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Sample records for protein mrna expression

  1. Oscillatory kinetics of gene expression: Protein conversion and slow mRNA transport

    SciTech Connect

    Zhdanov, V. P.

    2009-06-15

    The negative feedback between mRNA and regulatory-protein production may result in oscillations in the kinetics of gene expression if the mRNA-protein interplay includes protein conversion. Using a mean-field kinetic model, we show that such oscillations can be amplified due to limitations of the mRNA transport between the nucleus and cytoplasm. This effect may be dramatic for the mRNA population in the nucleus.

  2. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

  3. Effect of taurine on mRNA expression of thioredoxin interacting protein in Caco-2 cells.

    PubMed

    Gondo, Yusuke; Satsu, Hideo; Ishimoto, Yoko; Iwamoto, Taku; Shimizu, Makoto

    2012-09-28

    Taurine (2-aminoethanesulfonic acid), a sulfur-containing β-amino acid, plays an important role in several essential biological processes; although, the underlying mechanisms for these regulatory functions remain to be elucidated, especially at the genetic level. We investigated the effects of taurine on the gene expression profile in Caco-2 cells using DNA microarray. Taurine increased the mRNA expression of thioredoxin interacting protein (TXNIP), which is involved in various metabolisms and diseases. β-Alanine or γ-aminobutyric acid (GABA), which are structurally or functionally related to taurine, did not increase TXNIP mRNA expression. These suggest the expression of TXNIP mRNA is induced specifically by taurine. β-Alanine is also known to be a substrate of taurine transporter (TAUT) and competitively inhibits taurine uptake. Inhibition of taurine uptake by β-alanine eliminated the up-regulation of TXNIP, which suggests TAUT is involved in inducing TXNIP mRNA expression. The up-regulation of TXNIP mRNA expression by taurine was also observed at the protein level. Furthermore, taurine significantly increased TXNIP promoter activity. Our present study demonstrated the taurine-specific phenomenon of TXNIP up-regulation, which sheds light on the physiological function of taurine. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. p73 expression is regulated by ribosomal protein RPL26 through mRNA translation and protein stability

    PubMed Central

    Yan, Wensheng; Chen, Xinbin

    2016-01-01

    p73, a p53 family tumor suppressor, is regulated by multiple mechanisms, including transcription and mRNA and protein stability. However, whether p73 expression is regulated via mRNA translation has not been explored. To test this, we examined whether ribosomal protein 26 (RPL26) plays a role in p73 expression. Here, we showed that p73 expression is controlled by RPL26 via protein stability and mRNA translation. To examine whether MDM2 mediates RPL26 to regulate p73 protein stability, we generated multiple MDM2-knockout cell lines by CRISPR-cas9. We found that in the absence of MDM2, the half-life of p73 protein is markedly increased. Interestingly, we also found that RPL26 is still capable of regulating p73 expression, albeit to a lesser extent, in MDM2-KO cells compared to that in isogenic control cells, suggesting that RPL26 regulates p73 expression via multiple mechanisms. Indeed, we found that RPL26 is necessary for efficient assembly of polysomes on p73 mRNA and de novo synthesis of p73 protein. Consistently, we found that RPL26 directly binds to p73 3′ untranslated region (3′UTR) and that RPL26 is necessary for efficient expression of an eGFP reporter that carries p73 3′UTR. We also found that RPL26 interacts with cap-binding protein eIF4E and enhances the association of eIF4E with p73 mRNA, leading to increased p73 mRNA translation. Finally, we showed that knockdown of RPL26 promotes, whereas ectopic expression of RPL26 inhibits, cell growth in a TAp73-dependent manner. Together, our data indicate that RPL26 regulates p73 expression via two distinct mechanisms: protein stability and mRNA translation. PMID:27825141

  5. Eosinophil cationic protein mRNA expression in children with bronchial asthma.

    PubMed

    Yu, H Y; Li, X Y; Cai, Z F; Li, L; Shi, X Z; Song, H X; Liu, X J

    2015-11-13

    Studies have shown that eosinophils are closely related to pathogenesis of bronchial asthma. Eosinophils release eosinophil cationic protein (ECP), which plays an important role in infection and allergic reactions. Serum ECP mRNA expression in children with bronchial asthma has not been adequately investigated. We analyzed serum ECP mRNA expression in 63 children with bronchial asthma and 21 healthy children by using reverse-transcriptase polymerase chain reaction to understand the role of ECP in children with bronchial asthma. The children with bronchial asthma were segregated into acute-phase and stable-phase groups, based on the severity of the illness. Serum ECP mRNA expression in children with bronchial asthma (0.375 ± 0.04) was significantly higher than that in healthy controls (0.20 ± 0.02; P < 0.05). Additionally, children in the acute-phase group showed higher ECP mRNA expression level (0.44 ± 0.06) than those in the stable-phase (0.31 ± 0.03) and healthy control groups (0.20 ± 0.02; P < 0.05), while the level in the stable-phase (0.31 ± 0.03) was markedly higher than that in the healthy control group (0.20 ± 0.02; P < 0.05). Detection of serum ECP mRNA expression level has possible applications in the diagnosis and treatment of children with bronchial asthma.

  6. Myxovirus Resistance Protein A mRNA Expression Kinetics in Multiple Sclerosis Patients Treated with IFNβ

    PubMed Central

    Libertinova, Jana; Meluzinova, Eva; Tomek, Ales; Horakova, Dana; Kovarova, Ivana; Matoska, Vaclav; Kumstyrova, Simona; Zajac, Miroslav; Hyncicova, Eva; Liskova, Petra; Houzvickova, Eva; Martinkovic, Lukas; Bojar, Martin; Havrdova, Eva; Marusic, Petr

    2017-01-01

    Introduction Interferon-β (IFNß) is the first-line treatment for relapsing-remitting multiple sclerosis. Myxovirus resistance protein A (MxA) is a marker of IFNß bioactivity, which may be reduced by neutralizing antibodies (NAbs) against IFNß. The aim of the study was to analyze the kinetics of MxA mRNA expression during long-term IFNβ treatment and assess its predictive value. Methods A prospective, observational, open-label, non-randomized study was designed in multiple sclerosis patients starting IFNß treatment. MxA mRNA was assessed prior to initiation of IFNß therapy and every three months subsequently. NAbs were assessed every six months. Assessment of relapses was scheduled every three months during 24 months of follow up. The disease activity was correlated to the pretreatment baseline MxA mRNA value. In NAb negative patients, clinical status was correlated to MxA mRNA values. Results 119 patients were consecutively enrolled and 107 were included in the final analysis. There was no correlation of MxA mRNA expression levels between baseline and month three. Using survival analysis, none of the selected baseline MxA mRNA cut off points allowed prediction of time to first relapse on the treatment. In NAb negative patients, mean MxA mRNA levels did not significantly differ in patients irrespective of relapse status. Conclusion Baseline MxA mRNA does not predict the response to IFNß treatment or the clinical status of the disease and the level of MxA mRNA does not correlate with disease activity in NAb negative patients. PMID:28081207

  7. Heterogeneous expression of protein and mRNA in pyruvate dehydrogenase deficiency.

    PubMed Central

    Wexler, I D; Kerr, D S; Ho, L; Lusk, M M; Pepin, R A; Javed, A A; Mole, J E; Jesse, B W; Thekkumkara, T J; Pons, G

    1988-01-01

    Deficiency of pyruvate dehydrogenase [pyruvate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1], the first component of the pyruvate dehydrogenase complex, is associated with lactic acidosis and central nervous system dysfunction. Using both specific antibodies to pyruvate dehydrogenase and cDNAs coding for its two alpha and beta subunits, we characterized pyruvate dehydrogenase deficiency in 11 patients. Three different patterns were found on immunologic and RNA blot analyses. (i) Seven patients had immunologically detectable crossreactive material for the alpha and beta proteins of pyruvate dehydrogenase. (ii) Two patients had no detectable crossreactive protein for either the alpha or beta subunit but had normal amounts of mRNA for both alpha and beta subunits. (iii) The remaining two patients also had no detectable crossreactive protein but had diminished amounts of mRNA for the alpha subunit of pyruvate dehydrogenase only. These results indicate that loss of pyruvate dehydrogenase activity may be associated with either absent or catalytically inactive proteins, and in those cases in which this enzyme is absent, mRNA for one of the subunits may also be missing. When mRNA for one of the subunits is lacking, both protein subunits are absent, suggesting that a mutation affecting the expression of one of the subunit proteins causes the remaining uncomplexed subunit to be unstable. The results show that several different mutations account for the molecular heterogeneity of pyruvate dehydrogenase deficiency. Images PMID:3140238

  8. Sprouty4 mRNA variants and protein expressions in breast and lung-derived cells

    PubMed Central

    Doriguzzi, Angelina; Salhi, Jihen; Sutterlüty-Fall, Hedwig

    2016-01-01

    Sprouty proteins are modulators of mitogen-induced signalling processes and are therefore hypothesized to affect malignant diseases. As a member of the Sprouty family, Sprouty4 has been previously shown to function as a tumour suppressor in lung and breast cancer. The present study analysed the expression of two known Sprouty4 splice variants in cells established from malignant and normal lung and breast tissues using semi-quantitative reverse transcription-polymerase chain reaction and immunoblotting. The results indicated that the expression of the two messenger RNA (mRNA) variants was reduced in the cells derived from malignant tissue in comparison to the normal counterparts. Although the expression of the two splice variants were associated in both tissue types, on average, the relative expression of the longer variant was slightly increased in malignant cells compared with normal tissues. Notably, the protein levels reflected the expression observed at the mRNA level only in breast-derived cells. Contrarily, with regards to the measured mRNA levels, Sprouty4 protein was disproportional augmented in lung cells known to harbour the mutated K-Ras gene. PMID:27895786

  9. Optimization of mRNA design for protein expression in the crustacean Daphnia magna.

    PubMed

    Törner, Kerstin; Nakanishi, Takashi; Matsuura, Tomoaki; Kato, Yasuhiko; Watanabe, Hajime

    2014-08-01

    The water flea Daphnia is a new model organism for ecological, evolutionary, and toxicological genomics. Detailed functional analysis of genes newly discovered through genomic approaches often requires overexpression of the identified protein. In the present study, we report the microinjection of in vitro-synthesized RNAs into the eggs as a method for overexpressing ubiquitous proteins in Daphnia magna. We injected a 1.3-kb mRNA that coded for the red fluorescent protein (DsRed2) flanked by UTRs from the ubiquitously expressed elongation factor 1α-1 (EF1α-1) into D. magna embryos. DsRed2 fluorescence in the embryos was measured 24 h after microinjection. Unexpectedly, the reporter RNA containing the 522-bp full-length EF1α-1 3' UTR failed to induce fluorescence. To assess reporter expression, the length of the 3' UTR that potentially contained negative regulatory elements of protein expression, including AU-rich regions and Musashi binding elements, was serially reduced from the 3' end. Assessing all injected RNA alternatives, mRNA containing the first 60 bp of the 3' UTR gave rise to the highest fluorescence, 14 times the Daphnia auto-fluorescence. In contrast, mRNA lacking the entire 3' UTR hardly induced any change in fluorescence intensity. This is the first evaluation of UTRs of mRNAs delivered into Daphnia embryos by microinjection for overexpressing proteins. The mRNA with truncated 3' UTRs of Daphnia EF1α-1 will be useful not only for gain-of-function analyses but also for labeling proteins and organelles with fluorescent proteins in Daphnia.

  10. CD44 in canine leukemia: analysis of mRNA and protein expression in peripheral blood.

    PubMed

    Gelain, M E; Martini, V; Giantin, M; Aricò, A; Poggi, A; Aresu, L; Riondato, F; Dacasto, M; Comazzi, S

    2014-05-15

    Hyaluronan receptor CD44 mediates interaction between cells and extracellular matrix. The expression of standard form and its variants is dysregulated in human leukemias and is associated with metastasis and prognosis. The aim of this work is the evaluation of CD44 mRNA and protein expression in canine leukemia. Peripheral blood from 20 acute leukemias (AL) (10 acute lymphoblastic, 6 acute myeloid and 4 acute undifferentiated leukemias), 21 chronic lymphocytic leukemias (CLL) and thirteen healthy dogs were collected. The mRNA expression of all CD44 variants presenting exons 1-5 and/or 16-20 (CD44_ex1-5 and CD44_ex16-20) and CD44 protein were determined by real-time RT-PCR and flow cytometry, using the mean fluorescent index (MFI), respectively. CD44 MFI was significantly higher in leukemic samples compared to controls and a higher expression was found in AL in respect with CLL. No significant differences were found when considering different phenotypic subtypes of AL and CLL. CD44_ex1-5 mRNA expression was significantly higher in AL compared to controls, whereas there was no difference in CLL compared to controls and AL. CD44_es16-20 showed the same trend, but without differences among groups. The high CD44 expression found in canine leukemias could be considered a step toward the definition of their molecular features. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. mRNA Expression of Ovine Angiopoietin-like Protein 4 Gene in Adipose Tissues.

    PubMed

    Zhang, Jing; Jing, Jiong-Jie; Jia, Xia-Li; Qiao, Li-Ying; Liu, Jian-Hua; Liang, Chen; Liu, Wen-Zhong

    2016-05-01

    Angiopoietin-like protein 4 (ANGPTL4) is involved in a variety of functions, including lipoprotein metabolism and angiogenesis. To reveal the role of ANGPTL4 in fat metabolism of sheep, ovine ANGPTL4 mRNA expression was analyzed in seven adipose tissues from two breeds with distinct tail types. Forty-eight animals with the gender ratio of 1:1 for both Guangling Large Tailed (GLT) and Small Tailed Han (STH) sheep were slaughtered at 2, 4, 6, 8, 10, and 12 months of age, respectively. Adipose tissues were collected from greater and lesser omental, subcutaneous, retroperitoneal, perirenal, mesenteric, and tail fats. Ontogenetic mRNA expression of ANGPTL4 in these adipose tissues from GTL and STH was studied by quantitative real time polymerase chain reaction. The results showed that ANGPTL4 mRNA expressed in all adipose tissues studied with the highest in subcutaneous and the lowest in mesenteric fat depots. Months of age, tissue and breed are the main factors that significantly influence the mRNA expression. These results provide new insights into ovine ANGPTL4 gene expression and clues for its function mechanism.

  12. mRNA Expression of Ovine Angiopoietin-like Protein 4 Gene in Adipose Tissues

    PubMed Central

    Zhang, Jing; Jing, Jiong-Jie; Jia, Xia-Li; Qiao, Li-Ying; Liu, Jian-Hua; Liang, Chen; Liu, Wen-Zhong

    2016-01-01

    Angiopoietin-like protein 4 (ANGPTL4) is involved in a variety of functions, including lipoprotein metabolism and angiogenesis. To reveal the role of ANGPTL4 in fat metabolism of sheep, ovine ANGPTL4 mRNA expression was analyzed in seven adipose tissues from two breeds with distinct tail types. Forty-eight animals with the gender ratio of 1:1 for both Guangling Large Tailed (GLT) and Small Tailed Han (STH) sheep were slaughtered at 2, 4, 6, 8, 10, and 12 months of age, respectively. Adipose tissues were collected from greater and lesser omental, subcutaneous, retroperitoneal, perirenal, mesenteric, and tail fats. Ontogenetic mRNA expression of ANGPTL4 in these adipose tissues from GTL and STH was studied by quantitative real time polymerase chain reaction. The results showed that ANGPTL4 mRNA expressed in all adipose tissues studied with the highest in subcutaneous and the lowest in mesenteric fat depots. Months of age, tissue and breed are the main factors that significantly influence the mRNA expression. These results provide new insights into ovine ANGPTL4 gene expression and clues for its function mechanism. PMID:26954186

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

    PubMed

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

    1993-01-01

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

  14. TLR2 and TLR4 polymorphisms influence mRNA and protein expression in colorectal cancer

    PubMed Central

    Proença, Marcela Alcântara; de Oliveira, Juliana Garcia; Cadamuro, Aline Cristina Targa; Succi, Maysa; Netinho, João Gomes; Goloni-Bertolo, Eny Maria; Pavarino, Érika Cristina; Silva, Ana Elizabete

    2015-01-01

    AIM: To evaluate the effect of promoter region polymorphisms of toll-like receptor (TLR)2-196 to -174del and TLR4-1607T/C (rs10759932) on mRNA and protein expression in tumor tissue and of TLR4+896A/G (rs4986790) on colorectal cancer (CRC) risk. METHODS: The TLR2-196 to -174del polymorphism was investigated using allele-specific polymerase chain reaction (PCR) and the TLR4-1607T/C and TLR4+896A/G by PCR-restriction fragment length polymorphism (RFLP). We genotyped 434 DNA samples from 194 CRC patients and 240 healthy individuals. The mRNA relative quantification (RQ) was performed in 40 tumor tissue samples by quantitative PCR TaqMan assay, using specific probes for TLR2 and TLR4 genes, and ACTB and GAPDH reference genes were used as endogenous controls. Protein expression was analyzed by immunohistochemistry with specific primary antibodies. RESULTS: No association was found for TLR4-1607T/C and TLR4+896A/G by three statistical models (log-additive, dominant and recessive). However, based on dominant and log-additive models, the polymorphic variant TLR2-196 to -174del was associated with increased CRC risk [dominant: odds ratio (OR) = 1.72, 95%CI: 1.03-2.89; P = 0.038 and log-additive: OR =1.59, 95%CI: 1.02-2.48; P = 0.039]. TLR2 mRNA expression was increased in tumor tissue (RQ = 2.36) when compared to adjacent normal tissue (RQ = 1; P < 0.0001), whereas the TLR4 mRNA showed a basal expression (RQ = 0.74 vs RQ = 1, P = 0.452). Immunohistochemistry analysis of TLR2 and TLR4 protein expression was concordant with the findings of mRNA expression. In addition, the TLR2-196 to -174del variant carriers showed mRNA relative expression 2.19 times higher than wild-genotype carriers. The TLR2 protein expression was also higher for the TLR2-196 to -174del variant carriers [117 ± 10 arbitrary unit (a.u.) vs 95 ± 4 a.u., P = 0.03]. However, for the TLR4 -1607T/C polymorphism no significant difference was found for both mRNA (P = 0.56) and protein expression (P = 0

  15. Control of Cytokine mRNA Expression by RNA-binding Proteins and microRNAs

    PubMed Central

    Palanisamy, V.; Jakymiw, A.; Van Tubergen, E.A.; D’Silva, N.J.; Kirkwood, K.L.

    2012-01-01

    Cytokines are critical mediators of inflammation and host defenses. Regulation of cytokines can occur at various stages of gene expression, including transcription, mRNA export, and post- transcriptional and translational levels. Among these modes of regulation, post-transcriptional regulation has been shown to play a vital role in controlling the expression of cytokines by modulating mRNA stability. The stability of cytokine mRNAs, including TNFα, IL-6, and IL-8, has been reported to be altered by the presence of AU-rich elements (AREs) located in the 3′-untranslated regions (3′UTRs) of the mRNAs. Numerous RNA-binding proteins and microRNAs bind to these 3′UTRs to regulate the stability and/or translation of the mRNAs. Thus, this paper describes the cooperative function between RNA-binding proteins and miRNAs and how they regulate AU-rich elements containing cytokine mRNA stability/degradation and translation. These mRNA control mechanisms can potentially influence inflammation as it relates to oral biology, including periodontal diseases and oral pharyngeal cancer progression. PMID:22302144

  16. Increased protein and mRNA expression of resistin after dexamethasone administration.

    PubMed

    Sasayama, D; Hori, H; Nakamura, S; Yamamoto, N; Hattori, K; Teraishi, T; Ota, M; Kunugi, H

    2015-06-01

    Synthetic glucocorticoids such as dexamethasone are widely used to treat a variety of inflammatory and autoimmune conditions, but they may induce adverse events including hyperglycemia. To shed light on the effect and action mechanism of dexamethasone, we examined the alterations of gene expression levels caused by dexamethasone.Microarray analysis was performed on whole blood collected from 24 physically healthy subjects at baseline and after dexamethasone administration. The expression levels of resistin mRNA were found to be significantly increased after the dexamethasone administration. In a separate sample of 12 subjects, we examined plasma resistin protein levels and found that they were increased after dexamethasone administration. Furthermore, the plasma mRNA and protein levels of resistin were significantly higher in individuals who carried the A allele of RETN single nucleotide polymorphism rs3219175 than in those who did not carry the allele. There was no significant interaction between the genotype and dexamethasone administration. No significant correlation was found between plasma levels of cortisol and resistin. Consistent with previous studies, the genotype of RETN rs3219175 was a strong determinant of resistin levels. The present study showed that oral administration of dexamethasone increases the protein and mRNA levels of resistin irrespective of the rs3219175 genotype. © Georg Thieme Verlag KG Stuttgart · New York.

  17. Expression of connexin 43 mRNA and protein in developing follicles of prepubertal porcine ovaries

    USGS Publications Warehouse

    Melton, C.M.; Zaunbrecher, G.M.; Yoshizaki, G.; Patio, R.; Whisnant, S.; Rendon, A.; Lee, V.H.

    2001-01-01

    A major form of cell-cell communication is mediated by gap junctions, aggregations of intercellular channels composed of connexins (Cxs), which are responsible for exchange of low molecular weight (< 1200 Da) cytosolic materials. These channels are a growing family of related proteins. This study was designed to determine the ontogeny of connexin 43 (Cx43) during early stages of follicular development in prepubertal porcine ovaries. A partial-length (412 base) cDNA clone was obtained from mature porcine ovaries and determined to have 98% identity with published porcine Cx43. Northern blot analysis demonstrated a 4.3-kb mRNA in total RNA isolated from prepubertal and adult porcine ovaries. In-situ hybridization revealed that Cx43 mRNA was detectable in granulosa cells of primary follicles but undetectable in dormant primordial follicles. The intensity of the signal increased with follicular growth and was greatest in the large antral follicles. Immunohistochemical evaluation indicated that Cx43 protein expression correlated with the presence of Cx43 mRNA. These results indicate that substantial amounts of Cx43 are first expressed in granulosa cells following activation of follicular development and that this expression increases throughout follicular growth and maturation. These findings suggest an association between the enhancement of intercellular gap-junctional communication and onset of follicular growth. ?? 2001 Elsevier Science Inc. All rights reserved.

  18. Organ specific expression of thyroid hormone receptor mRNA and protein in different human tissues.

    PubMed

    Shahrara, S; Drvota, V; Sylvén, C

    1999-10-01

    The major physiologic effect of thyroid hormone is thought to be initiated by the binding of T3 to the DNA binding thyroid hormone receptor (TR). The aim of this study has been to characterize the organ specific expression of thyroid hormone receptor mRNA, as well as its protein distribution and molecular weight in man. Determination of TRalpha1, alpha2, beta1 and beta2 mRNA molecular size was performed using Northern blot analysis in the human heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. TRalpha1, alpha2 and beta1 protein expression was characterized by Western blot analysis of human tissues. TRalpha1 mRNA of 4.9 kb was detected in all 8 tissues analyzed, with varying abundance in the various tissues. TRalpha2 mRNA was detected in 2 different sizes, with higher intensity at 5.7 and lower intensity at 3.2 kb. There were, however, multiple TRbeta1 mRNA of 8, 2 and 1 kb detected. TRbeta1 transcripts of 2 kb and 1 kb showed an organ specific pattern of expression. Multiple TRbeta2 mRNA of 6.6, 5.2, 2.5 and 2.4 kb were detected, as well as a unique 1 kb transcript, in the heart. TRbeta2 transcripts also displayed tissue specific expression. Western blot analysis displayed a single band of 48 kD for TRalpha1. The abundance of the TRalpha1 immunoreactive band was highest in the heart, brain, kidney and skeletal muscle, and lowest in the liver, placenta and lung, while no signals were detected in the spleen. The TRalpha2 specific antibody detected a band of 58 kD in all the tissues analyzed. The relative intensity of the immunoreactive TRalpha2 band detected was highest in the placenta and lung, with a medium concentration range in skeletal muscle, the heart and kidney. The TRalpha2 protein concentration was lowest in the spleen, liver and brain. Human TRbeta1 protein was detected as 55 and 52 kD bands, as well as a unique band of 45 kD in heart. The 52 kD band was detected in all tissues except the kidney and spleen. The 55 kD band was not

  19. Increases in mRNA and DREAM protein expression in the rat spinal cord after formalin induced pain.

    PubMed

    Long, Idris; Suppian, Rapeah; Ismail, Zalina

    2011-03-01

    Downstream Regulatory Element Antagonist Modulator (DREAM) protein modulates pain by regulating prodynorphin gene transcription. Therefore, we investigate the changes of mRNA and DREAM protein in relation to the mRNA and prodynorphin protein expression on the ipsilateral side of the rat spinal cord after formalin injection (acute pain model). DREAM like immunoreactivity (DLI) was not significantly different between C and F groups. However, we detected the upregulation of mean relative DREAM protein level in the nuclear but not in the cytoplasmic extract in the F group. These effects were consistent with the upregulation of the relative DREAM mRNA level. Prodynorphin like immunoreactivity (PLI) expression increased but the relative prodynorphin mRNA level remained unchanged. In conclusion, we suggest that upregulation of DREAM mRNA and protein expression in the nuclear compartment probably has functional consequences other than just the repression of prodynorphin gene. It is likely that these mechanisms are important in the modulation of pain.

  20. Induction of Ski protein expression upon luteinization in rat granulosa cells without a change in its mRNA expression.

    PubMed

    Kim, Hyun; Yamanouchi, Keitaro; Matsuwaki, Takashi; Nishihara, Masugi

    2012-01-01

    The Ski protein is implicated in the proliferation/differentiation of a variety of cells. We previously reported that the Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. However, granulosa cells cannot only undergo apoptosis but can alternatively differentiate into luteal cells. It is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to determine the localization of the Ski protein in the rat ovary during luteinization to examine if Ski might play a role in this process. In order to examine the Ski protein expression during the progression of luteinization, follicular growth was induced in immature female rats by administration of equine chorionic gonadotropin, and luteinization was induced by human chorionic gonadotropin treatment to mimic the luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in the preovulatory follicle, Ski protein expression was induced in response to the LH surge and was maintained after formation of the corpus luteum (CL). Although the Ski protein is absent from the granulosa cells of the preovulatory follicle, its mRNA (c-ski) was expressed, and the level of c-ski mRNA was unchanged even after the LH surge. The combined results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated posttranscriptionally.

  1. Circadian control of mRNA polyadenylation dynamics regulates rhythmic protein expression

    PubMed Central

    Kojima, Shihoko; Sher-Chen, Elaine L.; Green, Carla B.

    2012-01-01

    Poly(A) tails are 3′ modifications of eukaryotic mRNAs that are important in the control of translation and mRNA stability. We identified hundreds of mouse liver mRNAs that exhibit robust circadian rhythms in the length of their poly(A) tails. Approximately 80% of these are primarily the result of nuclear adenylation coupled with rhythmic transcription. However, unique decay kinetics distinguish these mRNAs from other mRNAs that are transcribed rhythmically but do not exhibit poly(A) tail rhythms. The remaining 20% are uncoupled from transcription and exhibit poly(A) tail rhythms even though the steady-state mRNA levels are not rhythmic. These are under the control of rhythmic cytoplasmic polyadenylation, regulated at least in some cases by cytoplasmic polyadenylation element-binding proteins (CPEBs). Importantly, we found that the rhythmicity in poly(A) tail length is closely correlated with rhythmic protein expression, with a several-hour delay between the time of longest tail and the time of highest protein level. Our study demonstrates that the circadian clock regulates the dynamic polyadenylation status of mRNAs, which can result in rhythmic protein expression independent of the steady-state levels of the message. PMID:23249735

  2. Bcl-2/Bax protein and mRNA expression in yak (Bos grunniens) placentomes.

    PubMed

    Fan, JiangFeng; Yu, SiJiu; Cui, Yan; Xu, Gengquan; Wang, Libin; Pan, Yangyang; He, Honghong

    2017-07-29

    Placental function is complex and influenced by various factors; furthermore, it depends on a delicate balance between cell proliferation, cell differentiation, and cell death. Bcl-2 and Bax proteins are key apoptosis regulators and are considered to play an important role in the maintenance of both dynamic balance and integrity of many tissues. Changes in Bcl-2 and Bax expressions have been described during different developmental stages in normal human placentas. Studies furthermore indicated several pathological placental changes to be related to abnormal Bcl-2 and Bax expressions. In the present study, we investigated both expression and distribution of Bcl-2 and Bax in yak placentas. For this, we collected placentas of 35 yaks at different stages of pregnancy as well as cotyledonary villi of four postpartum yaks. Protein and mRNA expressions of both Bcl-2 and Bax were investigated via immunohistochemistry, Western blot, and real-time PCR. Immunoreactive Bcl-2 protein was mainly localized near the fetal villous trophoblast at various gestational stages and post-partum. The Positive Index (PI) of Bcl-2 protein expression significantly decreased with increasing gestational age. Early during pregnancy (≤2 months), the Bax protein was widely distributed in the fetal villous trophoblast layer, the maternal caruncular crypt epithelium, and the stroma. Subsequently, the Bax protein distribution gradually concentrated in the fetal villous trophoblast layer. The staining intensity of Bax increased from the 3rd month to the prepartum of gestation. The PI reached a minimum of 9.4 ± 2.2 in fetal chorionic villi (FCV) and 1.3 ± 0.8 in maternal caruncular crypts (MCC) of the three months group. Both Bcl-2 and Bax had maximum immunoreactivity in the fetal villous trophoblast layer of placentas collected form postpartum yaks (with PIs of 36.6 ± 5.7 and 38.2 ± 4.8, respectively). Protein and mRNA expression of Bcl-2 and Bax investigated via Western blot and real

  3. Effect of running training on uncoupling protein mRNA expression in rat brown adipose tissue

    NASA Astrophysics Data System (ADS)

    Yamashita, Hitoshi; Yamamoto, Mikio; Sato, Yuzo; Izawa, Tetsuya; Komabayashi, Takao; Saito, Daizo; Ohno, Hideki

    1993-03-01

    The effect was investigated of endurance training on the expression of uncoupling protein (UCP) mRNA in brown adipose tissue (BAT) of rats. The exercised rats were trained on a rodent treadmill for 5 days per week and a total of 9 weeks. After the training programme, a marked decrease in BAT mass was found in terms of weight or weight per unit body weight; there was a corresponding decrease in DNA content and a downward trend in RNA and glycogen levels. The UCP mRNA was present at a markedly decreased level in BAT of trained animals. In consideration of the reduced levels of mRNAs for hormone-sensitive lipase and acylCoA synthetase, the brown adipose tissue investigated appeared to be in a relatively atrophied and thermogenically quiescent state.

  4. Exercise training does not increase muscle FNDC5 protein or mRNA expression in pigs.

    PubMed

    Fain, John N; Company, Joseph M; Booth, Frank W; Laughlin, M Harold; Padilla, Jaume; Jenkins, Nathan T; Bahouth, Suleiman W; Sacks, Harold S

    2013-10-01

    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. 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. 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. 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. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. 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

  6. UU/UA dinucleotide frequency reduction in coding regions results in increased mRNA stability and protein expression.

    PubMed

    Al-Saif, Maher; Khabar, Khalid S A

    2012-05-01

    UU and UA dinucleotides are rare in mammalian genes and may offer natural selection against endoribonuclease-mediated mRNA decay. This study hypothesized that reducing UU and UA (UW) dinucleotides in the mRNA-coding sequence, including the codons and the dicodon boundaries, may promote resistance to mRNA decay, thereby increasing protein production. Indeed, protein expression from UW-reduced coding regions of enhanced green fluorescent protein (EGFP), luciferase, interferon-α, and hepatitis B surface antigen (HBsAg) was higher when compared to the wild-type protein expression. The steady-state level of UW-reduced EGFP mRNA was higher and the mRNA half-life was also longer. Ectopic expression of the endoribonuclease, RNase L, did not reduce the wild type or UW-reduced mRNA. A mutant form of the mRNA decay-promoting protein, tristetraprolin (TTP/ZFP36), which has a point mutation in the zinc-finger domain (C124R), was used. The wild-type EGFP mRNA but not the UW-reduced mRNA responded to the dominant negative action of the C124R ZFP36/TTP mutant. The results indicate the efficacy of the described rational approach to formulate a general scheme for boosting recombinant protein production in mammalian cells.

  7. [Expression of heat shock protein 70 and its mRNA in career exposure to manganese].

    PubMed

    Chen, Wenwen; Shao, Hua; Chi, Mingfeng; Zhang, Zhihu; Shan, Yongle; Zou, Wei

    2015-10-01

    To analyze the expression levels of heat shock protein70 (HSPs70) and HSPs70 mRNA in different exposure to manganese, and research the neuroprotective effect on the career exposure to manganese. From 2008 to 2009, with cross-sectional study design, and in a locomotive and rolling stock works, by stratified random sampling method, the exposed sample consisted of 180 welders from different welding shops and 100 unexposed in the last three years, non-welder controls with age-matched workers of similar socioeconomic status from the same industry. The control workers had not been exposed to neurotoxic chemicals. The mRNA expressions of four different metabolic enzyme were detected by SYBR Green I quantitative real-time polymerase chain reaction. The expression levels of the two enzymes mRNA in different exposure to manganese were analyzed. The expressions of HSPs70 were detected by Western blot. The concentration of air manganese was determined by GFAAS. The average concentration of 8 h time (8h-TWA) was used to express the level of individual exposure to manganese, according to the air manganese workplace occupational exposure limit (8h-TWA=0.15 mg/m3), the exposed group is divided into high exposed group (>0.15 mg/m3) and low exposure group (<0.15 mg/m3). The individuals exposed to manganese dose of exposed group ((0.25±0.31) mg/m3) was higher than the control group ((0.06±0.02) mg/m3) (t=6.15, P=0.001); individuals exposed to manganese dose of high exposure group for (0.42±0.34) mg/m3, which was higher than low exposure group (0.09±0.07) mg/m3 (t=9.80, P=0.001). HSPs70 mRNA and protein of exposure group (5.65±0.21, 3.26±0.15) were higher than the reference group (0.41±0.03, 1.32±0.12) (t=18.91, t=8.68, P=0.001). HSP70 mRNA and protein of high exposure group (6.48±0.37, 3.67±0.26) were higher than the low exposure group (5.15±0.23, 3.02±0.19) (t=3.24, t=2.01, P=0.003, P=0.043). The expression of peripheral blood lymphocytes HSPs70 level and HSPs70 mRNA

  8. Antagonistic regulation of mRNA expression and splicing by CELF and MBNL proteins

    PubMed Central

    Wang, Eric T.; Ward, Amanda J.; Cherone, Jennifer M.; Giudice, Jimena; Wang, Thomas T.; Treacy, Daniel J.; Lambert, Nicole J.; Freese, Peter; Saxena, Tanvi; Cooper, Thomas A.; Burge, Christopher B.

    2015-01-01

    RNA binding proteins of the conserved CUGBP1, Elav-like factor (CELF) family contribute to heart and skeletal muscle development and are implicated in myotonic dystrophy (DM). To understand their genome-wide functions, we analyzed the transcriptome dynamics following induction of CELF1 or CELF2 in adult mouse heart and of CELF1 in muscle by RNA-seq, complemented by crosslinking/immunoprecipitation-sequencing (CLIP-seq) analysis of mouse cells and tissues to distinguish direct from indirect regulatory targets. We identified hundreds of mRNAs bound in their 3′ UTRs by both CELF1 and the developmentally induced MBNL1 protein, a threefold greater overlap in target messages than expected, including messages involved in development and cell differentiation. The extent of 3′ UTR binding by CELF1 and MBNL1 predicted the degree of mRNA repression or stabilization, respectively, following CELF1 induction. However, CELF1's RNA binding specificity in vitro was not detectably altered by coincubation with recombinant MBNL1. These findings support a model in which CELF and MBNL proteins bind independently to mRNAs but functionally compete to specify down-regulation or localization/stabilization, respectively, of hundreds of mRNA targets. Expression of many alternative 3′ UTR isoforms was altered following CELF1 induction, with 3′ UTR binding associated with down-regulation of isoforms and genes. The splicing of hundreds of alternative exons was oppositely regulated by these proteins, confirming an additional layer of regulatory antagonism previously observed in a handful of cases. The regulatory relationships between CELFs and MBNLs in control of both mRNA abundance and splicing appear to have evolved to enhance developmental transitions in major classes of heart and muscle genes. PMID:25883322

  9. Tobamovirus infection is independent of HSP101 mRNA induction and protein expression.

    PubMed

    Carr, Tyrell; Wang, Yongzeng; Huang, Zhonglian; Yeakley, Joanne M; Fan, Jian-Bing; Whitham, Steven A

    2006-10-01

    Heat shock protein 101 (HSP101) has been implicated in tobamovirus infections by virtue of its ability to enhance translation of mRNAs possessing the 5'Omega-leader of Tobacco mosaic virus (TMV). Enhanced translation is mediated by HSP101 binding to a CAA-repeat motif in TMV Omega leader. CAA repeat sequences are present in the 5' leaders of other tobamoviruses including Oilseed rape mosaic virus (ORMV), which infects Arabidopsis thaliana. HSP101 is one of eight HSP100 gene family members encoded by the A. thaliana genome, and of these, HSP101 and HSP98.7 are predicted to encode proteins localized to the cytoplasm where they could potentially interact with TMV RNA. Analysis of the expression of the HSP100s showed that only HSP101 mRNA transcripts were induced significantly by ORMV in A. thaliana. The induction of HSP101 mRNA was also correlated with an increase in its protein levels and was independent of defense-related signaling pathways involving salicylic acid, jasmonic acid, or ethylene. A. thaliana mutants lacking HSP101, HSP98.7, or both supported wild-type levels of ORMV replication and movement. Similar results were obtained for TMV infection in Nicotiana benthamiana plants silenced for HSP101, demonstrating that HSP101 is not necessary for efficient tobamovirus infection.

  10. On a stochastic gene expression with pre-mRNA, mRNA and protein contribution.

    PubMed

    Rudnicki, Ryszard; Tomski, Andrzej

    2015-12-21

    In this paper we develop a model of stochastic gene expression, which is an extension of the model investigated in the paper [T. Lipniacki, P. Paszek, A. Marciniak-Czochra, A.R. Brasier, M. Kimmel, Transcriptional stochasticity in gene expression, J. Theor. Biol. 238 (2006) 348-367]. In our model, stochastic effects still originate from random fluctuations in gene activity status, but we precede mRNA production by the formation of pre-mRNA, which enriches classical transcription phase. We obtain a stochastically regulated system of ordinary differential equations (ODEs) describing evolution of pre-mRNA, mRNA and protein levels. We perform mathematical analysis of a long-time behavior of this stochastic process, identified as a piece-wise deterministic Markov process (PDMP). We check exact results using numerical simulations for the distributions of all three types of particles. Moreover, we investigate the deterministic (adiabatic) limit state of the process, when depending on parameters it can exhibit two specific types of behavior: bistability and the existence of the limit cycle. The latter one is not present when only two kinds of gene expression products are considered.

  11. The Drosophila Tis11 Protein and Its Effects on mRNA Expression in Flies*

    PubMed Central

    Choi, Youn-Jeong; Lai, Wi S.; Fedic, Robert; Stumpo, Deborah J.; Huang, Weichun; Li, Leping; Perera, Lalith; Brewer, Brandy Y.; Wilson, Gerald M.; Mason, James M.; Blackshear, Perry J.

    2014-01-01

    Members of the mammalian tristetraprolin family of CCCH tandem zinc finger proteins can bind to certain AU-rich elements (AREs) in mRNAs, leading to their deadenylation and destabilization. Mammals express three or four members of this family, but Drosophila melanogaster and other insects appear to contain a single gene, Tis11. We found that recombinant Drosophila Tis11 protein could bind to ARE-containing RNA oligonucleotides with low nanomolar affinity. Remarkably, co-expression in mammalian cells with “target” RNAs demonstrated that Tis11 could promote destabilization of ARE-containing mRNAs and that this was partially dependent on a conserved C-terminal sequence resembling the mammalian NOT1 binding domain. Drosophila Tis11 promoted both deadenylation and decay of a target transcript in this heterologous cell system. We used chromosome deletion/duplication and P element insertion to produce two types of Tis11 deficiency in adult flies, both of which were viable and fertile. To address the hypothesis that Tis11 deficiency would lead to the abnormal accumulation of potential target transcripts, we analyzed gene expression in adult flies by deep mRNA sequencing. We identified 69 transcripts from 56 genes that were significantly up-regulated more than 1.5-fold in both types of Tis11-deficient flies. Ten of the up-regulated transcripts encoded probable proteases, but many other functional classes of proteins were represented. Many of the up-regulated transcripts contained potential binding sites for tristetraprolin family member proteins that were conserved in other Drosophila species. Tis11 is thus an ARE-binding, mRNA-destabilizing protein that may play a role in post-transcriptional gene expression in Drosophila and other insects. PMID:25342740

  12. Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis.

    PubMed

    Prabhu, Sridurga Mithra; Meistrich, Marvin L; McLaughlin, Eileen A; Roman, Shaun D; Warne, Sam; Mendis, Sirisha; Itman, Catherine; Loveland, Kate Lakoski

    2006-03-01

    Germ cell proliferation, migration and survival during all stages of spermatogenesis are affected by stem cell factor signalling through the c-Kit receptor, the expression and function of which are vital for normal male reproductive function. The present study comprehensively describes the c-Kit mRNA and protein cellular expression profiles in germ cells of the postnatal and adult rodent testis, revealing their significant elevation in synthesis at the onset of spermatogenesis. Real-time PCR analysis for both mice and rats matched the cellular mRNA expression profile where examined. Localization studies in normal mouse testes indicated that both c-Kit mRNA and protein are first detectable in differentiating spermatogonia. In addition, all spermatogonia isolated from 8-day-old mice displayed detectable c-Kit mRNA, but 30-50% of these lacked protein expression. The c-Kit mRNA and protein profile in normal rat testes indicated expression in gonocytes, in addition to differentiating spermatogonia. However, in the irradiated adult rat testes, in which undifferentiated spermatogonia are the only germ cell type, mRNA was also detected in the absence of protein. This persisted at 3 days and 1 and 2 weeks following treatment with gonadotrophin-releasing hormone (GnRH) antagonist to stimulate spermatogenesis recovery. By 4 weeks of GnRH antagonist treatment, accompanying the emergence of differentiating spermatogonia, both mRNA and protein were detected. Based on these observations, we propose that c-Kit mRNA and protein synthesis are regulated separately, possibly by influences linked to testis maturation and circulating hormone levels.

  13. Comprehensive expression analysis of FSHD candidate genes at the mRNA and protein level.

    PubMed

    Klooster, Rinse; Straasheijm, Kirsten; Shah, Bharati; Sowden, Janet; Frants, Rune; Thornton, Charles; Tawil, Rabi; van der Maarel, Silvère

    2009-12-01

    In facioscapulohumeral muscular dystrophy (FSHD) the majority of patients carry a D4Z4 macrosatellite repeat contraction in the subtelomere of chromosome 4q. Several disease mechanisms have been proposed to explain how repeat contraction causes muscular dystrophy. All proposed mechanisms foresee a change from a closed to a more open chromatin structure followed by loss of control over expression of genes in or proximal to D4Z4. Initially, a distance and residual repeat size-dependent upregulation of the candidate genes FRG2, FRG1 and ANT1 was observed, but most successive expression studies failed to support transcriptional upregulation of 4qter genes. Moreover, chromatin studies do not provide evidence for a cis-spreading mechanism operating at 4qter in FSHD. In part, this inconsistency may be explained by differences in the techniques used, and the use of RNA samples obtained from different muscle groups. The aim of this study is to comprehensively and uniformly study the expression of the FSHD candidate genes FRG1, FRG2, CRYM, ANT1, ALP, PITX1 and LRP2BP at the RNA and protein level in identically processed primary myoblasts, myotubes and quadriceps muscle. Expression was compared between samples obtained from FSHD patients and normal controls with samples from myotonic dystrophy type 1 patients as disease controls. No consistent changes in RNA or protein expression levels were observed between the samples. The one exception was a selective increase in FRG2 mRNA expression in FSHD myotubes. This study provides further evidence that there is no demonstrable consistent, large magnitude, overexpression of any of the FSHD candidate genes.

  14. Expression and cellular localization of hepcidin mRNA and protein in normal rat brain.

    PubMed

    Raha-Chowdhury, Ruma; Raha, Animesh Alexander; Forostyak, Serhiy; Zhao, Jing-Wei; Stott, Simon Russell William; Bomford, Adrian

    2015-04-21

    Hepcidin is a peptide hormone belonging to the defensin family of cationic antimicrobial molecules that has an essential role in systemic iron homeostasis. The peptide is synthesised by hepatocytes and transported in the circulation to target tissues where it regulates the iron export function of the ferrous iron permease, ferroportin. In the brain hepcidin protein has been identified using immuno-histochemistry and mRNA by real-time PCR but not by in situ hybridisation raising the question of whether there is measurable transcription of the hepcidin gene in the central nervous system. Alternatively hepcidin could be transported as a hormone to the brain via the circulation. By RT-PCR hepcidin mRNA was present at low level throughout normal rat brain while in situ hybridisation to detect low-abundant mRNA revealed that transcripts were restricted to endothelium of blood vessels and choroid plexus. In contrast, hepcidin protein analysed by immuno-histochemistry was highly expressed in blood vessels, in endothelium and in pericytes. Hepcidin was also present in glial cells and in the olfactory bulb, sub-ventricular zone and dentate gyrus, areas where neurogenesis and synaptic plasticity are maintained throughout adult life. The hepcidin species identified by Western blotting in sub-ventricular zone, cortex and hippocampus migrated as a ~2.8 kDa band, identical in size to hepcidin present in normal rat serum suggesting that hepcidin in brain was the full-length biologically active 25 amino acid peptide. Hepcidin co-localised with ferroportin in ependymal cells of the sub-ventricular zone and in the corpus callosum consistent with a regulatory role in iron metabolism at these sites. Hepcidin protein was widely expressed in brain parenchyma while levels of hepcidin gene transcription appeared to be below the limits of detection of the in situ hybridisation probes. This disparity suggests that not all hepcidin in the brain is transcribed in situ and may originate in part

  15. Cloning and mRNA expression of the Ca2+-binding DREAM protein in the pituitary.

    PubMed

    Leclerc, Gilles M; Leclerc, Guy J; Shorte, Spencer L; Stephen Frawley, L; Boockfor, Fredric R

    2002-10-15

    It is well recognized that the level of intracellular calcium governs several cellular processes such as gene expression and secretion in the pituitary. Recently, a novel gene has been identified in neuroendocrine cells that encodes DREAM, a calcium-binding protein that acts as a transcriptional repressor by binding specific downstream regulatory elements (DRE) on DNA. To explore the possibility that DREAM may be expressed in the rat pituitary and may function in endocrine activity, we analyzed its mRNA expression by RT-PCR. Using oligonucleotide primers derived from the mouse DREAM cDNA, we amplified, cloned, and characterized a 852-bp RT-PCR product from rat pituitary tissue. Two splice variants of the rat DREAM gene differing by four nucleotides (tetramer ACAG) were identified. The ACAG(+) variant (ORF1) consisted of 768bp encoding a protein of 256 residues with an estimated molecular weight of 29.5kDa. Amino acid sequence analysis of ORF1 indicated 92.6% and 98.1% identity to the DREAM gene product from human and mouse, respectively. The second variant, ACAG(-) (ORF2), was 567-bp long and was predicted to encode a peptide of 189 residues with a molecular mass of about 20.8kDa. To determine which endocrine pituitary cells were expressing DREAM, we evaluated several different clonal populations containing cells that expressed specific pituitary hormones. We found that both DREAM splice variants were expressed in each pituitary cell types examined, which included the mammotropes (MMQ cells), somatotropes (GC cells), mammosomatotropes (GH(3) cells), gonadotropes (LbetaT2 cells), thyrotropes (TalphaT1 cells), and corticotropes (AtT-20 cells). Interestingly, the levels of the two variants differed between the cell types tested with the ACAG(+) variant comprising about two-thirds of the DREAM expression for the mammotropes, somatotropes, mammosomatotropes, and corticotropes as compared to less than one-half for the thyrotropes and the gonadotropes. Our initial

  16. Expression of klotho mRNA and protein in rat brain parenchyma from early postnatal development into adulthood.

    PubMed

    Clinton, Sarah M; Glover, Matthew E; Maltare, Astha; Laszczyk, Ann M; Mehi, Stephen J; Simmons, Rebecca K; King, Gwendalyn D

    2013-08-21

    Without the age-regulating protein klotho, mouse lifespan is shortened and the rapid onset of age-related disorders occurs. Conversely, overexpression of klotho extends mouse lifespan. Klotho is most abundant in kidney and expressed in a limited number of other organs, including the brain, where klotho levels are highest in choroid plexus. Reports vary on where klotho is expressed within the brain parenchyma, and no data is available as to whether klotho levels change across postnatal development. We used in situ hybridization to map klotho mRNA expression in the developing and adult rat brain and report moderate, widespread expression across grey matter regions. mRNA expression levels in cortex, hippocampus, caudate putamen, and amygdala decreased during the second week of life and then gradually rose to adult levels by postnatal day 21. Immunohistochemistry revealed a protein expression pattern similar to the mRNA results, with klotho protein expressed widely throughout the brain. Klotho protein co-localized with both the neuronal marker NeuN, as well as, oligodendrocyte marker olig2. These results provide the first anatomical localization of klotho mRNA and protein in rat brain parenchyma and demonstrate that klotho levels vary during early postnatal development.

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

    SciTech Connect

    Higgins, G.A.; Lewis, D.A.; Bahmanyar, S.; Goldgaber, D.; Gajdusek, D.C.; Young, W.G.; Morrison, J.H.; Wilson, M.C.

    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 mRNA in the cornu Ammonis field 3 and parasubiculum are equivalent. These findings suggest that within certain neuronal subpopulations cell type-specific regulation of amyloid-..beta..-protein gene expression may be altered in Alzheimer disease.

  18. Changes in glial fibrillary acidic protein mRNA expression after corticospinal axotomy in the adult hamster.

    PubMed

    Kost-Mikucki, S A; Oblinger, M M

    1991-02-01

    We examined changes in the expression of glial fibrillary acidic protein (GFAP) mRNA during Wallerian degeneration in the corticospinal system of the adult Golden hamster following axotomy. GFAP is the product of a type III intermediate filament (IF) gene that is expressed specifically in mature astrocytes. A well-studied component of a complex response termed reactive astrogliosis that occurs after various types of CNS injury is the increased production of astrocytic processes filled with GFAP-containing IFs. While increased expression of GFAP during reactive astrogliosis has been well established at the protein level, little is known about whether or not changes in GFAP mRNA levels occur after CNS injury. In the present study we used in situ hybridization methods to examine this issue. A 35S-labeled mouse GFAP cDNA probe was used for in situ hybridizations of sections of the brain stem obtained 2, 7, and 14 days after unilateral transections of the corticospinal tract in the caudal medulla. Film as well as emulsion autoradiography showed a dramatic increase in GFAP mRNA labeling associated with the degenerating corticospinal tract. GFAP mRNA levels were already dramatically increased in the injured corticospinal tract by 2 days post axotomy and remained elevated at 14 days. Interestingly, in addition to the robust increase in GFAP mRNA levels specifically associated with the degenerating tract, a diffuse increase in GFAP mRNA labeling was observed throughout the grey matter of the brain stem at 2 days post-axotomy, but not after this time. Immunoblotting and immunocytochemical experiments verified that the increased GFAP mRNA levels in the degenerating corticospinal system were accompanied by an increased expression of the protein. These results demonstrate that an increase in GFAP mRNA levels occurs during Wallerian degeneration in the CNS and suggest that increased expression of the GFAP gene is a major contributor to CNS scarring that results after direct

  19. Expression of AEG-1 mRNA and protein in colorectal cancer patients and colon cancer cell lines.

    PubMed

    Gnosa, Sebastian; Shen, Yang-Mei; Wang, Chao-Jie; Zhang, Hong; Stratmann, Johannes; Arbman, Gunnar; Sun, Xiao-Feng

    2012-05-29

    Astrocyte elevated gene 1 (AEG-1), an important oncogene, has been shown to be overexpressed in several types of cancers. In colorectal cancer (CRC), the protein level of AEG-1 is up-regulated in tumour tissue compared to normal mucosa, showing prognostic significance. Since little is known about the transcriptional level of AEG-1 expression and its biological pathway in CRC the aim of the present study was to examine the relationship of AEG-1 mRNA expression, the protein level and clinicopathological variables as well as its biology pathway in CRC. The mRNA expression of AEG-1 was analysed by qPCR in fresh frozen patient samples including 156 primary tumours, along with the corresponding normal mucosa, and in five colon cancer cell lines, SW480, SW620, KM12C, KM12SM and KM12L4a. AEG-1 protein expression was investigated by immunohistochemistry in paraffin-embedded materials from 74 distant normal mucosa, 107 adjacent mucosa, 158 primary tumour, 35 lymph node metastasis and 9 liver metastasis samples. In addition, the AEG-1 protein expression was elucidated in the cell lines by Western blot. The lymph node metastatic cell line SW620 had a significantly higher AEG-1 mRNA (0.27 ± 0.02) expression compared to the primary tumour cell line SW480 (0.17 ± 0.04, p = 0.026). AEG-1 expression at the mRNA level and/or the protein level was significantly up-regulated gradually from normal mucosa to primary CRC, and then to lymph node metastasis and finally to liver metastasis (p < 0.05). There were significant associations of AEG-1 mRNA expression with tumour location (p = 0.047), as well as mRNA and protein expression with the tumour stage (p < 0.03). Furthermore AEG-1 protein expression was positively related to biological variables including NF-κB, p73, Rad50 and apoptosis (p < 0.05). AEG-1 is up-regulated, at the mRNA and the protein level, during CRC development and aggressiveness, and is related to tumour location and stage. It may play its

  20. Increase in uncoupling protein-2 mRNA expression by BRL49653 and bromopalmitate in human adipocytes.

    PubMed

    Viguerie-Bascands, N; Saulnier-Blache, J S; Dandine, M; Dauzats, M; Daviaud, D; Langin, D

    1999-03-05

    Uncoupling protein-2 (UCP2) is a novel mitochondrial protein that may be involved in the control of energy expenditure. We have previously reported an upregulation of adipose tissue UCP2 mRNA expression during fasting in humans. Analysis of changes in metabolic parameters suggested that fatty acids may be associated with the increased UCP2 mRNA level. Culture of human adipose tissue explants was used to study in vitro regulation of adipocyte UCP2 gene expression. A 48-h treatment with BRL49653 and bromopalmitate, two potent activators of PPARgamma, resulted in a dose-dependent increase in UCP2 mRNA levels. The induction by BRL49653 was rapid (from 6 h) and maintained up to 5 days. TNFalpha provoked a 2-fold decrease in UCP2 mRNA levels. Human recombinant leptin did not affect UCP2 mRNA expression. The data support the hypothesis that fatty acids are involved in the control of adipocyte UCP2 mRNA expression in humans. Copyright 1999 Academic Press.

  1. Perinuclear Mlp proteins downregulate gene expression in response to a defect in mRNA export.

    PubMed

    Vinciguerra, Patrizia; Iglesias, Nahid; Camblong, Jurgi; Zenklusen, Daniel; Stutz, Françoise

    2005-02-23

    The mRNA export adaptor Yra1p/REF contributes to nascent mRNP assembly and recruitment of the export receptor Mex67p. yra1 mutants exhibit mRNA export defects and a decrease in LacZ reporter and certain endogenous transcripts. The loss of Mlp1p/Mlp2p, two TPR-like proteins attached to nuclear pores, rescues LacZ mRNA levels and increases their appearance in the cytoplasm, without restoring bulk poly(A)+ RNA export. Chromatin immunoprecipitation, FISH and pulse-chase experiments indicate that Mlps downregulate LacZ mRNA synthesis in a yra1 mutant strain. Microarray analyses reveal that Mlp2p also reduces a subset of cellular transcripts in the yra1 mutant. Finally, we show that Yra1p genetically interacts with the shuttling mRNA-binding protein Nab2p and that loss of Mlps rescues the growth defect of yra1 and nab2 but not other mRNA export mutants. We propose that Nab2p and Yra1p are required for proper mRNP docking to the Mlp platform. Defects in Yra1p prevent mRNPs from crossing the Mlp gate and this block negatively feeds back on the transcription of a subset of genes, suggesting that Mlps link mRNA transcription and export.

  2. Breast Cancer Resistance Protein Abundance, but Not mRNA Expression, Correlates With Estrone-3-Sulfate Transport in Caco-2.

    PubMed

    Harwood, Matthew D; Neuhoff, Sibylle; Rostami-Hodjegan, Amin; Warhurst, Geoffrey

    2016-04-01

    Transporter mRNA and protein expression data are used to extrapolate in vitro transporter kinetics to in vivo drug disposition predictions. Breast cancer resistance protein (BCRP) possesses broad substrate specificity; therefore, understanding BCRP expression-activity relationships are necessary for the translation to in vivo. Bidirectional transport of estrone-3-sulfate (E-3-S), a BCRP probe, was evaluated with respect to relative BCRP mRNA expression and absolute protein abundance in 10- and 29-day cultured Caco-2 cells. BCRP mRNA expression was quantified by real-time PCR against a housekeeper gene, Cyclophilin A. The BCRP protein abundance in total membrane fractions was quantified by targeted proteomics, and [(3)H]-E-3-S bidirectional transport was determined in the presence or absence of Ko143, a potent BCRP inhibitor. BCRP mRNA expression was 1.5-fold higher in 29- versus 10-day cultured cells (n = 3), whereas a 2.4-fold lower (p < 0.001) BCRP protein abundance was observed in 29- versus 10-day cultured cells (1.28 ± 0.33 and 3.06 ± 0.22 fmol/μg protein, n = 6, respectively). This correlated to a 2.45-fold lower (p < 0.01) efflux ratio for E-3-S in 29- versus 10-day cultured cells (8.97 ± 2.51 and 3.32 ± 0.66, n = 6, respectively). Caco-2 cell BCRP protein abundance, but not mRNA levels, correlates with BCRP activity, suggesting that extrapolation strategies incorporating BCRP protein abundance-activity relationships may be more successful.

  3. Pattern of expression of transforming growth factor-beta 4 mRNA and protein in the developing chicken embryo.

    PubMed

    Jakowlew, S B; Ciment, G; Tuan, R S; Sporn, M B; Roberts, A B

    1992-12-01

    Expression of TGF-beta 4 mRNA and protein was studied in the developing chicken embryo using specific cDNA probes and antibodies for chicken TGF-beta 4. Expression of TGF-beta 4 mRNA was detected by day 4 of incubation (Hamburger and Hamilton stage 22, E4) by RNA Northern blot analysis and increased with developmental age until day 12 of incubation (stage 38, E12) where it was detected in every embryonic tissue examined, with expression being highest in smooth muscle and lowest in the kidney. The steady-state level of expression of TGF-beta 4 mRNA remained relatively constant in most embryonic tissues through day 19 (stage 45, E19). In situ hybridization analysis detected TGF-beta 4 mRNA as early as the "definitive primitive streak" stage (stage 4); during neurulation (stage 10), TGF-beta 4 mRNA was detected in all three germ layers, including neuroectoderm. Following neurulation, TGF-beta 4 mRNA was detected in the neural tube, notochord, ectoderm, endoderm, sclerotome, and myotome, but not dermotome at stage 16. By day 6 of incubation (stage 29, E6), TGF-beta 4 mRNA was localized in several tissues including heart, lung, and gizzard. Immunohistochemical staining analysis also showed expression of TGF-beta 4 protein in all three germ layers as early as stage 4 in various cell types in qualitatively similar locations as TGF-beta 4 mRNA. These results suggest that TGF-beta 4 may play an important role in the development of many tissues in the chicken.

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

    PubMed Central

    Szybka, Malgorzata; Malachowska, Beata; Fendler, Wojciech; Potemski, Piotr; Piaskowski, Sylwester; Jaskolski, Dariusz; Papierz, Wielislaw; Skowronski, Wieslaw; Och, Waldemar; Kordek, Radzislaw

    2014-01-01

    Reduced expression of TP53 by promoter methylation has been reported in several neoplasms. It remains unclear whether TP53 promoter methylation is associated with reduced transcriptional and protein expression in glioblastoma (GB). The aim of our work was to study the impact of TP53 methylation and mutations on TP53 mRNA level and protein expression in 42 molecularly characterized primary GB tumors. We also evaluate the impact of all molecular alterations on the overall patient survival. The frequency of TP53 promoter methylation was found in 21.4%. To the best of our knowledge, this is the first report showing such high frequency of TP53 promoter methylation in primary GB. There was no relation between TP53 promoter methylation and TP53 mRNA level (p=0.5722) and between TP53 promoter methylation and TP53 protein expression (p=0.2045). No significant associations were found between TP53 mRNA expression and mutation of TP53 gene (p=0.9076). However, significant association between TP53 mutation and TP53 protein expression was found (p=0.0016). Our data suggest that in primary GB TP53 promoter methylation does not play a role in silencing of TP53 transcriptional and protein expression and is probably regulated by other genetic and epigenetic mechanisms associated with genes involved in the TP53 pathway. PMID:24506545

  5. Expression and localization of the cystic fibrosis transmembrane conductance regulator mRNA and its protein in rat brain.

    PubMed

    Mulberg, A E; Resta, L P; Wiedner, E B; Altschuler, S M; Jefferson, D M; Broussard, D L

    1995-07-01

    In previous studies we have characterized the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in clathrin-coated vesicles derived from bovine brain and in neurons of rat brain. In this study we have further characterized the expression of the CFTR protein mRNA and protein in rat brain with reverse transcriptase polymerase chain reaction amplification (RT-PCR), in situ hybridization, and immunocytochemistry. The expression of CFTR mRNA and protein in discrete areas of brain, including the hypothalamus, thalamus, and amygdaloid nuclei, which are involved in regulation of appetite and resting energy expenditure, is identical. The presence of CFTR in neurons localized to these regions of brain controlling homeostasis and energy expenditure may elucidate the pathogenesis of other nonpulmonary and gastrointestinal manifestations which commonly are observed in children with cystic fibrosis. Dysregulation of normal neuropeptide vesicle trafficking by mutant CFTR in brain may serve as a pathogenic mechanism for disruption of homeostasis.

  6. Expression and localization of the cystic fibrosis transmembrane conductance regulator mRNA and its protein in rat brain.

    PubMed Central

    Mulberg, A E; Resta, L P; Wiedner, E B; Altschuler, S M; Jefferson, D M; Broussard, D L

    1995-01-01

    In previous studies we have characterized the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in clathrin-coated vesicles derived from bovine brain and in neurons of rat brain. In this study we have further characterized the expression of the CFTR protein mRNA and protein in rat brain with reverse transcriptase polymerase chain reaction amplification (RT-PCR), in situ hybridization, and immunocytochemistry. The expression of CFTR mRNA and protein in discrete areas of brain, including the hypothalamus, thalamus, and amygdaloid nuclei, which are involved in regulation of appetite and resting energy expenditure, is identical. The presence of CFTR in neurons localized to these regions of brain controlling homeostasis and energy expenditure may elucidate the pathogenesis of other nonpulmonary and gastrointestinal manifestations which commonly are observed in children with cystic fibrosis. Dysregulation of normal neuropeptide vesicle trafficking by mutant CFTR in brain may serve as a pathogenic mechanism for disruption of homeostasis. Images PMID:7542288

  7. Specific responses in rat small intestinal epithelial mRNA expression and protein levels during chemotherapeutic damage and regeneration.

    PubMed

    Verburg, Melissa; Renes, Ingrid B; Van Nispen, Danielle J P M; Ferdinandusse, Sacha; Jorritsma, Marieke; Büller, Hans A; Einerhand, Alexandra W C; Dekker, Jan

    2002-11-01

    The rapidly dividing small intestinal epithelium is very sensitive to the cytostatic drug methotrexate. We investigated the regulation of epithelial gene expression in rat jejunum during methotrexate-induced damage and regeneration. Ten differentiation markers were localized on tissue sections and quantified at mRNA and protein levels relative to control levels. We analyzed correlations in temporal expression patterns between markers. mRNA expression of enterocyte and goblet cell markers decreased significantly during damage for a specific period. Of these, sucrase-isomaltase (-62%) and CPS (-82%) were correlated. Correlations were also found between lactase (-76%) and SGLT1 (-77%) and between I-FABP (-52%) and L-FABP (-45%). Decreases in GLUT5 (-53%), MUC2 (-43%), and TFF3 (-54%) mRNAs occurred independently of any of the other markers. In contrast, lysozyme mRNA present in Paneth cells increased (+76%). At the protein level, qualitative and quantitative changes were in agreement with mRNA expression, except for Muc2 (+115%) and TFF3 (+81%), which increased significantly during damage, following independent patterns. During regeneration, expression of each marker returned to control levels. The enhanced expression of cytoprotective molecules (Muc2, TFF3, lysozyme) during damage represents maintenance of goblet cell and Paneth cell functions, most likely to protect the epithelium. Decreased expression of enterocyte-specific markers represents decreased enterocyte function, of which fatty acid transporters were least affected.

  8. Heat stress stimulates hepcidin mRNA expression and C/EBPα protein expression in aged rodent liver.

    PubMed

    Bloomer, Steven A; Kregel, Kevin C; Brown, Kyle E

    2014-01-01

    Elevations in hepatic iron content occur with aging and physiological stressors, which may promote oxidative injury to the liver. Since dysregulation of the iron regulatory hormone, hepcidin, can cause iron accumulation, our goal was to characterize the regulation of hepcidin in young (6 mo) and old (24 mo) Fischer 344 rats exposed to environmental heat stress. Liver and blood samples were taken in the control condition and after heating. Hepcidin expression did not differ between young and old rats in the control condition, despite higher levels of hepatic iron and IL-6 mRNA in the latter. Following heat stress, pSTAT3 increased in both groups, but C/EBPα and hepcidin mRNA increased only in old rats. Despite this, serum iron decreased in both age groups 2 h after heat stress, suggesting hepcidin-independent hypoferremia in the young rats. The differential regulation of hepcidin between young and old rats after hyperthermia may be due to the enhanced expression of C/EBPα protein in old rats. These data support the concept of "inflammaging" and suggest that repeated exposures to stressors may contribute to the development of anemia in older individuals.

  9. Expression of statherin mRNA and protein in nasal and vaginal secretions.

    PubMed

    Sakurada, Koichi; Akutsu, Tomoko; Watanabe, Ken; Fujinami, Yoshihito; Yoshino, Mineo

    2011-11-01

    Nasal secretion has been regarded as one of the most difficult body fluids to identify and is especially difficult to discriminate from vaginal secretions and saliva. At present, few specific markers are known for nasal secretions. The aim of this study is to find a new approach for the identification of nasal secretions. We examined expression levels of statherin and histatin, peptides which are commonly found in saliva, in nasal and vaginal secretions by real-time RT-PCR and ELISA assays. Statherin mRNA was highly expressed in all nasal samples (dCt value=-1.49±1.10, n=8) and was detected even in 1-day-old 0.1-μL stains. However, the stability of mRNA in nasal stains was significantly (P<0.01) lower than in saliva. Low levels of statherin mRNA were detected in 4 of the 17 vaginal samples (dCt value=11.65-14.72). Histatin mRNA was not detected in any nasal or vaginal samples, although it was highly expressed in all saliva samples. ELISA assays with anti-statherin goat polyclonal antibody showed that statherin peptide was detected in all nasal and saliva samples even after dilution of more than 1000-fold. The statherin peptide was not detected in any vaginal samples, including samples that expressed low levels of statherin mRNA. The amount of statherin peptide in vaginal samples might be less than the limit of detection of this assay. In the present study, statherin was highly expressed in nasal secretions, but histatin was not. These markers may be useful for discriminating nasal secretions from vaginal secretions and saliva. However, the usefulness of these markers in practical forensic case samples has not yet been examined. Therefore, further research is required to establish the utility of these assays for identification of nasal secretions.

  10. Arsenic Induces Polyadenylation of Canonical Histone mRNA by Down-regulating Stem-Loop-binding Protein Gene Expression*

    PubMed Central

    Brocato, Jason; Fang, Lei; Chervona, Yana; Chen, Danqi; Kiok, Kathrin; Sun, Hong; Tseng, Hsiang-Chi; Xu, Dazhong; Shamy, Magdy; Jin, Chunyuan; Costa, Max

    2014-01-01

    The replication-dependent histone genes are the only metazoan genes whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3′-end. Instead, the histone mRNAs display a stem-loop structure at their 3′-end. Stem-loop-binding protein (SLBP) binds the stem-loop and regulates canonical histone mRNA metabolism. Here we report that exposure to arsenic, a carcinogenic metal, decreased cellular levels of SLBP by inducing its proteasomal degradation and inhibiting SLBP transcription via epigenetic mechanisms. Notably, arsenic exposure dramatically increased polyadenylation of canonical histone H3.1 mRNA possibly through down-regulation of SLBP expression. The polyadenylated H3.1 mRNA induced by arsenic was not susceptible to normal degradation that occurs at the end of S phase, resulting in continued presence into mitosis, increased total H3.1 mRNA, and increased H3 protein levels. Excess expression of canonical histones have been shown to increase sensitivity to DNA damage as well as increase the frequency of missing chromosomes and induce genomic instability. Thus, polyadenylation of canonical histone mRNA following arsenic exposure may contribute to arsenic-induced carcinogenesis. PMID:25266719

  11. Expression of fragile X mental retardation protein and Fmr1 mRNA during folliculogenesis in the rat.

    PubMed

    Ferder, Ianina; Parborell, Fernanda; Sundblad, Victoria; Chiauzzi, Violeta; Gómez, Karina; Charreau, Eduardo H; Tesone, Marta; Dain, Liliana

    2013-04-01

    Fragile X mental retardation protein (FMRP) belongs to a small family of RNA-binding proteins. Its absence or inactivity is responsible for fragile X syndrome, the most common cause of inherited mental retardation. Despite its ubiquitous expression, FMRP function and expression remain almost understudied in non-neuronal tissues, though previous studies on germline development during oogenesis may suggest a special function of this protein also in ovarian tissue. In addition, the well-documented association of FMR1 premutation state with fragile X-related premature ovarian insufficiency adds interest to the role of FMRP in ovarian physiology. The aim of the present work was to investigate the expression of Fmr1 mRNA and its protein, FMRP, at different stages of rat follicular development. By immunohistochemical studies we demonstrated FMRP expression in granulosa, theca and germ cells in all stages of follicular development. In addition, changes in Fmr1 expression, both at the protein and mRNA levels, were observed. FMRP levels increased upon follicular development while preantral and early antral follicles presented similar levels of Fmr1 transcripts with decreased expression in preovulatory follicles. These observations suggest that Fmr1 expression in the ovary is regulated at different and perhaps independent levels. In addition, our results show expression of at least four different isoforms of FMRP during all stages of follicular growth with expression patterns that differ from those observed in brain and testis. Our study shows a regulated expression of Fmr1, both at mRNA and protein levels, during rat follicular development.

  12. The Andes hantavirus NSs protein is expressed from the viral small mRNA by a leaky scanning mechanism.

    PubMed

    Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P; Pino, Karla; Tischler, Nicole D; Ohlmann, Théophile; Darlix, Jean-Luc; López-Lastra, Marcelo

    2012-02-01

    The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism.

