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Sample records for affecting mrna expression

  1. 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

  2. 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

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

    PubMed Central

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

    2013-01-01

    Variants within the gene cluster encoding α3, α5, and β4 nicotinic receptor subunits are major risk factors for substance dependence. The strongest impact on risk is associated with variation in the CHRNA5 gene, where at least two mechanisms are at work: amino acid variation and altered mRNA expression levels. The risk allele of the non-synonymous variant (rs16969968; D398N) primarily occurs on the haplotype containing the low mRNA expression allele. In populations of European ancestry, there are approximately 50 highly correlated variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster and the adjacent PSMA4 gene region that are associated with CHRNA5 mRNA levels. It is not clear which of these variants contribute to the changes in CHRNA5 transcript level. Because populations of African ancestry have reduced linkage disequilibrium among variants spanning this gene cluster, eQTL mapping in subjects of African ancestry could potentially aid in defining the functional variants that affect CHRNA5 mRNA levels. We performed quantitative allele specific gene expression using frontal cortices derived from 49 subjects of African ancestry and 111 subjects of European ancestry. This method measures allele-specific transcript levels in the same individual, which eliminates other biological variation that occurs when comparing expression levels between different samples. This analysis confirmed that substance dependence associated variants have a direct cis-regulatory effect on CHRNA5 transcript levels in human frontal cortices of African and European ancestry and identified 10 highly correlated variants, located in a 9 kb region, that are potential functional variants modifying CHRNA5 mRNA expression levels. PMID:24303001

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

    PubMed

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

    2013-01-01

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

  5. Amphotericin B severely affects expression and activity of the endothelial constitutive nitric oxide synthase involving altered mRNA stability

    PubMed Central

    Suschek, Christoph Viktor; Bonmann, Eckhard; Kleinert, Hartmut; Wenzel, Michael; Mahotka, Csaba; Kolb, Hubert; Förstermann, Ulrich; Gerharz, Claus-Dieter; Kolb-Bachofen, Victoria

    2000-01-01

    The therapeutic use of the antifungal drug amphotericin B (AmB) is limited due to severe side effects like glomerular vasoconstriction and risk of renal failure during AmB administration. As nitric oxide (NO) has substantial functions in renal autoregulation, we have determined the effects of AmB on endothelial constitutive NO synthase (ecNOS) expression and activity in human and rat endothelial cell cultures.AmB used at concentrations of 0.6 to 1.25 μg ml−1 led to increases in ecNOS mRNA and protein expression as well as NO production. This was the result of an increased ecNOS mRNA half-life. In contrast, incubation of cells with higher albeit subtoxic concentrations of AmB (2.5–5.0 μg ml−1) resulted in a decrease or respectively in completely abolished ecNOS mRNA and protein expression with a strongly reduced or inhibited ecNOS activity, due to a decrease of ecNOS mRNA half-life. None of the AmB concentrations affected promoter activity as found with a reporter gene construct stably transfected into ECV304 cells.Thus, our experiments show a concentration-dependent biphasic effect of AmB on expression and activity of ecNOS, an effect best explained by AmB influencing ecNOS mRNA stability. In view of the known renal accumulation of this drug the results reported here could help to elucidate its renal toxicity. PMID:11015297

  6. Anguillicola crassus infection affects mRNA expression levels in gas gland tissue of European yellow and silver eel

    PubMed Central

    Schneebauer, Gabriel; Dirks, Ron P.

    2017-01-01

    Using Illumina sequencing, we investigated transcriptional changes caused by the nematode Anguillicola crassus within yellow and silver eels by comparing swimbladder samples of uninfected yellow with infected yellow eels, and uninfected silver with infected silver eels, respectively. In yellow eel gas gland, the infection caused a modification of steady state mRNA levels of 1675 genes, most of them being upregulated. Functional annotation analysis based on GO terms was used to categorize identified genes with regard to swimbladder metabolism or response to the infection. In yellow eels, the most prominent category was ‘immune response’, including various inflammatory components, complement proteins, and immunoglobulins. The elevated expression of several glucose and monocarboxylate transporters indicated an attempt to maintain the level of glucose metabolism, even in due to the infection thickened swimbladder tissue. In silver eel swimbladder tissue, on the contrary, the mRNA levels of only 291 genes were affected. Genes in the categories ‘glucose metabolism’ and ‘ROS metabolism’ barely responded to the infection and even the reaction of the immune system was much less pronounced compared to infected yellow eels. However, in the category ‘extracellular matrix’, the mRNA levels of several mucin genes were strongly elevated, suggesting increased mucus production as a defense reaction against the parasite. The present study revealed a strong reaction to an Anguillicola crassus infection on mRNA expression levels in swimbladder tissue of yellow eels, whereas in silver eels the changes ware almost negligible. A possible explanation for this difference is that the silvering process requires so much energy that there is not much scope to cope with the additional challenge of a nematode infection. Another possible explanation could be that gas-secreting activity of the silver eel swimbladder was largely reduced, which could coincide with a reduced

  7. Anguillicola crassus infection affects mRNA expression levels in gas gland tissue of European yellow and silver eel.

    PubMed

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

    2017-01-01

    Using Illumina sequencing, we investigated transcriptional changes caused by the nematode Anguillicola crassus within yellow and silver eels by comparing swimbladder samples of uninfected yellow with infected yellow eels, and uninfected silver with infected silver eels, respectively. In yellow eel gas gland, the infection caused a modification of steady state mRNA levels of 1675 genes, most of them being upregulated. Functional annotation analysis based on GO terms was used to categorize identified genes with regard to swimbladder metabolism or response to the infection. In yellow eels, the most prominent category was 'immune response', including various inflammatory components, complement proteins, and immunoglobulins. The elevated expression of several glucose and monocarboxylate transporters indicated an attempt to maintain the level of glucose metabolism, even in due to the infection thickened swimbladder tissue. In silver eel swimbladder tissue, on the contrary, the mRNA levels of only 291 genes were affected. Genes in the categories 'glucose metabolism' and 'ROS metabolism' barely responded to the infection and even the reaction of the immune system was much less pronounced compared to infected yellow eels. However, in the category 'extracellular matrix', the mRNA levels of several mucin genes were strongly elevated, suggesting increased mucus production as a defense reaction against the parasite. The present study revealed a strong reaction to an Anguillicola crassus infection on mRNA expression levels in swimbladder tissue of yellow eels, whereas in silver eels the changes ware almost negligible. A possible explanation for this difference is that the silvering process requires so much energy that there is not much scope to cope with the additional challenge of a nematode infection. Another possible explanation could be that gas-secreting activity of the silver eel swimbladder was largely reduced, which could coincide with a reduced responsiveness to other

  8. 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.

  9. 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.

  10. In vitro neuropeptide Y mRNA expressing model for screening essences that may affect appetite using Rolf B1.T cells.

    PubMed

    Chen, Shiau-Wei; Wu, Po-Ju; Chiang, Been-Huang

    2012-08-15

    Neuropeptide Y (NPY) is the most important appetite regulator. This study aimed to establish an in vitro NPY mRNA expression model for screening essences to determine if they are an appetite stimulator or inhibitor. We cultured the olfactory nerve cells Rolf B1.T for 2 days and then treated the cells with the known appetite inhibitor limonene and stimulator linalool. It was found that linalool could significantly stimulate NPY mRNA expression in 10 min, and limonene had the opposite effect. Similar results were also found in primary olfactory ensheathing cells isolated from rats. Further clinical trials using human subjects found that, when 10 min of treatment was applied, linalool indeed increased the serum NPY level in human peripheral blood. Limonene, on the other hand, decreased the serum NPY level. Thus, NPY mRNA expression in Rolf B1.T cells could be used as an in vitro model for screening essences that may affect appetite.

  11. High intensity interval training favourably affects antioxidant and inflammation mRNA expression in early-stage chronic kidney disease.

    PubMed

    Tucker, Patrick S; Briskey, David R; Scanlan, Aaron T; Coombes, Jeff S; Dalbo, Vincent J

    2015-12-01

    Increased levels of oxidative stress and inflammation have been linked to the progression of chronic kidney disease. To reduce oxidative stress and inflammation related to chronic kidney disease, chronic aerobic exercise is often recommended. Data suggests high intensity interval training may be more beneficial than traditional aerobic exercise. However, appraisals of differing modes of exercise, along with explanations of mechanisms responsible for observed effects, are lacking. This study assessed effects of eight weeks of high intensity interval training (85% VO2max), versus low intensity exercise (45-50% VO2max) and sedentary behaviour, in an animal model of early-stage chronic kidney disease. We examined kidney-specific mRNA expression of genes related to endogenous antioxidant enzyme activity (glutathione peroxidase 1; Gpx1, superoxide dismutase 1; Sod1, and catalase; Cat) and inflammation (kidney injury molecule 1; Kim1 and tumour necrosis factor receptor super family 1b; Tnfrsf1b), as well as plasma F2-isoprostanes, a marker of lipid peroxidation. Compared to sedentary behaviour, high intensity interval training resulted in increased mRNA expression of Sod1 (p=0.01) and Cat (p<0.001). Compared to low intensity exercise, high intensity interval training resulted in increased mRNA expression of Cat (p<0.001) and Tnfrsf1b (p=0.047). In this study, high intensity interval training was superior to sedentary behaviour and low intensity exercise as high intensity interval training beneficially influenced expression of genes related to endogenous antioxidant enzyme activity and inflammation.

  12. Selenium Deficiency Affects the mRNA Expression of Inflammatory Factors and Selenoprotein Genes in the Kidneys of Broiler Chicks.

    PubMed

    Zhang, Jiu-Li; Xu, Bo; Huang, Xiao-Dan; Gao, Yu-Hong; Chen, Yu; Shan, An-Shan

    2016-05-01

    The aim of this study was to investigate the influence of Se deficiency on the transcription of inflammatory factors and selenoprotein genes in the kidneys of broiler chicks. One hundred fifty 1-day-old broiler chicks were randomly assigned to two groups fed with either a low-Se diet (L group, 0.033 mg/kg Se) or an adequate Se diet (C group, 0.2 mg/kg Se). The levels of uric acid (UA) and creatinine (Cr) in the serum and the mRNA levels of 6 inflammatory factors and 25 selenoprotein genes in the kidneys were measured as the clinical signs of Se deficiency occurred at 20 days old. The results indicated that the contents of UA and Cr in the serum increased in L group (p < 0.05), and the mRNA levels of the inflammatory factors (NF-κB, iNOS, COX-2, and TNF-α) increased in L group (p < 0.05). Meanwhile, the mRNA levels of PTGEs and HO-1 were not changed. In addition, 25 selenoprotein transcripts displayed ubiquitous expression in the kidneys of the chicks. The mRNA levels of 14 selenoprotein genes (Dio1, Dio2, GPx3, Sepp1, SelH, SelI, SelK, Sepn1, SelO, SelW, Sep15, SelT, SelU, and SelS) decreased, and 9 selenoprotein genes (GPx1, GPx2, GPx4, SelPb, Txnrd1, Txnrd2, Txnrd3, SPS2, and SelM) increased in L group (p < 0.05), but the Dio3 and Sepx1 mRNA levels did not change. The results indicated that Se deficiency resulted in kidney dysfunction, activation of the NF-κB pathway, and a change in selenoprotein gene expression. The changes of inflammatory factor and selenoprotein gene expression levels were directly related to the abnormal renal functions induced by Se deficiency.

  13. Selective COX-2 inhibition affects fatty acids, but not COX mRNA expression in patients with FAP

    PubMed Central

    Larsen, Laila N.; Fausa, Olav; Bratlie, Jorunn; Høstmark, Arne T.; Aabakken, Lars

    2010-01-01

    Familial adenomatous polyposis (FAP) provides a model for sporadic colorectal cancer development. Cyclooxygenase (COX) inhibition may ameliorate polyp development, but rofecoxib was withdrawn due to cardiovascular side effects. Although this selective COX-2 inhibitor, like diet, may alter the fatty acid and eicosanoid pattern, data on the potential alteration in tissues after use, are scarce. The aims were to study if rofecoxib might influence the fatty acid distribution in serum phospholipids and duodenal lesions, mRNA for COX-1 and COX-2 in leucocytes and duodenal lesions, and finally plasma levels of PGE2 in a randomized, double-blind, placebo controlled study (n = 38). Significant reductions were found for essential fatty acid index both in serum phospholipids (P = 0.01, 95% CI = −0.9; −0.1), and in duodenal lesions (P = 0.04, 95 CI % = −0.9; −0.1) after treatment. No treatment effects were found on the COX mRNA expression, or in the plasma PGE2 levels. Dietary AA/EPA ratio was inversely associated with all the indicators of EFA status (all P < 0.01). These findings suggest that the effects of COX chemoprevention should be further investigated in FAP and that dietary needs should be included in the treatment of FAP. PMID:20593240

  14. 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

  15. Quantitative mapping shows that serotonin rather than dopamine receptor mRNA expressions are affected after repeated intermittent administration of MDMA in rat brain.

    PubMed

    Kindlundh-Högberg, Anna M S; Svenningsson, Per; Schiöth, Helgi B

    2006-09-01

    Ecstasy, (+/-)-3,4-methylenedioxy-metamphetamine (MDMA), is a popular recreational drug among young people. The present study aims to mimic MDMA intake among adolescents at dance clubs, taking repeated doses in the same evening on an intermittent basis. Male Sprague-Dawley rats received either 3x1 or 3x5 mg/kg/day (3 h apart) every seventh day during 4 weeks. We used real-time RT-PCR to determine the gene expression of serotonin 5HT1A, 5HT1B, 5HT2A, 5HT2C, 5HT3, 5HT6 receptors and dopamine D1, D2, D3 receptors in seven brain nuclei. The highest dose of MDMA extensively increased the 5HT1B-receptor mRNA in the cortex, caudate putamen, nucleus accumbens, and hypothalamus. The 5HT2A-receptor mRNA was reduced at the highest MDMA dose in the cortex. The 5HT2C mRNA was significantly increased in a dose-dependent manner in the cortex and the hypothalamus, as well as the 5HT3-receptor mRNA was in the hypothalamus. The 5HT6 mRNA level was increased in the forebrain cortex and the amygdala. Dopamine receptor mRNAs were only affected in the hypothalamus. In conclusion, this study provides evidence for a unique implication of serotonin rather than dopamine receptor mRNA levels, in response to repeated intermittent MDMA administration. We therefore suggest that serotonin regulated functions also primarily underlie repeated MDMA intake at rave parties.

  16. The mRNA expression and histological integrity in rat forebrain motor and sensory regions are minimally affected by acrylamide exposure through drinking water

    SciTech Connect

    Bowyer, John F.; Latendresse, John R.; Delongchamp, Robert R.; Warbritton, Alan R.; Thomas, Monzy; Divine, Becky; Doerge, Daniel R.

    2009-11-01

    A study was undertaken to determine whether alterations in the gene expression or overt histological signs of neurotoxicity in selected regions of the forebrain might occur from acrylamide exposure via drinking water. Gene expression at the mRNA level was evaluated by cDNA array and/or RT-PCR analysis in the striatum, substantia nigra and parietal cortex of rat after a 2-week acrylamide exposure. The highest dose tested (maximally tolerated) of approximately 44 mg/kg/day resulted in a significant decreased body weight, sluggishness, and locomotor activity reduction. These physiological effects were not accompanied by prominent changes in gene expression in the forebrain. All the expression changes seen in the 1200 genes that were evaluated in the three brain regions were <= 1.5-fold, and most not significant. Very few, if any, statistically significant changes were seen in mRNA levels of the more than 50 genes directly related to the cholinergic, noradrenergic, GABAergic or glutamatergic neurotransmitter systems in the striatum, substantia nigra or parietal cortex. All the expression changes observed in genes related to dopaminergic function were less than 1.5-fold and not statistically significant and the 5HT1b receptor was the only serotonin-related gene affected. Therefore, gene expression changes were few and modest in basal ganglia and sensory cortex at a time when the behavioral manifestations of acrylamide toxicity had become prominent. No histological evidence of axonal, dendritic or neuronal cell body damage was found in the forebrain due to the acrylamide exposure. As well, microglial activation was not present. These findings are consistent with the absence of expression changes in genes related to changes in neuroinflammation or neurotoxicity. Over all, these data suggest that oral ingestion of acrylamide in drinking water or food, even at maximally tolerable levels, induced neither marked changes in gene expression nor neurotoxicity in the motor and

  17. The mRNA expression and histological integrity in rat forebrain motor and sensory regions are minimally affected by acrylamide exposure through drinking water.

    PubMed

    Bowyer, John F; Latendresse, John R; Delongchamp, Robert R; Warbritton, Alan R; Thomas, Monzy; Divine, Becky; Doerge, Daniel R

    2009-11-01

    A study was undertaken to determine whether alterations in the gene expression or overt histological signs of neurotoxicity in selected regions of the forebrain might occur from acrylamide exposure via drinking water. Gene expression at the mRNA level was evaluated by cDNA array and/or RT-PCR analysis in the striatum, substantia nigra and parietal cortex of rat after a 2-week acrylamide exposure. The highest dose tested (maximally tolerated) of approximately 44 mg/kg/day resulted in a significant decreased body weight, sluggishness, and locomotor activity reduction. These physiological effects were not accompanied by prominent changes in gene expression in the forebrain. All the expression changes seen in the 1200 genes that were evaluated in the three brain regions were < or =1.5-fold, and most not significant. Very few, if any, statistically significant changes were seen in mRNA levels of the more than 50 genes directly related to the cholinergic, noradrenergic, GABAergic or glutamatergic neurotransmitter systems in the striatum, substantia nigra or parietal cortex. All the expression changes observed in genes related to dopaminergic function were less than 1.5-fold and not statistically significant and the 5HT1b receptor was the only serotonin-related gene affected. Therefore, gene expression changes were few and modest in basal ganglia and sensory cortex at a time when the behavioral manifestations of acrylamide toxicity had become prominent. No histological evidence of axonal, dendritic or neuronal cell body damage was found in the forebrain due to the acrylamide exposure. As well, microglial activation was not present. These findings are consistent with the absence of expression changes in genes related to changes in neuroinflammation or neurotoxicity. Over all, these data suggest that oral ingestion of acrylamide in drinking water or food, even at maximally tolerable levels, induced neither marked changes in gene expression nor neurotoxicity in the motor and

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

    PubMed

    Mukhopadhyay, Debdip; Priya, Pooja; Chattopadhyay, Ansuman

    2015-09-01

    Sodium fluoride (NaF), used as pesticides and for industrial purposes are deposited in the water bodies and therefore affects its biota. Zebrafish exposed to NaF in laboratory condition showed hyperactivity and frequent surfacing activity, somersaulting and vertical swimming pattern as compared to the control group. Reactive oxygen species level was elevated and glutathione level was depleted along with increased malondialdehyde content in the brain. Levels of glutathione-s-transferase (GST), catalase (CAT) and superoxide dismutase were also elevated in the treatment groups. Expression of mRNA of nuclear factor erythroid 2 related factor 2 (Nrf2) and its inhibitor Kelch-like ECH-associated protein 1 (Keap1) during stress condition were observed along with Gst, Cat, NADPH: quinone oxidoreductase 1(Nqo1) and p38. Except Keap1, all other genes exhibited elevated expression. Nrf2/Keap1 proteins had similar expression pattern as their corresponding mRNA. The findings in this study might help to understand the molecular mechanism of fluoride induced neurotoxicity in fish. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. 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.

  20. Altered expression of hyaluronan synthase and hyaluronidase mRNA may affect hyaluronic acid distribution in keloid disease compared with normal skin.

    PubMed

    Sidgwick, Gary P; Iqbal, Syed A; Bayat, Ardeshir

    2013-05-01

    Keloid disease (KD) is a fibroproliferative disorder characterised partly by an altered extracellular matrix (ECM) profile. In fetal scarring, hyaluronic acid (HA) expression is increased, but is reduced in KD tissue compared with normal skin (NS). The expression of Hyaluronan Synthase (HAS) and hyaluronidase (HYAL) in KD and NS tissue were investigated for the first time using a range of techniques. Hyaluronan synthase and HYAL mRNA expression were significantly increased in NS tissue compared with KD tissue (P < 0.05). Immunohistological analysis of tissue indicated an accumulation of HAS and HYAL protein expression in KD compared with NS due to the thicker epidermis. No differences were observed in mRNA or protein expression in KD and NS fibroblasts. Reduced expression of HAS and HYAL may alter HA synthesis, degradation and accumulation in KD. Better understanding of the role of HA in KD may lead to novel therapeutic approaches to address the resulting ECM imbalance.

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

    PubMed

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

    2016-04-01

    Lasers emit light beams with specific characteristics, in which wavelength, frequency, power, fluence, and emission mode properties determine the photophysical, photochemical, and photobiological responses. Low-intensity lasers could induce free radical generation in biological tissues and cause alterations in macromolecules, such as DNA. Thus, the aim of this work was to evaluate excision repair cross-complementing group 1 (ERCC1) and excision repair cross-complementing group 2 (ERCC2) messenger RNA (mRNA) expression in biological tissues exposed to low-intensity lasers. Wistar rat (n = 28, 4 for each group) skin and muscle were exposed to low-intensity red (660 nm) and near-infrared (880 nm) lasers at different fluences (25, 50, and 100 J/cm(2)), and samples of these tissues were withdrawn for RNA extraction, cDNA synthesis, and gene expression evaluation by quantitative polymerase chain reaction. Laser exposure was in continuous wave and power of 100 mW. Data show that ERCC1 and ERCC2 mRNA expressions decrease in skin (p < 0.001) exposed to near-infrared laser, but increase in muscle tissue (p < 0.001). ERCC1 mRNA expression does not alter (p > 0.05), but ERCC2 mRNA expression decreases in skin (p < 0.001) and increases in muscle tissue (p < 0.001) exposed to red laser. Our results show that ERCC1 and ERCC2 mRNA expression is differently altered in skin and muscle tissue exposed to low-intensity lasers depending on wavelengths and fluences used in therapeutic protocols.

  2. Insulin-Regulated Srebp-1c and Pck1 mRNA Expression in Primary Hepatocytes from Zucker Fatty but Not Lean Rats Is Affected by Feeding Conditions

    PubMed Central

    Zhang, Yan; Chen, Wei; Li, Rui; Li, Yang; Ge, Yuebin; Chen, Guoxun

    2011-01-01

    Insulin regulates the transcription of genes for hepatic glucose and lipid metabolism. We hypothesized that this action may be impaired in hepatocytes from insulin resistant animals. Primary hepatocytes from insulin sensitive Zucker lean (ZL) and insulin resistant Zucker fatty (ZF) rats in ad libitum or after an overnight fasting were isolated, cultured and treated with insulin and other compounds for analysis of gene expression using real-time PCR. The mRNA levels of one insulin-induced (Srebp-1c) and one insulin-suppressed (Pck1) genes in response to insulin, glucagon, and compactin treatments in hepatocytes from ad libitum ZL and ZF rats were analyzed. Additionally, the effects of insulin and T1317 on their levels in hepatocytes from ad libitum or fasted ZL or ZF rats were compared. The mRNA levels of Srebp-1c, Fas, and Scd1, but not that of Insr, Gck and Pck1, were higher in freshly isolated hepatocytes from ad libitum ZF than that from ZL rats. These patterns of Srebp-1c and Pck1 mRNA levels remained in primary hepatocyte cultured in vitro. Insulin's ability to regulate Srebp-1c and Pck1 expression was diminished in hepatocytes from ad libitum ZF, but not ZL rats. Glucagon or compactin suppressed Srebp-1c mRNA expression in lean, but not fatty hepatocytes. However, glucagon induced Pck1 mRNA expression similarly in hepatocytes from ad libitum ZL and ZF rats. Insulin caused the same dose-dependent increase of Akt phosphorylation in hepatocytes from ad libitum ZL and ZF rats. It synergized with T1317 to induce Srebp-1c, and suppressed Pck1 mRNA levels in hepatocytes from fasted, but not that from ad libitum ZF rats. We demonstrated that insulin was unable to regulate its downstream genes' mRNA expression in hepatocytes from ad libitum ZF rats. This impairment can be partially restored in hepatocytes from ZF rats after an overnight fasting, a phenomenon that deserves further investigation. PMID:21731709

  3. High Intensity Interval Training Favourably Affects Angiotensinogen mRNA Expression and Markers of Cardiorenal Health in a Rat Model of Early-Stage Chronic Kidney Disease

    PubMed Central

    Tucker, Patrick S.; Scanlan, Aaron T.; Dalbo, Vincent J.

    2015-01-01

    The majority of CKD-related complications stem from cardiovascular pathologies such as hypertension. To help reduce cardiovascular complications, aerobic exercise is often prescribed. Emerging evidence suggests high intensity interval training (HIIT) may be more beneficial than traditional aerobic exercise. However, appraisals of varying forms of aerobic exercise, along with descriptions of mechanisms responsible for health-related improvements, are lacking. This study examined the effects of 8 weeks of HIIT (85% VO2max), versus low intensity aerobic exercise (LIT; 45–50% VO2max) and sedentary behaviour (SED), in an animal model of early-stage CKD. Tissue-specific mRNA expression of RAAS-related genes and CKD-related clinical markers were examined. Compared to SED, HIIT resulted in increased plasma albumin (p = 0.001), reduced remnant kidney weight (p = 0.028), and reduced kidney weight-body weight ratios (p = 0.045). Compared to LIT, HIIT resulted in reduced Agt mRNA expression (p = 0.035), reduced plasma LDL (p = 0.001), triglycerides (p = 0.029), and total cholesterol (p = 0.002), increased plasma albumin (p = 0.047), reduced remnant kidney weight (p = 0.005), and reduced kidney weight-body weight ratios (p = 0.048). These results suggest HIIT is a more potent regulator of several markers that describe and influence health in CKD. PMID:26090382

  4. 3'-UTR SNP rs2229611 in G6PC1 affects mRNA stability, expression and Glycogen Storage Disease type-Ia risk.

    PubMed

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

    2017-08-01

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

  5. In utero and lactational exposure to low-dose genistein-vinclozolin mixture affects the development and growth factor mRNA expression of the submandibular salivary gland in immature female rats.

    PubMed

    Kouidhi, Wided; Desmetz, Catherine; Nahdi, Afef; Bergès, Raymond; Cravedi, Jean-Pierre; Auger, Jacques; El May, Michèle; Canivenc-Lavier, Marie Chantal

    2012-06-01

    It has been suggested that hormonally controlled submandibular salivary gland (SSG) development and secretions may be affected by endocrine disruptor compounds. We investigated the effects of oral gestation-lactation exposure to 1 mg/kg body weight daily dose of the estrogenic soy-isoflavone genistein and/or the anti-androgenic food contaminant vinclozolin in female rats. The SSGs of female offspring were collected at postnatal day 35 to study gland morphogenesis and mRNA expression of sex-hormone receptors and endocrine growth factors as sex-dependent biomarkers. Because of high expression in neonatal SSG, mRNA expression of transforming growth factor α was also studied. Exposure to genistein, vinclozolin, or a genistein+vinclozolin mixture resulted in significantly lower numbers of striated ducts linked to an increase in their area and lower acinar proliferation (Ki-67-positive nuclei). Exposure to the mixture had the highest significant effects, which were particularly associated with repression of epidermal growth factor, nerve growth factor, and transforming growth factor α expression. In conclusion, early exposure to low doses of genistein and vinclozolin can affect glandular structure and endocrine gene mRNA expression in prepubertal SSG in female rats, and the effects are potentialized by the genistein+vinclozolin mixture. Our study provides the first evidence that SSG are targeted by both estrogenic and anti-androgenic disrupting compounds and are more sensitive to mixtures.

  6. FOXP3 mRNA expression at 6 months of age is higher in infants who develop atopic dermatitis, but is not affected by giving probiotics from birth.

    PubMed

    Taylor, Angie L; Hale, Jasmine; Hales, Belinda J; Dunstan, Janet A; Thomas, Wayne R; Prescott, Susan L

    2007-02-01

    Factors that influence immune regulation in early life may be implicated in the rise in allergic disease, including reduced microbial burden. The aim of the study was to examine the infant regulatory T-cell function in relation to (a) probiotic supplementation for the first 6 months of life and (b) the subsequent development of an early allergic phenotype. Two hundred and thirty-one allergic, pregnant women were recruited into a randomized, controlled trial. The infants received either a probiotic or placebo daily for the first 6 months of life. One hundred and seventy-eight children completed the study, with blood samples available from 118 (60 placebo; 58 probiotic). CD4(+)CD25(+)CTLA4(+)T-regulatory phenotype and allergen-induced FOXP3 mRNA expression were compared in relation to this intervention as well as according to evidence of early disease (atopic dermatitis). The administration of probiotics was not associated with any significant differences in the proportion of circulating CD4(+)CD25(+)CTLA4(+)cells, or in the resting expression of FOXP3. There were also no relationships between these parameters and patterns of gut colonization, and this probiotic did not reduce the risk of atopic dermatitis. Children who developed atopic dermatitis (n = 36/118) had significantly higher induced FOXP3 expression following stimulation with both house dust mite (HDM) (p = 0.017) and ovalbumin (OVA) allergens (p = 0.021) than those that did not develop atopic dermatitis. Although this relationship was seen in both the probiotic and placebo groups, it was more pronounced in the probiotic group. However, regression analysis demonstrated that higher allergen-induced FOXP3 expression was predicted by the presence of atopic dermatitis (p = 0.018) rather than probiotics supplementation (p = 0.217). The higher levels of allergen-induced FOXP3 in atopic dermatitis suggest activation of these compensatory mechanisms rather than a primary defect in this pathway. Probiotic

  7. Oxygen concentration and cysteamine supplementation during in vitro production of buffalo (Bubalus bubalis) embryos affect mRNA expression of BCL-2, BCL-XL, MCL-1, BAX and BID.

    PubMed

    Elamaran, G; Singh, K P; Singh, M K; Singla, S K; Chauhan, M S; Manik, R S; Palta, P

    2012-12-01

    This study examined the effects of O(2) concentration (5% vs 20%) during in vitro maturation (IVM), fertilization (IVF) and culture (IVC) or supplementation of IVM and IVC media with cysteamine (50 and 100 μm, respectively; IVM, IVF and IVC carried out in 20% O(2)), on blastocyst rate and relative mRNA abundance of some apoptosis-related genes measured by real-time qPCR in immature and in vitro-matured buffalo oocytes and in embryos at 2-, 4-, 8- to 16-cell, morula and blastocyst stages. The blastocyst rate was significantly higher (p < 0.05) while the percentage of TUNEL-positive cells was significantly lower (p < 0.05) under 5% O(2) than that under 20% O(2). The mRNA expression of anti-apoptotic genes BCL-2 and MCL-1 was significantly higher (p < 0.05) and that of pro-apoptotic genes BAX and BID was lower (p < 0.05) under 5% O(2) than that under 20% O(2) concentration at many embryonic stages. Following cysteamine supplementation, the blastocyst rate and the relative mRNA abundance of BCL-XL and MCL-1 was significantly higher (p < 0.05) and that of BAX but not BID was lower (p < 0.05) at many stages of embryonic development, although it did not affect the percentage of TUNEL positive cells in the blastocysts significantly. The mRNA expression pattern of these genes during embryonic development was different in 5% vs 20% O(2) groups and in cysteamine supplemented vs controls. At the 8- to 16-cell stage, where developmental block occurs in buffalo, the relative mRNA abundance of BCL-2 and MCL-1 was highest under 5% O(2) concentration and that of BAX and BID was highest (p < 0.05) under 20% O(2) concentration. These results suggest that one of the mechanisms through which beneficial effects of low O(2) concentration and cysteamine supplementation are mediated during in vitro embryo production is through an increase in the expression of anti-apoptotic and a decrease in the expression of pro-apoptotic genes.

  8. Increase in flavan-3-ols by silencing flavonol synthase mRNA affects the transcript expression and activity levels of antioxidant enzymes in tobacco.

    PubMed

    Mahajan, M; Joshi, R; Gulati, A; Yadav, S K

    2012-09-01

    Flavonoids are plant secondary metabolites widespread throughout the plant kingdom involved in many physiological and biochemical functions. Amongst the flavonoids, flavan-3-ols (catechin and epicatechin) are known for their direct free radical scavenging activity in vitro, but studies on their antioxidant potential and interaction with antioxidant enzymes in vivo are lacking. Here, the flavonoid pathway was engineered by silencing a gene encoding flavonol synthase (FLS) in tobacco to direct the flow of metabolites towards production of flavan-3-ols. FLS silencing reduced flavonol content 17-53%, while it increased catechin and epicatechin content 51-93% and 18-27%, respectively. The silenced lines showed a significant increase in expression of genes for dihydroflavonol reductase and anthocyanidin synthase, a downstream gene towards epicatechin production, with no significant change in expression of other genes of the flavonoid pathway. Effects of accumulation of flavan-3-ols in FLS silenced lines on transcript level and activities of antioxidant enzymes were studied. Transcripts of the antioxidant enzymes glutathione reductase (GR), ascorbate peroxidase (APx), and catalase (CAT) increased, while glutathione-S-transferase (GST), decreased in FLS silenced lines. Enhanced activity of all the antioxidant enzymes was observed in silenced tobacco lines. To validate the affect of flavan-3-ols on the antioxidant system, in vitro experiments were conducted with tobacco seedlings exposed to two concentrations of catechin (10  and 50 μm) for 2 days. In vitro exposed seedlings produced similar levels of transcripts and activity of antioxidant enzymes as FLS silenced seedlings. Results suggest that flavan-3-ols (catechin) might be increasing activity of GR, Apx and CAT by elevating their mRNAs levels. Since these enzymes are involved in scavenging of reactive oxygen species, this strategy would help in tailoring crops for enhanced catechin production as well as making

  9. Grape polyphenols affect mRNA expression of PGHS-2, TIS11b and FOXO3 in endometrium of heifers under ACTH-induced stress.

    PubMed

    Colitti, M; Sgorlon, S; Stradaioli, G; Farinacci, M; Gabai, G; Stefanon, B

    2007-10-15

    Stress activates the hypothalamo-pituitary-adrenal axis leading to enhanced glucocorticoid secretion and concurrently disrupts ovarian cycle. Plant polyphenols are known to posses antioxidant and anti-inflammatory proprieties. This could be of interest for ovarian cycle when stressing conditions lead to progesterone enhancement and hamper normal reproduction activity. The present study examined whether ovarian follicular development and progesterone secretory pattern are affected by exogenous ACTH administration in heifers. Moreover, the effect of grape polyphenols in endometrium of heifers, under adrenocorticotropic hormone challenge, is evaluated in terms of transcriptional patterns of genes related to inflammation, oxidative stress and endometrial functions. At day 14 of synchronized estrous cycle, Holstein Friesian heifers received injections of either saline (CTR group) or adrenocorticotropic hormone (ACT group) agonist every 12 h for 7 days. Another group (POL group) of animals received the same treatment plus an oral supplementation of 15 g/day of grape skin extract. Cortisol and progesterone were analysed in the blood samples collected at days 0, 3, 6, 9, 12, 14, 17, 21, 24 of the estrous cycle. Endometrial biopsies were collected at diestrus (day 18) and at estrus and a panel of gene expressions were quantified by real-time PCR. ACTH administration increased both cortisol (P<0.001) and progesterone concentrations (P<0.01) compared to CTR group. PGHS-2 was significantly (P<0.01) up-regulated in the POL group compared to ACT and CTR groups at diestrus and at estrus. FOXO3 and TIS11b were down-regulated in the CTR group compared to ACT and POL groups. The PGHS-2, SOD2 (P<0.05), FOXO3 and TIS11b (P<0.10) genes were down-regulated at estrus in all groups compared to diestrus. An interesting role of polyphenols in modulating the expression levels of PGHS-2 in endometrial tissue and on the activation of TIS11b and SOD2 through c-AMP-dependent signalling was

  10. Prenatal ethanol increases ethanol intake throughout adolescence, alters ethanol-mediated aversive learning, and affects μ but not δ or κ opioid receptor mRNA expression.

    PubMed

    Fabio, María Carolina; Macchione, Ana Fabiola; Nizhnikov, Michael E; Pautassi, Ricardo Marcos

    2015-06-01

    Animal models of prenatal ethanol exposure (PEE) have indicated a facilitatory effect of PEE on adolescent ethanol intake, but few studies have assessed the effects of moderate PEE throughout adolescence. The mechanisms underlying this facilitatory effect remain largely unknown. In the present study, we analysed ethanol intake in male and female Wistar rats with or without PEE (2.0 g/kg, gestational days 17-20) from postnatal days 37 to 62. The results revealed greater ethanol consumption in PEE rats than in controls, which persisted throughout adolescence. By the end of testing, ethanol ingestion in PEE rats was nearly 6.0 g/kg. PEE was associated with insensitivity to ethanol-induced aversion. PEE and control rats were further analysed for levels of μ, δ and κ opioid receptor mRNA in the infralimbic cortex, nucleus accumbens shell, and ventral tegmental area. Similar levels of mRNA were observed across most areas and opioid receptors, but μ receptor mRNA in the ventral tegmental area was significantly increased by PEE. Unlike previous studies that assessed the effects of PEE on ethanol intake close to birth, or in only a few sessions during adolescence, the present study observed a facilitatory effect of PEE that lasted throughout adolescence. PEE was associated with insensitivity to the aversive effect of ethanol, and increased levels of μ opioid receptor transcripts. PEE is a prominent vulnerability factor that probably favors the engagement of adolescents in risky trajectories of ethanol use. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  11. 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

  12. Propionate induces mRNA expression of gluconeogenic genes in bovine calf hepatocytes.

    PubMed

    Zhang, Qian; Koser, Stephanie L; Donkin, Shawn S

    2016-05-01

    Hepatocytes monolayers from neonatal calves were used to determine the responses of the cytosolic phosphoenolpyruvate carboxykinase (PCK1) mRNA expression to propionate and direct hormonal cues including cyclic AMP (cAMP), dexamethasone, and insulin. The responses of other key gluconeogenic genes, including mitochondrial phosphoenolpyruvate carboxykinase (PCK2), pyruvate carboxylase (PC), and glucose-6-phosphotase (G6PC), were also measured. Expression of PCK1 was linearly induced with increasing propionate concentrations in media and 2.5 mM propionate increased PCK1 mRNA at 3 and 6h of incubation; however, the induction disappeared at 12 and 24 h. The induction of PCK1 mRNA by propionate was mimicked by 1 mM cAMP, or in combination with 5 µM dexamethasone, but not by dexamethasone alone. The induction of PCK1 mRNA by propionate or cAMP was eliminated by addition of 100 nM insulin. Additionally, expression of PCK2 and PC mRNA was also induced by propionate in a concentration-dependent manner. Consistent with PCK1, propionate-stimulated PCK2 and PC mRNA expression was inhibited by insulin. Expression of G6PC mRNA was neither affected by propionate nor cAMP, dexamethasone, insulin, or their combinations. These findings demonstrate that propionate can directly regulate its own metabolism in bovine calf hepatocytes through upregulation of PCK1, PCK2, and PC mRNA expression.

  13. Short-term hypertonic exposure enhances in vitro follicle growth and meiotic competence of enclosed oocytes while modestly affecting mRNA expression of aquaporin and steroidogenic genes in the domestic cat model.

    PubMed

    Songsasen, N; Thongkittidilok, C; Yamamizu, K; Wildt, D E; Comizzoli, P

    2017-03-01

    Using the domestic cat as a non-rodent, larger animal model, the objective was to determine the impact of a brief incubation in a hypertonic microenvironment on (1) ovarian follicle and oocyte growth in vitro, (2) developmental capacity of the resident oocyte, and (3) expression of aquaporin (AQP) genes in parallel with genes involved in regulation of folliculogenesis. In Study 1: Secondary or early antral follicles encapsulated in 0.5% alginate were allocated to one of three treatment groups: 1) culture in standard medium at 290 mOsm for 15 d (Control); 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for 15 d (Hypertonic-1h); or 3) incubation in 350 mOsm medium for 24 h followed by incubation in standard medium for additional 14 d (Hypertonic-24h). After measuring follicle and oocyte diameters on Day 15, in vitro-grown oocytes were incubated for 24 h before assessing nuclear status. In Study 2: secondary or early antral follicles were subjected to one of the three treatments: 1) culture in standard medium at 290 mOsm for 48 h; 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for additional 47 h; or 3) incubation in 350 mOsm medium for 24 h followed by culture in standard medium for additional 24 h. At the end of the culture period, all follicles were assessed for mRNA level of Cyp17a1, Cyp19a1, Star, Aqp1, 3, 5, 7 and 8 as well as Fshr using qPCR. Freshly collected follicles also were subjected to gene expression analysis and served as the 'Non-cultured control'. Hypertonic-24h follicles grew larger (P < 0.05) than the control, whereas those in Hypertonic-1h group exhibited intermediate growth, especially when the culture started at the early antral stage. Oocytes in the Hypertonic-24h group were larger and resumed meiosis at a higher rate than in the other treatments. In vitro culture affected (P < 0.05) mRNA expression of Cyp19a1, Star, Aqp1, and Aqp7 in both the secondary and early

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

    PubMed

    Nakashima, Yukiko; Takahashi, Satoru

    2014-08-22

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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. Cyclin-Dependent Kinase 7 Controls mRNA Synthesis by Affecting Stability of Preinitiation Complexes, Leading to Altered Gene Expression, Cell Cycle Progression, and Survival of Tumor Cells

    PubMed Central

    Kelso, Timothy W. R.; Baumgart, Karen; Eickhoff, Jan; Albert, Thomas; Antrecht, Claudia; Lemcke, Sarah; Klebl, Bert

    2014-01-01

    Cyclin-dependent kinase 7 (CDK7) activates cell cycle CDKs and is a member of the general transcription factor TFIIH. Although there is substantial evidence for an active role of CDK7 in mRNA synthesis and associated processes, the degree of its influence on global and gene-specific transcription in mammalian species is unclear. In the current study, we utilize two novel inhibitors with high specificity for CDK7 to demonstrate a restricted but robust impact of CDK7 on gene transcription in vivo and in in vitro-reconstituted reactions. We distinguish between relative low- and high-dose responses and relate them to distinct molecular mechanisms and altered physiological responses. Low inhibitor doses cause rapid clearance of paused RNA polymerase II (RNAPII) molecules and sufficed to cause genome-wide alterations in gene expression, delays in cell cycle progression at both the G1/S and G2/M checkpoints, and diminished survival of human tumor cells. Higher doses and prolonged inhibition led to strong reductions in RNAPII carboxyl-terminal domain (CTD) phosphorylation, eventual activation of the p53 program, and increased cell death. Together, our data reason for a quantitative contribution of CDK7 to mRNA synthesis, which is critical for cellular homeostasis. PMID:25047832

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver.

