Kim, Kyungsub; Sim, Se-Hoon; Jeon, Che Ok; Lee, Younghoon; Lee, Kangseok

    2011-02-01

    RNase III, a double-stranded RNA-specific endoribonuclease, degrades bdm mRNA via cleavage at specific sites. To better understand the mechanism of cleavage site selection by RNase III, we performed a genetic screen for sequences containing mutations at the bdm RNA cleavage sites that resulted in altered mRNA stability using a transcriptional bdm'-'cat fusion construct. While most of the isolated mutants showed the increased bdm'-'cat mRNA stability that resulted from the inability of RNase III to cleave the mutated sequences, one mutant sequence (wt-L) displayed in vivo RNA stability similar to that of the wild-type sequence. In vivo and in vitro analyses of the wt-L RNA substrate showed that it was cut only once on the RNA strand to the 5'-terminus by RNase III, while the binding constant of RNase III to this mutant substrate was moderately increased. A base substitution at the uncleaved RNase III cleavage site in wt-L mutant RNA found in another mutant lowered the RNA-binding affinity by 11-fold and abolished the hydrolysis of scissile bonds by RNase III. Our results show that base substitutions at sites forming the scissile bonds are sufficient to alter RNA cleavage as well as the binding activity of RNase III. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

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

    PubMed

    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. Copyright © 2015. Published by Elsevier Inc.

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

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

    Pham, Hung; Department of Medicine, Veterans Affair Greater Los Angeles Healthcare System, Los Angeles, CA 90073; Ekaterina Rodriguez, C.

    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 prostaglandinmore » 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.« less

  16. Wheel running alters patterns of uncontrollable stress-induced cfos mRNA expression in rat dorsal striatum direct and indirect pathways: a possible role for plasticity in adenosine receptors

    PubMed Central

    Clark, Peter J.; Ghasem, Parsa R.; Mika, Agnieszka; Day, Heidi E.; Herrera, Jonathan J.; Greenwood, Benjamin N.; Fleshner, Monika

    2014-01-01

    Emerging evidence indicates that adenosine is a major regulator of striatum activity, in part, through the antagonistic modulation of dopaminergic function. Exercise can influence adenosine and dopamine activity, which may subsequently promote plasticity in striatum adenosine and dopamine systems. Such changes could alter activity of medium spiny neurons and impact striatum function. The purpose of this study was two-fold. The first was to characterize the effect of long-term wheel running on adenosine 1 (A1R), adenosine 2A (A2AR), dopamine 1 (D1R), and dopamine 2 (D2R) receptor mRNA expression in adult rat dorsal and ventral striatum structures using in situ hybridization. The second was to determine if changes to adenosine and dopamine receptor mRNA from running are associated with altered cfos mRNA induction in dynorphin- (direct pathway) and enkephalin- (indirect pathway) expressing neurons of the dorsal striatum following stress exposure. We report that chronic running, as well as acute uncontrollable stress, reduced A1R and A2AR mRNA levels in the dorsal and ventral striatum. Running also modestly elevated D2R mRNA levels in striatum regions. Finally, stress-induced cfos was potentiated in dynorphin and attenuated in enkephalin expressing neurons of running rats. These data suggest striatum adenosine and dopamine systems are targets for neuroplasticity from exercise, which may contribute to changes in direct and indirect pathway activity. These findings may have implications for striatum mediated motor and cognitive processes, as well as exercise facilitated stress-resistance. PMID:25017571

  17. The induced RNA-binding protein, HuR, targets 3'-UTR region of IL-6 mRNA and enhances its stabilization in periodontitis.

    PubMed

    Ouhara, K; Munenaga, S; Kajiya, M; Takeda, K; Matsuda, S; Sato, Y; Hamamoto, Y; Iwata, T; Yamasaki, S; Akutagawa, K; Mizuno, N; Fujita, T; Sugiyama, E; Kurihara, H

    2018-06-01

    RNA-binding proteins (RBPs) regulate mRNA stability by binding to the 3'-untranslated region (UTR) region of mRNA. Human antigen-R (HuR), one of the RBPs, is involved in the progression of diseases, such as rheumatoid arthritis, diabetes mellitus and some inflammatory diseases. Interleukin (IL)-6 is a major inflammatory cytokine regulated by HuR binding to mRNA. Periodontal disease (PD) is also an inflammatory disease caused by elevations in IL-6 following an infection by periodontopathogenic bacteria. The involvement of HuR in the progression of PD was assessed using in-vitro and in-vivo experiments. Immunohistochemistry of inflamed periodontal tissue showed strong staining of HuR in the epithelium and connective tissue. HuR mRNA and protein level was increased following stimulation with Porphyromonas gingivalis (Pg), one of the periodontopathogenic bacteria, lipopolysacchride (LPS)-derived from Pg (PgLPS) and tumour necrosis factor (TNF)-α in OBA-9, an immortalized human gingival epithelial cell. The luciferase activity of 3'-UTR of IL-6 mRNA was increased by TNF-α, Pg and PgLPS in OBA-9. Luciferase activity was also increased in HuR-over-expressing OBA-9 following a bacterial stimulation. Down-regulation of HuR by siRNA resulted in a decrease in mRNA expression and production of IL-6. In contrast, the over-expression of HuR increased IL-6 mRNA expression and production in OBA-9. The HuR inhibitor, quercetin, suppressed Pg-induced HuR mRNA expression and IL-6 production in OBA-9. An oral inoculation with quercetin also inhibited bone resorption in ligature-induced periodontitis model mice as a result of down-regulation of IL-6. These results show that HuR modulates inflammatory responses by regulating IL-6. © 2018 British Society for Immunology.

  18. Regulation of bone sialoprotein mRNA by steroid hormones

    PubMed Central

    1989-01-01

    In this report we demonstrate an increase in the steady-state level of bone sialoprotein (BSP) mRNA in rat calvaria and a rat osteosarcoma cell line (ROS 17/2.8) after treatment with the synthetic glucocorticoid, dexamethasone. In contrast, 1.25-dihydroxyvitamin D3 reduced the amount of BSP mRNA in calvaria and inhibited the dexamethasone induction in ROS 17/2.8 cells. The increase in BSP mRNA is most likely due to an increase in the transcriptional rate. The stability of mRNA was unchanged after dexamethasone treatment with a half-life of approximately 5 h. Nuclear transcription experiments with nuclei isolated from ROS 17/2.8 cells showed an increased BSP mRNA synthesis in cells treated with dexamethasone. PMID:2592421

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

    PubMed

    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. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

    PubMed Central

    Aitken, Colin Echeverría; Beznosková, Petra; Vlčkova, Vladislava; Chiu, Wen-Ling; Zhou, Fujun; Valášek, Leoš Shivaya; Hinnebusch, Alan G; Lorsch, Jon R

    2016-01-01

    Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA. DOI: http://dx.doi.org/10.7554/eLife.20934.001 PMID:27782884

  1. Role of mRNA Methylation in Prostate Cancer

    DTIC Science & Technology

    2015-02-01

    position of adenosine (m6A) is a post-transcriptional modification of mRNA. However, little is known regarding the biological meanings of this epigenetic ...its level is altered in various cancer cell lines. FTO, the fat mass and obesity associated gene, was recently shown as an m6A demethylase. FTO gene...mRNA. However, little is known regarding the biological meanings of this epigenetic regulation of mRNA. Recent technological advances have made it

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

    USGS Publications Warehouse

    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.

  3. A U-Rich Element in the 5′ Untranslated Region Is Necessary for the Translation of p27 mRNA

    PubMed Central

    Millard, S. Sean; Vidal, Anxo; Markus, Maurice; Koff, Andrew

    2000-01-01

    Increased translation of p27 mRNA correlates with withdrawal of cells from the cell cycle. This raised the possibility that antimitogenic signals might mediate their effects on p27 expression by altering complexes that formed on p27 mRNA, regulating its translation. In this report, we identify a U-rich sequence in the 5′ untranslated region (5′UTR) of p27 mRNA that is necessary for efficient translation in proliferating and nonproliferating cells. We show that a number of factors bind to the 5′UTR in vitro in a manner dependent on the U-rich element, and their availability in the cytosol is controlled in a growth- and cell cycle-dependent fashion. One of these factors is HuR, a protein previously implicated in mRNA stability, transport, and translation. Another is hnRNP C1 and C2, proteins implicated in mRNA processing and the translation of a specific subset of mRNAs expressed in differentiated cells. In lovastatin-treated MDA468 cells, the mobility of the associated hnRNP C1 and C2 proteins changed, and this correlated with increased p27 expression. Together, these data suggest that the U-rich dependent RNP complex on the 5′UTR may regulate the translation of p27 mRNA and may be a target of antimitogenic signals. PMID:10913178

  4. RNase MRP cleaves the CLB2 mRNA to promote cell cycle progression: novel method of mRNA degradation.

    PubMed

    Gill, Tina; Cai, Ti; Aulds, Jason; Wierzbicki, Sara; Schmitt, Mark E

    2004-02-01

    RNase mitochondrial RNA processing (RNase MRP) mutants have been shown to have an exit-from-mitosis defect that is caused by an increase in CLB2 mRNA levels, leading to increased Clb2p (B-cyclin) levels and a resulting late anaphase delay. Here we describe the molecular defect behind this delay. CLB2 mRNA normally disappears rapidly as cells complete mitosis, but the level remains high in RNase MRP mutants. This is in direct contrast to other exit-from-mitosis mutants and is the result of an increase in CLB2 mRNA stability. We found that highly purified RNase MRP cleaved the 5' untranslated region (UTR) of the CLB2 mRNA in several places in an in vitro assay. In vivo, we identified RNase MRP-dependent cleavage products on the CLB2 mRNA that closely matched in vitro products. Disposal of these products was dependent on the 5'-->3' exoribonuclease Xrn1 and not the exosome. Our results demonstrate that the endoribonuclease RNase MRP specifically cleaves the CLB2 mRNA in its 5'-UTR to allow rapid 5' to 3' degradation by the Xrn1 nuclease. Degradation of the CLB2 mRNA by the RNase MRP endonuclease provides a novel way to regulate the cell cycle that complements the protein degradation machinery. In addition, these results denote a new mechanism of mRNA degradation not seen before in the yeast Saccharomyces cerevisiae.

  5. Aiding and Abetting Cancer: mRNA export and the nuclear pore

    PubMed Central

    Culjkovic-Kraljacic, Biljana; Borden, Katherine L.B

    2013-01-01

    mRNA export is a critical step in gene expression. Export of transcripts can be modulated in response to cellular signaling or stress. Consistently, mRNA export is dysregulated in primary human specimens derived from many different forms of cancer. Aberrant expression of export factors can alter export of specific transcripts encoding proteins involved in proliferation, survival and oncogenesis. These specific factors, which are not used for bulk mRNA export, are obvious therapeutic targets. Indeed, given the emerging role of mRNA export in cancer, it is not surprising that efforts to target different aspects of this pathway have reached the clinical trial stage. Thus, like transcription and translation, mRNA export may also play a critical role in cancer genesis and maintenance. PMID:23582887

  6. Amicoumacin A inhibits translation by stabilizing mRNA interaction with the ribosome

    PubMed Central

    Polikanov, Yury S.; Osterman, Ilya A.; Szal, Teresa; Tashlitsky, Vadim N.; Serebryakova, Marina V.; Kusochek, Pavel; Bulkley, David; Malanicheva, Irina A.; Efimenko, Tatyana A.; Efremenkova, Olga V.; Konevega, Andrey L.; Shaw, Karen J.; Bogdanov, Alexey A.; Rodnina, Marina V.; Dontsova, Olga A.; Mankin, Alexander S.; Steitz, Thomas A.; Sergiev, Petr V.

    2014-01-01

    SUMMARY We demonstrate that the antibiotic amicoumacin A (AMI) whose cellular target was unknown, is a potent inhibitor of protein synthesis. Resistance mutations in helix 24 of the 16S rRNA mapped the AMI binding site to the small ribosomal subunit. The crystal structure of bacterial ribosome in complex with AMI solved at 2.4 Å resolution revealed that the antibiotic makes contacts with universally conserved nucleotides of 16S rRNA in the E site and the mRNA backbone. Simultaneous interactions of AMI with 16S rRNA and mRNA and the in vivo experimental evidence suggest that it may inhibit the progression of the ribosome along mRNA. Consistent with this proposal, binding of AMI interferes with translocation in vitro. The inhibitory action of AMI can be partly compensated by mutations in the translation elongation factor G. PMID:25306919

  7. Rift Valley fever virus NS{sub S} gene expression correlates with a defect in nuclear mRNA export

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

    Copeland, Anna Maria; Van Deusen, Nicole M.; Schmaljohn, Connie S., E-mail: Connie.s.schmaljohn.civ@mail.mil

    We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NS{sub S} gene, but not the N, G{sub N} or NS{sub M} genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NS{sub S}, confirming that expression of NS{sub S} is likely responsible for this phenomenon. - Highlights: • Riftmore » Valley fever virus (RVFV) infection alters the localization of host mRNA. • mRNA accumulates in the nuclei of RVFV-infected but not mock-infected cells. • NS{sub S} is likely responsible for mRNA relocalization to the nucleus.« less

  8. Ways and means of eukaryotic mRNA decay.

    PubMed

    Balagopal, Vidya; Fluch, Lydia; Nissan, Tracy

    2012-06-01

    Messenger RNA degradation is an important point of control for gene expression. Genome-wide studies on mRNA stability have demonstrated its importance in adaptation and stress response. Most of the key players in mRNA decay appear to have been identified. The study of these proteins brings insight into the mechanism of general and specific targeting of transcripts for degradation. Recruitment and assembly of mRNP complexes enhance and bring specificity to mRNA decay. mRNP complexes can form larger structures that have been found to be ubiquitous in nature. Discovery of P-Bodies, an archetype of this sort of aggregates, has generated interest in the question of where mRNA degrades. This is currently an open question under extensive investigation. This review will discuss in detail the recent developments in the regulation of mRNA decay focusing on yeast as a model system. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein.

    PubMed

    Chen, Linan; Dumelie, Jason G; Li, Xiao; Cheng, Matthew Hk; Yang, Zhiyong; Laver, John D; Siddiqui, Najeeb U; Westwood, J Timothy; Morris, Quaid; Lipshitz, Howard D; Smibert, Craig A

    2014-01-07

    Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown. To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug's target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes. Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism.

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

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

    Egloff, Caroline; Crump, Doug, E-mail: doug.crump@ec.gc.ca; Porter, Emily

    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 highestmore » 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

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

    PubMed

    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. Optimal Down Regulation of mRNA Translation

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

  14. Altered Sex Hormone Concentrations and Gonadal mRNA Expression Levels of Activin Signaling Factors in Hatchling Alligators From a Contaminated Florida Lake

    PubMed Central

    MOORE, BRANDON C.; KOHNO, SATOMI; COOK, ROBERT W.; ALVERS, ASHLEY L.; HAMLIN, HEATHER J.; WOODRUFF, TERESA K.; GUILLETTE, LOUIS J.

    2014-01-01

    Activins and estrogens participate in regulating the breakdown of ovarian germ cell nests and follicle assembly in mammals. In 1994, our group reported elevated frequencies of abnormal, multioocytic ovarian follicles in 6 month old, environmental contaminant-exposed female alligators after gonadotropin challenge. Here, we investigated if maternal contribution of endocrine disrupting contaminants to the egg subsequently alters estrogen/inhibin/activin signaling in hatchling female offspring, putatively predisposing an increased frequency of multioocytic follicle formation. We quantified basal and exogenous gonadotropin-stimulated concentrations of circulating plasma steroid hormones and ovarian activin signaling factor mRNA abundance in hatchling alligators from the same contaminated (Lake Apopka) and reference (Lake Woodruff) Florida lakes, as examined in 1994. Basal circulating plasma estradiol and testosterone concentrations were greater in alligators from the contaminated environment, whereas activin/inhibin βA subunit and follistatin mRNA abundances were lower than values measured in ovaries from reference lake animals. Challenged, contaminant-exposed animals showed a more robust increase in plasma estradiol concentration following an acute follicle stimulating hormone (FSH) challenge compared with reference site alligators. Aromatase and follistatin mRNA levels increased in response to an extended FSH challenge in the reference site animals, but not in the contaminant-exposed animals. In hatchling alligators, ovarian follicles have not yet formed; therefore, these endocrine differences are likely to affect subsequent ovarian development, including ovarian follicle assembly. PMID:20166196

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

    PubMed

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. 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

    PubMed Central

    Sala, Claudia; Forti, Francesca; Magnoni, Francesca; Ghisotti, Daniela

    2008-01-01

    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 and translation initiation are

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

    PubMed Central

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

    2015-01-01

    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 for 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 pH dependent, suggestive of general acid-base catalysis. pH-rate curves indicate that wild-type RelE operates with the pKa 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 pKa 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 pKa shift and facilitate a common catalytic mechanism by uncommon means furthers our understanding of other atypical enzymatic active sites. PMID:26535789

  18. Changes in beta-actin mRNA expression in remodeling canine myocardium.

    PubMed

    Carlyle, W C; Toher, C A; Vandervelde, J R; McDonald, K M; Homans, D C; Cohn, J N

    1996-01-01

    Beta-actin, a cytoskeletal protein important in the maintenance of cytoarchitecture, has long been thought to be expressed constitutively in myocardial tissue. As such, beta-actin mRNA has been used as a control gene in a wide range of experiments. However, we have uncovered consistent changes in beta-actin mRNA expression in canine myocardium remodeling as a result of insult to the left ventricle. The experimental canine models used were either DC shock damage to the left ventricle or volume overload resulting from severe mitral regurgitation. The remodeling process in both canine models is characterized by an increase in left ventricular mass. PCR amplification using primers designed to selectively amplify the 3' end and a portion of the 3' untranslated region of beta-actin mRNA resulted in the generation of a 297 base pair product predominant only in normal canine myocardium and a 472 base pair product that became increasingly prominent from 1 to 30 days after DC shock damage to the left ventricle and from 10 to 90 days after creation of mitral regurgitation. Northern analysis showed a three-fold increase in beta-actin mRNA after either DC shock or creation of mitral regurgitation. Western analysis revealed an early increase in beta-actin protein followed by an apparent decrease to below baseline levels. These observations suggest that changes in beta-actin mRNA expression accompany the structural alterations that occur in response to myocardial damage. Whether or not the changes in beta-actin mRNA expression play a role in mediating these structural alterations remains to be determined.

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

    PubMed Central

    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

  20. mRNA interactome capture in mammalian cells.

    PubMed

    Kastelic, Nicolai; Landthaler, Markus

    2017-08-15

    Throughout their entire life cycle, mRNAs are associated with RNA-binding proteins (RBPs), forming ribonucleoprotein (RNP) complexes with highly dynamic compositions. Their interplay is one key to control gene regulatory mechanisms from mRNA synthesis to decay. To assay the global scope of RNA-protein interactions, we and others have published a method combining crosslinking with highly stringent oligo(dT) affinity purification to enrich proteins associated with polyadenylated RNA (poly(A)+ RNA). Identification of the poly(A)+ RNA-bound proteome (also: mRNA interactome capture) has by now been applied to a diversity of cell lines and model organisms, uncovering comprehensive repertoires of RBPs and hundreds of novel RBP candidates. In addition to determining the RBP catalog in a given biological system, mRNA interactome capture allows the examination of changes in protein-mRNA interactions in response to internal and external stimuli, altered cellular programs and disease. Copyright © 2017. Published by Elsevier Inc.

  1. Serum-deprivation stimulates cap-binding by PARN at the expense of eIF4E, consistent with the observed decrease in mRNA stability

    PubMed Central

    Seal, Ruth; Temperley, Richard; Wilusz, Jeffrey; Lightowlers, Robert N.; Chrzanowska-Lightowlers, Zofia M. A.

    2005-01-01

    PARN, a poly(A)-specific ribonuclease, binds the 5′ cap-structure of mRNA and initiates deadenylation-dependent decay. Eukaryotic initiation factor 4E (eIF4E) also binds to the cap structure, an interaction that is critical for initiating cap-dependent translation. The stability of various mRNA transcripts in human cell lines is reduced under conditions of serum starvation as determined by both functional and chemical half-lives. Serum starvation also leads to enhanced cap association by PARN. In contrast, the 5′ cap occupancy by eIF4E decreases under serum-deprivation, as does the translation of reporter transcripts. Further, we show that PARN is a phosphoprotein and that this modification can be modulated by serum status. Taken together, these data are consistent with a natural competition existing at the 5′ cap structure between PARN and eIF4E that may be regulated by changes in post-translational modifications. These phosphorylation-induced changes in the interplay of PARN and eIF4E may determine whether the mRNA is translated or decayed. PMID:15653638

  2. Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein

    PubMed Central

    2014-01-01

    Background Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown. Results To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug’s target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes. Conclusions Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism. PMID:24393533

  3. Chaperone Hsp27 Modulates AUF1 Proteolysis and AU-Rich Element-Mediated mRNA Degradation▿

    PubMed Central

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

    2011-01-01

    AUF1 is an AU-rich element (ARE)-binding protein that recruits translation initiation factors, molecular chaperones, and mRNA degradation enzymes to the ARE for mRNA destruction. We recently found chaperone Hsp27 to be an AUF1-associated ARE-binding protein required for tumor necrosis factor alpha (TNF-α) mRNA degradation in monocytes. Hsp27 is a multifunctional protein that participates in ubiquitination of proteins for their degradation by proteasomes. A variety of extracellular stimuli promote Hsp27 phosphorylation on three serine residues—Ser15, Ser78, and Ser82—by a number of kinases, including the mitogen-activated protein (MAP) pathway kinases p38 and MK2. Activating either kinase stabilizes ARE mRNAs. Likewise, ectopic expression of phosphomimetic mutant forms of Hsp27 stabilizes reporter ARE mRNAs. Here, we continued to examine the contributions of Hsp27 to mRNA degradation. As AUF1 is ubiquitinated and degraded by proteasomes, we addressed the hypothesis that Hsp27 phosphorylation controls AUF1 levels to modulate ARE mRNA degradation. Indeed, selected phosphomimetic mutants of Hsp27 promote proteolysis of AUF1 in a proteasome-dependent fashion and render ARE mRNAs more stable. Our results suggest that the p38 MAP kinase (MAPK)-MK2–Hsp27 signaling axis may target AUF1 destruction by proteasomes, thereby promoting ARE mRNA stabilization. PMID:21245386

  4. The Phosphorolytic Exoribonucleases Polynucleotide Phosphorylase and RNase PH Stabilize sRNAs and Facilitate Regulation of Their mRNA Targets

    PubMed Central

    2016-01-01

    ABSTRACT Gene regulation by base pairing between small noncoding RNAs (sRNAs) and their mRNA targets is an important mechanism that allows bacteria to maintain homeostasis and respond to dynamic environments. In Gram-negative bacteria, sRNA pairing and regulation are mediated by several RNA-binding proteins, including the sRNA chaperone Hfq and polynucleotide phosphorylase (PNPase). PNPase and its homolog RNase PH together represent the two 3′ to 5′ phosphorolytic exoribonucleases found in Escherichia coli; however, the role of RNase PH in sRNA regulation has not yet been explored and reported. Here, we have examined in detail how PNPase and RNase PH interact to support sRNA stability, activity, and base pairing in exponential and stationary growth conditions. Our results indicate that these proteins facilitate the stability and regulatory function of the sRNAs RyhB, CyaR, and MicA during exponential growth. PNPase further appears to contribute to pairing between RyhB and its mRNA targets. During stationary growth, each sRNA responded differently to the absence or presence of PNPase and RNase PH. Finally, our results suggest that PNPase and RNase PH stabilize only Hfq-bound sRNAs. Taken together, these results confirm and extend previous findings that PNPase participates in sRNA regulation and reveal that RNase PH serves a similar, albeit more limited, role as well. These proteins may, therefore, act to protect sRNAs from spurious degradation while also facilitating regulatory pairing with their targets. IMPORTANCE In many bacteria, Hfq-dependent base-pairing sRNAs facilitate rapid changes in gene expression that are critical for maintaining homeostasis and responding to stress and environmental changes. While a role for Hfq in this process was identified more than 2 decades ago, the identity and function of the other proteins required for Hfq-dependent regulation by sRNAs have not been resolved. Here, we demonstrate that PNPase and RNase PH, the two

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Rift Valley fever virus NSS gene expression correlates with a defect in nuclear mRNA export.

    PubMed

    Copeland, Anna Maria; Van Deusen, Nicole M; Schmaljohn, Connie S

    2015-12-01

    We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NSS gene, but not the N, GN or NSM genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NSS, confirming that expression of NSS is likely responsible for this phenomenon. Published by Elsevier Inc.

  7. 3'-UTR SNP rs2229611 in G6PC1 affects mRNA stability, expression and Glycogen Storage Disease type-Ia risk.

    PubMed

    Karthi, Sellamuthu; Rajeshwari, Mohan; Francis, Amirtharaj; Saravanan, Matheshwaran; Varalakshmi, Perumal; Houlden, Henry; Thangaraj, Kumarasamy; Ashokkumar, Balasubramaniem

    2017-08-01

    The frequency of rs2229611, previously reported in Chinese, Caucasians, Japanese and Hispanics, was investigated for the first time in Indian ethnicity. We analyzed its role in the progression of Glycogen Storage Disease type-Ia (GSD-Ia) and breast cancer. Genotype data on rs2229611 revealed that the risk of GSD-Ia was higher (P=0.0195) with CC compared to TT/TC genotypes, whereas no such correlation was observed with breast cancer cases. We observed a strong linkage disequilibrium (LD) among rs2229611 and other disease causing G6PC1 variants (|D'|=1, r 2 =1). Functional validation performed in HepG2 cells using luciferase constructs showed significant (P<0.05) decrease in expression than wild-type 3'-UTR due to curtailed mRNA stability. Furthermore, AU-rich elements (AREs) mediated regulation of G6PC1 expression characterized using 3'-UTR deletion constructs showed a prominent decrease in mRNA stability. We then examined whether miRNAs are involved in controlling G6PC1 expression using pmirGLO-UTR constructs, with evidence of more distinct inhibition in the reporter function with rs2229611. These data suggests that rs2229611 is a crucial regulatory SNP which in homozygous state leads to a more aggressive disease phenotype in GSD-Ia patients. The implication of this result is significant in predicting disease onset, progression and response to disease modifying treatments in patients with GSD-Ia. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. A Novel Post-translational Modification of Nucleolin, SUMOylation at Lys-294, Mediates Arsenite-induced Cell Death by Regulating gadd45α mRNA Stability*

    PubMed Central

    Zhang, Dongyun; Liang, Yuguang; Xie, Qipeng; Gao, Guangxun; Wei, Jinlong; Huang, Haishan; Li, Jingxia; Gao, Jimin; Huang, Chuanshu

    2015-01-01

    Nucleolin is a ubiquitously expressed protein and participates in many important biological processes, such as cell cycle regulation and ribosomal biogenesis. The activity of nucleolin is regulated by intracellular localization and post-translational modifications, including phosphorylation, methylation, and ADP-ribosylation. Small ubiquitin-like modifier (SUMO) is a category of recently verified forms of post-translational modifications and exerts various effects on the target proteins. In the studies reported here, we discovered SUMOylational modification of human nucleolin protein at Lys-294, which facilitated the mRNA binding property of nucleolin by maintaining its nuclear localization. In response to arsenic exposure, nucleolin-SUMO was induced and promoted its binding with gadd45α mRNA, which increased gadd45α mRNA stability and protein expression, subsequently causing GADD45α-mediated cell death. On the other hand, ectopic expression of Mn-SOD attenuated the arsenite-generated superoxide radical level, abrogated nucleolin-SUMO, and in turn inhibited arsenite-induced apoptosis by reducing GADD45α expression. Collectively, our results for the first time demonstrate that nucleolin-SUMO at K294R plays a critical role in its nucleus sequestration and gadd45α mRNA binding activity. This novel biological function of nucleolin is distinct from its conventional role as a proto-oncogene. Therefore, our findings here not only reveal a new modification of nucleolin protein and its novel functional paradigm in mRNA metabolism but also expand our understanding of the dichotomous roles of nucleolin in terms of cancer development, which are dependent on multiple intracellular conditions and consequently the appropriate regulations of its modifications, including SUMOylation. PMID:25561743

  9. A new regulatory pathway of mRNA export by an F-box protein, Mdm30.

    PubMed

    Durairaj, Geetha; Lahudkar, Shweta; Bhaumik, Sukesh R

    2014-02-01

    Mdm30, an F-box protein in yeast, has been recently shown to promote mRNA export. However, it remains unknown how Mdm30 facilitates mRNA export. Here, we show that Mdm30 targets the Sub2 component of the TREX (Transcription/Export) complex for ubiquitylation and subsequent proteasomal degradation. Such a targeted degradation of Sub2 enhances the recruitment of the mRNA export adaptor, Yra1, to the active genes to promote mRNA export. Together, these results elucidate that Mdm30 promotes mRNA export by lowering Sub2's stability and consequently enhancing Yra1 recruitment, thus illuminating new regulatory mechanisms of mRNA export by Mdm30.

  10. A Novel de novo CDH1 Germline Variant Aids in the Classification of C-terminal E-cadherin Alterations Predicted to Escape Nonsense-Mediated mRNA Decay.

    PubMed

    Krempely, Kate; Karam, Rachid

    2018-05-24

    Most truncating CDH1 pathogenic alterations confer an elevated lifetime risk of diffuse gastric cancer and lobular breast cancer. However, transcripts containing carboxyl-terminal (C-terminal) premature stop codons have been demonstrated to escape the nonsense-mediated mRNA decay (NMD) pathway, and gastric and breast cancer risks associated with these truncations should be carefully evaluated. A female patient underwent multigene panel testing due to a personal history of invasive lobular breast cancer diagnosed at age 54, which identified the germline CDH1 nonsense alteration, c.2506G>T (p.E836*), in the last exon of the gene. Subsequent parental testing for the alteration was negative and additional short tandem repeat analysis confirmed the familial relationships and the de novo occurrence in the proband. Based on the de novo occurrence, clinical history, and rarity in general population databases, this alteration was classified as a likely pathogenic variant. This is the most C-terminal pathogenic alteration reported to date. Additionally, this alteration contributed to the classification of six other upstream CDH1 C-terminal truncating variants as pathogenic or likely pathogenic. Identifying the most distal pathogenic alteration provides evidence to classify other C-terminal truncating variants as either pathogenic or benign, a fundamental step to offering pre-symptomatic screening and prophylactic procedures to the appropriate patients. Cold Spring Harbor Laboratory Press.

  11. Differential utilization of decapping enzymes in mammalian mRNA decay pathways

    PubMed Central

    Li, You; Song, Mangen; Kiledjian, Megerditch

    2011-01-01

    mRNA decapping is a crucial step in the regulation of mRNA stability and gene expression. Dcp2 is an mRNA decapping enzyme that has been widely studied. We recently reported the presence of a second mammalian cytoplasmic decapping enzyme, Nudt16. Here we address the differential utilization of the two decapping enzymes in specified mRNA decay processes. Using mouse embryonic fibroblast (MEF) cell lines derived from a hypomorphic knockout of the Dcp2 gene with undetectable levels of Dcp2 or MEF cell lines harboring a Nudt16-directed shRNA to generate reduced levels of Nudt16, we demonstrate the distinct roles for Dcp2 and Nudt16 in nonsense-mediated mRNA decay (NMD), decay of ARE-containing mRNA and miRNA-mediated silencing. Our results indicated that NMD preferentially utilizes Dcp2 rather than Nudt16; Dcp2 and Nudt16 are redundant in miRNA-mediated silencing; and Dcp2 and Nudt16 are differentially utilized for ARE-mRNA decay. These data demonstrate that the two distinct decapping enzymes can uniquely function in specific mRNA decay processes in mammalian cells. PMID:21224379

  12. Cell type-specific regulation of beta2-adrenoceptor mRNA by agonists.

    PubMed

    Danner, S; Lohse, M J

    1997-07-16

    Prolonged agonist stimulation of beta2-adrenoceptors results in receptor down-regulation which is often paralleled by a reduction of the corresponding mRNA. In this study, we investigated the agonist-dependent regulation of beta2-adrenoceptor mRNA in DDT1-MF2 smooth muscle cells and C6 glioma cells. In DDT1-MF2 cells the half-life of the mRNA was 12 h in monolayer compared to 2 h in suspension cultures. Under both conditions, the agonist isoproterenol reduced this half-life by a factor of 2. In contrast, in C6 glioma cells isoproterenol had no effect on the mRNA stability, even though it reduced mRNA levels by approximately 50%. Isoproterenol-induced downregulation of beta2-adrenoceptor mRNA was completely blocked in C6 cells by the presence of a protein synthesis inhibitor, while this was not so in DDT1-MF2-cells. These data show that beta2-adrenoceptor downregulation occurs via cell-type specific mechanisms.

  13. Altered Fibroblast Growth Factor Receptor 4 Stability Promotes Prostate Cancer Progression1

    PubMed Central

    Wang, Jianghua; Yu, Wendong; Cai, Yi; Ren, Chengxi; Ittmann, Michael M

    2008-01-01

    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. PMID:18670643

  14. Selective decline of Nogo mRNA in the aging brain.

    PubMed

    Trifunovski, Alexandra; Josephson, Anna; Bickford, Paula C; Olson, Lars; Brené, Stefan

    2006-06-26

    The Nogo system has recently been implicated not only in regeneration but also in modulating plasticity. One reason for declining memory functions in aging may be altered plasticity in the aged hippocampus and cortex cerebri. Therefore, we have examined the levels of mRNA encoding Nogo, OMgp and MAG, as well as the receptor components NgR, Lingo-1 and Troy in cortex and hippocampus of young (4 months), middle aged (16 months) and old (24 months) Fisher 344 rats. No significant changes of receptor components or the ligands OMgp or MAG were observed. Nogo mRNA, however, was significantly decreased in hippocampal subregions of aged animals. The specific decrease of Nogo mRNA levels in hippocampus and possibly cortex cerebri may relate to age-dependent decline of brain plasticity.

  15. Elevation of D4 dopamine receptor mRNA in postmortem schizophrenic brain.

    PubMed

    Stefanis, N C; Bresnick, J N; Kerwin, R W; Schofield, W N; McAllister, G

    1998-01-01

    The D4 dopamine (DA) receptor has been proposed to be a target for the development of a novel antipsychotic drug based on its pharmacological and distribution profile. There is much interest in whether D4 DA receptor levels are altered in schizophrenia, but the lack of an available receptor subtype-specific radioligand made this difficult to quantitate. In this study, we examined whether D4 mRNA levels are altered in different brain regions of schizophrenics compared to controls. Ribonuclease protection assays were carried out on total RNA samples isolated postmortem from frontal cortex and caudate brain regions of schizophrenics and matched controls. 32P-labelled RNA probes to the D4 DA receptor and to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), were hybridised with the RNA samples, digested with ribonucleases to remove unhybridised probe, and separated on 6% sequencing gels. Densitometer analysis on the subsequent autoradiogams was used to calculate the relative optical density of D4 mRNA compared to G3PDH mRNA. Statistical analysis of the data revealed a 3-fold higher level (P<0.011) of D4 mRNA in the frontal cortex of schizophrenics compared to controls. No increase was seen in caudate. D4 receptors could play a role in mediating dopaminergic activity in frontal cortex, an activity which may be malfunctioning in schizophrenia.

  16. Effect of surface charge alteration on stability of L-asparaginase II from Escherichia sp.

    PubMed

    Vidya, Jalaja; Ushasree, Mrudula Vasudevan; Pandey, Ashok

    2014-03-05

    Escherichia coli L-asparaginases have great significance in the treatment of leukemia. Consequently, there is considerable interest in engineering this enzyme for improving its stability. In this work, the effect of surface charge on the stability of the enzyme l-asparaginase II was studied by site-directed mutagenesis of the cloned ansB gene from Escherichia sp. Replacement of two positively charged residues (K139 and K207) on the surface loops with neutral and reverse charges resulted in altered thermo stability in designed variants. Neutral charge substitutions (K139A and K207A) retained greater tolerance and stability followed by negative charge substitutions (K139D and K207D) compared to control mutant K139R and wild enzyme. From the results, it was concluded that the optimization of surface charge contributed much to the thermal properties of proteins without affecting the structure. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Osteoblast fibronectin mRNA, protein synthesis, and matrix are unchanged after exposure to microgravity

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.; Gilbertson, V.

    1999-01-01

    The well-defined osteoblast line, MC3T3-E1 was used to examine fibronectin (FN) mRNA levels, protein synthesis, and extracellular FN matrix accumulation after growth activation in spaceflight. These osteoblasts produce FN extracellular matrix (ECM) known to regulate adhesion, differentiation, and function in adherent cells. Changes in bone ECM and osteoblast cell shape occur in spaceflight. To determine whether altered FN matrix is a factor in causing these changes in spaceflight, quiescent osteoblasts were launched into microgravity and were then sera activated with and without a 1-gravity field. Synthesis of FN mRNA, protein, and matrix were measured after activation in microgravity. FN mRNA synthesis is significantly reduced in microgravity (0-G) when compared to ground (GR) osteoblasts flown in a centrifuge simulating earth's gravity (1-G) field 2.5 h after activation. However, 27.5 h after activation there were no significant differences in mRNA synthesis. A small but significant reduction of FN protein was found in the 0-G samples 2.5 h after activation. Total FN protein 27.5 h after activation showed no significant difference between any of the gravity conditions, however, there was a fourfold increase in absolute amount of protein synthesized during the incubation. Using immunofluorescence, we found no significant differences in the amount or in the orientation of the FN matrix after 27.5 h in microgravity. These results demonstrate that FN is made by sera-activated osteoblasts even during exposure to microgravity. These data also suggest that after a total period of 43 h of spaceflight FN transcription, translation, or altered matrix assembly is not responsible for the altered cell shape or altered matrix formation of osteoblasts.

  18. The cellular growth rate controls overall mRNA turnover, and modulates either transcription or degradation rates of particular gene regulons.

    PubMed

    García-Martínez, José; Delgado-Ramos, Lidia; Ayala, Guillermo; Pelechano, Vicent; Medina, Daniel A; Carrasco, Fany; González, Ramón; Andrés-León, Eduardo; Steinmetz, Lars; Warringer, Jonas; Chávez, Sebastián; Pérez-Ortín, José E

    2016-05-05

    We analyzed 80 different genomic experiments, and found a positive correlation between both RNA polymerase II transcription and mRNA degradation with growth rates in yeast. Thus, in spite of the marked variation in mRNA turnover, the total mRNA concentration remained approximately constant. Some genes, however, regulated their mRNA concentration by uncoupling mRNA stability from the transcription rate. Ribosome-related genes modulated their transcription rates to increase mRNA levels under fast growth. In contrast, mitochondria-related and stress-induced genes lowered mRNA levels by reducing mRNA stability or the transcription rate, respectively. We also detected these regulations within the heterogeneity of a wild-type cell population growing in optimal conditions. The transcriptomic analysis of sorted microcolonies confirmed that the growth rate dictates alternative expression programs by modulating transcription and mRNA decay.The regulation of overall mRNA turnover keeps a constant ratio between mRNA decay and the dilution of [mRNA] caused by cellular growth. This regulation minimizes the indiscriminate transmission of mRNAs from mother to daughter cells, and favors the response capacity of the latter to physiological signals and environmental changes. We also conclude that, by uncoupling mRNA synthesis from decay, cells control the mRNA abundance of those gene regulons that characterize fast and slow growth. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

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

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

    Fu, Zidong Donna; Klaassen, Curtis D., E-mail: cklaasse@kumc.edu

    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.more » 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.« less

  1. Maintenance of CCL5 mRNA stores by post-effector and memory CD8 T cells is dependent on transcription and is coupled to increased mRNA stability.

    PubMed

    Marçais, Antoine; Tomkowiak, Martine; Walzer, Thierry; Coupet, Charles-Antoine; Ravel-Chapuis, Aymeric; Marvel, Jacqueline

    2006-10-01

    Immunological memory is associated with the display of improved effector functions by cells of the adaptive immune system. The storage of untranslated mRNA coding for the CCL5 chemokine by CD8 memory cells is a new process supporting the immediate display of an effector function. Here, we show that, after induction during the primary response, high CCL5 mRNA levels are specifically preserved in CD8 T cells. We have investigated the mechanisms involved in the long-term maintenance of CCL5 mRNA levels by memory CD8 T cells. We demonstrate that the CCL5 mRNA half-life is increased in memory CD8 T cells and that these cells constitutively transcribe ccl5 gene. By inhibiting ccl5 transcription using IL-4, we demonstrate the essential role of transcription in the maintenance of CCL5 mRNA stores. Finally, we show that these stores are spontaneously reconstituted when the inhibitory signal is removed, indicating that the transcription of ccl5 is a default feature of memory CD8 T cells imprinted in their genetic program.

  2. 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

    ERIC Educational Resources Information Center

    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…

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

    PubMed

    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.

  4. Regulated Expression of a Calmodulin Isoform Alters Growth and Development in Potato

    NASA Technical Reports Server (NTRS)

    Poovaiah, B. W.; Takezawa, D.; An, G.; Han, T.-J.

    1996-01-01

    A transgene approach was taken to study the consequences of altered expression of a calmodutin iso-form on plant growth and development. Eight genomic clones of potato calmodulin (PCM 1 to 8) have been isolated and characterized. Among the potato calmodulin isoforms studied, PCM 1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM 1 fused to the CAMV 35S promoter. Transgenic plants showing a moderate increase in PCM 1 MRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM 1 MRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM 1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM 1 MRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM 1 protein in transgenic plants, indicating that the expression of both MRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM 1 alters growth and development in potato plants.

  5. Global Maps of ProQ Binding In Vivo Reveal Target Recognition via RNA Structure and Stability Control at mRNA 3' Ends.

    PubMed

    Holmqvist, Erik; Li, Lei; Bischler, Thorsten; Barquist, Lars; Vogel, Jörg

    2018-05-15

    The conserved RNA-binding protein ProQ has emerged as the centerpiece of a previously unknown third large network of post-transcriptional control in enterobacteria. Here, we have used in vivo UV crosslinking and RNA sequencing (CLIP-seq) to map hundreds of ProQ binding sites in Salmonella enterica and Escherichia coli. Our analysis of these binding sites, many of which are conserved, suggests that ProQ recognizes its cellular targets through RNA structural motifs found in small RNAs (sRNAs) and at the 3' end of mRNAs. Using the cspE mRNA as a model for 3' end targeting, we reveal a function for ProQ in protecting mRNA against exoribonucleolytic activity. Taken together, our results underpin the notion that ProQ governs a post-transcriptional network distinct from those of the well-characterized sRNA-binding proteins, CsrA and Hfq, and suggest a previously unrecognized, sRNA-independent role of ProQ in stabilizing mRNAs. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Exaptive origins of regulated mRNA decay in eukaryotes

    PubMed Central

    Hamid, Fursham M.

    2016-01-01

    Eukaryotic gene expression is extensively controlled at the level of mRNA stability and the mechanisms underlying this regulation are markedly different from their archaeal and bacterial counterparts. We propose that two such mechanisms, nonsense‐mediated decay (NMD) and motif‐specific transcript destabilization by CCCH‐type zinc finger RNA‐binding proteins, originated as a part of cellular defense against RNA pathogens. These branches of the mRNA turnover pathway might have been used by primeval eukaryotes alongside RNA interference to distinguish their own messages from those of RNA viruses and retrotransposable elements. We further hypothesize that the subsequent advent of “professional” innate and adaptive immunity systems allowed NMD and the motif‐triggered mechanisms to be efficiently repurposed for regulation of endogenous cellular transcripts. This scenario explains the rapid emergence of archetypical mRNA destabilization pathways in eukaryotes and argues that other aspects of post‐transcriptional gene regulation in this lineage might have been derived through a similar exaptation route. PMID:27438915

  7. 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.

    PubMed

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

    2016-01-01

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

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

    PubMed

    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. HuR binding to cytoplasmic mRNA is perturbed by heat shock

    PubMed Central

    Gallouzi, Imed-Eddine; Brennan, Christopher M.; Stenberg, Myrna G.; Swanson, Maurice S.; Eversole, Ashley; Maizels, Nancy; Steitz, Joan A.

    2000-01-01

    AU-rich elements (AREs) located in the 3′ untranslated region target the mRNAs encoding many protooncoproteins, cytokines, and lymphokines for rapid degradation. HuR, a ubiquitously expressed member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, binds ARE sequences and selectively stabilizes ARE-containing reporter mRNAs when overexpressed in transiently transfected cells. HuR appears predominantly nucleoplasmic but has been shown to shuttle between the nucleus and cytoplasm via a novel shuttling sequence HNS. We report generation of a mouse monoclonal antibody 3A2 that both immunoblots and immunoprecipitates HuR protein; it recognizes an epitope located in the first of HuR's three RNA recognition motifs. This antibody was used to probe HuR interactions with mRNA before and after heat shock, a condition that has been reported to stabilize ARE-containing mRNAs. At 37°C, approximately one-third of the cytoplasmic HuR appears polysome associated, and in vivo UV crosslinking reveals that HuR interactions with poly(A)+ RNA are predominantly cytoplasmic rather than nuclear. This comprises evidence that HuR directly interacts with mRNA in vivo. After heat shock, 12–15% of HuR accumulates in discrete foci in the cytoplasm, but surprisingly the majority of HuR crosslinks instead to nuclear poly(A)+ RNA, whose levels are dramatically increased in the stressed cells. This behavior of HuR differs from that of another ARE-binding protein, hnRNP D, which has been implicated as an effector of mRNA decay rather than mRNA stabilization and of the general pre-RNA-binding protein hnRNP A1. We interpret these differences to mean that the temporal association of HuR with ARE-containing mRNAs is different from that of these other two proteins. PMID:10737787

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

    PubMed Central

    Brumann, M.; Matz, M.; Kusmenkov, T.; Stegmaier, J.; Biberthaler, P.; Kanz, K.-G.; Mutschler, W.; Bogner, V.

    2014-01-01

    Background. Fulminant changes in cytokine receptor signalling might provoke severe pathological alterations after multiple trauma. The aim of this study was to evaluate the posttraumatic imbalance of the innate immune system with a special focus on the STAT/SOCS family. Methods. 20 polytraumatized patients were included. Blood samples were drawn 0 h–72 h after trauma; mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3 were quantified by qPCR. Results. IL-10 mRNA expression increased significantly in the early posttraumatic period. STAT 3 mRNA expressions showed a significant maximum at 6 h after trauma. SOCS 1 levels significantly decreased 6 h–72 h after trauma. SOCS 3 levels were significantly higher in nonsurvivors 6 h after trauma. Conclusion. We present a serial, sequential investigation in human neutrophil granulocytes of major trauma patients evaluating mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3. Posttraumatically, immune disorder was accompanied by a significant increase of IL-10 and STAT 3 mRNA expression, whereas SOCS 1 mRNA levels decreased after injury. We could demonstrate that death after trauma was associated with higher SOCS 3 mRNA levels already at 6 h after trauma. To support our results, further investigations have to evaluate protein levels of STAT/SOCS family in terms of posttraumatic immune imbalance. PMID:24648661

  11. Postnatal handling does not normalize hypothalamic corticotropin-releasing factor mRNA levels in animals prenatally exposed to ethanol.

    PubMed

    Gabriel, Kara I; Glavas, Maria M; Ellis, Linda; Weinberg, Joanne

    2005-06-09

    Postnatal handling has been shown to attenuate some of the deficits in developmental outcome observed following prenatal ethanol exposure (E) although it appears to be ineffective at ameliorating the hypothalamic-pituitary-adrenal (HPA) hyperresponsiveness to stressors that has been observed in adult E animals. However, the effects of postnatal handling on central regulation of HPA activity in E animals, particularly with regard to alterations in steady-state hypothalamic corticotropin-releasing factor (CRF) activity, have not been examined. In the present study, offspring from E, pair-fed (PF), and ad-libitum-fed control (C) groups were exposed to daily handling during the first 2 weeks of life (H) or were left entirely undisturbed until weaning (NH). Basal CRF and arginine vasopressin (AVP) mRNA in the parvocellular portion of the paraventricular nucleus (pPVN) of the hypothalamus were assessed at 90-110 days of age. Prenatal ethanol exposure resulted in elevated basal pPVN CRF mRNA levels compared to those in ad-libitum-fed controls. Handling altered CRF mRNA levels in a sex-specific and prenatal treatment-specific manner. Females showed no significant effects of handling. In contrast, handling decreased CRF mRNA levels in PF and C but not E males compared to their NH counterparts. There were no effects of prenatal ethanol or postnatal handling on AVP mRNA levels. These findings indicate that prenatal ethanol exposure results in elevated basal CRF mRNA levels in adulthood and that handling appears to be ineffective in normalizing those elevations, supporting the suggestion that altered basal HPA regulation in E animals may, at least in part, underlie their HPA hyperresponsiveness to stressors.

  12. Rational Manipulation of mRNA Folding Free Energy Allows Rheostat Control of Pneumolysin Production by Streptococcus pneumoniae

    PubMed Central

    Amaral, Fábio E.; Parker, Dane; Randis, Tara M.; Kulkarni, Ritwij; Prince, Alice S.; Shirasu-Hiza, Mimi M.; Ratner, Adam J.

    2015-01-01

    The contribution of specific factors to bacterial virulence is generally investigated through creation of genetic “knockouts” that are then compared to wild-type strains or complemented mutants. This paradigm is useful to understand the effect of presence vs. absence of a specific gene product but cannot account for concentration-dependent effects, such as may occur with some bacterial toxins. In order to assess threshold and dose-response effects of virulence factors, robust systems for tunable expression are required. Recent evidence suggests that the folding free energy (ΔG) of the 5’ end of mRNA transcripts can have a significant effect on translation efficiency and overall protein abundance. Here we demonstrate that rational alteration of 5’ mRNA folding free energy by introduction of synonymous mutations allows for predictable changes in pneumolysin (PLY) expression by Streptococcus pneumoniae without the need for chemical inducers or heterologous promoters. We created a panel of isogenic S. pneumoniae strains, differing only in synonymous (silent) mutations at the 5’ end of the PLY mRNA that are predicted to alter ΔG. Such manipulation allows rheostat-like control of PLY production and alters the cytotoxicity of whole S. pneumoniae on primary and immortalized human cells. These studies provide proof-of-principle for further investigation of mRNA ΔG manipulation as a tool in studies of bacterial pathogenesis. PMID:25798590

  13. Inhibition and Avoidance of mRNA Degradation by RNA Viruses

    PubMed Central

    Moon, Stephanie L.; Barnhart, Michael D.; Wilusz, Jeffrey

    2012-01-01

    The cellular mRNA decay machinery plays a major role in regulating the quality and quantity of gene expression in cells. This machinery involves multiple enzymes and pathways that converge to promote the exonucleolytic decay of mRNAs. The transcripts made by RNA viruses are susceptible to degradation by this machinery and, in fact, can be actively targeted. Thus, to maintain gene expression and replication, RNA viruses have evolved a number of strategies to avoid and/or inactivate aspects of the cellular mRNA decay machinery. Recent work uncovering the mechanisms used by RNA viruses to maintain the stability of their transcripts is described below. PMID:22626865

  14. Regulated mRNA Decay in Arabidopsis: A global analysis of differential control by hormones and the circadian clock

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

    Green, Pamela J.

    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 analyzemore » mRNA decay genome wide. Thus, the research was hugely successful beyond our objectives.« less

  15. Severe neurocognitive and growth disorders due to variation in THOC2, an essential component of nuclear mRNA export machinery.

    PubMed

    Kumar, Raman; Gardner, Alison; Homan, Claire C; Douglas, Evelyn; Mefford, Heather; Wieczorek, Dagmar; Lüdecke, Hermann-Josef; Stark, Zornitza; Sadedin, Simon; Nowak, Catherine Bearce; Douglas, Jessica; Parsons, Gretchen; Mark, Paul; Loidi, Lourdes; Herman, Gail E; Mihalic Mosher, Theresa; Gillespie, Meredith K; Brady, Lauren; Tarnopolsky, Mark; Madrigal, Irene; Eiris, Jesús; Domènech Salgado, Laura; Rabionet, Raquel; Strom, Tim M; Ishihara, Naoko; Inagaki, Hidehito; Kurahashi, Hiroki; Dudding-Byth, Tracy; Palmer, Elizabeth E; Field, Michael; Gecz, Jozef

    2018-05-31

    Highly conserved TREX-mediated mRNA export is emerging as a key pathway in neuronal development and differentiation. TREX subunit variants cause neurodevelopmental disorders (NDDs) by interfering with mRNA export from the cell nucleus to the cytoplasm. Previously we implicated four missense variants in the X-linked THOC2 gene in intellectual disability (ID). We now report an additional six affected individuals from five unrelated families with two de novo and three maternally-inherited pathogenic or likely pathogenic variants in THOC2 extending the genotypic and phenotypic spectrum. These comprise three rare missense THOC2 variants that affect evolutionarily conserved amino acid residues and reduce protein stability and two with canonical splice-site THOC2 variants that result in C-terminally truncated THOC2 proteins. We present detailed clinical assessment and functional studies on a de novo variant in a female with an epileptic encephalopathy and discuss an additional four families with rare variants in THOC2 with supportive evidence for pathogenicity. Severe neurocognitive features, including movement and seizure disorders were observed in this cohort. Taken together our data show that even subtle alterations to the canonical molecular pathways such as mRNA export, otherwise essential for cellular life, can be compatible with life, but lead to NDDs in humans. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  16. Regulated expression of a calmodulin isoform alters growth and development in potato.

    PubMed

    Poovaiah, B W; Takezawa, D; An, G; Han, T J

    1996-01-01

    A transgene approach was taken to study the consequences of altered expression of a calmodulin isoform on plant growth and development. Eight genomic clones of potato calmodulin (PCM1 to 8) have been isolated and characterized (Takezawa et al., 1995). Among the potato calmodulin isoforms studied, PCM1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM1 fused to the CaMV 35S promoter. Transgenic plants showing a moderate increase in PCM1 mRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM1 mRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM1 mRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM1 protein in transgenic plants, indicating that the expression of both mRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM1 alters growth and development in potato plants.

  17. Colonization by non-pathogenic bacteria alters mRNA expression of cytochromes P450 in originally germ-free mice.

    PubMed

    Jourová, L; Anzenbacher, P; Lišková, B; Matušková, Z; Hermanová, P; Hudcovic, T; Kozáková, H; Hrnčířová, L; Anzenbacherová, E

    2017-11-01

    Gut microbiota provides a wide range of beneficial function for the host and has an immense effect on the host's health state. It has also been shown that gut microbiome is often involved in the biotransformation of xenobiotics; however, the molecular mechanisms of the interaction between the gut bacteria and the metabolism of drugs by the host are still unclear. To investigate the effect of microbial colonization on messenger RNA (mRNA) expression of liver cytochromes P450 (CYPs), the main drug-metabolizing enzymes, we used germ-free (GF) mice, lacking the intestinal flora and mice monocolonized by non-pathogenic bacteria Lactobacillus plantarum NIZO2877 or probiotic bacteria Escherichia coli Nissle 1917 compared to specific pathogen-free (SPF) mice. Our results show that the mRNA expression of Cyp1a2 and Cyp2e1 was significantly increased, while the expression of Cyp3a11 mRNA was decreased under GF conditions compared to the SPF mice. The both bacteria L. plantarum NIZO2877 and E. coli Nissle 1917 given to the GF mice decreased the level of Cyp1a2 mRNA and normalized it to the control level. On the other hand, the colonization by these bacteria had no effect on the expression of Cyp3a11 mRNA in the liver of the GF mice (which remained decreased). Surprisingly, monocolonization with chosen bacterial strains has shown a different effect on the expression of Cyp2e1 mRNA in GF mice. Increased level of Cyp2e1 expression observed in the GF mice was found also in mice colonized by L. plantarum NIZO2877 ; however, the colonization with probiotic E. coli Nissle 1917 caused a decrease in Cyp2e1 expression and partially restored the SPF mice conditions.

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

    NASA Astrophysics Data System (ADS)

    Dai, Zhongquan; Wu, Feng; Qu, Lina

    Abstract Spaceflight induces muscle atrophy but mechanism is not well understood. Here, we quantified microRNAs (miRNAs) and mRNA shifts of rat soleus after 7, 14 and 28 days tail suspension (TS). Microarray data revealed that TS altered 23 miRNAs and 1313 mRNAs at least 2-fold change. QRT-PCR confirmed changes of miRNAs and mRNAs related to muscle atrophy. MiR-214, miR-486-5p and miR-320 family decreased, but Let-7e increased. Actn3 and myh4 displayed abundant upregulation and a3galt2 downregulated. Predicted targeted genes (whyz, ywhaz and SFRP2) of altered miRNAs decreased. Further analysis of gene functional annotation confirmed consistency of alteration profile between miRNAs and mRNA and enrichment of main clusters in regulation of muscle metabolism. Our results highlight the importance of miR-214, miR-486-5p, miR-320 and Let-7e in muscle atrophy process induced by microgravity.

  19. Individuals with chronic ankle instability exhibit dynamic postural stability deficits and altered unilateral landing biomechanics: A systematic review.

    PubMed

    Simpson, Jeffrey D; Stewart, Ethan M; Macias, David M; Chander, Harish; Knight, Adam C

    2018-06-13

    To evaluate the literature regarding unilateral landing biomechanics and dynamic postural stability in individuals with and without chronic ankle instability (CAI). Four online databases (PubMed, ScienceDirect, Scopus, and SportDiscus) were searched from the earliest records to 31 January 2018, as well as reference sections of related journal articles, to complete the systematic search. Studies investigating the influence of CAI on unilateral landing biomechanics and dynamic postural stability were systematically reviewed and evaluated. Twenty articles met the criteria and were included in the systematic review. Individuals with CAI were found to have deficits in dynamic postural stability on the affected limb with medium to large effect sizes and altered lower extremity kinematics, most notably in the ankle and knee, with medium to large effect sizes. Additionally, greater loading rates and peak ground reaction forces, in addition to reductions in ankle muscle activity were also found in individuals with CAI during unilateral jump-landing tasks. Individuals with CAI demonstrate dynamic postural stability deficits, lower extremity kinematic alterations, and reduced neuromuscular control during unilateral jump-landings. These are likely factors that contribute recurrent lateral ankle sprain injuries during dynamic activity in individuals with CAI. Copyright © 2018 Elsevier Ltd. All rights reserved.

  20. Exaptive origins of regulated mRNA decay in eukaryotes.

    PubMed

    Hamid, Fursham M; Makeyev, Eugene V

    2016-09-01

    Eukaryotic gene expression is extensively controlled at the level of mRNA stability and the mechanisms underlying this regulation are markedly different from their archaeal and bacterial counterparts. We propose that two such mechanisms, nonsense-mediated decay (NMD) and motif-specific transcript destabilization by CCCH-type zinc finger RNA-binding proteins, originated as a part of cellular defense against RNA pathogens. These branches of the mRNA turnover pathway might have been used by primeval eukaryotes alongside RNA interference to distinguish their own messages from those of RNA viruses and retrotransposable elements. We further hypothesize that the subsequent advent of "professional" innate and adaptive immunity systems allowed NMD and the motif-triggered mechanisms to be efficiently repurposed for regulation of endogenous cellular transcripts. This scenario explains the rapid emergence of archetypical mRNA destabilization pathways in eukaryotes and argues that other aspects of post-transcriptional gene regulation in this lineage might have been derived through a similar exaptation route. © 2016 The Authors BioEssays Published by WILEY Periodicals, Inc.

  1. Stability of medicines after repackaging into multicompartment compliance aids: eight criteria for detection of visual alteration.

    PubMed

    Albert, Valerie; Lanz, Michael; Imanidis, Georgios; Hersberger, Kurt E; Arnet, Isabelle

    2017-01-01

    Multicompartment compliance aids (MCA) are widely used by patients. They support the management of medication and reduce unintentional nonadherence. MCA are filled with medicines unpacked from their original packaging. Swiss pharmacists currently provide MCA for 1-2 weeks, although little and controversial information exists on the stability of repackaged medicines. We aimed to validate the usefulness of a simple screening method capable of detecting visual stability problems with repackaged medicines. We selected eight criteria for solid formulations from The International Pharmacopoeia : (1) rough surface, (2) chipping, (3) cracking, (4) capping, (5) mottling, (6) discoloration, (7) swelling, and (8) crushing. A selection of 24 critical medicines was repackaged in three different MCA (Pharmis ® , SureMed™, and self-produced blister) and stored at room temperature for 4 weeks. Pharmis ® was additionally stored at accelerated conditions. Appearance was scored weekly. Six alterations (rough surface, cracking, mottling, discoloration, swelling, and crushing) were observed at accelerated conditions. No alteration was observed at room temperature, except for the chipping of tablets that had been stuck to cold seal glue. The eight criteria can detect alterations of the appearance of oral solid medicines repackaged in MCA. In the absence of specific guidelines, they can serve as a simple screening method in community pharmacies for identifying medicines unsuitable for repackaging.

  2. Membrane-association of mRNA decapping factors is independent of stress in budding yeast

    PubMed Central

    Huch, Susanne; Gommlich, Jessie; Muppavarapu, Mridula; Beckham, Carla; Nissan, Tracy

    2016-01-01

    Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation. PMID:27146487

  3. Membrane-association of mRNA decapping factors is independent of stress in budding yeast.

    PubMed

    Huch, Susanne; Gommlich, Jessie; Muppavarapu, Mridula; Beckham, Carla; Nissan, Tracy

    2016-05-05

    Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation.