  13. The Andes Hantavirus NSs Protein Is Expressed from the Viral Small mRNA by a Leaky Scanning Mechanism

    PubMed Central

    Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P.; Pino, Karla; Tischler, Nicole D.; Ohlmann, Théophile; Darlix, Jean-Luc

    2012-01-01

    The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism. PMID:22156529

  14. Pyruvate dehydrogenase complex: mRNA and protein expression patterns of E1α subunit genes in human spermatogenesis.

    PubMed

    Pinheiro, Ana; Silva, Maria João; Graça, Inês; Silva, Joaquina; Sá, Rosália; Sousa, Mário; Barros, Alberto; Tavares de Almeida, Isabel; Rivera, Isabel

    2012-09-10

    During spermatogenesis, germ cells undergo a complex process of cell differentiation and morphological restructuring, which depends on the coordinated expression of different genes. Some vital examples are those involved in cell energy metabolism, namely the genes encoding the E1α subunit of pyruvate dehydrogenase complex: the somatic PDHA1 (X-linked) and the testis-specific PDHA2 (autosomal). There are no data related to the study at the RNA and protein levels of PDHA genes during human spermatogenesis. The present study aimed to describe the mRNA and protein expression patterns of the human PDHA genes during spermatogenesis. Expression profiles of the PDHA1 and PDHA2 genes were characterized using different human tissues and cells. Diploid and haploid germ cells fractions were obtained from testis tissues. The mRNA profiles were analyzed by quantitative RT-PCR, whereas the protein profiles were evaluated by immunohistochemistry, western blotting and two-dimensional electrophoresis. Expression of the PDHA1 gene was found in all somatic cells, whereas expression of PDHA2 gene was restricted to germ cells. The switch from X-linked to autosomic gene expression occurred in spermatocytes. Data suggest the activation of PDHA2 gene expression is most probably a mechanism to ensure the continued expression of the protein, thus allowing germ cell viability and functionality.

  15. mRNA and protein expression levels of four candidate genes for ear size in Erhualian and Large White pigs.

    PubMed

    Zhang, L C; Liang, J; Pu, L; Zhang, Y B; Wang, L G; Liu, X; Yan, H; Wang, L X

    2017-04-13

    Porcine ear size is an important characteristic for distinguishing among pig breeds. In a previous genome-wide association study of porcine ear size, LEM domain-containing 3 (LEMD3), methionine sulfoxide reductase B3 (MSRB3), high mobility group AT-hook 2 (HMGA2), and Wnt inhibitory factor 1 (WIF1) were implicated as important candidate genes for ear size. This study investigated the expression levels of four candidate genes for ear size in Erhualian and Large White pigs. Ten Erhualian pigs with large ears and eight Large White pigs with small ears at 60 days of age were examined. The mRNA expression levels of the four candidate genes were quantified by real-time polymerase chain reaction. WIF1 mRNA expression was significantly higher in Large White than in Erhualian pigs (P < 0.05), whereas the expression levels of the other three genes were not significantly different between the two breeds. The protein expression levels of the four genes were analyzed using western blot. WIF1 protein expression was significantly higher in Large White than in Erhualian pigs (P < 0.01), whereas MSRB3 protein expression was significantly higher in Erhualian than in Large White pigs (P < 0.05). There were no significant differences between the two breeds in residual protein expression. These results suggest that WIF1 is the main causal gene for ear size in pigs.

  16. Estrogen and exercise interact to regulate brain-derived neurotrophic factor mRNA and protein expression in the hippocampus.

    PubMed

    Berchtold, N C; Kesslak, J P; Pike, C J; Adlard, P A; Cotman, C W

    2001-12-01

    We investigated the possibility that estrogen and exercise interact in the hippocampus and regulate brain-derived neurotrophic factor (BDNF), a molecule increasingly recognized for its role in plasticity and neuron function. An important aspect of this study is to examine the effect of different time intervals between estrogen loss and estrogen replacement intervention. We demonstrate that in the intact female rat, physical activity increases hippocampal BDNF mRNA and protein levels. However, the exercise effect on BDNF up-regulation is reduced in the absence of estrogen, in a time-dependent manner. In addition, voluntary activity itself is stimulated by the presence of estrogen. In exercising animals, estrogen deprivation reduced voluntary activity levels, while estrogen replacement restored activity to normal levels. In sedentary animals, estrogen deprivation (ovariectomy) decreased baseline BDNF mRNA and protein, which were restored by estrogen replacement. Despite reduced activity levels in the ovariectomized condition, exercise increased BDNF mRNA levels in the hippocampus after short-term (3 weeks) estrogen deprivation. However, long-term estrogen-deprivation blunted the exercise effect. After 7 weeks of estrogen deprivation, exercise alone no longer affected either BDNF mRNA or protein levels. However, exercise in combination with long-term estrogen replacement increased BDNF protein above the effects of estrogen replacement alone. Interestingly, protein levels across all conditions correlated most closely with mRNA levels in the dentate gyrus, suggesting that expression of mRNA in this hippocampal region may be the major contributor to the hippocampal BDNF protein pool. The interaction of estrogen, physical activity and hippocampal BDNF is likely to be an important issue for maintenance of brain health, plasticity and general well-being, particularly in women.

  17. Differential expression of equine myosin heavy-chain mRNA and protein isoforms in a limb muscle.

    PubMed

    Eizema, Karin; van den Burg, Maarten; Kiri, Arpna; Dingboom, Elizabeth G; van Oudheusden, Hans; Goldspink, Geoffrey; Weijs, Wim A

    2003-09-01

    The horse is one of the few animals kept and bred for its athletic performance and is therefore an interesting model for human sports performance. The regulation of the development of equine locomotion in the first year of life, and the influence of early training on later performance, are largely unknown. The major structural protein in skeletal muscle, myosin heavy-chain (MyHC), is believed to be primarily transcriptionally controlled. To investigate the expression of the MyHC genes at the transcriptional level, we isolated cDNAs encoding the equine MyHC isoforms type 1 (slow), type 2a (fast oxidative), and type 2d/x (fast glycolytic). cDNAs encoding the 2b gene were not identified. The mRNA expression was compared to the protein expression on a fiber-to-fiber basis using in situ hybridization (non-radioactive) and immunohistochemistry. Marked differences were detected between the expression of MyHC transcripts and MyHC protein isoforms in adult equine gluteus medius muscle. Mismatches were primarily due to the presence of hybrid fibers expressing two fast (2ad) MyHC protein isoforms, but only one fast (mainly 2a) MyHC RNA isoform. This discrepancy was most likely not due to differential mRNA expression of myonuclei.

  18. Protein synthesis inhibitors enhance the expression of mRNAs for early inducible inflammatory genes via mRNA stabilization.

    PubMed

    Yamazaki, Soh; Takeshige, Koichiro

    2008-02-01

    Expression of inflammatory genes is regulated at multiple steps, including transcriptional activation and mRNA stabilization. During an investigation into the requirement of de novo protein synthesis for the induction of inflammatory genes, it was revealed that protein synthesis inhibitors unexpectedly potentiated the induction of mRNAs for primary response genes, while the inhibitors suppressed the induction of secondary inducible genes as previously described. Stimulus-induced nuclear translocation and promoter recruitment of NF-kappaB, which is responsible for the transcriptional activation of many inflammatory genes, were largely unaffected by the inhibitors. Instead, these inhibitors prolonged the half-lives of all of the primary inducible mRNAs tested. Thus, these findings emphasize the important contribution of regulated mRNA longevity to gene expression induced by pro-inflammatory stimulation.

  19. Abnormal Glucocorticoid Receptor mRNA and Protein Isoform Expression in the Prefrontal Cortex in Psychiatric Illness

    PubMed Central

    Sinclair, Duncan; Tsai, Shan Yuan; Woon, Heng Giap; Weickert, Cynthia Shannon

    2011-01-01

    Stress has been implicated in the onset and illness course of schizophrenia and bipolar disorder. The effects of stress in these disorders may be mediated by abnormalities of the hypothalamic–pituitary–adrenal axis, and its corticosteroid receptors. We investigated mRNA expression of the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), and protein expression of multiple GRα isoforms, in the prefrontal cortex of 37 schizophrenia cases and 37 matched controls. Quantitative real-time PCR, western blotting, and luciferase assays were employed. In multiple regression analysis, schizophrenia diagnosis was a significant predictor of total GR mRNA expression (p<0.05), which was decreased (11.4%) in schizophrenia cases relative to controls. No significant effect of diagnosis on MR mRNA was detected. At the protein level, no significant predictors of total GRα protein or the full-length GRα isoform were identified. However, schizophrenia diagnosis was a strong predictor (p<0.0005) of the abundance of a truncated ∼50 kDa GRα protein isoform, putative GRα-D1, which was increased in schizophrenia cases (80.4%) relative to controls. This finding was replicated in a second cohort of 35 schizophrenia cases, 34 bipolar disorder cases, and 35 controls, in which both schizophrenia and bipolar disorder diagnoses were significant predictors of putative GRα-D1 abundance (p<0.05 and p=0.005, respectively). Full-length GRα was increased in bipolar disorder relative to schizophrenia cases. Luciferase assays demonstrated that the GRα-D1 isoform can activate transcription at glucocorticoid response elements. These findings confirm total GR mRNA reductions in schizophrenia and provide the first evidence of GR protein isoform abnormalities in schizophrenia and bipolar disorder. PMID:21881570

  20. Divergent expression patterns of SATB1 mRNA and SATB1 protein in colorectal cancer and normal tissues.

    PubMed

    Kowalczyk, Anna E; Godlewski, Janusz; Krazinski, Bartlomiej E; Kiewisz, Jolanta; Sliwinska-Jewsiewicka, Agnieszka; Kwiatkowski, Przemyslaw; Pula, Bartosz; Dziegiel, Piotr; Janiszewski, Jacek; Wierzbicki, Piotr M; Kmiec, Zbigniew

    2015-06-01

    Special AT-rich sequence-binding protein 1 (SATB1) is a 'genome organizer,' and it has been proposed as a factor that affects the development and progression of various human neoplasms, including colorectal cancer (CRC). This study aimed to compare SATB1 expression in a group of CRC patients and healthy subjects at the mRNA and protein levels. We collected paired tumor tissue and unchanged mucosa of the large intestine from 102 CRC patients as well as 53 biopsies of normal colon mucosa obtained from healthy patients during screening colonoscopy. Tissue samples were quantified for SATB1 mRNA by quantitative PCR, while SATB1 protein expression was determined by Western blotting and immunohistochemistry. SATB1 mRNA level in tumor tissues was over twofolds lower than in samples of corresponding unchanged tissues and fourfolds lower than in biopsies of healthy colon mucosa. Western blotting analysis revealed that SATB1 protein content in tumor and unchanged tissues of CRC patients was over sixfold and fivefolds higher than in biopsies of healthy colon mucosa, respectively. Immunohistochemical staining demonstrated higher nuclear and cytoplasmic SATB1 reactivity in the tumor tissue compared to unchanged mucosa of CRC patients. Despite these differences, SATB1 mRNA, protein, and immunoreactivity levels did not correlate with patients' clinicopathological data and their overall survival, but the latter analysis was limited by a relatively short period of follow-up. In conclusion, we suggest that some as yet unidentified posttranscriptional mechanisms that regulate SATB1 expression may be altered in the CRC tissue.

  1. Effect of acidic ribosomal phosphoprotein mRNA 5'-untranslated region on gene expression and protein accumulation.

    PubMed

    Bermejo, B; Remacha, M; Ortiz-Reyes, B; Santos, C; Ballesta, J P

    1994-02-11

    Constructions were made from genes encoding ribosomal acidic phosphoproteins YP1 beta (L44') and YP2 beta (L45) from Saccharomyces cerevisiae in which different parts of the 5'-untranslated regions were included. The constructs were inserted into centromeric plasmids under the control of the GAL1 promoter and expressed in yeast strains in which the genes coding for each acidic protein family, P1 and P2, had been disrupted. Deletions in the 5' region of the two genes have been found to oppositely affect their expression. Deletion of most of this region strongly stimulates the expression of YP2 beta (L45), increasing the translation efficiency of the mRNA, and generating a 6-fold excess of protein in the cell. A similar deletion in the rpYP1 beta gene represses the expression of the protein, reducing drastically the amount of the mRNA in the cell. The overexpression of rpYP2 beta affects the cell growth by inhibiting protein synthesis at the level of initiation. Reduction of the YP2 beta(L45) overproduction by growing in controlled concentrations of glucose abolishes the inhibitory effect. The excess protein, probably as a high molecular weight complex, apparently interferes with the joining of the 60 S subunit to the initiation complex generating the accumulation of polysome half-mers. In addition, the results indicate the existence of a regulatory mechanism by which each one of the two acidic proteins controls the expression of the other polypeptide. YP1 beta(L44') represses the expression of YP2 beta(L45), while this protein stimulates the expression of YP1 beta(L44').

  2. Human skeletal muscle creatine transporter mRNA and protein expression in healthy, young males and females.

    PubMed

    Murphy, Robyn M; Tunstall, Rebecca J; Mehan, Kate A; Cameron-Smith, David; McKenna, Michael J; Spriet, Lawrence L; Hargreaves, Mark; Snow, Rodney J

    2003-02-01

    The present study investigated whether there were any differences between males and females in respect to creatine transporter (CreaT) gene expression and/or total creatine (TCr) content in human vastus lateralis muscle. Skeletal muscle obtained from young healthy male (n = 13, age: 23.2 +/- 5.0 years) and female subjects (n = 12, age: 21.7 +/- 4.3 years) was analyzed for CreaT mRNA, CreaT protein and TCr content. Total CreaT protein content in the muscle was similar (p > 0.05) between the sexes. Two bands (approximately 55 and 73 kDa) of the CreaT protein were detected in all muscle samples. Both the 55 and the 73 kDa bands were present in similar (p > 0.05) amounts in males compared with females. The 73 kDa band was in greater abundance (p < 0.05) than the 55 kDa band, irrespective of gender. In addition, CreaT mRNA expression relative to beta-actin mRNA and the TCr content (males: 117.8 +/- 2.2, females: 125.3 +/- 4.3 mmol.kg(-1) dry mass) were also unaffected (p > 0.05) by gender. These data demonstrate that gender does not influence skeletal muscle TCr content and CreaT gene expression in young human subjects.

  3. Effect of silicon dioxide on expression of poly (ADP-ribose) polymerase mRNA and protein.

    PubMed

    Gao, Ai; Song, Shanshan; Wang, Danlin; Peng, Wei; Tian, Lin

    2009-07-01

    Silicon dioxide induces acute injury and chronic pulmonary fibrosis. International Agency for Research on Cancer (IARC) listed it as a human carcinogen in 1996. However, the molecular mechanisms to induce cancer are not understood yet. The content of poly (ADP-ribose) polymerases (PARP) mRNA and protein in Hela cells treated with concentrations of silicon dioxide up to 400microg/ml was determined by real-time fluorogenetic quantitative PCR (RQ-PCR) and immunofluorescence assay, respectively. MTT assay was used to determine cell viability. The results showed that viability at 400microg/ml silica was significantly decreased but not at lower concentrations. The protein content of gamma-H2AX in silica-treated group was significantly higher than the controls. The PARP mRNA and protein levels were significantly reduced with a dose response manner from the lowest silicon dioxide level. Our findings suggested that silicon dioxide increased the expression of gamma-H2AX and inhibited the expression of PARP mRNA and protein in Hela cells.

  4. Molecular Cloning, mRNA Expression, and Localization of the G-protein Subunit Galphaq in Sheep Testis and Epididymis

    PubMed Central

    Li, Zhen; Lu, Jieli; Sun, Xiaowei; Pang, Quanhai; Zhao, Yiwen

    2016-01-01

    The reproductive function of G-protein subunit Galphaq (GNAQ), a member of the G protein alpha subunit family, has been extensively studied in humans and rats. However, no data is available on its status in ruminants. The objectives of this study were to evaluate the expression pattern of the GNAQ in the testis and epididymis of sheep by polymerase chain reaction (PCR). The mRNA expression levels were detected by real-time fluorescent quantitative PCR, and cellular localization of GNAQ in the testis and epididymis was examined by immunohistochemistry. Additionally, GNAQ protein was qualitatively evaluated via western blot, with the results indicating that similarities between GNAQ mRNA levels from sheep was highly conserved with those observed in Bos taurus and Sus scrofa. Our results also indicated that GNAQ exists in the caput and cauda epididymis of sheep, while GNAQ in the testis and epididymis was localized to Leydig cells, spermatogonial stem cells, spermatocytes, Sertoli cells, spermatid, principal cells, and epididymis interstitial cells. The concentrations of GNAQ mRNA and protein in the caput and cauda epididymis were significantly greater than those observed in the corpus epididymis (p<0.01) and testis (p<0.05). Our results indicated that GNAQ exists at high concentrations in the caput and cauda epididymis of sheep, suggesting that GNAQ may play an important role in gonad development and sperm maturation. PMID:27004818

  5. Synergistic Effect of Caffeine and Glucocorticoids on Expression of Surfactant Protein B (SP-B) mRNA

    PubMed Central

    Fehrholz, Markus; Bersani, Iliana; Kramer, Boris W.; Speer, Christian P.; Kunzmann, Steffen

    2012-01-01

    Administration of glucocorticoids and caffeine is a common therapeutic intervention in the neonatal period, but possible interactions between these substances are still unclear. The present study investigated the effect of caffeine and different glucocorticoids on expression of surfactant protein (SP)-B, crucial for the physiological function of pulmonary surfactant. We measured expression levels of SP-B, various SP-B transcription factors including erythroblastic leukemia viral oncogene homolog 4 (ErbB4) and thyroid transcription factor-1 (TTF-1), as well as the glucocorticoid receptor (GR) after administering different doses of glucocorticoids, caffeine, cAMP, or the phosphodiesterase-4 inhibitor rolipram in the human airway epithelial cell line NCI-H441. Administration of dexamethasone (1 µM) or caffeine (5 mM) stimulated SP-B mRNA expression with a maximal of 38.8±11.1-fold and 5.2±1.4-fold increase, respectively. Synergistic induction was achieved after co-administration of dexamethasone (1 mM) in combination with caffeine (10 mM) (206±59.7-fold increase, p<0.0001) or cAMP (1 mM) (213±111-fold increase, p = 0.0108). SP-B mRNA was synergistically induced also by administration of caffeine with hydrocortisone (87.9±39.0), prednisolone (154±66.8), and betamethasone (123±6.4). Rolipram also induced SP-B mRNA (64.9±21.0-fold increase). We detected a higher expression of ErbB4 and GR mRNA (7.0- and 1.7-fold increase, respectively), whereas TTF-1, Jun B, c-Jun, SP1, SP3, and HNF-3α mRNA expression was predominantly unchanged. In accordance with mRNA data, mature SP-B was induced significantly by dexamethasone with caffeine (13.8±9.0-fold increase, p = 0.0134). We found a synergistic upregulation of SP-B mRNA expression induced by co-administration of various glucocorticoids and caffeine, achieved by accumulation of intracellular cAMP. This effect was mediated by a caffeine-dependent phosphodiesterase inhibition and by upregulation of both ErbB4 and

  6. Actinidia DRM1--an intrinsically disordered protein whose mRNA expression is inversely correlated with spring budbreak in kiwifruit.

    PubMed

    Wood, Marion; Rae, Georgina M; Wu, Rong-Mei; Walton, Eric F; Xue, Bin; Hellens, Roger P; Uversky, Vladimir N

    2013-01-01

    Intrinsically disordered proteins (IDPs) are a relatively recently defined class of proteins which, under native conditions, lack a unique tertiary structure whilst maintaining essential biological functions. Functional classification of IDPs have implicated such proteins as being involved in various physiological processes including transcription and translation regulation, signal transduction and protein modification. Actinidia DRM1 (Ade DORMANCY ASSOCIATED GENE 1), represents a robust dormancy marker whose mRNA transcript expression exhibits a strong inverse correlation with the onset of growth following periods of physiological dormancy. Bioinformatic analyses suggest that DRM1 is plant specific and highly conserved at both the nucleotide and protein levels. It is predicted to be an intrinsically disordered protein with two distinct highly conserved domains. Several Actinidia DRM1 homologues, which align into two distinct Actinidia-specific families, Type I and Type II, have been identified. No candidates for the Arabidopsis DRM1-Homologue (AtDRM2) an additional family member, has been identified in Actinidia.

  7. Towards a multi protein and mRNA expression of biological predictive and distinguish model for post stroke depression

    PubMed Central

    Yue, Yingying; Jiang, Haitang; Liu, Rui; Yin, Yingying; Zhang, Yuqun; Liang, Jinfeng; Li, Shenghua; Wang, Jun; Lu, Jianxin; Geng, Deqin; Wu, Aiqin; Yuan, Yonggui

    2016-01-01

    Previous studies suggest that neurotrophic factors participate in the development of stroke and depression. So we investigated the utility of these biomarkers as predictive and distinguish model for post stroke depression (PSD). 159 individuals including PSD, stroke without depression (Non-PSD), major depressive disorder (MDD) and normal control groups were recruited and examined the protein and mRNA expression levels of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptors (VEGFR2), placental growth factor (PIGF), insulin-like growth factor (IGF-1) and insulin-like growth factor receptors (IGF-1R). The chi-square test was used to evaluate categorical variable, while nonparametric test and one-way analysis of variance were applied to continuous variables of general characteristics, clinical and biological changes. In order to explore the predictive and distinguish role of these factors in PSD, discriminant analysis and receiver operating characteristic curve were calculated. The four groups had statistical differences in these neurotrophic factors (all P < 0.05) except VEGF concentration and IGF-1R mRNA (P = 0.776, P = 0.102 respectively). We identified these mRNA expression and protein analytes with general predictive performance for PSD and Non-PSD groups [area under the curve (AUC): 0.805, 95% CI, 0.704-0.907, P < 0.001]. Importantly, there is an excellent predictive performance (AUC: 0.984, 95% CI, 0.964-1.000, P < 0.001) to differentiate PSD patients from MDD patients. This was the first study to explore the changes of neurotrophic factors family in PSD patients, the results intriguingly demonstrated that the combination of protein and mRNA expression of biological factors could use as a predictive and discriminant model for PSD. PMID:27527872

  8. Expression and physiological role of CCN4/Wnt-induced secreted protein 1 mRNA splicing variants in chondrocytes.

    PubMed

    Yanagita, Takeshi; Kubota, Satoshi; Kawaki, Harumi; Kawata, Kazumi; Kondo, Seiji; Takano-Yamamoto, Teruko; Tanaka, Shinji; Takigawa, Masaharu

    2007-04-01

    CCN4/Wnt-induced secreted protein 1 (WISP1) is one of the CCN (CTGF/Cyr61/Nov) family proteins. CCN members have typical structures composed of four conserved cysteine-rich modules and their variants lacking certain modules, generated by alternative splicing or gene mutations, have been described in various pathological conditions. Several previous reports described a CCN4/WISP1 variant (WISP1v) lacking the second module in a few malignancies, but no information concerning the production of WISP1 variants in normal tissue is currently available. The expression of CCN4/WISP1 mRNA and its variants were analyzed in a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, and primary rabbit growth cartilage (RGC) chondrocytes. First, we found WISP1v and a novel variant of WISP1 (WISP1vx) to be expressed in HCS-2/8, as well as full-length WISP1 mRNA. This new variant was lacking the coding regions for the second and third modules and a small part of the first module. To monitor the expression of CCN4/WISP1 mRNA along chondrocyte differentiation, RGC cells were cultured and sampled until they were mineralized. As a result, we identified a WISP1v ortholog in normal RGC cells. Interestingly, the WISP1v mRNA level increased dramatically along with terminal differentiation. Furthermore, overexpression of WISP1v provoked expression of an alkaline phosphatase gene that is a marker of terminal differentiation in HCS-2/8 cells. These findings indicate that WISP1v thus plays a critical role in chondrocyte differentiation toward endochondral ossification, whereas HCS-2/8-specific WISP1vx may be associated with the transformed phenotypes of chondrosarcomas.

  9. Expression and Presence of OPG and RANKL mRNA and Protein in Human Periodontal Ligament with Orthodontic Force

    PubMed Central

    Otero, Liliana; García, Dabeiba Adriana; Wilches-Buitrago, Liseth

    2016-01-01

    OBJECTIVE The objective of this study is to investigate the expression and concentration of ligand receptor activator of NFkB (RANKL) and osteoprotegerin (OPG) in human periodontal ligament (hPDL) with orthodontic forces of different magnitudes. METHODS Right premolars in 32 patients were loaded with 4oz or 7oz of orthodontic force for 7 days. Left first premolars were not loaded. After 7 days, premolars were extracted for treatment as indicated. OPG and RANKL mRNA expressions were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and ELISA was used to assess OPG and RANKL protein concentration in compression and tension sides of PDL. Data were subjected to analysis of variance and Tukey tests. RESULTS There was statistically significant difference in RANKL concentration on comparing control teeth with tension and compression sides of the experimental teeth (P < 0.0001). The expression of mRNA RANKL was increased in the tension and compression sides with 4oz (P < 0.0001). OPG did not show statistically significant association with any group. Changes in RANKL/OPG protein ratio in experimental and control groups showed statistically significant difference (P < 0.0001). CONCLUSIONS RANKL protein levels are elevated in hPDL loaded with orthodontic forces, suggesting that RANKL protein contributes to bone modeling in response to the initial placement of orthodontic force. PMID:26823650

  10. In vivo expression of mRNA for the Ca++-binding protein SPARC (osteonectin) revealed by in situ hybridization

    PubMed Central

    1987-01-01

    In situ hybridization is used to survey the tissue-specific and developmental expression of the cloned mouse gene Sparc, coding for a protein homologous to the bovine Ca++-binding protein, osteonectin. High levels of SPARC RNA are found in osteoblasts and odontoblasts. In addition, high grain counts are associated with a variety of other cell types in the embryo and newborn mouse, including parietal endoderm, deciduum, whisker follicles (connective tissue sheath), peripheral nerve trunk, skin (dermis), and stomach (submucosa). Spatially restricted but high levels of SPARC mRNA are also seen in the adult adrenal glands, testis, and ovary. This pattern of differential gene expression demands a reassessment of the function originally proposed for osteonectin, and predicts a much wider role for the protein in a variety of biological processes. PMID:2440898

  11. Luteotropic and luteolytic factors regulate mRNA and protein expression of progesterone receptor isoforms A and B in the bovine endometrium.

    PubMed

    Rekawiecki, Robert; Kowalik, Magdalena Karolina; Kotwica, Jan

    2014-12-17

    The aim of the present study was to examine the effects of luteotropic and luteolytic factors on the mRNA and protein levels of progesterone receptor isoforms A (PGRA) and B (PGRB) in the bovine endometrium. Endometrial slices from Days 6-10 and 17-20 of the oestrous cycle were treated with LH (100ngmL-1), oestradiol (E2; 1×10-8M), prostaglandin (PG) E2 (1×10-6M) and PGF2? (1×10-6M) and the nitric oxide donor NONOate (1×10-4M); these treatments lasted for 6h for mRNA expression analysis and 24h for protein expression analysis. On Days 6-10 of the oestrous cycle PGRAB (PGRAB; the entire PGRA mRNA sequence is common to the PGRB mRNA sequence) mRNA expression in endometrial slices was enhanced by E2 treatment (PPGRB mRNA expression was increased by LH (PPPPGRAB mRNA expression increased after E2 (P2 (PPGRB mRNA expression was increased by PGE2 (P2? (PPPPPP2? (P2 (P2? (P<0.001). These data suggest that luteotropic and luteolytic factors affect PGRA and PGRB mRNA and protein levels, and this may regulate the effects of progesterone on endometrial cells.

  12. Resveratrol Decreases TXNIP mRNA and Protein Nuclear Expressions With an Arterial Function Improvement in Old Mice.

    PubMed

    Bedarida, Tatiana; Baron, Stephanie; Vibert, Françoise; Ayer, Audrey; Henrion, Daniel; Thioulouse, Elizabeth; Marchiol, Carmen; Beaudeux, Jean-Louis; Cottart, Charles-Henry; Nivet-Antoine, Valerie

    2016-06-01

    Aging leads to a high prevalence of glucose intolerance and cardiovascular diseases, with oxidative stress playing a potential role. Resveratrol has shown promising effects on glucose tolerance and tends to improve endothelial function in elderly patients. Thioredoxin-interacting protein (TXNIP) was recently proposed as a potential link connecting glucose metabolism to oxidative stress. Here, we investigated the resveratrol-induced improvement of arterial aging phenotype in old mice and the expression of aortic TXNIP. Using an in vivo model of old mice with or without 3-month resveratrol treatment, we investigated the effects of resveratrol on age-related impairments from a cardiovascular Doppler analysis, to a molecular level, by studying inflammation and oxidative stress factors. We found a dual effect of resveratrol, with a decrease of age-related glucose intolerance and oxidative stress imbalance leading to reduced matrix remodeling that forestalls arterial aging phenotype in terms of intima-media thickness and arterial distensibility. These results provide the first evidence that aortic TXNIP mRNA and protein nuclear expressions are increased in the arterial aging and decreased by resveratrol treatment. In conclusion, we demonstrated that resveratrol helped to restore several aging impaired processes in old mice, with a decrease of aortic TXNIP mRNA and protein nuclear expressions. © The Author 2015. 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.

  13. Strain differences in cytochrome P450 mRNA and protein expression, and enzymatic activity among Sprague Dawley, Wistar, Brown Norway and Dark Agouti rats.

    PubMed

    Nishiyama, Yoshihiro; Nakayama, Shouta M M; Watanabe, Kensuke P; Kawai, Yusuke K; Ohno, Marumi; Ikenaka, Yoshinori; Ishizuka, Mayumi

    2016-05-03

    Rat cytochrome P450 (CYP) exhibits inter-strain differences, but their analysis has been scattered across studies under different conditions. To identify these strain differences in CYP more comprehensively, mRNA expression, protein expression and metabolic activity among Wistar (WI), Sprague Dawley (SD), Dark Agouti (DA) and Brown Norway (BN) rats were compared. The mRNA level and enzymatic activity of CYP1A1 were highest in SD rats. The rank order of Cyp3a2 mRNA expression mirrored its protein expression, i.e., DA>BN>SD>WI, and was similar to the CYP3A2-dependent warfarin metabolic activity, i.e., DA>SD>BN>WI. These results suggest that the strain differences in CYP3A2 enzymatic activity are caused by differences in mRNA expression. Cyp2b1 mRNA levels, which were higher in DA rats, did not correlate with its protein expression or enzymatic activity. This suggests that the strain differences in enzymatic activity are not related to Cyp2b1 mRNA expression. In conclusion, WI rats tended to have the lowest CYP1A1, 2B1 and 3A2 mRNA expression, protein expression and enzymatic activity among the strains. In addition, SD rats had the highest CYP1A1 mRNA expression and activity, while DA rats had higher CYP2B1 and CYP3A2 mRNA and protein expression. These inter-strain differences in CYP could influence pharmacokinetic considerations in preclinical toxicological studies.

  14. ProteoMirExpress: inferring microRNA and protein-centered regulatory networks from high-throughput proteomic and mRNA expression data.

    PubMed

    Qin, Jing; Li, Mulin Jun; Wang, Panwen; Wong, Nai Sum; Wong, Maria P; Xia, Zhengyuan; Tsao, George S W; Zhang, Michael Q; Wang, Junwen

    2013-11-01

    MicroRNAs (miRNAs) regulate gene expression through translational repression and RNA degradation. Recently developed high-throughput proteomic methods measure gene expression changes at protein level and therefore can reveal the direct effects of miRNAs' translational repression. Here, we present a web server, ProteoMirExpress, that integrates proteomic and mRNA expression data together to infer miRNA-centered regulatory networks. With both types of high-throughput data from the users, ProteoMirExpress is able to discover not only miRNA targets that have decreased mRNA, but also subgroups of targets with suppressed proteins whose mRNAs are not significantly changed or with decreased mRNA whose proteins are not significantly changed, which are usually ignored by most current methods. Furthermore, both direct and indirect targets of miRNAs can be detected. Therefore, ProteoMirExpress provides more comprehensive miRNA-centered regulatory networks. We used several published data to assess the quality of our inferred networks and prove the value of our server. ProteoMirExpress is available online, with free access to academic users.

  15. Delayed inflammatory mRNA and protein expression after spinal cord injury

    PubMed Central

    2011-01-01

    Background Spinal cord injury (SCI) induces secondary tissue damage that is associated with inflammation. We have previously demonstrated that inflammation-related gene expression after SCI occurs in two waves - an initial cluster that is acutely and transiently up-regulated within 24 hours, and a more delayed cluster that peaks between 72 hours and 7 days. Here we extend the microarray analysis of these gene clusters up to 6 months post-SCI. Methods Adult male rats were subjected to mild, moderate or severe spinal cord contusion injury at T9 using a well-characterized weight-drop model. Tissue from the lesion epicenter was obtained 4 hours, 24 hours, 7 days, 28 days, 3 months or 6 months post-injury and processed for microarray analysis and protein expression. Results Anchor gene analysis using C1qB revealed a cluster of genes that showed elevated expression through 6 months post-injury, including galectin-3, p22PHOX, gp91PHOX, CD53 and progranulin. The expression of these genes occurred primarily in microglia/macrophage cells and was confirmed at the protein level using both immunohistochemistry and western blotting. As p22PHOX and gp91PHOX are components of the NADPH oxidase enzyme, enzymatic activity and its role in SCI were assessed and NADPH oxidase activity was found to be significantly up-regulated through 6 months post-injury. Further, treating rats with the nonspecific, irreversible NADPH oxidase inhibitor diphenylene iodinium (DPI) reduced both lesion volume and expression of chronic gene cluster proteins one month after trauma. Conclusions These data demonstrate that inflammation-related genes are chronically up-regulated after SCI and may contribute to further tissue loss. PMID:21975064

  16. Patterns of mRNA and protein expression during minus-lens compensation and recovery in tree shrew sclera

    PubMed Central

    Gao, Hong; Frost, Michael R.; Norton, Thomas T.

    2011-01-01

    Purpose To increase our understanding of the mechanisms that remodel the sclera during the development of lens-induced myopia, when the sclera responds to putative “go” signals of retinal origin, and during recovery from lens-induced myopia, when the sclera responds to retinally-derived “stop” signals. Methods Seven groups of tree shrews were used to examine mRNA levels during minus lens compensation and recovery. Starting 24 days after eye opening (days of visual experience [VE]) lens compensation animals wore a monocular –5D lens for 1, 4, or 11 days. Recovery animals wore the –5D lens for 11 days, which was then removed for 1 or 4 days. Normal animals were examined at 24 and 38 days of VE. All groups contained 8 animals. Scleral mRNA levels were examined in the treated and contralateral control eyes with quantitative real-time polymerase chain reaction (qPCR) for 27 genes divided into four categories: 1) signaling molecules, 2) matricellular proteins, 3) metalloproteinases (MPs) and tissue inhibitors of metalloproteinases (TIMPs), and 4) cell adhesion and other proteins. Four groups (n=5 per group) were used to examine protein levels. One group wore a –5D lens for 4 days. A second group recovered for 4 days after 11 days of −5D lens treatment. Two groups were used to examine age-matched normal protein levels at 28 and 39 days of VE. The levels of six scleral proteins that showed differential mRNA expression were examined with quantitative western blots. Results Nineteen of the genes showed differential (treated eye versus control eye) expression of mRNA levels in at least one group of animals. Which genes showed differential expression differed after 1 and 4 days of compensation and after 1 or 4 days of recovery. The mRNA level for one gene, a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), was upregulated in the treated eyes after 1 day of compensation. After 4 days, transforming growth factor beta receptor 3 (TGFBR3

  17. Comparison of immunomodulator mRNA and protein expression in the lungs of Stachybotrys chartarum spore-exposed mice.

    PubMed

    Hudson, B; Flemming, J; Sun, G; Rand, T G

    2005-08-13

    Stachybotrys chartarum is an important toxigenic fungus that has been associated with respiratory disease onset in animals and humans. It can be separated into macrocyclic trichothecene-producing and nonproducing chemotypes based on secondary metabolite production. However, effects of spores of the two chemotypes on lung inflammatory responses are poorly understood. In this study, real-time reverse-transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA) were used to investigate time-course (1, 3, 6, 24, and 48 h post-instillation [PI]) relationships in mice intratracheally exposed to 300 spores/g body weight of a macrocyclic trichothecene-producing (JS 58-17) and a nonproducing (JS 58-06) S. chartarum isolate and of Cladosporium cladosporioides. There were marked differences in the magnitude and temporal patterns of mouse lung immune responses to intratracheal exposure to spores of these species at this spore dose. Both macrophage inflammatory protein 2 (MIP-2) and surfactant protein-D (SP-D) mRNA expression were significantly upregulated in lungs of JS 58-17-treated animals compared to that of all other treatment animals at 6 and 24 h PI. Heightened mRNA expression of these immunomodulators combined with comparatively depressed MIP-2 and tumor necrosis factor (TNF)-a protein expression suggests that the action of macrocyclic trichothecenes sequestered in 58-17 spores is involved. Interestingly, TNF-a protein expression in all spore treatment animal groups was also significantly increased over that in saline controls. Similarities in expression among all spore treatment animals suggest that chemicals other than toxic secondary metabolites, and possibly spore-sequestered 1,3-beta-D-glucan, may contribute to lung pathogenesis.

  18. Effect of Gestational Age on mRNA and Protein Expression of Polyspecific Organic Cation Transporters during Pregnancy

    PubMed Central

    Lee, Nora; Hebert, Mary F.; Prasad, Bhagwat; Easterling, Thomas R.; Kelly, Edward J.; Unadkat, Jashvant D.

    2013-01-01

    Polyspecific organic cation (OC) transporters play important roles in the disposition of clinically used drugs, including drugs used during pregnancy. Pregnancy is known to alter the expression of drug-metabolizing enzymes and transporters, but its specific effect on OC transporters has not been well defined. Using quantitative polymerase chain reaction and liquid chromatography coupled with tandem mass spectrometry targeted proteomics, we determined the effect of pregnancy and gestational age on mRNA and protein expression of major OC transporters in the kidney, liver, and placenta in mice with timed pregnancies. Human organic cation transporter 3 (hOCT3) expression was further investigated in human placentas from the first and second trimesters and at term. Our results showed that pregnancy had a marginal effect on renal mouse organic cation transporter 1/2 (mOct1/2) expression but significantly reduced mouse multidrug and toxin extrusion transporter 1 (mMate1) expression by 20%–40%. Hepatic expression of mOct1 and mMate1 was minimally affected by pregnancy. Human and mouse placentas predominantly expressed OCT3 with little expression of OCT1/2, MATE1/2, and plasma membrane monoamine transporter (PMAT). The hOCT3 protein in first and second trimester and term placentas was quantified to be 0.23 ± 0.033, 0.38 ± 0.072, and 0.36 ± 0.099 fmol/μg membrane protein, respectively. In contrast with the moderate increase in hOCT3 protein during human pregnancy, mOct3 expression in the mouse placenta was highly dependent on gestational age. Compared with gestational day (gd) 10, placental mOct3 mRNA increased by 37-fold and 46-fold at gd 15 and 19, leading to a 56-fold and 128-fold increase in mOct3 protein, respectively. Our study provides new insights into the effect of pregnancy on the expression of polyspecific OC transporters and supports an important role of OCT3 in OC transport at the placental barrier. PMID:24101703

  19. Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

    PubMed Central

    Widelitz, R B; Magun, B E; Gerner, E W

    1986-01-01

    A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis. Images PMID:3785158

  20. Prognostic impact of HER3 based on protein and mRNA expression in high-grade serous ovarian carcinoma.

    PubMed

    Unger, Ulrike; Denkert, Carsten; Braicu, Ioana; Sehouli, Jalid; Dietel, Manfred; Loibl, Sibylle; Darb-Esfahani, Silvia

    2017-02-01

    HER3 is a member of the epidermal growth factor family and was predominantly described as a negative prognostic factor in various solid tumors as well as in ovarian cancer. In this study, we investigated HER3 on protein and mRNA expression in histologically defined subtypes of ovarian cancer looking for an influence on patient's survival. Altogether, we examined HER3 in ovarian high-grade serous (HGSC, n = 320), low-grade serous (LGSC, n = 55), endometrioid (EC, n = 33), and clear cell (CCC, n = 48) carcinomas using immunohistochemistry (IHC) and quantitative real-time reverse transcription PCR (qRT-PCR). Univariate and multivariate analyses were performed to explore the association between HER3 and overall survival (OS) as well as progression-free survival (PFS). In HGSC, high HER3 mRNA expression was a favorable prognostic factor for PFS (P = 0.008) and OS (P = 0.052), while for high HER3 protein expression, a trend towards better survival was seen (OS P = 0.064; PFS P = 0.099). A subgroup of HGSC with negative HER3 staining and negative HER3 mRNA levels showed most unfavorable OS and PFS (P = 0.002 and P = 0.004, respectively). Using the multivariate Cox regression model, HER3 was predictive for prolonged PFS (HR, 0.48; 95% CI, 0.26-0.88; P = 0.018). All in all, we cannot confirm the reported negative prognostic impact of HER3 expression in high-grade serous ovarian carcinoma and moreover find a rather positive prognostic implication of HER3 in this major ovarian cancer histological subtype.

  1. Effect of hyperosmotic conditions on flavin-containing monooxygenase activity, protein and mRNA expression in rat kidney

    PubMed Central

    Rodríguez-Fuentes, Gabriela; Coburn, Cary; Currás-Collazo, Margarita; Guillén, Gabriel; Schlenk, Daniel

    2010-01-01

    Flavin-containing monooxigenases (FMOs) are a polymorphic family of drug and pesticide metabolizing enzymes, found in the smooth endoplasmatic reticulum that catalyze the oxidation of soft nucleophilic heteroatom substances to their respective oxides. Previous studies in euryhaline fishes have indicated induction of FMO expression and activity in vivo under hyperosmotic conditions. In this study we evaluated the effect of hypersaline conditions in rat kidney. Male Sprague–Dawley rats were injected intraperitoneal with 3.5 M NaCl at a doses ranging from 0.3 cm3/100 g to 0.6 cm3/100 g in two separate treatments. Three hours after injection, FMO activities and FMO1 protein was examined in the first experiment, and the expression of FMO1 mRNA was measured in the second experiment from kidneys after treatment with NaCl. A positive significant correlation was found between FMO1 protein expression and plasma osmolarity (p < 0.05, r = 0.6193). Methyl-p-tolyl sulfide oxidase showed a statistically significant increase in FMO activity, and a positive correlation was observed between plasma osmolarity and production of FMO1-derived (R)-methyl-p-tolyl sulfoxide (p < 0.05, r = 0.6736). Expression of FMO1 mRNA was also positively correlated with plasma osmolality (p < 0.05, r = 0.8428). Similar to studies in fish, these results suggest that expression and activities of FMOs may be influenced by hyperosmotic conditions in the kidney of rats. PMID:19429252

  2. Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation.

    PubMed

    Pearce, Robin E; Gaedigk, Roger; Twist, Greyson P; Dai, Hongying; Riffel, Amanda K; Leeder, J Steven; Gaedigk, Andrea

    2016-07-01

    Although CYP2B6 catalyzes the biotransformation of many drugs used clinically for children and adults, information regarding the effects of development on CYP2B6 expression and activity are scarce. Utilizing a large panel of human liver samples (201 donors: 24 fetal, 141 pediatric, and 36 adult), we quantified CYP2B6 mRNA and protein expression levels, characterized CYP2B6 (bupropion hydroxylase) activity in human liver microsomes (HLMs), and performed an extensive genotype analysis to differentiate CYP2B6 haplotypes such that the impact of genetic variation on these parameters could be assessed. Fetal livers contained extremely low levels of CYP2B6 mRNA relative to postnatal samples and fetal HLMs did not appear to catalyze bupropion hydroxylation; however, fetal CYP2B6 protein levels were not significantly different from postnatal levels. Considerable interindividual variation in CYP2B6 mRNA expression, protein levels, and activity was observed in postnatal HLMs (mRNA, ∼40,000-fold; protein, ∼300-fold; activity, ∼600-fold). The extremely wide range of interindividual variability in CYP2B6 expression and activity was significantly associated with age (P < 0.01) following log transformation of the data. Our data suggest that CYP2B6 activity appears as early as the first day of life, increases through infancy, and by 1 year of age, CYP2B6 levels and activity may approach those of adults. Surprisingly, CYP2B6 interindividual variability was not significantly associated with genetic variation in CYP2B6, nor was it associated with differences in gender or ethnicity, suggesting that factors other than these are largely responsible for the wide range of variability in CYP2B6 expression and activity observed among a large group of individuals/samples.

  3. Brazilein inhibits survivin protein and mRNA expression and induces apoptosis in hepatocellular carcinoma HepG2 cells.

    PubMed

    Zhong, X; Wu, B; Pan, Y J; Zheng, S

    2009-01-01

    Hepatocellular carcinomas represent the third leading cause of cancer-related deaths worldwide. Survivin, a structurally unique member of the inhibitor of apoptosis protein (IAP) family, is overexpressed in a wide range of malignancies, including hepatocellular carcinoma. Due to its involvement in cancer progression and treatment resistance, survivin is currently undergoing extensive investigation as a novel intervention target to induce apoptosis in cancer cells by phytochemicals or synthetic agents. Brazilein, a compound obtained in a large amount from the dried heartwood of Caesalpinia sappan Linn., which has long been used in traditional medicine in China, has some pharmacological activities. Human hepatocellular carcinoma HepG2 cells were treated with brazilein and analyzed for survivin protein and mRNA levels by Western blotting and real-time RT-PCR, respectively. Brazilein treatment of cells for 48 h at 5 and 10 microg/ml doses resulted in significantly decrease in survivin protein expression. We also observed that brazilein caused a strong decrease in survivin mRNA expression. In other studies, down-regulation of survivin by brazilein was associated with a strong and prominent caspases-9 and -3 activation as well as PARP cleavage. It was also shown that brazilein induced a strong apoptotic cell death, as shown by DNA ladder assay, and growth inhibition of HepG2 cells. Further studies are needed to investigate in vivo effect of brazilein on survivin expression and associated biological effects in hepatocellular carcinoma that could provide useful information for brazilein efficacy in the prevention/intervention of human hepatocellular carcinoma.

  4. p19INK4d mRNA and protein expression as new prognostic factors in ovarian cancer patients

    PubMed Central

    Felisiak-Golabek, Anna; Dansonka-Mieszkowska, Agnieszka; Rzepecka, Iwona K; Szafron, Lukasz; Kwiatkowska, Ewa; Konopka, Bozena; Podgorska, Agnieszka; Rembiszewska, Alina; Kupryjanczyk, Jolanta

    2013-01-01

    p19INK4d (CDKN2D) is a negative regulator of the cell cycle. Little is known of its role in cancer development and prognosis. We aimed to evaluate the clinical significance of p19INK4d expression in ovarian carcinomas with respect to the TP53 accumulation status, as well as the frequency of CDKN2D mutations. p19INK4d and TP53 expression was evaluated immunohistochemically in 445 ovarian carcinomas: 246 patients were treated with platinum–cyclophosphamide (PC/PAC), while 199 were treated with taxane–platinum agents (TP). CDKN2D gene expression (mRNA) was examined in 106 carcinomas, while CDKN2D mutations in 68 tumors. Uni- and multivariate statistical analyses (logistic regression and the Cox proportional hazards model) were performed for patient groups divided according to the chemotherapeutic regimen administered, and in subgroups with and without TP53 accumulation. High p19INK4d expression increased the risk of death, but only in patients with the TP53-negative carcinomas (HR 1.61, P = 0.049 for PC/PAC-treated patients, HR 2.00, P = 0.015 for TP-treated patients). This result was confirmed by the mRNA analysis (HR 4.24, P = 0.001 for TP-treated group). High p19INK4d protein expression associated with adverse clinicopathological factors. We found no alterations in the CDKN2D gene; the c.90C>G (p.R30R; rs1968445) polymorphism was detected in 10% of tumors. Our results suggest that p19INK4d expression is a poor prognostic factor in ovarian cancer patients. Analyses of tumor groups according to the TP53 accumulation status facilitate the identification of cancer biomarkers. PMID:24022213

  5. 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

    2017-03-15

    Although tumor protein D52 (TPD) 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 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 TPD53 and 54. 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. 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 RNA immunoprecipitation 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 TGF-b and EGF decreased the binding ability of these factors, resulted 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 herein report the specific interactions between these elements in the post-transcriptional regulation of the TPD52 gene.

  6. Actinidia DRM1 - An Intrinsically Disordered Protein Whose mRNA Expression Is Inversely Correlated with Spring Budbreak in Kiwifruit

    PubMed Central

    Wood, Marion; Rae, Georgina M.; Wu, Rong-Mei; Walton, Eric F.; Xue, Bin; Hellens, Roger P.; Uversky, Vladimir N.

    2013-01-01

    Intrinsically disordered proteins (IDPs) are a relatively recently defined class of proteins which, under native conditions, lack a unique tertiary structure whilst maintaining essential biological functions. Functional classification of IDPs have implicated such proteins as being involved in various physiological processes including transcription and translation regulation, signal transduction and protein modification. Actinidia DRM1 (Ade DORMANCY ASSOCIATED GENE 1), represents a robust dormancy marker whose mRNA transcript expression exhibits a strong inverse correlation with the onset of growth following periods of physiological dormancy. Bioinformatic analyses suggest that DRM1 is plant specific and highly conserved at both the nucleotide and protein levels. It is predicted to be an intrinsically disordered protein with two distinct highly conserved domains. Several Actinidia DRM1 homologues, which align into two distinct Actinidia-specific families, Type I and Type II, have been identified. No candidates for the Arabidopsis DRM1-Homologue (AtDRM2) an additional family member, has been identified in Actinidia. PMID:23516402

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

    PubMed Central

    1993-01-01

    We have previously shown that nerve growth factor (NGF) selectively stabilizes the GAP-43 mRNA in PC12 cells. To study the cellular mechanisms for this post-transcriptional control and to determine the contribution of mRNA stability to GAP-43 gene expression, we examined the effects of several agents that affect PC12 cell differentiation on the level of induction and rate of degradation of the GAP-43 mRNA. The NGF-mediated increase in GAP-43 mRNA levels and neurite outgrowth was mimicked by the phorbol ester TPA, but not by dibutyryl cAMP or the calcium ionophore A12783. Downregulation of protein kinase C (PKC) by high doses of phorbol esters or selective PKC inhibitors prevented the induction of this mRNA by NGF, suggesting that NGF and TPA act through a common PKC-dependent pathway. In mRNA decay studies, phorbol esters caused a selective 6-fold increase in the half-life of the GAP-43 mRNA, which accounts for most of the induction of this mRNA by TPA. The phorbol ester-induced stabilization of GAP-43 mRNA was blocked by the protein kinase inhibitor polymyxin B and was partially inhibited by dexamethasone, an agent that blocks GAP-43 expression and neuronal differentiation in PC12 cells. In contrast, the rates of degradation and the levels of the GAP-43 mRNA in control and TPA-treated cells were not affected by cycloheximide treatment. Thus, changes in GAP-43 mRNA turnover do not appear to require continuous protein synthesis. In conclusion, these data suggest that PKC activity regulates the levels of the GAP-43 mRNA in PC12 cells through a novel, translation- independent mRNA stabilization mechanism. PMID:8436593

  8. Promoter methylation and mRNA expression of HLA-G in relation to HLA-G protein expression in colorectal cancer.

    PubMed

    Swets, Marloes; Seneby, Lina; Boot, Arnoud; van Wezel, Tom; Gelderblom, Hans; van de Velde, Cornelis J H; van den Elsen, Peter J; Kuppen, Peter J K

    2016-09-01

    Expression of human leukocyte antigen-G (HLA-G) is a suggested mechanism used by tumor cells to escape from host immune recognition and destruction. Advances in the field have made it evident that HLA-G is expressed in different types of malignancies including colorectal cancer (CRC). We analyzed HLA-G expression in 21 low passage CRC cell lines. The level of DNA methylation of the HLA-G gene and the presence of mRNA encoding HLA-G was measured. Moreover, HLA-G protein expression was determined by flow cytometry and immunohistochemistry (IHC). IHC was performed with three different monoclonal antibodies (mAbs) (4H84, MEM-G/1 and MEM-G/2). In addition, HLA-G protein expression was measured in matching primary tumor tissues. RNA analysis using RT-PCR followed by sequencing in 6 samples indicated strong homology of the PCR product with HLA-G3 in 5 samples. In accordance, in none of the cell lines, HLA-G1 expression was detected by flow-cytometry. Furthermore, no association between HLA-G DNA methylation patterns and HLA-G mRNA expression was observed. In addition, different immunohistochemical staining profiles among various anti-HLA-G mAbs were observed. In conclusion, the results of this study show that the HLA-G3 isoform was expressed in some of the CRC cell lines irrespective of the level of DNA methylation of HLA-G.

  9. Transgenic Expression of Human LAMA5 Suppresses Murine Lama5 mRNA and Laminin α5 Protein Deposition

    PubMed Central

    Steenhard, Brooke M.; Zelenchuk, Adrian; Stroganova, Larysa; Isom, Kathryn; St. John, Patricia L.; Andrews, Glen K.; Peterson, Kenneth R.; Abrahamson, Dale R.

    2011-01-01

    Laminin α5 is required for kidney glomerular basement membrane (GBM) assembly, and mice with targeted deletions of the Lama5 gene fail to form glomeruli. As a tool to begin to understand factors regulating the expression of the LAMA5 gene, we generated transgenic mice carrying the human LAMA5 locus in a bacterial artificial chromosome. These mice deposited human laminin α5 protein into basement membranes in heart, liver, spleen and kidney. Here, we characterized two lines of transgenics; Line 13 expressed ∼6 times more LAMA5 than Line 25. Mice from both lines were healthy, and kidney function and morphology were normal. Examination of developing glomeruli from fetal LAMA5 transgenics showed that the human transgene was expressed at the correct stage of glomerular development, and deposited into the nascent GBM simultaneously with mouse laminin α5. Expression of human LAMA5 did not affect the timing of the mouse laminin α1–α5 isoform switch, or that for mouse laminin β1–β2. Immunoelectron microscopy showed that human laminin α5 originated in both glomerular endothelial cells and podocytes, known to be origins for mouse laminin α5 normally. Notably, in neonatal transgenics expressing the highest levels of human LAMA5, there was a striking reduction of mouse laminin α5 protein in kidney basement membranes compared to wildtype, and significantly lower levels of mouse Lama5 mRNA. This suggests the presence in kidney of a laminin expression monitor, which may be important for regulating the overall production of basement membrane protein. PMID:21915268

  10. Higher expression of mRNA and protein of insulin-like growth factor binding protein-3 in old rat penile tissues: implications for erectile dysfunction.

    PubMed

    Pu, Xiao-Yong; Zheng, Xiang-Guang; Zhang, Yan; Xiao, Heng-Jun; Xu, Zhan-Ping; Liu, Jiu-Ming; Wang, Huai-Peng; Wen, An-Ming; Zhou, Xiang-Xue; Wu, Yi-Long

    2011-08-01

    Previous studies have confirmed the gene transfer of insulin-like growth factor-1 (IGF-1) and the IGF-1 protein can improve the erectile function in aging rats. IGF binding protein (BP)-3 can regulates the availability of IGF-I. The higher expression of IGFBP-3 may play an important role in erectile dysfunction (ED). The study aimed to investigate the mRNA and protein expression of IGFBP-3 in young and old rat penile tissues and assess the alteration of the penile structure and the NO-guanosine 3',5'-cyclic-monophosphate (cGMP) signaling pathways-related marker in ED associated with aging. The main outcome measures for this study were the expression of IGFBP-3, morphological changes, NO-cGMP signaling pathways-related marker, erectile responses were determined. Traditional reverse transcriptase polymerase chain reaction (RT-PCR) and real-time PCR were performed to examine the mRNA expression of the IGFBP-3. The Western blot was used to confirm the protein expression. Immunohistochemistry was also performed to identify the cellular localization of the encoded protein. The percentage of smooth muscle in corpus cavernosum tissue, the activity of nitric oxide synthase (NOS), and concentration of cGMP in penile tissue were also analyzed. The expression levels of IGFBP-3 of mRNA and protein were greatly increased in aging rats compared with young control rats, which is confirmed by traditional RT-PCR, real-time PCR, and Western blot (P < 0.01, respectively). Increased IGFBP-3 protein was localized to the epithelium of the urethra, penile endothelium, and smooth muscle in the corpus cavernosum. Significant depletion of the smooth muscle density relative to the connective tissue was also observed in the penis of the aged rats, and the lower activity of NOS and lower concentration of cGMP was also demonstrated accompanied with a significant reduction in the intracavernous pressure. Our data suggest that the increased mRNA and protein expression of IGFBP-3 in old rats may

  11. Sex-dependent expression of mRNA encoding a major egg protein in the gonochoric coral Galaxea fascicularis

    NASA Astrophysics Data System (ADS)

    Hayakawa, H.; Nakano, Y.; Andoh, T.; Watanabe, T.

    2005-11-01

    A cDNA encoding a major egg protein was cloned in Galaxea fascicularis, a hermatypic coral with a gonochoric breeding system, and gene expression at the transcriptional level was compared between female and functional male colonies. In an electrophoretic analysis, four soluble proteins were present in high abundance in the female egg, but not in the pseudo-eggs of functional males. Partial amino acid sequences of one of the major proteins named GfEP-1 (88 kDa) were determined, and a cDNA fragment of about 2 kb containing a partial GfEP-1 sequence was isolated. The deduced amino acid sequence exhibited sequence similarities to vertebrate and invertebrate vitellogenins. GfEP-1 transcripts were detected in both sexes 0 1 month before spawning. However, the mRNA levels were significantly higher in females than in functional males. The expression of GfEP-1 may be utilized in sexing and also monitoring effects of environmental and anthropogenic factors on vitellogenesis and sex determination.

  12. [In vitro effect of total flavones of Fructus Chorspondiatis on expression of collagen type I and type III mRNA and protein of cultured rat cardiac fibroblasts].

    PubMed

    Bao, Jun-Ping; Jin, Ming; Yang, Yu-Min; Gao, Xiao-Hui; Shu, Liang; Xing, Hui-Hui; Jia, Lei

    2014-01-01

    This study aims to investigate the effect of total flavones of Fructus Chorspondiatis (TFFC) on the mRNA and protein expression of collagen type I and III of rat cardiac fibroblasts (CFs) induced by angiotensin II (Ang II), and explore its anti-myocardial fibrosis molecular mechanism. Neonatal rat CFs were prepared from Sprague-Dawley rats (1-3 d after birth). The expression of collagen type I and III mRNA and protein were measured by RT-PCR and Western blotting, respectively. The study showed that stimulation of neonatal rat CFs with 100 nmol.L-1 of Ang II for 72 h resulted in a significant increase of the expression of collagen type I and III mRNA and protein. The changes on the expression level were blocked by TFFC. The results demonstrated that TFFC can inhibit myocardial fibrosis induced by Ang II in rats, which is probably associated with the collagen type I and III mRNA and protein levels up-regulated by Ang II, and TFFC was shown to decrease the expression levels of collagen type I and III mRNA and protein.

  13. Glyceraldehyde 3-phosphate dehydrogenase protein and mRNA are both differentially expressed in adult chickens but not chick embryos.

    PubMed Central

    Milner, R J; Brow, M D; Cleveland, D W; Shinnick, T M; Sutcliffe, J G

    1983-01-01

    We have determined the 679 nucleotide sequence of a cDNA clone which, by hybridization-translation experiments, corresponds to a 36K chick brain protein. Our studies provide a partial amino acid sequence for this protein, identifying it as chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Antisera raised against purified chicken GAPDH reacted with a 36K protein present in chick brain extracts and estimated to be the fourth most prevalent protein, as determined by either Coomassie Blue staining or by in vitro translation of chick brain mRNA. The amounts of GAPDH mRNA in chick brain, liver and muscle and adult chicken brain are similar, whereas the relative amount of adult chicken muscle GPDH mRNA is greatly elevated and that of adult liver lowered. The GAPDH protein levels showed a similar variation between tissues, suggesting that the levels of GAPDH protein are largely regulated by the amount of available GAPDH mRNA. The chicken GAPDH clone does not hybridize to rat mRNA, even though GAPDH is one of the most evolutionarily conserved proteins, indicating that selection pressures are heavier at the primary protein sequence level than at the nucleic acid sequence level for this gene, a situation contrasting to that of the tubulins. Images PMID:6687938

  14. Morphine-induced place preference affects mRNA expression of G protein α subunits in rat brain.

    PubMed

    Zelek-Molik, Agnieszka; Bielawski, Adam; Kreiner, Grzegorz; Popik, Piotr; Vetulani, Jerzy; Nalepa, Irena

    2012-01-01

    The conditioned place preference (CPP) test is an animal model serving to assess addictive potential of drugs in which environmental cues become associated with the subjective effects of drugs of abuse. Morphine, a known addictive drug, is an agonist of opioid receptors that couple to the G(i/o) family of guanine nucleotide-binding proteins (GP). We have recently found that chronic treatment with morphine affects mRNA levels of GPs that are not coupled to opioid receptors (OR). Therefore, in this study, we investigated the influence of morphine-induced CPP on mRNA expression of the Gα subunits, G(i/o), G(s), G(q/11), and G(12), in the rat prefrontal cortex (PFC) and nucleus accumbens (NAc) using standard PCR techniques. CPP and NO-CPP experiments were conducted; Wistar rats were either subjected to the standard CPP procedure or were injected with morphine (or saline) in their home cage. All rats were decapitated 24 h after the last injection. We found that mRNA levels of Gα(q), Gα(11) and Gα(12) were increased after morphine in non-conditioned treatment in the PFC but remained unchanged in the NAc. In rats showing conditioned place preference to morphine, levels of Gα(i2) in the PFC and levels of Gα(oA) in the NAc were diminished by ≈58% and ≈30%, respectively (p < 0.05 vs. saline), but levels of Gα(s-l) in NAc were increased (≈60%, p = 0.05). Our data indicate that only G(i/o) and G(s) were specifically changed in animals after morphine-induced CPP, thus suggesting that the effect was related to learning environmental cues associated with morphine.

  15. Imbalanced Expression of Vcan mRNA Splice Form Proteins Alters Heart Morphology and Cellular Protein Profiles

    PubMed Central

    Burns, Tara A.; Dours-Zimmermann, Maria T.; Zimmermann, Dieter R.; Krug, Edward L.; Comte-Walters, Susana; Reyes, Leticia; Davis, Monica A.; Schey, Kevin L.; Schwacke, John H.; Kern, Christine B.; Mjaatvedt, Corey H.

    2014-01-01

    The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart’s pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican’s expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan(tm1Zim), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan(tm1Zim) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles. PMID:24586547

  16. Expression of NK1 receptor at the protein and mRNA level in the porcine female reproductive system.

    PubMed

    Bukowski, R

    2014-01-01

    The presence and distribution of substance P (SP) receptor NK1 was studied in the ovary, the oviduct and the uterus (uterine horn and cervix) of the domestic pig using the methods of molecular biology (RT-PCR and immunoblot) and immunohistochemistry. The expression of NK1 receptor at mRNA level was confirmed with RT-PCR in all the studied parts of the porcine female reproductive system by the presence of 525 bp PCR product and at the protein level by the detection of 46 kDa protein band in immunoblot. Immunohistochemical staining revealed the cellular distribution of NK1 receptor protein. In the ovary NKI receptor was present in the wall of arterial blood vessels, as well as in ovarian follicles of different stages of development. In the tubular organs the NK1 receptor immunohistochemical stainings were observed in the wall of the arterial blood vessels, in the muscular membrane, as well as in the mucosal epithelium. The study confirmed the presence of NK1 receptor in the tissues of the porcine female reproductive tract which clearly points to the possibility that SP can influence porcine ovary, oviduct and uterus.

  17. Correlative mRNA and protein expression of middle and inner ear inflammatory cytokines during mouse acute otitis media.

    PubMed

    Trune, Dennis R; Kempton, Beth; Hausman, Frances A; Larrain, Barbara E; MacArthur, Carol J

    2015-08-01

    Although the inner ear has long been reported to be susceptible to middle ear disease, little is known of the inflammatory mechanisms that might cause permanent sensorineural hearing loss. Recent studies have shown inner ear tissues are capable of expressing inflammatory cytokines during otitis media. However, little quantitative information is available concerning cytokine gene expression in the inner ear and the protein products that result. Therefore, this study was conducted of mouse middle and inner ear during acute otitis media to measure the relationship between inflammatory cytokine genes and their protein products with quantitative RT-PCR and ELISA, respectively. Balb/c mice were inoculated transtympanically with heat-killed Haemophilus influenzae and middle and inner ear tissues collected for either quantitative RT-PCR microarrays or ELISA multiplex arrays. mRNA for several cytokine genes was significantly increased in both the middle and inner ear at 6 h. In the inner ear, these included MIP-2 (448 fold), IL-6 (126 fold), IL-1β (7.8 fold), IL-10 (10.7 fold), TNFα (1.8 fold), and IL-1α (1.5 fold). The 24 h samples showed a similar pattern of gene expression, although generally at lower levels. In parallel, the ELISA showed the related cytokines were present in the inner ear at concentrations higher by 2-122 fold higher at 18 h, declining slightly from there at 24 h. Immunohistochemistry with antibodies to a number of these cytokines demonstrated they occurred in greater amounts in the inner ear tissues. These findings demonstrate considerable inflammatory gene expression and gene products in the inner ear following acute otitis media. These higher cytokine levels suggest one potential mechanism for the permanent hearing loss seen in some cases of acute and chronic otitis media.

  18. Correlative mRNA and Protein Expression of Middle and Inner Ear Inflammatory Cytokines during Mouse Acute Otitis Media

    PubMed Central

    Trune, Dennis R.; Kempton, Beth; Hausman, Frances A.; Larrain, Barbara E.; MacArthur, Carol J.

    2015-01-01

    Although the inner ear has long been reported to be susceptible to middle ear disease, little is known of the inflammatory mechanisms that might cause permanent sensorineural hearing loss. Recent studies have shown inner ear tissues are capable of expressing inflammatory cytokines during otitis media. However, little quantitative information is available concerning cytokine gene expression in the inner ear and the protein products that result. Therefore, this study was conducted of mouse middle and inner ear during acute otitis media to measure the relationship between inflammatory cytokine genes and their protein products with quantitative RT-PCR and ELISA, respectively. Balb/c mice were inoculated transtympanically with heat-killed Haemophilus influenzae and middle and inner ear tissues collected for either quantitative RT-PCR microarrays or ELISA multiplex arrays. mRNA for several cytokine genes was significantly increased in both the middle and inner ear at 6 hours. In the inner ear, these included MIP-2 (448 fold), IL-6 (126 fold), IL-1β (7.8 fold), IL-10 (10.7 fold), TNFα (1.8 fold), and IL-1α (1.5 fold). The 24 hour samples showed a similar pattern of gene expression, although generally at lower levels. In parallel, the ELISA showed the related cytokines were present in the inner ear at concentrations higher by 2 to 122 fold higher at 18 hours, declining slightly from there at 24 hours. Immunohistochemistry with antibodies to a number of these cytokines demonstrated they occurred in greater amounts in the inner ear tissues. These findings demonstrate considerable inflammatory gene expression and gene products in the inner ear following acute otitis media. These higher cytokine levels suggest one potential mechanism for the permanent hearing loss seen in some cases of acute and chronic otitis media. PMID:25922207

  19. Integration of MicroRNA, mRNA, and Protein Expression Data for the Identification of Cancer-Related MicroRNAs

    PubMed Central

    Seo, Jiyoun; Jin, Daeyong; Choi, Chan-Hun; Lee, Hyunju

    2017-01-01

    MicroRNAs (miRNAs) are responsible for the regulation of target genes involved in various biological processes, and may play oncogenic or tumor suppressive roles. Many studies have investigated the relationships between miRNAs and their target genes, using mRNA and miRNA expression data. However, mRNA expression levels do not necessarily represent the exact gene expression profiles, since protein translation may be regulated in several different ways. Despite this, large-scale protein expression data have been integrated rarely when predicting gene-miRNA relationships. This study explores two approaches for the investigation of gene-miRNA relationships by integrating mRNA expression and protein expression data. First, miRNAs were ranked according to their effects on cancer development. We calculated influence scores for each miRNA, based on the number of significant mRNA-miRNA and protein-miRNA correlations. Furthermore, we constructed modules containing mRNAs, proteins, and miRNAs, in which these three molecular types are highly correlated. The regulatory interactions between miRNA and genes in these modules have been validated based on the direct regulations, indirect regulations, and co-regulations through transcription factors. We applied our approaches to glioblastomas (GBMs), ranked miRNAs depending on their effects on GBM, and obtained 52 GBM-related modules. Compared with the miRNA rankings and modules constructed using only mRNA expression data, the rankings and modules constructed using mRNA and protein expression data were shown to have better performance. Additionally, we experimentally verified that miR-504, highly ranked and included in the identified modules, plays a suppressive role in GBM development. We demonstrated that the integration of both expression profiles allows a more precise analysis of gene-miRNA interactions and the identification of a higher number of cancer-related miRNAs and regulatory mechanisms. PMID:28056026

  20. Sequence and developmental expression of mRNA coding for a gap junction protein in Xenopus

    PubMed Central

    1988-01-01

    Cloned complementary DNAs representing the complete coding sequence for an embryonic gap junction protein in the frog Xenopus laevis have been isolated and sequenced. The cDNAs hybridize with an RNA of 1.5 kb that is first detected in gastrulating embryos and accumulates throughout gastrulation and neurulation. By the tailbud stage, the highest abundance of the transcript is found in the region containing ventroposterior endoderm and the rudiment of the liver. In the adult, transcripts are present in the lungs, alimentary tract organs, and kidneys, but are not detected in the brain, heart, body wall and skeletal muscles, spleen, or ovary. The gene encoding this embryonic gap junction protein is present in only one or a few copies in the frog genome. In vitro translation of RNA synthesized from the cDNA template produces a 30-kD protein, as predicted by the coding sequence. This product has extensive sequence similarity to mammalian gap junction proteins in its putative transmembrane and extracellular domains, but has diverged substantially in two of its intracellular domains. PMID:2843548

  1. OIL FLY ASH AND VANADIUM DIMINISH NRAMP-2MRNA AND PROTEIN EXPRESSION IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    The capacity of Nramp2 to transport iron and its ubiquitous expression make it a likely candidate for transferrin-independent uptake of iron in peripheral tissues. Airway epithelial cells increase both mRNA and expression of that isoform of Nramp-2 without an iron response ele...

  2. Expression of mRNA of apolipoprotein E, apolipoprotein A-IV, and matricellular proteins in the myocardium and intensity of fibroplastic processes during experimental hypercholesterolemia.

    PubMed

    Lushnikova, E L; Nepomnyashchikh, L M; Pichigin, V I; Klinnikova, M G; Nepomnyashchikh, R D; Sergeevichev, D S

    2013-12-01

    The expression of mRNA of matricellular proteins (osteopontin, and lumican), apolipoproteins E and A-IV, and microsomal triglyceride transfer protein, and the intensity of fibroplastic processes were studied in the myocardium of rats during experimental chronic hypercholesterolemia. We have found that the development of chronic hypercholesterolemia was followed by an increase in volume density of interstitial connective tissue in the myocardium reflecting the activation of fibroplastic processes. A slight positive correlation was observed between the connective tissue density in the myocardium and expression of osteopontin mRNA (r=0.408) and lumican mRNA (r=0.470). Myocardium remodeling during hypercholesterolemia is realized against the background of increased expression of apolipoproteins E and A-IV mRNA and microsomal triglyceride transfer protein mRNA involved in transport and metabolism of lipoproteins in several tissues and probably play a pivotal role in the regulation of lipoprotein transport and metabolism in the myocardium. We concluded that the increase in the expression of apolipoproteins (E and A-IV) and microsomal triglyceride transfer protein play adaptive and compensatory role and is related to the increase in lipoprotein utilization by macrophages.

  3. The Expression Level of mRNA, Protein, and DNA Methylation Status of FOSL2 of Uyghur in XinJiang in Type 2 Diabetes

    PubMed Central

    Cao, Guolei; Wang, Xiaoli

    2016-01-01

    Objective. We investigated the expression levels of both FOSL2 mRNA and protein as well as evaluating DNA methylation in the blood of type 2 diabetes mellitus (T2DM) Uyghur patients from Xinjiang. This study also evaluated whether FOSL2 gene expression had demonstrated any associations with clinical and biochemical indicators of T2DM. Methods. One hundred Uyghur subjects where divided into two groups, T2DM and nonimpaired glucose tolerance (NGT) groups. DNA methylation of FOSL2 was also analyzed by MassARRAY Spectrometry and methylation data of individual units were generated by the EpiTyper v1.0.5 software. The expression levels of FOS-like antigen 2 (FOSL2) and the protein expression levels were analyzed. Results. Significant differences were observed in mRNA and protein levels when compared with the NGT group, while methylation rates of eight CpG units within the FOSL2 gene were higher in the T2DM group. Methylation of CpG sites was found to inversely correlate with expression of other markers. Conclusions. Results show that a correlation between mRNA, protein, and DNA methylation of FOSL2 gene exists among T2DM patients from Uyghur. FOSL2 protein and mRNA were downregulated and the DNA became hypermethylated, all of which may be involved in T2DM pathogenesis in this population. PMID:28050569

  4. Expression of mRNA Encoding the LH Receptor (LHR) and LHR Binding Protein in Granulosa Cells from Nelore (Bos indicus) Heifers Around Follicle Deviation.