    PubMed

    Atger, Florian; Gobet, Cédric; Marquis, Julien; Martin, Eva; Wang, Jingkui; Weger, Benjamin; Lefebvre, Grégory; Descombes, Patrick; Naef, Felix; Gachon, Frédéric

    2015-11-24

    Diurnal oscillations of gene expression are a hallmark of rhythmic physiology across most living organisms. Such oscillations are controlled by the interplay between the circadian clock and feeding rhythms. Although rhythmic mRNA accumulation has been extensively studied, comparatively less is known about their transcription and translation. Here, we quantified simultaneously temporal transcription, accumulation, and translation of mouse liver mRNAs under physiological light-dark conditions and ad libitum or night-restricted feeding in WT and brain and muscle Arnt-like 1 (Bmal1)-deficient animals. We found that rhythmic transcription predominantly drives rhythmic mRNA accumulation and translation for a majority of genes. Comparison of wild-type and Bmal1 KO mice shows that circadian clock and feeding rhythms have broad impact on rhythmic gene expression, Bmal1 deletion affecting surprisingly both transcriptional and posttranscriptional levels. Translation efficiency is differentially regulated during the diurnal cycle for genes with 5'-Terminal Oligo Pyrimidine tract (5'-TOP) sequences and for genes involved in mitochondrial activity, many harboring a Translation Initiator of Short 5'-UTR (TISU) motif. The increased translation efficiency of 5'-TOP and TISU genes is mainly driven by feeding rhythms but Bmal1 deletion also affects amplitude and phase of translation, including TISU genes. Together this study emphasizes the complex interconnections between circadian and feeding rhythms at several steps ultimately determining rhythmic gene expression and translation.

  7. Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver

    PubMed Central

    Atger, Florian; Gobet, Cédric; Marquis, Julien; Martin, Eva; Wang, Jingkui; Weger, Benjamin; Lefebvre, Grégory; Descombes, Patrick; Naef, Felix; Gachon, Frédéric

    2015-01-01

    Diurnal oscillations of gene expression are a hallmark of rhythmic physiology across most living organisms. Such oscillations are controlled by the interplay between the circadian clock and feeding rhythms. Although rhythmic mRNA accumulation has been extensively studied, comparatively less is known about their transcription and translation. Here, we quantified simultaneously temporal transcription, accumulation, and translation of mouse liver mRNAs under physiological light–dark conditions and ad libitum or night-restricted feeding in WT and brain and muscle Arnt-like 1 (Bmal1)-deficient animals. We found that rhythmic transcription predominantly drives rhythmic mRNA accumulation and translation for a majority of genes. Comparison of wild-type and Bmal1 KO mice shows that circadian clock and feeding rhythms have broad impact on rhythmic gene expression, Bmal1 deletion affecting surprisingly both transcriptional and posttranscriptional levels. Translation efficiency is differentially regulated during the diurnal cycle for genes with 5′-Terminal Oligo Pyrimidine tract (5′-TOP) sequences and for genes involved in mitochondrial activity, many harboring a Translation Initiator of Short 5′-UTR (TISU) motif. The increased translation efficiency of 5′-TOP and TISU genes is mainly driven by feeding rhythms but Bmal1 deletion also affects amplitude and phase of translation, including TISU genes. Together this study emphasizes the complex interconnections between circadian and feeding rhythms at several steps ultimately determining rhythmic gene expression and translation. PMID:26554015

  8. 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.

  9. Decoding Children's Expressions of Affect.

    ERIC Educational Resources Information Center

    Feinman, Joel A.; Feldman, Robert S.

    1982-01-01

    Mothers' ability to decode their children's nonverbal expressions of four affects (happiness, sadness, fear, and anger) was contrasted with the decoding ability of a matched group of nonmothers. Results indicate that mothers were accurately able to decode expressions of happiness but had relative difficulty with decoding expressions of sadness,…

  10. Pamidronate, farnesyl transferase, and geranylgeranyl transferase-I inhibitors affects cell proliferation, apoptosis, and OPG/RANKL mRNA expression in stromal cells of giant cell tumor of bone.

    PubMed

    Lau, Carol P Y; Huang, Lin; Tsui, Stephen K W; Ng, Patrick K S; Leung, Pui-Yin; Kumta, Shekhar M

    2011-03-01

    Giant cell tumor (GCT) is the most common nonmalignant primary bone tumor reported in Hong Kong. It usually affects young adults between the ages of 20 and 40. This tumor is well known for its potential to recur following treatment. To date no effective adjuvant therapy exists for GCT. Our project aimed to study the effects of pamidronate (PAM), farnesyl transferase inhibitor (FTI-277), geranylgeranyl transferase inhibitor (GGTI-298), and their combinations on GCT stromal cells (SC). Individual treatment with PAM, FTI-277, and GGTI-298, inhibited the cell viability and proliferation of GCT SC in a dose-dependent way. Combination of FTI-277 with GGTI-298 caused synergistic effects in reducing cell viability, and its combination index was 0.49, indicating a strong synergism. Moreover, the combination of FTI-277 with GGTI-298 synergistically enhanced cell apoptosis and activated caspase-3/7, -8, and -9 activities. PAM induced cell-cycle arrest at the S-phase. The combination of PAM with GGTI-298 significantly increased OPG/RANKL mRNA ratio and activated caspase-3/7 activity. Our findings support that the combination of bisphosphonates with GGTIs or FTIs with GGTIs may be used as potential adjuvants in the treatment of GCT of bone. Copyright © 2010 Orthopaedic Research Society.

  11. 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

  12. 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

  13. Long-term treatment with moisturizers affects the mRNA levels of genes involved in keratinocyte differentiation and desquamation.

    PubMed

    Buraczewska, Izabela; Berne, Berit; Lindberg, Magnus; Lodén, Marie; Törmä, Hans

    2009-02-01

    In a recent study, we showed that long-term treatment with two different moisturizers affected TEWL in opposite directions. Therefore, we decided to examine the effect of these moisturizers on the cellular and molecular level. In a randomized controlled study on 20 volunteers, epidermal mRNA expression of genes essential for keratinocyte differentiation and desquamation after a 7-week treatment with two moisturizers was analyzed. Treatment with one test moisturizer increased gene expression of involucrin, transglutaminase 1, kallikrein 5, and kallikrein 7, while the other moisturizer affected only expression of cyclin-dependent kinase inhibitor 1A. Thus, moisturizers are able to modify the skin barrier function and change the mRNA expression of certain epidermal genes. Since the type of influence depends on the composition of the moisturizer, these should be tailored in accordance with the requirement of the barrier of each individual patient, which merits further investigations.

  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. Salt stress affects mRNA editing in soybean chloroplasts.

    PubMed

    Rodrigues, Nureyev F; Fonseca, Guilherme C da; Kulcheski, Franceli R; Margis, Rogério

    2017-03-02

    Soybean, a crop known by its economic and nutritional importance, has been the subject of several studies that assess the impact and the effective plant responses to abiotic stresses. Salt stress is one of the main environmental stresses and negatively impacts crop growth and yield. In this work, the RNA editing process in the chloroplast of soybean plants was evaluated in response to a salt stress. Bioinformatics approach using sRNA and mRNA libraries were employed to detect specific sites showing differences in editing efficiency. RT-qPCR was used to measure editing efficiency at selected sites. We observed that transcripts of NDHA, NDHB, RPS14 and RPS16 genes presented differences in coverage and editing rates between control and salt-treated libraries. RT-qPCR assays demonstrated an increase in editing efficiency of selected genes. The salt stress enhanced the RNA editing process in transcripts, indicating responses to components of the electron transfer chain, photosystem and translation complexes. These increases can be a response to keep the homeostasis of chloroplast protein functions in response to salt stress.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. Chemically modified tetracyclines selectively inhibit IL-6 expression in osteoblasts by decreasing mRNA stability.

    PubMed

    Kirkwood, Keith; Martin, Thomas; Andreadis, Stelios T; Kim, Young Joon

    2003-11-01

    In bone biology, interleukin (IL)-6 is an autocrine/paracrine cytokine which can induce osteoclasts formation and activation to help mediate inflammatory bone destruction. Previous studies have shown that tetracycline and its derivatives have potentially beneficial therapeutic effects in the prevention and treatment of metabolic bone diseases by modulating osteoblast and osteoclast activities. Our previous studies indicated that non-antimicrobial chemically modified tetracyclines (CMTs) can dose-dependently inhibit IL-1 beta-induced IL-6 secretion in osteoblastic cells. In the present study, we explored the molecular mechanisms underlying the ability of doxycycline analogs CMT-8 and its non-chelating pyrazole derivative, CMT-5 to affect IL-6 gene expression in murine osteoblasts. Steady-state IL-6 mRNA was decreased with CMT-8 (ca. 50%) but not by CMT-5 when stimulated by IL-1 beta. CMT-8 regulation of IL-1 beta-induced IL-6 gene expression was further explored. CMT-8 did not affect IL-6 promoter activity in reporter gene assays. However, the IL-6 mRNA stability was decreased in the presence of CMT-8. These effects require de novo protein synthesis as they were inhibited by cycloheximide. Western blot analysis indicated that CMT-8 did not affect p38 mitogen-activated protein kinase, c-jun NH(2)-terminal kinases, or extracellular signal-regulated kinases (1 and 2) phosphorylation in response to IL-1 beta. These data suggest that CMT-8 can modulate inhibit IL-1 beta-induced IL-6 expression in MC3T3-E1 cells at the post-transcriptional level affecting IL-6 mRNA stability. These observations may offer a novel molecular basis for this treatment of metabolic bone diseases that are mediated by IL-6.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. [Expression and significance of ERalpha mRNA of residents exposed to arsenic via drinking water].

    PubMed

    Han, Xiaohong; Guo, Zhiwei; Yang, Weihong; Wu, Kegong; Li, Yanhong; Xia, Yajuan

    2014-05-01

    To detect ERbeta mRNA expression of subjects exposed to different arsenic drinking water, and to analyze the potential relationship between their abnormal expression and heart injury caused by arsenic in order to study the endocrine disturbing effect of arsenic. Molecular epidemiological method was used. The study subjects included a total of 273 residents exposed to arsenic via drinking water,and they were divided into four groups according to arsenic concentration, and their blood ERbeta mRNA expression was detected by real-time RT-PCR. The level of ERbeta mRNA expression rose with increasing of water As and urine As (r = 0.159, 0.21, P < 0.05). The prevalence of Q-Tc interval prolongation aggravated with increasing of water As(chi2 = 4.35, P = 0.037), while according with the variation of ERbeta mRNA expression. Tp-Te interval prolonged with increasing of water As (r = 0.199, P = 0.023), as well prolonged with increasing of ERbeta mRNA expression (r = 0.205, P = 0.019). The prevalence of arrhythmia was almost according with the variation of ERbeta mRNA expression. chronic arsenic exposure can potentially disturb ERbeta mRNA expression, as well as there are possible relationship between ERbeta mRNA abnormal expression and Q-Tc interval prolongation and Tp-Te interval prolonged caused by arsenic.

  12. REDUCED NITRIC OXIDE PRODUCTION AND INOS MRNA EXPRESSION IN IFN-G STIMULATED CHICKEN MACROPHAGES TRANSFECTED WITH INOS SIRNAS

    USDA-ARS?s Scientific Manuscript database

    Utilizing RNA interference technology with siRNA in the HD-11 macrophage cell line, we determined how the inhibition or knock-down of the iNOS (inducible nitric oxide synthase) gene affected IFN-y' induced macrophage production of nitric oxide (NO) and mRNA expression of genes involved in this biolo...

  13. 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.

  14. Expression of APOBEC3B mRNA in Primary Breast Cancer of Japanese Women

    PubMed Central

    Tokunaga, Eriko; Yamashita, Nami; Tanaka, Kimihiro; Inoue, Yuka; Akiyoshi, Sayuri; Saeki, Hiroshi; Oki, Eiji; Kitao, Hiroyuki; Maehara, Yoshihiko

    2016-01-01

    Recent studies have identified the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3B (APOBEC3B) as a source of mutations in various malignancies. APOBEC3B is overexpressed in several human cancer types, including breast cancer. In this study, we analyzed APOBEC3B mRNA expression in 305 primary breast cancers of Japanese women using quantitative reverse transcription-PCR, and investigated the relationships between the APOBEC3B mRNA expression and clinicopathological characteristics, prognosis, and TP53 mutations. The expression of APOBEC3B mRNA was detected in 277 tumors and not detected in 28 tumors. High APOBEC3B mRNA expression was significantly correlated with ER- and PR-negativity, high grade and high Ki67 index. The APOBEC3B mRNA expression was highest in the triple-negative and lowest in the hormone receptor-positive/HER2-negative subtypes. The TP53 gene was more frequently mutated in the tumors with high APOBEC3B mRNA expression. High APOBEC3B mRNA expression was significantly associated with poor recurrence-free survival in all cases and the ER-positive cases. These findings were almost consistent with the previous reports from the Western countries. In conclusion, high APOBEC3B mRNA expression was related to the aggressive phenotypes of breast cancer, high frequency of TP53 mutation and poor prognosis, especially in ER-positive tumors. PMID:27977754

  15. Olive Leaf Extract Elevates Hepatic PPAR α mRNA Expression and Improves Serum Lipid Profiles in Ovariectomized Rats.

    PubMed

    Yoon, Leena; Liu, Ya-Nan; Park, Hyunjin; Kim, Hyun-Sook

    2015-07-01

    We hypothesized that olive leaf extract might alleviate dyslipidemia resulting from estrogen deficiency. Serum lipid profile and mRNA expression of the related genes in the liver and adipose tissue were analyzed after providing olive leaf extract (200 or 400 mg/kg body weight; n=7 for each group) to ovariectomized rats for 10 weeks. After 10 weeks' administration, the rats in the olive leaf extract-administered groups showed significantly lower levels of serum triglyceride and very-low-density lipoprotein (VLDL)-cholesterol compared with the rats in the control group, whereas the administration of olive leaf extract did not significantly change the elevated low-density lipoprotein cholesterol levels. In addition, administration of high dose of olive leaf extract significantly decreased the liver triglyceride and increased serum estradiol levels. mRNA expressions of peroxisome proliferator-activated receptor alpha (PPAR α) and acyl-CoA oxidase (ACO) were not affected by ovariectomy, however, administration of olive leaf extract significantly increased both PPAR α and ACO mRNA expression. Expression of adiponectin mRNA in adipose tissue was significantly decreased in the ovariectomized control group. Rats administered low-dose olive leaf extract showed significantly elevated adiponectin mRNA expression compared with rats in the ovariectomized control group. Even though dose-dependent effects were not observed in most of the measurements, these results suggest that genes involved in lipid metabolism may be regulated by olive leaf extract administration in ovariectomized rats.

  16. Relationship Between the DPD and TS mRNA Expression and the Response to S-1-Based Chemotherapy and Prognosis in Patients with Advanced Gastric Cancer.

    PubMed

    Shen, Xiao-Ming; Zhou, Chong; Lian, Lian; Li, Li-Qun; Li, Wei; Tao, Min

    2015-04-01

    The aim was to determine changes in dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) mRNAs in the blood of advanced gastric cancer (AGC) patients to see whether these enzymes affected the patients' response to S-1-based chemotherapy and prognosis. For this purpose, pretreatment DPD/TS mRNA expressions were determined in 40 AGC patients using RT-PCR. The patients were then administered with S-1-based regimen (S-1 + cisplatin) and toxicities were recorded. The relationship between the DPD/TS mRNA expressions and the chemotherapy response, drug resistance, and prognosis was analyzed. The data show that DPD mRNA expression correlated significantly with Lauren type while TS mRNA expression correlated with distant metastasis. Patients with higher DPD and/or TS mRNA expression(s) showed poor response, while those with low DPD mRNA expression showed better response to the chemotherapy. Pooled analysis showed that the patients with low DPD/TS mRNA expressions had better therapeutic response. The incidence of bone marrow suppression, diarrhea, and oral mucositis was high in patients with low DPD mRNA expression. Median overall survival (OS) in 40 patients was 13.5 months. It was 17 months for low and 10 months for high DPD (P = 0.044) and TS mRNA expression (P = 0.047). Pooled analysis showed that the patients with both low DPD/TS mRNA expressions had longer OS (P = 0.001). In conclusion, the detection of DPD and/or TS mRNA expression can be used to predict the response to S-1-based chemotherapy, drug resistance, and prognosis in AGC patients as well as to help guide the individualized treatment of gastric cancer.

  17. 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.

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

    PubMed Central

    2015-01-01

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

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

    PubMed

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

    2014-12-23

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

  20. Analysis of the sequence and embryonic expression of chicken neurofibromin mRNA.

    PubMed

    Schafer, G L; Ciment, G; Stocker, K M; Baizer, L

    1993-04-01

    Neurofibromatosis type 1 (NF1) is a common inherited disorder that primarily affects tissues derived from the neural crest. Recent identification and characterization of the human NF1 gene has revealed that it encodes a protein (now called neurofibromin) that is similar in sequence to the ras-GTPase activator protein (or ras-GAP), suggesting that neurofibromin may be a component of cellular signal transduction pathways regulating cellular proliferation and/or differentiation. To initiate investigations on the role of the NF1 gene product in embryonic development, we have isolated a partial cDNA for chicken neurofibromin. Sequence analysis reveals that the predicted amino acid sequence is highly conserved between chick and human. The chicken cDNA hybridizes to a 12.5-kb transcript on RNA blots, a mol wt similar to that reported for the human and murine mRNAs. Ribonuclease protection assays indicate that NF1 mRNA is expressed in a variety of tissues in the chick embryo; this is confirmed by in situ hybridization analysis. NF1 mRNA expression is detectable as early as embryonic stage 18 in the neural plate. This pattern of expression may suggest a role for neurofibromin during normal development, including that of the nervous system.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-09-01

    Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells. © 2016 Hollerer et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  7. COX-2 mRNA expression in esophageal squamous cell carcinoma (ESCC) and effect by NSAID.

    PubMed

    Liu, X; Li, P; Zhang, S-T; You, H; Jia, J-D; Yu, Z-L

    2008-01-01

    To investigate cyclooxygenase-2 (COX-2) mRNA expression in human esophageal squamous cell carcinoma and the effect of a non-steroidal anti-inflammatory drug (NSAID) on it, in order to explore the mechanism of COX-2 in esophageal squamous cell carcinoma (ESCC) carcinogenesis and the ability of NSAID to prevent or treat ESCC. Frozen specimens of human ESCC and adjacent normal esophageal squamous epithelium pairs (n = 22) were examined for COX-2 mRNA expression by reverse-transcription polymerase chain reaction (RT-PCR). After incubation with aspirin (a non-selective COX inhibitor) or Nimesulide (a selective COX-2 inhibitor), the proliferation status of two human esophageal squamous cancer cell lines, EC-9706 and EC-109, was quantified by 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide assay. The expression of COX-2 mRNA in these cells was detected by RT-PCR. COX-2 mRNA was expressed in 12 of 22 (54.5%) ESCC tissue samples, but it was undetectable in all the specimens of adjacent normal esophageal squamous epithelium COX-2 mRNA expression. Both aspirin (5-20 mmol/L) and Nimesulide (0.1-0.8 mmol/L) inhibited EC-9706 cell line proliferation and suppressed its COX-2 mRNA expression dose-dependently. However, only aspirin (5-20 mmol/L) could inhibit proliferation in the EC-109 cell line and suppress COX-2 mRNA expression. Nimesulide (0.1-0.8 mmol/L) could neither inhibit EC-109 cell growth nor suppress COX-2 mRNA expression. COX-2 mRNA expression is a frequent phenomenon in human ESCC tissue samples and plays an important role in the carcinogenesis of ESCC. NSAID may be useful in the chemoprevention and therapy of human ESCC and its effects are likely to be mediated by modulating COX-2 activity.

  8. Control of mammalian gene expression by selective mRNA export.

    PubMed

    Wickramasinghe, Vihandha O; Laskey, Ronald A

    2015-07-01

    Nuclear export of mRNAs is a crucial step in the regulation of gene expression, linking transcription in the nucleus to translation in the cytoplasm. Although important components of the mRNA export machinery are well characterized, such as transcription-export complexes TREX and TREX-2, recent work has shown that, in some instances, mammalian mRNA export can be selective and can regulate crucial biological processes such as DNA repair, gene expression, maintenance of pluripotency, haematopoiesis, proliferation and cell survival. Such findings show that mRNA export is an unexpected, yet potentially important, mechanism for the control of gene expression and of the mammalian transcriptome.

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

    PubMed

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

    2016-05-25

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

  10. Maternal overnutrition enhances mRNA expression of adipogenic markers and collagen deposition in skeletal muscle of beef cattle fetuses.

    PubMed

    Duarte, M S; Gionbelli, M P; Paulino, P V R; Serão, N V L; Nascimento, C S; Botelho, M E; Martins, T S; Filho, S C V; Dodson, M V; Guimarães, S E F; Du, M

    2014-09-01

    Twenty-four pregnant Nellore cows were randomly assigned into 2 feeding level groups (control [CTL]; fed 1.0 times the maintenance requirement; n = 12; and overnourished [ON]; fed at 1.5 times the maintenance requirement; n = 12) to evaluate effects of maternal overnutrition on fetal skeletal muscle development. Cows were slaughtered at 135, 190, and 240 d of gestation and samples of fetal LM were collected for analysis of mRNA expression analysis and for histological evaluation of collagen content and number of muscle cells. There was no interaction between gestational period and maternal nutrition for the variables evaluated (P > 0.05). The mRNA expression of Cadherin-associated protein, β 1 (β-catenin) tended to be greater in fetuses from ON cows (P = 0.08), while myogenic differentiation 1 (MyoD; P = 0.56), myogenin (MyoG; P = 0.70), and the number of muscle cells (P = 0.90) were not affected by maternal overnutrition. Gestational period did not affect the mRNA expression of β-catenin (P = 0.60) and MyoG (P = 0.21). The mRNA expression of MyoD tended to increase with days of gestation (P = 0.06). The mRNA expression of zinc finger protein 423 (Zfp423; P < 0.0001), C/EBPα (P = 0.01), and PPARγ (P < 0.0001) were enhanced in ON fetuses. No effects of days of gestation were observed for mRNA expression of Zfp423 (P = 0.75) and C/EBPα (P = 0.48). The mRNA expression of PPARγ in fetuses at 190 d of gestation tended to be greater than those at 135 and 240 d of gestation (P = 0.06). The mRNA expression of transforming growth factor β (TGF-β; P < 0.0001), collagen type III, α I (COL3A1; P < 0.0001), and collagen content (P = 0.01) were increased in ON fetuses. Gestational period did not affect the mRNA expression of collagen type I, α I (COL1A1; P = 0.65). The mRNA expression of COL3A1 (P = 0.09) in fetuses at 190 d of gestation tended to be greater than fetuses at 135 and 240 d of gestation. The mRNA expression of TGF-β in fetuses at 190 d of gestation was

  11. Transcription Expression and Clinical Significance of Dishevelled-3 mRNA and δ-Catenin mRNA in Pleural Effusions from Patients with Lung Cancer

    PubMed Central

    Li, Xiao-Yan; Liu, Shu-Li; Cha, Na; Zhao, Yu-Jie; Wang, Shao-Cheng; Li, Wei-Nan; Wang, En-Hua; Wu, Guang-Ping

    2012-01-01

    Objective. To evaluate diagnostic utility of Dishevelled-3 (DVL-3) mRNA and δ-catenin mRNA expression in pleural effusions of patients with lung cancer. Methods. DVL-3 mRNA and δ-catenin mRNA levels were assessed by performing RT-PCR on pleural effusion specimens from patients with lung cancer (n = 75) and with lung benign disease (n = 51). Results. The expressions of DVL-3 mRNA and δ-catenin mRNA were significantly higher in malignant than in benign lung disease (P < 0.01) and were obviously higher than cytology in adenocarcinoma (P < 0.01). In single use, DVL-3 mRNA had the highest specificity (94.1%) and PPV (95.7%), whereas δ-catenin mRNA had the highest sensitivity (92.0%) and NPV (88.5%). When combinations of markers were evaluated together, DVL-3 mRNA and δ-catenin mRNA gave a high-diagnostic performance: sensitivity of 100.0%, NPV of 100.0%, and accuracy of 96.0%, respectively. Conclusion. As molecular markers of detecting pleural micrometastasis, DVL-3 mRNA and δ-catenin mRNA are helpful to diagnose the cancer cells in pleural effusions of patients with lung cancer. PMID:22461838

  12. Effects of social isolation on mRNA expression for corticotrophin-releasing hormone receptors in prairie voles.

    PubMed

    Pournajafi-Nazarloo, Hossein; Partoo, Leila; Yee, Jason; Stevenson, Jennifer; Sanzenbacher, Lisa; Kenkel, William; Mohsenpour, Seyed Ramezan; Hashimoto, Kozo; Carter, C Sue

    2011-07-01

    Previous studies have demonstrated that various type of stressors modulate messenger ribonucleic acid (mRNA) for type 1 corticotropin-releasing hormone (CRH) receptor (CRH-R1 mRNA) and type 2 CRH receptor (CRH-R2 mRNA). The purpose of this study was to explore the effect of social isolation stress of varying durations on the CRH, CRH-R1 and CRH-R2 mRNAs expression in the hypothalamus, hippocampus and pituitary of socially monogamous female and male prairie voles (Microtus ochrogaster). Isolation for 1h (single isolation) or 1h of isolation every day for 4 weeks (repeated isolation) was followed by a significant increase in plasma corticosterone levels. Single or repeated isolation increased hypothalamic CRH mRNA expression, but no changes in CRH-R1 mRNA in the hypothalamus were observed. Continuous isolation for 4 weeks (chronic isolation) showed no effect on hypothalamic CRH or CRH-R1 mRNAs in female or male animals. However, hypothalamic CRH-R2 mRNA was significantly reduced in voles exposed to chronic isolation. Single or repeated isolation, but not chronic isolation, significantly increased CRH-R1 mRNA and decreased CRH-R2 mRNA in the pituitary. Despite elevated CRH mRNA expression, CRH-R1 and CRH-R2 mRNAs were not modulated in the hippocampus following single or repeated isolation. Although, chronic isolation did not affect hippocampal CRH or CRH-R1 mRNAs, it did increase CRH-R2 mRNA expression in females and males. The results of the present study in prairie voles suggest that social isolation has receptor subtype and species-specific consequences for the modulation of gene expression for CRH and its receptors in brain and pituitary. Previous studies have revealed a female-biased increase in oxytocin in response to chronic isolation; however, we did not find a sex difference in CRH or its receptors following single, repeated or chronic social isolation, suggesting that sexually dimorphic processes beyond the CRH system, possibly involving vasopressin, might

  13. Acquisition of and withdrawal from cocaine self-administration regulates 5-HT mRNA expression in rat striatum.

    PubMed

    Neumaier, John F; McDevitt, Ross A; Polis, Ilham Y; Parsons, Loren H

    2009-10-01

    This study investigated how different stages of cocaine self-administration in rats affect the expression of two serotonin receptors in dorsal and ventral striatum, the 5-HT(1B) and 5-HT(6) subtypes, which have both been implicated in mediating some aspects of cocaine-related behaviors. In the first experiment, rats were trained to work for saccharin (oral) or cocaine (i.v.) reinforcers. We found that continuous access to cocaine for 23 days did not change the level of 5-HT(1B) mRNA expression compared to control animals receiving saccharin. However, a single cocaine session, given either by self-administration or non-contingently, increased 5-HT(1B) mRNA in dorsal striatum, whereas forced abstinence for two weeks after cocaine reduced 5-HT(1B) mRNA expression in the same subregion. 5-HT(6) mRNA was not changed by any of these treatments. A follow-up experiment investigated the effects of limited versus extended access to cocaine as well as forced abstinence, and we found that 14 days of forced abstinence significantly reduced 5-HT(1B) mRNA throughout the dorsal and ventral striatum compared to no withdrawal. These results suggest that the influence of 5-HT(1B) receptors in striatal projection neurons may be increased during cocaine acquisition and reduced after forced abstinence and may therefore be targets for pharmacological intervention in addiction.

  14. Comparative mRNA Expression Profiles of Riboflavin Biosynthesis Genes in Lactobacilli Isolated from Human Feces and Fermented Bamboo Shoots.

    PubMed

    Thakur, Kiran; Tomar, Sudhir K; Wei, Zhao-Jun

    2017-01-01

    With the aim to bioprospect potent riboflavin producing lactobacilli, the present study was carried out to evaluate the relative mRNA expression of riboflavin biosynthesis genes namely Rib 1, Rib 2, Rib 3, and Rib 4 from potent riboflavin producers obtained from our previous studies. All the four genes were successfully cloned and sequenced for further analysis by in silico procedures. As studied by non-denaturing Polyacrylamide gel electrophoresis, no difference in size of all the four genes among those of various lactobacilli was observed. The relative fold increase in mRNA expression in Rib 1, Rib 2, Rib 3, and Rib 4 genes has been observed to be 10-, 1-, 0.7-, and 8.5-fold, respectively. Due to increase in relative mRNA expression for all the Rib genes as well as phenotypic production attribute, KTLF1 strain was used further for expression studies in milk and whey. The fold increase in mRNA expression for all the four Rib genes was higher at 12 and 18 h in milk and whey respectively. After exposure to roseoflavin, resistant variant of KTLF1 showed considerable increase in expression of all the targets genes. This is the first ever study to compare the mRNA expression of riboflavin biosynthesis pathway genes in lactobacilli and it also under lines the effect of media and harvesting time which significantly affect the expression of rib genes. The use of roseoflavin-resistant strains capable of synthesizing riboflavin in milk and whey paves a way for an exciting and economically viable biotechnological approach to develop novel riboflavin bio-enriched functional foods.

  15. Comparative mRNA Expression Profiles of Riboflavin Biosynthesis Genes in Lactobacilli Isolated from Human Feces and Fermented Bamboo Shoots

    PubMed Central

    Thakur, Kiran; Tomar, Sudhir K.; Wei, Zhao-Jun

    2017-01-01

    With the aim to bioprospect potent riboflavin producing lactobacilli, the present study was carried out to evaluate the relative mRNA expression of riboflavin biosynthesis genes namely Rib 1, Rib 2, Rib 3, and Rib 4 from potent riboflavin producers obtained from our previous studies. All the four genes were successfully cloned and sequenced for further analysis by in silico procedures. As studied by non-denaturing Polyacrylamide gel electrophoresis, no difference in size of all the four genes among those of various lactobacilli was observed. The relative fold increase in mRNA expression in Rib 1, Rib 2, Rib 3, and Rib 4 genes has been observed to be 10-, 1-, 0.7-, and 8.5-fold, respectively. Due to increase in relative mRNA expression for all the Rib genes as well as phenotypic production attribute, KTLF1 strain was used further for expression studies in milk and whey. The fold increase in mRNA expression for all the four Rib genes was higher at 12 and 18 h in milk and whey respectively. After exposure to roseoflavin, resistant variant of KTLF1 showed considerable increase in expression of all the targets genes. This is the first ever study to compare the mRNA expression of riboflavin biosynthesis pathway genes in lactobacilli and it also under lines the effect of media and harvesting time which significantly affect the expression of rib genes. The use of roseoflavin-resistant strains capable of synthesizing riboflavin in milk and whey paves a way for an exciting and economically viable biotechnological approach to develop novel riboflavin bio-enriched functional foods. PMID:28367143

  16. Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

    PubMed Central

    Larsson, S; Hotchkiss, G; Andäng, M; Nyholm, T; Inzunza, J; Jansson, I; Ahrlund-Richter, L

    1994-01-01

    We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels. Images PMID:8036151

  17. Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

    PubMed

    Larsson, S; Hotchkiss, G; Andäng, M; Nyholm, T; Inzunza, J; Jansson, I; Ahrlund-Richter, L

    1994-06-25

    We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels.

  18. Fucoxanthin regulates adipocytokine mRNA expression in white adipose tissue of diabetic/obese KK-Ay mice.

    PubMed

    Hosokawa, Masashi; Miyashita, Tatsuya; Nishikawa, Sho; Emi, Shingo; Tsukui, Takayuki; Beppu, Fumiaki; Okada, Tomoko; Miyashita, Kazuo

    2010-12-01

    Fucoxanthin, a marine carotenoid found in edible brown seaweeds, attenuates white adipose tissue (WAT) weight gain and hyperglycemia in diabetic/obese KK-A(y) mice, although it does not affect these parameters in lean C57BL/6J mice. In perigonadal and mesenteric WATs of KK-A(y) mice fed fucoxanthin, mRNA expression levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α), which are considered to induce insulin resistance, were markedly reduced compared to control mice. In contrast to KK-A(y) mice, fucoxanthin did not alter MCP-1 and TNF-α mRNA expression levels in the WAT of lean C57BL/6J mice. Interleukin-6 (IL-6) and plasminogen activator inhibitor-1 mRNA expression levels in WAT were also decreased by fucoxanthin in KK-A(y) mice. In differentiating 3T3-F442A adipocytes, fucoxanthinol, which is a fucoxanthin metabolite found in WAT, attenuated TNF-α-induced MCP-1 and IL-6 mRNA overexpression and protein secretion into the culture medium. In addition, fucoxanthinol decreased TNF-α, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) mRNA expression in RAW264.7 macrophage-like cells stimulated by palmitic acid. These findings indicate that fucoxanthin regulates mRNA expression of inflammatory adipocytokines involved in insulin resistance, iNOS, and COX-2 in WAT and has specific effects on diabetic/obese KK-A(y) mice, but not on lean C57BL/6J mice. Copyright © 2010 Elsevier Inc. All rights reserved.

  19. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    SciTech Connect

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M. )

    1990-08-01

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.

  20. Enhanced expression of IL-10 in contrast to IL-12B mRNA in poultry with experimental coccidiosis.

    PubMed

    Haritova, A M; Stanilova, S A

    2012-11-01

    Interleukin (IL)-12 and IL-10 are immunoregulatory cytokines with an antagonistic effect on the T-helper (Th)1/Th2 cytokine balance and they provide a functional link between innate resistance and the adaptive immune response. This investigation was conducted to determine the expression of IL-10 and IL-12B mRNA levels in chickens' gut mucosa infected with Eimeria tenella and in sulfachlorpyrazine-sodium treated animals after infection. Broiler chickens were randomly allocated in three groups: healthy untreated control; infected untreated animals and infected, treated with sulfachlorpyrazine sodium chickens 6 days after the challenge with an E. tenella. Quantitative real time PCR analysis was performed using specific primer pairs and probes for IL-10 and IL-12B. The expression of IL-10 mRNA was greater in the duodenum then in the caecum and the liver of healthy chickens. E. tenella infection led to significant up-regulation of IL-10 mRNA in the caecum, followed by mRNA in the liver. A significant down regulation was observed mainly in the caecum after the treatment with sulfachlorpyrazine. In contrast, IL-12B expression in all investigated tissues remained insignificantly affected in the studied groups of animals. Distinct up-regulation of IL-10 mRNA, after the challenge with E. tenella, in the caecum can be attributed to the tissue tropism of Eimeria spp. The production of IL-12 is regulated by negative feedback through IL-10 which explains lack of increase in IL-12B mRNA. Sulfonamide treatment resulted in clinical improvement and restoration of IL-10 mRNA to the levels observed in healthy chickens. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. 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

  2. Regulation of glutamic acid decarboxylase mRNA expression in rat brain after sertraline treatment.

    PubMed

    Giardino, L; Zanni, M; Bettelli, C; Savina, M A; Calzà, L

    1996-09-26

    We now investigated the effect of chronic treatment with sertraline on glutamic acid decarboxylase mRNA expression in different rat brain areas by means of in situ hybridization. We found a reduced glutamic acid decarboxylase mRNA expression in the prefrontal cortex, accumbens nucleus, olfactory tubercle and reticular nucleus of the thalamus. The involvement of presynaptic modulation of gamma-amino-butyric acid transmission in the anxiolytic effect of sertraline is discussed.

  3. 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.

  4. 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

  5. Riboflavin and ultraviolet illumination affects selected platelet mRNA transcript amounts differently.

    PubMed

    Klein-Bosgoed, Christa; Schubert, Peter; Devine, Dana V

    2016-09-01

    Pathogen inactivation (PI) techniques use ultraviolet (UV) illumination with or without a photosensitizer to destroy pathogen RNA and DNA. Although lacking a nucleus and innate DNA transcription, platelets (PLTs) contain RNA and can synthesize proteins. The impact of PI on PLT protein synthesis and function is unknown; altered synthesis may affect overall PLT quality. In this study we determine to what extent PLT RNA is affected by PI. In a pool-and-split design, paired apheresis PLT concentrates were treated with riboflavin and UV illumination or were left untreated. PLT total RNA and mRNA amounts specific for glycoproteins (GP)IIIa, GPIIb, and GPIb; α-granule proteins PLT factor (PF)4; osteonectin and thrombospondin (TSP); and housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using absorbance and quantitative polymerase chain reaction. After treatment, amounts of all analyzed mRNAs were significantly reduced (p < 0.05), but to different degrees. For GAPDH and PF4, transcripts appeared less susceptible to the treatment, with 70% remaining 1 hour after UV illumination. For GPIIIa and TSP, less than 15% remained after treatment. There was a correlation (R(2)  = 0.85) between transcript length and amount of mRNA remaining 1 hour after treatment. Total RNA demonstrated a life span equal to the PLT life span of 10 to 11 days. This is the first report of the impact of riboflavin and UV illumination on PLT mRNA. Results suggest that all mRNA present in PLTs is affected by the treatment although the degree of the effect varies among transcripts. © 2016 AABB.

  6. Localization and differential regulation of angiotensinogen mRNA expression in the vessel wall.

    PubMed Central

    Naftilan, A J; Zuo, W M; Inglefinger, J; Ryan, T J; Pratt, R E; Dzau, V J

    1991-01-01

    Recent data demonstrate the existence of a vascular renin angiotensin system. In this study we examine the localization of angiotensinogen mRNA in the blood vessel wall of two rat strains, the Wistar and Wistar Kyoto (WKY), as well as the regulation of vascular angiotensinogen mRNA expression by dietary sodium. Northern blot analysis and in situ hybridization histochemistry demonstrate that in both strains angiotensinogen mRNA is detected in the aortic medial smooth muscle layer as well as the periaortic fat. In WKY rats fed a 1.6% sodium diet, angiotensinogen mRNA concentration is 2.6-fold higher in the periaortic fat than in the smooth muscle, as analyzed by quantitative slot blot hybridization. Angiotensinogen mRNA expression in the medial smooth muscle layer is sodium regulated. After 5 d of a low (0.02%) sodium diet, smooth muscle angiotensinogen mRNA levels increase 3.2-fold (P less than 0.005) as compared with the 1.6% sodium diet. In contrast, angiotensinogen mRNA level in the periaortic fat is not influenced by sodium diet. In summary, our data demonstrate regional (smooth muscle vs. periaortic fat) differential regulation of angiotensinogen mRNA levels in the blood vessel wall by sodium. This regional differential regulation by sodium may have important physiological implications. Images PMID:2010543

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

    PubMed

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

    2012-09-01

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

  8. 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

  9. Gonadal mRNA expression levels of TGFbeta superfamily signaling factors correspond with post-hatching morphological development in American alligators.