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

    PubMed

    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. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. The Association between Splenocyte Apoptosis and Alterations of Bax, Bcl-2 and Caspase-3 mRNA Expression, and Oxidative Stress Induced by Dietary Nickel Chloride in Broilers

    PubMed Central

    Huang, Jianying; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wu, Bangyuan

    2013-01-01

    Two hundred and forty avian broilers were equally divided into four groups, and raised with a corn-soybean basal diet or the same diet supplemented with 300, 600, 900 mg/kg NiCl2 for 42 days. Numbers or percentages of apoptotic splenocytes by flow cytometry (FCM) and TUNEL were higher (p < 0.05 or p < 0.01) in the 300, 600 and 900 mg/kg groups than those in the control group. Results measured by qRT-PCR and ELISA showed that mRNA expression and contents were significantly higher (p < 0.05 or p < 0.01) in Bax and Caspase-3, and were significantly lower (p < 0.05 or p < 0.01) in Bcl-2 of the 300, 600 and 900 mg/kg groups. Also, the SOD, CAT and GSH-Px activities, and the ability to inhibit hydroxyl radical, and GSH contents were significantly decreased (p < 0.05 or p < 0.01), and MDA contents were increased (p < 0.05 or p < 0.01) in all groups. In conclusion, dietary NiCl2 in excess of 300 mg/kg caused apoptosis, altered Bax, Bcl-2 and Caspase-3 mRNA expression levels and contents, and induced oxidative stress in the spleen. Also, splenocyte apoptosis was closely related to the alternations of Bax, Bcl-2 and Caspase-3 mRNA expression, and oxidative damage. The splenic immunity and blood filtration functions were impaired in broilers. PMID:24351749

  6. Stool metatranscriptomics: A technical guideline for mRNA stabilisation and isolation.

    PubMed

    Reck, Michael; Tomasch, Jürgen; Deng, Zhiluo; Jarek, Michael; Husemann, Peter; Wagner-Döbler, Irene

    2015-07-04

    The complex microbiome of the gut has an enormous impact on human health. Analysis of the transcriptional activity of microorganisms through mRNA sequencing (metatranscriptomics) opens a completely new window into their activity in vivo, but it is highly challenging due to numerous technical and bioinformatical obstacles. Here we present an optimized pipeline for extraction of high quality mRNA from stool samples. Comparison of three commercially available RNA extraction kits with the method of Zoetendal revealed that the Powermicrobiome Kit (MoBio) performed best with respect to RNA yield and purity. Next, the influence of the stabilization reagent during sample storage for up to 15 days was studied. RIN analysis and qRT-PCR of spiked-in and indigenous genes revealed that RNA Later preserved mRNA integrity most efficiently, while samples conserved in RNA Protect showed substantial mRNA decay. Using the optimized pipeline developed here, recovery rates for spiked-in E.coli cells expressing fluorescing proteins were 8.7-9.7% for SuperfolderGFP and 14.7-17.8% for mCherry. The mRNA of stabilized stool samples as well as of snap-frozen controls was sequenced with Illumina Hiseq, yielding on average 74 million reads per sample. PCoA analysis, taxonomic classification using Kraken and functional classification using bwa showed that the transcriptomes of samples conserved in RNA Later were unchanged for up to 6 days even at room temperature, while RNA Protect was inefficient for storage durations exceeding 24 h. However, our data indicate that RNA Later introduces a bias which is then maintained throughout storage, while RNA Protect conserved samples are initially more similar to the snap frozen controls. RNA Later conserved samples had a reduced abundance of e.g. Prevotellaceae transcripts and were depleted for e.g. COG category "Carbohydrate transport and metabolism". Since the overall similarity between all stool transcriptional profiles studied here was >0.92, these

  7. Kinetic measures of restabilisation during volitional stepping reveal age-related alterations in the control of mediolateral dynamic stability.

    PubMed

    Singer, Jonathan C; McIlroy, William E; Prentice, Stephen D

    2014-11-07

    Research examining age-related changes in dynamic stability during stepping has recognised the importance of the restabilisation phase, subsequent to foot-contact. While regulation of the net ground reaction force (GRFnet) line of action is believed to influence dynamic stability during steady-state locomotion, such control during restabilisation remains unknown. This work explored the origins of age-related decline in mediolateral dynamic stability by examining the line of action of GRFnet relative to the centre of mass (COM) during restabilisation following voluntary stepping. Healthy younger and older adults (n=20 per group) performed three single-step tasks (varying speed and step placement), altering the challenge to stability control. Age-related differences in magnitude and intertrial variability of the angle of divergence of GRFnet line of action relative to the COM were quantified, along with the peak mediolateral and vertical GRFnet components. The angle of divergence was further examined at discrete points during restabilisation, to uncover events of potential importance to stability control. Older adults exhibited a reduced angle of divergence throughout restabilisation. Temporal and spatial constraints on stepping increased the magnitude and intertrial variability of the angle of divergence, although not differentially among the older adults. Analysis of the time-varying angle of divergence revealed age-related reductions in magnitude, with increases in timing and intertrial timing variability during the later phase of restabilisation. This work further supports the idea that age-related challenges in lateral stability control emerge during restabilisation. Age-related alterations during the later phase of restabilisation may signify challenges with reactive control. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Investigation of mRNA quadruplex formation in Escherichia coli.

    PubMed

    Wieland, Markus; Hartig, Jörg S

    2009-01-01

    The protocol presented here allows for the investigation of the formation of unusual nucleic acid structures in the 5'-untranslated region (UTR) of bacteria by correlating gene expression levels to the in vitro stability of the respective structure. In particular, we describe the introduction of G-quadruplex forming sequences close to the ribosome-binding site (RBS) on the mRNA of a reporter gene and the subsequent read-out of the expression levels. Insertion of a stable secondary structure results in the cloaking of RBS and eventually reduced gene expression levels. The structures and stability of the introduced sequences are further characterized by circular dichroism (CD) spectroscopy and thermal melting experiments. The extent of inhibition is then correlated to the stability of the respective quadruplex structure, allowing judgement of whether factors other than thermodynamic stability affect the formation of a given quadruplex sequence in vivo. Measuring gene expression levels takes 2 d including cloning; CD experiments take 5 hours per experiment.

  9. HUMAN LIVER FATTY ACID BINDING PROTEIN (L-FABP) T94A VARIANT ALTERS STRUCTURE, STABILITY, AND INTERACTION WITH FIBRATES

    PubMed Central

    Martin, Gregory G.; McIntosh, Avery L.; Huang, Huan; Gupta, Shipra; Atshaves, Barbara P.; Landrock, Kerstin K.; Landrock, Danilo; Kier, Ann B.; Schroeder, Friedhelm

    2014-01-01

    Although the human L-FABP T94A variant arises from the most commonly occurring SNP in the entire FABP family, there is a complete lack of understanding regarding the role of this polymorphism in human disease. It has been hypothesized that the T94A substitution results in complete loss of ligand binding ability and function analogous to L-FABP gene ablation. This possibility was addressed using recombinant human WT T94T and T94A variant L-FABP and cultured primary human hepatocytes. Non-conservative replacement of the medium sized, polar, uncharged T residue by a smaller, nonpolar, aliphatic A residue at position 94 of human L-FABP significantly increased L-FABP protein α-helical structure at the expense of β-sheet and concomitantly decreased thermal stability. T94A did not alter binding affinities for PPARα agonist ligands (phytanic acid, fenofibrate, fenofibric acid). While T94A did not alter the impact of phytanic acid and only slightly altered that of fenofibrate on human L-FABP secondary structure, the active metabolite fenofibric acid altered T94A secondary structure much more than that of WT T94T L-FABP. Finally, in cultured primary human hepatocytes the T94A variant exhibited significantly reduced fibrate-mediated induction of PPARα-regulated proteins such as L-FABP, FATP5, and PPARα itself. Thus, while T94A substitution did not alter the affinity of human L-FABP for PPARα agonist ligands, it significantly altered human L-FABP structure, stability, as well as conformational and functional response to fibrate. PMID:24299557

  10. Natural antisense transcript-targeted regulation of inducible nitric oxide synthase mRNA levels.

    PubMed

    Yoshigai, Emi; Hara, Takafumi; Araki, Yoshiro; Tanaka, Yoshito; Oishi, Masaharu; Tokuhara, Katsuji; Kaibori, Masaki; Okumura, Tadayoshi; Kwon, A-Hon; Nishizawa, Mikio

    2013-04-01

    Natural antisense transcripts (asRNAs) are frequently transcribed from mammalian genes. Recently, we found that non-coding asRNAs are transcribed from the 3' untranslated region (3'UTR) of the rat and mouse genes encoding inducible nitric oxide synthase (iNOS), which catalyzes the production of the inflammatory mediator nitric oxide. The iNOS asRNA stabilizes iNOS mRNA by interacting with the mRNA 3'UTR. Furthermore, single-stranded 'sense' oligonucleotides corresponding to the iNOS mRNA sequence were found to reduce iNOS mRNA levels by interfering with mRNA-asRNA interactions in rat hepatocytes. This method was named natural antisense transcript-targeted regulation (NATRE) technology. In this study, we detected human iNOS asRNA expressed in hepatocarcinoma and colon carcinoma tissues. The human iNOS asRNA harbored a sequence complementary to an evolutionarily conserved region of the iNOS mRNA 3'UTR. When introduced into hepatocytes, iNOS sense oligonucleotides that were modified by substitution with partial phosphorothioate bonds and locked nucleic acids or 2'-O-methyl nucleic acids greatly reduced levels of iNOS mRNA and iNOS protein. Moreover, sense oligonucleotides and short interfering RNAs decreased iNOS mRNA to comparable levels. These results suggest that NATRE technology using iNOS sense oligonucleotides could potentially be used to treat human inflammatory diseases and cancers by reducing iNOS mRNA levels. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    PubMed

    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.

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

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

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

    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 mapsmore » 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.« less

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

    PubMed

    Fraser, Christopher S

    2015-07-01

    The process of peptide bond synthesis by ribosomes is conserved between species, but the initiation step differs greatly between the three kingdoms of life. This is illustrated by the evolution of roughly an order of magnitude more initiation factor mass found in humans compared with bacteria. Eukaryotic initiation of translation is comprised of a number of sub-steps: (i) recruitment of an mRNA and initiator methionyl-tRNA to the 40S ribosomal subunit; (ii) migration of the 40S subunit along the 5' UTR to locate the initiation codon; and (iii) recruitment of the 60S subunit to form the 80S initiation complex. Although the mechanism and regulation of initiation has been studied for decades, many aspects of the pathway remain unclear. In this review, I will focus discussion on what is known about the mechanism of mRNA selection and its recruitment to the 40S subunit. I will summarize how the 43S preinitiation complex (PIC) is formed and stabilized by interactions between its components. I will discuss what is known about the mechanism of mRNA selection by the eukaryotic initiation factor 4F (eIF4F) complex and how the selected mRNA is recruited to the 43S PIC. The regulation of this process by secondary structure located in the 5' UTR of an mRNA will also be discussed. Finally, I present a possible kinetic model with which to explain the process of mRNA selection and recruitment to the eukaryotic ribosome. Copyright © 2015 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

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

    PubMed

    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 < 0.001) exposed to near-infrared laser, but increase in muscle tissue (p < 0.001). ERCC1 mRNA expression does not alter (p > 0.05), but ERCC2 mRNA expression decreases in skin (p < 0.001) and increases in muscle tissue (p < 0.001) exposed to red 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.

  15. Development, food intake, and ethinylestradiol influence hepatic triglyceride lipase and LDL-receptor mRNA levels in rats.

    PubMed

    Staels, B; Jansen, H; van Tol, A; Stahnke, G; Will, H; Verhoeven, G; Auwerx, J

    1990-07-01

    The influence of development and ethinylestradiol on low density lipoprotein (LDL)-receptor mRNA and hepatic triglyceride lipase (HTGL) activity and mRNA levels was studied in rat liver and intestine. Intestinal LDL-receptor mRNA levels are maximal in the perinatal period, whereas liver LDL-receptor and HTGL mRNA levels are highest after weaning in adult life. All mRNA levels reach a maximum between day 15 and 20 when rats still consume a lipid-rich diet, and increase twofold during weaning. Liver and intestinal LDL-receptor mRNA levels are not influenced by ovariectomy, but increase after ethinylestradiol treatment. Liver LDL-receptor mRNA shows a dose-dependent increase after ethinylestradiol and a sevenfold rise in liver LDL-receptor mRNA is attained with a dose of 2000 micrograms/day. Intestinal LDL-receptor mRNA increases slightly more than twofold after ethinylestradiol and this increase is not dose-dependent. Changes in LDL-receptor mRNA are independent of changes in food intake induced by ethinylestradiol treatment, since they are still observed after pair-feeding. The ethinylestradiol-induced increases in LDL-receptor mRNA levels are reflected by decreased serum apoB levels. HTGL mRNA levels increase after ovariectomy and show a dose-dependent decrease after ethinylestradiol. Pair-feeding abolishes the increase seen after ovariectomy, while the estrogen-mediated decrease is attenuated. These alterations in HTGL mRNA are reflected by similar changes in liver HTGL activity.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

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

  17. Induction of cysteine-rich motor neuron 1 mRNA expression in vascular endothelial cells.

    PubMed

    Nakashima, Yukiko; Takahashi, Satoru

    2014-08-22

    Cysteine-rich motor neuron 1 (CRIM1) is expressed in vascular endothelial cells and plays a crucial role in angiogenesis. In this study, we investigated the expression of CRIM1 mRNA in human umbilical vein endothelial cells (HUVECs). CRIM1 mRNA levels were not altered in vascular endothelial growth factor (VEGF)-stimulated monolayer HUVECs or in cells in collagen gels without VEGF. In contrast, the expression of CRIM1 mRNA was elevated in VEGF-stimulated cells in collagen gels. The increase in CRIM1 mRNA expression was observed even at 2h when HUVECs did not form tubular structures in collagen gels. Extracellular signal-regulated kinase (Erk) 1/2, Akt and focal adhesion kinase (FAK) were activated by VEGF in HUVECs. The VEGF-induced expression of CRIM1 mRNA was significantly abrogated by PD98059 or PF562271, but was not affected by LY294002. These results demonstrate that CRIM1 is an early response gene in the presence of both angiogenic stimulation (VEGF) and environmental (extracellular matrix) factors, and Erk and FAK might be involved in the upregulation of CRIM1 mRNA expression in vascular endothelial cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. IL-17A acts via p38 MAPK to increase stability of TNF-alpha-induced IL-8 mRNA in human ASM.

    PubMed

    Henness, Sheridan; van Thoor, Eveline; Ge, Qi; Armour, Carol L; Hughes, J Margaret; Ammit, Alaina J

    2006-06-01

    Human airway smooth muscle (ASM) plays an immunomodulatory role in asthma. Recently, IL-17A has become of increasing interest in asthma, being found at elevated levels in asthmatic airways and emerging as playing an important role in airway neutrophilia. IL-17A predominantly exerts its neutrophil orchestrating role indirectly via the induction of cytokines by resident airway structural cells. Here, we perform an in vitro study to show that although IL-17A did not induce secretion of the CXC chemokine IL-8 from ASM cells, IL-17A significantly potentiates TNF-alpha-induced IL-8 protein secretion and gene expression in a concentration- and time-dependent manner (P < 0.05). Levels of IL-8 protein produced after 24 h of incubation with TNF-alpha were enhanced 2.7-fold in the presence of IL-17A, and conditioned media significantly enhanced neutrophil chemotaxis in vitro. As IL-17A had no effect on the activity of NF-kappaB, a key transcriptional regulator of IL-8 gene expression, we then examined whether IL-17A acts at the posttranscriptional level. We found that IL-17A significantly augmented TNF-alpha-induced IL-8 mRNA stability. Interestingly, this enhanced stability occurred via a p38 MAPK-dependent pathway. The decay of IL-8 mRNA transcripts proceeded at a significantly faster rate when cells were pretreated with the p38 MAPK inhibitor SB-203580 (-0.05763 +/- 0.01964, t(1/2) = 12.0 h), compared with vehicle (-0.01030 +/- 0.007963, t(1/2) = 67.3 h) [results are expressed as decay constant (means +/- SE) and half-life (t(1/2) in h): P < 0.05]. Collectively, these results demonstrate that IL-17A amplifies the synthetic function of ASM cells, acting via a p38 MAPK-dependent posttranscriptional pathway to augment TNF-alpha-induced secretion of the potent neutrophil chemoattractant IL-8 from ASM cells.

  19. Rainfall and hydrological stability alter the impact of top predators on food web structure and function.

    PubMed

    Marino, Nicholas A C; Srivastava, Diane S; MacDonald, A Andrew M; Leal, Juliana S; Campos, Alice B A; Farjalla, Vinicius F

    2017-02-01

    Climate change will alter the distribution of rainfall, with potential consequences for the hydrological dynamics of aquatic habitats. Hydrological stability can be an important determinant of diversity in temporary aquatic habitats, affecting species persistence and the importance of predation on community dynamics. As such, prey are not only affected by drought-induced mortality but also the risk of predation [a non-consumptive effect (NCE)] and actual consumption by predators [a consumptive effect (CE)]. Climate-induced changes in rainfall may directly, or via altered hydrological stability, affect predator-prey interactions and their cascading effects on the food web, but this has rarely been explored, especially in natural food webs. To address this question, we performed a field experiment using tank bromeliads and their aquatic food web, composed of predatory damselfly larvae, macroinvertebrate prey and bacteria. We manipulated the presence and consumption ability of damselfly larvae under three rainfall scenarios (ambient, few large rainfall events and several small rainfall events), recorded the hydrological dynamics within bromeliads and examined the effects on macroinvertebrate colonization, nutrient cycling and bacterial biomass and turnover. Despite our large perturbations of rainfall, rainfall scenario had no effect on the hydrological dynamics of bromeliads. As a result, macroinvertebrate colonization and nutrient cycling depended on the hydrological stability of bromeliads, with no direct effect of rainfall or predation. In contrast, rainfall scenario determined the direction of the indirect effects of predators on bacteria, driven by both predator CEs and NCEs. These results suggest that rainfall and the hydrological stability of bromeliads had indirect effects on the food web through changes in the CEs and NCEs of predators. We suggest that future studies should consider the importance of the variability in hydrological dynamics among habitats as

  20. Polysome Fractionation to Analyze mRNA Distribution Profiles.

    PubMed

    Panda, Amaresh C; Martindale, Jennifer L; Gorospe, Myriam

    2017-02-05

    Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al. , 2014a and 2014b; Abdelmohsen et al. , 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ribosomes with a given mRNA. It provides valuable information about the translational status of that mRNA, depending on the number of ribosomes with which they are associated, and identifies mRNAs that are not translated (Panda et al. , 2016). mRNAs associated with many ribosomes form large polysomes that are predicted to be actively translated, while mRNAs associated with few or no ribosomes are expected to be translated poorly if at all. In sum, polysome fractionation analysis allows the direct determination of translation efficiencies at the level of the whole transcriptome as well as individual mRNAs.

  1. Tristetraprolin Inhibits Ras-dependent Tumor Vascularization by Inducing Vascular Endothelial Growth Factor mRNA Degradation

    PubMed Central

    Essafi-Benkhadir, Khadija; Onesto, Cercina; Stebe, Emmanuelle; Moroni, Christoph

    2007-01-01

    Vascular endothelial growth factor (VEGF) is one of the most important regulators of physiological and pathological angiogenesis. Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway and overexpression of VEGF are common denominators of tumors from different origins. We have established a new link between these two fundamental observations converging on VEGF mRNA stability. In this complex phenomenon, tristetraprolin (TTP), an adenylate and uridylate-rich element-associated protein that binds to VEGF mRNA 3′-untranslated region, plays a key role by inducing VEGF mRNA degradation, thus maintaining basal VEGF mRNA amounts in normal cells. ERKs activation results in the accumulation of TTP mRNA. However, ERKs reduce the VEGF mRNA-destabilizing effect of TTP, leading to an increase in VEGF expression that favors the angiogenic switch. Moreover, TTP decreases RasVal12-dependent VEGF expression and development of vascularized tumors in nude mice. As a consequence, TTP might represent a novel antiangiogenic and antitumor agent acting through its destabilizing activity on VEGF mRNA. Determination of TTP and ERKs status would provide useful information for the evaluation of the angiogenic potential in human tumors. PMID:17855506

  2. Long-distance transport of mRNA via parenchyma cells and phloem across the host-parasite junction in Cuscuta.

    PubMed

    David-Schwartz, Rakefet; Runo, Steven; Townsley, Brad; Machuka, Jesse; Sinha, Neelima

    2008-01-01

    It has been shown that the parasitic plant dodder (Cuscuta pentagona) establishes a continuous vascular system through which water and nutrients are drawn. Along with solutes, viruses and proteins, mRNA transcripts are transported from the host to the parasite. The path of the transcripts and their stability in the parasite have yet to be revealed. To discover the route of mRNA transportation, the in situ reverse transcriptase-polymerase chain reaction (RT-PCR) technique was used to locally amplify host transcript within parasitic tissue. The stability of host mRNA molecules was also checked by monitoring specific transcripts along the growing dodder thread. Four mRNAs, alpha and beta subunits of PYROPHOSPHATE (PPi)-DEPENDENT PHOSPHOFRUCTOKINASE (LePFP), the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and GIBBERELLIC ACID INSENSITIVE (LeGAI), were found to move from host (tomato (Solanum lycopersicum)) to dodder. LePFP mRNA was localized to the dodder parenchyma cells and to the phloem. LePFP transcripts were found in the growing dodder stem up to 30 cm from the tomato-dodder connection. These results suggest that mRNA molecules are transferred from host to parasite via symplastic connections between parenchyma cells, move towards the phloem, and are stable for a long distance in the parasite. This may allow developmental coordination between the parasite and its host.

  3. A Case for Microtubule Vulnerability in Amyotrophic Lateral Sclerosis: Altered Dynamics During Disease.

    PubMed

    Clark, Jayden A; Yeaman, Elise J; Blizzard, Catherine A; Chuckowree, Jyoti A; Dickson, Tracey C

    2016-01-01

    Amyotrophic lateral sclerosis (ALS) is an aggressive multifactorial disease converging on a common pathology: the degeneration of motor neurons (MNs), their axons and neuromuscular synapses. This vulnerability and dysfunction of MNs highlights the dependency of these large cells on their intracellular machinery. Neuronal microtubules (MTs) are intracellular structures that facilitate a myriad of vital neuronal functions, including activity dependent axonal transport. In ALS, it is becoming increasingly apparent that MTs are likely to be a critical component of this disease. Not only are disruptions in this intracellular machinery present in the vast majority of seemingly sporadic cases, recent research has revealed that mutation to a microtubule protein, the tubulin isoform TUBA4A, is sufficient to cause a familial, albeit rare, form of disease. In both sporadic and familial disease, studies have provided evidence that microtubule mediated deficits in axonal transport are the tipping point for MN survivability. Axonal transport deficits would lead to abnormal mitochondrial recycling, decreased vesicle and mRNA transport and limited signaling of key survival factors from the neurons peripheral synapses, causing the characteristic peripheral "die back". This disruption to microtubule dependant transport in ALS has been shown to result from alterations in the phenomenon of microtubule dynamic instability: the rapid growth and shrinkage of microtubule polymers. This is accomplished primarily due to aberrant alterations to microtubule associated proteins (MAPs) that regulate microtubule stability. Indeed, the current literature would argue that microtubule stability, particularly alterations in their dynamics, may be the initial driving force behind many familial and sporadic insults in ALS. Pharmacological stabilization of the microtubule network offers an attractive therapeutic strategy in ALS; indeed it has shown promise in many neurological disorders, ALS included

  4. Altered monocyte cyclo-oxygenase response in non-obese diabetic mice.

    PubMed

    Beyan, H; Buckley, L R; Bustin, S A; Yousaf, N; Pozzilli, P; Leslie, R D

    2009-02-01

    Monocytes infiltrate islets in non-obese diabetic (NOD) mice. Activated monocyte/macrophages express cyclo-oxygenase-2 (COX-2) promoting prostaglandin-E(2) (PGE(2)) secretion, while COX-1 expression is constitutive. We investigated in female NOD mice: (i) natural history of monocyte COX expression basally and following lipopolysaccharide (LPS) stimulation; (ii) impact of COX-2 specific inhibitor (Vioxx) on PGE(2), insulitis and diabetes. CD11b(+) monocytes were analysed for COX mRNA expression from NOD (n = 48) and C57BL/6 control (n = 18) mice. NOD mice were treated with either Vioxx (total dose 80 mg/kg) (n = 29) or methylcellulose as control (n = 29) administered by gavage at 4 weeks until diabetes developed or age 30 weeks. In all groups, basal monocyte COX mRNA and PGE(2) secretion were normal, while following LPS, after 5 weeks of age monocyte/macrophage COX-1 mRNA decreased (P < 0.01) and COX-2 mRNA increased (P < 0.01). However, diabetic NOD mice had reduced COX mRNA response (P = 0.03). Vioxx administration influenced neither PGE(2), insulitis nor diabetes. We demonstrate an isoform switch in monocyte/macrophage COX mRNA expression following LPS, which is altered in diabetic NOD mice as in human diabetes. However, Vioxx failed to affect insulitis or diabetes. We conclude that monocyte responses are altered in diabetic NOD mice but COX-2 expression is unlikely to be critical to disease risk.

  5. Alternative 3' UTRs Modify the Localization, Regulatory Potential, Stability, and Plasticity of mRNAs in Neuronal Compartments.

    PubMed

    Tushev, Georgi; Glock, Caspar; Heumüller, Maximilian; Biever, Anne; Jovanovic, Marko; Schuman, Erin M

    2018-05-02

    Neurons localize mRNAs near synapses where their translation can be regulated by synaptic demand and activity. Differences in the 3' UTRs of mRNAs can change their localization, stability, and translational regulation. Using 3' end RNA sequencing of microdissected rat brain slices, we discovered a huge diversity in mRNA 3' UTRs, with many transcripts showing enrichment for a particular 3' UTR isoform in either somata or the neuropil. The 3' UTR isoforms of localized transcripts are significantly longer than the 3' UTRs of non-localized transcripts and often code for proteins associated with axons, dendrites, and synapses. Surprisingly, long 3' UTRs add not only new, but also duplicate regulatory elements. The neuropil-enriched 3' UTR isoforms have significantly longer half-lives than somata-enriched isoforms. Finally, the 3' UTR isoforms can be significantly altered by enhanced activity. Most of the 3' UTR plasticity is transcription dependent, but intriguing examples of changes that are consistent with altered stability, trafficking between compartments, or local "remodeling" remain. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Low-level lasers on microRNA and uncoupling protein 2 mRNA levels in human breast cancer cells

    NASA Astrophysics Data System (ADS)

    Canuto, K. S.; Teixeira, A. F.; Rodrigues, J. A.; Paoli, F.; Nogueira, E. M.; Mencalha, A. L.; Fonseca, A. S.

    2017-06-01

    MicroRNA is short non-coding RNA and is a mediator of post-transcriptional regulation of gene expression. In addition, uncoupling proteins (UCPs) regulate thermogenesis, metabolic and energy balance, and decrease reactive oxygen species production. Both microRNA and UCP2 expression can be altered in cancer cells. At low power, laser wavelength, frequency, fluence and emission mode deternube photobiological responses, which are the basis of low-level laser therapy. There are few studies on miRNA and UCP mRNA levels after low-level laser exposure on cancer cells. In this work, we evaluate the micrRNA (mir-106b and mir-15a) and UCP2 mRNA levels in human breast cancer cells exposed to low-level lasers. MDA-MB-231 human breast cancer cells were exposed to low-level red and infrared lasers, total RNA was extracted for cDNA synthesis and mRNA levels by real time quantitative polymerase chain reaction were evaluated. Data show that mir-106b and mir-15a relative levels are not altered, but UCP2 mRNA relative levels are increased in MDA-MB-231 human breast cancer cells exposed to low-level red and infrared lasers at fluences used in therapeutic protocols.

  7. Alterations of type IV collagen alpha chains in patients with chronic acquired glomerulopathies: mRNA levels, protein expression and urinary loss.

    PubMed

    Sanna-Cherchi, Simone; Carnevali, Maria Luisa; Martorana, Davide; Cravedi, Paolo; Maggiore, Umberto; Alinovi, Rossella; Bovino, Achiropita; Mattei, Silvia; Orlandini, Guido; Gatti, Rita; Savi, Mario; Sado, Yoshikazu; Neri, Tauro M; Allegri, Landino

    2007-01-01

    Type IV collagen is a major structural component of the normal kidney glomerulus. However, its role in chronic acquired glomerulopathies has been only partially elucidated. Urinary levels of col(IV)alpha1, col(IV)alpha3 and col(IV)alpha5 collagen chains were analyzed in 107 patients with chronic acquired glomerulopathies. In a subgroup of 33 patients, tissue mRNA levels, protein expression and urinary excretion were evaluated for all col(IV)alpha chains, from col(IV)alpha1 to col(IV)alpha5. The renal specimens were examined to get a semiquantitative score of the acute and chronic activity of the histological lesions. Urines obtained from 13 healthy subjects and 10 normal renal tissue samples were used as controls. Urinary levels of col(IV)alpha1, col(IV)alpha3, col(IV)alpha5 chains were significantly higher in patients than in controls [p < 0.01 for all], while only col(IV)alpha1 and col(IV)alpha3 urinary excretion correlated with the degree of chronic histological damage [col(IV)alpha1 R = 0.44, p < 0.001; col(IV)alpha3: R = 0.47, p < 0.001]. Compared with controls, patients showed a renal expression of mRNA for col(IV)alpha5 chain significantly higher [p = 0.001], while having a significantly lower protein expression of col(IV)alpha3, col(IV)alpha4 and col(IV)alpha5 chains [p < 0.01 for all]. Patients with chronic acquired glomerulopathies show important alterations in the col(IV)alpha chain network mimicking some molecular features of the X-linked Alport's syndrome. Further studies are needed to show whether urinary levels of the col(IV)alpha chains may be used as markers for monitoring renal injury. Copyright 2007 S. Karger AG, Basel.

  8. Circadian clock-dependent and -independent posttranscriptional regulation underlies temporal mRNA accumulation in mouse liver

    PubMed Central

    Wang, Jingkui; Yeung, Jake; Gobet, Cédric; Sobel, Jonathan; Lück, Sarah; Molina, Nacho; Naef, Felix

    2018-01-01

    The mammalian circadian clock coordinates physiology with environmental cycles through the regulation of daily oscillations of gene expression. Thousands of transcripts exhibit rhythmic accumulations across mouse tissues, as determined by the balance of their synthesis and degradation. While diurnally rhythmic transcription regulation is well studied and often thought to be the main factor generating rhythmic mRNA accumulation, the extent of rhythmic posttranscriptional regulation is debated, and the kinetic parameters (e.g., half-lives), as well as the underlying regulators (e.g., mRNA-binding proteins) are relatively unexplored. Here, we developed a quantitative model for cyclic accumulations of pre-mRNA and mRNA from total RNA-seq data, and applied it to mouse liver. This allowed us to identify that about 20% of mRNA rhythms were driven by rhythmic mRNA degradation, and another 15% of mRNAs regulated by both rhythmic transcription and mRNA degradation. The method could also estimate mRNA half-lives and processing times in intact mouse liver. We then showed that, depending on mRNA half-life, rhythmic mRNA degradation can either amplify or tune phases of mRNA rhythms. By comparing mRNA rhythms in wild-type and Bmal1−/− animals, we found that the rhythmic degradation of many transcripts did not depend on a functional BMAL1. Interestingly clock-dependent and -independent degradation rhythms peaked at distinct times of day. We further predicted mRNA-binding proteins (mRBPs) that were implicated in the posttranscriptional regulation of mRNAs, either through stabilizing or destabilizing activities. Together, our results demonstrate how posttranscriptional regulation temporally shapes rhythmic mRNA accumulation in mouse liver. PMID:29432155

  9. Phosphorylation of poly(rC) binding protein 1 (PCBP1) contributes to stabilization of mu opioid receptor (MOR) mRNA via interaction with AU-rich element RNA-binding protein 1 (AUF1) and poly A binding protein (PABP)

    PubMed Central

    Hwang, Cheol Kyu; Wagley, Yadav; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

    2016-01-01

    Gene regulation at the post-transcriptional level is frequently based on cis- and trans-acting factors on target mRNAs. We found a C-rich element (CRE) in mu-opioid receptor (MOR) 3′-untranslated region (UTR) to which poly (rC) binding protein 1 (PCBP1) binds, resulting in MOR mRNA stabilization. RNA immunoprecipitation and RNA EMSA revealed the formation of PCBP1-RNA complexes at the element. Knockdown of PCBP1 decreased MOR mRNA half-life and protein expression. Stimulation by forskolin increased cytoplasmic localization of PCBP1 and PCBP1/MOR 3′-UTR interactions via increased serine phosphorylation that was blocked by protein kinase A (PKA) or (phosphatidyl inositol-3) PI3-kinase inhibitors. The forskolin treatment also enhanced serine- and tyrosine-phosphorylation of AU-rich element binding protein (AUF1), concurrent with its increased binding to the CRE, and led to an increased interaction of poly A binding protein (PABP) with the CRE and poly(A) sites. AUF1 phosphorylation also led to an increased interaction with PCBP1. These findings suggest that a single co-regulator, PCBP1, plays a crucial role in stabilizing MOR mRNA, and is induced by PKA signaling by conforming to AUF1 and PABP. PMID:27836661

  10. Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis

    DOE PAGES

    Xiao, Chaowen; Zhang, Tian; Zheng, Yunzhen; ...

    2015-11-02

    Here, xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis ( Arabidopsis thaliana) xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall-integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolatedmore » xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes, such as MAP70-5 and CLASP, and receptor genes, such as HERK1 and WAK1, were changed in xxt1 xxt2 plants and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish, to our knowledge, new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis.« less

  11. Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis.

    PubMed

    Xiao, Chaowen; Zhang, Tian; Zheng, Yunzhen; Cosgrove, Daniel J; Anderson, Charles T

    2016-01-01

    Xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis (Arabidopsis thaliana) xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall-integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolated xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes, such as MAP70-5 and CLASP, and receptor genes, such as HERK1 and WAK1, were changed in xxt1 xxt2 plants and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish, to our knowledge, new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

  12. Three-dimensional geophysical mapping of shallow water saturated altered rocks at Mount Baker, Washington: Implications for slope stability

    NASA Astrophysics Data System (ADS)

    Finn, Carol A.; Deszcz-Pan, Maryla; Ball, Jessica L.; Bloss, Benjamin J.; Minsley, Burke J.

    2018-05-01

    Water-saturated hydrothermal alteration reduces the strength of volcanic edifices, increasing the potential for catastrophic sector collapses that can lead to far traveled and destructive debris flows. Intense hydrothermal alteration significantly lowers the resistivity and magnetization of volcanic rock and therefore hydrothermally altered rocks can be identified with helicopter electromagnetic and magnetic measurements. Geophysical models constrained by rock properties and geologic mapping show that intensely altered rock is restricted to two small (500 m diameter), >150 m thick regions around Sherman Crater and Dorr Fumarole Field at Mount Baker, Washington. This distribution of alteration contrasts with much thicker and widespread alteration encompassing the summits of Mounts Adams and Rainier prior to the 5600 year old Osceola collapse, which is most likely due to extreme erosion and the limited duration of summit magmatism at Mount Baker. In addition, the models suggest that the upper 300 m of rock contains water which could help to lubricate potential debris flows. Slope stability modeling incorporating the geophysically modeled distribution of alteration and water indicates that the most likely and largest ( 0.1 km3) collapses are from the east side of Sherman Crater. Alteration at Dorr Fumarole Field raises the collapse hazard there, but not significantly because of its lower slope angles. Geochemistry and analogs from other volcanoes suggest a model for the edifice hydrothermal system.

  13. Three-dimensional geophysical mapping of shallow water saturated altered rocks at Mount Baker, Washington: Implications for slope stability

    USGS Publications Warehouse

    Finn, Carol A.; Deszcz-Pan, Maria; Ball, Jessica L.; Bloss, Benjamin J.; Minsley, Burke J.

    2018-01-01

    Water-saturated hydrothermal alteration reduces the strength of volcanic edifices, increasing the potential for catastrophic sector collapses that can lead to far traveled and destructive debris flows. Intense hydrothermal alteration significantly lowers the resistivity and magnetization of volcanic rock and therefore hydrothermally altered rocks can be identified with helicopter electromagnetic and magnetic measurements. Geophysical models constrained by rock properties and geologic mapping show that intensely altered rock is restricted to two small (500 m diameter), >150 m thick regions around Sherman Crater and Dorr Fumarole Field at Mount Baker, Washington. This distribution of alteration contrasts with much thicker and widespread alteration encompassing the summits of Mounts Adams and Rainier prior to the 5600 year old Osceola collapse, which is most likely due to extreme erosion and the limited duration of summit magmatism at Mount Baker. In addition, the models suggest that the upper ~300 m of rock contains water which could help to lubricate potential debris flows. Slope stability modeling incorporating the geophysically modeled distribution of alteration and water indicates that the most likely and largest (~0.1 km3) collapses are from the east side of Sherman Crater. Alteration at Dorr Fumarole Field raises the collapse hazard there, but not significantly because of its lower slope angles. Geochemistry and analogs from other volcanoes suggest a model for the edifice hydrothermal system.

  14. Viral and Cellular mRNA Translation in Coronavirus-Infected Cells

    PubMed Central

    Nakagawa, K.; Lokugamage, K.G.; Makino, S.

    2017-01-01

    Coronaviruses have large positive-strand RNA genomes that are 5′ capped and 3′ polyadenylated. The 5′-terminal two-thirds of the genome contain two open reading frames (ORFs), 1a and 1b, that together make up the viral replicase gene and encode two large polyproteins that are processed by viral proteases into 15–16 nonstructural proteins, most of them being involved in viral RNA synthesis. ORFs located in the 3′-terminal one-third of the genome encode structural and accessory proteins and are expressed from a set of 5′ leader-containing subgenomic mRNAs that are synthesized by a process called discontinuous transcription. Coronavirus protein synthesis not only involves cap-dependent translation mechanisms but also employs regulatory mechanisms, such as ribosomal frameshifting. Coronavirus replication is known to affect cellular translation, involving activation of stress-induced signaling pathways, and employing viral proteins that affect cellular mRNA translation and RNA stability. This chapter describes our current understanding of the mechanisms involved in coronavirus mRNA translation and changes in host mRNA translation observed in coronavirus-infected cells. PMID:27712623

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

    PubMed Central

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

    2011-01-01

    Familial dysautonomia (FD) is caused by an intronic splice mutation in the IKBKAP gene that leads to partial skipping of exon 20 and tissue-specific reduction in I-κ-B kinase complex associated protein/ elongation protein 1 (IKAP/ELP-1) expression. Kinetin (6-furfurylaminopurine) has been shown to improve splicing and increase wild-type IKBKAP mRNA and IKAP protein expression in FD cell lines and carriers. To determine if oral kinetin treatment could alter mRNA splicing in FD subjects and was tolerable, we administered kinetin to eight FD individuals homozygous for the splice mutation. Subjects received 23.5 mg/Kg/day for 28 days. An increase in wild-type IKBKAP mRNA expression in leukocytes was noted after eight days in six of eight individuals; after 28 days the mean increase as compared to baseline was significant (p=0.002). We have demonstrated that kinetin is tolerable in this medically fragile population. Not only did kinetin produce the desired effect on splicing in FD patients, but also that effect appears to improve with time despite lack of dose change. This is the first report of a drug that produces in vivo mRNA splicing changes in individuals with FD and supports future long-term trials to determine if kinetin will prove therapeutic in FD patients. PMID:21775922

  16. Depletion of mRNA export regulator DBP5/DDX19, GLE1 or IPPK that is a key enzyme for the production of IP6, resulting in differentially altered cytoplasmic mRNA expression and specific cell defect

    PubMed Central

    Okamura, Masumi; Yamanaka, Yasutaka; Shigemoto, Maki; Kitadani, Yuya; Kobayashi, Yuhko; Kambe, Taiho; Nagao, Masaya; Kobayashi, Issei; Okumura, Katsuzumi

    2018-01-01

    DBP5, also known as DDX19, GLE1 and inositol hexakisphosphate (IP6) function in messenger RNA (mRNA) export at the cytoplasmic surface of the nuclear pore complex in eukaryotic cells. DBP5 is a DEAD-box RNA helicase, and its activity is stimulated by interactions with GLE1 and IP6. In addition, these three factors also have unique role(s). To investigate how these factors influenced the cytoplasmic mRNA expression and cell phenotype change, we performed RNA microarray analysis to detect the effect and function of DBP5, GLE1 and IP6 on the cytoplasmic mRNA expression. The expression of some cytoplasmic mRNA subsets (e.g. cell cycle, DNA replication) was commonly suppressed by the knock-down of DBP5, GLE1 and IPPK (IP6 synthetic enzyme). The GLE1 knock-down selectively reduced the cytoplasmic mRNA expression required for mitotic progression, results in an abnormal spindle phenotype and caused the delay of mitotic process. Meanwhile, G1/S cell cycle arrest was observed in DBP5 and IPPK knock-down cells. Several factors that function in immune response were also down-regulated in DBP5 or IPPK knock-down cells. Thereby, IFNβ-1 mRNA transcription evoked by poly(I:C) treatment was suppressed. These results imply that DBP5, GLE1 and IP6 have a conserved and individual function in the cytoplasmic mRNA expression. Variations in phenotype are due to the difference in each function of DBP5, GLE1 and IPPK in intracellular mRNA metabolism. PMID:29746542

  17. Bioinspired Nanocomplex for Spatiotemporal Imaging of Sequential mRNA Expression in Differentiating Neural Stem Cells

    PubMed Central

    2015-01-01

    Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions. PMID:25494492

  18. Bioinspired nanocomplex for spatiotemporal imaging of sequential mRNA expression in differentiating neural stem cells.

    PubMed

    Wang, Zhe; Zhang, Ruili; Wang, Zhongliang; Wang, He-Fang; Wang, Yu; Zhao, Jun; Wang, Fu; Li, Weitao; Niu, Gang; Kiesewetter, Dale O; Chen, Xiaoyuan

    2014-12-23

    Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions.

  19. Interconnections between mRNA degradation and RDR-dependent siRNA production in mRNA turnover in plants.

    PubMed

    Tsuzuki, Masayuki; Motomura, Kazuki; Kumakura, Naoyoshi; Takeda, Atsushi

    2017-03-01

    Accumulation of an mRNA species is determined by the balance between the synthesis and the degradation of the mRNA. Individual mRNA molecules are selectively and actively degraded through RNA degradation pathways, which include 5'-3' mRNA degradation pathway, 3'-5' mRNA degradation pathway, and RNA-dependent RNA polymerase-mediated mRNA degradation pathway. Recent studies have revealed that these RNA degradation pathways compete with each other in mRNA turnover in plants and that plants have a hidden layer of non-coding small-interfering RNA production from a set of mRNAs. In this review, we summarize the current information about plant mRNA degradation pathways in mRNA turnover and discuss the potential roles of a novel class of the endogenous siRNAs derived from plant mRNAs.

  20. Increased expression of ADAM 9 and ADAM 15 mRNA in pancreatic cancer.

    PubMed

    Yamada, Daisuke; Ohuchida, Kenoki; Mizumoto, Kazuhiro; Ohhashi, Seiji; Yu, Jun; Egami, Takuya; Fujita, Hayato; Nagai, Eishi; Tanaka, Masao

    2007-01-01

    A disintegrin and metalloproteases (ADAMs) comprise a multifunctional family of membrane-anchored proteins. ADAM 9 and ADAM 15 are involved in cell migration and invasion. Expression of ADAM 9 and ADAM 15 was reported to be altered in several types of cancer. Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure the expression of ADAM 9 mRNA in bulk pancreatic tissues. Results showed no significant difference in the expression of ADAM 9 mRNA between pancreatic cancer and non-neoplastic pancreas. Primary cultured pancreatic fibroblasts also expressed ADAM 9 mRNA. Therefore, a laser microdissection and pressure catapulting technique was employed to isolate cancer cells from tumor tissues. The expression of ADAM 9 and ADAM 15 mRNA was measured in microdissected samples (cancer cells, n = 11; normal epithelial cells, n = 13 for ADAM 9; cancer cells, n = 9; normal epithelial cells, n = 9 for ADAM 15). Pancreatic cancer cells expressed significantly higher levels of ADAM 9 and ADAM 15 mRNA than did normal pancreatic epithelial cells (p = 0.016 for ADAM 9; p = 0.004 for ADAM 15). ADAM 9 and ADAM 15 are involved in pancreatic cancer. Microdissection-based analysis appears to be indispensable for the accurate analysis of the expression of certain ADAM family members in pancreatic cancer.

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

    PubMed

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Age-related Differences in Dystrophin: Impact on Force Transfer Proteins, Membrane Integrity, and Neuromuscular Junction Stability.

    PubMed

    Hughes, David C; Marcotte, George R; Marshall, Andrea G; West, Daniel W D; Baehr, Leslie M; Wallace, Marita A; Saleh, Perrie M; Bodine, Sue C; Baar, Keith

    2017-05-01

    The loss of muscle strength with age has been studied from the perspective of a decline in muscle mass and neuromuscular junction (NMJ) stability. A third potential factor is force transmission. The purpose of this study was to determine the changes in the force transfer apparatus within aging muscle and the impact on membrane integrity and NMJ stability. We measured an age-related loss of dystrophin protein that was greatest in the flexor muscles. The loss of dystrophin protein occurred despite a twofold increase in dystrophin mRNA. Importantly, this disparity could be explained by the four- to fivefold upregulation of the dystromir miR-31. To compensate for the loss of dystrophin protein, aged muscle contained increased α-sarcoglycan, syntrophin, sarcospan, laminin, β1-integrin, desmuslin, and the Z-line proteins α-actinin and desmin. In spite of the adaptive increase in other force transfer proteins, over the 48 hours following lengthening contractions, the old muscles showed more signs of impaired membrane integrity (fourfold increase in immunoglobulin G-positive fibers and 70% greater dysferlin mRNA) and NMJ instability (14- to 96-fold increases in Runx1, AchRδ, and myogenin mRNA). Overall, these data suggest that age-dependent alterations in dystrophin leave the muscle membrane and NMJ more susceptible to contraction-induced damage even before changes in muscle mass are obvious. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Neutrophil-derived alpha defensins control inflammation by inhibiting macrophage mRNA translation

    PubMed Central

    Tomlinson, Gareth H.; Miles, Katherine; Smith, Richard W. P.; Rossi, Adriano G.; Hiemstra, Pieter S.; van ’t Wout, Emily F. A.; Dean, Jonathan L. E.; Gray, Nicola K.; Lu, Wuyuan; Gray, Mohini

    2016-01-01

    Neutrophils are the first and most numerous cells to arrive at the site of an inflammatory insult and are among the first to die. We previously reported that alpha defensins, released from apoptotic human neutrophils, augmented the antimicrobial capacity of macrophages while also inhibiting the biosynthesis of proinflammatory cytokines. In vivo, alpha defensin administration protected mice from inflammation, induced by thioglychollate-induced peritonitis or following infection with Salmonella enterica serovar Typhimurium. We have now dissected the antiinflammatory mechanism of action of the most abundant neutrophil alpha defensin, Human Neutrophil Peptide 1 (HNP1). Herein we show that HNP1 enters macrophages and inhibits protein translation without inducing the unfolded-protein response or affecting mRNA stability. In a cell-free in vitro translation system, HNP1 powerfully inhibited both cap-dependent and cap-independent mRNA translation while maintaining mRNA polysomal association. This is, to our knowledge, the first demonstration of a peptide released from one cell type (neutrophils) directly regulating mRNA translation in another (macrophages). By preventing protein translation, HNP1 functions as a “molecular brake” on macrophage-driven inflammation, ensuring both pathogen clearance and the resolution of inflammation with minimal bystander tissue damage. PMID:27044108

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

    PubMed

    Souza, K de Picoli; Nunes, M T

    2014-08-01

    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.

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

    PubMed Central

    de Picoli Souza, K.; Nunes, M.T.

    2014-01-01

    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. PMID:25098716

  6. Identification of Suitable Reference Genes for mRNA Studies in Bone Marrow in a Mouse Model of Hematopoietic Stem Cell Transplantation.

    PubMed

    Li, H; Chen, C; Yao, H; Li, X; Yang, N; Qiao, J; Xu, K; Zeng, L

    2016-10-01

    Bone marrow micro-environment changes during hematopoietic stem cell transplantation (HSCT) with subsequent alteration of genes expression. Quantitative polymerase chain reaction (q-PCR) is a reliable and reproducible technique for the analysis of gene expression. To obtain more accurate results, it is essential to find a reference during HSCT. However, which gene is suitable during HSCT remains unclear. This study aimed to identify suitable reference genes for mRNA studies in bone marrow after HSCT. C57BL/6 mice were treated with either total body irradiation (group T) or busulfan/cyclophosphamide (BU/CY) (group B) followed by infusion of bone marrow cells. Normal mice without treatments were served as a control. All samples (group T + group B + control) were defined as group G. On days 7, 14, and 21 after transplantation, transcription levels of 7 candidate genes, ACTB, B2M, GAPDH, HMBS, HPRT, SDHA, and YWHAZ, in bone marrow cells were measured by use of real-time quantitative PCR. The expression stability of these 7 candidate reference genes were analyzed by 2 statistical software programs, GeNorm and NormFinder. Our results showed that ACTB displayed the highest expression in group G, with lowest expression of PSDHA in group T and HPRT in groups B and G. Analysis of expression stability by use of GeNorm or NormFinder demonstrated that expression of B2M in bone marrow were much more stable during HSCT, compared with other candidate genes including commonly used reference genes GAPDH and ACTB. ACTB could be used as a suitable reference gene for mRNA studies in bone marrow after HSCT. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Common pathological mutations in PQBP1 induce nonsense-mediated mRNA decay and enhance exclusion of the mutant exon.

    PubMed

    Musante, Luciana; Kunde, Stella-Amrei; Sulistio, Tina O; Fischer, Ute; Grimme, Astrid; Frints, Suzanna G M; Schwartz, Charles E; Martínez, Francisco; Romano, Corrado; Ropers, Hans-Hilger; Kalscheuer, Vera M

    2010-01-01

    The polyglutamine binding protein 1 (PQBP1) gene plays an important role in X-linked mental retardation (XLMR). Nine of the thirteen PQBP1 mutations known to date affect the AG hexamer in exon 4 and cause frameshifts introducing premature termination codons (PTCs). However, the phenotype in this group of patients is variable. To investigate the pathology of these PQBP1 mutations, we evaluated their consequences on mRNA and protein expression. RT-PCRs revealed mutation-specific reduction of PQBP1 mRNAs carrying the PTCs that can be partially restored by blocking translation, thus indicating a role for the nonsense-mediated mRNA decay pathway. In addition, these mutations resulted in altered levels of PQBP1 transcripts that skipped exon 4, probably as a result of altering important splicing motifs via nonsense-associated altered splicing (NAS). This hypothesis is supported by transfection experiments using wild-type and mutant PQBP1 minigenes. Moreover, we show that a truncated PQBP1 protein is indeed present in the patients. Remarkably, patients with insertion/deletion mutations in the AG hexamer express significantly increased levels of a PQBP1 isoform, which is very likely encoded by the transcripts without exon 4, confirming the findings at the mRNA level. Our study provides significant insight into the early events contributing to the pathogenesis of the PQBP1 related XLMR disease.

  8. Genetic analyses of Per.C6 cell clones producing a therapeutic monoclonal antibody regarding productivity and long-term stability.

    PubMed

    Tsuruta, Lilian Rumi; Lopes Dos Santos, Mariana; Yeda, Fernanda Perez; Okamoto, Oswaldo Keith; Moro, Ana Maria

    2016-12-01

    Genetic characterization of protein-producing clones represents additional value to cell line development. In the present study, ten Per.C6 clones producing a Rebmab100 monoclonal antibody were selected using two cloning methods: six clones originated from limiting dilution cloning and four by the automated colony picker ClonePix FL. A stability program was performed for 50 generations, including 4 batches distributed along the timeframe to determine specific productivity (Qp) maintenance. Four stable clones (two from limiting dilution and two from ClonePix FL) were further evaluated. The relative mRNA expression levels of both heavy chain (HC) and light chain (LC) genes were verified at generations 0, 30-35, and 50-55 of the stability program. At generations 0 and 30-35, LC gene expression level was higher than HC gene, whereas at generation 50-55, the opposite prevailed. A high correlation was observed between Qp and HC or LC mRNA expression level for all clones at each generation analyzed along the continuous culture. The mRNA stability study was performed at steady-state culture. The LC gene displayed a higher half-life and lower decay constant than HC gene, accounting for the higher observed expression level of LC mRNA in comparison to HC mRNA. Clone R6 was highlighted due its high Qp, mRNA expression levels, and mRNA stability. Besides the benefits of applying genetic characterization for the selection of stable and high-producing clones, the present study shows for the first time the correlation between Qp and HC or LC expression levels and also mRNA stability in clones derived from human cell line Per.C6(®).

  9. Comparison of Glomerular and Podocyte mRNA Profiles in Streptozotocin-Induced Diabetes

    PubMed Central

    Fu, Jia; Wei, Chengguo; Lee, Kyung; Zhang, Weijia; He, Wu; Chuang, Peter

    2016-01-01

    Evaluating the mRNA profile of podocytes in the diabetic kidney may indicate genes involved in the pathogenesis of diabetic nephropathy. To determine if the podocyte-specific gene information contained in mRNA profiles of the whole glomerulus of the diabetic kidney accurately reflects gene expression in the isolated podocytes, we crossed Nos3−/− IRG mice with podocin-rtTA and TetON-Cre mice for enhanced green fluorescent protein labeling of podocytes before diabetic injury. Diabetes was induced by streptozotocin, and mRNA profiles of isolated glomeruli and sorted podocytes from diabetic and control mice were examined 10 weeks later. Expression of podocyte-specific markers in glomeruli was downregulated in diabetic mice compared with controls. However, expression of these markers was not altered in sorted podocytes from diabetic mice. When mRNA levels of glomeruli were corrected for podocyte number per glomerulus, the differences in podocyte marker expression disappeared. Analysis of the differentially expressed genes in diabetic mice also revealed distinct upregulated pathways in the glomeruli (mitochondrial function, oxidative stress) and in podocytes (actin organization). In conclusion, our data suggest reduced expression of podocyte markers in glomeruli is a secondary effect of reduced podocyte number, thus podocyte-specific gene expression detected in the whole glomerulus may not represent that in podocytes in the diabetic kidney. PMID:26264855

  10. Maternal Nanos-Dependent RNA Stabilization in the Primordial Germ Cells of Drosophila Embryos.

    PubMed

    Sugimori, Seiko; Kumata, Yuji; Kobayashi, Satoru

    2018-01-01

    Nanos (Nos) is an evolutionary conserved protein expressed in the germline of various animal species. In Drosophila, maternal Nos protein is essential for germline development. In the germline progenitors, or the primordial germ cells (PGCs), Nos binds to the 3' UTR of target mRNAs to repress their translation. In contrast to this prevailing role of Nos, here we report that the 3' UTR of CG32425 mRNA mediates Nos-dependent RNA stabilization in PGCs. We found that the level of mRNA expressed from a reporter gene fused to the CG32425 3' UTR was significantly reduced in PGCs lacking maternal Nos (nos PGCs) as compared with normal PGCs. By deleting the CG32425 3' UTR, we identified the region required for mRNA stabilization, which includes Nos-binding sites. In normal embryos, CG32425 mRNA was maternally supplied into PGCs and remained in this cell type during embryogenesis. However, as expected from our reporter assay, the levels of CG32425 mRNA and its protein product expressed in nos PGCs were lower than in normal PGCs. Thus, we propose that Nos protein has dual functions in translational repression and stabilization of specific RNAs to ensure proper germline development. © 2017 Japanese Society of Developmental Biologists.

  11. Gnrh mRNA expression in the brain of cooperatively breeding female Damaraland mole-rats.

    PubMed

    Voigt, Cornelia; Bennett, Nigel C

    2017-04-01

    The Damaraland mole-rat ( Fukomys damarensis ) is a eusocial, subterranean rodent, in which breeding is limited to a single reproductive pair within each colony. Non-reproductive females, while in the confines of the colony, exhibit socially induced infertility. Anovulation is thought to be caused by a disruption in the normal gonadotropin-releasing hormone (GNRH) secretion from the hypothalamus. To assess whether social suppression is associated with altered Gnrh mRNA expression in the brain, we investigated the distribution and gene expression levels by means of in situ hybridization in female breeders and non-breeders from field captured colonies of the Damaraland mole-rat. We found expression of Gnrh mRNA as a loose network in several forebrain areas of female Damaraland mole-rats with the majority of labelling in the preoptic and anterior hypothalamus. The distribution matched previous findings using immunocytochemistry in this and other social mole-rat species. Quantification of the hybridisation signal revealed no difference between breeding and non-breeding females in the average optical density of the hybridization signal and the size of the total area covered by Gnrh mRNA. However, analysis along the rostro-caudal axis revealed significantly elevated Gnrh mRNA expression in the rostral preoptic region of breeders compared to non-breeders, whereas the latter had increased Gnrh mRNA expression at the caudal level of the anterior hypothalamus. This study indicates that social suppression affects the expression of Gnrh mRNA in female Damaraland mole-rats. Furthermore, differential regulation occurs within different neuron subpopulations. © 2017 Society for Reproduction and Fertility.

  12. Photoelectrochemical Stability and Alteration Products of n-Type Single-Crystal ZnO Photoanodes

    DOE PAGES

    Paulauskas, I. E.; Jellison, G. E.; Boatner, L. A.; ...

    2011-01-01

    The photoelectrochemical stability and surface-alteration characteristics of doped and undoped n-type ZnO single-crystal photoanode electrodes were investigated. The single-crystal ZnO photoanode properties were analyzed using current-voltage measurements plus spectral and time-dependent quantum-yield methods. These measurements revealed a distinct anodic peak and an accompanying cathodic surface degradation process at negative potentials. The features of this peak depended on time and the NaOH concentration in the electrolyte, but were independent of the presence of electrode illumination. Current measurements performed at the peak indicate that charging and discharging effects are apparently taking place at the semiconductor/electrolyte interface. This result is consistent with themore » significant reactive degradation that takes place on the ZnO single crystal photoanode surface and that ultimately leads to the reduction of the ZnO surface to Zn metal. The resulting Zn-metal reaction products create unusual, dendrite-like, surface alteration structural features that were analyzed using x-ray diffraction, energy-dispersive analysis, and scanning electron microscopy. ZnO doping methods were found to be effective in increasing the n-type character of the crystals. Higher doping levels result in smaller depletion widths and lower quantum yields, since the minority carrier diffusion lengths are very short in these materials.« less

  13. Up-regulated EMMPRIN/CD147 protein expression might play a role in colorectal carcinogenesis and its subsequent progression without an alteration of its glycosylation and mRNA level.

    PubMed

    Zheng, Hua-chuan; Wang, Wei; Xu, Xiao-yan; Xia, Pu; Yu, Miao; Sugiyama, Toshiro; Takano, Yasuo

    2011-04-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN) was reported to involve in the invasion and metastasis of malignancies by regulating the expression of vascular endothelial growth factor (VEGF) in stromal and cancer cells. The study aimed to clarify the role of EMMPRIN expression in tumorigenesis and progression of colorectal carcinomas (CRC). EMMPRIN expression was examined on tissue microarray containing colorectal carcinomas, adenoma and non-neoplastic mucosa (NNM) by immunohistochemistry and in situ hybridization (ISH). Colorectal carcinoma cell lines (DLD-1, HCT-15, SW480 and WiDr) and tissues were studied for EMMPRIN expression by Western blot or RT-PCR, followed by sequencing. All carcinoma cell lines showed EMMPRIN expression at both mRNA and protein levels. Two synonymous mutations were found in carcinoma cell lines at codon109 (GCT → GCC: Ala) or 179 (GAT → GAC: Asp). Frozen CRC tissues displayed higher EMMPRIN expression than paired NNM (P < 0.05). EMMPRIN expression was immunohistochemically stronger in colorectal high-grade adenoma, adenocarcinoma and metastatic carcinoma than non-neoplastic superficial epithelium and low-grade adenoma (P < 0.05). In contrast, its mRNA level was similar from colorectal NNM, adenoma to adenocarcinoma by ISH, in line with the findings of RT-PCR (P > 0.05). Immunohistochemically, EMMPRIN expression was positively correlated with tumor size, depth of invasion, vascular or lymphatic invasion, grade of infiltration (INF), ki-67 and VEGF expression of CRCs (P < 0.05). Among them, depth of invasion was an independent associated factor for EMMPRIN expression in CRCs (P < 0.05). Up-regulated EMMPRIN protein expression might contribute to colorectal carcinogenesis without the alteration of its glycosylation and mRNA level. Aberrant EMMPRIN protein expression might promote growth or invasion of CRCs possibly through increased ki-67 expression and inducible angiogenesis via up-regulating VEGF expression.

  14. Limb segment vibration modulates spinal reflex excitability and muscle mRNA expression after spinal cord injury

    PubMed Central

    Chang, Shuo-Hsiu; Tseng, Shih-Chiao; McHenry, Colleen L.; Littmann, Andrew E.; Suneja, Manish; Shields, Richard K.

    2012-01-01

    Objective We investigated the effect of various doses of vertical oscillation (vibration) on soleus H-reflex amplitude and post-activation depression in individuals with and without SCI. We also explored the acute effect of short-term limb vibration on skeletal muscle mRNA expression of genes associated with spinal plasticity. Methods Six healthy adults and five chronic complete SCI subjects received vibratory stimulation of their tibia over three different gravitational accelerations (0.3g, 0.6g, and 1.2g) at a fixed frequency (30 Hz). Soleus H-reflexes were measured before, during, and after vibration. Two additional chronic complete SCI subjects had soleus muscle biopsies 3 h following a single bout of vibration. Results H-reflex amplitude was depressed over 83% in both groups during vibration. This vibratory-induced inhibition lasted over 2 min in the control group, but not in the SCI group. Post-activation depression was modulated during the long-lasting vibratory inhibition. A single bout of mechanical oscillation altered mRNA expression from selected genes associated with synaptic plasticity. Conclusions Vibration of the lower leg inhibits the H-reflex amplitude, influences post-activation depression, and alters skeletal muscle mRNA expression of genes associated with synaptic plasticity. Significance Limb segment vibration may offer a long term method to reduce spinal reflex excitability after SCI. PMID:21963319

  15. mRNA Cancer Vaccines-Messages that Prevail.