    PubMed

    Ereno, R L; Loureiro, B; Castilho, A C S; Machado, M F; Pegorer, M F; Satrapa, R A; Nogueira, M F G; Buratini, J; Barros, C M

    2015-12-01

    The time at which follicles acquire LHR in bovine granulosa cells is the subject of some controversy among researchers. The main objective of the present study was to assess the mRNA expression of LHR and LRBP (mRNA protein binding), a post-transcriptional suppressor of LHR mRNA expression, in granulosa cells from the two largest follicles around the expected time of follicle deviation in Nelore heifers. First, the interval between ovulation and follicle deviation in 20 Nelore heifers was determined (2.3 ± 0.2 days after ovulation). Ovulation was hormonally synchronized, and then, heifers were slaughtered on days 2, 2.5 and 3 after ovulation (before, during and after, respectively, the expected time of follicle deviation), and granulosa cells from the two largest follicles were collected. The mRNA abundance of an LHR fragment common to all isoforms (total LHR) and LRBP was assessed by real-time RT-PCR, and LHR alternative transcripts were assessed by semiquantitative RT-PCR followed by electrophoresis. LHR mRNA expression was not detected before the expected time of deviation. Total LHR mRNA abundance was greater in the largest follicle and increased from day 2.5 to 3. In contrast, LRBP mRNA was detected starting on day 2 and was more expressed in the second largest follicle on days 2.5 and 3. The present data suggest that the expression of LHR mRNA in bovine granulosa cells is established after follicle deviation and that the lower abundance of LRBP mRNA after the expected time of deviation may contribute to greater expression of LHR in the bovine dominant follicle. © 2015 Blackwell Verlag GmbH.

  5. Evaluation of DNA methylation and mRNA expression of heat shock proteins in thermal manipulated chicken.

    PubMed

    Vinoth, A; Thirunalasundari, T; Shanmugam, M; Uthrakumar, A; Suji, S; Rajkumar, U

    2017-08-25

    Thermal manipulation during embryogenesis has been demonstrated to enhance the thermotolerance capacity of broilers through epigenetic modifications. Heat shock proteins (HSPs) are induced in response to stress for guarding cells against damage. The present study investigates the effect of thermal conditioning during embryogenesis and thermal challenge at 42 days of age on HSP gene and protein expression, DNA methylation and in vitro luciferase assay in brain tissue of Naked Neck (NN) and Punjab Broiler-2 (PB-2) chicken. On the 15th day of incubation, fertile eggs from two breeds, NN and PB-2, were randomly divided in to two groups: control (C)-eggs were incubated under standard incubation conditions, and thermal conditioning (TC)-eggs were exposed to higher incubation temperature (40.5°C) for 3 h on the 15th, 16th, and 17th days of incubation. The chicks obtained from each group were further subdivided and reared under different environmental conditions from the 15th to the 42nd day as normal [N; 25 ± 1 °C, 70% relative humidity (RH)] and heat exposed (HE; 35 ± 1 °C, 50% RH) resulting in four treatment groups (CN, CHE, TCN, and TCHE). The results revealed that HSP promoter activity was stronger in CHE, which had lesser methylation and higher gene expression. The activity of promoter region was lesser in TCHE birds that were thermally manipulated at the embryonic stage, thus reflecting their stress-free condition. This was confirmed by the lower level of mRNA expression of all the HSP genes. In conclusion, thermal conditioning during embryogenesis has a positive impact and improves chicken thermotolerance capacity in postnatal life.

  6. [Expressions and clinical significance of high mobility group box-1 mRNA and protein in laryngeal squamous cell carcinoma tissues and serum].

    PubMed

    Guo, Ying; Liu, Yong; Tan, Ping-qing; Li, Guo; Su, Zhong-wu; Tian, Yong-quan; Qiu, Yuan-zheng

    2012-06-01

    To investigate the mRNA and protein expressions of high mobility group box-1 (HMGB1) in the tumor tissues and sera of patients with laryngeal squamous cell carcinoma (LSCC) and their clinical significance. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were used to detected the expressions of HMGB1 mRNA and protein in the tumors and adjacent normal epithelial tissues in 30 patients with LSCC. Serum HMGB1 protein levels in the patients with LSCC and in 10 healthy volunteers were detected with enzyme-linked immunosorbent adsorption experiment (ELISA). RT-PCR demonstrated that the mean relative mRNA expression levels of HMGB1 (HMGB1/GAPDH) in LSCC tissues and in adjacent normal epithelial tissues were 1.25 ± 0.12 and 0.32 ± 0.04, respectively (t = 40.27, P < 0.05). Western blot revealed that the mean relative protein expression levels of HMGB1 (HMGB1/β-actin) were 1.29 ± 0.10 and 0.34 ± 0.03 (t = 49.84, P < 0.05), respectively. Both mRNA and protein expression levels of HMGB1 were associated with T stage, clinical stage, lymph node metastasis status and smoking (all P < 0.05), but no significant correlation with age, alcohol consumption and primary tumor grade and location (all P > 0.05). Mean serum HMGB1 protein levels in patients with LSCC and healthy volunteers were (24.80 ± 14.08) ng/ml and (23.58 ± 14.69) ng/ml (t = 0.37, P > 0.05). Both mRNA and protein expressions of HMGB1 were obviously elevated in LSCC, which were associated closely with T stage, clinical stage and lymph node metastasis.

  7. Disorders in barrier protein mRNA expression and placenta secretory activity under the influence of polychlorinated biphenyls in vitro.

    PubMed

    Wojciechowska, A; Mlynarczuk, J; Kotwica, J

    2017-02-01

    Pregnancy disorders are often correlated with the presence of organic pollutants in the tissues of living bodies. The aim of this study was to investigate the effects (over 24 and 48 hours) of polychlorinated biphenyls (PCBs) 153, 126, and 77 at doses of 1, 10, and 100 ng/mL on barrier function and secretory activity in cow placentome sections collected during the second trimester of pregnancy. None of the PCBs affected the viability of the sections (P > 0.05). Polychlorinated biphenyl 153 decreased (P < 0.05) connexin 26 (Cx 26) mRNA expression, and all three PCBs reduced (P < 0.05) Cx 43 mRNA expression. Cx 32 mRNA expression showed a downward trend (P > 0.05) under the influence of PCBs 126 and 77. Moreover, PCBs 153 and 126 increased keratin 8 (KRT8) mRNA expression, whereas all PCBs decreased (P < 0.05) placenta specific protein 1 (PLAC-1) mRNA expression without changing (P > 0.05) hypoxia inducible factor 1α (HIF1α) mRNA expression. Concomitantly, PCBs 153 and 126 stimulated (P < 0.05) cyclooxygenase 2 (COX-2) mRNA expression, all PCBs increased (P < 0.05) prostaglandin E2 synthase (PGES) mRNA expression, and PCBs 126 and 77 increased prostaglandin E2 (PGE2) secretion. All three PCBs decreased (P < 0.05) prostaglandin F2α synthase (PGFS) mRNA expression and prostaglandin F2α (PGF2α) secretion. In addition, all three PCBs increased (P < 0.05) neurophysin I/oxytocin (NP-I/OT) mRNA expression and OT secretion but did not affect peptidyl-glycine-α-amidating monooxygenase (PGA) mRNA expression (P > 0.05). Moreover, the PCBs increased (P < 0.05) estradiol (E2) secretion, whereas progesterone (P4) secretion remained unchanged (P > 0.05). These changes could affect trophoblast invasion and uterine contractility and thus impact the course of gestation and/or fetal development in the cow.

  8. FGFR1 mRNA and protein expression, not gene copy number, predict FGFR TKI sensitivity across all lung cancer histologies.

    PubMed

    Wynes, Murry W; Hinz, Trista K; Gao, Dexiang; Martini, Michael; Marek, Lindsay A; Ware, Kathryn E; Edwards, Michael G; Böhm, Diana; Perner, Sven; Helfrich, Barbara A; Dziadziuszko, Rafal; Jassem, Jacek; Wojtylak, Szymon; Sejda, Aleksandra; Gozgit, Joseph M; Bunn, Paul A; Camidge, D Ross; Tan, Aik-Choon; Hirsch, Fred R; Heasley, Lynn E

    2014-06-15

    FGFR1 gene copy number (GCN) is being evaluated as a biomarker for FGFR tyrosine kinase inhibitor (TKI) response in squamous cell lung cancers (SCC). The exclusive use of FGFR1 GCN for predicting FGFR TKI sensitivity assumes increased GCN is the only mechanism for biologically relevant increases in FGFR1 signaling. Herein, we tested whether FGFR1 mRNA and protein expression may serve as better biomarkers of FGFR TKI sensitivity in lung cancer. Histologically diverse lung cancer cell lines were submitted to assays for ponatinib sensitivity, a potent FGFR TKI. A tissue microarray composed of resected lung tumors was submitted to FGFR1 GCN, and mRNA analyses and the results were validated with The Cancer Genome Atlas (TCGA) lung cancer data. Among 58 cell lines, 14 exhibited ponatinib sensitivity (IC50 values ≤ 50 nmol/L) that correlated with FGFR1 mRNA and protein expression, but not with FGFR1 GCN or histology. Moreover, ponatinib sensitivity associated with mRNA expression of the ligands, FGF2 and FGF9. In resected tumors, 22% of adenocarcinomas and 28% of SCCs expressed high FGFR1 mRNA. Importantly, only 46% of SCCs with increased FGFR1 GCN expressed high mRNA. Lung cancer TCGA data validated these findings and unveiled overlap of FGFR1 mRNA positivity with KRAS and PIK3CA mutations. FGFR1 dependency is frequent across various lung cancer histologies, and FGFR1 mRNA may serve as a better biomarker of FGFR TKI response in lung cancer than FGFR1 GCN. The study provides important and timely insight into clinical testing of FGFR TKIs in lung cancer and other solid tumor types. ©2014 American Association for Cancer Research.

  9. Posttranscriptional regulation of hsp70 expression in human cells: effects of heat shock, inhibition of protein synthesis, and adenovirus infection on translation and mRNA stability.

    PubMed Central

    Theodorakis, N G; Morimoto, R I

    1987-01-01

    We have examined the posttranscriptional regulation of hsp70 gene expression in two human cell lines, HeLa and 293 cells, which constitutively express high levels of HSP70. HSP70 mRNA translates with high efficiency in both control and heat-shocked cells. Therefore, heat shock is not required for the efficient translation of HSP70 mRNA. Rather, the main effect of heat shock on translation is to suppress the translatability of non-heat shock mRNAs. Heat shock, however, has a marked effect on the stability of HSP70 mRNA; in non-heat-shocked cells the half-life of HSP70 mRNA is approximately 50 min, and its stability increases at least 10-fold upon heat shock. Moreover, HSP70 mRNA is more stable in cells treated with protein synthesis inhibitors, suggesting that a heat shock-sensitive labile protein regulates its turnover. An additional effect on posttranscriptional regulation of hsp70 expression can be found in adenovirus-infected cells, in which HSP70 mRNA levels decline precipititously late during infection although hsp70 transcription continues unabated. Images PMID:3437893

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

    PubMed

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

    2005-09-12

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

  11. The effect of GABA stimulation on GABAA receptor subunit protein and mRNA expression in rat cultured cerebellar granule cells.

    PubMed Central

    Platt, K. P.; Zwartjes, R. E.; Bristow, D. R.

    1996-01-01

    1. After 8 days in vitro, rat cerebellar granule cells were exposed to 1 mM gamma-aminobutyric acid (GABA) for periods of 1, 2, 4, 6, 8 and 10 days. The effect of the GABA exposure on GABAA receptor alpha 1, alpha 6 and beta 2,3 subunit protein expression and alpha 1 and alpha 6 subunit steady-state mRNA levels, was examined using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. 2. GABA exposure for 2 days decreased alpha 1 (35 +/- 10%, mean +/- s.e.mean), beta 2,3 (21 +/- 9%) and alpha 6 (28 +/- 10%) subunit protein expression compared to control levels. The GABA-mediated reduction in alpha 1 subunit expression after 2 days treatment was abolished in the presence of the GABAA receptor antagonist, Ru 5135 (10 microM). 3. GABA exposure for 8 days increased alpha 1 (26 +/- 10%, mean +/- s.e.mean) and beta 2,3 (56 +/- 23%) subunit protein expression over control levels, whereas alpha 6 subunit protein expression remained below control levels (by 38 +/- 10%). However, after 10 days GABA exposure, alpha 6 subunit protein expression was also increased over control levels by 65 +/- 29% (mean +/- s.e.mean). 4. GABA exposure did not change the alpha 1 or alpha 6 subunit steady-state mRNA levels over and 8 day period, nor did it alter the expression of cyclophilin mRNA over 1-8 days. 5. These results suggest that chronic GABA exposure of rat cerebellar granule cells has a bi-phasic effect on GABAA receptor subunit expression that is independent of changes to mRNA levels. Therefore, the regulation of the GABAA receptor expression by chronic agonist treatment appears to involve post-transcriptional and/or post-translational processes. Images Figure 1 Figure 3 Figure 4 PMID:8968548

  12. Effects of cold-water immersion on VEGF mRNA and protein expression in heart and skeletal muscles of rats.

    PubMed

    Kim, J C; Yi, H K; Hwang, P H; Yoon, J S; Kim, H J; Kawano, F; Ohira, Y; Kim, C K

    2005-04-01

    The effects of cold exposure on gene and protein expression of vascular endothelial growth factor (VEGF), in heart and skeletal muscles, were studied in male adult Wistar rats. Cold immersion was accomplished by submerging the rats in shoulder-deep water maintained at approximately 18 degrees C, either acutely (1 h) or chronically (1 h day(-1), 5 days week(-1) for 20 weeks). The expressions of VEGF mRNA and protein in heart, gastrocnemius, and soleus muscles were examined by Northern and Western blotting and competitive-polymerase chain reaction techniques. The expressions of VEGF mRNA and protein were markedly increased in cardiac muscle of the cold-immersed group, particularly in the 1-hour exposure group, whereas VEGF mRNA and protein in gastrocnemius were decreased significantly after an acute exposure. Although the protein level in gastrocnemius remained low in the chronically exposed group, the expression of mRNA of VEGF(165) with chronic exposure in this group returned to the control level and that of VEGF(206) was 15% greater than that in controls. The expression of mRNA for VEGF(165) in soleus was also lowered by acute cold exposure, although that for VEGF(206) was stable. However, VEGF protein was increased by 50%. After 20 weeks, all of these parameters were increased over the levels found in the controls. These results suggest that the VEGF gene may be a major regulatory factor in cardiac and skeletal muscle adaptation to the cold environment stimulating angiogenesis and thermogenesis.

  13. Comparison of the ovarian and uterine reproductive parameters, and the ovarian mRNA and protein expression of LHR and FSHR between the prepubertal and adult female cats.

    PubMed

    Mehl, N S; Khalid, M; Srisuwatanasagul, S; Swangchan-Uthai, T; Sirivaidyapong, S

    2017-04-01

    This study aimed to evaluate and compare the ovarian and uterine characteristics along with the ovarian mRNA and protein expression of LHR and FSHR between the pre-pubertal and adult female cats. The uterine horns and ovaries were collected from pre-pubertal and adult female cats at their follicular, luteal and interoestrous stages of the oestrous cycle (n = 6/group). Endometrial and myometrial thickness, uterine gland diameter, ovarian weight and type of follicles were analysed. The mRNA and protein expression of LHR and FSHR was analysed by IHC and qPCR, respectively. The ovarian weight of pre-pubertal cats was significantly lower than that of adult cats. No differences were recorded in the numbers of primordial and primary follicles between the study groups, while adult luteal cats had significantly lower numbers of antral follicles compared to pre-pubertal cats. No differences in the ovarian expression of FSHR mRNA, LHR protein or mRNA were found between the pre-pubertal and adult cats, but significantly lower FSHR protein expression was found in pre-pubertal cats compared to adult luteal cats. © 2017 Blackwell Verlag GmbH.

  14. Effects of breeds and dietary protein levels on the growth performance, energy expenditure and expression of avUCP mRNA in chickens.

    PubMed

    Li, Qihua; Xu, Zhiqiang; Liu, L; Yu, Hongxin; Rong, Hua; Tao, Linli; Zhang, Xi; Chen, Xiaobo; Gu, Dahai; Fan, Yueyuan; Li, Xiaoqin; Ge, Changrong; Tian, Yunbo; Jia, Junjing

    2013-04-01

    The physiological mechanisms of thermogenesis, energy balance and energy expenditure are poorly understood in poultry. The aim of this study was designed to investigate the physiological roles of avian uncoupling protein (avUCP) regulating in energy balance and thermogenesis by using three chicken breeds of existence striking genetic difference and feeding with different dietary protein levels. Three chicken breeds including broilers, hybrid chickens, and non-selection Wuding chickens were used in this study. Total 150 chicks of 1 day of age, with 50 from each breed were reared under standard conditions on starter diets to 30 days. At 30 days of age, forty chicks from each breed chicks were divided into two groups. One group was fed low protein diet (LP, 17.0 %), and the other group was fed high protein diet (HP, 19.5 %) for 60 days. Wuding chickens showed the lowest feed conversion efficiency (FCE) and the highest expressions of avUCP mRNA association with high plasma T3 and insulin concentrations. Hybrid chickens showed the lowest expressions of avUCP mRNA association with high FCE and energy efficiency. Expressions of avUCP mRNA association with diet-induced thermogenesis (DIT) were only observed in broiler and hybrid chickens. The expressions of avUCP mRNA were positive association with plasma insulin, T3 and NEFA concentrations. Age influence on the expression of avUCP mRNA were observed only for hybrid and broiler chickens. It seems that both roles of avUCP regulation thermogenesis and lipid utilisation as fuel were observed in the present study response to variation in dietary protein and breeds.

  15. MEIS1 intronic risk haplotype associated with restless legs syndrome affects its mRNA and protein expression levels

    PubMed Central

    Xiong, Lan; Catoire, Hélène; Dion, Patrick; Gaspar, Claudia; Lafrenière, Ronald G.; Girard, Simon L.; Levchenko, Anastasia; Rivière, Jean-Baptiste; Fiori, Laura; St-Onge, Judith; Bachand, Isabelle; Thibodeau, Pascale; Allen, Richard; Earley, Christopher; Turecki, Gustavo; Montplaisir, Jacques; Rouleau, Guy A.

    2009-01-01

    Restless legs syndrome (RLS) is a common neurological disorder characterized by an irresistible urge to move the legs at night, which is often accompanied by unpleasant sensations. A recent genomewide association study identified an association between RLS and intronic markers from the MEIS1 gene. Comparative genomic analysis indicates that MEIS1 is the only gene encompassed in this evolutionarily conserved chromosomal segment, i.e. a conservation synteny block, from mammals to fish. We carried out a series of experiments to delineate the role of MEIS1 in RLS pathogenesis and the underlying genetic mechanism. We sequenced all 13 MEIS1 exons and their splice junctions in 285 RLS probands with confirmed clinical diagnosis and did not identify any causative coding or exon–intron junction mutations. We found no evidence of structural variation or disease-associated haplotype differential splicing. However, sequencing of conserved regions of MEIS1 introns 8 and 9 identified a novel single nucleotide polymorphism (C13B_2) significantly associated with RLS (allelic association, P = 1.81E−07). We detected a significant decrease in MEIS1 mRNA expression by quantitative real-time polymerase chain reaction in lymphoblastoid cell lines (LCLs) and brain tissues from RLS patients homozygous for the intronic RLS risk haplotype, compared with those homozygous for the non-risk haplotype. Finally, we found significantly decreased MEIS1 protein levels in the same batch of LCLs and brain tissues from the homozygous carriers of the risk haplotype, compared with the homozygous non-carriers. Therefore, these data suggest that reduced expression of the MEIS1 gene, possibly through intronic cis-regulatory element(s), predisposes to RLS. PMID:19126776

  16. In phyllodes tumors of the breast expression of SPARC (osteonectin/BM40) mRNA by in situ hybridization correlates with protein expression by immunohistochemistry and is associated with tumor progression.

    PubMed

    Kim, Nah Ihm; Kim, Ga-Eon; Lee, Ji Shin; Park, Min Ho

    2017-01-01

    Secreted protein acidic and rich in cysteine (SPARC) plays an essential role in tumor invasion and metastasis. The present work was undertaken to detect expression of SPARC mRNA in phyllodes tumors (PTs) and its association with SPARC protein expression. This study also evaluated expression of SPARC mRNA and its correlation between grade and clinical behavior of PTs. In addition, we assessed in PTs the association of expression of SPARC with that of matrix metalloproteinase (MMP)-2 and of MMP-9. SPARC mRNA expression was determined by RNAscope in situ hybridization (ISH) in 50 benign, 22 borderline, and 10 malignant PTs using a tissue microarray. Furthermore, we applied immunohistochemistry (IHC) to examine expression of SPARC, MMP-2, and MMP-9. SPARC mRNA appeared to be concentrated mainly in the stromal compartment of PTs. IHC staining patterns of SPARC protein showed concordance with SPARC mRNA ISH results. Stromal SPARC expression increased continuously as PTs progress from benign through borderline to malignant PTs, both at mRNA (using ISH) (P = 0.044) and protein level (using IHC) (P = 0.000). The recurrence percentage was higher in the stromal SPARC mRNA or protein-positive group than in the SPARC-negative group but this difference was not statistically significant. Stromal SPARC mRNA and protein expression was associated with PT grade and correlated with MMP-2 expression. These results indicate that SPARC-mediated degradation of the extracellular matrix, and its possible association with MMPs, might contribute to progression of PTs.

  17. Enhanced expression of mRNA for FK506-binding protein 5 in bone marrow CD34 positive cells in patients with rheumatoid arthritis.

    PubMed

    Matsushita, R; Hashimoto, A; Tomita, T; Yoshitawa, H; Tanaka, S; Endo, H; Hirohata, S

    2010-01-01

    Recent studies have disclosed that several genes are up-regulated in bone marrow (BM) mononuclear cells from rheumatoid arthritis (RA) patients. However, it remains unclear whether such abnormalities result from systemic inflammation or from abnormalities at stem cell level. The current study therefore examined the expression of several representative genes, including amphiregulin (AREG), chemokine receptor 4 (CXCR4), and FK506-binding protein 5 (FKBP5) in RA BM CD34+ cells. BM samples were obtained from 52 patients with RA and 35 patients with osteroarthritis (OA) during joint operations. CD34+ cells were purified from the BM mononuclear cells by positive selection with magnetic beads. The mRNA expression for AREG, CXCR4, and FKBP5 was measured using quantitative real-time PCR. The expression of mRNA for FKBP5, but not that of AREG or CXCR4, was significantly higher in RA BM CD34+ cells than in OA BM CD34+ cells. The FKBP5 mRNA expression level was not correlated with serum CRP or treatment. In addition, tumour necrosis factor-alpha did not enhance the expression of FKBP5 mRNA in BM CD34+ cells from healthy donors. The results suggest that the enhanced expression of FKBP5 in BM CD34+ cells might be an intrinsic abnormality of RA BM CD34+ cells, whereas the enhanced expression of AREG and CXCR4 in BM mononuclear cells might be secondary to systemic inflammation.

  18. Cellular protein and mRNA expression of β1 nicotinic acetylcholine receptor (nAChR) subunit in brain, skeletal muscle and placenta.

    PubMed

    Aishah, Atqiya; Hinton, Tina; Machaalani, Rita

    2017-01-30

    The β1 nicotinic acetylcholine receptor (nAChR) subunit is a muscle type subunit of this family and as such, is found predominantly in muscle. Recent reports document its expression in other tissues and cell lines including adrenal glands, carcinomas, lung and brain. However, the majority of studies were of tissue lysates, thus the cellular distribution was not determined. This study aimed to determine the cellular distribution of the β1 nAChR subunit in the brain, at both the mRNA and protein levels, using non-radioactive in situ hybridization (ISH) and immunohistochemistry (IHC), respectively, and to compare it to two muscle tissue types, skeletal and placenta. Tissue was formalin fixed and paraffin embedded (all tissue types) and frozen (placenta) from humans. Additional control tissue from the piglet and mouse brain were also studied, as was mRNA for the α3 nAChR and N-methyl-d-aspartate receptor 1 (NR1) subunit. We found no β1 nAChR subunit mRNA expression in the human and piglet brain despite strong protein expression. Some signal was seen in the mouse brain but considered inconclusive given the probes designed were not of 100% homology to the mouse. In the skeletal muscle and placenta tissues, β1 nAChR subunit mRNA expression was prominent and mirrored protein expression. No α3 nAChR or NR1 mRNA was seen in the skeletal muscle, as expected, although both subunit mRNAs were present in the placenta. This study concludes that further experiments are required to conclusively state that the β1 nAChR subunit is expressed in the human, piglet and mouse brain.

  19. Joint cytokine quantification in two rodent arthritis models: kinetics of expression, correlation of mRNA and protein levels and response to prednisolone treatment

    PubMed Central

    RIOJA, I; BUSH, K A; BUCKTON, J B; DICKSON, M C; LIFE, P F

    2004-01-01

    Biomarker quantification in disease tissues from animal models of rheumatoid arthritis (RA) can help to provide insights into the mechanisms of action of novel therapeutic agents. In this study we validated the kinetics of IL-1β, TNF-α and IL-6 mRNA and protein expression levels in joints from DBA/1OlaHsd murine collagen-induced arthritis (CIA) and Lewis rat Streptococcal cell wall (SCW)-induced arthritis by real-time polymerase chain reaction (PCR) TaqMan® and Enzyme-linked immunosorbent assay (ELISA). Prednisolone was used as a reference to investigate any correlation between clinical response and cytokine levels at selected time-points. To our knowledge this is the first report showing a close pattern of expression between mRNA and protein for IL-1β and IL-6, but not for TNF-α, in these two models of RA. The kinetics of expression for these biomarkers suggested that the optimal sampling time-points to study the effect of compounds on both inflammation and cytokine levels were day 4 postonset in CIA and day 3 after i.v challenge in SCW-induced arthritis. Prednisolone reduced joint swelling through a mechanism associated with a reduction in IL-1β and IL-6 protein and mRNA expression levels. At the investigated time points, protein levels for TNF-α in arthritic joints were lower than the lower limit of detection of the ELISA, whereas mRNA levels for this cytokine were reliably detected. These observations suggest that RT-PCR TaqMan® is a sensitive technique that can be successfully applied to the quantification of mRNA levels in rodent joints from experimental arthritis models providing insights into mechanisms of action of novel anti-inflammatory drugs. PMID:15196245

  20. C-reactive protein induces interleukin-6 and thrombospondin-1 protein and mRNA expression through activation of nuclear factor-ĸB in HK-2 cells.

    PubMed

    Wang, Hai-rong; Chen, De-liang; Zhao, Mingming; Shu, Shao-wu; Xiong, Shi-xi; Gan, Xue-dong; Chao, Sheng-ping; Cao, Jian-lei

    2012-01-01

    Although C-reactive protein (CRP) is significantly increased in patients with diabetic nephropathy, whether CRP exerts direct proinflammatory effects on human renal tubular epithelial cells (HK-2 cells) is still unclear. HK-2 cells were incubated with purified CRP at clinically relevant concentrations (0, 5, 10, 20 and 40 μg/ml). The protein and transcript levels of thrombospondin-1 (TSP-1) and interleukin-6 (IL-6) were determined by ELISA and RT-PCR. Phosphorylation of p38MAPK was investigated through Western blot analysis in HK-2 cells induced by CRP. The activation of nuclear factor-kappa B (NF-κB) was studied via EMSA. A specific p38MAPK inhibitor (SB203580) and an NF-κB inhibitor (PDTC; pyrrolidine dithiocarbamate) were used to analyze the signal transduction in CRP induction. To explore the direct or indirect role of CRP in HK-2 cells, IL-6 or TSP-1 antibodies were used. The expression of IL-6, TSP-1 and transforming growth factor-β(1 )(TGF-β(1)) were determined through Western blot analysis in HK-2 cells. In HK-2 cells, purified CRP significantly induced protein release and mRNA expression of IL-6 and TSP-1 in a dose- and time-dependent manner. TGF-β(1) protein was overexpressed in HK-2 cells induced by CRP, which cannot be inhibited by IL-6 or TSP-1 antibodies. CRP triggered phosphorylation of p38MAPK and activation of NF-κB-mediated signal transduction. SB203580 (5 μM) and PDTC (50 μM) efficiently suppressed those effects of CRP in HK-2 cells. CRP induces IL-6 and TSP-1 protein release and mRNA expression from HK-2 cells via activation of the p38MAPK and NF-κB signaling pathways and TGF-β(1) was highly expressed in HK-2 cells, suggesting that CRP plays an important role in the propagation and prolongation of inflammation in renal fibrosis. Copyright © 2012 S. Karger AG, Basel.

  1. [Effects of 2000 μW/cm2; electromagnetic radiation on expression of immunoreactive protein and mRNA of NMDA receptor 2A subunit in rats hippocampus].

    PubMed

    Li, Yu-hong; Lu, Guo-bing; Shi, Chang-hua; Zhang, Zhuo; Xu, Qian

    2011-01-01

    To evaluate the effects of electromagnetic irradiation of 2000 μW/cm(2); exposure on mRNA and protein expression levels of immunoreactive protein and mRNA of NMDA receptor 2A subunit in rats hippocampal, and to explore the mechanism of electromagnetic irradiation induced learning and memory impairment. Rats were randomly divided into normal control group, sham-radiated group, and 1 h/d, 2 h/d, and 3 h/d radiation groups. The rats in the radiation groups were fixed after microwave exposure of 2000 μW/cm(2);, then their learning and memory abilities were tested by Morris water maze experiment, the change of NR2A protein in hippocampal neurons of each group of rats were measured with immunohistochmistry and Western blot techniques, and the expression of NR2A mRNA in hippocampus were determined by RT-PCR. Compared with the normal control group, each index of the sham-radiated group has no significant change (P>0.05), while the latency of rats of radiated group in Morris water maze test were significantly longer (P<0.05). In the radiation group, the hippocampal neurons of rats showing evident reduction in the ratio of NR2A positive cells, irregular, and arrayed in disorder. Moreover, the expession of NR2A protein and its mRNA in hippocampal neurons were significant decreased (P<0.05). Electromagnetic irradiation of 2000 μW/cm(2); exposure can impair the learning and memory abilities of rats possibly through a mechanism correlated with the lower expression of NR2A protein and its mRNA in hippocampus.

  2. Increased expression and localization of the RNA-binding protein HuD and GAP-43 mRNA to cytoplasmic granules in DRG neurons during nerve regeneration.

    PubMed

    Anderson, K D; Merhege, M A; Morin, M; Bolognani, F; Perrone-Bizzozero, N I

    2003-09-01

    The neuronal-specific RNA-binding protein, HuD, binds to a U-rich regulatory element of the 3' untranslated region (3' UTR) of the GAP-43 mRNA and delays the onset of its degradation. We have recently shown that overexpression of HuD in embryonic rat cortical cells accelerated the time course of normal neurite outgrowth and resulted in a twofold increase in GAP-43 mRNA levels. Given this evidence, we sought to investigate the involvement of HuD during nerve regeneration. It is known that HuD protein and GAP-43 mRNA are expressed in the dorsal root ganglia (DRG) of adult rat and that GAP-43 is upregulated in DRG neurons during regeneration. In this study, we examined the expression patterns and levels of HuD and GAP-43 mRNA in DRG neurons following sciatic nerve injury using a combination of in situ hybridization, immunocytochemistry, and quantitative RT-PCR. GAP-43 and HuD expression increased in the ipsilateral DRG during the first 3 weeks of regeneration, with peak values seen at 7 days postcrush. At this time point, the levels of HuD and GAP-43 mRNAs in the ipsilateral DRG increased by twofold and sixfold, respectively, relative to the contralateral DRG. Not only were the temporal patterns of expression of HuD protein and GAP-43 mRNA similar, but also they were found to colocalize in the cytoplasm of DRG neurons. Moreover, both molecules were distributed in cytoplasmic granules containing ribosomal RNA. In conclusion, our results suggest that HuD is involved in the upregulation of GAP-43 expression observed at early stages of peripheral nerve regeneration.

  3. Gastric acid induces mucosal H2S release in rats by upregulating mRNA and protein expression of cystathionine gamma lyase.

    PubMed

    Mard, Seyyed Ali; Veisi, Ali; Ahangarpour, Akram; Gharib-Naseri, Mohammad Kazem

    2015-11-01

    It is well known that hydrogen sulfide (H2S) protects the gastric mucosa against gastric acid and other noxious stimulants by several mechanisms but until now the effect of gastric acid on H2S production has not been evaluated. This study was performed to determine the effect of basal and stimulated gastric acid secretion on mRNA and protein expression of cystathionine gamma lyase (CSE) and cystathionine beta synthase (CBS), and on mucosal release of H2S in rats. Seventy-two male rats were randomly assigned into 9 groups (8 in each)-control, distention, and pentagastrin-induced gastric acid secretion groups. The effects of 15% alcohol solution, propargylglycine (PAG), L-NAME, and pantoprazole were also investigated. Under anesthesia, animals underwent tracheostomy and midline laparotomy. A catheter was inserted into the stomach through the duodenum for gastric washout. At the end of the experiments, the animals were killed and the gastric mucosa was collected to measure H2S concentration and to quantify mRNA expression of CSE and CBS by quantitative real-time PCR, and expression of their proteins by western blot. Basal and stimulated gastric acid secretion increased mucosal levels of H2S, and mRNA and protein expression of CSE. Pantoprazole and L-NAME reversed H2S release and restored protein expression of CSE to the control level. Pantoprazole, but not propargylglycine, pretreatment inhibited the elevated level of protein expression of eNOS in response to distention-induced gastric acid secretion. Our findings indicated that NO mediated the stimulatory effect of gastric acid on H2S release and protein expression of CSE.

  4. The autoimmunity-associated BLK haplotype exhibits cis-regulatory effects on mRNA and protein expression that are prominently observed in B cells early in development

    PubMed Central

    Simpfendorfer, Kim R.; Olsson, Lina M.; Manjarrez Orduño, Nataly; Khalili, Houman; Simeone, Alyssa M.; Katz, Matthew S.; Lee, Annette T.; Diamond, Betty; Gregersen, Peter K.

    2012-01-01

    The gene B lymphocyte kinase (BLK) is associated with rheumatoid arthritis, systemic lupus erythematosus and several other autoimmune disorders. The disease risk haplotype is known to be associated with reduced expression of BLK mRNA transcript in human B cell lines; however, little is known about cis-regulation of BLK message or protein levels in native cell types. Here, we show that in primary human B lymphocytes, cis-regulatory effects of disease-associated single nucleotide polymorphisms in BLK are restricted to naïve and transitional B cells. Cis-regulatory effects are not observed in adult B cells in later stages of differentiation. Allelic expression bias was also identified in primary human T cells from adult peripheral and umbilical cord blood (UCB), thymus and tonsil, although mRNA levels were reduced compared with B cells. Allelic regulation of Blk expression at the protein level was confirmed in UCB B cell subsets by intracellular staining and flow cytometry. Blk protein expression in CD4+ and CD8+ T cells was documented by western blot analysis; however, differences in protein expression levels by BLK genotype were not observed in any T cell subset. Blk allele expression differences at the protein level are thus restricted to early B cells, indicating that the involvement of Blk in the risk for autoimmune disease likely acts during the very early stages of B cell development. PMID:22678060

  5. Correlation of mRNA and protein levels: Cell type-specific gene expression of cluster designation antigens in the prostate

    PubMed Central

    Pascal, Laura E; True, Lawrence D; Campbell, David S; Deutsch, Eric W; Risk, Michael; Coleman, Ilsa M; Eichner, Lillian J; Nelson, Peter S; Liu, Alvin Y

    2008-01-01

    Background: Expression levels of mRNA and protein by cell types exhibit a range of correlations for different genes. In this study, we compared levels of mRNA abundance for several cluster designation (CD) genes determined by gene arrays using magnetic sorted and laser-capture microdissected human prostate cells with levels of expression of the respective CD proteins determined by immunohistochemical staining in the major cell types of the prostate – basal epithelial, luminal epithelial, stromal fibromuscular, and endothelial – and for prostate precursor/stem cells and prostate carcinoma cells. Immunohistochemical stains of prostate tissues from more than 50 patients were scored for informative CD antigen expression and compared with cell-type specific transcriptomes. Results: Concordance between gene and protein expression findings based on 'present' vs. 'absent' calls ranged from 46 to 68%. Correlation of expression levels was poor to moderate (Pearson correlations ranged from 0 to 0.63). Divergence between the two data types was most frequently seen for genes whose array signals exceeded background (> 50) but lacked immunoreactivity by immunostaining. This could be due to multiple factors, e.g. low levels of protein expression, technological sensitivities, sample processing, probe set definition or anatomical origin of tissue and actual biological differences between transcript and protein abundance. Conclusion: Agreement between these two very different methodologies has great implications for their respective use in both molecular studies and clinical trials employing molecular biomarkers. PMID:18501003

  6. The Minor Allele of rs7574865 in the STAT4 Gene Is Associated with Increased mRNA and Protein Expression

    PubMed Central

    Lamana, Amalia; López-Santalla, Mercedes; Castillo-González, Raquel; Ortiz, Ana María; Martín, Javier; García-Vicuña, Rosario; González-Álvaro, Isidoro

    2015-01-01

    Objective The T allele of rs7574865 in STAT4 confers risk of developing autoimmune disorders. However, its functional significance remains unclear. Here we analyze how rs7574865 affects the transcription of STAT4 and its protein expression. Methods We studied 201 patients (80% female; median age, 54 years; median disease duration, 5.4 months) from PEARL study. Demographic, clinical, laboratory and therapeutic data were collected at each visit. IL-6 serum levels were measured by enzyme immune assay. The rs7574865 was genotyped using TaqMan probes. The expression levels of STAT4 mRNA were determined at 182 visits from 69 patients using quantitative real-time polymerase chain reaction. STAT4 protein was assessed by western blot in 62 samples from 34 patients. To determine the effect of different variables on the expression of STAT4 mRNA and protein, we performed multivariate longitudinal analyses using generalized linear models. Results After adjustment for age, disease activity and glucocorticoid dose as confounders, the presence of at least one copy of the T allele of rs7574865 was significantly associated with higher levels of STAT4 mRNA. Similarly, TT patients showed significantly higher levels of STAT4 protein than GG patients. IL-6 induced STAT4 and STAT5 phosphorylation in peripheral blood lymphocytes. Patients carrying at least one T allele of rs7574865 displayed lower levels of serum IL-6 compared to GG homozygous; by contrast the production of C-reactive protein was similar in both populations. Conclusion Our data suggest that the presence of the rs7574865 T allele enhances STAT4 mRNA transcription and protein expression. It may enhance the signaling of molecules depending on the STAT4 pathway. PMID:26569609

  7. The role of de novo protein synthesis and SIRT1 in ER stress-induced Atf4 and Chop mRNA expression in mammalian cells.

    PubMed

    Chan, Stanley M H; Zhao, Xuechan; Elfowiris, Abdulsalam; Ratnam, Cherubina; Herbert, Terence P

    2017-07-01

    Endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR) have been implicated in the pathogenesis of many common human diseases. Integral to the UPR and an important determinant in cell fate is the expression of the pro-apoptotic transcription factor C/EBP homologous protein (CHOP). This is promoted by activating transcription factor 4 (ATF4) whose expression is rapidly up-regulated in response to ER stress through an eIF2α phosphorylation-dependent increase in protein synthesis. Our data demonstrates that this ER stress-induced increase in ATF4 and CHOP expression is initiated by an increase in Atf4 and Chop mRNA, which is also dependent upon eIF2α phosphorylation. Despite being dependent on eIF2α phosphorylation, we provide evidence that these increases in Atf4 and Chop mRNA expression may occur independently of de novo protein synthesis. Moreover, we show that ER stress-induced Chop mRNA expression is exacerbated by Sirtuin-1 (SIRT1) inhibition indicating that changes in the energy status of the cell may play an important role in its regulation. This work highlights and extends previous findings, and provides important new insights into the mechanism of ER stress-induced expression of Atf4 and Chop mRNA that clearly warrants further investigation. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  8. Regulation of protein and mRNA expression of the mTORC1 repressor REDD1 in response to leucine and serum.

    PubMed

    Black, Adam J; Gordon, Bradley S; Dennis, Michael D; Jefferson, Leonard S; Kimball, Scot R

    2016-12-01

    Expression of the mTORC1 repressor, Regulated in DNA Damage and Development 1 (REDD1), is elevated in skeletal muscle during various catabolic conditions including fasting, hindlimb immobilization, and sepsis. Conversely, REDD1 expression is suppressed by anabolic stimuli such as resistance exercise or nutrient consumption following a fast. Though it is known that nutrient consumption reduces REDD1 expression, it is largely unknown how nutrients and hormones individually contribute to the reduction in REDD1 expression. Therefore, the purpose of the present study was to determine how nutrients and hormones individually regulate REDD1 expression. HeLa cells were deprived of leucine or serum for 10 hours, after which either leucine or serum was reintroduced to cell culture medium for 60 minutes. Re-supplementation of either leucine or serum resulted in a reduction in REDD1 protein levels by 34.8 ± 5.8% and 54.1 ± 3.4%, respectively, compared to the deprived conditions. Re-supplementation of leucine or serum to deprived cells also led to a reduction in REDD1 mRNA content by 49.1% ± 2.7% and 65.0 ± 1.4%, respectively, compared to the deprived conditions. Interestingly, rates of REDD1 protein degradation were unaffected by either leucine or serum re-supplementation, as assessed in cells treated with cycloheximide to block protein synthesis. Likewise, addition of leucine or serum to cells treated with Actinomycin D to inhibit gene transcription failed to alter the rate of REDD1 mRNA degradation. The data indicate that the leucine or serum-induced suppression of REDD1 expression occurs independent of changes in the rate of degradation of either the REDD1 protein or mRNA. Thus, the leucine- or serum-induced suppression likely occurs through alternative mechanism(s) such as reduced REDD1 gene transcription and/or mRNA translation.

  9. Quantification of heat shock protein mRNA expression in warm and cold anoxic turtles (Trachemys scripta) using an external RNA control for normalization.

    PubMed

    Stecyk, Jonathan A W; Couturier, Christine S; Fagernes, Cathrine E; Ellefsen, Stian; Nilsson, Göran E

    2012-03-01

    The mRNA expression of heat-shock protein 90 (HSP90) and heat-shock cognate 70 (HSC70) was examined in cardiac chambers and telencephalon of warm- (21°C) and cold-acclimated (5°C) turtles (Trachemys scripta) exposed to normoxia, prolonged anoxia or anoxia followed by reoxygenation. Additionally, the suitability of total RNA as well as mRNA from β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cyclophilin A (PPIA) for normalizing gene expression data was assessed, as compared to the use of an external RNA control. Measurements of HSP90 and HSC70 mRNA expression revealed that anoxia and reoxygenation have tissue- and gene-specific effects. By and large, the alterations support previous investigations on HSP protein abundance in the anoxic turtle heart and brain, as well as the hypothesized roles of HSP90 and HSC70 during stress and non-stress conditions. However, more prominent was a substantially increased HSP90 and HSC70 mRNA expression in the cardiac chambers with cold acclimation. The finding provides support for the notion that cold temperature induces a number of adaptations in tissues of anoxia-tolerant vertebrates that precondition them for winter anoxia. β-actin, GAPDH and PPIA mRNA expression and total RNA also varied with oxygenation state and acclimation temperature in a tissue- and gene-specific manner, as well as among tissue types, thus disqualifying them as suitable for real-time RT-PCR normalization. Thus, the present data highlights the advantages of normalizing real-time RT-PCR data to an external RNA control, an approach that also allows inter-tissue and potentially inter-species comparisons of target gene expression. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Platelet-derived growth factor activity and mRNA expression in healing vascular grafts in baboons. Association in vivo of platelet-derived growth factor mRNA and protein with cellular proliferation.

    PubMed Central

    Golden, M A; Au, Y P; Kirkman, T R; Wilcox, J N; Raines, E W; Ross, R; Clowes, A W

    1991-01-01

    In a baboon graft model of arterial intimal thickening, smooth muscle cells (SMC) have been observed to proliferate underneath an intact monolayer of endothelium and in the absence of platelet adherence. Because platelets are not present and therefore cannot be a major source of growth stimulus, we have proposed that the vascular wall cells in the graft intima express mitogens and regulate SMC proliferation. To test this hypothesis, we assayed the grafts for mitogenic activity and expression of growth factor genes. Segments of healing graft and of normal artery, when perfused ex vivo, released mitogenic activity into the perfusate. The graft released more mitogen than the normal arterial segment, and some of the activity was inhibitable with an antibody to human platelet-derived growth factor (PDGF). In addition, Northern analysis of total RNA demonstrated higher expression of PDGF-A chain mRNA in the graft intima compared to normal artery. PDGF-B chain mRNA was barely detectable in both tissues. PDGF mRNA levels within the graft interstices were not measured. In situ hybridization of 7.5- or 12-wk grafts indicated that some luminal endothelial cells and adjacent intimal SMC contained PDGF-A chain mRNA. By thymidine autoradiography, intimal SMC were observed to be proliferating in the inner third of the intima. These data demonstrate a difference in the pattern of PDGF transcript expression and luminal perfusate activity in graft as compared with control arteries. The association of intimal smooth muscle cell proliferation with intimal PDGF mRNA expression and release of PDGF-like protein supports the hypothesis that factors from cells that have grown into the graft or populated its surface rather than platelets may regulate intimal smooth muscle cell proliferation in this model. Images PMID:1825089

  11. Short-term changes of mRNA expression of various inflammatory factors and milk proteins in mammary tissue during LPS-induced mastitis.

    PubMed

    Schmitz, S; Pfaffl, M W; Meyer, H H D; Bruckmaier, R M

    2004-03-01

    During mammary gland infection, non-specific responses are the predominant ones. The goal of this study was to investigate the mRNA expression of various soluble immune components and of the major milk proteins during the acute phase of mammary inflammation. Five healthy lactating cows were intramammary infused in one quarter with 100 microg Escherichia coli-endotoxin (lipopolysaccharide, LPS) and the contralateral quarter with saline (9 g/l) serving as control. Mammary biopsy samples of both quarters were taken immediately before and at 3, 6, 9 and 12 h after infusion and mRNA expression of various factors was quantified via real-time RT-PCR. Blood samples for determination of leukocyte number were taken simultaneously with the biopsy samples and rectal temperature was measured at 1-h intervals. Rectal temperature increased until 5h (P < 0.05) after LPS administration and remained elevated until 9 h after LPS inoculation. Blood leukocyte number decreased (P < 0.05) from 0 to 3 h from 7.7 +/- 1.1 x 10(9)l(-1) to 5.7 +/- 1.0 x 10(9)l(-1) and thereafter recovered to pre-treatment levels until 12 h after LPS challenge. In LPS-treated quarters, tumor necrosis factor-alpha and cyclooxygenase-2-mRNA expression increased (P < 0.05) to highest values at 3h after LPS challenge. Lactoferrin, lysozyme, inducible nitric oxide synthase increased (P < 0.05) and peaked at 6 h after challenge, and platelet-activating factor acetylhydrolase-mRNA expression tended to increase (P = 0.07). mRNA expression of insulin-like growth factor-I and of alphaS1-casein (CN), alphaS2-CN, beta-CN and beta-lactoglobulin did not change significantly, whereas mRNA expression of 5-lipoxygenase and alpha-lactalbumin decreased (P < 0.05) in both quarters and that of kappa-CN only in the LPS quarter. mRNA expression of some investigated factors (tumor necrosis factor-alpha, lysozyme, 5-lipoxygenase, alpha-lactalbumin) changed in control quarters, however in all respective factors less than in the LPS

  12. mRNA stability plays a major role in regulating the temperature-specific expression of a Tetrahymena thermophila surface protein

    SciTech Connect

    Love, H.D. Jr.; Allen-Nash, A.; Zhao, Q.; Bannon, G.A.

    1988-01-01

    Synthesis of the serotype H3 (SerH3) surface antigen is temperature dependent and responds within 1 h to a change in incubation conditions. Recently, a Tetrahymena thermophila cDNA clone has been shown to be homologous to a portion of the SerH3 mRNA, and it was shown that the cellular levels of this RNA rapidly decreased when cells were shifted from 30 to 41/sup 0/C. These observations indicate that synthesis of the SerH3 protein is highly regulated in response to temperature and led us to initiate studies to determine the mechanism(s) by which SerH3 gene expression is controlled. Using pC6 as a hybridization probe for the SerH3 mRNA, they have determined that (i) the level of SerH3 protein synthesis is directly correlated with the amount of SerH3 message available for translation; (ii) there is, at most, a twofold difference between the relative transcription rates of SerH3 genes at 30 and 40/sup 0/C; (iii) the SerH3 mRNA half-life in cells incubated at 30/sup 0/C is greater than 1 h, whereas the half-life in cells incubated at 40/sup 0/C is only -- 3 min. These results demonstrate that Tetrahymena SerH3 surface protein expression is regulated by mRNA abundance. Furthermore, the major mechanism controlling mRNA abundance is a dramatic temperature-dependent change in SerH3 mRNA stability.

  13. Inhibition of CYP3A mRNA and protein expression, and enzymatic activity, by enrofloxacin in chickens.

    PubMed

    Hu, H-F; Gao, Z-X; Cheng, Y-Y

    2010-12-01

    This study was to investigate the effect of enrofloxacin (EF) on CYP3A in chicken by using quantitative reverse transcription-polymerase chain reaction and immunodetected. The treated chickens were given 5, 25 and 125 mg/kg of EF while the control chickens were treated with the same volume saline. There was no significant difference between the low dose group and controls in the concentration of hepatic microsome protein and total CYP content, while the middle and high dose EF caused the down regulation. Depression of the CYP3A activity, mRNA and protein were observed in treated chickens, and the inhibition degree was different from each group. It was concluded that EF caused the inhibition of CYP3A both in genetic transcription and protein levels. But the inhibition metabolism still needs further researches. © 2010 Blackwell Publishing Ltd.

  14. Effects of quercetin on CDK4 mRNA and protein expression in A549 cells infected by H1N1

    PubMed Central

    WAN, QIAOFENG; WANG, HAO; LIN, YUAN; GU, LIGANG; HAN, MEI; YANG, ZHIWEI; ZHANG, YANLI; MA, RUI; WANG, LI; WANG, ZHISHENG

    2013-01-01

    This study was conducted to investigate the effects of quercetin on the expression of cyclin-dependent kinase (CDK4) mRNA and protein in A549 lung epithelial tumor cells infected by H1N1. First, the Thiazolyl Blue Tetrazolium Bromide (MTT) method was used to determine H1N1 virulence, quercetin cytotoxicity and inhibition of the cytopathic effect of H1N1 on A549 cells by quercetin. Subsequently, 100 TCID50 H1N1 was used to infect A549 cells for 2 h prior to culture in maintenance media containing 10 mg/l quercetin. After 4, 12, 24 and 48 h of culture, the cells were collected and total RNA and protein were extracted. Fluorescent quantitative polymerase chain reaction and western blot analysis were then performed to assess the expression of CDK4 mRNA and protein. The experiment demonstrated that the TCID50 of H1N1 in A549 cells was 10−4.75, the maximum non-toxic concentration of quercetin in A549 cells was 30–60 mg/l and the minimum effective concentration of quercetin for the inhibition of the H1N1 cytopathic effect on A549 cells was 10 mg/l. The results indicated that quercetin may significantly inhibit CDK4 mRNA and protein overexpression caused by H1N1 within 4–48 h. In conclusion, quercetin may protect against H1N1 infection by effectively reducing the mRNA and protein expression of CDK4 caused by H1N1 infection. PMID:24649026

  15. Parathyroid hormone regulates osterix and Runx2 mRNA expression predominantly through protein kinase A signaling in osteoblast-like cells.

    PubMed

    Wang, B L; Dai, C L; Quan, J X; Zhu, Z F; Zheng, F; Zhang, H X; Guo, S Y; Guo, G; Zhang, J Y; Qiu, M C

    2006-02-01

    Runt-related transcription factor 2 (Runx2) and osterix are osteoblast-specific transcription factors essential for the development of osteoblastic cells and bone formation. PTH given intermittently has anabolic effects on bone; however, the exact role remains to be understood completely. The purpose of this study was both to investigate whether PTH regulates Runx2 as well as osterix expression and to identify the signaling used. Using RT-PCR, we confirmed that PTH (1-34) regulated Runx2 and osterix mRNA expression, in rat osteoblast-like cell line UMR 106, in a dose- and time-dependent manner. PTH in low concentrations stimulated both Runx2 and osterix mRNA expression while that in high concentrations did not. Forskolin, an adenylate cyclase activator, also enhanced Runx2 and osterix transcription, and the stimulatory effects of PTH and forskolin were blocked by the pre-treatment of the cells with H-89, a protein kinase A (PKA) inhibitor. In contrast, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) had no effect on Runx2 transcription, but induced an increase in osterix mRNA level at the concentration of 500 nM at 12 h after treatment. Moreover, pre-treatment of the cells with calphostin C, a PKC-specific inhibitor, reduced the increase in osterix transcripts enhanced by PTH and PMA 12 h after treatment. However, these inhibitory effects were not sustained for longer terms. These observations demonstrate that PTH stimulates Runx2 and osterix expression in vitro, at least in part, at transcriptional level. Induction of Runx2 mRNA is mediated through the activation of cAMP/PKA signal transduction. In the case of osterix, although the increase in mRNA level is predominantly mediated via cAMP/PKA signaling, PKC activation might also be involved in this process.

  16. Status epilepticus decreases glutamate receptor 2 mRNA and protein expression in hippocampal pyramidal cells before neuronal death

    PubMed Central

    Grooms, Sonja Y.; Opitz, Thoralf; Bennett, Michael V. L.; Zukin, R. Suzanne

    2000-01-01

    Kainic acid (KA)-induced status epilepticus in adult rats leads to delayed, selective death of pyramidal neurons in the hippocampal CA1 and CA3. Death is preceded by down-regulation of glutamate receptor 2 (GluR2) mRNA and protein [the subunit that limits Ca2+ permeability of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors] in CA1 and CA3, as indicated by in situ hybridization, immunolabeling, and quantitative Western blotting. GluR1 mRNA and protein are unchanged or slightly increased before cell death. These changes could lead to formation of GluR2-lacking, Ca2+-permeable AMPA receptors and increased toxicity of endogenous glutamate. GluR2 immunolabeling is unchanged in granule cells of the dentate gyrus, which are resistant to seizure-induced death. Thus, formation of Ca2+-permeable AMPA receptors may be a critical mediator of delayed neurodegeneration after status epilepticus. PMID:10725374

  17. [Expression of mRNA and protein of Klotho gene in placental tissue of macrosomia and its relationship with birth weight of neonates].

    PubMed

    Shao, W J; Wang, D X; Wan, Q Y; Zhang, M M; Chen, M M; Song, W W

    2016-06-25

    To explore the the expression of Klotho mRNA and protein in placenta of macrosomia and its relationship with the birth weight of neonates. The cases were from November 2014 to March 2015 in Shengjing Hospital of China Medical University, divided into 4 groups: the gestational diabetes with macrosomia group (GM), the gestational diabetes with normal birth weight group (GN), the normal pregnancy with macrosomia group (NM) and the normal pregnancy with normal birth weight group (NN). Klotho mRNA and protein expression in the placenta were detected by immunohistochemistry SP method, real-time fluorescent quantitative PCR and western blot, respectively, and were compared among the 4 groups. (1) Immunohistochemical detection showed the positive rate of Klotho protein was significantly higher in the placenta of GM (93%,28/30) than in the GN (73%,22/30; P<0.05). The positive rate was significantly higher in the placenta of NM (97%,29/30) than in the NN (80%,24/30; P<0.05). (2) Real-time fluorescent quantitative PCR showed the Klotho mRNA expression was significantly higher in the placenta of GM (4.3 ± 3.1) than in the GN (2.1 ± 2.4; P<0.05). The Klotho mRNA expression was also significantly higher in the placenta of NM (4.8± 3.4) than in the NN (2.6± 3.3; P<0.05). (3) Western blot showed the Klotho protein expression was significantly higher in the placenta of GM (1.27±0.90) than in the GN (0.64±0.24; P<0.05). It was also significantly higher in the placenta of NM (2.51±3.52) than in the NN (0.77±0.37; P<0.05). (4) There were no significant differences in the expression of Klotho mRNA and protein between GM and NM, GN and NN (P>0.05). The up-regulation of Klotho gene may be associated with macrosomia. The relationship is not affected by the complication of gestational diabetes.

  18. Expression of heat-shock protein 72 mRNA in relation to heart rate variability of Sahiwal and Karan-Fries in different temperature-humidity indices

    PubMed Central

    Mayengbam, Prava; Tolenkhomba, T. C.; Upadhyay, R. C.

    2016-01-01

    Aim: To investigate the effect of temperature-humidity index (THI) on the expression pattern of heat-shock protein 72 (HSP72) mRNA of Sahiwal and Karan-Fries (KF) cattle in different THIs. Materials and Methods: Five different periods of a year were selected based on combinations of Tmax/Tmin, viz., P1: <20°C/<10°C; P2: >20°C/<10°C, P3: <30°C/<15°C; P4: >35°C/<20°C, and P5: >35°C/>20°C. The THI was calculated from the records of temperature and relative humidity in different periods. Heart rate variability (HRV) was calculated from electrocardiogram records in different periods. HSP72 mRNA expression was estimated by reverse transcription polymerase chain reaction. Results: The THI recorded during P1, P2, P3, P4, and P5 were 55.5, 60.3, 70.1, 74.5, and 79.0, respectively. THI in P4 and P5 were stressful to animals. HSP72 mRNA expression increased during cold stress in P1 in Sahiwal and heat stress in P4 and P5 in both Sahiwal and KF. Sahiwal maintained increased HSP72 mRNA expression longer than KF without causing a significant change in HRV. Conclusion: Both low THI in winter and high THI in summer increased HSP72 mRNA of Sahiwal and KF without significant change in HRV. Thermotolerance of Sahiwal could be due to the maintenance of higher HSP72 expression longer than KF in prolonged heat stress in summer. PMID:27847412

  19. Discordant mRNA and protein expression of CXCR4 under in vitro CoCl2-induced hypoxic conditions.

    PubMed

    Tang, Mingjun; Yang, Ying; Yu, Jingzhi; Wu, Nandan; Chen, Pei; Xu, Lijun; Wang, Qiyun; Xu, Zhuojun; Ge, Jian; Yu, Keming; Zhuang, Jing

    2017-03-04

    Cobalt chloride (CoCl2) has long been accepted as a suitable in vitro hypoxia-mimetic agent. The gene CXCR4, which encodes a chemokine receptor, plays a key role in hypoxic retinal disease. Here, we investigated the mRNA and protein expression of CXCR4 in WERI-Rb1 retinoblastoma cells and human umbilical vein endothelial cells (HUVECs) under CoCl2-induced hypoxic conditions, by means of real-time PCR and western blot. We found that CoCl2-induced hypoxia profoundly increased CXCR4 expression at the mRNA level, but not at the protein level, at 12, 24, 48 and 72 h in these cells. Interestingly, this result differed from observations of 1% O2 hypoxic conditions. Additionally, luciferase assays demonstrated that CoCl2-induced hypoxia significantly increased transcription at the CXCR4 promoter. In order to compare our in vitro findings with the effects of hypoxia in vivo, an OIR (Oxygen-induced retinopathy) rat model was constructed. However, both CXCR4 mRNA and protein levels in OIR rats were significantly increased compared to controls. Thus taken together, our findings suggest that the relationship between CXCR4 mRNA and protein expression is not strictly linear under in vitro CoCl2-induced hypoxic conditions. through comparative in vitro and in vivo experiments, this study implies that CoCl2 is an imperfect simulation of hypoxia in retinal disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. The regulation of exon-specific brain-derived neurotrophic factor mRNA expression by protein kinase C in rat cultured dorsal root ganglion neurons.

    PubMed

    Morioka, Norimitsu; Yoshida, Yosuke; Nakamura, Yoki; Hidaka, Nobue; Hisaoka-Nakashima, Kazue; Nakata, Yoshihiro

    2013-05-06

    Although brain-derived neurotrophic factor (BDNF) is localized in primary sensory neurons and has crucial roles in nociceptive transduction, the mechanisms involved in regulation of BDNF exon-specific mRNA expression in dorsal root ganglion (DRG) neurons have yet to be determined. Rat primary cultures of DRG neurons were stimulated with phorbol-12-myristate-13-acetate (PMA), a potent activator of protein kinase C (PKC), which resulted in the robust expression of both BDNF mRNA and protein. Among each BDNF mRNA exon, it was found that exons I, IV and VI were especially induced after PMA stimulation. The induction of these exons was significantly blocked by Gö6983 (a broad spectrum PKC inhibitor), Gö6976 (a conventional PKCs and PKCμ inhibitor), and rottlerin (a PKCδ inhibitor), but not by a PKCε inhibitor. The effect of PMA on exons I and VI was blocked by either U0126 (a MAP kinase kinase (MEK) inhibitor) or SB202190 (a p38 inhibitor), and PMA's effect on exon IV was inhibited by U0126 but not by SB202190. Furthermore, the activation of cAMP-responsive element-binding protein (CREB) was associated with the induction of exons I and IV, and the activation of nuclear factor-κB (NF-κB) contributed to the induction of exons I, IV and VI. These results show that the activation of PKCs induces the expression of BDNF mRNA exons I, IV and VI through exon-specific mechanisms, including extracellular signal-regulated kinase, p38, CREB and NF-κB, in cultured DRG neurons. These data suggest multiple pathways in the expression of BDNF in nociceptive sensory neurons.

  1. ER stress increases StarD5 expression by stabilizing its mRNA and leads to relocalization of its protein from the nucleus to the membranes

    PubMed Central

    Rodriguez-Agudo, Daniel; Calderon-Dominguez, Maria; Medina, Miguel Angel; Ren, Shunlin; Gil, Gregorio; Pandak, William M.

    2012-01-01

    StarD5 belongs to the StarD4 subfamily of steroidogenic acute regulatory lipid transfer (START) domain proteins. In macrophages, StarD5 is found in the cytosol and maintains a loose association with the Golgi. Like StarD1 and StarD4, StarD5 is known to bind cholesterol. However, its function and regulation remain poorly defined. Recently, it has been shown that its mRNA expression is induced in response to different inducers of endoplasmic reticulum (ER) stress. However, the molecular mechanism(s) involved in the induction of StarD5 expression during ER stress is not known. Here we show that in 3T3-L1 cells, the ER stressor thapsigargin increases intracellular free cholesterol due to an increase in HMG-CoA reductase expression. Activation of StarD5 expression is mediated by the transcriptional ER stress factor XBP-1. Additionally, the induction of ER stress stabilizes the StarD5 mRNA. Furthermore, StarD5 protein is mainly localized in the nucleus, and upon ER stress, it redistributes away from the nucleus, localizing prominently to the cytosol and membranes. These results reveal the increase in StarD5 expression and protein redistribution during the cell protective phase of the ER stress, suggesting a role for StarD5 in cholesterol metabolism during the ER stress response. PMID:23053693

  2. Cloning of a nitrate reductase inactivator (NRI) cDNA from Spinacia oleracea L. and expression of mRNA and protein of NRI in cultured spinach cells.

    PubMed

    Sonoda, Masatoshi; Ide, Hiroaki; Nakayama, Shinya; Sasaki, Asako; Kitazaki, Shinei; Sato, Takahide; Nakagawa, Hiroki

    2003-04-01

    The spinach ( Spinacia oleracea L. (cv. Hoyo) nitrate reductase inactivator (NRI) is a novel protein that irreversibly inactivates NR. Using degenerate primers based on an N-terminal amino acid sequence of NRI purified from spinach leaves and a cDNA library, we isolated a full-length NRI cDNA from spinach that contains an open reading frame encoding 479 amino acid residues. This protein shares 67.4% and 51.1-68.3% amino acid sequence similarities with a nucleotide pyrophosphatase (EC 3.6.1.9) from rice and three types of the nucleotide pyrophosphatase-like protein from Arabidopsis thaliana, respectively. Immunoblot analysis revealed that NRI was constitutively expressed in suspension-cultured spinach cells; however, its expression level is quite low in 1-day-subcultured cells. Moreover, northern blot analysis indicated that this expression was regulated at the mRNA level. These results suggest that NRI functions in mature cells.

  3. Regulation of AQP6 mRNA and protein expression in rats in response to altered acid-base or water balance.

    PubMed

    Promeneur, D; Kwon, T H; Yasui, M; Kim, G H; Frøkiaer, J; Knepper, M A; Agre, P; Nielsen, S

    2000-12-01

    In the rat, aquaporin-6 (AQP6) is mainly localized in intercalated cells (ICs) in collecting ducts, where it is exclusively associated with intracellular vesicles. In this study, we examined whether AQP6 protein and mRNA expression were regulated in the inner medulla or inner stripe of the outer medulla. Rats treated with dietary alkali or acid load for 7 days with a fixed daily water intake revealed appropriate changes in urine pH but unchanged urine output. AQP6 protein and mRNA abundance were increased in alkali-loaded rats (187 +/- 18 and 151 +/- 17% of control, respectively), whereas no changes were observed in acid-loaded rats. Immunohistochemistry revealed increased IC AQP6 labeling in alkali-loaded rats but not in acid-loaded rats. In contrast, administration of NH(4)Cl in the drinking water for 2 wk (free access to water) revealed a significant increase in AQP6 protein abundance (194 +/- 9% of control), but this was associated with increased water intake. Combined, this suggests that AQP6 expression was not affected by acid loading per se but rather was in response to changes in water intake. Consistent with this, water loading for 48 h was associated with increased AQP6 protein abundance, compared with thirsted rats. Moreover, rats with lithium-induced nephrogenic diabetes insipidus had a threefold increase in both AQP6 protein and mRNA expression. Overall, these results suggest that AQP6 expression in collecting duct ICs is regulated by altered acid/alkali load or water balance. Thus AQP6 may contribute to maintenance of acid-base homeostasis and water balance.

  4. Bovine DNase I: gene organization, mRNA expression, and changes in the topological distribution of the protein during apoptosis in lens epithelial cells.

    PubMed

    De María, Alicia; Arruti, Cristina

    2003-12-19

    Genomic DNA sequencing and alignment with the known DNase I mRNA showed that the bovine gene consists of 9 exons and that only the last 8 encode the protein, since initial ATG was found at exon II. RT-PCR was used to identify DNase I mRNA in lens epithelium in vivo and in cultured epithelial cells. We found DNase I transcripts having the same nucleotide sequence as the pancreas form and others lacking almost all exon V. The lens protein presented a slightly higher relative molecular weight than the pancreatic enzyme. Lens DNase I was located in secretory pathway organelles and excluded from the nucleus. Nevertheless, in apoptotic lens epithelial cells in vitro, DNase I translocated to the nucleus and co-localized with TUNEL positive chromatin aggregates. These results indicate that cells in the lens epithelium constitutively express DNase I, and suggest a direct involvement of this nuclease in the final phases of chromatin degradation.

  5. Effects of Saturated Long-chain Fatty Acid on mRNA Expression of Genes Associated with Milk Fat and Protein Biosynthesis in Bovine Mammary Epithelial Cells.

    PubMed

    Qi, Lizhi; Yan, Sumei; Sheng, Ran; Zhao, Yanli; Guo, Xiaoyu

    2014-03-01

    This study was conducted to determine the effects of saturated long-chain fatty acids (LCFA) on cell proliferation and triacylglycerol (TAG) content, as well as mRNA expression of αs1-casein (CSN1S1) and genes associated with lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows, and were passaged twice. Then cells were cultured with different levels of palmitate or stearate (0, 200, 300, 400, 500, and 600 μM) for 48 h and fetal bovine serum in the culture solution was replaced with fatty acid-free BSA (1 g/L). The results showed that cell proliferation tended to be increased quadratically with increasing addition of stearate. Treatments with palmitate or stearate induced an increase in TAG contents at 0 to 600 μM in a concentration-dependent manner, and the addition of 600 μM was less effective in improving TAG accumulation. The expression of acetyl-coenzyme A carboxylase alpha, fatty acid synthase and fatty acid-binding protein 3 was inhibited when palmitate or stearate were added in culture medium, whereas cluster of differentiation 36 and CSN1S1 mRNA abundance was increased in a concentration-dependent manner. The mRNA expressions of peroxisome proliferator-activated receptor gamma, mammalian target of rapamycin and signal transducer and activator of transcription 5 with palmitate or stearate had no significant differences relative to the control. These results implied that certain concentrations of saturated LCFA could stimulate cell proliferation and the accumulation of TAG, whereas a reduction may occur with the addition of an overdose of saturated LCFA. Saturated LCFA could up-regulate CSN1S1 mRNA abundance, but further studies are necessary to elucidate the mechanism for regulating milk fat and protein synthesis.

  6. The effect of select seminal plasma proteins on endometrial mRNA cytokine expression in mares susceptible to persistent mating-induced endometritis.

    PubMed

    Fedorka, C E; Scoggin, K E; Woodward, E M; Squires, E L; Ball, B A; Troedsson, Mht

    2017-02-01

    In the horse, breeding induces a transient endometrial inflammation. A subset of mares are unable to resolve this inflammation, and they are considered susceptible to persistent mating-induced endometritis PMIE Select seminal plasma proteins cysteine-rich secretory protein-3 (CRISP-3) and lactoferrin have been shown to affect the innate immune response to sperm in vitro. The objective of this study was to determine whether the addition of CRISP-3 and lactoferrin at the time of insemination had an effect on the mRNA expression of endometrial cytokines in susceptible mares after breeding. Six mares classified as susceptible to PMIE were inseminated during four consecutive oestrous cycles with treatments in randomized order of: 1 mg/ml CRISP-3, 150 μg/ml lactoferrin, seminal plasma (positive control) or lactated Ringer's solution (LRS; negative control) to a total volume of 10 ml combined with 1 × 10(9) spermatozoa pooled from two stallions. Six hours after treatment, an endometrial biopsy was obtained for qPCR analysis of selected genes associated with inflammation (pro-inflammatory cytokines interleukin (IL)-1β, IL-8, tumour necrosis factor (TNF)-α, interferon (INF)-γ, anti-inflammatory cytokines IL-1RN and IL-10, and inflammatory-modulating cytokine IL-6). Seminal plasma treatment increased the mRNA expression of IL-1β (p = .019) and IL-8 (p = .0068), while suppressing the mRNA expression of TNF (p = .0013). Lactoferrin also suppressed the mRNA expression of TNF (p = .0013). In conclusion, exogenous lactoferrin may be considered as one modulator of the complex series of events resulting in the poorly regulated pro-inflammatory response seen in susceptible mares.

  7. Alpha1-chimaerin, a Rac1 GTPase-activating protein, is expressed at reduced mRNA levels in the brain of Alzheimer's disease patients

    PubMed Central

    Kato, Tomoko; Konishi, Yoshihiro; Shimohama, Shun; Beach, Thomas G.; Akatsu, Hiroyasu; Tooyama, Ikuo

    2015-01-01

    Alpha1-chimaerin is a GTPase-activating protein (GAP) for Rac1, a member of the Rho small GTPase family, whose action leads to the inactivation of Rac1. Rac1 activity is upregulated in Alzheimer's disease, but little is known about the role of α1-chimaerin. In this study, we investigated the expression and localization of α1-chimaerin mRNA in postmortem human brains from patients with Alzheimer's disease and control subjects. In situ hybridization studies demonstrated that α1-chimaerin was expressed by neurons in the neo-cortex of the temporal lobe and the hippocampus of both controls and Alzheimer's disease cases, with the signal intensity dramatically decreased in patients with Alzheimer's disease. Real-time PCR analysis confirmed a significant reduction of α1-chimaerin mRNA expression in the temporal cortex of Alzheimer's disease cases. In contrast, α2-chimaerin mRNA levels showed no significant difference between the groups. The present study showed reduced α1-chimaerin expression in the brain of Alzheimer's disease cases, suggesting a role in the upregulation of Rac1 activity during the disease process. PMID:25676811

  8. DECREASED EXPRESSION OF ErbB4 AND TYROSINE HYDROXYLASE mRNA AND PROTEIN IN THE VENTRAL MIDBRAIN OF AGED RATS

    PubMed Central

    DICKERSON, J. W.; HEMMERLE, A. M.; NUMAN, S.; LUNDGREN, K. H.; SEROOGY, K. B.