    PubMed

    Moore, B C; Hamlin, H J; Botteri, N L; Guillette, L J

    2010-01-01

    Paracrine factor signaling regulates many aspects of vertebrate gonadal development. We investigated key ovarian and testicular morphological markers of the American alligator (Alligator mississippiensis) during the first 5 months post-hatching and correlated gonadal development with mRNA expression levels of a suite of regulatory factors. In both sexes, we observed significant morphology changes, including ovarian follicle assembly and meiotic progression of testicular germ cells. Concomitant with these changes were sexually dimorphic and ontogenetically variable mRNA expressions. In ovaries, FOXL2, aromatase, and follistatin mRNA expression was greater than in testes at all ages. At one week after hatching, we observed ovarian medullary remodeling in association with elevated activin/inhibin beta A subunit, follistatin, and aromatase mRNA expressions. Three and 5 months following hatching and concomitant with follicle assembly, ovaries showed increased mRNA expression levels of GDF9 and the mitotic factor PCNA. In testes, the activin/inhibin alpha and beta B subunit transcript levels were greater than in ovaries at all ages. Elevated testicular expression of GDF9 mRNA levels at 5 months after hatching aligned with increased spermatogenic activity. We propose that the mRNA expression levels and concomitant morphological changes observed here affect the establishment of alligator reproductive health and later fertility. (c) 2010 S. Karger AG, Basel.

  10. Gonadal mRNA Expression Levels of TGFβ Superfamily Signaling Factors Correspond with Post-Hatching Morphological Development in American Alligators

    PubMed Central

    Moore, B.C.; Hamlin, H.J.; Botteri, N.L.; Guillette, L.J.

    2010-01-01

    Paracrine factor signaling regulates many aspects of vertebrate gonadal development. We investigated key ovarian and testicular morphological markers of the American alligator (Alligator mississippiensis) during the first 5 months post-hatching and correlated gonadal development with mRNA expression levels of a suite of regulatory factors. In both sexes, we observed significant morphology changes, including ovarian follicle assembly and meiotic progression of testicular germ cells. Concomitant with these changes were sexually dimorphic and ontogenetically variable mRNA expressions. In ovaries, FOXL2, aromatase, and follistatin mRNA expression was greater than in testes at all ages. At one week after hatching, we observed ovarian medullary remodeling in association with elevated activin/inhibin βA subunit, follistatin, and aromatase mRNA expressions. Three and 5 months following hatching and concomitant with follicle assembly, ovaries showed increased mRNA expression levels of GDF9 and the mitotic factor PCNA. In testes, the activin/inhibin α and βB subunit transcript levels were greater than in ovaries at all ages. Elevated testicular expression of GDF9 mRNA levels at 5 months after hatching aligned with increased spermatogenic activity. We propose that the mRNA expression levels and concomitant morphological changes observed here affect the establishment of alligator reproductive health and later fertility. PMID:20110644

  11. TGF beta 2 mRNA expression and pregnancy failure in mice.

    PubMed

    Gorivodsky, M; Torchinsky, A; Zemliak, I; Savion, S; Fein, A; Toder, V

    1999-08-01

    We describe here a pattern of transforming growth factor (TGF) beta2 mRNA expression at the fetomaternal interface in mice with high rate of resorptions as well as its expression following maternal immunopotentiation. TGF beta 2 mRNA expression was evaluated in the uteroplacental units of mice with spontaneous (CBA/J x DBA/2J mouse combination) or cyclophosphamide (CP)-induced pregnancy loss. The effect of immunopotentiation on TGF beta 2 mRNA expression was determined in CP-treated females who underwent nonspecific immunostimulation with xenogeneic (rat) leukocytes. A quantitative analysis of TGF beta 2 mRNA level was performed using RNase protection assay. Distribution of TGF beta 2 mRNA transcripts at the fetomaternal interface was studied by in situ hybridization analysis. RNase protection analysis revealed four TGF beta 2 specific mRNA forms (330, 270, 230, and 170 bp) in the uteroplacental units of mice with either normal or decreased reproductive performance. A significant decrease (about 50%) in the level of TGF beta 2 mRNA was registered in the uteroplacental unit of mice with pregnancy loss as compared to the control mice. TGF beta 2 transcripts were abundant in the uterine epithelium and stroma. A specific hybridization signal was detected also in metrial gland cells and it was found to be substantially lower in CP-treated as compared to intact mice. In the resorbing uteroplacental unit, the expression of TGF beta 2 mRNA was completely lost in the uterine epithelium, and the number of TGF beta 2 mRNA-positive metrial gland cells was lower as compared to the control. Immunopotentiation decreased the resorption rate in mice with CP-induced pregnancy loss and caused a dramatic increase in TGF beta 2 mRNA expression: the level of TGF beta 2 mRNA was found to be higher by 2.0-3.2 fold in the uteroplacental unit of immunized as compared to nonimmunized CP-treated mice. These data suggest that distortion of TGF beta 2 expression at the fetomaternal interface

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

    PubMed

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

    1998-12-10

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

  13. Hypothalamic expression of NPY mRNA, vasopressin mRNA and CRF mRNA in response to food restriction and central administration of the orexigenic peptide GHRP-6.

    PubMed

    Johnstone, Louise E; Srisawat, Rungrudee; Kumarnsit, Ekkasit; Leng, Gareth

    2005-03-01

    In this study, we examined the effects of restricted feeding and of central administration of an orexigenic ghrelin agonist GHRP-6 on peptide mRNA expression in the hypothalamus. We compared rats fed ad libitum with rats that were allowed food for only 2?h every day, and treated with a continuous chronic i.c.v. infusion of GHRP-6 or vehicle. Ad libitum fed rats exposed to GHRP-6 increased their food intake and body weight over 6 days, but, at the end of this period, neuropeptide Y mRNA expression in the arcuate nucleus was not different to that in control rats. By contrast, expression of neuropeptide Y mRNA in the arcuate nucleus was elevated in food-restricted rats, consistent with the interpretation that increased expression reflects increased hunger. However, neuropeptide Y mRNA expression was no greater in food-restricted rats infused with GHRP-6 than in food-restricted rats infused with vehicle; thus if the drive to eat was stronger in rats infused with GHRP-6, this was not reflected by higher levels of neuropeptide Y mRNA expression. Expression of vasopressin mRNA and corticotrophin releasing factor (CRF) mRNA in the paraventricular nucleus (PVN) was not changed by food restriction. GHRP-6 infusion increased CRF mRNA expression in ad libitum rats only.

  14. Local anesthetics inhibit tissue factor expression in activated monocytes via inhibition of tissue factor mRNA synthesis.

    PubMed

    Kim, Ji-Eun; Kim, Ki Jun; Ahn, Wonsik; Han, Kyou-Sup; Kim, Hyun Kyung

    2011-01-01

    Local anesthetics have been reported to have anticoagulant properties, but the mechanisms responsible for this action are poorly understood. Here, we evaluated the in vitro effects of 3 local anesthetics--lidocaine, ropivacaine, and bupivacaine--on the tissue factor expression by monocytes. Monocytes from peripheral blood were stimulated with lipopolysaccharide (LPS) in the presence or absence of local anesthetics. All 3 local anesthetics inhibited the expression of tissue factor antigen and tissue factor activity in LPS-stimulated monocytes in a dose- and time-dependent manner and reduced tissue factor messenger RNA (mRNA) expression in endothelial cells and a monocytic cell line. None of the 3 drugs induced apoptosis or affected the viability of monocytes. Our findings that local anesthetics inhibited the tissue factor induction in activated monocytes by inhibiting tissue factor mRNA level may demonstrate the feasibility of using local anesthetics in hypercoagulable and inflammatory conditions.

  15. Testosterone Regulates NUCB2 mRNA Expression in Male Mouse Hypothalamus and Pituitary Gland.

    PubMed

    Seon, Sojeong; Jeon, Daun; Kim, Heejeong; Chung, Yiwa; Choi, Narae; Yang, Hyunwon

    2017-03-01

    Nesfatin-1/NUCB2 is known to take part in the control of the appetite and energy metabolism. Recently, many reports have shown nesfatin-1/NUCB2 expression and function in various organs. We previously demonstrated that nesfatin-1/NUCB2 expression level is higher in the pituitary gland compared to other organs and its expression is regulated by 17β-estradiol and progesterone secreted from the ovary. However, currently no data exist on the expression of nesfatin-1/NUCB2 and its regulation mechanism in the pituitary of male mouse. Therefore, we examined whether nesfatin-1/NUCB2 is expressed in the male mouse pituitary and if its expression is regulated by testosterone. As a result of PCR and western blotting, we found that a large amount of nesfatin-1/NUCB2 was expressed in the pituitary and hypothalamus. The NUCB2 mRNA expression level in the pituitary was decreased after castration, but not in the hypothalamus. In addition, its mRNA expression level in the pituitary was increased after testosterone treatment in the castrated mice, whereas, the expression level in the hypothalamus was significantly decreased after the treatment with testosterone. The in vitro experiment to elucidate the direct effect of testosterone on NUCB2 mRNA expression showed that NUCB2 mRNA expression was significantly decreased with testosterone in cultured hypothalamus tissue, but increased with testosterone in cultured pituitary gland. The present study demonstrated that nesfatin-1/NUCB2 was highly expressed in the male mouse pituitary and was regulated by testosterone. This data suggests that reproductive-endocrine regulation through hypothalamus-pituitary-testis axis may contribute to NUCB2 mRNA expression in the mouse hypothalamus and pituitary gland.

  16. Testosterone Regulates NUCB2 mRNA Expression in Male Mouse Hypothalamus and Pituitary Gland

    PubMed Central

    Seon, Sojeong; Jeon, Daun; Kim, Heejeong; Chung, Yiwa; Choi, Narae; Yang, Hyunwon

    2017-01-01

    ABSTRACT Nesfatin-1/NUCB2 is known to take part in the control of the appetite and energy metabolism. Recently, many reports have shown nesfatin-1/NUCB2 expression and function in various organs. We previously demonstrated that nesfatin-1/NUCB2 expression level is higher in the pituitary gland compared to other organs and its expression is regulated by 17β-estradiol and progesterone secreted from the ovary. However, currently no data exist on the expression of nesfatin-1/NUCB2 and its regulation mechanism in the pituitary of male mouse. Therefore, we examined whether nesfatin-1/NUCB2 is expressed in the male mouse pituitary and if its expression is regulated by testosterone. As a result of PCR and western blotting, we found that a large amount of nesfatin-1/NUCB2 was expressed in the pituitary and hypothalamus. The NUCB2 mRNA expression level in the pituitary was decreased after castration, but not in the hypothalamus. In addition, its mRNA expression level in the pituitary was increased after testosterone treatment in the castrated mice, whereas, the expression level in the hypothalamus was significantly decreased after the treatment with testosterone. The in vitro experiment to elucidate the direct effect of testosterone on NUCB2 mRNA expression showed that NUCB2 mRNA expression was significantly decreased with testosterone in cultured hypothalamus tissue, but increased with testosterone in cultured pituitary gland. The present study demonstrated that nesfatin-1/NUCB2 was highly expressed in the male mouse pituitary and was regulated by testosterone. This data suggests that reproductive-endocrine regulation through hypothalamus-pituitary-testis axis may contribute to NUCB2 mRNA expression in the mouse hypothalamus and pituitary gland. PMID:28484746

  17. 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.

  18. Development × environment interactions control tph2 mRNA expression.

    PubMed

    Lukkes, J L; Kopelman, J M; Donner, N C; Hale, M W; Lowry, C A

    2013-05-01

    Adverse early life experience is thought to increase an individual's susceptibility to mental health disorders, including anxiety and affective disorders, later in life. Our previous studies have shown that post-weaning social isolation of female rats during a critical period of development sensitizes an anxiety-related serotonergic dorsal raphe nucleus (DR) system in adulthood. Therefore, we investigated how post-weaning social isolation, in combination with a challenge with the anxiogenic drug, N-methyl-beta-carboline-3-carboxamide (FG-7142; a partial inverse agonist at the benzodiazepine allosteric site on the GABAA receptor), affects home cage behavior and serotonergic gene expression in the DR of female rats using in situ hybridization histochemistry. Juvenile female rats were reared in isolation or groups of three for a 3-week period from weaning (postnatal day (PD) 21 to mid-adolescence (PD42)), after which all rats were group-reared for an additional 16 days until adulthood. Among vehicle-treated rats, isolation-reared rats had decreased rodent tryptophan hydroxylase 2 (tph2) mRNA expression in ventral and ventrolateral subdivisions of the DR, a pattern observed previously in a rat model of panic disorder. Isolation-reared rats, but not group-reared rats, responded to FG-7142 with increased duration of vigilance and arousal behaviors. In addition, FG-7142 decreased tph2 expression, measured 4h following treatment, in multiple subregions of the DR of group-reared rats but had no effect in isolation-reared rats. No treatment effects were observed on 5-HT1A receptor or serotonin transporter gene expression. These data suggest that adolescent social isolation alters tph2 expression in specific subregions of the DR and alters the effects of stress-related stimuli on behavior and serotonergic systems.

  19. Development x environment interactions control tph2 mRNA expression

    PubMed Central

    Lukkes, Jodi L.; Kopelman, Jared M.; Donner, Nina C.; Hale, Matthew W.; Lowry, Christopher A.

    2013-01-01

    Adverse early life experience is thought to increase an individual's susceptibility to mental health disorders, including anxiety and affective disorders, later in life. Our previous studies have shown that post-weaning social isolation of female rats during a critical period of development sensitizes an anxiety-related serotonergic dorsal raphe nucleus (DR) system in adulthood. Therefore, we investigated how post-weaning social isolation, in combination with a challenge with the anxiogenic drug, N-methyl-beta-carboline-3-carboxamide (FG-7142; a partial inverse agonist at the benzodiazepine allosteric site on the GABAA receptor), affects home cage behavior and serotonergic gene expression in the DR of female rats using in situ hybridization histochemistry. Juvenile female rats were reared in isolation or groups of three for a 3-week period from weaning (postnatal day (PD) 21 to mid-adolescence (PD42)), after which all rats were group-reared for an additional 16 days until adulthood. Among vehicle-treated rats, isolation-reared rats had decreased tryptophan hydroxylase 2 (tph2) mRNA expression in ventral and ventrolateral subdivisions of the DR, a pattern observed previously in a rat model of panic disorder. Isolation-reared rats, but not group-reared rats, responded to FG-7142 with increased duration of vigilance and arousal behaviors. In addition, FG-7142 decreased tph2 expression, measured 4 h following treatment, in multiple subregions of the DR of group-reared rats but had no effect in isolation-reared rats. No treatment effects were observed on 5-HT1A receptor or serotonin transporter gene expression. These data suggest that adolescent social isolation alters tph2 expression in specific subregions of the DR and alters the effects of stress-related stimuli on behavior and serotonergic systems. PMID:23403177

  20. BENZO(A)PYRENE DECREASES BRAIN AND OVARIAN AROMATASE mRNA EXPRESSION IN FUNDULUS HETEROCLITUS

    PubMed Central

    Dong, Wu; Wang, Lu; Thornton, Cammi; Scheffler, Brian E.; Willett, Kristine L.

    2008-01-01

    The higher molecular weight polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BaP) are typically associated with genotoxicity, however, newer evidence suggests that these compounds may also act as endocrine system disruptors. We hypothesized that altered expression of the P450 enzyme aromatase genes could be a target for reproductive or developmental dysfunction caused by BaP exposure. Aromatase is at least partially responsible for estrogen homeostasis by converting androgens into estrogens. In fish, there are two isoforms of aromatase, a predominantly ovarian form, CYP19A1, and a brain form, CYP19A2. CYP19 mRNA expression was measured following BaP exposure (0, 10, 100 µg/L waterborne for 10 or 15 days) in Fundulus adults, juveniles and embryos by in situ hybridization. The CYP19A1 expression was significantly decreased after BaP exposure in the 3 month old Fundulus immature oocytes, but BaP did not affect CYP19A1 expression at any stage in adult oocytes. In embryo brains, BaP significantly decreased CYP19A2 compared to controls by 3.6-fold at 14 days post-fertilization. In adults, CYP19A2 expression was decreased significantly in the pituitary and hypothalamus (81% and 85% of controls, respectively). Promoter regions of Fundulus CYP19s were cloned, and putative response elements in the CYP19A1 and CYP19A2 promoters such as CRE, AhR and ERE may be involved in BaP-mediated changes in CYP19 expression. In order to compare the mechanism of BaP-mediated inhibition with that of a known aromatase inhibitor, fish were also exposed to fadrozole (20 and 100 µg/L). Fadrozole did not significantly decrease the mRNA expression in embryos or adult Fundulus. However, aromatase enzyme activity was significantly decreased in adult ovary and brain tissues. These studies provide a greater molecular understanding of the mechanisms of action of BaP and its potential to impact reproduction or development. PMID:18571745

  1. Altered EphA5 mRNA expression in rat brain with a single methamphetamine treatment.

    PubMed

    Numachi, Yohtaro; Yoshida, Sumiko; Yamashita, Motoyasu; Fujiyama, Ko; Toda, Shigenobu; Matsuoka, Hiroo; Kajii, Yasushi; Nishikawa, Toru

    2007-09-07

    Methamphetamine is a potent and indirect dopaminergic agonist which can cause chronic brain dysfunctions including drug abuse, drug dependence and drug-induced psychosis. Methamphetamine is known to trigger molecular mechanisms involved in associative learning and memory, and thereby alter patterns of synaptic connectivity. The persistent risk of relapse in methamphetamine abuse, dependence and psychosis may be caused by such alterations in synaptic connectivity. EphA5 receptors constitute large families of tyrosine kinase receptor and are expressed almost exclusively in the nervous system, especially in the limbic structures. Recent studies suggest EphA5 to be important in the topographic projection, development, and plasticity of limbic structures, and to be involved in dopaminergic neurotransmission. We used in situ hybridization to examine whether methamphetamine alters EphA5 mRNA expression in the brains of adult male Wister rats. EphA5 mRNA was widely distributed in the medial frontal cortex, cingulate cortex, piriform cortex, hippocampus, habenular nucleus and amygdala. Compared to baseline expression at 0h, EphA5 mRNA was significantly decreased (by 20%) in the medial frontal cortex at 24h, significantly increased (by 30%) in the amygdala at 9 and 24h, significantly but transiently decreased (by 30%) in the habenular nucleus at 1h after a single injection of methamphetamine. Methamphetamine did not change EphA5 mRNA expression in the cingulate cortex, piriform cortex or hippocampus. Our results that methamphetamine altered EphA5 mRNA expression in rat brain suggest methamphetamine could affect patterns of synaptic connectivity, which might be responsible for methamphetamine-induced chronic brain dysfunctions.

  2. 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.

  3. 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

  4. Deciphering the rules by which dynamics of mRNA secondary structure affect translation efficiency in Saccharomyces cerevisiae.

    PubMed

    Mao, Yuanhui; Liu, HuiLing; Liu, Yanlin; Tao, Shiheng

    2014-04-01

    Messenger RNA (mRNA) secondary structure decreases the elongation rate, as ribosomes must unwind every structure they encounter during translation. Therefore, the strength of mRNA secondary structure is assumed to be reduced in highly translated mRNAs. However, previous studies in vitro reported a positive correlation between mRNA folding strength and protein abundance. The counterintuitive finding suggests that mRNA secondary structure affects translation efficiency in an undetermined manner. Here, we analyzed the folding behavior of mRNA during translation and its effect on translation efficiency. We simulated translation process based on a novel computational model, taking into account the interactions among ribosomes, codon usage and mRNA secondary structures. We showed that mRNA secondary structure shortens ribosomal distance through the dynamics of folding strength. Notably, when adjacent ribosomes are close, mRNA secondary structures between them disappear, and codon usage determines the elongation rate. More importantly, our results showed that the combined effect of mRNA secondary structure and codon usage in highly translated mRNAs causes a short ribosomal distance in structural regions, which in turn eliminates the structures during translation, leading to a high elongation rate. Together, these findings reveal how the dynamics of mRNA secondary structure coupling with codon usage affect translation efficiency.

  5. 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.

  6. Sodium regulation of angiotensinogen mRNA expression in rat kidney cortex and medulla.

    PubMed Central

    Ingelfinger, J R; Pratt, R E; Ellison, K; Dzau, V J

    1986-01-01

    Rat liver angiotensinogen cDNA (pRang 3) and mouse renin cDNA (pDD-1D2) were used to identify angiotensinogen and renin mRNA sequences in rat kidney cortex and medulla in rats on high and low salt diet. Angiotensinogen mRNA sequences were present in renal cortex and medulla in apparently equal proportions, whereas renin mRNA sequences were found primarily in renal cortex. Average relative signal of rat liver to whole kidney angiotensinogen mRNA was 100:3. Densitometric analysis of Northern blots demonstrated that renal cortical angiotensinogen mRNA concentrations increased 3.5-fold (P less than 0.001) and medulla, 1.5-fold (P less than 0.005) on low sodium compared with high sodium diet, whereas renal cortex renin mRNA levels increased 6.8-fold (P less than 0.0005). Dietary sodium did not significantly influence liver angiotensinogen mRNA levels. These findings provide evidence for sodium regulation of renal renin and angiotensinogen mRNA expressions, which supports potential existence of an intrarenally regulated RAS and suggest that different factors regulate renal and hepatic angiotensinogen. Images PMID:3533999

  7. Stem-loop RNA labeling can affect nuclear and cytoplasmic mRNA processing.

    PubMed

    Heinrich, Stephanie; Sidler, Corinne L; Azzalin, Claus M; Weis, Karsten

    2017-02-01

    The binding of sequence-specific RNA-interacting proteins, such as the bacteriophage MS2 or PP7 coat proteins, to their corresponding target sequences has been extremely useful and widely used to visualize single mRNAs in vivo. However, introduction of MS2 stem-loops into yeast mRNAs has recently been shown to lead to the accumulation of RNA fragments, suggesting that the loops impair mRNA decay. This result was questioned, because fragment occurrence was mainly assessed using ensemble methods, and their cellular localization and its implications had not been addressed on a single transcript level. Here, we demonstrate that the introduction of either MS2 stem-loops (MS2SL) or PP7 stem-loops (PP7SL) can affect the processing and subcellular localization of mRNA. We use single-molecule fluorescence in situ hybridization (smFISH) to determine the localization of three independent mRNAs tagged with the stem-loop labeling systems in glucose-rich and glucose starvation conditions. Transcripts containing MS2SL or PP7SL display aberrant localization in both the nucleus and the cytoplasm. These defects are most prominent in glucose starvation conditions, with nuclear mRNA processing being altered and stem-loop fragments abnormally enriching in processing bodies (PBs). The mislocalization of SL-containing RNAs is independent of the presence of the MS2 or PP7 coat protein (MCP or PCP).

  8. Alkaline decontamination of sputum specimens adversely affects stability of mycobacterial mRNA.

    PubMed Central

    Desjardin, L E; Perkins, M D; Teixeira, L; Cave, M D; Eisenach, K D

    1996-01-01

    Reverse transcriptase PCR (RT-PCR) is an important tool for Mycobacterium tuberculosis research and diagnostics. A standard procedure using N-acetyl-L-cysteine (NALC) and NaOH has been widely adopted for digestion and decontamination of sputum specimens for mycobacterial culture. The objective of this study was to determine the compatibility of this method with the recovery of RNA for RT-PCR assays. Nineteen sputum specimens were collected from smear-positive, pretreatment tuberculosis patients. After homogenization with NALC and glass beads, specimens were further processed by the addition of either NaOH, as per the standard decontamination protocol, or phosphate buffer. RNA was prepared by using a modified guanidine-phenol extraction method developed specifically for sputum sediments. DNA was isolated from the same specimens. Reverse transcriptions of alpha antigen (85B protein) mRNA and 16S rRNA were performed together, and aliquots were removed for separate PCRs. In all specimens, the 85B mRNA target was greatly diminished by treatment with NaOH; however, the 16S rRNA target remained unaffected. Storing sputum specimens for 48 h at 4 degrees C before processing did not seem to affect the integrity or yield of RNA; however, some degradation occurred by 72 h. Data suggest that the NaOH-NALC method for processing sputum samples is not suitable for detecting mRNA targets in RT-PCR assays. PMID:8880495

  9. Expression of vascular endothelial growth factor mRNA in non-small-cell lung carcinomas

    PubMed Central

    Fontanini, G; Boldrini, L; Chinè, S; Pisaturo, F; Basolo, F; Calcinai, A; Lucchi, M; Mussi, A; Angeletti, C A; Bevilacqua, G

    1999-01-01

    The vascular endothelial growth factor (VEGF) has been shown to be strictly related to vascular permeability and endothelial cell growth under physiological and pathological conditions. In tumour development and progression, VEGF plays a pivotal role in the development of the tumoral vascular network, and useful information in the progression of human cancer can be obtained by analysing the vascular endothelial growth factor expression of the tumours. In this study, we investigated the vascular endothelial growth factor transcript expression in non-small-cell lung carcinomas to evaluate the significance of this factor in a group of cancers in which the vascular pattern has been shown to significantly affect progression. Surgical samples of 42 patients with NSCLC were studied using reverse transcription polymerase chain reaction (PCR) analysis and in situ hybridization. Thirty-three out of 42 cases (78.6%) showed VEGF transcript expression predominantly as transcripts for the secretory forms of VEGF (isoforms 121 and 165). In situ hybridization, performed on 24 out of 42 samples, showed that the VEGF transcript expression was in several cases present in the cytoplasm both of neoplastic and normal cells, even if the VEGF mRNA was less expressed in the corresponding non-tumoral part. The VEGF 121 expression was associated with hilar and/or mediastinal nodal involvement (P = 0.02), and, taken together, the VEGF isoforms were shown to significantly influence overall (P = 0.02) and disease-free survival (P = 0.03). As a regulator of tumour angiogenesis, VEGF may represent a useful indicator of progression and poor prognosis in non-small-cell lung carcinomas. © 1999 Cancer Research Campaign PMID:9888482

  10. [The expression of human telomerase reverse transcriptase mRNA and its significance in acute leukemia].

    PubMed

    Meng, Xiao-Li; Lin, Mao-Fang; Jin, Jie

    2003-02-01

    To investigate the expression of hTERT mRNA in bone marrow mononuclear cells (MNCs) from acute leukemia patients, the method of semi-quntitative RT-PCR was used to examine the expression of hTERT mRNA in marrow MNCs, and the telomerase activity of marrow MNCs was determined with the method of TRAP-PCR-ELISA by using a commercial kit. The results indicated that the expression of hTERT mRNA of marrow MNCs in 30 untreated AL patients was markedly higher than that in 12 CR cases (0.71 +/- 0.34 vs 0.43 +/- 0.25, P < 0.05) and 6 normal volunteers (0.71 +/- 0.34 vs 0.22 +/- 0.21, P < 0.01), respectively. Telomerase activity of marrow MNCs in 30 untreated AL patients was significantly higher than that in 12 CR cases (0.235 +/- 0.395 vs 0.012 +/- 0.015, P = 0.007). Moreover, there was a positive correlation between the hTERT mRNA synthesis and telomerase activity in AL cells (r = 0.421, P < 0.01). The pencentage of blast cells in marrow smear of the untreated AL patients was positively correlated with both the expression of hTERT mRNA and the telomerase activity of bone marrow MNCs (r = 0.457, P < 0.05 and r = 0.411, P < 0.05), respectively. It is concluded that the expression of hTERT mRNA in bone marrow MNCs from untreated AL patients was correlated with their telomerase activity. It is suggested that the expression of hTERT mRNA leukemic cells indicates their higher proliferation ability.

  11. Differential Patterns of Synaptotagmin7 mRNA Expression in Rats with Kainate- and Pilocarpine-Induced Seizures

    PubMed Central

    Glavan, Gordana; See, Ronald Eugene; Živin, Marko

    2012-01-01

    Previous studies in rat models of neurodegenerative disorders have shown disregulation of striatal synaptotagmin7 mRNA. Here we explored the expression of synaptotagmin7 mRNA in the brains of rats with seizures triggered by the glutamatergic agonist kainate (10 mg/kg) or by the muscarinic agonist pilocarpine (30 mg/kg) in LiCl (3 mEq/kg) pre-treated (24 h) rats, in a time-course experiment (30 min - 1 day). After kainate-induced seizures, synaptotagmin7 mRNA levels were transiently and uniformly increased throughout the dorsal and ventral striatum (accumbens) at 8 and 12 h, but not at 24 h, followed at 24 h by somewhat variable upregulation within different parts of the cerebral cortex, amigdala and thalamic nuclei, the hippocampus and the lateral septum. By contrast, after LiCl/pilocarpine-induced seizures, there was a more prolonged increase of striatal Synaptotagmin7 mRNA levels (at 8, 12 and 24 h), but only in the ventromedial striatum, while in some other of the aforementioned brain regions there was a decline to below the basal levels. After systemic post-treatment with muscarinic antagonist scopolamine in a dose of 2 mg/kg the seizures were either extinguished or attenuated. In scopolamine post-treated animals with extinguished seizures the striatal synaptotagmin7 mRNA levels (at 12 h after the onset of seizures) were not different from the levels in control animals without seizures, while in rats with attenuated seizures, the upregulation closely resembled kainate seizures-like pattern of striatal upregulation. In the dose of 1 mg/kg, scopolamine did not significantly affect the progression of pilocarpine-induced seizures or pilocarpine seizures-like pattern of striatal upregulation of synaptotagmin7 mRNA. In control experiments, equivalent doses of scopolamine per se did not affect the expression of synaptotagmin7 mRNA. We conclude that here described differential time course and pattern of synaptotagmin7 mRNA expression imply regional differences of

  12. Expression of a streptomycete leaderless mRNA encoding chloramphenicol acetyltransferase in Escherichia coli.

    PubMed Central

    Wu, C J; Janssen, G R

    1997-01-01

    The chloramphenicol acetyltransferase (cat) gene from Streptomyces acrimycini encodes a leaderless mRNA. Expression of the cat coding sequence as a leaderless mRNA from a modified lac promoter resulted in chloramphenicol resistance in Escherichia coli. Transcript mapping with nuclease S1 confirmed that the 5' end of the cat message initiated at the A of the AUG translational start codon. Site-directed mutagenesis of the lac promoter or the cat start codon abolished chloramphenicol resistance, indicating that E. coli initiated translation at the 5' terminal AUG of the cat leaderless mRNA. Addition of 5'-AUGC-3' to the 5' end of the cat mRNA resulted in translation occurring also from the reading frame defined by the added AUG triplet, suggesting that a 5'-terminal start codon is an important recognition feature for initiation and establishing reading frame during translation of leaderless mRNA. Addition of an untranslated leader and Shine-Dalgarno sequence to the cat coding sequence increased cat expression in a cat:lacZ fusion; however, the level of expression was significantly lower than when a fragment of the bacteriophage lambda cI gene, also encoding a leaderless mRNA, was fused to lacZ. These results indicate that in the absence of an untranslated leader and Shine-Dalgarno sequence, the streptomycete cat mRNA is translated by E. coli; however, the cat translation signals, or other features of the cat mRNA, provide for only a low level of expression in E. coli. PMID:9352935

  13. Neuropeptide Y mRNA expression levels following chronic olanzapine, clozapine and haloperidol administration in rats.

    PubMed

    Huang, X-F; Deng, Chao; Zavitsanou, Katerina

    2006-06-01

    Using quantitative in situ hybridization, this study examined regional changes in rat brain mRNA levels encoding neuropeptide Y (NPY) following olanzapine, clozapine and haloperidol administration (1.2, 1.5 and 2.0 mg/kg, oral) for 36 days. The NPY mRNA expression levels and patterns were examined after the last drug administration at both time points enabling the measurement of immediate effect at 2h and the effects after 48 h of drug administration. It was found that all these drugs had an immediate effect on NPY mRNA expression, while virtually all these changes normalized 48 h after the drug treatments. A similarity in altered NPY mRNA expression patterns was seen between the olanzapine and clozapine groups; however, haloperidol was very different. Olanzapine and clozapine administration decreased NPY mRNA levels in the nucleus accumbens, striatum and anterior cingulate cortex (from -60% to -77%, p<0.05). Haloperidol decreased NPY mRNA expression in the amygdala and hippocampus (-69%, -64%, p<0.05). In the lateral septal nucleus, NPY mRNA levels significantly decreased in the olanzapine group (-66%, p<0.05), a trend toward a decrease was observed in the clozapine group, and no change was found in the haloperidol treated group. These results suggest that the different effects of atypical and typical antipsychotics on NPY systems may reflect the neural chemical mechanisms responsible for the differences between these drugs in their effects in treating positive and negative symptoms of schizophrenia. The immediate decrease of NPY mRNA levels suggests an immediate reduction of NPY biosynthesis in response to these drugs.

  14. mRNA secondary structure engineering of Thermobifida fusca endoglucanase (Cel6A) for enhanced expression in Escherichia coli.

    PubMed

    Ali, Imran; Asghar, Rehana; Ahmed, Sajjad; Sajjad, Muhammad; Tariq, Muhammad; Waheed Akhtar, M

    2015-03-01

    The sequence and structure of mRNA plays an important role in solubility and expression of the translated protein. To divulge the role of mRNA secondary structure and its thermodynamics in the expression level of the recombinant endoglucanase in Escherichia coli, 5'-end of the mRNA was thermodynamically optimized. Molecular engineering was done by introducing two silent synonymous mutations at positions +5 (UCU with UCC) and +7 (UUC with UUU) of the 5'-end of mRNA to relieve hybridization with ribosomal binding site. Two variants of glycoside hydrolase family six endoglucanase, wild type (cel6A.wt) and mutant (cel6A.mut) from Thermobifida fusca were expressed and characterized in E. coli using T7 promoter-based expression vector; pET22b(+). Enhanced expression level of engineered construct (Cel6A.mut) with ∆G = -2.7 kcal mol(-1)was observed. It showed up to ~45 % higher expression as compared to the wild type construct (Cel6A.wt) having ∆G = -7.8 kcal mol(-1) and ~25 % expression to the total cell proteins. Heterologous protein was purified by heating the recombinant E. coli BL21 (DE3) CodonPlus at 60 °C. The optimum pH for enzyme activity was six and optimum temperature was 60 °C. Maximum activity was observed 4.5 Umg(-1) on CMC. Hydrolytic activity was also observed on insoluble substrates, i.e. RAC (2.8 Umg(-1)), alkali treated bagass (1.7 Umg(-1)), filter paper (1.2 Umg(-1)) and BMCC (0.3 Umg(-1)). Metal ions affect endoglucanase activity in different ways. Only Fe(2+) exhibited 20.8 % stimulatory effects on enzyme activity. Enzyme activity was profoundly inhibited by Hg2(+) (91.8 %).

  15. Cell-type-specific signatures of microRNAs on target mRNA expression.

    PubMed

    Sood, Pranidhi; Krek, Azra; Zavolan, Mihaela; Macino, Giuseppe; Rajewsky, Nikolaus

    2006-02-21

    Although it is known that the human genome contains hundreds of microRNA (miRNA) genes and that each miRNA can regulate a large number of mRNA targets, the overall effect of miRNAs on mRNA tissue profiles has not been systematically elucidated. Here, we show that predicted human mRNA targets of several highly tissue-specific miRNAs are typically expressed in the same tissue as the miRNA but at significantly lower levels than in tissues where the miRNA is not present. Conversely, highly expressed genes are often enriched in mRNAs that do not have the recognition motifs for the miRNAs expressed in these tissues. Together, our data support the hypothesis that miRNA expression broadly contributes to tissue specificity of mRNA expression in many human tissues. Based on these insights, we apply a computational tool to directly correlate 3' UTR motifs with changes in mRNA levels upon miRNA overexpression or knockdown. We show that this tool can identify functionally important 3' UTR motifs without cross-species comparison.

  16. 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.

  17. Cytochrome P450IA mRNA expression in feral Hudson River tomcod

    SciTech Connect

    Kreamer, G.L.; Squibb, K.; Gioeli, D.; Garte, S.J.; Wirgin, I. )

    1991-06-01

    The authors sought to determine if levels of cytochrome P450IA gene expression are environmentally induced in feral populations of Hudson River tomcod, a cancer prone fish, and whether laboratory exposure of tomcod to artificially spiked and naturally contaminated Hudson sediments can elicit a significant response. Using Northern blot analysis, they found levels of P450IA mRNA in tomcod collected from two Hudson River sites higher than those in tomcod from a river in Maine. Depuration of environmentally induced Hudson tomcod P450IA mRNA was rapid, with an initial detectable decline in P450 gene expression by 8 hr and basal levels reached by 5 days. Intraperitoneal injection of {beta}-napthoflavone in depurated Hudson tomcod resulted in a 15-fold induction of P450 gene expression within 26 hr. Exposure of depurated Hudson tomcod to natural sediment spiked with two PAHs resulted in a 7-fold induction of P450 gene expression. Exposure of depurated tomcod to sediment from a contaminated Hudson site also resulted in a 7- to 15-fold induction of P450IA mRNA expression. Northern blot analysis revealed a second polymorphic cytochrome P450IA mRNA band in some tomcod which was also detected by Southern blot analysis. Induction of cytochrome P450IA mRNA in Atlantic tomcod may provide a sensitive biomarker of environmentally relevant concentrations of some pollutants in the Hudson and other northeastern tidal rivers.

  18. 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

  19. SOD mRNA and MDA expression in rectus femoris muscle of rats with different eccentric exercise programs and time points.

    PubMed

    Zhao, Heng; Liu, Jiani; Pan, Shinong; Sun, Yingwei; Li, Qi; Li, Fei; Ma, Li; Guo, Qiyong

    2013-01-01

    Although superoxide dismutase (SOD) and malondialdehyde (MDA) affect Delayed Onset Muscle Soreness (DOMS), their effects are unclear in rectus femoris muscles (RFM) of rats with different eccentric exercise programs and time points. The purpose of this study is to investigate the effects of the various eccentric exercise programs at different time points on the SOD mRNA expression and MDA using rat as the animal model. 248 male rats were randomly divided into 4 groups: control group (CTL, n = 8), once-only exercise group (OEG, n = 80), continuous exercise group (CEG, n = 80), and intermittent exercise group (IEG, n = 80). Each exercise group was divided into 10 subgroups that exercised 0.5 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, or 168 h. Rats were sacrificed and their SOD mRNA expression, and MDA concentrations of skeletal muscle tissue were measured. The specimen in all eccentric exercise programs showed increased RFM SOD1 mRNA expression levels at 0.5 h (P<0.05), and decreased RFM SOD3 mRNA expression at 0.5 h (P<0.05). The continuous eccentric exercise (CE) significantly enhanced muscle SOD2 mRNA level at 0.5 h (P<0.05). After once-only eccentric exercise (OE), SOD1, SOD2, and SOD3 mRNA expression significantly increased at 96 h, whereas MDA concentrations decreased at 96 h. After CE, the correlation coefficients of SOD1, SOD2, SOD3 mRNA expression levels and MDA concentrations were -0.814, -0.763, -0.845 (all P<0.05) at 12 h. Regular eccentric exercise, especially CE could enhance SOD1 and SOD2 mRNA expression in acute stage and the SOD2 mRNA expression correlates to MDA concentration in vivo, which may improve the oxidative adaption ability of skeletal muscles.

  20. Rapamycin inhibits human renal epithelial cell proliferation: effect on cyclin D3 mRNA expression and stability.

    PubMed

    Pallet, Nicolas; Thervet, Eric; Le Corre, Delphine; Knebelmann, Bertrand; Nusbaum, Patrick; Tomkiewicz, Céline; Meria, Paul; Flinois, Jean-Pierre; Beaune, Philippe; Legendre, Christophe; Anglicheau, Dany

    2005-06-01

    Recent data have suggested that rapamycin use during the initial period after transplantation is associated with prolonged delayed graft function (DGF). Because of the known effects of rapamycin in other cell types, we speculated that this action may be secondary to human renal epithelial cells (HRECs) inhibition of proliferation. Primary cultures of HRECs were incubated with various concentrations of rapamycin. Cell proliferation was evaluated by cytotoxicity assays. The cell cycle was analyzed by flow cytometry. Protein expression levels were assessed by Western blot. Cyclin D3 mRNA levels were measured by quantitative real-time polymerase chain reaction (PCR). The transcriptional activity of the cyclin D3 gene was evaluated using transient transfection. Rapamycin exerted a significant concentration-dependent antiproliferative effect on growing HRECs by inhibiting the G(1) to S transition. The p70(S6) kinase pathway leading to cell cycle progression was found to be active, and low concentrations of rapamycin dramatically reduced p70(S6) kinase phosphorylation. Rapamycin completely inhibited the increase in cyclin D3 protein expression and mRNA accumulation induced by fetal calf serum, but did not affect cyclin E or cdk-inhibitor expression levels. This regulation of cyclin D3 protein expression is mainly due to a destabilization of its mRNA. Rapamycin reduced the mRNA half-life by 26% (4.8 +/- 1.3 hours vs. 6.5 +/- 1.0 hours, P < 0.001). Rapamycin inhibits the proliferative response of HRECs to mitogenic stimuli, and causes cell cycle arrest in the early G(1) phase, not only by a nonspecific process due to inhibition of the p70(S6k) pathway, but also by a direct effect on cyclin D3 mRNA stability.