    PubMed

    Grunwitz, Christian; Kranz, Lena M

    2017-01-01

    During the last decade, mRNA became increasingly recognized as a versatile tool for the development of new innovative therapeutics. Especially for vaccine development, mRNA is of outstanding interest and numerous clinical trials have been initiated. Strikingly, all of these studies have proven that large-scale GMP production of mRNA is feasible and concordantly report a favorable safety profile of mRNA vaccines. Induction of T-cell immunity is a multi-faceted process comprising antigen acquisition, antigen processing and presentation, as well as immune stimulation. The effectiveness of mRNA vaccines is critically dependent on making the antigen(s) of interest available to professional antigen-presenting cells, especially DCs. Efficient delivery of mRNA into DCs in vivo remains a major challenge in the mRNA vaccine field. This review summarizes the principles of mRNA vaccines and highlights the importance of in vivo mRNA delivery and recent advances in harnessing their therapeutic potential.

  16. Principles of mRNA transport in yeast.

    PubMed

    Heym, Roland Gerhard; Niessing, Dierk

    2012-06-01

    mRNA localization and localized translation is a common mechanism by which cellular asymmetry is achieved. In higher eukaryotes the mRNA transport machinery is required for such diverse processes as stem cell division and neuronal plasticity. Because mRNA localization in metazoans is highly complex, studies at the molecular level have proven to be cumbersome. However, active mRNA transport has also been reported in fungi including Saccharomyces cerevisiae, Ustilago maydis and Candida albicans, in which these events are less difficult to study. Amongst them, budding yeast S. cerevisiae has yielded mechanistic insights that exceed our understanding of other mRNA localization events to date. In contrast to most reviews, we refrain here from summarizing mRNA localization events from different organisms. Instead we give an in-depth account of ASH1 mRNA localization in budding yeast. This approach is particularly suited to providing a more holistic view of the interconnection between the individual steps of mRNA localization, from transcriptional events to cytoplasmic mRNA transport and localized translation. Because of our advanced mechanistic understanding of mRNA localization in yeast, the present review may also be informative for scientists working, for example, on mRNA localization in embryogenesis or in neurons.

  17. In What Ways Do Synthetic Nucleotides and Natural Base Lesions Alter the Structural Stability of G-Quadruplex Nucleic Acids?

    PubMed Central

    2017-01-01

    Synthetic analogs of natural nucleotides have long been utilized for structural studies of canonical and noncanonical nucleic acids, including the extensively investigated polymorphic G-quadruplexes (GQs). Dependence on the sequence and nucleotide modifications of the folding landscape of GQs has been reviewed by several recent studies. Here, an overview is compiled on the thermodynamic stability of the modified GQ folds and on how the stereochemical preferences of more than 70 synthetic and natural derivatives of nucleotides substituting for natural ones determine the stability as well as the conformation. Groups of nucleotide analogs only stabilize or only destabilize the GQ, while the majority of analogs alter the GQ stability in both ways. This depends on the preferred syn or anti N-glycosidic linkage of the modified building blocks, the position of substitution, and the folding architecture of the native GQ. Natural base lesions and epigenetic modifications of GQs explored so far also stabilize or destabilize the GQ assemblies. Learning the effect of synthetic nucleotide analogs on the stability of GQs can assist in engineering a required stable GQ topology, and exploring the in vitro action of the single and clustered natural base damage on GQ architectures may provide indications for the cellular events. PMID:29181193

  18. Resource-driven changes to host population stability alter the evolution of virulence and transmission.

    PubMed

    Hite, Jessica L; Cressler, Clayton E

    2018-05-05

    What drives the evolution of parasite life-history traits? Recent studies suggest that linking within- and between-host processes can provide key insight into both disease dynamics and parasite evolution. Still, it remains difficult to understand how to pinpoint the critical factors connecting these cross-scale feedbacks, particularly under non-equilibrium conditions; many natural host populations inherently fluctuate and parasites themselves can strongly alter the stability of host populations. Here, we develop a general model framework that mechanistically links resources to parasite evolution across a gradient of stable and unstable conditions. First, we dynamically link resources and between-host processes (host density, stability, transmission) to virulence evolution, using a 'non-nested' model. Then, we consider a 'nested' model where population-level processes (transmission and virulence) depend on resource-driven changes to individual-level (within-host) processes (energetics, immune function, parasite production). Contrary to 'non-nested' model predictions, the 'nested' model reveals complex effects of host population dynamics on parasite evolution, including regions of evolutionary bistability; evolution can push parasites towards strongly or weakly stabilizing strategies. This bistability results from dynamic feedbacks between resource-driven changes to host density, host immune function and parasite production. Together, these results highlight how cross-scale feedbacks can provide key insights into the structuring role of parasites and parasite evolution.This article is part of the theme issue 'Anthropogenic resource subsidies and host-parasite dynamics in wildlife'. © 2018 The Author(s).

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

    NASA Astrophysics Data System (ADS)

    Gu, Wei

    1990-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  3. Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis1[OPEN

    PubMed Central

    Xiao, Chaowen; Zhang, Tian; Zheng, Yunzhen

    2016-01-01

    Xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis (Arabidopsis thaliana) xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall-integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolated xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes, such as MAP70-5 and CLASP, and receptor genes, such as HERK1 and WAK1, were changed in xxt1 xxt2 plants and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish, to our knowledge, new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis. PMID:26527657

  4. Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA*

    PubMed Central

    Woo, Ho-Hyung; Baker, Terri; Laszlo, Csaba; Chambers, Setsuko K.

    2013-01-01

    CSF-1 mRNA 3′UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3′UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3′UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of

  5. Mutations That Affect the Efficiency of Translation of mRNA for the cII Gene of Coliphage Lambda

    PubMed Central

    Dul, Ed; Mahoney, Michael E.; Wulff, Daniel L.

    1987-01-01

    Starting with the λ pRE- strain λctr1 cy3008, which forms clear plaques, we have isolated two mutant strains, λdya2 ctr1 cy3008 and λ dya3 ctr1 cy3008, that form plaques with very slightly turbid centers. The dya2 and dya3 mutations lie in the region of overlap between the PRE promoter and the ribosome recognition region of the cII gene, and have nucleotide alterations at positions -1 and +5 of pRE, and alterations of cII mRNA at -16 and -21 nucleotides before the initial AUG codon of the gene. Both mutations destabilize a stem structure that may be formed by cII mRNA, and dya2 also changes the sequence on cII mRNA that is complementary to the 3'-end of 16 S rRNA from 5'-UAAGGA-3' to 5'-UGAGGA-3'.—The dya2 and dya3 mutations, along with the ctr1 mutation, which destabilizes either of two alternate stem structures which may be formed by cII mRNA (these being more stable stem structures than the one affected by dya2 and dya3), were tested for their ability to reverse two cII- mutations that are characterized by inefficient translation of cII mRNA. These are cII3088, an A → G mutation four bases before the initial AUG codon, and cII3059 , a GUU → GAU (Val2 → Asp) second codon mutation. It was found that ctr1 completely reverses the translation defects of these two mutations, while dya2 partially reverses these translation defects. The dya3 mutation has no effect on translation efficiency under any condition tested. However neither the ctr1 mutation nor the dya2 mutation has much effect on translation efficiency in an otherwise cII+ background, indicating that other factors must limit the rate of translation of cII mRNA under these conditions. PMID:2953647

  6. mTOR referees memory and disease through mRNA repression and competition.

    PubMed

    Raab-Graham, Kimberly F; Niere, Farr

    2017-06-01

    Mammalian target of rapamycin (mTOR) activity is required for memory and is dysregulated in disease. Activation of mTOR promotes protein synthesis; however, new studies are demonstrating that mTOR activity also represses the translation of mRNAs. Almost three decades ago, Kandel and colleagues hypothesised that memory was due to the induction of positive regulators and removal of negative constraints. Are these negative constraints repressed mRNAs that code for proteins that block memory formation? Herein, we will discuss the mRNAs coded by putative memory suppressors, how activation/inactivation of mTOR repress protein expression at the synapse, how mTOR activity regulates RNA binding proteins, mRNA stability, and translation, and what the possible implications of mRNA repression are to memory and neurodegenerative disorders. © 2017 Federation of European Biochemical Societies.

  7. Multiple lipopolysaccharide (LPS) injections alter interleukin 6 (IL-6), IL-7, IL-10 and IL-6 and IL-7 receptor mRNA in CNS and spleen.

    PubMed

    Szot, Patricia; Franklin, Allyn; Figlewicz, Dianne P; Beuca, Timothy Petru; Bullock, Kristin; Hansen, Kim; Banks, William A; Raskind, Murray A; Peskind, Elaine R

    2017-07-04

    Neuroinflammation is proposed to be an important component in the development of several central nervous system (CNS) disorders including depression, Alzheimer's disease, Parkinson's disease, and traumatic brain injury. However, exactly how neuroinflammation leads to, or contributes to, these central disorders is unclear. The objective of the study was to examine and compare the expression of mRNAs for interleukin-6 (IL-6), IL-7, IL-10 and the receptors for IL-6 (IL-6R) and IL-7 (IL-7R) using in situ hybridization in discrete brain regions and in the spleen after multiple injections of 3mg/kg lipopolysaccharide (LPS), a model of neuroinflammation. In the spleen, LPS significantly elevated IL-6 mRNA expression, then IL-10 mRNA, with no effect on IL-7 or IL-7R mRNA, while significantly decreasing IL-6R mRNA expression. In the CNS, LPS administration had the greatest effect on IL-6 and IL-6R mRNA. LPS increased IL-6 mRNA expression only in non-neuronal cells throughout the brain, but significantly elevated IL-6R mRNA in neuronal populations, where observed, except the cerebellum. LPS resulted in variable effects on IL-10 mRNA, and had no effect on IL-7 or IL-7R mRNA expression. These studies indicate that LPS-induced neuroinflammation has substantial but variable effects on the regional and cellular patterns of CNS IL-6, IL-7 and IL-10, and for IL-6R and IL-7R mRNA expression. It is apparent that administration of LPS can affect non-neuronal and neuronal cells in the brain. Further research is required to determine how CNS inflammatory changes associated with IL-6, IL-10 and IL-6R could in turn contribute to the development of CNS neurological disorders. Published by Elsevier Ltd.

  8. Mesenchymal stem cells cannot affect mRNA expression of toll-like receptors in different tissues during sepsis.

    PubMed

    Pedrazza, Leonardo; Pereira, Talita Carneiro Brandão; Abujamra, Ana Lucia; Nunes, Fernanda Bordignon; Bogo, Maurício Reis; de Oliveira, Jarbas Rodrigues

    2017-07-01

    Experimental animal models and human clinical studies support a crucial role for TLRs in infectious diseases. The aim of this study was to test the ability of MSCs, which have immunomodulatory effects, of altering the mRNA expression of toll-like receptors during a experimental model of sepsis in different tissues. Three experimental groups (male C57BL/6 mice) were formed for the test: control group, untreated septic group and septic group treated with MSCs (1 × 10 6 cells/animal). Lungs, cortex, kidney, liver and colon tissue were dissected after 12 h of sepsis induction and TLR2/3/4/9 mRNA were evaluated by RT-qPCR. We observed a decrease of TLR2 and 9 mRNA expression in the liver of the sepsis group, while TLR3 was decreased in the lung and liver. No change was found between the sepsis group and the sepsis + MSC group. In this model of experimental sepsis the MSCs were unable to modify the mRNA expression of the different toll-like receptors evaluated.

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

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

    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 eukaryoticmore » 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?« less

  10. Enhanced levels of scrapie responsive gene mRNA in BSE-infected mouse brain.

    PubMed

    Dandoy-Dron, F; Benboudjema, L; Guillo, F; Jaegly, A; Jasmin, C; Dormont, D; Tovey, M G; Dron, M

    2000-03-10

    The expression of the mRNA of nine scrapie responsive genes was analyzed in the brains of FVB/N mice infected with bovine spongiform encephalopathy (BSE). The RNA transcripts of eight genes were overexpressed to a comparable extent in both BSE-infected and scrapie-infected mice, indicating a common series of pathogenic events in the two transmissible spongiform encephalopathies (TSEs). In contrast, the serine proteinase inhibitor spi 2, an analogue of the human alpha-1 antichymotrypsin gene, was overexpressed to a greater extent in the brains of scrapie-infected animals than in animals infected with BSE, reflecting either an agent specific or a mouse strain specific response. The levels of spi 2 mRNA were increased during the course of scrapie prior to the onset of clinical signs of the disease and the increase reached 11 to 45 fold relative to uninfected controls in terminally ill mice. Spi 2, in common with four of the other scrapie responsive genes studied, is known to be associated with pro-inflammatory processes. These observations underline the importance of cell reactivity in TSE. In addition, scrg2 mRNA the level of which is enhanced in TSE-infected mouse brain, was identified as a previously unrecognized long transcript of the murine aldolase C gene. However, the level of the principal aldolase C mRNA is unaffected in TSE. The increased representation of the longer transcript in the late stage of the disease may reflect changes in mRNA processing and/or stability in reactive astrocytes or in damaged Purkinje cells.

  11. Sodium lauryl sulphate alters the mRNA expression of lipid-metabolizing enzymes and PPAR signalling in normal human skin in vivo.

    PubMed

    Törmä, Hans; Berne, Berit

    2009-12-01

    Detergents irritate skin and affect skin barrier homeostasis. In this study, healthy skin was exposed to 1% sodium lauryl sulphate (SLS) in water for 24 h. Biopsies were taken 6 h to 8 days post exposure. Lipid patterns were stained in situ and real-time polymerase chain reaction (PCR) was used to examine mRNA expression of enzymes synthesizing barrier lipids, peroxisome proliferator-activated receptors (PPAR) and lipoxygenases. The lipid pattern was disorganized from 6 h to 3 days after SLS exposure. Concomitant changes in mRNA expression included: (i) reduction, followed by induction, of ceramide-generating beta-glucocerebrosidase, (ii) increase on day 1 of two other enzymes for ceramide biosynthesis and (iii) persistent reduction of acetyl-CoA carboxylase-B, a key enzyme in fatty acid synthesis. Surprisingly, the rate-limiting enzyme in cholesterol synthesis, HMG-CoA reductase, was unaltered. Among putative regulators of barrier lipids synthesis, PPARalpha and PPARgamma exhibited reduced mRNA expression, while PPARbeta/delta and LXRbeta were unaltered. Epidermal lipoxygenase-3, which may generate PPARalpha agonists, exhibited reduced expression. In conclusion, SLS induces reorganization of lipids in the stratum corneum, which play a role in detergents' destruction of the barrier. The changes in mRNA expression of enzymes involved in synthesizing barrier lipids are probably important for the restoration of the barrier.

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

    PubMed Central

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

    2014-01-01

    Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs1, enhances the function of transfer RNA and ribosomal RNA by stabilizing RNA structure2–8. mRNAs 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 center9,10. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological was unclear. Here we present a comprehensive analysis of pseudouridylation in yeast 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 100 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 disease11–13. PMID:25192136

  13. Four weeks one-leg training and high fat diet does not alter PPARalpha protein or mRNA expression in human skeletal muscle.

    PubMed

    Helge, J W; Bentley, D; Schjerling, P; Willer, M; Gibala, M J; Franch, J; Tapia-Laliena, M A; Daugaard, J R; Andersen, J L

    2007-09-01

    Fatty acid metabolism is influenced by training and diet with exercise training mediating this through activation of nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in skeletal muscle. This study investigated the effect of training and high fat or normal diet on PPARalpha expression in human skeletal muscle. Thirteen men trained one leg (T) four weeks (31.5 h in total), while the other leg (UT) served as control. During the 4 weeks six subjects consumed high fat (FAT) diet and seven subjects maintained a normal (CHO) diet. Biopsies were obtained from vastus lateralis muscle in both legs before and after training. After the biopsy, one-leg extension exercise was performed in random order with both legs 30 min at 95% of workload max. A training effect was evident as citrate synthase activity increased (P < 0.05) by 15% in the trained, but not the control leg in both groups. During exercise respiratory exchange ratio was lower in FAT (0.86 +/- 0.01, 0.83 +/- 0.01, mean +/- SEM) than CHO (0.96 +/- 0.02, 0.94 +/- 0.03) and in UT than T legs, respectively. The PPARalpha protein (144 +/- 44, 104 +/- 28, 79 +/- 15, 79 +/- 14, % of pre level) and PPARalpha mRNA (69 +/- [2, 2], 78 +/- [7, 6], 92 +/- [22, 18], 106 +/- [21, 18], % of pre level, geometric mean +/- SEM) expression remained unchanged by diet and training in FAT (UT, T) and CHO (UT, T), respectively. After the training and diet CS, HAD, PPARalpha, UCP2, UCP3 and mFABP mRNA content remained unchanged, whereas GLUT4 mRNA was lower in both groups and LDHA mRNA was lower (P < 0.05) only in FAT. 4 weeks one leg knee extensor training did not affect PPARalpha protein or mRNA expression. Furthermore, higher fat oxidation during exercise after fat rich diet was not accompanied by an increased PPARalpha protein or mRNA expression after 4 weeks.

  14. Translational efficiency of poliovirus mRNA: mapping inhibitory cis-acting elements within the 5' noncoding region.

    PubMed Central

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

    1988-01-01

    Poliovirus mRNA contains a long 5' noncoding region of about 750 nucleotides (the exact number varies among the three virus serotypes), which contains several AUG codons upstream of the major initiator AUG. Unlike most eucaryotic mRNAs, poliovirus does not contain a m7GpppX (where X is any nucleotide) cap structure at its 5' end and is translated by a cap-independent mechanism. To study the manner by which poliovirus mRNA is expressed, we examined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. In this paper we report striking translation system-specific differences in the ability of the altered mRNAs to be translated. The results suggest the existence of an inhibitory cis-acting element(s) within the 5' noncoding region of poliovirus (between nucleotides 70 and 381) which restricts mRNA translation in reticulocyte lysate, wheat germ extract, and Xenopus oocytes, but not in HeLa cell extracts. In addition, we show that HeLa cell extracts contain a trans-acting factor(s) that overcomes this restriction. Images PMID:2836606

  15. Epithelial response to high-grain diets involves alteration in nutrient transporters and Na+/K+-ATPase mRNA expression in rumen and colon of goats.

    PubMed

    Metzler-Zebeli, B U; Hollmann, M; Sabitzer, S; Podstatzky-Lichtenstein, L; Klein, D; Zebeli, Q

    2013-09-01

    Emerging evidence at the mRNA level indicates that feeding high-grain diets to ruminants leads to coordinated changes in the molecular response of the rumen epithelium. Yet, epithelial adaptation of the hindgut to increasing dietary grain levels has not been established in ruminants. Therefore, the objective of this study was to characterize alterations in mRNA expression associated with nutrient transport and electrochemical gradients in rumen and colon epithelium, and rumen morphology in growing goats fed different grain levels. Goats (n = 6) were fed diets with increasing levels of 0, 30, or 60% barley grain for 6 wk. Goats were euthanized 2 h after their last feeding, and digesta and tissue samples of the cranial part of the ventral rumen and proximal colon were collected. Goats fed the 60% grain diet exhibited a lower ruminal and colonic pH (P < 0.01) and a greater colonic total VFA concentration (P < 0.05) compared with those fed the 0 and 30% grain diets. As response to the decreased ruminal pH, goats fed the 60% grain diet had a greater (P < 0.05) keratinization and thicker stratum corneum of the rumen epithelium than goats fed the 0 and 30% grain diets. The 60% grain diet upregulated (P < 0.05) MCT1 expression by 45% and downregulated (P < 0.05) the expression of MCT4 and SGLT1 by 28 and 50%, respectively, in rumen epithelium compared with the 0 and 30% grain diets. Accordingly, goats fed the 60% grain diet had a greater (P < 0.05) expression of MCT1 and ATP1A1 in colon epithelium than goats fed the 0 and 30% grain diets. Regression analyses showed negative relationships (R(2) = 0.35 to 0.87, P < 0.05) of MCT1 and ATP1A1 expression in rumen and colon epithelium and thickening of ruminal stratum corneum to decreasing luminal pH values, suggesting greater mRNA expression at lower pH. In contrast, MCT4 expression in rumen epithelium positively correlated to luminal pH (R(2) = 0.95, P < 0.01). In conclusion, results of this model study indicated that with the

  16. Interference of a speB 5' untranslated region partial deletion with mRNA degradation in Streptococcus pyogenes.

    PubMed

    Chen, Z; Mashburn-Warren, L; Merritt, J; Federle, M J; Kreth, J

    2017-10-01

    The 5' untranslated region (5' UTR) of an mRNA molecule embeds important determinants that modify its stability and translation efficiency. In Streptococcus pyogenes, a strict human pathogen, a gene encoding a secreted protease (speB) has a large 5' UTR with unknown functions. Here we describe that a partial deletion of the speB 5' UTR caused a general accumulation of mRNA in the stationary phase, and that the mRNA accumulation was due to retarded mRNA degradation. The phenotype was observed in several M serotypes harboring the partial deletion of the speB 5' UTR. The phenotype was triggered by the production of the truncated speB 5' UTR, but not by the disruption of the intact speB 5' UTR. RNase Y, a major endoribonuclease, was previously shown to play a central role in bulk mRNA turnover in stationary phase. However, in contrast to our expectations, we observed a weaker interaction between the truncated speB 5' UTR and RNase Y compared with the wild-type, which suggests that other unidentified RNA degrading components are required for the pleiotropic effects observed from the speB UTR truncation. Our study demonstrates how S. pyogenes uses distinct mRNA degradation schemes in exponential and stationary growth phases. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. The Structure and Function of the Rous Sarcoma virus RNA Stability Element

    PubMed Central

    Withers, Johanna B.; Beemon, Karen L.

    2013-01-01

    For simple retroviruses, such as the Rous sarcoma virus (RSV), post-transcriptional control elements regulate viral RNA splicing, export, stability, and packaging into virions. These RNA sequences interact with cellular host proteins to regulate and facilitate productive viral infections. One such element, known as the RSV stability element (RSE), is required for maintaining stability of the full-length unspliced RNA. This viral RNA serves as the mRNA for the Gag and Pol proteins and also as the genome packaged in progeny virions. When the RSE is deleted from the viral RNA, the unspliced RNA becomes unstable and is degraded in a Upf1-dependent manner. Current evidence suggests that the RSE inhibits recognition of the viral gag termination codon by the nonsense-mediated mRNA decay (NMD) pathway. We believe that the RSE acts as an insulator to NMD, thereby preventing at least one of the required functional steps that target an mRNA for degradation. Here, we discuss the history of the RSE and the current model of how the RSE is interacting with cellular NMD factors. PMID:21769913

  18. Coupled Evolution of Transcription and mRNA Degradation

    PubMed Central

    Dori-Bachash, Mally; Shema, Efrat; Tirosh, Itay

    2011-01-01

    mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor binding motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast. PMID:21811398

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

    PubMed

    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.

  20. hnRNP-Q1 represses nascent axon growth in cortical neurons by inhibiting Gap-43 mRNA translation

    PubMed Central

    Williams, Kathryn R.; McAninch, Damian S.; Stefanovic, Snezana; Xing, Lei; Allen, Megan; Li, Wenqi; Feng, Yue; Mihailescu, Mihaela Rita; Bassell, Gary J.

    2016-01-01

    Posttranscriptional regulation of gene expression by mRNA-binding proteins is critical for neuronal development and function. hnRNP-Q1 is an mRNA-binding protein that regulates mRNA processing events, including translational repression. hnRNP-Q1 is highly expressed in brain tissue, suggesting a function in regulating genes critical for neuronal development. In this study, we have identified Growth-associated protein 43 (Gap-43) mRNA as a novel target of hnRNP-Q1 and have demonstrated that hnRNP-Q1 represses Gap-43 mRNA translation and consequently GAP-43 function. GAP-43 is a neuronal protein that regulates actin dynamics in growth cones and facilitates axonal growth. Previous studies have identified factors that regulate Gap-43 mRNA stability and localization, but it remains unclear whether Gap-43 mRNA translation is also regulated. Our results reveal that hnRNP-Q1 knockdown increased nascent axon length, total neurite length, and neurite number in mouse embryonic cortical neurons and enhanced Neuro2a cell process extension; these phenotypes were rescued by GAP-43 knockdown. Additionally, we have identified a G-quadruplex structure in the 5′ untranslated region of Gap-43 mRNA that directly interacts with hnRNP-Q1 as a means to inhibit Gap-43 mRNA translation. Therefore hnRNP-Q1–mediated repression of Gap-43 mRNA translation provides an additional mechanism for regulating GAP-43 expression and function and may be critical for neuronal development. PMID:26658614

  1. Icaritin Inhibits Collagen Degradation-Related Factors and Facilitates Collagen Accumulation in Atherosclerotic Lesions: A Potential Action for Plaque Stabilization

    PubMed Central

    Zhang, Zong-Kang; Li, Jie; Yan, De-Xin; Leung, Wing-Nang; Zhang, Bao-Ting

    2016-01-01

    Most acute coronary syndromes result from rupture of vulnerable atherosclerotic plaques. The collagen content of plaques may critically affect plaque stability. This study tested whether Icaritin (ICT), an intestinal metabolite of Epimedium-derived flavonoids, could alter the collagen synthesis/degradation balance in atherosclerotic lesions. Rabbits were fed with an atherogenic diet for four months. Oral administration of ICT (10 mg·kg−1·day−1) was started after two months of an atherogenic diet and lasted for two months. The collagen degradation-related parameters, including macrophages accumulation, content and activity of interstitial collagenase-1 (MMP-1), and the collagen synthesis-related parameters, including amount and distribution of smooth muscle cells (SMC) and collagen mRNA/protein levels, were evaluated in the aorta. ICT reduced plasma lipid levels, inhibited macrophage accumulation, lowered MMP-1 mRNA and protein expression, and suppressed proteolytic activity of pro-MMP-1 and MMP-1 in the aorta. ICT changed the distribution of the SMCs towards the fibrous cap of lesions without increasing the amount of SMCs. Higher collagen protein content in lesions and aorta homogenates was observed with ICT treatment compared with the atherogenic diet only, without altered collagen mRNA level. These results suggest that ICT could inhibit the collagen degradation-related factors and facilitate collagen accumulation in atherosclerotic lesions, indicating a new potential of ICT in atherosclerotic plaques. PMID:26828485

  2. Axonal transport of TDP-43 mRNA granules in neurons is impaired by ALS-causing mutations

    PubMed Central

    Carrasco, Monica A.; Williams, Luis A.; Winborn, Christina S.; Han, Steve S. W.; Kiskinis, Evangelos; Winborn, Brett; Freibaum, Brian D.; Kanagaraj, Anderson; Clare, Alison J.; Badders, Nisha M.; Bilican, Bilada; Chaum, Edward; Chandran, Siddharthan; Shaw, Christopher E.; Eggan, Kevin C.; Maniatis, Tom; Taylor, J. Paul

    2014-01-01

    Summary The RNA binding protein TDP-43 regulates RNA metabolism at multiple levels, including transcription, RNA splicing, and mRNA stability. TDP-43 is a major component of the cytoplasmic inclusions characteristic of amyotrophic lateral sclerosis and some types of frontotemporal lobar degeneration. The importance of TDP-43 in disease is underscored by the fact that dominant missense mutations are sufficient to cause disease, although the role of TDP-43 in pathogenesis is unknown. Here we show that TDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons in vitro and in vivo and facilitate delivery of target mRNA to distal neuronal compartments. TDP-43 mutations impair this mRNA transport function in vivo and in vitro, including in stem cell-derived motor neurons from ALS patients bearing any one of three different TDP-43 ALS-causing mutations. Thus, TDP43 mutations that cause ALS lead to partial loss of a novel cytoplasmic function of TDP-43. PMID:24507191

  3. The Human T-Lymphotropic Virus Type 1 Tax Protein Inhibits Nonsense-Mediated mRNA Decay by Interacting with INT6/EIF3E and UPF1

    PubMed Central

    Mocquet, Vincent; Neusiedler, Julia; Rende, Francesca; Cluet, David; Robin, Jean-Philippe; Terme, Jean-Michel; Duc Dodon, Madeleine; Wittmann, Jürgen; Morris, Christelle; Le Hir, Hervé; Ciminale, Vincenzo

    2012-01-01

    In this report, we analyzed whether the degradation of mRNAs by the nonsense-mediated mRNA decay (NMD) pathway was affected in human T-lymphotropic virus type 1 (HTLV-1)-infected cells. This pathway was indeed strongly inhibited in C91PL, HUT102, and MT2 cells, and such an effect was also observed by the sole expression of the Tax protein in Jurkat and HeLa cells. In line with this activity, Tax binds INT6/EIF3E (here called INT6), which is a subunit of the translation initiation factor eukaryotic initiation factor 3 (eIF3) required for efficient NMD, as well as the NMD core factor upstream frameshift protein 1 (UPF1). It was also observed that Tax expression alters the morphology of processing bodies (P-bodies), the cytoplasmic structures which concentrate RNA degradation factors. The presence of UPF1 in these subcellular compartments was increased by Tax, whereas that of INT6 was decreased. In line with these effects, the level of the phosphorylated form of UPF1 was increased in the presence of Tax. Analysis of several mutants of the viral protein showed that the interaction with INT6 is necessary for NMD inhibition. The alteration of mRNA stability was observed to affect viral transcripts, such as that coding for the HTLV-1 basic leucine zipper factor (HBZ), and also several cellular mRNAs sensitive to the NMD pathway. Our data indicate that the effect of Tax on viral and cellular gene expression is not restricted to transcriptional control but can also involve posttranscriptional regulation. PMID:22553336

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

    PubMed

    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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Prenatal arsenic exposure alters the programming of the glucocorticoid signaling system during embryonic development

    PubMed Central

    Caldwell, Katharine E.; Labrecque, Matthew T.; Solomon, Benjamin R.; Ali, Abdulmehdi; Allan, Andrea M.

    2015-01-01

    The glucocorticoid system, which plays a critical role in a host of cellular functions including mood disorders and learning and memory, has been reported to be disrupted by arsenic. In previous work we have developed and characterized a prenatal moderate arsenic exposure (50 ppb) model and identified several deficits in learning and memory and mood disorders, as well as alterations within the glucocorticoid receptor signaling system in the adolescent mouse. In these present studies we assessed the effects of arsenic on the glucocorticoid receptor (GR) pathway in both the placenta and the fetal brain in response at two critical periods, embryonic days 14 and 18. The focus of these studies was on the 11β-hydroxysteroid dehydrogenase enzymes (11β-HSD1 and 11β-HSD2) which play a key role in glucorticoid synthesis, as well as the expression and set point of the GR negative feedback regulation. Negative feedback regulation is established early in development. At E14 we found arsenic exposure significantly decreased expression of both protein and message in brain of GR and the 11β-HSD1, while 11β-HSD2 enzyme protein levels were increased but mRNA levels were decreased in the brain. These changes in brain protein continued into the E18 time point, but mRNA levels were no longer significantly altered. Placental HSD11B2 mRNA was not altered by arsenic treatment but protein levels were elevated at E14. GR placental protein levels were decreased at E18 in the arsenic exposed condition. This suggests that arsenic exposure may alter GR expression levels as a consequence of a prolonged developmental imbalance between 11β-HSD1 and 11β-HSD2 protein expression despite decreased 11HSDB2 mRNA. The suppression of GR and the failure to turn down 11β-HSD2 protein expression during fetal development may lead to an altered set point for GR signaling throughout adulthood. To our knowledge, these studies are the first to demonstrate that gestational exposure to moderate levels of

  6. Creatine uptake in mouse hearts with genetically altered creatine levels

    PubMed Central

    Hove, Michiel ten; Makinen, Kimmo; Sebag-Montefiore, Liam; Hunyor, Imre; Fischer, Alexandra; Wallis, Julie; Isbrandt, Dirk; Lygate, Craig; Neubauer, Stefan

    2008-01-01

    Creatine plays an important role in energy metabolism in the heart. Cardiomyocytes accumulate creatine via a specific creatine transporter (CrT), the capacity of which is reduced in the failing heart, resulting in lower myocardial creatine concentration. Therefore, to gain insight into how the CrT is regulated, we studied two mouse models of severely altered myocardial creatine levels. Cardiac creatine uptake levels were measured in isolated hearts from creatine-free guanidinoacetate-N-methyl transferase knock out (GAMT−/−) mice and from mice overexpressing the myocardial CrT (CrT-OE) using 14C-radiolabeled creatine. CrT mRNA levels were measured using real time RT-PCR and creatine levels with HPLC. Hearts from GAMT−/− mice showed a 7-fold increase in Vmax of creatine uptake and a 1.4-fold increase in CrT mRNA levels. The increase in Cr uptake and in CrT mRNA levels, however, was almost completely prevented when mice were fed a creatine supplemented diet, indicating that creatine uptake is subject to negative feedback regulation. Cardiac creatine uptake levels in CrT-OE mice were increased on average 2.7-fold, showing a considerable variation, in line with a similar variation in creatine content. Total CrT mRNA levels correlated well with myocardial creatine content (r = 0.67; p < 0.0001) but endogenous CrT mRNA levels did not correlate at all with myocardial creatine content (r = 0.01; p = 0.96). This study shows that creatine uptake can be massively upregulated in the heart, by almost an order of magnitude and that this upregulation is subject to feedback inhibition. In addition, our results strongly suggest that CrT activity is predominantly regulated by mechanisms other than alterations in gene expression. PMID:18602925

  7. Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells.

    PubMed

    Voynow, J A; Young, L R; Wang, Y; Horger, T; Rose, M C; Fischer, B M

    1999-05-01

    Chronic neutrophil-predominant inflammation and hypersecretion of mucus are common pathophysiological features of cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma. Neutrophils release elastase, a serine protease, that causes increased mucin production and secretion. The molecular mechanisms of elastase-induced mucin production are unknown. We hypothesized that as part of this mechanism, elastase upregulates expression of a major respiratory mucin gene, MUC5AC. A549, a human lung carcinoma cell line that expresses MUC5AC mRNA and protein, and normal human bronchial epithelial cells in an air-liquid interface culture were stimulated with neutrophil elastase. Neutrophil elastase increased MUC5AC mRNA levels in a time-dependent manner in both cell culture systems. Neutrophil elastase treatment also increased MUC5AC protein levels in A549 cells. The mechanism of MUC5AC gene regulation by elastase was determined in A549 cells. The induction of MUC5AC gene expression required serine protease activity; other classes of proteases had no effect on MUC5AC gene expression. Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability. This is the first report of mucin gene regulation by this mechanism.

  8. Analysis of miRNA and mRNA Expression Profiles Highlights Alterations in Ionizing Radiation Response of Human Lymphocytes under Modeled Microgravity

    PubMed Central

    Casara, Silvia; Sales, Gabriele; Lanfranchi, Gerolamo; Celotti, Lucia; Mognato, Maddalena

    2012-01-01

    Background Ionizing radiation (IR) can be extremely harmful for human cells since an improper DNA-damage response (DDR) to IR can contribute to carcinogenesis initiation. Perturbations in DDR pathway can originate from alteration in the functionality of the microRNA-mediated gene regulation, being microRNAs (miRNAs) small noncoding RNA that act as post-transcriptional regulators of gene expression. In this study we gained insight into the role of miRNAs in the regulation of DDR to IR under microgravity, a condition of weightlessness experienced by astronauts during space missions, which could have a synergistic action on cells, increasing the risk of radiation exposure. Methodology/Principal Findings We analyzed miRNA expression profile of human peripheral blood lymphocytes (PBL) incubated for 4 and 24 h in normal gravity (1 g) and in modeled microgravity (MMG) during the repair time after irradiation with 0.2 and 2Gy of γ-rays. Our results show that MMG alters miRNA expression signature of irradiated PBL by decreasing the number of radio-responsive miRNAs. Moreover, let-7i*, miR-7, miR-7-1*, miR-27a, miR-144, miR-200a, miR-598, miR-650 are deregulated by the combined action of radiation and MMG. Integrated analyses of miRNA and mRNA expression profiles, carried out on PBL of the same donors, identified significant miRNA-mRNA anti-correlations of DDR pathway. Gene Ontology analysis reports that the biological category of “Response to DNA damage” is enriched when PBL are incubated in 1 g but not in MMG. Moreover, some anti-correlated genes of p53-pathway show a different expression level between 1 g and MMG. Functional validation assays using luciferase reporter constructs confirmed miRNA-mRNA interactions derived from target prediction analyses. Conclusions/Significance On the whole, by integrating the transcriptome and microRNome, we provide evidence that modeled microgravity can affects the DNA-damage response to IR in human PBL. PMID:22347458

  9. A spontaneous tRNA suppressor of a mutation in the Chlamydomonas reinhardtii nuclear MCD1 gene required for stability of the chloroplast petD mRNA

    PubMed Central

    Murakami, Shinya; Kuehnle, Katrin; Stern, David B.

    2005-01-01

    Numerous nuclear gene products are required for the correct expression of organellar genes. One such gene in the green alga Chlamydomonas reinhardtii is MCD1, whose product is required for stability of the chloroplast-encoded petD mRNA. In mcd1 mutants, which are non-photosynthetic, petD mRNA is degraded by a 5′–3′ exonuclease activity, resulting in a failure to synthesize its product, subunit IV of the cytochrome b 6/f complex. Here, we report the sequence of the wild-type MCD1 gene, which encodes a large and novel putative protein. Analysis of three mutant alleles showed that two harbored large deletions, but that one allele, mcd1-2, had a single base change resulting in a nonsense codon near the N-terminus. This same mutant allele can be suppressed by a second-site mutation in the nuclear MCD2 gene, whereas mcd2-1 cannot suppress the deletion in mcd1-1 (Esposito,D. Higgs,D.C. Drager,R.G. Stern, D.B. and Girard-Bascou,J. (2001) Curr. Genet., 39, 40–48). We report the cloning of mcd2-1, and show that the mutation lies in a tRNASer(CGA), which has been modified to translate the nonsense codon in mcd1-2. We discuss how the existence of a large tRNASer gene family may permit this suppression without pleiotropic consequences. PMID:15947135

  10. Reduced transcript stabilization restricts TNF-alpha expression in RAW264.7 macrophages infected with pathogenic mycobacteria: evidence for an involvement of lipomannan.

    PubMed

    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.

  11. Bupropion and naltrexone combination alters high fructose corn syrup self-administration and gene expression in rats.

    PubMed

    Levy, AnneMarie; Daniels, Stephen; Hudson, Roger; Horman, Thomas; Flynn, Amanda; Zhou, Yan; Leri, Francesco

    2018-06-01

    Contrave ® is an adjunct pharmacotherapy for obesity that contains bupropion (BUP) and naltrexone (NTX). To further explore the psychopharmacology of this drug combination, male Sprague-Dawley rats were implanted with subcutaneous osmotic mini-pumps releasing: 40 mg/kg/day BUP, 4 mg/kg/day NTX, or 40 + 4 mg/kg/day BUP and NTX (BN). During 12 days of exposure, the animals were tested on operant intraoral self-administration (IOSA) of high fructose corn syrup (HFCS) on continuous (FR1) and progressive ratio (PR) schedules, on home cage drinking of HFCS, and on HFCS taste reactivity. Locomotion activity was also assessed. At the conclusion of the study, mRNA expression of genes involved in reward processing, appetite and mood were quantified. It was found that BN produced effects that could largely be ascribed to either BUP or NTX independently. More specifically, BN-induced reductions of HFCS IOSA on a FR1 schedule and home cage drinking, as well as alterations of MOR and POMC mRNA in the nucleus accumbens core and hypothalamus respectively, were attributable to NTX; while alterations of hippocampal BDNF mRNA was attributable to BUP. But, there was also some evidence of drug synergy: only BN caused persistent reductions of HFCS IOSA and drinking; BN produced the least gain of body weight; and only BN-treated rats displayed altered D2R mRNA in the caudate-putamen. Taken together, these observations support the use of BUP + NTX as a mean to alter consumption of sugars and reducing their impact on brain systems involved in reward, appetite and mood. Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. Stability of gametocyte-specific Pfs25-mRNA in dried blood spots on filter paper subjected to different storage conditions

    PubMed Central

    2012-01-01

    Background Real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) is a sensitive method for detection of sub-microscopic gametocytaemia by measuring gametocyte-specific mRNA. Performing analysis on fresh whole blood samples is often not feasible in remote and resource-poor areas. Convenient methods for sample storage and transport are urgently needed. Methods Real-time QT-NASBA was performed on whole blood spiked with a dilution series of purified in-vitro cultivated gametocytes. The blood was either freshly processed or spotted on filter papers. Gametocyte detection sensitivity for QT-NASBA was determined and controlled by microscopy. Dried blood spot (DBS) samples were subjected to five different storage conditions and the loss of sensitivity over time was investigated. A formula to approximate the loss of Pfs25-mRNA due to different storage conditions and time was developed. Results Pfs25-mRNA was measured in time to positivity (TTP) and correlated well with the microscopic counts and the theoretical concentrations of the dilution series. TTP results constantly indicated higher amounts of RNA in filter paper samples extracted after 24 hours than in immediately extracted fresh blood. Among investigated storage conditions freezing at −20°C performed best with 98.7% of the Pfs25-mRNA still detectable at day 28 compared to fresh blood samples. After 92 days, the RNA detection rate was only slightly decreased to 92.9%. Samples stored at 37°C showed most decay with only 64.5% of Pfs25-mRNA detectable after one month. The calculated theoretical detection limit for 24 h-old DBS filter paper samples was 0.0095 (95% CI: 0.0025 to 0.0380) per μl. Conclusions The results suggest that the application of DBS filter papers for quantification of Plasmodium falciparum gametocytes with real-time QT-NASBA is practical and recommendable. This method proved sensitive enough for detection of sub-microscopic densities even after prolonged storage. Decay rates

  13. Immediate Translation of Formin DIAPH1 mRNA after Its Exiting the Nucleus Is Required for Its Perinuclear Localization in Fibroblasts

    PubMed Central

    Liao, Guoning; Liu, Gang

    2013-01-01

    DIAPH1 is a formin protein which promotes actin polymerization, stabilizes microtubules and consequently is involved in cytoskeleton dynamics, cell migration and differentiation. In contrast to the relatively well-understood signaling cascades that regulate DIAPH1 activity, its spatial regulation of biogenesis is not understood. A recent report showed that synthesis of DIAPH1 is confined in the perinuclear ER compartment through translation-dependent mRNA targeting. However, the underlying mechanism of DIAPH1 local synthesis is yet to be elucidated. Here, we provide evidence to demonstrate that the 5′-cap-mediated immediate translation of DIAPH1 mRNA upon exiting nucleus is required for localizing the mRNA in the perinuclear ER compartment. This is supported by data: 1) Delayed translation of DIAPH1 mRNA resulted in loss of perinuclear localization of the mRNA; 2) Once delocalized, DIAPH1 mRNA could not be retargeted to the perinuclear region; and 3) The translation of DIAPH1 mRNA is 5′-cap dependent. These results provide new insights into the novel mechanism of DIAPH1 local synthesis. In addition, these findings have led to the development of new approaches for manipulating DIAPH1 mRNA localization and local protein synthesis in cells for functional studies. Furthermore, a correlation of DIAPH1 mRNA and DIAPH1 protein localization has been demonstrated using a new method to quantify the intracellular distribution of protein. PMID:23840831

  14. Self-amplifying mRNA vaccines.

    PubMed

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Reversible inhibition of PSD-95 mRNA translation by miR-125a, FMRP phosphorylation and mGluR signaling

    PubMed Central

    Muddashetty, Ravi S.; Nalavadi, Vijayalaxmi C.; Gross, Christina; Yao, Xiaodi; Xing, Lei; Laur, Oskar; Warren, Stephen T.; Bassell, Gary J.

    2011-01-01

    Summary The molecular mechanism how RISC and microRNAs selectively and reversibly regulate mRNA translation in response to receptor signaling is unknown but could provide a means for temporal and spatial control of translation. Here we show that miR-125a targeting PSD-95 mRNA allows reversible inhibition of translation and regulation by mGluR signaling. Inhibition of miR-125a increased PSD-95 levels in dendrites and altered dendritic spine morphology. Bidirectional control of PSD-95 expression depends on miR-125a and FMRP phosphorylation status. miR-125a levels at synapses and its association with AGO2 is reduced in Fmr1 KO. FMRP phosphorylation promotes the formation of an AGO2-miR-125a inhibitory complex on PSD-95 mRNA, whereas mGluR signaling of translation requires FMRP dephosphorylation and release of AGO2 from the mRNA. These findings reveal a novel mechanism whereby FMRP phosphorylation provides a reversible switch for AGO2 and microRNA to selectively regulate mRNA translation at synapses in response to receptor activation. PMID:21658607

  16. Wig1 prevents cellular senescence by regulating p21 mRNA decay through control of RISC recruitment

    PubMed Central

    Kim, Bong Cho; Lee, Hyung Chul; Lee, Je-Jung; Choi, Chang-Min; Kim, Dong-Kwan; Lee, Jae Cheol; Ko, Young-Gyu; Lee, Jae-Seon

    2012-01-01

    Premature senescence, a key strategy used to suppress carcinogenesis, can be driven by p53/p21 proteins in response to various stresses. Here, we demonstrate that Wig1 plays a critical role in this process through regulation of p21 mRNA stability. Wig1 controls the association of Argonaute2 (Ago2), a central component of the RNA-induced silencing complex (RISC), with target p21 mRNA via binding of the stem-loop structure near the microRNA (miRNA) target site. Depletion of Wig1 prohibited miRNA-mediated p21 mRNA decay and resulted in premature senescence. Wig1 plays an essential role in cell proliferation, as demonstrated in tumour xenografts in mice, and Wig1 and p21 mRNA levels are inversely correlated in human normal and cancer tissues. Together, our data indicate a novel role of Wig1 in RISC target accessibility, which is a key step in RNA-mediated gene silencing. In addition, these findings indicate that fine-tuning of p21 levels by Wig1 is essential for the prevention of cellular senescence. PMID:23085987

  17. Wig1 prevents cellular senescence by regulating p21 mRNA decay through control of RISC recruitment.

    PubMed

    Kim, Bong Cho; Lee, Hyung Chul; Lee, Je-Jung; Choi, Chang-Min; Kim, Dong-Kwan; Lee, Jae Cheol; Ko, Young-Gyu; Lee, Jae-Seon

    2012-11-14

    Premature senescence, a key strategy used to suppress carcinogenesis, can be driven by p53/p21 proteins in response to various stresses. Here, we demonstrate that Wig1 plays a critical role in this process through regulation of p21 mRNA stability. Wig1 controls the association of Argonaute2 (Ago2), a central component of the RNA-induced silencing complex (RISC), with target p21 mRNA via binding of the stem-loop structure near the microRNA (miRNA) target site. Depletion of Wig1 prohibited miRNA-mediated p21 mRNA decay and resulted in premature senescence. Wig1 plays an essential role in cell proliferation, as demonstrated in tumour xenografts in mice, and Wig1 and p21 mRNA levels are inversely correlated in human normal and cancer tissues. Together, our data indicate a novel role of Wig1 in RISC target accessibility, which is a key step in RNA-mediated gene silencing. In addition, these findings indicate that fine-tuning of p21 levels by Wig1 is essential for the prevention of cellular senescence.

  18. WEREWOLF, a Regulator of Root Hair Pattern Formation, Controls Flowering Time through the Regulation of FT mRNA Stability1[C][W][OA

    PubMed Central

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

    2011-01-01

    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. PMID:21653190

  19. Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

    DTIC Science & Technology

    2016-09-07

    sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

  20. Expression of dopamine D2 receptor and choline acetyltransferase mRNA in the dopamine deafferented rat caudate-putamen.

    PubMed

    Brené, S; Lindefors, N; Herrera-Marschitz, M; Persson, H

    1990-01-01

    In situ hybridization was used to study dopamine D2 receptor (D2R) and choline acetyltransferase (ChAT) mRNA expression in neurons of the rat forebrain, both on control animals and after a unilateral 6-hydroxydopamine (6-OHDA) lesion of midbrain dopamine neurons. D2R mRNA expressing neurons were seen in regions which are known to be heavily innervated by midbrain dopamine fibers such as caudate-putamen, nucleus accumbens and olfactory tubercle. ChAT mRNA expressing neurons were seen in caudate-putamen, nucleus accumbens and septal regions including vertical limb of the diagonal band. In caudate-putamen, approximately 55% of the medium sized neurons, which is the predominating neuronal cell-size in this region, were specifically labeled with the D2R probe. In addition, approximately 95% of the large size neurons in caudate-putamen were specifically labeled with both the D2R and ChAT probes, suggesting that most cholinergic neurons in the caudate-putamen express D2R mRNA. After a unilateral lesion of midbrain dopamine neurons, no change in the level of either D2R or ChAT mRNA were seen in the large size intrinsic cholinergic neurons in caudate-putamen. Similarly, no evidence was obtained for altered levels of D2R mRNA in medium size neurons in medial caudate-putamen, or nucleus accumbens. However, an increase in the number of medium size neurons expressing D2R mRNA was observed in the lateral part of the dopamine deafferented caudate-putamen. Thus, it appears that midbrain dopamine deafferentation causes an increase in D2R mRNA expression in a subpopulation of medium size neurons in the lateral caudate-putamen.

  1. mRNA export: threading the needle

    PubMed Central

    Gaouar, Ouassila; Germain, Hugo

    2013-01-01

    After mRNA biogenesis, several proteins interact with the messenger to ensure its proper export to the cytoplasm. Some of these proteins will bind RNA early on, at the onset of transcription by RNA polymerase II holoenzyme, while others will join later for downstream processing steps, such as poly-adenylation or splicing, or may direct mRNA ribonucleoprotein particle migration to the nucleopore. We recently discovered that Arabidopsis plant knockout for the protein MOS11 (MODIFIER OF SNC1, 11) partially suppresses autoimmune responses observed in the TNL-type [TIR/NBS/LRR (Toll-interleukin-like receptor/nucleotide-binding site/C-terminal leucine-rich repeat)] R gene gain-of-function variant snc1 (suppressor of npr1-1, constitutive 1). This suppression of resistance to pathogens appears to be caused by a decrease in nuclear mRNA export in mos11-1 snc1 plants. In humans, the putative ortholog of MOS11, CIP29 (29-kDa cytokine-induced protein), interacts with three proteins that are also involved in mRNA export: DDX39 (DEAD-box RNA helicase), TAF15 of the FUS family (FUSED IN SARCOMA), and ALY (ALWAYS EARLY), a protein implicated in mRNA export in mammalian systems. These proteins have received very little attention in plants. Here, we will discuss their particularities and role in mRNA export and biotic stress. PMID:23526740

  2. Identification of sperm mRNA biomarkers associated with testis injury during preclinical testing of pharmaceutical compounds

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

    Dere, Edward

    The human testis is sensitive to toxicant-induced injury but current methods for detecting adverse effects are limited, insensitive and unreliable. Animal studies use sensitive histopathological endpoints to assess toxicity, but require testicular tissue that is not available during human clinical trials. More sensitive and reliable molecular biomarkers of testicular injury are needed to better monitor testicular toxicity in both clinical and preclinical. Adult male Wistar Han rats were exposed for 4 weeks to compounds previously associated with testicular injury, including cisplatin (0, 0.2, 0.3, or 0.4 mg/kg/day), BI665915 (0, 20, 70, 100 mg/kg/d), BI665636 (0, 20, 100 mg/kg/d) or BI163538more » (0, 70, 150, 300 mg/kg/d) to evaluate reproductive toxicity and assess changes in sperm mRNA levels. None of the compounds resulted in any significant changes in body, testis or epididymis weights, nor were there decreases in testicular homogenization resistant spermatid head counts. Histopathological evaluation found that only BI665915 treatment caused any testicular effects, including minor germ cell loss and disorganization of the seminiferous tubule epithelium, and an increase in the number of retained spermatid heads. A custom PCR-array panel was used to assess induced changes in sperm mRNA. BI665915 treatment resulted in a significant increase in clusterin (Clu) levels and decreases in GTPase, IMAP family member 4 (Gimap4), prostaglandin D2 synthase (Ptgds) and transmembrane protein with EGF like and two follistatin like domains 1 (Tmeff1) levels. Correlation analysis between transcript levels and quantitative histopathological endpoints found a modest association between Clu with retained spermatid heads. These results demonstrate that sperm mRNA levels are sensitive molecular indicators of testicular injury that can potentially be translated into a clinical setting. - Highlights: • Testing of pharmaceutical compounds identified altered sperm mRNA transcripts

  3. Post-transcriptional regulation tends to attenuate the mRNA noise and to increase the mRNA gain

    NASA Astrophysics Data System (ADS)

    Shi, Changhong; Wang, Shuqiang; Zhou, Tianshou; Jiang, Yiguo

    2015-10-01

    Post-transcriptional regulation is ubiquitous in prokaryotic and eukaryotic cells, but how it impacts gene expression remains to be fully explored. Here, we analyze a simple gene model in which we assume that mRNAs are produced in a constitutive manner but are regulated post-transcriptionally by a decapping enzyme that switches between the active state and the inactive state. We derive the analytical mRNA distribution governed by a chemical master equation, which can be well used to analyze the mechanism of how post-transcription regulation influences the mRNA expression level including the mRNA noise. We demonstrate that the mean mRNA level in the stochastic case is always higher than that in the deterministic case due to the stochastic effect of the enzyme, but the size of the increased part depends mainly on the switching rates between two enzyme states. More interesting is that we find that in contrast to transcriptional regulation, post-transcriptional regulation tends to attenuate noise in mRNA. Our results provide insight into the role of post-transcriptional regulation in controlling the transcriptional noise.

  4. CELFish ways to modulate mRNA decay

    PubMed Central

    St. Louis, Irina Vlasova; Dickson, Alexa M.; Bohjanen, Paul R.; Wilusz, Carol J.

    2013-01-01

    The CELF family of RNA-binding proteins regulates many steps of mRNA metabolism. Although their best characterized function is in pre-mRNA splice site choice, CELF family members are also powerful modulators of mRNA decay. In this review we focus on the different modes of regulation that CELF proteins employ to mediate mRNA decay by binding to GU-rich elements. After starting with an overview of the importance of CELF proteins during development and disease pathogenesis, we then review the mRNA networks and cellular pathways these proteins regulate and the mechanisms by which they influence mRNA decay. Finally, we discuss how CELF protein activity is modulated during development and in response to cellular signals. We conclude by highlighting the priorities for new experiments in this field. PMID:23328451

  5. 3'-UTR-located inverted Alu repeats facilitate mRNA translational repression and stress granule accumulation

    PubMed Central

    Fitzpatrick, Terry; Huang, Sui

    2012-01-01

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

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

    NASA Technical Reports Server (NTRS)

    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.

  7. Differential conserted activity induced regulation of Nogo receptors (1-3), LOTUS and Nogo mRNA in mouse brain.

    PubMed

    Karlsson, Tobias E; Koczy, Josefin; Brené, Stefan; Olson, Lars; Josephson, Anna

    2013-01-01

    Nogo Receptor 1 (NgR1) mRNA is downregulated in hippocampal and cortical regions by increased neuronal activity such as a kainic acid challenge or by exposing rats to running wheels. Plastic changes in cerebral cortex in response to loss of specific sensory inputs caused by spinal cord injury are also associated with downregulation of NgR1 mRNA. Here we investigate the possible regulation by neuronal activity of the homologous receptors NgR2 and NgR3 as well as the endogenous NgR1 antagonist LOTUS and the ligand Nogo. The investigated genes respond to kainic acid by gene-specific, concerted alterations of transcript levels, suggesting a role in the regulation of synaptic plasticity, Downregulation of NgR1, coupled to upregulation of the NgR1 antagonist LOTUS, paired with upregulation of NgR2 and 3 in the dentate gyrus suggest a temporary decrease of Nogo/OMgp sensitivity while CSPG and MAG sensitivity could remain. It is suggested that these activity-synchronized temporary alterations may serve to allow structural alterations at the level of local synaptic circuitry in gray matter, while maintaining white matter pathways and that subsequent upregulation of Nogo-A and NgR1 transcript levels signals the end of such a temporarily opened window of plasticity.

  8. Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells.

    PubMed

    Safina, Alfiya; Garcia, Henry; Commane, Mairead; Guryanova, Olga; Degan, Seamus; Kolesnikova, Kateryna; Gurova, Katerina V

    2013-08-01

    Facilitates chromatin transcription (FACT) is a chromatin remodeling complex with two subunits: SSRP1 and SPT16. Mechanisms controlling FACT levels are of interest, since the complex is not expressed in most differentiated cells, but is frequently upregulated in cancer, particularly in poorly differentiated, aggressive tumors. Moreover, inhibition of FACT expression or function in tumor cells interferes with their survival. Here we demonstrate that SSRP1 and SPT16 protein levels decline upon induction of cellular differentiation or senescence in vitro and that similar declines in protein levels for both SSRP1 and SPT16 occur upon RNAi-mediated knockdown of either SSRP1 or SPT16. The interdependence of SSRP1 and SPT16 protein levels was found to be due to their association with SSRP1 and SPT16 mRNAs, which stabilizes the proteins. In particular, presence of SSRP1 mRNA is critical for SPT16 protein stability. In addition, binding of SSRP1 and SPT16 mRNAs to the FACT complex increases the stability and efficiency of translation of the mRNAs. These data support a model in which the FACT complex is stable when SSRP1 mRNA is present, but quickly degrades when SSRP1 mRNA levels drop. In the absence of FACT complex, SSRP1 and SPT16 mRNAs are unstable and inefficiently translated, making reactivation of FACT function unlikely in normal cells. Thus, we have described a complex and unusual mode of regulation controlling cellular FACT levels that results in amplified and stringent control of FACT activity. The FACT dependence of tumor cells suggests that mechanisms controlling FACT levels could be targeted for anticancer therapy.

  9. Altered native stability is the dominant basis for susceptibility of α1-antitrypsin mutants to polymerization

    PubMed Central

    Irving, James A.; Haq, Imran; Dickens, Jennifer A.; Faull, Sarah V.; Lomas, David A.

    2014-01-01

    Serpins are protease inhibitors whose most stable state is achieved upon transition of a central 5-stranded β-sheet to a 6-stranded form. Mutations, low pH, denaturants and elevated temperatures promote this transition, which can result in a growing polymer chain of inactive molecules. Different types of polymer are possible, but, experimentally only heat has been shown to generate polymers in vitro consistent with ex vivo pathological specimens. Many mutations that alter the rate of heat-induced polymerization have been described, but interpretation is problematic because discrimination is lacking between the effect of global changes in native stability and specific effects on structural mechanism. We show that the temperature midpoint (Tm) of thermal denaturation reflects the transition of α1-antitrypsin to the polymerization intermediate, and determine the relationship with fixed-temperature polymerization half-times (t0.5) in the presence of stabilizing additives [TMAO (trimethylamine N-oxide), sucrose and sodium sulfate], point mutations and disulfide bonds. Combined with a retrospective analysis of 31 mutants characterized in the literature, the results of the present study show that global changes to native state stability are the predominant basis for the effects of mutations and osmolytes on heat-induced polymerization, summarized by the equation: ln(t0.5,mutant/t0.5,wild-type)=0.34×ΔTm. It is deviations from this relationship that hold key information about the polymerization process. PMID:24552432

  10. Extensive alterations of the whole-blood transcriptome are associated with body mass index: results of an mRNA profiling study involving two large population-based cohorts.

    PubMed

    Homuth, Georg; Wahl, Simone; Müller, Christian; Schurmann, Claudia; Mäder, Ulrike; Blankenberg, Stefan; Carstensen, Maren; Dörr, Marcus; Endlich, Karlhans; Englbrecht, Christian; Felix, Stephan B; Gieger, Christian; Grallert, Harald; Herder, Christian; Illig, Thomas; Kruppa, Jochen; Marzi, Carola S; Mayerle, Julia; Meitinger, Thomas; Metspalu, Andres; Nauck, Matthias; Peters, Annette; Rathmann, Wolfgang; Reinmaa, Eva; Rettig, Rainer; Roden, Michael; Schillert, Arne; Schramm, Katharina; Steil, Leif; Strauch, Konstantin; Teumer, Alexander; Völzke, Henry; Wallaschofski, Henri; Wild, Philipp S; Ziegler, Andreas; Völker, Uwe; Prokisch, Holger; Zeller, Tanja

    2015-10-15

    Obesity, defined as pathologically increased body mass index (BMI), is strongly related to an increased risk for numerous common cardiovascular and metabolic diseases. It is particularly associated with insulin resistance, hyperglycemia, and systemic oxidative stress and represents the most important risk factor for type 2 diabetes (T2D). However, the pathophysiological mechanisms underlying these associations are still not completely understood. Therefore, in order to identify potentially disease-relevant BMI-associated gene expression signatures, a transcriptome-wide association study (TWAS) on BMI was performed. Whole-blood mRNA levels determined by array-based transcriptional profiling were correlated with BMI in two large independent population-based cohort studies (KORA F4 and SHIP-TREND) comprising a total of 1977 individuals. Extensive alterations of the whole-blood transcriptome were associated with BMI: More than 3500 transcripts exhibited significant positive or negative BMI-correlation. Three major whole-blood gene expression signatures associated with increased BMI were identified. The three signatures suggested: i) a ratio shift from mature erythrocytes towards reticulocytes, ii) decreased expression of several genes essentially involved in the transmission and amplification of the insulin signal, and iii) reduced expression of several key genes involved in the defence against reactive oxygen species (ROS). Whereas the first signature confirms published results, the other two provide possible mechanistic explanations for well-known epidemiological findings under conditions of increased BMI, namely attenuated insulin signaling and increased oxidative stress. The putatively causative BMI-dependent down-regulation of the expression of numerous genes on the mRNA level represents a novel finding. BMI-associated negative transcriptional regulation of insulin signaling and oxidative stress management provide new insights into the pathogenesis of metabolic

  11. Determining if an mRNA is a Substrate of Nonsense-Mediated mRNA Decay in Saccharomyces cerevisiae.

    PubMed

    Johansson, Marcus J O

    2017-01-01

    Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic quality control mechanism which triggers decay of mRNAs harboring premature translation termination codons. In this chapter, I describe methods for monitoring the influence of NMD on mRNA abundance and decay rates in Saccharomyces cerevisiae. The descriptions include detailed methods for growing yeast cells, total RNA isolation, and Northern blotting. Although the chapter focuses on NMD, the methods can be easily adapted to assess the effect of other mRNA decay pathways.