    2009-01-01

    Decreased availability or efficacy of neurotrophic factors may underlie an increased susceptibility of mesencephalic dopaminergic cells to age-related degeneration. Neuregulins (NRGs) are pleotrophic growth factors for many cell types including mesencephalic dopamine cells in culture and in vivo. The functional NRG receptor ErbB4 is expressed by virtually all midbrain dopamine neurons. To determine if levels of the NRG receptor are maintained during aging in the dopaminergic ventral mesencephalon, expression of ErbB4 mRNA and protein was examined in young (3 months), middle-aged (18 months), and old (24–25 months) Brown Norway/Fischer 344 F1 rats. ErbB4 mRNA levels in the substantia nigra pars compacta (SNpc), but not the adjacent ventral tegmental area (VTA) or subtantia nigra pars lateralis (SNl), were significantly reduced in the middle-aged and old animals when compared to young rats. Protein expression of ErbB4 in the ventral midbrain was significantly decreased in the old rats when compared to the young rats. Expression of tyrosine hydroxylase (TH) mRNA levels were significantly reduced in the old rats when compared to young animals in the SNpc, but not in the VTA or SNl. Tyrosine hydroxylase protein levels in the ventral midbrain were also decreased in the old animals when compared to the young animals. These data demonstrate a progressive decline of ErbB4 expression, coinciding with a loss of the dopamine-synthesizing enzyme TH, in the ventral midbrain of aged rats, particularly in the SNpc. These findings may implicate a role for diminished NRG/ErbB4 trophic support in dopamine-related neurodegenerative disorders of aging such as Parkinson’s disease. PMID:19505538

  9. Expression of BRI2 mRNA and protein in normal human brain and familial British dementia: its relevance to the pathogenesis of disease

    PubMed Central

    Lashley, T.; Revesz, T.; Plant, G.; Bandopadhyay, R.; Lees, A. J.; Frangione, B.; Wood, N. W.; de Silva, R.; Ghiso, J.; Rostagno, A.; Holton, J. L.

    2009-01-01

    Introduction Two different disease-specific mutations in the BRI2 gene, situated on chromosome 13, have been identified as giving rise to familial British dementia (FBD) and familial Danish dementia (FDD). Each mutation results in extension of the open reading frame generating the disease-specific precursor proteins which are cleaved by furin-like proteolysis releasing the amyloidogenic C-terminal peptides ABri and ADan in FBD and FDD, respectively. Material and methods To understand the mechanism of the formation of amyloid lesions in FBD, we studied the origin of the precursor proteins and furin in the human brain. We used control brains, cases of sporadic Alzheimer’s disease (AD), variant AD with cotton wool plaques and FBD to study BRI2 mRNA expression using in situ hybridization. Furin and BRI2 protein expression was investigated using Western blotting and immunohistochemistry. Results BRI2 mRNA and BRI2 protein are widely expressed primarily by neurones and glia and are deposited in the amyloid lesions in FBD. They were, however, not expressed by cerebrovascular components. Furin expression showed a similar pattern except that it was also present in cerebrovascular smooth muscle cells. Conclusions These findings suggest that neurones and glia and are a major source of BRI2 protein and that in FBD, the mutated precursor protein may undergo furin cleavage within neurones to produce the amyloid peptide ABri. The failure to demonstrate BRI2 in blood vessels under the conditions tested suggests that vascular amyloid peptide production does not contribute significantly to cerebral amyloid angiopathy (CAA) in FBD and FDD, lending indirect support to the drainage hypothesis of CAA. PMID:18282158

  10. Expression of BRI2 mRNA and protein in normal human brain and familial British dementia: its relevance to the pathogenesis of disease.

    PubMed

    Lashley, T; Revesz, T; Plant, G; Bandopadhyay, R; Lees, A J; Frangione, B; Wood, N W; de Silva, R; Ghiso, J; Rostagno, A; Holton, J L

    2008-10-01

    Two different disease-specific mutations in the BRI2 gene, situated on chromosome 13, have been identified as giving rise to familial British dementia (FBD) and familial Danish dementia (FDD). Each mutation results in extension of the open reading frame generating the disease-specific precursor proteins which are cleaved by furin-like proteolysis releasing the amyloidogenic C-terminal peptides ABri and ADan in FBD and FDD, respectively. To understand the mechanism of the formation of amyloid lesions in FBD, we studied the origin of the precursor proteins and furin in the human brain. We used control brains, cases of sporadic Alzheimer's disease (AD), variant AD with cotton wool plaques and FBD to study BRI2 mRNA expression using in situ hybridization. Furin and BRI2 protein expression was investigated using Western blotting and immunohistochemistry. BRI2 mRNA and BRI2 protein are widely expressed primarily by neurones and glia and are deposited in the amyloid lesions in FBD. They were, however, not expressed by cerebrovascular components. Furin expression showed a similar pattern except that it was also present in cerebrovascular smooth muscle cells. These findings suggest that neurones and glia and are a major source of BRI2 protein and that in FBD, the mutated precursor protein may undergo furin cleavage within neurones to produce the amyloid peptide ABri. The failure to demonstrate BRI2 in blood vessels under the conditions tested suggests that vascular amyloid peptide production does not contribute significantly to cerebral amyloid angiopathy (CAA) in FBD and FDD, lending indirect support to the drainage hypothesis of CAA.

  11. Human Immunodeficiency Virus Tat-Activated Expression of Poliovirus Protein 2A Inhibits mRNA Translation

    NASA Astrophysics Data System (ADS)

    Sun, Xiao-Hong; Baltimore, David

    1989-04-01

    To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was contransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.

  12. Association of time-dependent changes in mu opioid receptor mRNA, but not BDNF, TrkB, or MeCP2 mRNA and protein expression in the rat nucleus accumbens with incubation of heroin craving

    PubMed Central

    Theberge, Florence R. M.; Pickens, Charles L.; Goldart, Evan; Fanous, Sanya; Hope, Bruce T.; Liu, Qing-Rong

    2013-01-01

    Rationale and objectives Responding to heroin cues progressively increases after cessation of heroin self-administration (incubation of heroin craving). We investigated whether this incubation is associated with time-dependent changes in brain-derived neurotrophic factor (BDNF) and methyl-CpG binding protein 2 (MeCP2) signaling and mu opioid receptor (MOR) expression in nucleus accumbens (NAc), dorsal striatum (DS), and medial pre-frontal cortex (mPFC). We also investigated the effect of the preferential MOR antagonist naloxone on cue-induced heroin seeking during abstinence. Methods We trained rats to self-administer heroin or saline for 9–10 days and then dissected the NAc, DS, and mPFC at different abstinence days and measured mRNA and protein levels of BDNF, TrkB, and MeCP2, as well as MOR mRNA (Oprm1). In other groups, we assessed cue-induced heroin seeking in extinction tests after 1, 11, and 30 abstinence days, and naloxone’s (0–1.0 mg/kg) effect on extinction responding after 1 and 15 days. Results Cue-induced heroin seeking progressively increased or incubated during abstinence. This incubation was not associated with changes in BDNF, TrkB, or MeCP2 mRNA or protein levels in NAc, DS, or mPFC; additionally, no molecular changes were observed after extinction tests on day 11. In NAc, but not DS or mPFC, MOR mRNA decreased on abstinence day 1 and returned to basal levels over time. Naloxone significantly decreased cue-induced heroin seeking after 15 abstinence days but not 1 day. Conclusions Results suggest a role of MOR in incubation of heroin craving. As previous studies implicated NAc BDNF in incubation of cocaine craving, our data suggest that different mechanisms contribute to incubation of heroin versus cocaine craving. PMID:22790874

  13. [Influence of ablactation rats' high calcium intake to the expression of uncoupling protein 2 mRNA after adulthood obesity induction].

    PubMed

    Xu, Shudi; Zhang, Keyuan; Zheng, Shutao; Wang, Zhong; Yu, Libo; Ding, Hiong

    2014-01-01

    To study the influence of early high calcium intake to adulthood obesity through detection the expression of uncoupling protein 2 ( UCP2) mRNA in muscle by a reverse transcription polymerase chain reaction ( RT-PCR) technique. 120 male Wistar rats were divided randomly into normal control group, high dose calcium group, medium dose calcium group and low doses calcium group, with basic diet and high calcium diet for 4 weeks. After intervention, blood fat was compared. Subsequently, all rats were fed basic diet for 3 weeks and blood fat was compared. Then, normal feed rats were randomly divided into normal controls and obesity induction group. Obesity induction group, high dose calcium group, medium dose calcium group and low doses calcium group were fed high-fat food. After 8 weeks, RT-PCR was used for analysis After obesity induction, weight the expression of UCP2 mRNA in muscle. growth of three high calcium groups were below obesity induction group, weight growth of low doses and high doses of high calcium groups were no difference with normal control group. Serum triglyceride levels of high dose high calcium group were significantly lower than obesity induction group and no difference with normal control group. Expression level of UCP2 mRNA of obesity induction group and high doses of high calcium group were obviously lower than normal controls, low dose and medium dose high calcium group, medium dose high calcium group' expression was significantly higher than normal control group. In rats' early life high calcium intake can continue to affect adulthood obesity induced by high-fat feed, increase expression level of UCP2 mRNA, improve the disorder of blood fat metabolism.

  14. mRNA expression of the DNA replication-initiation proteins in epithelial dysplasia and squamous cell carcinoma of the tongue.

    PubMed

    Li, Jian-na; Feng, Chong-jin; Lu, Yong-jun; Li, Hui-jun; Tu, Zheng; Liao, Gui-qing; Liang, Chun

    2008-12-30

    The tongue squamous cell carcinomas (SCCs) are characterized by high mitotic activity, and early detection is desirable. Overexpression of the DNA replication-initiation proteins has been associated with dysplasia and malignancy. Our aim was to determine whether these proteins are useful biomarkers for assessing the development of tongue SCC. We analyzed the mRNA expression of CDC6, CDT1, MCM2 and CDC45 in formalin-fixed, paraffin-embedded benign and malignant tongue tissues using quantitative real-time PCR followed by statistical analysis. We found that the expression levels are significantly higher in malignant SCC than mild precancerous epithelial dysplasia, and the expression levels in general increase with increasing grade of precancerous lesions from mild, moderate to severe epithelial dysplasia. CDC6 and CDC45 expression is dependent of the dysplasia grade and lymph node status. CDT1 expression is higher in severe dysplasia than in mild and moderate dysplasia. MCM2 expression is dependent of the dysplasia grade, lymph node status and clinical stage. The expression of the four genes is independent of tumor size or histological grade. A simple linear regression analysis revealed a linear increase in the mRNA levels of the four genes from the mild to severe dysplasia and SCC. A strong association was established between CDC6 and CDT1, and between MCM2 and CDC45 expression. The nonparametric receiver operating characteristic analysis suggested that MCM2 and CDC45 had a higher accuracy than CDC6 and CDT1 for distinguishing dysplasia from tongue SCC. These proteins can be used as biomarkers to distinguish precancerous dysplasia from SCC and are useful for early detection and diagnosis of SCC as an adjunct to clinicopathological parameters.

  15. Chicken growth-associated protein (GAP)-43: primary structure and regulated expression of mRNA during embryogenesis.

    PubMed

    Baizer, L; Alkan, S; Stocker, K; Ciment, G

    1990-01-01

    Growth-associated protein (GAP)-43 is a neuron-specific phosphoprotein whose expression is associated with axonal outgrowth during neuronal development and regeneration. In order to investigate the expression of this gene product in the early developing nervous system we have isolated and sequenced a cDNA for chicken GAP-43. The predicted amino acid sequence for chicken GAP-43 displays extensive similarity to that of the mammalian protein, particularly in the amino-terminal region, to which functional domains of the protein have been assigned. The cDNA hybridizes with two RNAs of differing molecular weights on Northern blots; both appear to be regulated similarly. These RNAs first appear in the brain on embryonic day 3 (E3), suggesting that GAP-43 begins to be expressed when neuroblasts become post-mitotic. In situ hybridization analysis reveals that GAP-43 RNA is expressed by several neural structures in the chick embryo, including derivatives of the neural tube, neural crest, and neuroectodermal placodes.

  16. PrP mRNA and protein expression in brain and PrPc in CSF in Creutzfeldt-Jakob disease MM1 and VV2

    PubMed Central

    Llorens, Franc; Ansoleaga, Belén; Garcia-Esparcia, Paula; Zafar, Saima; Grau-Rivera, Oriol; López-González, Irene; Blanco, Rosi; Carmona, Margarita; Yagüe, Jordi; Nos, Carlos; del Río, José Antonio; Gelpí, Ellen; Zerr, Inga; Ferrer, Isidre

    2013-01-01

    Creutzfeldt-Jakob disease (CJD) is a heterogenic neurodegenerative disorder associated with abnormal post-translational processing of cellular prion protein (PrPc). CJD displays distinctive clinical and pathological features which correlate with the genotype at the codon 129 (methionine or valine: M or V respectively) in the prion protein gene and with size of the protease-resistant core of the abnormal prion protein PrPsc (type 1: 20/21 kDa and type 2: 19 kDa). MM1 and VV2 are the most common sporadic CJD (sCJD) subtypes. PrP mRNA expression levels in the frontal cortex and cerebellum are reduced in sCJD in a form subtype-dependent. Total PrP protein levels and PrPsc levels in the frontal cortex and cerebellum accumulate differentially in sCJD MM1 and sCJD VV2 with no relation between PrPsc deposition and spongiform degeneration and neuron loss, but with microgliosis, and IL6 and TNF-α response. In the CSF, reduced PrPc, the only form present in this compartment, occurs in sCJD MM1 and VV2. PrP mRNA expression is also reduced in the frontal cortex in advanced stages of Alzheimer disease, Lewy body disease, progressive supranuclear palsy, and frontotemporal lobe degeneration, but PrPc levels in brain varies from one disease to another. Reduced PrPc levels in CSF correlate with PrP mRNA expression in brain, which in turn reflects severity of degeneration in sCJD. PMID:24047819

  17. PrP mRNA and protein expression in brain and PrP(c) in CSF in Creutzfeldt-Jakob disease MM1 and VV2.

    PubMed

    Llorens, Franc; Ansoleaga, Belén; Garcia-Esparcia, Paula; Zafar, Saima; Grau-Rivera, Oriol; López-González, Irene; Blanco, Rosi; Carmona, Margarita; Yagüe, Jordi; Nos, Carlos; Del Río, José Antonio; Gelpí, Ellen; Zerr, Inga; Ferrer, Isidre

    2013-01-01

    Creutzfeldt-Jakob disease (CJD) is a heterogenic neurodegenerative disorder associated with abnormal post-translational processing of cellular prion protein (PrP(c)). CJD displays distinctive clinical and pathological features which correlate with the genotype at the codon 129 (methionine or valine: M or V respectively) in the prion protein gene and with size of the protease-resistant core of the abnormal prion protein PrP(sc) (type 1: 20/21 kDa and type 2: 19 kDa). MM1 and VV2 are the most common sporadic CJD (sCJD) subtypes. PrP mRNA expression levels in the frontal cortex and cerebellum are reduced in sCJD in a form subtype-dependent. Total PrP protein levels and PrP(sc) levels in the frontal cortex and cerebellum accumulate differentially in sCJD MM1 and sCJD VV2 with no relation between PrP(sc) deposition and spongiform degeneration and neuron loss, but with microgliosis, and IL6 and TNF-α response. In the CSF, reduced PrP(c), the only form present in this compartment, occurs in sCJD MM1 and VV2. PrP mRNA expression is also reduced in the frontal cortex in advanced stages of Alzheimer disease, Lewy body disease, progressive supranuclear palsy, and frontotemporal lobe degeneration, but PrP(c) levels in brain varies from one disease to another. Reduced PrP(c) levels in CSF correlate with PrP mRNA expression in brain, which in turn reflects severity of degeneration in sCJD.

  18. Effects of nitrite stress on mRNA expression of antioxidant enzymes, immune-related genes and apoptosis-related proteins in Marsupenaeus japonicus.

    PubMed

    Zheng, Jinbin; Mao, Yong; Su, Yongquan; Wang, Jun

    2016-11-01

    Nitrite accumulation in aquaculture systems is a potential risk factor that may trigger stress responses in aquatic organisms. However, the mechanisms regulating the responses of shrimp to nitrite stress remain unclear. In this study, full-length cDNA sequences of two apoptosis-related genes, caspase-3 and defender against apoptotic death (DAD-1), were cloned from Marsupenaeus japonicus for the first time, and their expression levels and tissue distribution were analyzed by quantitative real-time PCR (qRT-PCR). The full lengths of Mjcaspase-3 and MjDAD-1 were 1203 bp and 640 bp respectively, with deduced amino acid (AA) sequences of 321 and 114 AA. Mjcaspase-3 was predominantly expressed in haemocytes and weakly expressed in the seven other tissues tested. MjDAD-1 was mainly expressed in the defense and digestive tissues, especially in the hepatopancreas and hemocytes. To explore the influence of nitrite stress on the genetic response of antioxidant enzymes, immune-related genes and apoptosis-related proteins, the mRNA expression profiles of MjCAT, MjMnSOD, Mj-ilys, Mj-sty, Mjcaspase-3 and MjDAD-1 in response to nitrite stress were analyzed by qRT-PCR. The mRNA levels of MjCAT, MjMnSOD, Mj-ilys, Mj-sty, Mjcaspase-3 and MjDAD-1 show both time- and dose-dependent changes in response to nitrite stress. The mRNA expression levels of MjCAT and MjSOD peaked at 6 h for all nitrite concentrations tested (p < 0.05) and the up-regulated of MjCAT and MjSOD exhibited a positive correlation with the nitrite concentration. The mRNA expression levels of Mj-ilys and Mj-sty gradually decreased during the experiment period. Mjcaspase-3 mRNA level reached a maximum at 6 h (p < 0.05), and MjDAD-1 reached its peak at 12 h and 48 h in 10 mg/L and 20 mg/L nitrite, respectively. In addition, CAT and SOD activity showed changes in response to nitrite stress that mirrored the induced expression of MjCAT and MjMnSOD, and prolonged nitrite exposure reduced the activity of CAT. This

  19. Chronic Administration of Bacopa Monniera Increases BDNF Protein and mRNA Expressions: A Study in Chronic Unpredictable Stress Induced Animal Model of Depression

    PubMed Central

    Banerjee, Ritabrata; Hazra, Somoday; Ghosh, Anup Kumar

    2014-01-01

    Objective The present study aimed to investigate whether graded doses of Bacopa Monniera (BM) extract could produce antidepressant-like effects in chronic unpredictable stress (CUS) induced depression in rats and its possible mechanism(s). Methods Rats were subjected to an experimental setting of CUS. The effect of BM extract treatment in CUS-induced depression was examined using behavioral tests including the sucrose consumption, open field test and shuttle box escape test. The mechanism underlying the antidepressant-like action of BM extract was examined by measuring brain-derived neurotrophic factor (BDNF) protein and mRNA expression in brain tissues of CUS-exposed rats. Results Exposure to CUS for 4 weeks caused depression-like behavior in rats, as indicated by significant decreases in sucrose consumption, locomotor activity and escape latency. In addition, it was found that BDNF protein and mRNA levels in the hippocampus and frontal cortex were lower in CUS-treated rats, as compared to controls. Daily administration of the graded doses of BM extract during the 4-week period of CUS significantly suppressed behavioral changes and attenuated the CUS-induced decrease in BDNF protein and mRNA levels in the hippocampus and frontal cortex. Conclusion The results suggest that BM extract alleviates depression induced by CUS. Present study also confirms that 80-120 mg/kg doses of BM extract have significantly higher antidepressant-like activity. PMID:25110503

  20. Age determines the magnitudes of angiotensin II-induced contractions, mRNA, and protein expression of angiotensin type 1 receptors in rat carotid arteries.

    PubMed

    Vamos, Zoltan; Cseplo, Peter; Ivic, Ivan; Matics, Robert; Hamar, Janos; Koller, Akos

    2014-05-01

    In this study, we hypothesized that aging alters angiotensin II (Ang II)-induced vasomotor responses and expression of vascular mRNA and protein angiotensin type 1 receptor (AT1R). Thus, carotid arteries were isolated from the following age groups of rats: 8 days, 2-9 months, 12-20 months, and 20-30 months, and their vasomotor responses were measured in a myograph after repeated administrations of Ang II. Vascular relative AT1R mRNA level was determined by quantitative reverse-transcriptase polymerase chain reaction and the AT1R protein density was measured by Western blot. Contractions to the first administration of Ang II increased from 8 days to 6 months and then they decreased to 30 months. In general, second administration of Ang II elicited reduced contractions, but they also increased from 8 days until 2 months and then they decreased to 30 months. Similarly the AT1R mRNA level increased from 8 days to 12 months and then decreased to 30 months. Similarly the AT1R protein density increased from 8 days until 16 months and then they decreased to 30 months. The pattern of these changes correlated with functional vasomotor data. We conclude that aging (newborn to senescence) has substantial effects on Ang II-induced vasomotor responses and AT1R signaling suggesting the importance of genetic programs.

  1. Correlation of mRNA expression and protein abundance affected by multiple sequence features related to translational efficiency in Desulfovibrio vulgaris: A quantitative analysis

    SciTech Connect

    Nie, Lei; Wu, Gang; Zhang, Weiwen

    2006-12-01

    The modest correlation between mRNA expression and protein abundance in large scale datasets is explained in part by experimental challenges, such as technological limitations, and in part by fundamental biological factors in the transcription and translation processes. Among various factors affecting the mRNA-protein correlation, the roles of biological factors related to translation are poorly understood. In this study, using experimental mRNA expression and protein abundance data collected from Desulfovibrio vulgaris by DNA microarray and LC-MS/MS proteomic analysis, we quantitatively examined the effects of several translational-efficiency-related sequence features on mRNA-protein correlation. Three classes of sequence features were investigated according to different translational stages: (1) initiation: Shine-Dalgarno sequences, start codon identity and start codon context; (2) elongation: codon usage and amino acid usage; and (3) termination: stop codon identity and stop codon context. Surprisingly, although it is widely accepted that translation initiation is a rate-limiting step for translation, our results showed that the mRNA-protein correlation was affected the most by the features at elongation stages, codon usage and amino acid composition (7.4-12.6% and 5.3-9.3% of the total variation of mRNA-protein correlation, respectively), followed by stop codon context and the Shine-Dalgarno sequence (2.5-4.2% and 2.3%, respectively). Taken together, all sequence features contributed to 18.4-21.8% of the total variation of mRNA-protein correlation. As the first comprehensive quantitative analysis of the mRNA-protein correlation in bacterial D. vulgaris, our results suggest that the traditional view of the relative importance of various sequence features in prokaryotic protein translation might be questionable.

  2. Correlation between Heart-type Fatty Acid-binding Protein Gene Polymorphism and mRNA Expression with Intramuscular Fat in Baicheng-oil Chicken

    PubMed Central

    Wang, Yong; He, Jianzhong; Yang, Wenxuan; Muhantay, Gemenggul; Chen, Ying; Xing, Jinming; Liu, Jianzhu

    2015-01-01

    This study aims to determine the polymorphism and mRNA expression pattern of the heart-type fatty acid-binding protein (H-FABP) gene and their association with intramuscular fat (IMF) content in the breast and leg muscles of Baicheng oil chicken (BOC). A total of 720 chickens, including 240 black Baicheng oil chicken (BBOC), 240 silky Baicheng oil chicken (SBOC), and 240 white Baicheng oil chicken (WBOC) were raised. Three genotypes of H-FABP gene second extron following AA, AB, and BB were detected by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) strategy. The G939A site created AA genotype and G956A site created BB genotype. The content of IMF in AA genotype in breast muscle of BBOC was significantly higher than that of AB (p = 0.0176) and the genotype in leg muscle of WBOC was significantly higher than that of AB (p = 0.0145). The G939A site could be taken as genetic marker for higher IMF content selecting for breast muscle of BBOC and leg muscle of WBOC. The relative mRNA expression of H-FABP was measured by real-time PCR at 30, 60, 90, and 120 d. The IMF content significantly increased with age in both muscles. The mRNA expression level of H-FABP significantly decreased with age in both muscles of the three types of chickens. Moreover, a significant negative correlation between H-FABP abundance and IMF content in the leg muscles of WBOC (p = 0.035) was observed. The mRNA expression of H-FABP negatively correlated with the IMF content in both breast and leg muscles of BOC sat slaughter time. PMID:26323394

  3. Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity.

    PubMed Central

    Purcell, D F; Martin, M A

    1993-01-01

    Multiple RNA splicing sites exist within human immunodeficiency virus type 1 (HIV-1) genomic RNA, and these sites enable the synthesis of many mRNAs for each of several viral proteins. We evaluated the biological significance of the alternatively spliced mRNA species during productive HIV-1 infections of peripheral blood lymphocytes and human T-cell lines to determine the potential role of alternative RNA splicing in the regulation of HIV-1 replication and infection. First, we used a semiquantitative polymerase chain reaction of cDNAs that were radiolabeled for gel analysis to determine the relative abundance of the diverse array of alternatively spliced HIV-1 mRNAs. The predominant rev, tat, vpr, and env RNAs contained a minimum of noncoding sequence, but the predominant nef mRNAs were incompletely spliced and invariably included noncoding exons. Second, the effect of altered RNA processing was measured following mutagenesis of the major 5' splice donor and several cryptic, constitutive, and competing 3' splice acceptor motifs of HIV-1NL4-3. Mutations that ablated constitutive splice sites led to the activation of new cryptic sites; some of these preserved biological function. Mutations that ablated competing splice acceptor sites caused marked alterations in the pool of virus-derived mRNAs and, in some instances, in virus infectivity and/or the profile of virus proteins. The redundant RNA splicing signals in the HIV-1 genome and alternatively spliced mRNAs provides a mechanism for regulating the relative proportions of HIV-1 proteins and, in some cases, viral infectivity. Images PMID:8411338

  4. Intraovarian expression of GnRH-1 and gonadotropin mRNA and protein levels in Siberian hamsters during the estrus cycle and photoperiod induced regression/recrudescence

    PubMed Central

    Shahed, Asha; Young, Kelly A.

    2010-01-01

    The hypothalamic-pituitary-gonadal (HPG) axis is the key reproductive regulator in vertebrates. While gonadotropin releasing hormone (GnRH), follicle stimulating (FSH), and luteinizing (LH) hormones are primarily produced in the hypothalamus and pituitary, they can be synthesized in the gonads, suggesting an intraovarian GnRH-gonadotropin axis. Because these hormones are critical for follicle maturation and steroidogenesis, we hypothesized that this intraovarian axis may be important in photoperiod-induced ovarian regression/recrudescence in seasonal breeders. Thus, we investigated GnRH-1 and gonadotropin mRNA and protein expression in Siberian hamster ovaries during (1) the estrous cycle; where ovaries from cycling long day hamsters (LD;16L:8D) were collected at proestrus, estrus, diestrus I, and diestrus II and (2) during photoperiod induced regression/ recrudescence; where ovaries were collected from hamsters exposed to 14wks of LD, short days (SD;8L:16D), or 8wks post-transfer to LD after 14wks SD (PT). GnRH-1, LHβ, FSHβ, and common α subunit mRNA expression was observed in cycling ovaries. GnRH-1 expression peaked at diestrus I compared to other stages (p<0.05). FSHβ and LHβ mRNA levels peaked at proestrus and diestrus I (p<0.05), with no change in the α subunit across the cycle (p>0.05). SD exposure decreased ovarian mass and plasma estradiol concentrations (p<0.05) and increased GnRH-1, LHβ, FSHβ, and α subunit mRNA expression as compared to LD and, except for LH, compared to PT (p<0.05). GnRH and gonadotropin protein was also dynamically expressed across the estrous cycle and photoperiod exposure. The presence of cycling intraovarian GnRH-1 and gonadotropin mRNA suggests that these hormones may be locally involved in ovarian maintenance during SD regression and/or could potentially serve to prime ovaries for rapid recrudescence. PMID:20955709

  5. Differential expression of p53, p63 and p73 protein and mRNA for DMBA-induced hamster buccal-pouch squamous-cell carcinomas

    PubMed Central

    Chen, Yuk-Kwan; Huse, Shue-Sang; Lin, Li-Min

    2004-01-01

    Abnormalities in the p53 gene are regarded as the most consistent of the genetic abnormalities associated with oral squamous-cell carcinoma. Two related members of the p53 gene family, p73 and p63, have shown remarkable structural similarity to p53, suggesting possible functional and biological interactions. The purpose of this study was to investigate the differential expression of p73, p63 and p53 genes for DMBA-induced hamster buccal-pouch squamous-cell carcinoma. Immunohistochemical analysis for protein expression and reverse transcriptase-polymerase chain reaction (RT-PCR) for mRNA expression were performed for 40 samples of hamster buccal pouches, the total being separated into one experimental group (15-week DMBA-treated; 20 animals) and two control groups (untreated and mineral oil-treated; 10 animals each). Using immunohistochemical techniques, nuclear staining of p53 and p73 proteins was detected in a subset of hamster buccal-pouch tissue specimens treated with DMBA for a period of 15 weeks, whereas p63 proteins were noted for all of the 20 hamster buccal-pouch tissue specimens treated with DMBA for 15 weeks as well as for all of the untreated and mineral oil-treated hamster buccal-pouch tissue specimens. Differential expression of p63, p73 and p53 protein for the experimental group was as follows: p63+/p73+/p53+ (n = 14; 70%); p63+/p73+/p53− (n = 2; 10%); p63+/p73−/p53− (n = 4; 20%) and p63+/p73−/p53− (untreated [n = 10] and mineral oil-treated mucosa [n = 10]; 100% each). Upon RT-PCR, ΔNp63mRNA was detected within all of the 20 hamster buccal-pouch tissue specimens treated with DMBA for 15 weeks, whereas expression of TAp63 was not detected. Furthermore, p73 mRNA was identified for 16 of the hamster buccal-pouch tissue specimens treated with DMBA for 15 weeks, whereas p53 mRNA was noted for 14 15-week DMBA-treated pouches. The proportional (percentage) expression of ΔNp63, p73 and p53 mRNA for the hamster buccal-pouch tissue specimens

  6. MUC1-ARF—A Novel MUC1 Protein That Resides in the Nucleus and Is Expressed by Alternate Reading Frame Translation of MUC1 mRNA

    PubMed Central

    Pichinuk, Edward; Garbar, Christian; Bensussan, Armand; Meeker, Alan; Ziv, Ravit; Zehavi, Tania; Smorodinsky, Nechama I.; Hilkens, John; Hanisch, Franz-Georg; Rubinstein, Daniel B.; Wreschner, Daniel H.

    2016-01-01

    Translation of mRNA in alternate reading frames (ARF) is a naturally occurring process heretofore underappreciated as a generator of protein diversity. The MUC1 gene encodes MUC1-TM, a signal-transducing trans-membrane protein highly expressed in human malignancies. Here we show that an AUG codon downstream to the MUC1-TM initiation codon initiates an alternate reading frame thereby generating a novel protein, MUC1-ARF. MUC1-ARF, like its MUC1-TM 'parent’ protein, contains a tandem repeat (VNTR) domain. However, the amino acid sequence of the MUC1-ARF tandem repeat as well as N- and C- sequences flanking it differ entirely from those of MUC1-TM. In vitro protein synthesis assays and extensive immunohistochemical as well as western blot analyses with MUC1-ARF specific monoclonal antibodies confirmed MUC1-ARF expression. Rather than being expressed at the cell membrane like MUC1-TM, immunostaining showed that MUC1-ARF protein localizes mainly in the nucleus: Immunohistochemical analyses of MUC1-expressing tissues demonstrated MUC1-ARF expression in the nuclei of secretory luminal epithelial cells. MUC1-ARF expression varies in different malignancies. While the malignant epithelial cells of pancreatic cancer show limited expression, in breast cancer tissue MUC1-ARF demonstrates strong nuclear expression. Proinflammatory cytokines upregulate expression of MUC1-ARF protein and co-immunoprecipitation analyses demonstrate association of MUC1-ARF with SH3 domain-containing proteins. Mass spectrometry performed on proteins coprecipitating with MUC1-ARF demonstrated Glucose-6-phosphate 1-dehydrogenase (G6PD) and Dynamin 2 (DNM2). These studies not only reveal that the MUC1 gene generates a previously unidentified MUC1-ARF protein, they also show that just like its ‘parent’ MUC1-TM protein, MUC1-ARF is apparently linked to signaling and malignancy, yet a definitive link to these processes and the roles it plays awaits a precise identification of its molecular functions

  7. MUC1-ARF-A Novel MUC1 Protein That Resides in the Nucleus and Is Expressed by Alternate Reading Frame Translation of MUC1 mRNA.

    PubMed

    Chalick, Michael; Jacobi, Oded; Pichinuk, Edward; Garbar, Christian; Bensussan, Armand; Meeker, Alan; Ziv, Ravit; Zehavi, Tania; Smorodinsky, Nechama I; Hilkens, John; Hanisch, Franz-Georg; Rubinstein, Daniel B; Wreschner, Daniel H

    2016-01-01

    Translation of mRNA in alternate reading frames (ARF) is a naturally occurring process heretofore underappreciated as a generator of protein diversity. The MUC1 gene encodes MUC1-TM, a signal-transducing trans-membrane protein highly expressed in human malignancies. Here we show that an AUG codon downstream to the MUC1-TM initiation codon initiates an alternate reading frame thereby generating a novel protein, MUC1-ARF. MUC1-ARF, like its MUC1-TM 'parent' protein, contains a tandem repeat (VNTR) domain. However, the amino acid sequence of the MUC1-ARF tandem repeat as well as N- and C- sequences flanking it differ entirely from those of MUC1-TM. In vitro protein synthesis assays and extensive immunohistochemical as well as western blot analyses with MUC1-ARF specific monoclonal antibodies confirmed MUC1-ARF expression. Rather than being expressed at the cell membrane like MUC1-TM, immunostaining showed that MUC1-ARF protein localizes mainly in the nucleus: Immunohistochemical analyses of MUC1-expressing tissues demonstrated MUC1-ARF expression in the nuclei of secretory luminal epithelial cells. MUC1-ARF expression varies in different malignancies. While the malignant epithelial cells of pancreatic cancer show limited expression, in breast cancer tissue MUC1-ARF demonstrates strong nuclear expression. Proinflammatory cytokines upregulate expression of MUC1-ARF protein and co-immunoprecipitation analyses demonstrate association of MUC1-ARF with SH3 domain-containing proteins. Mass spectrometry performed on proteins coprecipitating with MUC1-ARF demonstrated Glucose-6-phosphate 1-dehydrogenase (G6PD) and Dynamin 2 (DNM2). These studies not only reveal that the MUC1 gene generates a previously unidentified MUC1-ARF protein, they also show that just like its 'parent' MUC1-TM protein, MUC1-ARF is apparently linked to signaling and malignancy, yet a definitive link to these processes and the roles it plays awaits a precise identification of its molecular functions

  8. Growth hormone stimulates protein synthesis in bovine skeletal muscle cells without altering insulin-like growth factor-I mRNA expression.

    PubMed

    Ge, X; Yu, J; Jiang, H

    2012-04-01

    Growth hormone is a major stimulator of skeletal muscle growth in animals, including cattle. In this study, we determined whether GH stimulates skeletal muscle growth in cattle by direct stimulation of proliferation or fusion of myoblasts, by direct stimulation of protein synthesis, or by direct inhibition of protein degradation in myotubes. We also determined whether these direct effects of GH are mediated by IGF-I produced by myoblasts or myotubes. Satellite cells were isolated from cattle skeletal muscle and were allowed to proliferate as myoblasts or induced to fuse into myotubes in culture. Growth hormone at 10 and 100 ng/mL increased protein synthesis in myotubes (P < 0.05), but had no effect on protein degradation in myotubes or proliferation of myoblasts (P > 0.05). Insulin-like growth factor-I at 50 and 500 ng/mL stimulated protein synthesis (P < 0.01), and this effect of IGF-I was much greater than that of GH (P < 0.05). Besides stimulating protein synthesis, IGF-I at 50 and 500 ng/mL also inhibited protein degradation in myotubes (P < 0.01), and IGF-I at 500 ng/mL stimulated proliferation of myoblasts (P < 0.05). Neither GH nor IGF-I had effects on fusion of myoblasts into myotubes (P > 0.1). These data indicate that GH and IGF-I have largely different direct effects on bovine muscle cells. Growth hormone at 10 and 100 ng/mL had no effect on IGF-I mRNA expression in either myoblasts or myotubes (P > 0.1). This lack of effect was not because the cultured myoblasts or myotubes were not responsive to GH; GH receptor mRNA was detectable in them and the expression of the cytokine-inducible SH2-containing protein (CISH) gene, a well-established GH target gene, was increased by GH in bovine myoblasts (P < 0.05). Overall, the data suggest that GH stimulates skeletal muscle growth in cattle in part through stimulation of protein synthesis in the muscle and that this stimulation is not mediated through increased IGF-I mRNA expression in the muscle.

  9. Nav1.7 protein and mRNA expression in the dorsal root ganglia of rats with chronic neuropathic pain.

    PubMed

    Liu, Chao; Cao, Jing; Ren, Xiuhua; Zang, Weidong

    2012-07-15

    Neuropathic pain was produced by chronic constriction injury of the sciatic nerve in rats. Behavioral tests showed that the thresholds for thermal and mechanical hyperalgesia were significantly reduced in neuropathic pain rats 3-28 days following model induction. The results of immunohistochemistry, western blot assays and reverse transcription-PCR showed that Nav1.7 protein and mRNA expression was significantly increased in the injured dorsal root ganglia. These findings indicated that Nav1.7 might play an important role in the model of chronic neuropathic pain.

  10. Motor Skills Training Improves Sensorimotor Dysfunction and Increases Microtubule-Associated Protein 2 mRNA Expression in Rats with Intracerebral Hemorrhage.

    PubMed

    Tamakoshi, Keigo; Kawanaka, Kentaro; Onishi, Hideaki; Takamatsu, Yasuyuki; Ishida, Kazuto

    2016-08-01

    In this study, we examined the effects of motor skills training on the sensorimotor function and the expression of genes associated with synaptic plasticity after intracerebral hemorrhage (ICH) in rats. Male Wistar rats were subjected to ICH or sham operation. ICH was caused by the injection of collagenase into the left striatum. Rats were randomly assigned to no training, acrobatic training, and sham groups. The acrobatic group performed 5 types of acrobatic tasks from 4 to 28 days after surgery. The forelimb sensorimotor function was evaluated over time using forepaw grasping, forelimb placing, and postural instability tests. At 14 and 29 days after the lesion, we analyzed the mRNA expression levels of microtubule-associated protein 2 (MAP2), brain-derived neurotrophic factor, and growth-associated protein 43 in the bilateral sensorimotor cortex (forelimb area) by real-time reverse transcription-polymerase chain reaction. Motor skills training in ICH rats improved the sensorimotor dysfunction significantly from the early phase. The mRNA expression level of MAP2 was upregulated in the ipsilesional sensorimotor cortex by motor skills training at 29 days after the lesion. Our results suggest that sensorimotor functional recovery following motor skills training after ICH is promoted by dendritic growth in the ipsilesional sensorimotor cortex. Copyright © 2016 National Stroke Association. Published by Elsevier Inc. All rights reserved.

  11. In vitro gene expression and mRNA translocation from transformed walnut (Juglans regia) rootstocks expressing DsRED fluorescent protein to wild-type scions.

    PubMed

    Liu, Xiaochen; Walawage, Sriema L; Leslie, Charles A; Dandekar, Abhaya M; Tricoli, David M; Hu, Hengkang; Huang, Youjun; Zhang, Jiaqi; Xv, Chuanmei; Huang, Jianqin; Zhang, Qixiang

    2017-06-01

    An in vitro grafting method was developed for examining gene translocation from rootstock to scion in walnut. Results showed the DsRED gene itself was not translocated but expressed mRNA was. Grafting is widely used in plants, especially in fruit and nut crops. Selected rootstocks can control scion growth and physiological traits, including shortening of the juvenile phase and controlling tree size. Rootstocks also can provide improved soil adaptation and pathogen resistance. Development of genetically modified (GM) fruit crops has progressed recently, but commercial cultivation is still limited due to the time required for evaluation and issues with deregulation. In this study, we evaluated the stability of DsRED marker gene expression in in vitro walnut shoots and examined translocation of the gene and its mRNA from transformed rootstock to wild-type scion. Results show that DsRED was expressed uniformly in transformed tissue-cultured shoots. When used as in vitro rootstocks, these had good graft affinity with wild-type control scion. PCR and qRT-PCR analysis showed that the DsRED gene was not transported from rootstock to scion, but the transcribed mRNA was translocated. This result provides further evidence of gene signal transport from rootstock to scion in fruit and nut crops.

  12. Intestinal immune function, antioxidant status and tight junction proteins mRNA expression in young grass carp (Ctenopharyngodon idella) fed riboflavin deficient diet.

    PubMed

    Chen, Liang; Feng, Lin; Jiang, Wei-Dan; Jiang, Jun; Wu, Pei; Zhao, Juan; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Liu, Yang

    2015-11-01

    This study investigated the effects of riboflavin on intestinal immunity, tight junctions and antioxidant status of young grass carp (Ctenopharyngodon idella). Fish were fed diets containing graded levels of riboflavin (0.63-10.04 mg/kg diet) for 8 weeks. The study indicated that riboflavin deficiency decreased lysozyme, acid phosphatase, copper/zinc superoxide dismutase, glutathione reductase and glutathione peroxidase activities, and contents of complement component 3 and reduced glutathione in the intestine of fish (P < 0.05). Meanwhile, riboflavin deficiency increased reactive oxygen species, malondialdehyde and protein carbonyl contents and catalase activity (P < 0.05) in the intestine of fish. Furthermore, real-time polymerase chain reaction analysis was used to investigate mRNA expression patterns and found that the mRNA levels of interleukin 10 and transforming growth factor β1, Occludin, zonula occludens 1, Claudin-b and Claudin-c, inhibitor protein κBα, target of rapamycin, ribosomal S6 protein kinase 1 and NF-E2-related factor 2, copper/zinc superoxide dismutase, glutathione peroxidase and glutathione reductase were decreased (P < 0.05) in the intestine of fish fed riboflavin-deficient diet. Conversely, the mRNA levels of tumor necrosis factor α, interleukin 1β, interleukin 8, nuclear factor kappa B p65, Ikappa B kinase β, Ikappa B kinase γ, Kelch-like-ECH-associated protein 1b, p38 mitogen-activated protein kinase, myosin light chain kinase and Claudin-12 were increased (P < 0.05) in the intestine of fish fed riboflavin-deficient diet. In conclusion, riboflavin deficiency decreased immunity and structural integrity of fish intestine. The optimum riboflavin level for intestinal acid phosphatase activity of young grass carp was estimated to be 6.65 mg/kg diet.

  13. [Effects of arsenic trioxide or retinoic acid on mRNA and protein expression of tissue factor and thrombomodulin and procoagulant activity in NB4 cells].

    PubMed

    Zhang, Xiao-Hui; Hu, Yu; Hong, Mei; Xia, Ling-Hui; Guo, Tao; Shen, Guan-Xin; Wei, Wen-Ning; Song, Shan-Jun

    2007-04-01

    To investigate the effect of arsenic trioxide (As(2)O(3)) or all-trans retinoic acid (ATRA) on the mRNA and protein expression of tissue factor (TF) and thrombomodulin (TM) and procoagulant activity (PCA) in NB4 cells. The NB4 cells were cultured in vitro and treated with As(2)O(3) or ATRA, expression of TF and TM antigen, and PCA change of treated NB4 cells were detected with ELISA, TF and TM mRNA transcription on the NB4 cells was assayed with reversed transcription polymerase chain reaction (RT-PCR). The results showed that 1 micromol/L As(2)O(3) and 1 micromol/L ATRA both gradually downregulated the expression of TF antigen and mRNA on NB4 cells, a human promyelocytic leukemia cell line, in time-dependent manner, as compared with control. The levels of TF antigen expression in AS(2)O(3) group were 13.3 +/- 1.8, 8.6 +/- 1.9, 10.8 +/- 1.5, 2.0 +/- 0.6 and 2.6 +/- 0.9 ng/10(7) respectively; while the levels of TF antigen expression in ATRA group were 12.4 +/- 1.1, 11.3 +/- 1.8, 5.7 +/- 1.7, 2.8 +/- 0.8 and 2.0 +/- 0.6 ng/10(7) at 24, 48, 72, 96 and 120 hours respectively (P<0.05). The procoagulant activity (PCA) of NB4 cells was decreased, blood coagulation times were 123.5 +/- 10.5, 156.3 +/- 11.6, 179.3 +/- 15.3, 248.9 +/- 20.1, 312.0 +/- 29.8 seconds in As(2)O(3) groups, respectively; 76.4 +/- 5.6, 146.8 +/- 10.9, 198.2 +/- 15.6, 265.8 +/- 20.6 and 363.8 +/- 31.9 seconds in ATRA groups respectively at 24, 48, 72, 96 and 120 hours (P<0.05). ATRA upregulated TM antigen expression on NB4 cells. It is concluded that the As(2)O(3) and ATRA decrease mRNA transcription of TF, downregulate expression of TF and reduce procoagulant activity in NB4 cells. The TM transcription and expression upregulated by ATRA may alleviate dysfunction of coagulation in APL.

  14. mRNA expression and protein distribution of fibronectin splice variants and high-molecular weight tenascin-C in different phases of human fracture healing.

    PubMed

    Kilian, O; Dahse, R; Alt, V; Zardi, L; Hentschel, J; Schnettler, R; Kosmehl, H

    2008-08-01

    Fracture healing is a reparative physiological process, which proceeds in stages, each characterized by the predominant tissue in the fracture gap. The tissue matrix is continuously reorganized by cell migration, proliferation, and differentiation. Adhesive proteins such as fibronectin and tenascin transmit information between matrix and cells. As a result of alternative splicing of pre-RNA, EDA + fibronectin, EDB + fibronectin, and high-molecular weight (hm) tenascin-C are generated. By definition, EDB + fibronectin is an oncofetal protein because it is extremely rare in normal adult tissue and plasma, whereas it is expressed in fetal and tumor tissues and during wound healing. In this study, we for the first time describe EDA + fibronectin, EDB + fibronectin, and hm tenascin-C expression in human fracture gap tissue during various stages of differentiation. We demonstrate mRNA expression of all three splice variants in the initial fibrin matrix with upregulation in the enchondral ossification/osteoid and woven bone stages. Of all variants, EDA + fibronectin mRNA has the highest concentration in all stages. For the analysis, we used LightCycler-based relative mRNA quantification and immunohistochemistry. Our data demonstrate that EDA + fibronectin and hm tenascin-C show a diffuse distribution pattern in fracture gap connective tissue, while EDB + fibronectin is focally concentrated in osteoblastic cells at the margins of woven bone. EDA + fibronectin and hm tenascin represent markers for active granulation processes, whereas EDB + fibronectin is specific for cells forming the enchondral and osteoid matrix. The possibility of stimulating fracture healing by EDB + fibronectin-cytokine complexes should be tested in further investigations.

  15. Steroidogenic acute regulatory protein in eels: cDNA cloning and effects of ACTH and seawater transfer on its mRNA expression.

    PubMed

    Li, Yuan-You; Inoue, Koji; Takei, Yoshio

    2003-02-01

    Steroidogenic acute regulatory protein (StAR) is a key molecule for steroid production by translocating cholesterol from the outer to inner mitochondrial membrane. Two cDNAs of different length encoding StAR was cloned from the head kidney of the eel (Anguilla japonica). In the 3'-untranslated region (UTR) of the longer cDNA, two putative polyadenylation signals were found. The shorter one differed from the longer one solely by the lack of middle of 3'-UTR including the first polyadenylation signal. Reverse transcription-polymerase chain reaction (RT-PCR) that differentiates the two mRNAs showed that the ratio of the two was highly variable among individuals, and no preferential expression was detected between freshwater and seawater eels. The predicted protein consists of 285 amino acid residues with 64-83% identity to other StARs thus far obtained. RT-PCR analyses revealed that eel StAR mRNA was expressed abundantly in the head kidney and gonad, and faintly in the brain; but no expression was detected in the gill, heart, liver, intestine, kidney and skeletal muscle. Plasma cortisol concentration increased, but StAR mRNA content in the head kidney did not change, 3 and 24 h after transfer of freshwater eels to seawater, indicating that the transcriptional regulation of StAR may not be involved in cortisol production after seawater transfer. However, ACTH elevated both plasma cortisol and StAR mRNA levels in the head kidney 1.5 and 4.5 h after injection. Thus, the steroidogenic effect of ACTH is mediated by increased StAR production as observed in mammals.

  16. Expression of insulin-like growth factor I receptors at mRNA and protein levels during metamorphosis of Japanese flounder (Paralichthys olivaceus).

    PubMed

    Zhang, Junling; Shi, Zhiyi; Cheng, Qi; Chen, Xiaowu

    2011-08-01

    Insulin-like growth factor I (IGF-I) is an important regulator of fish growth and development, and its biological actions are initiated by binding to IGF-I receptor (IGF-IR). Our previous study has revealed that IGF-I could play an important role during metamorphosis of Japanese flounder, Paralichthys olivaceus. The analysis of IGF-IR expression thus helps further elucidate the IGF-I regulation of metamorphic processes. In this study, the spatial-temporal expression of two distinct IGF-IR mRNAs was investigated by real-time RT-PCR. The spatial distribution of two IGF-IR mRNAs in adult tissues is largely overlapped, but they exhibit distinct temporal expression patterns during larval development. A remarkable decrease in IGF-IR-2 mRNA was detected during metamorphosis. In contrast, a significant increase in IGF-IR-1 mRNA was determined from pre-metamorphosis to metamorphic completion. These indicate that they may play different function roles during the flounder metamorphosis. The levels and localization of IGF-IR proteins during larval development were further studied by Western blotting and immunohistochemistry. Immunoreactive IGF-IRs were detected throughout larval development, and the IGF-IR proteins displayed a relatively abundant expression during metamorphosis. Moreover, the IGF-IR proteins appeared in key tissues, such as thickened skin beneath the migrating eye, developing intestine, gills and kidney during metamorphosis. These results further suggest that the IGF-I system may be involved in metamorphic development of Japanese flounder. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Resveratrol induces antioxidant and heat shock protein mRNA expression in response to heat stress in black-boned chickens.

    PubMed

    Liu, L L; He, J H; Xie, H B; Yang, Y S; Li, J C; Zou, Y

    2014-01-01

    This study investigated the effects of dietary resveratrol at 0, 200, 400, or 600 mg/kg of diet on the performance, immune organ growth index, serum parameters, and expression levels of heat shock protein (Hsp) 27, Hsp70, and Hsp90 mRNA in the bursa of Fabricius, thymus, and spleen of 42-d-old female black-boned chickens exposed to heat stress at 37 ± 2°C for 15 d. The results showed that heat stress reduced daily feed intake and BW gain; decreased serum glutathione (GSH), growth hormone, and insulin-like growth factor-1 levels; and inhibited GSH peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) activities compared with birds subjected to thermo-neutral circumstances. Chickens that were fed diets supplemented with resveratrol exhibited a linear increase in feed intake and BW gain (P < 0.001); serum GSH, growth hormone, and insulin-like growth factor-1 levels (P ≤ 0.01); and GSH-Px, SOD, and CAT activities (P < 0.001) compared with chickens that were fed diets without resveratrol during heat stress. In contrast, serum malonaldehyde concentrations were decreased (P < 0.001) in the chickens fed a resveratrol-supplemented diet. Heat stress also reduced (P < 0.05) the growth index of the bursa of Fabricus and spleen; however, it had no effect on the growth index of the thymus. The growth index of the bursa of Fabricius and spleen increased (P < 0.05) upon heat stress and coincided with an increase in supplemental resveratrol levels. The expression of Hsp27, Hsp70, and Hsp90 mRNA in the bursa of Fabricius and spleen were increased (P < 0.01), but those of Hsp27 and Hsp90 mRNA in thymus were decreased (P < 0.01) under heat stress compared with no heat stress. Resveratrol attenuated the heat stress-induced overexpression of Hsp27, Hsp70, and Hsp90 mRNA in the bursa of Fabricius and spleen and increased the low expression of Hsp27 and Hsp90 mRNA in thymus upon heat stress. The results suggest that supplemental resveratrol improves growth performance

  18. Estrogen secreting adrenal adenocarcinoma in an 18-month-old boy: aromatase activity, protein expression, mRNA and utilization of gonadal type promoter.

    PubMed

    Watanabe, T; Yasuda, T; Noda, H; Wada, K; Kazukawa, I; Someya, T; Minamitani, K; Minagawa, M; Wataki, K; Matsunaga, T; Ohnuma, N; Kohno, Y; Harada, N

    2000-12-01

    We examined clinical, endocrinological and molecular biological aspects of an estrogen-secreting adrenal carcinoma in an 18-month-old male to clarify the pathogenesis of this condition. An 18-month-old boy was referred for evaluation of progressive bilateral gynecomastia and appearance of pubic hair. The patient had elevated plasma estradiol (349 pg/ml) and testosterone (260 ng/dl) levels that completely suppressed FSH and LH levels, and was subsequently diagnosed with an adrenal tumor on the right side. After removal of a 300-g adenocarcinoma, gynecomastia regressed and essentially normal hormone levels were restored. Aromatase activity in the tumor tissue determined by the 3H-water method was 71.0-104.4 pmol/min/mg protein. High levels of aromatase protein and mRNA in the tumor tissue were also demonstrated, while neither aromatase activity nor protein was detected in normal adrenal glands. To investigate the regulation of aromatase expression in the adrenal carcinoma, we examined the usage of alternate promoters responsible for aromatase gene transcription. In the present case, the amounts of aromatase mRNA utilizing gonadal types of exon 1c (1.3) and 1d (II) were significantly higher than those that using other exon 1s. This result suggested that the utilization of a gonadal-type exon 1 might be involved in the over-production of aromatase in estrogen-secreting adrenal carcinoma.

  19. Protein and mRNA expression of Shh, Smo and Gli1 and inhibition by cyclopamine in hepatocytes of rats with chronic fluorosis.

    PubMed

    Zhao, Lina; Yu, Yanni; Deng, Chaonan

    2014-03-03

    In order to investigate the Sonic hedgehog (Shh) signaling pathway and the effect of cyclopamine in rat hepatocytes with chronic fluorosis, 48 Wistar rats were randomly divided into 4 groups. The control group was provided with tap water in which the fluorine concentration was <1mg/L, while the remaining three groups were provided with water containing sodium fluoride (NaF) at a concentration of 50mg/L. After 6 months, the blocking and blocking control groups were injected intraperitoneally once every 2 days for 6 days with 10mg/kg cyclopamine or dimethyl sulfoxide, respectively. The urinary and skeletal fluoride contents were determined by the ion selective electrode method. Levels of aspartate transaminase (AST), alanine transaminase (ALT), total protein (TP) and albumin (Alb) in the serum were determined by using autobiochemical machine. Histological changes in liver tissue were evaluated with Hematoxylin & Eeosin (H&E) staining using light microscopy. The protein and mRNA expression of Shh, Smo and Gli1 in hepatocytes of experimental animals was determined by immunohistochemistry (IHC), Western blotting (Wb) and Real-time quantitative PCR (RT-qPCR). Fluoride content of the urine and bone was increased in the fluorosis and blocking groups compared to those in the control group (P<0.05), while fluoride content in the blocking group was decreased compared to the fluorosis and blocking control groups (P<0.05). The expression of Shh, Smo and Gli1 at the mRNA and protein levels was significantly increased in hepatocytes from the fluorosis and blocking control groups compared with the control group, and expression in the blocking group was lower than that of the fluorosis and blocking control groups. The difference between any two groups was considered to be statistically significant (P<0.05). Taken together, our study indicates that the expression of Shh, Smo and Gli1 at the protein and mRNA level in hepatocytes of rats with chronic fluorosis can be increased by

  20. Cinnamaldehyde up-regulates the mRNA expression level of TRPV1 receptor potential ion channel protein and its function in primary rat DRG neurons in vitro.

    PubMed

    Sui, Feng; Lin, Na; Guo, Jian-You; Zhang, Chang-Bin; Du, Xin-Liang; Zhao, Bao-Sheng; Liu, Hong-Bin; Yang, Na; Li, Lan-Fang; Guo, Shu-Ying; Huo, Hai-Ru; Jiang, Ting-Liang

    2010-01-01

    Cinnamaldehyde (1) is a pharmacologically active ingredient isolated from cassia twig (Ramulus Cinnamomi), which is commonly used in herbal remedies to treat fever-related diseases. Both TRPV1 and TRPM8 ion channel proteins are abundantly expressed in sensory neurons, and are assumed to act as a thermosensor, with the former mediating the feeling of warmth and the latter the feeling of cold in the body. Both of them have recently been reported to be involved in thermoregulation. The purpose of this paper is to further uncover the antipyretic mechanisms of 1 by investigating its effects on the mRNA expression levels and functions of both TRPV1 and TRPM8. The results showed that 1 could up-regulate the mRNA expression levels of TRPV1 at both 37 and 39 degrees C, and its calcium-mediating function was significantly increased at 39 degrees C, all of which could not be blocked by pretreatment of the neuronal cells with ruthenium red, a general transient receptor potential (TRP) blocker, indicating that the action of 1 was achieved through a non-TRPA1 channel pathway. In conclusion, the findings in our in vitro studies might account for part of the peripheral molecular mechanisms for the antipyretic action of 1.

  1. Balancing Arc synthesis, mRNA decay, and proteasomal degradation: maximal protein expression triggered by rapid eye movement sleep-like bursts of muscarinic cholinergic receptor stimulation.

    PubMed

    Soulé, Jonathan; Alme, Maria; Myrum, Craig; Schubert, Manja; Kanhema, Tambudzai; Bramham, Clive R

    2012-06-22

    Cholinergic signaling induces Arc/Arg3.1, an immediate early gene crucial for synaptic plasticity. However, the molecular mechanisms that dictate Arc mRNA and protein dynamics during and after cholinergic epochs are little understood. Using human SH-SY5Y neuroblastoma cells, we show that muscarinic cholinergic receptor (mAchR) stimulation triggers Arc synthesis, whereas translation-dependent RNA decay and proteasomal degradation strictly limit the amount and duration of Arc expression. Chronic application of the mAchR agonist, carbachol (Cch), induces Arc transcription via ERK signaling and release of calcium from IP(3)-sensitive stores. Arc translation requires ERK activation, but not changes in intracellular calcium. Proteasomal degradation of Arc (half-life ∼37 min) was enhanced by thapsigargin, an inhibitor of the endoplasmic calcium-ATPase pump. Similar mechanisms of Arc protein regulation were observed in cultured rat hippocampal slices. Functionally, we studied the impact of cholinergic epoch duration and temporal pattern on Arc protein expression. Acute Cch treatment (as short as 2 min) induces transient, moderate Arc expression, whereas continuous treatment of more than 30 min induces maximal expression, followed by rapid decline. Cholinergic activity associated with rapid eye movement sleep may function to facilitate long term synaptic plasticity and memory. Employing a paradigm designed to mimic intermittent rapid eye movement sleep epochs, we show that application of Cch in a series of short bursts generates persistent and maximal Arc protein expression. The results demonstrate dynamic, multifaceted control of Arc synthesis during mAchR signaling, and implicate cholinergic epoch duration and repetition as critical determinants of Arc expression and function in synaptic plasticity and behavior.

  2. Expression of progesterone receptor membrane component 1, serpine mRNA binding protein 1 and nuclear progesterone receptor isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy.

    PubMed

    Slonina, Dominika; Kowalik, Magdalena K; Kotwica, Jan

    2012-01-01

    The aim of this study was to investigate the (1) expression of progesterone membrane component 1 (PGRMC1), serpine mRNA binding protein 1 (SERBP1) and progesterone receptor (PR) mRNA and (2) protein expression levels of PGRMC1, SERBP1 and PR isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy. Uteri from cows on days 1-5, 6-10, 11-16 and 17-21 of the estrous cycle and weeks 3-5, 6-8 and 9-12 of pregnancy were used (n=5-6 per period). There were no changes (P>0.05) in PGRMC1 mRNA expression during the estrous cycle, while expression of SERBP1 and PR mRNA was the lowest (P<0.05) on days 11-16 relative to other days of the cycle. The highest mRNA expression of PGRMC1, SERBP1 and PR was found during pregnancy. There were no changes (P>0.05) in SERBP1 protein expression in cycling and pregnant cows, while the highest (P<0.05) PGRMC1 protein expression was found during weeks 3-5 of pregnancy. Similar protein expression profiles for PRA and PRB were found, and protein levels were highest on days 1-5 of the estrous cycle. From day 6 of the cycle, PRA and PRB protein expression decreased and were maintained at this lower level during pregnancy. In conclusion, our study assessed mRNA and protein expression levels of PGRMC1, SERBP1 and PR in the bovine myometrium during the estrous cycle and the first trimester of pregnancy. It is possible that progesterone (P4) affects myometrial function in a genomic and nongenomic manner.

  3. Lupin protein isolate versus casein modifies cholesterol excretion and mRNA expression of intestinal sterol transporters in a pig model

    PubMed Central

    2014-01-01

    Background Lupin proteins exert hypocholesterolemic effects in man and animals, although the underlying mechanism remains uncertain. Herein we investigated whether lupin proteins compared to casein modulate sterol excretion and mRNA expression of intestinal sterol transporters by use of pigs as an animal model with similar lipid metabolism as humans, and cellular cholesterol-uptake by Caco-2 cells. Methods Two groups of pigs were fed cholesterol-containing diets with either 230 g/kg of lupin protein isolate from L. angustifolius or 230 g/kg casein, for 4 weeks. Faeces were collected quantitatively over a 5 d period for analysis of neutral sterols and bile acids by gas chromatographically methods. The mRNA abundances of intestinal lipid transporters were analysed by real-time RT-PCR. Cholesterol-uptake studies were performed with Caco-2 cells that were incubated with lupin conglutin γ, phytate, ezetimibe or albumin in the presence of labelled [4-14C]-cholesterol. Results Pigs fed the lupin protein isolate revealed lower cholesterol concentrations in total plasma, LDL and HDL than pigs fed casein (P < 0.05). Analysis of faeces revealed a higher output of cholesterol in pigs that were fed lupin protein isolate compared to pigs that received casein (+57.1%; P < 0.05). Relative mRNA concentrations of intestinal sterol transporters involved in cholesterol absorption (Niemann-Pick C1-like 1, scavenger receptor class B, type 1) were lower in pigs fed lupin protein isolate than in those who received casein (P < 0.05). In vitro data showed that phytate was capable of reducing the uptake of labelled [4-14C]-cholesterol into the Caco-2 cells to the same extend as ezetimibe when compared to control (−20.5% vs. −21.1%; P < 0.05). Conclusions Data reveal that the cholesterol-lowering effect of lupin protein isolate is attributable to an increased faecal output of cholesterol and a reduced intestinal uptake of cholesterol. The findings indicate phytate as a

  4. Short Communication: Effect of heat stress on heat-shock protein (Hsp60) mRNA expression in rainbow trout Oncorhynchus mykiss.

    PubMed

    Shi, H N; Liu, Z; Zhang, J P; Kang, Y J; Wang, J F; Huang, J Q; Wang, W M

    2015-05-18

    The enhanced expression of heat shock proteins (hsps) in organisms can be detected in response to many kinds of stressor. For fish, high temperature is an important stressor, and hsp expression is associated with differences in environmental temperature. In this study, rainbow trout (Oncorhynchus mykiss) that were accustomed to an aquatic temperature of 18°C were exposed to an elevated temperature (25°C), and hsp60 expression in the gill, liver, spleen, heart, and head kidney was quantified using real-time polymerase chain reaction in unstressed and heat-stressed animals. The fish responded to heat stress in a time- and tissue-specific manner. Cardiac hsp60 mRNA levels were largely unchanged, and the greatest induction of hsp60 in heat-stressed animals was recorded in the liver, suggesting that protein damage and the consequent requirement for the Hsp60 protein are probably greater in hepatic tissue. Therefore, fish must be provided with optimal temperature conditions in order to realize their potential growth and maximize fish farm profits.

  5. Increased mRNA expression of a laminin-binding protein in human colon carcinoma: Complete sequence of a full-length cDNA encoding the protein

    SciTech Connect

    Yow, Hsiukang; Wong, Jau Min; Chen, Hai Shiene; Lee, C.; Steele, G.D. Jr.; Chen, Lanbo

    1988-09-01

    Reliable markers to distinguish human colon carcinoma from normal colonic epithelium are needed particularly for poorly differentiated tumors where no useful marker is currently available. To search for markers the authors constructed cDNA libraries from human colon carcinoma cell lines and screened for clones that hybridize to a greater degree with mRNAs of colon carcinomas than with their normal counterparts. Here they report one such cDNA clone that hybridizes with a 1.2-kilobase (kb) mRNA, the level of which is /approx/9-fold greater in colon carcinoma than in adjacent normal colonic epithelium. Blot hybridization of total RNA from a variety of human colon carcinoma cell lines shows that the level of this 1.2-kb mRNA in poorly differentiated colon carcinomas is as high as or higher than that in well-differentiated carcinomas. Molecular cloning and complete sequencing of cDNA corresponding to the full-length open reading frame of this 1.2-kb mRNA unexpectedly show it to contain all the partial cDNA sequence encoding 135 amino acid residues previously reported for a human laminin receptor. The deduced amino acid sequence suggests that this putative laminin-binding protein from human colon carcinomas consists of 295 amino acid residues with interesting features. There is an unusual C-terminal 70-amino acid segment, which is trypsin-resistant and highly negatively charged.

  6. Modeling rotavirus-like particles production in a baculovirus expression vector system: Infection kinetics, baculovirus DNA replication, mRNA synthesis and protein production.

    PubMed

    Roldão, António; Vieira, Helena L A; Charpilienne, Annie; Poncet, Didier; Roy, Polly; Carrondo, Manuel J T; Alves, Paula M; Oliveira, R

    2007-03-10

    Rotavirus is the most common cause of severe diarrhoea in children worldwide, responsible for more than half a million deaths in children per year. Rotavirus-like particles (Rota VLPs) are excellent vaccine candidates against rotavirus infection, since they are non-infectious, highly immunogenic, amenable to large-scale production and safer to produce than those based on attenuated viruses. This work focuses on the analysis and modeling of the major events taking place inside Spodoptera frugiperda (Sf-9) cells infected by recombinant baculovirus that may be critical for the expression of rotavirus viral proteins (VPs). For model validation, experiments were performed adopting either a co-infection strategy, using three monocistronic recombinant baculovirus each one coding for viral proteins VP(2), VP(6) and VP(7), or single-infection strategies using a multigene baculovirus coding for the three proteins of interest. A characteristic viral DNA (vDNA) replication rate of 0.19+/-0.01 h(-1) was obtained irrespective of the monocistronic or multigene vector employed, and synthesis of progeny virus was found to be negligible in comparison to intracellular vDNA concentrations. The timeframe for vDNA, mRNA and VP synthesis tends to decrease with increasing multiplicity of infection (MOI) due to the metabolic burden effect. The protein synthesis rates could be ranked according to the gene size in the multigene experiments but not in the co-infection experiments. The model exhibits acceptable prediction power of the dynamics of intracellular vDNA replication, mRNA synthesis and VP production for the three proteins involved. This model is intended to be the basis for future Rota VLPs process optimisation and also a means to evaluating different baculovirus constructs for Rota VLPs production.

  7. Penicillin-Binding Protein 5 Sequence Alteration and Levels of plp5 mRNA Expression in Clinical Isolates of Enterococcus faecium with Different Levels of Ampicillin Resistance.

    PubMed

    Belhaj, Mondher; Boutiba-Ben Boubaker, Ilhem; Slim, Amin

    2016-04-01

    Eighty-two nonduplicated ampicillin-resistant Enterococcus faecium (AREF) isolates from clinical infections at the Charles Nicolle Hospital of Tunisia were investigated. They were collected from January 2001 to December 2009. Genetic relationship between them was studied using pulsed-field gel electrophoresis. The amino acid sequence difference variations of the C-terminal part of penicillin-binding protein 5 (PBP5) versus levels of expressed mRNA were investigated by polymerase chain reaction (PCR), sequencing, and real-time PCR quantification of (PBP5), respectively. No β-lactamase activity was detected and none of our strains showed resistance to glycopeptides, which retain their therapeutic efficiency against enterococcal infections in our hospital. Pattern analysis of the strains revealed six main clones disseminating in different wards. Sequence data revealed the existence of 19 different plp5 alleles with a difference in 16 amino acid positions spanning from residue 414 to 632. Each allele presented at least five amino acid substitutions (His-470→Gln, Asn-496→Lys, Ala-499→Thr, Glu-525→Asp, and Glu-629→Val). No correlation between amino acid sequence polymorphism of PBP5 and levels of ampicillin resistance was detected. The levels of plp5 mRNA expression varied between strains and did not always correlate with levels of ampicillin resistance in clinical AREF.

  8. Reduction in mRNA and protein expression of a nicotinic acetylcholine receptor α8 subunit is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.

    PubMed

    Zhang, Yixi; Wang, Xin; Yang, Baojun; Hu, Yuanyuan; Huang, Lixin; Bass, Chris; Liu, Zewen

    2015-11-01

    Target-site resistance is commonly caused by qualitative changes in insecticide target-receptors and few studies have implicated quantitative changes in insecticide targets in resistance. Here we show that resistance to imidacloprid in a selected strain of Nilaparvata lugens is associated with a reduction in expression levels of the nicotinic acetylcholine receptor (nAChR) subunit Nlα8. Synergism bioassays of the selected strain suggested resistance was conferred, in part, by a target-site mechanism. Sequencing of N. lugens nAChR subunit genes identified no mutations associated with resistance, however, a decrease in mRNA and protein levels of Nlα8 was observed during selection. RNA interference knockdown of Nlα8 decreased the sensitivity of N. lugens to imidacloprid, demonstrating that a decrease in Nlα8 expression is sufficient to confer resistance in vivo. Radioligand binding assays revealed that the affinity of the high-affinity imidacloprid-binding site of native nAChRs was reduced by selection, and reducing the amount of Nlα8 cRNA injected into Xenopus oocytes significantly decreased imidacloprid potency on recombinant receptors. Taken together, these results provide strong evidence that a decrease in Nlα8 levels confers resistance to imidacloprid in N. lugens, and thus provides a rare example of target-site resistance associated with a quantitative rather than qualitative change. In insects, target-site mutations often cause high resistance to insecticides, such as neonicotinoids acting on nicotinic acetylcholine receptors (nAChRs). Here we found that a quantitative change in target-protein level, decrease in mRNA and protein levels of Nlα8, contributed importantly to imidacloprid resistance in Nilaparvata lugens. This finding provides a new target-site mechanism of insecticide resistance. © 2015 International Society for Neurochemistry.

  9. Angiotensin II-induced protein kinase D activates the ATF/CREB family of transcription factors and promotes StAR mRNA expression.