  1. Chemokines mRNA expression in relation to the Macrophage Migration Inhibitory Factor (MIF) mRNA and Vascular Endothelial Growth Factor (VEGF) mRNA expression in the microenvironment of endometrial cancer tissue and normal endometrium: a pilot study.

    PubMed

    Giannice, Raffaella; Erreni, Marco; Allavena, Paola; Buscaglia, Mauro; Tozzi, Roberto

    2013-11-01

    Tumor microenvironment inflammatory cells play a major role in cancer progression. Among these, the Tumor Associated Macrophages (TAMs) infiltration depends on the kind of chemokine, cytokines and growth factors secreted by the tumor cells and by the stroma in response to the cancer invasion. TAMs have been found to promote anti-tumor response in early stages and to stimulate neovascularization and metastases in advanced disease. In the microenvironment chemo-attractants of many human cancers, MIF and VEGF correlate with an increased TAMs recruitment. In addition, MIF enhances tumor cells metastases by modulating the immune responses and by promoting the angiogenesis related to VEGF. On the contrary the inhibition of MIF can lead to cell cycle arrest and apoptosis. Some chemokines (e.g. CXCL12, CXCL11, CXCL8) and their receptors, thanks to their ability to modulate migration and proliferation, are involved in the angiogenetic process. In this study we compared the expression of MIF mRNA with VEGF mRNA expression and with mRNA expression of other chemokines related to neo-angiogenesis, such as CXCL12, CXCL11, CXCL8 and CXCR4, in human endometrial cancer tissue (EC) and normal endometrium (NE). Fresh samples of EC tissue and NE were extracted from 15 patients with FIGO stage I-III undergoing primary surgery. Some of the tissue was sent for histology and part of it was treated with RNA later and stored at -80°C. Four patients dropped out. A significant up-regulation of MIF mRNA in EC tissue versus NE samples (P=0.01) was observed in all 11 patients. The MIF mRNA over-expression was coincident with a VEGF mRNA overexpression in 54% of patients (P=NS). MIF mRNA was inversely related to CXCL12 mRNA expression (P=0.01). MIF over-expression was significantly related to low grading G1-2 (P=0.01), endometrial type I (P=0.05), no lymphovascular spaces invasion (P=0.01) and 3years DFS (P=0.01). As reported in previous studies on patients with breast cancer, our data suggest

  2. 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

  3. 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.

  4. Decoding Children's Expressions of Affect.

    ERIC Educational Resources Information Center

    Feinman, Joel A.; Feldman, Robert S.

    Mothers' ability to decode the emotional expressions of their male and female children was compared to the decoding ability of non-mothers. Happiness, sadness, fear and anger were induced in children in situations that varied in terms of spontaneous and role-played encoding modes. It was hypothesized that mothers would be more accurate decoders of…

  5. Expression of type IV collagen alpha 3 and alpha 4 chain mRNA in X-linked Alport syndrome.

    PubMed

    Nakanishi, K; Yoskikawa, N; Iijima, K; Nakamura, H

    1996-06-01

    X-Linked Alport syndrome is caused by mutations in the COL4A5 gene encoding the Type IV collagen alpha 5 chain (alpha 5(IV)). The authors' recent immunohistochemical study demonstrated abnormal expression of alpha 3(IV) and alpha 4(IV), as well as of alpha 5(IV), in patients with this syndrome, and a correlation between abnormal alpha 3(IV) and alpha 4(IV) expression and severity of the disease. The mechanism linking alpha 5(IV) mutations with abnormal alpha 3(IV) and alpha 4(IV) expression is unknown. To examine alpha 3(IV) and alpha 4(IV) mRNA expression in renal cortical tissues of patients with X-linked Alport syndrome, a nonradioisotopic, semiquantitative reverse transcription-polymerase chain reaction assay (alpha 3(IV) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alpha 4(IV), and GAPDH coamplification) was performed. There were no significant differences among severely affected male (N = 3), mildly affected male (N = 2), and female (N = 1) X-linked Alport patients and control subjects (N = 2) with respect to alpha 3(IV) and alpha 4(IV) mRNA expression in renal cortical tissue. These findings indicate that alpha 3(IV) and alpha 4(IV) transcription is not turned off in X-linked Alport syndrome and suggest that abnormal expression of alpha 3(IV) and alpha 4(IV) proteins in this syndrome may be the result of failure of incorporation of alpha 3(IV) and alpha 4(IV) into the glomerular basement membrane.

  6. OPIATE EXPOSURE AND WITHDRAWAL DYNAMICALLY REGULATE mRNA EXPRESSION IN THE SEROTONERGIC DORSAL RAPHE NUCLEUS

    PubMed Central

    Lunden, Jason; Kirby, Lynn G.

    2013-01-01

    Previous results from our lab suggest that hypofunctioning of the serotonergic (5-HT) dorsal raphe nucleus (DRN) is involved in stress-induced opiate reinstatement. To further investigate the effects of morphine dependence and withdrawal on the 5-HT DRN system, we measured gene expression at the level of mRNA in the DRN during a model of morphine dependence, withdrawal and post withdrawal stress exposure in rats. Morphine pellets were implanted for 72h and then either removed or animals were injected with naloxone to produce spontaneous or precipitated withdrawal, respectively. Animals exposed to these conditions exhibited withdrawal symptoms including weight loss, wet dog shakes and jumping behavior. Gene expression for brain-derived neurotrophic factor (BDNF), TrkB, corticotrophin releasing-factor (CRF)-R1, CRF-R2, GABAA-α1, μ-opioid receptor (MOR), 5-HT1A, tryptophan hydroxylase2 and the 5-HT transporter was then measured using quantitative real-time PCR at multiple time-points across the model of morphine exposure, withdrawal and post withdrawal stress. Expression levels of BDNF, TrkB and CRF-R1 mRNA were decreased during both morphine exposure and following seven days of withdrawal. CRF-R2 mRNA expression was elevated after seven days of withdrawal. 5-HT1A receptor mRNA expression was decreased following 3 hours of morphine exposure, while TPH2 mRNA expression was decreased after seven days of withdrawal with swim stress. There were no changes in the expression of GABAA-α1, MOR or 5-HT transporter mRNA. Collectively these results suggest that alterations in neurotrophin support, CRF-dependent stress signaling, 5-HT synthesis and release may underlie 5-HT DRN hypofunction that can potentially lead to stress-induced opiate relapse. PMID:24055683

  7. 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

  8. Fasting induces preproghrelin mRNA expression in the brain and gut of zebrafish, Danio rerio.

    PubMed

    Amole, Navpreet; Unniappan, Suraj

    2009-03-01

    Ghrelin is a gut/brain hormone with a unique acyl modification and various biological functions in fish and mammals. In addition to its possible role as a circulating orexigenic factor, ghrelin has been shown to regulate several other physiological processes in fish in a species-specific manner. The objectives of this project were to identify the zebrafish ghrelin gene organization, study tissue specific preproghrelin mRNA expression and investigate the fasting induced changes in the expression of preproghrelin mRNA in zebrafish. Our reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed the predicted ghrelin sequence available in the GenBank and identified preproghrelin mRNA expression in several tissues including the brain, gut, ovary, testis, heart and gill. PCR using genomic DNA identified that the ghrelin gene in zebrafish is comprised of four exons and three introns. Quantitative (real-time) PCR studies indicate that there is a significant increase in preproghrelin mRNA expression in the brain and gut of zebrafish fasted for 3, 5 and 7 days when compared to the expression in ad libitum fed fish. Refeeding after a 7 day fast caused a significant and dramatic decrease in preproghrelin mRNA expression in the gut and brain of zebrafish. An increase in the expression of preproghrelin mRNA during fasting, and its decrease following refeeding suggests an orexigenic role for ghrelin in zebrafish. Overall, our results provide evidence for a highly conserved structure and biological actions of ghrelin in zebrafish. Further studies are required to identify the tissue specific functions of ghrelin in zebrafish.

  9. Developmental expression pattern of Hspb8 mRNA in the mouse brain: analysis through online databases.

    PubMed

    García-Lax, Noelia; Tomás-Roca, Laura; Marín, Faustino

    2012-03-01

    Hspb8 is a member of the Hspb family of chaperone-like proteins. It is involved in several neural disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, hereditary distal motor neuropathy, and Charcot-Marie-Tooth's disease. In this work, we aimed to characterize its expression pattern in the mouse brain, by using the information available at online databases of high-throughput in situ hybridization. Therefore, we downloaded and analyzed the image series from these databases showing Hspb8 mRNA expression from embryonic to adult and aging stages. In early gestational embryos, Hspb8 was expressed in the hippocampal anlagen and in the ventricular layer of rhombomere 4. At perinatal stages, there appeared transitory expression in the dentate gyrus and the cerebellar cortex. From perinatal to aging stages, the neurons of the mesencephalic trigeminal nucleus and cranial motor nuclei displayed stable and strong Hspb8 expression. Additionally, along these stages there was moderate and relatively homogenous expression in the anterodorsal thalamic, lateral mammillary, arcuate hypothalamic and medial habenular nuclei, and in the locus coeruleus. In its turn, the basal ganglia, cerebellar inner granular layer and diverse sensory and reticular formation nuclei of the hindbrain contained scattered cells with strong expression. In conclusion, Hspb8 mRNA is constitutively expressed in specific brain structures across ontogeny, so that eventually they could be affected by the malfunction or deregulation of this molecule. Copyright © 2011 Wiley Periodicals, Inc.

  10. 3-Nitropropionic acid modifies neurotrophin mRNA expression in the mouse striatum: 18S-rRNA is a reliable control gene for studies of the striatum.

    PubMed

    Espíndola, S; Vilches-Flores, A; Hernández-Echeagaray, E

    2012-10-01

    The aim of the present study was to determine the changes in the mRNA levels of neurotrophins and their receptors in the striatal tissue of mice treated with 3-nitropropionic acid (3-NP). At 1 and 48 h after the last drug administration, the mRNA expression of nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 as well as their receptors p75, TrkA, TrkB and TrkC, was evaluated using semi-quantitative (semi-Q) and real-time RT-PCR. β-actin mRNA and ribosomal 18S (18S rRNA) were tested as internal controls. 3-NP treatment did not affect mRNA expression of all neurotrophins and their respective receptors equally. Also, differences in neurotrophin and receptor mRNA expression were observed between semi-Q and real-time RT-PCR. Real-time RT-PCR was more accurate in evaluating the mRNA expression of the neurotrophins than semi-Q, and 18S rRNA was more reliable than β-actin as an internal control. Neurotrophins and their receptors expression is differentially affected by neuronal damage produced by inhibition of mitochondrial respiration with 3-NP treatment in low, sub-chronic doses in vivo.

  11. mRNA expression profiling of neonatal rats after 16-day spaceflight

    NASA Astrophysics Data System (ADS)

    Miyake, M.; Ijiri, K.

    Some studies pointed out that postnatal development is the key to realize generation change of mammalians in space. For example, functional changes and hypoplasia in some organs after spaceflight during postnatal development were reported. Though profiling mRNA expression is useful to evaluate what happened in animals, these studies after spaceflight are limited to specific organs for understanding the relationship between phenotype and gene. The organ-wide analysis of mRNA expression is important to evaluate the condition of each animal, and it can find new phenomenon and help precise understanding for effect induced by spaceflight. In this experiment, we analyzed mRNA expression of liver, spleen and intestine in neonatal rats after 16-day spaceflight by Space Shuttle Columbia (STS-90).

  12. Detection of MDR1 mRNA expression with optimized gold nanoparticle beacon

    NASA Astrophysics Data System (ADS)

    Zhou, Qiumei; Qian, Zhiyu; Gu, Yueqing

    2016-03-01

    MDR1 (multidrug resistance gene) mRNA expression is a promising biomarker for the prediction of doxorubicin resistance in clinic. However, the traditional technical process in clinic is complicated and cannot perform the real-time detection mRNA in living single cells. In this study, the expression of MDR1 mRNA was analyzed based on optimized gold nanoparticle beacon in tumor cells. Firstly, gold nanoparticle (AuNP) was modified by thiol-PEG, and the MDR1 beacon sequence was screened and optimized using a BLAST bioinformatics strategy. Then, optimized MDR1 molecular beacons were characterized by transmission electron microscope, UV-vis and fluorescence spectroscopies. The cytotoxicity of MDR1 molecular beacon on L-02, K562 and K562/Adr cells were investigated by MTT assay, suggesting that MDR1 molecular beacon was low inherent cytotoxicity. Dark field microscope was used to investigate the cellular uptake of hDAuNP beacon assisted with ultrasound. Finally, laser scanning confocal microscope images showed that there was a significant difference in MDR1 mRNA expression in K562 and K562/Adr cells, which was consistent with the results of q-PCR measurement. In summary, optimized MDR1 molecular beacon designed in this study is a reliable strategy for detection MDR1 mRNA expression in living tumor cells, and will be a promising strategy for in guiding patient treatment and management in individualized medication.

  13. Induction of mRNA expression of osteogenesis-related genes by guaiacol in human dental pulp cells.

    PubMed

    Kato, Takashi; Shirayama, Kumiko; Tsutsui, Takeo W; Tsutsui, Takeki

    2010-07-01

    To investigate the stimulating effect of endodontic medications on the mRNA expression of some osteogenesis-related genes associated with reparative dentinogenesis and hard-tissue formation, human dental pulp cells (D824 cells) were treated with calcium hydroxide (Ca (OH)(2)), formocresol, or guaiacol. The effect on growth was determined by growth curves of D824 cells treated for 1-3 days with 0.03-0.3 mM Ca (OH)(2), 0.0007%-0.0014% formocresol, or 0.24-2.43 mM guaiacol. The mitotic activity of individual cells and the mRNA expression of the osteogenesis-related genes for alkaline phosphatase (ALP), type I collagen (COL-1), and bone sialoprotein (BSP) in the cells treated for 24 h with the same concentrations of the medications as described above were determined by colony-forming efficiency and by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, respectively. Cellular growth and mitotic activity were scarcely affected by Ca (OH)(2), but were significantly reduced by formocresol or guaiacol. The mRNA expression of the osteogenesis-related genes was little affected by Ca (OH)(2) or formocresol, but was significantly enhanced by guaiacol. The results indicate that guaiacol may stimulate the mRNA expression of genes associated with reparative dentinogenesis and hard-tissue formation in human dental pulp cells, suggesting that the novel property of guaiacol provides new insights into the utilization of guaiacol in endodontic therapy.

  14. Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

    PubMed Central

    Retta, Saverio Francesco; Cassarà, Georgia; D'Amato, Monica; Alessandro, Riccardo; Pellegrino, Maurizio; Degani, Simona; De Leo, Giacomo; Silengo, Lorenzo; Tarone, Guido

    2001-01-01

    There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of β1-null GD25 cells ectopically expressing the β1A integrin subunit, we provide evidence for the existence of a cross talk between β1 and αV integrins that affects the ratio of αVβ3 and αVβ5 integrin cell surface levels. In particular, we demonstrate that a down-regulation of αVβ3 and an up-regulation of αVβ5 occur as a consequence of β1A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms β1B and β1D, as well as two β1 cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (β1TR) or only its “variable” region (β1COM), we show that the effects of β1 over αV integrins take place irrespective of the type of β1 isoform, but require the presence of the “common” region of the β1 cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby β1 integrins exert their trans-acting functions, we have found that the down-regulation of αVβ3 is due to a decreased β3 subunit mRNA stability, whereas the up-regulation of αVβ5 is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability. PMID:11598197

  15. Effects of Ginseng and Echinacea on Cytokine mRNA Expression in Rats

    PubMed Central

    Uluışık, Deniz; Keskin, Ercan

    2012-01-01

    The aim of the study was to determine the effect of ginseng and echinacea on the mRNA expression of IL-10, TNF-α, and TGF-β1 in healthy rats. Six-week-old male Fischer 344 rats (n = 48) were used. The animals were divided into three equal groups, as follows: control (C); ginseng (G); echinacea (E). While the C group was fed a standard rat diet (Purina) ad libitum for a period of 40 days, the G and E groups animals received the same diet containing 0.5 g/kg of Panax ginseng root powder and 0.75 g/kg of Echinacea purpurea root powder, respectively. Blood samples were obtained from 8 rats in each group after 20 and 40 days of treatment, and the mRNA expression of IL-10, TNF-α, and TGF-β1 was determined. After 20 days of treatment, the expression of IL-10 mRNA in the G group was different from the C group (P < 0.05); however, after 40 days of treatment, there was no difference between the groups. There was no difference after 20 and 40 days of treatment between the groups with respect to the expression of TGF-β1 mRNA. After 20 days of treatment, the expression of TNF-α mRNA in the E group was higher (P < 0.05) than the C group. After 40 days of treatment, the expression of TNF-α mRNA was similar in all of the groups. Based on the current study, the increase in expression of IL-10 mRNA in the G group and the increase in expression of TNF-α mRNA in the E group support the use of these plants for purposes of modulating the immune system. However, a more detailed study regarding the effects of ginseng and echinacea on these cytokines and other cytokines is needed. PMID:22666172

  16. TLRs and NODs mRNA expression pattern in healthy mouse eye

    PubMed Central

    Rodríguez-Martínez, S; Cancino-Díaz, M E; Jiménez-Zamudio, L; García-Latorre, E; Cancino-Díaz, J C

    2005-01-01

    Aims: To look for TLR and NOD mRNA expression in the healthy eye and in other immune privileged and non-immune privileged mouse organs. Methods: Semiquantitative RT-PCR was performed to look for TLR1–9 and NOD1 and NOD2 mRNA expressions in the whole eye, in the anterior (AP) and posterior (PP) portions of the eye, in corneal fibroblasts (CF) and in ovary, brain, testis, heart, lung, and spleen. Results: All the TLR mRNAs were expressed in the whole eye of Balb/c mice. NIH and C57BL/6 did not express TLR9 and TLR8, respectively. NIH expressed higher levels of TLR1, 2, 3, and 6 than the other strains. C57BL/6 expressed the lowest levels of all TLRs. TLR9, 5, and 4 were the less expressed in all strains. All TLRs were expressed in Balb/c PP and TLR1 was not expressed in AP. In NIH and Balb/c CF the majority of TLRs were overexpressed with LPS. In testis, expression of most TLRs was absent. Non-immune privileged organs expressed most of the TLRs. All the organs expressed NOD1 and NOD2. In PP NOD2 was not expressed. Conclusion: TLRs and NODs are expressed in the eye, and could have an important role in the innate immunity. PMID:15965176

  17. Fibronectin 1 mRNA expression correlates with advanced disease in renal cancer

    PubMed Central

    2010-01-01

    Background Fibronectin 1 (FN1) is a glycoprotein involved in cellular adhesion and migration processes. The aim of this study was to elucidate the role of FN1 in development of renal cell cancer (RCC) and to determine a prognostic relevance for optimal clinical management. Methods 212 renal tissue samples (109 RCC, 86 corresponding tissues from adjacent normal renal tissue and 17 oncocytomas) were collected from patients undergoing surgery for renal tumors and subjected to total RNA extraction. Detection of FN1 mRNA expression was performed using quantitative real time PCR, three endogenous controls, renal proximal tubular epithelial cells (RPTEC) as biological control and the ΔΔCt method for calculation of relative quantities. Results Mean tissue specific FN1 mRNA expression was found to be increased approximately seven fold comparing RCC and corresponding kidney control tissues (p < 0.001; ANOVA). Furthermore, tissue specific mean FN1 expression was increased approx. 11 fold in clear cell compared to papillary RCC (p = 9×10-5; Wilcoxon rank sum test). Patients with advanced disease had higher FN1 expression when compared to organ-confined disease (p < 0.001; Wilcoxon rank sum test). Applying subgroup analysis we found a significantly higher FN1 mRNA expression between organ-confined and advanced disease in the papillary and not in the clear cell RCC group (p = 0.02 vs. p = 0.2; Wilcoxon rank sum test). There was an increased expression in RCC compared to oncocytoma (p = 0.016; ANOVA). Conclusions To our knowledge, this is the first study to show that FN1 mRNA expression is higher in RCC compared to normal renal tissue. FN1 mRNA expression might serve as a marker for RCC aggressiveness, indicating early systemic progression particularly for patients with papillary RCC. PMID:20860816

  18. Fibronectin 1 mRNA expression correlates with advanced disease in renal cancer.

    PubMed

    Waalkes, Sandra; Atschekzei, Faranaz; Kramer, Mario W; Hennenlotter, Jörg; Vetter, Gesa; Becker, Jan U; Stenzl, Arnulf; Merseburger, Axel S; Schrader, Andres J; Kuczyk, Markus A; Serth, Jürgen

    2010-09-22

    Fibronectin 1 (FN1) is a glycoprotein involved in cellular adhesion and migration processes. The aim of this study was to elucidate the role of FN1 in development of renal cell cancer (RCC) and to determine a prognostic relevance for optimal clinical management. 212 renal tissue samples (109 RCC, 86 corresponding tissues from adjacent normal renal tissue and 17 oncocytomas) were collected from patients undergoing surgery for renal tumors and subjected to total RNA extraction. Detection of FN1 mRNA expression was performed using quantitative real time PCR, three endogenous controls, renal proximal tubular epithelial cells (RPTEC) as biological control and the ΔΔCt method for calculation of relative quantities. Mean tissue specific FN1 mRNA expression was found to be increased approximately seven fold comparing RCC and corresponding kidney control tissues (p < 0.001; ANOVA). Furthermore, tissue specific mean FN1 expression was increased approx. 11 fold in clear cell compared to papillary RCC (p = 9×10-5; Wilcoxon rank sum test). Patients with advanced disease had higher FN1 expression when compared to organ-confined disease (p < 0.001; Wilcoxon rank sum test). Applying subgroup analysis we found a significantly higher FN1 mRNA expression between organ-confined and advanced disease in the papillary and not in the clear cell RCC group (p = 0.02 vs. p = 0.2; Wilcoxon rank sum test). There was an increased expression in RCC compared to oncocytoma (p = 0.016; ANOVA). To our knowledge, this is the first study to show that FN1 mRNA expression is higher in RCC compared to normal renal tissue. FN1 mRNA expression might serve as a marker for RCC aggressiveness, indicating early systemic progression particularly for patients with papillary RCC.

  19. 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

  20. Effect of gamma radiation on the expression of mRNA growth factors in glycerol cryopreserved human amniotic membrane.

    PubMed

    Yatim, Rusidah Mat; Kannan, Thirumulu Ponnuraj; Ab Hamid, Suzina Sheikh

    2016-12-01

    Human amniotic membrane (HAM) due to its high biocompatibility, low immunogenicity, anti-microbial, anti-viral properties as well as the presence of growth factors has been used in various clinical applications. The growth factors play an important role in wound healing. The current study aimed to explore the effect of 15 kGy gamma radiation dose on selected growth factors and receptors mRNA present in HAM. Eight growth factors, namely, EGF, HGF, KGF, TGF-α, TGF-β1, TGF-β2, TGF-β3 and bFGF and two growth factor receptors, HGFR and KGFR were evaluated in this study. The total RNA was extracted and converted to complimentary DNA using commercial kits. Subsequently, the mRNA expressions of these growth factors were evaluated using real-time PCR and the results were statistically analyzed using REST-MCS software. This study confirmed the presence of these mRNA growth factors and receptors in fresh, glycerol cryopreserved and irradiated glycerol cryopreserved HAM. In glycerol cryopreserved HAM, the results showed up-regulation of HGF and bFGF and down-regulation of EGF, HGFR, KGF, KGFR, TGF-α, TGF-β1, TGF-β2 and TGF-β3 relative to the fresh HAM which acted as the control, whereas in irradiated glycerol cryopreserved HAM, the results showed up-regulation of EGF, HGF, KGF, KGFR, TGF-β1, TGF-β2 and TGF-β3 and down-regulation of HGFR, TGF-α and bFGF relative to the glycerol cryopreserved HAM which acted as the control. However, these mRNA expressions did not show any statistical significant difference compared to the control groups. This study concluded that a dose of 15 kGy of gamma radiation did not affect the mRNA expression for the growth factors' and receptors' in the glycerol cryopreserved HAM.

  1. Cytokine mRNA expressions after racing at a high altitude and at sea level in horses with exercise-induced pulmonary hemorrhage.

    PubMed

    Saulez, Montague N; Godfroid, Jacques; Bosman, Anamarie; Stiltner, Jackie L; Breathnach, Cormac C; Horohov, David W

    2010-04-01

    To determine concentrations of cytokine mRNA in horses with exercise-induced pulmonary hemorrhage (EIPH) after racing. 97 Thoroughbreds. Following tracheobronchoscopy, the severity of EIPH was graded (scale of 0 to 4), and venous blood samples were collected from 10 horses in each grade. After RNA isolation and cDNA synthesis, real-time PCR assay was conducted to detect cytokinespecific mRNA for interleukin (IL)-1, IL-6, and IL-10; interferon (INF)-gamma; and tumor necrosis factor (TNF)-alpha. Neither location nor grade of EIPH affected the expression of IL-1 and INF-gamma. There was significantly greater overall expression of IL-6 mRNA at sea level, with significantly more IL-6 expressed in horses with grade 4 EIPH than in horses with grade 0, 1, or 2 EIPH. At a high altitude, no difference was detected for IL-6 expression among the various EIPH grades. There was significantly greater overall expression of TNF-alpha mRNA at a high altitude; however, there was no difference within the various grades of EIPH. Expression of IL-10 was significantly affected by grade of EIPH because horses with grade 3 EIPH expressed significantly more IL-10 mRNA than did horses with grade 0 or 2 EIPH; this expression was not affected by location. At sea level, increased IL-6 expression was associated with more severe EIPH, and altitude may affect gene expressions of the proinflammatory cytokine TNF-alpha and anti-inflammatory cytokine IL-6. Studies on protein concentrations of cytokine expression are needed. The pathophysiologic importance of these findings remains to be explained.

  2. Leptin and cholecystokinin in Schizothorax prenanti: molecular cloning, tissue expression, and mRNA expression responses to periprandial changes and fasting.

    PubMed

    Yuan, Dengyue; Wang, Tao; Zhou, Chaowei; Lin, Fangjun; Chen, Hu; Wu, Hongwei; Wei, Rongbin; Xin, Zhiming; Li, Zhiqiong

    2014-08-01

    In the present study, full-length cDNA sequences of leptin and cholecystokinin (CCK) were cloned from Schizothorax prenanti (S. prenanti), and applied real-time quantitative PCR to characterize the tissue distribution, and appetite regulatory effects of leptin and CCK in S. prenanti. The S. prenanti leptin and CCK full-length cDNA sequences were 1121 bp and 776 bp in length, encoding the peptide of 171 and 123 amino acid residues, respectively. Tissue distribution analysis showed that leptin mRNA was mainly expressed in the liver of S. prenanti. CCK was widely expressed, with the highest levels of expression in the hypothalamus, myelencephalon, telencephalon and foregut of S. prenanti. The CCK mRNA expression was highly elevated after feeding, whereas the leptin mRNA expression was not affected by single meal. These results suggested that CCK is a postprandial satiety signal in S. prenanti, but leptin might not be. In present study, leptin and CCK gene expression were both decreased after fasting and increased after refeeding, which suggested leptin and CCK might be involved in regulation of appetite in S. prenanti. This study provides an essential groundwork to further elucidate the appetite regulatory systems of leptin and CCK in S. prenanti as well as in other teleosts.

  3. TSG101, a tumor susceptibility gene, bidirectionally modulates cell invasion through regulating MMP-9 mRNA expression.

    PubMed

    Sai, Xu Bin; Makiyama, Tomohiko; Sakane, Hiroshi; Horii, Yukimi; Hiraishi, Hideyuki; Shirataki, Hiromichi

    2015-11-25

    Tumor susceptibility gene 101 (TSG101) was initially identified in fibroblasts as a tumor suppressor gene but subsequent studies show that TSG101 also functions as a tumor-enhancing gene in some epithelial tumor cells. Although previous studies have unraveled diverse biological functions of TSG101, the precise mechanism by which TSG101 is involved in carcinogenesis and tumor progression in a bidirectional and multifaceted manner remains unclear. To reveal the mechanism underlying bidirectional modulation of cell invasion by TSG101, we used RNA interference to examine whether TSG101 depletion bidirectionally modulated matrix metalloproteinase (MMP)-9 expression in different cell types. TSG101 depletion promoted cell invasion of HT1080 cells but contrarily reduced cell invasion of HeLaS3 cells. In HT1080 cells, TSG101 depletion increased both baseline and phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 secretion through enhancing MMP-9 mRNA expression, but did not affect the expression or activation of MMP-2. In contrast, TSG101 depletion decreased PMA-induced MMP-9 secretion through reducing MMP-9 mRNA expression in HeLaS3 cells. TSG101 depletion had little impact on the signaling pathways required for the activation of transcription of MMP-9 or MMP-9 mRNA stability in either cell line. TSG101 bidirectionally modulates cell invasion through regulating MMP-9 mRNA expression in different cell types. Our results provide a mechanistic context for the role of TSG101 in cell invasion as a multifaceted gene.

  4. Expression and stability of c-sis mRNA in human glioblastoma cells

    SciTech Connect

    Press, R.D.; Samols, D.; Goldthwait, D.A.

    1988-07-26

    The production of platelet-derived growth factor like (PDGF-like) material by glioblastomas may be involved in the conversion of normal cells to tumor cells. In an investigation of this problem, the authors have examined some of the properties of the platelet-derived growth factor B-chain mRNA (c-sis mRNA) by a sensitive and quantitative RNA-RNA solution hybridization method. In 5 out of 8 human glioblastoma cell lines, c-sis mRNA was present, and in the line with the highest level, there were approximately 4-10 molecules per cell. The half-lives of the c-sis mRNA in two glioblastoma cell lines were 2.6 and 3.4 h, while in human umbilical vein endothelial (HUVE) and bladder carcinoma (T24) cells they were 1.6 and 2.5 h, respectively. Inhibiting protein synthesis produced no significant alteration of the c-sis mRNA half-lives in the glioblastoma or HUVE cells. The A-U-rich sequence at the 3' end of the c-sis mRNA therefore does not appear to affect the mRNA stability in the presence of cycloheximide as it does in other transcripts. The similarity of the c-sis mRNA half-lives in normal and tumor cells suggests that regulation of stability of c-sis mRNA is not a major factor in tumorigenesis in the glioblastoma cell lines examined.

  5. Deregulated expression of VHL mRNA variants in papillary thyroid cancer.

    PubMed

    Baldini, Enke; Tuccilli, Chiara; Arlot-Bonnemains, Yannick; Chesnel, Frank; Sorrenti, Salvatore; De Vito, Corrado; Catania, Antonio; D'Armiento, Eleonora; Antonelli, Alessandro; Fallahi, Poupak; Watutantrige-Fernando, Sara; Tartaglia, Francesco; Barollo, Susi; Mian, Caterina; Bononi, Marco; Arceri, Stefano; Mascagni, Domenico; Vergine, Massimo; Pironi, Daniele; Monti, Massimo; Filippini, Angelo; Ulisse, Salvatore

    2017-03-05

    Recent findings demonstrated that a subset of papillary thyroid cancers (PTCs) is characterized by reduced expression of the von Hippel-Lindau (VHL) tumor suppressor gene, and that lowest levels associated with more aggressive PTCs. In the present study, the levels of the two VHL mRNA splicing variants, VHL-213 (V1) and VHL-172 (V2), were measured in a series of 96 PTC and corresponding normal matched tissues by means of quantitative RT-PCR. Variations in the mRNA levels were correlated with patients' clinicopathological parameters and disease-free interval (DFI). The analysis of VHL mRNA in tumor tissues, compared to normal matched tissues, revealed that its expression was either up- or down-regulated in the majority of PTC. In particular, V1 and V2 mRNA levels were altered, respectively, in 78 (81.3%) and 65 (67.7%) out of the 96 PTCs analyzed. A significant positive correlation between the two mRNA variants was observed (p < 0.001). Univariate analysis documented the lack of association between each variant and clinicopathological parameters such as age, tumor size, histology, TNM stage, lymph node metastases, and BRAF mutational status. However, a strong correlation was found between altered V1 or V2 mRNA levels and DFI. Multivariate regression analysis indicated higher V1 mRNA values, along with lymph node metastases at diagnosis, as independent prognostic factors predicting DFI. In conclusion, the data reported demonstrate that VHL gene expression is deregulated in the majority of PTC tissues. Of particular interest is the apparent protective role exerted by VHL transcripts against PTC recurrences.

  6. Influence of Olmesartan on Sirtuin 1 mRNA Expression in 5/6 Nephrectomized Spontaneously Hypertensive Rats.

    PubMed

    Takata, Tomoaki; Munemura, Chishio; Fukui, Takeaki; Fukuda, Satoko; Murawaki, Yoshikazu

    2015-06-01

    Recent studies revealed that sirtuin 1 (SIRT1) has a relation to the mechanism of transforming growth factor-beta (TGF-beta) mediated apoptosis in glomerular mesangial cells and plays an important role in blood pressure regulation. It has been suggested that SIRT1 contributes to the renoprotective effect of angiotensin receptor blocker (ARB), but this has not yet become clearly recognized. In this study, we examined the relationship between SIRT1 and the therapeutic effect of olmesartan on renal injury in nephrectomized spontaneously hypertensive rats (SHRs). Male Wistar rats and 5/6 nephrectomized (5/6Nx) SHRs were assigned to 5 groups as follows: group A, Wistar rats; group B, Wistar rats administered high-dose olmesartan (15 mg/kg/day); group C, 5/6Nx SHRs; group D, 5/6Nx SHRs administered low-dose (3 mg/kg/day) olmesartan; and group E, 5/6Nx SHRs administered high-dose olmesartan. The drugs were administered for 12 weeks. Blood pressure and urinary protein excretion were measured every 4 weeks. Serum creatinine, glomerular sclerosis, SIRT1 mRNA level, TGF-beta mRNA level and klotho mRNA level were measured at the end of the examination. Systolic blood pressure, urinary protein excretion, serum creatinine and glomerular sclerosis in Wistar rats were significantly lower than that of 5/6Nx SHRs. Among 5/6Nx SHRs, high doses of olmesartan significantly decreased urinary protein excretion, serum creatinine and glomerular sclerosis compared to the non-treated and low-dose olmesartan groups. Expression of SIRT1 and klotho mRNA were significantly downregulated in 5/6Nx SHRs; however, olmesartan did not attribute to any change in gene expression. Expression of TGF-beta mRNA was significantly increased in 5/6Nx SHRs, and olmesartan did not affect the level of TGF-beta mRNA expression. Expression of SIRT1 is decreased in 5/6Nx SHRs compared to Wistar rats. Olmesartan suppressed glomerular sclerosis, but did not increase the expression of SIRT1, suggesting that the

  7. 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.

  8. 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

  9. 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.

  10. CYP1A mRNA expression in redeye mullets (Liza haematocheila) from Bohai Bay, China.

    PubMed

    An, Lihui; Hu, Jianying; Yang, Min; Zheng, Binghui; Wei, An; Shang, Jingjing; Zhao, Xingru

    2011-04-01

    Induction of cytochrome P4501A (CYP1A) has been used as a biomarker in fish for monitoring aromatic and organic contaminants. In this study, a partial of CYP1A gene in redeye mullet (Liza haematocheila) was isolated and sequenced, and then a real-time quantitative reverse-transcription polymerase chain reaction assay was developed for quantification of CYP1A mRNA normalized to β-actin. The developed method was applied to detect CYP1A mRNA expression in redeye mullets collected from Nandaihe (reference site) and Dashentang (impacted site) in Bohai Bay, China. CYP1A mRNA expression values were significantly elevated in redeye mullets from Dashentang compared to a reference site--Nandaihe, which was correlated with the contents of different environmentally relevant pollutants in tissues, particularly with PCBs and PBDEs.

  11. Altered placental glutathione peroxidase mRNA expression in preeclampsia according to the presence or absence of labor.

    PubMed

    Roland-Zejly, L; Moisan, V; St-Pierre, I; Bilodeau, J-F

    2011-02-01

    Increased placental oxidative stress in preeclampsia (PE) has been associated in part to a decrease in glutathione peroxidase (GPX) antioxidant activity. However, it is not clear if GPX mRNA expression is affected in PE, and how the presence of labor may impair this expression. In this study, we characterized by quantitative PCR, in situ hybridization and immunohistochemistry, the expression of four GPX (GPX1 to 4) in the placenta of normotensive (NP; n = 23) and PE pregnancies (n = 25) according to mode of delivery: vaginal delivery (with labor) or cesarean (without labor); the tissue layer: amnion-chorion (AC) and villi; and the sampling site: peri-insertion or peripheral. Concomitantly, oxidative stress markers mRNA expression, HSP70 and HO-1 were measured. All GPX mRNA and protein were detected in all layers of the placenta and sampling sites. In absence of labor, GPX1 is more expressed near the umbilical cord than at the periphery of the villi (p = 0.037). At the periphery of AC membranes, GPX2 was more expressed in PE than in controls in presence of labor (p = 0.037). Interestingly, GPX4 mRNA level was clearly deficient in the PE villi in presence or absence of labor (p < 0.0473). Also, the GPX4 expression in PE was lower than controls in AC membranes in presence of labor (p = 0.0007). Oxidative stress markers, HSP70 and HO-1, were higher in PE placental membranes than in controls in absence of labor (p < 0.011). HSP70 was also upregulated in PE placental membranes in presence of labor (p = 0.034). Correlations between stress markers and GPX mRNA expression were mostly present in AC membranes in presence of labor in NP. Most of the latter correlations were lost in PE. In conclusion, our results suggest that the reported decrease in GPx activity and increased oxidative stress in PE placental villi may be attributed in part to GPX4 independently of the presence or absence of labor. Copyright © 2010 Elsevier Ltd. All rights reserved.

  12. ERCC1 mRNA expression as a postoperative prognostic marker in extrahepatic bile duct cancer.

    PubMed

    Uemura, Shuichiro; Kuramochi, Hidekazu; Higuchi, Ryota; Nakajima, Go; Yamamoto, Masakazu

    2014-12-01

    No reliable biomarker for biliary tract cancer has yet been identified because of the varying composition of the cancer type, differences in tumor location, a mixture of curative and non-curative operations, and differences in operative methods. Fifty extrahepatic bile duct cancer patients, pathologically diagnosed with papillary or tubular adenocarcinoma who underwent a pancreatoduodenectomy with R0 resection, were included in the study. Messenger RNA (mRNA) expression levels were measured from formalin-fixed, paraffin-embedded samples by real-time reverse-transcription polymerase chain reaction. The preliminary analysis selected ten patients who have survived more than 5 years (LS group) and ten who had a relapse within 2 years (SS group). mRNA expression of seven target genes was examined, but only excision repair cross-complementing 1 (ERCC1) mRNA levels showed a significant difference between the LS and SS groups (median ERCC1: LS 26.5 vs. SS 9.7; p = 0.0073). The median survival time of patients with high ERCC1 levels was significantly longer than in patients with low ERCC1 levels (p = 0.0105). Thirty more patients with identical backgrounds were added to the study, and ERCC1 mRNA levels were measured in all 50 patients. Those with high ERCC1 mRNA levels had a significantly greater overall survival (OS) time compared with those with low ERCC1 levels (MST: 174 vs. 86 M; p = 0.048). Multivariate analysis found that an absence of lymph node metastases and high ERCC1 expression were significantly associated with improved OS. ERCC1 mRNA expression appears to be a useful prognostic biomarker for extrahepatic bile duct cancer with R0 resection.

  13. Comparison of mRNA features affecting translation initiation and reinitiation

    PubMed Central

    Osterman, Ilya A.; Evfratov, Sergey A.; Sergiev, Petr V.; Dontsova, Olga A.

    2013-01-01

    Regulation of gene expression at the level of translation accounts for up to three orders of magnitude in its efficiency. We systematically compared the impact of several mRNA features on translation initiation at the first gene in an operon with those for the second gene. Experiments were done in a system with internal control based on dual cerulean and red (CER/RFP) fluorescent proteins. We demonstrated significant differences in the efficiency of Shine Dalgarno sequences acting at the leading gene and at the following genes in an operon. The majority of frequent intercistronic arrangements possess medium SD dependence, medium dependence on the preceding cistron translation and efficient stimulation by A/U-rich sequences. The second cistron starting immediately after preceding cistron stop codon displays unusually high dependence on the SD sequence. PMID:23093605

  14. Predicting gene targets of perturbations via network-based filtering of mRNA expression compendia

    PubMed Central

    Cosgrove, Elissa J.; Zhou, Yingchun; Gardner, Timothy S.; Kolaczyk, Eric D.