  12. Dose-dependent effects of morphine exposure on mRNA and microRNA (miR) expression in hippocampus of stressed neonatal mice.

    PubMed

    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, 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

  13. Dose-Dependent Effects of Morphine Exposure on mRNA and microRNA (miR) Expression in Hippocampus of Stressed Neonatal Mice

    PubMed Central

    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, 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

  14. Influence of chronic undernutrition and leptin on GOAT mRNA levels in rat stomach mucosa.

    PubMed

    González, C Ruth; Vázquez, María J; López, Miguel; Diéguez, Carlos

    2008-12-01

    The most unique feature of ghrelin is the acyl-modification of a hydroxyl group of the Ser3 in the N-terminus. The Ser3 is commonly modified by n-octanoic acid in vertebrates being needed for its biological effects, at least in terms of feeding. Therefore, a critical question regarding the role of ghrelin was to characterize the mechanism involved in its acylation. The acyltransferase that catalyzes ghrelin octanoylation has been recently identified and named ghrelin O-acyltransferase (GOAT). The aim of this study was to clarify the physiological implications of GOAT in the regulation of energy balance, by assessing the effect of undernutrition, as well as fasting in adult male rats. We have determined GOAT mRNA expression levels by real time-PCR in the stomach mucosa. Our results show that chronic food restriction led to an increase in GOAT mRNA, particularly following long-term chronic malnutrition (21 days). Furthermore, following 48 h complete fasting, a situation with high-circulating ghrelin levels, we found similar mRNA expression of GOAT in fed and fasted rats; exogenous leptin administration markedly increase GOAT mRNA levels in the stomach mucosa of fasted rats. These findings suggest that increased GOAT mRNA levels may have a role in mediating the physiological responses to chronic undernutrition and could represent an adaptive response to prevent long-lasting alterations in energy balance and body weight homeostasis. Furthermore, our data also offer mechanistic insights into the reason why during fasting acylated ghrelin levels are not increased at a time when a marked increase in an orexigenic signal as important as acylated ghrelin will be expected.

  15. Pharmacogenetic Inhibition of eIF4E-Dependent Mmp9 mRNA Translation Reverses Fragile X Syndrome-like Phenotypes

    PubMed Central

    Gkogkas, Christos G.; Khoutorsky, Arkady; Cao, Ruifeng; Jafarnejad, Seyed Mehdi; Prager-Khoutorsky, Masha; Giannakas, Nikolaos; Kaminari, Archontia; Fragkouli, Apostolia; Nader, Karim; Price, Theodore J.; Konicek, Bruce W.; Graff, Jeremy R.; Tzinia, Athina K.; Lacaille, Jean-Claude; Sonenberg, Nahum

    2015-01-01

    SUMMARY Fragile X syndrome (FXS) is the leading genetic cause of autism. Mutations in Fmr1 (fragile X mental retardation 1 gene) engender exaggerated translation resulting in dendritic spine dysmorphogenesis, synaptic plasticity alterations, and behavioral deficits in mice, which are reminiscent of FXS pheno-types. Using postmortem brains from FXS patients and Fmr1 knockout mice (Fmr1 −/y), we show that phosphorylation of the mRNA 5′ cap binding protein, eukaryotic initiation factor 4E (eIF4E), is elevated concomitant with increased expression of matrix metalloproteinase 9 (MMP-9) protein. Genetic or pharmacological reduction of eIF4E phosphorylation rescued core behavioral deficits, synaptic plasticity alterations, and dendritic spine morphology defects via reducing exaggerated translation of Mmp9 mRNA in Fmr1 −/y mice, whereas MMP-9 overexpression produced several FXS-like phenotypes. These results uncover a mechanism of regulation of synaptic function by translational control of Mmp-9 in FXS, which opens the possibility of new treatment avenues for the diverse neurological and psychiatric aspects of FXS. PMID:25466251

  16. Pharmacogenetic inhibition of eIF4E-dependent Mmp9 mRNA translation reverses fragile X syndrome-like phenotypes.

    PubMed

    Gkogkas, Christos G; Khoutorsky, Arkady; Cao, Ruifeng; Jafarnejad, Seyed Mehdi; Prager-Khoutorsky, Masha; Giannakas, Nikolaos; Kaminari, Archontia; Fragkouli, Apostolia; Nader, Karim; Price, Theodore J; Konicek, Bruce W; Graff, Jeremy R; Tzinia, Athina K; Lacaille, Jean-Claude; Sonenberg, Nahum

    2014-12-11

    Fragile X syndrome (FXS) is the leading genetic cause of autism. Mutations in Fmr1 (fragile X mental retardation 1 gene) engender exaggerated translation resulting in dendritic spine dysmorphogenesis, synaptic plasticity alterations, and behavioral deficits in mice, which are reminiscent of FXS phenotypes. Using postmortem brains from FXS patients and Fmr1 knockout mice (Fmr1(-/y)), we show that phosphorylation of the mRNA 5' cap binding protein, eukaryotic initiation factor 4E (eIF4E), is elevated concomitant with increased expression of matrix metalloproteinase 9 (MMP-9) protein. Genetic or pharmacological reduction of eIF4E phosphorylation rescued core behavioral deficits, synaptic plasticity alterations, and dendritic spine morphology defects via reducing exaggerated translation of Mmp9 mRNA in Fmr1(-/y) mice, whereas MMP-9 overexpression produced several FXS-like phenotypes. These results uncover a mechanism of regulation of synaptic function by translational control of Mmp-9 in FXS, which opens the possibility of new treatment avenues for the diverse neurological and psychiatric aspects of FXS. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Long-distance transport of Gibberellic Acid Insensitive mRNA in Nicotiana benthamiana

    PubMed Central

    2013-01-01

    Background The Gibberellic Acid (GA) signal is governed by the GAI (Gibberellic Acid Insensitive) repressor, which is characterized by a highly conserved N-terminal DELLA domain. Deletion of the DELLA domain results in constitutive suppression of GA signaling. As the GAI transcript is transportable in phloem elements, a Δ-DELLA GAI (gai) transgenic stock plant can reduce the stature of a scion through transport of gai mRNA from the stock. However, little is known about the characteristics of a scion on a gai stock. Results Arabidopsis Δ-DELLA GAI (gai) was fused with a T7 epitope tag and expressed under the control of a companion cell-specific expression promoter, Commelina yellow mottle virus promoter (CoYMVp), to enhance transport in the phloem. The CoYMVp:Atgai-T7 (CgT) transgenic Nicotiana benthamiana exhibited a dwarf phenotype and lower sensitivity to GA enhancement of shoot stature. A wild-type (WT) scion on a CgT stock contained both Atgai-T7 mRNA and the translated product. Microarray analysis to clarify the effect of the CgT stock on the gene expression pattern in the scion clearly revealed that the WT scions on CgT stocks had fewer genes whose expression was altered in response to GA treatment. An apple rootstock variety, Malus prunifolia, integrating CoYMVp:Atgai moderately reduced the tree height of the apple cultivar scion. Conclusions Our results demonstrate that Atgai mRNA can move from companion cells to sieve tubes and that the translated product remains at the sites to which it is transported, resulting in attenuation of GA responses by reducing the expression of many genes. The induction of semi-dwarfism in an apple cultivar on root stock harbouring Atgai suggests that long-distance transport of mRNA from grafts would be applicable to horticulture crops. PMID:24144190

  18. LC3-mediated fibronectin mRNA translation induces fibrosarcoma growth by increasing connective tissue growth factor

    PubMed Central

    Ying, Lihua; Lau, Agatha; Alvira, Cristina M.; West, Robert; Cann, Gordon M.; Zhou, Bin; Kinnear, Caroline; Jan, Eric; Sarnow, Peter; Van de Rijn, Matt; Rabinovitch, Marlene

    2009-01-01

    Summary Previously, we related fibronectin (Fn1) mRNA translation to an interaction between an AU-rich element in the Fn1 3′ UTR and light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. Since human fibrosarcoma (HT1080) cells produce little fibronectin and LC3, we used these cells to investigate how LC3-mediated Fn1 mRNA translation might alter tumor growth. Transfection of HT1080 cells with LC3 enhanced fibronectin mRNA translation. Using polysome analysis and RNA-binding assays, we show that elevated levels of translation depend on an interaction between a triple arginine motif in LC3 and the AU-rich element in Fn1 mRNA. Wild-type but not mutant LC3 accelerated HT1080 cell growth in culture and when implanted in SCID mice. Comparison of WT LC3 with vector-transfected HT1080 cells revealed increased fibronectin-dependent proliferation, adhesion and invasion. Microarray analysis of genes differentially expressed in WT and vector-transfected control cells indicated enhanced expression of connective tissue growth factor (CTGF). Using siRNA, we show that enhanced expression of CTGF is fibronectin dependent and that LC3-mediated adhesion, invasion and proliferation are CTGF dependent. Expression profiling of soft tissue tumors revealed increased expression of both LC3 and CTGF in some locally invasive tumor types. PMID:19366727

  19. Rous Sarcoma Virus RNA Stability Element Inhibits Deadenylation of mRNAs with Long 3′UTRs

    PubMed Central

    Balagopal, Vidya; Beemon, Karen L.

    2017-01-01

    All retroviruses use their full-length primary transcript as the major mRNA for Group-specific antigen (Gag) capsid proteins. This results in a long 3′ untranslated region (UTR) downstream of the termination codon. In the case of Rous sarcoma virus (RSV), there is a 7 kb 3′UTR downstream of the gag terminator, containing the pol, env, and src genes. mRNAs containing long 3′UTRs, like those with premature termination codons, are frequently recognized by the cellular nonsense-mediated mRNA decay (NMD) machinery and targeted for degradation. To prevent this, RSV has evolved an RNA stability element (RSE) in the RNA immediately downstream of the gag termination codon. This 400-nt RNA sequence stabilizes premature termination codons (PTCs) in gag. It also stabilizes globin mRNAs with long 3′UTRs, when placed downstream of the termination codon. It is not clear how the RSE stabilizes the mRNA and prevents decay. We show here that the presence of RSE inhibits deadenylation severely. In addition, the RSE also impairs decapping (DCP2) and 5′-3′ exonucleolytic (XRN1) function in knockdown experiments in human cells. PMID:28763028

  20. Neonatal paternal deprivation impairs social recognition and alters levels of oxytocin and estrogen receptor α mRNA expression in the MeA and NAcc, and serum oxytocin in mandarin voles.

    PubMed

    Cao, Yan; Wu, Ruiyong; Tai, Fadao; Zhang, Xia; Yu, Peng; An, Xiaolei; Qiao, Xufeng; Hao, Ping

    2014-01-01

    Paternal care is necessary for the healthy development of social behavior in monogamous rodents and social recognition underpins social behavior in these animals. The effects of paternal care on the development of social recognition and underlying neuroendocrine mechanisms, especially the involvement of oxytocin and estrogen pathways, remain poorly understood. We investigated the effects of paternal deprivation (PD: father was removed from neonatal pups and mother alone raised the offspring) on social recognition in mandarin voles (Microtus mandarinus), a socially monogamous rodent. Paternal deprivation was found to inhibit the development of social recognition in female and male offspring according to a habituation-dishabituation paradigm. Paternal deprivation resulted in increased inactivity and reduced investigation during new encounters with other animals. Paternal deprivation reduced oxytocin receptor (OTR) and estrogen receptor α (ERα) mRNA expression in the medial amygdala and nucleus accumbens. Paternal deprivation reduced serum oxytocin (OT) concentration in females, but had no effect on males. Our results provide substantial evidence that paternal deprivation inhibits the development of social recognition in female and male mandarin voles and alters social behavior later in life. This is possibly the result of altered expression of central OTR and ERα and serum OT levels caused by paternal deprivation. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Altered cell function in microgravity

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, Millie

    1991-01-01

    The paper overviews published results from investigations of changes in basic biological parameters taking place as a result of spaceflight exposure. These include changes in the rates of the DNA, mRNA, and protein biosyntheses; changes in the growth rate of an organism; and alterations in the cytoskeleton structure, differentiation, hormone accumulation, and collagen matrix secretion. These results, obtained both in complex biological organisms and on cultured cells, suggest that a basic cellular function is influenced and changed by microgravity. Many of the above mentioned changes are also found to take place in aging cells.

  2. Altered gene expression in conjunctival squamous cell carcinoma.

    PubMed

    Mahale, Alka; Alkatan, Hind; Alwadani, Saeed; Othman, Maha; Suarez, Maria J; Price, Antoinette; Al-Hussain, Hailah; Jastaneiah, Sabah; Yu, Wayne; Maktabi, Azza; Deepak, Edward P; Eberhart, Charles G; Asnaghi, Laura

    2016-05-01

    Conjunctival squamous cell carcinoma is a malignancy of the ocular surface. The molecular drivers responsible for the development and progression of this disease are not well understood. We therefore compared the transcriptional profiles of eight snap-frozen conjunctival squamous cell carcinomas and one in situ lesion with normal conjunctival specimens in order to identify diagnostic markers or therapeutic targets. RNA was analyzed using oligonucleotide microarrays, and a wide range of transcripts with altered expression identified, including many dysregulated in carcinomas arising at other sites. Among the upregulated genes, we observed more than 30-fold induction of the matrix metalloproteinases, MMP-9 and MMP-11, as well as a prominent increase in the mRNA level of a calcium-binding protein important for the intracellular calcium signaling, S100A2, which was induced over 20-fold in the tumor cohort. Clusterin was the most downregulated gene, with an approximately 180-fold reduction in the mRNA expression. These alterations were all confirmed by qPCR in the samples used for initial microarray analysis. In addition, immunohistochemical analysis confirmed the overexpression of MMP-11 and S100A2, as well as reductions in clusterin, in several independent in situ carcinomas of conjunctiva. These data identify a number of alterations, including upregulation of MMP-9, MMP-11, and S100A2, as well as downregulation of clusterin, associated with epithelial tumorigenesis in the ocular surface.

  3. Regulation of Egr-1, VIP, and Shh mRNA and Egr-1 protein in the mouse retina by light and image quality.

    PubMed

    Brand, Christine; Burkhardt, Eva; Schaeffel, Frank; Choi, Jeong Won; Feldkaemper, Marita Pauline

    2005-04-28

    To analyze mRNA expression changes of Egr-1, VIP, and Shh under different light and treatment conditions in mice. The mRNA expression levels of the three genes and additionally the Egr-1 protein expression were compared in form deprived eyes and eyes with normal vision. Moreover, the influence of dark to light and light to dark transitions and of changes in retinal illumination on mRNA levels was investigated. Form deprivation of mice was induced by fitting frosted diffusers over one eye and an attentuation matched neutral density (ND) filter over the other eye. To measure the effects of retinal illumination changes on mRNA expression, animals were bilaterally fitted with different ND filters. Semiquantitative real-time RT-PCR was used to measure the mRNA levels and immunohistochemistry was applied to localize and detect Egr-1 protein. The expression levels of both Egr-1 mRNA and protein were reduced in form deprived eyes compared to their fellow eyes after 30 min and 1 h, respectively. Egr-1 mRNA was strikingly upregulated both after dark to light and light to dark transitions, whereas minor changes in retinal illumination by covering the eyes with neutral density filters did not alter Egr-1 mRNA expression. In mice, the mRNA levels of VIP and Shh were not affected by form deprivation, but they were found to be regulated depending on the time of day. Both Egr-1 mRNA and protein expression levels were strongly regulated by light, especially by transitions between light and darkness. Image contrast may exert an additional influence on mRNA and protein expression of Egr-1, particularly in the cells in the ganglion cell layer and in bipolar cells.

  4. [Effects of lipopolysaccharides extracted from Porphyromonas endodontalis on the expression of IL-1beta mRNA and IL-6 mRNA in osteoblasts].

    PubMed

    Yang, Di; Li, Ren; Qiu, Li-Hong; Li, Chen

    2009-04-01

    To quantify the IL-1 beta mRNA and IL-6 mRNA expression induced by lipopolysaccharides (LPS)extracted from Porphyromonas endodontalis(P.e) in osteoblasts, and to relate P.e-LPS to bone absorption pathogenesis in lesions of chronical apical periodontitis. MG63 was treated with different concentrations of P.e-LPS(0-50 microg/mL) for different hours(0-24h). The expression of IL-1 beta mRNA and IL-6 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR).Statistical analysis was performed using one- way ANOVA and Dunnett t test with SPSS11.0 software package. The level of IL-1 beta mRNA and IL-6 mRNA increased significantly after treatment with P.e-LPS at more than 5 microg/mL (P<0.01)and for more than 1 hour (P<0.01), which indicated that P.e-LPS induced osteoblasts to express IL-1 beta mRNA and IL-6 mRNA in dose and time dependent manners. P.e-LPS may promote bone resorption in lesions of chronical apical periodontitis by inducing IL-1 beta mRNA and IL-6 mRNA expression in osteoblasts.

  5. [The expressions of HSP 70 mRNA and c-fos mRNA in the skeletal muscle and cardiac muscle of rabbits by electrocuted].

    PubMed

    Wang, Ye; Liu, Min; Cheng, Wei-bo; He, Gui-qiong; Li, Fan; Liao, Zhi-gang

    2008-08-01

    To study the changes of HSP 70 mRNA and c-fos mRNA expression and to find a method to differentiate antemortem from postmortem electrocution. Fifteen New Zealand rabbits were randomly divided into three groups, the antemortem electrocution group, the postmortem electrocution group, and the control group. Each group consists of five rabbits. The levels of HSP 70 mRNA and c-fos mRNA in skeletal muscle and cardiac muscle were examined with quantitative fluorescent RT-PCR. The levels of HSP 70 mRNA and c-fos mRNA in the antemortem electrocution group increased significantly (P<0.05), compared with that of the postmortem electrocution group. The changes of HSP 70 mRNA and c-fos mRNA expression in skeletal muscle and cardiac muscle can be used as an indicator to distinguish antemortem from postmortem electrocution.

  6. Altered expression of alternatively spliced isoforms of the mRNA NMDAR1 receptor in the visual cortex of strabismic cats.

    PubMed

    Yin, Z Q; Deng, Z M; Crewther, S G; Crewther, D P

    2001-11-20

    Although much has been written about the role of the NMDA receptor's role in experience dependent visual plasticity, the function of the NMDAR1 receptor subunit in the post-plasticity stage of development is still not well understood. However, in the well studied model of strabismic amblyopia where binocularity is reduced, but where most primary visual cortex neurons can be driven by one or other eye, the density of expression of NMDAR1 receptor protein is significantly reduced, compared to normals. This study aims to identify which of eight isoforms of the spliced heterogeneous variants of the NMDAR1 mRNA receptor gene are associated with this decrease in expression as a means of elucidating possible function. A series of digoxygenin-labelled oligonucleotide probes based on the human gene sequence have been used for in situ hybridization (ISH) of sections from the striate cortex of four adult cats. The probes were used to uniquely detect the expression of alternatively spliced mRNA variants in 66,487 cells from sections from the area centralis projection of two normal cats and two cats made esotropic as kittens by tenotomy at two weeks of age. As expected, total NMDAR1 mRNA isoform expression was significantly lower in the striate cortex of strabismic compared to normal cats. The proportion of cortical cells expressing the R1-a, R1-b, and R1-1 isoforms in strabismic animals was decreased while the proportion expressing R1-3 was increased, especially in layers V and VI. No significant difference in expression of the R1-2 and R1-4 isoforms was seen comparing strabismic and normal cats. These results confirm our previous findings and suggest that transcriptional inhibition of specific isoforms of NMDAR1 mRNA may underlie the change in receptor expression. This preferential reduction in the proportion of neurons bearing particular NMDAR1 isoforms, i.e. isoforms R1-a and b, and R1-1 with partial compensation through the expression of the R1-3 isoform, is more likely

  7. CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

    PubMed

    Busi, Florent; Cresteil, Thierry

    2005-09-01

    The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

  8. Glucocorticoid-induced changes in glucocorticoid receptor mRNA and protein expression in the human placenta as a potential factor for altering fetal growth and development.

    PubMed

    Bivol, Svetlana; Owen, Suzzanne J; Rose'Meyer, Roselyn B

    2016-02-05

    Glucocorticoids (GCs) control essential metabolic processes in virtually every cell in the body and play a vital role in the development of fetal tissues and organ systems. The biological actions of GCs are mediated via glucocorticoid receptors (GRs), the cytoplasmic transcription factors that regulate the transcription of genes involved in placental and fetal growth and development. Several experimental studies have demonstrated that fetal exposure to high maternal GC levels early in gestation is associated with adverse fetal outcomes, including low birthweight, intrauterine growth restriction and anatomical and structural abnormalities that may increase the risk of cardiovascular, metabolic and neuroendocrine disorders in adulthood. The response of the fetus to GCs is dependent on gender, with female fetuses becoming hypersensitive to changes in GC levels whereas male fetuses develop GC resistance in the environment of high maternal GCs. In this paper we review GR function and the physiological and pathological effects of GCs on fetal development. We propose that GC-induced changes in the placental structure and function, including alterations in the expression of GR mRNA and protein levels, may play role in inhibiting in utero fetal growth.

  9. NMD3 regulates both mRNA and rRNA nuclear export in African trypanosomes via an XPOI-linked pathway

    PubMed Central

    Bühlmann, Melanie; Walrad, Pegine; Rico, Eva; Ivens, Alasdair; Capewell, Paul; Naguleswaran, Arunasalam; Roditi, Isabel; Matthews, Keith R.

    2015-01-01

    Trypanosomes mostly regulate gene expression through post-transcriptional mechanisms, particularly mRNA stability. However, much mRNA degradation is cytoplasmic such that mRNA nuclear export must represent an important level of regulation. Ribosomal RNAs must also be exported from the nucleus and the trypanosome orthologue of NMD3 has been confirmed to be involved in rRNA processing and export, matching its function in other organisms. Surprisingly, we found that TbNMD3 depletion also generates mRNA accumulation of procyclin-associated genes (PAGs), these being co-transcribed by RNA polymerase I with the procyclin surface antigen genes expressed on trypanosome insect forms. By whole transcriptome RNA-seq analysis of TbNMD3-depleted cells we confirm the regulation of the PAG transcripts by TbNMD3 and using reporter constructs reveal that PAG1 regulation is mediated by its 5′UTR. Dissection of the mechanism of regulation demonstrates that it is not dependent upon translational inhibition mediated by TbNMD3 depletion nor enhanced transcription. However, depletion of the nuclear export factors XPO1 or MEX67 recapitulates the effects of TbNMD3 depletion on PAG mRNAs and mRNAs accumulated in the nucleus of TbNMD3-depleted cells. These results invoke a novel RNA regulatory mechanism involving the NMD3-dependent nuclear export of mRNA cargos, suggesting a shared platform for mRNA and rRNA export. PMID:25873624

  10. Elevated GRIA1 mRNA expression in Layer II/III and V pyramidal cells of the DLPFC in schizophrenia

    PubMed Central

    O’Connor, J.A.; Hemby, S.E.

    2012-01-01

    The functional integrity of the dorsolateral prefrontal cortex (DLPFC) is altered in schizophrenia leading to profound deficits in working memory and cognition. Growing evidence indicates that dysregulation of glutamate signaling may be a significant contributor to the pathophysiology mediating these effects; however, the contribution of NMDA and AMPA receptors in the mediation of this deficit remains unclear. The equivocality of data regarding ionotropic glutamate receptor alterations of subunit expression in the DLPFC of schizophrenics is likely reflective of subtle alterations in the cellular and molecular composition of specific neuronal populations within the region. Given previous evidence of Layer II/III and V pyramidal cell alterations in schizophrenia and the significant influence of subunit composition on NMDA and AMPA receptor function, laser capture microdissection combined with quantitative PCR was used to examine the expression of AMPA (GRIA1-4) and NMDA (GRIN1, 2A and 2B) subunit mRNA levels in Layer II/III and Layer V pyramidal cells in the DLPFC. Comparisons were made between individuals diagnosed with schizophrenia, bipolar disorder, major depressive disorder and controls (n=15/group). All subunits were expressed at detectable levels in both cell populations for all diseases as well as for the control group. Interestingly, GRIA1 mRNA was significantly increased in both cell types in the schizophrenia group compare to controls, while similar trends were observed in major depressive disorder (Layers II/III and V) and bipolar disorder (Layer V). These data suggest that increased GRIA1 subunit expression may contribute to schizophrenia pathology. PMID:17942280

  11. Differential Conserted Activity Induced Regulation of Nogo Receptors (1–3), LOTUS and Nogo mRNA in Mouse Brain

    PubMed Central

    Karlsson, Tobias E.; Koczy, Josefin; Brené, Stefan; Olson, Lars; Josephson, Anna

    2013-01-01

    Nogo Receptor 1 (NgR1) mRNA is downregulated in hippocampal and cortical regions by increased neuronal activity such as a kainic acid challenge or by exposing rats to running wheels. Plastic changes in cerebral cortex in response to loss of specific sensory inputs caused by spinal cord injury are also associated with downregulation of NgR1 mRNA. Here we investigate the possible regulation by neuronal activity of the homologous receptors NgR2 and NgR3 as well as the endogenous NgR1 antagonist LOTUS and the ligand Nogo. The investigated genes respond to kainic acid by gene-specific, concerted alterations of transcript levels, suggesting a role in the regulation of synaptic plasticity, Downregulation of NgR1, coupled to upregulation of the NgR1 antagonist LOTUS, paired with upregulation of NgR2 and 3 in the dentate gyrus suggest a temporary decrease of Nogo/OMgp sensitivity while CSPG and MAG sensitivity could remain. It is suggested that these activity-synchronized temporary alterations may serve to allow structural alterations at the level of local synaptic circuitry in gray matter, while maintaining white matter pathways and that subsequent upregulation of Nogo-A and NgR1 transcript levels signals the end of such a temporarily opened window of plasticity. PMID:23593344

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

    PubMed Central

    Ferrero, Rut; Torres, Magdalena

    2002-01-01

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

  13. Replacement of the yeast TRP4 3' untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail.

    PubMed Central

    Düvel, Katrin; Valerius, Oliver; Mangus, David A; Jacobson, Allan; Braus, Gerhard H

    2002-01-01

    The mRNA poly(A) tail serves different purposes, including the facilitation of nuclear export, mRNA stabilization, efficient translation, and, finally, specific degradation. The posttranscriptional addition of a poly(A) tail depends on sequence motifs in the 3' untranslated region (3' UTR) of the mRNA and a complex trans-acting protein machinery. In this study, we have replaced the 3' UTR of the yeast TRP4 gene with sequences encoding a hammerhead ribozyme that efficiently cleaves itself in vivo. Expression of the TRP4-ribozyme allele resulted in the accumulation of a nonpolyadenylated mRNA. Cells expressing the TRP4-ribozyme mRNA showed a reduced growth rate due to a reduction in Trp4p enzyme activity. The reduction in enzyme activity was not caused by inefficient mRNA export from the nucleus or mRNA destabilization. Rather, analyses of mRNA association with polyribosomes indicate that translation of the ribozyme-containing mRNA is impaired. This translational defect allows sufficient synthesis of Trp4p to support growth of trp4 cells, but is, nevertheless, of such magnitude as to activate the general control network of amino acid biosynthesis. PMID:12003493

  14. Embryonal carcinoma cell induction of miRNA and mRNA changes in co-cultured prostate stromal fibromuscular cells

    PubMed Central

    VÊNCIO, ENEIDA F.; PASCAL, LAURA E.; PAGE, LAURA S.; DENYER, GARETH; WANG, AMY J.; RUOHOLA-BAKER, HANNELE; ZHANG, SHILE; WANG, KAI; GALAS, DAVID J.; LIU, ALVIN Y.

    2014-01-01

    The prostate stromal mesenchyme controls organ-specific development. In cancer, the stromal compartment shows altered gene expression compared to non-cancer. The lineage relationship between cancer-associated stromal cells and normal tissue stromal cells is not known. Nor is the cause underlying the expression difference. Previously, the embryonal carcinoma (EC) cell line, NCCIT, was used by us to study the stromal induction property. In the current study, stromal cells from non-cancer (NP) and cancer (CP) were isolated from tissue specimens and co-cultured with NCCIT cells in a trans-well format to preclude heterotypic cell contact. After 3 days, the stromal cells were analyzed by gene arrays for microRNA (miRNA) and mRNA expression. In co-culture, NCCIT cells were found to alter the miRNA and mRNA expression of NP stromal cells to one like that of CP stromal cells. In contrast, NCCIT had no significant effect on the gene expression of CP stromal cells. We conclude that the gene expression changes in stromal cells can be induced by diffusible factors synthesized by EC cells, and suggest that cancer-associated stromal cells represent a more primitive or less differentiated stromal cell type. PMID:20945389

  15. Contributions of testosterone and territory ownership to sexually-motivated behaviors and mRNA expression in the medial preoptic area of male European starlings

    PubMed Central

    Spool, Jeremy A.; Stevenson, Sharon A.; Angyal, Caroline S.; Riters, Lauren V.

    2016-01-01

    Animals integrate social information with their internal endocrine state to control the timing of behavior, but how these signals are integrated in the brain is not understood. The medial preoptic area (mPOA) may play an integrative role in the control of courtship behavior, as it receives projections from multiple sensory systems, and is central to the hormonal control of courtship behavior across vertebrates. Additionally, data from many species implicate opioid and dopaminergic systems in the mPOA in the control of male courtship behavior. We used European starlings to test the hypothesis that testosterone (T) and social status (in the form of territory possession) interact to control the timing of courtship behavior by modulating steroid hormone-, opioid- and dopaminergic-related gene expression in the mPOA. We found that only males given both T and a nesting territory produced high rates of courtship behavior in response to a female. T treatment altered patterns of gene expression in the mPOA by increasing androgen receptor, aromatase, mu-opioid receptor and preproenkephalin mRNA and decreasing tyrosine hydroxylase mRNA expression. Territory possession did not alter mRNA expression in the mPOA, despite the finding that only birds with both T and a nesting territory produced courtship behavior. We propose that T prepares the mPOA to respond to the presence of a female with high rates of courtship song by altering gene expression, but that activity in the mPOA is under a continuous (i.e. tonic) inhibition until a male starling obtains a nesting territory. PMID:27633459

  16. Sensitivity of mRNA Translation

    PubMed Central

    Poker, Gilad; Margaliot, Michael; Tuller, Tamir

    2015-01-01

    Using the dynamic mean-field approximation of the totally asymmetric simple exclusion process (TASEP), we investigate the effect of small changes in the initiation, elongation, and termination rates along the mRNA strand on the steady-state protein translation rate. We show that the sensitivity of mRNA translation is equal to the sensitivity of the maximal eigenvalue of a symmetric, nonnegative, tridiagonal, and irreducible matrix. This leads to new analytical results as well as efficient numerical schemes that are applicable for large-scale models. Our results show that in the usual endogenous case, when initiation is more rate-limiting than elongation, the sensitivity of the translation rate to small mutations rapidly increases towards the 5′ end of the ORF. When the initiation rate is high, as may be the case for highly expressed and/or heterologous optimized genes, the maximal sensitivity is with respect to the elongation rates at the middle of the mRNA strand. We also show that the maximal possible effect of a small increase/decrease in any of the rates along the mRNA is an increase/decrease of the same magnitude in the translation rate. These results are in agreement with previous molecular evolutionary and synthetic biology experimental studies. PMID:26238363

  17. APP mRNA splicing is upregulated in the brain of biglycan transgenic mice.

    PubMed

    Bjelik, Annamária; Pákáski, Magdolna; Bereczki, Erika; Gonda, Szilvia; Juhász, Anna; Rimanóczy, Agnes; Zana, Marianna; Janka, Zoltán; Sántha, Miklós; Kálmán, János

    2007-01-01

    Many of the risk factors for cerebrovascular disease and atherosclerosis also increase the risk of Alzheimer's disease, characterized by the cerebral deposition of beta-amyloid plaques resulting from the abnormal processing of the transmembrane amyloid precursor protein (APP). The initiating event of cholesterol-induced atherosclerosis is the retention and accumulation of atherogenic apolipoprotein B (apoB) together with low-density lipoproteins in the vascular intima. Biglycan, a member of the small leucine-rich protein family, was suspected of contributing to this process. The individual and combined overexpressions of biglycan and apoB-100 were therefore examined on the cortical APP mRNA levels of transgenic mice by means of semiquantitative PCR. As compared with the control littermates, transgenic biglycan mice had significantly increased cortical APP695 (122%) and APP770 (157%) mRNA levels, while the double transgenic (apoB(+/-)xbiglycan(+/-)) mice did not exhibit any changes. These results provide the first experimental evidence that the atherogenic risk factor biglycan alters APP splicing and may participate in the pathogenesis of both Alzheimer and vascular dementias.

  18. Cis-Regulatory Variants Affect CHRNA5 mRNA Expression in Populations of African and European Ancestry

    PubMed Central

    Wang, Jen-Chyong; Spiegel, Noah; Bertelsen, Sarah; Le, Nhung; McKenna, Nicholas; Budde, John P.; Harari, Oscar; Kapoor, Manav; Brooks, Andrew; Hancock, Dana; Tischfield, Jay; Foroud, Tatiana; Bierut, Laura J.; Steinbach, Joe Henry; Edenberg, Howard J.; Traynor, Bryan J.; Goate, Alison M.

    2013-01-01

    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. PMID:24303001

  19. Dietary nickel chloride induces oxidative stress, apoptosis and alters Bax/Bcl-2 and caspase-3 mRNA expression in the cecal tonsil of broilers.

    PubMed

    Wu, Bangyuan; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Huang, Jianying

    2014-01-01

    The purpose of this study was to investigate the effects of dietary NiCl2 on antioxidant function, apoptosis, and the protein expression, mRNA expression and contents of the bcl-2, bax and caspase-3 in the cecal tonsil of broilers. 280 one-day-old avian broilers were divided into four groups and fed on a corn-soybean basal diet as control diet or the same basal diet supplemented with 300, 600 and 900 mg/kg of NiCl2 for 42 days. The activities of SOD, CAT and GSH-Px, and the ability to inhibit hydroxy radical, and GSH content were significantly decreased in all experimental groups. MDA content was significantly increased. The protein expression, mRNA expression and contents of bcl-2 were decreased, and bax and caspase-3 were increased in all experimental groups. The percentages of apoptotic lymphocytes were significantly increased. In conclusion, dietary NiCl2 in excess of 300 mg/kg caused oxidative stress, and then induced decreased the protein expression, mRNA expression and the contents of bcl-2, and increased protein expression, mRNA expression and the contents of bax and caspase-3 proteins in the cecal tonsil. The local intestinal mucosal immunity could finally be impaired due to the oxidative stress and apoptosis in the cecal tonsil caused by NiCl2. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. The effects of estradiol and selective estrogen receptor modulators on gene expression and messenger RNA stability in immortalized sheep endometrial stromal cells and human endometrial adenocarcinoma cells.

    PubMed

    Farnell, Yuhua Z; Ing, Nancy H

    2003-03-01

    The purpose of this study was to identify an endometrial cell line that maintained the E2 up-regulation of estrogen receptor (ER) mRNA by enhanced message stability and to assess its dependence on ER protein. Estradiol (E2) effects on gene expression were measured in three cell lines: one immortalized from sheep endometrial stroma (ST) and two from human endometrial adenocarcinomas (Ishikawa and ECC-1). E2 up-regulated ER mRNA levels in ST and Ishikawa cells, but down-regulated ER mRNA levels in ECC-1 cells. E2 up-regulated progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and transforming growth factor-alpha (TGF-alpha) in both Ishikawa and ECC-1 cells. The selective estrogen receptor modulator ICI 182,780 antagonized the E2-induced up-regulation of ER and/or PR mRNA levels in all three cells, while another, GW 5638, antagonized the up-regulation of PR mRNA in Ishikawa and ECC-1 cells. In mechanistic studies, E2 had no effect on ER mRNA stability in ST cells and it destabilized ER mRNA in ECC-1 cells. Thus, Ishikawa cells appear to be the most physiologically relevant cell line in which to study the up-regulation of ER mRNA levels by enhanced mRNA stability. Its antagonism by ICI 182,780 reveals that ER protein is involved in this E2 response.

  1. A snoRNA modulates mRNA 3′ end processing and regulates the expression of a subset of mRNAs

    PubMed Central

    Huang, Chunliu; Shi, Junjie; Guo, Yibin; Huang, Weijun; Huang, Shanshan; Ming, Siqi; Wu, Xingui; Zhang, Rui; Ding, Junjun; Zhao, Wei; Jia, Jie; Huang, Xi; Xiang, Andy Peng

    2017-01-01

    Abstract mRNA 3′ end processing is an essential step in gene expression. It is well established that canonical eukaryotic pre-mRNA 3′ processing is carried out within a macromolecular machinery consisting of dozens of trans-acting proteins. However, it is unknown whether RNAs play any role in this process. Unexpectedly, we found that a subset of small nucleolar RNAs (snoRNAs) are associated with the mammalian mRNA 3′ processing complex. These snoRNAs primarily interact with Fip1, a component of cleavage and polyadenylation specificity factor (CPSF). We have functionally characterized one of these snoRNAs and our results demonstrated that the U/A-rich SNORD50A inhibits mRNA 3′ processing by blocking the Fip1-poly(A) site (PAS) interaction. Consistently, SNORD50A depletion altered the Fip1–RNA interaction landscape and changed the alternative polyadenylation (APA) profiles and/or transcript levels of a subset of genes. Taken together, our data revealed a novel function for snoRNAs and provided the first evidence that non-coding RNAs may play an important role in regulating mRNA 3′ processing. PMID:28911119

  2. GDF-15 gene expression alterations in human lymphoblastoid cells and peripheral blood lymphocytes following exposure to ionizing radiation

    PubMed Central

    Li, Shuang; Zhang, Qing-Zhao; Zhang, De-Qin; Feng, Jiang-Bin; Luo, Qun; Lu, Xue; Wang, Xin-Ru; Li, Kun-Peng; Chen, De-Qing; Mu, Xiao-Feng; Gao, Ling; Liu, Qing-Jie

    2017-01-01

    The identification of rapid, sensitive and high-throughput biomarkers is imperative in order to identify individuals harmed by radiation accidents, and accurately evaluate the absorbed doses of radiation. DNA microarrays have previously been used to evaluate the alterations in growth/differentiation factor 15 (GDF15) gene expression in AHH-1 human lymphoblastoid cells, following exposure to γ-rays. The present study aimed to characterize the relationship between the dose of ionizing radiation and the produced effects in GDF-15 gene expression in AHH-1 cells and human peripheral blood lymphocytes (HPBLs). GDF-15 mRNA and protein expression levels following exposure to γ-rays and neutron radiation were assessed by reverse transcription-quantitative polymerase chain reaction and western blot analysis in AHH-1 cells. In addition, alterations in GDF-15 gene expression in HPBLs following ex vivo irradiation were evaluated. The present results demonstrated that GDF-15 mRNA and protein expression levels in AHH-1 cells were significantly upregulated following exposure to γ-ray doses ranging between 1 and 10 Gy, regardless of the dose rate. A total of 48 h following exposure to neutron radiation, a dose-response relationship was identified in AHH-1 cells at γ-ray doses between 0.4 and 1.6 Gy. GDF-15 mRNA levels in HPBLs were significantly upregulated following exposure to γ-ray doses between 1 and 8 Gy, within 4–48 h following irradiation. These results suggested that significant time- and dose-dependent alterations in GDF-15 mRNA and protein expression occur in AHH-1 cells and HPBLs in the early phases following exposure to ionizing radiation. In conclusion, alterations in GDF-15 gene expression may have potential as a biomarker to evaluate radiation exposure. PMID:28440431

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

    PubMed Central

    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

  4. Expression of CD30 mRNA, CD30L mRNA and a variant form of CD30 mRNA in restimulated peripheral blood mononuclear cells (PBMC) of patients with helminthic infections resembling a Th2 disease

    PubMed Central

    Kilwinski, J; Berger, T; Mpalaskas, J; Reuter, S; Flick, W; Kern, P

    1999-01-01

    It has been proposed that CD30, a member of the tumour necrosis factor (TNF) receptor superfamily, is preferentially up-regulated on Th2-type human T cells. In order to investigate a correlation between infection with Echinococcus multilocularis and CD30 expression, we analysed regulation of CD30 mRNA, a variant form of CD30 mRNA (CD30v) and CD30 ligand (CD30L) mRNA expression on PBMC from patients with alveolar echinococcosis (AE) using reverse transcriptase-polymerase chain reaction (RT-PCR). In PBMC of patients with AE as well as healthy donors, spontaneous expression of CD30L mRNA and the CD30v mRNA could be detected. However, the intact form of CD30 mRNA could be detected neither in freshly isolated PBMC of patients nor in PBMC of healthy individuals. Expression of CD30L mRNA and the variant form of CD30 mRNA was frequently detected at individual time points during 72 h of culture of PBMC stimulated with crude Echinococcus antigen. In contrast to CD30v or CD30L mRNA expression, induction of CD30 mRNA expression was detected only in three out of six (50%) healthy donors and in 10 out of 21 (48%) patients with alveolar echinococcosis after 72 h of incubation. As a control, mitogenic stimulation of PBMC of both healthy individuals and infected patients led to expression of intact CD30 mRNA within 24 h of culture. These data demonstrate the different expression of two different forms of CD30 mRNA in PBMC of human individuals. The specific induction of CD30 expression is correlated only in rare cases with the clinical status of patients with AE, indicating the lack of a general induction of CD30 mRNA in this Th2-type-dominated helminthic disease. The data provide further evidence that the CD30 receptor is not an exclusive marker for a Th2-type response. PMID:9933429

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

    PubMed Central

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

    2016-01-01

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

  6. PNPase autocontrols its expression by degrading a double-stranded structure in the pnp mRNA leader

    PubMed Central

    Jarrige, Anne-Charlotte; Mathy, Nathalie; Portier, Claude

    2001-01-01

    Polynucleotide phosphorylase synthesis is autocontrolled at a post-transcriptional level in an RNase III-dependent mechanism. RNase III cleaves a long stem–loop in the pnp leader, which triggers pnp mRNA instability, resulting in a decrease in the synthesis of polynucleotide phosphorylase. The staggered cleavage by RNase III removes the upper part of the stem–loop structure, creating a duplex with a short 3′ extension. Mutations or high temperatures, which destabilize the cleaved stem–loop, decrease expression of pnp, while mutations that stabilize the stem increase expression. We propose that the dangling 3′ end of the duplex created by RNase III constitutes a target for polynucleotide phosphorylase, which binds to and degrades the upstream half of this duplex, hence inducing pnp mRNA instability. Consistent with this interpretation, a pnp mRNA starting at the downstream RNase III processing site exhibits a very low level of expression, regardless of the presence of polynucleotide phosphorylase. Moreover, using an in vitro synthesized pnp leader transcript, it is shown that polynucleotide phosphorylase is able to digest the duplex formed after RNase III cleavage. PMID:11726520

  7. Poly A tail length analysis of in vitro transcribed mRNA by LC-MS.

    PubMed

    Beverly, Michael; Hagen, Caitlin; Slack, Olga

    2018-02-01

    The 3'-polyadenosine (poly A) tail of in vitro transcribed (IVT) mRNA was studied using liquid chromatography coupled to mass spectrometry (LC-MS). Poly A tails were cleaved from the mRNA using ribonuclease T1 followed by isolation with dT magnetic beads. Extracted tails were then analyzed by LC-MS which provided tail length information at single-nucleotide resolution. A 2100-nt mRNA with plasmid-encoded poly A tail lengths of either 27, 64, 100, or 117 nucleotides was used for these studies as enzymatically added poly A tails showed significant length heterogeneity. The number of As observed in the tails closely matched Sanger sequencing results of the DNA template, and even minor plasmid populations with sequence variations were detected. When the plasmid sequence contained a discreet number of poly As in the tail, analysis revealed a distribution that included tails longer than the encoded tail lengths. These observations were consistent with transcriptional slippage of T7 RNAP taking place within a poly A sequence. The type of RNAP did not alter the observed tail distribution, and comparison of T3, T7, and SP6 showed all three RNAPs produced equivalent tail length distributions. The addition of a sequence at the 3' end of the poly A tail did, however, produce narrower tail length distributions which supports a previously described model of slippage where the 3' end can be locked in place by having a G or C after the poly nucleotide region. Graphical abstract Determination of mRNA poly A tail length using magnetic beads and LC-MS.

  8. Influence of developmental stage and genotype on liver mRNA levels among wild, domesticated, and hybrid rainbow trout (Oncorhynchus mykiss).

    PubMed

    White, Samantha L; Sakhrani, Dionne; Danzmann, Roy G; Devlin, Robert H

    2013-10-02

    Release of domesticated strains of fish into nature may pose a threat to wild populations with respect to their evolved genetic structure and fitness. Understanding alterations that have occurred in both physiology and genetics as a consequence of domestication can assist in evaluating the risks posed by introgression of domesticated genomes into wild genetic backgrounds, however the molecular causes of these consequences are currently poorly defined. The present study has examined levels of mRNA in fast-growing pure domesticated (D), slow-growing age-matched pure wild (Wa), slow-growing size-matched pure wild (Ws), and first generation hybrid cross (W/D) rainbow trout (Oncorhynchus mykiss) to investigate the influence of genotype (domesticated vs. wild, and their interactions in hybrids) and developmental stage (age- or size-matched animals) on genetic responses (i.e. dominant vs. recessive) and specific physiological pathways. Highly significant differences in mRNA levels were found between domesticated and wild-type rainbow trout genotypes (321 mRNAs), with many mRNAs in the wild-domesticated hybrid progeny showing intermediate levels. Differences were also found between age-matched and size-matched wild-type trout groups (64 mRNAs), with unique mRNA differences for each of the wild-type groups when compared to domesticated trout (Wa: 114 mRNAs, Ws: 88 mRNAs), illustrating an influence of fish developmental stage affecting findings when used as comparator groups to other genotypes. Analysis of differentially expressed mRNAs (found for both wild-type trout to domesticated comparisons) among the genotypes indicates that 34.8% are regulated consistent with an additive genetic model, whereas 39.1% and 26.1% show a recessive or dominant mode of regulation, respectively. These molecular data are largely consistent with phenotypic data (growth and behavioural assessments) assessed in domesticated and wild trout strains. The present molecular data are concordant with

  9. Characterization of rat calcitonin mRNA.

    PubMed Central

    Amara, S G; David, D N; Rosenfeld, M G; Roos, B A; Evans, R M

    1980-01-01

    A chimeric plasmic containing cDNA complementary to rat calcitonin mRNA has been constructed. Partial sequence analysis shows that the insert contains a nucleotide sequence encoding the complete amino acid sequence of calcitonin. Two basic amino acids precede and three basic amino acids follow the hormone sequence, suggesting that calcitonin is generated by the proteolytic cleavage of a larger precursor in a manner analogous to that of other small polypeptide hormones. The COOH-terminal proline, known to be amidated in the secreted hormone, is followed by a glycine in the precursor. The cloned calcitonin DNA was used to characterize the expression of calcitonin mRNA. Cytoplasmic mRNAs from calcitonin-producing rat medullary thyroid carcinoma lines and from normal rat thyroid glands contain a single species, 1050 nucleotides long, whch hybridizes to the cloned calcitonin cDNA. The concentration of calcitonin mRNA sequences is greater in those tumors that produce larger amounts of immunoreactive calcitonin. RNAs from other endocrine tissues, including anterior and neurointermediate lobes of rat pituitary, contain no detectable calcitonin mRNA. Images PMID:6933496

  10. Effects of Age on Bone mRNA Levels of Sclerostin and Other Genes Relevant to Bone Metabolism in Humans

    PubMed Central

    Roforth, Matthew M.; Fujita, Koji; McGregor, Ulrike I.; Kirmani, Salman; McCready, Louise K.; Peterson, James M.; Drake, Matthew T.; Monroe, David G.; Khosla, Sundeep

    2013-01-01

    Although aging is associated with a decline in bone formation in humans, the molecular pathways contributing to this decline remain unclear. Several previous clinical studies have shown that circulating sclerostin levels increase with age, raising the possibility that increased production of sclerostin by osteocytes leads to the age-related impairment in bone formation. Thus, in the present study, we examined circulating sclerostin levels as well as bone mRNA levels of sclerostin using quantitative polymerase chain reaction (QPCR) analyses in needle bone biopsies from young (mean age, 30.0 years) versus old (mean age, 72.9 years) women. In addition, we analyzed the expression of genes in a number of pathways known to be altered with skeletal aging, based largely on studies in mice. While serum sclerostin levels were 46% higher (p < 0.01) in the old as compared to the young women, bone sclerostin mRNA levels were no different between the two groups (p = 0.845). However, genes related to notch signaling were significantly upregulated (p = 0.003 when analyzed as a group) in the biopsies from the old women. In an additional analysis of 118 genes including those from genome-wide association studies related to bone density and/or fracture, BMP/TGFβ family genes, selected growth factors and nuclear receptors, and Wnt/Wnt-related genes, we found that mRNA levels of the Wnt inhibitor, SFRP1, were significantly increased (by 1.6-fold, p = 0.0004, false discovery rate [q] = 0.04) in the biopsies from the old as compared to the young women. Our findings thus indicate that despite increases in circulating sclerostin levels, bone sclerostin mRNA levels do not increase in elderly women. However, aging is associated with alterations in several key pathways and genes in humans that may contribute to the observed impairment in bone formation. These include notch signaling, which represents a potential therapeutic target for increasing bone formation in humans. Our studies further

  11. Regulation of diacylglycerol acyltransferase 2 protein stability by gp78-associated endoplasmic-reticulum-associated degradation.

    PubMed

    Choi, Kwangman; Kim, Hyeongki; Kang, Hyunju; Lee, So-Young; Lee, Sang Jun; Back, Sung Hoon; Lee, Seo Hyun; Kim, M Sun; Lee, Jeong Eun; Park, Ju Young; Kim, Jiye; Kim, Sunhong; Song, Jae-Hyung; Choi, Yura; Lee, Suui; Lee, Hyun-Jun; Kim, Jong Heon; Cho, Sungchan

    2014-07-01

    Triacylglycerol (TG) is the major form of stored energy in eukaryotic organisms and is synthesized by diacylglycerol acyltransferase (DGAT) in the endoplasmic reticulum (ER). DGAT2, one of the two DGAT enzymes, is barely detectable in cells, even though its mRNA transcripts are maintained at considerable levels. However, little is known about how DGAT2 expression is altered by protein stability. DGAT2 was highly unstable in cells and was rapidly degraded by proteasomes in an ubiquitin-dependent manner. Deletion mutation analysis identified transmembrane domain 1 (TMD1) as a protein degradation signal. TMD1 is also important for ER localization of DGAT2. Moreover, DGAT2 interacted with p97/VCP, a crucial component of the ER-associated degradation (ERAD) pathway, and polyubiquitinated DGAT2 accumulated following treatment with an ERAD inhibitor. Furthermore, gp78, an E3 ligase involved in ERAD, regulates the degradation of DGAT2 through direct interactions and ubiquitination. Consequently, the stabilization of DGAT2 increased the number of lipid droplets in hepatic cells. Therefore, DGAT2 is regulated by gp78-associated ERAD at the post-translational level. © 2014 FEBS.

  12. Hsp70 Is a Novel Posttranscriptional Regulator of Gene Expression That Binds and Stabilizes Selected mRNAs Containing AU-Rich Elements

    PubMed Central

    Kishor, Aparna; Tandukar, Bishal; Ly, Yann V.; Toth, Eric A.; Suarez, Yvelisse; Brewer, Gary

    2013-01-01

    The AU-rich elements (AREs) encoded within many mRNA 3′ untranslated regions (3′UTRs) are targets for factors that control transcript longevity and translational efficiency. Hsp70, best known as a protein chaperone with well-defined peptide-refolding properties, is known to interact with ARE-like RNA substrates in vitro. Here, we show that cofactor-free preparations of Hsp70 form direct, high-affinity complexes with ARE substrates based on specific recognition of U-rich sequences by both the ATP- and peptide-binding domains. Suppressing Hsp70 in HeLa cells destabilized an ARE reporter mRNA, indicating a novel ARE-directed mRNA-stabilizing role for this protein. Hsp70 also bound and stabilized endogenous ARE-containing mRNAs encoding vascular endothelial growth factor (VEGF) and Cox-2, which involved a mechanism that was unaffected by an inhibitor of its protein chaperone function. Hsp70 recognition and stabilization of VEGF mRNA was mediated by an ARE-like sequence in the proximal 3′UTR. Finally, stabilization of VEGF mRNA coincided with the accumulation of Hsp70 protein in HL60 promyelocytic leukemia cells recovering from acute thermal stress. We propose that the binding and stabilization of selected ARE-containing mRNAs may contribute to the cytoprotective effects of Hsp70 following cellular stress but may also provide a novel mechanism linking constitutively elevated Hsp70 expression to the development of aggressive neoplastic phenotypes. PMID:23109422

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

    PubMed

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

    2014-01-01

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

  14. Dynamic Proteomics Emphasizes the Importance of Selective mRNA Translation and Protein Turnover during Arabidopsis Seed Germination*

    PubMed Central

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

    2014-01-01

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

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

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

    Higgins, G.A.; Lewis, D.A.; Bahmanyar, S.

    1988-02-01

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

  16. Species dispersal rates alter diversity and ecosystem stability in pond metacommunities.

    PubMed

    Howeth, Jennifer G; Leibold, Mathew A

    2010-09-01

    Metacommunity theory suggests that relationships between diversity and ecosystem stability can be determined by the rate of species dispersal among local communities. The predicted relationships, however, may depend upon the relative strength of local environmental processes and disturbance. Here we evaluate the role of dispersal frequency and local predation perturbations in affecting patterns of diversity and stability in pond plankton metacommunities. Pond metacommunities were composed of three mesocosm communities: one of the three communities maintained constant "press" predation from a selective predator, bluegill sunfish (Lepomis macrochirus); the second community maintained "press" conditions without predation; and the third community experienced recurrent "pulsed" predation from bluegill sunfish. The triads of pond communities were connected at either no, low (0.7%/d), or high (20%/d) planktonic dispersal. Richness and composition of zooplankton and stability of plankton biomass and ecosystem productivity were measured at local and regional spatial scales. Dispersal significantly affected diversity such that local and regional biotas at the low dispersal rate maintained the greatest number of species. The unimodal local dispersal-diversity relationship was predator-dependent, however, as selective press predation excluded species regardless of dispersal. Further, there was no effect of dispersal on beta diversity because predation generated local conditions that selected for distinct community assemblages. Spatial and temporal ecosystem stability responded to dispersal frequency but not predation. Low dispersal destabilized the spatial stability of producer biomass but stabilized temporal ecosystem productivity. The results indicate that selective predation can prevent species augmentation from mass effects but has no apparent influence on stability. Dispersal rates, in contrast, can have significant effects on both species diversity and ecosystem

  17. Striatal output markers do not alter in response to circling behaviour in 6-OHDA lesioned rats produced by acute or chronic administration of the monoamine uptake inhibitor BTS 74 398.

    PubMed

    Lane, E L; Cheetham, S; Jenner, P

    2008-01-01

    The monoamine uptake inhibitor BTS 74 398 induces ipsilateral circling in 6-hydroxydopamine (6-OHDA) lesioned rats without induction of abnormal motor behaviours associated with L-dopa administration. We examined whether this was reflected in the expression of peptide mRNA in the direct and indirect striatal output pathways.6-OHDA lesioning of the nigrostriatal pathway increased striatal expression of PPE-A mRNA and decreased levels of PPT mRNA with PPE-B mRNA expression remaining unchanged. Acute L-dopa administration normalised PPE-A mRNA and elevated PPT mRNA while PPE-B mRNA expression remained unchanged. Acute administration of BTS 74 398 did not alter striatal peptide mRNA levels. Following chronic treatment with L-dopa, PPE-A mRNA expression in the lesioned striatum continued to be normalised and PPT mRNA was increased compared to the intact side. PPE-B mRNA expression was also markedly increased relative to the non-lesioned striatum. Chronic BTS 74 398 administration did not alter mRNA expression in the 6-OHDA lesioned striatum although small increases in PPT mRNA expression in the intact and sham lesioned striatum were observed. The failure of BTS 74 398 to induce changes in striatal neuropeptide mRNA correlated with its failure to induce abnormal motor behaviours or behavioural sensitisation but does not explain how it produces a reversal of motor deficits. An action in another area of the brain appears likely and may explain the subsequent failure of BTS 74 398 and related compounds to exert anti-parkinsonian actions in man.

  18. miR-128 inhibits telomerase activity by targeting TERT mRNA

    PubMed Central

    Guzman, Herlinda; Sanders, Katie; Idica, Adam; Bochnakian, Aurore; Jury, Douglas; Daugaard, Iben; Zisoulis, Dimitrios G; Pedersen, Irene Munk

    2018-01-01

    Telomerase is a unique cellular reverse transcriptase (RT) essential for maintaining telomere stability and required for the unlimited proliferation of cancer cells. The limiting determinant of telomerase activity is the catalytic component TERT, and TERT expression is closely correlated with telomerase activity and cancer initiation and disease progression. For this reason the regulation of TERT levels in the cell is of great importance. microRNAs (miRs) function as an additional regulatory level in cells, crucial for defining expression boundaries, proper cell fate decisions, cell cycle control, genome integrity, cell death and metastasis. We performed an anti-miR library screen to identity novel miRs, which participate in the control of telomerase. We identified the tumor suppressor miR (miR-128) as a novel endogenous telomerase inhibitor and determined that miR-128 significantly reduces the mRNA and protein levels of Tert in a panel of cancer cell lines. We further evaluated the mechanism by which miR-128 regulates TERT and demonstrated that miR-128 interacts directly with the coding sequence of TERT mRNA in both HeLa cells and teratoma cells. Interestingly, the functional miR-128 binding site in TERT mRNA, is conserved between TERT and the other cellular reverse transcriptase encoded by Long Interspersed Elements-1 (LINE-1 or L1), which can also contribute to the oncogenic phenotype of cancer. This finding supports the novel idea that miRs may function in parallel pathways to inhibit tumorigenesis, by regulating a group of enzymes (such as RT) by targeting conserved binding sites in the coding region of both enzymes. PMID:29568354

  19. Conserved small mRNA with an unique, extended Shine-Dalgarno sequence

    PubMed Central

    Hahn, Julia; Migur, Anzhela; von Boeselager, Raphael Freiherr; Kubatova, Nina; Kubareva, Elena; Schwalbe, Harald

    2017-01-01

    ABSTRACT Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3′-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level. PMID:27834614

  20. mRNA Molecules Containing Murine Leukemia Virus Packaging Signals Are Encapsidated as Dimers

    PubMed Central

    Hibbert, Catherine S.; Mirro, Jane; Rein, Alan

    2004-01-01

    Prior work by others has shown that insertion of ψ (i.e., leader) sequences from the Moloney murine leukemia virus (MLV) genome into the 3′ untranslated region of a nonviral mRNA leads to the specific encapsidation of this RNA in MLV particles. We now report that these RNAs are, like genomic RNAs, encapsidated as dimers. These dimers have the same thermostability as MLV genomic RNA dimers; like them, these dimers are more stable if isolated from mature virions than from immature virions. We characterized encapsidated mRNAs containing deletions or truncations of MLV ψ or with ψ sequences from MLV-related acute transforming viruses. The results indicate that the dimeric linkage in genomic RNA can be completely attributed to the ψ region of the genome. While this conclusion agrees with earlier electron microscopic studies on mature MLV dimers, it is the first evidence as to the site of the linkage in immature dimers for any retrovirus. Since the Ψ+ mRNA is not encapsidated as well as genomic RNA, it is only present in a minority of virions. The fact that it is nevertheless dimeric argues strongly that two of these molecules are packaged into particles together. We also found that the kissing loop is unnecessary for this coencapsidation or for the stability of mature dimers but makes a major contribution to the stability of immature dimers. Our results are consistent with the hypothesis that the packaging signal involves a dimeric structure in which the RNAs are joined by intermolecular interactions between GACG loops. PMID:15452213

  1. A nonimprinted Prader-Willi Syndrome (PWS)-region gene regulates a different chromosomal domain in trans but the imprinted pws loci do not alter genome-wide mRNA levels.

    PubMed

    Stefan, Mihaela; Portis, Toni; Longnecker, Richard; Nicholls, Robert D

    2005-05-01

    Prader-Willi syndrome (PWS) is a complex neurobehavioral disorder that results from loss of function of 10 clustered, paternally expressed genes in a 1.5-Mb region of chromosome 15q11-q13. Many of the primary PWS region genes appear to have nuclear RNA regulatory functions, suggesting that multiple genetic pathways could be secondarily affected in PWS. Using a transgenic mouse model of PWS (TgPWS) with an approximately 4-Mb chromosome 7C deletion of paternal origin that models the neonatal phenotype of the human syndrome we compared by oligonucleotide microarrays expression levels of approximately 12,000 genes and ESTs in TgPWS and wild-type brain. Hybridization data were processed with two distinct statistical algorithms and revealed a dramatically reduced expression of 4 imprinted genes within the deletion region in TgPWS mice, with 2 nonimprinted, codeleted genes reduced twofold. However, only 3 genes outside the deletion were significantly altered in TgPWS mouse brain, with approximately 1.5-fold up-regulation of mRNA levels. Remarkably, these genes map to a single chromosome domain (18B3), and by quantitative RT-PCR we show that 8 genes in this domain are up-regulated in TgPWS brain. These 18B3 genes were up-regulated in an equivalent manner in Angelman syndrome mouse (TgAS) brain, which has the same deletion but of maternal origin. Therefore, the trans-regulation of the chromosome 18B3 domain is due to decreased expression of a nonimprinted gene within the TgPWS/AS mouse deletion in mouse chromosome 7C. Most surprisingly, since 48-60% of the genome was screened, it appears that the imprinted mouse PWS loci do not widely regulate mRNA levels of other genes and may regulate RNA structure.