    PubMed

    Olala, Lawrence O; Choudhary, Vivek; Johnson, Maribeth H; Bollag, Wendy B

    2014-07-01

    Aldosterone synthesis is initiated upon the transport of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol is hydrolyzed to pregnenolone. This process is the rate-limiting step in acute aldosterone production and is mediated by the steroidogenic acute regulatory (StAR) protein. We have previously shown that angiotensin II (AngII) activation of the serine/threonine protein kinase D (PKD) promotes acute aldosterone production in bovine adrenal glomerulosa cells, but the mechanism remains unclear. Thus, the purpose of this study was to determine the downstream signaling effectors of AngII-stimulated PKD activity. Our results demonstrate that overexpression of the constitutively active serine-to-glutamate PKD mutant enhances, whereas the dominant-negative serine-to-alanine PKD mutant inhibits, AngII-induced StAR mRNA expression relative to the vector control. PKD has been shown to phosphorylate members of the activating transcription factor (ATF)/cAMP response element binding protein (CREB) family of leucine zipper transcription factors, which have been shown previously to bind the StAR proximal promoter and induce StAR mRNA expression. In primary glomerulosa cells, AngII induces ATF-2 and CREB phosphorylation in a time-dependent manner. Furthermore, overexpression of the constitutively active PKD mutant enhances the AngII-elicited phosphorylation of ATF-2 and CREB, and the dominant-negative mutant inhibits this response. Furthermore, the constitutively active PKD mutant increases the binding of phosphorylated CREB to the StAR promoter. Thus, these data provide insight into the previously reported role of PKD in AngII-induced acute aldosterone production, providing a mechanism by which PKD may be mediating steroidogenesis in primary bovine adrenal glomerulosa cells.

  10. Suppressed expression of insulin-like growth factor binding protein-1 mRNA in the endometrium: a molecular mechanism associating endometrial cancer with its risk factors.

    PubMed

    Rutanen, E M; Nyman, T; Lehtovirta, P; Ammälä, M; Pekonen, F

    1994-11-01

    The insulin-like growth factor (IGF) system is thought to function as a mediator of steroid hormone actions in the endometrium. IGFs (IGF-I and IGF-II) are also potent mitogens in endometrial cancer. The biological actions of IGFs are modulated by specific binding proteins (IGFBP)--6 cloned and sequenced so far--which may either inhibit or enhance the effects of IGF at the cellular level. In the endometrium, IGFBP-1 gene expression is stimulated by progesterone and inhibited by insulin, while IGFBP-1 inhibits the mitogenic action of IGF-I. In this study, we used a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to investigate IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5 and IGFBP-6 gene expression in endometrial cancer tissues. Endometrial cancer tissue samples were collected from 20 women (aged 54-79 yrs) with stage I to II well-differentiated endometrial adenocarcinoma. Samples of normal endometrium (n = 14) obtained from women undergoing tubal ligation in various phases of the menstrual cycle, and normal early-pregnancy endometrium (decidua) were studied for comparison. In endometrial cancer tissues, the IGFBP-1 mRNA was undetectable or minimally expressed when studied by RT-PCR. The mean (+ SD) levels of IGFBP-2 and IGFBP-4 and IGFBP-5 mRNAs in endometrial cancer tissues did not differ from those in normal endometrium, in which no cyclic variation was observed, suggesting that the genes encoding IGFBP-2, IGFBP-4 and IGFBP-5 are not hormonally regulated in the endometrium. The IGFBP-6 mRNA expression showed a significant cyclic variation in normal endometrium, with low levels in late-proliferative and early- to mid-secretory phases and high expression in late-secretory and early-proliferative phases. In endometrial cancer tissues, the mean IGFBP-6 mRNA level was similar to that in cycling endometrium during the peri-ovulatory period. In summary, a continuous stimulation of the endometrial epithelial cells by IGFs with suppressed IGFBP-1 expression

  11. HIF1A and EPAS1 mRNA and protein expression during in vitro culture of human primary term cytotrophoblasts and effect of oxygen tension on their expression.

    PubMed

    Depoix, Christophe Louis; Flabat, Olivier; Debiève, Frédéric; Hubinont, Corinne

    2016-09-01

    During the first trimester of pregnancy, placenta formation probably occurs in a low-oxygen environment necessary to protect cytotrophoblasts from oxidative stress and to allow proper gene regulation. Transcription factors involved in gene regulation under low oxygen tension are the hypoxia-inducible factors, mainly HIF1A, EPAS1 and their dimerization partner HIF1B. Little is known about their expression during in vitro culture of cytotrophoblasts under chronic hypoxia. We assessed HIF1A and EPAS1 expression in a 4-day in vitro culture of primary term cytotrophoblasts under 21% O2 and 2.5% O2. Copy numbers and relative mRNA expression were assessed by real-time quantitative polymerase chain reaction. Protein levels were quantified by immunoblot and densitometric analysis. In undifferentiated cytotrophoblasts, EPAS1 transcripts were four times more abundant than HIF1A transcripts (2.14e(7) and 5e(6)copies/μg total RNA, respectively). During cell culture, HIF1A mRNA expression increased after 24h and then decreased to stay stable. The expression was even lower when cells were grown under 2.5% O2. EPAS1 mRNA expression increased during cytotrophoblast differentiation. The expression was higher when cells were under 21% O2 than when they were under 2.5% O2. Interestingly, HIF1A, but not EPAS1, was detected in the nuclei of undifferentiated cytotrophoblasts, and in the nuclei of cytotrophoblasts that grew under 21% O2. During cytotrophoblast differentiation, no variation in HIF1A protein levels was detected. To the contrary, EPAS1 protein level increased during differentiation, and oxygen tension had no effect on EPAS1 protein level. In conclusion, HIF1A and EPAS1 expression was not inhibited by chronic hypoxia during in vitro cytotrophoblast differentiation.

  12. Effect of oxytocin on expression of cytosolic phospholipase A2 mRNA and protein in ovine endometrial tissue in vivo.

    PubMed

    Burns, P D; Graf, G A; Hayes, S H; Silvia, W J

    2000-11-01

    The induction of endometrial prostaglandin (PG) F2alpha synthesis by oxytocin is dependent upon activation of phospholipase (PL) A2 and mobilization of arachidonic acid. The objective of this study was to determine if oxytocin stimulates PGF2alpha synthesis by inducing synthesis of cytosolic PLA2 (cPLA2). In Experiment 1, 15 ovariectomized ewes were given progesterone and estradiol to simulate an estrous cycle. Ewes were then given an injection of oxytocin on Day 14 of the simulated estrous cycle. Jugular blood samples were collected and assayed for 13,14-dihydro-15-keto-prostaglandin F2alpha (PGFM). Uteri were collected at 0, 7.5, 25, 90, or 240 min postinjection (n = 3 ewes/time point). Total RNA was isolated from caruncular endometrium and subjected to dot-blot analysis. Oxytocin induced a rapid and transient increase in serum PGFM (P < 0.01). However, endometrial concentrations of cPLA2 mRNA did not change following oxytocin administration (P > 0.10). In Experiment 2, 11 ovary-intact ewes were given oxytocin (n = 5) or saline (n = 6) on Day 15 after estrus. Jugular blood samples were collected and assayed for serum concentrations of PGFM. Uteri were collected at 15 min postinjection. Homogenates were prepared from caruncular endometrium and subjected to Western blot analysis. Concentrations of PGFM were higher in oxytocin treated ewes compared to saline treated ewes at 15 min postinjection (P < 0.01). Endometrial concentrations of cPLA2 protein were greater in the cytosolic than in the microsomal fraction (P < 0.01). Oxytocin did not affect the amount of cPLA2 protein in either fraction (P > 0.10). In conclusion, oxytocin did not effect expression of either cPLA2 mRNA or protein in ovine endometrium. Oxytocin may stimulate PGF2alpha synthesis by activating cPLA2 protein that is already present in an inactive form.

  13. Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine. Implications for cystic fibrosis transmembrane conductance regulator.

    PubMed Central

    Markert, T; Vaandrager, A B; Gambaryan, S; Pöhler, D; Häusler, C; Walter, U; De Jonge, H R; Jarchau, T; Lohmann, S M

    1995-01-01

    Certain pathogenic bacteria produce a family of heat stable enterotoxins (STa) which activate intestinal guanylyl cyclases, increase cGMP, and elicit life-threatening secretory diarrhea. The intracellular effector of cGMP actions has not been clarified. Recently we cloned the cDNA for a rat intestinal type II cGMP dependent protein kinase (cGK II) which is highly enriched in intestinal mucosa. Here we show that cGK II mRNA and protein are restricted to the intestinal segments from the duodenum to the proximal colon, with the highest amounts of cGK II protein in duodenum and jejunum. cGK II mRNA and protein decreased along the villus to crypt axis in the small intestine, whereas substantial amounts of both were found in the crypts of cecum. In intestinal epithelia, cGK II was specifically localized in the apical membrane, a major site of ion transport regulation. In contrast to cGK II, cGK I was localized in smooth muscle cells of the villus lamina propria. Short circuit current (ISC), a measure of Cl- secretion, was increased to a similar extent by STa and by 8-Br-cGMP, a selective activator of cGK, except in distal colon and in monolayers of T84 human colon carcinoma cells in which cGK II was not detected. In human and mouse intestine, the cyclic nucleotide-regulated Cl- conductance can be exclusively accounted for by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Viewed collectively, the data suggest that cGK II is the mediator of STa and cGMP effects on Cl- transport in intestinal-epithelia. Images PMID:7543493

  14. Changes in the mRNA expression of structural proteins, hormone synthesis and secretion from bovine placentome sections after DDT and DDE treatment.

    PubMed

    Wojciechowska, A; Mlynarczuk, J; Kotwica, J

    2017-01-15

    Disorders in the barrier function and secretory activity of the placenta can be caused by xenobiotics (XB) present in the environment and their accumulation in tissues of living organisms. Thus, the aim of this study was to investigate the effect of 1,1,1-trichloro-2,2,-bis-4-chlorophenyl-ethane (DDT) and its metabolite 1,1-dichloro-2,2-bis-4-chlorophenyl-ethene (DDE) (for 24 or 48h) at doses of 1, 10 or 100ng/ml on the function of cow placentome sections in the second trimester of pregnancy. DDT and DDE affected neither (P>0.05) the viability nor hypoxia inducible factor 1 (HIF1α) mRNA expression of the sections. XB decreased (P<0.05) connexin (Cx) 26, 32, 43 and placenta-specific 1 (PLAC-1) mRNA expression but did not affect (P>0.05) keratin 8 (KRT8) mRNA expression. DDT and DDE also reduced (P<0.05) prostaglandin F2α (PGF2α) synthase (PGFS) mRNA expression, while DDT increased (P<0.05) prostaglandin E2 (PGE2) synthase (PGES) mRNA expression. Neither cyclooxygenase 2 (COX-2) mRNA expression nor PGF2α and PGE2 secretion were affected. Both DDT and DDE increased (P<0.05) neurophysin I/oxytocin (NP1/OT) mRNA expression and oxytocin (OT), oestradiol (E2) and progesterone (P4) secretion while DDT stimulated only 3β-hydroxysteroid dehydrogenase (3βHSD) and cholesterol side-chain cleavage enzyme (CYP11A1) mRNA expression (P<0.05). In summary, DDT and DDE impaired the barrier function and secretory activity of the placenta. Thus, these compounds can disrupt trophoblast invasion, myometrium contractility and gas/nutrient exchange throughout pregnancy in cows.

  15. Production of a Locus- and Allele-Specific Monoclonal Antibody for the Characterization of SLA-1*0401 mRNA and Protein Expression Levels in MHC-Defined Microminipigs.

    PubMed

    Kametani, Yoshie; Ohshima, Shino; Miyamoto, Asuka; Shigenari, Atsuko; Takasu, Masaki; Imaeda, Noriaki; Matsubara, Tatsuya; Tanaka, Masafumi; Shiina, Takashi; Kamiguchi, Hiroshi; Suzuki, Ryuji; Kitagawa, Hitoshi; Kulski, Jerzy K; Hirayama, Noriaki; Inoko, Hidetoshi; Ando, Asako

    2016-01-01

    The class I major histocompatibility complex (MHC) presents self-developed peptides to specific T cells to induce cytotoxity against infection. The MHC proteins are encoded by multiple loci that express numerous alleles to preserve the variability of the antigen-presenting ability in each species. The mechanism regulating MHC mRNA and protein expression at each locus is difficult to analyze because of the structural and sequence similarities between alleles. In this study, we examined the correlation between the mRNA and surface protein expression of swine leukocyte antigen (SLA)-1*0401 after the stimulation of peripheral blood mononuclear cells (PBMCs) by Staphylococcus aureus superantigen toxic shock syndrome toxin-1 (TSST-1). We prepared a monoclonal antibody (mAb) against a domain composed of Y102, L103 and L109 in the α2 domain. The Hp-16.0 haplotype swine possess only SLA-1*0401, which has the mAb epitope, while other haplotypes possess 0 to 3 SLA classical class I loci with the mAb epitopes. When PBMCs from SLA-1*0401 homozygous pigs were stimulated, the SLA-1*0401 mRNA expression level increased until 24 hrs and decreased at 48 hrs. The kinetics of the interferon regulatory transcription factor-1 (IRF-1) mRNA level were similar to those of the SLA-1*0401 mRNA. However, the surface protein expression level continued to increase until 72 hrs. Similar results were observed in the Hp-10.0 pigs with three mAb epitopes. These results suggest that TSST-1 stimulation induced both mRNA and surface protein expression of class I SLA in the swine PBMCs differentially and that the surface protein level was sustained independently of mRNA regulation.

  16. Production of a Locus- and Allele-Specific Monoclonal Antibody for the Characterization of SLA-1*0401 mRNA and Protein Expression Levels in MHC-Defined Microminipigs

    PubMed Central

    Kametani, Yoshie; Ohshima, Shino; Miyamoto, Asuka; Shigenari, Atsuko; Takasu, Masaki; Imaeda, Noriaki; Matsubara, Tatsuya; Tanaka, Masafumi; Shiina, Takashi; Kamiguchi, Hiroshi; Suzuki, Ryuji; Kitagawa, Hitoshi; Kulski, Jerzy K.; Hirayama, Noriaki; Inoko, Hidetoshi; Ando, Asako

    2016-01-01

    The class I major histocompatibility complex (MHC) presents self-developed peptides to specific T cells to induce cytotoxity against infection. The MHC proteins are encoded by multiple loci that express numerous alleles to preserve the variability of the antigen-presenting ability in each species. The mechanism regulating MHC mRNA and protein expression at each locus is difficult to analyze because of the structural and sequence similarities between alleles. In this study, we examined the correlation between the mRNA and surface protein expression of swine leukocyte antigen (SLA)-1*0401 after the stimulation of peripheral blood mononuclear cells (PBMCs) by Staphylococcus aureus superantigen toxic shock syndrome toxin-1 (TSST-1). We prepared a monoclonal antibody (mAb) against a domain composed of Y102, L103 and L109 in the α2 domain. The Hp-16.0 haplotype swine possess only SLA-1*0401, which has the mAb epitope, while other haplotypes possess 0 to 3 SLA classical class I loci with the mAb epitopes. When PBMCs from SLA-1*0401 homozygous pigs were stimulated, the SLA-1*0401 mRNA expression level increased until 24 hrs and decreased at 48 hrs. The kinetics of the interferon regulatory transcription factor-1 (IRF-1) mRNA level were similar to those of the SLA-1*0401 mRNA. However, the surface protein expression level continued to increase until 72 hrs. Similar results were observed in the Hp-10.0 pigs with three mAb epitopes. These results suggest that TSST-1 stimulation induced both mRNA and surface protein expression of class I SLA in the swine PBMCs differentially and that the surface protein level was sustained independently of mRNA regulation. PMID:27760184

  17. mRNA Expression and activity of ion-transporting proteins in gills of the blue crab Callinectes sapidus: effects of waterborne copper.

    PubMed

    Martins, Camila M G; Almeida, Daniela Volcan; Marins, Luis Fernando Fernandes; Bianchini, Adalto

    2011-01-01

    Waterborne Cu effects on the transcription of genes encoding ion-transporting proteins and the activities of these proteins were evaluated in gills of the blue crab Callinectes sapidus acclimated to diluted (2‰) and full (30‰) seawater. Crabs were exposed (96 h) to an environmentally relevant concentration of dissolved Cu (0.78 µM) and had their posterior (osmoregulating) gills dissected for enzymatic and molecular analysis. Endpoints analyzed were the activity of key enzymes involved in crab osmoregulation (sodium-potassium adenosine triphosphatase [Na(+)/K(+)-ATPase], hydrogen adenosine triphosphatase [H(+)-ATPase], and carbonic anhydrase [CA]) and the mRNA expression of genes encoding these enzymes and the sodium-potassium-chloride (Na(+)/K(+)/2Cl⁻) cotransporter. Copper effects were observed only in crabs acclimated to diluted seawater (hyperosmoregulating crabs) and were associated with an inhibition of the expression of mRNA of genes encoding the Na(+)/K(+)-ATPase and the Na(+)/K(+)/2Cl⁻ cotransporter. However, Cu did not affect Na(+)/K(+)-ATPase activity, indicating that the gene transcription is downregulated before a significant inhibition of the enzyme activity can be observed. This also suggests the existence of a compensatory response of this enzyme to prevent osmoregulatory disturbances after short-term exposure to environmentally relevant Cu concentrations. These findings suggest that Cu is a potential ionoregulatory toxicant in blue crabs C. sapidus acclimated to low salinity. The lack of Cu effect on blue crabs acclimated to full seawater would be due to the reduced ion uptake needed for the regulation of the hemolymph osmotic concentration in full seawater (30‰). Also, this could be explained considering the lower bioavailability of toxic Cu (free ion) associated with the higher ionic content and dissolved organic matter concentration in high salinity (30‰) than in diluted seawater (2‰).

  18. Expression of P2X4R mRNA and protein in rats with hypobaric hypoxia-induced pulmonary hypertension.

    PubMed

    Ohata, Yuichiro; Ogata, Sho; Nakanishi, Kuniaki; Kanazawa, Fumiko; Uenoyama, Maki; Hiroi, Sadayuki; Tominaga, Susumu; Kawai, Toshiaki

    2011-01-01

    The experimental pulmonary hypertension that develops in hypobaric hypoxia is characterized by structural remodeling of the heart. The P2X4 receptor (P2X4R) controls vascular tone and vessel remodeling in several blood vessels, and it has emerged as a key factor in the enhancement of cardiovascular performance. To study the possible effects of hypobaric hypoxia on the P2X4R-synthesis system, 150 male Wistar rats were housed in a chamber at the equivalent of the 5,500 m altitude level for 21 days. After 14 days' exposure to hypobaric hypoxia, pulmonary arterial pressure (PAP) was significantly increased. In the right ventricle (RV) of the heart, P2X4R expression was significantly increased on days 1 and 14 (mRNA) and on days 7 and 21 (protein) of hypobaric hypoxic exposure. Immunohistochemical staining for P2X4R protein became more intense in RV in the late phase of exposure. These changes in P2X4R synthesis in RV occurred alongside the increase in PAP. In addition, P2X1R and P2Y2R mRNA levels in the RV were significantly increased on days 1, 14, and 21, and day 5, respectively, of exposure. The level of P2X1R protein in the RV was significantly increased on day 21 of exposure. Conceivably, P2 receptors, including P2X4R and P2X1R, might play roles in modulating the RV hypertrophy that occurs due to pulmonary hypertension in hypobaric hypoxia. All rights are reserved to the Japanese Circulation Society.

  19. Preserved Expression of mRNA Coding von Willebrand Factor-Cleaving Protease ADAMTS13 by Selenite and Activated Protein C.

    PubMed

    Ekaney, Michael L; Bockmeyer, Clemens L; Sossdorf, Maik; Reuken, Philipp A; Conradi, Florian; Schuerholz, Tobias; Blaess, Markus F; Friedman, Scott L; Lösche, Wolfgang; Bauer, Michael; Claus, Ralf A

    2015-04-03

    In sepsis, the severity-dependent decrease of von Willebrand factor (VWF)-inactivating protease, a disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), results in platelet aggregation and consumption, leading to sepsis-associated thrombotic microangiopathy (TMA) and organ failure. Previous reports assessing its functional deficiency have pinpointed involvement of autoantibodies or mutations to propagate thrombotic thrombocytopenic purpura (TTP). However, mechanisms of acquired ADAMTS13 deficiency during host response remain unclear. To enhance understanding of ADAMTS13 deficiency in sepsis, we evaluated changes in expression of mRNA coding ADAMTS13 during septic conditions using primary cellular sources of the protease. We hypothesized that proinflammatory cytokines and constituents of serum from septic patients affect the transcriptional level of ADAMTS13 in vitro, and previously recommended therapeutic agents as adjunctive therapy for sepsis interact therewith. Cultured hepatic stellate cells (HSCs), endothelial cells (HMEC) and human precision-cut liver slices as an ex vivo model were stimulated with sepsis prototypic cytokines, bacterial endotoxin and pooled serum obtained from septic patients. Stimulation resulted in a significant decrease in ADAMTS13 mRNA between 10% and 80% of basal transcriptional rates. Costimulation of selenite or recombinant activated protein C (APC) with serum prevented ADAMTS13 decrease in HSCs and increased ADAMTS13 transcripts in HMEC. In archived clinical samples, the activity of ADAMTS13 in septic patients treated with APC (n = 5) increased with an accompanying decrease in VWF propeptide as surrogate for improved endothelial function. In conclusion, proinflammatory conditions of sepsis repress mRNA coding ADAMTS13 and the ameliorating effect by selenite and APC may support the concept for identification of beneficial mechanisms triggered by these drugs at a molecular level.

  20. Knockdown of BC200 RNA expression reduces cell migration and invasion by destabilizing mRNA for calcium-binding protein S100A11.

    PubMed

    Shin, Heegwon; Lee, Jungmin; Kim, Youngmi; Jang, Seonghui; Lee, Yunhee; Kim, Semi; Lee, Younghoon

    2017-03-01

    Although BC200 RNA is best known as a neuron-specific non-coding RNA, it is overexpressed in various cancer cells. BC200 RNA was recently shown to contribute to metastasis in several cancer cell lines, but the underlying mechanism was not understood in detail. To examine this mechanism, we knocked down BC200 RNA in cancer cells, which overexpress the RNA, and examined cell motility, profiling of ribosome footprints, and the correlation between cell motility changes and genes exhibiting altered ribosome profiles. We found that BC200 RNA knockdown reduced cell migration and invasion, suggesting that BC200 RNA promotes cell motility. Our ribosome profiling analysis identified 29 genes whose ribosomal occupations were altered more than 2-fold by BC200 RNA knockdown. Many (> 30%) of them were directly or indirectly related to cancer progression. Among them, we focused on S100A11 (which showed a reduced ribosome footprint) because its expression was previously shown to increase cellular motility. S100A11 was decreased at both the mRNA and protein levels following knockdown of BC200 RNA. An actinomycin-chase experiment showed that BC200 RNA knockdown significantly decreased the stability of the S100A11 mRNA without changing its transcription rate, suggesting that the down-regulation of S100A11 was mainly caused by destabilization of its mRNA. Finally, we showed that the BC200 RNA-knockdown-induced decrease in cell motility was mainly mediated by S100A11. Together, our results show that BC200 RNA promotes cell motility by stabilizing S100A11 transcripts.

  1. Preserved Expression of mRNA Coding von Willebrand Factor–Cleaving Protease ADAMTS13 by Selenite and Activated Protein C

    PubMed Central

    Ekaney, Michael L; Bockmeyer, Clemens L; Sossdorf, Maik; Reuken, Philipp A; Conradi, Florian; Schuerholz, Tobias; Blaess, Markus F; Friedman, Scott L; Lösche, Wolfgang; Bauer, Michael; Claus, Ralf A

    2015-01-01

    In sepsis, the severity-dependent decrease of von Willebrand factor (VWF)–inactivating protease, a disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), results in platelet aggregation and consumption, leading to sepsis-associated thrombotic microangiopathy (TMA) and organ failure. Previous reports assessing its functional deficiency have pinpointed involvement of autoantibodies or mutations to propagate thrombotic thrombocytopenic purpura (TTP). However, mechanisms of acquired ADAMTS13 deficiency during host response remain unclear. To enhance understanding of ADAMTS13 deficiency in sepsis, we evaluated changes in expression of mRNA coding ADAMTS13 during septic conditions using primary cellular sources of the protease. We hypothesized that proinflammatory cytokines and constituents of serum from septic patients affect the transcriptional level of ADAMTS13 in vitro, and previously recommended therapeutic agents as adjunctive therapy for sepsis interact therewith. Cultured hepatic stellate cells (HSCs), endothelial cells (HMEC) and human precision-cut liver slices as an ex vivo model were stimulated with sepsis prototypic cytokines, bacterial endotoxin and pooled serum obtained from septic patients. Stimulation resulted in a significant decrease in ADAMTS13 mRNA between 10% and 80% of basal transcriptional rates. Costimulation of selenite or recombinant activated protein C (APC) with serum prevented ADAMTS13 decrease in HSCs and increased ADAMTS13 transcripts in HMEC. In archived clinical samples, the activity of ADAMTS13 in septic patients treated with APC (n = 5) increased with an accompanying decrease in VWF propeptide as surrogate for improved endothelial function. In conclusion, proinflammatory conditions of sepsis repress mRNA coding ADAMTS13 and the ameliorating effect by selenite and APC may support the concept for identification of beneficial mechanisms triggered by these drugs at a molecular level. PMID:25860876

  2. High lib mRNA expression in breast carcinomas.

    PubMed

    Satoh, Kazuki; Hata, Mitsumi; Yokota, Hiroshi

    2004-06-30

    Lib, first identified as a novel beta-amyloid responsive gene in rat astrocytes, has an extracellular domain of 15 leucine-rich repeats (LRRs) followed by a transmembrane domain and a short cytoplasmic region. It is a distinctly inducible gene and is thought to play a key role in inflammatory states via the LRR extracellular motif, an ideal structural framework for protein-protein and protein-matrix interactions. To evaluate potential roles of Lib, we screened various tumors for Lib expression. Lib mRNA expression was high and uniquely expressed in breast tumor tissues, compared to paired normal breast tissues. Lib mRNA was localized in the ductal carcinoma cells and Lib protein displayed a homophilic association on the surface of cultured cells. These data suggest that Lib may play a role in the progression of breast carcinomas and may be a diagnostic marker for breast tumors.

  3. Ribosomal protein S6 is highly expressed in non-Hodgkin lymphoma and associates with mRNA containing a 5' terminal oligopyrimidine tract.

    PubMed

    Hagner, P R; Mazan-Mamczarz, K; Dai, B; Balzer, E M; Corl, S; Martin, S S; Zhao, X F; Gartenhaus, R B

    2011-03-31

    The molecular mechanism(s) linking tumorigenesis and morphological alterations in the nucleolus are presently coming into focus. The nucleolus is the cellular organelle in which the formation of ribosomal subunits occurs. Ribosomal biogenesis occurs through the transcription of ribosomal RNA (rRNA), rRNA processing and production of ribosomal proteins. An error in any of these processes may lead to deregulated cellular translation, evident in multiple cancers and 'ribosomopathies'. Deregulated protein synthesis may be achieved through the overexpression of ribosomal proteins as seen in primary leukemic blasts with elevated levels of ribosomal proteins S11 and S14. In this study, we demonstrate that ribosomal protein S6 (RPS6) is highly expressed in primary diffuse large B-cell lymphoma (DLBCL) samples. Genetic modulation of RPS6 protein levels with specifically targeted short hairpin RNA (shRNA) lentiviruses led to a decrease in the actively proliferating population of cells compared with control shRNA. Low-dose rapamycin treatments have been shown to affect the translation of 5' terminal oligopyrimidine (5' TOP) tract mRNA, which encodes the translational machinery, implicating RPS6 in 5' TOP translation. Recently, it was shown that disruption of 40S ribosomal biogenesis through specific small inhibitory RNA knockdown of RPS6 defined RPS6 as a critical regulator of 5' TOP translation. For the first time, we show that RPS6 associates with multiple mRNAs containing a 5' TOP tract. These findings expand our understanding of the mechanism(s) involved in ribosomal biogenesis and deregulated protein synthesis in DLBCL.

  4. Aestivation Induces Changes in the mRNA Expression Levels and Protein Abundance of Two Isoforms of Urea Transporters in the Gills of the African Lungfish, Protopterus annectens.

    PubMed

    Chng, You R; Ong, Jasmine L Y; Ching, Biyun; Chen, Xiu L; Hiong, Kum C; Wong, Wai P; Chew, Shit F; Lam, Siew H; Ip, Yuen K

    2017-01-01

    The African lungfish, Protopterus annectens, is ammonotelic in water despite being ureogenic. When it aestivates in mucus cocoon on land, ammonia is detoxified to urea. During the maintenance phase of aestivation, urea accumulates in the body, which is subsequently excreted upon arousal. Urea excretion involves urea transporters (UT/Ut). This study aimed to clone and sequence the ut isoforms from the gills of P. annectens, and to test the hypothesis that the mRNA and/or protein expression levels of ut/Ut isoforms could vary in the gills of P. annectens during the induction, maintenance, and arousal phases of aestivation. Two isoforms of ut, ut-a2a and ut-a2b, were obtained from the gills of P. annectens. ut-a2a consisted of 1227 bp and coded for 408 amino acids with an estimated molecular mass of 44.7 kDa, while ut-a2b consisted of 1392 bp and coded for 464 amino acids with an estimated molecular mass of 51.2 kDa. Ut-a2a and Ut-a2b of P. annectens had a closer phylogenetic relationship with Ut/UT of tetrapods than Ut of fishes. While the mRNA expression pattern of ut-a2a and ut-a2b across various tissues of P. annectens differed, the transcript levels of ut-a2a and ut-a2b in the gills were comparable, indicating that they might be equally important for branchial urea excretion during the initial arousal phase of aestivation. During the maintenance phase of aestivation, the transcript level of ut-a2a increased significantly, but the protein abundance of Ut-a2a remained unchanged in the gills of P. annectens. This could be an adaptive feature to prepare for an increase in the production of Ut-a2a upon arousal. Indeed, arousal led to a significant increase in the branchial Ut-a2a protein abundance. Although the transcript level of ut-a2b remained unchanged, there were significant increases in the protein abundance of Ut-a2b in the gills of P. annectens throughout the three phases of aestivation. The increase in the protein abundance of Ut-a2b during the maintenance

  5. Aestivation Induces Changes in the mRNA Expression Levels and Protein Abundance of Two Isoforms of Urea Transporters in the Gills of the African Lungfish, Protopterus annectens

    PubMed Central

    Chng, You R.; Ong, Jasmine L. Y.; Ching, Biyun; Chen, Xiu L.; Hiong, Kum C.; Wong, Wai P.; Chew, Shit F.; Lam, Siew H.; Ip, Yuen K.

    2017-01-01

    The African lungfish, Protopterus annectens, is ammonotelic in water despite being ureogenic. When it aestivates in mucus cocoon on land, ammonia is detoxified to urea. During the maintenance phase of aestivation, urea accumulates in the body, which is subsequently excreted upon arousal. Urea excretion involves urea transporters (UT/Ut). This study aimed to clone and sequence the ut isoforms from the gills of P. annectens, and to test the hypothesis that the mRNA and/or protein expression levels of ut/Ut isoforms could vary in the gills of P. annectens during the induction, maintenance, and arousal phases of aestivation. Two isoforms of ut, ut-a2a and ut-a2b, were obtained from the gills of P. annectens. ut-a2a consisted of 1227 bp and coded for 408 amino acids with an estimated molecular mass of 44.7 kDa, while ut-a2b consisted of 1392 bp and coded for 464 amino acids with an estimated molecular mass of 51.2 kDa. Ut-a2a and Ut-a2b of P. annectens had a closer phylogenetic relationship with Ut/UT of tetrapods than Ut of fishes. While the mRNA expression pattern of ut-a2a and ut-a2b across various tissues of P. annectens differed, the transcript levels of ut-a2a and ut-a2b in the gills were comparable, indicating that they might be equally important for branchial urea excretion during the initial arousal phase of aestivation. During the maintenance phase of aestivation, the transcript level of ut-a2a increased significantly, but the protein abundance of Ut-a2a remained unchanged in the gills of P. annectens. This could be an adaptive feature to prepare for an increase in the production of Ut-a2a upon arousal. Indeed, arousal led to a significant increase in the branchial Ut-a2a protein abundance. Although the transcript level of ut-a2b remained unchanged, there were significant increases in the protein abundance of Ut-a2b in the gills of P. annectens throughout the three phases of aestivation. The increase in the protein abundance of Ut-a2b during the maintenance

  6. Radish (Raphanus sativus L. leaf) ethanol extract inhibits protein and mRNA expression of ErbB(2) and ErbB(3) in MDA-MB-231 human breast cancer cells.

    PubMed

    Kim, Woo Kyoung; Kim, Ji Hae; Jeong, Da Hee; Chun, Young Hee; Kim, Sun Hee; Cho, Kang Jin; Chang, Moon-Jeong

    2011-08-01

    In this study, we investigated the effects of the ethanol extract of aerial parts of Raphanus sativus L. (ERL) on breast cancer cell proliferation and gene expression associated with cell proliferation and apoptosis in MDA-MB-231 human breast cancer cells. The MDA-MB-231 cells were cultured in the presence or absence of various concentrations (100, 200, or 300 µg/mL) of ERL. ERL significantly decreased cell proliferation after 48 h of incubation (P < 0.05). The protein and mRNA expression of ErbB(2) were decreased significantly in a dose-dependent manner (P < 0.05). The protein expression of ErbB(3) was decreased significantly at an ERL concentration of 300 µg/mL (P < 0.05), and mRNA expression of ErbB(3) was decreased significantly in a dose-dependent manner (P < 0.05). The protein expression of Akt was decreased significantly at the ERL concentration of 200 µg/mL (P < 0.05), and the protein expression of pAkt was decreased significantly in a dose-dependent manner (P < 0.05). The mRNA expression of Akt was decreased significantly at the ERL concentration of 200 µg/mL ERL (P < 0.05). The protein and mRNA expression of Bax were increased significantly at ERL concentrations of 200 µg/mL or higher (P < 0.05). The protein expression of Bcl(2) was increased significantly at ERL concentrations of 100 µg/mL or higher (P < 0.05), and mRNA expression of Bcl(2) was increased significantly at an ERL concentration of 300 µg/mL (P < 0.05). In conclusion, we suggest that Raphanus sativus, L. inhibits cell proliferation via the ErbB-Akt pathway in MDA-MB-231 cells.

  7. Peroxisome Proliferator Activated Receptors Alpha, Beta, and Gamma mRNA and protein expression in human fetal tissues

    EPA Science Inventory

    Peroxisome proliferator-activated receptors (PPARs) regulate lipid and glucose homeostasis, are targets of pharmaceuticals, and are also activated by environmental contaminants. Almost nothing is known about expression of PPARs during human fetal development. This study examine...

  8. Peroxisome Proliferator Activated Receptors Alpha, Beta, and Gamma mRNA and protein expression in human fetal tissues

    EPA Science Inventory

    Peroxisome proliferator-activated receptors (PPARs) regulate lipid and glucose homeostasis, are targets of pharmaceuticals, and are also activated by environmental contaminants. Almost nothing is known about expression of PPARs during human fetal development. This study examine...

  9. Muscle Glycogen Depletion Following 75-km of Cycling Is Not Linked to Increased Muscle IL-6, IL-8, and MCP-1 mRNA Expression and Protein Content

    PubMed Central

    Nieman, David C.; Zwetsloot, Kevin A.; Lomiwes, Dominic D.; Meaney, Mary P.; Hurst, Roger D.

    2016-01-01

    The cytokine response to heavy exertion varies widely for unknown reasons, and this study evaluated the relative importance of glycogen depletion, muscle damage, and stress hormone changes on blood and muscle cytokine measures. Cyclists (N = 20) participated in a 75-km cycling time trial (168 ± 26.0 min), with blood and vastus lateralis muscle samples collected before and after. Muscle glycogen decreased 77.2 ± 17.4%, muscle IL-6, IL-8, and MCP-1 mRNA increased 18.5 ± 2.8−, 45.3 ± 7.8−, and 8.25 ± 1.75-fold, and muscle IL-6, IL-8, and MCP-1 protein increased 70.5 ± 14.1%, 347 ± 68.1%, and 148 ± 21.3%, respectively (all, P < 0.001). Serum myoglobin and cortisol increased 32.1 ± 3.3 to 242 ± 48.3 mg/mL, and 295 ± 27.6 to 784 ± 63.5 nmol/L, respectively (both P < 0.001). Plasma IL-6, IL-8, and MCP-1 increased 0.42 ± 0.07 to 18.5 ± 3.8, 4.07 ± 0.37 to 17.0 ± 1.8, and 96.5 ± 3.7 to 240 ± 21.6 pg/mL, respectively (all P < 0.001). Increases in muscle IL-6, IL-8, and MCP-1 mRNA were unrelated to any of the outcome measures. Muscle glycogen depletion was related to change in plasma IL-6 (r = 0.462, P = 0.040), with change in myoglobin related to plasma IL-8 (r = 0.582, P = 0.007) and plasma MCP-1 (r = 0.457, P = 0.043), and muscle MCP-1 protein (r = 0.588, P = 0.017); cortisol was related to plasma IL-8 (r = 0.613, P = 0.004), muscle IL-8 protein (r = 0.681, P = 0.004), and plasma MCP-1 (r = 0.442, P = 0.050). In summary, this study showed that muscle IL-6, IL-8, and MCP-1 mRNA expression after 75-km cycling was unrelated to glycogen depletion and muscle damage, with change in muscle glycogen related to plasma IL-6, and changes in serum myoglobin and cortisol related to the chemotactic cytokines IL-8 and MCP-1. PMID:27729872

  10. Mismatch repair mRNA and protein expression in intestinal adenocarcinoma in sika deer (Cervus nippon) resembling heritable non-polyposis colorectal cancer in man.

    PubMed

    Jahns, H; Browne, J A

    2015-01-01

    Intestinal adenocarcinomas seen in an inbred herd of farmed sika deer (Cervus nippon) morphologically resembled human hereditary non-polyposis colorectal cancer (HNPCC). Features common to both included multiple de novo sites of tumourigenesis in the proximal colon, sessile and non-polyposis mucosal changes, the frequent finding of mucinous type adenocarcinoma, lymphocyte infiltration into the neoplastic tubules and Crohn's-like lymphoid follicles at the deep margin of the tumour. HNPCC is defined by a germline mutation of mismatch repair (MMR) genes resulting in their inactivation and loss of expression. To test the hypothesis that similar MMR gene inactivation occurs in the deer tumours, the expression of the four most important MMR genes, MSH2, MLH1, MSH6 and PMS2, was examined at the mRNA level by reverse transcriptase polymerase chain reaction (n = 12) and at the protein level by immunohistochemistry (n = 40) in tumour and control tissues. All four genes were expressed equally in normal and neoplastic tissues, so MMR gene inactivation could not be implicated in the carcinogenesis of this tumour in sika deer. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Expression of mRNA and protein of IL-18 and its receptor in human follicular granulosa cells.

    PubMed

    Salmassi, A; Fattahi, A; Nouri, M; Hedderich, J; Schmutzler, A G

    2017-04-01

    There is no information available about the IL-18 receptor in ovarian follicles, so the present study attempts to demonstrate the expression of IL-18 and its receptor in human granulosa cells (GCs). To evaluate the concentration of IL-18 in serum and follicular fluid (FF), we collected serum and FF from 102 women undergoing oocyte retrieval. Also, to detect expression of IL-18 and its receptor by luteinized GCs, these cells were pooled six times from a total of twenty individual patients with 5-16 follicles each. The IL-18 concentration was determined by ELISA and the expression of IL-18 and its receptor by immunocytochemistry and reverse transcription polymerase chain reaction. Our results showed that the median IL-18 concentration in serum, 159.27 pg/ml (IQR 121.41-210.1), was significantly higher than in FF, 142.1 pg/ml (IQR 95.7-176.5), p < 0.001. Moreover, we found that IL-18 and its receptor are expressed by GCs. The presence of IL-18 in FF and the expression of IL-18 and its receptor by GCs suggest an important role for this cytokine in ovarian function.

  12. Asymptotic behavior of distributions of mRNA and protein levels in a model of stochastic gene expression

    NASA Astrophysics Data System (ADS)

    Bobrowski, Adam; Lipniacki, Tomasz; Pichór, Katarzyna; Rudnicki, Ryszard

    2007-09-01

    The paper is devoted to a stochastic process introduced in the recent paper by Lipniacki et al. [T. Lipniacki, P. Paszek, A. Marciniak-Czochra, A.RE Brasier, M. Kimmel, Transcriptional stochasticity in gene expression, JE Theor. Biol. 238 (2006) 348-367] in modelling gene expression in eukaryotes. Starting from the full generator of the process we show that its distributions satisfy a (Fokker-Planck-type) system of partial differential equations. Then, we construct a c0 Markov semigroup in L1 space corresponding to this system. The main result of the paper is asymptotic stability of the involved semigroup in the set of densities.

  13. Immunolabelling, histochemistry and in situ hybridisation in human skeletal muscle fibres to detect myosin heavy chain expression at the protein and mRNA level

    PubMed Central

    SERRANO, A. L.; PÉREZ, MARGARITA; LUCÍA, A.; CHICHARRO, J. L.; QUIROZ-ROTHE, E.; RIVERO, J. L. L.

    2001-01-01

    The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in vastus lateralis muscle biopsies of 15 young men (with an average age of 22 y) by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies, myofibrillar ATPase (mATPase) histochemistry and in situ hybridisation with probes specific for MHC β-slow, MHC-IIA and MHC-IIX. The characterisation of a large number of individual fibres was compared and correlated on a fibre-to-fibre basis. The panel of monoclonal antibodies used in the study allowed classification of human skeletal muscle fibres into 5 categories according to the MHC isoform they express at the protein level, types I, I+IIA, IIA, IIAX and IIX. Hybrid fibres coexpressing two isoforms represented a considerable proportion of the fibre composition (about 14%) and were clearly underestimated by mATPase histochemistry. For a very high percentage of fibres there was a precise correspondence between the MHC protein isoforms and mRNA transcripts. The integrated methods used demonstrate a high degree of precision of the immunohistochemical procedure used for the identification and quantification of human skeletal muscle fibre types. The monoclonal antibody S5-8H2 is particularly useful for identifying hybrid IIAX fibres. This protocol offers new prospects for muscle fibre classification in human experimental studies. PMID:11554510

  14. Rapid, sequential activation of mitogen-activated protein kinases and transcription factors precedes proinflammatory cytokine mRNA expression in spleens of mice exposed to the trichothecene vomitoxin.

    PubMed

    Zhou, Hui-Ren; Islam, Zahidul; Pestka, James J

    2003-03-01

    Since proinflammatory cytokine mRNA expression is induced within lymphoid tissue in vivo by the trichothecene vomitoxin (VT) in a rapid (1-2 h) and transient (4-8 h) fashion, it was hypothesized that mitogen-activated protein kinases (MAPKs) and transcription factors associated upstream with gene transcription of these cytokines are activated prior to or within these time windows. To test this hypothesis, mice were first treated with a single oral dose of VT and then analyzed for MAPK phosphorylation in the spleen. As little as 1 mg/kg of VT induced JNK 1/2, ERK 1/2, and p38 phosphorylation with maximal effects being observed at 5 to 100 mg/kg of VT. VT transiently induced JNK and p38 phosphorylation over a 60-min time period with peak effects being observed at 15 and 30 min, respectively. In contrast, ERK remained phosphorylated from 15 to 120 min. Next, the binding of activating protein 1 (AP-1), CCAAT enhancer-binding protein (C/EBP), CRE-binding protein (CREB), and nuclear factor-kappaB (NF-kappaB) was measured by electrophoretic mobility shift assay (EMSA) using four different consensus transcriptional control motifs at 0, 0.5, 1.5, 4, and 8 h after oral exposure to 25 mg/kg of VT. AP-1 binding activity was differentially elevated from 0.5 h to 8 h, whereas C/EBP binding was elevated only at 0.5 h. CREB binding decreased slightly at 0.5 h but gradually increased, reaching a maximum at 4 h. NF-kappaB binding was increased only slightly at 4 and 8 h. The specificities of AP-1, C/EBP, CREB, and NF-kappaB for relevant DNA motifs were verified by competition assays, using an excess of unlabeled consensus and mutant oligonucleotides. Supershift EMSAs and Western blot analysis identified specific VT-inducible DNA binding proteins for AP-1 (cJun, phospho c-jun, JunB, and JunD), C/EBP (C/EBPbeta), CREB (CREB-1 and ATF-2), and NF-kappaB (p50 and cRel). Finally, when the effects of oral VT exposure on proinflammatory gene expression were assessed at 3, 6, and 9 h

  15. Effect of electroacupuncture on the mRNA and protein expression of Rho-A and Rho-associated kinase II in spinal cord injury rats

    PubMed Central

    Min, You-jiang; Ding, Li-li-qiang; Cheng, Li-hong; Xiao, Wei-ping; He, Xing-wei; Zhang, Hui; Min, Zhi-yun; Pei, Jia

    2017-01-01

    Electroacupuncture is beneficial for the recovery of spinal cord injury, but the underlying mechanism is unclear. The Rho/Rho-associated kinase (ROCK) signaling pathway regulates the actin cytoskeleton by controlling the adhesive and migratory behaviors of cells that could inhibit neurite regrowth after neural injury and consequently hinder the recovery from spinal cord injury. Therefore, we hypothesized electroacupuncture could affect the Rho/ROCK signaling pathway to promote the recovery of spinal cord injury. In our experiments, the spinal cord injury in adult Sprague-Dawley rats was caused by an impact device. Those rats were subjected to electroacupuncture at Yaoyangguan (GV3), Dazhui (GV14), Zusanli (ST36) and Ciliao (BL32) and/or monosialoganglioside treatment. Behavioral scores revealed that the hindlimb motor functions improved with those treatments. Real-time quantitative polymerase chain reaction, fluorescence in situ hybridization and western blot assay showed that electroacupuncture suppressed the mRNA and protein expression of Rho-A and Rho-associated kinase II (ROCKII) of injured spinal cord. Although monosialoganglioside promoted the recovery of hindlimb motor function, monosialoganglioside did not affect the expression of Rho-A and ROCKII. However, electroacupuncture combined with monosialoganglioside did not further improve the motor function or suppress the expression of Rho-A and ROCKII. Our data suggested that the electroacupuncture could specifically inhibit the activation of the Rho/ROCK signaling pathway thus partially contributing to the repair of injured spinal cord. Monosialoganglioside could promote the motor function but did not suppress expression of RhoA and ROCKII. There was no synergistic effect of electroacupuncture combined with monosialoganglioside. PMID:28400811

  16. Enhanced protein expression by internal ribosomal entry site-driven mRNA translation as a novel approach for in vitro loading of dendritic cells with antigens.

    PubMed

    Tan, Xiaohua; Wan, Yonghong

    2008-01-01

    Transfection of dendritic cells (DCs) with messenger RNAs (mRNAs) of tumor-associated antigens (TAAs) is a promising strategy for cancer vaccines. TAA mRNA can be generated by in vitro transcription using DNA encoding the TAA gene as a template. A cap analog is usually added upon in vitro transcription to stabilize mRNA and enhance the efficiency of mRNA translation. However, the inclusion of the cap analog correlates with significantly lower-yield mRNA transcription, potentially leading to an expensive vaccine manufacturing process. To solve this problem, we present a novel approach in which DNA templates are modified with an internal ribosomal entry site (IRES) sequence inserted upstream of the gene of interest to replace the use of the cap analog. The presence of IRES greatly enhanced transcription for the mRNA in vitro compared with the cap analog. Also, higher transgene expression was achieved using luciferase (Luc) mRNA with IRES than using capped Luc mRNA to transfect DCs. Immunization of mice with DCs transfected with IRES-containing mRNA encoding chicken ovalbumin (OVA) induced significant levels of antigen-specific interferon gamma-producing CD8(+) T cells and in vivo killing of antigen-bearing cells. Consistently, mice immunized with IRES-containing OVA mRNA-transfected DCs were protected from pulmonary metastasis of melanoma cells injected intravenously. We suggest that IRES can be used for the production of larger quantities of mRNA and that such IRES-containing mRNAs may be useful for DC-based antitumor immunotherapy.

  17. The activity, protein, and mRNA expression of CYP2E1 and CYP3A1 in rats after exposure to acute and chronic high altitude hypoxia.

    PubMed

    Li, Xiangyang; Wang, Xuejun; Li, Yongping; Zhu, Junbo; Su, Xiaodong; Yao, Xingchen; Fan, Xueru; Duan, Yabin

    2014-12-01

    The effects of exposure to acute and chronic high altitude hypoxia on the activity and expression of CYP2E1 and CYP3A1 were examined in rats. Rats were divided into low altitude (LA, 400 m), acute moderate altitude hypoxia (AMH, 2800 m), chronic moderate altitude hypoxia (CMH, 2800 m), acute high altitude hypoxia (AHH, 4300 m), and chronic high altitude hypoxia groups (CHH, 4300 m). Probe drugs were administrated orally to all five groups. Then the serum concentration of probe drug and its metabolite was determined by RP-HPLC. The activity of CYP2E1 and CYP3A1 was evaluated using the ratio of the metabolite to chlorzoxazone and testosterone, respectively. ELISA and real-time PCR were used to analyze the protein and mRNA expression of CYP2E1 and CYP3A1 in liver microsomes, respectively. Chronic high altitude hypoxia caused significant decreases in the activity and protein and mRNA expression of rat CYP2E1 and CYP3A1 in vivo. Acute high altitude hypoxia was not found to change the activity, protein or mRNA expression of rat CYP2E1 or CYP3A1. This study showed significant changes in the activity and protein and mRNA expression of CYP2E1 or CYP3A1 in rats after exposure to chronic high altitude hypoxia.

  18. Attenuation of the seizure-induced expression of BDNF mRNA in adult rat brain by an inhibitor of calcium/calmodulin-dependent protein kinases.

    PubMed

    Murray, K D; Hayes, V Y; Gall, C M; Isackson, P J

    1998-01-01

    We have examined the potential involvement of calcium/calmodulin-dependent protein kinases in the regulation of brain-derived neurotrophic factor mRNA in vivo following kainic acid (kainate)-induced seizure activity by in situ hybridization. KN-62, a specific inhibitor of calcium/calmodulin-dependent protein kinase type II and IV, blocked the characteristic induction of brain-derived neurotrophic factor mRNA seen following seizure activity. This blockade was specific to calcium/calmodulin-dependent protein kinase type II and IV as inhibitors of both protein kinase C and cAMP-dependent protein kinase had no effect. Inhibition of brain-derived neurotrophic factor mRNA increases varied between brain regions; an almost complete inhibition was seen throughout cortical regions, whereas only partial inhibitory effects were noted within hippocampus. A similar inhibition of increased c-fos mRNA was observed throughout cortical, hippocampal and diencephalic regions. The two predominant brain-derived neurotrophic factor transcripts induced by kainate, containing exons I or III, were differentially affected by KN-62. The cortical induction of exon I was blocked by KN-62, whereas exon III was not, providing additional evidence for the differential regulation of individual brain-derived neurotrophic factor transcripts and demonstrating that inhibition of brain-derived neurotrophic factor induction was not due to general blockade of seizure activity throughout the neocortex. These data implicate calcium/calmodulin-dependent protein kinase type II or IV in the regulation of brain-derived neurotrophic factor mRNA in vivo and suggest regionally specific mechanisms occur throughout the brain.

  19. [Effects of qiwei granule on the protein and mRNA expressions of renal tissue transforming growth factor-beta1 in KK-Ay mice with spontaneous type 2 diabetes mellitus].

    PubMed

    Li, Min-zhou; Gao, Yan-bin; Ma, Ming-fei

    2012-12-01

    To study the effects of Qiwei Granule (QWG) on the protein and mRNA expressions of renal tissue transforming growth factor beta1 (TGF-beta1) in KK-Ay mice with spontaneous type 2 diabetes mellitus (T2DM). Spontaneous T2DM KK-Ay mice model was adopted. Forty-five male mice were randomly divided into three groups, i. e., the model group, the Chinese medicine group, and the Western medicine group, 15 in each group. Fifteen male C57BL/6J mice were set up as the normal control group. The mice in the Chinese medicine group and the Western medicine group were administered intragastrically with QWG (at the daily dose of 20 g/kg) and valsartan (at the daily dose of 10 mg/kg), and the treatment lasted for 12 successive weeks. The pathological changes of the kidney were observed using HE staining, PAS, and Masson staining. The protein and mRNA expressions of TGF-beta1, were detected using immunohistochemical method and Real-time fluorescent quantitative PCR. The renal pathological changes of mice in the model group showed hypertrophic glomeruli, widened mesenteric matrix, increased mesangial cells, vacuolar renal tubular epithelial cells, tubular ectasia, and foci atrophy. Necrosis was occasionally seen. More protein cast, mesenchymal infiltration of inflammatory cells, and interstitial fibrosis could be seen. The protein and mRNA expressions of TGF-beta1 increased more in the model group than in the normal control group. After treatment by QWG and valsartan, the renal pathological changes were obviously alleviated, and the protein and mRNA expressions of TGF-beta1 were obviously lowered (P<0.05). By inhibiting the protein and mRNA expressions of TGF-beta1, QWG could play a role in preventing and curing diabetic nephropathy.

  20. Effects of chronic noise on mRNA and protein expression of CRF family molecules and its relationship with p-tau in the rat prefrontal cortex.

    PubMed

    Gai, Zhihui; Li, Kang; Sun, Huanrui; She, Xiaojun; Cui, Bo; Wang, Rui

    2016-09-15

    Chronic noise exposure has been associated with Alzheimer's disease (AD)-like pathological changes, such as tau hyperphosphorylation and β-amyloid peptide accumulation in the prefrontal cortex (PFC). Corticotropin-releasing factor (CRF) is the central driving force in the stress response and a regulator of tau phosphorylation via binding to CRF receptors (CRFR). Little is known about the CRF system in relation to noise-induced AD-like changes in the PFC. The aim of this study was to explore the effects of chronic noise exposure on the CRF system in the PFC of rats and its relationship to tau phosphorylation. Male Wistar rats were randomly divided into control and noise exposure groups. The CRF system was evaluated following chronic noise exposure (95dB sound pressure level white noise, 4h/day×30days). Chronic noise significantly accelerated the progressive overproduction of corticosterone and upregulated CRF and CRFR1 mRNA and protein, both of which persisted 7-14days after noise exposure. In contrast, CRFR2 was elevated 3-7days following the last stimulus. Double-labeling immunofluorescence co-localized p-tau with CRF in PFC neurons. The results suggest that chronic noise exposure elevates the expression of the CRF system, which may contribute to AD-like changes. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Effect of Potato Virus Y on the NADP-Malic Enzyme from Nicotiana tabacum L.: mRNA, Expressed Protein and Activity

    PubMed Central

    Doubnerová, Veronika; Müller, Karel; Čeřovská, Noemi; Synková, Helena; Spoustová, Petra; Ryšlavá, Helena

    2009-01-01

    The effect of biotic stress induced by viral infection (Potato virus Y, strain NTN and O) on NADP-malic enzyme (EC 1.1.1.40) in tobacco plants (Nicotiana tabacum L., cv. Petit Havana, SR1) was tested at the transcriptional, translational and activity level. The increase of enzyme activity in infected leaves was correlated with the increased amount of expressed protein and with mRNA of cytosolic NADP-ME isoform. Transcription of the chloroplastic enzyme was not influenced by viral infection. The increase of the enzyme activity was also detected in stems and roots of infected plants. The effect of viral infection induced by Potato virus Y, NTN strain, causing more severe symptoms, was compared with the effect induced by milder strain PVYO. The observed increase in NADP-malic enzyme activity in all parts of the studied plants was higher in the case of PVYNTN strain than in the case of strain PVYO. The relevance of NADP-malic enzyme in plants under stress conditions was discussed. PMID:20111689

  2. 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.

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

    PubMed

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

    2000-09-01

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

  4. Hodgkin's lymphoma cell lines express a fusion protein encoded by intergenically spliced mRNA for the multilectin receptor DEC-205 (CD205) and a novel C-type lectin receptor DCL-1.

    PubMed

    Kato, Masato; Khan, Seema; Gonzalez, Nelson; O'Neill, Brian P; McDonald, Kylie J; Cooper, Ben J; Angel, Nicola Z; Hart, Derek N J

    2003-09-05

    Classic Hodgkin's lymphoma (HL) tissue contains a small population of morphologically distinct malignant cells called Hodgkin and Reed-Sternberg (HRS) cells, associated with the development of HL. Using 3'-rapid amplification of cDNA ends (RACE) we identified an alternative mRNA for the DEC-205 multilectin receptor in the HRS cell line L428. Sequence analysis revealed that the mRNA encodes a fusion protein between DEC-205 and a novel C-type lectin DCL-1. Although the 7.5-kb DEC-205 and 4.2-kb DCL-1 mRNA were expressed independently in myeloid and B lymphoid cell lines, the DEC-205/DCL-1 fusion mRNA (9.5 kb) predominated in the HRS cell lines (L428, KM-H2, and HDLM-2). The DEC-205 and DCL-1 genes comprising 35 and 6 exons, respectively, are juxtaposed on chromosome band 2q24 and separated by only 5.4 kb. We determined the DCL-1 transcription initiation site within the intervening sequence by 5'-RACE, confirming that DCL-1 is an independent gene. Two DEC-205/DCL-1 fusion mRNA variants may result from cotranscription of DEC-205 and DCL-1, followed by splicing DEC-205 exon 35 or 34-35 along with DCL-1 exon 1. The resulting reading frames encode the DEC-205 ectodomain plus the DCL-1 ectodomain, the transmembrane, and the cytoplasmic domain. Using DCL-1 cytoplasmic domain-specific polyclonal and DEC-205 monoclonal antibodies for immunoprecipitation/Western blot analysis, we showed that the fusion mRNA is translated into a DEC-205/DCL-1 fusion protein, expressed in the HRS cell lines. These results imply an unusual transcriptional control mechanism in HRS cells, which cotranscribe an mRNA containing DEC-205 and DCL-1 prior to generating the intergenically spliced mRNA to produce a DEC-205/DCL-1 fusion protein.

  5. ADAM-17 and TIMP3 protein and mRNA expression in spinal cord white matter of rats with acute experimental autoimmune encephalomyelitis.

    PubMed

    Plumb, Jonnie; Cross, Alison K; Surr, Jessica; Haddock, Gail; Smith, Terence; Bunning, Rowena A D; Woodroofe, M Nicola

    2005-07-01

    Tumour necrosis factor (TNF) is a major immunomodulatory and proinflammatory cytokine implicated in the pathogenesis of multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE). ADAM-17 cleaves membrane-bound TNF into its soluble form. The distribution and level of ADAM-17 expression within spinal cords of Lewis rats with EAE was investigated. ADAM-17 was associated with endothelial cells in the naïve and pre-disease spinal cords. In peak disease astrocytic and inflammatory cells expressed ADAM-17. Upregulation of ADAM-17 mRNA expression was coupled with a decrease in mRNA levels of its inhibitor TIMP3 suggesting a role for ADAM-17 in EAE pathogenesis.

  6. Insulin-like growth factor mRNA binding protein 3 (IMP3) is differentially expressed in benign and malignant follicular patterned thyroid tumors.

    PubMed

    Slosar, Magdalena; Vohra, Poonam; Prasad, Manju; Fischer, Andrew; Quinlan, Robert; Khan, Ashraf

    2009-01-01

    Insulin-like growth factor mRNA binding protein 3 (IMP3) is an mRNA-binding protein that regulates transcription of insulin-like growth factor II affecting cell proliferation during embryogenesis. It is highly expressed in carcinomas of the pancreas, stomach, colon, rectum, kidneys, uterine cervix, lung, and ovary. The purpose of our study was to evaluate IMP3 expression in thyroid follicular lesions, to determine whether it has a role in differentiating among these lesions, and to understand their biological relationships. We immunostained 219 thyroid lesions selected from our surgical pathology archives including 14 hyperplastic colloid nodules (CN), 19 Hashimoto's thyroiditis (HT), two Graves disease (GD), ten Hürthle cell adenoma (HCA), 20 follicular adenoma (FA), 37 conventional papillary thyroid carcinoma (PTC), 60 follicular variant of papillary carcinoma (FVPC), 19 Hürthle cell carcinoma (HCC), 32 follicular carcinoma (FC), and six poorly differentiated/anaplastic carcinoma. Immunohistochemistry was performed on formalin-fixed sections using monoclonal antibody to IMP3. Clinicopathological data were also reviewed. In all cases, residual thyroid tissue, CN, HT, GD, HCA, and FA were completely negative for IMP3 staining. Of the 60 FVPC, 23 tumors (38%) were positive for IMP3, with 13 of these (22%) showing very strong staining (3+). Of the 32 FC, 22 tumors (69%) were positive, with seven (22%) showing very strong staining (3+). Furthermore, 33 out of 37 cases (89%) of PTC were negative for IMP3. In all four PTC cases that did stain positive, staining was weak-moderate (1-2+). Similarly, 15 out of 19 cases (79%) of HCC were negative. No significant correlation was found between pathologic tumor characteristics and IMP3 expression in differentiated follicular pattern thyroid carcinoma. With 100% specificity and 69% sensitivity for FC as compared to FA and 100% specificity for FVPC, again compared to FA, IMP3 has the potential to be diagnostically useful in

  7. Rapid evolution and gene expression: a rapidly evolving Mendelian trait that silences field crickets has widespread effects on mRNA and protein expression.

    PubMed

    Pascoal, S; Liu, X; Ly, T; Fang, Y; Rockliffe, N; Paterson, S; Shirran, S L; Botting, C H; Bailey, N W

    2016-06-01

    A major advance in modern evolutionary biology is the ability to start linking phenotypic evolution in the wild with genomic changes that underlie that evolution. We capitalized on a rapidly evolving Hawaiian population of crickets (Teleogryllus oceanicus) to test hypotheses about the genomic consequences of a recent Mendelian mutation of large effect which disrupts the development of sound-producing structures on male forewings. The resulting silent phenotype, flatwing, persists because of natural selection imposed by an acoustically orienting parasitoid, but it interferes with mate attraction. We examined gene expression differences in developing wing buds of wild-type and flatwing male crickets using RNA-seq and quantitative proteomics. Most differentially expressed (DE) transcripts were down-regulated in flatwing males (625 up vs. 1716 down), whereas up- and down-regulated proteins were equally represented (30 up and 34 down). Differences between morphs were clearly not restricted to a single pathway, and we recovered annotations associated with a broad array of functions that would not be predicted a priori. Using a candidate gene detection test based on homology, we identified 30% of putative Drosophila wing development genes in the cricket transcriptome, but only 10% were DE. In addition to wing-related annotations, endocrine pathways and several biological processes such as reproduction, immunity and locomotion were DE in the mutant crickets at both biological levels. Our results illuminate the breadth of genetic pathways that are potentially affected in the early stages of adaptation. © 2016 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2016 European Society For Evolutionary Biology.

  8. Human peripheral blood CD4+ and CD8+ T cells express Th1-like cytokine mRNA and proteins following in vitro stimulation with heat-inactivated Brucella abortus.

    PubMed Central

    Zaitseva, M B; Golding, H; Betts, M; Yamauchi, A; Bloom, E T; Butler, L E; Stevan, L; Golding, B

    1995-01-01

    Defining the pattern of lymphokine production associated with Brucella abortus is critical for advancing the development of B. abortus as a vaccine carrier. In the present study we investigated the ability of heat-inactivated B. abortus or lipopolysaccharide from B. abortus to induce lymphokine production from purified human T cells in vitro. Gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-5 induction was assayed by mRNA-specific PCR and by enzyme-linked immunosorbent assay and bioassay for protein production. Following depletion of monocytes and B cells, B. abortus increased IFN-gamma and IL-2 mRNA expression in purified T cells compared with expression in unstimulated cells. In contrast, no IL-5 mRNA expression and only transient low-level IL-4 mRNA expression and no IL-4 protein secretion were detected. Phytohemagglutinin or phorbol myristate acetate plus ionomycin induced mRNA and protein for all these cytokines. Similar results were obtained with LPS purified from B. abortus. Removal of NK cells did not reduce lymphokine production, and enriched NK cells did not express IFN-gamma mRNA or secrete IFN-gamma protein in response to B. abortus, indicating that NK cells were not the responding population. Both CD4+ and CD8+ populations produced IFN-gamma and IL-2 in response to B. abortus. Preincubation of resting T cells with B. abortus or LPS from B. abortus for 7 days induced their differentiation into Th1-like cells as judged by their subsequent lymphokine response to phorbol myristate acetate plus ionomycin. These results suggest that B. abortus can induce differentiation of Th0 into Th1-type cells. PMID:7790090

  9. Expression and localization of progesterone receptor membrane component 1 and 2 and serpine mRNA binding protein 1 in the bovine corpus luteum during the estrous cycle and the first trimester of pregnancy.

    PubMed

    Kowalik, Magdalena K; Rekawiecki, Robert; Kotwica, Jan

    2014-11-01

    The aim of this study was to evaluate the mRNA and protein expression and the localization of progesterone receptor membrane component 1 (PGRMC1), PGRMC2, and the PGRMC1 partner serpine mRNA binding protein 1 (SERBP1) in the bovine CL on Days 2 to 5, 6 to 10, 11 to 16, and 17 to 20 of the estrous cycle as well as during Weeks 3 to 5, 6 to 8, and 9 to 12 of pregnancy (n = 5-6 per each period). The highest levels of PGRMC1 and PGRMC2 mRNA expression were found on Days 6 to 16 (P < 0.05) and 11 to 16, respectively, of the estrous cycle and during pregnancy (P < 0.001). The level of PGRMC1 protein was the highest (P < 0.05) on Days 11 to 16 of the estrous cycle compared with the other stages of the estrous cycle and pregnancy, whereas PGRMC2 protein expression (P < 0.001) was the highest on Days 17 to 20 and also during pregnancy. The mRNA expression of SERBP1 was increased (P < 0.05) on Days 11 to 16, whereas the level of its protein product was decreased (P < 0.05) on Days 6 to 10 of the estrous cycle and was at its lowest (P < 0.001) on Days 17 to 20. In pregnant cows, the patterns of SERBP1 mRNA and protein expression remained constant and were comparable with those observed during the estrous cycle. Progesterone receptor membrane component 1 and PGRMC2 localized to both large and small luteal cells, whereas SERBP1 was observed mainly in small luteal cells and much less frequently in large luteal cells. All proteins were also localized in the endothelial cells of blood vessels. The data obtained indicate the variable expression of PGRMC1, PGRMC2, and SERBP1 mRNA and protein in the bovine CL and suggest that progesterone may regulate CL function via its membrane receptors during both the estrous cycle and pregnancy. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. An NXF1 mRNA with a retained intron is expressed in hippocampal and neocortical neurons and is translated into a protein that functions as an Nxf1 cofactor

    PubMed Central

    Li, Ying; Bor, Yeou-cherng; Fitzgerald, Mark P.; Lee, Kevin S.; Rekosh, David; Hammarskjold, Marie-Louise

    2016-01-01

    The Nxf1 protein is a major nuclear export receptor for the transport of mRNA, and it also is essential for export of retroviral mRNAs with retained introns. In the latter case, it binds to RNA elements known as constitutive transport elements (CTEs) and functions in conjunction with a cofactor known as Nxt1. The NXF1 gene also regulates expression of its own intron-containing RNA through the use of a functional CTE within intron 10. mRNA containing this intron is exported to the cytoplasm, where it can be translated into the 356–amino acid short Nxf1(sNxf1) protein, despite the fact that it is a prime candidate for nonsense-mediated decay (NMD). Here we demonstrate that sNxf1 is highly expressed in nuclei and dendrites of hippocampal and neocortical neurons in rodent brain. Additionally, we show that sNxf1 localizes in RNA granules in neurites of differentiated N2a mouse neuroblastoma cells, where it shows partial colocalization with Staufen2 isoform SS, a protein known to play a role in dendritic mRNA trafficking. We also show that sNxf1 forms heterodimers in conjunction with the full-length Nxf1 and that sNxf1 can replace Nxt1 to enhance the expression of CTE-containing mRNA and promote its association with polyribosomes. PMID:27708137

  11. Expression of progesterone receptor membrane component (PGRMC) 1 and 2, serpine mRNA binding protein 1 (SERBP1) and nuclear progesterone receptor (PGR) in the bovine endometrium during the estrous cycle and the first trimester of pregnancy.

    PubMed

    Kowalik, Magdalena K; Slonina, Dominika; Rekawiecki, Robert; Kotwica, Jan

    2013-03-01

    Progesterone (P4) is involved in the regulation of essential reproductive functions affecting the target cells through both nuclear progesterone receptors (PGRs) and membrane progesterone receptors. The aim of this study was to determine the mRNA and protein expression for PGRMC1, PGRMC2, SERBP1 and PGR within the bovine endometrium during the estrous cycle and the first trimester of pregnancy. There were no changes in PGRMC1 and PGRMC2 mRNA and protein expression during the estrous cycle, however, mRNA levels of PGRMC1 and PGRMC2 were increased (P<0.001) in pregnant animals. SERBP1 mRNA expression was increased (P<0.05), while the level of this protein was decreased (P<0.05) on days 11-16 of the estrous cycle. The expression of PGR mRNA was higher (P<0.01) on days 17-20 compared to days 6-10 and 11-16 of the estrous cycle and pregnancy. PGR-A and PGR-B protein levels were elevated on days 1-5 and 17-20 of the estrous cycle as compared to other stages of the cycle and during pregnancy. In conclusion, our results indicate that P4 may influence endometrial cells through both genomic and nongenomic way. This mechanism may contribute to the regulation of the estrous cycle and provide protection during pregnancy. Copyright © 2013 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  12. [Effects of 18beta-glycyrrhizic acid and 18alpha-glycyrrhizic acid on mRNA and protein expression of cytochrome P450 3A in cultured rat primary hepatocyte].

    PubMed

    Wang, Yuguang; Yang, Minghui; Ma, Zengchun; Liang, Qiande; Tan, Hongling; Xiao, Chengrong; Gao, Yue

    2009-02-01

    To study the effects of 18beta-glycyrrhizic acid and 18alpha-glycyrrhizic acid on mRNA and protein expression of cytochrome P450 3A in cultured rat primary hepatocyte. Rat heaptocyte were isolated by two-step in situ collagenase perfusion method; the hepatocytes were seeded in dishes coated with type I rat tail collagen, culture medium was added and changed daily after gelation, the effect of 18beta-glycyrrhizic acid and 18alpha-glycyrrhizic acid on the expression of CYP3A was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot respectively. Treatment with 18beta-glycyrrhizic acid of primary rat hepatocytes resulted in marked up-regulation of CYP3A1 expression at both mRNA and protein levels in a concentration dependent manner, while exposure to 18alpha-glycyrrhizic acid of primary rat hepatocytes resulted in marked down-regulation of CYP3A1 expression at both mRNA and protein levels in a concentration dependent manmer. 18beta-glycyrrhizic acid and 18alpha-glycyrrhizic acid up-regulate and down-regulate CYP3A1 expression at the transcriptive levels.

  13. Feeding a DHA-enriched diet increases skeletal muscle protein synthesis in growing pigs: association with increased skeletal muscle insulin action and local mRNA expression of insulin-like growth factor 1.

    PubMed

    Wei, Hong-Kui; Zhou, Yuanfei; Jiang, Shuzhong; Tao, Ya-Xiong; Sun, Haiqing; Peng, Jian; Jiang, Siwen

    2013-08-01

    Dietary n-3 PUFA have been demonstrated to promote muscle growth in growing animals. In the present study, fractional protein synthesis rates (FSR) in the skeletal muscle of growing pigs fed a DHA-enriched (DE) diet (DE treatment) or a soyabean oil (SO) diet (SO treatment) were evaluated in the fed and feed-deprived states. Feeding-induced increases in muscle FSR, as well as the activation of the mammalian target of rapamycin and protein kinase B, were higher in the DE treatment as indicated by the positive interaction between diet and feeding. In the fed state, the activation of eIF4E-binding protein 1 in the skeletal muscle of pigs on the DE diet was higher than that in pigs on the SO diet (P<0·05). Feeding the DE diet increased muscle insulin-like growth factor 1 (IGF-1) expression (P<0·05) and insulin action (as demonstrated by increased insulin receptor (IR) phosphorylation, P<0·05), resulting in increased IR substrate 1 activation in the fed state. However, no difference in plasma IGF-1 concentration or hepatic IGF-1 expression between the two treatments was associated. The increased IGF-1 expression in the DE treatment was associated with increased mRNA expression of the signal transducer and activator of transcription 5A and decreased mRNA expression of protein tyrosine phosphatase, non-receptor type 3 in skeletal muscle. Moreover, mRNA expression of protein tyrosine phosphatase, non-receptor type 1 (PTPN1), the activation of PTPN1 and the activation of NF-κB in muscle were significantly lower in the DE treatment (P<0·05). The results of the present study suggest that feeding a DE diet increased feeding-induced muscle protein synthesis in growing pigs, and muscle IGF-1 expression and insulin action were involved in this action.