    2008-01-01

    Motivation: DNA microarrays are routinely applied to study diseased or drug-treated cell populations. A critical challenge is distinguishing the genes directly affected by these perturbations from the hundreds of genes that are indirectly affected. Here, we developed a sparse simultaneous equation model (SSEM) of mRNA expression data and applied Lasso regression to estimate the model parameters, thus constructing a network model of gene interaction effects. This inferred network model was then used to filter data from a given experimental condition of interest and predict the genes directly targeted by that perturbation. Results: Our proposed SSEM–Lasso method demonstrated substantial improvement in sensitivity compared with other tested methods for predicting the targets of perturbations in both simulated datasets and microarray compendia. In simulated data, for two different network types, and over a wide range of signal-to-noise ratios, our algorithm demonstrated a 167% increase in sensitivity on average for the top 100 ranked genes, compared with the next best method. Our method also performed well in identifying targets of genetic perturbations in microarray compendia, with up to a 24% improvement in sensitivity on average for the top 100 ranked genes. The overall performance of our network-filtering method shows promise for identifying the direct targets of genetic dysregulation in cancer and disease from expression profiles. Availability: Microarray data are available at the Many Microbe Microarrays Database (M3D, http://m3d.bu.edu). Algorithm scripts are available at the Gardner Lab website (http://gardnerlab.bu.edu/SSEMLasso). Contact: kolaczyk@math.bu.edu Supplementary information: Supplementary Data are available at Bioinformatics on line. PMID:18779235

  15. An integrated analysis of differential miRNA and mRNA expressions in human gallstones.

    PubMed

    Yang, Bin; Liu, Bin; Bi, Pinduan; Wu, Tao; Wang, Qiang; Zhang, Jie

    2015-04-01

    Gallstone disease, including cholesterol precipitation in bile, increased bile salt hydrophobicity and gallbladder inflammation. Here, we investigated miRNA and mRNA involved in the formation of gallstones, and explored the molecular mechanisms in the development of gallstones. Differentially expressed 17 miRNAs and 525 mRNA were identified based on Illumina sequencing from gallbladder mucosa of patients with or without gallstones, and were validated by randomly selected 6 miRNAs and 8 genes using quantitative RT-PCR. 114 miRNA target genes were identified, whose functions and regulating pathways were related to gallstones. The differentially expressed genes were enriched upon lipoprotein binding and some metabolic pathways, and differentially expressed miRNAs enriched upon ABC transportation and cancer related pathways. A molecular regulatory network consisting of 17 differentially expressed miRNAs, inclusive of their target genes, was constructed. miR-210 and its potential target gene ATP11A were found to be differentially expressed in both miRNA and mRNA profiles. ATP11A was a direct target of miR-210, which was predicted to regulate the ABC-transporters pathway. The expression levels of ATP11A in the gallstone showed inverse correlation with miR-210 expression, and up-regulation of miR-210 could reduce ATP11A expression in HGBEC. This is the first report that indicates the existence of differences in miRNA and mRNA expression in patients with or without gallstones. Our data shed light on further investigating the mechanisms of gallstone formation.

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

    SciTech Connect

    Maquat, Lynne

    2002-12-01

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

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

    PubMed

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

    2007-11-01

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

  19. 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

  20. Selecting Reliable mRNA Expression Measurements Across Platforms Improves Downstream Analysis.

    PubMed

    Tong, Pan; Diao, Lixia; Shen, Li; Li, Lerong; Heymach, John Victor; Girard, Luc; Minna, John D; Coombes, Kevin R; Byers, Lauren Averett; Wang, Jing

    2016-01-01

    With increasing use of publicly available gene expression data sets, the quality of the expression data is a critical issue for downstream analysis, gene signature development, and cross-validation of data sets. Thus, identifying reliable expression measurements by leveraging multiple mRNA expression platforms is an important analytical task. In this study, we propose a statistical framework for selecting reliable measurements between platforms by modeling the correlations of mRNA expression levels using a beta-mixture model. The model-based selection provides an effective and objective way to separate good probes from probes with low quality, thereby improving the efficiency and accuracy of the analysis. The proposed method can be used to compare two microarray technologies or microarray and RNA sequencing measurements. We tested the approach in two matched profiling data sets, using microarray gene expression measurements from the same samples profiled on both Affymetrix and Illumina platforms. We also applied the algorithm to mRNA expression data to compare Affymetrix microarray data with RNA sequencing measurements. The algorithm successfully identified probes/genes with reliable measurements. Removing the unreliable measurements resulted in significant improvements for gene signature development and functional annotations.

  1. Selecting Reliable mRNA Expression Measurements Across Platforms Improves Downstream Analysis

    PubMed Central

    Tong, Pan; Diao, Lixia; Shen, Li; Li, Lerong; Heymach, John Victor; Girard, Luc; Minna, John D.; Coombes, Kevin R.; Byers, Lauren Averett; Wang, Jing

    2016-01-01

    With increasing use of publicly available gene expression data sets, the quality of the expression data is a critical issue for downstream analysis, gene signature development, and cross-validation of data sets. Thus, identifying reliable expression measurements by leveraging multiple mRNA expression platforms is an important analytical task. In this study, we propose a statistical framework for selecting reliable measurements between platforms by modeling the correlations of mRNA expression levels using a beta-mixture model. The model-based selection provides an effective and objective way to separate good probes from probes with low quality, thereby improving the efficiency and accuracy of the analysis. The proposed method can be used to compare two microarray technologies or microarray and RNA sequencing measurements. We tested the approach in two matched profiling data sets, using microarray gene expression measurements from the same samples profiled on both Affymetrix and Illumina platforms. We also applied the algorithm to mRNA expression data to compare Affymetrix microarray data with RNA sequencing measurements. The algorithm successfully identified probes/genes with reliable measurements. Removing the unreliable measurements resulted in significant improvements for gene signature development and functional annotations. PMID:27199546

  2. EXPRESSION OF AHR AND ARNT MRNA IN CULTURED HUMAN ENDOMETRIAL EXPLANTS EXPOSED TO TCDD

    EPA Science Inventory

    Expression of AhR and ARNT mRNA in cultured human endometrial explants exposed to TCDD.

    Pitt JA, Feng L, Abbott BD, Schmid J, Batt RE, Costich TG, Koury ST, Bofinger DP.

    Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC 27599, USA.

    Endom...

  3. EXPRESSION OF AHR AND ARNT MRNA IN CULTURED HUMAN ENDOMETRIAL EXPLANTS EXPOSED TO TCDD

    EPA Science Inventory

    Expression of AhR and ARNT mRNA in cultured human endometrial explants exposed to TCDD.

    Pitt JA, Feng L, Abbott BD, Schmid J, Batt RE, Costich TG, Koury ST, Bofinger DP.

    Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC 27599, USA.

    Endom...

  4. Use of mRNA expression signatures to discover small molecule inhibitors of skeletal muscle atrophy

    PubMed Central

    Adams, Christopher M.; Ebert, Scott M.; Dyle, Michael C.

    2017-01-01

    Purpose of review Here, we discuss a recently developed experimental strategy for discovering small molecules with potential to prevent and treat skeletal muscle atrophy. Recent findings Muscle atrophy involves and requires widespread changes in skeletal muscle gene expression, which generate complex but measurable patterns of positive and negative changes in skeletal muscle mRNA levels (a.k.a. mRNA expression signatures of muscle atrophy). Many bioactive small molecules generate their own characteristic mRNA expression signatures, and by identifying small molecules whose signatures approximate mirror images of muscle atrophy signatures, one may identify small molecules with potential to prevent and/or reverse muscle atrophy. Unlike a conventional drug discovery approach, this strategy does not rely on a predefined molecular target but rather exploits the complexity of muscle atrophy to identify small molecules that counter the entire spectrum of pathological changes in atrophic muscle. We discuss how this strategy has been used to identify two natural compounds, ursolic acid and tomatidine, that reduce muscle atrophy and improve skeletal muscle function. Summary Discovery strategies based on mRNA expression signatures can elucidate new approaches for preserving and restoring muscle mass and function. PMID:25807353

  5. 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

  6. Periodontal ligament cells under intermittent tensile stress regulate mRNA expression of osteoprotegerin and tissue inhibitor of matrix metalloprotease-1 and -2.

    PubMed

    Tsuji, Kiyomi; Uno, Keiji; Zhang, Gui Xia; Tamura, Masato

    2004-01-01

    We studied the mRNA expression of osteoprotegerin (OPG), receptor activator of NF-kappa B ligand (RANKL), tissue inhibitor of matrix metalloprotease (TIMP)-1 and -2, and matrix metalloprotease (MMP)-1 and -2 by human periodontal ligament (PDL) cells under intermittent tensile stress using a Flexercell Strain Unit. Analysis by reverse transcriptase-polymerase chain reaction showed that mechanical force upregulated OPG mRNA. We also demonstrated that the protein concentration of OPG in conditioned medium increased upon loading with tensile stress, as determined by enzyme-linked immunosorbent assay. TIMP-1 and -2 mRNA levels also increased, whereas levels of RANKL, MMP-1, and MMP-2 mRNA were barely affected. We further examined the effect of loading with tensile stress and addition of Salmonella abortus equi lipopolysaccharide (LPS) on the mRNA expression of PDL cells. The amount of OPG mRNA induced by mechanical strain was found to decrease with the addition of LPS to cultures. The induction of OPG mRNA expression by stretching was inhibited in the presence of indomethacin or genistein, whereas TIMP-1 mRNA expression induced by stretching was inhibited by the addition of cycloheximide, suggesting that tensile stress regulates cyclooxygenase activities, tyrosine phosphorylation, and de novo protein synthesis in PDL cells through the induction of OPG and TIMP-1 mRNA expression. These results provide evidence that the mechanical stimulus of stretching is responsible for the observed regulation of bone resorption and tissue degradation in PDL tissue.

  7. 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.

  8. Expression and regulation of Icer mRNA in the Syrian hamster pineal gland.

    PubMed

    Diaz, Elena; Garidou, Marie-Laure; Dardente, Hugues; Salingre, Anthony; Pévet, Paul; Simonneaux, Valérie

    2003-04-10

    Inducible-cAMP early repressor (ICER) is a potent inhibitor of CRE (cAMP-related element)-driven gene transcription. In the rat pineal gland, it has been proposed to be part of the mechanisms involved in the shutting down of the transcription of the gene coding for arylalkylamine N-acetyltransferase (AA-NAT, the melatonin rhythm-generating enzyme). In this study, we report that ICER is expressed in the pineal gland of the photoperiodic rodent Syrian hamster although with some difference compared to the rat. In the Syrian hamster pineal, Icer mRNA levels, low at daytime, displayed a 20-fold increase during the night. Nighttime administration of a beta-adrenergic antagonist, propranolol, significantly reduced Icer mRNA levels although daytime administration of a beta-adrenergic agonist, isoproterenol, was unable to raise the low amount of Icer mRNA. These observations indicate that Icer mRNA expression is induced by the clock-driven norepinephrine release and further suggest that this stimulation is restricted to nighttime, as already observed for Aa-nat gene transcription. Furthermore, we found that the daily profile of Icer mRNA displayed photoperiodic variation with a lengthening of the nocturnal peak in short versus long photoperiod. These data indicate that ICER may be involved in both daily and seasonal regulation of melatonin synthesis in the Syrian hamster.

  9. BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells.

    PubMed

    Awe, Jason P; Crespo, Agustin Vega; Li, You; Kiledjian, Megerditch; Byrne, James A

    2013-02-06

    The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through an NF-κB-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells. We tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules - B18R, BX795, and BAY11 - were used in immunocytochemical and proliferation-based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4. We found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels. For the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule-based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics.

  10. mRNA Blood Expression Patterns In New Onset Idiopathic Pediatric Epilepsy

    PubMed Central

    Greiner, Hansel M; Horn, Paul S; Holland, Katherine; Collins, James; Hershey, Andrew D; Glauser, Tracy A

    2012-01-01

    Purpose Determine if blood mRNA expression patterns in children with newly diagnosed untreated idiopathic epilepsy are different than those in healthy controls. Determine the differential expression patterns between epilepsy subjects with generalized onset or partial onset seizures compared to healthy controls. Methods Whole blood was obtained from otherwise healthy pediatric subjects with newly diagnosed untreated idiopathic epilepsy along with healthy pediatric controls. mRNA was isolated and hybridized to Affymetrix HGU 133 2.0+ microarrays. Analysis was performed using Genespring. Differentially expressed gene lists resulted from comparison of i) epilepsy and control groups and ii) seizure type subgroups with controls.. Tissue expression and gene ontology analysis was performed using DAVID. Key findings Thirty-seven epilepsy patients and 28 controls were included. Overall, 575 genes were differentially expressed in subjects with epilepsy compared to controls. The generalized seizure subgroup versus control (GvC) gene list and the partial seizure subgroup versus control (PvC) gene list were different (p < 0.05). Tissue expression analysis identified almost half of the genes in GvC and PvC as brain based. Functional group analysis identified several biologically relevant pathways. In GvC, these included mitochondria and lymphocyte activation. In PvC, apoptosis, inflammatory defense and cell motion pathways were identified. Significance A unique, biologically meaningful mRNA expression pattern is detectable in whole blood of pediatric subjects with new onset and untreated epilepsy. This analysis finds many similar pathways to those identified in brain studies examining lesional intractable epilepsy. Blood mRNA expression patterns show promise as a target for biomarker development in pediatric epilepsy. PMID:23167847

  11. BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells

    PubMed Central

    2013-01-01

    Introduction The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through an NF-κB-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells. Methods We tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules - B18R, BX795, and BAY11 - were used in immunocytochemical and proliferation-based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4. Results We found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels. Conclusions For the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule-based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics. PMID:23388106

  12. 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.

  13. Effect of cortisol on neurophysin I/oxytocin and peptidyl glycine-alpha-amidating mono-oxygenase mRNA expression in bovine luteal and granulosa cells.

    PubMed

    Ziolkowska, A; Mlynarczuk, J; Kotwica, J

    2013-01-01

    Cortisol stimulates the synthesis and secretion of oxytocin (OT) from bovine granulosa and luteal cells, but the molecular mechanisms of cortisol action remain unknown. In this study, granulosa cells or luteal cells from days 1-5 and 11-15 of the oestrous cycle were incubated for 4 or 8 h with cortisol (1 x 10(-5), 1 x 10(-7) M). After testing cell viability and hormone secretion (OT, progesterone, estradiol), we studied the effect of cortisol on mRNA expression for precursor of OT (NP-I/OT) and peptidyl glycine-alpha-amidating mono-oxygenase (PGA). The influence of RU 486 (1 x 10(-5) M), a progesterone receptor blocker and inhibitor of the glucocorticosteroid receptor (GR), on the expression for both genes was tested. Cortisol increased the mRNA expression for NP-I/OT and PGA in granulosa cells and stimulated the expression for NP-I/OT mRNA in luteal cells obtained from days 1-5 and days 11-15 of the oestrous cycle. Expression for PGA mRNA was increased only in luteal cells from days 11-15 of the oestrous cycle. In addition, RU 486 blocked the cortisol-stimulated mRNA expression for NP-I/OT and PGA in both types of cells. These data suggest that cortisol affects OT synthesis and secretion in bovine ovarian cells, by acting on the expression of key genes, that may impair ovary

  14. 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.

  15. 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.

  16. 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

  17. Regulation of bovine pyruvate carboxylase mRNA and promoter expression by thermal stress.

    PubMed

    White, H M; Koser, S L; Donkin, S S

    2012-09-01

    Pyruvate carboxylase (PC) catalyzes the rate-limiting step in gluconeogenesis from lactate and is a determinant of tricarboxylic acid cycle carbon flux. Bovine PC 5' untranslated region (UTR) mRNA variants are the products of a single PC gene containing 3 promoter regions (P3, P2, and P1, 5' to 3') that are responsive to physiological and nutritional stressors. The objective of this study was to determine the direct effects of thermal stress on PC mRNA and gene expression in bovine hepatocyte monolayer cultures, rat hepatoma (H4IIE) cells, and Madin-Darby bovine kidney epithelial (MDBK) cells. Hepatocytes were isolated from 3 Holstein bull calves and used to prepare monolayer cultures. Rat hepatoma cells and MDBK cells were obtained from American Type Culture Collection, Manassas, VA. Beginning 24 h after initial seeding, cells were subjected to either 37°C (control) or 42°C (thermal stress) for 24 h. Treatments were applied in triplicate in a minimum of 3 independent cell preparations. For bovine primary hepatocytes, endogenous expression of bovine PC mRNA increased (P < 0.1) with 24 h of thermal stress (1.31 vs. 2.79 ± 0.49, arbitrary units, control vs. thermal stress, respectively), but there was no change (P ≥ 0.1) in cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) mRNA expression. Similarly, exposure of MDBK cells to thermal stress increased (P < 0.1) expression of bovine PC mRNA without altering (P ≥ 0.1) PEPCK-C mRNA expression. Conversely, there was no effect (P ≥ 0.1) of thermal stress on endogenous rat PC (0.47 vs. 0.30 ± 0.08, control vs. thermal stress) or PEPCK-C (1.61 vs. 1.20 ± 0.48, arbitrary units, control vs. thermal stress, respectively) mRNA expressions in H4IIE cells. To further investigate the regulation of PC, H4IIE cells were transiently transfected with bovine promoter-luciferase constructs containing either P1, P2, or P3, and exposed to thermal stress for 23 h. Activity of P1 was suppressed (P < 0.1) 5-fold, activity of P2

  18. Genome-Wide Screening of mRNA Expression in Leprosy Patients

    PubMed Central

    Belone, Andrea de Faria F.; Rosa, Patrícia S.; Trombone, Ana P. F.; Fachin, Luciana R. V.; Guidella, Cássio C.; Ura, Somei; Barreto, Jaison A.; Pinilla, Mabel G.; de Carvalho, Alex F.; Carraro, Dirce M.; Soares, Fernando A.; Soares, Cleverson T.

    2015-01-01

    Leprosy, an infectious disease caused by Mycobacterium leprae, affects millions of people worldwide. However, little is known regarding its molecular pathophysiological mechanisms. In this study, a comprehensive assessment of human mRNA was performed on leprosy skin lesions by using DNA chip microarrays, which included the entire spectrum of the disease along with its reactional states. Sixty-six samples from leprotic lesions (10TT, 10BT, 10BB, 10BL, 4LL, 14R1, and 10R2) and nine skin biopsies from healthy individuals were used as controls (CC) (ages ranged from 06 to 83 years, 48 were male and 29 female). The evaluation identified 1580 differentially expressed mRNAs [Fold Change (FC) ≥ 2.0, p ≤ 0.05] in diseased lesions vs. healthy controls. Some of these genes were observed in all forms of the disease (CD2, CD27, chit1, FA2H, FAM26F, GZMB, MMP9, SLAMF7, UBD) and others were exclusive to reactional forms (Type “1” reaction: GPNMB, IL1B, MICAL2, FOXQ1; Type “2” reaction: AKR1B10, FAM180B, FOXQ1, NNMT, NR1D1, PTX3, TNFRSF25). In literature, these mRNAs have been associated with numerous pathophysiological processes and signaling pathways and are present in a large number of diseases. The role of these mRNAs maybe studied in the context of developing new diagnostic markers and therapeutic targets for leprosy. PMID:26635870

  19. Change of dopamine receptor mRNA expression in lymphocyte of schizophrenic patients

    PubMed Central

    Kwak, Yong T; Koo, Min-Seong; Choi, Chul-Hee; Sunwoo, IN

    2001-01-01

    Background Though the dysfunction of central dopaminergic system has been proposed, the etiology or pathogenesis of schizophrenia is still uncertain partly due to limited accessibility to dopamine receptor. The purpose of this study was to define whether or not the easily accessible dopamine receptors of peripheral lymphocytes can be the peripheral markers of schizophrenia. Results 44 drug-medicated schizophrenics for more than 3 years, 28 drug-free schizophrenics for more than 3 months, 15 drug-naïve schizophrenic patients, and 31 healthy persons were enrolled. Sequential reverse transcription and quantitative polymerase chain reaction of the mRNA were used to investigate the expression of D3 and D5 dopamine receptors in peripheral lymphocytes. The gene expression of dopamine receptors was compared in each group. After taking antipsychotics in drug-free and drug-naïve patients, the dopamine receptors of peripheral lymphocytes were sequentially studied 2nd week and 8th week after medication. In drug-free schizophrenics, D3 dopamine receptor mRNA expression of peripheral lymphocytes significantly increased compared to that of controls and drug-medicated schizophrenics, and D5 dopamine receptor mRNA expression increased compared to that of drug-medicated schizophrenics. After taking antipsychotics, mRNA of dopamine receptors peaked at 2nd week, after which it decreases but the level was above baseline one at 8th week. Drug-free and drug-naïve patients were divided into two groups according to dopamine receptor expression before medications, and the group of patients with increased dopamine receptor expression had more severe psychiatric symptoms. Conclusions These results reveal that the molecular biologically-determined dopamine receptors of peripheral lymphocytes are reactive, and that increased expression of dopamine receptor in peripheral lymphocyte has possible clinical significance for subgrouping of schizophrenis. PMID:11252158

  20. Influence of beta-blockers on the myocardial mRNA expressions of circadian clock- and metabolism-related genes.

    PubMed

    Ushijima, Kentarou; Maekawa, Tomohiro; Ishikawa-Kobayashi, Eiko; Ando, Hitoshi; Shiga, Tsuyoshi; Fujimura, Akio

    2013-01-01

    Daily rhythms are regulated by a master clock-system in the suprachiasmatic nucleus and by a peripheral clock-system in each organ. Because norepinephrine is one of the timekeepers for the myocardial circadian clock that influences cardiac metabolism, it is speculated that a beta-blocker may affect the circadian clock and metabolism in heart tissue. In this study, thirty mg/kg/day of propranolol (a lipophilic beta-blocker) or atenolol (a hydrophilic beta-blocker) was given orally to Wistar rats for 4 weeks. The mRNA expressions of Bmal1 and E4BP4 in heart tissue were suppressed by the beta-blockers. However, the mRNA expressions of these clock genes in the suprachiasmatic nucleus were unchanged. Myocardial mRNA expressions of lactate dehydrogenase a and pyruvate dehydrogenase kinase 4 were also suppressed by the beta-blockers. In addition, ATP content in heart tissue was significantly elevated by the beta-blockers throughout 24 hours. The effects of propranolol and atenolol did not differ significantly. This study showed for the first time that a beta-blocker affects myocardial clock gene expression. Propranolol and atenolol increased ATP content in heart tissue throughout 24 hours. The influences of beta-blockers may be negligible on the SCN, and may be independent of lipid solubility on heart tissue. It is well known that these drugs exert a protective effect against myocardial ischemia, which may be mediated by an increase in the preservation of myocardial ATP. Copyright © 2013 American Society of Hypertension. Published by Elsevier Inc. All rights reserved.

  1. Audio-visual affective expression recognition

    NASA Astrophysics Data System (ADS)

    Huang, Thomas S.; Zeng, Zhihong

    2007-11-01

    Automatic affective expression recognition has attracted more and more attention of researchers from different disciplines, which will significantly contribute to a new paradigm for human computer interaction (affect-sensitive interfaces, socially intelligent environments) and advance the research in the affect-related fields including psychology, psychiatry, and education. Multimodal information integration is a process that enables human to assess affective states robustly and flexibly. In order to understand the richness and subtleness of human emotion behavior, the computer should be able to integrate information from multiple sensors. We introduce in this paper our efforts toward machine understanding of audio-visual affective behavior, based on both deliberate and spontaneous displays. Some promising methods are presented to integrate information from both audio and visual modalities. Our experiments show the advantage of audio-visual fusion in affective expression recognition over audio-only or visual-only approaches.

  2. Epigenetic Regulation of Dopamine Transporter mRNA Expression in Human Neuroblastoma Cells

    PubMed Central

    Green, Ashley L.; Hossain, Muhammad M.; Tee, Siew C.; Zarbl, Helmut; Guo, Grace L.; Richardson, Jason R.

    2016-01-01

    The dopamine transporter (DAT) is a key regulator of dopaminergic neurotransmission. As such, proper regulation of DAT expression is important to maintain homeostasis, and disruption of DAT expression can lead to neurobehavioral dysfunction. Based on genomic features within the promoter of the DAT gene, there is potential for DAT expression to be regulated through epigenetic mechanisms, including DNA methylation and histone acetylation. However, the relative contribution of these mechanisms to DAT expression has not been empirically determined. Using pharmacologic and genetic approaches, we demonstrate that inhibition of DNA methyltransferase (DNMT) activity increased DAT mRNA approximately 1.5–2 fold. This effect was confirmed by siRNA knockdown of DNMT1. Likewise, the histone deacetylase (HDAC) inhibitors valproate and butyrate also increased DAT mRNA expression, but the response was much more robust with expression increasing over tenfold. Genetic knockdown of HDAC1 by siRNA also increased DAT expression, but not to the extent seen with pharmacological inhibition, suggesting additional isoforms of HDAC or other targets may contribute to the observed effect. Together, these data identify the relative contribution of DNMTs and HDACs in regulating expression. These finding may aid in understanding the mechanistic basis for changes in DAT expression in normal and pathophysiological states. PMID:25963949

  3. 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.

  4. Expression of SART-1 mRNA in canine squamous cell carcinomas.

    PubMed

    Takaishi, Yumi; Yoshida, Yukari; Nakagaki, Kazuhide; Fujita, Michio; Taniguchi, Akiko; Orima, Hiromitsu

    2008-12-01

    SART-1, a squamous cell carcinoma (SCC) antigen recognized by cytotoxic T lymphocytes, has been useful in human cancer therapy. The SART-1(259) peptide is a potential candidate for vaccine. The present study examined an orthologue of the mRNA coding this peptide in canine SCCs. Specimens were obtained from seven canine patients with SCC, and the mRNA was isolated from the samples. The SART-1 and beta-actin genes were amplified by reverse-transcription polymerase chain reaction, using the isolated mRNA as a template. Canine SART-1 was amplified in six of the seven specimens, while beta-actin was detected in all the samples. In dogs, carcinomas expressing SART-1 could be a target for cytotoxic T lymphocyte mediated immunotherapy.

  5. Abnormal expression of ENaC and SGK1 mRNA induced by dietary sodium in Dahl salt-sensitively hypertensive rats.

    PubMed

    Aoi, Wataru; Niisato, Naomi; Sawabe, Yukinori; Miyazaki, Hiroaki; Tokuda, Shinsaku; Nishio, Kyosuke; Yoshikawa, Toshikazu; Marunaka, Yoshinori

    2007-10-01

    Epithelial sodium channel (ENaC) plays a crucial role in controlling sodium reabsorption in the kidney keeping the normal blood pressure. We previously reported that the expression of ENaC mRNA in the kidney of Dahl salt-sensitive (DS) rats was abnormally regulated by aldosterone, however it is unknown if dietary sodium affects the expression of ENaC and serum and glucocorticoid-regulated kinase 1 (SGK1), which plays an important role in ENaC activation, in DS rats. In the present study, we investigated whether dietary sodium abnormally affects the expression of ENaC and SGK1 mRNA in DS rats. DS and Dahl salt-resistant (DR) rats (8 weeks old) were divided into three different groups, respectively: (1) low sodium diet (0.005% NaCl), (2) normal sodium diet (0.3% NaCl), and (3) high sodium diet (8% NaCl). The high sodium diet for 4 weeks in DS rats elevated the systolic blood pressure, but did not in any other groups. The expression of alpha-ENaC mRNA in DS rats was abnormally increased by high sodium diet in contrast to DR rats, while it was normally increased by low sodium diet in DS rats similar to DR rats. The expression of beta- and gamma-ENaC mRNA in DS rats was also abnormally increased by high sodium diet unlike DR rats. The expression of SGK1 mRNA was elevated by high sodium diet in DS rats, but it was decreased in DR rats. These observations indicate that the expression of ENaC and SGK1 mRNA is abnormally regulated by dietary sodium in salt-sensitively hypertensive rats, and that this abnormal expression would be one of the factors causing salt-sensitive hypertension.

  6. Adrenocorticotropin receptors: Functional expression from rat adrenal mRNA in Xenopus laevis oocytes

    SciTech Connect

    Mertz, L.M.; Catt, K.J. )

    1991-10-01

    The adrenocorticotropin (ACTH) receptor, which binds corticotropin and stimulates adenylate cyclase and steroidogenesis in adrenocortical cells, was expressed in Xenopus laevis oocytes microinjected with rat adrenal poly(A){sup +} RNA. Expression of the ACTH receptor in individual stage 5 and 6 oocytes was monitored by radioimmunoassay of ligand-stimulated cAMP production. Injection of 5-40 ng of adrenal mRNA caused dose-dependent increases in ACTH-responsive cAMP production. Size fractionation of rat adrenal poly(A){sup +}RNA by sucrose density-gradient centrifugation revealed that mRNA encoding the ACTH receptor was present in the 1.1-to 2.0-kilobase fraction. These data indicate that ACTH receptors can be expressed from adrenal mRNA in Xenopus oocytes and are fully functional in terms of ligand specificity and signal generation. The extracellular cAMP response to ACTH is a sensitive and convenient index of receptor expression. This system should permit more complete characterization and expression cloning of the ACTH receptor.

  7. Maternal mRNA expression levels of H19 are inversely associated with risk of macrosomia

    PubMed Central

    Jiang, Hua; Yu, Yang; Xun, Pengcheng; Zhang, Jun; Luo, Guanghua

    2014-01-01

    Introduction To investigate the associations between the mRNA levels of H19 in term placenta and risk of macrosomia. Material and methods Term placentas were collected from 37 macrosomia and 37 matched neonates with normal birth weight (controls) born in Changzhou Women and Children Health Hospital, Jiangsu province, P. R. China from March 1 to June 30, 2008. The mRNA levels of H19 in those placentas were measured by real-time polymerase chain reaction (PCR). Simple and multiple logistic regression models were used to explore the risk factors in the development of macrosomia. All analyses were performed using Stata 10.0 (StataCorp, College Station, Texas, USA). Results The average H19 mRNA level of the macrosomia group was 1.450 ±0.456 while in the control group it was 2.080 ±1.296. Based on the result of Student's t test, there was a significant difference in H19 mRNA level between the macrosomia group and the control group (p = 0.008). After controlling for potential confounders, the multivariable adjusted odds ratio (OR) of macrosomia for those in the highest tertile of H19 mRNA level was 0.12 (95% CI: 0.02–0.59) when compared to those in the lowest tertile (p for linear trend = 0.009). Conclusions The term placental H19 mRNA levels were inversely related to the occurrence of macrosomia. Our findings suggest that the low expression of H19 mRNA may contribute to the development of macrosomia. PMID:25097584

  8. Maternal mRNA expression levels of H19 are inversely associated with risk of macrosomia.

    PubMed

    Jiang, Hua; Yu, Yang; Xun, Pengcheng; Zhang, Jun; Luo, Guanghua; Wang, Qiuwei

    2014-06-29

    To investigate the associations between the mRNA levels of H19 in term placenta and risk of macrosomia. Term placentas were collected from 37 macrosomia and 37 matched neonates with normal birth weight (controls) born in Changzhou Women and Children Health Hospital, Jiangsu province, P. R. China from March 1 to June 30, 2008. The mRNA levels of H19 in those placentas were measured by real-time polymerase chain reaction (PCR). Simple and multiple logistic regression models were used to explore the risk factors in the development of macrosomia. All analyses were performed using Stata 10.0 (StataCorp, College Station, Texas, USA). The average H19 mRNA level of the macrosomia group was 1.450 ±0.456 while in the control group it was 2.080 ±1.296. Based on the result of Student's t test, there was a significant difference in H19 mRNA level between the macrosomia group and the control group (p = 0.008). After controlling for potential confounders, the multivariable adjusted odds ratio (OR) of macrosomia for those in the highest tertile of H19 mRNA level was 0.12 (95% CI: 0.02-0.59) when compared to those in the lowest tertile (p for linear trend = 0.009). The term placental H19 mRNA levels were inversely related to the occurrence of macrosomia. Our findings suggest that the low expression of H19 mRNA may contribute to the development of macrosomia.

  9. A genome-wide survey demonstrates widespread non-linear mRNA in expressed sequences from multiple species

    PubMed Central

    Dixon, Richard J.; Eperon, Ian C.; Hall, Laurence; Samani, Nilesh J.

    2005-01-01

    We describe here the results of the first genome-wide survey of candidate exon repetition events in expressed sequences from human, mouse, rat, chicken, zebrafish and fly. Exon repetition is a rare event, reported in <10 genes, in which one or more exons is tandemly duplicated in mRNA but not in the gene. To identify candidates, we analysed database sequences for mRNA transcripts in which the order of the spliced exons does not follow the linear genomic order of the individual gene [events we term rearrangements or repetition in exon order (RREO)]. Using a computational approach, we have identified 245 genes in mammals that produce RREO events. RREO in mRNA occurs predominantly in the coding regions of genes. However, exon 1 is never involved. Analysis of the open reading frames suggests that this process may increase protein diversity and regulate protein expression via nonsense-mediated RNA decay. The sizes of the exons and introns involved around these events suggest a gene model structure that may facilitate non-linear splicing. These findings imply that RREO affects a significant subset of genes within a genome and suggests that non-linear information encoded within the genomes of complex organisms could contribute to phenotypic variation. PMID:16237125

  10. Staufen Recruitment into Stress Granules Does Not Affect Early mRNA Transport in Oligodendrocytes

    PubMed Central

    Thomas, María G.; Tosar, Leandro J. Martinez; Loschi, Mariela; Pasquini, Juana M.; Correale, Jorge; Kindler, Stefan; Boccaccio, Graciela L.

    2005-01-01

    Staufen is a conserved double-stranded RNA-binding protein required for mRNA localization in Drosophila oocytes and embryos. The mammalian homologues Staufen 1 and Staufen 2 have been implicated in dendritic RNA targeting in neurons. Here we show that in rodent oligodendrocytes, these two proteins are present in two independent sets of RNA granules located at the distal myelinating processes. A third kind of RNA granules lacks Staufen and contains major myelin mRNAs. Myelin Staufen granules associate with microfilaments and microtubules, and their subcellular distribution is affected by polysome-disrupting drugs. Under oxidative stress, both Staufen 1 and Staufen 2 are recruited into stress granules (SGs), which are stress-induced organelles containing transiently silenced messengers. Staufen SGs contain the poly(A)-binding protein (PABP), the RNA-binding proteins HuR and TIAR, and small but not large ribosomal subunits. Staufen recruitment into perinuclear SGs is paralleled by a similar change in the overall localization of polyadenylated RNA. Under the same conditions, the distribution of recently transcribed and exported mRNAs is not affected. Our results indicate that Staufen 1 and Staufen 2 are novel and ubiquitous SG components and suggest that Staufen RNPs are involved in repositioning of most polysomal mRNAs, but not of recently synthesized transcripts, during the stress response. PMID:15525674

  11. 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').

  12. Embryonic and Postnatal Expression of Aryl Hydrocarbon Receptor mRNA in Mouse Brain

    PubMed Central

    Kimura, Eiki; Tohyama, Chiharu

    2017-01-01

    Aryl hydrocarbon receptor (AhR), a member of the basic helix-loop-helix-Per-Arnt-Sim transcription factor family, plays a critical role in the developing nervous system of invertebrates and vertebrates. Dioxin, a ubiquitous environmental pollutant, avidly binds to this receptor, and maternal exposure to dioxin has been shown to impair higher brain functions and dendritic morphogenesis, possibly via an AhR-dependent mechanism. However, there is little information on AhR expression in the developing mammalian brain. To address this issue, the present study analyzed AhR mRNA expression in the brains of embryonic, juvenile, and adult mice by reverse transcription (RT)-PCR and in situ hybridization. In early brain development (embryonic day 12.5), AhR transcript was detected in the innermost cortical layer. The mRNA was also expressed in the hippocampus, cerebral cortex, cerebellum, olfactory bulb, and rostral migratory stream on embryonic day 18.5, postnatal days 3, 7, and 14, and in 12-week-old (adult) mice. Hippocampal expression was abundant in the CA1 and CA3 pyramidal and dentate gyrus granule cell layers, where expression level of AhR mRNA in 12-week old is higher than that in 7-day old. These results reveal temporal and spatial patterns of AhR mRNA expression in the mouse brain, providing the information that may contribute to the elucidation of the physiologic and toxicologic significance of AhR in the developing brain. PMID:28223923

  13. Expression of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase-3 and VEGF mRNA in rat liver, lung and heart: effect of methyl tertbutyl ether.

    PubMed

    Minchenko, D O; Kundieva, A V; Tsuchihara, K; Yavorovsky, O P; Paustovsky, Y O; Esumi, H; Minchenko, O H

    2009-01-01

    The main goal of this work was investigation of the effect of methyl tertbutyl ether, ecologically dangerous chemical compound, on the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB-3) and vascular endothelial growth factor (VEGF) mRNA in different rat organs. Expression of PFKFB-3 and VEGF is a hypoxia inducible factor (HIF)-dependent process which significantly increases under hypoxia, in malignant tumors and other pathology. In this study we have shown that PFKFB-3 and VEGF mRNA expression in the liver, lung, and heart changes in rats, treated with methyl tertbutyl ether for two months, in organ-specific manner. Expression of alternative splice variants of PFKFB-3 mRNA as well as VEGF mRNA also changes in organ-specific manner in rats, treated with methyl tertbutyl ether. The effect of methyl tertbutyl ether on the expression of PFKFB-3 and VEGF mRNA and its alternative splice variants is dose-dependent. Results of this investigation clearly demonstrated that methyl tertbutyl ether affects the expression of PFKFB-3, a key regulatory enzyme of glycolysis, as well as VEGF, very important factor of angiogenesis, in an organ-specific and dose-dependent manner.

  14. NITRIC OXIDE PRODUCTION AND iNOS mRNA EXPRESSION IN IFN-8-STIMULATED CHICKEN MACROPHAGES TRANSFECTED WITH iNOS siRNAs

    USDA-ARS?s Scientific Manuscript database

    Utilizing RNA interference technology with siRNA in the HD-11 macrophage cell line, we determined how the knock-down of the iNOS (inducible nitric oxide synthase) gene affected IFN-' induced macrophage production of nitric oxide (NO) and mRNA expression of genes involved in this biological pathway i...

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

    PubMed

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

    2014-06-01

    Multiple system atrophy (MSA) is a progressive neurodegenerative disease presenting clinically with parkinsonian, cerebellar, and autonomic features. α-Synuclein (αsyn), encoded by the gene SNCA, is the main constituent of glial cytoplasmic inclusion (GCI) found in oligodendrocytes in MSA, but the methods of its accumulation have not been established. The aim of this study is to investigate alterations in regional and cellular SNCA mRNA expression in MSA as a possible substrate for GCI formation. Quantitative reverse transcription polymerase chain reaction (qPCR) was performed on postmortem brain samples from 15 MSA, 5 IPD, and 5 control cases to investigate regional expression in the frontal and occipital regions, dorsal putamen, pontine base, and cerebellum. For cellular expression analysis, neurons and oligodendrocytes were isolated by laser-capture microdissection from five MSA and five control cases. SNCA mRNA expression was not significantly different between the MSA, IPD and control cases in all regions (multilevel model, P = 0.14). After adjusting for group effect, the highest expression was found in the occipital cortex while the lowest was in the putamen (multilevel model, P < 0.0001). At the cellular level, MSA oligodendrocytes expressed more SNCA than control oligodendrocytes and expression in MSA neurons was slightly lower than that in controls, however, these results did not reach statistical significance. We have demonstrated regional variations in SNCA expression, which is higher in cortical than subcortical regions. This study is the first to demonstrate SNCA mRNA expression by oligodendrocytes in human postmortem tissue using qPCR and, although not statistically significant, could suggest that this may be increased in MSA compared to controls.