  2. Stress exposure alters brain mRNA expression of the genes involved in insulin signalling, an effect modified by a high fat/high fructose diet and cinnamon supplement

    PubMed Central

    Qin, Bolin; Arvy, Nathalie; Poulet, Laurent; Batandier, Cécile; Roussel, Anne-Marie; Anderson, Richard A.

    2018-01-01

    In occidental societies, high fat and high sugar diets often coincide with episodes of stress. The association is likely to modify brain energy control. Brain insulin signalling is rarely studied in stressed individuals consuming high fat diets. Furthermore the effects of cinnamon supplement are not known in these conditions. Therefore, we exposed rats, over a 12-week period, to a control (C) or a high fat/high fructose (HF/HFr) diet that induces peripheral insulin resistance. A cinnamon supplement (C+CN and HF/HFr +CN) was added or not. After diet exposure, one group of rats was exposed to a 30-min restraint followed by a 10-min open-field test, their combination featuring a moderate stressor, the other rats staying unstressed in their home cages. The insulin signalling in hippocampus and frontal cortex was studied through the mRNA expression of the following genes: insulin receptor (Ir), insulin receptor substrate (Irs1), glucose transporters (Glut1 and Glut3), glycogen synthase (Gys1) and their modulators, Akt1 and Pten. In C rats, stress enhanced the expression of Ir, Irs1, Glut1, Gys1 and Akt1 mRNA. In C+CN rats, stress induced an increase in Pten but a decrease in Gys1 mRNA expression. In HF/HFr rats, stress was associated with an increase in Pten mRNA expression. In HF/HFr+CN rats, stress increased Pten mRNA expression but also decreased Gys1 mRNA expression. This suggests that a single moderate stress favours energy refilling mechanisms, an effect blunted by a previous HF/HFr diet and cinnamon supplement. PMID:29813096

  3. Stress exposure alters brain mRNA expression of the genes involved in insulin signalling, an effect modified by a high fat/high fructose diet and cinnamon supplement.

    PubMed

    Canini, Frédéric; Qin, Bolin; Arvy, Nathalie; Poulet, Laurent; Batandier, Cécile; Roussel, Anne-Marie; Anderson, Richard A

    2018-01-01

    In occidental societies, high fat and high sugar diets often coincide with episodes of stress. The association is likely to modify brain energy control. Brain insulin signalling is rarely studied in stressed individuals consuming high fat diets. Furthermore the effects of cinnamon supplement are not known in these conditions. Therefore, we exposed rats, over a 12-week period, to a control (C) or a high fat/high fructose (HF/HFr) diet that induces peripheral insulin resistance. A cinnamon supplement (C+CN and HF/HFr +CN) was added or not. After diet exposure, one group of rats was exposed to a 30-min restraint followed by a 10-min open-field test, their combination featuring a moderate stressor, the other rats staying unstressed in their home cages. The insulin signalling in hippocampus and frontal cortex was studied through the mRNA expression of the following genes: insulin receptor (Ir), insulin receptor substrate (Irs1), glucose transporters (Glut1 and Glut3), glycogen synthase (Gys1) and their modulators, Akt1 and Pten. In C rats, stress enhanced the expression of Ir, Irs1, Glut1, Gys1 and Akt1 mRNA. In C+CN rats, stress induced an increase in Pten but a decrease in Gys1 mRNA expression. In HF/HFr rats, stress was associated with an increase in Pten mRNA expression. In HF/HFr+CN rats, stress increased Pten mRNA expression but also decreased Gys1 mRNA expression. This suggests that a single moderate stress favours energy refilling mechanisms, an effect blunted by a previous HF/HFr diet and cinnamon supplement.

  4. Contributions of testosterone and territory ownership to sexually-motivated behaviors and mRNA expression in the medial preoptic area of male European starlings.

    PubMed

    Spool, Jeremy A; Stevenson, Sharon A; Angyal, Caroline S; Riters, Lauren V

    2016-11-01

    Animals integrate social information with their internal endocrine state to control the timing of behavior, but how these signals are integrated in the brain is not understood. The medial preoptic area (mPOA) may play an integrative role in the control of courtship behavior, as it receives projections from multiple sensory systems, and is central to the hormonal control of courtship behavior across vertebrates. Additionally, data from many species implicate opioid and dopaminergic systems in the mPOA in the control of male courtship behavior. We used European starlings to test the hypothesis that testosterone (T) and social status (in the form of territory possession) interact to control the timing of courtship behavior by modulating steroid hormone-, opioid- and dopaminergic-related gene expression in the mPOA. We found that only males given both T and a nesting territory produced high rates of courtship behavior in response to a female. T treatment altered patterns of gene expression in the mPOA by increasing androgen receptor, aromatase, mu-opioid receptor and preproenkephalin mRNA and decreasing tyrosine hydroxylase mRNA expression. Territory possession did not alter mRNA expression in the mPOA, despite the finding that only birds with both T and a nesting territory produced courtship behavior. We propose that T prepares the mPOA to respond to the presence of a female with high rates of courtship song by altering gene expression, but that activity in the mPOA is under a continuous (i.e. tonic) inhibition until a male starling obtains a nesting territory. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Impact of obesity on 7,12-dimethylbenz[a]anthracene-induced altered ovarian connexin gap junction proteins in female mice

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

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Nteeba, Jackson, E-mail: nteeba@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    The ovarian gap junction proteins alpha 4 (GJA4 or connexin 37; CX37), alpha 1 (GJA1 or connexin 43; CX43) and gamma 1 (GJC1 or connexin 45; CX45) are involved in cell communication and folliculogenesis. 7,12-dimethylbenz[a]anthracene (DMBA) alters Cx37 and Cx43 expression in cultured neonatal rat ovaries. Additionally, obesity has an additive effect on DMBA-induced ovarian cell death and follicle depletion, thus, we investigated in vivo impacts of obesity and DMBA on CX protein levels. Ovaries were collected from lean and obese mice aged 6, 12, 18, or 24 wks. A subset of 18 wk old mice (lean and obese) weremore » dosed with sesame oil or DMBA (1 mg/kg; ip) for 14 days and ovaries collected 3 days thereafter. Cx43 and Cx45 mRNA and protein levels decreased (P < 0.05) after 18 wks while Cx37 mRNA and protein levels decreased (P < 0.05) after 24 wks in obese ovaries. Cx37 mRNA and antral follicle protein staining intensity were reduced (P < 0.05) by obesity while total CX37 protein was reduced (P < 0.05) in DMBA exposed obese ovaries. Cx43 mRNA and total protein levels were decreased (P < 0.05) by DMBA in both lean and obese ovaries while basal protein staining intensity was reduced (P < 0.05) in obese controls. Cx45 mRNA, total protein and protein staining intensity level were decreased (P < 0.05) by obesity. These data support that obesity temporally alters gap junction protein expression and that DMBA-induced ovotoxicity may involve reduced gap junction protein function. - Highlights: • Ovarian gap junction proteins are affected by ovarian aging and obesity. • DMBA exposure negatively impacts gap junction proteins. • Altered gap junction proteins may contribute to infertility.« less

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

    PubMed

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

    2016-09-01

    Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells. © 2016 Hollerer et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  7. Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells.

    PubMed

    Kawasaki, Shunsuke; Fujita, Yoshihiko; Nagaike, Takashi; Tomita, Kozo; Saito, Hirohide

    2017-07-07

    Synthetic biology has great potential for future therapeutic applications including autonomous cell programming through the detection of protein signals and the production of desired outputs. Synthetic RNA devices are promising for this purpose. However, the number of available devices is limited due to the difficulty in the detection of endogenous proteins within a cell. Here, we show a strategy to construct synthetic mRNA devices that detect endogenous proteins in living cells, control translation and distinguish cell types. We engineered protein-binding aptamers that have increased stability in the secondary structures of their active conformation. The designed devices can efficiently respond to target proteins including human LIN28A and U1A proteins, while the original aptamers failed to do so. Moreover, mRNA delivery of an LIN28A-responsive device into human induced pluripotent stem cells (hiPSCs) revealed that we can distinguish living hiPSCs and differentiated cells by quantifying endogenous LIN28A protein expression level. Thus, our endogenous protein-driven RNA devices determine live-cell states and program mammalian cells based on intracellular protein information. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    PubMed Central

    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

  9. Spaceflight has compartment- and gene-specific effects on mRNA levels for bone matrix proteins in rat femur

    NASA Technical Reports Server (NTRS)

    Evans, G. L.; Morey-Holton, E.; Turner, R. T.

    1998-01-01

    In the present study, we evaluated the possibility that the abnormal bone matrix produced during spaceflight may be associated with reduced expression of bone matrix protein genes. To test this possibility, we investigated the effects of a 14-day spaceflight (SLS-2 experiment) on steady-state mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), osteocalcin, osteonectin, and prepro-alpha(1) subunit of type I collagen in the major bone compartments of rat femur. There were pronounced site-specific differences in the steady-state levels of expression of the mRNAs for the three bone matrix proteins and GAPDH in normal weight-bearing rats, and these relationships were altered after spaceflight. Specifically, spaceflight resulted in decreases in mRNA levels for GAPDH (decreased in proximal metaphysis), osteocalcin (decreased in proximal metaphysis), osteonectin (decreased in proximal and distal metaphysis), and collagen (decreased in proximal and distal metaphysis) compared with ground controls. There were no changes in mRNA levels for matrix proteins or GAPDH in the shaft and distal epiphysis. These results demonstrate that spaceflight leads to site- and gene-specific decreases in mRNA levels for bone matrix proteins. These findings are consistent with the hypothesis that spaceflight-induced decreases in bone formation are caused by concomitant decreases in expression of genes for bone matrix proteins.

  10. 3'UTR AU-Rich Elements (AREs) and the RNA-Binding Protein Tristetraprolin (TTP) Are Not Required for the LPS-Mediated Destabilization of Phospholipase-Cβ-2 mRNA in Murine Macrophages.

    PubMed

    Shukla, Smita; Elson, Genie; Blackshear, Perry J; Lutz, Carol S; Leibovich, S Joseph

    2017-04-01

    We have shown previously that bacterial lipopolysaccharide (LPS)-mediated suppression of phospholipase-Cβ-2 (PLCβ-2) expression is involved in M1 (inflammatory) to M2-like (wound healing) phenotypic switching of macrophages triggered by adenosine. This suppression is mediated post-transcriptionally by destabilization of PLCβ-2 mRNA (messenger ribonucleic acid). To investigate the mechanism of this LPS-mediated destabilization, we examined the roles of RNA-binding agents including microRNAs and RNA-binding proteins that are involved in regulating stability of mRNAs encoding growth factors, inflammatory mediators, and proto-oncogenes. Adenylate and uridylate (AU)-rich elements (AREs) in 3'UTRs are specific recognition sites for RNA-binding proteins including tristetraprolin (TTP), HuR, and AUF1 and for microRNAs that are involved in regulating mRNA stability. In this study, we investigated the role of TTP and AREs in regulating PLCβ-2 mRNA stability. The 3'UTR of the PLCβ-2 gene was inserted into the pLightswitch luciferase reporter plasmid and transfected into RAW264.7 cells. LPS suppressed luciferase expression from this reporter. Luciferase expression from mutant 3'UTR constructs lacking AREs was similarly downregulated, suggesting that these regions are not required for LPS-mediated suppression of PLCβ-2. TTP was rapidly upregulated in both primary murine macrophages and RAW264.7 cells in response to LPS. Suppression of PLCβ-2 by LPS was examined using macrophages from mice lacking TTP (TTP -/- ). LPS suppressed PLCβ-2 expression to the same extent in wild type (WT) and TTP -/- macrophages. Also, the rate of decay of PLCβ-2 mRNA in LPS-treated macrophages following transcriptional blockade was similar in WT and TTP -/- macrophages, clearly indicating that TTP is not involved in LPS-mediated destabilization of PLCβ-2 mRNA in macrophages.

  11. A large-scale forest fragmentation experiment: the Stability of Altered Forest Ecosystems Project.

    PubMed

    Ewers, Robert M; Didham, Raphael K; Fahrig, Lenore; Ferraz, Gonçalo; Hector, Andy; Holt, Robert D; Kapos, Valerie; Reynolds, Glen; Sinun, Waidi; Snaddon, Jake L; Turner, Edgar C

    2011-11-27

    Opportunities to conduct large-scale field experiments are rare, but provide a unique opportunity to reveal the complex processes that operate within natural ecosystems. Here, we review the design of existing, large-scale forest fragmentation experiments. Based on this review, we develop a design for the Stability of Altered Forest Ecosystems (SAFE) Project, a new forest fragmentation experiment to be located in the lowland tropical forests of Borneo (Sabah, Malaysia). The SAFE Project represents an advance on existing experiments in that it: (i) allows discrimination of the effects of landscape-level forest cover from patch-level processes; (ii) is designed to facilitate the unification of a wide range of data types on ecological patterns and processes that operate over a wide range of spatial scales; (iii) has greater replication than existing experiments; (iv) incorporates an experimental manipulation of riparian corridors; and (v) embeds the experimentally fragmented landscape within a wider gradient of land-use intensity than do existing projects. The SAFE Project represents an opportunity for ecologists across disciplines to participate in a large initiative designed to generate a broad understanding of the ecological impacts of tropical forest modification.

  12. A large-scale forest fragmentation experiment: the Stability of Altered Forest Ecosystems Project

    PubMed Central

    Ewers, Robert M.; Didham, Raphael K.; Fahrig, Lenore; Ferraz, Gonçalo; Hector, Andy; Holt, Robert D.; Kapos, Valerie; Reynolds, Glen; Sinun, Waidi; Snaddon, Jake L.; Turner, Edgar C.

    2011-01-01

    Opportunities to conduct large-scale field experiments are rare, but provide a unique opportunity to reveal the complex processes that operate within natural ecosystems. Here, we review the design of existing, large-scale forest fragmentation experiments. Based on this review, we develop a design for the Stability of Altered Forest Ecosystems (SAFE) Project, a new forest fragmentation experiment to be located in the lowland tropical forests of Borneo (Sabah, Malaysia). The SAFE Project represents an advance on existing experiments in that it: (i) allows discrimination of the effects of landscape-level forest cover from patch-level processes; (ii) is designed to facilitate the unification of a wide range of data types on ecological patterns and processes that operate over a wide range of spatial scales; (iii) has greater replication than existing experiments; (iv) incorporates an experimental manipulation of riparian corridors; and (v) embeds the experimentally fragmented landscape within a wider gradient of land-use intensity than do existing projects. The SAFE Project represents an opportunity for ecologists across disciplines to participate in a large initiative designed to generate a broad understanding of the ecological impacts of tropical forest modification. PMID:22006969

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

    PubMed Central

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

    2011-01-01

    Insulin-like growth factor-1 (IGF-1) signaling is important for the maintenance of plaque stability in atherosclerosis due to its effects on vascular smooth muscle cell (vSMC) phenotype. To investigate this hypothesis, we studied the effects of the highly inflammatory milieu of the atherosclerotic plaque on IGF-1 signaling and stability-related phenotypic parameters of murine vSMCs in vitro, and the effects of IGF-1 supplementation on plaque phenotype in an atherosclerotic mouse model. M1-polarized, macrophage-conditioned medium inhibited IGF-1 signaling by ablating IGF-1 and increasing IGF-binding protein 3, increased vSMC apoptosis, and decreased proliferation. Expression of α-actin and col3a1 genes was strongly attenuated by macrophage-conditioned medium, whereas expression of matrix-degrading enzymes was increased. Importantly, all of these effects could be corrected by supplementation with IGF-1. In vivo, treatment with the stable IGF-1 analog Long R3 IGF-1 in apolipoprotein E knockout mice reduced stenosis and core size, and doubled cap/core ratio in early atherosclerosis. In advanced plaques, Long R3 IGF-1 increased the vSMC content of the plaque by more than twofold and significantly reduced the rate of intraplaque hemorrhage. We believe that IGF-1 in atherosclerotic plaques may have a role in preventing plaque instability, not only by modulating smooth muscle cell turnover, but also by altering smooth muscle cell phenotype. PMID:21281823

  14. Mutants of the Paf1 Complex Alter Phenotypic Expression of the Yeast Prion [PSI+

    PubMed Central

    Strawn, Lisa A.; Lin, Changyi A.; Tank, Elizabeth M.H.; Osman, Morwan M.; Simpson, Sarah A.

    2009-01-01

    The yeast [PSI+] prion is an epigenetic modifier of translation termination fidelity that causes nonsense suppression. The prion [PSI+] forms when the translation termination factor Sup35p adopts a self-propagating conformation. The presence of the [PSI+] prion modulates survivability in a variety of growth conditions. Nonsense suppression is essential for many [PSI+]-mediated phenotypes, but many do not appear to be due to read-through of a single stop codon, but instead are multigenic traits. We hypothesized that other global mechanisms act in concert with [PSI+] to influence [PSI+]-mediated phenotypes. We have identified one such global regulator, the Paf1 complex (Paf1C). Paf1C is conserved in eukaryotes and has been implicated in several aspects of transcriptional and posttranscriptional regulation. Mutations in Ctr9p and other Paf1C components reduced [PSI+]-mediated nonsense suppression. The CTR9 deletion also alters nonsense suppression afforded by other genetic mutations but not always to the same extent as the effects on [PSI+]-mediated read-through. Our data suggest that the Paf1 complex influences mRNA translatability but not solely through changes in transcript stability or abundance. Finally, we demonstrate that the CTR9 deletion alters several [PSI+]-dependent phenotypes. This provides one example of how [PSI+] and genetic modifiers can interact to uncover and regulate phenotypic variability. PMID:19225160

  15. The poly(A) tail length of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of milk.

    PubMed Central

    Kuraishi, T; Sun, Y; Aoki, F; Imakawa, K; Sakai, S

    2000-01-01

    The length of casein mRNA from the lactating mouse mammary gland, as assessed on Northern blots, is shorter after weaning, but is elongated following the removal of milk. In order to investigate this phenomenon, the molecular structures of beta- and gamma-casein mRNAs were analysed. The coding and non-coding regions of the two forms were the same length, but the long form of casein mRNA had a longer poly(A) tail than the short form (P<0.05). In order to examine the stability of casein mRNA under identical conditions, casein mRNAs with the long and short poly(A) tails were incubated in the rabbit reticulocyte lysate (RRL) cell-free translation system. Casein mRNA with the long poly(A) tail had a longer half-life than that with the short tail (P<0.05). The beta- and gamma-casein mRNAs were first degraded into 0.92 and 0.81 kb fragments respectively. With undegraded mRNA, the poly(A) tail shortening by exoribonuclease was not observed until the end of the incubation. Northern blot analysis showed that casein mRNA with the long poly(A) tail was protected efficiently from endoribonucleases. We conclude that the length of the poly(A) tail of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of the gland's milk, and we show that the longer poly(A) tail potentially protects the mRNA from degradation by endoribonucleases. PMID:10749689

  16. Noise stress changes mRNA expressions of corticotropin-releasing hormone, its receptors in amygdala, and anxiety-related behaviors.

    PubMed

    Eraslan, Evren; Akyazi, Ibrahim; Erg L-Ekiz, Elif; Matur, Erdal

    2015-01-01

    Noise is a psychological, environmental stressor that activates limbic sites in the brain. Limbic sites such as the amygdala and the amygdaloid corticotropin-releasing hormone (CRH) system play an important role in integrating stress response. We investigated the association between noise exposures, CRH-related molecules in the amygdala, and behavioral alterations. In total 54 Sprague-Dawley rats were divided into the following three groups: Control (CON), acute noise exposure (ANE), and chronic noise exposure (CNE). The ANE group was exposed to 100 dB white noise only once in 4 h and the CNE group was exposed to the same for 4 h per day for 30 days. Expression profiles of CRH and its receptors CRH-R1 and CRH-R2 were analyzed by quantitative real-time polymerase chain reaction (qPCR). The same stress procedure was applied to the ANE and CNE groups for behavior testing. The anxiety responses of the animals after acute and chronic stress exposure were measured in the defensive withdrawal test. CNE upregulated CRH and CRH-R1 mRNA levels but downregulated CRH-R2 mRNA levels. ANE led to a decrease in both CRH-R1 and CRH-R2 expression. In the defensive withdrawal test, while the ANE increased, CNE reduced anxiety-like behaviors. The present study shows that the exposure of rats to white noise (100 dB) leads to behavioral alterations and molecule-specific changes in the CRH system. Behavioral alterations can be related to these molecular changes in the amygdala.

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

    PubMed Central

    Kabani, Sarah; Fenn, Katelyn; Ross, Alan; Ivens, Al; Smith, Terry K; Ghazal, Peter; Matthews, Keith

    2009-01-01

    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. PMID:19747379

  18. Experiment K-6-11. Actin mRNA and cytochrome c mRNA concentrations in the tricepts brachia muscle of rats

    NASA Technical Reports Server (NTRS)

    Booth, F. W.; Morrison, P. R.; Thomason, D. B.; Oganov, V. S.

    1990-01-01

    It is well known that some skeletal muscles atrophy as a result of weightlessness (Steffen and Musacchia 1986) and as a result of hindlimb suspension (Tischler et al., 1985, Thomason et al., 1987). Because the content of protein is determined by the rates of protein synthesis and degradation, a decrease in protein synthesis rate, or an increase in the protein degradation, or changes in both could produce the atrophy. Indeed, an increased protein degradation (Tischler et al., 1985) and a decreased protein synthesis (Thomason et al., 1988) have been observed in skeletal muscles of suspended hindlimbs of rats. Any decrease in protein synthesis rate could be caused by decreases in mRNA concentrations. Such decreases in the concentration and content of alpha-actin mRNA and cytochrome c mRNA have been noted in skeletal muscles of hindlimb suspended rats (Babij and Booth, 1988). From these findings researchers hypothesized that alpha-actin mRNA and cytochrome c mRNA would decrease in the triceps brachia muscle of Cosmos 1887 rats.

  19. Amphiphysin I but not dynamin I nor synaptojanin mRNA expression increased after repeated methamphetamine administration in the rat cerebrum and cerebellum.

    PubMed

    Hamamura, Mitsuko; Okouchi, Jiro; Ozawa, Hidetoshi; Kimuro, Yoshihiko; Iwaki, Akiko; Fukumaki, Yasuyuki

    2013-07-01

    Dopamine increases/decreases synaptic vesicle recycling and in schizophrenia the proteins/mRNA is decreased. We isolated cDNA clone, similar to amphiphysin 1 (vesicle protein) mRNA from the neocortex of rats injected repeatedly with methamphetamine using polymerase chain reaction (PCR) differential display. This clone is highly homologous to the 3' region of the human amphiphysin gene. PCR extension study using a primer specific for the rat amphiphysin 1 gene and a primer located within the clone revealed that it is the 3' UTR region of the rat amphiphysin 1 gene. Furthermore, in situ hybridization revealed that amphiphysin 1 mRNA is expressed in the cerebrum, medial thalamus, hippocampus and cerebellum. In the cerebellum, amphiphysin mRNA expression was confined to upper granule cell layer. Repeated methamphetamine administration increased amphiphysin I mRNA expression in both anterior part of the cerebrum, and the cerebellum. However, the repeated administration did not alter mRNA expression of the other vesicle proteins, synaptotagmin I, synapsin I, synaptojanin and dynamin I, we conclude that the repeated administration selectively increased amphiphysin 1 mRNA expression. Thus, amphiphysin 1 does not work as synaptic recycling, but it is suggested, as a part of pathogenesis of brain tissue injury (under Ca²⁺ and Mg²⁺ devoid environment) in repeated methamphetamine-injected states, the gene regulate actin-asssembly, learning, cell stress signaling and cell polarity.

  20. APC alterations are frequently involved in the pathogenesis of acinar cell carcinoma of the pancreas, mainly through gene loss and promoter hypermethylation.

    PubMed

    Furlan, Daniela; Sahnane, Nora; Bernasconi, Barbara; Frattini, Milo; Tibiletti, Maria Grazia; Molinari, Francesca; Marando, Alessandro; Zhang, Lizhi; Vanoli, Alessandro; Casnedi, Selenia; Adsay, Volkan; Notohara, Kenji; Albarello, Luca; Asioli, Sofia; Sessa, Fausto; Capella, Carlo; La Rosa, Stefano

    2014-05-01

    Genetic and epigenetic alterations involved in the pathogenesis of pancreatic acinar cell carcinomas (ACCs) are poorly characterized, including the frequency and role of gene-specific hypermethylation, chromosome aberrations, and copy number alterations (CNAs). A subset of ACCs is known to show alterations in the APC/β-catenin pathway which includes mutations of APC gene. However, it is not known whether, in addition to mutation, loss of APC gene function can occur through alternative genetic and epigenetic mechanisms such as gene loss or promoter methylation. We investigated the global methylation profile of 34 tumor suppressor genes, CNAs of 52 chromosomal regions, and APC gene alterations (mutation, methylation, and loss) together with APC mRNA level in 45 ACCs and related peritumoral pancreatic tissues using methylation-specific multiplex ligation probe amplification (MS-MLPA), fluorescence in situ hybridization (FISH), mutation analysis, and reverse transcription-droplet digital PCR. ACCs did not show an extensive global gene hypermethylation profile. RASSF1 and APC were the only two genes frequently methylated. APC mutations were found in only 7 % of cases, while APC loss and methylation were more frequently observed (48 and 56 % of ACCs, respectively). APC mRNA low levels were found in 58 % of cases and correlated with CNAs. In conclusion, ACCs do not show extensive global gene hypermethylation. APC alterations are frequently involved in the pathogenesis of ACCs mainly through gene loss and promoter hypermethylation, along with reduction of APC mRNA levels.

  1. A Cellular High-Throughput Screening Approach for Therapeutic trans-Cleaving Ribozymes and RNAi against Arbitrary mRNA Disease Targets

    PubMed Central

    Yau, Edwin H.; Butler, Mark C.; Sullivan, Jack M.

    2016-01-01

    Major bottlenecks in development of therapeutic post transcriptional gene silencing (PTGS) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease. Methods for rapid and reliable cellular testing of PTGS agents are needed to identify potent lead candidates for optimization. Our goal was to develop a means of rapid assessment of many RNA agents to identify a lead candidate for a given mRNA associated with a disease state. We developed a rapid human cell-based screening platform to test efficacy of hammerhead ribozyme (hhRz) or RNA interference (RNAi) constructs, using a model retinal degeneration target, human rod opsin (RHO) mRNA. The focus is on RNA Drug Discovery for diverse retinal degeneration targets. To validate the approach, candidate hhRzs were tested against NUH↓ cleavage sites (N=G,C,A,U; H=C,A,U) within the target mRNA of secreted alkaline phosphatase (SEAP), a model gene expression reporter, based upon in silico predictions of mRNA accessibility. HhRzs were embedded in a larger stable adenoviral VAI RNA scaffold for high cellular expression, cytoplasmic trafficking, and stability. Most hhRz expression plasmids exerted statistically significant knockdown of extracellular SEAP enzyme activity when readily assayed by a fluorescence enzyme assay intended for high throughput screening (HTS). Kinetics of PTGS knockdown of cellular targets is measureable in live cells with the SEAP reporter. The validated SEAP HTS platform was transposed to identify lead PTGS agents against a model hereditary retinal degeneration target, RHO mRNA. Two approaches were used to physically fuse the model retinal gene target mRNA to the SEAP reporter mRNA. The most expedient way to evaluate a

  2. 3′UTR AU-rich elements (AREs) and the RNA-binding protein Tristetraprolin (TTP) are not required for the LPS-mediated destabilization of phospholipase-Cβ-2 mRNA in murine macrophages

    PubMed Central

    Shukla, Smita; Elson, Genie; Blackshear, Perry J.; Lutz, Carol S.; Leibovich, S. Joseph

    2017-01-01

    We have shown previously that bacterial lipopolysaccharide (LPS)-mediated suppression of Phospholipase-Cβ-2 (PLCβ-2) expression is involved in M1 (inflammatory) to M2-like (wound healing) phenotypic switching of macrophages triggered by adenosine. This suppression is mediated post-transcriptionally by destabilization of PLCβ-2 mRNA. To investigate the mechanism of this LPS-mediated destabilization, we examined the roles of RNA-binding agents including microRNAs and RNA-binding proteins that are involved in regulating stability of mRNAs encoding growth factors, inflammatory mediators and proto-oncogenes. Adenylate and Uridylate (AU)-rich elements (AREs) in 3′UTRs are specific recognition sites for RNA-binding proteins including Tristetraprolin (TTP), HuR and AUF1, and for microRNAs that are involved in regulating mRNA stability. In this study, we investigated the role of TTP and AREs in regulating PLCβ-2 mRNA stability. The 3′UTR of the PLCβ-2 gene was inserted into the pLightswitch luciferase reporter plasmid and transfected into RAW264.7 cells. LPS suppressed Luciferase expression from this reporter. Luciferase expression from mutant 3′UTR constructs lacking AREs was similarly down-regulated, suggesting that these regions are not required for LPS-mediated suppression of PLCβ-2. TTP was rapidly upregulated in both primary murine macrophages and RAW264.7 cells in response to LPS. Suppression of PLCβ-2 by LPS was examined using macrophages from mice lacking TTP. LPS suppressed PLCβ-2 expression to the same extent in wild type and TTP−/− macrophages. Also, the rate of decay of PLCβ-2 mRNA in LPS-treated macrophages following transcriptional blockade was similar in wild type and TTP−/− macrophages, clearly indicating that TTP is not involved in LPS-mediated destabilization of PLCβ-2 mRNA in macrophages. PMID:28124257

  3. Low-level infrared laser modulates muscle repair and chromosome stabilization genes in myoblasts.

    PubMed

    da Silva Neto Trajano, Larissa Alexsandra; Stumbo, Ana Carolina; da Silva, Camila Luna; Mencalha, Andre Luiz; Fonseca, Adenilson S

    2016-08-01

    Infrared laser therapy is used for skeletal muscle repair based on its biostimulative effect on satellite cells. However, shortening of telomere length limits regenerative potential in satellite cells, which occurs after each cell division cycle. Also, laser therapy could be more effective on non-physiologic tissues. This study evaluated low-level infrared laser exposure effects on mRNA expression from muscle injury repair and telomere stabilization genes in myoblasts in normal and stressful conditions. Laser fluences were those used in clinical protocols. C2C12 myoblast cultures were exposed to low-level infrared laser (10, 35, and 70 J/cm(2)) in standard or normal (10 %) and reduced (2 %) fetal bovine serum concentrations; total RNA was extracted for mRNA expression evaluation from muscle injury repair (MyoD and Pax7) and chromosome stabilization (TRF1 and TRF2) genes by real time quantitative polymerization chain reaction. Data show that low-level infrared laser increases the expression of MyoD and Pax7 in 10 J/cm(2) fluence, TRF1 expression in all fluences, and TRF2 expression in 70 J/cm(2) fluence in both 10 and 2 % fetal bovine serum. Low-level infrared laser increases mRNA expression from genes related to muscle repair and telomere stabilization in myoblasts in standard or normal and stressful conditions.

  4. A retained intron in the 3'-UTR of Calm3 mRNA mediates its Staufen2- and activity-dependent localization to neuronal dendrites.

    PubMed

    Sharangdhar, Tejaswini; Sugimoto, Yoichiro; Heraud-Farlow, Jacqueline; Fernández-Moya, Sandra M; Ehses, Janina; Ruiz de Los Mozos, Igor; Ule, Jernej; Kiebler, Michael A

    2017-10-01

    Dendritic localization and hence local mRNA translation contributes to synaptic plasticity in neurons. Staufen2 (Stau2) is a well-known neuronal double-stranded RNA-binding protein (dsRBP) that has been implicated in dendritic mRNA localization. The specificity of Stau2 binding to its target mRNAs remains elusive. Using individual-nucleotide resolution CLIP (iCLIP), we identified significantly enriched Stau2 binding to the 3'-UTRs of 356 transcripts. In 28 (7.9%) of those, binding occurred to a retained intron in their 3'-UTR The strongest bound 3'-UTR intron was present in the longest isoform of Calmodulin 3 ( Calm3 L ) mRNA Calm3 L 3'-UTR contains six Stau2 crosslink clusters, four of which are in this retained 3'-UTR intron. The Calm3 L mRNA localized to neuronal dendrites, while lack of the 3'-UTR intron impaired its dendritic localization. Importantly, Stau2 mediates this dendritic localization via the 3'-UTR intron, without affecting its stability. Also, NMDA-mediated synaptic activity specifically promoted the dendritic mRNA localization of the Calm3 L isoform, while inhibition of synaptic activity reduced it substantially. Together, our results identify the retained intron as a critical element in recruiting Stau2, which then allows for the localization of Calm3 L mRNA to distal dendrites. © 2017 The Authors.

  5. Incorporation of mRNA in Lamellar Lipid Matrices for Parenteral Administration.

    PubMed

    Ziller, Antje; Nogueira, Sara S; Hühn, Eva; Funari, Sergio S; Brezesinski, Gerald; Hartmann, Hermann; Sahin, Ugur; Haas, Heinrich; Langguth, Peter

    2018-02-05

    Insertion of high molecular weight messenger RNA (mRNA) into lyotropic lipid phases as model systems for controlled release formulations for the mRNA was investigated. Low fractions of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used as an anchor to load the mRNA into a lamellar lipid matrix. Dispersions of zwitterionic lipid in the aqueous phase in the presence of increasing fractions of mRNA and cationic lipid were prepared, and the molecular organization was investigated as a function of mRNA and cationic lipid fraction. Insertion of both cationic lipid and mRNA was clearly proven from the physicochemical characteristics. The d-spacing of the lipid bilayers, as determined by small-angle X-ray scattering (SAXS) measurements, responded sensitively to the amount of inserted DOTAP and mRNA. A concise model of the insertion of the mRNA in the lipid matrices was derived, indicating that the mRNA was accommodated in the aqueous slab between lipid bilayers. Depending on the DOTAP and mRNA fraction, a different excess of water was present in this slab. Results from further physicochemical characterization, including determination of free and bound mRNA, zeta potential, and calorimetry data, were in line with this assumption. The structure of these concentrated lipid/mRNA preparations was maintained upon dilution. The functionality of the inserted mRNA was proven by cell culture experiments using C2C12 murine myoblast cells with the luciferase-encoding mRNA. The described lipid phases as carriers for the mRNA may be applicable for different routes of local administration, where control of the release kinetics and the form of the released mRNA (bound or free) is required.

  6. Alteration of lung atrial natriuretic peptide receptors in genetic cardiomyopathy.

    PubMed

    Mukaddam-Daher, S; Tremblay, J; Fujio, N; Koch, C; Jankowski, M; Quillen, E W; Gutkowska, J

    1996-07-01

    These studies were designed to characterize the atrial natriuretic peptide (ANF) receptor subtypes [guanylyl cyclase natriuretic peptide receptors (NPR-A, NPR-B) and NPR-C] in lungs of normal hamsters and to evaluate alterations in receptor kinetics in genetic cardiomyopathy (CMO), a model of human congestive heart failure. Lung membranes were obtained from normal and CMO 200-to 230-day-old hamsters. Cross-linking and competitive binding receptor assays using 125I-labeled human ANF showed that lung membranes exhibit NPR, mainly guanylyl cyclase NPR-A and clearance NPR-C receptors. Stimulation of guanylyl cyclase by ANF and C-type natriuretic peptide (CNP) confirmed the presence of NPR-A and NPR-B. The maximum binding capacity of total ANF binding sites (442 +/- 68 vs. 271 +/- 57 fmol/mg protein, P < 0.05) was reduced, but dissociation constant (0.26 +/- 0.04 vs. 0.41 +/- 0.08 nM) was not altered in CMO animals. Similar reductions were observed in the binding sites for brain natriuretic peptide (BNP; 438 +/- 83 vs. 236 +/- 53 fmol/mg protein) and CNP (321 +/- 80 vs. 165 +/- 56 fmol/mg protein, P < 0.05) which may reflect a decline in NPR-A and NPR-B and/or NPR-C. Acid wash improved binding of 125I-labeled rat ANF to lung membranes of both normal and CMO hamsters, but the tendency towards reduced binding in CMO hamsters did not reach statistical significance, implying that downregulation may not have been due only to prior occupancy of the receptors. Transcripts of NPR-A, NPR-B, and NPR-C receptors in hamster lungs were detected by quantitative polymerase chain reaction. Compared with normal controls, the CMO hamster lung NPR-A mRNA was reduced by 50%, but NPR-B mRNA and NPR-C mRNA were not altered. Moreover, CMO hamster lungs showed less activation of guanylyl cyclase by ANF. These studies demonstrate that lung NPR are downregulated in hamster CMO.

  7. T-lymphocyte cytokine mRNA expression in cystic echinococcosis.

    PubMed

    Fauser, S; Kern, P

    1997-04-01

    In the present study we investigated cytokine mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with cystic echinococcosis (CE) after stimulation with different antigens. By using reverse transcriptase polymerase chain reaction (RT-PCR) we could demonstrate that restimulation with crude Echinococcus granulosus antigen (Eg-Ag) induced or enhanced Th2 cytokine mRNA expression, especially IL-5 (by using antigen from sheep cyst fluid) in 23 out of 26 investigated CE patients and IL-10 (by using antigen from camel cyst fluid) in 10 out of 10 investigated CE patients. In contrast, IL-5 mRNA expression was absent in PBMC of healthy controls after Eg-Ag stimulation. To determine the specificity of this reaction we stimulated PBMC from 11 CE patients with crude Echinococcus multilocularis antigen (Em-Ag) and PBMC from 8 CE patients with Toxocara canis antigen (Tc-Ag). We found that the PBMC of patients showed a similar mRNA cytokine pattern on stimulation with Em-Ag when compared with Eg-Ag stimulation. The cytokine mRNA pattern on stimulation with Tc-Ag, however, resembled the cytokine mRNA pattern of unstimulated PBMC. Furthermore, the stimulation of PBMC with crude Mycobacterium tuberculosis antigen (H37Ra) and purified protein derivative (PPD) of M. tuberculosis revealed distinct IL-5 mRNA expression in all investigated CE patients, whereas in healthy controls IL-5 mRNA expression was very weak or totally absent. Thus, our results indicate an induction of Th2 cytokine mRNA expression in CE patients, which is frequently observed in parasite infections. Interestingly, this response persists after stimulation with tuberculosis antigens, which normally induce Th1 response.

  8. mRNA levels of circadian clock components Bmal1 and Per2 alter independently from dosing time-dependent efficacy of combination treatment with valsartan and amlodipine in spontaneously hypertensive rats.

    PubMed

    Potucek, Peter; Radik, Michal; Doka, Gabriel; Kralova, Eva; Krenek, Peter; Klimas, Jan

    2017-01-01

    Chronopharmacological effects of antihypertensives play a role in the outcome of hypertension therapy. However, studies produce contradictory findings when combination of valsartan plus amlodipine (VA) is applied. Here, we hypothesized different efficacy of morning versus evening dosing of VA in spontaneously hypertensive rats (SHR) and the involvement of circadian clock genes Bmal1 and Per2. We tested the therapy outcome in short-term and also long-term settings. SHRs aged between 8 and 10 weeks were treated with 10 mg/kg of valsartan and 4 mg/kg of amlodipine, either in the morning or in the evening with treatment duration 1 or 6 weeks and compared with parallel placebo groups. After short-term treatment, only morning dosing resulted in significant blood pressure (BP) control (measured by tail-cuff method) when compared to placebo, while after long-term treatment, both dosing groups gained similar superior results in BP control against placebo. However, mRNA levels of Bmal1 and Per2 (measured by RT-PCR) exhibited an independent pattern, with similar alterations in left and right ventricle, kidney as well as in aorta predominantly in groups with evening dosing in both, short-term and also long-term settings. This was accompanied by increased cardiac mRNA expression of plasminogen activator inhibitor-1. In summary, morning dosing proved to be advantageous due to earlier onset of antihypertensive action; however, long-term treatment was demonstrated to be effective regardless of administration time. Our findings also suggest that combination of VA may serve as an independent modulator of circadian clock and might influence disease progression beyond the primary BP lowering effect.

  9. Widespread promoter-mediated coordination of transcription and mRNA degradation

    PubMed Central

    2012-01-01

    Background Previous work showed that mRNA degradation is coordinated with transcription in yeast, and in several genes the control of mRNA degradation was linked to promoter elements through two different mechanisms. Here we show at the genomic scale that the coordination of transcription and mRNA degradation is promoter-dependent in yeast and is also observed in humans. Results We first demonstrate that swapping upstream cis-regulatory sequences between two yeast species affects both transcription and mRNA degradation and suggest that while some cis-regulatory elements control either transcription or degradation, multiple other elements enhance both processes. Second, we show that adjacent yeast genes that share a promoter (through divergent orientation) have increased similarity in their patterns of mRNA degradation, providing independent evidence for the promoter-mediated coupling of transcription to mRNA degradation. Finally, analysis of the differences in mRNA degradation rates between mammalian cell types or mammalian species suggests a similar coordination between transcription and mRNA degradation in humans. Conclusions Our results extend previous studies and suggest a pervasive promoter-mediated coordination between transcription and mRNA degradation in yeast. The diverse genes and regulatory elements associated with this coordination suggest that it is generated by a global mechanism of gene regulation and modulated by gene-specific mechanisms. The observation of a similar coupling in mammals raises the possibility that coupling of transcription and mRNA degradation may reflect an evolutionarily conserved phenomenon in gene regulation. PMID:23237624

  10. Impact of prebiotics on metabolic and behavioral alterations in a mouse model of metabolic syndrome.

    PubMed

    de Cossío, Lourdes Fernández; Fourrier, Célia; Sauvant, Julie; Everard, Amandine; Capuron, Lucile; Cani, Patrice D; Layé, Sophie; Castanon, Nathalie

    2017-08-01

    Mounting evidence shows that the gut microbiota, an important player within the gut-brain communication axis, can affect metabolism, inflammation, brain function and behavior. Interestingly, gut microbiota composition is known to be altered in patients with metabolic syndrome (MetS), who also often display neuropsychiatric symptoms. The use of prebiotics, which beneficially alters the microbiota, may therefore be a promising way to potentially improve physical and mental health in MetS patients. This hypothesis was tested in a mouse model of MetS, namely the obese and type-2 diabetic db/db mice, which display emotional and cognitive alterations associated with changes in gut microbiota composition and hippocampal inflammation compared to their lean db/+ littermates. We assessed the impact of chronic administration (8weeks) of prebiotics (oligofructose) on both metabolic (body weight, food intake, glucose homeostasis) and behavioral (increased anxiety-like behavior and impaired spatial memory) alterations characterizing db/db mice, as well as related neurobiological correlates, with particular attention to neuroinflammatory processes. Prebiotic administration improved excessive food intake and glycemic dysregulations (glucose tolerance and insulin resistance) in db/db mice. This was accompanied by an increase of plasma anti-inflammatory cytokine IL-10 levels and hypothalamic mRNA expression of the anorexigenic cytokine IL-1β, whereas unbalanced mRNA expression of hypothalamic orexigenic (NPY) and anorexigenic (CART, POMC) peptides was unchanged. We also detected signs of improved blood-brain-barrier integrity in the hypothalamus of oligofructose-treated db/db mice (normalized expression of tight junction proteins ZO-1 and occludin). On the contrary, prebiotic administration did not improve behavioral alterations and associated reduction of hippocampal neurogenesis displayed by db/db mice, despite normalization of increased hippocampal IL-6 mRNA expression. Of note

  11. Expression of calmodulin mRNA in rat olfactory neuroepithelium.

    PubMed

    Biffo, S; Goren, T; Khew-Goodall, Y S; Miara, J; Margolis, F L

    1991-04-01

    A calmodulin (CaM) cDNA was isolated by differential hybridization screening of a lambda gt10 library prepared from rat olfactory mucosa. This cDNA fragment, containing most of the open reading frame of the rat CaMI gene, was subcloned and used to characterize steady-state expression of CaM mRNA in rat olfactory neuroepithelium and bulb. Within the bulb mitral cells are the primary neuronal population expressing CaM mRNA. The major CaM mRNA expressed in the olfactory mucosa is 1.7 kb with smaller contributions from mRNAs of 4.0 and 1.4 kb. CaM mRNA was primarily associated with the olfactory neurons and, despite the cellular complexity of the tissue and the known involvement of CaM in diverse cellular processes, was only minimally evident in sustentacular cells, gland cells or respiratory epithelium. Following bulbectomy CaM mRNA declines in the olfactory neuroepithelium as does olfactory marker protein (OMP) mRNA. In contrast to the latter, CaM mRNA makes a partial recovery by one month after surgery. These results, coupled with those from in situ hybridization, indicate that CaM mRNA is expressed in both mature and immature olfactory neurons. The program regulating CaM gene expression in olfactory neurons is distinct from those controlling expression of B50/GAP43 in immature, or OMP in mature, neurons respectively.

  12. Structural and functional analysis of mRNA export regulation by the nuclear pore complex.

    PubMed

    Lin, Daniel H; Correia, Ana R; Cai, Sarah W; Huber, Ferdinand M; Jette, Claudia A; Hoelz, André

    2018-06-13

    The nuclear pore complex (NPC) controls the passage of macromolecules between the nucleus and cytoplasm, but how the NPC directly participates in macromolecular transport remains poorly understood. In the final step of mRNA export, the DEAD-box helicase DDX19 is activated by the nucleoporins Gle1, Nup214, and Nup42 to remove Nxf1•Nxt1 from mRNAs. Here, we report crystal structures of Gle1•Nup42 from three organisms that reveal an evolutionarily conserved binding mode. Biochemical reconstitution of the DDX19 ATPase cycle establishes that human DDX19 activation does not require IP 6 , unlike its fungal homologs, and that Gle1 stability affects DDX19 activation. Mutations linked to motor neuron diseases cause decreased Gle1 thermostability, implicating nucleoporin misfolding as a disease determinant. Crystal structures of human Gle1•Nup42•DDX19 reveal the structural rearrangements in DDX19 from an auto-inhibited to an RNA-binding competent state. Together, our results provide the foundation for further mechanistic analyses of mRNA export in humans.

  13. METHOD FOR STABILIZING KLYSTRONS

    DOEpatents

    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.

  14. Correlation between mutations and mRNA expression of APC and MUTYH genes: new insight into hereditary colorectal polyposis predisposition.

    PubMed

    Aceto, Gitana Maria; Fantini, Fabiana; De Iure, Sabrina; Di Nicola, Marta; Palka, Giandomenico; Valanzano, Rosa; Di Gregorio, Patrizia; Stigliano, Vittoria; Genuardi, Maurizio; Battista, Pasquale; Cama, Alessandro; Curia, Maria Cristina

    2015-10-28

    Transcript dosage imbalance may influence the transcriptome. To gain insight into the role of altered gene expression in hereditary colorectal polyposis predisposition, in the present study we analyzed absolute and allele-specific expression (ASE) of adenomatous polyposis coli (APC) and mutY Homolog (MUTYH) genes. We analyzed DNA and RNA extracted from peripheral blood mononuclear cells (PBMC) of 49 familial polyposis patients and 42 healthy blood donors selected according similar gender and age. Patients were studied for germline alterations in both genes using dHPLC, MLPA and automated sequencing. APC and MUTYH mRNA expression levels were investigated by quantitative Real-Time PCR (qRT-PCR) analysis using TaqMan assay and by ASE assays using dHPLC-based primer extension. Twenty out of 49 patients showed germline mutations: 14 in APC gene and six in MUTYH gene. Twenty-nine patients did not show mutations in both genes. Results from qRT-PCR indicated that gene expression of both APC and MUTYH was reduced in patients analyzed. In particular, a significant reduction in APC expression was observed in patients without APC germline mutation vs control group (P < 0.05) while APC expression in the mutation carrier patients, although lower compared to control individuals, did not show statistical significance. On the other hand a significant reduced MUTYH expression was detected in patients with MUTYH mutations vs control group (P < 0.05). Altered ASE of APC was detected in four out of eight APC mutation carriers. In particular one case showed a complete loss of one allele. Among APC mutation negative cases, 4 out of 13 showed a moderate ASE. ASE of MUTYH did not show any altered expression in the cases analyzed. Spearman's Rho Test analysis showed a positive and significant correlation between APC and MUTYH genes both in cases and in controls (P = 0.020 and P < 0.001). APC and MUTYH showed a reduced germline expression, not always corresponding to gene

  15. Whole-genome analysis of mRNA decay in Plasmodium falciparum reveals a global lengthening of mRNA half-life during the intra-erythrocytic development cycle.

    PubMed

    Shock, Jennifer L; Fischer, Kael F; DeRisi, Joseph L

    2007-01-01

    The rate of mRNA decay is an essential element of post-transcriptional regulation in all organisms. Previously, studies in several organisms found that the specific half-life of each mRNA is precisely related to its physiologic role, and plays an important role in determining levels of gene expression. We used a genome-wide approach to characterize mRNA decay in Plasmodium falciparum. We found that, globally, rates of mRNA decay increase dramatically during the asexual intra-erythrocytic developmental cycle. During the ring stage of the cycle, the average mRNA half-life was 9.5 min, but this was extended to an average of 65 min during the late schizont stage of development. Thus, a major determinant of mRNA decay rate appears to be linked to the stage of intra-erythrocytic development. Furthermore, we found specific variations in decay patterns superimposed upon the dominant trend of progressive half-life lengthening. These variations in decay pattern were frequently enriched for genes with specific cellular functions or processes. Elucidation of Plasmodium mRNA decay rates provides a key element for deciphering mechanisms of genetic control in this parasite, by complementing and extending previous mRNA abundance studies. Our results indicate that progressive stage-dependent decreases in mRNA decay rate function are a major determinant of mRNA accumulation during the schizont stage of intra-erythrocytic development. This type of genome-wide change in mRNA decay rate has not been observed in any other organism to date, and indicates that post-transcriptional regulation may be the dominant mechanism of gene regulation in P. falciparum.

  16. Early postnatal maternal separation causes alterations in the expression of β3-adrenergic receptor in rat adipose tissue suggesting long-term influence on obesity

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

    Miki, Takanori, E-mail: mikit@med.kagawa-u.ac.jp; Liu, Jun-Qian; Ohta, Ken-ichi

    Highlights: •High-fat diet intake following maternal separation did not cause body weight gain. •However, levels of metabolism-related molecules in adipose tissue were altered. •Increased levels of prohibitin mRNA in white fat were observed. •Attenuated levels of β3-adrenergic receptor mRNA were observed in brown fat. •Such alterations in adipose tissue may contribute to obesity later in life. -- Abstract: The effects of early postnatal maternal deprivation on the biological characteristics of the adipose tissue later in life were investigated in the present study. Sprague–Dawley rats were classified as either maternal deprivation (MD) or mother-reared control (MRC) groups. MD was achieved bymore » separating the rat pups from their mothers for 3 h each day during the 10–15 postnatal days. mRNA levels of mitochondrial uncoupling protein 1 (UCP-1), β3-adrenergic receptor (β3-AR), and prohibitin (PHB) in the brown and white adipose tissue were determined using real-time RT-PCR analysis. UCP-1, which is mediated through β3-AR, is closely involved in the energy metabolism and expenditure. PHB is highly expressed in the proliferating tissues/cells. At 10 weeks of age, the body weight of the MRC and MD rats was similar. However, the levels of the key molecules in the adipose tissue were substantially altered. There was a significant increase in the expression of PHB mRNA in the white adipose tissue, while the β3-AR mRNA expression decreased significantly, and the UCP-1 mRNA expression remained unchanged in the brown adipose tissue. Given that these molecules influence the mitochondrial metabolism, our study indicates that early postnatal maternal deprivation can influence the fate of adipose tissue proliferation, presumably leading to obesity later in life.« less

  17. The Arabidopsis THO/TREX component TEX1 functionally interacts with MOS11 and modulates mRNA export and alternative splicing events.

    PubMed

    Sørensen, Brian B; Ehrnsberger, Hans F; Esposito, Silvia; Pfab, Alexander; Bruckmann, Astrid; Hauptmann, Judith; Meister, Gunter; Merkl, Rainer; Schubert, Thomas; Längst, Gernot; Melzer, Michael; Grasser, Marion; Grasser, Klaus D

    2017-02-01

    We identify proteins that associate with the THO core complex, and show that the TEX1 and MOS11 components functionally interact, affecting mRNA export and splicing as well as plant development. TREX (TRanscription-EXport) is a multiprotein complex that plays a central role in the coordination of synthesis, processing and nuclear export of mRNAs. Using targeted proteomics, we identified proteins that associate with the THO core complex of Arabidopsis TREX. In addition to the RNA helicase UAP56 and the mRNA export factors ALY2-4 and MOS11 we detected interactions with the mRNA export complex TREX-2 and multiple spliceosomal components. Plants defective in the THO component TEX1 or in the mRNA export factor MOS11 (orthologue of human CIP29) are mildly affected. However, tex1 mos11 double-mutant plants show marked defects in vegetative and reproductive development. In tex1 plants, the levels of tasiRNAs are reduced, while miR173 levels are decreased in mos11 mutants. In nuclei of mos11 cells increased mRNA accumulation was observed, while no mRNA export defect was detected with tex1 cells. Nevertheless, in tex1 mos11 double-mutants, the mRNA export defect was clearly enhanced relative to mos11. The subnuclear distribution of TEX1 substantially overlaps with that of splicing-related SR proteins and in tex1 plants the ratio of certain alternative splicing events is altered. Our results demonstrate that Arabidopsis TEX1 and MOS11 are involved in distinct steps of the biogenesis of mRNAs and small RNAs, and that they interact regarding some aspects, but act independently in others.

  18. WIG1 is crucial for AGO2-mediated ACOT7 mRNA silencing via miRNA-dependent and -independent mechanisms.

    PubMed

    Lee, Hyung Chul; Jung, Seung Hee; Hwang, Hyun Jung; Kang, Donghee; De, Supriyo; Dudekula, Dawood B; Martindale, Jennifer L; Park, Byungkyu; Park, Seung Kuk; Lee, Eun Kyung; Lee, Jeong-Hwa; Jeong, Sunjoo; Han, Kyungsook; Park, Heon Joo; Ko, Young-Gyu; Gorospe, Myriam; Lee, Jae-Seon

    2017-06-20

    RNA-binding proteins (RBPs) are involved in mRNA splicing, maturation, transport, translation, storage and turnover. Here, we identified ACOT7 mRNA as a novel target of human WIG1. ACOT7 mRNA decay was triggered by the microRNA miR-9 in a WIG1-dependent manner via classic recruitment of Argonaute 2 (AGO2). Interestingly, AGO2 was also recruited to ACOT7 mRNA in a WIG1-dependent manner in the absence of miR-9, which indicates an alternative model whereby WIG1 controls AGO2-mediated gene silencing. The WIG1-AGO2 complex attenuated translation initiation via an interaction with translation initiation factor 5B (eIF5B). These results were confirmed using a WIG1 tethering system based on the MS2 bacteriophage coat protein and a reporter construct containing an MS2-binding site, and by immunoprecipitation of WIG1 and detection of WIG1-associated proteins using liquid chromatography-tandem mass spectrometry. We also identified WIG1-binding motifs using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation analyses. Altogether, our data indicate that WIG1 governs the miRNA-dependent and the miRNA-independent recruitment of AGO2 to lower the stability of and suppress the translation of ACOT7 mRNA. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Role of RNase Y in Clostridium perfringens mRNA Decay and Processing.

    PubMed

    Obana, Nozomu; Nakamura, Kouji; Nomura, Nobuhiko

    2017-01-15

    RNase Y is a major endoribonuclease that plays a crucial role in mRNA degradation and processing. We study the role of RNase Y in the Gram-positive anaerobic pathogen Clostridium perfringens, which until now has not been well understood. Our study implies an important role for RNase Y-mediated RNA degradation and processing in virulence gene expression and the physiological development of the organism. We began by constructing an RNase Y conditional knockdown strain in order to observe the importance of RNase Y on growth and virulence. Our resulting transcriptome analysis shows that RNase Y affects the expression of many genes, including toxin-producing genes. We provide data to show that RNase Y depletion repressed several toxin genes in C. perfringens and involved the virR-virS two-component system. We also observe evidence that RNase Y is indispensable for processing and stabilizing the transcripts of colA (encoding a major toxin collagenase) and pilA2 (encoding a major pilin component of the type IV pili). Posttranscriptional regulation of colA is known to be mediated by cleavage in the 5' untranslated region (5'UTR), and we observe that RNase Y depletion diminishes colA 5'UTR processing. We show that RNase Y is also involved in the posttranscriptional stabilization of pilA2 mRNA, which is thought to be important for host cell adherence and biofilm formation. RNases have important roles in RNA degradation and turnover in all organisms. C. perfringens is a Gram-positive anaerobic spore-forming bacterial pathogen that produces numerous extracellular enzymes and toxins, and it is linked to digestive disorders and disease. A highly conserved endoribonuclease, RNase Y, affects the expression of hundreds of genes, including toxin genes, and studying these effects is useful for understanding C. perfringens specifically and RNases generally. Moreover, RNase Y is involved in processing specific transcripts, and we observed that this processing in C. perfringens results

  20. Plasticity of cardiovascular function in snapping turtle embryos (Chelydra serpentina): chronic hypoxia alters autonomic regulation and gene expression.

    PubMed

    Eme, John; Rhen, Turk; Tate, Kevin B; Gruchalla, Kathryn; Kohl, Zachary F; Slay, Christopher E; Crossley, Dane A

    2013-06-01

    Reptile embryos tolerate large decreases in the concentration of ambient oxygen. However, we do not fully understand the mechanisms that underlie embryonic cardiovascular short- or long-term responses to hypoxia in most species. We therefore measured cardiac growth and function in snapping turtle embryos incubated under normoxic (N21; 21% O₂) or chronic hypoxic conditions (H10; 10% O₂). We determined heart rate (fH) and mean arterial pressure (Pm) in acute normoxic (21% O₂) and acute hypoxic (10% O₂) conditions, as well as embryonic responses to cholinergic, adrenergic, and ganglionic pharmacological blockade. Compared with N21 embryos, chronic H10 embryos had smaller bodies and relatively larger hearts and were hypotensive, tachycardic, and following autonomic neural blockade showed reduced intrinsic fH at 90% of incubation. Unlike other reptile embryos, cholinergic and ganglionic receptor blockade both increased fH. β-Adrenergic receptor blockade with propranolol decreased fH, and α-adrenergic blockade with phentolamine decreased Pm. We also measured cardiac mRNA expression. Cholinergic tone was reduced in H10 embryos, but cholinergic receptor (Chrm2) mRNA levels were unchanged. However, expression of adrenergic receptor mRNA (Adrb1, Adra1a, Adra2c) and growth factor mRNA (Igf1, Igf2, Igf2r, Pdgfb) was lowered in H10 embryos. Hypoxia altered the balance between cholinergic receptors, α-adrenoreceptor and β-adrenoreceptor function, which was reflected in altered intrinsic fH and adrenergic receptor mRNA levels. This is the first study to link gene expression with morphological and cardioregulatory plasticity in a developing reptile embryo.

  1. Tissue-specific expression and regulation by 1,25(OH)2D3 of chick protein kinase inhibitor (PKI) mRNA.

    PubMed

    Marchetto, G S; Henry, H L

    1997-02-01

    The heat-stable protein kinase inhibitor (PKI) protein is a specific and potent competitive inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKA). Previously, it has been shown that vitamin D status affects chick kidney PKI activity: a 5- to 10-fold increase in PKI activity was observed in kidneys of chronically vitamin D-deficient chicks and treatment with 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) in cultured kidney cells resulted in a 95% decrease in PKI activity. The authors have recently cloned the cDNA for chick kidney PKI and have used the coding sequence to study the regulation of PKI mRNA. Northern analysis showed the expression of two PKI messages, which are 2.7 and 3.3 kb in size. These mRNAs are expressed in brain, muscle, testis, and kidney, but not in pancreas, liver, or intestine. PKI mRNA steady-state levels are downregulated by 47% in kidneys from vitamin D-replete chicks as compared to vitamin D-deficient chicks. PKI mRNA levels in brain, muscle, and testis are not affected by vitamin D status. Treatment of primary chick kidney cultures treated with 10(-7) M 1,25(OH)2D3 for 24h resulted in a 20-30% decrease in PKI mRNA. 1,25(OH)2D3 treatment does not affect the stability of PKI mRNA as determined by treatment of cell cultures with actinomycin D. This study shows that 1,25(OH)2D3 directly and tissue-specifically downregulates PKI mRNA in the chick kidney.

  2. Staufen-mediated mRNA decay.

    PubMed

    Park, Eonyoung; Maquat, Lynne E

    2013-01-01

    Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'-UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base pairing of 3'-UTR sequences or by intermolecular base pairing of 3'-UTR sequences with a long-noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Because both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1; SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. Copyright © 2013 John Wiley & Sons, Ltd.

  3. Staufen-mediated mRNA decay

    PubMed Central

    Park, Eonyoung; Maquat, Lynne E.

    2013-01-01

    Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base-pairing of 3'UTR sequences or by intermolecular base-pairing of 3'UTR sequences with a long noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Since both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1, SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. PMID:23681777

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

    PubMed

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

    2016-10-01

    F toxicity to immune system, especially to macrophage, has been studied a lot recently. Nuclear factor-kappa B (NF-κB), as a transcription factor, plays a central role in immune and inflammatory responses via the regulation of downstream gene expression. Recent studies indicated that fluoride effect on inflammatory cytokine secretion, however, the molecular mechanism was less understood. In our study, peritoneal macrophages (PMs) were divided several groups and were administrated sodium fluoride (NaF, 50, 100, 200, 400, 800 μM) and/or lipopolysaccharide (LPS, 30 ng/mg). The mRNA expression of p65, inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) in macrophages exposed to fluoride was determined by quantitative real-time RT-PCR respectively. The translocation of NF-κB from cytoplasm to nucleus, which in a way reflects NF-κB activity, was demonstrated by Immunofluorescence and ELISA. Our results showed that fluoride had a dose-dependent effect on NF-κB activity, which coincided with LPS-induced mRNA expression of its downstream genes, iNOS and IL-1β. Fluoride alone causes no effect on gene expression. However, the mRNA expression of TNF-α showed non-NF-κB-dependent manner. Therefore, we come to the conclusion that fluoride can regulate LPS-induced mRNA expression of iNOS and IL-1β via NF-κB pathway in mouse peritoneal macrophages. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Maternal BMI and gestational diabetes alter placental lipid transporters and fatty acid composition.