  14. Over-Expression of CDC25B and LAMC2 mRNA and Protein in Esophageal Squamous Cell Carcinomas and Pre-Malignant Lesions in Subjects from a High-Risk Population in China

    PubMed Central

    Shou, Jian-Zhong; Hu, Nan; Takikita, Mikiko; Roth, Mark J; Johnson, Laura Lee; Giffen, Carol; Wang, Quan-Hong; Wang, Chaoyu; Wang, Yuan; Su, Hua; Kong, Li-Hui; Emmert-Buck, Michael R; Goldstein, Alisa M; Hewitt, Stephen M; Taylor, Philip R

    2009-01-01

    Molecular events associated with the initiation and progression of esophageal squamous cell carcinoma (ESCC) remain poorly understood, but likely hold the key to effective early detection approaches for this almost invariably fatal cancer. CDC25B and LAMC2 are two promising early detection candidates emerging from new molecular studies of ESCC. To further elucidate the role of these two genes in esophageal carcinogenesis, we performed a series of studies to: (i) confirm RNA over-expression; (ii) establish the prevalence of protein over-expression; (iii) relate protein over-expression to survival; and (iv) explore their potential as early detection biomarkers. Results of these studies indicated that CDC25B mRNA was over-expressed (≥2-fold over-expression in tumor compared to normal) in 64% of the 73 ESCC cases evaluated, while LAMC2 mRNA was over-expressed in 89% of cases. CDC25B protein expression was categorized as positive in 59% (144/243) of ESCC cases on a tumor tissue microarray, and non-negative LAMC2 patterns of protein expression were observed in 82% (225/275) of cases. Multivariate-adjusted proportional hazard regression models showed no association between CDC25B protein expression score and risk of death (Hazard Ratio [HR] for each unit increase in expression score = 1.00, P=0.90), however, several of the LAMC2 protein expression patterns strongly predicted survival. Using the cytoplasmic pattern as the reference (the pattern with the lowest mortality), cases with a diffuse pattern had a 254% increased risk of death (HR=3.52, P=0.007), cases with no LAMC2 expression had a 169% increased risk of death (HR=2.69, P=0.009), and cases with a peripheral pattern had a 130% greater risk of death (HR=2.30, P=0.02). CDC25B protein expression scores in subjects with esophageal biopsies diagnosed as normal (n=35), dysplastic (n=23), or ESCC (n=32) increased significantly with morphologic progression. For LAMC2, all normal and dysplastic patients had a continuous

  15. Effects of high environmental ammonia on branchial ammonia excretion rates and tissue Rh-protein mRNA expression levels in seawater acclimated Dungeness crab Metacarcinus magister.

    PubMed

    Martin, Michael; Fehsenfeld, Sandra; Sourial, Mary M; Weihrauch, Dirk

    2011-10-01

    In the present study of the marine Dungeness crabs Metacarcinus magister, the long term effects of high environmental ammonia (HEA) on hemolymph ammonia and urea concentrations, branchial ammonia excretion rates and mRNA expression levels of the crustacean Rh-like ammonia transporter (RhMM), H(+)-ATPase (subunit B), Na(+)/K(+)-ATPase (α-subunit) and Na(+)/H(+)-exchanger (NHE) were investigated. Under control conditions, the crabs' hemolymph exhibited a total ammonia concentration of 179.3±14.5μmol L(-1), while urea accounted for 467.2±33.5μmol L(-1), respectively. Both anterior and posterior gills were capable of excreting ammonia against a 16-fold inwardly directed gradient. Under control conditions, mRNA expression levels of RhMM were high in the gills in contrast to very low expression levels in all other tissues investigated, including the antennal gland, hepatopancreas, and skeletal muscle. After exposure to 1mmol L(-1) NH(4)Cl, hemolymph ammonia increased within the first 12h to ca. 500µmol L(-1) and crabs were able the keep this hemolymph ammonia level for at least 4 days. During this initial period, branchial RhMM and H(+)-ATPase (subunit B) mRNA expression levels roughly doubled. After 14 days of HEA exposure, hemolymph ammonia raised up to environmental levels, whereas urea levels increased by ca. 30%. At the same time, whole animal ammonia and urea excretion vanished. Additionally, branchial RhMM, H(+)-ATPase, Na(+)/K(+)-ATPase and NHE mRNA levels decreased significantly after long term HEA exposure, whereas expression levels of RhMM in the internal tissues increased substantially. Interestingly, crabs acclimated to HEA showed no mortality even after 4 weeks of HEA exposure. This suggests that M. magister possesses a highly adaptive mechanism to cope with elevated ammonia concentrations in its body fluids, including an up-regulation of an Rh-like ammonia transporter in the internal tissues and excretion or storage of waste nitrogen in a so far

  16. Endothelin-1 and endothelin receptor mRNA expression in normal and atherosclerotic human arteries.

    PubMed

    Winkles, J A; Alberts, G F; Brogi, E; Libby, P

    1993-03-31

    Endothelin-1 (ET-1) is a potent vasoconstrictor peptide implicated in a number of human diseases including atherosclerosis. ET-1 binds to two distinct G protein-coupled receptors, known as the ETA and ETB receptor subtypes. In this study, we have examined ET-1, ETA and ETB mRNA expression levels in human vascular cells cultured in vitro and in normal and atherosclerotic human arteries. The results indicate that (a) ET-1 mRNA is constitutively expressed by endothelial cells but not by smooth muscle cells, (b) endothelial cells express only ETB mRNA but smooth muscle cells co-express ETA and ETB mRNA, and (c) in comparison to normal aorta, ET-1 mRNA expression is elevated and endothelin receptor mRNA expression is repressed in atherosclerotic lesions.

  17. Cytokine mRNA expression in postischemic/reperfused myocardium.

    PubMed Central

    Herskowitz, A.; Choi, S.; Ansari, A. A.; Wesselingh, S.

    1995-01-01

    While the role of cytokines in mediating injury during hind limb skeletal muscle ischemia followed by reperfusion has recently been described, the role of cytokines in myocardial infarction and ischemia/reperfusion have remained relatively unexplored. We hypothesize that cytokines play an important role in the regulation of postischemic myocardial inflammation. This study reports the temporal sequence of proinflammatory cytokine gene expression in postischemic/reperfused myocardium and localizes interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha)-protein by immunostaining. Rats were subjected to either permanent left anterior descending (LAD) occlusion or to 35 minutes of LAD occlusion followed by reperfusion and sacrificed up to 7 days later. Rat-specific oligonucleotide probes were used to semiquantitatively assess the relative expression of mRNA for TNF-alpha, IL-1 beta, IL-2, IL-6, interferon-gamma (IFN-gamma), and transforming growth factor-beta 1 (TGF-beta 1) utilizing the reverse transcriptase-polymerase chain reaction amplification technique. Increased cardiac mRNA levels for all cytokines except IL-6 and IFN-gamma were measurable within 15 to 30 minutes of LAD occlusion and increased levels were generally sustained for 3 hours. During early reperfusion, mRNA levels for IL-6 and TGF-beta 1 were significantly reduced compared with permanent LAD occlusion. In both groups, cytokine mRNA levels all returned to baseline levels at 24 hours, while IL-1 beta, TNF-alpha, and TGF-beta 1 mRNA levels again rose significantly at 7 days only in animals with permanent LAD occlusion. Immunostaining for IL-1 beta and TNF-alpha protein revealed two patterns of reactivity: 1) microvascular staining for both IL-1 beta and TNF-alpha protein only in postischemic reperfused myocardium in early post-reperfusion time points; and 2) staining of infiltrating macrophages in healing infarct zones which was most prominent at 7 days after permanent LAD occlusion

  18. Up-Regulation of mRNA Ventricular PRNP Prion Protein Gene Expression in Air Pollution Highly Exposed Young Urbanites: Endoplasmic Reticulum Stress, Glucose Regulated Protein 78, and Nanosized Particles

    PubMed Central

    Villarreal-Calderon, Rodolfo; Franco-Lira, Maricela; González-Maciel, Angélica; Reynoso-Robles, Rafael; Harritt, Lou; Pérez-Guillé, Beatriz; Ferreira-Azevedo, Lara; Drecktrah, Dan; Zhu, Hongtu; Sun, Qiang; Torres-Jardón, Ricardo; Aragón-Flores, Mariana; Calderón-Garcidueñas, Ana; Diaz, Philippe; Calderón-Garcidueñas, Lilian

    2013-01-01

    Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM) vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER) stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.97 ± 6.8 years with a lifetime of low (n:4) vs. high (n:26) air pollution exposures. Light microscopy and transmission electron microscopy studies were carried out in human ventricles, and electron microscopy studies were also done in 5 young, highly exposed Mexico City dogs. There was significant left ventricular PRNP and bi-ventricular GRP78 mRNA up-regulation in Mexico City young urbanites vs. controls. PRNP up-regulation in the left ventricle was significantly different from the right, p < 0.0001, and there was a strong left ventricular PRNP and GRP78 correlation (p = 0.0005). Marked abnormalities in capillary endothelial cells, numerous nanosized particles in myocardial ER and in abnormal mitochondria characterized the highly exposed ventricles. Early and sustained cardiac ER stress could result in detrimental irreversible consequences in urban children, and while highly complex systems maintain myocardial homeostasis, failure to compensate for chronic myocardial inflammation, oxidative and ER stress, and particles damaging myocardial organelles may prime the development of pathophysiological cardiovascular states in young urbanites. Nanosized PM could play a key cardiac myocyte toxicity role. PMID:24287918

  19. Up-regulation of mRNA ventricular PRNP prion protein gene expression in air pollution highly exposed young urbanites: endoplasmic reticulum stress, glucose regulated protein 78, and nanosized particles.

    PubMed

    Villarreal-Calderon, Rodolfo; Franco-Lira, Maricela; González-Maciel, Angélica; Reynoso-Robles, Rafael; Harritt, Lou; Pérez-Guillé, Beatriz; Ferreira-Azevedo, Lara; Drecktrah, Dan; Zhu, Hongtu; Sun, Qiang; Torres-Jardón, Ricardo; Aragón-Flores, Mariana; Calderón-Garcidueñas, Ana; Diaz, Philippe; Calderón-Garcidueñas, Lilian

    2013-11-28

    Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM) vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER) stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.97 ± 6.8 years with a lifetime of low (n:4) vs. high (n:26) air pollution exposures. Light microscopy and transmission electron microscopy studies were carried out in human ventricles, and electron microscopy studies were also done in 5 young, highly exposed Mexico City dogs. There was significant left ventricular PRNP and bi-ventricular GRP78 mRNA up-regulation in Mexico City young urbanites vs. controls. PRNP up-regulation in the left ventricle was significantly different from the right, p < 0.0001, and there was a strong left ventricular PRNP and GRP78 correlation (p = 0.0005). Marked abnormalities in capillary endothelial cells, numerous nanosized particles in myocardial ER and in abnormal mitochondria characterized the highly exposed ventricles. Early and sustained cardiac ER stress could result in detrimental irreversible consequences in urban children, and while highly complex systems maintain myocardial homeostasis, failure to compensate for chronic myocardial inflammation, oxidative and ER stress, and particles damaging myocardial organelles may prime the development of pathophysiological cardiovascular states in young urbanites. Nanosized PM could play a key cardiac myocyte toxicity role.

  20. Association of expression of selenoprotein P in mRNA and protein levels with metabolic syndrome in subjects with cardiovascular disease: Results of the Selenegene study.

    PubMed

    Gharipour, Mojgan; Sadeghi, Masoumeh; Salehi, Mansour; Behmanesh, Mehrdad; Khosravi, Elham; Dianatkhah, Minoo; Haghjoo Javanmard, Shaghayegh; Razavi, Rouzbeh; Gharipour, Amin

    2017-03-01

    Selenoprotein P (SeP) is involved in transporting selenium from the liver to target tissues. Because SeP confers protection against disease by reducing chronic oxidative stress, the present study aimed to assess the level of SeP in the serum of patients with metabolic syndrome (MetS) with a history of cardiovascular disease (CVD). A cross-sectional study was conducted in 63 and 71 subjects with and without MetS in the presence of documented CVD. All demographic, anthropometric and cardiometabolic variables (lipids, blood glucose, blood pressure) were assessed. Lifestyle-related factors and personal history and familial CVD risk factors were recorded. The expression of SELP in mRNA and protein levels in the serum was measured, and MetS was determined using ATPIII criteria. Binary logistic regression analysis demonstrated MetS and SeP to be dependent and independent variables, respectively. Mean of systolic and diastolic blood pressure, triglyceride, high-density lipoprotein-cholesterol, fasting blood sugar, body mass index and waist circumference were higher among subjects with MetS (p = 0.05). The mean of selenium was higher among subjects with MetS, whereas the mean of SeP was lower among subjects with MetS (p < 0.001). In the unadjusted model, the SeP had decreased odds for MetS [odds ratio (OR) = 0.995; 95% confidence interval (CI) = 0.989-1.00] (p < 0.04). Furthermore, the association between MetS and SeP levels remained marginally significant even after adjusting for potential confounders such as age, gender, family history, smoking status and nutrition. SeP and waist circumference show a significant relationship (OR =0.995; 95% CI = 0.990-1.00) (p < 0.033). We have demonstrated a significant decrease in circulating SeP levels according to MetS status in patients with documented cardiovascular disease. Copyright © 2017 John Wiley & Sons, Ltd.

  1. mRNA expression profiles for corticotrophin-releasing hormone, urocortin, CRH-binding protein and CRH receptors in human term gestational tissues determined by real-time quantitative RT-PCR.

    PubMed

    Sehringer, B; Zahradnik, H P; Simon, M; Ziegler, R; Noethling, C; Schaefer, W R

    2004-04-01

    Increasing maternal plasma levels of corticotrophin-releasing hormone (CRH) during the last weeks of pregnancy suggest that this stress hormone plays an important role in the control of human parturition. Little is known about the quantitative contribution of gestational tissues (other than placenta) to intrauterine formation of CRH, urocortin and CRH-binding protein (CRH-BP), or about the distribution of CRH receptors within the uterus. We have investigated the mRNA expression of CRH, urocortin, CRH-BP and CRH receptors 1 and 2 (CRH-R1 and -R2) in gestational tissues by real-time RT-PCR. Placenta, myometrium and choriodecidua were collected after uncomplicated pregnancies at term, before the onset of labour. Distribution of CRH-R1 and CRH-R2 protein was also investigated by immunostaining with receptor subtype-specific antibodies. The placenta was identified as the main site of CRH and CRH-BP mRNA expression, displaying mRNA levels >1000 and >20 times higher than those found in the myometrium and choriodecidua respectively (P<0.05 in each case). mRNA expression of urocortin was low in all tissues investigated. Myometrium and choriodecidua expressed relevant amounts of both receptor subtypes, whereas the CRH receptor population in placenta consisted mainly of CRH-R2. The high expression of CRH in placenta and the substantial expression of CRH receptors in choriodecidua and myometrium suggested that CRH derived from placenta exerts direct or indirect actions on these tissues. Neither CRH produced by myometrium or choriodecidua nor urocortin from other intrauterine sources seem to play a major role in the control of labour.

  2. Increased expression of PGRN protein in follicular fluid and mRNA in granulosa cells in overweight patients with polycystic ovary syndrome.

    PubMed

    Zhou, Danni; Li, Saijiao; Li, Wei; Yin, Tailang; Xu, Wangming; Zhang, Jun; Yang, Jing

    2017-09-19

    Obesity and low-grade chronic inflammation play critical roles in pathological process of PCOS. PGRN is an adipokine and was recently reported that it could induce a low-grade chronic inflammatory state and plays a functional role in insulin resistance associated with obesity. The overall goal of the present study was to evaluate the levels of PGRN in follicular fluid (FF) and the expression of PGRN in granulosa cells (GCs) with respect to the quality of the oocytes both in patients with PCOS and in the normal ovary during COH cycles. Ninety-two patients underwent IVF-ET were divided into four groups based on body mass index: normal-weight PCOS group; overweight PCOS group; non-overweight control group and overweight control group. FF samples and GCs were collected at the time of oocyte retrieval. The PGRN, TNF-α, IL-6 and MCP-1 levels were measured by ELISA, and the mRNA expression of PGRN in GCs was detected by real-time polymerase chain reaction. Analysis of PGRN expression revealed that PGRN levels in FF and the mRNA expression of PGRN in GCs were higher in patients with PCOS than in control patients, and higher in overweight patients than in the normal-weight patients; PGRN in FF of PCOS was positively correlate with basal testosterone and FF TNF-α,but negative correlation with retrieved oocytes number CONCLUSION: This study suggests that PGRN may be a crucial determinant of fertilization success for PCOS patients. Copyright © 2017. Published by Elsevier B.V.

  3. Transfection efficiency and transgene expression kinetics of mRNA delivered in naked and nanoparticle format.

    PubMed

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

    2013-03-28

    Transfection efficiencies and transgene expression kinetics of messenger RNA (mRNA), an emerging class of nucleic acid-based therapeutics, have been poorly characterized. In this study, we evaluated transfection efficiencies of mRNA delivered in naked and nanoparticle format in vitro and in vivo using GFP and luciferase as reporters. While mRNA nanoparticles transfect primary human and mouse dendritic cells (DCs) efficiently in vitro, naked mRNA could not produce any detectable gene product. The protein expression of nanoparticle-mediated transfection in vitro peaks rapidly within 5-7h and decays in a biphasic manner. In vivo, naked mRNA is more efficient than mRNA nanoparticles when administered subcutaneously. In contrast, mRNA nanoparticle performs better when administered intranasally and intravenously. Gene expression is most transient when delivered intravenously in nanoparticle format with an apparent half-life of 1.4h and lasts less than 24h, and most sustained when delivered in the naked format subcutaneously at the base of tail with an apparent half-life of 18h and persists for at least 6days. Notably, exponential decreases in protein expression are consistently observed post-delivery of mRNA in vivo regardless of the mode of delivery (naked or nanoparticle) or the site of administration. This study elucidates the performance of mRNA transfection and suggests a niche for mRNA therapeutics when predictable in vivo transgene expression kinetics is imperative.

  4. Low HIP1R mRNA and protein expression are associated with worse survival in diffuse large B-cell lymphoma patients treated with R-CHOP.

    PubMed

    Wong, Kah Keng; Ch'ng, Ewe Seng; Loo, Suet Kee; Husin, Azlan; Muruzabal, María Arestin; Møller, Michael B; Pedersen, Lars M; Pomposo, María Puente; Gaafar, Ayman; Banham, Alison H; Green, Tina M; Lawrie, Charles H

    2015-12-01

    Huntingtin-interacting protein 1-related (HIP1R) is an endocytic protein involved in receptor trafficking, including regulating cell surface expression of receptor tyrosine kinases. We have previously shown that low HIP1R protein expression was associated with poorer survival in diffuse large B-cell lymphoma (DLBCL) patients from Denmark treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). In this multicenter study, we extend these findings and validate the prognostic and subtyping utility of HIP1R expression at both transcript and protein level. Using data mining on three independent transcriptomic datasets of DLBCL, HIP1R transcript was preferentially expressed in germinal center B-cell (GCB)-like DLBCL subtype (P<0.01 in all three datasets), and lower expression was correlated with worse overall survival (OS; P<0.01) and progression-free survival (PFS; P<0.05) in a microarray-profiled DLBCL dataset. At the protein level examined by immunohistochemistry, HIP1R expression at 30% cut-off was associated with GCB-DLBCL molecular subtype (P=0.0004; n=42), and predictive of OS (P=0.0006) and PFS (P=0.0230) in de novo DLBCL patients treated with R-CHOP (n=73). Cases with high FOXP1 and low HIP1R expression frequency (FOXP1(hi)/HIP1R(lo) phenotype) exhibited poorer OS (P=0.0038) and PFS (P=0.0134). Multivariate analysis showed that HIP1R<30% or FOXP1(hi)/HIP1R(lo) subgroup of patients exhibited inferior OS and PFS (P<0.05) independently of the International Prognostic Index. We conclude that HIP1R expression is strongly indicative of survival when utilized on its own or in combination with FOXP1, and the molecule is potentially applicable for subtyping of DLBCL cases. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. The correlation of TRPM1 (Melastatin) mRNA expression with microphthalmia-associated transcription factor (MITF) and other melanogenesis-related proteins in normal and pathological skin, hair follicles and melanocytic nevi

    PubMed Central

    Lu, Song; Slominski, Andrzej; Yang, Sung-Eun; Sheehan, Christine; Ross, Jeffrey; Carlson, J. Andrew

    2010-01-01

    Background Melastatin (TRPM1), a.k.a. transient receptor potential cation channel, subfamily M, member 1 (TRPM-1) regulates melanocyte differentiation and proliferation. TRPM1 is transcriptionally regulated by the essential melanocyte transcription factor MITF (microphthalmia-associated transcription factor). For the most part, MITF expression is preserved during melanoma progression, while TRPM1 mRNA expression decreases or is completely lost. The loss of TRPM1 is associated with melanomas that are more aggressive. Objective To assess the relationship between TRPM1 mRNA expression and the expression of MITF and nine other markers of melanocytes and melanin-related proteins by immunohistochemistry in normal skin, scars, hair follicles and ordinary melanocytic nevi. Methods Samples of normal skin (n = 102; from tumor excisions and plastic procedures), scars (n = 5; from re-excision specimens) and compound melanocytic nevi (n = 4) were evaluated for the presence of TRPM1 mRNA transcripts as detected by chromogenic in situ hybridization (CISH). Immunohistochemical techniques were used to detect melanin-related proteins including: MITF, S100 protein, Mart-1, tyrosinase, Mel5, HMB45, tyrosinase-related protein-1 (TRP1), TRP2 and α-melanocyte stimulating hormone (αMSH). The labeling index (LI) was defined as the number of intraepidermal cells expressing mRNA or protein per one hundred basal keratinocytes. Results A wide range of LI was found for all markers (0–33 positive cells/100 keratinocytes). When these LI were compared, no significant differences in the expression of MITF, S100, Mart1, tyrosinase proteins and TRPM1 mRNA were identified. The LI for TRPM1 mRNA expression ranged from 74% of that for MITF to 86% for tyrosinase. The LI for TRP-1, TRP-2 and Mel5 was similar to that of TRPM1, while HMB-45 had a significantly lower LI than all other markers. TRPM1 mRNA correlated most tightly with MITF and tyrosinase expression (r = 0.81 and 0.68, respectively, both p

  6. Effects of Ethanol on the Expression Level of Various BDNF mRNA Isoforms and Their Encoded Protein in the Hippocampus of Adult and Embryonic Rats.

    PubMed

    Shojaei, Shahla; Ghavami, Saeid; Panjehshahin, Mohammad Reza; Owji, Ali Akbar

    2015-12-21

    We aimed to compare the effects of oral ethanol (Eth) alone or combined with the phytoestrogen resveratrol (Rsv) on the expression of various brain-derived neurotrophic factor (BDNF) transcripts and the encoded protein pro-BDNF in the hippocampus of pregnant and embryonic rats. A low (0.25 g/kg body weight (BW)/day) dose of Eth produced an increase in the expression of BDNF exons I, III and IV and a decrease in that of the exon IX in embryos, but failed to affect BDNF transcript and pro-BDNF protein expression in adults. However, co-administration of Eth 0.25 g/kg·BW/day and Rsv led to increased expression of BDNF exons I, III and IV and to a small but significant increase in the level of pro-BDNF protein in maternal rats. A high (2.5 g/kg·BW/day) dose of Eth increased the expression of BDNF exons III and IV in embryos, but it decreased the expression of exon IX containing BDNF mRNAs in the maternal rats. While the high dose of Eth alone reduced the level of pro-BDNF in adults, it failed to change the levels of pro-BDNF in embryos. Eth differentially affects the expression pattern of BDNF transcripts and levels of pro-BDNF in the hippocampus of both adult and embryonic rats.

  7. Effects of Ethanol on the Expression Level of Various BDNF mRNA Isoforms and Their Encoded Protein in the Hippocampus of Adult and Embryonic Rats

    PubMed Central

    Shojaei, Shahla; Ghavami, Saeid; Panjehshahin, Mohammad Reza; Owji, Ali Akbar

    2015-01-01

    We aimed to compare the effects of oral ethanol (Eth) alone or combined with the phytoestrogen resveratrol (Rsv) on the expression of various brain-derived neurotrophic factor (BDNF) transcripts and the encoded protein pro-BDNF in the hippocampus of pregnant and embryonic rats. A low (0.25 g/kg body weight (BW)/day) dose of Eth produced an increase in the expression of BDNF exons I, III and IV and a decrease in that of the exon IX in embryos, but failed to affect BDNF transcript and pro-BDNF protein expression in adults. However, co-administration of Eth 0.25 g/kg·BW/day and Rsv led to increased expression of BDNF exons I, III and IV and to a small but significant increase in the level of pro-BDNF protein in maternal rats. A high (2.5 g/kg·BW/day) dose of Eth increased the expression of BDNF exons III and IV in embryos, but it decreased the expression of exon IX containing BDNF mRNAs in the maternal rats. While the high dose of Eth alone reduced the level of pro-BDNF in adults, it failed to change the levels of pro-BDNF in embryos. Eth differentially affects the expression pattern of BDNF transcripts and levels of pro-BDNF in the hippocampus of both adult and embryonic rats. PMID:26703578

  8. 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.

  9. Insulin-like growth factor II in human adrenal and pheochromocytomas and Wilms tumors: expression at the mRNA and protein level

    SciTech Connect

    Haselbacher, G.K.; Irminger, J.C.; Zapf, J.; Ziegler, W.H.; Humbel, R.E.

    1987-02-01

    Two forms of insulin-like growth factor (IGF) II with molecular masses of 10 and 7.5 kDa, respectively, were found in tumor tissue from human adrenal pheochromocytomas. The tumors contained 5.3-7.1 ..mu..g of immunoreactive IGF-II per g of tissue, which is about 20 times more than in adrenal medulla. The total bioactive IGF measured by radioimmunoassay in the pheochromocytomas exceeded that in normal liver or kidney, which contained only the 7.5-kDa IGF-II species, by a factor of approx.100. By contrast, the amount of IGF-I was just measurable and did not vary significantly between tumor and normal tissue. The high amounts of IGF-II in the pheochromocytomas were not reflected, however, by a corresponding increase of mRNA. The opposite situations was found in Wilms tumors, where IGF-II content was in the same range as in nontumor tissues despite increased expression of IGF-II mRNA.

  10. Effect of polysaccharides extract of rhizoma atractylodis macrocephalae on thymus, spleen and cardiac indexes, caspase-3 activity ratio, Smac/DIABLO and HtrA2/Omi protein and mRNA expression levels in aged rats.

    PubMed

    Guo, Ling; Sun, Yong Le; Wang, Ai Hong; Xu, Chong En; Zhang, Meng Yuan

    2012-10-01

    This study was designed to determine the possible protective effect of polysaccharides extract of rhizoma atractylodis macrocephalae on heart function in aged rats. Polysaccharides extract of rhizoma atractylodis macrocephalae was administered to aged rats. Results showed that thymus, spleen and cardiac indexs were significantly increased, whereas caspase-3 activity ratio, Smac/DIABLO and HtrA2/Omi protein expression, Smac/DIABLO and HtrA2/Omi mRNA expression levels were markedly reduced. It can be concluded that polysaccharides extract of rhizoma atractylodis macrocephalae may enhance immunity and improve heart function in aged rats.

  11. In contrast to their murine counterparts, normal human keratinocytes and human epidermoid cell lines A431 and HaCaT fail to express IL-10 mRNA and protein

    PubMed Central

    TEUNISSEN, M B M; KOOMEN, C W; JANSEN, J; DE WAAL MALEFYT, R; SCHMITT, E; VAN DEN WIJNGAARD, R M J G J; DAS, P K; BOS, J D

    1997-01-01

    In mice, keratinocyte-derived IL-10 is up-regulated by ultraviolet-B (UVB) radiation and plays a major role in UVB-induced immunosuppression. The present study was designed to examine whether a comparable phenomenon can be detected in man. Freshly isolated or cultured normal human keratinocytes (NHK) and keratinocyte cell lines A431 and HaCaT were stimulated with graded doses of UVB (up to 200 J/m2) or with a variety of other stimuli. RNA was extracted at various time points post-stimulation and analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) using four different IL-10-specific primer pairs and RNA from monocytes or T cells as positive controls. We failed to detect IL-10 mRNA in NHK from 40 different donors (breast, abdomen, leg, scalp, foreskin) and in A431 and HaCaT cells, irrespective of the stimulation used and despite successful stimulation. Supernatants of NHK, A431 and HaCaT cultures were negative for IL-10 protein, as tested by four different ELISAs and a bioassay. Murine keratinocytes, stimulated under comparable conditions and tested by the same techniques, displayed a strong expression of IL-10 mRNA and protein. Remarkably, an IL-10 mRNA signal could be detected in NHK after a second round of PCR amplification. Because NHK suspensions are contaminated with Langerhans cells, melanocytes and possibly fibroblasts, we tested pure populations of each individual cell type to determine the origin of this IL-10 mRNA. Our results clearly indicate that NHK, Langerhans cells and fibroblasts fail to express IL-10 and that melanocytes are the principal source of IL-10 mRNA in normal human epidermis. PMID:9010278

  12. Several Cis-regulatory Elements Control mRNA Stability, Translation Efficiency, and Expression Pattern of Prrxl1 (Paired Related Homeobox Protein-like 1)*

    PubMed Central

    Regadas, Isabel; Matos, Mariana Raimundo; Monteiro, Filipe Almeida; Gómez-Skarmeta, José Luis; Lima, Deolinda; Bessa, José; Casares, Fernando; Reguenga, Carlos

    2013-01-01

    The homeodomain transcription factor Prrxl1/DRG11 has emerged as a crucial molecule in the establishment of the pain circuitry, in particular spinal cord targeting of dorsal root ganglia (DRG) axons and differentiation of nociceptive glutamatergic spinal cord neurons. Despite Prrxl1 importance in the establishment of the DRG-spinal nociceptive circuit, the molecular mechanisms that regulate its expression along development remain largely unknown. Here, we show that Prrxl1 transcription is regulated by three alternative promoters (named P1, P2, and P3), which control the expression of three distinct Prrxl1 5′-UTR variants, named 5′-UTR-A, 5′-UTR-B, and 5′-UTR-C. These 5′-UTR sequences confer distinct mRNA stability and translation efficiency to the Prrxl1 transcript. The most conserved promoter (P3) contains a TATA-box and displays in vivo enhancer activity in a pattern that overlaps with the zebrafish Prrxl1 homologue, drgx. Regulatory modules present in this sequence were identified and characterized, including a binding site for Phox2b. Concomitantly, we demonstrate that zebrafish Phox2b is required for the expression of drgx in the facial, glossopharyngeal, and vagal cranial ganglia. PMID:24214975

  13. Discrepancy between mRNA and Protein Expression of Neutrophil Gelatinase-Associated Lipocalin in Bronchial Epithelium Induced by Sulfur Mustard

    PubMed Central

    Ebrahimi, Majid; Roudkenar, Mehryar Habibi; Imani Fooladi, Abbas Ali; Halabian, Raheleh; Ghanei, Mostafa; Kondo, Hisatake; Nourani, Mohammad Reza

    2010-01-01

    Sulfur mustard (SM) is a potent vesicant that has been employed as a chemical weapon in various conflicts during the 20th century. More recently, mustard was used in the Iraq conflict against Iranian troops and civilians. At the present time there are more than 40.000 people suffering from pulmonary lesions special bronchiolitis obliterans (BOs) due to mustard gas. SM increases the endogenous production of reactive oxygen species (ROS). Neutrophil Gelatinase-associated Lipocalin 2 (Lcn2, NGAL) is a member of the lipocalin superfamily for which a variety of functions such as cellular protection against oxidative stress have been reported. Ten normal and Twenty SM-induced COPD patient individuals were studied. Assessment of NGAL expressions in healthy and the patients endobrinchial biopsies were performed by semiquantitative RT-PCR, real-time RT-PCR, and Immunohistochemistry analysis. While Normal control samples expressed same level of mRNA NGAL, expression level of mRNA-NGAL was upregulated about 1.4- to 9.8-folds compared to normal samples. No significant immunoreactivity was revealed in both samples. As we are aware this is the first report of induction of NGAL in patients exposed to SM. NGAL may play an important role in cellular protection against oxidative stress toxicity induced by mustard gas in airway wall of patients. PMID:20508729

  14. The effects of electroacupuncture on TH1/TH2 cytokine mRNA expression and mitogen-activated protein kinase signaling pathways in the splenic T cells of traumatized rats.

    PubMed

    Wang, Kun; Wu, Huaxing; Wang, Guonian; Li, Mingming; Zhang, Zhaodi; Gu, Guangying

    2009-11-01

    Surgical trauma contributes to postoperative immune suppression, which is associated with an increased susceptibility to subsequent infections. Electroacupuncture (EA) can alleviate pain and exert immunoregulatory effects. However, the mechanism underlying the immnuomodulation effects of EA is not fully elucidated. Therefore, we investigated the effects of EA on T helper (Th)1/Th2 cytokine production and mRNA expression and evaluated the signaling regulatory mechanism of EA effects. Rats were divided into four groups (n = 24 each): control, trauma, trauma (T) + sham EA, and T + EA. EA was applied to Zusanli (ST36) and Lanwei (Extra37) acupoints at 20 min after surgery for 30 min, and then performed once a day on postoperative days 1-5. Splenic T cells were isolated and the production and mRNA expression of interleukin (IL)-2, interferon-gamma, IL-4, and IL-10 were assayed. The activation of mitogen-activated protein kinase and the DNA binding activity of nuclear factor (NF)-kappaB and activator protein (AP)-1 were examined. Paw withdrawal threshold and paw withdrawal latency were significantly increased in the T + EA group compared with the trauma group from postoperative day 1 (paw withdrawal threshold: 5.8 +/- 0.7 vs 3.0 +/- 0.7 g; paw withdrawal latency: 7.0 +/- 0.8 vs 4.5 +/- 0.5 s; P < 0.001) to day 5 (9.0 +/- 0.6 vs 5.5 +/- 0.6 g; 12.0 +/- 1.3 vs 7.0 +/- 0.8 s; P < 0.001). Th1 cytokine (IL-2 and interferon-gamma) production and mRNA expression in splenic T cells of traumatized rats were significantly decreased on postoperative day 3 (P < 0.001, trauma group versus control group), whereas Th2 cytokine (IL-4 and IL-10) production and mRNA expression were increased (P < 0.001). This was accompanied with a significant depression in the activity of extracellular-regulated protein kinase (ERK)1/2, p38, NF-kappaB, and AP-1 (P < 0.001, trauma group versus control group). EA administration increased Th1 cytokine protein and mRNA expression, suppressed Th2 cytokine

  15. Imaging mRNA and protein interactions within neurons

    PubMed Central

    Eliscovich, Carolina; Shenoy, Shailesh M.

    2017-01-01

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

  16. Exercise and adrenaline increase PGC-1α mRNA expression in rat adipose tissue

    PubMed Central

    Sutherland, Lindsey N; Bomhof, Marc R; Capozzi, Lauren C; Basaraba, Susan A U; Wright, David C

    2009-01-01

    The purpose of the present investigation was to explore the effects of exercise and adrenaline on the mRNA expression of PGC-1α, a master regulator of mitochondrial biogenesis, in rat abdominal adipose tissue. We hypothesized that (1) exercise training would increase PGC-1α mRNA expression in association with increases in mitochondrial marker enzymes, (2) adrenaline would increase PGC-1α mRNA expression and (3) the effect of exercise on PGC-1α mRNA expression in white adipose tissue would be attenuated by a β-blocker. Two hours of daily swim training for 4 weeks led to increases in mitochondrial marker proteins and PGC-1α mRNA expression in epididymal and retroperitoneal fat depots. Additionally, a single 2 h bout of exercise led to increases in PGC-1α mRNA expression immediately following exercise cessation. Adrenaline treatment of adipose tissue organ cultures led to dose-dependent increases in PGC-1α mRNA expression. A supra-physiological concentration of adrenaline increased PGC-1α mRNA expression in epididymal but not retroperitoneal adipose tissue. β-Blockade attenuated the effects of an acute bout of exercise on PGC-1α mRNA expression in epididymal but not retroperitoneal fat pads. In summary, this is the first investigation to demonstrate that exercise training, an acute bout of exercise and adrenaline all increase PGC-1α mRNA expression in rat white adipose tissue. Furthermore it would appear that increases in circulating catecholamine levels may be one potential mechanism mediating exercise induced increases in PGC-1α mRNA expression in rat abdominal adipose tissue. PMID:19221126

  17. BAX/BCL-2 mRNA and protein expression in human breast MCF-7 cells exposed to drug vehicles-methanol and dimethyl sulfoxide (DMSO) for 24 hrs

    PubMed Central

    Adefolaju, Gbenga Anthony; Theron, Kathrine E; Hosie, Margot Jill

    2015-01-01

    Background: Methanol and DMSO are commonly used as carrier solvents for lipophilic chemicals in in-vitro experiments. However, very little information is available regarding the effects of these solvents on the expression of pro and anti-apoptotic genes and proteins. Materials and Methods: In this study, we examined the cytotoxic effects of methanol and dimethylsulfoxide at 0.5% (final concentrations recommended for in-vitro toxicity assays) on human breast cancer MCF-7 cells. We also investigated the effects of these solvents on the mRNA and immunocytochemical expression of apoptotic proteins BAX and BCL-2. Results: The results of neutral red cell viability assay showed that methanol and DMSO concentrations of 0.5% exhibited no cytotoxic effects on MCF-7 cells following a 24 hour exposure. Gene expression and Immunofluorescence results showed that methanol but not DMSO reduced the expression of the BAX pro-apoptotic protein, while both solvents did not alter the expression of the BCL-2 oncoprotein. Conclusion: Our results suggest that while methanol concentrations at 0.5% may be appropriate for in vitro toxicity studies in human breast cancer MCF-7 cells, it could alter the results of gene and protein expression experiments. PMID:26229223

  18. High Brain Ammonia Tolerance and Down-Regulation of Na+:K+:2Cl- Cotransporter 1b mRNA and Protein Expression in the Brain of the Swamp Eel, Monopterus albus, Exposed to Environmental Ammonia or Terrestrial Conditions

    PubMed Central

    Ip, Yuen K.; Hou, Zhisheng; Chen, Xiu L.; Ong, Jasmine L. Y.; Chng, You R.; Ching, Biyun; Hiong, Kum C.; Chew, Shit F.

    2013-01-01

    Na+:K+:2Cl- cotransporter 1 (NKCC1) has been implicated in mediating ischemia-, trauma- or ammonia-induced astrocyte swelling/brain edema in mammals. This study aimed to determine the effects of ammonia or terrestrial exposure on ammonia concentrations in the plasma and brain, and the mRNA expression and protein abundance of nkcc/Nkcc in the brain, of the swamp eel Monopterusalbus. Ammonia exposure led to a greater increase in the ammonia concentration in the brain of M. albus than terrestrial exposure. The brain ammonia concentration of M. albus reached 4.5 µmol g-1 and 2.7 µmol g-1 after 6 days of exposure to 50 mmol l-1 NH4Cl and terrestrial conditions, respectively. The full cDNA coding sequence of nkcc1b from M. albus brain comprised 3276 bp and coded for 1092 amino acids with an estimated molecular mass of 119.6 kDa. A molecular characterization indicated that it could be activated through phosphorylation and/or glycosylation by osmotic and/or oxidative stresses. Ammonia exposure for 1 day or 6 days led to significant decreases in the nkcc1b mRNA expression and Nkcc1b protein abundance in the brain of M. albus. In comparison, a significant decrease in nkcc1b mRNA expression was observed in the brain of M. albus only after 6 days of terrestrial exposure, but both 1 day and 6 days of terrestrial exposure resulted in significant decreases in the protein abundance of Nkcc1b. These results are novel because it has been established in mammals that ammonia up-regulates NKCC1 expression in astrocytes and NKCC1 plays an important role in ammonia-induced astrocyte swelling and brain edema. By contrast, our results indicate for the first time that M. albus is able to down-regulate the mRNA and protein expression of nkcc1b/Nkcc1b in the brain when confronted with ammonia toxicity, which could be one of the contributing factors to its extraordinarily high brain ammonia tolerance. PMID:24069137

  19. High brain ammonia tolerance and down-regulation of Na+:K+:2Cl(-) Cotransporter 1b mRNA and protein expression in the brain of the Swamp Eel, Monopterus albus, exposed to environmental ammonia or terrestrial conditions.

    PubMed

    Ip, Yuen K; Hou, Zhisheng; Chen, Xiu L; Ong, Jasmine L Y; Chng, You R; Ching, Biyun; Hiong, Kum C; Chew, Shit F

    2013-01-01

    Na(+):K(+):2Cl(-) cotransporter 1 (NKCC1) has been implicated in mediating ischemia-, trauma- or ammonia-induced astrocyte swelling/brain edema in mammals. This study aimed to determine the effects of ammonia or terrestrial exposure on ammonia concentrations in the plasma and brain, and the mRNA expression and protein abundance of nkcc/Nkcc in the brain, of the swamp eel Monopterusalbus. Ammonia exposure led to a greater increase in the ammonia concentration in the brain of M. albus than terrestrial exposure. The brain ammonia concentration of M. albus reached 4.5 µmol g(-1) and 2.7 µmol g(-1) after 6 days of exposure to 50 mmol l(-1) NH4Cl and terrestrial conditions, respectively. The full cDNA coding sequence of nkcc1b from M. albus brain comprised 3276 bp and coded for 1092 amino acids with an estimated molecular mass of 119.6 kDa. A molecular characterization indicated that it could be activated through phosphorylation and/or glycosylation by osmotic and/or oxidative stresses. Ammonia exposure for 1 day or 6 days led to significant decreases in the nkcc1b mRNA expression and Nkcc1b protein abundance in the brain of M. albus. In comparison, a significant decrease in nkcc1b mRNA expression was observed in the brain of M. albus only after 6 days of terrestrial exposure, but both 1 day and 6 days of terrestrial exposure resulted in significant decreases in the protein abundance of Nkcc1b. These results are novel because it has been established in mammals that ammonia up-regulates NKCC1 expression in astrocytes and NKCC1 plays an important role in ammonia-induced astrocyte swelling and brain edema. By contrast, our results indicate for the first time that M. albus is able to down-regulate the mRNA and protein expression of nkcc1b/Nkcc1b in the brain when confronted with ammonia toxicity, which could be one of the contributing factors to its extraordinarily high brain ammonia tolerance.

  20. Quantitative analysis of CDKN2A methylation, mRNA, and p16(INK4a) protein expression in children and adolescents with Burkitt lymphoma: biological and clinical implications.

    PubMed

    Robaina, Marcela Cristina S; Faccion, Roberta Soares; Arruda, Viviane Oliveira; de Rezende, Lidia Maria Magalhães; Vasconcelos, Gisele Moledo; Apa, Alexandre Gustavo; Bacchi, Carlos E; Klumb, Claudete Esteves

    2015-02-01

    CDKN2A is a tumor suppressor gene critical in the cell cycle regulation. Little is known regarding the role of CDKN2A methylation in the pathogenesis of Burkitt lymphoma (BL). CDKN2A methylation was investigated using pyrosequencing in 51 tumor samples. p16(INK4a) mRNA and protein levels were measured using real-time PCR and immunohistochemistry, respectively. CDKN2A methylation was detectable in 72% cases. Nuclear expression of p16(INK4a) was not detected in 41% cases. There was an association between methylation and absence of CDKN2A mRNA (P=0.003). In conclusion, CDKN2A methylation occurs at a high frequency suggesting a role in BL pathogenesis and potential therapeutic implications.

  1. Dietary riboflavin deficiency decreases immunity and antioxidant capacity, and changes tight junction proteins and related signaling molecules mRNA expression in the gills of young grass carp (Ctenopharyngodon idella).

    PubMed

    Chen, Liang; Feng, Lin; Jiang, Wei-Dan; Jiang, Jun; Wu, Pei; Zhao, Juan; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Liu, Yang

    2015-08-01

    This study investigated the effects of dietary riboflavin on the growth, gill immunity, tight junction proteins, antioxidant system and related signaling molecules mRNA expression of young grass carp (Ctenopharyngodon idella). Fish were fed six diets containing graded levels of riboflavin (0.63-10.04 mg/kg diet) for 8 weeks. The study indicated that riboflavin deficiency decreased lysozyme and acid phosphatase activities, and complement component 3 content in the gills of fish (P < 0.05). Moreover, riboflavin deficiency caused oxidative damage, which might be partly due to decrease copper, zinc superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and glutathione-S-transferase activities and reduced glutathione content in the gills of fish (P < 0.05). Furthermore, the relative mRNA levels of antimicrobial peptides (liver expressed antimicrobial peptide 2 and Hepcidin), anti-inflammatory cytokines (interleukin 10 and transforming growth factor β1), tight junction proteins (Occludin, zonula occludens 1, Claudin-c and Claudin-3), signaling molecules (inhibitor of κBα, target of rapamycin and NF-E2-related factor 2) and antioxidant enzymes (copper, zinc superoxide dismutase and glutathione reductase) were significantly decreased (P < 0.05) in the gills of fish fed riboflavin-deficient diet. Conversely, the mRNA levels of pro-inflammatory cytokines (tumor necrosis factor α, interleukin 8, interferon γ2, and interleukin 1β), signaling molecules (nuclear factor kappa B p65, IκB kinase β, IκB kinase γ, Kelch-like-ECH-associated protein 1b and myosin light chain kinase) and tight junction protein Claudin-12 were significantly increased (P < 0.05) in the gills of fish fed riboflavin-deficient diet. In addition, this study indicated for the first time that young fish fed a riboflavin-deficient diet exhibited anorexia and poor growth. In conclusion, riboflavin deficiency decreased growth and gill immunity, impaired gill antioxidant system, as

  2. Fragile X mental retardation protein control of neuronal mRNA metabolism: Insights into mRNA stability.

    PubMed

    De Rubeis, Silvia; Bagni, Claudia

    2010-01-01

    The fragile X mental retardation protein (FMRP) is an RNA binding protein that has an essential role in neurons. From the soma to the synapse, FMRP is associated with a specific subset of messenger RNAs and controls their posttranscriptional fates, i.e., dendritic localization and local translation. Because FMRP target mRNAs encode important neuronal proteins, the deregulation of their expression in the absence of FMRP leads to a strong impairment of synaptic function. Here, we review emerging evidence indicating a critical role for FMRP in the control of mRNA stability. To date, two mRNAs have been identified as being regulated in this manner: PSD-95 mRNA, encoding a scaffolding protein, and Nxf1 mRNA, encoding a general export factor. Moreover, expression studies suggest that the turnover of other neuronal mRNAs, including those encoding for the GABA(A) receptors subunits, could be affected by the loss of FMRP. According to the specific target and/or cellular context, FMRP could influence mRNA stability in the brain.

  3. Effect of growth hormone on steroid concentrations and mRNA expression of their receptor, and selected egg-specific protein genes in the chicken oviduct during pause in laying induced by fasting.

    PubMed

    Socha, J K; Sechman, A; Mika, M; Hrabia, A

    2017-10-01

    This study was undertaken to examine the effect of growth hormone (GH) treatment during pause in laying on (1) the concentration of steroids in blood plasma and oviduct tissues, (2) the expression of mRNA of steroid receptors, and (3) the mRNA expression of selected egg-specific proteins in the chicken oviduct. A pause in egg laying was induced by food deprivation for 5 d, followed by feeding every other day, and then feeding daily from Day 10 onward. Birds were divided into three groups: control (n = 18) fed ad libitum, subjected to pause in laying (n = 18), and subjected to pause in laying and injected every day with 200 μg/kg BW of chicken GH (chGH; n = 18). The oviduct was isolated from hens of each group on Days 6 (when the oviduct was regressed), 13 (during oviduct recrudescence), and 17 or 20 (rejuvenated oviduct) of the experiment. Fasting caused a decrease in plasma concentrations of progesterone (P4), testosterone, and estradiol on Day 6 and a reduction in tissue concentrations of these steroids on Days 6 and 13. Fasting also caused an increased relative expression of estrogen receptor α and β (ERα, ERβ) and progesterone receptor (PR) in the magnum and shell gland on Day 6, increased ERα and PR in the magnum on Days 13 and 17 or 20, and increased androgen receptor (AR) mRNA in the magnum on Days 6 and 13 and in the shell gland on Day 13. A fasting-induced elevation in ovocalyxin-36 mRNA expression on Day 6 and a decrease in avidin mRNA on Days 6 and 13 and in ovocleidin-116 on Day 13 were also observed (P < 0.05 to P < 0.001). Administration of chGH abolished the fasting-induced decrease in the concentration of steroids in plasma and tissues. Furthermore, chGH enhanced the effect of fasting on mRNA expression of PR, ERα, and avidin in the magnum on Day 6, and ERα in the shell gland on Day 13. The gene expression of ovalbumin on Days 6 and 13, ovocalyxin-36 and ovocleidin-116 on Day 6 was decreased in chGH-treated chickens. In contrast, the

  4. The Unique Catalytic Subunit of Sperm cAMP-dependent Protein Kinase Is the Product of an Alternative Cα mRNA Expressed Specifically in Spermatogenic Cells

    PubMed Central

    Agustin, Jovenal T. San; Wilkerson, Curtis G.; Witman, George B.

    2000-01-01

    cAMP-dependent protein kinase has a central role in the control of mammalian sperm capacitation and motility. Previous protein biochemical studies indicated that the only cAMP-dependent protein kinase catalytic subunit (C) in ovine sperm is an unusual isoform, termed Cs, whose amino terminus differs from those of published C isoforms of other species. Isolation and sequencing of cDNA clones encoding ovine Cs and Cα1 (the predominant somatic isoform) now reveal that Cs is the product of an alternative transcript of the Cα gene. Cs cDNA clones from murine and human testes also were isolated and sequenced, indicating that Cs is of ancient origin and widespread in mammals. In the mouse, Cs transcripts were detected only in testis and not in any other tissue examined, including ciliated tissues and ovaries. Finally, immunohistochemistry of the testis shows that Cs first appears in pachytene spermatocytes. This is the first demonstration of a cell type–specific expression for any C isoform. The conservation of Cs throughout mammalian evolution suggests that the unique structure of Cs is important in the subunit's localization or function within the sperm. PMID:10982398

  5. p53-Regulated Networks of Protein, mRNA, miRNA, and lncRNA Expression Revealed by Integrated Pulsed Stable Isotope Labeling With Amino Acids in Cell Culture (pSILAC) and Next Generation Sequencing (NGS) Analyses*

    PubMed Central

    Hünten, Sabine; Kaller, Markus; Drepper, Friedel; Oeljeklaus, Silke; Bonfert, Thomas; Erhard, Florian; Dueck, Anne; Eichner, Norbert; Friedel, Caroline C.; Meister, Gunter; Zimmer, Ralf; Warscheid, Bettina; Hermeking, Heiko

    2015-01-01

    We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics (pulsed stable isotope labeling with amino acids in cell culture/pSILAC) in the colorectal cancer cell line SW480. This was combined with mRNA and noncoding RNA expression analyses by next generation sequencing (RNA-, miR-Seq). Furthermore, genome-wide DNA binding of p53 was analyzed by chromatin-immunoprecipitation (ChIP-Seq). Thereby, we identified differentially regulated proteins (542 up, 569 down), mRNAs (1258 up, 415 down), miRNAs (111 up, 95 down) and lncRNAs (270 up, 123 down). Changes in protein and mRNA expression levels showed a positive correlation (r = 0.50, p < 0.0001). In total, we detected 133 direct p53 target genes that were differentially expressed and displayed p53 occupancy in the vicinity of their promoter. More transcriptionally induced genes displayed occupied p53 binding sites (4.3% mRNAs, 7.2% miRNAs, 6.3% lncRNAs, 5.9% proteins) than repressed genes (2.4% mRNAs, 3.2% miRNAs, 0.8% lncRNAs, 1.9% proteins), suggesting indirect mechanisms of repression. Around 50% of the down-regulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3′-UTRs. Moreover, proteins repressed by p53 significantly overlapped with those previously shown to be repressed by miR-34a. We confirmed up-regulation of the novel direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and showed that ectopic LINC01021 expression inhibits proliferation in SW480 cells. Furthermore, KLF12, HMGB1 and CIT mRNAs were confirmed as direct targets of the p53-induced miR-34a, miR-205 and miR-486–5p, respectively. In line with the loss of p53 function during tumor progression, elevated expression of KLF12, HMGB1 and CIT was detected in advanced stages of cancer. In conclusion, the integration of multiple omics methods allowed the comprehensive identification of direct and indirect effectors of p53 that provide new insights and leads into the

  6. Cadherin-11 mRNA and protein expression in ovarian tumors of different malignancy: No evidence of oncogenic or tumor-suppressive function

    PubMed Central

    VON BÜLOW, CHARLOTTE; OLIVEIRA-FERRER, LETICIA; LÖNING, THOMAS; TRILLSCH, FABIAN; MAHNER, SVEN; MILDE-LANGOSCH, KARIN

    2015-01-01

    Cadherin-11 (CDH11, OB-cadherin) is a mesenchymal cadherin found to be upregulated in various types of tumors and implicated in tumor progression and metastasis. In order to determine the role of CDH11 expression in ovarian tumors, we performed a combined reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blot analysis and immunohistochemical study on a large cohort of benign, borderline and invasive ovarian tumors. The RT-qPCR and western blot analysis demonstrated that the CDH11 expression was high in benign cystadenomas and decreased with increasing malignancy. This may be explained by the different tumor-stroma ratios, since immunohistochemistry revealed strong staining of stromal cells, particularly vascular smooth muscle cells and endothelial cells, but only weak cytoplasmic or nuclear immunoreactivity of cancer cells. Within the group of invasive carcinomas, high CDH11 protein expression, as detected by western blot analysis, was found to be significantly correlated with advanced stage and nodal involvement. However, the recurrence-free and overall survival analyses did not reveal any prognostic or predictive significance. In conclusion, in contrast to other tumor types, CDH11 does not play an important role in ovarian cancer progression. PMID:26623052

  7. High level of ezrin mRNA expression in an osteosarcoma biopsy sample with lung metastasis.

    PubMed

    Ogino, Wakako; Takeshima, Yasuhiro; Mori, Takeshi; Yanai, Tomoko; Hayakawa, Akira; Akisue, Toshihiro; Kurosaka, Masahiro; Matsuo, Masafumi

    2007-07-01

    Osteosarcoma (OS) remains a life-threatening malignancy and its molecular character is not fully understood. Ezrin is a cytoskeleton linker protein involved in regulating the growth and metastatic capacity of cancer cells. However, the correlation between ezrin mRNA expression and clinical severity has not yet been examined in OS biopsy samples. Furthermore, recent evidence has demonstrated that the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression is increased in human cancers of various origins, but this has not yet been examined in OS cells. To clarify the correlation between the clinical severity and the levels of ezrin and GAPDH mRNA expression, we quantified these mRNA levels in 4 pediatric OS biopsy samples using real-time quantitative polymerase chain reaction. Among these 4 samples, ezrin mRNA expression was approximately 5-fold higher in a case with lung metastasis compared with the other cases without metastasis, suggesting an association between the ezrin mRNA expression level and metastasis. On the other hand, the GAPDH mRNA expression level was not related to the clinical severity. This is the first report to demonstrate a high level of ezrin mRNA expression in an OS biopsy sample with lung metastasis.

  8. Effects of dietary soybean isoflavones on non-specific immune responses and hepatic antioxidant abilities and mRNA expression of two heat shock proteins (HSPs) in juvenile golden pompano Trachinotus ovatus under pH stress.

    PubMed

    Zhou, Chuanpeng; Lin, Heizhao; Huang, Zhong; Wang, Jun; Wang, Yun; Yu, Wei

    2015-12-01

    This study determined the effect of dietary soybean isoflavones on non-specific immunity and on mRNA expression of two HSPs in juvenile golden pompano Trachinotus ovatus under pH stress. Six diets were formulated to contain 0, 10, 20, 40, 60 and 80 mg/kg of soybean isoflavones. Each diet was fed to triplicate groups of fish in cylindrical tanks. After 56 days of feeding, 15 fish per tank were exposed to pH stress (pH ≈ 9.2) for 24 h. Serum total protein (TP), respiratory burst activity (RBA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), lysozyme (LYZ), complement 3 (C3), complement 4 (C4), cortisol, hepatic total antioxidant capacity (T-AOC), superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT) and the relative mRNA expression of heat shock protein 70 (HSP70) and 90 (HSP90) were investigated. The results showed that after pH stress, serum TP, RBA, LYZ, C4, hepatic T-AOC and CAT levels were significantly reduced (P < 0.05) while serum ALT, hepatic MDA and HSP70 and HSP90 mRNA expression levels were significantly increased (P < 0.05). On the other hand, supplementation with soybean isoflavones significantly reduced levels of serum ALT (20, 40, 60 mg/kg soybean isoflavones groups) and hepatic MDA (40, 60 and 80 mg/kg soybean isoflavones groups). Supplemented groups had increased serum TP content (40 mg/kg soybean isoflavones groups), RBA (20 and 40 mg/kg soybean isoflavones groups), LYZ (40 and 60 mg/kg soybean isoflavones groups), C3(20, 40, 60 and 80 mg/kg soybean isoflavones groups), hepatic SOD activity (40, 60 and 80 mg/kg soybean isoflavones groups) as well as increased relative mRNA expression of hepatic HSP70 (40, 60 and 80 mg/kg soybean isoflavones groups) and HSP90 (40 and 60 mg/kg soybean isoflavones groups) (P < 0.05). These results indicate that ingestion of a basal diet supplemented with 40-60 mg/kg soybean isoflavones could enhance resistance against pH stress in T. Ovatus to some degree.

  9. Differential expression of IGF-1 mRNA isoforms in colorectal carcinoma and normal colon tissue.

    PubMed

    Kasprzak, Aldona; Szaflarski, Witold; Szmeja, Jacek; Andrzejewska, Małgorzata; Przybyszewska, Wiesława; Kaczmarek, Elżbieta; Koczorowska, Maria; Kościński, Tomasz; Zabel, Maciej; Drews, Michał

    2013-01-01

    The insulin-like growth factor (IGF)-1 gene consists of 6 exons resulting in the expression of 6 variant forms of mRNA (IA, IB, IC, IIA, IIB and IIC) due to an alternative splicing. The mechanisms of IGF-1 gene splicing and the role of local expression manifested by IGF-1 mRNA variants in colorectal carcinoma (CRC) have not been extensively investigated. Therefore, the aim of our study was to analyse the expression of IGF-1 mRNA isoforms [A, B, C, P1 (class I) and P2 (class II)], as well as the protein expression in CRC and control samples isolated from 28 patients. The expression of Ki-67 was also analysed and clinical data were obtained. For this purpose, we used quantitative real-time PCR (qPCR) and immunocytochemistry. The expression of mRNAs coding for all splicing isoforms of IGF-1 was observed in every tissue sample studied, with a significantly lower expression noted in the CRC as compared to the control samples. The cytoplasmic expression of IGF-1 protein was found in 50% of the CRC and in ~40% of the non-tumor tissues; however, no significant quantitative inter-group differences were observed. The expression of the IGF-1 gene in the 2 groups of tissues was controlled by the P1 and P2 promoters in a similar manner. No significant differences were detected in the expression of the IGF-1 A and B isoforms; however, their expression was significantly higher compared to that of isoform C. No significant differences were observed between the expression of Ki-67 mRNA in the CRC and control tissue even though the expression of the Ki-67 protein was higher in the CRC compared to the control samples. Ki-67 protein expression was associated with the macroscopic and microscopic aspects of CRC. A significant positive correlation was found between the local production of total mRNA and isoform A and the expression of Ki-67 mRNA, although only in the non-tumor tissues. In CRC samples, the local expression of the total IGF-1 mRNA and all splicing isoforms of IGF-1 mRNA

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

    PubMed

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

    2015-03-01

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

  11. Expression of brain derived neurotrophic factor, activity-regulated cytoskeleton protein mRNA, and enhancement of adult hippocampal neurogenesis in rats after sub-chronic and chronic treatment with the triple monoamine re-uptake inhibitor tesofensine.

    PubMed

    Larsen, Marianne H; Rosenbrock, Holger; Sams-Dodd, Frank; Mikkelsen, Jens D

    2007-01-26

    The changes of gene expression resulting from long-term exposure to monoamine antidepressant drugs in experimental animals are key to understanding the mechanisms of action of this class of drugs in man. Many of these genes and their products are either relevant biomarkers or directly involved in structural changes that are perhaps necessary for the antidepressant effect. Tesofensine is a novel triple monoamine reuptake inhibitor that acts to increase noradrenaline, serotonin, and dopamine neurotransmission. This study was undertaken to examine the effect of sub-chronic (5 days) and chronic (14 days) administration of Tesofensine on the expression of brain derived neurotrophic factor (BDNF) and activity-regulated cytoskeleton protein (Arc) in the rat hippocampus. Furthermore, hippocampi from the same animals were used to investigate the effect on cell proliferation by means of Ki-67- and NeuroD-immunoreactivity. We find that chronic, but not sub-chronic treatment with Tesofensine increases BDNF mRNA in the CA3 region of the hippocampus (35%), and Arc mRNA in the CA1 of the hippocampus (65%). Furthermore, the number of Ki-67- and neuroD-positive cells increased after chronic, but not sub-chronic treatment. This study shows that Tesofensine enhances hippocampal gene expression and new cell formation indicative for an antidepressant potential of this novel drug substance.

  12. Enhanced SRSF5 Protein Expression Reinforces Lamin A mRNA Production in HeLa Cells and Fibroblasts of Progeria Patients.

    PubMed

    Vautrot, Valentin; Aigueperse, Christelle; Oillo-Blanloeil, Florence; Hupont, Sébastien; Stevenin, James; Branlant, Christiane; Behm-Ansmant, Isabelle

    2016-03-01

    The Hutchinson Gilford Progeria Syndrome (HGPS) is a rare genetic disease leading to accelerated aging. Three mutations of the LMNA gene leading to HGPS were identified. The more frequent ones, c.1824C>T and c.1822G>A, enhance the use of the intron 11 progerin 5'splice site (5'SS) instead of the LMNA 5'SS, leading to the production of the truncated dominant negative progerin. The less frequent c.1868C>G mutation creates a novel 5'SS (LAΔ35 5'SS), inducing the production of another truncated LMNA protein (LAΔ35). Our data show that the progerin 5'SS is used at low yield in the absence of HGPS mutation, whereas utilization of the LAΔ35 5'SS is dependent upon the presence of the c.1868C>G mutation. In the perspective to correct HGPS splicing defects, we investigated whether SR proteins can modify the relative yields of utilization of intron 11 5'SSs. By in cellulo and in vitro assays, we identified SRSF5 as a direct key regulator increasing the utilization of the LMNA 5'SS in the presence of the HGPS mutations. Enhanced SRSF5 expression in dermal fibroblasts of HGPS patients as well as PDGF-BB stimulation of these cells decreased the utilization of the progerin 5'SS, and improves nuclear morphology, opening new therapeutic perspectives for premature aging.

  13. The mRNA and Proteins Expression Levels Analysis of TC-1 Cells Immune Response to H9N2 Avian Influenza Virus

    PubMed Central

    Liu, Jiyuan; Li, Ning; Meng, Dan; Hao, Mengchan; Wei, Liangmeng; Chai, Tongjie

    2016-01-01

    Since 1994, the H9N2 avian influenza virus (AIV) has spread widely in mainland China, causing great economic losses to the poultry industry there. Subsequently, it was found that the H9N2 AIV had the ability to infect mammals, which gave rise to great panic. In order to investigate the immune response of a host infected with H9N2 AIV, TC-1 cells were set as a model in this research. Quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay methods were used to study the expression changes of pattern recognition receptors (PRRs), inflammatory cytokines, and chemokines in AIV-infected TC-1 cells. Our research found that TC-1 cells had similar susceptibility to both CK/SD/w3 (A/Chicken/Shandong/W3/2012) and CK/SD/w4 (A/Chicken/Shandong/W4/2012) H9N2 isolates, while the CK/SD/w3 isolate had a stronger capability of replication in the TC-1 cells. At the same time, the expression of PRRs (melanoma differentiation-associated gene 5, MDA-5), cytokines [interleukin (IL)-1β and IL-6], and chemokines [regulated on activation, normal T cell expressed and secreted (RANTES) and interferon-γ-induced protein-10 kDa (IP-10)] were significantly up-regulated. These results indicated that MDA-5, IL-1β, IL-6, RANTES, and IP-10 might play important roles in the host immune response to H9N2 AIV infection. This study provided useful information for further understanding the interaction between H9N2 virus infection and host immunity, and had certain guiding significance for the prevention and treatment of this disease. PMID:27446066

  14. Isolation and nucleotide sequence analysis of the of Rhinella arenarum β-catenin: an mRNA and protein expression study during the larval stages of the digestive tract development.

    PubMed

    Galetto, C D; Izaguirre, M F; Bessone, V; Casco, V H

    2012-12-15

    β-catenin interacts with several proteins mediating key biological processes, such as cadherin-mediated cell-cell adhesion as well as signal transduction. This work was done to establish the molecular basis and regulation of the formation pattern of cadherin/β-catenin-mediated adherens junctions, using an animal model of unknown gene sequence, the toad Rhinella arenarum. A Rhinella arenarum β-catenin homolog was isolated from larval tissue, their sequence compared and analyzed with those of eight other vertebrates using bioinformatics tools. The mRNA and protein expression levels of β-catenin were determined during the development of Rhinella arenarum digestive tract both by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and immunohistochemistry-morphometry respectively. Using Xenopus laevis frog specific primers, a fragment 539 bp of Rhinella arenarum toad β-catenin cDNA was obtained and sequenced. The resulting putative sequence of 177 amino acids showed high similarity at the amino acid level (97%) when compared to other six vertebrates (Xenopus laevis, Xenopus tropicalis, Mus musculus, Rattus norvegicus, Bos taurus and Homo sapiens), with sequences and structural domains characteristic of catenins. Subsequently, using primers specifically designed for Rhinella arenarum nucleotide sequence, β-catenin-mRNA increasing levels were found during the Rhinella arenarum metamorphosis. Finally, increasing β-catenin protein expression during development has confirmed the specificity the detection of Rhinella arenarum β-catenin. Summarizing, we have isolated and sequenced a β-catenin-homologue sequence from the Rhinella arenarum toad, which is highly conserved between species, and following we have detected β-catenin mRNA and protein levels during their digestive tract development.

  15. Asparagine reduces the mRNA expression of muscle atrophy markers via regulating protein kinase B (Akt), AMP-activated protein kinase α, toll-like receptor 4 and nucleotide-binding oligomerisation domain protein signalling in weaning piglets after lipopolysaccharide challenge.

    PubMed

    Wang, Xiuying; Liu, Yulan; Wang, Shuhui; Pi, Dingan; Leng, Weibo; Zhu, Huiling; Zhang, Jing; Shi, Haifeng; Li, Shuang; Lin, Xi; Odle, Jack

    2016-10-01

    Pro-inflammatory cytokines are critical in mechanisms of muscle atrophy. In addition, asparagine (Asn) is necessary for protein synthesis in mammalian cells. We hypothesised that Asn could attenuate lipopolysaccharide (LPS)-induced muscle atrophy in a piglet model. Piglets were allotted to four treatments (non-challenged control, LPS-challenged control, LPS+0·5 % Asn and LPS+1·0 % Asn). On day 21, the piglets were injected with LPS or saline. At 4 h post injection, piglet blood and muscle samples were collected. Asn increased protein and RNA content in muscles, and decreased mRNA expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). However, Asn had no effect on the protein abundance of MAFbx and MuRF1. In addition, Asn decreased muscle AMP-activated protein kinase (AMPK) α phosphorylation, but increased muscle protein kinase B (Akt) and Forkhead Box O (FOXO) 1 phosphorylation. Moreover, Asn decreased the concentrations of TNF-α, cortisol and glucagon in plasma, and TNF-α mRNA expression in muscles. Finally, Asn decreased mRNA abundance of muscle toll-like receptor (TLR) 4 and nucleotide-binding oligomerisation domain protein (NOD) signalling-related genes, and regulated their negative regulators. The beneficial effects of Asn on muscle atrophy may be associated with the following: (1) inhibiting muscle protein degradation via activating Akt and inactivating AMPKα and FOXO1; and (2) decreasing the expression of muscle pro-inflammatory cytokines via inhibiting TLR4 and NOD signalling pathways by modulation of their negative regulators.

  16. Molecular Characterization of Aquaporin 1 and Aquaporin 3 from the Gills of the African Lungfish, Protopterus annectens, and Changes in Their Branchial mRNA Expression Levels and Protein Abundance during Three Phases of Aestivation

    PubMed Central

    Chng, You R.; Ong, Jasmine L. Y.; Ching, Biyun; Chen, Xiu L.; Hiong, Kum C.; Wong, Wai P.; Chew, Shit F.; Lam, Siew H.; Ip, Yuen K.

    2016-01-01

    African lungfishes can undergo long periods of aestivation on land during drought. During aestivation, lungfishes are confronted with desiccation and dehydration, and their gills become non-functional and covered with a thick layer of dried mucus. Aquaporins (Aqps) are a superfamily of integral membrane proteins which generally facilitate the permeation of water through plasma membranes. This study aimed to obtain the complete cDNA coding sequences of aqp1 and aqp3 from the gills of Protopterus annectens, and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Dendrogramic analyses of the deduced Aqp1 and Aqp3 amino acid sequences of P. annectens revealed their close relationships with those of Latimeria chalumnae and tetrapods. During the induction phase, there were significant decreases in the transcript levels of aqp1 and aqp3 in the gills of P. annectens, but the branchial Aqp1 and Aqp3 protein abundance remained unchanged. As changes in transcription might precede changes in translation, this could be regarded as an adaptive response to decrease the protein abundance of Aqp1 and Aqp3 in the subsequent maintenance phase of aestivation. As expected, the branchial transcript levels and protein abundance of aqp1/Aqp1 and aqp3/Aqp3 were significantly down-regulated during the maintenance phase, probably attributable to the shutdown of branchial functions and the cessation of volume regulation of branchial epithelial cells. Additionally, these changes could reduce the loss of water through branchial epithelial surfaces, supplementing the anti-desiccating property of the dried mucus. Upon arousal, it was essential for the lungfish to restore branchial functions. Indeed, the protein abundance of Aqp1 recovered partially, with complete recovery of mRNA expression level and protein abundance of Aqp3, in the gills of P. annectens after 3 days of arousal. These results provide insights into

  17. Molecular Characterization of Aquaporin 1 and Aquaporin 3 from the Gills of the African Lungfish, Protopterus annectens, and Changes in Their Branchial mRNA Expression Levels and Protein Abundance during Three Phases of Aestivation.