  16. Integrated analysis of microRNA and mRNA expression profiles in HBx-expressing hepatic cells

    PubMed Central

    Chen, Ruo-Chan; Wang, Juan; Kuang, Xu-Yuan; Peng, Fang; Fu, Yong-Ming; Huang, Yan; Li, Ning; Fan, Xue-Gong

    2017-01-01

    AIM To identify the miRNA-mRNA regulatory network in hepatitis B virus X (HBx)-expressing hepatic cells. METHODS A stable HBx-expressing human liver cell line L02 was established. The mRNA and miRNA expression profiles of L02/HBx and L02/pcDNA liver cells were identified by RNA-sequencing analysis. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed to investigate the function of candidate biomarkers, and the relationship between miRNA and mRNA was studied by network analysis. RESULTS Compared with L02/pcDNA cells, 742 unregulated genes and 501 downregulated genes were determined as differentially expressed in L02/HBx cells. Gene ontology analysis suggested that the differentially expressed genes were relevant to different biological processes. Concurrently, 22 differential miRNAs were also determined in L02/HBx cells. Furthermore, integrated analysis of miRNA and mRNA expression profiles identified a core miRNA-mRNA regulatory network that is correlated with the carcinogenic role of HBx. CONCLUSION Collectively, the miRNA-mRNA network-based analysis could be useful to elucidate the potential role of HBx in liver cell malignant transformation and shed light on the underlying molecular mechanism and novel therapy targets for hepatocellular carcinoma. PMID:28348484

  17. 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

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

    PubMed

    Conde, Patricia; Acosta-Saavedra, Leonor C; Goytia-Acevedo, Raquel C; Calderon-Aranda, Emma S

    2007-04-01

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

  19. Oxygen tension affects lubricin expression in chondrocytes.

    PubMed

    Hatta, Taku; Kishimoto, Koshi N; Okuno, Hiroshi; Itoi, Eiji

    2014-10-01

    We assessed the effects of oxygen tension on lubricin expression in bovine chondrocytes and cartilage explants and a role for hypoxia-inducible transcription factor (HIF)-1α in regulating lubricin expression was investigated using a murine chondroprogenitor cell line, ATDC5, and bovine chondrocytes isolated from superficial and middle/deep zones of femoral cartilage. ATDC5 cells and bovine chondrocytes were cultured in micromass under different oxygen tensions (21%, 5%, and 1%). ATDC5 cells and middle/deep zone chondrocytes that initially had low lubricin expression levels were also cultured with or without transforming growth factor (TGF)-β1. Quantitative reverse transcription (RT)-PCR was used to determine lubricin and chondrogenic marker gene mRNA levels and immunohistochemistry was used to assess lubricin protein expression. Explant cartilage plugs cultured under different oxygen tensions were also subjected to immunohistological analysis for lubricin. HIF-1α gene silencing was achieved by electroporatic transfer into ATDC5 cells. A low oxygen tension reduced lubricin gene expression levels in bovine superficial chondrocytes, TGF-β1-treated middle/deep zone chondrocytes, and TGF-β1-treated ATDC5 cells. Lubricin expression in explant cartilage was also suppressed under hypoxia. HIF-1α gene silencing in ATDC5 cells attenuated the lubricin expression response to the oxygen tension. These results corroborate with previous studies that the oxygen tension regulates lubricin gene expression and suggest that HIF-1α plays an important role in this regulation. The normal distribution of lubricin in articular cartilage may be due to the hypoxic oxygen environment of cartilage as it is an avascular tissue. An oxygen tension gradient may be a key factor for engineering cartilage tissue with a layered morphology.

  20. Does inbreeding affect gene expression in birds?

    PubMed

    Hansson, Bengt; Naurin, Sara; Hasselquist, Dennis

    2014-09-01

    Inbreeding increases homozygosity, exposes genome-wide recessive deleterious alleles and often reduces fitness. The physiological and reproductive consequences of inbreeding may be manifested already during gene regulation, but the degree to which inbreeding influences gene expression is unknown in most organisms, including in birds. To evaluate the pattern of inbreeding-affected gene expression over the genome and in relation to sex, we performed a transcriptome-wide gene expression (10 695 genes) study of brain tissue of 10-day-old inbred and outbred, male and female zebra finches. We found significantly lower gene expression in females compared with males at Z-linked genes, confirming that dosage compensation is incomplete in female birds. However, inbreeding did not affect gene expression at autosomal or sex-linked genes, neither in males nor in females. Analyses of single genes again found a clear sex-biased expression at Z-linked genes, whereas only a single gene was significantly affected by inbreeding. The weak effect of inbreeding on gene expression in zebra finches contrasts to the situation, for example, in Drosophila where inbreeding has been found to influence gene expression more generally and at stress-related genes in particular.

  1. 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

  2. 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

  3. The mRNA Decay Pathway Regulates the Expression of the Flo11 Adhesin and Biofilm Formation in Saccharomyces cerevisiae

    PubMed Central

    Lo, Tricia L.; Qu, Yue; Uwamahoro, Nathalie; Quenault, Tara; Beilharz, Traude H.; Traven, Ana

    2012-01-01

    Regulation of the FLO11 adhesin is a model for gene expression control by extracellular signals and developmental switches. We establish that the major mRNA decay pathway regulates FLO11 expression. mRNA deadenylation of transcriptional repressors of FLO11 by the exonuclease Ccr4 keeps their levels low, thereby allowing FLO11 transcription. PMID:22595243

  4. 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.

  5. 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

  6. Cytokine mRNA expression in lesions in cats with chronic gingivostomatitis.

    PubMed

    Harley, R; Helps, C R; Harbour, D A; Gruffydd-Jones, T J; Day, M J

    1999-07-01

    Semiquantitative reverse transcription-PCR assays were developed to measure feline interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-10, and IL-12 (p35 & p40); gamma interferon (IFN-gamma); and glyceraldehyde-3-phosphate dehydrogenase mRNA concentrations in biopsies of feline oral mucosa. Biopsies were collected from 30 cats with chronic gingivostomatitis (diseased) prior to each cat receiving one of four treatments. In 23 cases replicate biopsies were collected 3 months after treatment commenced. Biopsies were also analyzed from 11 cats without clinical disease (nondiseased). Expression of IL-2, IL-10, IL-12 (p35 and p40), and IFN-gamma was detected in most nondiseased biopsies, while IL-6 was detected in a minority, and IL-4 and IL-5 were both undetectable. Compared to nondiseased cats, the diseased population showed a significant increase in the relative mRNA expression of IL-2, IL-4, IL-6, IL-10, IL-12 (p35 and p40), and IFN-gamma. In contrast, IL-5 mRNA expression was unchanged and was only detected in one case. No significant relationship was demonstrable between the change in relative expression of specific cytokine mRNA and the change in clinical severity of the local mucosal lesions over the treatment period. The results demonstrate that the normal feline oral mucosa is biased towards a predominantly (Th) type 1 profile of cytokine expression and that during the development of lesions seen in feline chronic gingivostomatitis there is a shift in the cytokine profile from a type 1 to a mixed type 1 and type 2 response.

  7. Cytokine mRNA Expression in Lesions in Cats with Chronic Gingivostomatitis

    PubMed Central

    Harley, R.; Helps, C. R.; Harbour, D. A.; Gruffydd-Jones, T. J.; Day, M. J.

    1999-01-01

    Semiquantitative reverse transcription-PCR assays were developed to measure feline interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-10, and IL-12 (p35 & p40); gamma interferon (IFN-γ); and glyceraldehyde-3-phosphate dehydrogenase mRNA concentrations in biopsies of feline oral mucosa. Biopsies were collected from 30 cats with chronic gingivostomatitis (diseased) prior to each cat receiving one of four treatments. In 23 cases replicate biopsies were collected 3 months after treatment commenced. Biopsies were also analyzed from 11 cats without clinical disease (nondiseased). Expression of IL-2, IL-10, IL-12 (p35 and p40), and IFN-γ was detected in most nondiseased biopsies, while IL-6 was detected in a minority, and IL-4 and IL-5 were both undetectable. Compared to nondiseased cats, the diseased population showed a significant increase in the relative mRNA expression of IL-2, IL-4, IL-6, IL-10, IL-12 (p35 and p40), and IFN-γ. In contrast, IL-5 mRNA expression was unchanged and was only detected in one case. No significant relationship was demonstrable between the change in relative expression of specific cytokine mRNA and the change in clinical severity of the local mucosal lesions over the treatment period. The results demonstrate that the normal feline oral mucosa is biased towards a predominantly (Th) type 1 profile of cytokine expression and that during the development of lesions seen in feline chronic gingivostomatitis there is a shift in the cytokine profile from a type 1 to a mixed type 1 and type 2 response. PMID:10391845

  8. Elevated tph2 mRNA Expression in a Rat Model of Chronic Anxiety

    PubMed Central

    Donner, Nina C.; Johnson, Philip L.; Fitz, Stephanie D.; Kellen, Karen E.; Shekhar, Anantha; Lowry, Christopher A.

    2015-01-01

    Background Allelic variations in TPH2, the gene encoding tryptophan hydroxylase 2, the rate-limiting enzyme for brain serotonin (5-HT) biosynthesis, may be genetic predictors of panic disorder and panic responses to panicogenic challenges in healthy volunteers. To test the hypothesis that tph2 mRNA is altered in chronic anxiety states, we measured tph2 expression in an established rat model of panic disorder. Methods We implanted 16 adult, male rats with bilateral guide cannulae and then primed them with daily injections of the corticotropin-releasing factor (CRF) receptor agonist, urocortin 1 (UCN1, 6 fmoles/100 nl per side, n = 8) or vehicle (n = 8) into the basolateral amygdaloid complex (BL) for 5 consecutive days. Anxiety-like behavior was assessed, 24 hr prior to and 48 hr following priming, in the social interaction (SI) test. A third group (n = 7) served as undisturbed home cage controls. All rats were killed 3 days after the last intra-BL injection to analyze tph2 and slc6a4 (gene encoding the serotonin transporter, SERT) mRNA expression in the dorsal raphe nucleus (DR), the main source of serotonergic projections to anxiety-related brain regions, using in situ hybridization histochemistry. Results UCN1 priming increased anxiety-related behavior in the SI test compared to vehicle-injected controls and elevated tph2, but not slc6a4, mRNA expression in DR subregions, including the ventrolateral DR/ventrolateral periaqueductal gray (DRVL/VLPAG), a subregion previously implicated in control of panic-related physiologic responses. Tph2 mRNA expression in the DRVL/VLPAG was correlated with increased anxiety-related behavior. Conclusion Our data support the hypothesis that chronic anxiety states are associated with dysregulated tph2 expression. PMID:22511363

  9. Naproxen sodium decreases prostaglandins secretion from cultured human endometrial stromal cells modulating metabolizing enzymes mRNA expression.

    PubMed

    Carrarelli, Patrizia; Funghi, Lucia; Bruni, Simone; Luisi, Stefano; Arcuri, Felice; Petraglia, Felice

    2016-01-01

    Dysmenorrhea, defined as painful cramps occurring immediately before or during the menstrual period, is a common symptom of different gynecological diseases. An acute uterine inflammatory response driven by prostaglandins (PGs) is responsible for painful symptoms. Progesterone withdrawal is responsible for activation of cyclooxygenase (COX-2) enzyme and decrease of hydroxyprostaglandin dehydrogenase (HPDG) with consequent increased secretion of PGs secretion, inducing uterine contractility and pain. The most widely used drugs for the treatment of pelvic pain associated with menstrual cycle are non steroidal anti-inflammatory drugs (NSAIDs). The uterine site of action of these drugs is still not defined and the present study evaluated the effect of naproxen sodium in cultured human endometrial stromal cells (HESC) collected from healthy women. PGE2 release was measured by ELISA; COX-2 and HPDG mRNA expression were assessed by qRT-PCR. Naproxen sodium did not affect HESC vitality. Naproxen sodium significantly decreased PGE2 secretion (p < 0.01) and COX-2 mRNA expression (p < 0.01). TNF-α induced PGE2 release was reduced in presence of naproxen sodium (p < 0.05), in association with decreased COX-2 and increased HPDG mRNAs expression. Naproxen sodium decreases endometrial PGE2 release induced by inflammatory stimulus acting on endometrial COX-2 and HPDG expression, suggesting endometrial synthesis of prostaglandins as a possible target for reduction of uterine inflammatory mechanism in dysmenorrhea.

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

    PubMed

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

    2015-12-01

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

  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. Chronic nicotine and smoking treatment increases dopamine transporter mRNA expression in the rat midbrain.

    PubMed

    Li, Shupeng; Kim, Kun Yang; Kim, Jong Hun; Kim, Jin Hoi; Park, Moon Seok; Bahk, Jong Yoon; Kim, Myeong Ok

    2004-06-03

    Previous pharmacokinetics and electrophysiological results indicated an important role of nicotine in the modulation of dopamine transporter (DAT). To elucidate the expression changes of DAT on chronic nicotine and smoke administration, the effects of nicotine and passive cigarette smoke on DAT mRNA expression in the ventral tegmental area (VTA) and the substantia nigra (SN) area were examined using in situ hybridization and RNase protection assay. The results showed that chronic nicotine and smoke exposure highly unregulated DAT mRNA in the VTA and SN areas, including the dorsal part of substantia nigra pars compacta. Smoke for 30 min showed the highest increasing effect, whereas nicotine and smoke for 10 min only had slightly increasing effects. However, smoke for 1 h showed an increasing effect to a lesser extent than 30 min. These results revealed a new aspect of nicotine's modulation on the DAT, and may have important roles in neuropsychological disorders related to the midbrain abnormalities such as drugs addiction.

  13. Increased GAD67 mRNA expression in cerebellar interneurons in autism: implications for Purkinje cell dysfunction.

    PubMed

    Yip, Jane; Soghomonian, Jean-Jacques; Blatt, Gene J

    2008-02-15

    It has been widely reported that in autism, the number of Purkinje cells (PCs) is decreased, and recently, decreased expression of glutamic acid decarboxylase 67 (GAD67) mRNA in Purkinje cells also has been observed. However, the autism literature has not addressed key GABAergic inputs into Purkinje cells. Inhibitory basket and stellate cell interneurons in the molecular layer of the cerebellar cortex provide direct key GABAergic input into Purkinje cells and could potently influence the output of Purkinje cells to deep cerebellar nuclei. We investigated the capacity for interneuronal synthesis of gamma-amino butyric acid (GABA) in both types of interneurons that innervate the remaining PCs in the posterolateral cerebellar hemisphere in autism. The level of GAD67 mRNA, one of the isoforms of the key synthesizing enzymes for GABA, was quantified at the single-cell level using in situ hybridization in brains of autistic and aged-matched controls. The National Institutes of Health imaging system showed that expression of GAD67 mRNA in basket cells was significantly up-regulated, by 28%, in eight autistic brains compared with that in eight control brains (mean +/- SEM pixels per cell, 1.03 +/- 0.05 versus 0.69 +/- 0.05, respectively; P < 0.0001 by independent t test). Stellate cells showed a trend toward a small increase in GAD67 mRNA levels, but this did not reach significance. The results suggest that basket cells likely provide increased GABAergic feed-forward inhibition to PCs in autism, directly affecting PC output to target neurons in the dentate nucleus and potentially disrupting its modulatory role in key motor and/or cognitive behaviors in autistic individuals.

  14. Glutamine and glutamic acid enhance thyroid-stimulating hormone β subunit mRNA expression in the rat pars tuberalis.

    PubMed

    Aizawa, Sayaka; Sakai, Takafumi; Sakata, Ichiro

    2012-03-01

    Thyroid-stimulating hormone (TSH)-producing cells of the pars tuberalis (PT) display distinct characteristics that differ from those of the pars distalis (PD). The mRNA expression of TSHβ and αGSU in PT has a circadian rhythm and is inhibited by melatonin via melatonin receptor type 1; however, the detailed regulatory mechanism for TSHβ expression in the PT remains unclear. To identify the factors that affect PT, a microarray analysis was performed on laser-captured PT tissue to screen for genes coding for receptors that are abundantly expressed in the PT. In the PT, we found high expression of the KA2, which is an ionotropic glutamic acid receptor (iGluR). In addition, the amino acid transporter A2 (ATA2), also known as the glutamine transporter, and glutaminase (GLS), as well as GLS2, were highly expressed in the PT compared to the PD. We examined the effects of glutamine and glutamic acid on TSHβ expression and αGSU expression in PT slice cultures. l-Glutamine and l-glutamic acid significantly stimulated TSHβ expression in PT slices after 2- and 4-h treatments, and the effect of l-glutamic acid was stronger than that of l-glutamine. In contrast, treatment with glutamine and glutamic acid did not affect αGSU expression in the PT or the expression of TSHβ or αGSU in the PD. These results strongly suggest that glutamine is taken up by PT cells through ATA2 and that glutamic acid locally converted from glutamine by Gls induces TSHβ expression via the KA2 in an autocrine and/or paracrine manner in the PT.

  15. 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.

  16. Progressive APOBEC3B mRNA expression in distant breast cancer metastases

    PubMed Central

    Dalm, Simone U.; de Weerd, Vanja; Moelans, Cathy B.; ter Hoeve, Natalie; van Diest, Paul J.; Martens, John W. M.; van Deurzen, Carolien H. M.

    2017-01-01

    Background APOBEC3B was recently identified as a gain-of-function enzymatic source of mutagenesis, which may offer novel therapeutic options with molecules that specifically target this enzyme. In primary breast cancer, APOBEC3B mRNA is deregulated in a substantial proportion of cases and its expression is associated with poor prognosis. However, its expression in breast cancer metastases, which are the main causes of breast cancer-related death, remained to be elucidated. Patients and methods RNA was isolated from 55 primary breast cancers and paired metastases, including regional lymph node (N = 20) and distant metastases (N = 35). APOBEC3B mRNA levels were measured by RT-qPCR. Expression levels of the primary tumors and corresponding metastases were compared, including subgroup analysis by estrogen receptor (ER/ESR1) status. Results Overall, APOBEC3B mRNA levels of distant metastases were significantly higher as compared to the corresponding primary breast tumor (P = 0.0015), an effect that was not seen for loco-regional lymph node metastases (P = 0.23). Subgroup analysis by ER-status showed that increased APOBEC3B levels in distant metastases were restricted to metastases arising from ER-positive primary breast cancers (P = 0.002). However, regarding ER-negative primary tumors, only loco-regional lymph node metastases showed increased APOBEC3B expression when compared to the corresponding primary tumor (P = 0.028). Conclusion APOBEC3B mRNA levels are significantly higher in breast cancer metastases as compared to the corresponding ER-positive primary tumors. This suggests a potential role for APOBEC3B in luminal breast cancer progression, and consequently, a promising role for anti-APOBEC3B therapies in advanced stages of this frequent form of breast cancer. PMID:28141868

  17. Analysis of myosin heavy chain mRNA expression by RT-PCR

    NASA Technical Reports Server (NTRS)

    Wright, C.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

    1997-01-01

    An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.

  18. Analysis of myosin heavy chain mRNA expression by RT-PCR

    NASA Technical Reports Server (NTRS)

    Wright, C.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

    1997-01-01

    An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.

  19. Reduced mRNA Expression of PTGDS in Peripheral Blood Mononuclear Cells of Rapid-Cycling Bipolar Disorder Patients Compared with Healthy Control Subjects

    PubMed Central

    Peijs, Lone; Kessing, Lars Vedel; Vinberg, Maj

    2015-01-01

    Background: Disturbances related to the arachidonic acid cascade and prostaglandin metabolism may be involved in the pathophysiology of bipolar disorder, as supported by a recent genome-wide association study meta-analysis; however, evidence from clinical studies on a transcriptional level is lacking. Two enzymes in the arachidonic acid cascade are the prostaglandin D synthase (PTGDS), which catalyzes the conversion of prostaglandin H2 to prostaglandin D2 (PGD2), and the aldo-keto reductase family 1 member C3 (AKR1C3), which catalyzes the reduction of PGD2. We aimed to test the hypothesis that mRNA expression of PTGDS and AKR1C3 is deregulated in rapid-cycling disorder patients in a euthymic or current affective state compared with healthy control subjects, and that expression alters with affective states. Methods: PTGDS and AKR1C3 mRNA expression in peripheral blood mononuclear cells was measured in 37 rapid-cycling bipolar disorder patients and 40 age- and gender-matched healthy control subjects using reverse transcription quantitative real-time polymerase chain reaction. Repeated measurements of PTGDS and AKR1C3 mRNA expression were obtained in various affective states during 6–12 months and compared with repeated measurements in healthy control subjects. Results: Adjusted for age and gender, PTGDS mRNA expression was down-regulated in rapid-cycling bipolar disorder patients in a euthymic, depressive, and manic/hypomanic state compared with healthy control subjects. No difference in PTGDS mRNA expression was observed between affective states. AKR1C3 mRNA expression did not differ between bipolar disorder patients in any affective state or in comparison with healthy control subjects. Conclusions: The results suggest a role for aberrantly-regulated PTGDS mRNA expression in rapid-cycling bipolar disorder. The sample size was limited; replication of the findings in larger, independent samples is warranted to further explore the role of the arachidonic acid cascade

  20. Conditioned Fear Inhibits c-fos mRNA Expression in the Central Extended Amygdala

    PubMed Central

    Day, Heidi E.W.; Kryskow, Elisa M.; Nyhuis, Tara J.; Herlihy, Lauren; Campeau, Serge

    2008-01-01

    We have shown previously that unconditioned stressors inhibit neurons of the lateral/capsular division of the central nucleus of the amygdala (CEAl/c) and oval division of the bed nucleus of the stria terminalis (BSTov), which form part of the central extended amygdala. The current study investigated whether conditioned fear inhibits c-fos mRNA expression in these regions. Male rats were trained either to associate a visual stimulus (light) with footshock or were exposed to the light alone. After training, animals were replaced in the apparatus, and 2 hours later injected remotely, via a catheter, with amphetamine (2 mg/kg i.p.), to induce c-fos mRNA and allow inhibition of expression to be measured. The rats were then presented with 15 visual stimuli over a 30 minute period. As expected, fear conditioned animals that were not injected with amphetamine, had extremely low levels of c-fos mRNA in the central extended amygdala. In contrast, animals that were trained with the light alone (no fear conditioning) and were injected with amphetamine had high levels of c-fos mRNA in the CEAl/c and BSTov. Animals that underwent fear-conditioning, and were re-exposed to the conditioned stimulus after amphetamine injection had significantly reduced levels of c-fos mRNA in both the BSTov and CEAl/c, compared to the non-conditioned animals. These data suggest that conditioned fear can inhibit neurons of the central extended amygdala. Because these neurons are GABAergic, and project to the medial CEA (an amygdaloid output region), this may be a novel mechanism whereby conditioned fear potentiates amygdaloid output. PMID:18634767

  1. Developmental and tissue-specific expression of prosaposin mRNA in murine tissues.

    PubMed Central

    Sun, Y.; Witte, D. P.; Grabowski, G. A.

    1994-01-01

    Prosaposin is a multifunctional locus in humans and mice that encodes in tandem and in the same reading frame four glycoprotein activators, or saposins, of lysosomal hydrolases. These ubiquitously expressed glycoproteins and the precursor, prosaposin, have been proposed to function in glycosphingolipid catabolic pathways and glycolipid transport. To characterize the temporal and spatial expression of the prosaposin locus, prenatal and postnatal mouse tissues were screened by in situ hybridization with a mouse antisense riboprobe for prosaposin. Prenatally, prosaposin mRNA was expressed differentially in the placenta and prominently in the decidua basalis and capsularis where expression was gestational age dependent. No other region of high-level expression was detectable in the prenatal mouse. In comparison, high-level differential expression of prosaposin was clearly evident postnatally in a variety of organs, including secretory epithelial cells of the choroid plexus, ependymal lining, upper trachea, esophagus, cortical tubules of the kidney, sertoli cells of the testes and epididymis. Discrete localization of prosaposin mRNA expression was also found in neurons of the cerebral cortex, cerebellar cortex, and lateral columns of the spinal cord as well as in hepatocytes of the mature liver. Very high levels of expression were found in specialized tissues including the Harderian glands and macrophages of lymph nodes, lungs, splenic tissue, and thymus. These studies indicate that the expression of the prosaposin locus, a presumed "housekeeping" gene, is under tissue- and cell-specific differential control. The spatial organization of expression suggests a role for this locus in the expression of glycosphingolipid-storage diseases. Images Figure 2 PMID:7992842

  2. Hormone and metabolic factors associated with leptin mRNA expression in pre- and postmenopausal women.

    PubMed

    Fajardo, Martha E; Malacara, Juan M; Martínez-Rodríguez, Herminia G; Barrera-Saldaña, Hugo A

    2004-06-01

    Recent information has extended leptin's action, beyond the control of appetite, to various sites of metabolic regulation. To better understand leptin's role we studied its production in subcutaneous and visceral fat compartments before and after menopause. During elective abdominal surgery, biopsies of subcutaneous and omental tissues were taken from 20 women at pre- (BMI 28.4 +/- 4.5 kg/m2) and 10 at postmenopause (BMI 30.6 +/- 7.7 kg/m2). In both groups serum leptin levels were similar, and highly correlated with BMI. In subcutaneous adipose tissue, leptin mRNA expression was significantly higher in pre- than in postmenopausal women (50.4 +/- 20.5 amol/microg total RNA versus 34.5 +/- 24.9 amol/microg total RNA, respectively). Leptin mRNA expression in subcutaneous tissue was independently correlated with fasting glucose (R = 0.89, P < 0.006) at premenopause, and with serum estradiol (R = 0.77, P < 0.04) at postmenopause. Leptin mRNA expression in visceral fat was correlated with DHEAS (R = 0.86, P < 0.001), at premenopause. These results indicate that in both compartments, leptin production is sensitive to different but overlapping stimuli, conveying information about energy availability to central and peripheral sites under different conditions of estrogen exposure.

  3. 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.

  4. Nonsense codons can reduce the abundance of nuclear mRNA without affecting the abundance of pre-mRNA or the half-life of cytoplasmic mRNA.

    PubMed Central

    Cheng, J; Maquat, L E

    1993-01-01

    The abundance of the mRNA for human triosephosphate isomerase (TPI) is decreased to approximately 20% of normal by frameshift and nonsense mutations that cause translation to terminate at a nonsense codon within the first three-fourths of the reading frame. Results of previous studies inhibiting RNA synthesis with actinomycin D suggested that the decrease is not attributable to an increased rate of cytoplasmic mRNA decay. However, the step in TPI RNA metabolism that is altered was not defined, and the use of actinomycin D, in affecting all polymerase II-transcribed genes, could result in artifactual conclusions. In data presented here, the nonsense codon-mediated reduction in the level of TPI mRNA is shown to be characteristic of both nuclear and cytoplasmic fractions of the cell, indicating that the altered metabolic step is nucleus associated. Neither aberrancies in gene transcription nor aberrancies in RNA splicing appear to contribute to the reduction since there were no accompanying changes in the amount of nuclear run-on transcription, the level of any of the six introns in TPI pre-mRNA, or the size of processed mRNA in the nucleus. Deletion of all splice sites that reside downstream of a nonsense codon does not abrogate the reduction, indicating that the reduction takes place independently of the splicing of a downstream intron. Experiments that placed TPI gene expression under the control of the human c-fos promoter, which can be transiently activated by the addition of serum to serum-deprived cells, verified that there is no detectable effect of a nonsense codon on the turnover of cytoplasmic mRNA. Images PMID:8441420

  5. Distinct prognostic values of four-Notch-receptor mRNA expression in ovarian cancer.

    PubMed

    Zhou, Xinling; Teng, Lingling; Wang, Min

    2016-05-01

    Notch signaling pathway includes ligands and Notch receptors, which are frequently deregulated in several human malignancies including ovarian cancer. Aberrant activation of Notch signaling has been linked to ovarian carcinogenesis and progression. In the current study, we used the "Kaplan-Meier plotter" (KM plotter) database, in which updated gene expression data and survival information from a total of 1306 ovarian cancer patients were used to access the prognostic value of four Notch receptors in ovarian cancer patients. Hazard ratio (HR), 95 % confidence intervals, and log-rank P were calculated. Notch1 messenger RNA (mRNA) high expression was not found to be correlated to overall survival (OS) for all ovarian cancer, as well as in serous and endometrioid cancer patients followed for 20 years. However, Notch1 mRNA high expression is significantly associated with worsen OS in TP53 wild-type ovarian cancer patients, while it is significantly associated with better OS in TP53 mutation-type ovarian cancer patients. Notch2 mRNA high expression was found to be significantly correlated to worsen OS for all ovarian cancer patients, as well as in grade II ovarian cancer patients. Notch3 mRNA high expression was found to be significantly correlated to better OS for all ovarian cancer patients, but not in serous cancer patients and endometrioid cancer patients. Notch4 mRNA high expression was not found to be significantly correlated to OS for all ovarian cancer patients, serous cancer patients, and endometrioid cancer patients. These results indicate that there are distinct prognostic values of four Notch receptors in ovarian cancer. This information will be useful for better understanding of the heterogeneity and complexity in the molecular biology of ovarian cancer and for developing tools to more accurately predict their prognosis. Based on our results, Notch1 could be a potential drug target of TP53 wild-type ovarian cancer and Notch2 could be a potential drug

  6. Neuronal function of the mRNA decapping complex determines survival of Caenorhabditis elegans at high temperature through temporal regulation of heterochronic gene expression.

    PubMed

    Borbolis, Fivos; Flessa, Christina-Maria; Roumelioti, Fani; Diallinas, George; Stravopodis, Dimitrios J; Syntichaki, Popi

    2017-03-01

    In response to adverse environmental cues, Caenorhabditis elegans larvae can temporarily arrest development at the second moult and form dauers, a diapause stage that allows for long-term survival. This process is largely regulated by certain evolutionarily conserved signal transduction pathways, but it is also affected by miRNA-mediated post-transcriptional control of gene expression. The 5'-3' mRNA decay mechanism contributes to miRNA-mediated silencing of target mRNAs in many organisms but how it affects developmental decisions during normal or stress conditions is largely unknown. Here, we show that loss of the mRNA decapping complex activity acting in the 5'-3' mRNA decay pathway inhibits dauer formation at the stressful high temperature of 27.5°C, and instead promotes early developmental arrest. Our genetic data suggest that this arrest phenotype correlates with dysregulation of heterochronic gene expression and an aberrant stabilization of lin-14 mRNA at early larval stages. Restoration of neuronal dcap-1 activity was sufficient to rescue growth phenotypes of dcap-1 mutants at both high and normal temperatures, implying the involvement of common developmental timing mechanisms. Our work unveils the crucial role of 5'-3' mRNA degradation in proper regulation of heterochronic gene expression programmes, which proved to be essential for survival under stressful conditions. © 2017 The Authors.

  7. Neuronal function of the mRNA decapping complex determines survival of Caenorhabditis elegans at high temperature through temporal regulation of heterochronic gene expression

    PubMed Central

    Borbolis, Fivos; Flessa, Christina-Maria; Roumelioti, Fani; Diallinas, George; Stravopodis, Dimitrios J.

    2017-01-01

    In response to adverse environmental cues, Caenorhabditis elegans larvae can temporarily arrest development at the second moult and form dauers, a diapause stage that allows for long-term survival. This process is largely regulated by certain evolutionarily conserved signal transduction pathways, but it is also affected by miRNA-mediated post-transcriptional control of gene expression. The 5′–3′ mRNA decay mechanism contributes to miRNA-mediated silencing of target mRNAs in many organisms but how it affects developmental decisions during normal or stress conditions is largely unknown. Here, we show that loss of the mRNA decapping complex activity acting in the 5′–3′ mRNA decay pathway inhibits dauer formation at the stressful high temperature of 27.5°C, and instead promotes early developmental arrest. Our genetic data suggest that this arrest phenotype correlates with dysregulation of heterochronic gene expression and an aberrant stabilization of lin-14 mRNA at early larval stages. Restoration of neuronal dcap-1 activity was sufficient to rescue growth phenotypes of dcap-1 mutants at both high and normal temperatures, implying the involvement of common developmental timing mechanisms. Our work unveils the crucial role of 5′–3′ mRNA degradation in proper regulation of heterochronic gene expression programmes, which proved to be essential for survival under stressful conditions. PMID:28250105

  8. High Salt Diet Affects Renal Sodium Excretion and ERRα Expression.

    PubMed

    Wang, Dan; Wang, Yang; Liu, Fu-Qiang; Yuan, Zu-Yi; Mu, Jian-Jun

    2016-04-01

    Kidneys regulate the balance of water and sodium and therefore are related to blood pressure. It is unclear whether estrogen-related receptor α (ERRα), an orphan nuclear receptor and transcription factor highly expressed in kidneys, affects the reabsorption of water and sodium. The aim of this study was to determine whether changes in the expressions of ERRα, Na⁺/K⁺-ATPase and epithelial sodium channel (ENaC) proteins affected the reabsorption of water and sodium in kidneys of Dahl salt-sensitive (DS) rats. SS.13BN rats, 98% homologous to the DS rats, were used as a normotensive control group. The 24 h urinary sodium excretion of the DS and SS.13BN rats increased after the 6-week high salt diet intervention, while sodium excretion was increased in DS rats with daidzein (agonist of ERRα) treatment. ERRα expression was decreased, while β- and γ-ENaC mRNA expressions were increased upon high sodium diet treatment in the DS rats. In the chromatin immunoprecipitation (CHIP) assay, positive PCR signals were obtained in samples treated with anti-ERRα antibody. The transcriptional activity of ERRα was decreased upon high salt diet intervention. ERRα reduced the expressions of β- and γ-ENaC by binding to the ENaC promoter, thereby increased Na+ reabsorption. Therefore, ERRα might be one of the factors causing salt-sensitive hypertension.

  9. High Salt Diet Affects Renal Sodium Excretion and ERRα Expression

    PubMed Central

    Wang, Dan; Wang, Yang; Liu, Fu-Qiang; Yuan, Zu-Yi; Mu, Jian-Jun

    2016-01-01

    Kidneys regulate the balance of water and sodium and therefore are related to blood pressure. It is unclear whether estrogen-related receptor α (ERRα), an orphan nuclear receptor and transcription factor highly expressed in kidneys, affects the reabsorption of water and sodium. The aim of this study was to determine whether changes in the expressions of ERRα, Na+/K+-ATPase and epithelial sodium channel (ENaC) proteins affected the reabsorption of water and sodium in kidneys of Dahl salt-sensitive (DS) rats. SS.13BN rats, 98% homologous to the DS rats, were used as a normotensive control group. The 24 h urinary sodium excretion of the DS and SS.13BN rats increased after the 6-week high salt diet intervention, while sodium excretion was increased in DS rats with daidzein (agonist of ERRα) treatment. ERRα expression was decreased, while β- and γ-ENaC mRNA expressions were increased upon high sodium diet treatment in the DS rats. In the chromatin immunoprecipitation (CHIP) assay, positive PCR signals were obtained in samples treated with anti-ERRα antibody. The transcriptional activity of ERRα was decreased upon high salt diet intervention. ERRα reduced the expressions of β- and γ-ENaC by binding to the ENaC promoter, thereby increased Na+ reabsorption. Therefore, ERRα might be one of the factors causing salt-sensitive hypertension. PMID:27043552

  10. 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.

  11. Sequencing of mRNA identifies re-expression of fetal splice variants in cardiac hypertrophy.

    PubMed

    Ames, E G; Lawson, M J; Mackey, A J; Holmes, J W

    2013-09-01

    Cardiac hypertrophy has been well-characterized at the level of transcription. During cardiac hypertrophy, genes normally expressed primarily during fetal heart development are re-expressed, and this fetal gene program is believed to be a critical component of the hypertrophic process. Recently, alternative splicing of mRNA transcripts has been shown to be temporally regulated during heart development, leading us to consider whether fetal patterns of splicing also reappear during hypertrophy. We hypothesized that patterns of alternative splicing occurring during heart development are recapitulated during cardiac hypertrophy. Here we present a study of isoform expression during pressure-overload cardiac hypertrophy induced by 10 days of transverse aortic constriction (TAC) in rats and in developing fetal rat hearts compared to sham-operated adult rat hearts, using high-throughput sequencing of poly(A) tail mRNA. We find a striking degree of overlap between the isoforms expressed differentially in fetal and pressure-overloaded hearts compared to control: forty-four percent of the isoforms with significantly altered expression in TAC hearts are also expressed at significantly different levels in fetal hearts compared to control (P<0.001). The isoforms that are shared between hypertrophy and fetal heart development are significantly enriched for genes involved in cytoskeletal organization, RNA processing, developmental processes, and metabolic enzymes. Our data strongly support the concept that mRNA splicing patterns normally associated with heart development recur as part of the hypertrophic response to pressure overload. These findings suggest that cardiac hypertrophy shares post-transcriptional as well as transcriptional regulatory mechanisms with fetal heart development. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. 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

  13. DDR2 polymorphisms and mRNA expression in lung cancers of Japanese patients.

    PubMed

    Sasaki, Hidefumi; Shitara, Masayuki; Yokota, Keisuke; Okuda, Katsuhiro; Hikosaka, Yu; Moriyama, Satoru; Yano, Motoki; Fujii, Yoshitaka

    2012-07-01

    Discoidin domain receptor 2, DDR2, is a tyrosine kinase receptor for fibrillar collagen that is involved in postnatal development, tissue repair and primary and metastatic cancer progression. Recently, mutations in the DDR2 kinase gene were identified in squamous cell lung cancer from large-scale Sanger sequencing. The present study investigated the DDR2 gene mutations and mRNA expression in surgically treated non-small cell lung cancer (NSCLC) of squamous histology cases. The presence or absence of DDR2 mutations at the kinase and discoidin domain was analyzed by direct sequencing. In this cohort, DDR2 mutations were not observed in the 166 patients with lung cancer, although DDR2 polymorphisms were observed (H136H, n=14) at the discoidin domain. mRNA levels of DDR2 in lung tumor samples and the adjacent normal lung samples were simultaneously analyzed. DDR2 mRNA levels were significantly decreased in tumor samples compared with normal lung samples. However, the DDR2 mRNA levels were elevated in the DDR2 polymorphism cases.

  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. 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.

  16. 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

  17. [Effects of endothelial lipase on mRNA expression of adhesion molecule of human umbilical vascular endothelial cells].

    PubMed

    Fang, Yu-qiang; Huang, Lan; Zhao, Xiao-hui; Yin, Yang-guang; Kang, Hua-li; Deng, Meng-yang

    2007-12-01

    To explore the relationship between human umbilical vascular endothelial cells (HUVECs) and endothelial lipase (EL), and the effect of EL on expression of endothelial cell adhesion molecule (ICAM). HUVECs was treated with tumor necrosis factor-alpha(TNF-alpha) 10 microg/L and the mRNA of adhesion molecules [intercellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1) and E-selectin] were detected by reverse transcription-polymerase chain reaction (RT-PCR). Then the effect of 50 microg/L anti-endothelial lipase (anti-EL) antibody on the influence of TNF-alpha on these adhesion molecules was observed. After being treated with TNF-alpha, the mRNA of adhesion molecules expressed by HUVECs were significant up-regulated, there was significant difference compared with control group (all P<0.01). These effects of TNF-alpha were significantly abolished by 50 microg/L anti-EL antibody (P<0.05 or P<0.01). EL can affect the expression of adhesion molecules on endothelial cell adhesion molecule. This effect of EL may play a role in the pathophysiologic process in the pathogenesis progress of atherosclerosis.

  18. Seasonal relationship between gonadotropin, growth hormone, and estrogen receptor mRNA expression in the pituitary gland of largemouth bass.

    PubMed

    Martyniuk, Christopher J; Kroll, Kevin J; Porak, Wesley F; Steward, Cheree; Grier, Harry J; Denslow, Nancy D

    2009-09-15

    The objectives of this study were to investigate the seasonal changes in pituitary gonadotropins, growth hormone (GH), and estrogen receptor (ER) isoform mRNA in wild female and male largemouth bass (LMB) (Micropterus salmoides) from an unpolluted habitat to better understand reproductive physiology in this ecologically important species. Female pituitary luteinizing hormone (LH) beta subunit and follicle stimulating hormone (FSH) beta subunit mRNA showed significant seasonal variation with levels peaking from January to April and were lowest from May to August. Male LMB showed more variation in gonadotropin subunit expression from month to month. Females had approximately 2-3 times higher gonadotropin mRNA levels in the pituitary when compared to males. All three gonadotropin mRNAs in females were positively correlated to gonadosomatic index (GSI), but only LHbeta mRNA was correlated to GSI in males. Gonadotropin mRNA expression also increased with increasing oocyte and sperm maturation. Gonadotropin beta subunit mRNA expression was positively correlated to GH mRNA in both sexes. The expression of all three ER isoforms was significantly correlated to each other in both sexes. The concurrent increase in all three ER mRNA isoforms with increasing gonadotropin mRNA in females and males suggests a prominent role for E2 feedback on pituitary gonadotropin synthesis in both sexes and that each of the three ER isoforms are likely to play a role in the pituitary during teleost reproduction.

  19. Seasonal Relationship between Gonadotropin, Growth Hormone, and Estrogen Receptor mRNA Expression in the Pituitary Gland of Largemouth Bass

    PubMed Central

    Martyniuk, Christopher J; Kroll, Kevin J.; Porak, Wesley F.; Steward, Cheree; Grier, Harry J.; Denslow, Nancy D.