    PubMed

    Segura, Maria Teresa; Demmelmair, Hans; Krauss-Etschmann, Susanne; Nathan, Petra; Dehmel, Stefan; Padilla, Maria Carmen; Rueda, Ricardo; Koletzko, Berthold; Campoy, Cristina

    2017-09-01

    Placental fatty acid (FA) uptake and metabolism depend on maternal supply which may be altered when women have a high pre-pregnancy body mass index (BMI) or develop gestational diabetes (GDM). Consequently, an impaired FA transport to the fetus may negatively affect fetal development. While placental adaptation of maternal-fetal glucose transfer in mild GDM has been described, knowledge on placental FA acid metabolism and possible adaptations in response to maternal obesity or GDM is lacking. We aimed to analyze the FA composition and the expression of key genes involved in FA uptake and metabolism in placentas from women with pre-pregnancy normal weight (18.5 ≤ BMI<25 kg/m2), overweight (25 ≤ BMI<30 kg/m 2 ), obesity (BMI ≥ 30 kg/m 2 ), and lean pregnant women with GDM. Placental FA content was determined by gas liquid chromatography. Placental mRNA expression of FA transport proteins (FATP1, FATP4, FATP6), FA binding proteins (FABP3, FABP4, FABP7), FA translocase (FAT/CD36) and enzymes (Endothelial lipase (EL) and lipoprotein lipase (LPL)) were quantified by qRT-PCR. High pre-pregnancy BMI and GDM were associated with decreased placental FATP1, FATP4, EL and increased FAT/CD36 and FATP6 expressions. LPL mRNA levels and placental total FA content were similar among groups. Specific FA, including some long-chain polyunsaturated FA, were altered. Our results demonstrate that high pre-pregnancy BMI or GDM independently alter mRNA expression levels of genes involved in FA uptake and metabolism and the placental FA profile, which could affect fetal development and long-term health. Copyright © 2017. Published by Elsevier Ltd.

  6. 1,2-Dibromo-4-(1,2-dibromoethyl)-cyclohexane and tris(methylphenyl) phosphate cause significant effects on development, mRNA expression, and circulating bile acid concentrations in chicken embryos

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

    Crump, Doug, E-mail: doug.crump@ec.gc.ca; Porter, Emily; Egloff, Caroline

    1,2-Dibromo-4-(1,2-dibromoethyl)-cyclohexane (DBE-DBCH; formerly abbreviated as TBECH) and tris(methylphenyl) phosphate (TMPP; formerly abbreviated as TCP) are additive flame retardants that are detected in the environment and biota. A recent avian in vitro screening study of 16 flame retardants identified DBE-DBCH and TMPP as important chemicals for follow-up in ovo evaluation based on their effects on cytotoxicity and mRNA expression in avian hepatocytes. In this study, technical mixtures of DBE-DBCH and TMPP were injected into the air cell of chicken embryos at concentrations ranging from 0 to 54,900 ng/g and from 0 to 261,400 ng/g, respectively, to determine effects on pipping success,more » development, hepatic mRNA expression, thyroid hormone levels, and circulating bile acid concentrations. Both compounds were detectable in embryos at pipping and the β-DBE-DBCH isomer was depleted more rapidly than the α-isomer in tissue samples. DBE-DBCH had limited effects on the endpoints measured, with the exception of the up-regulation of two phase I metabolizing enzymes, CYP3A37 and CYP2H1. TMPP exposure caused embryonic deformities, altered growth, increased liver somatic index (LSI) and plasma bile acid concentrations, and altered mRNA expression levels of genes associated with xenobiotic and lipid metabolism and the thyroid hormone pathway. Overall, TMPP elicited more adverse molecular and phenotypic effects than DBE-DBCH albeit at concentrations several orders of magnitude greater than those detected in the environment. The increase in plasma bile acid concentrations was a useful phenotypic anchor as it was associated with a concomitant increase in LSI, discoloration of the liver tissue, and modulation of hepatic genes involved with xenobiotic and lipid metabolism. - Highlights: • DBE-DBCH and TMPP are not embryolethal to chicken embryos. • TMPP caused deformities, morphometric alterations, and increased plasma bile acids. • DBE-DBCH and TMPP altered mRNA

  7. mRNA vaccines — a new era in vaccinology

    PubMed Central

    Pardi, Norbert; Hogan, Michael J.; Porter, Frederick W.; Weissman, Drew

    2018-01-01

    mRNA vaccines represent a promising alternative to conventional vaccine approaches because of their high potency, capacity for rapid development and potential for low-cost manufacture and safe administration. However, their application has until recently been restricted by the instability and inefficient in vivo delivery of mRNA. Recent technological advances have now largely overcome these issues, and multiple mRNA vaccine platforms against infectious diseases and several types of cancer have demonstrated encouraging results in both animal models and humans. This Review provides a detailed overview of mRNA vaccines and considers future directions and challenges in advancing this promising vaccine platform to widespread therapeutic use. PMID:29326426

  8. Alterations in Bronchial Airway miRNA Expression for Lung Cancer Detection.

    PubMed

    Pavel, Ana B; Campbell, Joshua D; Liu, Gang; Elashoff, David; Dubinett, Steven; Smith, Kate; Whitney, Duncan; Lenburg, Marc E; Spira, Avrum

    2017-11-01

    We have previously shown that gene expression alterations in normal-appearing bronchial epithelial cells can serve as a lung cancer detection biomarker in smokers. Given that miRNAs regulate airway gene expression responses to smoking, we evaluated whether miRNA expression is also altered in the bronchial epithelium of smokers with lung cancer. Using epithelial brushings from the mainstem bronchus of patients undergoing bronchoscopy for suspected lung cancer (as part of the AEGIS-1/2 clinical trials), we profiled miRNA expression via small-RNA sequencing from 347 current and former smokers for which gene expression data were also available. Patients were followed for one year postbronchoscopy until a final diagnosis of lung cancer ( n = 194) or benign disease ( n = 153) was made. Following removal of 6 low-quality samples, we used 138 patients (AEGIS-1) as a discovery set to identify four miRNAs (miR-146a-5p, miR-324-5p, miR-223-3p, and miR-223-5p) that were downregulated in the bronchial airway of lung cancer patients (ANOVA P < 0.002, FDR < 0.2). The expression of these miRNAs is significantly more negatively correlated with the expression of their mRNA targets than with the expression of other nontarget genes (K-S P < 0.05). Furthermore, these mRNA targets are enriched among genes whose expression is elevated in cancer patients (GSEA FDR < 0.001). Finally, we found that the addition of miR-146a-5p to an existing mRNA biomarker for lung cancer significantly improves its performance (AUC) in the 203 samples (AEGIS-1/2) serving an independent test set (DeLong P < 0.05). Our findings suggest that there are miRNAs whose expression is altered in the cytologically normal bronchial epithelium of smokers with lung cancer, and that they may regulate cancer-associated gene expression differences. Cancer Prev Res; 10(11); 651-9. ©2017 AACR . ©2017 American Association for Cancer Research.

  9. Sequencing of mRNA identifies re-expression of fetal splice variants in cardiac hypertrophy

    PubMed Central

    Ames, EG; Lawson, MJ; Mackey, AJ; Holmes, JW

    2013-01-01

    Cardiac hypertrophy has been well-characterized at the level of transcription. During cardiac hypertrophy, genes normally expressed primarily during fetal heart development are reexpressed, and this fetal gene program is believed to be a critical component of the hypertrophic process. Recently, alternative splicing of mRNA transcripts has been shown to be temporally regulated during heart development, leading us to consider whether fetal patterns of splicing also reappear during hypertrophy. We hypothesized that patterns of alternative splicing occurring during heart development are recapitulated during cardiac hypertrophy. Here we present a study of isoform expression during pressure-overload cardiac hypertrophy induced by 10 days of transverse aortic constriction (TAC) in rats and in developing fetal rat hearts compared to sham-operated adult rat hearts, using high-throughput sequencing of poly(A) tail mRNA. We find a striking degree of overlap between the isoforms expressed differentially in fetal and pressure-overloaded hearts compared to control: forty-four percent of the isoforms with significantly altered expression in TAC hearts are also expressed at significantly different levels in fetal hearts compared to control (P < 0.001). The isoforms that are shared between hypertrophy and fetal heart development are significantly enriched for genes involved in cytoskeletal organization, RNA processing, developmental processes, and metabolic enzymes. Our data strongly support the concept that mRNA splicing patterns normally associated with heart development recur as part of the hypertrophic response to pressure overload. These findings suggest that cardiac hypertrophy shares post-transcriptional as well as transcriptional regulatory mechanisms with fetal heart development. PMID:23688780

  10. [Impacts of the formula of Suoquanwan(SQW) on expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency].

    PubMed

    Cao, Hong-Ying; Wu, Qing-He; Huang, Ping; He, Jin-Yang

    2009-06-01

    To observe the impacts of the formula of Suoquanwan (SQW) on the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency. The model rats were induced by adenine (250 mg/kg) for 4 weeks, then treated respectively with SQW or dDAVP. The expression of AQP-2 mRNA and AVPR-V2 mRNA in kidney of Yang-deficiency model by realtime fluorescence quantitative PCR method were investigated. In model rats, the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney decreased, dDAVP and SQW high dose could increased the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney. The others had no influence on the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney. SQW can increase the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency.

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

    PubMed

    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

  12. Small Molecule Inhibitors of Staphylococcus aureus RnpA Alter Cellular mRNA Turnover, Exhibit Antimicrobial Activity, and Attenuate Pathogenesis

    PubMed Central

    Olson, Patrick D.; Kuechenmeister, Lisa J.; Anderson, Kelsi L.; Daily, Sonja; Beenken, Karen E.; Roux, Christelle M.; Reniere, Michelle L.; Lewis, Tami L.; Weiss, William J.; Pulse, Mark; Nguyen, Phung; Simecka, Jerry W.; Morrison, John M.; Sayood, Khalid; Asojo, Oluwatoyin A.; Smeltzer, Mark S.; Skaar, Eric P.; Dunman, Paul M.

    2011-01-01

    Methicillin-resistant Staphylococcus aureus is estimated to cause more U.S. deaths annually than HIV/AIDS. The emergence of hypervirulent and multidrug-resistant strains has further amplified public health concern and accentuated the need for new classes of antibiotics. RNA degradation is a required cellular process that could be exploited for novel antimicrobial drug development. However, such discovery efforts have been hindered because components of the Gram-positive RNA turnover machinery are incompletely defined. In the current study we found that the essential S. aureus protein, RnpA, catalyzes rRNA and mRNA digestion in vitro. Exploiting this activity, high through-put and secondary screening assays identified a small molecule inhibitor of RnpA-mediated in vitro RNA degradation. This agent was shown to limit cellular mRNA degradation and exhibited antimicrobial activity against predominant methicillin-resistant S. aureus (MRSA) lineages circulating throughout the U.S., vancomycin intermediate susceptible S. aureus (VISA), vancomycin resistant S. aureus (VRSA) and other Gram-positive bacterial pathogens with high RnpA amino acid conservation. We also found that this RnpA-inhibitor ameliorates disease in a systemic mouse infection model and has antimicrobial activity against biofilm-associated S. aureus. Taken together, these findings indicate that RnpA, either alone, as a component of the RNase P holoenzyme, and/or as a member of a more elaborate complex, may play a role in S. aureus RNA degradation and provide proof of principle for RNA catabolism-based antimicrobial therapy. PMID:21347352

  13. Evidence for Alterations in Stimulatory G proteins and Oxytocin Levels in Children with Autism

    PubMed Central

    Jacobson, Jill D.; Ellerbeck, Kathryn A.; Kelly, Kelsie A.; Fleming, Kandace K.; Jamison, T. Rene; Coffey, Charles W.; Smith, Catherine M.; Reese, R. Matthew; Sands, Scott A.

    2014-01-01

    The neurotransmitter oxytocin plays an important role in social affiliation. Low oxytocin levels and defects in the oxytocin receptor have been reported in childhood autism. However, little is known about oxytocin’s post-receptor signaling pathways in autism. Oxytocin signals via stimulatory and inhibitory G proteins. c-fos mRNA expression has been used as a marker of OT signaling as well as of G protein signaling. Herein, we hypothesized that oxytocin and its signaling pathways would be altered in children with autism. We measured plasma oxytocin levels by ELISA, G-protein and c-fos mRNA by PCR, and G proteins by immunoblot in cultured peripheral blood mononuclear cells (PBMCs) in children with autism and in age-matched controls. Males with autism displayed elevated oxytocin levels compared to controls (p<0.05). Children with autism displayed significantly higher mRNA for stimulatory G proteins compared to controls (p<0.05). Oxytocin levels correlated strongly positively with c-fos mRNA levels, but only in control participants (p<0.01). Oxytocin, G-protein, and c-fos mRNA levels correlated inversely with measures of social and emotional behaviors, but only in control participants. These data suggest that children with autism may exhibit a dysregulation in oxytocin and/or its signaling pathways. PMID:24485488

  14. 6-Thioguanine alters the structure and stability of duplex DNA and inhibits quadruplex DNA formation.

    PubMed

    Marathias, V M; Sawicki, M J; Bolton, P H

    1999-07-15

    The ability to chemically synthesize biomolecules has opened up the opportunity to observe changes in structure and activity that occur upon single atom substitution. In favorable cases this can provide information about the roles of individual atoms. The substitution of 6-thioguanine (6SG) for guanine is a potentially very useful single atom substitution as 6SG has optical, photocrosslinking, metal ion binding and other properties of potential utility. In addition, 6-mercaptopurine is a clinically important pro-drug that is activated by conversion into 6SG by cells. The results presented here indicate that the presence of 6SG blocks the formation of quadruplex DNA. The presence of 6SG alters the structure and lowers the thermal stability of duplex DNA, but duplex DNA can be formed in the presence of 6SG. These results indicate that some of the cytotoxic activity of 6SG may be due to disruption of the quadruplex structures formed by telomere and other DNAs. This additional mode of action is consistent with the delayed onset of cytotoxicity.

  15. Staufen 2 regulates mGluR long-term depression and Map1b mRNA distribution in hippocampal neurons.

    PubMed

    Lebeau, Geneviève; Miller, Linda C; Tartas, Maylis; McAdam, Robyn; Laplante, Isabel; Badeaux, Frédérique; DesGroseillers, Luc; Sossin, Wayne S; Lacaille, Jean-Claude

    2011-01-01

    The two members of the Staufen family of RNA-binding proteins, Stau1 and Stau2, are present in distinct ribonucleoprotein complexes and associate with different mRNAs. Stau1 is required for protein synthesis-dependent long-term potentiation (L-LTP) in hippocampal pyramidal cells. However, the role of Stau2 in synaptic plasticity remains unexplored. We found that unlike Stau1, Stau2 is not required for L-LTP. In contrast, Stau2, but not Stau1, is necessary for DHPG-induced protein synthesis-dependent long-term depression (mGluR-LTD). While Stau2 is involved in early development of spines, its down-regulation does not alter spine morphology or spontaneous miniature synaptic activity in older cultures where LTD occurs. In addition, Stau2, but not Stau1, knockdown reduces the dendritic localization of Map1b mRNA, a specific transcript involved in mGluR-LTD. Moreover, mGluR stimulation with DHPG induces Map1b, but not Map2, mRNA dissociation from mRNA granules containing Stau2 and the ribosomal protein P0. This dissociation was not observed in cells in which Stau2 was depleted. Finally, Stau2 knockdown reduces basal Map1b protein expression in dendrites and prevents DHPG-induced increases in dendritic Map1b protein level. We suggest a role for Stau2 in the generation and regulation of Map1b mRNA containing granules that are required for mGluR-LTD.

  16. WNT2B2 mRNA, up-regulated in primary gastric cancer, is a positive regulator of the WNT- beta-catenin-TCF signaling pathway.

    PubMed

    Katoh, M; Kirikoshi, H; Terasaki, H; Shiokawa, K

    2001-12-21

    Genetic alterations of WNT signaling molecules lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway. We have previously cloned and characterized WNT2B/WNT13 gene on human chromosome 1p13, which is homologous to proto-oncogene WNT2 on human chromosome 7q31. WNT2B1 and WNT2B2 mRNAs, generated from the WNT2B gene due to alternative splicing of the alternative promoter type, encode almost identical polypeptides with divergence in the N-terminal region. WNT2B2 mRNA rather than WNT2B1 mRNA is preferentially expressed in NT2 cells with the potential of neuronal differentiation. Here, we describe our investigations of expression of WNT2B mRNAs in various types of human primary cancer. Matched tumor/normal expression array analysis revealed that WNT2B mRNAs were significantly up-regulated in 2 of 8 cases of primary gastric cancer. WNT2B2 mRNA rather than WNT2B1 mRNA was found to be preferentially up-regulated in a case of primary gastric cancer (signet ring cell carcinoma). Function of WNT2B1 mRNA and that of WNT2B2 mRNA were investigated by using Xenopus axis duplication assay. Injection of synthetic WNT2B1 mRNA into the ventral marginal zone of fertilized Xenopus eggs at the 4-cell stage did not induce axis duplication. In contrast, ventral injection of synthetic WNT2B2 mRNA induced axis duplication in 90% of embryos (complete axis duplication, 24%). These results strongly suggest that WNT2B2 up-regulation in some cases of gastric cancer might lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway.

  17. The C. elegans neural editome reveals an ADAR target mRNA required for proper chemotaxis

    PubMed Central

    Deffit, Sarah N; Yee, Brian A; Manning, Aidan C; Rajendren, Suba; Vadlamani, Pranathi; Wheeler, Emily C; Domissy, Alain; Washburn, Michael C

    2017-01-01

    ADAR proteins alter gene expression both by catalyzing adenosine (A) to inosine (I) RNA editing and binding to regulatory elements in target RNAs. Loss of ADARs affects neuronal function in all animals studied to date. Caenorhabditis elegans lacking ADARs exhibit reduced chemotaxis, but the targets responsible for this phenotype remain unknown. To identify critical neural ADAR targets in C. elegans, we performed an unbiased assessment of the effects of ADR-2, the only A-to-I editing enzyme in C. elegans, on the neural transcriptome. Development and implementation of publicly available software, SAILOR, identified 7361 A-to-I editing events across the neural transcriptome. Intersecting the neural editome with adr-2 associated gene expression changes, revealed an edited mRNA, clec-41, whose neural expression is dependent on deamination. Restoring clec-41 expression in adr-2 deficient neural cells rescued the chemotaxis defect, providing the first evidence that neuronal phenotypes of ADAR mutants can be caused by altered gene expression. PMID:28925356

  18. Adrenal neuropeptide Y mRNA but not preproenkephalin mRNA induction by stress is impaired by aging in Fischer 344 rats.

    PubMed

    Silverstein, J H; Beasley, J; Mizuno, T M; London, E; Mobbs, C V

    1998-04-01

    Relatively few molecular markers of stress have been studied in aged individuals. Interactions of age and stress on adrenal neuropeptide Y (NPY) and preproenkephalin (ppENK) expression have not been reported. The purpose of these studies was to characterize the adrenal NPY and ppENK responses to stress using a common stressor, physical restraint for 2 h, in Fischer 344 rats at 7, 16 and 23 months of age. Northern blot techniques were used to evaluate induction by stress of adrenal NPY mRNA and adrenal ppENK mRNA. Two humoral responses to stress, serum glucose and corticosterone, were measured to corroborate that a stress response occurred. We observed that the induction by stress of adrenal NPY mRNA is impaired with age but the stress-induced elevation of adrenal ppENK mRNA, blood glucose, and corticosterone show no evidence of age-related impairments.

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

    PubMed

    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. Copyright © 2015, American Association for the Advancement of Science.

  20. Induced expression of mRNA for IL-5, IL-6, TNF-alpha, MIP-2 and IFN-gamma in immunologically activated rat peritoneal mast cells: inhibition by dexamethasone and cyclosporin A.

    PubMed

    Williams, C M; Coleman, J W

    1995-10-01

    We examined the capacity of purified rat peritoneal connective tissue-type mast cells (PMC) to express mRNA for several cytokines. Stimulation of PMC with anti-IgE for 4 hr induced the expression of mRNA encoding interleukin-5 (IL-5), IL-6, tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Unstimulated PMC expressed detectable mRNA for TNF-alpha but not for the other four cytokines. Incubation of PMC with cyclosporin A (CsA) or dexamethasone (DEX), each at 10(-6) M for 24 hr, significantly inhibited the induced expression of mRNA for each of the five cytokines, and also inhibited release of biologically active TNF-alpha. Throughout these experiments mRNA levels of the housekeeping gene G3PDH were not altered by stimulation with anti-IgE or incubation with CsA or DEX. We conclude that immunological activation of rat PMC induces gene expression of several cytokines and that expression of these genes can be inhibited by immunosuppressive drugs.

  1. Induced expression of mRNA for IL-5, IL-6, TNF-alpha, MIP-2 and IFN-gamma in immunologically activated rat peritoneal mast cells: inhibition by dexamethasone and cyclosporin A.

    PubMed Central

    Williams, C M; Coleman, J W

    1995-01-01

    We examined the capacity of purified rat peritoneal connective tissue-type mast cells (PMC) to express mRNA for several cytokines. Stimulation of PMC with anti-IgE for 4 hr induced the expression of mRNA encoding interleukin-5 (IL-5), IL-6, tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Unstimulated PMC expressed detectable mRNA for TNF-alpha but not for the other four cytokines. Incubation of PMC with cyclosporin A (CsA) or dexamethasone (DEX), each at 10(-6) M for 24 hr, significantly inhibited the induced expression of mRNA for each of the five cytokines, and also inhibited release of biologically active TNF-alpha. Throughout these experiments mRNA levels of the housekeeping gene G3PDH were not altered by stimulation with anti-IgE or incubation with CsA or DEX. We conclude that immunological activation of rat PMC induces gene expression of several cytokines and that expression of these genes can be inhibited by immunosuppressive drugs. Images Figure 1 Figure 2 Figure 3 PMID:7490125

  2. Volcano collapse promoted by hydrothermal alteration and edifice shape, Mount Rainier, Washington

    USGS Publications Warehouse

    Reid, M.E.; Sisson, T.W.; Brien, D.L.

    2001-01-01

    Catastrophic collapses of steep volcano flanks threaten many populated regions, and understanding factors that promote collapse could save lives and property. Large collapses of hydrothermally altered parts of Mount Rainier have generated far-traveled debris flows; future flows would threaten densely populated parts of the Puget Sound region. We evaluate edifice collapse hazards at Mount Rainier using a new three-dimensional slope stability method incorporating detailed geologic mapping and subsurface geophysical imaging to determine distributions of strong (fresh) and weak (altered) rock. Quantitative three-dimensional slope stability calculations reveal that sizeable flank collapse (>0.1 km3) is promoted by voluminous, weak, hydrothermally altered rock situated high on steep slopes. These conditions exist only on Mount Rainier's upper west slope, consistent with the Holocene debris-flow history. Widespread alteration on lower flanks or concealed in regions of gentle slope high on the edifice does not greatly facilitate collapse. Our quantitative stability assessment method can also provide useful hazard predictions using reconnaissance geologic information and is a potentially rapid and inexpensive new tool for aiding volcano hazard assessments.

  3. Neuroleptic Drugs and PACAP Differentially Affect the mRNA Expression of Genes Encoding PAC1/VPAC Type Receptors.

    PubMed

    Jóźwiak-Bębenista, Marta; Kowalczyk, Edward

    2017-04-01

    Several lines of evidence suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide playing an important role as a neuromodulator. It has been indicated that PACAP is associated with mental diseases, and that regulation of the PACAPergic signals could be a potential target for the treatment of such psychiatric states as schizophrenia. Recent studies have suggested that action of neuroleptic drugs is mediated not only by dopaminergic and serotonergic neurotransmission, but also via neuropeptides which may act both as neurotransmitters and as neuromodulators. The present study examines whether currently-used neuroleptics influence the action of PACAP receptors, whose expression is altered in a schizophrenic patient. Real-time polymerase chain reaction (PCR) was used to examine the effects of haloperidol, olanzapine and amisulpride on the expression of genes coding PAC1/VPAC type receptors in the T98G glioblastoma cell line, as an example of an in vitro model of glial cells. PAC1 mRNA expression fell after 24-h incubation with haloperidol or olanzapine; however the effect was not maintained after 72 h, and haloperidol even up-regulated PAC1 mRNA expression in a dose-dependent manner. All the examined drugs decreased VPAC2 mRNA expression, especially after 72-h incubation. Haloperidol (typical neuroleptic) was distinctly more potent than atypical neuroleptic drugs (olanzapine and amisulpride). In addition, PACAP increased PAC1 and VPAC2 mRNA expression. In conclusion, our findings suggest PACAP receptors may be involved in the mechanism of typical and atypical neuroleptic drugs.

  4. Adrenal-dependent and -independent stress-induced Per1 mRNA in hypothalamic paraventricular nucleus and prefrontal cortex of male and female rats.

    PubMed

    Chun, Lauren E; Christensen, Jenny; Woodruff, Elizabeth R; Morton, Sarah J; Hinds, Laura R; Spencer, Robert L

    2018-01-01

    Oscillating clock gene expression gives rise to a molecular clock that is present not only in the body's master circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), but also in extra-SCN brain regions. These extra-SCN molecular clocks depend on the SCN for entrainment to a light:dark cycle. The SCN has limited neural efferents, so it may entrain extra-SCN molecular clocks through its well-established circadian control of glucocorticoid hormone secretion. Glucocorticoids can regulate the normal rhythmic expression of clock genes in some extra-SCN tissues. Untimely stress-induced glucocorticoid secretion may compromise extra-SCN molecular clock function. We examined whether acute restraint stress during the rat's inactive phase can rapidly (within 30 min) alter clock gene (Per1, Per2, Bmal1) and cFos mRNA (in situ hybridization) in the SCN, hypothalamic paraventricular nucleus (PVN), and prefrontal cortex (PFC) of male and female rats (6 rats per treatment group). Restraint stress increased Per1 and cFos mRNA in the PVN and PFC of both sexes. Stress also increased cFos mRNA in the SCN of male rats, but not when subsequently tested during their active phase. We also examined in male rats whether endogenous glucocorticoids are necessary for stress-induced Per1 mRNA (6-7 rats per treatment group). Adrenalectomy attenuated stress-induced Per1 mRNA in the PVN and ventral orbital cortex, but not in the medial PFC. These data indicate that increased Per1 mRNA may be a means by which extra-SCN molecular clocks adapt to environmental stimuli (e.g. stress), and in the PFC this effect is largely independent of glucocorticoids.

  5. No neuronal loss, but alterations of the GDNF system in asymptomatic diverticulosis.

    PubMed

    Barrenschee, Martina; Wedel, Thilo; Lange, Christina; Hohmeier, Ines; Cossais, François; Ebsen, Michael; Vogel, Ilka; Böttner, Martina

    2017-01-01

    Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor known to promote the survival and maintenance of neurons not only in the developing but also in the adult enteric nervous system. As diverticular disease (DD) is associated with reduced myenteric neurons, alterations of the GDNF system were studied in asymptomatic diverticulosis (diverticulosis) and DD. Morphometric analysis for quantifying myenteric ganglia and neurons were assessed in colonic full-thickness sections of patients with diverticulosis and controls. Samples of tunica muscularis (TM) and laser-microdissected myenteric ganglia from patients with diverticulosis, DD and controls were analyzed for mRNA expression levels of GDNF, GFRA1, and RET by RT-qPCR. Myenteric protein expression of both receptors was quantified by fluorescence-immunohistochemistry of patients with diverticulosis, DD, and controls. Although no myenteric morphometric alterations were found in patients with diverticulosis, GDNF, GFRA1 and RET mRNA expression was down-regulated in the TM of patients with diverticulosis as well as DD. Furthermore GFRA1 and RET myenteric plexus mRNA expression of patients with diverticulosis and DD was down-regulated, whereas GDNF remained unaltered. Myenteric immunoreactivity of the receptors GFRα1 and RET was decreased in both asymptomatic diverticulosis and DD patients. Our data provide evidence for an impaired GDNF system at gene and protein level not only in DD but also during early stages of diverticula formation. Thus, the results strengthen the idea of a disturbed GDNF-responsiveness as contributive factor for a primary enteric neuropathy involved in the pathogenesis and disturbed intestinal motility observed in DD.

  6. Altered Kinematics and Time to Stabilization During Drop-Jump Landings in Individuals With or Without Functional Ankle Instability

    PubMed Central

    Wright, Cynthia J.; Arnold, Brent L.; Ross, Scott E.

    2016-01-01

    Context It has been proposed that altered dynamic-control strategies during functional activity such as jump landings may partially explain recurrent instability in individuals with functional ankle instability (FAI). Objective To capture jump-landing time to stabilization (TTS) and ankle motion using a multisegment foot model among FAI, coper, and healthy control individuals. Design Cross-sectional study. Setting Laboratory. Patients or Other Participants Participants were 23 individuals with a history of at least 1 ankle sprain and at least 2 episodes of giving way in the past year (FAI), 23 individuals with a history of a single ankle sprain and no subsequent episodes of instability (copers), and 23 individuals with no history of ankle sprain or instability in their lifetime (controls). Participants were matched for age, height, and weight (age = 23.3 ± 3.8 years, height = 1.71 ± 0.09 m, weight = 69.0 ± 13.7 kg). Intervention(s) Ten single-legged drop jumps were recorded using a 12-camera Vicon MX motion-capture system and a strain-gauge force plate. Main Outcome Measures Mediolateral (ML) and anteroposterior (AP) TTS in seconds, as well as forefoot and hindfoot sagittal- and frontal-plane angles at jump-landing initial contact and at the point of maximum vertical ground reaction force were calculated. Results For the forefoot and hindfoot in the sagittal plane, group differences were present at initial contact (forefoot: P = .043, hindfoot: P = .004). At the hindfoot, individuals with FAI displayed more dorsiflexion than the control and coper groups. Time to stabilization differed among groups (AP TTS: P < .001; ML TTS: P = .040). Anteroposterior TTS was longer in the coper group than in the FAI or control groups, and ML TTS was longer in the FAI group than in the control group. Conclusions During jump landings, copers showed differences in sagittal-plane control, including less plantar flexion at initial contact and increased AP sway during stabilization

  7. Substance P induced preprotachykinin-a mRNA, neutral endopeptidase mRNA and substance P in cultured normal fibroblasts.

    PubMed

    Bae, Sang-Jae; Matsunaga, Yoshitaka; Takenaka, Motoi; Tanaka, Yoichi; Hamazaki, Yoichiro; Shimizu, Kazuhiro; Katayama, Ichiro

    2002-04-01

    In certain skin diseases, stress can modulate the induction and/or progression of cutaneous manifestations. However, little is known about the circuit in neuroendocrine and in the immune systems of the skin. To address this question, we have analyzed the regulatory mechanisms of autocrine induction of substance P (SP) by cultured normal human fibroblasts that compose the major population of the skin and might augment stress-induced skin inflammatory responses. In nonstimulated conditions, normal fibroblasts express a moderate amount of preprotachykinin-A (PPT-A), a precursor of SP mRNA, and exogenous SP significantly upregulated PPT-A mRNA expression. Maximum response of SP peptide and SP mRNA in fibroblasts was observed 1-3 h after stimulation with SP. In contrast, the expression of neutral endopeptidase (NEP), a cell surface peptide with hydrolyzing activity of SP, was increased in fibroblasts stimulated with SP after 24 h. The administration of NEP inhibitor (phosphoramidon) to the fibroblasts induced higher SP production. In addition, the neurokinin (NK) receptor antagonists (spantide, FK224 and FK888) and protein synthesis inhibitor (cycloheximide) inhibited SP production by 30-40% of control response. In immunostaining study, specific cytoplasmic staining of SP was observed in fibroblasts stimulated with SP. Finally, we confirmed that the nucleotide sequence of the PPT-A expressed in fibroblasts perfectly corresponded to the gene bank human PPT-A cDNA. This is the first report that SP mRNA, NEP mRNA and SP peptide can be induced by normal human skin fibroblasts in response to exogenous SP, and that fibroblast-derived SP might play an important role in the induction and acceleration of certain cutaneous diseases. Copyright 2002 S. Karger AG, Basel

  8. 3,4-Methylenedioxymethamphetamine (MDMA), but not morphine, alters APP processing in the rat brain.

    PubMed

    Kálmán, János; Bjelik, Annamária; Hugyecz, Marietta; Tímár, Júlia; Gyarmati, Zsuzsanna; Zana, Marianna; Fürst, Zsuzsanna; Janka, Zoltán; Rakonczay, Zoltán; Horváth, Zoltán; Pákáski, Magdolna

    2007-04-01

    The abuse of drugs such as opioids and 3,4-methylenedioxymethamphetamine (MDMA or 'ecstasy') can have detrimental effects on the cognitive functions, but the exact molecular mechanism whereby these drugs promote neurodegeneration remains to be elucidated. The major purpose of the present pilot study was to determine whether the chronic in-vivo administration of morphine (10 mg/kg) or MDMA (1 mg/kg) to rats can alter the expression and processing of amyloid precursor protein (APP), the central molecule in the proposed pathomechanism of Alzheimer's disease. MDMA treatment significantly decreased the production of APP in the cytosolic fraction of the brain cortex. A concomitant 25% increase was found both in the beta-secretase (BACE) and APP mRNA levels (108%). In contrast, in the applied single dosage chronic morphine treatment did not influence either the APP and BACE protein levels or the APP mRNA production. These results indicate that the chronic use of 'ecstasy', but not morphine, may be harmful via a novel mode of action, i.e. by altering the APP expression and processing in the brain.

  9. The RDE-10/RDE-11 complex triggers RNAi-induced mRNA degradation by association with target mRNA in C. elegans

    PubMed Central

    Yang, Huan; Zhang, Ying; Vallandingham, Jim; Li, Hau; Florens, Laurence; Mak, Ho Yi

    2012-01-01

    The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans. PMID:22508728

  10. The RDE-10/RDE-11 complex triggers RNAi-induced mRNA degradation by association with target mRNA in C. elegans.

    PubMed

    Yang, Huan; Zhang, Ying; Vallandingham, Jim; Li, Hua; Li, Hau; Florens, Laurence; Mak, Ho Yi

    2012-04-15

    The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans.

  11. Tools for translation: non-viral materials for therapeutic mRNA delivery

    NASA Astrophysics Data System (ADS)

    Hajj, Khalid A.; Whitehead, Kathryn A.

    2017-10-01

    In recent years, messenger RNA (mRNA) has come into the spotlight as a versatile therapeutic with the potential to prevent and treat a staggering range of diseases. Billions of dollars have been invested in the commercial development of mRNA drugs, with ongoing clinical trials focused on vaccines (for example, influenza and Zika viruses) and cancer immunotherapy (for example, myeloma, leukaemia and glioblastoma). Although significant progress has been made in the design of in vitro-transcribed mRNA that retains potency while minimizing unwanted immune responses, the widespread use of mRNA drugs requires the development of safe and effective drug delivery vehicles. In this Review, we provide an overview of the field of mRNA therapeutics and describe recent advances in the development of synthetic materials that encapsulate and deliver mRNA payloads.

  12. Endogenous CNS Expression of Neurotensin and Neurotensin Receptors Is Altered during the Postpartum Period in Outbred Mice

    PubMed Central

    Driessen, Terri M.; Zhao, Changjiu; Whittlinger, Anna; Williams, Horecia; Gammie, Stephen C.

    2014-01-01

    Neurotensin (NT) is a neuropeptide identical in mice and humans that is produced and released in many CNS regions associated with maternal behavior. NT has been linked to aspects of maternal care and previous studies have indirectly suggested that endogenous NT signaling is altered in the postpartum period. In the present study, we directly examine whether NT and its receptors exhibit altered gene expression in maternal relative to virgin outbred mice using real time quantitative PCR (qPCR) across multiple brain regions. We also examine NT protein levels using anti-NT antibodies and immunohistochemistry in specific brain regions. In the medial preoptic area (MPOA), which is critical for maternal behaviors, mRNA of NT and NT receptor 3 (Sort1) were significantly up-regulated in postpartum mice compared to virgins. NT mRNA was also elevated in postpartum females in the bed nucleus of the stria terminalis dorsal. However, in the lateral septum, NT mRNA was down-regulated in postpartum females. In the paraventricular nucleus of the hypothalamus (PVN), Ntsr1 expression was down-regulated in postpartum females. Neurotensin receptor 2 (Ntsr2) expression was not altered in any brain region tested. In terms of protein expression, NT immunohistochemistry results indicated that NT labeling was elevated in the postpartum brain in the MPOA, lateral hypothalamus, and two subregions of PVN. Together, these findings indicate that endogenous changes occur in NT and its receptors across multiple brain regions, and these likely support the emergence of some maternal behaviors. PMID:24416154

  13. p21WAF1/CIP1 Upregulation through the Stress Granule-Associated Protein CUGBP1 Confers Resistance to Bortezomib-Mediated Apoptosis

    PubMed Central

    Gareau, Cristina; Fournier, Marie-Josée; Filion, Christine; Coudert, Laetitia; Martel, David; Labelle, Yves; Mazroui, Rachid

    2011-01-01

    Background p21WAF1/CIP1 is a well known cyclin-dependent kinase inhibitor induced by various stress stimuli. Depending on the stress applied, p21 upregulation can either promote apoptosis or prevent against apoptotic injury. The stress-mediated induction of p21 involves not only its transcriptional activation but also its posttranscriptional regulation, mainly through stabilization of p21 mRNA levels. We have previously reported that the proteasome inhibitor MG132 induces the stabilization of p21 mRNA, which correlates with the formation of cytoplasmic RNA stress granules. The mechanism underlying p21 mRNA stabilization, however, remains unknown. Methodology/Principal Findings We identified the stress granules component CUGBP1 as a factor required for p21 mRNA stabilization following treatment with bortezomib ( =  PS-341/Velcade). This peptide boronate inhibitor of the 26S proteasome is very efficient for the treatment of myelomas and other hematological tumors. However, solid tumors are sometimes refractory to bortezomib treatment. We found that depleting CUGBP1 in cancer cells prevents bortezomib-mediated p21 upregulation. FISH experiments combined to mRNA stability assays show that this effect is largely due to a mistargeting of p21 mRNA in stress granules leading to its degradation. Altering the expression of p21 itself, either by depleting CUGBP1 or p21, promotes bortezomib-mediated apoptosis. Conclusions/Significance We propose that one key mechanism by which apoptosis is inhibited upon treatment with chemotherapeutic drugs might involve upregulation of the p21 protein through CUGBP1. PMID:21637851

  14. Regulatory RNA binding proteins contribute to the transcriptome-wide splicing alterations in human cellular senescence.

    PubMed

    Dong, Qiongye; Wei, Lei; Zhang, Michael Q; Wang, Xiaowo

    2018-06-24

    Dysregulation of mRNA splicing has been observed in certain cellular senescence process. However, the common splicing alterations on the whole transcriptome shared by various types of senescence are poorly understood. In order to systematically identify senescence-associated transcriptomic changes in genome-wide scale, we collected RNA sequencing datasets of different human cell types with a variety of senescence-inducing methods from public databases and performed meta-analysis. First, we discovered that a group of RNA binding proteins were consistently down-regulated in diverse senescent samples and identified 406 senescence-associated common differential splicing events. Then, eight differentially expressed RNA binding proteins were predicted to regulate these senescence-associated splicing alterations through an enrichment analysis of their RNA binding information, including motif scanning and enhanced cross-linking immunoprecipitation data. In addition, we constructed the splicing regulatory modules that might contribute to senescence-associated biological processes. Finally, it was confirmed that knockdown of the predicted senescence-associated potential splicing regulators through shRNAs in HepG2 cell line could result in senescence-like splicing changes. Taken together, our work demonstrated a broad range of common changes in mRNA splicing switches and detected their central regulatory RNA binding proteins during senescence. These findings would help to better understand the coordinating splicing alterations in cellular senescence.

  15. A sense oligonucleotide to inducible nitric oxide synthase mRNA increases the survival rate of rats in septic shock.

    PubMed

    Okuyama, Tetsuya; Nakatake, Richi; Kaibori, Masaki; Okumura, Tadayoshi; Kon, Masanori; Nishizawa, Mikio

    2018-01-30

    Natural antisense transcripts (asRNAs) that do not encode proteins are transcribed from rat, mouse, and human genes, encoding inducible nitric oxide synthase (iNOS), which catalyzes the production of the inflammatory mediator nitric oxide (NO). In septic shock, NO is excessively produced in hepatocytes and macrophages. The iNOS asRNA interacts with and stabilizes iNOS mRNA. We found that single-stranded 'sense' oligonucleotides corresponding to the iNOS mRNA sequence reduced iNOS mRNA levels by interfering with the mRNA-asRNA interactions in rat hepatocytes. The iNOS sense oligonucleotides that were substituted with phosphorothioate bonds and locked nucleic acids efficiently decreased the levels of iNOS mRNA and iNOS protein. In this study, the gene expression patterns in the livers of two endotoxemia model rats with acute liver failure were compared. Next, we optimized the sequence and modification of the iNOS sense oligonucleotides in interleukin 1β-treated rat hepatocytes. When a sense oligonucleotide was simultaneously administered with d-galactosamine and bacterial lipopolysaccharide (LPS) to rats, their survival rate significantly increased compared to the rats administered d-galactosamine and LPS alone. In the livers of the sense oligonucleotide-administered rats, apoptosis in the hepatocytes markedly decreased. These results suggest that natural antisense transcript-targeted regulation technology using iNOS sense oligonucleotides may be used to treat human inflammatory diseases, such as sepsis and septic shock. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Differential stability of host mRNAs in Friend erythroleukemia cells infected with herpes simplex virus type 1

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

    Mayman, B.A.; Nishioka, Y.

    1985-01-01

    The consequences of herpes simplex virus type 1 infection on cellular macromolecules were investigated in Friend erythroleukemia cells. The patterns of protein synthesis, examined by polyacrylamide gel electrophoresis, demonstrated that by 4 h postinfection the synthesis of many host proteins, with the exception of histones, was inhibited. Examination of the steady-state level of histone H3 mRNA by molecular hybridization of total RNA to a cloned mouse histone H3 complementary DNA probe demonstrated that the ratio of histone H3 mRNA to total RNA remained unchanged for the first 4 h postinfection. In contrast, the steady-state levels of globin and actin mRNAsmore » decreased progressively at early intervals postinfection. Studies on RNA synthesis in isolated nuclei demonstrated that the transcription of the histone H3 gene was inhibited to approximately the same extent as that of actin gene. It was concluded that the stabilization of preexisting histone H3 mRNA was responsible for the persistence of H3 mRNA and histone protein synthesis in herpes simplex virus type 1-infected Friend erythroleukemia cells. The possible mechanisms influencing the differential stability of host mRNAs during the course of productive infection with herpes simplex virus type 1 are discussed.« less

  17. Prolonged endoplasmic reticulum stress alters placental morphology and causes low birth weight

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

    Kawakami, Takashige, E-mail: tkawakami@ph.bunri-u.ac.jp; Yoshimi, Masaki; Kadota, Yoshito

    The role of endoplasmic reticulum (ER) stress in pregnancy remains largely unknown. Pregnant mice were subcutaneously administered tunicamycin (Tun), an ER stressor, as a single dose [0, 50, and 100 μg Tun/kg/body weight (BW)] on gestation days (GDs) 8.5, 12.5, and 15.5. A high incidence (75%) of preterm delivery was observed only in the group treated with Tun 100 μg/kg BW at GD 15.5, indicating that pregnant mice during late gestation are more susceptible to ER stress on preterm delivery. We further examined whether prolonged in utero exposure to ER stress affects fetal development. Pregnant mice were subcutaneously administered amore » dose of 0, 20, 40, and 60 μg Tun/kg from GD 12.5 to 16.5. Tun treatment decreased the placental and fetal weights in a dose-dependent manner. Histological evaluation showed the formation of a cluster of spongiotrophoblast cells in the labyrinth zone of the placenta of Tun-treated mice. The glycogen content of the fetal liver and placenta from Tun-treated mice was lower than that from control mice. Tun treatment decreased mRNA expression of Slc2a1/glucose transporter 1 (GLUT1), which is a major transporter for glucose, but increased placental mRNA levels of Slc2a3/GLUT3. Moreover, maternal exposure to Tun resulted in a decrease in vascular endothelial growth factor receptor-1 (VEGFR-1), VEGFR-2, and placental growth factor. These results suggest that excessive and exogenous ER stress may induce functional abnormalities in the placenta, at least in part, with altered GLUT and vascular-related gene expression, resulting in low infant birth weight. - Highlights: • Maternal exposure to excessive ER stress induced preterm birth and IUGR. • Prolonged excessive ER stress altered the formation of the placental labyrinth. • ER stress decreased GLUT1 mRNA expression in the placenta, but increased GLUT3. • ER stress-induced IUGR causes decreased glycogen and altered glucose transport.« less

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

    PubMed

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. A novel homozygous stop-codon mutation in human HFE responsible for nonsense-mediated mRNA decay.

    PubMed

    Padula, Maria Carmela; Martelli, Giuseppe; Larocca, Marilena; Rossano, Rocco; Olivieri, Attilio

    2014-09-01

    HFE-hemochromatosis (HH) is an autosomal disease characterized by excessive iron absorption. Homozygotes for H63D variant, and still less H63D heterozygotes, generally do not express HH phenotype. The data collected in our previous study in the province of Matera (Basilicata, Italy) underlined that some H63D carriers showed altered iron metabolism, without additional factors. In this study, we selected a cohort of 10/22 H63D carriers with severe biochemical iron overload (BIO). Additional analysis was performed for studying HFE exons, exon-intron boundaries, and untranslated regions (UTRs) by performing DNA extraction, PCR amplification and sequencing. The results showed a novel substitution (NM_000410.3:c.847C>T) in a patient exon 4 (GenBankJQ478433); it introduces a premature stop-codon (PTC). RNA extraction and reverse-transcription were also performed. Quantitative real-time PCR was carried out for verifying if our aberrant mRNA is targeted for nonsense-mediated mRNA decay (NMD); we observed that patient HFE mRNA was expressed much less than calibrator, suggesting that the mutated HFE protein cannot play its role in iron metabolism regulation, resulting in proband BIO. Our finding is the first evidence of a variation responsible for a PTC in iron cycle genes. The genotype-phenotype correlation observed in our cases could be related to the additional mutation. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Coupling mRNA processing with transcription in time and space

    PubMed Central

    Bentley, David L.

    2015-01-01

    Maturation of mRNA precursors often occurs simultaneously with their synthesis by RNA polymerase II (Pol II). The co-transcriptional nature of mRNA processing has permitted the evolution of coupling mechanisms that coordinate transcription with mRNA capping, splicing, editing and 3′ end formation. Recent experiments using sophisticated new methods for analysis of nascent RNA have provided important insights into the relative amount of co-transcriptional and post-transcriptional processing, the relationship between mRNA elongation and processing, and the role of the Pol II carboxy-terminal domain (CTD) in regulating these processes. PMID:24514444

  1. Lower expression of glutamic acid decarboxylase 67 in the prefrontal cortex in schizophrenia: contribution of altered regulation by Zif268.

    PubMed

    Kimoto, Sohei; Bazmi, H Holly; Lewis, David A

    2014-09-01

    Cognitive deficits of schizophrenia may be due at least in part to lower expression of the 67-kDa isoform of glutamic acid decarboxylase (GAD67), a key enzyme for GABA synthesis, in the dorsolateral prefrontal cortex of individuals with schizophrenia. However, little is known about the molecular regulation of lower cortical GAD67 levels in schizophrenia. The GAD67 promoter region contains a conserved Zif268 binding site, and Zif268 activation is accompanied by increased GAD67 expression. Thus, altered expression of the immediate early gene Zif268 may contribute to lower levels of GAD67 mRNA in the dorsolateral prefrontal cortex in schizophrenia. The authors used polymerase chain reaction to quantify GAD67 and Zif268 mRNA levels in dorsolateral prefrontal cortex area 9 from 62 matched pairs of schizophrenia and healthy comparison subjects, and in situ hybridization to assess Zif268 expression at laminar and cellular levels of resolution. The effects of potentially confounding variables were assessed in human subjects, and the effects of antipsychotic treatments were tested in antipsychotic-exposed monkeys. The specificity of the Zif268 findings was assessed by quantifying mRNA levels for other immediate early genes. GAD67 and Zif268 mRNA levels were significantly lower and were positively correlated in the schizophrenia subjects. Both Zif268 mRNA-positive neuron density and Zif268 mRNA levels per neuron were significantly lower in the schizophrenia subjects. These findings were robust to the effects of the confounding variables examined and differed from other immediate early genes. Deficient Zif268 mRNA expression may contribute to lower cortical GAD67 levels in schizophrenia, suggesting a potential mechanistic basis for altered cortical GABA synthesis and impaired cognition in schizophrenia.

  2. MRNA and miRNA expression patterns associated to pathways linked to metal mixture health effects.

    PubMed

    Martínez-Pacheco, M; Hidalgo-Miranda, A; Romero-Córdoba, S; Valverde, M; Rojas, E

    2014-01-10

    Metals are a threat to human health by increasing disease risk. Experimental data have linked altered miRNA expression with exposure to some metals. MiRNAs comprise a large family of non-coding single-stranded molecules that primarily function to negatively regulate gene expression post-transcriptionally. Although several human populations are exposed to low concentrations of As, Cd and Pb as a mixture, most toxicology research focuses on the individual effects that these metals exert. Thus, this study aims to evaluate global miRNA and mRNA expression changes induced by a metal mixture containing NaAsO2, CdCl2, Pb(C2H3O2)2·3H2O and to predict possible metal-associated disease development under these conditions. Our results show that this metal mixture results in a miRNA expression profile that may be responsible for the mRNA expression changes observed under experimental conditions in which coding proteins are involved in cellular processes, including cell death, growth and proliferation related to the metal-associated inflammatory response and cancer. © 2013 Elsevier B.V. All rights reserved.

  3. Renal C-type natriuretic peptide and natriuretic peptide receptor B mRNA expression are affected by water deprivation in the Spinifex Hopping mouse.

    PubMed

    Heimeier, Rachel A; Donald, John A

    2003-11-01

    This study investigated the effect of water deprivation on the expression of C-type natriuretic peptide (CNP) and natriuretic peptide receptor B (NPR-B) mRNA, and the ability of NPR-B to generate cGMP in the Spinifex Hopping mouse, Notomys alexis. This rodent is a native of central and western Australia that is well adapted to survive in arid environments. Initially, CNP and NPR-B cDNAs (partial for NPR-B) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. RT-PCR analysis showed CNP mRNA expression in the kidney, proximal and distal colon and small intestine, whilst NPR-B mRNA expression was found in the kidney, proximal and distal colon and the atria. Using a semi-quantitative multiplex PCR technique, the expression of renal CNP and NPR-B mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control hopping mice (access to water). Water deprivation significantly decreased the relative levels of CNP and NPR-B mRNA expression in both the 7- and 14-day water-deprived hopping mice, when compared to control hopping mice. In contrast, the ability of CNP to stimulate cGMP production was significantly increased after 14 days of water deprivation. This study shows that alterations in the renal CNP/NPR-B system may be an important physiological adjustment when water is scarce.

  4. Genome wide assessment of mRNA in astrocyte protrusions by direct RNA sequencing reveals mRNA localization for the intermediate filament protein nestin.

    PubMed

    Thomsen, Rune; Pallesen, Jonatan; Daugaard, Tina F; Børglum, Anders D; Nielsen, Anders L

    2013-11-01

    Subcellular RNA localization plays an important role in development, cell differentiation, and cell migration. For a comprehensive description of the population of protrusion localized mRNAs in astrocytes we separated protrusions from cell bodies in a Boyden chamber and performed high-throughput direct RNA sequencing. The mRNAs with localization in astrocyte protrusions encode proteins belonging to a variety of functional groups indicating involvement of RNA localization for a palette of cellular functions. The mRNA encoding the intermediate filament protein Nestin was among the identified mRNAs. By RT-qPCR and RNA FISH analysis we confirmed Nestin mRNA localization in cell protrusions and also protrusion localization of Nestin protein. Nestin mRNA localization was dependent of Fragile X mental retardation syndrome proteins Fmrp and Fxr1, and the Nestin 3'-UTR was sufficient to mediate protrusion mRNA localization. The mRNAs for two other intermediate filament proteins in astrocytes, Gfap and Vimentin, have moderate and no protrusion localization, respectively, showing that individual intermediate filament components have different localization mechanisms. The correlated localization of Nestin mRNA with Nestin protein in cell protrusions indicates the presence of a regulatory mechanism at the mRNA localization level for the Nestin intermediate filament protein with potential importance for astrocyte functions during brain development and maintenance. Copyright © 2013 Wiley Periodicals, Inc.

  5. Transition from shod to barefoot alters dynamic stability during running.

    PubMed

    Ekizos, Antonis; Santuz, Alessandro; Arampatzis, Adamantios

    2017-07-01

    Barefoot running recently received increased attention, with controversial results regarding its effects on injury risk and performance. Numerous studies examined the kinetic and kinematic changes between the shod and the barefoot condition. Intrinsic parameters such as the local dynamic stability could provide new insight regarding neuromuscular control when immediately transitioning from one running condition to the other. We investigated the local dynamic stability during the change from shod to barefoot running. We further measured biomechanical parameters to examine the mechanisms governing this transition. Twenty habitually shod, young and healthy participants ran on a pressure plate-equipped treadmill and alternated between shod and barefoot running. We calculated the largest Lyapunov exponents as a measure of errors in the control of the movement. Biomechanical parameters were also collected. Local dynamic stability decreased significantly (d=0.41; 2.1%) during barefoot running indicating worse control over the movement. We measured higher cadence (d=0.35; 2.2%) and total flight time (d=0.58; 19%), lower total contact time (d=0.58; -5%), total vertical displacement (d=0.39; -4%), and vertical impulse (d=1.32; 11%) over the two minutes when running barefoot. The strike index changed significantly (d=1.29; 237%) towards the front of the foot. Immediate transition from shod to the barefoot condition resulted in an increased instability and indicates a worst control over the movement. The increased instability was associated with biomechanical changes (i.e. foot strike patterns) of the participants in the barefoot condition. Possible reasons why this instability arises, might be traced in the stance phase and particularly in the push-off. The decreased stability might affect injury risk and performance. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Expression of beta 3-adrenoceptor mRNA in rat tissues.

    PubMed

    Evans, B A; Papaioannou, M; Bonazzi, V R; Summers, R J

    1996-01-01

    1. This study examines the expression of beta 3-adrenoceptor messenger RNA (beta 3-AR mRNA) in rat tissues to allow comparison with atypical beta-adrenoceptors determined by functional and radioligand binding techniques. 2. A reverse transcription/polymerase chain reaction protocol has been developed for determining the relative amounts of beta 3-AR mRNA in rat tissues. 3. Measurement of adipsin and uncoupling protein (UCP) mRNA was used to examine all tissues for the presence of white and brown adipose tissue which may contribute beta 3-AR mRNA. 4. The beta 3-AR mRNA is expressed at high levels in brown and white adipose tissue, stomach fundus, the longitudinal/circular smooth muscle of both colon and ileum, and colon submucosa. There was substantial expression of adipsin in colon submucosa and moderate expression in fundus, suggesting that in these regions at least some of the beta 3-AR signal may be contributed by fat. Pylorus and colon mucosa showed moderate levels of beta 3-AR mRNA with lower levels of adipsin. Ileum mucosa and submucosa showed low but readily detectable levels of beta 3-AR. 5. Expression of adipsin in rat skeletal muscles coupled to very low levels of beta 3-AR mRNA indicates that the observed beta 3-AR may be due to the presence of intrinsic fat. beta 3-AR mRNA was virtually undetectable in heart, lung and liver. These results raise the possibility that the atypical beta-AR demonstrated by functional and/or binding studies in muscle and in heart is not the beta 3-AR. 6. By use of two different sets of primers for amplification of beta 3-AR cDNA, no evidence was found for differential splicing of the mRNA in any of the tissues examined. 7. The detection of beta 3-AR mRNA in the gut mucosa and submucosa suggests that in addition to its established roles in lipolysis, thermogenesis and regulation of gut motility beta 3-AR may subserve other functions in the gastrointestinal tract. The absence of beta 3-AR mRNA in rat heart or its presence with

  7. Comparative Analysis of mRNA Isoform Expression in Cardiac Hypertrophy and Development Reveals Multiple Post-Transcriptional Regulatory Modules

    PubMed Central

    Park, Ji Yeon; Li, Wencheng; Zheng, Dinghai; Zhai, Peiyong; Zhao, Yun; Matsuda, Takahisa; Vatner, Stephen F.; Sadoshima, Junichi; Tian, Bin

    2011-01-01

    Cardiac hypertrophy is enlargement of the heart in response to physiological or pathological stimuli, chiefly involving growth of myocytes in size rather than in number. Previous studies have shown that the expression pattern of a group of genes in hypertrophied heart induced by pressure overload resembles that at the embryonic stage of heart development, a phenomenon known as activation of the “fetal gene program”. Here, using a genome-wide approach we systematically defined genes and pathways regulated in short- and long-term cardiac hypertrophy conditions using mice with transverse aortic constriction (TAC), and compared them with those regulated at different stages of embryonic and postnatal development. In addition, exon-level analysis revealed widespread mRNA isoform changes during cardiac hypertrophy resulting from alternative usage of terminal or internal exons, some of which are also developmentally regulated and may be attributable to decreased expression of Fox-1 protein in cardiac hypertrophy. Genes with functions in certain pathways, such as cell adhesion and cell morphology, are more likely to be regulated by alternative splicing. Moreover, we found 3′UTRs of mRNAs were generally shortened through alternative cleavage and polyadenylation in hypertrophy, and microRNA target genes were generally de-repressed, suggesting coordinated mechanisms to increase mRNA stability and protein production during hypertrophy. Taken together, our results comprehensively delineated gene and mRNA isoform regulation events in cardiac hypertrophy and revealed their relations to those in development, and suggested that modulation of mRNA isoform expression plays an importance role in heart remodeling under pressure overload. PMID:21799842

  8. Low power lasers on genomic stability.

    PubMed

    Trajano, Larissa Alexsandra da Silva Neto; Sergio, Luiz Philippe da Silva; Stumbo, Ana Carolina; Mencalha, Andre Luiz; Fonseca, Adenilson de Souza da

    2018-03-01

    Exposure of cells to genotoxic agents causes modifications in DNA, resulting to alterations in the genome. To reduce genomic instability, cells have DNA damage responses in which DNA repair proteins remove these lesions. Excessive free radicals cause DNA damages, repaired by base excision repair and nucleotide excision repair pathways. When non-oxidative lesions occur, genomic stability is maintained through checkpoints in which the cell cycle stops and DNA repair occurs. Telomere shortening is related to the development of various diseases, such as cancer. Low power lasers are used for treatment of a number of diseases, but they are also suggested to cause DNA damages at sub-lethal levels and alter transcript levels from DNA repair genes. This review focuses on genomic and telomere stabilization modulation as possible targets to improve therapeutic protocols based on low power lasers. Several studies have been carried out to evaluate the laser-induced effects on genome and telomere stabilization suggesting that exposure to these lasers modulates DNA repair mechanisms, telomere maintenance and genomic stabilization. Although the mechanisms are not well understood yet, low power lasers could be effective against DNA harmful agents by induction of DNA repair mechanisms and modulation of telomere maintenance and genomic stability. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. mRNA transcripts as molecular biomarkers in medicine and nutrition

    PubMed Central

    Sunde, Roger A.

    2010-01-01

    In medicine, mRNA transcripts are being developed as molecular biomarkers for the diagnosis and treatment of a number of diseases. These biomarkers offer early and more accurate prediction and diagnosis of disease and disease progression, and ability to identify individuals at risk. Use of microarrays also offers opportunity to identify orthogonal (uncorrelated) biomarkers not known to be linked with conventional biomarkers. Investigators are increasingly using blood as a surrogate tissue for biopsy and analysis; total RNA isolated from whole blood is predominantly from erythroid cells, and whole blood mRNA share more than 80% of the transcriptome with major tissues. Thus blood mRNA biomarkers for individualized disease prediction and diagnosis are an exciting area in medicine; mRNA biomarkers in nutrition have potential application that parallel these opportunities. Assessment of selenium (Se) status and requirements is one area where tissue mRNA levels have been used successfully. Selenoprotein-H and selenoprotein-W as well as glutathione peroxidase-1 (Gpx1) mRNAs are highly down-regulated in Se deficiency in rat liver, and the minimum dietary Se requirement is 0.06–0.07 μg Se/g based on these biomarkers, similar to requirements determined using conventional biomarkers. Blood Gpx1 mRNA can also be used to determine Se requirements in rats, showing that blood mRNA has potential for assessment of nutrient status. Future research is needed to develop mRNA biomarker panels for all nutrients that will discriminate between deficient, marginal, adequate, and supernutritional individuals and populations, and differentiate between individuals that will benefit versus be adversely affected by nutrient supplementation. PMID:20303730

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

    PubMed

    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

  11. Relationship between mRNA secondary structure and sequence variability in Chloroplast genes: possible life history implications.