    PubMed

    Chng, You R; Ong, Jasmine L Y; Ching, Biyun; Chen, Xiu L; Hiong, Kum C; Wong, Wai P; Chew, Shit F; Lam, Siew H; Ip, Yuen K

    2016-01-01

    African lungfishes can undergo long periods of aestivation on land during drought. During aestivation, lungfishes are confronted with desiccation and dehydration, and their gills become non-functional and covered with a thick layer of dried mucus. Aquaporins (Aqps) are a superfamily of integral membrane proteins which generally facilitate the permeation of water through plasma membranes. This study aimed to obtain the complete cDNA coding sequences of aqp1 and aqp3 from the gills of Protopterus annectens, and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Dendrogramic analyses of the deduced Aqp1 and Aqp3 amino acid sequences of P. annectens revealed their close relationships with those of Latimeria chalumnae and tetrapods. During the induction phase, there were significant decreases in the transcript levels of aqp1 and aqp3 in the gills of P. annectens, but the branchial Aqp1 and Aqp3 protein abundance remained unchanged. As changes in transcription might precede changes in translation, this could be regarded as an adaptive response to decrease the protein abundance of Aqp1 and Aqp3 in the subsequent maintenance phase of aestivation. As expected, the branchial transcript levels and protein abundance of aqp1/Aqp1 and aqp3/Aqp3 were significantly down-regulated during the maintenance phase, probably attributable to the shutdown of branchial functions and the cessation of volume regulation of branchial epithelial cells. Additionally, these changes could reduce the loss of water through branchial epithelial surfaces, supplementing the anti-desiccating property of the dried mucus. Upon arousal, it was essential for the lungfish to restore branchial functions. Indeed, the protein abundance of Aqp1 recovered partially, with complete recovery of mRNA expression level and protein abundance of Aqp3, in the gills of P. annectens after 3 days of arousal. These results provide insights into

  18. A multi-omic analysis of an Enterococcus faecium mutant reveals specific genetic mutations and dramatic changes in mRNA and protein expression

    PubMed Central

    2013-01-01

    Background For a long time, Enterococcus faecium was considered a harmless commensal of the mammalian gastrointestinal (GI) tract and was used as a probiotic in fermented foods. In recent decades, E. faecium has been recognised as an opportunistic pathogen that causes diseases such as neonatal meningitis, urinary tract infections, bacteremia, bacterial endocarditis and diverticulitis. E. faecium could be taken into space with astronauts and exposed to the space environment. Thus, it is necessary to observe the phenotypic and molecular changes of E. faecium after spaceflight. Results An E. faecium mutant with biochemical features that are different from those of the wild-type strain was obtained from subculture after flight on the SHENZHOU-8 spacecraft. To understand the underlying mechanism causing these changes, the whole genomes of both the mutant and the WT strains were sequenced using Illumina technology. The genomic comparison revealed that dprA, a recombination-mediator gene, and arpU, a gene associated with cell wall growth, were mutated. Comparative transcriptomic and proteomic analyses showed that differentially expressed genes or proteins were involved with replication, recombination, repair, cell wall biogenesis, glycometabolism, lipid metabolism, amino acid metabolism, predicted general function and energy production/conversion. Conclusion This study analysed the comprehensive genomic, transcriptomic and proteomic changes of an E. faecium mutant from subcultures that were loaded on the SHENZHOU-8 spacecraft. The implications of these gene mutations and expression changes and their underlying mechanisms should be investigated in the future. We hope that the current exploration of multiple “-omics” analyses of this E. faecium mutant will provide clues for future studies on this opportunistic pathogen. PMID:24373636

  19. GLUT3 protein and mRNA in autopsy muscle specimens

    NASA Technical Reports Server (NTRS)

    Stuart, C. A.; Wen, G.; Jiang, J.

    1999-01-01

    GLUT3 is expressed in rat muscle, but this glucose transporter protein has not been identified previously in adult human skeletal muscle. We quantified the rapidity of disappearance of mRNA and protein from human skeletal muscle at room temperature and at 4 degrees C. Fifty percent of the immunologically detectable GLUT3 protein disappeared by 1 hour at 20 degrees C and by 2 hours at 4 degrees C. mRNA for GLUT3 was decreased 50% by 2.2 hours at 20 degrees C and by 24 hours at 4 degrees C. Half of the measurable mRNAs for GLUT4, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), alpha-actin, and beta-myosin disappeared by 0.8 to 2.1 hours at 20 degrees C and by 5.0 to 16.6 hours at 4 degrees C. Previous conclusions that GLUT3 is not expressed in human muscle were likely drawn because of artifacts related to degradation of GLUT3 protein in the specimens prior to study. Because of the rapid degradation of protein and mRNA, autopsy specimens of muscle must be obtained within 6 hours of death, and even then, protein and mRNA data will likely dramatically underestimate their expression in fresh muscle. Some previously published conclusions and recommendations regarding autopsy specimens are not stringent enough to consistently yield useful protein and mRNA.

  20. GLUT3 protein and mRNA in autopsy muscle specimens

    NASA Technical Reports Server (NTRS)

    Stuart, C. A.; Wen, G.; Jiang, J.

    1999-01-01

    GLUT3 is expressed in rat muscle, but this glucose transporter protein has not been identified previously in adult human skeletal muscle. We quantified the rapidity of disappearance of mRNA and protein from human skeletal muscle at room temperature and at 4 degrees C. Fifty percent of the immunologically detectable GLUT3 protein disappeared by 1 hour at 20 degrees C and by 2 hours at 4 degrees C. mRNA for GLUT3 was decreased 50% by 2.2 hours at 20 degrees C and by 24 hours at 4 degrees C. Half of the measurable mRNAs for GLUT4, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), alpha-actin, and beta-myosin disappeared by 0.8 to 2.1 hours at 20 degrees C and by 5.0 to 16.6 hours at 4 degrees C. Previous conclusions that GLUT3 is not expressed in human muscle were likely drawn because of artifacts related to degradation of GLUT3 protein in the specimens prior to study. Because of the rapid degradation of protein and mRNA, autopsy specimens of muscle must be obtained within 6 hours of death, and even then, protein and mRNA data will likely dramatically underestimate their expression in fresh muscle. Some previously published conclusions and recommendations regarding autopsy specimens are not stringent enough to consistently yield useful protein and mRNA.

  1. RRM1, TUBB3, TOP2A, CYP19A1, CYP2D6: Difference between mRNA and protein expression in predicting prognosis of breast cancer patients.

    PubMed

    Xu, Ying-Chun; Zhang, Feng-Chun; Li, Jun-Jian; Dai, Jia-Qi; Liu, Qiang; Tang, Lei; Ma, Yue; Xu, Qi; Lin, Xiao-Lin; Fan, Hong-Bin; Wang, Hong-Xia

    2015-10-01

    The study investigated the clinical significance of RRM1 (ribonucleoside reductase subunit M1), TUBB3 (tubulin-β-III), TOP2A (DNA topoisomerase II), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1) and CYP2D6 (cytochrome P450, family 2, subfamily D, polypeptide 6) for the diagnosis and possible predictive roles in breast cancer. Tissue microarray detected the expression of RRM1, tubulin-β-III, Topo IIα, CYP19A1 and CYP2D6 protein in breast cancer tissue and tissue adjacent to tumors (TATs). In addition, a publically available tool, was used to assess the prognostic value of their gene expression in breast cancer (http://kmplot.com). Analysis for relapse-free survival (RFS), disease-free survival (DFS) and overall survival (OS) was performed. Cytoplasmic RRM1, tubulin-β-III, CYP19A1 and Topo IIα staining were significantly higher in breast cancer tissues compared with TATs (P<0.050). Significant correlation occurred between RRM1 expression with pathological classification (P=0.018), lymph node involvement (P=0.035) and ER status (P=0.003). Tubulin-β-III and CYP2D6 expression correlated significantly with tumor grade (P=0.021 for tubulin-β-III and P=0.029 for CYP2D6, respectively). Cox analysis showed that the protein expression of CYP2D6, CYP19A1, RRM1, Topo IIα or tubulin-β-III was not an independent prognostic factor. A significant association occurred between RFS and TUBB3, TOP2A, CYP19A1, and CYP2D6 mRNA expression. With CYP19A1 (P<0.001) and CYP2D6 (P<0.001), a high expression was associated with good clinical outcome. Conversely, a low expression of TUBB3 (P<0.001) and TOP2A (P<0.001) was associated with good clinical outcome. TUBB3 (P=0.0004) and TOP2A (P<0.001) were significant prognostic factors in predicting the patient OS. The expression of RRM1, tubulin-β-III, Topo IIα and CYP19A1 in tumor tissues was significantly higher than that in TATs. TUBB3, TOP2A, CYP19A1 and CYP2D6 gene expression, but not protein expression, was

  2. In utero and postnatal exposure to a high-protein or high-carbohydrate diet leads to differences in adipose tissue mRNA expression and blood metabolites in kittens.

    PubMed

    Vester, Brittany M; Liu, Kari J; Keel, Tonya L; Graves, Thomas K; Swanson, Kelly S

    2009-10-01

    The objective of the present study was to measure the differences in body composition, adipose tissue gene expression, blood metabolite and hormone concentrations, and insulin sensitivity in kittens exposed to high-protein (HP) or high-carbohydrate (HC) nutrition in utero and through the growth period. Eight dams were randomised onto two test diets, and fed the diets throughout gestation and lactation. Male offspring were evaluated for 9 months. Kittens were weaned at 2 months of age onto the same treatment diet as the dam and were allowed to consume diets ad libitum. The HC diet contained 34.3 % crude protein (CP), 19.2 % fat and 30.8 % digestible carbohydrate, while the HP diet contained 52.9 % CP, 23.5 % fat and 10.8 % digestible carbohydrate. Blood samples were collected at 6 months after birth. Body composition was determined at 2 and 8 months of age and an intravenous glucose tolerance test, neutering and adipose tissue biopsy conducted at 8 months of age. Physical activity was quantified at 6 and 9 months. Energy intake, DM intake and body weight were not different between groups. At 2 months, blood TAG were greater (P < 0.05) in kittens fed the HP diet. At 8 months, blood leptin was higher (P < 0.05) in kittens fed the HC diet, while chemokine receptor 5, hormone-sensitive lipase, uncoupling protein 2, leptin and insulin receptor mRNA were greater (P < 0.05) in kittens fed the HP diet. The present results demonstrate some of the changes in blood metabolites and hormones, physical activity and mRNA abundance that occur with feeding high protein levels to kittens.

  3. Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism

    PubMed Central

    Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K.; Lehtonen, Jukka Y.A.

    2016-01-01

    As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3′-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. PMID:26681690

  4. Histone gene expression and histone mRNA 3' end structure in Caenorhabditis elegans.

    PubMed

    Keall, Rebecca; Whitelaw, Sandra; Pettitt, Jonathan; Müller, Berndt

    2007-06-14

    Histone protein synthesis is essential for cell proliferation and required for the packaging of DNA into chromatin. In animals, histone proteins are provided by the expression of multicopy replication-dependent histone genes. Histone mRNAs that are processed by a histone-specific mechanism to end after a highly conserved RNA hairpin element, and lack a poly(A) tail. In vertebrates and Drosophila, their expression is dependent on HBP/SLBP that binds to the RNA hairpin element. We showed previously that these cis and trans acting regulators of histone gene expression are conserved in C. elegans. Here we report the results of an investigation of the histone mRNA 3' end structure and of histone gene expression during C. elegans development. Sequence analysis of replication-dependent histone genes revealed the presence of several highly conserved sequence elements in the 3' untranslated region of histone pre-mRNAs, including an RNA hairpin element and a polyadenylation signal. To determine whether in C. elegans histone mRNA 3' end formation occurs at this polyadenylation signal and results in polyadenylated histone mRNA, we investigated the mRNA 3' end structure of histone mRNA. Using poly(A) selection, RNAse protection and sequencing of histone mRNA ends, we determined that a majority of C. elegans histone mRNAs lack a poly(A) tail and end three to six nucleotides after the hairpin structure, after an A or a U, and have a 3' OH group. RNAi knock down of CDL-1, the C. elegans HBP/SLBP, does not significantly affect histone mRNA levels but severely depletes histone protein levels. Histone gene expression varies during development and is reduced in L3 animals compared to L1 animals and adults. In adults, histone gene expression is restricted to the germ line, where cell division occurs. Our findings indicate that the expression of C. elegans histone genes is subject to control mechanisms similar to the ones in other animals: the structure of C. elegans histone mRNA 3

  5. RNA-Binding Proteins Revisited - The Emerging Arabidopsis mRNA Interactome.

    PubMed

    Köster, Tino; Marondedze, Claudius; Meyer, Katja; Staiger, Dorothee

    2017-04-12

    RNA-protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture - where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

  6. Expression and clinicopathological significance of Mel-18 and Bmi-1 mRNA in gastric carcinoma.

    PubMed

    Lu, You-Wei; Li, Jin; Guo, Wei-Jian

    2010-11-08

    The Polycomb group (PcG) genes are a class of regulators responsible for maintaining homeotic gene expression throughout cell division. PcG expression is deregulated in some types of human cancer. Both Bmi-1 and Mel-18 are of the key PcG proteins. We investigate the expression and clinicopathological roles of Mel-18 and Bmi-1 mRNA in gastric cancer. The expression of Mel-18 and Bmi-1 in a series of 71 gastric cancer tissues and paired normal mucosal tissues distant from the tumorous lesion was assayed by quantitative real time RT-PCR. The correlation between Mel-18 and Bmi-1 mRNA expression, and between Mel-18 or Bmi-1 mRNA level and clinicopathological characteristics were analyzed. Expression of Mel-18 and Bmi-1 genes was variably detected, but overexpression of Bmi-1 mRNA and decreased expression of Mel-18 mRNA were the most frequent alteration. In addition, the expression of Bmi-1 and Mel-18 mRNA inversely correlates in gastric tumors. Moreover, a significant positive correlation between Bmi-1 overexpression and tumor size, depth of invasion, or lymph node metastasis, and a significant negative correlation between Mel-18 low-expression with lymph node metastasis or the clinical stage were observed. Our data suggest that Mel-18 and Bmi-1 may play crucial but opposite roles in gastric cancer. Decreased Mel-18 and increased Bmi-1 mRNA expression was associated with the carcinogenesis and progression of gastric cancer. It is possible to list Bmi-1 and Mel-18 as biomarkers for predicting the prognosis of gastric cancer.

  7. Expression and clinicopathological significance of Mel-18 and Bmi-1 mRNA in gastric carcinoma

    PubMed Central

    2010-01-01

    Background The Polycomb group (PcG) genes are a class of regulators responsible for maintaining homeotic gene expression throughout cell division. PcG expression is deregulated in some types of human cancer. Both Bmi-1 and Mel-18 are of the key PcG proteins. We investigate the expression and clinicopathological roles of Mel-18 and Bmi-1 mRNA in gastric cancer. Methods The expression of Mel-18 and Bmi-1 in a series of 71 gastric cancer tissues and paired normal mucosal tissues distant from the tumorous lesion was assayed by quantitative real time RT-PCR. The correlation between Mel-18 and Bmi-1 mRNA expression, and between Mel-18 or Bmi-1 mRNA level and clinicopathological characteristics were analyzed. Results Expression of Mel-18 and Bmi-1 genes was variably detected, but overexpression of Bmi-1 mRNA and decreased expression of Mel-18 mRNA were the most frequent alteration. In addition, the expression of Bmi-1 and Mel-18 mRNA inversely correlates in gastric tumors. Moreover, a significant positive correlation between Bmi-1 overexpression and tumor size, depth of invasion, or lymph node metastasis, and a significant negative correlation between Mel-18 low-expression with lymph node metastasis or the clinical stage were observed. Conclusion Our data suggest that Mel-18 and Bmi-1 may play crucial but opposite roles in gastric cancer. Decreased Mel-18 and increased Bmi-1 mRNA expression was associated with the carcinogenesis and progression of gastric cancer. It is possible to list Bmi-1 and Mel-18 as biomarkers for predicting the prognosis of gastric cancer. PMID:21059209

  8. Jueming Prescription reduces body weight by increasing the mRNA expressions of beta3-adrenergic receptor and uncoupling protein-2 in adipose tissue of diet-induced obese rats.

    PubMed

    Yang, Ling; Lu, Kun; Wen, Xiu-ying; Liu, Hao; Chen, Ai-ping; Xu, Ming-wang; Zhang, Hong; Yu, Jie

    2012-10-01

    To investigate the antiobesity effect of Jueming Prescription (JMP), a Chinese herbal medicine formula, and its influence on mRNA expressions of beta3 adrenergic receptor (beta3-AR) and uncoupling protein-2 (UCP-2) in adipose tissue of diet-induced obese rats. Fifty male Sprague-Dawley rats were randomly divided into the normal control group (n =8) that was on a standard chow diet, and the obese model group (n =42) that was on a diet of high fat chow. Two weeks after the high fat diet, 29 obese rats in the obese model group were further randomly divided into 3 groups: the untreated obese model group (n =9), the metformin group (n =10, metformin 300 mg kg⁻¹ day)⁻¹, and the JMP group (n =10, JMP 4 g kg⁻¹ day⁻¹). After 8-week treatment, body weight, wet weight of visceral fat, and percentage of body fat (PBF) were measured. The levels of fasting blood glucose, serum lipids, and insulin were assessed, and insulin sensitivity index (ISI) was calculated. The adipose tissue section was stained with hematoxylin-Eosin, and the cellular diameter and quantity of adipocytes were evaluated by light microscopy. The mRNA expressions of beta3-AR and UCP-2 from the peri-renal fat tissue were determined by real-time reverse transcription polymerase chain reaction (RT-PCR). Compared with the obese model group, treatment with JMP resulted in significantly lower body weight, wet weight of visceral fat, PBF, and diameter of adipocytes, and significantly higher level of high-density lipoprotein cholesterol, ISI (all P<0.01), JMP increased the mRNA expressions of beta3-AR and UCP-2 from perirenal fat tissue (P <0.05, P<0.01). JMP could reduce body weight and adipocyte size; and the effect was associated with the up-regulation of beta3-AR and UCP-2 expressions in the adipose tissue and improvement of insulin sensitivity.

  9. Huntington's Disease Protein Huntingtin Associates with its own mRNA.

    PubMed

    Culver, Brady P; DeClercq, Josh; Dolgalev, Igor; Yu, Man Shan; Ma, Bin; Heguy, Adriana; Tanese, Naoko

    2016-01-01

    The Huntington's disease (HD) protein huntingtin (Htt) plays a role in multiple cellular pathways. Deregulation of one or more of these pathways by the mutant Htt protein has been suggested to contribute to the disease pathogenesis. Our recent discovery-based proteomics studies have uncovered RNA binding proteins and translation factors associated with the endogenous Htt protein purified from mouse brains, suggesting a potential new role for Htt in RNA transport and translation. To investigate how Htt might affect RNA metabolism we set out to purify and analyze RNA associated with Htt. RNA was extracted from immunopurified Htt-containing protein complexes and analyzed by microarrays and RNA-Seq. Surprisingly, the most enriched mRNA that co-purified with Htt was Htt mRNA itself. The association of Htt protein and Htt mRNA was detected independent of intact ribosomes suggesting that it is not an RNA undergoing translation. Furthermore, we identified the recently reported mis-spliced Htt mRNA encoding a truncated protein comprised of exon 1 and a portion of the downstream intron in the immunoprecipitates containing mutant Htt protein. We show that Htt protein co-localizes with Htt mRNA and that wild-type Htt reduces expression of a reporter construct harboring the Htt 3' UTR. HD protein is found in a complex with its own mRNA and RNA binding proteins and translation factors. Htt may be involved in modulating its expression through post-transcriptional pathways. It is possible that Htt shares mechanistic properties similar to RNA binding proteins such as TDP-43 and FUS implicated in other neurodegenerative diseases.

  10. Huntington’s Disease Protein Huntingtin Associates with its own mRNA

    PubMed Central

    Culver, Brady P.; DeClercq, Josh; Dolgalev, Igor; Yu, Man Shan; Ma, Bin; Heguy, Adriana; Tanese, Naoko

    2016-01-01

    Background: The Huntington’s disease (HD) protein huntingtin (Htt) plays a role in multiple cellular pathways. Deregulation of one or more of these pathways by the mutant Htt protein has been suggested to contribute to the disease pathogenesis. Our recent discovery-based proteomics studies have uncovered RNA binding proteins and translation factors associated with the endogenous Htt protein purified from mouse brains, suggesting a potential new role for Htt in RNA transport and translation. Objective: To investigate how Htt might affect RNA metabolism we set out to purify and analyze RNA associated with Htt. Methods: RNA was extracted from immunopurified Htt-containing protein complexes and analyzed by microarrays and RNA-Seq. Results: Surprisingly, the most enriched mRNA that co-purified with Htt was Htt mRNA itself. The association of Htt protein and Htt mRNA was detected independent of intact ribosomes suggesting that it is not an RNA undergoing translation. Furthermore, we identified the recently reported mis-spliced Htt mRNA encoding a truncated protein comprised of exon 1 and a portion of the downstream intron in the immunoprecipitates containing mutant Htt protein. We show that Htt protein co-localizes with Htt mRNA and that wild-type Htt reduces expression of a reporter construct harboring the Htt 3’ UTR. Conclusions: HD protein is found in a complex with its own mRNA and RNA binding proteins and translation factors. Htt may be involved in modulating its expression through post-transcriptional pathways. It is possible that Htt shares mechanistic properties similar to RNA binding proteins such as TDP-43 and FUS implicated in other neurodegenerative diseases. PMID:26891106

  11. Molecular characterization of myostatin from the skeletal muscle of the African lungfish, Protopterus annectens, and changes in its mRNA and protein expression levels during three phases of aestivation.

    PubMed

    Ong, Jasmine L Y; Chng, You R; Ching, Biyun; Chen, Xiu L; Hiong, Kum C; Wong, Wai P; Chew, Shit F; Ip, Yuen K

    2017-05-01

    African lungfishes can aestivate and remain torpid without food and water for years, but disuse muscle atrophy is not prominent during aestivation. This study aimed to clone myostatin (mstn/Mstn), a factor associated with disuse muscle atrophy in mammals, from the skeletal muscle of the African lungfish Protopterus annectens, and to determine its mRNA expression level and protein abundance therein during the induction, maintenance, and arousal phases of aestivation. The complete coding cDNA sequence of mstn comprised 1128 bp, encoding for 376 amino acids with an estimated molecular mass of 42.9 kDa. It was grouped together with Mstn/MSTN of coelacanth and tetrapods in a clade separated from teleost Mstn. After 6 days (the induction phase) of aestivation, the mstn transcript level in the muscle increased significantly, while the protein abundance of Mstn remained comparable to the control. Following that, a significant increase in the expression levels of mstn/Mstn occurred on day 12 (the early maintenance phase) of aestivation. After 6 months of aestivation (the prolonged maintenance phase), the expression levels of mstn/Mstn returned to control levels, indicating the possible impediment of a drastic increase in muscle degradation to prevent muscle atrophy. During 1-3 days of arousal from aestivation, the expression levels of mstn/Mstn in the muscle remained comparable to the control. Hence, tissue reconstruction/regeneration of certain organs might not involve the mobilization of amino acids from the muscle during the early arousal. These results provide insights into how aestivating P. annectens regulates the expression of mstn/Mstn possibly to ameliorate disuse muscle atrophy.

  12. The ticking tail: daily oscillations in mRNA poly(A) tail length drive circadian cycles in protein synthesis.

    PubMed

    Gotic, Ivana; Schibler, Ueli

    2012-12-15

    In this issue of Genes & Development, Kojima and colleagues (pp. 2724-2736) examined the impact of mRNA poly(A) tail length on circadian gene expression. Their study demonstrates how dynamic changes in transcript poly(A) tail length can lead to rhythmic protein expression, irrespective of whether mRNA accumulation is circadian or constitutive.

  13. Differential protein occupancy profiling of the mRNA transcriptome

    PubMed Central

    2014-01-01

    Background RNA-binding proteins (RBPs) mediate mRNA biogenesis, translation and decay. We recently developed an approach to profile transcriptome-wide RBP contacts on polyadenylated transcripts by next-generation sequencing. A comparison of such profiles from different biological conditions has the power to unravel dynamic changes in protein-contacted cis-regulatory mRNA regions without a priori knowledge of the regulatory protein component. Results We compared protein occupancy profiles of polyadenylated transcripts in MCF7 and HEK293 cells. Briefly, we developed a bioinformatics workflow to identify differential crosslinking sites in cDNA reads of 4-thiouridine crosslinked polyadenylated RNA samples. We identified 30,000 differential crosslinking sites between MCF7 and HEK293 cells at an estimated false discovery rate of 10%. 73% of all reported differential protein-RNA contact sites cannot be explained by local changes in exon usage as indicated by complementary RNA-seq data. The majority of differentially crosslinked positions are located in 3′ UTRs, show distinct secondary-structure characteristics and overlap with binding sites of known RBPs, such as ELAVL1. Importantly, mRNA transcripts with the most significant occupancy changes show elongated mRNA half-lives in MCF7 cells. Conclusions We present a global comparison of protein occupancy profiles from different cell types, and provide evidence for altered mRNA metabolism as a result of differential protein-RNA contacts. Additionally, we introduce POPPI, a bioinformatics workflow for the analysis of protein occupancy profiling experiments. Our work demonstrates the value of protein occupancy profiling for assessing cis-regulatory RNA sequence space and its dynamics in growth, development and disease. PMID:24417896

  14. Tia1 dependent regulation of mRNA subcellular location and translation controls p53 expression in B cells.

    PubMed

    Díaz-Muñoz, Manuel D; Kiselev, Vladimir Yu; Novère, Nicolas Le; Curk, Tomaz; Ule, Jernej; Turner, Martin

    2017-09-13

    Post-transcriptional regulation of cellular mRNA is essential for protein synthesis. Here we describe the importance of mRNA translational repression and mRNA subcellular location for protein expression during B lymphocyte activation and the DNA damage response. Cytoplasmic RNA granules are formed upon cell activation with mitogens, including stress granules that contain the RNA binding protein Tia1. Tia1 binds to a subset of transcripts involved in cell stress, including p53 mRNA, and controls translational silencing and RNA granule localization. DNA damage promotes mRNA relocation and translation in part due to dissociation of Tia1 from its mRNA targets. Upon DNA damage, p53 mRNA is released from stress granules and associates with polyribosomes to increase protein synthesis in a CAP-independent manner. Global analysis of cellular mRNA abundance and translation indicates that this is an extended ATM-dependent mechanism to increase protein expression of key modulators of the DNA damage response.Sequestering mRNA in cytoplasmic stress granules is a mechanism for translational repression. Here the authors find that p53 mRNA, present in stress granules in activated B lymphocytes, is released upon DNA damage and is translated in a CAP-independent manner.

  15. Amount and source of dietary copper affects small intestine morphology, duodenal lipid peroxidation, hepatic oxidative stress,and mRNA expression of hepatic copper regulatory proteins in weanling pigs.

    PubMed

    Fry, R S; Ashwell, M S; Lloyd, K E; O'Nan, A T; Flowers, W L; Stewart, K R; Spears, J W

    2012-09-01

    Thirty weanling, crossbred barrows (SUS SCROFA) were used to determine the effects of amount and source of dietary Cu on small intestinal morphology and lipid peroxidation, Cu metabolism, and mRNA expression of proteins involved in hepatic Cu homeostasis. At 21 d of age, pigs were stratified by BW (6.33 ± 0.23 kg) and allocated to 1 of the following dietary treatments: i) control (no supplemental Cu; 6.7 mg Cu/kg), ii) 225 mg supplemental Cu/kg diet from Cu sulfate (CuSO(4)), or iii) 225 mg supplemental Cu/kg diet from tribasic Cu chloride (TBCC). Pigs were housed 2 pigs per pen and were fed a 3-phase diet regimen until d 35 or 36 of the study. During harvest, bile and liver were obtained for mineral analysis, and liver samples were also obtained for analysis of liver glutathione (GSH) and mRNA expression of Cu regulatory proteins. Segments of duodenum, proximal jejunum, and ileum were obtained for mucosal morphology, and duodenal mucosal scrapings were collected from all pigs for analysis of malondialdehyde (MDA). Duodenal villus height was reduced in CuSO(4) pigs compared with control (P = 0.001) and TBCC (P = 0.03) pigs. Villus height in the proximal jejunum of CuSO(4) pigs was reduced (P = 0.03) compared with control pigs, but ileal villus height was not affected (P = 0.82) by treatment. Duodenal MDA concentrations were greater (P = 0.03) in CuSO(4) pigs and tended to be greater (P = 0.10) in pigs supplemented with TBCC compared with control pigs. Liver Cu was greater (P = 0.01) in CuSO(4) vs. control pigs, and tended (P = 0.07) to be greater in TBCC pigs than control pigs. Bile Cu concentrations were greater (P < 0.001) in CuSO(4) and TBCC pigs vs. controls and were also greater (P = 0.04) in TBCC vs. CuSO(4) pigs. Total liver GSH concentrations were less (P = 0.02) in pigs fed diets supplemented with CuSO(4) vs. pigs fed control diets but total liver GSH did not differ (P = 0.11) between control and TBCC pigs. Hepatic mRNA of cytochrome c oxidase assembly

  16. A novel cis-acting element from the 3'UTR of DNA damage-binding protein 2 mRNA links transcriptional and post-transcriptional regulation of gene expression.

    PubMed

    Melanson, Brian D; Cabrita, Miguel A; Bose, Reetesh; Hamill, Jeffrey D; Pan, Elysia; Brochu, Christian; Marcellus, Kristen A; Zhao, Tong T; Holcik, Martin; McKay, Bruce C

    2013-06-01

    The DNA damage-binding protein 2 (DDB2) is an adapter protein that can direct a modular Cul4-DDB1-RING E3 Ligase complex to sites of ultraviolet light-induced DNA damage to ubiquitinate substrates during nucleotide excision repair. The DDB2 transcript is ultraviolet-inducible; therefore, its regulation is likely important for its function. Curiously, the DDB2 mRNA is reportedly short-lived, but the transcript does not contain any previously characterized cis-acting determinants of mRNA stability in its 3' untranslated region (3'UTR). Here, we used a tetracycline regulated d2EGFP reporter construct containing specific 3'UTR sequences from DDB2 to identify novel cis-acting elements that regulate mRNA stability. Synthetic 3'UTRs corresponding to sequences as short as 25 nucleotides from the central region of the 3'UTR of DDB2 were sufficient to accelerate decay of the heterologous reporter mRNA. Conversely, these same 3'UTRs led to more rapid induction of the reporter mRNA, export of the message to the cytoplasm and the subsequent accumulation of the encoded reporter protein, indicating that this newly identified cis-acting element affects transcriptional and post-transciptional processes. These results provide clear evidence that nuclear and cytoplasmic processing of the DDB2 mRNA is inextricably linked.

  17. A novel cis-acting element from the 3′UTR of DNA damage-binding protein 2 mRNA links transcriptional and post-transcriptional regulation of gene expression

    PubMed Central

    Melanson, Brian D.; Cabrita, Miguel A.; Bose, Reetesh; Hamill, Jeffrey D.; Pan, Elysia; Brochu, Christian; Marcellus, Kristen A.; Zhao, Tong T.; Holcik, Martin; McKay, Bruce C.

    2013-01-01

    The DNA damage-binding protein 2 (DDB2) is an adapter protein that can direct a modular Cul4-DDB1-RING E3 Ligase complex to sites of ultraviolet light-induced DNA damage to ubiquitinate substrates during nucleotide excision repair. The DDB2 transcript is ultraviolet-inducible; therefore, its regulation is likely important for its function. Curiously, the DDB2 mRNA is reportedly short-lived, but the transcript does not contain any previously characterized cis-acting determinants of mRNA stability in its 3′ untranslated region (3′UTR). Here, we used a tetracycline regulated d2EGFP reporter construct containing specific 3′UTR sequences from DDB2 to identify novel cis-acting elements that regulate mRNA stability. Synthetic 3′UTRs corresponding to sequences as short as 25 nucleotides from the central region of the 3′UTR of DDB2 were sufficient to accelerate decay of the heterologous reporter mRNA. Conversely, these same 3′UTRs led to more rapid induction of the reporter mRNA, export of the message to the cytoplasm and the subsequent accumulation of the encoded reporter protein, indicating that this newly identified cis-acting element affects transcriptional and post-transciptional processes. These results provide clear evidence that nuclear and cytoplasmic processing of the DDB2 mRNA is inextricably linked. PMID:23605047

  18. Transcriptional bursting explains the noise–versus–mean relationship in mRNA and protein levels

    SciTech Connect

    Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai; Razooky, Brandon S.; Simpson, Michael L.; Raj, Arjun; Weinberger, Leor S.

    2016-07-28

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: that increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.

  19. Transcriptional bursting explains the noise–versus–mean relationship in mRNA and protein levels

    DOE PAGES

    Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai; ...

    2016-07-28

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

  20. Developmental expression of parvalbumin mRNA in the cerebral cortex and hippocampus of the rat.

    PubMed

    de Lecea, L; del Río, J A; Soriano, E

    1995-08-01

    Parvalbumin (PARV) belongs to the family of calcium-binding proteins bearing the EF hand domain. Immunocytochemical studies in the cerebral cortex have demonstrated that neurons containing PARV include two types of GABAergic interneurons, namely, basket and axo-axonic chandelier cells. The present study examines the onset and pattern of PARV mRNA expression during the development of rat neocortex and hippocampus by means of 'in situ' hybridization with an oligonucleotide probe corresponding to rat PARV cDNA. In animals aged P0-P6 no signal was detected above background in neocortex or hippocampus. At P8, a few cortical cells displayed a number of silver grains just above background levels. By P10 PARV mRNA-expressing cells in the neocortex were detected almost exclusively in layer V of somatosensory, frontal and cingulate cortices. At P12 PARV mRNA was mainly detected in layers IV, V and VIa. By P14 there was a marked overall increase in the entire neocortex, including layer II-III, both in the number of cells and in their intensity of labelling. Further maturation in the pattern of PARV mRNA concentration was observed between P16 and P21. In the hippocampus low hybridization was observed at P10-P12. In subsequent stages both the number of positive cells and the intensity of labelling increased steadily. No clear-cut radial gradients for the expression of PARV mRNA were observed in the hippocampal region. Our results show that the developmental radial gradient followed by PARV mRNA expression in the neocortex does not follow an 'inside-out' gradient, consistent with previous immunocytochemical findings. Taken together, these data indicate that the developmental sequence followed by the PARV protein directly reflects mRNA abundance and suggest that PARV mRNA expression correlates with the functional maturation of cortical interneurons.

  1. Protein functional features are reflected in the patterns of mRNA translation speed.

    PubMed

    López, Daniel; Pazos, Florencio

    2015-07-09

    The degeneracy of the genetic code makes it possible for the same amino acid string to be coded by different messenger RNA (mRNA) sequences. These "synonymous mRNAs" may differ largely in a number of aspects related to their overall translational efficiency, such as secondary structure content and availability of the encoded transfer RNAs (tRNAs). Consequently, they may render different yields of the translated polypeptides. These mRNA features related to translation efficiency are also playing a role locally, resulting in a non-uniform translation speed along the mRNA, which has been previously related to some protein structural features and also used to explain some dramatic effects of "silent" single-nucleotide-polymorphisms (SNPs). In this work we perform the first large scale analysis of the relationship between three experimental proxies of mRNA local translation efficiency and the local features of the corresponding encoded proteins. We found that a number of protein functional and structural features are reflected in the patterns of ribosome occupancy, secondary structure and tRNA availability along the mRNA. One or more of these proxies of translation speed have distinctive patterns around the mRNA regions coding for certain protein local features. In some cases the three patterns follow a similar trend. We also show specific examples where these patterns of translation speed point to the protein's important structural and functional features. This support the idea that the genome not only codes the protein functional features as sequences of amino acids, but also as subtle patterns of mRNA properties which, probably through local effects on the translation speed, have some consequence on the final polypeptide. These results open the possibility of predicting a protein's functional regions based on a single genomic sequence, and have implications for heterologous protein expression and fine-tuning protein function.

  2. Tualang Honey Protects against BPA-Induced Morphological Abnormalities and Disruption of ERα, ERβ, and C3 mRNA and Protein Expressions in the Uterus of Rats

    PubMed Central

    Mohamad Zaid, Siti Sarah; Kassim, Normadiah M.; Othman, Shatrah

    2015-01-01

    Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) that can disrupt the normal functions of the reproductive system. The objective of the study is to investigate the potential protective effects of Tualang honey against BPA-induced uterine toxicity in pubertal rats. The rats were administered with BPA by oral gavage over a period of six weeks. Uterine toxicity in BPA-exposed rats was determined by the degree of the morphological abnormalities, increased lipid peroxidation, and dysregulated expression and distribution of ERα, ERβ, and C3 as compared to the control rats. Concurrent treatment of rats with BPA and Tualang honey significantly improved the uterine morphological abnormalities, reduced lipid peroxidation, and normalized ERα, ERβ, and C3 expressions and distribution. There were no abnormal changes observed in rats treated with Tualang honey alone, comparable with the control rats. In conclusion, Tualang honey has potential roles in protecting the uterus from BPA-induced toxicity, possibly accounted for by its phytochemical properties. PMID:26788107

  3. Vibrational force alters mRNA expression in osteoblasts.

    PubMed

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

    1997-05-01

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

  4. 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.

  5. 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.

  6. Robust changes in expression of brain-derived neurotrophic factor (BDNF) mRNA and protein across the brain do not translate to detectable changes in BDNF levels in CSF or plasma.

    PubMed

    Lanz, Thomas A; Bove, Susan E; Pilsmaker, Catherine D; Mariga, Abigail; Drummond, Elena M; Cadelina, Gregory W; Adamowicz, Wendy O; Swetter, Brentt J; Carmel, Sharon; Dumin, Jo Ann; Kleiman, Robin J

    2012-09-01

    Adult rats were treated acutely with peripheral kainic acid (KA), and changes in brain-derived neurotrophic factor (BDNF) mRNA and protein were tracked over time across multiple brain regions. Despite robust elevation in both mRNA and protein in multiple brain regions, plasma BDNF was unchanged and cerebrospinal fluid (CSF) BDNF levels remained undetectable. Primary neurons were then treated with KA. BDNF was similarly elevated within neurons, but was undetectable in neuronal media. Thus, while deficits in BDNF signaling have been implicated in a number of diseases, these data suggest that extracellular concentrations of BDNF may not be a facile biomarker for changes in neurons.

  7. Molecular mechanism of teratogenic effects induced by the fungicide triadimefon: Study of the expression of TGF-{beta} mRNA and TGF-{beta} and CRABPI proteins during rat in vitro development

    SciTech Connect

    Di Renzo, F.; Corsini, E.; Broccia, M.L.; Marinovich, M.; Galli, C.L.; Giavini, E.; Menegola, E.

    2009-01-01

    Azole derivatives are teratogenic in rats and mice in vitro and in vivo. The postulated mechanism for the dysmorphogenetic effects is the inhibition of retinoic acid (RA)-degrading enzyme CYP26. Azole-related abnormalities are confined to structures controlled by RA, especially the neural crest cells, hindbrain, cranial nerves, and craniofacial structures, through a complex signal cascade. The aim of this work is to study the expression of signal molecules activated by RA (TGF-{beta}s) or involved in the modulation of cellular RA concentrations (CRABPI). E9.5 (9.5 day post coitum old embryos) rat embryos, exposed in vitro to triadimefon (FON) for 24 h, were examined or cultured in normal serum for extra 4, 16, and 24 h. RT-PCR was performed to quantify TGF-{beta}1, TGF-{beta}2, TGF-{beta}3, TGF-{beta}RI, TGF-{beta}RII, and TGF-{beta}RIII mRNA in the hindbrain after 24 h of culture. TGF-{beta}1, TGF-{beta}2, and TGF-{beta}RI were found significantly decreased by FON exposure, and consequently their protein expression was analyzed by Western blot and immunohistochemistry. In both controls and FON-exposed embryos, TGF-{beta}1 and TGF-{beta}RI were detected at 24 and 24 + 4 h; TGF-{beta}2 was present only at 24 h. Only TGF-{beta}1 was expressed at the level of hindbrain and branchial tissues. After quantization, TGF-{beta}1 was reduced in the FON group. The expression of CRABPI was observed at all developmental stages. However, in FON-exposed embryos, it was increased at 24 and 24 + 4 h. The hindbrain distribution of CRABPI-positive cells was abnormal in FON-exposed embryos. The results show that the two RA-related molecules (TGF-{beta}1 and CRABPI) are altered by FON exposure in vitro.

  8. Prolyl carboxypeptidase mRNA expression in the mouse brain.

    PubMed

    Jeong, Jin Kwon; Diano, Sabrina

    2014-01-13

    Prolyl carboxypeptidase (PRCP), a serine protease, is widely expressed in the body including liver, lung, kidney and brain, with a variety of known substrates such as plasma prekallikrein, bradykinin, angiotensins II and III, and α-MSH, suggesting its role in the processing of tissue-specific substrates. In the brain, PRCP has been shown to inactivate hypothalamic α-MSH, thus modulating melanocortin signaling in the control of energy metabolism. While its expression pattern has been reported in the hypothalamus, little is known on the distribution of PRCP throughout the mouse brain. This study was undertaken to determine PRCP expression in the mouse brain. Radioactive in situ hybridization was performed to determine endogenous PRCP mRNA expression. In addition, using a gene-trap mouse model for PRCP deletion, X-gal staining was performed to further determine PRCP distribution. Results from both approaches showed that PRCP gene is broadly expressed in the brain.

  9. The pentatricopeptide repeat MTSF1 protein stabilizes the nad4 mRNA in Arabidopsis mitochondria

    PubMed Central

    Haïli, Nawel; Arnal, Nadège; Quadrado, Martine; Amiar, Souad; Tcherkez, Guillaume; Dahan, Jennifer; Briozzo, Pierre; Colas des Francs-Small, Catherine; Vrielynck, Nathalie; Mireau, Hakim

    2013-01-01

    Gene expression in plant mitochondria involves a complex collaboration of transcription initiation and termination, as well as subsequent mRNA processing to produce mature mRNAs. In this study, we describe the function of the Arabidopsis mitochondrial stability factor 1 (MTSF1) gene and show that it encodes a pentatricopeptide repeat protein essential for the 3′-processing of mitochondrial nad4 mRNA and its stability. The nad4 mRNA is highly destabilized in Arabidopsis mtsf1 mutant plants, which consequently accumulates low amounts of a truncated form of respiratory complex I. Biochemical and genetic analyses demonstrated that MTSF1 binds with high affinity to the last 20 nucleotides of nad4 mRNA. Our data support a model for MTSF1 functioning in which its association with the last nucleotides of the nad4 3′ untranslated region stabilizes nad4 mRNA. Additionally, strict conservation of the MTSF1-binding sites strongly suggests that the protective function of MTSF1 on nad4 mRNA is conserved in dicots. These results demonstrate that the mRNA stabilization process initially identified in plastids, whereby proteins bound to RNA extremities constitute barriers to exoribonuclease progression occur in plant mitochondria to protect and concomitantly define the 3′ end of mature mitochondrial mRNAs. Our study also reveals that short RNA molecules corresponding to pentatricopeptide repeat-binding sites accumulate also in plant mitochondria. PMID:23658225

  10. Acute digoxin loading reduces ABCA8A mRNA expression in the mouse liver.

    PubMed

    Wakaumi, Michi; Ishibashi, Kenichi; Ando, Hitoshi; Kasanuki, Hiroshi; Tsuruoka, Shuichi

    2005-12-01

    Human ABCA8, a new member of the ATP binding cassette (ABC) transporter family, transports certain lipophilic drugs, such as digoxin. To investigate the roles of this transporter, we cloned a mouse homologue of ABCA8, from a mouse heart cDNA library, named ABCA8a. The deduced mouse ABCA8a protein is 66% identical with that of human ABCA8 and possesses features common to the ABC superfamily. It was found that ABCA8a was mainly expressed in the liver and heart, similar to human ABCA8. We further evaluated the effect of acute digoxin (a substrate for ABCA8) intoxication on the mRNA expression of ABCA8 using northern blotting with a 3' non-coding region as a probe to avoid cross-hybridization with other ABCA genes. Following acute digoxin infusion, the mRNA expression of ABCA8 was significantly reduced in the liver 12-24 h after injection (14.7% of vehicle treatment), but not in the heart and kidney. Real-time quantitative polymerase chain reaction analysis confirmed the reduction in ABCA8a mRNA. Similar reductions in ABCA5, ABCA7, ABCA8b and ABCA9 mRNA were also observed. A comparable amount of digitoxin did not affect ABCA8a mRNA expression in the liver. The results suggest that ABCA8 may play a role in digoxin metabolism in the liver.

  11. Expression of Npas4 mRNA in Telencephalic Areas of Adult and Postnatal Mouse Brain

    PubMed Central

    Damborsky, Joanne C.; Slaton, G. Simona; Winzer-Serhan, Ursula H.

    2015-01-01

    The transcription factor neuronal PAS domain-containing protein 4 (Npas4) is an inducible immediate early gene which regulates the formation of inhibitory synapses, and could have a significant regulatory role during cortical circuit formation. However, little is known about basal Npas4 mRNA expression during postnatal development. Here, postnatal and adult mouse brain sections were processed for isotopic in situ hybridization using an Npas4 specific cRNA antisense probe. In adults, Npas4 mRNA was found in the telencephalon with very restricted or no expression in diencephalon or mesencephalon. In most telencephalic areas, including the anterior olfactory nucleus (AON), piriform cortex, neocortex, hippocampus, dorsal caudate putamen (CPu), septum and basolateral amygdala nucleus (BLA), basal Npas4 expression was detected in scattered cells which exhibited strong hybridization signal. In embryonic and neonatal brain sections, Npas4 mRNA expression signals were very low. Starting at postnatal day 5 (P5), transcripts for Npas4 were detected in the AON, CPu and piriform cortex. At P8, additional Npas4 hybridization was found in CA1 and CA3 pyramidal layer, and in primary motor cortex. By P13, robust mRNA expression was located in layers IV and VI of all sensory cortices, frontal cortex and cingulate cortex. After onset of expression, postnatal spatial mRNA distribution was similar to that in adults, with the exception of the CPu, where Npas4 transcripts became gradually restricted to the most dorsal part. In conclusion, the spatial distribution of Npas4 mRNA is mostly restricted to telencephalic areas, and the temporal expression increases with developmental age during postnatal development, which seem to correlate with the onset of activity-driven excitatory transmission. PMID:26633966

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

    SciTech Connect

    Dalgaard, Louise T.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer UCP2 mRNA levels are decreased in islets of Langerhans from glucokinase deficient mice. Black-Right-Pointing-Pointer UCP2 mRNA up-regulation by glucose is dependent on glucokinase. Black-Right-Pointing-Pointer Absence of UCP2 increases GSIS of glucokinase heterozygous pancreatic islets. Black-Right-Pointing-Pointer This may protect glucokinase deficient mice from hyperglycemic damages. -- Abstract: Uncoupling Protein 2 (UCP2) is expressed in the pancreatic {beta}-cell, where it partially uncouples the mitochondrial proton gradient, decreasing both ATP-production and glucose-stimulated insulin secretion (GSIS). Increased glucose levels up-regulate UCP2 mRNA and protein levels, but the mechanism for UCP2 up-regulation in response to increased glucose is unknown. The aim was to examine the effects of glucokinase (GK) deficiency on UCP2 mRNA levels and to characterize the interaction between UCP2 and GK with regard to glucose-stimulated insulin secretion in pancreatic islets. UCP2 mRNA expression was reduced in GK+/- islets and GK heterozygosity prevented glucose-induced up-regulation of islet UCP2 mRNA. In contrast to UCP2 protein function UCP2 mRNA regulation was not dependent on superoxide generation, but rather on products of glucose metabolism, because MnTBAP, a superoxide dismutase mimetic, did not prevent the glucose-induced up-regulation of UCP2. Glucose-stimulated insulin secretion was increased in UCP2-/- and GK+/- islets compared with GK+/- islets and UCP2 deficiency improved glucose tolerance of GK+/- mice. Accordingly, UCP2 deficiency increased ATP-levels of GK+/- mice. Thus, the compensatory down-regulation of UCP2 is involved in preserving the insulin secretory capacity of GK mutant mice and might also be implicated in limiting disease progression in MODY2 patients.

  13. Tissue-specific mRNA expression profiling in grape berry tissues

    PubMed Central

    Grimplet, Jerome; Deluc, Laurent G; Tillett, Richard L; Wheatley, Matthew D; Schlauch, Karen A; Cramer, Grant R; Cushman, John C

    2007-01-01

    transport processes. Seeds, which supply essential resources for embryo development, showed higher mRNA abundance of genes encoding phenylpropanoid biosynthetic enzymes, seed storage proteins, and late embryogenesis abundant proteins. Water-deficit stress affected the mRNA abundance of 13% of the genes with differential expression patterns occurring mainly in the pulp and skin. In pulp and seed tissues transcript abundance in most functional categories declined in water-deficit stressed vines relative to well-watered vines with transcripts for storage proteins and novel (no-hit) functional assignments being over represented. In the skin of berries from water-deficit stressed vines, however, transcripts from several functional categories including general phenypropanoid and ethylene metabolism, pathogenesis-related responses, energy, and interaction with the environment were significantly over-represented. Conclusion These results revealed novel insights into the tissue-specific expression mRNA expression patterns of an extensive repertoire of genes expressed in berry tissues. This work also establishes an extensive catalogue of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern tissue-specific expression patterns associated with tissue differentiation within berries. These results also confirmed that water-deficit stress has a profound effect on mRNA expression patterns particularly associated with the biosynthesis of aroma and color metabolites within skin and pulp tissues that ultimately impact wine quality. PMID:17584945

  14. Soya phytoestrogens change cortical and hippocampal expression of BDNF mRNA in male rats.

    PubMed

    File, Sandra E; Hartley, David E; Alom, Nazmul; Rattray, Marcus

    2003-02-27

    Adult male hooded Lister rats were either fed a diet containing 150 microg/g soya phytoestrogens or a soya-free diet for 18 days. This concentration of phytoestrogens should have been sufficient to occupy the oestrogen-beta, but not the oestrogen-alpha, receptors. Using in situ hybridisation, significant reductions were found in brain-derived neurotrophic factor (BDNF) mRNA expression in the CA3 and CA4 region of the hippocampus and in the cerebral cortex in the rats fed the diet containing phytoestrogens, compared with those on the soya-free diet. No changes in glutamic acid decarboxylase-67 or glial fibrillary acidic protein mRNA were found. This suggests a role for oestrogen-beta receptors in regulating BDNF mRNA expression.

  15. 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.

  16. Constitutive and allergen-induced expression of eotaxin mRNA in the guinea pig lung

    PubMed Central

    1995-01-01

    Eotaxin is a member of the C-C family of chemokines and is related during antigen challenge in a guinea pig model of allergic airway inflammation (asthma). Consistent with its putative role in eosinophilic inflammation, eotaxin induces the selective infiltration of eosinophils when injected into the lung and skin. Using a guinea pig lung cDNA library, we have cloned full-length eotaxin cDNA. The cDNA encodes a protein of 96 amino acids, including a putative 23-amino acid hydrophobic leader sequence, followed by 73 amino acids composing the mature active eotaxin protein. The protein-coding region of this cDNA is 73, 71, 50, and 48% identical in nucleic acid sequence to those of human macrophage chemoattractant protein (MCP) 3, MCP-1, macrophage inflammatory protein (MIP) 1 alpha, and RANTES, respectively. Analysis of genomic DNA suggested that there is a single eotaxin gene in guinea pig which is apparently conserved in mice. High constitutive levels of eotaxin mRNA expression were observed in the lung, while the intestines, stomach, spleen, liver, heart, thymus, testes, and kidney expressed lower levels. To determine if eotaxin mRNA levels are elevated during allergen-induced eosinophilic airway inflammation, ovalbumin (OVA)-sensitized guinea pigs were challenged with aerosolized antigen. Compared with the lungs from saline-challenged animals, eotaxin mRNA levels increased sixfold within 3 h and returned to baseline by 6 h. Thus, eotaxin mRNA levels are increased in response to allergen challenge during the late phase response. The identification of constitutive eotaxin mRNA expression in multiple tissues suggests that in addition to regulating airway eosinophilia, eotaxin is likely to be involved in eosinophil recruitment into other tissues as well as in baseline tissue homing. PMID:7869037

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

    PubMed Central

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

    2016-01-01

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

  18. Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism.

    PubMed

    Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K; Lehtonen, Jukka Y A

    2016-04-20

    As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3'-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Differential expression of Ro/SSA 60 kDa and La/SSB, but not Ro/SSA 52 kDa, mRNA and protein in minor salivary glands from patients with primary Sjögren's syndrome.

    PubMed

    Barcellos, Karin S A; Nonogaki, Suely; Enokihara, Milvia M S S; Teixeira, Miriam S; Andrade, Luís E C

    2007-06-01

    To analyze the protein and messenger RNA (mRNA) expression of La/SSB, Ro/SSA 60, and Ro/SSA 52 antigens in minor salivary glands (MSG) from patients with primary Sjögren's syndrome (pSS). La/SSB, Ro/SSA 60, and Ro/SSA 52 protein expression was studied by immunohistochemistry in MSG from 26 patients with pSS and 16 controls. mRNA expression was determined by real-time polymerase chain reaction in MSG of 10 patients with pSS and 7 controls. La/SSB and Ro/SSA 60, but not Ro/SSA 52, mRNA expression was higher in samples from patients with pSS compared to controls (p < 0.05). La/SSB protein had higher expression in the cytoplasm of ductal cells than in the cytoplasm of mucous acinar cells in patients with pSS (p = 0.013) but not in controls. Ro/SSA 60 had higher expression in the cytoplasm of ductal cells than in the cytoplasm of serous acinar cells in patients with pSS (p = 0.006) but not in controls. The Ro/SSA 52 expression pattern was similar in patients and controls. There was no association between circulating autoantibodies to Ro/SSA or La/SSB and the aberrant expression of the cognate autoantigens. The increased Ro/SSA 60 and La/SSB mRNA expression in MSG of patients with pSS as well as the differential Ro/SSA 60 and La/SSB protein expression in ductal cells of MSG in patients with pSS suggest that these these 2 autoantigens, but not Ro/SSA 52, are probably involved in triggering and maintaining the tissue-specific autoimmune response in pSS MSG and may contribute to the antigen-driven immune response and local autoantibody production in pSS.

  20. The utility of protein and mRNA correlation

    SciTech Connect

    Payne, Samuel H.

    2015-01-01

    Transcriptomic, proteomic and metabolomic measurements are revolutionizing the way we model and predict cellular behavior, and multi-omic comparisons are being published with increased regularity. Some have expected a trivial and predictable correlation between mRNA and protein; however the manifest complexity of biological regulation suggests a more nuanced relationship. Indeed, observing this lack of strict correlation provides clues for new research topics, and has the potential for transformative biological insight.

  1. Differences in expression of retinal pigment epithelium mRNA between normal canines

    PubMed Central

    2004-01-01

    Abstract A reference database of differences in mRNA expression in normal healthy canine retinal pigment epithelium (RPE) has been established. This database identifies non-informative differences in mRNA expression that can be used in screening canine RPE for mutations associated with clinical effects on vision. Complementary DNA (cDNA) pools were prepared from mRNA harvested from RPE, amplified by PCR, and used in a subtractive hybridization protocol (representational differential analysis) to identify differences in RPE mRNA expression between canines. The effect of relatedness of the test canines on the frequency of occurrence of differences was evaluated by using 2 unrelated canines for comparison with 2 female sibling canines of blue heeler/bull terrier lineage. Differentially expressed cDNA species were cloned, sequenced, and identified by comparison to public database entries. The most frequently observed differentially expressed sequence from the unrelated canine comparison was cDNA with 21 base pairs (bp) identical to the human epithelial membrane protein 1 gene (present in 8 of 20 clones). Different clones from the same-sex sibling RPE contained repetitions of several short sequence motifs including the human epithelial membrane protein 1 (4 of 25 clones). Other prevalent differences between sibling RPE included sequences similar to a chicken genetic marker sequence motif (5 of 25), and 6 clones with homology to porcine major histocompatibility loci. In addition to identifying several repetitively occurring, noninformative, differentially expressed RPE mRNA species, the findings confirm that fewer differences occurred between siblings, highlighting the importance of using closely related subjects in representational difference analysis studies. PMID:15352545

  2. mRNA Distribution and Heterologous Expression of Orphan Cytochrome P450 20A1

    PubMed Central

    Stark, Katarina; Wu, Zhong-Liu; Bartleson, Cheryl J.; Guengerich, F. Peter

    2015-01-01

    Cytochrome P450 (P450) 20A1 is one of the so-called “orphan” P450s without assigned biological function. mRNA expression was detected in human liver and extrahepatic expression was noted in several human brain regions, including substantia nigra, hippocampus, and amygdala, using conventional polymerase chain reaction and RNA dot blot analysis. Adult human liver contained 3-fold higher overall mRNA levels than whole brain, although specific regions (i.e., hippocampus and substantia nigra) exhibited higher mRNA expression levels than liver. Orthologous full-length and truncated transcripts of P450 20A1 were transcribed and sequenced from rat liver, heart, and brain. In rat, the concentrations of full-length transcripts were 3–4 fold higher in brain and heart than liver. In situ hybridization of rat whole brain sections showed a similar mRNA expression pattern as observed for human P450 20A1, indicating expression in substantia nigra, hippocampus, and amygdala. A number of N-terminal modifications of the codon-optimized human P450 20A1 sequence were prepared and expressed in Escherichia coli, and two of the truncated derivatives showed characteristic P450 spectra (200–280 nmol P450/l). Although the recombinant enzyme system oxidized NADPH, no catalytic activity was observed with the heterologously expressed protein when a number of potential steroids and biogenic amines were surveyed as potential substrates. The function of P450 20A1 remains unknown; however, the sites of mRNA expression in human brain and the conservation among species may suggest possible neurophysiological function. PMID:18541694

  3. TDP-43 suppresses tau expression via promoting its mRNA instability.

    PubMed

    Gu, Jianlan; Wu, Feng; Xu, Wen; Shi, Jianhua; Hu, Wen; Jin, Nana; Qian, Wei; Wang, Xinglong; Iqbal, Khalid; Gong, Cheng-Xin; Liu, Fei

    2017-06-02

    In the brains of individuals with Alzheimer's disease (AD) and chronic traumatic encephalopathy, tau pathology is accompanied usually by intracellular aggregation of transactive response DNA-binding protein 43 (TDP-43). However, the role of TDP-43 in tau pathogenesis is not understood. Here, we investigated the role of TDP-43 in tau expression in vitro and in vivo. We found that TDP-43 suppressed tau expression by promoting its mRNA instability through the UG repeats of its 3΄-untranslated region (3΄-UTR). The C-terminal region of TDP-43 was required for this function. Neurodegenerative diseases-causing TDP-43 mutations affected tau mRNA instability differentially, in that some promoted and others did not significantly affect tau mRNA instability. The expression levels of tau and TDP-43 were inverse in the frontal cortex and the cerebellum. Accompanied with cytoplasmic accumulation of TDP-43, tau expression was elevated in TDP-43M337V transgenic mouse brains. The level of TDP-43, which is decreased in AD brains, was found to correlate negatively with the tau level in human brain. Our findings indicate that TDP-43 suppresses tau expression by promoting the instability of its mRNA. Down-regulation of TDP-43 may be involved in the tau pathology in AD and related neurodegenerative disorders. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. TDP-43 suppresses tau expression via promoting its mRNA instability

    PubMed Central

    Gu, Jianlan; Wu, Feng; Xu, Wen; Shi, Jianhua; Hu, Wen; Jin, Nana; Qian, Wei; Wang, Xinglong; Iqbal, Khalid; Gong, Cheng-Xin

    2017-01-01

    Abstract In the brains of individuals with Alzheimer's disease (AD) and chronic traumatic encephalopathy, tau pathology is accompanied usually by intracellular aggregation of transactive response DNA-binding protein 43 (TDP-43). However, the role of TDP-43 in tau pathogenesis is not understood. Here, we investigated the role of TDP-43 in tau expression in vitro and in vivo. We found that TDP-43 suppressed tau expression by promoting its mRNA instability through the UG repeats of its 3΄-untranslated region (3΄-UTR). The C-terminal region of TDP-43 was required for this function. Neurodegenerative diseases-causing TDP-43 mutations affected tau mRNA instability differentially, in that some promoted and others did not significantly affect tau mRNA instability. The expression levels of tau and TDP-43 were inverse in the frontal cortex and the cerebellum. Accompanied with cytoplasmic accumulation of TDP-43, tau expression was elevated in TDP-43M337V transgenic mouse brains. The level of TDP-43, which is decreased in AD brains, was found to correlate negatively with the tau level in human brain. Our findings indicate that TDP-43 suppresses tau expression by promoting the instability of its mRNA. Down-regulation of TDP-43 may be involved in the tau pathology in AD and related neurodegenerative disorders. PMID:28335005

  5. Sequence and expression of ferredoxin mRNA in barley

    SciTech Connect

    Zielinski, R.; Funder, P.M.; Ling, V. )

    1990-05-01

    We have isolated and structurally characterized a full-length cDNA clone encoding ferredoxin from a {lambda}gt10 cDNA library prepared from barley leaf mRNA. The ferredoxin clone (pBFD-1) was fused head-to-head with a partial-length cDNA clone encoding calmodulin, and was fortuitously isolated by screening the library with a calmodulin-specific oligonucleotide probe. The mRNA sequence from which pBFD-1 was derived is expressed exclusively in the leaf tissues of 7-d old barley seedlings. Barley pre-ferredoxin has a predicted size of 15.3 kDal, of which 4.6 kDal are accounted for by the transit peptide. The polypeptide encoded by pBFD-1 is identical to wheat ferredoxin, and shares slightly more amino acid sequence similarity with spinach ferredoxin I than with ferredoxin II. Ferredoxin mRNA levels are rapidly increased 10-fold by white light in etiolated barley leaves.

  6. MRNA expression of genes regulating lipid metabolism in ringed seals (Pusa hispida) from differently polluted areas.

    PubMed

    Castelli, Martina Galatea; Rusten, Marte; Goksøyr, Anders; Routti, Heli

    2014-01-01

    There is a growing concern about the ability of persistent organic pollutants (POPs) to influence lipid metabolism. Although POPs are found at high concentrations in some populations of marine mammals, for example in the ringed seal (Pusa hispida) from the Baltic Sea, little is known about the effects of POPs on their lipid metabolism. An optimal regulation of lipid metabolism is crucial for ringed seals during the fasting/molting season. This is a physiologically stressful period, during which they rely on the energy stored in their fat reserves. The mRNA expression levels for seven genes involved in lipid metabolism were analyzed in liver and/or blubber tissue from molting ringed seals from the polluted Baltic Sea and a less polluted reference location, Svalbard (Norway). mRNA expression of genes encoding peroxisome proliferator-activated receptors (PPAR) α and γ and their target genes acyl-coenzyme A oxidase 1 (ACOX1) and cluster of differentiation 36 (CD36) were analyzed in liver. mRNA expression level of genes encoding PPARβ, PPARγ and their target genes encoding fatty acid binding protein 4 (FABP4) and adiponectin (ADIPOQ) were measured in inner and middle blubber layers. In addition, we evaluated the influence of molting status on hepatic mRNA expression of genes encoding PPARs and their target genes in ringed seals from Svalbard. Our results show higher mRNA expression of genes encoding hepatic PPARγ and adipose PPARβ, FABP4, and ADIPOQ in the Baltic seals compared to the Svalbard seals. A positive relationship between mRNA expressions of genes encoding hepatic PPARγ, adipose FABP4, adipose ADIPOQ and ΣPOP concentrations was observed. These findings suggest that lipid metabolism may be affected by contaminant exposure in the Baltic population. mRNA expression of genes encoding PPARβ, PPARγ, FABP4 and ADIPOQ were similar between the mid and inner adipose layer. Hepatic mRNA expression of genes encoding PPARα and PPARγ was higher in the pre

  7. Cellular differentiation state modulates the mRNA export activity of SR proteins.

    PubMed

    Botti, Valentina; McNicoll, François; Steiner, Michaela C; Richter, Florian M; Solovyeva, Anfisa; Wegener, Marius; Schwich, Oliver D; Poser, Ina; Zarnack, Kathi; Wittig, Ilka; Neugebauer, Karla M; Müller-McNicoll, Michaela

    2017-07-03

    SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining individual-resolution cross-linking and immunoprecipitation (iCLIP) and mass spectrometry, we show that elevated arginine methylation of SRSF5 and lower phosphorylation levels of cobound SRSF2 enhance shuttling of SRSF5 in P19 cells by modulating protein-protein and protein-RNA interactions. Moreover, SRSF5 is bound to pluripotency-specific transcripts such as Lin28a and Pou5f1/Oct4 in the cytoplasm. SRSF5 depletion reduces and overexpression increases their cytoplasmic mRNA levels, suggesting that enhanced mRNA export by SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19 cells leads to dramatically reduced SRSF5 shuttling. Our findings indicate that posttranslational modification of SR proteins underlies the regulation of their mRNA export activities and distinguishes pluripotent from differentiated cells. © 2017 Botti et al.

  8. Antisense oligodeoxynucleotides targeted to MAG mRNA profoundly alter BP and PLP mRNA expression in differentiating oligodendrocytes: a caution.

    PubMed

    Laszkiewicz, I; Wiggins, R C; Konat, G W

    1999-09-01

    The applicability of antisense technology to suppress the expression of myelin associated glycoprotein (MAG) in cultured oligodendrocytes was evaluated. Differentiating oligodendrocyte precursor cells obtained by the shake-off method were exposed to nine unmodified antisense oligodeoxynucleotides (ODNs) targeted to the first seven exons of MAG mRNA. After four days, steady-state levels of MAG, proteolipid protein (PLP) and basic protein (BP) mRNAs were determined by Northern blot analysis. Only ODN annealing to 599-618 nt of the MAG mRNA (the junction of exon 5 and 6) resulted in a significant, 75% decrease in the MAG mRNA level. Unexpectedly, six other anti-MAG ODNs which had no significant effect on the MAG message, greatly increased the level of BP mRNA. The highest upregulation of approximately 12 fold was observed with ODN annealing to 139-168 nt (junction of exon 3 and 4). On the other hand, the 997-1016 ODN decreased the levels of BP and PLP messages by 70-80%. The 599-618 ODN also decreased the PLP mRNA by 85%. The results demonstrate that antisense ODNs targeted to one gene may profoundly alter the expression of other genes, and hence, complicate functional analysis of the targeted protein.

  9. Identification of Nuclear and Cytoplasmic mRNA Targets for the Shuttling Protein SF2/ASF

    PubMed Central

    Sanford, Jeremy R.; Coutinho, Pedro; Hackett, Jamie A.; Wang, Xin; Ranahan, William; Caceres, Javier F.

    2008-01-01

    The serine and arginine-rich protein family (SR proteins) are highly conserved regulators of pre-mRNA splicing. SF2/ASF, a prototype member of the SR protein family, is a multifunctional RNA binding protein with roles in pre-mRNA splicing, mRNA export and mRNA translation. These observations suggest the intriguing hypothesis that SF2/ASF may couple splicing and translation of specific mRNA targets in vivo. Unfortunately the paucity of endogenous mRNA targets for SF2/ASF has hindered testing of this hypothesis. Here, we identify endogenous mRNAs directly cross-linked to SF2/ASF in different sub-cellular compartments. Cross-Linking Immunoprecipitation (CLIP) captures the in situ specificity of protein-RNA interaction and allows for the simultaneous identification of endogenous RNA targets as well as the locations of binding sites within the RNA transcript. Using the CLIP method we identified 326 binding sites for SF2/ASF in RNA transcripts from 180 protein coding genes. A purine-rich consensus motif was identified in binding sites located within exon sequences but not introns. Furthermore, 72 binding sites were occupied by SF2/ASF in different sub-cellular fractions suggesting that these binding sites may influence the splicing or translational control of endogenous mRNA targets. We demonstrate that ectopic expression of SF2/ASF regulates the splicing and polysome association of transcripts derived from the SFRS1, PABC1, NETO2 and ENSA genes. Taken together the data presented here indicate that SF2/ASF has the capacity to co-regulate the nuclear and cytoplasmic processing of specific mRNAs and provide further evidence that the nuclear history of an mRNA may influence its cytoplasmic fate. PMID:18841201

  10. Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines.

    PubMed Central

    Nørgaard, P.; Spang-Thomsen, M.; Poulsen, H. S.

    1996-01-01

    In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II mRNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF-beta receptor proteins beta-glycan mRNA was rapidly down-regulated and this effect was sustained throughout the 24 h observation period. RI and RII mRNAs were slightly increased 24 h after treatment. In one cell line sensitive to growth inhibition by TGF-beta, 1 but lacking beta-glycan expression, and one cell line expressing only beta-glycan and thus TGF-beta 1 -resistant, no autoregulation of mRNA of either TGF-beta receptor was demonstrated. The results suggest that TGF-beta 1 regulates the expression of its receptors, in particular beta-glycan, and that this effect is dependent on co-expression of beta-glycan, RI and RII. Images Figure 1 Figure 2 Figure 4 PMID:8624260

  11. Increased 8-hydroxy-2'-deoxyguanosine in plasma and decreased mRNA expression of human 8-oxoguanine DNA glycosylase 1, anti-oxidant enzymes, mitochondrial biogenesis-related proteins and glycolytic enzymes in leucocytes in patients with systemic lupus erythematosus.

    PubMed

    Lee, H-T; Lin, C-S; Lee, C-S; Tsai, C-Y; Wei, Y-H

    2014-04-01

    We measured plasma levels of the oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) and leucocyte mRNA expression levels of the genes encoding the 8-OHdG repair enzyme human 8-oxoguanine DNA glycosylase 1 (hOGG1), the anti-oxidant enzymes copper/zinc superoxide dismutase (Cu/ZnSOD), manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase-1 (GPx-1), GPx-4, glutathione reductase (GR) and glutathione synthetase (GS), the mitochondrial biogenesis-related proteins mtDNA-encoded ND 1 polypeptide (ND1), ND6, ATPase 6, mitochondrial transcription factor A (Tfam), nuclear respiratory factor 1(NRF-1), pyruvate dehydrogenase E1 component alpha subunit (PDHA1), pyruvate dehydrogenase kinase isoenzyme 1 (PDK-1) and hypoxia inducible factor-1α (HIF-1α) and the glycolytic enzymes hexokinase-II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase A (LDHa). We analysed their relevance to oxidative damage in 85 systemic lupus erythematosus (SLE) patients, four complicated SLE patients undergoing rituximab treatment and 45 healthy individuals. SLE patients had higher plasma 8-OHdG levels (P < 0·01) but lower leucocyte expression of the genes encoding hOGG1(P < 0·01), anti-oxidant enzymes (P < 0·05), mitochondrial biogenesis-related proteins (P < 0·05) and glycolytic enzymes (P < 0·05) than healthy individuals. The increase in plasma 8-OHdG was correlated positively with the elevation of leucocyte expression of the genes encoding hOGG1 (P < 0·05), anti-oxidant enzymes (P < 0·05), several mitochondrial biogenesis-related proteins (P < 0·05) and glycolytic enzymes (P < 0·05) in lupus patients. The patients, whose leucocyte mtDNA harboured D310 heteroplasmy, exhibited a positive correlation between the mtDNA copy number and expression of ND1, ND6 and ATPase 6 (P < 0·05) and a negative correlation between mt

  12. Cloning, comparative sequence analysis and mRNA expression of calcium-transporting genes in horses.

    PubMed

    Rourke, K M; Coe, S; Kohn, C W; Rosol, T J; Mendoza, F J; Toribio, R E

    2010-05-15

    Epithelial calcium transport occurs by paracellular and transcellular mechanisms. Transcellular transport in intestinal and renal epithelia involves several transport proteins, including transient receptor potential vanilloid member 5 (TRPV5), member 6 (TRPV6), calbindin D9k (CB9), calbindin D28k (CB28), sodium calcium exchanger 1 (NCX1), plasma membrane calcium ATPase 1 (PMCA1), and the vitamin D receptor (VDR). We are interested in the horse because of its unique calcium physiology (high blood calcium, high intestinal calcium absorption, high renal excretion of calcium, low vitamin D concentrations), and because horses often have dysregulated calcium balance with various diseases. We cloned the mRNA for equine TRPV5, TRPV6, CB9, CB28, NCX1, PMCA1, and VDR, performed comparative mRNA and protein sequence analysis, and quantified their mRNA expression in the kidney and gastrointestinal tract. Sequence homology for the mRNAs and proteins was high among mammals (>75%), with fish having the lowest homology (<75%). TRPV5, TRPV6, and CB9 expression was higher in the duodenum and proximal jejunum and followed a similar expression pattern. CB28 expression was greatest in the kidney. PMCA1 and NCX1 expression was similar throughout the intestine, but in the kidney PMCA1 expression was higher. Based on our findings, the proximal small intestine is the main site for transcellular calcium transport, with TRPV6 and CB9 serving as the main transport proteins. In the kidney, TRPV6, CB28, and PMCA1 are likely more important. The low VDR expression in the equine small intestine and kidney relative to the large intestine, together with the reported high intestinal absorption and renal excretion of calcium, and low vitamin D concentrations suggests that epithelial calcium transport in horses is not as dependent on vitamin D as in other species. Copyright 2010 Elsevier Inc. All rights reserved.