    2011-01-01

    The objectives of this study were to investigate the seasonal changes in pituitary gonadotropins, growth hormone (GH), and estrogen receptor (ER) isoform mRNA in wild female and male largemouth bass (LMB) (Micropterus salmoides) from an unpolluted habitat to better understand reproductive physiology in this ecologically important species. Female pituitary luteinizing hormone (LH) β subunit and follicle-stimulating hormone (FSH) β subunit mRNA showed significant seasonal variation with levels peaking from January to April and were lowest from May through August. Male LMB showed more variation in gonadotropin subunit expression from month to month. Females had approximately 2–3 times higher gonadotropin mRNA levels in the pituitary when compared to males. All three gonadotropin mRNAs in females were positively correlated to gonadosomatic index (GSI), but only LHβ mRNA was correlated to GSI in males. Gonadotropin mRNA expression also increased with increasing oocyte and sperm maturation. Gonadotropin β subunit mRNA expression was positively correlated to GH mRNA in both sexes. The expression of all three ER isoforms was significantly correlated to each other in both sexes. The concurrent increase in all three ER mRNA isoforms with increasing gonadotropin mRNA in females and males suggests a prominent role for E2 feedback on pituitary gonadotropin synthesis in both sexes and that each of the three ER isoforms are likely to play a role in the pituitary during teleost reproduction. PMID:19416730

  20. Stress and withdrawal from d-amphetamine alter 5-HT2A receptor mRNA expression in the prefrontal cortex.

    PubMed

    Murray, Ryan C; Hebbard, John C; Logan, Anna S; Vanchipurakel, Golda A; Gilbert, Yamiece E; Horner, Kristen A

    2014-01-24

    Psychostimulant withdrawal results in emotional, behavioral, and cognitive impairments, which may be exacerbated by stress. However, little is known about the neurochemical changes that occur when these two conditions are experienced concomitantly. 5-HT2A receptor (5-HT2AR) mRNA expression in the prefrontal cortex (PFC) is diminished following withdrawal from d-amphetamine (AMPH) and may underlie the emotional and cognitive impairments observed in psychostimulant withdrawal, but whether stress affects 5-HT2AR mRNA expression during psychostimulant withdrawal is unknown. The goal of this study was to examine the impact of forced swim test (FST) exposure during AMPH withdrawal on 5-HT2AR mRNA expression in PFC. Animals were treated 3 times a day for 4 days with escalating doses of AMPH (1-10mg/kg) and 24h or 4 days after the final injection, animals were subjected to FST. At 24h of withdrawal, AMPH-treated animals showed greater immobility in FST and at 4 days of withdrawal, AMPH-treated animals did not show immobility. At 24h of withdrawal, animals showed lower 5-HT2AR mRNA expression in the PFC relative to saline-treated animals, and exposure to FST did not further decrease expression in these animals. At 4 days of withdrawal, AMPH-treated animals showed greater 5-HT2AR mRNA expression relative to saline-treated animals in the PFC, an effect that was diminished by exposure to FST. These data indicate that stress and short-term AMPH withdrawal affect prefrontal 5-HT2AR mRNA expression to a similar degree, and stress experienced during long-term AMPH withdrawal can diminish the recovery of 5-HT2AR mRNA expression. Together, these data suggest that exposure to stress during extended AMPH withdrawal could prolong withdrawal-induced, 5-HT2AR mRNA expression which could be related to 5-HT2AR mediated deficits.

  1. Small, synthetic, GC-rich mRNA stem-loop modules 5′ proximal to the AUG start-codon predictably tune gene expression in yeast

    PubMed Central

    2013-01-01

    Background A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5′ UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Results Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5′ UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = −15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (

  2. Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast.

    PubMed

    Lamping, Erwin; Niimi, Masakazu; Cannon, Richard D

    2013-07-29

    A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5' UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5' UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = -15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = -4.4 kcal/mol) inhibited

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. Prognostic value of amphiregulin and epiregulin mRNA expression in metastatic colorectal cancer patients

    PubMed Central

    You, Zhai; Qiong, Qian; Jun, Zhou

    2016-01-01

    Epidermal growth factor receptor (EGFR) and its ligands amphiregulin (AREG) and epiregulin (EREG) play a central role in the development of colorectal cancer, but the prognostic values of AREG and EREG are controversial. We conducted a meta-analysis of studies that investigated AREG and/or EREG mRNA levels in primary tumors to determine their prognostic value in metastatic colorectal cancer (mCRC). In addition, RAS status was assessed. Relevant articles were identified by searching the EMBASE, PubMed, and Cochrane Library databases. Hazard ratios (HR) with 95% confidence intervals (CIs) were calculated using a random-effects model. Nine studies involving 2167 patients were included in this meta-analysis. High AREG expression was associated with longer overall survival (OS) and progression-free survival (PFS). High EREG expression was also associated with prolonged OS and PFS. In RAS wild-type (WT) patients who received anti-EGFR therapy, high AREG and EREG expression was associated with longer OS. Our results indicate that high AREG and EREG mRNA expression are independent favorable prognostic biomarkers in mCRC. The expression of these ligands should be considered when evaluating prognoses in RAS-WT patients receiving anti-EGFR therapy. PMID:27344184

  8. Regulation of selenoprotein mRNA expression by hormones and retinoic acid in bovine mammary cells.

    PubMed

    Bruzelius, Katharina; Sundler, Roger; Pagmantidis, Vasileios; Akesson, Björn

    2010-10-01

    Selenium is essential for maintaining many body functions through the actions of selenoproteins. To find factors regulating selenoprotein biosynthesis in the bovine mammary cell line MAC-T, the effects of supplementation with selenite and also with retinoic acid, insulin, hydrocortisone and prolactin on the mRNA expression of a number of selenoproteins were investigated. It was found that MAC-T cells express glutathione peroxidase (GPx) 1 and 4, thioredoxin reductase 1 and selenoprotein P, but not GPx 3, which is interesting considering that GPx 3 is one of the only few selenoproteins detected in milk so far. Addition of selenite to the cell culture resulted in a large increase in GPx 1 expression and an increase in selenoprotein P expression, which is similar to the findings made in other systems investigated. Increased mRNA levels of GPx 1 were also observed in cells treated with insulin and hydrocortisone or with retinoic acid. The expression of thioredoxin reductase 1 was increased in cells treated with retinoic acid, whereas that of selenoprotein P was decreased in cells exposed to insulin. The results indicate that several hormones, selenium, and retinoic acid regulate the biosynthesis of various selenoproteins differently in the bovine mammary cell. The possible implications of the findings for processes related to milk formation and mammary carcinogenesis will need additional investigation. Further study of the detailed mechanisms involved is also necessary. Copyright © 2010. Published by Elsevier GmbH.

  9. Integrative genomics analysis of genes with biallelic loss and its relation to the expression of mRNA and micro-RNA in esophageal squamous cell carcinoma.

    PubMed

    Hu, Nan; Wang, Chaoyu; Clifford, Robert J; Yang, Howard H; Su, Hua; Wang, Lemin; Wang, Yuan; Xu, Yi; Tang, Ze-Zhong; Ding, Ti; Zhang, Tongwu; Goldstein, Alisa M; Giffen, Carol; Lee, Maxwell P; Taylor, Philip R

    2015-09-26

    Genomic instability plays an important role in human cancers. We previously characterized genomic instability in esophageal squamous cell carcinomas (ESCC) in terms of loss of heterozygosity (LOH) and copy number (CN) changes in tumors. In the current study we focus on biallelic loss and its relation to expression of mRNA and miRNA in ESCC using results from 500 K SNP, mRNA, and miRNA arrays in 30 cases from a high-risk region of China. (i) Biallelic loss was uncommon but when it occurred it exhibited a consistent pattern: only 77 genes (<0.5%) showed biallelic loss in at least 10% of ESCC samples, but nearly all of these genes were concentrated on just four chromosomal arms (i.e., 42 genes on 3p, 14 genes on 9p, 10 genes on 5q, and seven genes on 4p). (ii) Biallelic loss was associated with lower mRNA expression: 52 of the 77 genes also had RNA expression data, and 41 (79%) showed lower expression levels in cases with biallelic loss compared to those without. (iii) The relation of biallelic loss to miRNA expression was less clear but appeared to favor higher miRNA levels: of 60 miRNA-target gene pairs, 34 pairs (57%) had higher miRNA expression with biallelic loss than without, while 26 pairs (43%) had lower miRNA expression. (iv) Finally, the effect of biallelic loss on the relation between miRNA and mRNA expression was complex. Biallelic loss was most commonly associated with a pattern of elevated miRNA and reduced mRNA (43%), but a pattern of both reduced miRNA and mRNA was also common (35%). Our results indicate that biallelic loss in ESCC is uncommon, but when it occurs it is localized to a few specific chromosome regions and is associated with reduced mRNA expression of affected genes. The effect of biallelic loss on miRNA expression and on the relation between miRNA and mRNA expressions was complex.

  10. 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.

  11. 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

  12. The effects of pilates exercise on lipid metabolism and inflammatory cytokines mRNA expression in female undergraduates

    PubMed Central

    Kim, Hyo-Jin; Kim, Jiyeon; Kim, Chang-Sun

    2014-01-01

    [Purpose] The purpose of the study was to verify the effects of Pilates exercise by observing the impact of 8 weeks of Pilates exercise on lipid metabolism and inflammatory cytokine mRNA expression in female undergraduates in their 20s who had no prior experience in Pilates exercise and had not exercised in the previous 6 months. [Methods] There were 18 subjects with no prior experience in Pilates exercise. The subjects were separated into the Pilates exercise group (n = 9) and the non-exercise control group (n = 9). The former performed Pilates exercise for 60-70 minutes over 8 weeks with a gradual strength increase of 9-16 in the Rating of Perceived Exercise (RPE). The body composition, creatine kinase in the bloodstream and lipid metabolism (TC, LDL-C, HDL-C, TG) were measured before and after the experiment and Real-Time PCR was used to investigate the mRNA expression of the inflammatory cytokines IL-6 and TNF-⍺. [Results] The creatine kinase (CK) in the blood had significant differences between the groups. The test group showed significant increase compared to the control group after 8 weeks of Pilates exercise (p = 0.007). Lipid analysis showed that the level of high-density lipoprotein cholesterol (HDL-C) was significantly different in the two groups (p = 0.049), with the Pilates exercise group exhibiting significantly higher levels compared to the control group. No significant differences were observed in the levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG). IL-6 mRNA expression did not show significant differences between the groups either. Timing and TNF-α mRNA expression showed significant effect in both the exercise and the control groups (p = 0.013) but no correlation. [Conclusion] It was found from the study that Pilates exercise for 8 weeks affected CK expression (the muscle damage marker) and induced positive changes in the levels of high-density lipoprotein. PMID:25566463

  13. The effects of pilates exercise on lipid metabolism and inflammatory cytokines mRNA expression in female undergraduates.

    PubMed

    Kim, Hyo-Jin; Kim, Jiyeon; Kim, Chang-Sun

    2014-09-01

    The purpose of the study was to verify the effects of Pilates exercise by observing the impact of 8 weeks of Pilates exercise on lipid metabolism and inflammatory cytokine mRNA expression in female undergraduates in their 20s who had no prior experience in Pilates exercise and had not exercised in the previous 6 months. There were 18 subjects with no prior experience in Pilates exercise. The subjects were separated into the Pilates exercise group (n = 9) and the non-exercise control group (n = 9). The former performed Pilates exercise for 60-70 minutes over 8 weeks with a gradual strength increase of 9-16 in the Rating of Perceived Exercise (RPE). The body composition, creatine kinase in the bloodstream and lipid metabolism (TC, LDL-C, HDL-C, TG) were measured before and after the experiment and Real-Time PCR was used to investigate the mRNA expression of the inflammatory cytokines IL-6 and TNF-⍺. The creatine kinase (CK) in the blood had significant differences between the groups. The test group showed significant increase compared to the control group after 8 weeks of Pilates exercise (p = 0.007). Lipid analysis showed that the level of high-density lipoprotein cholesterol (HDL-C) was significantly different in the two groups (p = 0.049), with the Pilates exercise group exhibiting significantly higher levels compared to the control group. No significant differences were observed in the levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG). IL-6 mRNA expression did not show significant differences between the groups either. Timing and TNF-α mRNA expression showed significant effect in both the exercise and the control groups (p = 0.013) but no correlation. It was found from the study that Pilates exercise for 8 weeks affected CK expression (the muscle damage marker) and induced positive changes in the levels of high-density lipoprotein.

  14. Effects of long-term smoking on the activity and mRNA expression of CYP isozymes in rats

    PubMed Central

    He, Xiao-Meng; Zhou, Ying; Xu, Ming-Zhen; Li, Yang; Li, Hu-Qun

    2015-01-01

    Background To investigate the effect of long-term smoking on the activity and mRNA expression of cytochrome P450 (CYP) enzymes. Methods Sprague-Dawley rats were exposed to passive smoking 6 cigarettes per day for 180 days. A cocktail solution which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg), chlorzoxazone (20 mg/kg) and midazolam (10 mg/kg) was given orally to rats. Blood samples were collected at pre-specified time points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The corresponding pharmacokinetic parameters were calculated by DAS 3.0. In addition, real-time RT-PCR was used to analyze the mRNA expression of CYP1A2, CYP2C11, CYP2E1 and CYP3A1 in rat liver. Results There were no significant influences of pharmacokinetic profiles of chlorzoxazone in long-term smoking pretreated rats. But many pharmacokinetic profiles of phenacetin, tolbutamide, and midazolam in long-term smoking pretreated rats were affected significantly (P<0.05). The results suggested that long-term smoking had significant inhibition effects on CYP2C11 and CYP3A1 while CYP1A2 enzyme activity was induced. Furthermore, Long-term smoking had no effects on rat CYP2E1. The mRNA expression results were consistent with the pharmacokinetic results. Conclusions Alterations of CYP450 enzyme activities may fasten or slow down excretion with corresponding influence on drug efficacy or toxicity in smokers compared to nonsmokers, which may lead to clinical failures of lung cancer therapy or toxicity in smokers. PMID:26623094

  15. Molecular Cloning, Characterization, and mRNA Expression of Hemocyanin Subunit in Oriental River Prawn Macrobrachium nipponense

    PubMed Central

    Kong, Youqin; Ding, Zhili; Sun, Shengming; Wang, Ligai; Ye, Jinyun

    2016-01-01

    Hemocyanin is a copper-containing protein with immune function against disease. In this study, a hemocyanin subunit named MnHc-1 was cloned from Macrobrachium nipponense. The full-length cDNA of MnHc-1 was 2,163 bp with a 2,028-bp open reading frame (ORF) encoding a polypeptide of 675 amino acids. The MnHc-1 mRNA was expressed in the hepatopancreas, gill, hemocytes, intestine, ovary, and stomach, with the highest level in the hepatopancreas. In the infection trial, the MnHc-1 mRNA transcripts in the hemocytes were significantly downregulated at 3 h after injection of Aeromonas hydrophila and then upregulated at 6 h and 12 h, followed by a gradual recovery from 24 to 48 h. The MnHc-1 transcriptional expression in the hepatopancreas was measured after M. nipponense were fed seven diets with 2.8, 12.2, 20.9, 29.8, 43.1, 78.9, and 157.1 mg Cu kg−1 for 8 weeks, respectively. The level of MnHc-1 mRNA was significantly higher in the prawns fed 43.1–157.1 mg Cu kg−1 diet than in that fed 2.8–29.8 mg Cu kg−1 diet. This study indicated that the MnHc-1 expression can be affected by dietary copper and the hemocyanin may potentially participate in the antibacterial defense of M. nipponense. PMID:27818993

  16. Estrogen-dependent changes in estrogen receptor-β mRNA expression in middle-aged female rat brain.

    PubMed

    Yamaguchi, Naoko; Yuri, Kazunari

    2014-01-16

    During aging, estrogen production and circulating levels of estrogen are markedly decreased in females. Although several differences exist in the process of reproductive aging between women and female rats, the results of many studies suggest that the female rat, especially the middle-aged or aged ovariectomized female, is an important animal model of hormone loss in women. In target tissues including the brain, the actions of estrogen are mediated mainly via the alpha and beta subtypes of the estrogen receptor (ER-α and ER-β). Estrogen treatment is known to change the expression of ER-α mRNA and protein in specific regions of the brain in middle-aged female rodents. In contrast, we do not know if estrogen regulates the expression of ER-β in the brain at this stage of life. In the present study, we performed in situ hybridization on brain sections of ovariectomized and estrogen-treated middle-aged female rats to reveal the effects of estrogen on the expression of ER-β throughout the brain. Our results showed that estrogen treatment decreased the number of ER-β mRNA-positive cells in the mitral cell and external plexiform layers of the olfactory bulb, central amygdaloid nucleus, medial geniculate nucleus, posterior hypothalamic nucleus, suprachiasmatic nucleus, and reticular part of the substantia nigra. As compared to the results of previous studies of young females, our data revealed that the regions in which expression of ER-β mRNA expression is affected by estrogen differ in middle age. These results suggest that the effects of estrogen on ER-β expression change with age. © 2013 Published by Elsevier B.V.

  17. 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.

  18. Combinatorial analysis of mRNA expression patterns in mouse embryos using hybridization chain reaction.

    PubMed

    Huss, David; Choi, Harry M T; Readhead, Carol; Fraser, Scott E; Pierce, Niles A; Lansford, Rusty

    2015-03-02

    Multiplexed fluorescent hybridization chain reaction (HCR) and advanced imaging techniques can be used to evaluate combinatorial gene expression patterns in whole mouse embryos with unprecedented spatial resolution. Using HCR, DNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled DNA HCR hairpins self-assemble into tethered fluorescent amplification polymers. Each target mRNA is detected by a probe set containing one or more DNA probes, with each probe carrying two HCR initiators. For multiplexed experiments, probe sets for different target mRNAs carry orthogonal initiators that trigger orthogonal DNA HCR amplification cascades labeled by spectrally distinct fluorophores. As a result, in situ amplification is performed for all targets simultaneously, and the duration of the experiment is independent of the number of target mRNAs. We have used multiplexed fluorescent in situ HCR and advanced imaging technologies to address questions of cell heterogeneity and tissue complexity in craniofacial patterning and anterior neural development. In the sample protocol presented here, we detect three different mRNA targets: Tg(egfp), encoding the enhanced green fluorescent protein (GFP) transgene (typically used as a control); Twist1, encoding a transcription factor involved in cell lineage determination and differentiation; and Pax2, encoding a transcription factor expressed in the mid-hindbrain region of the mouse embryo.

  19. Distinct prognostic values of S100 mRNA expression in breast cancer

    PubMed Central

    Zhang, Shizhen; Wang, Zhen; Liu, Weiwei; Lei, Rui; Shan, Jinlan; Li, Ling; Wang, Xiaochen

    2017-01-01

    S100 family genes encode low molecular weight, acidic-Ca2+ binding proteins implicating in a wide spectrum of biological processes. S100 family contains at least 20 members, most of which are frequently dysregulated in human malignancies including breast cancer. However, the prognostic roles of each individual S100, especially the mRNA level, in breast cancer patients remain elusive. In the current study, we used “The Kaplan-Meier plotter” (KM plotter) database to investigate the prognostic values of S100 mRNA expression in breast cancer. Our results indicated that high mRNA expression of S100A8, S100A9, S100A11 and S100P were found to be significantly correlated to worse outcome, while S100A1 and S100A6 were associated with better prognosis in all breast cancer patients. We further assessed the prognostic value of S100 in different intrinsic subtypes and clinicopathological features of breast cancer. The associated results will elucidate the role of S100 in breast cancer and may further lead the research to explore the S100-targeting reagents for treating breast cancer patients. PMID:28051137

  20. 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.

  1. The stability of an mRNA is influenced by its concentration: a potential physical mechanism to regulate gene expression.

    PubMed

    Nouaille, Sébastien; Mondeil, Sophie; Finoux, Anne-Laure; Moulis, Claire; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2017-09-07

    Changing mRNA stability is a major post-transcriptional way of controlling gene expression, particularly in newly encountered conditions. As the concentration of mRNA is the result of an equilibrium between transcription and degradation, it is generally assumed that at constant transcription, any change in mRNA concentration is the consequence of mRNA stabilization or destabilization. However, the literature reports many cases of opposite variations in mRNA concentration and stability in bacteria. Here, we analyzed the causal link between the concentration and stability of mRNA in two phylogenetically distant bacteria Escherichia coli and Lactococcus lactis. Using reporter mRNAs, we showed that modifying the stability of an mRNA had unpredictable effects, either higher or lower, on its concentration, whereas increasing its concentration systematically reduced stability. This inverse relationship between the concentration and stability of mRNA was generalized to native genes at the genome scale in both bacteria. Higher mRNA turnover in the case of higher concentrations appears to be a simple physical mechanism to regulate gene expression in the bacterial kingdom. The consequences for bacterial adaptation of this control of the stability of an mRNA by its concentration are discussed. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. TRPV4 regulates insulin mRNA expression and INS-1E cell death via ERK1/2 and NO-dependent mechanisms.

    PubMed

    Billert, M; Skrzypski, M; Sassek, M; Szczepankiewicz, D; Wojciechowicz, T; Mergler, S; Strowski, M Z; Nowak, K W

    2017-07-01

    TRPV4 is a Ca(2+)-permeable, nonselective cation channel. Recently, TRPV4 was implicated in controlling peripheral insulin sensitivity, insulin secretion and apoptosis of pancreatic beta cells. Here, we characterize the role and potential mechanisms of TRPV4 in regulating insulin mRNA expression and cell death in insulin producing INS-1E cells and rat pancreatic islets. TRPV4 protein production was downregulated by siRNA. Intracellular calcium level was measured using Fluo-3 AM. Gene expression was studied by real-time PCR. Phosphorylation of extracellular signal-regulated kinase (ERK1 and ERK2) was detected by Western blot. Nitric oxide (NO) production was assessed by chemiluminescent reaction. Reactive oxygen species (ROS) level was analysed using a fluorogenic dye (DCFDA). Cell death was evaluated by determination of cytoplasmic histone-associated DNA fragments. Downregulation of TRPV4 neither affected insulin mRNA expression nor INS-1E cell growth. By contrast, pharmacological TRPV4 activation by 100nmol/l GSK1016790A increased Ca(2+) levels in INS-1E cells and enhanced insulin mRNA expression after 1 and 3h, whereas a suppression of insulin mRNA expression was detected after 24h incubation. GSK1016790A increased ERK1/2 phosphorylation and NO production but not ROS production. Pharmacological blockade of ERK1/2 attenuated GSK1016790A-induced insulin mRNA expression. Inhibition of NO synthesis by l-NAME failed to affect insulin mRNA expression in GSK1016790A treated INS-1E cells. Furthermore, inhibition of NO production attenuated GSK1016790A-induced INS-1E cell death. In pancreatic islets, 100nmol/l GSK1016790A increased insulin mRNA levels after 3h without inducing cytotoxicity after 24h. In conclusion, TRPV4 differently regulates insulin mRNA expression in INS-1E cells via ERK1/2 and NO-dependent mechanisms. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. Neuropeptide Y in tiger puffer (Takifugu rubripes): distribution, cloning, characterization, and mRNA expression responses to prandial condition.

    PubMed

    Kamijo, Motoki; Kojima, Kenji; Maruyama, Keisuke; Konno, Norifumi; Motohashi, Eiji; Ikegami, Taro; Uchiyama, Minoru; Shioda, Seiji; Ando, Hironori; Matsuda, Kouhei

    2011-12-01

    Neuropeptide tyrosine (NPY) is a potent orexigenic neuropeptide implicated in feeding regulation in rodents. However, the involvement of NPY in feeding behavior has not well been studied in fish. Therefore, we investigated the role of NPY in food intake using a tiger puffer (Takifugu rubripes) model. We observed the distribution of NPY-like immunoreactivity in the brain. Neuronal cell bodies containing NPY were located in the telencephalon, hypothalamus, mesencephalon, and medulla oblongata, and their nerve fibers were also found throughout the brain. We cloned two cDNAs, encoding NPYa and NPYb orthologs, respectively, from the brain, and also confirmed two genes encoding these NPYs in the Takifugu genome database. We examined the distribution of these transcripts in the brain using real-time PCR. Levels of NPYa mRNA in the telencephalon, mesencephalon and hypothalamus were much higher than in the medulla oblongata and cerebellum, whereas levels of NPYb mRNA in the medulla oblongata were higher than in other regions. We also examined prandial effects on the expression level of these transcripts in the telencephalon and hypothalamus. NPYa mRNA levels in the hypothalamus, but not in the telencephalon, obtained from fish fasted for one week were higher than those in fish that had been fed normally. The level was decreased at 2 h after feeding. Levels of NPYb mRNA were not affected by prandial conditions. These results suggest that NPY is present throughout the brain, and that NPYa, but not NPYb, in the hypothalamus is involved in the feeding regulation in the tiger puffer.

  8. 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.

  9. Chondrogenic mRNA expression in prechondrogenic cells after blue laser irradiation.

    PubMed

    Kushibiki, Toshihiro; Tajiri, Takako; Ninomiya, Yoshihisa; Awazu, Kunio

    2010-03-08

    Low-level laser therapy (LLLT) has been used as a method for biostimulation. Cartilage develops through the differentiation of mesenchymal cells into chondrocytes, and differentiated chondrocytes in articular cartilage maintain cartilage homeostasis by synthesizing cartilage-specific extracellular matrix. The aim of this study is to evaluate the enhancement of chondrocyte differentiation and the expression levels of chondrogenic mRNA in prechondrogenic ATDC5 cells after laser irradiation. For chondrogenic induction, ATDC5 cells were irradiated with a blue laser (405 nm, continuous wave) at 100 mW/cm(2) for 180 s following incubation in chondrogenic differentiation medium. Differentiation after laser irradiation was quantitatively evaluated by the measurement of total collagen contents and chondrogenesis-related mRNAs. The total amount of collagen and mRNA levels of aggrecan, collagen type II, SOX-9, and DEC-1 were increased relative to those of a non-laser irradiated group after 14 days of laser irradiation. On the other hand, Ap-2alpha mRNA, a negative transcription factor of chondrogenesis, was dramatically decreased after laser irradiation. In addition, intracellular reactive oxygen species (ROS) were generated after laser irradiation. These results, for the first time, provide functional evidence that mRNA expression relating to chondrogenesis is increased, and Ap-2alpha is decreased immediately after laser irradiation. As this technique could readily be applied in situ to control the differentiation of cells at an implanted site within the body, this approach may have therapeutic potential for the restoration of damaged or diseased tissue.

  10. Zinc transporter mRNA expression in the RWPE-1 human prostate epithelial cell line.

    PubMed

    Albrecht, Amy L; Somji, Seema; Sens, Mary Ann; Sens, Donald A; Garrett, Scott H

    2008-08-01

    The human prostate gland undergoes a prominent alteration in Zn+2 homeostasis during the development of prostate cancer. The goal of the present study was to determine if the immortalized human prostate cell line (RWPE-1) could serve as a model system to study the role of zinc in prostate cancer. The study examined the expression of mRNA for 19 members of the zinc transporter gene family in normal prostate tissue, the prostate RWPE-1 cell line, and the LNCaP, DU-145 and PC-3 prostate cancer cell lines. The study demonstrated that the expression of the 19 zinc transporters was similar between the RWPE-1 cell line and the in situ prostate gland. Of the 19 zinc transporters, only 5 had levels that were different between the RWPE-1 cells and the tissue samples; all five being increased (ZnT-6, Zip-1, Zip-3A, Zip-10, and Zip-14). The response of the 19 transporters was also determined when the cell lines were exposed to 75 microM Zn+2 for 24 h. It was shown for the RWPE-1 cells that only 5 transporters responded to Zn+2 with mRNA for ZnT-1 and ZnT-2 being increased while mRNA for ZnT-7, Zip-7 and Zip-10 transporters were decreased. It was shown for the LNCaP, DU-145 and PC-3 cells that Zn+2 had no effect on the mRNA levels of all 19 transporters except for an induction of ZnT-1 in PC-3 cells. Overall, the study suggests that the RWPE-1 cells could be a valuable model for the study of the zinc transporter gene family in the prostate.

  11. Affective Scaffolds, Expressive Arts, and Cognition

    PubMed Central

    Maiese, Michelle

    2016-01-01

    Some theorists have argued that elements of the surrounding world play a crucial role in sustaining and amplifying both cognition and emotion. Such insights raise an interesting question about the relationship between cognitive and affective scaffolding: in addition to enabling the realization of specific affective states, can an affective niche also enable the realization of certain cognitive capacities? In order to gain a better understanding of this relationship between affective niches and cognition, I will examine the use of expressive arts in the context of psychotherapy and peacebuilding. In these settings, environmental resources and interpersonal scaffolds not only evoke emotion and encourage the adoption of particular bodily affective styles, but also support the development of capacities for self-awareness and interpersonal understanding. These affective scaffolds play a crucial role in therapy and peacebuilding, in fact, insofar as they facilitate the development of self-knowledge, enhance capacities associated with social cognition, and build positive rapport and trust among participants. I will argue that this is because affectivity is linked to the way that subjects frame and attend to their surroundings. Insofar as the regulation and modification of emotion goes hand in hand with opening up new interpretive frames and establishing new habits of mind, the creation of an affective niche can contribute significantly to various modes of cognition. PMID:27014164

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

    PubMed

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

    2015-10-01

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

  13. Expression and clinicopathological significance of Mel-18 mRNA in colorectal cancer.

    PubMed

    Tao, Ji; Liu, Yan-Long; Zhang, Gan; Ma, Yu-Yan; Cui, Bin-Bin; Yang, Yan-Mei

    2014-10-01

    Mel-18 is a member of the polycomb group (PcG) of proteins, which are chromatin regulatory factors that play an important role in oncogenesis. This study was designed to investigate the clinical and prognostic significance of Mel-18 in colorectal cancer (CRC) patients. For this purpose, expression of Mel-18 mRNA was evaluated in 82 primary CRC and paired noncancerous mucosa samples by qRT-PCR and Western blotting. We found that overall Mel-18 mRNA expression in the CRC tissue was significantly lower than in the noncancerous mucosal tissue (p = 0.007, Wilcoxon matched-pairs signed-ranks test). Mel-18 was conversely correlated with the pathological classifications (p = 0.003 for T, p < 0.001 for N, and p = 0.015 for M classifications, respectively) and clinical AJCC stage (p < 0.001). Furthermore, CRC patients with a higher level of Mel-18 showed prolonged disease-free survivals (DFS) (p < 0.001). In multivariate analysis, the diminished Mel-18 expression may be a risk factor for the patients' 3-year DFS (HR = 1.895; 95 % CI 1.032, 3.477; p = 0.039). It was therefore concluded that the lower Mel-18 expression might contribute to the CRC development/progression.

  14. Cellular expression of gut chitinase mRNA in the gastrointestinal tract of mice and chickens.

    PubMed

    Suzuki, Masako; Fujimoto, Wakako; Goto, Marie; Morimatsu, Masami; Syuto, Bunei; Iwanaga, Toshihiko

    2002-08-01

    Recently, the second mammalian chitinase, designated acidic mammalian chitinase (AMCase), has been identified in human, mouse, and cow. In contrast to the earlier identified macrophage-derived chitinase (chitotriosidase), this chitinase is richly expressed in the gastrointestinal (GI) tract, suggesting its role in digestion of chitin-containing foods as well as defense against chitin-coated microorganisms and parasites. This in situ hybridization study first revealed cellular localization of the gut-type chitinase in the mouse and chicken. In adult mice, the parotid gland, von Ebner's gland, and gastric chief cells, all of which are exocrine cells of the serous type, expressed the gut chitinase mRNA. In the chicken, oxyntico-peptic cells in glandular stomach (proventriculus) and hepatocytes expressed the chitinase mRNA. Because cattle produce the gut chitinase (chitin-binding protein b04) only in the liver, the gut chitinases in mammals and birds have three major sources of production, i.e., the salivary gland, stomach, and liver. During ontogenetic development, the expression level in the parotid gland and stomach of mice increased to the adult level before weaning, whereas in the stomach of chickens intense signals were detectable in embryos from incubation day 7.

  15. Effect of different culture systems on mRNA expression in developing rabbit embryos.

    PubMed

    Saenz-de-Juano, M D; Naturil-Alfonso, C; Vicente, J S; Marco-Jiménez, F

    2013-02-01

    The rate of zygotes in vitro developed to hatched blastocyst stage was evaluated between two different commercial media (TCM-199 and Ham's F10) and two different culture systems (renewal and non-renewal single medium) to determine the effects of culture conditions on rabbit embryo preimplantation development. The relative transcript abundances of OCT4, vascular endothelial growth factor (VEGF) and epidermal growth factor receptor 3 (erbB3) of resultant blastocysts were also analysed and compared with in vivo developed blastocysts. Results showed an important divergence in mRNA expression between embryos developed under in vivo and in vitro conditions despite there being no significant difference in hatching blastocyst rates between different culture systems and different media. For OCT4, transcript abundance of in vitro culture embryos differs from their in vivo chronological counterparts, but, when the medium is renewed, mRNA expression seemed similar to in vivo developed 4-day-old embryos. In addition, VEGF and erbB3 expression showed marked variation between different in vitro conditions. Therefore, the study of specific transcript abundance in rabbit blastocyst provides a more detailed description of which alterations in gene expression occur due in vitro conditions, and further studies should be carried out to reduce current limitations of long-term culture of rabbit pre-implantation embryos.

  16. HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 Is Required for Circadian Periodicity through the Promotion of Nucleo-Cytoplasmic mRNA Export in Arabidopsis[W][OPEN

    PubMed Central

    MacGregor, Dana R.; Gould, Peter; Foreman, Julia; Griffiths, Jayne; Bird, Susannah; Page, Rhiannon; Stewart, Kelly; Steel, Gavin; Young, Jack; Paszkiewicz, Konrad; Millar, Andrew J.; Halliday, Karen J.; Hall, Anthony J.; Penfield, Steven

    2013-01-01

    Cold acclimation has been shown to be attenuated by the degradation of the INDUCER OF CBF EXPRESSION1 protein by the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1). However, recent work has suggested that HOS1 may have a wider range of roles in plants than previously appreciated. Here, we show that hos1 mutants are affected in circadian clock function, exhibiting a long-period phenotype in a wide range of temperature and light environments. We demonstrate that hos1 mutants accumulate polyadenylated mRNA in the nucleus and that the circadian defect in hos1 is shared by multiple mutants with aberrant mRNA export, but not in a mutant attenuated in nucleo-cytoplasmic transport of microRNAs. As revealed by RNA sequencing, hos1 exhibits gross changes to the transcriptome with genes in multiple functional categories being affected. In addition, we show that hos1 and other previously described mutants with altered mRNA export affect cold signaling in a similar manner. Our data support a model in which altered mRNA export is important for the manifestation of hos1 circadian clock defects and suggest that HOS1 may indirectly affect cold signaling through disruption of the circadian clock. PMID:24254125

  17. HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 is required for circadian periodicity through the promotion of nucleo-cytoplasmic mRNA export in Arabidopsis.

    PubMed

    MacGregor, Dana R; Gould, Peter; Foreman, Julia; Griffiths, Jayne; Bird, Susannah; Page, Rhiannon; Stewart, Kelly; Steel, Gavin; Young, Jack; Paszkiewicz, Konrad; Millar, Andrew J; Halliday, Karen J; Hall, Anthony J; Penfield, Steven

    2013-11-01

    Cold acclimation has been shown to be attenuated by the degradation of the INDUCER OF CBF EXPRESSION1 protein by the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1). However, recent work has suggested that HOS1 may have a wider range of roles in plants than previously appreciated. Here, we show that hos1 mutants are affected in circadian clock function, exhibiting a long-period phenotype in a wide range of temperature and light environments. We demonstrate that hos1 mutants accumulate polyadenylated mRNA in the nucleus and that the circadian defect in hos1 is shared by multiple mutants with aberrant mRNA export, but not in a mutant attenuated in nucleo-cytoplasmic transport of microRNAs. As revealed by RNA sequencing, hos1 exhibits gross changes to the transcriptome with genes in multiple functional categories being affected. In addition, we show that hos1 and other previously described mutants with altered mRNA export affect cold signaling in a similar manner. Our data support a model in which altered mRNA export is important for the manifestation of hos1 circadian clock defects and suggest that HOS1 may indirectly affect cold signaling through disruption of the circadian clock.

  18. Differential cytokine mRNA expression in heterophils isolated from Salmonella-resistant and -susceptible chickens

    PubMed Central

    Swaggerty, Christina L; Kogut, Michael H; Ferro, Pamela J; Rothwell, Lisa; Pevzner, Igal Y; Kaiser, Pete

    2004-01-01

    We recently showed that increased in vitro heterophil functional efficiency translates to increased in vivo resistance to a systemic Salmonella enteritidis (SE) infection utilizing a parental pair of broiler chickens (lines A and B) and the F1 reciprocal crosses (C and D). Heterophils produce cytokines and modulate acute protection against Salmonella in young poultry. Therefore, we hypothesize that heterophils from SE-resistant chickens (A and D) have the ability to produce an up-regulated pro-inflammatory cytokine response compared to that of heterophils from SE-susceptible chickens (B and C). In this study, heterophils were isolated from day-old chickens and treated with either RPMI-1640 (as the control), or phagocytic agonists (SE, or SE opsonized with either normal chicken serum or immune serum against SE) and cytokine mRNA expression assessed using real-time quantitative reverse transcription–polymerase chain reaction. Heterophils from SE-resistant chickens (A and D) had significantly higher levels of pro-inflammatory cytokine (interleukin (IL)-6, IL-8, and IL-18) mRNA expression upon treatment with all agonists compared to heterophils from SE-susceptible lines (B and C). Further, heterophils from SE-resistant chickens had significantly decreased mRNA expression levels of transforming growth factor-β4, an anti-inflammatory cytokine, when compared to heterophils from SE-susceptible chickens. These data indicate cytokine gene expression in heterophils may be a useful parameter in determining resistance to Salmonella, as indicated by our previous in vivo SE studies. Therefore, heterophil functional efficiency and cytokine production may be useful biomarkers for poultry breeders to consider when developing new immunocompetent lines of birds. PMID:15312145

  19. Different structure and mRNA expression of Entamoeba invadens chitinases in the encystation and excystation.

    PubMed

    Makioka, Asao; Kumagai, Masahiro; Hiranuka, Kazushi; Kobayashi, Seiki; Takeuchi, Tsutomu

    2011-08-01

    Entamoeba histolytica forms chitin-walled cysts during encystation process, where formation of the cyst wall needs not only chitin synthase but also chitinase. During excystation, quadruplet amoebae emerge from the chitin-walled cysts by dissolving the wall, so that chitinase may be necessary for excystation process as well. There is, however, no report on chitinase expression during excystation. In this study, we used Entamoeba invadens, a reptilian amoeba, as a model for encystation and excystation of E. histolytica, and studied chitinase mRNA expression in those processes. Although expression of three E. invadens chitinases designated EiChit1, EiChit2, and EiChit3 during encystation has been reported, we identified another enzyme named as EiChit4 in the E. invadens genome database. Therefore, we investigated the primary structure and mRNA expression of these four chitinases of Ei in the excystation as well as the encystation by real-time reverse transcription polymerase chain reaction (RT-PCR). Like EiChit1, EiChit4 had an 8 × Cys chitin-binding domain (CBD) and a hydrophilic spacer between the CBD and catalytic domain, and was also closer to EiChit1 than EiChit2 and EiChit3 in the phylogenetic tree. During encystation, the expression of all four chitinases increased in the early phase; the increase in EiChit1 and EiChit4 was much higher than in EiChit2 and EiChit3. Then, the expression of all four chitinases sharply decreased in the later phase. In cysts, EiChit1 was most abundantly expressed and EiChit4 was at a lower level, while the expressions of EiChit2 and EiChit3 were virtually absent. Following the induction of excystation, mRNA levels of EiChit1 and EiChit4 in cysts 5 h after induction were significantly lower than those in cysts before induction, while those of EiChit2 and EiChit3 were remarkably higher than before induction. The mRNAs of only EiChit2 and EiChit3 remarkably increased when the excystation was induced in the presence of cytochalasin D

  20. Dietary supplementation of boron differentially alters expression of borate transporter (NaBCl) mRNA by jejunum and kidney of growing pigs.

    PubMed

    Liao, Shengfa F; Monegue, James S; Lindemann, Merlin D; Cromwell, Gary L; Matthews, James C

    2011-11-01

    Inorganic boron (B), in the form of various borates, is readily absorbed across gastrointestinal epithelia. Although there is no stated B requirement, dietary B supplementation is thought to positively affect animal growth and metabolism, including promotion of bone strength and cell proliferation. Because of effective homeostatic control of plasma B levels, primarly by renal excretion, B toxicity in animals and humans is rare. The mechanisms responsible for improved animal performance and borate homeostasis are incompletely understood. Although a Na+-coupled borate transporter (NaBC1) has been identified, the effect of dietary B supplementation on expression of NaBCl has not been evaluated. An experiment was conducted with growing pigs to determine if NaBC1 mRNA was expressed by small intestinal epithelia and kidney of growing barrows and whether dietary B (as borate) supplementation would affect expression of NaBC1 mRNA. A concomitant objective was to test the hypothesis that B supplementation of a phosphorus (P)-deficient diet would improve calcium, phosphorus, or nitrogen retention. Twenty-four crossbred growing barrows (body weight=74.0±9.8 kg) were selected and used in a randomized complete block design experiment with a total of eight blocks and three B supplementation treatments (n=8/treatment). A typical corn-soybean meal basal diet (calculated to contain 41 mg intrinsic B/kg) was formulated to meet or exceed nutrient requirements, except for P, and fed to all pigs for 12 days. The basal diet plus 0, 50, or 100 mg/kg of B (prilled sodium borate pentahydrate, Na₂B₄O₇·5H₂O) was then fed for 18 more days. Feces and urine were collected during days 6 to 16 of the B supplementation, and pigs were killed for collection of jejunal and ileal epithelia and kidney tissue. Real-time reverse transcription-PCR analysis revealed that NaBC1 mRNA was expressed by these tissues, a novel finding for jejunal and ileal epithelia. Boron supplementation increased

  1. Temporal expression of brain-derived neurotrophic factor (BDNF) mRNA in the rat hippocampus after treatment with selective and mixed monoaminergic antidepressants.