    PubMed

    Krishnan, Neeraja M; Seligmann, Hervé; Rao, Basuthkar J

    2008-01-28

    Synonymous sites are freer to vary because of redundancy in genetic code. Messenger RNA secondary structure restricts this freedom, as revealed by previous findings in mitochondrial genes that mutations at third codon position nucleotides in helices are more selected against than those in loops. This motivated us to explore the constraints imposed by mRNA secondary structure on evolutionary variability at all codon positions in general, in chloroplast systems. We found that the evolutionary variability and intrinsic secondary structure stability of these sequences share an inverse relationship. Simulations of most likely single nucleotide evolution in Psilotum nudum and Nephroselmis olivacea mRNAs, indicate that helix-forming propensities of mutated mRNAs are greater than those of the natural mRNAs for short sequences and vice-versa for long sequences. Moreover, helix-forming propensity estimated by the percentage of total mRNA in helices increases gradually with mRNA length, saturating beyond 1000 nucleotides. Protection levels of functionally important sites vary across plants and proteins: r-strategists minimize mutation costs in large genes; K-strategists do the opposite. Mrna length presumably predisposes shorter mRNAs to evolve under different constraints than longer mRNAs. The positive correlation between secondary structure protection and functional importance of sites suggests that some sites might be conserved due to packing-protection constraints at the nucleic acid level in addition to protein level constraints. Consequently, nucleic acid secondary structure a priori biases mutations. The converse (exposure of conserved sites) apparently occurs in a smaller number of cases, indicating a different evolutionary adaptive strategy in these plants. The differences between the protection levels of functionally important sites for r- and K-strategists reflect their respective molecular adaptive strategies. These converge with increasing domestication levels of

  12. mRNA localization: an orchestration of assembly, traffic and synthesis.

    PubMed

    Xing, Lei; Bassell, Gary J

    2013-01-01

    Asymmetrical mRNA localization and subsequent local translation provide efficient mechanisms for protein sorting in polarized cells. Defects in mRNA localization have been linked to developmental abnormalities and neurological diseases. Thus, it is critical to understand the machineries mediating and mechanisms underlying the asymmetrical distribution of mRNA and its regulation. The goal of this review is to summarize recent advances in the understanding of mRNA transport and localization, including the assembly and sorting of transport messenger ribonucleic protein (mRNP) granules, molecular mechanisms of active mRNP transport, cytoskeletal interactions and regulation of these events by extracellular signals. © 2012 John Wiley & Sons A/S.

  13. Reproductive experience alters prolactin receptor expression in mammary and hepatic tissues in female rats.

    PubMed

    Bridges, Robert S; Scanlan, Victoria F; Lee, Jong-O; Byrnes, Elizabeth M

    2011-08-01

    Recent studies have reported that reproductive experience in female rats alters prolactin (PRL) receptor gene expression in the brain as well as neural sensitivity to PRL. Given PRL's actions in nonneural tissues, that is, mammary tissue and liver, it was asked whether reproductive experience may also alter prolactin receptor (Prlr) gene expression in these tissues. Groups of age-matched female rats were generated with varying reproductive histories. Separate groups of primiparous (first lactation) and multiparous (second lactation) had mammary tissue and liver samples collected on Day 3 or 10 of lactation. A fifth group raised one litter to weaning and then resumed estrous cyclicity. This group and a final group of age-matched, virgin controls were killed on diestrus. Tissue was processed by quantitative PCR for expression rates of the long and short forms of Prlr mRNA as well as casein beta mRNA (mammary tissue only). Western blots were performed to quantify receptor protein content. Multiple lactations as well as lactation itself resulted in alterations in Prlr expression. Prlr gene expression in mammary tissue was increased in primiparous mothers compared with that in multiparous dams, whereas in the liver, Prlr expression was reduced during an initial lactation. In contrast, PRLR protein levels declined during lactation in mammary, but not hepatic, tissues. Overall, the results demonstrate that the prolactin receptor system is altered in nonneural tissues as a result of the female's reproductive history. The findings are discussed in the context of milk and bile production and PRL's possible role in breast cancer.

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

    PubMed

    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.

  15. Lower glutamic acid decarboxylase 65kD mRNA and protein levels in the prefrontal cortex in schizoaffective disorder but not schizophrenia

    PubMed Central

    Glausier, JR; Kimoto, S; Fish, KN; Lewis, DA

    2014-01-01

    Background Altered GABA signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in schizophrenia and schizoaffective disorder. PFC levels of the GABA-synthesizing enzyme glutamic acid decarboxylase 67kD (GAD67) has been consistently reported to be lower in these disorders, but the status of the second GABA-synthesizing enzyme, GAD65, remains unclear. Methods GAD65 mRNA levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. GAD65 relative protein levels were quantified in a subset of subject pairs by confocal immunofluorescence microscopy. Results Mean GAD65 mRNA levels were 13.6% lower in schizoaffective disorder subjects, but did not differ in schizophrenia subjects, relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein measures within schizoaffective disorder subjects was not attributable to factors commonly comorbid with the diagnosis. Conclusions In concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in subjects with schizoaffective disorder relative to subjects with schizophrenia, these findings may support an interpretation that GAD65 down-regulation provides a homeostatic response complementary to GAD67 down-regulation expression that serves to reduce inhibition in the face of lower PFC network activity. PMID:24993056

  16. Early social deprivation impairs pair bonding and alters serum corticosterone and the NAcc dopamine system in mandarin voles.

    PubMed

    Yu, Peng; An, Shucheng; Tai, Fadao; Wang, Jianli; Wu, Ruiyong; Wang, Bo

    2013-12-01

    Early life stress has a long-term negative impact on emotion, learning, memory and adult sexual behavior, and these deficits most likely impair pair bonding. Here, we investigated whether early social deprivation (ED) affects the formation of pair bonds in socially monogamous mandarin voles (Microtus mandarinus). In a partner preference test (PPT), ED-reared adult females and males did not show a preference for their partner, spent more time exploring the cage of an unfamiliar animal and directed high levels of aggression toward unfamiliar animals. In social interaction test, ED increased exploring behavior only in females, but increased movement around the partner and reduced inactivity in both males and females. Three days of cohabitation did not alter serum corticosterone levels in ED-reared males, but increased corticosterone levels in males that received bi-parental care (PC). Interestingly, serum corticosterone levels in ED- and PC-reared females declined after cohabitation. ED significantly increased basal serum corticosterone levels in males, but had no effect on females. ED significantly up-regulated the levels of dopamine and the mRNA expression of dopamine 1-type receptor (D1R) in the nucleus accumbens (NAcc) in females and males. ED suppressed dopamine 2-type receptor mRNA (D2R) expression in females, but increased this in males. After three days of cohabitation, levels of D1R mRNA and D2R mRNA expression changed in opposite directions in PC-reared voles, but in the same direction in ED-reared males, and only the expression of D2R mRNA increased in ED-reared females. Our results indicate that early social deprivation inhibits pair bonding at adulthood. This inhibition is possibly associated with sex-specific alterations in serum corticosterone, levels of dopamine and mRNA expression of two types of dopamine receptors in the NAcc. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Genome-wide mRNA processing in methanogenic archaea reveals post-transcriptional regulation of ribosomal protein synthesis

    PubMed Central

    Qi, Lei; Yue, Lei; Feng, Deqin; Qi, Fengxia

    2017-01-01

    Abstract Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an ‘all-or-none’ pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry. PMID:28520982

  18. Substance P activates ADAM9 mRNA expression and induces α-secretase-mediated amyloid precursor protein cleavage.

    PubMed

    Marolda, R; Ciotti, M T; Matrone, C; Possenti, R; Calissano, P; Cavallaro, S; Severini, C

    2012-04-01

    Altered levels of Substance P (SP), a neuropeptide endowed with neuroprotective and anti-apoptotic properties, were found in brain areas and spinal fluid of Alzheimer's disease (AD) patients. One of the hallmarks of AD is the abnormal extracellular deposition of neurotoxic beta amyloid (Aβ) peptides, derived from the proteolytic processing of amyloid precursor protein (APP). In the present study, we confirmed, the neurotrophic action of SP in cultured rat cerebellar granule cells (CGCs) and investigated its effects on APP metabolism. Incubation with low (5 mM) potassium induced apoptotic cell death of CGCs and amyloidogenic processing of APP, whereas treatment with SP (200 nM) reverted these effects via NK1 receptors. The non-amyloidogenic effect of SP consisted of reduction of Aβ(1-42), increase of sAPPα and enhanced α-secretase activity, without a significant change in steady-state levels of cellular APP. The intracellular mechanisms whereby SP alters APP metabolism were further investigated by measuring mRNA and/or steady-state protein levels of key enzymes involved with α-, β- and γ-secretase activity. Among them, Adam9, both at the mRNA and protein level, was the only enzyme to be significantly down-regulated following the induction of apoptosis (K5) and up-regulated after SP treatment. In addition to its neuroprotective properties, this study shows that SP is able to stimulate non-amyloidogenic APP processing, thereby reducing the possibility of generation of toxic Aβ peptides in brain. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. A Free-Choice High-Fat High-Sugar Diet Alters Day-Night Per2 Gene Expression in Reward-Related Brain Areas in Rats.

    PubMed

    Blancas-Velazquez, Aurea Susana; Unmehopa, Unga A; Eggels, Leslie; Koekkoek, Laura; Kalsbeek, Andries; Mendoza, Jorge; la Fleur, Susanne E

    2018-01-01

    Under normal light-dark conditions, nocturnal rodents consume most of their food during the dark period. Diets high in fat and sugar, however, may affect the day-night feeding rhythm resulting in a higher light phase intake. In vitro and in vivo studies showed that nutrients affect clock-gene expression. We therefore hypothesized that overconsuming fat and sugar alters clock-gene expression in brain structures important for feeding behavior. We determined the effects of a free-choice high-fat high-sugar (fcHFHS) diet on clock-gene expression in rat brain areas related to feeding and reward and compared them with chow-fed rats. Consuming a fcHFHS diet for 6 weeks disrupted day-night differences in Per2 mRNA expression in the nucleus accumbens (NAc) and lateral hypothalamus but not in the suprachiasmatic nucleus, habenula, and ventral tegmental area. Furthermore, short-term sugar drinking, but not fat feeding, upregulates Per2 mRNA expression in the NAc. The disruptions in day-night differences in NAc Per2 gene expression were not accompanied by altered day-night differences in the mRNA expression of peptides related to food intake. We conclude that the fcHFHS diet and acute sugar drinking affect Per2 gene expression in areas involved in food reward; however, this is not sufficient to alter the day-night pattern of food intake.

  20. A Free-Choice High-Fat High-Sugar Diet Alters Day–Night Per2 Gene Expression in Reward-Related Brain Areas in Rats

    PubMed Central

    Blancas-Velazquez, Aurea Susana; Unmehopa, Unga A.; Eggels, Leslie; Koekkoek, Laura; Kalsbeek, Andries; Mendoza, Jorge; la Fleur, Susanne E.

    2018-01-01

    Under normal light–dark conditions, nocturnal rodents consume most of their food during the dark period. Diets high in fat and sugar, however, may affect the day–night feeding rhythm resulting in a higher light phase intake. In vitro and in vivo studies showed that nutrients affect clock-gene expression. We therefore hypothesized that overconsuming fat and sugar alters clock-gene expression in brain structures important for feeding behavior. We determined the effects of a free-choice high-fat high-sugar (fcHFHS) diet on clock-gene expression in rat brain areas related to feeding and reward and compared them with chow-fed rats. Consuming a fcHFHS diet for 6 weeks disrupted day–night differences in Per2 mRNA expression in the nucleus accumbens (NAc) and lateral hypothalamus but not in the suprachiasmatic nucleus, habenula, and ventral tegmental area. Furthermore, short-term sugar drinking, but not fat feeding, upregulates Per2 mRNA expression in the NAc. The disruptions in day–night differences in NAc Per2 gene expression were not accompanied by altered day–night differences in the mRNA expression of peptides related to food intake. We conclude that the fcHFHS diet and acute sugar drinking affect Per2 gene expression in areas involved in food reward; however, this is not sufficient to alter the day–night pattern of food intake. PMID:29686649

  1. Alterations in the endocannabinoid system in the rat valproic acid model of autism.

    PubMed

    Kerr, D M; Downey, L; Conboy, M; Finn, D P; Roche, M

    2013-07-15

    The endocannabinoid system plays a crucial role in regulating emotionality and social behaviour, however it is unknown whether this system plays a role in symptoms associated with autism spectrum disorders. The current study evaluated if alterations in the endocannabinoid system accompany behavioural changes in the valproic acid (VPA) rat model of autism. Adolescent rats prenatally exposed to VPA exhibited impaired social investigatory behaviour, hypoalgesia and reduced lococmotor activity on exposure to a novel aversive arena. Levels of the endocananbinoids, anandamide (AEA) and 2-arachidonylglycerol (2-AG) in the hippocampus, frontal cortex or cerebellum were not altered in VPA- versus saline-exposed animals. However, the expression of mRNA for diacylglycerol lipase α, the enzyme primarily responsible for the synthesis of 2-AG, was reduced in the cerebellum of VPA-exposed rats. Furthermore, while the expression of mRNA for the 2-AG-catabolising enzyme monoacylglycerol lipase was reduced, the activity of this enzyme was increased, in the hippocampus of VPA-exposed animals. CB1 or CB2 receptor expression was not altered in any of the regions examined, however VPA-exposed rats exhibited reduced PPARα and GPR55 expression in the frontal cortex and PPARγ and GPR55 expression in the hippocampus, additional receptor targets of the endocannabinoids. Furthermore, tissue levels of the fatty acid amide hydrolase substrates, AEA, oleoylethanolamide and palmitoylethanolamide, were higher in the hippocampus of VPA-exposed rats immediately following social exposure. These data indicate that prenatal VPA exposure is associated with alterations in the brain's endocannabinoid system and support the hypothesis that endocannabinoid dysfunction may underlie behavioural abnormalities observed in autism spectrum disorders. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Adenovirus-mediated interference of FABP4 regulates mRNA expression of ADIPOQ, LEP and LEPR in bovine adipocytes.

    PubMed

    Wei, S; Zan, L S; Wang, H B; Cheng, G; Du, M; Jiang, Z; Hausman, G J; McFarland, D C; Dodson, M V

    2013-02-27

    Fatty acid binding protein 4 (FABP4) is an important adipocyte gene, with roles in fatty acid transport and fat deposition in animals as well as human metabolic syndrome. However, little is known about the functional regulation of FABP4 at the cellular level in bovine. We designed and selected an effective shRNA (small hairpin RNA) against bovine FABP4, constructed a corresponding adenovirus (AD-FABP4), and then detected its influence on mRNA expression of four differentiation-related genes (PPAR(y), CEBPA, CEBPB, and SREBF1) and three lipid metabolism-related genes (ADIPOQ, LEP and LEPR) of adipocytes. The FABP4 mRNA content, derived from bovine adipocytes, decreased by 41% (P < 0.01) after 24 h and 66% (P < 0.01) after 72 h of AD-FABP4 infection. However, lower mRNA content of FABP4 did not significantly alter levels of differentiation-related gene expression at 24 h following AD-FABP4 treatment of bovine-derived preadipocytes (P = 0.54, 0.78, 0.89, and 0.94, respectively). Meanwhile, knocking down (partially silencing) FABP4 significantly decreased ADIPOQ (P < 0.05) and LEP (P < 0.01) gene expression after 24 h of AD-FABP4 treatment, decreased ADIPOQ (P < 0.01) and LEP (P < 0.01) gene expression, but increased LEPR mRNA expression (P < 0.01) after a 72-h treatment of bovine preadipocytes. We conclude that FABP4 plays a role in fat deposition and metabolic syndrome by regulating lipid metabolism-related genes (such as ADIPOQ, LEP and LEPR), without affecting the ability of preadipocytes to differentiate into adipocytes.

  3. Altered Mitochondria, Protein Synthesis Machinery, and Purine Metabolism Are Molecular Contributors to the Pathogenesis of Creutzfeldt-Jakob Disease.

    PubMed

    Ansoleaga, Belén; Garcia-Esparcia, Paula; Llorens, Franc; Hernández-Ortega, Karina; Carmona Tech, Margarita; Antonio Del Rio, José; Zerr, Inga; Ferrer, Isidro

    2016-06-12

    Neuron loss, synaptic decline, and spongiform change are the hallmarks of sporadic Creutzfeldt-Jakob disease (sCJD), and may be related to deficiencies in mitochondria, energy metabolism, and protein synthesis. To investigate these relationships, we determined the expression levels of genes encoding subunits of the 5 protein complexes of the electron transport chain, proteins involved in energy metabolism, nucleolar and ribosomal proteins, and enzymes of purine metabolism in frontal cortex samples from 15 cases of sCJD MM1 and age-matched controls. We also assessed the protein expression levels of subunits of the respiratory chain, initiation and elongation translation factors of protein synthesis, and localization of selected mitochondrial components. We identified marked, generalized alterations of mRNA and protein expression of most subunits of all 5 mitochondrial respiratory chain complexes in sCJD cases. Expression of molecules involved in protein synthesis and purine metabolism were also altered in sCJD. These findings point to altered mRNA and protein expression of components of mitochondria, protein synthesis machinery, and purine metabolism as components of the pathogenesis of CJD. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

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

    PubMed

    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. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Arc/Arg3.1 mRNA expression reveals a subcellular trace of prior sound exposure in adult primary auditory cortex.

    PubMed

    Ivanova, T N; Matthews, A; Gross, C; Mappus, R C; Gollnick, C; Swanson, A; Bassell, G J; Liu, R C

    2011-05-05

    Acquiring the behavioral significance of sound has repeatedly been shown to correlate with long term changes in response properties of neurons in the adult primary auditory cortex. However, the molecular and cellular basis for such changes is still poorly understood. To address this, we have begun examining the auditory cortical expression of an activity-dependent effector immediate early gene (IEG) with documented roles in synaptic plasticity and memory consolidation in the hippocampus: Arc/Arg3.1. For initial characterization, we applied a repeated 10 min (24 h separation) sound exposure paradigm to determine the strength and consistency of sound-evoked Arc/Arg3.1 mRNA expression in the absence of explicit behavioral contingencies for the sound. We used 3D surface reconstruction methods in conjunction with fluorescent in situ hybridization (FISH) to assess the layer-specific subcellular compartmental expression of Arc/Arg3.1 mRNA. We unexpectedly found that both the intranuclear and cytoplasmic patterns of expression depended on the prior history of sound stimulation. Specifically, the percentage of neurons with expression only in the cytoplasm increased for repeated versus singular sound exposure, while intranuclear expression decreased. In contrast, the total cellular expression did not differ, consistent with prior IEG studies of primary auditory cortex. Our results were specific for cortical layers 3-6, as there was virtually no sound driven Arc/Arg3.1 mRNA in layers 1-2 immediately after stimulation. Our results are consistent with the kinetics and/or detectability of cortical subcellular Arc/Arg3.1 mRNA expression being altered by the initial exposure to the sound, suggesting exposure-induced modifications in the cytoplasmic Arc/Arg3.1 mRNA pool. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. Lysophosphatidic acid receptor mRNA levels in heart and white adipose tissue are associated with obesity in mice and humans.

    PubMed

    Brown, Amy; Hossain, Intekhab; Perez, Lester J; Nzirorera, Carine; Tozer, Kathleen; D'Souza, Kenneth; Trivedi, Purvi C; Aguiar, Christie; Yip, Alexandra M; Shea, Jennifer; Brunt, Keith R; Legare, Jean-Francois; Hassan, Ansar; Pulinilkunnil, Thomas; Kienesberger, Petra C

    2017-01-01

    Lysophosphatidic acid (LPA) receptor signaling has been implicated in cardiovascular and obesity-related metabolic disease. However, the distribution and regulation of LPA receptors in the myocardium and adipose tissue remain unclear. This study aimed to characterize the mRNA expression of LPA receptors (LPA1-6) in the murine and human myocardium and adipose tissue, and its regulation in response to obesity. LPA receptor mRNA levels were determined by qPCR in i) heart ventricles, isolated cardiomyocytes, and perigonadal adipose tissue from chow or high fat-high sucrose (HFHS)-fed male C57BL/6 mice, ii) 3T3-L1 adipocytes and HL-1 cardiomyocytes under conditions mimicking gluco/lipotoxicity, and iii) human atrial and subcutaneous adipose tissue from non-obese, pre-obese, and obese cardiac surgery patients. LPA1-6 were expressed in myocardium and white adipose tissue from mice and humans, except for LPA3, which was undetectable in murine adipocytes and human adipose tissue. Obesity was associated with increased LPA4, LPA5 and/or LPA6 levels in mice ventricles and cardiomyocytes, HL-1 cells exposed to high palmitate, and human atrial tissue. LPA4 and LPA5 mRNA levels in human atrial tissue correlated with measures of obesity. LPA5 mRNA levels were increased in HFHS-fed mice and insulin resistant adipocytes, yet were reduced in adipose tissue from obese patients. LPA4, LPA5, and LPA6 mRNA levels in human adipose tissue were negatively associated with measures of obesity and cardiac surgery outcomes. This study suggests that obesity leads to marked changes in LPA receptor expression in the murine and human heart and white adipose tissue that may alter LPA receptor signaling during obesity.

  7. Lysophosphatidic acid receptor mRNA levels in heart and white adipose tissue are associated with obesity in mice and humans

    PubMed Central

    Perez, Lester J.; Nzirorera, Carine; Tozer, Kathleen; D’Souza, Kenneth; Trivedi, Purvi C.; Aguiar, Christie; Yip, Alexandra M.; Shea, Jennifer; Brunt, Keith R.; Legare, Jean-Francois; Hassan, Ansar; Pulinilkunnil, Thomas

    2017-01-01

    Background Lysophosphatidic acid (LPA) receptor signaling has been implicated in cardiovascular and obesity-related metabolic disease. However, the distribution and regulation of LPA receptors in the myocardium and adipose tissue remain unclear. Objectives This study aimed to characterize the mRNA expression of LPA receptors (LPA1-6) in the murine and human myocardium and adipose tissue, and its regulation in response to obesity. Methods LPA receptor mRNA levels were determined by qPCR in i) heart ventricles, isolated cardiomyocytes, and perigonadal adipose tissue from chow or high fat-high sucrose (HFHS)-fed male C57BL/6 mice, ii) 3T3-L1 adipocytes and HL-1 cardiomyocytes under conditions mimicking gluco/lipotoxicity, and iii) human atrial and subcutaneous adipose tissue from non-obese, pre-obese, and obese cardiac surgery patients. Results LPA1-6 were expressed in myocardium and white adipose tissue from mice and humans, except for LPA3, which was undetectable in murine adipocytes and human adipose tissue. Obesity was associated with increased LPA4, LPA5 and/or LPA6 levels in mice ventricles and cardiomyocytes, HL-1 cells exposed to high palmitate, and human atrial tissue. LPA4 and LPA5 mRNA levels in human atrial tissue correlated with measures of obesity. LPA5 mRNA levels were increased in HFHS-fed mice and insulin resistant adipocytes, yet were reduced in adipose tissue from obese patients. LPA4, LPA5, and LPA6 mRNA levels in human adipose tissue were negatively associated with measures of obesity and cardiac surgery outcomes. This study suggests that obesity leads to marked changes in LPA receptor expression in the murine and human heart and white adipose tissue that may alter LPA receptor signaling during obesity. PMID:29236751

  8. mRNA of cytokines in bone marrow and bone biomarkers in response to propranolol in a nutritional growth retardation model.

    PubMed

    Tasat, Deborah R; Lezón, Christian E; Astort, Francisco; Pintos, Patricia M; Macri, Elisa V; Friedman, Silvia M; Boyer, Patricia M

    2014-10-01

    The aim of this study was to assess mRNA of IL-6, TNFα and IL-10 cytokines in bone marrow, possible mediators involved in altered bone remodeling with detrimental consequences on bone quality in NGR (Nutritional growth retardation) rats. Weanling male Wistar rats were assigned either to control (C) or experimental group (NGR) (n=20 each). C and NGR groups were assigned to 2 groups according to receiving saline solution (SS) or propranolol hydrochloride (P): C, C+P (CP), NGR or NGR+P (NGRP). For 4 weeks, NGR and NGRP rats received 80% of the amount of food consumed by C and CP, respectively, the previous day, corrected by body weight. P (7 mg/kg/day) was injected ip 5 days/week, for 4 weeks in CP and NGRP rats. Body weight and length were recorded. After 4 weeks, blood was drawn. Femurs were dissected for RNA isolation from bone marrow and mRNA of cytokines assays. Food restriction induced a significant negative effect on body growth in NGR and NGRP rats (p<0.001). P had no effects on zoometric parameters (p>0.05). CTX-I increased in NGR rats vs. C (p<0.001), but diminished in NGRP (p<0.01). Serum osteocalcin, PTH, calcium and phosphate levels remained unchanged between groups (p>0.05). In NGR, bone marrow IL-6 mRNA and IL-10 mRNA levels were low as compared to other groups (p<0.05). In contrast, bone marrow TNF-α mRNA levels were significantly high (p<0.05). This study provides evidences that NGR outcomes in a bone marrow proinflammatory microenvironment leading to unbalanced bone remodeling by enhancement of bone resorption reverted by propranolol. Copyright © 2014. Published by Elsevier Urban & Partner Sp. z o.o.

  9. Efficacy of Omega Fatty Acid Supplementation on mRNA Expression Level of Tumor Necrosis Factor Alpha in Patients with Gastric Adenocarcinoma.

    PubMed

    Hosseinzadeh, Asghar; Ardebili, Seyed Mojtaba Mohaddes

    2016-09-01

    Tumor necrosis factor alpha (TNF-α), a multifunctional cytokine, is involved in apoptosis, cell proliferation, cell survival, and inflammation. It plays a dual role in cancer development and progression. It has been revealed that polyunsaturated fatty acids (PUFAs) modulate the production and activity of TNF family cytokines. The objective of the present study was to evaluate the effect of PUFAs on messenger RNA expression levels of TNF-α in patients with gastric adenocarcinoma. Thirty-four chemotherapy-naive patients diagnosed with gastric adenocarcinoma were randomly divided into two groups. The first group (17 individuals) received cisplatin without supplements and the second group (17 individuals) received cisplatin plus orally administered PUFA supplements for 3 weeks, based on treatment strategies. The gastric biopsy samples were obtained from all participants before and after treatment, and TNF-α mRNA expression levels were evaluated by quantitative real-time PCR procedure. Our findings revealed that TNF-α mRNA expression is downregulated in group II, after receiving cisplatin and omega fatty acid supplement for 3 weeks. However, this difference is not statistically significant (p > 0.05). TNF-α mRNA expression did not show significant alteration in group I, after receiving cisplatin alone. Taken together, we concluded that omega fatty acids reduce TNF-α expression at the mRNA level in patients with gastric adenocarcinoma. These data suggest that TNF-α may act as a potential target for the therapy of human gastric adenocarcinoma.

  10. The neonatal ventral hippocampal lesion model of schizophrenia: effects on dopamine and GABA mRNA markers in the rat midbrain.

    PubMed

    Lipska, Barbara K; Lerman, Daniel N; Khaing, Zin Z; Weinberger, Daniel R

    2003-12-01

    The neonatal ventral hippocampal lesion in the rat has been used as a model of schizophrenia, a human disorder associated with changes in markers of dopamine and gamma-aminobutyric acid (GABA) circuits in various regions of the brain. We investigated whether alterations in mRNA markers related to the activity of midbrain dopaminergic and GABAergic neurons are associated with this model. We used in situ hybridization histochemistry to assess expression of mRNAs for dopamine transporter (DAT), tyrosine hydroxylase (TH) and glutamate decarboxylase-67 (GAD67) in the midbrain of adult rats with neonatal and adult ibotenic acid lesions of the ventral hippocampus. Neonatally lesioned rats showed in adulthood significantly reduced expression of DAT mRNA in the substantia nigra and the ventral tegmental area but no changes in the expression of TH and GAD67 mRNAs in these midbrain regions. Adult lesioned rats showed no changes in the expression of any of these genes. As the neonatal ventral hippocampal lesion reproduces many aspects of schizophrenia and is used as an animal model of this disorder, these results suggest that the reduction in DAT mRNA could result from developmental neuropathology in the ventral hippocampus and may thus represent a molecular substrate of the disease process.

  11. Consequences of metaphase II oocyte cryopreservation on mRNA content.

    PubMed

    Chamayou, S; Bonaventura, G; Alecci, C; Tibullo, D; Di Raimondo, F; Guglielmino, A; Barcellona, M L

    2011-04-01

    We studied the consequences of freezing/thawing processes on mRNA contents in MII oocytes after slow-freezing/rapid thawing (SF/RT) and vitrification/warming (V/W) protocols, and compared the results to fresh MII oocytes. We quantified the nuclear transcript mRNA responsible for the translation of proteins belonging either to trans-regulatory protein family or to functional structural proteins such as proteins involved in DNA structural organization (NAP1L1, TOP1, H1F0H1), chromosomal structure maintenance (SMC, SCC3, RAD21, SMC1A, SMC1B, STAG3, REC8), mitochondrial energetic pathways (ATP5GJ, SDHC), cell cycle regulation and processes (CLTA, MAPK6, CKS2) and staminal cell potency-development competence stage (DPPA3, OCT4, FOXJ2). Surplus MII oocytes were donated from patients in IVF cycles and divided in three groups of 15 oocytes. Group 1 was comprised of non-cryopreserved oocytes and Groups 2 and 3 underwent SF/RT and V/W procedures, respectively. There was an overall decrease of mRNA extracted from cryopreserved oocytes compared to control group. Only 39.4% of mRNA content were preserved after SF/RT while 63.3% of mRNA content were maintained after V/W. Oocyte cryopreservation is associated with molecular injury associated with the decrease of stored mRNA. However the V/W protocol is more conservative than SF/RT resulting in a level of mRNA sufficient to maintain biologic functions in the subsequent fertilized oocyte. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Olfactory discrimination ability and brain expression of c-fos, Gir and Glut1 mRNA are altered in n-3 fatty acid-depleted rats.

    PubMed

    Hichami, Aziz; Datiche, Frédérique; Ullah, Sana; Liénard, Fabienne; Chardigny, Jean-Michel; Cattarelli, Martine; Khan, Naim Akhtar

    2007-11-22

    The long-chain polyunsaturated n-3 fatty acids (n-3 PUFA), particularly docosahexaenoic acid (DHA), are abundantly present in the central nervous system and play an important role in cognitive functions such as learning and memory. We, therefore, investigated the effects of n-3 PUFA-depletion in rats (F2 generation) on the learning of an olfactory discrimination task, progressively acquired within a four-arm maze, and on the mRNA expression of some candidate genes, i.e., c-fos, Gir and glucose transporter (Glut1), which could reflect the level of cerebral activity. We observed that DHA contents were dramatically decreased in the olfactory bulb, the piriform cortex and the neocortex of n-3-depleted rats. Furthermore, the n-3 deficiency resulted in a mild olfactory learning impairment as these rats required more days to master the olfactory task compared to control rats. Real-time RT-PCR experiments revealed that the training induced the expression of c-fos mRNA in all the three regions of the brain whereas Gir and Glut1 mRNA were induced only in olfactory bulb and neocortex. However, such an increase was less marked in the n-3-deficient rats. Taken together, these results allow us to assume that the behavioural impairment in n-3-deficient rats is linked to the depletion of n-3 fatty acids in brain regions processing olfactory cues. Data are discussed in view of the possible role of some of these genes in learning-induced neuronal olfactory plasticity.

  13. Aggressive Encounters Alter the Activation of Serotonergic Neurons and the Expression of 5-HT1A mRNA in the Hamster Dorsal Raphe Nucleus

    PubMed Central

    Cooper, Matthew A.; Grober, Matthew S.; Nicholas, Christopher; Huhman, Kim L.

    2009-01-01

    Serotonergic (5-HT) neurons in the dorsal raphe nucleus (DRN) have been implicated in stress-induced changes in behavior. Previous research indicates that stressful stimuli activate 5-HT neurons in select subregions of the DRN. Uncontrollable stress is thought to sensitize 5-HT neurons in the DRN and allow for an exaggerated 5-HT response to future stimuli. In the current study, we tested the hypothesis that following aggressive encounters, losing male Syrian hamsters would exhibit increased c-Fos immunoreactivity in 5-HT DRN neurons compared to winners or controls. In addition, we tested the hypothesis that losers would have decreased 5-HT1A mRNA levels in the DRN compared to winners or controls. We found that a single 15-min aggressive encounter increased c-Fos expression in 5-HT and non-5-HT neurons in losers compared to winners and controls. The increased c-Fos expression in losers was restricted to ventral regions of the rostral DRN. We also found that four 5-min aggressive encounters reduced total 5-HT1A mRNA levels in the DRN in losers compared to winners and controls, and that differences in mRNA levels were not restricted to specific DRN subregions. These results suggest that social defeat activates neurons in select subregions of the DRN and reduces message for DRN 5-HT1A autoreceptors. Our results support the hypothesis that social stress can activate 5-HT neurons in the DRN, reduce 5-HT1A autoreceptor-mediated inhibition, and lead to hyperactivity of 5-HT neurons. PMID:19362123

  14. CCS mRNA transcripts and serum CCS protein as copper marker in adults suffering inflammatory processes.

    PubMed

    Araya, Magdalena; Gutiérrez, Ricardo; Arredondo, Miguel

    2014-08-01

    The chaperone to Zn-Cu superoxide dismutase (CCS) has been postulated as a candidate copper indicator, changing in a consistent manner in induced and recovered copper deficiency, in experimental cell and animal models. In real life people have various conditions that may modify molecules acting as acute phase proteins, such as serum ceruloplasmin and copper concentration and could alter CCS responses. With the hypothesis that CCS mRNA transcripts and protein would be different in individuals suffering inflammatory processes in comparison to healthy individuals, we assessed adult individuals who, although not ill had conditions known to induce variable degrees of inflammation. Screening of 600 adults resulted in two study groups, formed on the basis of their clinical history and levels of serum C reactive protein (CRP): Group 1 (n = 61, mean (range) CRP = 0.9 (0.3-2.0 mg/dL) and Group 2 (n = 150, mean (range) CRP = 6.1 (4.3-8.7 mg/dL). Results showed that mRNA transcripts relative abundance was not different for CCS, MTIIA, TNF-alpha and Cu-Zn-SOD by group (p > 0.05, one way Anova), nor between sexes (p > 0.05, one way Anova). Distribution of CCS mRNA transcripts and CCS protein in serum did not show any differences or trends. Results disproved our hypothesis that CCS abundance of transcripts and CCS protein would be different in individuals suffering inflammatory processes, adding further support to the idea that CCS may be a copper marker.

  15. Ancestral vinclozolin exposure alters the epigenetic transgenerational inheritance of sperm small noncoding RNAs.

    PubMed

    Schuster, Andrew; Skinner, Michael K; Yan, Wei

    Exposure to the agricultural fungicide vinclozolin during gestation promotes a higher incidence of various diseases in the subsequent unexposed F3 and F4 generations. This phenomenon is termed epigenetic transgenerational inheritance and has been shown to in part involve alterations in DNA methylation, but the role of other epigenetic mechanisms remains unknown. The current study investigated the alterations in small noncoding RNA (sncRNA) in the sperm from F3 generation control and vinclozolin lineage rats. Over 200 differentially expressed sncRNAs were identified and the tRNA-derived sncRNAs, namely 5' halves of mature tRNAs (5' halves), displayed the most dramatic changes. Gene targets of the altered miRNAs and tRNA 5' halves revealed associations between the altered sncRNAs and differentially DNA methylated regions. Dysregulated sncRNAs appear to correlate with mRNA profiles associated with the previously observed vinclozolin-induced disease phenotypes. Data suggest potential connections between sperm-borne RNAs and the vinclozolin-induced epigenetic transgenerational inheritance phenomenon.

  16. Reproductive Experience Alters Prolactin Receptor Expression in Mammary and Hepatic Tissues in Female Rats1

    PubMed Central

    Bridges, Robert S.; Scanlan, Victoria F.; Lee, Jong-O; Byrnes, Elizabeth M.

    2011-01-01

    Recent studies have reported that reproductive experience in female rats alters prolactin (PRL) receptor gene expression in the brain as well as neural sensitivity to PRL. Given PRL's actions in nonneural tissues, that is, mammary tissue and liver, it was asked whether reproductive experience may also alter prolactin receptor (Prlr) gene expression in these tissues. Groups of age-matched female rats were generated with varying reproductive histories. Separate groups of primiparous (first lactation) and multiparous (second lactation) had mammary tissue and liver samples collected on Day 3 or 10 of lactation. A fifth group raised one litter to weaning and then resumed estrous cyclicity. This group and a final group of age-matched, virgin controls were killed on diestrus. Tissue was processed by quantitative PCR for expression rates of the long and short forms of Prlr mRNA as well as casein beta mRNA (mammary tissue only). Western blots were performed to quantify receptor protein content. Multiple lactations as well as lactation itself resulted in alterations in Prlr expression. Prlr gene expression in mammary tissue was increased in primiparous mothers compared with that in multiparous dams, whereas in the liver, Prlr expression was reduced during an initial lactation. In contrast, PRLR protein levels declined during lactation in mammary, but not hepatic, tissues. Overall, the results demonstrate that the prolactin receptor system is altered in nonneural tissues as a result of the female's reproductive history. The findings are discussed in the context of milk and bile production and PRL's possible role in breast cancer. PMID:21508351

  17. Altered peroxisome-proliferator activated receptors expression in human endometrial cancer.

    PubMed

    Knapp, Paweł; Chabowski, Adrian; Błachnio-Zabielska, Agnieszka; Jarząbek, Katarzyna; Wołczyński, Sławomir

    2012-01-01

    Peroxisome proliferator-activated receptors (PPARs) belong to a family of nuclear hormone receptors acting as transcriptional factors, recently involved also in carcinogenesis. Present study was undertaken to evaluate the presence and subcellular localization of different PPAR isoforms (α, β, γ) in healthy endometrial tissue (n = 10) and endometrial carcinoma (FIGO I, endometrioides type, G1, n = 35). We sought to analyze PPARs mRNA content as well as protein immunohistochemical expression that was further quantified by Western Blot technique. For both PPARα and PPARβ, protein expression was significantly higher in endometrial cancers compared to normal endometrial mucosa. In opposite, PPARγ protein expression was lower in endometrial cancer cells. In each case, immunohistochemical reaction was confined to the perinuclear and/or nuclear region. At the transcriptional level, the content of mRNA of all PPAR subunits did not follow the protein pattern of changes. These results provide evidence for altered PPAR's protein expression and disregulation of posttranslational processes in endometrial cancers.

  18. Selective probing of mRNA expression levels within a living cell.

    PubMed

    Nawarathna, D; Turan, T; Wickramasinghe, H Kumar

    2009-08-24

    We report on a selective and nondestructive measurement of mRNA (messenger ribonucleic acid) expression levels within a living cell. We first modify an atomic force microscope tip to create a tapered nanoscale coaxial cable. Application of an ac (alternating potential) between the inner and outer electrodes of this cable creates a dielectrophoretic force attracting mRNA molecules toward the tip-end which is pretreated with gene specific primers. We selectively extracted and analyzed both high ( approximately 2500) and extremely low (11 0) copy number mRNA from a living cell mRNA in less than 10 s.

  19. Selective probing of mRNA expression levels within a living cell

    PubMed Central

    Nawarathna, D.; Turan, T.; Wickramasinghe, H. Kumar

    2009-01-01

    We report on a selective and nondestructive measurement of mRNA (messenger ribonucleic acid) expression levels within a living cell. We first modify an atomic force microscope tip to create a tapered nanoscale coaxial cable. Application of an ac (alternating potential) between the inner and outer electrodes of this cable creates a dielectrophoretic force attracting mRNA molecules toward the tip-end which is pretreated with gene specific primers. We selectively extracted and analyzed both high (∼2500) and extremely low (11¯0) copy number mRNA from a living cell mRNA in less than 10 s. PMID:19777090

  20. Experimental reconstitution of chronic ER stress in the liver reveals feedback suppression of BiP mRNA expression

    PubMed Central

    Gomez, Javier A; Rutkowski, D Thomas

    2016-01-01

    Endoplasmic reticulum (ER) stress is implicated in many chronic diseases, but very little is known about how the unfolded protein response (UPR) responds to persistent ER stress in vivo. Here, we experimentally reconstituted chronic ER stress in the mouse liver, using repeated injection of a low dose of the ER stressor tunicamycin. Paradoxically, this treatment led to feedback-mediated suppression of a select group of mRNAs, including those encoding the ER chaperones BiP and GRP94. This suppression was due to both silencing of the ATF6α pathway of UPR-dependent transcription and enhancement of mRNA degradation, possibly via regulated IRE1-dependent decay (RIDD). The suppression of mRNA encoding BiP was phenocopied by ectopic overexpression of BiP protein, and was also observed in obese mice. Our findings suggest that persistent cycles of UPR activation and deactivation create an altered, quasi-stable setpoint for UPR-dependent transcriptional regulation—an outcome that could be relevant to conditions such as metabolic syndrome. DOI: http://dx.doi.org/10.7554/eLife.20390.001 PMID:27938665

  1. Familial retinoblastoma due to intronic LINE-1 insertion causes aberrant and noncanonical mRNA splicing of the RB1 gene.

    PubMed

    Rodríguez-Martín, Carlos; Cidre, Florencia; Fernández-Teijeiro, Ana; Gómez-Mariano, Gema; de la Vega, Leticia; Ramos, Patricia; Zaballos, Ángel; Monzón, Sara; Alonso, Javier

    2016-05-01

    Retinoblastoma (RB, MIM 180200) is the paradigm of hereditary cancer. Individuals harboring a constitutional mutation in one allele of the RB1 gene have a high predisposition to develop RB. Here, we present the first case of familial RB caused by a de novo insertion of a full-length long interspersed element-1 (LINE-1) into intron 14 of the RB1 gene that caused a highly heterogeneous splicing pattern of RB1 mRNA. LINE-1 insertion was inferred by mRNA studies and full-length sequenced by massive parallel sequencing. Some of the aberrant mRNAs were produced by noncanonical acceptor splice sites, a new finding that up to date has not been described to occur upon LINE-1 retrotransposition. Our results clearly show that RNA-based strategies have the potential to detect disease-causing transposon insertions. It also confirms that the incorporation of new genetic approaches, such as massive parallel sequencing, contributes to characterize at the sequence level these unique and exceptional genetic alterations.

  2. Activity-Based Anorexia Alters the Expression of BDNF Transcripts in the Mesocorticolimbic Reward Circuit.

    PubMed

    Ho, Emily V; Klenotich, Stephanie J; McMurray, Matthew S; Dulawa, Stephanie C

    2016-01-01

    Anorexia nervosa (AN) is a complex eating disorder with severe dysregulation of appetitive behavior. The activity-based anorexia (ABA) paradigm is an animal model in which rodents exposed to both running wheels and scheduled feeding develop aspects of AN including paradoxical hypophagia, dramatic weight loss, and hyperactivity, while animals exposed to only one condition maintain normal body weight. Brain-derived neurotrophic factor (BDNF), an activity-dependent modulator of neuronal plasticity, is reduced in the serum of AN patients, and is a known regulator of feeding and weight maintenance. We assessed the effects of scheduled feeding, running wheel access, or both on the expression of BDNF transcripts within the mesocorticolimbic pathway. We also assessed the expression of neuronal cell adhesion molecule 1 (NCAM1) to explore the specificity of effects on BDNF within the mesocorticolimbic pathway. Scheduled feeding increased the levels of both transcripts in the hippocampus (HPC), increased NCAM1 mRNA expression in the ventral tegmental area (VTA), and decreased BDNF mRNA levels in the medial prefrontal cortex (mPFC). In addition, wheel running increased BDNF mRNA expression in the VTA. No changes in either transcript were observed in the nucleus accumbens (NAc). Furthermore, no changes in either transcript were induced by the combined scheduled feeding and wheel access condition. These data indicate that scheduled feeding or wheel running alter BDNF and NCAM1 expression levels in specific regions of the mesocorticolimbic pathway. These findings contribute to our current knowledge of the molecular alterations induced by ABA and may help elucidate possible mechanisms of AN pathology.

  3. Arc/Arg3.1 mRNA expression reveals a sub-cellular trace of prior sound exposure in adult primary auditory cortex

    PubMed Central

    Ivanova, Tamara; Matthews, Andrew; Gross, Christina; Mappus, Rudolph C.; Gollnick, Clare; Swanson, Andrew; Bassell, Gary J.; Liu, Robert C.

    2011-01-01

    Acquiring the behavioral significance of a sound has repeatedly been shown to correlate with long term changes in response properties of neurons in the adult primary auditory cortex. However, the molecular and cellular basis for such changes is still poorly understood. To address this, we have begun examining the auditory cortical expression of an activity-dependent effector immediate early gene (IEG) with documented roles in synaptic plasticity and memory consolidation in the hippocampus: Arc/Arg3.1. For initial characterization, we applied a repeated 10 minute (24 hour separation) sound exposure paradigm to determine the strength and consistency of sound-evoked Arc/Arg3.1 mRNA expression in the absence of explicit behavioral contingencies for the sound. We used 3D surface reconstruction methods in conjunction with fluorescent in-situ hybridization (FISH) to assess the layer-specific sub-cellular compartmental expression of Arc/Arg3.1 mRNA. We unexpectedly found that both the intranuclear and cytoplasmic patterns of expression depended on the prior history of sound stimulation. Specifically, the percentage of neurons with expression only in the cytoplasm increased for repeated versus singular sound exposure, while intranuclear expression decreased. In contrast, the total cellular expression did not differ, consistent with prior IEG studies of primary auditory cortex. Our results were specific for cortical layers 3–6, as there was virtually no sound driven Arc/Arg3.1 mRNA in layers 1–2 immediately after stimulation. Our results are consistent with the kinetics and/or detectability of cortical sub-cellular Arc/Arg3.1 mRNA expression being altered by the initial exposure to the sound, suggesting exposure-induced modifications in the cytoplasmic Arc/Arg3.1 mRNA pool. PMID:21334422

  4. Bicc1 Polymerization Regulates the Localization and Silencing of Bound mRNA

    PubMed Central

    Rothé, Benjamin; Leal-Esteban, Lucia; Bernet, Florian; Urfer, Séverine; Doerr, Nicholas; Weimbs, Thomas; Iwaszkiewicz, Justyna

    2015-01-01

    Loss of the RNA-binding protein Bicaudal-C (Bicc1) provokes renal and pancreatic cysts as well as ectopic Wnt/β-catenin signaling during visceral left-right patterning. Renal cysts are linked to defective silencing of Bicc1 target mRNAs, including adenylate cyclase 6 (AC6). RNA binding of Bicc1 is mediated by N-terminal KH domains, whereas a C-terminal sterile alpha motif (SAM) self-polymerizes in vitro and localizes Bicc1 in cytoplasmic foci in vivo. To assess a role for multimerization in silencing, we conducted structure modeling and then mutated the SAM domain residues which in this model were predicted to polymerize Bicc1 in a left-handed helix. We show that a SAM-SAM interface concentrates Bicc1 in cytoplasmic clusters to specifically localize and silence bound mRNA. In addition, defective polymerization decreases Bicc1 stability and thus indirectly attenuates inhibition of Dishevelled 2 in the Wnt/β-catenin pathway. Importantly, aberrant C-terminal extension of the SAM domain in bpk mutant Bicc1 phenocopied these defects. We conclude that polymerization is a novel disease-relevant mechanism both to stabilize Bicc1 and to present associated mRNAs in specific silencing platforms. PMID:26217012

  5. Expression of CGRP neurotransmitter and vascular genesis marker mRNA is age-dependent in superior cervical ganglia of senescence-accelerated prone mice.

    PubMed

    Mitsuoka, Kazuyuki; Kikutani, Takeshi; Miwa, Yoko; Sato, Iwao

    2018-01-18

    Calcitonin gene-related peptide (CGRP) is a neurotransmitter that is released from the superior cervical ganglion (SCG) and causes head and neck pain. The morphological properties of human SCG neurons, including neurotransmitter content, are altered during aging. However, morphological changes in CGRP in the SCG during aging are not known. Therefore, we investigated CGRP and other markers in the SCG during aging in an aging model of senescence-accelerated prone mouse (SAMP8) and senescence-accelerated resistant mice (SAMR1) using real-time RT-PCR mRNA analyses and in situ hybridization. The abundance of neurotransmitter (CGRP, NPY, TRPV1), vascular genesis marker (CD31, LYVE-1), and cytochrome C mRNA differed between 12-week-old and 24-week-old SAMP8 and SAMR1. Abundance of TRPV1, CD31 and cytochrome C mRNAs of SAMP8 decreased between 12- and 24-week-old. The ratio of CGRP mRNA positive cells and CGRP mRNA abundance levels of the SCG of aging mouse such as SAMP8 have already been also higher than that of SAMR1 at 12-week-old. The CGRP positive shrunken ganglion cells was increased from 12- to 24-weeks-old mouse in SAMR1 and SAMP8. The SCG primarily affected the internal and external carotid arteries, larynx thyroid gland, and pharyngeal muscle during aging. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Alterations to Melanocortinergic, GABAergic and Cannabinoid Neurotransmission Associated with Olanzapine-Induced Weight Gain

    PubMed Central

    Weston-Green, Katrina; Huang, Xu-Feng; Deng, Chao

    2012-01-01

    Background/Aim Second generation antipsychotics (SGAs) are used to treat schizophrenia but can cause serious metabolic side-effects, such as obesity and diabetes. This study examined the effects of low to high doses of olanzapine on appetite/metabolic regulatory signals in the hypothalamus and brainstem to elucidate the mechanisms underlying olanzapine-induced obesity. Methodology/Results Levels of pro-opiomelanocortin (POMC), neuropeptide Y (NPY) and glutamic acid decarboxylase (GAD65, enzyme for GABA synthesis) mRNA expression, and cannabinoid CB1 receptor (CB1R) binding density (using [3H]SR-141716A) were examined in the arcuate nucleus (Arc) and dorsal vagal complex (DVC) of female Sprague Dawley rats following 0.25, 0.5, 1.0 or 2.0 mg/kg olanzapine or vehicle (3×/day, 14-days). Consistent with its weight gain liability, olanzapine significantly decreased anorexigenic POMC and increased orexigenic NPY mRNA expression in a dose-sensitive manner in the Arc. GAD65 mRNA expression increased and CB1R binding density decreased in the Arc and DVC. Alterations to neurotransmission signals in the brain significantly correlated with body weight and adiposity. The minimum dosage threshold required to induce weight gain in the rat was 0.5 mg/kg olanzapine. Conclusions Olanzapine-induced weight gain is associated with reduced appetite-inhibiting POMC and increased NPY. This study also supports a role for the CB1R and GABA in the mechanisms underlying weight gain side-effects, possibly by altering POMC transmission. Metabolic dysfunction can be modelled in the female rat using low, clinically-comparable olanzapine doses when administered in-line with the half-life of the drug. PMID:22438946

  7. Genome-wide mRNA processing in methanogenic archaea reveals post-transcriptional regulation of ribosomal protein synthesis.

    PubMed

    Qi, Lei; Yue, Lei; Feng, Deqin; Qi, Fengxia; Li, Jie; Dong, Xiuzhu

    2017-07-07

    Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an 'all-or-none' pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

    PubMed Central

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

    2013-01-01

    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. PMID:23872491

  9. Conjunctival mucin mRNA expression in contact lens wear.

    PubMed

    Corrales, Rosa M; Galarreta, David; Herreras, Jose M; Saez, Victoria; Arranz, Isabel; González, Maria J; Mayo, Agustin; Calonge, Margarita; Chaves, Felipe J

    2009-09-01

    To investigate the influence of the water content in non-ionic hydrogel contact lenses (HCL) on the mRNA levels of human conjunctival mucin genes (MUCs). Sixteen healthy subjects with no history of contact lenses wear were selected and randomized into two equal groups. Group 1 subjects wore low water content (38%, Soflens 38) non-ionic HCLs. Group 2 wore high water content (66%, Soflens 66) non-ionic HCLs. Conjunctival impression cytology was applied to the superior bulbar conjunctiva of both eyes before, 6 months, and 1 year after HCL fitting, and 15 days after discontinuation of wearing. Total RNA was isolated, retrotranscribed, and amplified by conventional polymerase chain reaction (PCR) and by quantitative real time PCR to study the mRNA levels of MUCs and to analyze variations during the study period. Time- and HCL-dependent variations in mRNA expression were analyzed using Student's test. From the known MUCs, transcripts from MUC1, MUC2, MUC4, MUC5AC, MUC7, MUC13, MUC15, MUC16, and MUC17 genes were detected in all subjects before HCL fitting. Except for MUC2, the expression of some MUC genes significantly increased whereas others significantly decreased at either the 6- and 12-month period. Statistically significant differences between both HCL groups (p < 0.001) were found in the MUC4, MUC13, and MUC15 mRNA expression after 1 year of wear and after the 15 days without HCL wear. However, these differences were not clearly related to the water content of the lenses. Low and high water content non-ionic HCLs induced different changes in the mRNA levels of several MUCs, but the water content was not related to the changes. Recovery to basal levels of conjunctival MUC mRNA expression after wearing HCL lenses for a year takes longer than 15 days for some MUCs.

  10. Correlation between Fibrillin-1 Degradation and mRNA Downregulation and Myofibroblast Differentiation in Cultured Human Dental Pulp Tissue

    PubMed Central

    Yoshiba, Nagako; Yoshiba, Kunihiko; Ohkura, Naoto; Takei, Erika; Edanami, Naoki; Oda, Youhei; Hosoya, Akihiro; Nakamura, Hiroaki; Okiji, Takashi

    2015-01-01

    Myofibroblasts and extracellular matrix are important components in wound healing. Alpha-smooth muscle actin (α-SMA) is a marker of myofibroblasts. Fibrillin-1 is a major constituent of microfibrils and an extracellular-regulator of TGF-β1, an important cytokine in the transdifferentiation of resident fibroblasts into myofibroblasts. To study the correlation between changes in fibrillin-1 expression and myofibroblast differentiation, we examined alterations in fibrillin-1 and α-SMA expression in organotypic cultures of dental pulp in vitro. Extracted healthy human teeth were cut to 1-mm-thick slices and cultured for 7 days. In intact dental pulp, fibrillin-1 was broadly distributed, and α-SMA was observed in pericytes and vascular smooth muscle cells. After 7 days of culture, immunostaining for fibrillin-1 became faint concomitant with a downregulation in its mRNA levels. Furthermore, fibroblasts, odontoblasts and Schwann cells were immunoreactive for α-SMA with a significant increase in α-SMA mRNA expression. Double immunofluorescence staining was positive for pSmad2/3, central mediators of TGF-β signaling, and α-SMA. The administration of inhibitors for extracellular matrix proteases recovered fibrillin-1 immunostaining; moreover, fibroblasts lost their immunoreactivity for α-SMA along with a downregulation in α-SMA mRNA. These findings suggest that the expression of α-SMA is TGF-β1 dependent, and fibrillin-1 degradation and downregulation might be implicated in the differentiation of myofibroblasts in dental pulp wound healing. PMID:25805839

  11. Regulation of mRNA Trafficking by Nuclear Pore Complexes

    PubMed Central

    Bonnet, Amandine; Palancade, Benoit

    2014-01-01

    Over the last two decades, multiple studies have explored the mechanisms governing mRNA export out of the nucleus, a crucial step in eukaryotic gene expression. During transcription and processing, mRNAs are assembled into messenger ribonucleoparticles (mRNPs). mRNPs are then exported through nuclear pore complexes (NPCs), which are large multiprotein assemblies made of several copies of a limited number of nucleoporins. A considerable effort has been put into the dissection of mRNA export through NPCs at both cellular and molecular levels, revealing the conserved contributions of a subset of nucleoporins in this process, from yeast to vertebrates. Several reports have also demonstrated the ability of NPCs to sort out properly-processed mRNPs for entry into the nuclear export pathway. Importantly, changes in mRNA export have been associated with post-translational modifications of nucleoporins or changes in NPC composition, depending on cell cycle progression, development or exposure to stress. How NPC modifications also impact on cellular mRNA export in disease situations, notably upon viral infection, is discussed. PMID:25184662

  12. Altered JS-2 expression in colorectal cancers and its clinical pathological relevance.

    PubMed

    Lam, Alfred King-Yin; Gopalan, Vinod; Nassiri, Mohammad Reza; Kasim, Kais; Dissanayake, Jayampathy; Tang, Johnny Chuek-On; Smith, Robert Anthony

    2011-10-01

    JS-2 is a novel gene located at 5p15.2 and originally detected in primary oesophageal cancer. There is no study on the role of JS-2 in colorectal cancer. The aim of this study is to determine the gene copy number and expression of JS-2 in a large cohort of patients with colorectal tumours and correlate these to the clinicopathological features of the cancer patients. We evaluated the DNA copy number and mRNA expression of JS-2 in 176 colorectal tissues (116 adenocarcinomas, 30 adenomas and 30 non-neoplastic tissues) using real-time polymerase chain reaction. JS-2 expression was also evaluated in two colorectal cancer cell lines and a benign colorectal cell line. JS-2 amplification was noted in 35% of the colorectal adenocarcinomas. Significant differences in relative expression levels for JS-2 mRNA between different colorectal tissues were noted (p = 0.05). Distal colorectal adenocarcinoma had significantly higher copy number than proximal adenocarcinoma (p = 0.005). The relative expression level of JS-2 was different between colonic and rectal adenocarcinoma (p = 0.007). Mucinous adenocarcinoma showed higher JS-2 expression than non-mucinous adenocarcinoma (p = 0.02). Early T-stage cancers appear to have higher JS-2 copy number and lower expression of JS-2 mRNA than later stage cancers (p = 0.001 and 0.03 respectively). Colorectal cancer cell lines showed lower expression of JS-2 than the benign colorectal cell line. JS-2 copy number change and expression were shown for the first time to be altered in the carcinogenesis of colorectal cancer. In addition, genetic alteration of JS-2 was found to be related to location, pathological subtypes and staging of colorectal cancer. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  13. Short-term fasting alters cytochrome P450-mediated drug metabolism in humans.

    PubMed

    Lammers, Laureen A; Achterbergh, Roos; de Vries, Emmely M; van Nierop, F Samuel; Klümpen, Heinz-Josef; Soeters, Maarten R; Boelen, Anita; Romijn, Johannes A; Mathôt, Ron A A

    2015-06-01

    Experimental studies indicate that short-term fasting alters drug metabolism. However, the effects of short-term fasting on drug metabolism in humans need further investigation. Therefore, the aim of this study was to evaluate the effects of short-term fasting (36 h) on P450-mediated drug metabolism. In a randomized crossover study design, nine healthy subjects ingested a cocktail consisting of five P450-specific probe drugs [caffeine (CYP1A2), S-warfarin (CYP2C9), omeprazole (CYP2C19), metoprolol (CYP2D6), and midazolam (CYP3A4)] on two occasions (control study after an overnight fast and after 36 h of fasting). Blood samples were drawn for pharmacokinetic analysis using nonlinear mixed effects modeling. In addition, we studied in Wistar rats the effects of short-term fasting on hepatic mRNA expression of P450 isoforms corresponding with the five studied P450 enzymes in humans. In the healthy subjects, short-term fasting increased oral caffeine clearance by 20% (P = 0.03) and decreased oral S-warfarin clearance by 25% (P < 0.001). In rats, short-term fasting increased mRNA expression of the orthologs of human CYP1A2, CYP2C19, CYP2D6, and CYP3A4 (P < 0.05), and decreased the mRNA expression of the ortholog of CYP2C9 (P < 0.001) compared with the postabsorptive state. These results demonstrate that short-term fasting alters cytochrome P450-mediated drug metabolism in a nonuniform pattern. Therefore, short-term fasting is another factor affecting cytochrome P450-mediated drug metabolism in humans. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  14. Generation and Evaluation of Prophylactic mRNA Vaccines Against Allergy.

    PubMed

    Weiss, Richard; Scheiblhofer, Sandra; Thalhamer, Josef

    2017-01-01

    Due to the worldwide increase in allergies and a limited efficacy of therapeutic interventions, the need for prophylactic vaccination against allergies has been recognized. mRNA and DNA vaccines have demonstrated their high potential for preventing allergic sensitization by inducing an immunological bias that prevents TH2 sensitization. However, only mRNA vaccines fulfill the stringent safety requirements for vaccination of healthy children. In this chapter, we describe the generation of conventional as well as self-replicating mRNA vaccines and methods to test their prophylactic efficacy in animal models.

  15. Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells

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

    Jalava, A.M.; Heikkilae, J.E.; Akerman, K.E.O.

    1988-11-01

    The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc RNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC{sub 8}) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA andmore » DiC{sub 8} it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.« less

  16. PCB-associated changes in mRNA expression in killer whales (Orcinus orca) from the NE Pacific Ocean.

    PubMed

    Buckman, Andrea H; Veldhoen, Nik; Ellis, Graeme; Ford, John K B; Helbing, Caren C; Ross, Peter S

    2011-12-01

    Killer whales in the NE Pacific Ocean are among the world's most PCB-contaminated marine mammals, raising concerns about implications for their health. Sixteen health-related killer whale mRNA transcripts were analyzed in blubber biopsies collected from 35 free-ranging killer whales in British Columbia using real-time quantitative polymerase chain reaction. We observed PCB-related increases in the expression of five gene targets, including the aryl hydrocarbon receptor (AhR; r(2) = 0.83; p < 0.001), thyroid hormone α receptor (TRα; r(2) = 0.64; p < 0.001), estrogen α receptor (ERα; r(2) = 0.70; p < 0.001), interleukin 10 (IL-10; r(2) = 0.74 and 0.68, males and females, respectively; p < 0.001), and metallothionein 1 (MT1; r(2) = 0.58; p < 0.001). Best-fit models indicated that population (dietary preference), age, and sex were not confounding factors, except for IL-10, where males differed from females. While the population-level consequences are unclear, the PCB-associated alterations in mRNA abundance of such pivotal end points provide compelling evidence of adverse physiological effects of persistent environmental contaminants in these endangered killer whales.

  17. Localization of the mRNA for the dopamine D sub 2 receptor in the rat brain by in situ hybridization histochemistry

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

    Mengod, G.; Martinez-Mir, M.I.; Vilaro, M.T.