  13. Gastrointestinal hormone mRNA expression in human colonic adenocarcinomas, hepatic metastases and cell lines

    PubMed Central

    Monges, G; Biagini, P; Cantaloube, J F; De Micco, P; Parriaux, D; Seitz, J F; Delpero, J R; Hassoun, J

    1996-01-01

    Aims—(1) To investigate the expression of the four main hormones of the digestive tract by performing reverse transcription polymerase chain reaction (RT-PCR) on a series of samples, comprising tumoral and healthy colonic tissues, hepatic metastases and colonic cell line samples; and (2) to study the patterns of labelling obtained with serological and morphological markers. Methods—After extraction and reverse transcription, gastrin, somatostatin, cholecystokinin (CCK) and transforming growth factor α (TGFα) mRNAs were detected by PCR and nested PCR using specific primers. The corresponding proteins were detected by immunohistochemistry. Results—The cell lines expressed all four mRNAs. Gastrin mRNA was present in most tumoral and metastatic samples, while the somatostatin transcript was detected in all samples and was frequently overexpressed in the normal colon. TGFα mRNA was expressed systematically in tumours of the right and transverse colon, but not in those located in the left colon; the expression of CCK mRNA was systematically absent in the left colon. Conclusions—The data presented here shed some light on the transcriptional events involved in the production of the various hormones present in the gastrointestinal tract, in both healthy and tumoral tissues. The various mRNAs expressed in cell lines are therefore not systematically expressed in the human pathology. Images PMID:16696065

  14. Moisturizers change the mRNA expression of enzymes synthesizing skin barrier lipids.

    PubMed

    Buraczewska, Izabela; Berne, Berit; Lindberg, Magnus; Lodén, Marie; Törmä, Hans

    2009-09-01

    In a previous study, 7-week treatment of normal human skin with two test moisturizers, Complex cream and Hydrocarbon cream, was shown to affect mRNA expression of certain genes involved in keratinocyte differentiation. Moreover, the treatment altered transepidermal water loss (TEWL) in opposite directions. In the present study, the mRNA expression of genes important for formation of barrier lipids, i.e., cholesterol, free fatty acids and ceramides, was examined. Treatment with Hydrocarbon cream, which increased TEWL, also elevated the gene expression of GBA, SPTLC2, SMPD1, ALOX12B, ALOXE3, and HMGCS1. In addition, the expression of PPARG was decreased. On the other hand, Complex cream, which decreased TEWL, induced only the expression of PPARG, although not confirmed at the protein level. Furthermore, in the untreated skin, a correlation between the mRNA expression of PPARG and ACACB, and TEWL was found, suggesting that these genes are important for the skin barrier homeostasis. The observed changes further demonstrate that long-term treatment with certain moisturizers may induce dysfunctional skin barrier, and as a consequence several signaling pathways are altered.

  15. Circulating resistin protein and mRNA concentrations and clinical severity of coronary artery disease

    PubMed Central

    Sopic, Miron; Spasojevic-Kalimanovska, Vesna; Kalimanovska-Ostric, Dimitra; Andjelkovic, Kristina; Jelic-Ivanovic, Zorana

    2015-01-01

    Introduction Previous studies have implicated a strong link between circulating plasma resistin and coronary artery disease (CAD). The aim of this study was to evaluate the differences in peripheral blood mononuclear cells (PBMC) resistin mRNA and its plasma protein concentrations between the patients with CAD of different clinical severity. Material and methods This study included 33 healthy subjects as the control group (CG) and 77 patients requiring coronary angiography. Of the latter 30 was CAD negative whereas 47 were CAD positive [18 with stable angina pectoris (SAP) and 29 with acute coronary syndrome (ACS)]. Circulating resistin was measured by ELISA; PBMC resistin mRNA was determined by real-time PCR. Results Resistin protein was significantly higher in the ACS group compared to the CG (P = 0.001) and the CAD negative group (P = 0.018). Resistin mRNA expression did not vary across the study groups, despite the positive correlation seen with plasma resistin (ρ = 0.305, P = 0.008). In patients, plasma resistin and PBMC resistin mRNA negatively correlated with HDL-C (ρ = -0.404, P < 0.001 and ρ = -0.257, P = 0.032, respectively). Furthermore, the highest plasma resistin tertile showed the lowest HDL-C (P = 0.006). Plasma resistin was positively associated with serum creatinine (ρ = 0.353, P = 0.002). Conclusion Significant increase of plasma resistin in patients with ACS compared to CG and CAD negative patients was observed. Despite no change in PBMC resistin mRNA in different disease conditions a positive association between resistin mRNA and resistin plasma protein was evident. Both plasma resistin and PBMC resistin mRNA were negatively associated with plasma HDL-C, and plasma resistin positively with serum creatinine. PMID:26110037

  16. Circulating resistin protein and mRNA concentrations and clinical severity of coronary artery disease.

    PubMed

    Joksic, Jelena; Sopic, Miron; Spasojevic-Kalimanovska, Vesna; Kalimanovska-Ostric, Dimitra; Andjelkovic, Kristina; Jelic-Ivanovic, Zorana

    2015-01-01

    Previous studies have implicated a strong link between circulating plasma resistin and coronary artery disease (CAD). The aim of this study was to evaluate the differences in peripheral blood mononuclear cells (PBMC) resistin mRNA and its plasma protein concentrations between the patients with CAD of different clinical severity. This study included 33 healthy subjects as the control group (CG) and 77 patients requiring coronary angiography. Of the latter 30 was CAD negative whereas 47 were CAD positive [18 with stable angina pectoris (SAP) and 29 with acute coronary syndrome (ACS)]. Circulating resistin was measured by ELISA; PBMC resistin mRNA was determined by real-time PCR. Resistin protein was significantly higher in the ACS group compared to the CG (P=0.001) and the CAD negative group (P=0.018). Resistin mRNA expression did not vary across the study groups, despite the positive correlation seen with plasma resistin (ρ=0.305, P=0.008). In patients, plasma resistin and PBMC resistin mRNA negatively correlated with HDL-C (ρ=-0.404, P<0.001 and ρ=-0.257, P=0.032, respectively). Furthermore, the highest plasma resistin tertile showed the lowest HDL-C (P=0.006). Plasma resistin was positively associated with serum creatinine (ρ=0.353, P=0.002). Significant increase of plasma resistin in patients with ACS compared to CG and CAD negative patients was observed. Despite no change in PBMC resistin mRNA in different disease conditions a positive association between resistin mRNA and resistin plasma protein was evident. Both plasma resistin and PBMC resistin mRNA were negatively associated with plasma HDL-C, and plasma resistin positively with serum creatinine.

  17. Phenobarbital specifically reduces gap junction protein mRNA level in rat liver.

    PubMed

    Mesnil, M; Fitzgerald, D J; Yamasaki, H

    1988-01-01

    The gene expression of liver major gap junction (GJ) protein was studied in rats systemically administered phenobarbital, a rat liver tumor promoter. Using a GJ protein cDNA and northern blot analysis, the level of GJ protein mRNA in liver was observed to be markedly reduced at 4 and 11 wk of phenobarbital exposure (0.1% in drinking water). However, the level of GJ protein mRNA was not altered in kidney at 11 wk of exposure. In liver, phenobarbital did not induce expression of the neoplasm-associated marker genes glutathione S-transferase (placental form) and gamma-glutamyltranspeptidase, while in kidney the observed expression of these genes was not changed. These in vivo results indicate that phenobarbital reduces GJ protein gene expression specifically in rat liver without altering expression of genes often altered during liver carcinogenesis, and they support assigning a role for the impairment of gap junctional intercellular communication in phenobarbital-mediated liver tumor promotion.

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

    PubMed

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

    2005-03-15

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

  19. Protein expression-yeast.

    PubMed

    Nielsen, Klaus H

    2014-01-01

    Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline.

  20. Cistanches Herba aqueous extract affecting serum BGP and TRAP and bone marrow Smad1 mRNA, Smad5 mRNA, TGF-β1 mRNA and TIEG1 mRNA expression levels in osteoporosis disease.

    PubMed

    Liang, Hai-Dong; Yu, Fang; Tong, Zhi-Hong; Zhang, Hong-Quan; Liang, Wu

    2013-02-01

    We studied molecular mechanism of Cistanches Herba aqueous extract (CHAE) in ovariectomized (OVX) rats, as an experimental model of postmenopausal osteoporosis. Female rats were either sham-operated or bilaterally OVX; and at 60 days postoperatively. The OVX group (n = 8) received an ovariectomy and treatment with normal saline for 90 days commencing from 20th post ovariectomy day. The ovariectomized +CHAE (OVX + CHAE) group (n = 8) received an ovariectomy and were treated with Cistanches Herba aqueous extract of 100 mg/kg body weight daily for 90 days commencing from 22nd post ovariectomy day. The ovariectomy +CHAE (OVX + CHAE) group (n = 8) received an ovariectomy, and were treated with the of 200 mg/kg body weight daily for 90 days commencing from 20th post ovariectomy day. Serum BGP and TRAP, E2, FSH and LH level, bone marrow Smad1, Smad5, TGF-β1 and TIEG1 mRNA expression levels were examined. Results showed that serum BGP and TRAP, FSH and LH levels were significantly increased, whereas E2, Smad1, Smad5, TGF-β1 and TIEG1 mRNA and proteins expression levels were significantly decreased in OVX rats compared to sham rats. 90 days of CHAE treatment could significantly decrease serum BGP and TRAP, FSH and LH levels, and increase E2, Smad1, Smad5, TGF-β1 and TIEG1 mRNA and proteins expression levels in OVX rats. It can be concluded that CHAE play its protective effect against OVX-induced bone degeneration partly by regulating some bone metabolism related genes, e.g. Smad1, Smad5, TGF-β1 and TIEG1.

  1. Regulation of the growth hormone (GH) receptor and GH-binding protein mRNA

    SciTech Connect

    Kaji, Hidesuke; Ohashi, Shin-Ichirou; Abe, Hiromi; Chihara, Kazuo

    1994-12-31

    In fasting rats, a transient increase in growth hormone-binding protein (GHBP) mRNA levels was observed after 1 day, in muscle, heart, and liver, but not in fat tissues. The liver GH receptor (GHR) mRNA level was significantly increased after 1 day (but not after 5 days) of bovine GH (bGH) treatment in fed rats. Both the liver GHR mRNA level and the net increment of plasma IGF-I markedly decreased after 5 days of bGH administration in fasting rats. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in a different way and that the expression of GHBP mRNA is regulated differently between tissues, at least in rats. The results also suggest that refractoriness to GH in a sustained fasting state might be beneficial in preventing anabolic effects of GH. In humans, GHR mRNA in lymphocytes, from subjects with either GH-deficiency or acromegaly, could be detected by the reverse transcription-polymerase chain reaction method. In one patient with partial GH insensitivity, a heterozygous missense mutation (P561T) was identified in the cytoplasmic domain of GHR. 15 refs., 4 figs.

  2. The Role of Neuropeptide Y mRNA Expression Level in Distinguishing Different Types of Depression.

    PubMed

    Yue, Yingying; Jiang, Haitang; Yin, Yingying; Zhang, Yuqun; Liang, Jinfeng; Li, Shenghua; Wang, Jun; Lu, Jianxin; Geng, Deqin; Wu, Aiqin; Yuan, Yonggui

    2016-01-01

    Previous studies demonstrate that the protein of neuropeptide Y (NPY) is abnormal in depression patients, but the changes of NPY in different types of depression are unclear. This study was aimed to examine protein and mRNA expression levels of NPY in 159 cases with four groups including post-stroke depression (PSD) group, stroke without depression (Non-PSD) group, major depressive disorder (MDD) group and normal control (NC) group. The protein and gene expression analysis were performed by enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction-based methods. One way analysis of variance (ANOVA), chi-square tests and nonparametric test were used to evaluate general characteristics, clinical and biological materials. In order to explore the role of NPY in different types of depression, the partial correlations, binary logistic regression analysis and receiver operating characteristic (ROC) curve were calculated for PSD and MDD groups. There are significant differences of NPY protein (Fdf(3) = 5.167, P = 0.002) and mRNA expression levels ([Formula: see text] = 20.541, P < 0.001) among four groups. Bonferroni multiple comparisons found that the NPY protein was significantly decreased in PSD (FBonferroni = -7.133, P = 0.002) and Non-PSD group (FBonferroni = -5.612, P = 0.018) compared with NC group. However, contrasted with MDD group, the mRNA expression was increased in PSD and Non-PSD group by nonparametric test (all P < 0.05). In binary logistic analyses, NPY mRNA expression was independent predictors of PSD (odds ratio: 1.452, 95% CI, 1.081-1.951, P = 0.013). The ROC curve showed NPY mRNA had a general prognostic accuracy (area under the curve: 0.766, 95% CI, 0.656-0.876, P < 0.001). This is the first study to explore the distinguishing function of NPY in different types of depression. It will provide help in the identification of different subtypes of depression.

  3. The Role of Neuropeptide Y mRNA Expression Level in Distinguishing Different Types of Depression

    PubMed Central

    Yue, Yingying; Jiang, Haitang; Yin, Yingying; Zhang, Yuqun; Liang, Jinfeng; Li, Shenghua; Wang, Jun; Lu, Jianxin; Geng, Deqin; Wu, Aiqin; Yuan, Yonggui

    2016-01-01

    Previous studies demonstrate that the protein of neuropeptide Y (NPY) is abnormal in depression patients, but the changes of NPY in different types of depression are unclear. This study was aimed to examine protein and mRNA expression levels of NPY in 159 cases with four groups including post-stroke depression (PSD) group, stroke without depression (Non-PSD) group, major depressive disorder (MDD) group and normal control (NC) group. The protein and gene expression analysis were performed by enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction-based methods. One way analysis of variance (ANOVA), chi-square tests and nonparametric test were used to evaluate general characteristics, clinical and biological materials. In order to explore the role of NPY in different types of depression, the partial correlations, binary logistic regression analysis and receiver operating characteristic (ROC) curve were calculated for PSD and MDD groups. There are significant differences of NPY protein (Fdf(3) = 5.167, P = 0.002) and mRNA expression levels (χKruskal2-Wallis, df(3) = 20.541, P < 0.001) among four groups. Bonferroni multiple comparisons found that the NPY protein was significantly decreased in PSD (FBonferroni = −7.133, P = 0.002) and Non-PSD group (FBonferroni = −5.612, P = 0.018) compared with NC group. However, contrasted with MDD group, the mRNA expression was increased in PSD and Non-PSD group by nonparametric test (all P < 0.05). In binary logistic analyses, NPY mRNA expression was independent predictors of PSD (odds ratio: 1.452, 95% CI, 1.081–1.951, P = 0.013). The ROC curve showed NPY mRNA had a general prognostic accuracy (area under the curve: 0.766, 95% CI, 0.656–0.876, P < 0.001). This is the first study to explore the distinguishing function of NPY in different types of depression. It will provide help in the identification of different subtypes of depression. PMID:28082897

  4. Mycobacterium tuberculosis ESAT6 and CPF10 Induce Adenosine Deaminase 2 mRNA Expression in Monocyte-Derived Macrophages

    PubMed Central

    Bae, Mi Jung; Ryu, Suyeon; Kim, Ha-Jeong; Cha, Seung Ick

    2017-01-01

    Background Delayed hypersensitivity plays a large role in the pathogenesis of tuberculous pleural effusion (TPE). Macrophages infected with live Mycobacterium tuberculosis (MTB) increase the levels of adenosine deaminase2 (ADA2) in the pleural fluid of TPE patients. However, it is as yet unclear whether ADA2 can be produced by macrophages when challenged with MTB antigens alone. This study therefore evaluated the levels of ADA2 mRNA expression, using monocyte-derived macrophages (MDMs) stimulated with MTB antigens. Methods Purified monocytes from the peripheral blood mononuclear cells of healthy volunteers were differentiated into macrophages using granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF). The MDMs were stimulated with early secretory antigenic target protein 6 (ESAT6) and culture filtrate protein 10 (CFP10). The mRNA expression levels for the cat eye syndrome chromosome region, candidate 1 (CECR1) gene encoding ADA2 were then measured. Results CECR1 mRNA expression levels were significantly higher in MDMs stimulated with ESAT6 and CFP10, than in the unstimulated MDMs. When stimulated with ESAT6, M-CSF-treated MDMs showed more pronounced CECR1 mRNA expression than GM-CSF-treated MDMs. Interferon-γ decreased the ESAT6- and CFP10-induced CECR1 mRNA expression in MDMs. CECR1 mRNA expression levels were positively correlated with mRNA expression of tumor necrosis factor α and interleukin 10, respectively. Conclusion ADA2 mRNA expression increased when MDMs were stimulated with MTB antigens alone. This partly indicates that pleural fluid ADA levels could increase in patients with culture-negative TPE. Our results may be helpful in improving the understanding of TPE pathogenesis. PMID:28119750

  5. Mycobacterium tuberculosis ESAT6 and CPF10 Induce Adenosine Deaminase 2 mRNA Expression in Monocyte-Derived Macrophages.

    PubMed

    Bae, Mi Jung; Ryu, Suyeon; Kim, Ha-Jeong; Cha, Seung Ick; Kim, Chang Ho; Lee, Jaehee

    2017-01-01

    Delayed hypersensitivity plays a large role in the pathogenesis of tuberculous pleural effusion (TPE). Macrophages infected with live Mycobacterium tuberculosis (MTB) increase the levels of adenosine deaminase2 (ADA2) in the pleural fluid of TPE patients. However, it is as yet unclear whether ADA2 can be produced by macrophages when challenged with MTB antigens alone. This study therefore evaluated the levels of ADA2 mRNA expression, using monocyte-derived macrophages (MDMs) stimulated with MTB antigens. Purified monocytes from the peripheral blood mononuclear cells of healthy volunteers were differentiated into macrophages using granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF). The MDMs were stimulated with early secretory antigenic target protein 6 (ESAT6) and culture filtrate protein 10 (CFP10). The mRNA expression levels for the cat eye syndrome chromosome region, candidate 1 (CECR1) gene encoding ADA2 were then measured. CECR1 mRNA expression levels were significantly higher in MDMs stimulated with ESAT6 and CFP10, than in the unstimulated MDMs. When stimulated with ESAT6, M-CSF-treated MDMs showed more pronounced CECR1 mRNA expression than GM-CSF-treated MDMs. Interferon-γ decreased the ESAT6- and CFP10-induced CECR1 mRNA expression in MDMs. CECR1 mRNA expression levels were positively correlated with mRNA expression of tumor necrosis factor α and interleukin 10, respectively. ADA2 mRNA expression increased when MDMs were stimulated with MTB antigens alone. This partly indicates that pleural fluid ADA levels could increase in patients with culture-negative TPE. Our results may be helpful in improving the understanding of TPE pathogenesis.

  6. Characterizing exogenous mRNA delivery, trafficking, cytoplasmic release and RNA-protein correlations at the level of single cells.

    PubMed

    Kirschman, Jonathan L; Bhosle, Sushma; Vanover, Daryll; Blanchard, Emmeline L; Loomis, Kristin H; Zurla, Chiara; Murray, Kathryn; Lam, Blaine C; Santangelo, Philip J

    2017-07-07

    The use of synthetic messenger ribonucleic acid (mRNA) to express specific proteins is a highly promising therapeutic and vaccine approach that avoids many safety issues associated with viral or DNA-based systems. However, in order to optimize mRNA designs and delivery, technology advancements are required to study fundamental mechanisms of mRNA uptake and localization at the single-cell and tissue level. Here, we present a single RNA sensitive fluorescent labeling method which allows us to label and visualize synthetic mRNA without significantly affecting function. This approach enabled single cell characterization of mRNA uptake and release kinetics from endocytic compartments, the measurement of mRNA/protein correlations, and motivated the investigation of mRNA induced cellular stress, all important mechanisms influencing protein production. In addition, we demonstrated this approach can facilitate near-infrared imaging of mRNA localization in vivo and in ex-vivo tissue sections, which will facilitate mRNA trafficking studies in pre-clinical models. Overall, we demonstrate the ability to study fundamental mechanisms necessary to optimize delivery and therapeutic strategies, in order to design the next generation of novel mRNA therapeutics and vaccines. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. FKBP5, SERT and COMT mRNA expressions in the peripheral leukocytes during menstruation cycle in healthy reproductive females.

    PubMed

    Kinouchi, Sawako; Iga, Jun-Ichi; Ueno, Shu-Ichi; Yamauchi, Ken; Numata, Shusuke; Song, Hongwei; Sumitani, Satsuki; Shibuya-Tayoshi, Sumiko; Haku, Mari; Yasui, Toshiyuki; Irahara, Minoru; Morita, Kyoko; Rokutan, Kazuhito; Ohmori, Tetsuro

    2008-03-21

    There have been several evidences that the mRNA expressions in the peripheral leukocytes may indicate not only physical but also psychological states. The purpose of this study is whether the mRNA expressional changes in the leukocytes are related to the mental states across the menstrual cycle in reproductive healthy female subjects. Thirty-eight female subjects (22.4+/-1.4 year-old) were participated in this study at three menstruation cycle periods (menstrual, follicular and luteal phase). The FKBP5 (FK506-binding protein gene), SERT (serotonin transporter gene) and COMT (catechol-o-methyltransferase gene) mRNA expressions in the leukocytes were determined with hormonal data. The psychological changes were assessed with self-rating hospital anxiety and depression scale (HADS). Only one thirds of subjects (n=12) had regular menstrual cycles during the experiment. So we analyzed the data from these 12 subjects. The anxiety score of each subject was changed across the menstrual cycle (Friedman test: P<0.05). The FKBP5 mRNA expression was significantly lower in the follicular phase than in the other phases but no changes were seen in either SERT or COMT mRNA expressions among the phases. In conclusion, there are differences of HADS anxiety score and FKBP5 mRNA expression in the leukocytes across the menstrual cycle but there is no correlation between anxiety scores and FKBP5 mRNA.

  8. Identification of a Functionally Distinct Truncated BDNF mRNA Splice Variant and Protein in Trachemys scripta elegans

    PubMed Central

    Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

    2013-01-01

    Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein. PMID:23825634

  9. Cloning and expression analysis of prohibitin mRNA in canine mammary tumors

    PubMed Central

    MATSUYAMA, Satoshi; NAKANO, Yuko; NAKAMURA, Mieko; YAMAMOTO, Ryohei; SHIMADA, Terumasa; OHASHI, Fumihito; KUBO, Kihei

    2014-01-01

    Prohibitin is an antiproliferative protein that is a product of a putative tumor suppressor gene. However, there is little information on prohibitins in companion animals. In this study, we cloned canine prohibitin mRNA using RT-PCR and 3′-RACE (Rapid Amplification of cDNA Ends). The sequence was well conserved compared with those of other mammals, including human. The deduced amino acid sequence translated from the open reading frame completely corresponded to the human sequence. Canine prohibitin mRNA was expressed in all normal mammary and tumor samples examined. These results suggest that this protein plays a vital role in cell growth mechanisms and may be related to the occurrence of canine mammary tumors. PMID:25312047

  10. Cloning and expression analysis of prohibitin mRNA in canine mammary tumors.

    PubMed

    Matsuyama, Satoshi; Nakano, Yuko; Nakamura, Mieko; Yamamoto, Ryohei; Shimada, Terumasa; Ohashi, Fumihito; Kubo, Kihei

    2015-01-01

    Prohibitin is an antiproliferative protein that is a product of a putative tumor suppressor gene. However, there is little information on prohibitins in companion animals. In this study, we cloned canine prohibitin mRNA using RT-PCR and 3'-RACE (Rapid Amplification of cDNA Ends). The sequence was well conserved compared with those of other mammals, including human. The deduced amino acid sequence translated from the open reading frame completely corresponded to the human sequence. Canine prohibitin mRNA was expressed in all normal mammary and tumor samples examined. These results suggest that this protein plays a vital role in cell growth mechanisms and may be related to the occurrence of canine mammary tumors.

  11. Genome-wide analysis of microRNA and mRNA expression signatures in cancer

    PubMed Central

    Li, Ming-hui; Fu, Sheng-bo; Xiao, Hua-sheng

    2015-01-01

    Cancer is an extremely diverse and complex disease that results from various genetic and epigenetic changes such as DNA copy-number variations, mutations, and aberrant mRNA and/or protein expression caused by abnormal transcriptional regulation. The expression profiles of certain microRNAs (miRNAs) and messenger RNAs (mRNAs) are closely related to cancer progression stages. In the past few decades, DNA microarray and next-generation sequencing techniques have been widely applied to identify miRNA and mRNA signatures for cancers on a genome-wide scale and have provided meaningful insights into cancer diagnosis, prognosis and personalized medicine. In this review, we summarize the progress in genome-wide analysis of miRNAs and mRNAs as cancer biomarkers, highlighting their diagnostic and prognostic roles. PMID:26299954

  12. 14-3-3 protein binds to the low molecular weight neurofilament (NFL) mRNA 3' UTR.

    PubMed

    Ge, Wei-Wen; Volkening, Kathryn; Leystra-Lantz, Cheryl; Jaffe, Howard; Strong, Michael J

    2007-01-01

    We have previously reported that altered stability of low molecular weight neurofilament (NFL) mRNA in lumbar spinal cord homogenates in amyotrophic lateral sclerosis (ALS) is associated with altered expression of trans-acting 3' UTR mRNA binding proteins. We have identified two hexanucleotide motifs as the main cis elements and, using LC/MS/MS of peptide digests of NFL 3' UTR interacting proteins from human spinal cord, observed that 14-3-3 proteins interact with these motifs. 14-3-3 beta, zeta, tau, gamma, and eta isoforms were found to be expressed in human spinal cord. Each isoform was expressed in vitro and shown to interact with NFL 3' UTR mRNA. Mutation of one or both motifs resulted in decreased 14-3-3 interaction, changes in predicted mRNA structure or alteration in stability of the mRNA. These data show a novel interaction for 14-3-3 with NFL mRNA, and suggests that 14-3-3 may play a role in regulating NFL mRNA stability.

  13. Analysis of xanthine dehydrogenase mRNA levels in mutants affecting the expression of the rosy locus.

    PubMed Central

    Covington, M; Fleenor, D; Devlin, R B

    1984-01-01

    Xanthine dehydrogenase (XDH) mRNA levels were measured in a number of mutants and natural variants affecting XDH gene expression. Two variants, ry+4 and ry+10, contain cis-acting elements which map to a region flanking the 5' end of the XDH gene. Ry+4, which has 2-3 times more XDH protein than a wild type strain, has 3.2 times more XDH mRNA. Ry+10 has 50% of the wild type XDH level and 54% of the wild type XDH mRNA level. Three rosy mutants which map within the structural gene were also examined. Two of these had little if any XDH mRNA, but the third mutant had 1.3 times more XDH mRNA than wild type flies. Another mutant, ry2 , which contains no XDH protein and has a 9KB transposable element inserted into the XDH gene, has normal levels of XDH mRNA transcripts which are also the same size as those found in the wild type strain. Changes in XDH mRNA levels were measured during Drosophila development and found to parallel changes in the amount of XDH protein. In addition, there were no large changes in the size of XDH mRNA during development. Images PMID:6588363

  14. Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum

    PubMed Central

    Staudacher, Jonas J.; Naarmann-de Vries, Isabel S.; Ujvari, Stefanie J.; Klinger, Bertram; Kasim, Mumtaz; Benko, Edgar; Ostareck-Lederer, Antje; Ostareck, Dirk H.; Bondke Persson, Anja; Lorenzen, Stephan; Meier, Jochen C.; Blüthgen, Nils; Persson, Pontus B.; Henrion-Caude, Alexandra; Mrowka, Ralf; Fähling, Michael

    2015-01-01

    Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5′- and 3′-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5′- as well as 3′-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage. PMID:25753659

  15. Tristetraprolin (TTP): Interactions with mRNA and proteins, and current thoughts on mechanisms of action

    PubMed Central

    Brooks, Seth A.; Blackshear, Perry J.

    2013-01-01

    Changes in mRNA stability and translation are critical control points in the regulation of gene expression, particularly genes encoding growth factors, inflammatory mediators, and proto-oncogenes. Adenosine and uridine (AU)-rich elements (ARE), often located in the 3′ untranslated regions (3′UTR) of mRNAs, are known to target transcripts for rapid decay. They are also involved in the regulation of mRNA stability and translation in response to extracellular cues. This review focuses on one of the best characterized ARE binding proteins, tristetraprolin (TTP), the founding member of a small family of CCCH tandem zinc finger proteins. In this survey, we have reviewed the current status of TTP interactions with mRNA and proteins, and discussed current thinking about TTP's mechanism of action to promote mRNA decay. We also review the proposed regulation of TTP's functions by phosphorylation. Finally, we have discussed emerging evidence for TTP operating as a translational regulator. PMID:23428348

  16. Protein and mRNA levels of YKL-40 in high-grade glioma.

    PubMed

    Kazakova, M H; Staneva, D N; Koev, I G; Staikov, D G; Mateva, N; Timonov, P T; Miloshev, G A; Sarafian, V S

    2014-01-01

    Malignant gliomas are the most common type of primary malignant brain tumours, characterized by extreme proliferation and aggressive invasion. There is evidence for over-expression of the YKL40 gene in high-grade gliomas. The high serum levels of the glycoprotein are associated with poor prognosis of various inflammatory and tumour processes. We investigated the YKL40 mRNA level and protein expression in the tumour site and in the serum of high-grade glioma patients. The YKL-40 expression in 36 patients with glial tumours (astrocytoma grade III, glioblastoma) and 33 age-matched healthy persons was measured by gene analysis, immunohistochemistry and ELISA. YKL-40 serum levels in high-grade glioma patients compared to healthy subjects were significantly increased (P ≤ 0.05). A wide range of variability in YKL40 mRNA expression was found. YKL-40 staining in situ was more abundant in glioblastoma tissue than in anaplastic astrocytoma, with the lowest level in normal brain tissue. Our gene analysis revealed that in general, YKL40 mRNA in glioma patients was over-expressed versus normal brain. A significant correlation between YKL40 transcript and protein levels was observed (P ≤ 0.05). It could be speculated that the YKL-40 protein might contribute to glioblastomas' specific biological characteristics that distinguish them from grade III gliomas. A complex investigation of YKL40 expression was performed at the molecular and cellular levels in human high-grade gliomas. Serum YKL-40 concentrations increased with tumour grade and correlated positively with transcript rate, being the highest in glioblastoma. We provide evidence for a relationship between YKL40 expression and the malignancy of glial tumours.

  17. Activated Human T Cells Express Alternative mRNA Transcripts Encoding a Secreted Form of RANKL

    PubMed Central

    Walsh, NC; Alexander, KA; Manning, CA; Karmakar, S; Wang, JF; Weyand, CM; Pettit, AR; Gravallese, EM

    2013-01-01

    Receptor activator of nuclear factor-kappaB -ligand (RANKL), encoded by the gene TNFSF11, is required for osteoclastogenesis, and its expression is upregulated in pathologic bone loss. Transcript variants of TNFSF11 mRNA have been described that encode a membrane-bound and a putative secreted form of RANKL. We identify a TNFSF11 transcript variant that extends the originally identified transcript encoding secreted RANKL. We demonstrate that this TNFSF11 transcript variant is expressed by the human osteosarcoma cell line, Saos-2, and by both primary human T cells and Jurkat T cells. Of relevance to the production of RANKL in pathologic bone loss, expression of this secreted TNFSF11 transcript is upregulated in Jurkat T cells and primary human T cells upon activation. Furthermore, this transcript can be translated and secreted in Jurkat T cells in vitro and is able to support osteoclast differentiation. Our data highlight the complexity of the TNFSF11 genomic locus and demonstrate the potential for the expression of alternate mRNA transcripts encoding membrane-bound and secreted forms of RANKL. Implications of alternate mRNA transcripts encoding different RANKL protein isoforms should be carefully considered and specifically examined in future studies, particularly those implicating RANKL in pathologic bone loss. PMID:23698708

  18. Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans.

    PubMed

    Maxwell, Colin S; Antoshechkin, Igor; Kurhanewicz, Nicole; Belsky, Jason A; Baugh, L Ryan

    2012-10-01

    Nutrient availability profoundly influences gene expression. Many animal genes encode multiple transcript isoforms, yet the effect of nutrient availability on transcript isoform expression has not been studied in genome-wide fashion. When Caenorhabditis elegans larvae hatch without food, they arrest development in the first larval stage (L1 arrest). Starved larvae can survive L1 arrest for weeks, but growth and post-embryonic development are rapidly initiated in response to feeding. We used RNA-seq to characterize the transcriptome during L1 arrest and over time after feeding. Twenty-seven percent of detectable protein-coding genes were differentially expressed during recovery from L1 arrest, with the majority of changes initiating within the first hour, demonstrating widespread, acute effects of nutrient availability on gene expression. We used two independent approaches to track expression of individual exons and mRNA isoforms, and we connected changes in expression to functional consequences by mining a variety of databases. These two approaches identified an overlapping set of genes with alternative isoform expression, and they converged on common functional patterns. Genes affecting mRNA splicing and translation are regulated by alternative isoform expression, revealing post-transcriptional consequences of nutrient availability on gene regulation. We also found that phosphorylation sites are often alternatively expressed, revealing a common mode by which alternative isoform expression modifies protein function and signal transduction. Our results detail rich changes in C. elegans gene expression as larvae initiate growth and post-embryonic development, and they provide an excellent resource for ongoing investigation of transcriptional regulation and developmental physiology.

  19. mRNA expression profile of serotonin receptor subtypes and distribution of serotonergic terminations in marmoset brain

    PubMed Central

    Shukla, Rammohan; Watakabe, Akiya; Yamamori, Tetsuo

    2014-01-01

    To better understand serotonin function in the primate brain, we examined the mRNA expression patterns of all the 13 members of the serotonin receptor (5HTR) family, by in situ hybridization (ISH) and the distribution of serotonergic terminations by serotonin transporter (SERT) protein immunohistochemical analysis. Ten of the 13 5HTRs showed significant mRNA expressions in the marmoset brain. Our study shows several new features of the organization of serotonergic systems in the marmoset brain. (1) The thalamus expressed only a limited number of receptor subtypes compared with the cortex, hippocampus, and other subcortical regions. (2) In the cortex, there are layer-selective and area-selective mRNA expressions of 5HTRs. (3) Highly localized mRNA expressions of 5HT1F and 5HT3A were observed. (4) There was a conspicuous overlap of the mRNA expressions of receptor subtypes known to have somatodendritic localization of receptor proteins with dense serotonergic terminations in the visual cortex, the central lateral (CL) nucleus of the thalamus, the presubiculum, and the medial mammillary nucleus of the hypothalamus. This suggests a high correlation between serotonin availability and receptor expression at these locations. (5) The 5HTRs show differences in mRNA expression pattern between the marmoset and mouse cortices whereas the patterns of both the species were much similar in the hippocampus. We discuss the possible roles of 5HTRs in the marmoset brain revealed by the analysis of their overall mRNA expression patterns. PMID:24904298

  20. Apigenin prevents UVB-induced cyclooxygenase 2 expression: coupled mRNA stabilization and translational inhibition.

    PubMed

    Tong, Xin; Van Dross, Rukiyah T; Abu-Yousif, Adnan; Morrison, Aubrey R; Pelling, Jill C

    2007-01-01

    Cyclooxygenase 2 (COX-2) is a key enzyme in the conversion of arachidonic acid to prostaglandins, and COX-2 overexpression plays an important role in carcinogenesis. Exposure to UVB strongly increased COX-2 protein expression in mouse 308 keratinocytes, and this induction was inhibited by apigenin, a nonmutagenic bioflavonoid that has been shown to prevent mouse skin carcinogenesis induced by both chemical carcinogens and UV exposure. Our previous study suggested that one pathway by which apigenin inhibits UV-induced and basal COX-2 expression is through modulation of USF transcriptional activity in the 5' upstream region of the COX-2 gene. Here, we found that apigenin treatment also increased COX-2 mRNA stability, and the inhibitory effect of apigenin on UVB-induced luciferase reporter gene activity was dependent on the AU-rich element of the COX-2 3'-untranslated region. Furthermore, we identified two RNA-binding proteins, HuR and the T-cell-restricted intracellular antigen 1-related protein (TIAR), which were associated with endogenous COX-2 mRNA in 308 keratinocytes, and apigenin treatment increased their localization to cell cytoplasm. More importantly, reduction of HuR levels by small interfering RNA inhibited apigenin-mediated stabilization of COX-2 mRNA. Cells expressing reduced TIAR showed marked resistance to apigenin's ability to inhibit UVB-induced COX-2 expression. Taken together, these results indicate that in addition to transcriptional regulation, another mechanism by which apigenin prevents COX-2 expression is through mediating TIAR suppression of translation.

  1. Apigenin Prevents UVB-Induced Cyclooxygenase 2 Expression: Coupled mRNA Stabilization and Translational Inhibition▿

    PubMed Central

    Tong, Xin; Van Dross, Rukiyah T.; Abu-Yousif, Adnan; Morrison, Aubrey R.; Pelling, Jill C.

    2007-01-01

    Cyclooxygenase 2 (COX-2) is a key enzyme in the conversion of arachidonic acid to prostaglandins, and COX-2 overexpression plays an important role in carcinogenesis. Exposure to UVB strongly increased COX-2 protein expression in mouse 308 keratinocytes, and this induction was inhibited by apigenin, a nonmutagenic bioflavonoid that has been shown to prevent mouse skin carcinogenesis induced by both chemical carcinogens and UV exposure. Our previous study suggested that one pathway by which apigenin inhibits UV-induced and basal COX-2 expression is through modulation of USF transcriptional activity in the 5′ upstream region of the COX-2 gene. Here, we found that apigenin treatment also increased COX-2 mRNA stability, and the inhibitory effect of apigenin on UVB-induced luciferase reporter gene activity was dependent on the AU-rich element of the COX-2 3′-untranslated region. Furthermore, we identified two RNA-binding proteins, HuR and the T-cell-restricted intracellular antigen 1-related protein (TIAR), which were associated with endogenous COX-2 mRNA in 308 keratinocytes, and apigenin treatment increased their localization to cell cytoplasm. More importantly, reduction of HuR levels by small interfering RNA inhibited apigenin-mediated stabilization of COX-2 mRNA. Cells expressing reduced TIAR showed marked resistance to apigenin's ability to inhibit UVB-induced COX-2 expression. Taken together, these results indicate that in addition to transcriptional regulation, another mechanism by which apigenin prevents COX-2 expression is through mediating TIAR suppression of translation. PMID:17074806

  2. Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells

    PubMed Central

    Kawasaki, Shunsuke; Fujita, Yoshihiko; Nagaike, Takashi; Tomita, Kozo

    2017-01-01

    Abstract 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. PMID:28525643

  3. 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.

  4. Polynucleotide phosphorylase hinders mRNA degradation upon ribosomal protein S1 overexpression in Escherichia coli

    PubMed Central

    Briani, Federica; Curti, Serena; Rossi, Francesca; Carzaniga, Thomas; Mauri, Pierluigi; Dehò, Gianni

    2008-01-01

    The exoribonuclease polynucleotide phosphorylase (PNPase, encoded by pnp) is a major player in bacterial RNA decay. In Escherichia coli, PNPase expression is post-transcriptionally regulated at the level of mRNA stability. The primary transcript is very efficiently processed by the endonuclease RNase III at a specific site and the processed pnp mRNA is rapidly degraded in a PNPase-dependent manner. While investigating the PNPase autoregulation mechanism we found, by UV-cross-linking experiments, that the ribosomal protein S1 in crude extracts binds to the pnp-mRNA leader region. We assayed the potential role of S1 protein in pnp gene regulation by modulating S1 expression from depletion to overexpression. We found that S1 depletion led to a sharp decrease of the amount of pnp and other tested mRNAs, as detected by Northern blotting, whereas S1 overexpression caused a strong stabilization of pnp and the other transcripts. Surprisingly, mRNA stabilization depended on PNPase, as it was not observed in a pnp deletion strain. PNPase-dependent stabilization, however, was not detected by chemical decay assay of bulk mRNA. Overall, our data suggest that PNPase exonucleolytic activity may be modulated by the translation potential of the target mRNAs and that, upon ribosomal protein S1 overexpression, PNPase protects from degradation a set of full-length mRNAs. It thus appears that a single mRNA species may be differentially targeted to either decay or PNPase-dependent stabilization, thus preventing its depletion in conditions of fast turnover. PMID:18824515

  5. Polynucleotide phosphorylase hinders mRNA degradation upon ribosomal protein S1 overexpression in Escherichia coli.

    PubMed

    Briani, Federica; Curti, Serena; Rossi, Francesca; Carzaniga, Thomas; Mauri, Pierluigi; Dehò, Gianni

    2008-11-01

    The exoribonuclease polynucleotide phosphorylase (PNPase, encoded by pnp) is a major player in bacterial RNA decay. In Escherichia coli, PNPase expression is post-transcriptionally regulated at the level of mRNA stability. The primary transcript is very efficiently processed by the endonuclease RNase III at a specific site and the processed pnp mRNA is rapidly degraded in a PNPase-dependent manner. While investigating the PNPase autoregulation mechanism we found, by UV-cross-linking experiments, that the ribosomal protein S1 in crude extracts binds to the pnp-mRNA leader region. We assayed the potential role of S1 protein in pnp gene regulation by modulating S1 expression from depletion to overexpression. We found that S1 depletion led to a sharp decrease of the amount of pnp and other tested mRNAs, as detected by Northern blotting, whereas S1 overexpression caused a strong stabilization of pnp and the other transcripts. Surprisingly, mRNA stabilization depended on PNPase, as it was not observed in a pnp deletion strain. PNPase-dependent stabilization, however, was not detected by chemical decay assay of bulk mRNA. Overall, our data suggest that PNPase exonucleolytic activity may be modulated by the translation potential of the target mRNAs and that, upon ribosomal protein S1 overexpression, PNPase protects from degradation a set of full-length mRNAs. It thus appears that a single mRNA species may be differentially targeted to either decay or PNPase-dependent stabilization, thus preventing its depletion in conditions of fast turnover.

  6. Variations in mRNA and protein levels of Ikaros family members in pediatric T cell acute lymphoblastic leukemia

    PubMed Central

    Mitchell, Julie L.

    2016-01-01

    Background Pediatric T cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous disease in which the cells share phenotypic characteristics with normal human thymocytes. The Ikaros family of transcription factors includes five members that are required for normal T cell development and are implicated in leukemogenesis. The goal of this work was to correlate the pattern of expression of Ikaros family members with the phenotype of the T-ALL cells. Methods We obtained twenty-four samples from pediatric T-ALL patients and used multi-parameter flow cytometry to characterize each sample, comparing the phenotype of the leukemic cells with normal human thymocytes. Then, we defined the expression levels of each Ikaros family member to determine whether the mRNA levels or splicing or protein levels were similar to the normal patterns seen during human T cell development. Results Multi-parameter analysis of the phenotype of T-ALL cells revealed that each patient’s cells were unique and could not be readily correlated with stages of T cell development. Similarly, the pattern of Ikaros expression varied among patients. In most patients, Ikaros mRNA was the dominant family member expressed, but some patients’ cells contained mostly Helios, Aiolos, or Eos mRNA. Despite that most patients had elevated mRNA levels of Ikaros family members and unique patterns of mRNA splicing, most patients had significantly reduced protein levels of Ikaros and Aiolos. Conclusions Our analysis of the cell phenotype and Ikaros expression levels in T-ALL cells revealed the extent of heterogeneity among patients. While it is rarely possible to trace leukemic cells to their developmental origin, we found distinct patterns of Ikaros family mRNA levels in groups of patients. Further, mRNA and protein levels of Ikaros and Aiolos did not correlate, indicating that mRNA and protein levels are regulated via distinct mechanisms. PMID:27826566

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

    PubMed

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

    1998-11-20

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

  8. Anesthesia for euthanasia influences mRNA expression in healthy mice and after traumatic brain injury.

    PubMed

    Staib-Lasarzik, Irina; Kriege, Oliver; Timaru-Kast, Ralph; Pieter, Dana; Werner, Christian; Engelhard, Kristin; Thal, Serge C

    2014-10-01

    Tissue sampling for gene expression analysis is usually performed under general anesthesia. Anesthetics are known to modulate hemodynamics, receptor-mediated signaling cascades, and outcome parameters. The present study determined the influence of anesthetic paradigms typically used for euthanization and tissue sampling on cerebral mRNA expression in mice. Naïve mice and animals with acute traumatic brain injury induced by controlled cortical impact (CCI) were randomized to the following euthanasia protocols (n=10-11/group): no anesthesia (NA), 1 min of 4 vol% isoflurane in room air (ISO), 3 min of a combination of 5 mg/kg midazolam, 0.05 mg/kg fentanyl, and 0.5 mg/kg medetomidine intraperitoneally (COMB), or 3 min of 360 mg/kg chloral hydrate intraperitoneally (CH). mRNA expression of actin-1-related gene (Act1), FBJ murine osteosarcoma viral oncogene homolog B (FosB), tumor necrosis factor alpha (TNFα), heat shock protein beta-1 (HspB1), interleukin (IL)-6, tight junction protein 1 (ZO-1), IL-1ß, cyclophilin A, micro RNA 497 (miR497), and small cajal body-specific RNA 17 were determined by real-time polymerase chain reaction (PCR) in hippocampus samples. In naïve animals, Act1 expression was downregulated in the CH group compared with NA. FosB expression was downregulated in COMB and CH groups compared with NA. CCI reduced Act1 and FosB expression, whereas HspB1 and TNFα expression increased. After CCI, HspB1 expression was significantly higher in ISO, COMB, and CH groups, and TNFα expression was elevated in ISO and COMB groups. MiR497, IL-6, and IL-1ß were upregulated after CCI but not affected by anesthetics. Effects were independent of absolute mRNA copy numbers. The data demonstrate that a few minutes of anesthesia before tissue sampling are sufficient to induce immediate mRNA changes, which seem to predominate in the early-regulated gene cluster. Anesthesia-related effects on gene expression might explain limited reproduciblity of real

  9. Anesthesia for Euthanasia Influences mRNA Expression in Healthy Mice and after Traumatic Brain Injury

    PubMed Central

    Staib-Lasarzik, Irina; Kriege, Oliver; Timaru-Kast, Ralph; Pieter, Dana; Werner, Christian; Engelhard, Kristin

    2014-01-01

    Abstract Tissue sampling for gene expression analysis is usually performed under general anesthesia. Anesthetics are known to modulate hemodynamics, receptor-mediated signaling cascades, and outcome parameters. The present study determined the influence of anesthetic paradigms typically used for euthanization and tissue sampling on cerebral mRNA expression in mice. Naïve mice and animals with acute traumatic brain injury induced by controlled cortical impact (CCI) were randomized to the following euthanasia protocols (n=10–11/group): no anesthesia (NA), 1 min of 4 vol% isoflurane in room air (ISO), 3 min of a combination of 5 mg/kg midazolam, 0.05 mg/kg fentanyl, and 0.5 mg/kg medetomidine intraperitoneally (COMB), or 3 min of 360 mg/kg chloral hydrate intraperitoneally (CH). mRNA expression of actin-1-related gene (Act1), FBJ murine osteosarcoma viral oncogene homolog B (FosB), tumor necrosis factor alpha (TNFα), heat shock protein beta-1 (HspB1), interleukin (IL)-6, tight junction protein 1 (ZO-1), IL-1ß, cyclophilin A, micro RNA 497 (miR497), and small cajal body-specific RNA 17 were determined by real-time polymerase chain reaction (PCR) in hippocampus samples. In naïve animals, Act1 expression was downregulated in the CH group compared with NA. FosB expression was downregulated in COMB and CH groups compared with NA. CCI reduced Act1 and FosB expression, whereas HspB1 and TNFα expression increased. After CCI, HspB1 expression was significantly higher in ISO, COMB, and CH groups, and TNFα expression was elevated in ISO and COMB groups. MiR497, IL-6, and IL-1ß were upregulated after CCI but not affected by anesthetics. Effects were independent of absolute mRNA copy numbers. The data demonstrate that a few minutes of anesthesia before tissue sampling are sufficient to induce immediate mRNA changes, which seem to predominate in the early-regulated gene cluster. Anesthesia-related effects on gene expression might explain limited

  10. Analysis of global mRNA expression in human skeletal muscle during recovery from endurance exercise.

    PubMed

    Mahoney, D J; Parise, G; Melov, S; Safdar, A; Tarnopolsky, M A

    2005-09-01

    To search for novel transcriptional pathways that are activated in skeletal muscle after endurance exercise, we used cDNA microarrays to measure global mRNA expression after an exhaustive bout of high-intensity cycling (approximately 75 min). Healthy, young, sedentary males performed the cycling bout, and skeletal muscle biopsies were taken from the vastus lateralis before, and at 3 and 48 h after exercise. We examined mRNA expression in individual muscle samples from four subjects using cDNA microarrays, used repeated-measures significance analysis of microarray (SAM) to determine statistically significant expression changes, and confirmed selected results using real-time RT-PCR. In total, the expression of 118 genes significantly increased 3 h postcycling and 8 decreased. At 48 h, the expression of 29 genes significantly increased and 5 decreased. Many of these are potentially important novel genes involved in exercise recovery and adaptation, including several involved in 1) metabolism and mitochondrial biogenesis (FOXO1, PPARdelta, PPARgamma, nuclear receptor binding protein 2, IL-6 receptor, ribosomal protein L2, aminolevulinate delta-synthase 2); 2) the oxidant stress response (metalothioneins 1B, 1F, 1G, 1H, 1L, 2A, 3, interferon regulatory factor 1); and 3) electrolyte transport across membranes [Na+-K+-ATPase (beta3), SERCA3, chloride channel 4]. Others include genes involved in cell stress, proteolysis, apoptosis, growth, differentiation, and transcriptional activation, as well as all three nuclear receptor subfamily 4A family members (Nur77, Nurr1, and Nor1). This study is the first to characterize global mRNA expression during recovery from endurance exercise, and the results provide potential insight into 1) the transcriptional contributions to homeostatic recovery in human skeletal muscle after endurance exercise, and 2) the transcriptional contributions from a single bout of endurance exercise to the adaptive processes that occur after a period of

  11. 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.

  12. Combining miRNA and mRNA Expression Profiles in Wilms Tumor Subtypes

    PubMed Central

    Ludwig, Nicole; Werner, Tamara V.; Backes, Christina; Trampert, Patrick; Gessler, Manfred; Keller, Andreas; Lenhof, Hans-Peter; Graf, Norbert; Meese, Eckart

    2016-01-01

    Wilms tumor (WT) is the most common childhood renal cancer. Recent findings of mutations in microRNA (miRNA) processing proteins suggest a pivotal role of miRNAs in WT genesis. We performed miRNA expression profiling of 36 WTs of different subtypes and four normal kidney tissues using microarrays. Additionally, we determined the gene expression profile of 28 of these tumors to identify potentially correlated target genes and affected pathways. We identified 85 miRNAs and 2107 messenger RNAs (mRNA) differentially expressed in blastemal WT, and 266 miRNAs and 1267 mRNAs differentially expressed in regressive subtype. The hierarchical clustering of the samples, using either the miRNA or mRNA profile, showed the clear separation of WT from normal kidney samples, but the miRNA pattern yielded better separation of WT subtypes. A correlation analysis of the deregulated miRNA and mRNAs identified 13,026 miRNA/mRNA pairs with inversely correlated expression, of which 2844 are potential interactions of miRNA and their predicted mRNA targets. We found significant upregulation of miRNAs-183, -301a/b and -335 for the blastemal subtype, and miRNAs-181b, -223 and -630 for the regressive subtype. We found marked deregulation of miRNAs regulating epithelial to mesenchymal transition, especially in the blastemal subtype, and miRNAs influencing chemosensitivity, especially in regressive subtypes. Further research is needed to assess the influence of preoperative chemotherapy and tumor infiltrating lymphocytes on the miRNA and mRNA patterns in WT. PMID:27043538

  13. Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii

    PubMed Central

    Onishi, Masayuki; Pringle, John R.

    2016-01-01

    The unicellular green alga Chlamydomonas reinhardtii is a model organism that provides an opportunity to understand the evolution and functional biology of the lineage that includes the land plants, as well as aspects of the fundamental core biology conserved throughout the eukaryotic phylogeny. Although many tools are available to facilitate genetic, molecular biological, biochemical, and cell biological studies in Chlamydomonas, expression of unselected transgenes of interest (GOIs) has been challenging. In most methods used previously, the GOI and a selectable marker are expressed from two separate mRNAs, so that their concomitant expression is not guaranteed. In this study, we developed constructs that allow expression of an upstream GOI and downstream selectable marker from a single bicistronic mRNA. Although this approach in other systems has typically required a translation-enhancing element such as an internal ribosome entry site for the downstream marker, we found that a short stretch of unstructured junction sequence was sufficient to obtain adequate expression of the downstream gene, presumably through post-termination reinitiation. With this system, we obtained robust expression of both endogenous and heterologous GOIs, including fluorescent proteins and tagged fusion proteins, in the vast majority of transformants, thus eliminating the need for tedious secondary screening for GOI-expressing transformants. This improved efficiency should greatly facilitate a variety of genetic and cell-biological studies in Chlamydomonas and also enable new applications such as expression-based screens and large-scale production of foreign proteins. PMID:27770025

  14. 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.

  15. The increased mucosal mRNA expressions of complement C3 and interleukin-17 in inflammatory bowel disease

    PubMed Central

    Sugihara, T; Kobori, A; Imaeda, H; Tsujikawa, T; Amagase, K; Takeuchi, K; Fujiyama, Y; Andoh, A

    2010-01-01

    Recent studies have demonstrated that the complement system participates in the regulation of T cell functions. To address the local biosynthesis of complement components in inflammatory bowel disease (IBD) mucosa, we investigated C3 and interleukin (IL)-17 mRNA expression in mucosal samples obtained from patients with IBD. The molecular mechanisms underlying C3 induction were investigated in human colonic subepithelial myofibroblasts (SEMFs). IL-17 and C3 mRNA expressions in the IBD mucosa were evaluated by real-time polymerase chain reaction. The C3 levels in the supernatant were determined by enzyme-linked immunosorbent assay. IL-17 and C3 mRNA expressions were elevated significantly in the active lesions from ulcerative colitis (UC) and Crohn's disease (CD) patients. There was a significant positive correlation between IL-17 and C3 mRNA expression in the IBD mucosa. IL-17 stimulated a dose- and time-dependent increase in C3 mRNA expression and C3 secretion in colonic SEMFs. The C3 molecules secreted by colonic SEMFs were a 115-kDa α-chain linked to a 70-kDa β-chain by disulphide bonds, which was identical to serum C3. The IL-17-induced C3 mRNA expression was blocked by p42/44 mitogen-activated protein kinase (MAPK) inhibitors (PD98059 and U0216) and a p38 MAPK inhibitor (SB203580). Furthermore, IL-17-induced C3 mRNA expression was inhibited by an adenovirus containing a stable mutant form of IκBα. C3 and IL-17 mRNA expressions are enhanced, with a strong correlation, in the inflamed mucosa of IBD patients. Part of these clinical findings was considered to be mediated by the colonic SEMF response to IL-17. PMID:20089077

  16. Regulation of mRNA abundance in activated T lymphocytes: identification of mRNA species affected by the inhibition of protein synthesis.

    PubMed Central

    Coleclough, C; Kuhn, L; Lefkovits, I

    1990-01-01

    Inhibition of protein synthesis has often been observed to increase the concentration of mRNAs that encode proteins associated with the regulation of cell division. As two-dimensional gel electrophoresis permits the simultaneous monitoring of individual elements in large populations of gene products, we have used this technique to assess the effect of cycloheximide treatment on the mRNA complement of activated mouse T cells in an objective fashion. Two-dimensional gels of proteins generated by cell-free translation of mRNA from T-cell blasts display about 400 spots; only 5 of these are reproducibly enhanced by cycloheximide treatment and about 4 are diminished. The cDNA cloning vector lambda jac allows analysis of large arrays of molecular clones by cell-free expression, and we have used it in a sibling selection scheme to isolate a clone of one of the prominently induced mRNA species, which we refer to as chx1. chx1 mRNA concentration is increased by cycloheximide treatment of activated B cells, as well as T cells, and it is rapidly and transiently induced, in a cycloheximide-enhanced manner, upon serum stimulation of resting 3T3 fibroblastoid cells. The chx1 protein is hydrophilic, is slightly basic, and has patches of homology with the Jun-D gene product. The chx1 gene is remarkable in its lack of detectable introns and of strong bias against CpG dinucleotides. Images PMID:2308934

  17. 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

    SciTech Connect

    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 and 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.

  18. An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1

    PubMed Central

    AbuQattam, Ali; Gallego, José; Rodríguez-Navarro, Susana

    2016-01-01

    Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. Unlike most yeast genes, the SUS1 pre-mRNA of Saccharomyces cerevisiae contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. Using in silico analyses together with NMR spectroscopy, gel electrophoresis, and UV thermal denaturation experiments, we show that the downstream intron (I2) of SUS1 forms a weakly stable, 37-nucleotide stem–loop structure containing the branch site near its apical loop and the 3′ splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semiquantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. This work demonstrates that I2 structure is relevant for SUS1 expression, and that this effect is likely exerted through modulation of splicing. PMID:26546116

  19. An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1.

    PubMed

    AbuQattam, Ali; Gallego, José; Rodríguez-Navarro, Susana

    2016-01-01

    Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. Unlike most yeast genes, the SUS1 pre-mRNA of Saccharomyces cerevisiae contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. Using in silico analyses together with NMR spectroscopy, gel electrophoresis, and UV thermal denaturation experiments, we show that the downstream intron (I2) of SUS1 forms a weakly stable, 37-nucleotide stem-loop structure containing the branch site near its apical loop and the 3' splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semiquantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. This work demonstrates that I2 structure is relevant for SUS1 expression, and that this effect is likely exerted through modulation of splicing.

  20. Co-evolution of Bacterial Ribosomal Protein S15 with Diverse mRNA Regulatory Structures.

    PubMed

    Slinger, Betty L; Newman, Hunter; Lee, Younghan; Pei, Shermin; Meyer, Michelle M

    2015-12-01

    RNA-protein interactions are critical in many biological processes, yet how such interactions affect the evolution of both partners is still unknown. RNA and protein structures are impacted very differently by mechanisms of genomic change. While most protein families are identifiable at the nucleotide level across large phylogenetic distances, RNA families display far less nucleotide similarity and are often only shared by closely related bacterial species. Ribosomal protein S15 has two RNA binding functions. First, it is a ribosomal protein responsible for organizing the rRNA during ribosome assembly. Second, in many bacterial species S15 also interacts with a structured portion of its own transcript to negatively regulate gene expression. While the first interaction is conserved in most bacteria, the second is not. Four distinct mRNA structures interact with S15 to enable regulation, each of which appears to be independently derived in different groups of bacteria. With the goal of understanding how protein-binding specificity may influence the evolution of such RNA regulatory structures, we examine whether examples of these mRNA structures are able to interact with, and regulate in response to, S15 homologs from organisms containing distinct mRNA structures. We find that despite their shared RNA binding function in the rRNA, S15 homologs have distinct RNA recognition profiles. We present a model to explain the specificity patterns observed, and support this model by with further mutagenesis. After analyzing the patterns of conservation for the S15 protein coding sequences, we also identified amino acid changes that alter the binding specificity of an S15 homolog. In this work we demonstrate that homologous RNA-binding proteins have different specificity profiles, and minor changes to amino acid sequences, or to RNA structural motifs, can have large impacts on RNA-protein recognition.

  1. Co-evolution of Bacterial Ribosomal Protein S15 with Diverse mRNA Regulatory Structures

    PubMed Central

    Slinger, Betty L.; Newman, Hunter; Lee, Younghan; Pei, Shermin; Meyer, Michelle M.

    2015-01-01

    RNA-protein interactions are critical in many biological processes, yet how such interactions affect the evolution of both partners is still unknown. RNA and protein structures are impacted very differently by mechanisms of genomic change. While most protein families are identifiable at the nucleotide level across large phylogenetic distances, RNA families display far less nucleotide similarity and are often only shared by closely related bacterial species. Ribosomal protein S15 has two RNA binding functions. First, it is a ribosomal protein responsible for organizing the rRNA during ribosome assembly. Second, in many bacterial species S15 also interacts with a structured portion of its own transcript to negatively regulate gene expression. While the first interaction is conserved in most bacteria, the second is not. Four distinct mRNA structures interact with S15 to enable regulation, each of which appears to be independently derived in different groups of bacteria. With the goal of understanding how protein-binding specificity may influence the evolution of such RNA regulatory structures, we examine whether examples of these mRNA structures are able to interact with, and regulate in response to, S15 homologs from organisms containing distinct mRNA structures. We find that despite their shared RNA binding function in the rRNA, S15 homologs have distinct RNA recognition profiles. We present a model to explain the specificity patterns observed, and support this model by with further mutagenesis. After analyzing the patterns of conservation for the S15 protein coding sequences, we also identified amino acid changes that alter the binding specificity of an S15 homolog. In this work we demonstrate that homologous RNA-binding proteins have different specificity profiles, and minor changes to amino acid sequences, or to RNA structural motifs, can have large impacts on RNA-protein recognition. PMID:26675164

  2. Protein structure protection commits gene expression patterns.

    PubMed

    Chen, Jianping; Liang, Han; Fernández, Ariel

    2008-01-01

    Gene co-expressions often determine module-defining spatial and temporal concurrences of proteins. Yet, little effort has been devoted to tracing coordinating signals for expression correlations to the three-dimensional structures of gene products. We performed a global structure-based analysis of the yeast and human proteomes and contrasted this information against their respective transcriptome organizations obtained from comprehensive microarray data. We show that protein vulnerability quantifies dosage sensitivity for metabolic adaptation phases and tissue-specific patterns of mRNA expression, determining the extent of co-expression similarity of binding partners. The role of protein intrinsic disorder in transcriptome organization is also delineated by interrelating vulnerability, disorder propensity and co-expression patterns. Extremely vulnerable human proteins are shown to be subject to severe post-transcriptional regulation of their expression through significant micro-RNA targeting, making mRNA levels poor surrogates for protein-expression levels. By contrast, in yeast the expression of extremely under-wrapped proteins is likely regulated through protein aggregation. Thus, the 85 most vulnerable proteins in yeast include the five confirmed prions, while in human, the genes encoding extremely vulnerable proteins are predicted to be targeted by microRNAs. Hence, in both vastly different organisms protein vulnerability emerges as a structure-encoded signal for post-transcriptional regulation. Vulnerability of protein structure and the concurrent need to maintain structural integrity are shown to quantify dosage sensitivity, compelling gene expression patterns across tissue types and temporal adaptation phases in a quantifiable manner. Extremely vulnerable proteins impose additional constraints on gene expression: They are subject to high levels of regulation at the post-transcriptional level.

  3. A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export.

    PubMed

    Gong, Danyang; Kim, Yong Hoon; Xiao, Yuchen; Du, Yushen; Xie, Yafang; Lee, Kevin K; Feng, Jun; Farhat, Nisar; Zhao, Dawei; Shu, Sara; Dai, Xinghong; Chanda, Sumit K; Rana, Tariq M; Krogan, Nevan J; Sun, Ren; Wu, Ting-Ting

    2016-11-09

    Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication.

  4. Antipsychotics increase microtubule-associated protein 2 mRNA but not spinophilin mRNA in rat hippocampus and cortex.

    PubMed

    Law, Amanda J; Hutchinson, Louise J; Burnet, Philip W J; Harrison, Paul J

    2004-05-01

    Antipsychotic (neuroleptic) drugs induce structural alterations in synaptic terminals and changes in the expression of presynaptic protein genes. Whether there are also changes in corresponding postsynaptic (dendritic) markers has not been determined. We describe the effect of 14-day treatment with typical (haloperidol, chlorpromazine) or atypical (clozapine, olanzapine, risperidone) antipsychotics on the expression of two dendritic protein genes, microtubule-associated protein 2 (MAP2) and spinophilin, using in situ hybridization, in the rat hippocampus, retrosplenial, and occipitoparietal cortices. MAP2 mRNA was increased modestly in the dentate gyrus and retrosplenial cortex by chlorpromazine, risperidone, and olanzapine and in the occipitoparietal cortex by chlorpromazine, haloperidol, and risperidone. None of the antipsychotics affected spinophilin mRNA in any area. Overall, these results show a modulation of MAP2 gene expression, likely reflecting functional or structural changes in the dendritic tree in response to some typical and atypical antipsychotics. The lack of change in spinophilin mRNA suggests that dendritic spines are not affected selectively by the drugs. The data provide further evidence that antipsychotics regulate genes involved in synaptic structure and function. Such actions may underlie their long-term effects on neural plasticity in areas of the brain implicated in the pathology of schizophrenia. Copyright 2004 Wiley-Liss, Inc.

  5. Translational signatures and mRNA levels are highly correlated in human stably expressed genes.

    PubMed

    Line, Sergio R P; Liu, Xiaoming; de Souza, Ana Paula; Yu, Fuli

    2013-04-19

    Gene expression is one of the most relevant biological processes of living cells. Due to the relative small population sizes, it is predicted that human gene sequences are not strongly influenced by selection towards expression efficiency. One of the major problems in estimating to what extent gene characteristics can be selected to maximize expression efficiency is the wide variation that exists in RNA and protein levels among physiological states and different tissues. Analyses of datasets of stably expressed genes (i.e. with consistent expression between physiological states and tissues) would provide more accurate and reliable measurements of associations between variations of a specific gene characteristic and expression, and how distinct gene features work to optimize gene expression. Using a dataset of human genes with consistent expression between physiological states we selected gene sequence signatures related to translation that can predict about 42% of mRNA variation. The prediction can be increased to 51% when selecting genes that are stably expressed in more than 1 tissue. These genes are enriched for translation and ribosome biosynthesis processes and have higher translation efficiency scores, smaller coding sequences and 3' UTR sizes and lower folding energies when compared to other datasets. Additionally, the amino acid frequencies weighted by expression showed higher correlations with isoacceptor tRNA gene copy number, and smaller absolute correlation values with biosynthetic costs. Our results indicate that human gene sequence characteristics related to transcription and translation processes can co-evolve in an integrated manner in order to optimize gene expression.

  6. Isoeugenol destabilizes IL-8 mRNA expression in THP-1 cells through induction of the negative regulator of mRNA stability tristetraprolin.

    PubMed

    Galbiati, Valentina; Carne, Alice; Mitjans, Montserrat; Galli, Corrado Lodovico; Marinovich, Marina; Corsini, Emanuela

    2012-02-01

    We previously demonstrated in the human promyelocytic cell line THP-1 that all allergens tested, with the exception of the prohapten isoeugenol, induced a dose-related release of interleukin-8 (IL-8). In the present study, we investigated whether this abnormal behavior was regulated by the AU-rich element-binding proteins HuR and tristetraprolin (TTP) or by the downstream molecule suppressor of cytokine signaling (SOCS)-3. The contact allergens isoeugenol, diethylmaleate (DEM), and 2,4-dinitrochlorobenzene (DNCB), and the irritant salicylic acid were used as reference compounds. Chemicals were used at concentrations that induced a 20% decrease in cell viability as assessed by propidium iodide staining, namely 100 μg/ml (0.61 mM) for isoeugenol, 100 μg/ml (0.58 mM) for DEM, 3 μg/ml (14.8 μM) for DNCB, and 250 μg/ml (1.81 mM) for salicylic acid. Time course experiments of IL-8 mRNA expression and assessment of IL-8 mRNA half-life, indicated a decreased IL-8 mRNA stability in isoeugenol-treated cells. We could demonstrate that a combination and regulation of HuR and TTP following exposure to contact allergens resulted in a different modulation of IL-8 mRNA half-life and release. The increased expression of TTP in THP-1 cells treated with isoeugenol results in destabilization of the IL-8 mRNA, which can account for the lack of IL-8 release. In contrast, the strong allergen DNCB failing to up-regulate TTP, while inducing HuR, resulted in longer IL-8 mRNA half-life and protein release. SOCS-3 was induced only in isoeugenol-treated cells; however, its modulation did not rescue the lack of IL-8 release, indicating that it is unlikely to be involved in the lack of IL-8 production. Finally, the destabilization effect of isoeugenol on IL-8 mRNA expression together with SOCS-3 expression resulted in an anti-inflammatory effect, as demonstrated by the ability of isoeugenol to modulate LPS or ionomycin-induced cytokine release.

  7. Cytoplasmic-nuclear shuttling of the urokinase mRNA binding protein regulates message stability.

    PubMed

    Shetty, Sreerama

    2002-08-01

    Treatment of small airway epithelial (SAEC) cells or lung epithelial (Beas2B) cells with TNF-alpha or PMA induces urokinase-type plasminogen activator (uPA) expression. Treatment of these cells with TNF-alpha, PMA or cycloheximide but not TGF-beta increased steady-state expression of uPAmRNA. TNF-alpha, PMA or cycloheximide caused 8-10 fold extensions of the uPAmRNA half-life in SAEC or Beas2B cells treated with DRB, a transcriptional inhibitor. These findings suggest that uPA gene expression involves a post-transcriptional regulatory mechanism. Using gel mobility shift and UV cross-linking assays, we identified a 30 kDa uPA mRNA binding protein (uPA mRNABp) that selectively binds to a 66 nt protein binding fragment of uPA mRNA containing regulatory information for message stabilization. Binding of cytoplasmic uPA mRNABp to uPA mRNA was abolished after treatment with TNF-alpha but not TGF-beta. In addition, we found the accumulation of 30 kDa uPAmRNABp in the nuclear extracts of TNF-alpha but not TGF-beta treated cells. The uPA mRNABp starts moving to the nucleus from the cytoplasmic compartment as early as three hours after TNF-alpha treatment. Complete translocation is achieved between 12-24 h, which coincides with the maximal expression of uPA protein effected by cytokine stimulation. Treatment of Beas2B cells with NaF inhibited TNF-alpha-mediated translocation of uPA mRNABp from the cytoplasm to the nucleus and concomitant inhibition of uPA expression. TNF-alpha stabilizes uPA mRNA by translocating the uPA mRNABp from the cytoplasm to the nucleus. Our results demonstrate a novel mechanism governing uPA mRNA stability through shuttling of uPA mRNABp between the nucleus and cytoplasm. This newly identified pathway may have evolved to regulate uPA-mediated functions of the lung epithelium in inflamation or neoplasia.

  8. 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.

  9. Localized mRNA translation and protein association

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2014-08-01

    Recent direct observations of localization of mRNAs and proteins both in prokaryotic and eukaryotic cells can be related to slowdown of diffusion of these species due to macromolecular crowding and their ability to aggregate and form immobile or slowly mobile complexes. Here, a generic kinetic model describing both these factors is presented and comprehensively analyzed. Although the model is non-linear, an accurate self-consistent analytical solution of the corresponding reaction-diffusion equation has been constructed, the types of localized protein distributions have been explicitly shown, and the predicted kinetic regimes of gene expression have been classified.

  10. Reduction of polyhedrin mRNA and protein expression levels in Sf9 and Hi5 cell lines, but not in Sf21 cells, infected with Autographa californica multiple nucleopolyhedrovirus fp25k mutants.

    PubMed

    Cheng, Xin-Hua; Hillman, Christopher C; Zhang, Chuan-Xi; Cheng, Xiao-Wen

    2013-01-01

    During cell infection, the fp25k gene of baculoviruses frequently mutates, producing the few polyhedra (FP) per cell phenotype with reduced polyhedrin (polh) expression levels compared with wild-type baculoviruses. Here we report that the fp25k gene of the model baculovirus, Autographa cali