    1989-11-01

    {sup 32}P-labeled oligonucleotides derived from the coding region of rat dopamine D{sub 2} receptor cDNA were used as probes to localize cells in the rat brain that contain the mRNA coding for this receptor by using in situ hybridization histochemistry. The highest level of hybridization was found in the intermediate lobe of the pituitary gland. High mRNA content was observed in the anterior lobe of the pituitary gland, the nuclei caudate-putamen and accumbens, and the olfactory tubercle. Lower levels were seen in the substantia nigra pars compacta and the ventral tegmental area, as well as in the lateral mammillary body.more » In these areas the distribution was comparable to that of the dopamine D{sub 2} receptor binding sites as visualized by autoradiography using ({sup 3}H)SDZ 205-502 as a ligand. However, in some areas such as the olfactory bulb, neocortex, hippocampus, superior colliculus, and cerebellum, D{sub 2} receptors have been visualized but no significant hybridization signal could be detected. The mRNA coding for these receptors in these areas could be contained in cells outside those brain regions, be different from the one recognized by our probes, or be present at levels below the detection limits of our procedure. The possibility of visualizing and quantifying the mRNA coding for dopamine D{sub 2} receptor at the microscopic level will yield more information about the in vivo regulation of the synthesis of these receptor and their alteration following selective lesions or drug treatments.« less

  18. An endogenous RNA transcript antisense to CNG(alpha)1 cation channel mRNA.

    PubMed

    Cheng, Chin-Hung; Yew, David Tai-Wai; Kwan, Hiu-Yee; Zhou, Qing; Huang, Yu; Liu, Yong; Chan, Wing-Yee; Yao, Xiaoqiang

    2002-10-01

    CNG channels are cyclic nucleotide-gated Ca(2+)-permeable channels that are suggested to be involved in the activity-dependent alterations of synaptic strength that are thought to underlie information storage in the CNS. In this study, we isolated an endogenous RNA transcript antisense to CNG(alpha)1 mRNA. This transcript was capable of down-regulating the expression of sense CNG(alpha)1 in the Xenopus oocyte expression system. RT-PCR, Northern blot, and in situ hybridization analyses showed that the transcript was coexpressed with CNG(alpha)1 mRNA in many regions of human brain, notably in those regions that were involved in long-term potentiation and long-term depression, such as hippocampal CA1 and CA3, dentate gyrus, and cerebellar Purkinje layer. Comparison of expression patterns between adult and fetal cerebral cortex revealed that there were concurrent developmental changes in the expression levels of anti-CNG1 and CNG(alpha)1. Treatment of human glioma cell T98 with thyroid hormone T(3) caused a significant increase in anti-CNG1 expression and a parallel decrease in sense CNG(alpha)1 expression. These data suggest that the suppression of CNG(alpha)1 expression by anti-CNG1 may play an important role in neuronal functions, especially in synaptic plasticity and cortical development. Endogenous antisense RNA-mediated regulation may represent a new mechanism through which the activity of ion channels can be regulated in the human CNS.

  19. TSHB mRNA is linked to cholesterol metabolism in adipose tissue.

    PubMed

    Moreno-Navarrete, José María; Moreno, María; Ortega, Francisco; Xifra, Gemma; Hong, Shangyu; Asara, John M; Serrano, José C E; Jové, Mariona; Pissios, Pavlos; Blüher, Matthias; Ricart, Wifredo; Portero-Otin, Manuel; Fernández-Real, José Manuel

    2017-10-01

    Subclinical hypothyroidism is known to be associated with increased serum cholesterol. Since thyroid-stimulating hormone (TSH) exerts an inductor effect on cholesterol biosynthesis, we aimed to investigate the relationship between TSH mRNA and cholesterol metabolism in human adipose tissue (AT). Cross-sectionally, AT TSH-β ( TSHB ) mRNA was evaluated in 4 independent cohorts in association with serum total and LDL cholesterol, and AT lipidomics. Longitudinally, the effects of statins and of diet and exercise on AT TSHB mRNA were also examined. The bidirectional relationship between cholesterol and TSHB were studied in isolated human adipocytes. TSHB mRNA was consistently detected in AT from euthyroid subjects, and positively associated with serum total- and LDL-cholesterol, and with AT-specific cholesterol metabolism-associated lipids [arachidonoyl cholesteryl ester, C8-dihydroceramide, N -stearoyl-d-sphingosine, and GlcCer(18:0, 24:1)]. Reduction of cholesterol with statins and with diet and exercise interventions led to decreased TSHB mRNA in human AT, whereas excess cholesterol up-regulated TSHB mRNA in human adipocytes. In addition, recombinant human TSH α/β administration resulted in increased HMGCR mRNA levels in human adipocytes. In mice, subcutaneous AT Tshb expression levels correlated directly with circulating cholesterol levels. In summary, current results provide novel evidence of TSHB as a paracrine factor that is modulated in parallel with cholesterol metabolism in human AT.-Moreno-Navarrete, J. M., Moreno, M., Ortega, F., Xifra, G., Hong, S., Asara, J. M., Serrano, J. C. E., Jové, M., Pissios, P., Blüher, M., Ricart, W., Portero-Otin, M., Fernández-Real, J. M. TSHB mRNA is linked to cholesterol metabolism in adipose tissue. © FASEB.

  20. Preprotachykinin A mRNA expression in the rat brain during development.

    PubMed

    Brené, S; Lindefors, N; Friedman, W J; Persson, H

    1990-12-15

    Expression of preprotachykinin A (PPT-A) mRNA was analyzed by northern blots using mRNA prepared from rat brain at 12 different developmental stages ranging from embryonic day 15 (E15) to adult. A single PPT-A mRNA of 1.3 kb was detected throughout development. PPT-A mRNA was detected as early as E15 and an approximately 3-fold increase occurred at birth. This amount remained until 3 weeks of age when the level increased, reaching a peak at 5 weeks of age. Adult amounts were approximately 3-fold higher than the levels at birth. The distribution of PPT-A mRNA-expressing cells in rat brain was studied by in situ hybridization on sections from embryonic day 20, postnatal days 4 and 7 as well as adult. Cells expressing PPT-A mRNA were detected in the forebrain at all 4 ages analyzed. However, the hybridization pattern and the labeling intensity varied in different brain regions during development. In cingulate cortex, intense labeling was seen in numerous cells at embryonic day 20 and postnatal days 4 and 7, whereas in the adult cingulate cortex only a few scattered labeled cells were observed. In frontoparietal cortex labeled cells were found from postnatal day 4 to adult, with the highest density of labeled cells at P7. Developmental differences in both the distribution of PPT-A mRNA-expressing cells and the level of PPT-A mRNA expression were also found in caudate-putamen, lateral hypothalamus and amygdala. Thus, our results show several changes in PPT-A mRNA expression during ontogeny, indicating a region and time-specific regulation of PPT-A mRNA expression during brain maturation.

  1. Cellular localization of thrombopoietin mRNA in the liver by in situ hybridization.

    PubMed

    Nomura, S; Ogami, K; Kawamura, K; Tsukamoto, I; Kudo, Y; Kanakura, Y; Kitamura, Y; Miyazaki, H; Kato, T

    1997-07-01

    The expression of thrombopoietin (TPO) mRNA is observed in several tissues, including liver, kidney, brain, skeletal muscle, intestine, spleen, and bone marrow. Among these organs, the highest expression of TPO mRNA is detected in the liver. We identified cells producing TPO by means of in situ hybridization of adult rat liver using digoxigenin-11-UTP-labeled cRNA probes. We found that the cells expressing TPO mRNA also expressed serum albumin mRNA. TPO mRNA was detected in parenchymal cells (hepatocytes) but not in non-parenchymal cells (including endothelial cells, epithelial cells, and so forth). To determine the location of TPO expression in embryogenesis, sections of fetal mice were further analyzed by in situ hybridization. TPO mRNA was detected only in hepatocytes of fetal liver, which was also the major site of hematopoiesis. The expression of TPO mRNA in fetal liver was observed from 12.5 days postcoitus. Northern blot analysis showed that mouse liver transcribed the same size of TPO mRNA in the fetus and in the adult. These results clearly demonstrate that hepatocytes are the primary site of TPO production in the liver from fetus to adult.

  2. The modification of siRNA with 3' cholesterol to increase nuclease protection and suppression of native mRNA by select siRNA polyplexes.

    PubMed

    Ambardekar, Vishakha V; Han, Huai-Yun; Varney, Michelle L; Vinogradov, Serguei V; Singh, Rakesh K; Vetro, Joseph A

    2011-02-01

    Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3' cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC. Copyright © 2010 Elsevier Ltd. All rights reserved.

  3. Delta ribozyme has the ability to cleave in transan mRNA.

    PubMed Central

    Roy, G; Ananvoranich, S; Perreault, J P

    1999-01-01

    We report here the first demonstration of the cleavage of an mRNA in trans by delta ribozyme derived from the antigenomic version of the human hepatitis delta virus (HDV). We characterized potential delta ribozyme cleavage sites within HDV mRNA sequence (i.e. C/UGN6), using oligonucleotide binding shift assays and ribonuclease H hydrolysis. Ribozymes were synthesized based on the structural data and then tested for their ability to cleave the mRNA. Of the nine ribozymes examined, three specifically cleaved a derivative HDV mRNA. All three active ribozymes gave consistent indications that they cleaved single-stranded regions. Kinetic characterization of the ability of ribozymes to cleave both the full-length mRNA and either wild-type or mutant small model substrate suggests: (i) delta ribozyme has turnovers, that is to say, several mRNA molecules can be successively cleaved by one ribozyme molecule; and (ii) the substrate specificity of delta ribozyme cleavage is not restricted to C/UGN6. Specifically, substrates with a higher guanosine residue content upstream of the cleavage site (i.e. positions -4 to -2) were always cleaved more efficiently than wild-type substrate. This work shows that delta ribozyme constitutes a potential catalytic RNA for further gene-inactivation therapy. PMID:9927724

  4. Prenatal ethanol increases ethanol intake throughout adolescence, alters ethanol-mediated aversive learning, and affects μ but not δ or κ opioid receptor mRNA expression.

    PubMed

    Fabio, María Carolina; Macchione, Ana Fabiola; Nizhnikov, Michael E; Pautassi, Ricardo Marcos

    2015-06-01

    Animal models of prenatal ethanol exposure (PEE) have indicated a facilitatory effect of PEE on adolescent ethanol intake, but few studies have assessed the effects of moderate PEE throughout adolescence. The mechanisms underlying this facilitatory effect remain largely unknown. In the present study, we analysed ethanol intake in male and female Wistar rats with or without PEE (2.0 g/kg, gestational days 17-20) from postnatal days 37 to 62. The results revealed greater ethanol consumption in PEE rats than in controls, which persisted throughout adolescence. By the end of testing, ethanol ingestion in PEE rats was nearly 6.0 g/kg. PEE was associated with insensitivity to ethanol-induced aversion. PEE and control rats were further analysed for levels of μ, δ and κ opioid receptor mRNA in the infralimbic cortex, nucleus accumbens shell, and ventral tegmental area. Similar levels of mRNA were observed across most areas and opioid receptors, but μ receptor mRNA in the ventral tegmental area was significantly increased by PEE. Unlike previous studies that assessed the effects of PEE on ethanol intake close to birth, or in only a few sessions during adolescence, the present study observed a facilitatory effect of PEE that lasted throughout adolescence. PEE was associated with insensitivity to the aversive effect of ethanol, and increased levels of μ opioid receptor transcripts. PEE is a prominent vulnerability factor that probably favors the engagement of adolescents in risky trajectories of ethanol use. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  5. Dynamic postural stability during advancing pregnancy.

    PubMed

    McCrory, J L; Chambers, A J; Daftary, A; Redfern, M S

    2010-08-26

    Pregnant women are at an increased risk of experiencing a fall. Numerous anatomical, physiological, and hormonal alterations occur during pregnancy, but the influence of these factors on dynamic postural stability has not been explored. The purpose of this study was to examine dynamic postural stability in pregnant women during their second and third trimesters as well as in a group of non-pregnant control women. Eighty-one women (41 pregnant, 40 controls) participated stood on a force plate that translated anteroposteriorly at small, medium, and large magnitudes. Reaction time and center of pressure (COP) movement during the translations were analyzed. Trimester, perturbation direction, and perturbation magnitude were the independent variables in a mixed-model analysis of variance on each of the following dependent variables: reaction time, initial sway, total sway, and sway velocity. Reaction time to the perturbation was not significantly different between the groups. Initial sway, total sway, and sway velocity were significantly less during the third trimester than during the second trimester and when compared to the non-pregnant controls (P<0.05). No differences were found in any of the measures between the pregnant women in their second trimesters and the control group. Alterations in sway responses to perturbations are seen in the third trimester in healthy women with uncomplicated pregnancies. Further study is needed to examine the biomechanical and physiological reasons behind this altered dynamic postural stability. 2010 Elsevier Ltd. All rights reserved.

  6. Alterations in mRNA profiles of trastuzumab‑resistant Her‑2‑positive breast cancer.

    PubMed

    Zhao, Bin; Zhao, Yang; Sun, Yan; Niu, Haitao; Sheng, Long; Huang, Dongfang; Li, Li

    2018-05-07

    Breast cancer is one of the most common malignancies in women. Neoadjuvant trastuzumab therapy improves the prognosis of certain Her‑2‑positive breast cancer patients, however around two‑thirds of patients with Her‑2‑positive breast cancer do not benefit from Her‑2‑targeted therapy. To investigate the key mechanisms in trastuzumab resistance, potential biomarkers for neoadjuvant trastuzumab sensitivity were investigated using the gene expression omnibus (GEO) database for mRNA microarray data of Her‑2‑positive breast cancer patients who received neoadjuvant trastuzumab therapy. GEO profiles of 22 patients with a complete response and 48 patients with a partial response were identified in the GSE22358, GSE62327 and GSE66305 datasets. A total of 2,376, 1,000 and 1,152 differentially expressed genes in GSE22358, GSE62327 and GSE66305 datasets were demonstrated, respectively, utilizing GEO2R software. Furthermore, enriched gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways were analyzed using the Database for Annotation, Visualization and Integrated Discovery software. Subsequently, a protein‑protein interaction network was established using STRING software. The results demonstrated that low sex‑determining region Y‑box 11 and high Bcl‑2 expression may be employed as markers for neoadjuvant trastuzumab therapy for Her‑2‑positive breast cancer. More importantly, phosphoinositide 3‑kinase/Akt and angiogenesis pathways, which are known to be the key targets of trastuzumab, were activated at a lower level in the partial response patients, while the Wnt and estrogen receptor signaling pathways were activated in these patients. Therefore, combination therapy of trastuzumab and anti‑Wnt or hormone therapy may be a promising treatment modality and should be tested in further studies.

  7. The addition of body armor diminishes dynamic postural stability in military soldiers.

    PubMed

    Sell, Timothy C; Pederson, Jonathan J; Abt, John P; Nagai, Takashi; Deluzio, Jennifer; Wirt, Michael D; McCord, Larry J; Lephart, Scott M

    2013-01-01

    Poor postural stability has been identified as a risk factor for lower extremity musculoskeletal injury. The additional weight of body armor carried by Soldiers alters static postural stability and may predispose Soldiers to lower extremity musculoskeletal injuries. However, static postural stability tasks poorly replicate the dynamic military environment, which places considerable stress on the postural control system during tactical training and combat. Therefore, the purpose of this study was to examine the effects of body armor on dynamic postural stability during single-leg jump landings. Thirty-six 101st Airborne Division (Air Assault) Soldiers performed single-leg jump landings in the anterior direction with and without wearing body armor. The dynamic postural stability index and the individual stability indices (medial-lateral stability index, anterior-posterior stability index, and vertical stability index) were calculated for each condition. Paired sample t-tests were performed to determine differences between conditions. Significant differences existed for the medial-lateral stability index, anterior-posterior stability index, vertical stability index, and dynamic postural stability index (p < 0.05). The addition of body armor resulted in diminished dynamic postural stability, which may result in increased lower extremity injuries. Training programs should address the altered dynamic postural stability while wearing body armor in attempts to promote adaptations that will result in safer performance during dynamic tasks.

  8. Chronic gastrointestinal inflammation induces anxiety-like behavior and alters central nervous system biochemistry in mice.

    PubMed

    Bercik, Premysl; Verdu, Elena F; Foster, Jane A; Macri, Joseph; Potter, Murray; Huang, Xiaxing; Malinowski, Paul; Jackson, Wendy; Blennerhassett, Patricia; Neufeld, Karen A; Lu, Jun; Khan, Waliul I; Corthesy-Theulaz, Irene; Cherbut, Christine; Bergonzelli, Gabriela E; Collins, Stephen M

    2010-12-01

    Clinical and preclinical studies have associated gastrointestinal inflammation and infection with altered behavior. We investigated whether chronic gut inflammation alters behavior and brain biochemistry and examined underlying mechanisms. AKR mice were infected with the noninvasive parasite Trichuris muris and given etanercept, budesonide, or specific probiotics. Subdiaphragmatic vagotomy was performed in a subgroup of mice before infection. Gastrointestinal inflammation was assessed by histology and quantification of myeloperoxidase activity. Serum proteins were measured by proteomic analysis, circulating cytokines were measured by fluorescence activated cell sorting array, and serum tryptophan and kynurenine were measured by liquid chromatography. Behavior was assessed using light/dark preference and step-down tests. In situ hybridization was used to assess brain-derived neurotrophic factor (BDNF) expression in the brain. T muris caused mild to moderate colonic inflammation and anxiety-like behavior that was associated with decreased hippocampal BDNF messenger RNA (mRNA). Circulating tumor necrosis factor-α and interferon-γ, as well as the kynurenine and kynurenine/tryptophan ratio, were increased. Proteomic analysis showed altered levels of several proteins related to inflammation and neural function. Administration of etanercept, and to a lesser degree of budesonide, normalized behavior, reduced cytokine and kynurenine levels, but did not influence BDNF expression. The probiotic Bifidobacterium longum normalized behavior and BDNF mRNA but did not affect cytokine or kynurenine levels. Anxiety-like behavior was present in infected mice after vagotomy. Chronic gastrointestinal inflammation induces anxiety-like behavior and alters central nervous system biochemistry, which can be normalized by inflammation-dependent and -independent mechanisms, neither of which requires the integrity of the vagus nerve. Copyright © 2010 AGA Institute. Published by Elsevier Inc

  9. Exercise training does not increase muscle FNDC5 protein or mRNA expression in pigs

    PubMed Central

    Fain, John N.; Company, Joseph M.; Booth, Frank W.; Laughlin, M. Harold; Padilla, Jaume; Jenkins, Nathan T.; Bahouth, Suleiman W.; Sacks, Harold S.

    2013-01-01

    Background Exercise training elevates circulating irisin and induces the expression of the FNDC5 gene in skeletal muscles of mice. Our objective was to determine whether exercise training also increases FNDC5 protein or mRNA expression in the skeletal muscles of pigs as well as plasma irisin. Methods Castrated male pigs of the Rapacz familial hypercholesterolemic (FHM) strain and normal (Yucatan miniature) pigs were sacrificed after 16–20 weeks of exercise training. Samples of cardiac muscle, deltoid and triceps brachii muscle, subcutaneous and epicardial fat were obtained and FNDC5 mRNA, along with that of 6 other genes, was measured in all tissues of FHM pigs by reverse transcription polymerase chain reaction. FNDC protein in deltoid and triceps brachii was determined by Western blotting in both FHM and normal pigs. Citrate synthase activity was measured in the muscle samples of all pigs as an index of exercise training. Irisin was measured by an ELISA assay. Results There was no statistically significant effect of exercise training on FNDC5 gene expression in epicardial or subcutaneous fat, deltoid muscle, triceps brachii muscle or heart muscle. Exercise-training elevated circulating levels of irisin in the FHM pigs and citrate synthase activity in deltoid and triceps brachii muscle. A similar increase in citrate synthase activity was seen in muscle extracts of exercise-trained normal pigs but there was no alteration in circulating irisin. Conclusion Exercise training in pigs does not increase FNDC5 mRNA or protein in the deltoid or triceps brachii of FHM or normal pigs while increasing circulating irisin only in the FHM pigs. These data indicate that the response to exercise training in normal pigs is not comparable to that seen in mice. PMID:23831442

  10. Assessing mRNA nuclear export in mammalian cells by microinjection.

    PubMed

    Lee, Eliza S; Palazzo, Alexander F

    2017-08-15

    The nuclear export of mRNAs is an important yet little understood part of eukaryotic gene expression. One of the easiest methods for monitoring mRNA export in mammalian tissue culture cells is through the microinjection of DNA plasmids into the nucleus and monitoring the distribution of the transcribed product over time. Here we describe how to setup a microscope equipped with a micromanipulator used in cell microinjections, and we explain how to perform a nuclear mRNA export assay and obtain the nuclear export rate for any given mRNA. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Amyloid precursor protein mRNA levels in Alzheimer's disease brain.

    PubMed

    Preece, Paul; Virley, David J; Costandi, Moheb; Coombes, Robert; Moss, Stephen J; Mudge, Anne W; Jazin, Elena; Cairns, Nigel J

    2004-03-17

    Insoluble beta-amyloid deposits in Alzheimer's disease (AD) brain are proteolytically derived from the membrane bound amyloid precursor protein (APP). The APP gene is differentially spliced to produce isoforms that can be classified into those containing a Kunitz-type serine protease inhibitor domain (K(+), APP(751), APP(770), APRP(365) and APRP(563)), and those without (K(-), APP(695) and APP(714)). Given the hypothesis that Abeta is a result of aberrant catabolism of APP, differential expression of mRNA isoforms containing protease inhibitors might play an active role in the pathology of AD. We took 513 cerebral cortex samples from 90 AD and 81 control brains and quantified the mRNA isoforms of APP with TaqMan real-time RT-PCR. After adjustment for age at death, brain pH and gender we found a change in the ratio of KPI(+) to KPI(-) mRNA isoforms of APP. Three separate probes, designed to recognise only KPI(+) mRNA species, gave increases of between 28% and 50% in AD brains relative to controls (p=0.002). There was no change in the mRNA levels of KPI-(APP 695) (p=0.898). Therefore, whilst KPI-mRNA levels remained stable the KPI(+) species increased specifically in the AD brains.

  12. Effects of clofibric acid alone and in combination with 17β-estradiol on mRNA abundance in primary hepatocytes isolated from rainbow trout.

    PubMed

    Sovadinová, I; Liedtke, A; Schirmer, K

    2014-09-01

    Clofibric acid (CA) is the active substance of lipid lowering drugs. It is resistant to degradation, polar in nature, and has been found ubiquitously in the aquatic environment. Though CA is classified as a peroxisomal proliferator in rodents and is considered as a potential endocrine disruptor, little information exists on the effects of CA in aquatic organisms, such as fish. In the present study, we examined the mRNA levels of peroxisome proliferator- and estrogen-sensitive genes on the exposure of primary rainbow trout (Oncorhynchus mykiss) hepatocytes to CA alone and in combination with the natural female sex hormone, 17β-estradiol (E2). Our results demonstrate that rainbow trout hepatocytes are relatively refractory to the effects of CA on the PPAR signaling pathway and lipid metabolism. Moreover, CA did not show recognizable estrogenic activity, but after the induction of vitellogenesis by E2, CA significantly reduced vitellogenin (VTG) mRNA abundance. Apparently, the indirect repression of VTG transcription, independent of estrogen receptors, occurred. The mechanism is not yet clearly understood but may involve disruption of the stabilization of VTG mRNA known to be induced by E2. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Nutritional status alters saccharin intake and sweet receptor mRNA expression in rat taste buds.

    PubMed

    Chen, Ke; Yan, Jianqun; Suo, Yi; Li, Jinrong; Wang, Qian; Lv, Bo

    2010-04-14

    Sweet taste usually signifies the presence of caloric food. It is commonly accepted that a close association exists among sweet taste perception, preference, and nutritional status. However, the mechanisms involved remain unknown. To investigate whether nutritional status affects the preference for palatable solutions and alters sweet taste receptor gene expression in rats, we measured saccharin intake and preference using a two-bottle preference test, and changes in body weight, plasma leptin levels, and gene expression for the sweet taste receptor in taste buds in high-fat diet-induced obese rats and chronically diet-restricted rats. We found that the consumption and preference ratios for 0.01 and 0.04 M saccharin were significantly lower in the high-fat diet-induced obese rats than in the normal diet rats, while the serum leptin levels were markedly increased in obese rats. Consistent with the changes in saccharin intake, the gene expression level of the sweet taste receptor T1R3 was significantly decreased in the high-fat diet-induced obese rats compared with the control rats. By contrast, the chronically diet-restricted rats showed remarkably enhanced consumption and preference for 0.04 M saccharin. The serum leptin concentration was decreased, and the gene expression of the leptin receptor was markedly increased in the taste buds. In conclusion, our results suggest that nutritional status alters saccharin preference and the expression of T1R3 in taste buds. These processes may be involved in the mechanisms underlying the modulation of peripheral sweet taste sensitivity, in which leptin plays a role. Copyright 2010 Elsevier B.V. All rights reserved.

  14. Saccharomyces cerevisiae Boulardii Reduces the Deoxynivalenol-Induced Alteration of the Intestinal Transcriptome.

    PubMed

    Alassane-Kpembi, Imourana; Pinton, Philippe; Hupé, Jean-François; Neves, Manon; Lippi, Yannick; Combes, Sylvie; Castex, Mathieu; Oswald, Isabelle P

    2018-05-15

    Type B trichothecene mycotoxin deoxynivalenol (DON) is one of the most frequently occurring food contaminants. By inducing trans-activation of a number of pro-inflammatory cytokines and increasing the stability of their mRNA, trichothecene can impair intestinal health. Several yeast products, especially Saccharomyces cerevisiae , have the potential for improving the enteric health of piglets, but little is known about the mechanisms by which the administration of yeast counteracts the DON-induced intestinal alterations. Using a pig jejunum explant model, a whole-transcriptome analysis was performed to decipher the early response of the small intestine to the deleterious effects of DON after administration of S. cerevisiae boulardii strain CNCM I-1079. Compared to the control condition, no differentially expressed gene (DE) was observed after treatment by yeast only. By contrast, 3619 probes-corresponding to 2771 genes-were differentially expressed following exposure to DON, and 32 signaling pathways were identified from the IPA software functional analysis of the set of DE genes. When the intestinal explants were treated with S. cerevisiae boulardii prior to DON exposure, the number of DE genes decreased by half (1718 probes corresponding to 1384 genes). Prototypical inflammation signaling pathways triggered by DON, including NF-κB and p38 MAPK, were reversed, although the yeast demonstrated limited efficacy toward some other pathways. S. cerevisiae boulardii also restored the lipid metabolism signaling pathway, and reversed the down-regulation of the antioxidant action of vitamin C signaling pathway. The latter effect could reduce the burden of DON-induced oxidative stress. Altogether, the results show that S. cerevisiae boulardii reduces the DON-induced alteration of intestinal transcriptome, and point to new mechanisms for the healing of tissue injury by yeast.

  15. Saccharomyces cerevisiae boulardii Reduces the Deoxynivalenol-Induced Alteration of the Intestinal Transcriptome

    PubMed Central

    Alassane-Kpembi, Imourana; Hupé, Jean-François; Neves, Manon; Lippi, Yannick; Combes, Sylvie; Castex, Mathieu

    2018-01-01

    Type B trichothecene mycotoxin deoxynivalenol (DON) is one of the most frequently occurring food contaminants. By inducing trans-activation of a number of pro-inflammatory cytokines and increasing the stability of their mRNA, trichothecene can impair intestinal health. Several yeast products, especially Saccharomyces cerevisiae, have the potential for improving the enteric health of piglets, but little is known about the mechanisms by which the administration of yeast counteracts the DON-induced intestinal alterations. Using a pig jejunum explant model, a whole-transcriptome analysis was performed to decipher the early response of the small intestine to the deleterious effects of DON after administration of S. cerevisiae boulardii strain CNCM I-1079. Compared to the control condition, no differentially expressed gene (DE) was observed after treatment by yeast only. By contrast, 3619 probes—corresponding to 2771 genes—were differentially expressed following exposure to DON, and 32 signaling pathways were identified from the IPA software functional analysis of the set of DE genes. When the intestinal explants were treated with S. cerevisiae boulardii prior to DON exposure, the number of DE genes decreased by half (1718 probes corresponding to 1384 genes). Prototypical inflammation signaling pathways triggered by DON, including NF-κB and p38 MAPK, were reversed, although the yeast demonstrated limited efficacy toward some other pathways. S. cerevisiae boulardii also restored the lipid metabolism signaling pathway, and reversed the down-regulation of the antioxidant action of vitamin C signaling pathway. The latter effect could reduce the burden of DON-induced oxidative stress. Altogether, the results show that S. cerevisiae boulardii reduces the DON-induced alteration of intestinal transcriptome, and point to new mechanisms for the healing of tissue injury by yeast. PMID:29762474

  16. Translational induction of heat shock transcription factor σ32: evidence for a built-in RNA thermosensor

    PubMed Central

    Morita, Miyo Terao; Tanaka, Yoshiyuki; Kodama, Takashi S.; Kyogoku, Yoshimasa; Yanagi, Hideki; Yura, Takashi

    1999-01-01

    Induction of heat shock proteins in Escherichia coli is primarily caused by increased cellular levels of the heat shock σ-factor σ32 encoded by the rpoH gene. Increased σ32 levels result from both enhanced synthesis and stabilization. Previous work indicated that σ32 synthesis is induced at the translational level and is mediated by the mRNA secondary structure formed within the 5′-coding sequence of rpoH, including the translation initiation region. To understand the mechanism of heat induction of σ32 synthesis further, we analyzed expression of rpoH–lacZ gene fusions with altered stability of mRNA structure before and after heat shock. A clear correlation was found between the stability and expression or the extent of heat induction. Temperature-melting profiles of mRNAs with or without mutations correlated well with the expression patterns of fusion genes carrying the corresponding mutations in vivo. Furthermore, temperature dependence of mRNA–30S ribosome–tRNAfMet complex formation with wild-type or mutant mRNAs in vitro agreed well with that of the expression of gene fusions in vivo. Our results support a novel mechanism in which partial melting of mRNA secondary structure at high temperature enhances ribosome entry and translational initiation without involvement of other cellular components, that is, intrinsic mRNA stability controls synthesis of a transcriptional regulator. PMID:10090722

  17. Geophysical methods for monitoring soil stabilization processes

    NASA Astrophysics Data System (ADS)

    Saneiyan, Sina; Ntarlagiannis, Dimitrios; Werkema, D. Dale; Ustra, Andréa

    2018-01-01

    Soil stabilization involves methods used to turn unconsolidated and unstable soil into a stiffer, consolidated medium that could support engineered structures, alter permeability, change subsurface flow, or immobilize contamination through mineral precipitation. Among the variety of available methods carbonate precipitation is a very promising one, especially when it is being induced through common soil borne microbes (MICP - microbial induced carbonate precipitation). Such microbial mediated precipitation has the added benefit of not harming the environment as other methods can be environmentally detrimental. Carbonate precipitation, typically in the form of calcite, is a naturally occurring process that can be manipulated to deliver the expected soil strengthening results or permeability changes. This study investigates the ability of spectral induced polarization and shear-wave velocity for monitoring calcite driven soil strengthening processes. The results support the use of these geophysical methods as soil strengthening characterization and long term monitoring tools, which is a requirement for viable soil stabilization projects. Both tested methods are sensitive to calcite precipitation, with SIP offering additional information related to long term stability of precipitated carbonate. Carbonate precipitation has been confirmed with direct methods, such as direct sampling and scanning electron microscopy (SEM). This study advances our understanding of soil strengthening processes and permeability alterations, and is a crucial step for the use of geophysical methods as monitoring tools in microbial induced soil alterations through carbonate precipitation.

  18. 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.

    PubMed

    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.

  19. Drug metabolism alterations in nonalcoholic fatty liver disease

    PubMed Central

    Merrell, Matthew D.; Cherrington, Nathan J.

    2013-01-01

    Drug-metabolizing enzymes play a vital role in the elimination of the majority of therapeutic drugs. The major organ involved in drug metabolism is the liver. Chronic liver diseases have been identified as a potential source of significant interindividual variation in metabolism. Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the United States, affecting between 60 and 90 million Americans, yet the vast majority of NAFLD patients are undiagnosed. NAFLD encompasses a spectrum of pathologies, ranging from steatosis to nonalcoholic steatohepatitis and fibrosis. Numerous animal studies have investigated the effects of NAFLD on hepatic gene expression, observing significant alterations in mRNA, protein, and activity levels. Information on the effects of NAFLD in human patients is limited, though several significant investigations have recently been published. Significant alterations in the activity of drug-metabolizing enzymes may affect the clearance of therapeutic drugs, with the potential to result in adverse drug reactions. With the enormous prevalence of NAFLD, it is conceivable that every drug currently on the market is being given to patients with NAFLD. The current review is intended to present the results from both animal models and human patients, summarizing the observed alterations in the expression and activity of the phase I and II drug-metabolizing enzymes. PMID:21612324

  20. Aging alters circadian regulation of redox in Drosophila

    PubMed Central

    Klichko, Vladimir I.; Chow, Eileen S.; Kotwica-Rolinska, Joanna; Orr, William C.; Giebultowicz, Jadwiga M.; Radyuk, Svetlana N.

    2015-01-01

    Circadian coordination of metabolism, physiology, and neural functions contributes to healthy aging and disease prevention. Clock genes govern the daily rhythmic expression of target genes whose activities underlie such broad physiological parameters as maintenance of redox homeostasis. Previously, we reported that glutathione (GSH) biosynthesis is controlled by the circadian system via effects of the clock genes on expression of the catalytic (Gclc) and modulatory (Gclm) subunits comprising the glutamate cysteine ligase (GCL) holoenzyme. The objective of this study was to determine whether and how aging, which leads to weakened circadian oscillations, affects the daily profiles of redox-active biomolecules. We found that fly aging is associated with altered profiles of Gclc and Gclm expression at both the mRNA and protein levels. Analysis of free aminothiols and GCL activity revealed that aging abolishes daily oscillations in GSH levels and alters the activity of glutathione biosynthetic pathways. Unlike GSH, its precursors and products of catabolism, methionine, cysteine and cysteinyl-glycine, were not rhythmic in young or old flies, while rhythms of the glutathione oxidation product, GSSG, were detectable. We conclude that the temporal regulation of GSH biosynthesis is altered in the aging organism and that age-related loss of circadian modulation of pathways involved in glutathione production is likely to impair temporal redox homeostasis. PMID:25806044

  1. Phosphorylation Regulates NCC Stability and Transporter Activity In Vivo

    PubMed Central

    Yang, Sung-Sen; Fang, Yu-Wei; Tseng, Min-Hua; Chu, Pei-Yi; Yu, I-Shing; Wu, Han-Chung; Lin, Shu-Wha; Chau, Tom; Uchida, Shinichi; Sasaki, Sei; Lin, Yuh-Feng; Sytwu, Huey-Kang

    2013-01-01

    A T60M mutation in the thiazide-sensitive sodium chloride cotransporter (NCC) is common in patients with Gitelman’s syndrome (GS). This mutation prevents Ste20-related proline and alanine-rich kinase (SPAK)/oxidative stress responsive kinase-1 (OSR1)–mediated phosphorylation of NCC and alters NCC transporter activity in vitro. Here, we examined the physiologic effects of NCC phosphorylation in vivo using a novel Ncc T58M (human T60M) knock-in mouse model. NccT58M/T58M mice exhibited typical features of GS with a blunted response to thiazide diuretics. Despite expressing normal levels of Ncc mRNA, these mice had lower levels of total Ncc and p-Ncc protein that did not change with a low-salt diet that increased p-Spak. In contrast to wild-type Ncc, which localized to the apical membrane of distal convoluted tubule cells, T58M Ncc localized primarily to the cytosolic region and caused an increase in late distal convoluted tubule volume. In MDCK cells, exogenous expression of phosphorylation-defective NCC mutants reduced total protein expression levels and membrane stability. Furthermore, our analysis found diminished total urine NCC excretion in a cohort of GS patients with homozygous NCC T60M mutations. When Wnk4D561A/+ mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/Osr1-Ncc, were crossed with NccT58M/T58M mice, total Ncc and p-Ncc protein levels decreased and the GS phenotype persisted over the hypertensive phenotype. Overall, these data suggest that SPAK-mediated phosphorylation of NCC at T60 regulates NCC stability and function, and defective phosphorylation at this residue corrects the phenotype of pseudohypoaldosteronism type II. PMID:23833262

  2. Phosphorylation regulates NCC stability and transporter activity in vivo.

    PubMed

    Yang, Sung-Sen; Fang, Yu-Wei; Tseng, Min-Hua; Chu, Pei-Yi; Yu, I-Shing; Wu, Han-Chung; Lin, Shu-Wha; Chau, Tom; Uchida, Shinichi; Sasaki, Sei; Lin, Yuh-Feng; Sytwu, Huey-Kang; Lin, Shih-Hua

    2013-10-01

    A T60M mutation in the thiazide-sensitive sodium chloride cotransporter (NCC) is common in patients with Gitelman's syndrome (GS). This mutation prevents Ste20-related proline and alanine-rich kinase (SPAK)/oxidative stress responsive kinase-1 (OSR1)-mediated phosphorylation of NCC and alters NCC transporter activity in vitro. Here, we examined the physiologic effects of NCC phosphorylation in vivo using a novel Ncc T58M (human T60M) knock-in mouse model. Ncc(T58M/T58M) mice exhibited typical features of GS with a blunted response to thiazide diuretics. Despite expressing normal levels of Ncc mRNA, these mice had lower levels of total Ncc and p-Ncc protein that did not change with a low-salt diet that increased p-Spak. In contrast to wild-type Ncc, which localized to the apical membrane of distal convoluted tubule cells, T58M Ncc localized primarily to the cytosolic region and caused an increase in late distal convoluted tubule volume. In MDCK cells, exogenous expression of phosphorylation-defective NCC mutants reduced total protein expression levels and membrane stability. Furthermore, our analysis found diminished total urine NCC excretion in a cohort of GS patients with homozygous NCC T60M mutations. When Wnk4(D561A/+) mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/Osr1-Ncc, were crossed with Ncc(T58M/T58M) mice, total Ncc and p-Ncc protein levels decreased and the GS phenotype persisted over the hypertensive phenotype. Overall, these data suggest that SPAK-mediated phosphorylation of NCC at T60 regulates NCC stability and function, and defective phosphorylation at this residue corrects the phenotype of pseudohypoaldosteronism type II.

  3. Single step production of Cas9 mRNA for zygote injection.

    PubMed

    Redel, Bethany K; Beaton, Benjamin P; Spate, Lee D; Benne, Joshua A; Murphy, Stephanie L; O'Gorman, Chad W; Spate, Anna M; Prather, Randall S; Wells, Kevin D

    2018-03-01

    Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

  4. Magnesium Induced Nucleophile Activation in the Guanylyltransferase mRNA Capping Enzyme

    PubMed Central

    Swift, Robert V.; Ong, Chau D.; Amaro, Rommie E.

    2012-01-01

    The messenger RNA guanylyltransferase, or mRNA capping enzyme, co-transcriptionally caps the 5′-end of nascent mRNA with GMP during the second in a set of three enzymatic reactions that result in the formation of an N7-methyl guanosine cap during mRNA maturation. The mRNA capping enzyme is characterized, in part, by a conserved lysine nucleophile that attacks the alpha-phosphorous atom of GTP, forming a lysine-GMP intermediate. Experiments have firmly established that magnesium is required for efficient intermediate formation, but have provided little insight into the requirement’s molecular origins. Using empirical and thermodynamic integration pKa estimates, along with conventional MD simulations, we show that magnesium binding likely activates the lysine nucleophile by increasing its acidity and by biasing the deprotonated nucleophile into conformations conducive to intermediate formation. These results provide additional functional understanding of an important enzyme in the mRNA transcript life cycle and allow functional analogies to be drawn that affect our understanding of the metal dependence of related superfamily members. PMID:23205906

  5. Interleukin-1 homologues IL-1F7b and IL-18 contain functional mRNA instability elements within the coding region responsive to lipopolysaccharide

    PubMed Central

    2004-01-01

    IL-1F7b, a novel homologue of the IL-1 (interleukin 1) family, was discovered by computational cloning. We demonstrated that IL-1F7b shares critical amino acid residues with IL-18 and binds to the IL-18-binding protein enhancing its ability to inhibit IL-18-induced interferon-γ. We also showed that low levels of IL-1F7b are constitutively present intracellularly in human blood monocytes. In this study, we demonstrate that similar to IL-18, both mRNA and intracellular protein expression of IL-1F7b are up-regulated by LPS (lipopolysaccharide) in human monocytes. In stable transfectants of murine RAW264.7 macrophage cells, there was no IL-1F7b protein expression despite a highly active CMV promoter. We found that IL-1F7b-specific mRNA was rapidly degraded in transfected cells, via a 3′-UTR (untranslated region)-independent control of IL-1F7b transcript stability. After LPS stimulation, there was a rapid transient increase in IL-1F7b-specific mRNA and concomitant protein levels. Using sequence alignment, we found a conserved ten-nucleotide homology box within the open reading frame of IL-F7b, which is flanking the coding region instability elements of some selective genes. In-frame deletion of downstream exon 5 from the full-length IL-1F7b cDNA markedly increased the levels of IL-1F7b mRNA. A similar coding region element is located in IL-18. When transfected into RAW264.7 macrophages, IL-18 mRNA was also unstable unless treated with LPS. These results indicate that both IL-1F7b and IL-18 mRNA contain functional instability determinants within their coding region, which influence mRNA decay as a novel mechanism to regulate the expression of IL-1 family members. PMID:15046617

  6. Subchronic cadmium exposure upregulates the mRNA level of genes associated to hepatic lipid metabolism in adult female CD1 mice.

    PubMed

    Zhang, Jun; Wang, Yan; Fu, Lin; Feng, Yu-Jie; Ji, Yan-Li; Wang, Hua; Xu, De-Xiang

    2018-07-01

    Cadmium (Cd) is a persistent environmental and occupational contaminant that accumulates in humans and shows adverse effects on health. Accumulating evidence reveals that environmental Cd exposure is associated with hepatic lipid accumulation and metabolic alterations in adult male mice. However, whether Cd exposure induces hepatic lipid accumulation and metabolic alterations in female mice remains poorly understood. In the present study, we aimed to investigate the effects of Cd exposure on insulin resistance, hepatic lipid accumulation and associated metabolic pathways. Female CD1 mice were administrated with CdCl 2 (10 and 100 mg l -1 ) by drinking water. We found that Cd exposure did not induce obesity, insulin resistance and hepatic lipid accumulation. By contrary, mice in the Cd-100 mg l -1 group presented a significant reduction of the glucose area under the curve during the glucose tolerance test. However, there was a significant elevation in the mRNA level of Fasn and Scd-1, which were critical genes during hepatic fatty acid synthesis. Moreover, hepatic Fabp1 and Fabp4, two genes for hepatic fatty acid uptake were upregulated in Cd-treated mice. Of interest, Lpl, a key gene for hepatic lipoprotein lysis, was also upregulated in Cd-treated mice. Collectively, our results suggest that Cd exposure upregulated mRNA level of genes related to hepatic lipid metabolism although there was no insulin resistance and hepatic lipid accumulation shown in the present study. Copyright © 2018 John Wiley & Sons, Ltd.

  7. Mutational Inactivation of Herpes Simplex Virus 1 MicroRNAs Identifies Viral mRNA Targets and Reveals Phenotypic Effects in Culture

    PubMed Central

    Flores, Omar; Nakayama, Sanae; Whisnant, Adam W.; Javanbakht, Hassan; Cullen, Bryan R.

    2013-01-01

    Herpes simplex virus 1 (HSV-1), a ubiquitous human pathogen, expresses several viral microRNAs (miRNAs). These, along with the latency-associated transcript, represent the only viral RNAs detectable in latently infected neuronal cells. Here, for the first time, we analyze which HSV-1 miRNAs are loaded into the RNA-induced silencing complex (RISC), the key effector of miRNA function. Only 9 of the 17 reported HSV-1 miRNAs, i.e., miR-H1 to miR-H8 plus miR-H11, were found to actually load into the RISC. Surprisingly, this analysis also revealed that HSV-1 miRNAs loaded into the RISC with efficiencies that differed widely; <1% of the miR-H1-3p miRNA detectable in HSV-1-infected cells was loaded into the RISC. Analysis of HSV-1 mutants individually lacking the viral miR-H2, miR-H3, or miR-H4 miRNA revealed that loss of these miRNAs affected the rate of replication of HSV-1 in neuronal cells but not in fibroblasts. Analysis of mRNA and protein expression, as well as assays mapping viral miRNA binding sites in infected cells, showed that endogenous HSV-1 miR-H2 binds to viral ICP0 mRNA and inhibits its expression, while endogenous miR-H4 inhibits the expression of the viral ICP34.5 gene. In contrast, no viral mRNA target for miR-H3 could be detected, even though miR-H3, like miR-H4, is perfectly complementary to ICP34.5 mRNA. Together, these data demonstrate that endogenous HSV-1 miRNA expression can significantly alter viral replication in culture, and they also identify two viral mRNA targets for miR-H2 and miR-H4 that can partially explain this phenotype. PMID:23536669

  8. Ancestral vinclozolin exposure alters the epigenetic transgenerational inheritance of sperm small noncoding RNAs

    PubMed Central

    Schuster, Andrew; Skinner, Michael K.; Yan, Wei

    2016-01-01

    Abstract Exposure to the agricultural fungicide vinclozolin during gestation promotes a higher incidence of various diseases in the subsequent unexposed F3 and F4 generations. This phenomenon is termed epigenetic transgenerational inheritance and has been shown to in part involve alterations in DNA methylation, but the role of other epigenetic mechanisms remains unknown. The current study investigated the alterations in small noncoding RNA (sncRNA) in the sperm from F3 generation control and vinclozolin lineage rats. Over 200 differentially expressed sncRNAs were identified and the tRNA-derived sncRNAs, namely 5′ halves of mature tRNAs (5′ halves), displayed the most dramatic changes. Gene targets of the altered miRNAs and tRNA 5′ halves revealed associations between the altered sncRNAs and differentially DNA methylated regions. Dysregulated sncRNAs appear to correlate with mRNA profiles associated with the previously observed vinclozolin-induced disease phenotypes. Data suggest potential connections between sperm-borne RNAs and the vinclozolin-induced epigenetic transgenerational inheritance phenomenon. PMID:27390623

  9. Kaposi's Sarcoma-Associated Herpesvirus G-Protein-Coupled Receptor Prevents AU-Rich-Element-Mediated mRNA Decay

    PubMed Central

    Corcoran, Jennifer A.; Khaperskyy, Denys A.; Johnston, Benjamin P.; King, Christine A.; Cyr, David P.; Olsthoorn, Alisha V.

    2012-01-01

    During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, host gene expression is severely restricted by a process of global mRNA degradation known as host shutoff, which rededicates translational machinery to the expression of viral proteins. A subset of host mRNAs is spared from shutoff, and a number of these contain cis-acting AU-rich elements (AREs) in their 3′ untranslated regions. AREs are found in labile mRNAs encoding cytokines, growth factors, and proto-oncogenes. Activation of the p38/MK2 signal transduction pathway reverses constitutive decay of ARE-mRNAs, resulting in increased protein production. The viral G-protein-coupled receptor (vGPCR) is thought to play an important role in promoting the secretion of angiogenic molecules from KSHV-infected cells during lytic replication, but to date it has not been clear how vGPCR circumvents host shutoff. Here, we demonstrate that vGPCR activates the p38/MK2 pathway and stabilizes ARE-mRNAs, augmenting the levels of their protein products. Using MK2-deficient cells, we demonstrate that MK2 is essential for maximal vGPCR-mediated ARE-mRNA stabilization. ARE-mRNAs are normally delivered to cytoplasmic ribonucleoprotein granules known as processing bodies (PBs) for translational silencing and decay. We demonstrate that PB formation is prevented during KSHV lytic replication or in response to vGPCR-mediated activation of RhoA subfamily GTPases. Together, these data show for the first time that vGPCR impacts gene expression at the posttranscriptional level, coordinating an attack on the host mRNA degradation machinery. By suppressing ARE-mRNA turnover, vGPCR may facilitate escape of certain target mRNAs from host shutoff and allow secretion of angiogenic factors from lytically infected cells. PMID:22696654

  10. Organizing Principles of Mammalian Nonsense-Mediated mRNA Decay

    PubMed Central

    Popp, Maximilian Wei-Lin; Maquat, Lynne E.

    2014-01-01

    Cells use messenger RNAs (mRNAs) to ensure the accurate dissemination of genetic information encoded by DNA. Given that mRNAs largely direct the synthesis of a critical effector of cellular phenotype, i.e., proteins, tight regulation of both the quality and quantity of mRNA is a prerequisite for effective cellular homeostasis. Here, we review nonsense-mediated mRNA decay (NMD), which is the best-characterized posttranscriptional quality control mechanism that cells have evolved in their cytoplasm to ensure transcriptome fidelity. We use protein quality control as a conceptual framework to organize what is known about NMD, highlighting overarching similarities between these two polymer quality control pathways, where the protein quality control and NMD pathways intersect, and how protein quality control can suggest new avenues for research into mRNA quality control. PMID:24274751

  11. A Genetic Selection For Neurospora crassa Mutants Altered in Their Light Regulation of Transcription

    PubMed Central

    Navarro-Sampedro, Laura; Yanofsky, Charles; Corrochano, Luis M.

    2008-01-01

    Transcription of the Neurospora crassa gene con-10 is induced during conidiation and following exposure of vegetative mycelia to light, but light activation is transient due to photoadaptation. We describe mutational analyses of photoadaptation using a N. crassa strain bearing a translational fusion of con-10, including its regulatory region, to a selectable bacterial gene conferring hygromycin resistance (hph). Growth of this strain was sensitive to hygromycin, upon continuous culture in the light. Five mutants were isolated that were resistant to hygromycin when cultured under constant light. Three mutant strains displayed elevated, sustained accumulation of con-10∷hph mRNA during continued light exposure, suggesting that they bear mutations that reduce or eliminate the presumed light-dependent repression mechanism that blocks con-10 transcription upon prolonged illumination. These mutations altered photoadaptation for only a specific group of genes (con-10 and con-6), suggesting that regulation of photoadaptation is relatively gene specific. The mutations increased light-dependent mRNA accumulation for genes al-1, al-2, and al-3, each required for carotenoid biosynthesis, resulting in a threefold increase in carotenoid accumulation following continuous light exposure. Identification of the altered gene or genes in these mutants may reveal novel proteins that participate in light regulation of gene transcription in fungi. PMID:18202366

  12. Heparanase mRNA expression and point mutation in hepatocellular carcinoma

    PubMed Central

    Chen, Xiao-Peng; Liu, Yin-Bib; Rui, Jing; Peng, Shu-You; Peng, Cheng-Hong; Zhou, Zi-Yan; Shi, Liang-Hui; Shen, Hong-Wei; Xu, Bin

    2004-01-01

    AIM: To explore the expression of heparanase mRNA and point mutation in hepatocellular carcinoma (HCC). METHODS: Reverse transcription polymerase chain reaction was used to measure the expression of heparanase mRNA in the primary tumor tissues and surrounding liver tissues of 33 HCC patients. T-A cloning and sequencing were used to detect whether there was any mutation in the amplified PCR products. RESULTS: The expression of heparanase mRNA was positive in 16 primary tumor tissues of HCC, and the positive rate was 48.5%, which was significantly higher than that in the surrounding liver parenchyma (P < 0.01). The positive rate for heparanase gene in high-tendency to metastatic recurrence group (71.4%, 10/14) was obviously higher than that in low-tendency to metastatic recurrence group (31.6%, 6/19) (P = 0.023). The positive rate for heparanase gene in patients with metastatic recurrence during postoperative follow-up (78.6%, 11/14) was also significantly higher than that in those without metastatic recurrence (21.4%, 3/14) (P = 0.003). Sequence analysis of the HPA PCR products was made in 7 patients, and 2-point mutations were found in 4 patients, one of which was sense mutation, neither base insertion nor deletion was detected. The mutation rate was 57.1% (4/7). CONCLUSION: The expression rate of heparanase mRNA increases in HCC, and HPA mRNA may be one of the reliable markers for the metastatic activity gained by the liver tumor cells and could be used clinically in predicting metastatic recurrence of HCC. Point mutation may be one of the causes for enhanced heparanase mRNA expression. PMID:15334672

  13. The Modification of siRNA with 3′ Cholesterol to Increase Nuclease Protection and Suppression of Native mRNA by Select siRNA Polyplexes

    PubMed Central

    Ambardekar, Vishakha V.; Han, Huai-Yun; Varney, Michelle L.; Vinogradov, Serguei V.; Singh, Rakesh K.; Vetro, Joseph A.

    2010-01-01

    Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3′ cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC. PMID:21047680

  14. Maternal supplementation with rumen-protected methionine increases prepartal plasma methionine concentration and alters hepatic mRNA abundance of 1-carbon, methionine, and transsulfuration pathways in neonatal Holstein calves.

    PubMed

    Jacometo, C B; Zhou, Z; Luchini, D; Corrêa, M N; Loor, J J

    2017-04-01

    An important mechanism of nutritional "programming" induced by supplementation with methyl donors during pregnancy is the alteration of mRNA abundance in the offspring. We investigated the effects of rumen-protected Met (RPM) on abundance of 17 genes in the 1-carbon, Met, and transsulfuration pathways in calf liver from cows fed the same basal diet without (control, CON) or with RPM at 0.08% of diet dry matter/d (MET) from -21 through +30 d around calving. Biopsies (n = 8 calves per diet) were harvested on d 4, 14, 28, and 50 of age. Cows fed RPM had greater plasma concentration of Met (17.8 vs. 28.2 μM) at -10 d from calving. However, no difference was present in colostrum yield and free AA concentrations. Greater abundance on d 4 and 14 of betaine-homocysteine S-methyltransferase 2 (BHMT2), adenosylhomocysteinase (AHCY; also known as SAHH), and cystathionine-β-synthase (CBS) in MET calves indicated alterations in Met, choline, and homocysteine metabolism. Those data agree with the greater abundance of methionine adenosyltransferase 1A (MAT1A) in MET calves. Along with CBS, the greater abundance of glutamate-cysteine ligase (GCLC) and glutathione reductase (GSR) on d 4 in MET calves indicated a short-term postnatal alteration in the use of homocysteine for taurine and glutathione synthesis (both are potent intracellular antioxidants). The striking 7-fold upregulation at d 50 versus 4 of cysteine sulfinic acid decarboxylase (CSAD), catalyzing the last step of taurine synthesis, in MET and CON calves underscores an important role of taurine during postnatal calf growth. The unique role of taurine in the young calf is further supported by the upregulation of CBS, GCLC, and GSR at d 50 versus 14 and 28 in MET and CON. Although betaine-homocysteine S-methyltransferase (BHMT) activity did not differ in MET and CON, it increased ∼50% at d 14 and 28 versus 4. A significant positive correlation (r = 0.79) was present between BHMT abundance and BHMT activity regardless

  15. Proteomic analysis of altered proteins in lymphoid organ of yellow head virus infected Penaeus monodon.

    PubMed

    Bourchookarn, Apichai; Havanapan, Phattara-Orn; Thongboonkerd, Visith; Krittanai, Chartchai

    2008-03-01

    A comparative proteomic analysis was employed to identify altered proteins in the yellow head virus (YHV) infected lymphoid organ (LO) of Penaeus monodon. At 24 h post-infection, the infected shrimps showed obvious signs of infection, while the control shrimps remained healthy. Two-dimensional electrophoresis of proteins extracted from the LO revealed significant alterations in abundance of several proteins in the infected group. Protein identification by MALDI-TOF MS and nanoLC-ESI-MS/MS revealed significant increase of transglutaminase, protein disulfide isomerase, ATP synthase beta subunit, V-ATPase subunit A, and hemocyanin fragments. A significant decrease was also identified for Rab GDP-dissociation inhibitor, 6-phosphogluconate dehydrogenase, actin, fast tropomyosin isoform, and hemolymph clottable protein. Some of these altered proteins were further investigated at the mRNA level using real-time RT-PCR, which confirmed the proteomic data. Identification of these altered proteins in the YHV-infected shrimps may provide novel insights into the molecular responses of P. monodon to YHV infection.

  16. Climate change alters stability and species potential interactions in a large marine ecosystem.

    PubMed

    Griffith, Gary P; Strutton, Peter G; Semmens, Jayson M

    2018-01-01

    We have little empirical evidence of how large-scale overlaps between large numbers of marine species may have altered in response to human impacts. Here, we synthesized all available distribution data (>1 million records) since 1992 for 61 species of the East Australian marine ecosystem, a global hot spot of ocean warming and continuing fisheries exploitation. Using a novel approach, we constructed networks of the annual changes in geographical overlaps between species. Using indices of changes in species overlap, we quantified changes in the ecosystem stability, species robustness, species sensitivity and structural keystone species. We then compared the species overlap indices with environmental and fisheries data to identify potential factors leading to the changes in distributional overlaps between species. We found that the structure of the ecosystem has changed with a decrease in asymmetrical geographical overlaps between species. This suggests that the ecosystem has become less stable and potentially more susceptible to environmental perturbations. Most species have shown a decrease in overlaps with other species. The greatest decrease in species overlap robustness and sensitivity to the loss of other species has occurred in the pelagic community. Some demersal species have become more robust and less sensitive. Pelagic structural keystone species, predominately the tunas and billfish, have been replaced by demersal fish species. The changes in species overlap were strongly correlated with regional oceanographic changes, in particular increasing ocean warming and the southward transport of warmer and saltier water with the East Australian Current, but less correlated with fisheries catch. Our study illustrates how large-scale multispecies distribution changes can help identify structural changes in marine ecosystems associated with climate change. © 2017 John Wiley & Sons Ltd.

  17. Methamphetamine and HIV-Tat alter murine cardiac DNA methylation and gene expression

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

    Koczor, Christopher A., E-mail: ckoczor@emory.edu; Fields, Earl; Jedrzejczak, Mark J.

    This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10 d, 3 mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change > 1.5, p < 0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides formore » calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. - Highlights: • HIV-1 Tat and methamphetamine (METH) alter cardiac gene expression and epigenetics. • METH impacts gene expression or epigenetics more significantly than Tat expression. • METH alters cardiac mitochondrial function and calcium signaling independent of Tat. • METH alters DNA methylation, expression, and protein

  18. Clinical values of AFP, GPC3 mRNA in peripheral blood for prediction of hepatocellular carcinoma recurrence following OLT: AFP, GPC3 mRNA for prediction of HCC.

    PubMed

    Wang, Yuliang; Shen, Zhongyang; Zhu, Zhijun; Han, Ruifa; Huai, Mingsheng

    2011-03-01

    Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Annually, about 200,000 patients died of HCC in China. Liver transplantation (LT) holds great theoretical appeal in treating HCC. However, the high recurrence rate after transplantation is the most important limiting factor for long-term survival. To assess the value of alpha-fetoprotein (AFP) messenger RNA (mRNA), Glypican-3 (GPC3) mRNA-expressing cells in the peripheral blood (PB) for prediction of HCC recurrence following orthotopic liver transplantation (OLT). 29 patients with HCC who underwent OLT with a minimum clinical follow-up of 12 months were included in this retrospective study. We detected AFP mRNA, GPC3 mRNA-expressing cells in the PB by TaqMan real-time reverse transcriptase-polymerase chain reaction (RT-PCR), pre-, intra- and post-operatively. The early recurrence of patients was evaluated. 8 (28%), 15 (52%), and 9 (31%) patients had AFP mRNA detected pre-, intra-, and post-operatively, respectively. With 12 months of follow-up, HCC recurred in 7 (24%) patients. Univariate analysis revealed that positive pre- and post-operative AFP mRNA, TNM stage as well as vascular invasion were significant predictors for the HCC recurrence. Multivariate analysis revealed that being positive for AFP mRNA pre-operatively remained a significant risk factor for HCC recurrence after OLT. GPC3 mRNA was expressed in all PB samples. There was no significant difference in the expression levels of GPC3 mRNA between the HCC and control groups. There were no significant differences in GPC3 mRNA expression values between those patients with and without tumor recurrence. The pre-operative detection of circulating AFP mRNA-expressing cells could be a useful predictor for HCC recurrence following OLT. GPC3 mRNA-expressing cells in PB seem to have no diagnostic value.

  19. Texture Analysis of Poly-Adenylated mRNA Staining Following Global Brain Ischemia and Reperfusion

    PubMed Central

    Szymanski, Jeffrey J.; Jamison, Jill T.; DeGracia, Donald J.

    2011-01-01

    Texture analysis provides a means to quantify complex changes in microscope images. We previously showed that cytoplasmic poly-adenylated mRNAs form mRNA granules in post-ischemic neurons and that these granules correlated with protein synthesis inhibition and hence cell death. Here we utilized the texture analysis software MaZda to quantify mRNA granules in photomicrographs of the pyramidal cell layer of rat hippocampal region CA3 around 1 hour of reperfusion after 10 min of normothermic global cerebral ischemia. At 1 hour reperfusion, we observed variations in the texture of mRNA granules amongst samples that were readily quantified by texture analysis. Individual sample variation was consistent with the interpretation that animal-to-animal variations in mRNA granules reflected the time-course of mRNA granule formation. We also used texture analysis to quantify the effect of cycloheximide, given either before or after brain ischemia, on mRNA granules. If administered before ischemia, cycloheximide inhibited mRNA granule formation, but if administered after ischemia did not prevent mRNA granulation, indicating mRNA granule formation is dependent on dissociation of polysomes. We conclude that texture analysis is an effective means for quantifying the complex morphological changes induced in neurons by brain ischemia and reperfusion. PMID:21477879

  20. Stability Testing of Herbal Drugs: Challenges, Regulatory Compliance and Perspectives.

    PubMed

    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. Copyright © 2016 John Wiley & Sons, Ltd.