    PubMed

    Larsen, Marianne H; Hay-Schmidt, Anders; Rønn, Lars C B; Mikkelsen, Jens D

    2008-01-14

    Strong evidence suggests that antidepressants work by induction of neuroplastic changes mediated through regulation of brain-derived neurotrophic factor (BDNF). This study was undertaken to investigate the time-course of the effect of three antidepressants; fluoxetine, imipramine and venlafaxine, which differentially affect monoamine reuptake, on BDNF mRNA expression in the hippocampus. The consequences of increased BDNF in the hippocampus are still indefinite. Here, we also determined the effects on the expression of two other genes (synaptophysin and growth-associated protein-43 (GAP-43)) known to be involved in synapse formation and axonal growth and likely regulated by BDNF. The effects were determined in rats after sub-chronic (7 days) and chronic (14 and 21 days) treatment using semi-quantitative in situ hybridisation. BDNF mRNA levels in the dentate gyrus (DG) were increased after treatment with venlafaxine (7, 14 and 21 days) and imipramine (14 and 21 days), but not after treatment with fluoxetine, indicating that stimulation of BDNF mRNA expression is dependent on the pharmacological profile and on the time-course of drug treatment. A transient increase in synaptophysin mRNA was observed after treatment with venlafaxine and fluoxetine whereas imipramine had no effect. In the CA3 region a reduction of GAP-43 mRNA was observed after treatment with imipramine (21 days) and fluoxetine (7 and 14 days). These results suggest that venlafaxine and imipramine, but not fluoxetine, induce neuroplastic effects in the hippocampus through stimulation of BDNF mRNA expression, and that the effect on BDNF is not directly translated into regulation of synaptophysin and GAP-43 mRNA.

  2. Alterations in Somatostatin mRNA Expression in the Dorsolateral Prefrontal Cortex of Subjects with Schizophrenia or Schizoaffective Disorder

    PubMed Central

    Morris, Harvey M.; Hashimoto, Takanori; Lewis, David A.

    2010-01-01

    Alterations in the inhibitory circuitry of the dorsolateral prefrontal cortex (DLPFC) in schizophrenia include reduced expression of the messenger RNA (mRNA) for somatostatin (SST), a neuropeptide present in a subpopulation of γ-aminobutyric acid (GABA) neurons. However, neither the cellular substrate nor the causal mechanisms for decreased SST mRNA levels in schizophrenia are known. We used in situ hybridization to quantify the compartmental, laminar, and cellular levels of SST mRNA expression in the DLPFC of 23 pairs of schizophrenia or schizoaffective disorder and control subjects. We also explored potential causal mechanisms by utilizing similar methods to analyze SST mRNA expression in 2 animal models. The expression of SST mRNA was significantly decreased in layers 2–superficial 6 of subjects with schizophrenia, but not in layer 1, deep 6 or the white matter. At the cellular level, both the density of cortical SST mRNA-positive neurons and the expression of SST mRNA per neuron were reduced in the subjects with schizophrenia. These alterations were not due to potential confounds and appeared to be a downstream consequence of impaired neurotrophin signaling through the trkB receptor. These findings support the hypothesis that a marked reduction in SST mRNA expression in a subset of GABA neurons contributes to DLPFC dysfunction in schizophrenia. PMID:18203698

  3. Identification of prostate cancer mRNA markers by averaged differential expression and their detection in biopsies, blood, and urine

    PubMed Central

    Bai, V. Uma; Kaseb, Ahmed; Tejwani, Sheela; Divine, George W.; Barrack, Evelyn R.; Menon, Mani; Pardee, Arthur B.; Reddy, G. Prem-Veer

    2007-01-01

    The advent of serum prostate-specific antigen (PSA) as a biomarker has enabled early detection of prostate cancer and, hence, improved clinical outcome. However, a low PSA is not a guarantee of disease-free status, and an elevated PSA is frequently associated with a negative biopsy. Therefore, our goal is to identify molecular markers that can detect prostate cancer with greater specificity in body fluids such as urine or blood. We used the RT-PCR differential display method to first identify mRNA transcripts differentially expressed in tumor vs. patient-matched nontumor prostate tissue. This analysis led to the identification of 44 mRNA transcripts that were expressed differentially in some but not all tumor specimens examined. To identify mRNA transcripts that are differentially expressed in most tumor specimens, we turned to differential display of pooled tissue samples, a technique we name averaged differential expression (ADE). We performed differential display of mRNA from patient-matched nontumor vs. tumor tissue, each pooled from 10 patients with various Gleason scores. Differentially expressed mRNA transcripts identified by ADE were fewer in number, but were expressed in a greater percentage of tumors (>75%) than those identified by differential display of mRNA from individual patient samples. Differential expression of these mRNA transcripts was also detected by RT-PCR in mRNA isolated from urine and blood samples of prostate cancer patients. Our findings demonstrate the principle that specific cDNA probes of frequently differentially expressed mRNA transcripts identified by ADE can be used for the detection of prostate cancer in urine and blood samples. PMID:17283334

  4. 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.

  5. 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

  6. Hyaluronan tetrasaccharides stimulate ceramide production through upregulated mRNA expression of ceramide synthesis-associated enzymes.

    PubMed

    Kage, Madoka; Tokudome, Yoshihiro

    2016-03-01

    It has been reported that hyaluronan has different physiological functions as suggested by variation in molecular weight. In addition, it has also been reported that CD44, the major hyaluronan receptor, was demonstrated to induce keratinocyte differentiation and lipid synthesis of cholesterol. We focus attention on the hyaluronan tetrasaccharides (HA4) which is the smallest unit of hyaluronan. We previously reported that HA4 induced keratinocyte differentiation and that CD44 may be involved. For the purpose of clarifying the influence of HA4 on ceramide synthesis, we evaluated both of these factors in keratinocytes in vitro and in vivo. The mRNA expression of ceramide synthesis-associated enzymes and intracellular ceramide content were evaluated after HA4 treatment in normal human epidermal keratinocytes. In addition, the ceramide increasing effect of HA4 on skin in UVA-irradiated hairless mice was assessed by water content of stratum corneum (SC) and transepidermal water loss (TEWL) methods. The mRNA expression of ceramide synthesis-associated enzymes and intracellular ceramide content after HA4 treatment were increased compared with the control. Furthermore, HA4 treatment increased water content of SC and decreased TEWL. These findings suggest that HA4 affected ceramide synthesis and is involved in the improvement of UV-induced skin damage.

  7. miRNA and mRNA expression analysis reveals potential sex-biased miRNA expression

    PubMed Central

    Guo, Li; Zhang, Qiang; Ma, Xiao; Wang, Jun; Liang, Tingming

    2017-01-01

    Recent studies suggest that mRNAs may be differentially expressed between males and females. This study aimed to perform expression analysis of mRNA and its main regulatory molecule, microRNA (miRNA), to discuss the potential sex-specific expression patterns using abnormal expression profiles from The Cancer Genome Atlas database. Generally, deregulated miRNAs and mRNAs had consistent expression between males and females, but some miRNAs may be oppositely expressed in specific diseases: up-regulated in one group and down-regulated in another. Studies of miRNA gene families and clusters further confirmed that these sequence or location related miRNAs might have opposing expression between sexes. The specific miRNA might have greater expression divergence across different groups, suggesting flexible expression across different individuals, especially in tumor samples. The typical analysis regardless of the sex will ignore or balance these sex-specific deregulated miRNAs. Compared with flexible miRNAs, their targets of mRNAs showed relative stable expression between males and females. These relevant results provide new insights into miRNA-mRNA interaction and sex difference. PMID:28045090

  8. [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

  9. Differential expression and clinical significance of glioblastoma mRNA expression profiles in Uyghur and Han patients in Xinjiang province.

    PubMed

    Liu, Liang; Li, Wenting; Xia, Haicheng; Zhu, Zhengquan; Luan, Xinping

    2014-11-24

    The aim of this study was to investigate differences in glioblastoma RNA gene expression profiles between Uyghur and Han patients in Xinjiang province and to screen and compare differentially expressed genes with respect to their clinical significance in the pathogenesis of high-grade glioma and their relationship to disease prognosis. Illumina HT-12mRNA expression profiles microarray was employed to measure the gene expression profiles of 6 patients with advanced glioma and to screen for differentially expressed genes. GO and KEGG analyses were performed on the differentially expressed genes using Web Gestalt software (P<0.05). Comparison of glioblastoma RNA expression profiles in the Uyghur and Han patients indicated that 1475 genes were significantly differentially expressed, of which 669 showed increased expression, while 807 showed decreased expression. One gene (STRC) corresponded to 2 transcripts, 1 of which showed increased expression and the other showed decreased expression. The differentially expressed genes participate in metabolic processes, biological regulation, stress response, and multi-cellular organic processes, including small GTPase regulatory signaling pathways, Ras signaling pathway, neuronal reactive protein regulation, and myelination of the central nervous system. The genes are also involved in tumor-related signaling pathways, including metabolic pathways, cancer pathways, MAPK signaling pathway, TGF-β signaling pathway, neurotrophic factor signal transduction pathway, and mTOR signaling pathway. Differentially expressed genes were screened by studying the gene expression profiles in glioblastoma from Uyghur and Han patients. The cellular function and location of these genes were further investigated. Based on related molecular markers of glioblastoma, the differences in the mechanism of initiation and development of glioblastoma between Uyghur and Han patients were investigated for polygenic interactions.

  10. 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.

  11. Changes in superoxide dismutase mRNA expression by streptozotocin-induced diabetes.

    PubMed Central

    Kamata, K.; Kobayashi, T.

    1996-01-01

    1. Experiments were designed to investigate the involvement of superoxide anions in the attenuated endothelium-dependent relaxation of the rat aorta from streptozotocin (STZ)-induced diabetic rats. 2. The endothelium-dependent relaxation responses to acetylcholine (ACh, 10(-7) M) in helical strips of the aorta precontracted with noradrenaline (NA, 5 x 10(-3) approximately 3 x 10(-7) M) were significantly decreased in STZ-induced diabetic rats. The recovery phase of the relaxation after single administration of ACh in the STZ-induced diabetic rats was more rapid than those in control vessels. 3. Preincubation of aortic strips with superoxide dismutase (SOD, 60 u ml-1) normalized the recovery phase of the relaxation of diabetic aorta after single administration of ACh, whereas catalase (150 u ml-1) or indomethacin (10(-5) M) had no effects on the relaxation. 4. SOD (180 u ml-1) caused relaxation in NA precontracted aortic strips and the degree of the SOD-induced relaxation was significantly greater in diabetic aorta as compared with age-matched control vessels. 5. When the changes in mRNA expressions of Mn-SOD or Cu-Zn-SOD were observed, Mn-SOD mRNA expression was markedly decreased, and Cu-Zn-SOD was slightly decreased in diabetic aorta. 6. These results suggest that the rapid destruction of NO by superoxide anions may occur in the STZ-induced diabetic rats, and this may be due to a decrease in mRNA expression of Mn-SOD or Cu-Zn-SOD. Images Figure 4 PMID:8894182

  12. 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

  13. GABAergic mRNA expression is upregulated in the prefrontal cortex of rats sensitized to methamphetamine.

    PubMed

    Wearne, Travis A; Parker, Lindsay M; Franklin, Jane L; Goodchild, Ann K; Cornish, Jennifer L

    2016-01-15

    Inhibitory gamma-aminobutyric acid (GABA)-mediated neurotransmission plays an important role in the regulation of the prefrontal cortex (PFC), with increasing evidence suggesting that dysfunctional GABAergic processing of the PFC may underlie certain deficits reported across psychotic disorders. Methamphetamine (METH) is a psychostimulant that induces chronic psychosis in a subset of users, with repeat administration producing a progressively increased vulnerability to psychotic relapse following subsequent drug administration (sensitization). The aim here was to investigate changes to GABAergic mRNA expression in the PFC of rats sensitized to METH using quantitative polymerase chain reaction (qPCR). Male Sprague-Dawley rats (n=12) underwent repeated methamphetamine (intraperitoneal (i.p.) or saline injections for 7 days. Following 14 days of withdrawal, rats were challenged with acute methamphetamine (1mg/kg i.p.) and RNA was isolated from the PFC to compare the relative mRNA expression of a range of GABA enzymes, transporters and receptors subunits. METH challenge resulted in a significant sensitized behavioral (locomotor) response in METH pre-treated animals compared with saline pre-treated controls. The mRNAs of transporters (GAT1 and GAT3), ionotropic GABAA receptor subunits (α3 and β1), together with the metabotropic GABAB1 receptor, were upregulated in the PFC of sensitized rats compared with saline controls. These findings indicate that GABAergic mRNA expression is significantly altered at the pre and postsynaptic level following sensitization to METH, with sensitization resulting in the transcriptional upregulation of several inhibitory genes. These changes likely have significant consequences on GABA-mediated neurotransmission in the PFC and may underlie certain symptoms conserved across psychotic disorders, such as executive dysfunction.

  14. 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.

  15. BCL6 mRNA Expression Level in Invasive Duct Carcinoma not otherwise Specified

    PubMed Central

    Badr, Eman; Masoud, Eman; Eldien, Marwa Serag

    2016-01-01

    Introduction B-Cell Lymphoma 6 (BCL6) has an oncogenic role in tumourigenesis of various malignancies. It represses genes involved in terminal differentiation and plays complementary role with Signal Transducer and Activator of Transcription 3 (STAT3) in triple-negative breast cancer cellular function. Aim To evaluate the expression of BCL6 in cancer breast and determine its correlation with the clinico-pathological features including the molecular subtype of breast carcinoma. Materials and Methods This prospective case control study was carried out on 150 patients, divided into 100 cases of invasive duct carcinoma not otherwise specified and 50 benign breast lesions including fibroadenoma and fibrocystic disease. Fresh tissues were excised, which were then subjected to RNA extraction. The BCL6 mRNA level was assessed using real-time reverse transcription Polymerase Chain Reaction (PCR). Results There was a significant higher levels of BCL6 mRNA in malignant cases compared to benign ones (p<0.001). The level of BCL6 mRNA was higher in cases showing advanced tumor stage (p<0.04), triple negative subtype and associated in situ component (p<0.001) compared to cases with an early stage, luminal or Her 2-neu positive subtypes and those lacking in situ component. Conclusion BCL6 is up-regulated in breast cancer and is associated with poor prognostic features such as advanced stage and triple negative molecular subtype. BCL6 inhibitors might be considered as targeted therapy for breast cancer. PMID:28208987

  16. Keratin14 mRNA expression in human pneumocytes during quiescence, repair and disease

    PubMed Central

    Confalonieri, Marco; Buratti, Emanuele; Grassi, Gabriele; Bussani, Rossana; Chilosi, Marco; Farra, Rossella; Abrami, Michela; Stuani, Cristiana; Salton, Francesco; Ficial, Miriam; Confalonieri, Paola; Zandonà, Lorenzo; Romano, Maurizio

    2017-01-01

    The lung alveoli slowly self-renew pneumocytes, but their facultative regeneration capacity is rapidly efficient after an injury, so fibrosis infrequently occurs. We recently observed Keratin 14 (KRT14) expression during diffuse alveolar damage (DAD), but not in controls. We wonder if KRT14 may be a marker of pneumocyte transition from quiescence to regeneration. Quantitative PCR and Western blot analyses highlighted the presence of KRT14 (mRNA and protein) only in human lung samples with DAD or interstitial lung disease (ILD). In the exponentially growing cell lines A549 and H441, the mRNA and protein levels of KRT14 peaked at day one after cell seeding and decreased at day two, opposite to what observed for the proliferation marker E2F1. The inverse relation of KRT14 versus E2F1 expression holds true also for other proliferative markers, such as cyclin E1 and cyclin D1. Of interest, we also found that E2F1 silencing caused cell cycle arrest and increased KRT14 expression, whilst E2F1 stimulation induced cell cycle progression and decreased KRT14. KRT14 also increased in proliferative pneumocytes (HPAEpiC) just before transdifferentiation. Overall, our results suggest that KRT14 is a viable biomarker of pneumocyte activation, and repair/regeneration. The involvement of KRT14 in regenerative process may suggest a novel pharmaceutical target to accelerate lung repair. PMID:28199407

  17. 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.

  18. Embedding mRNA stability in correlation analysis of time-series gene expression data.

    PubMed

    Farina, Lorenzo; De Santis, Alberto; Salvucci, Samanta; Morelli, Giorgio; Ruberti, Ida

    2008-08-01

    Current methods for the identification of putatively co-regulated genes directly from gene expression time profiles are based on the similarity of the time profile. Such association metrics, despite their central role in gene network inference and machine learning, have largely ignored the impact of dynamics or variation in mRNA stability. Here we introduce a simple, but powerful, new similarity metric called lead-lag R(2) that successfully accounts for the properties of gene dynamics, including varying mRNA degradation and delays. Using yeast cell-cycle time-series gene expression data, we demonstrate that the predictive power of lead-lag R(2) for the identification of co-regulated genes is significantly higher than that of standard similarity measures, thus allowing the selection of a large number of entirely new putatively co-regulated genes. Furthermore, the lead-lag metric can also be used to uncover the relationship between gene expression time-series and the dynamics of formation of multiple protein complexes. Remarkably, we found a high lead-lag R(2) value among genes coding for a transient complex.

  19. Phospholipase C-{delta}{sub 1} regulates interleukin-1{beta} and tumor necrosis factor-{alpha} mRNA expression

    SciTech Connect

    Chung, Eric; Jakinovich, Paul; Bae, Aekyung; Rebecchi, Mario

    2012-10-01

    Phospholipase C-{delta}{sub 1} (PLC{delta}{sub 1}) is a widely expressed highly active PLC isoform, modulated by Ca{sup 2+} that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLC{delta}{sub 1} modulated expression of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLC{delta}{sub 1} was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLC{delta}{sub 1} knockdown enhanced expression IL-1{beta} and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLC{delta}{sub 1} knock down caused persistently high Nf{kappa}b levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLC{delta}{sub 1} knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1{beta} and TNF-{alpha} mRNA's induced by PLC{delta}{sub 1} knockdown. Our results show that loss of PLC{delta}{sub 1} enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nf{kappa}b pathway. Our findings are consistent with the idea that PLC{delta}{sub 1} is a

  20. 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

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

    PubMed Central

    Donaldson, Michael E; Saville, Barry J

    2013-01-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense–antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis. PMID:23650872

  2. 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.

  3. Stability of facial affective expressions in schizophrenia.

    PubMed

    Fatouros-Bergman, H; Spang, J; Merten, J; Preisler, G; Werbart, A

    2012-01-01

    Thirty-two videorecorded interviews were conducted by two interviewers with eight patients diagnosed with schizophrenia. Each patient was interviewed four times: three weekly interviews by the first interviewer and one additional interview by the second interviewer. 64 selected sequences where the patients were speaking about psychotic experiences were scored for facial affective behaviour with Emotion Facial Action Coding System (EMFACS). In accordance with previous research, the results show that patients diagnosed with schizophrenia express negative facial affectivity. Facial affective behaviour seems not to be dependent on temporality, since within-subjects ANOVA revealed no substantial changes in the amount of affects displayed across the weekly interview occasions. Whereas previous findings found contempt to be the most frequent affect in patients, in the present material disgust was as common, but depended on the interviewer. The results suggest that facial affectivity in these patients is primarily dominated by the negative emotions of disgust and, to a lesser extent, contempt and implies that this seems to be a fairly stable feature.

  4. Regulation and Function of FTO mRNA Expression in Human Skeletal Muscle and Subcutaneous Adipose Tissue

    PubMed Central

    Grunnet, Louise G.; Nilsson, Emma; Ling, Charlotte; Hansen, Torben; Pedersen, Oluf; Groop, Leif; Vaag, Allan; Poulsen, Pernille

    2009-01-01

    OBJECTIVE Common variants in FTO (the fat mass– and obesity-associated gene) associate with obesity and type 2 diabetes. The regulation and biological function of FTO mRNA expression in target tissue is unknown. We investigated the genetic and nongenetic regulation of FTO mRNA in skeletal muscle and adipose tissue and their influence on in vivo glucose and fat metabolism. RESEARCH DESIGN AND METHODS The FTO rs9939609 polymorphism was genotyped in two twin cohorts: 1) 298 elderly twins aged 62–83 years with glucose tolerance ranging from normal to type 2 diabetes and 2) 196 young (25–32 years) and elderly (58–66 years) nondiabetic twins examined by a hyperinsulinemic-euglycemic clamp including indirect calorimetry. FTO mRNA expression was determined in subcutaneous adipose tissue (n = 226) and skeletal muscle biopsies (n = 158). RESULTS Heritability of FTO expression in both tissues was low, and FTO expression was not influenced by FTO rs9939609 genotype. FTO mRNA expression in skeletal muscle was regulated by age and sex, whereas age and BMI were predictors of adipose tissue FTO mRNA expression. FTO mRNA expression in adipose tissue was associated with an atherogenic lipid profile. In skeletal muscle, FTO mRNA expression was negatively associated to fat and positively to glucose oxidation rates as well as positively correlated with expression of genes involved in oxidative phosphorylation including PGC1α. CONCLUSIONS The heritability of FTO expression in adipose tissue and skeletal muscle is low and not influenced by obesity-associated FTO genotype. The age-dependent decline in FTO expression is associated with peripheral defects of glucose and fat metabolism. PMID:19587359

  5. Regulation and function of FTO mRNA expression in human skeletal muscle and subcutaneous adipose tissue.

    PubMed

    Grunnet, Louise G; Nilsson, Emma; Ling, Charlotte; Hansen, Torben; Pedersen, Oluf; Groop, Leif; Vaag, Allan; Poulsen, Pernille

    2009-10-01

    Common variants in FTO (the fat mass- and obesity-associated gene) associate with obesity and type 2 diabetes. The regulation and biological function of FTO mRNA expression in target tissue is unknown. We investigated the genetic and nongenetic regulation of FTO mRNA in skeletal muscle and adipose tissue and their influence on in vivo glucose and fat metabolism. The FTO rs9939609 polymorphism was genotyped in two twin cohorts: 1) 298 elderly twins aged 62-83 years with glucose tolerance ranging from normal to type 2 diabetes and 2) 196 young (25-32 years) and elderly (58-66 years) nondiabetic twins examined by a hyperinsulinemic-euglycemic clamp including indirect calorimetry. FTO mRNA expression was determined in subcutaneous adipose tissue (n = 226) and skeletal muscle biopsies (n = 158). Heritability of FTO expression in both tissues was low, and FTO expression was not influenced by FTO rs9939609 genotype. FTO mRNA expression in skeletal muscle was regulated by age and sex, whereas age and BMI were predictors of adipose tissue FTO mRNA expression. FTO mRNA expression in adipose tissue was associated with an atherogenic lipid profile. In skeletal muscle, FTO mRNA expression was negatively associated to fat and positively to glucose oxidation rates as well as positively correlated with expression of genes involved in oxidative phosphorylation including PGC1alpha. The heritability of FTO expression in adipose tissue and skeletal muscle is low and not influenced by obesity-associated FTO genotype. The age-dependent decline in FTO expression is associated with peripheral defects of glucose and fat metabolism.

  6. Leptin in Whales: Validation and Measurement of mRNA Expression by Absolute Quantitative Real-Time PCR

    PubMed Central

    Ball, Hope C.; Holmes, Robert K.; Londraville, Richard L.; Thewissen, Johannes G. M.; Duff, Robert Joel

    2013-01-01

    Leptin is the primary hormone in mammals that regulates adipose stores. Arctic adapted cetaceans maintain enormous adipose depots, suggesting possible modifications of leptin or receptor function. Determining expression of these genes is the first step to understanding the extreme physiology of these animals, and the uniqueness of these animals presents special challenges in estimating and comparing expression levels of mRNA transcripts. Here, we compare expression of two model genes, leptin and leptin-receptor gene-related product (OB-RGRP), using two quantitative real-time PCR (qPCR) methods: “relative” and “absolute”. To assess the expression of leptin and OB-RGRP in cetacean tissues, we first examined how relative expression of those genes might differ when normalized to four common endogenous control genes. We performed relative expression qPCR assays measuring the amplification of these two model target genes relative to amplification of 18S ribosomal RNA (18S), ubiquitously expressed transcript (Uxt), ribosomal protein 9 (Rs9) and ribosomal protein 15 (Rs15) endogenous controls. Results demonstrated significant differences in the expression of both genes when different control genes were employed; emphasizing a limitation of relative qPCR assays, especially in studies where differences in physiology and/or a lack of knowledge regarding levels and patterns of expression of common control genes may possibly affect data interpretation. To validate the absolute quantitative qPCR methods, we evaluated the effects of plasmid structure, the purity of the plasmid standard preparation and the influence of type of qPCR “background” material on qPCR amplification efficiencies and copy number determination of both model genes, in multiple tissues from one male bowhead whale. Results indicate that linear plasmids are more reliable than circular plasmid standards, no significant differences in copy number estimation based upon background material used, and

  7. Increased interleukin-1beta mRNA expression in skin biopsies of horses with Culicoides hypersensitivity following challenge with Culicoides nubeculosus extract.

    PubMed

    Kolm, Gabriela; Knapp, Elzbieta; Wagner, Regina; Klein, Dieter

    2006-09-15

    Interleukin-1beta (IL-1beta) is a primary cytokine of the skin that has a pivotal role in keratinocyte differentiation, epidermal wound healing and host defense. Pathological increase of cutaneous IL-1beta is associated with edema formation, epidermal hyperproliferation and atopic dermatitis in humans. However, in horses the role of cutaneous IL-1beta in edema formation and allergic skin disease has not been characterised so far. Particularly in Culicoides hypersensitivity (CHS), intradermal injection of Culicoides extract may be associated with enhanced transcription of local IL-1beta. To examine the mRNA expression of IL-1beta and its receptor antagonist IL-1RA in the skin of horses, biopsy specimens of horses affected and non-affected by CHS prior and following intradermal challenge with a commercial C. nubeculosus extract were examined. Our hypothesis was that cutaneous IL-1beta mRNA was significantly upregulated in horses with CHS in response to Culicoides allergen. Biopsies were taken from sites prior to and 4 h following intradermal challenge with C. nubeculosus extract. In order to obtain reliable data, real time PCR was performed and genes of interest were normalized using three different housekeeping genes, beta-actin, GAPDH, beta-2-microglobulin. No significant difference was detected in non-challenged cutaneous IL-1beta mRNA and IL-1RA mRNA levels between CHS affected and non-affected horses. Intradermal injection of C. nubeculosus extract resulted in local upregulation of IL-1beta mRNA both in horses with typical history, characteristic clinical signs for CHS and a positive intradermal skin test (IDT), and non-affected horses with a negative IDT. However, the difference in prior and post challenged site IL-1beta mRNA levels only reached statistical significance in the affected horses (p=0.01 versus 0.7). In contrast, IL-1RA mRNA levels did not demonstrate any modification following intradermal injection with C. nubeculosus in either group. In contrast

  8. Intronless mRNA transport elements may affect multiple steps of pre-mRNA processing.

    PubMed Central

    Huang, Y; Wimler, K M; Carmichael, G G

    1999-01-01

    We have reported recently that a small element within the mouse histone H2a-coding region permits efficient cytoplasmic accumulation of intronless beta-globin cDNA transcripts. This sequence lowers the levels of spliced products from intron-containing constructs and can functionally replace Rev and the Rev-responsive element (RRE) in the nuclear export of unspliced HIV-1-related mRNAs. In work reported here, we further investigate the molecular mechanisms by which this element might work. We demonstrate here through both in vivo and in vitro assays that, in addition to promoting mRNA nuclear export, this element acts as a polyadenylation enhancer and as a potent inhibitor of splicing. Surprisingly, two other described intronless mRNA transport elements (from the herpes simplex virus thymidine kinase gene and hepatitis B virus) appear to function in a similar manner. These findings prompt us to suggest that a general feature of intronless mRNA transport elements might be a collection of phenotypes, including the inhibition of splicing and the enhancement of both polyadenylation and mRNA export. PMID:10075934

  9. Time-course of 5-HT(6) receptor mRNA expression during memory consolidation and amnesia.

    PubMed

    Huerta-Rivas, A; Pérez-García, G; González-Espinosa, C; Meneses, A

    2010-01-01

    Growing evidence indicates that antagonists of the 5-hydroxytryptamine (serotonin) receptor(6) (5-HT(6)) improve memory and reverse amnesia although the mechanisms involved are poorly understood. Hence, in this paper RT-PCR was used to evaluate changes in mRNA expression of 5-HT(6) receptor in trained and untrained rats treated with the 5-HT(6) receptor antagonist SB-399885 and amnesic drugs scopolamine or dizocilpine. Changes in mRNA expression of 5-HT(6) receptor were investigated at different times in prefrontal cortex, hippocampus and striatum. Data indicated that memory in the Pavlovian/instrumental autoshaping task was a progressive process associated to reduced mRNA expression of 5-HT(6) receptor in the three structures examined. SB-399885 improved long-term memory at 48h, while the muscarinic receptor antagonist scopolamine or the non-competitive NMDA receptor antagonist dizocilpine impaired it at 24h. Autoshaping training and treatment with SB-399885 increased 5-HT(6) receptor mRNA expression in (maximum increase) prefrontal cortex and striatum, 24 or 48h. The scopolamine-induced amnesia suppressed 5-HT(6) receptor mRNA expression while the dizocilpine-induced amnesia did not modify 5-HT(6) receptor mRNA expression. SB-399885 and scopolamine or dizocilpine were able to reestablish memory and 5-HT(6) receptor mRNA expression. These data confirmed previous memory evidence and of more interest is the observation that training, SB-399885 and amnesic drugs modulated 5-HT(6) receptor mRNA expression in prefrontal cortex, hippocampus and striatum. Further investigation in different memory tasks, times and amnesia models together with more complex control groups might provide further clues.

  10. 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

  11. 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.

  12. 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

  13. Gonadotropin-releasing hormone mRNA and gonadotropin beta-subunit mRNA expression in the adult female rat exposed to ethanol in utero.

    PubMed

    Wilson, M E; Marshall, M T; Bollnow, M R; McGivern, R F; Handa, R J

    1995-10-01

    Previous studies have demonstrated that exposure of female rats to ethanol in utero results in long-term deficits in reproductive function, including a delayed onset of puberty and an early onset of acyclicity. In the present studies, we determined if changes in reproduction are correlated with changes in gonadotropin-releasing hormone (GnRH) mRNA expression in the brain or gonadotropin subunit mRNA expression in the anterior pituitary gland. We used in situ hybridization histochemical techniques to examine the density of GnRH mRNA and the distribution of GnRH mRNA-containing cells in the basal forebrain, and reverse transcription-polymerase chain reaction (RT-PCR) to quantitate the beta-subunit mRNA of luteinizing hormone (LH beta) and follicle-stimulating hormone (FSH beta) in the anterior pituitary gland of adult (3 months of age) fetal alcohol-exposed (FAE) female rats. For GnRH mRNA measurements, animals were gonadectomized 4 days before use. Three groups of animals were examined. FAE females were derived from pregnant dams fed a liquid diet containing 35% ethanol-derived calories from gestational day 14 until parturition. Dams of control animals were either pair-fed (PF) an isocaloric diet with sucrose substituted for ethanol or maintained on normal laboratory rat chow [chow-fed (CF)]. Serial blood samples taken by indwelling right atrial cannulae demonstrated significantly smaller pulses of LH (p < 0.05) and FSH (p < 0.05) in ovariectomized FAE females at 3 months of age, compared with PF and CF controls. Distribution of GnRH mRNA-containing cells was mapped throughout the forebrain, and the number of autoradiographic silver grains/cell was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Myometrial prostaglandin E2 synthetic enzyme mRNA expression: spatial and temporal variations with pregnancy and labour.

    PubMed

    Sooranna, S R; Grigsby, P L; Engineer, N; Liang, Z; Sun, K; Myatt, L; Johnson, M R

    2006-10-01

    We have investigated the hypothesis that the expression of the enzymes involved in PGE(2) synthesis in the human uterus is co-ordinated. We have studied (i) the mRNA expression of the enzymes involved in PGE(2) synthesis [phospholipases (cPLA(2) and sPLA(2)), prostaglandin H synthase (PGHS)-2 and PG E synthases (PGES-1 and -2)] and their relationship to the expression of inflammatory cytokines in samples of myometrium obtained from pregnant women undergoing caesarean section (LSCS) either before or after the onset of labour at or before term; and (ii) the effect of IL-1beta, IL-6, TNF-alpha, PGE(2) and stretch on PGE(2) enzyme mRNA expression. We found that cPLA(2), sPLA(2) and PGHS-2 mRNA expression were greater in labour samples; cPLA(2), sPLA(2), PGHS-2, PGES-1 and -2 mRNA expression were greater in lower- than upper-segment samples; and there was no effect of gestational age. PGHS-2 mRNA levels correlated with those of PGES-1, cPLA(2), IL-1beta and IL-8; PGES-1 mRNA levels correlated with those of IL-1beta, IL-8 and cPLA(2). In primary cultures of uterine myocytes, cPLA(2) mRNA expression was increased by IL-1beta and IL-6; PGHS-2 mRNA expression was increased by IL-1beta, PGE(2) and stretch; and PGES-1 mRNA expression was increased by IL-1beta only. These data show that labour is associated with increased expression of the enzymes involved in PGE(2) synthesis and their expression is greater in the lower uterine segment. The presence of associations between the levels of PGE(2) enzyme mRNA expression and the effects of IL-1beta suggest that their expression is co-ordinated and that IL-1beta is the responsible factor.

  15. RNA/DNA ratio and LPL and MyoD mRNA expressions in muscle of Oreochromis niloticus fed with elevated levels of palm oil

    NASA Astrophysics Data System (ADS)

    Ayisi, Christian Larbi; Zhao, Jinliang

    2016-02-01

    Palm oil is of great potential as one of the sustainable alternatives to fish oil (FO) in aquafeeds. In this present study, five isonitrogenous diets (32% crude protein) with elevated palm oil levels of 0%, 2%, 4%, 6% and 8% were used during an 8-week feeding trial to evaluate its effects on RNA/DNA ratio and lipoprotein lipase (LPL) and MyoD mRNA expressions in muscle of Oreochromis niloticus. The results showed that RNA, DNA content as well as ratio of RNA to DNA were significantly affected ( P < 0.05), in each case the highest was recorded in fish group subjected to 6% palm oil level. There was a strong positive correlation between nucleic acid concentration (RNA concentration and RNA: DNA ratio) and specific growth rate (SGR), protein efficiency ratio (PER), while a negative correlation existed between nucleic acid concentration (RNA concentration and RNA: DNA ratio) and feed conversion ratio (FCR). The mRNA expressions of LPL and MyoD in muscle were not significantly affected by the different palm oil levels, although the highest expression was observed in fish fed with 6% palm oil level. There also existed a strong positive correlation between the mRNA expression of LPL, MyoD and SGR, PER, while their correlation with FCR was negative. In conclusion, elevated palm oil affected the RNA, DNA concentration as well as RNA/DNA ratio significantly, although the mRNA expression of LPL and MyoD were not affected significantly by elevated palm oil levels.

  16. Isoenzyme-specific up-regulation of glutathione transferase and aldo-keto reductase mRNA expression by dietary quercetin in rat liver.

    PubMed

    Odbayar, Tseye-Oidov; Kimura, Toshinori; Tsushida, Tojiro; Ide, Takashi

    2009-05-01

    The impact of quercetin on the mRNA expression of hepatic enzymes involved in drug metabolism was evaluated with a DNA microarray and real-time PCR. Male Sprague-Dawley rats were fed an experimental diet containing either 0, 2.5, 5, 10, or 20 g/kg of quercetin for 15 days. The DNA microarray analysis of the gene expression profile in pooled RNA samples from rats fed diets containing 0, 5, and 20 g/kg of quercetin revealed genes of some isoenzymes of glutathione transferase (Gst) and aldo-keto reductase (Akr) to be activated by this flavonoid. Real-time PCR conducted with RNA samples from individual rats fed varying amounts of quercetin together with the microarray analysis showed that quercetin caused marked dose-dependent increases in the mRNA expression of Gsta3, Gstp1, and Gstt3. Some moderate increases were also noted in the mRNA expression of isoenzymes belonging to the Gstm class. Quercetin also dose-dependently increased the mRNA expression of Akr1b8 and Akr7a3. However, it did not affect the parameters of the other Gst and Akr isoenzymes. It is apparent that quercetin increases the mRNA expression of Gst and Akr involved in drug metabolism in an isoenzyme-specific manner. Inasmuch as Gst and Akr isoenzymes up-regulated in their gene expression are involved in the prevention and attenuation of cancer development, this consequence may account for the chemopreventive propensity of quercetin.

  17. Correlation of Apobec Mrna Expression with overall Survival and pd-l1 Expression in Urothelial Carcinoma

    PubMed Central

    Mullane, Stephanie A.; Werner, Lillian; Rosenberg, Jonathan; Signoretti, Sabina; Callea, Marcella; Choueiri, Toni K.; Freeman, Gordon J.; Bellmunt, Joaquim

    2016-01-01

    Metastatic urothelial carcinoma (mUC) has a very high mutational rate and is associated with an APOBEC mutation signature. We examined the correlation of APOBEC expression with overall survival (OS) and PD-L1 expression in a cohort of 73 mUC patients. mRNA expression of APOBEC3 family of genes (A3A, A3B, A3C, A3F_a, A3F_b, A3G, A3H) was measured using Nanostring. PD-L1 expression, evaluated by immunohistochemistry, on tumor infiltrating mononuclear cells (TIMCs) and tumor cells was scored from 0 to 4, with 2–4 being positive. Wilcoxon’s non-parametric tests assessed the association of APOBEC and PD-L1. The Cox regression model assessed the association of APOBEC with OS. All APOBEC genes were expressed in mUC. Increased A3A, A3D, and A3H expression associates with PD-L1 positive TIMCs (p = 0.0009, 0.009, 0.06). Decreased A3B expression was marginally associated with PD-L1 positive TIMCs expression (p = 0.05). Increased A3F_a and A3F_b expression was associated with increased expression of PD-L1 on tumor cells (p = 0.05). Increased expression of A3D and A3H was associated with longer OS (p = 0.0009). Specific APOBEC genes have different effects on mUC in terms of survival and PD-L1 expression. A3D and A3H may have the most important role in mUC as they are associated with OS and PD-L1 TIMC expression. PMID:27283319

  18. 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

  19. [Expression of the P450arom mRNA in the glandular tissue of the pubertal mammary hypertrophy].

    PubMed

    Lu, Jian-Jian; Teng, Li; Hu, Guo-Dong; Jin, Xiao-Lei; Zhang, Fei-Nan; Xu, Jia-Jie; Yang, Kai; Fan, Xin-Yu; Zhang, Bo

    2009-09-01

    To investigate the expression status of the P450arom mRNA in breast tissue of pubertal mammary hypertrophy and then explore the possible etiology of pubertal mammary hypertrophy. 15 patients were selected for pubertal mammary hypertrophy group. Breast hypertrophy tissue specimens were collected from the gland excised during reduction mammaplasty. 15 patients with pathologically simple fibroadenoma were used as another control group. Patient approval of participation in this study was obtained preoperatively. The expression of P450arom mRNA was detected by RT-PCR in all the cases above. There was no significant difference between the pubertal mammary hypertrophy groups and normal groups on the expression rates of P450arom mRNA. But among the positive cases, the expression of P450arom mRNA within breast tissue were 0.202 +/- 0.048 in pubertal mammary hypertrophy group; and 0.159 +/- 0.068 in normal group. There was significant difference between the pubertal mammary hypertrophy and normal groups (P < 0.05). The expression of P450arom mRNA in pubertal mammary hypertrophy are significantly higher than in normal mammary glandular tissue. The pubertal mammary hypertrophy may be related to the expression status of P450arom mRNA within breast tissue.

  20. 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.

  1. Effect of doxycycline and Lactobacillus probiotics on mRNA expression of ABCC2 in small intestines of chickens

    PubMed Central

    Milanova, A.; Pavlova, I.; Yordanova, V.; Danova, S.

    2016-01-01

    Probiotics and antibiotics are widely used in poultry and may alter drug bioavailability by affecting the expression of intestinal ATP-binding cassette (ABC) efflux transporters. Therefore the aim of the present investigation was to evaluate the effect of Lactobacilli probiotics, administered alone or in combination with doxycycline, on the expression of ABCB1 (gene, encoding P-glycoprotein), ABCC2 (gene, encoding multidrug resistance protein 2, MRP2) and ABCG2 (gene, encoding breast cancer resistance protein) mRNAs in chicken using RT-PCR. Duc one-day-old chicks (n=24) were divided equally in four groups: untreated control, probiotics supplemented group, probiotics plus doxycycline treated chickens and antibiotic administered group. Expression of ABCC2 mRNA was affected by doxycycline or by combination of Lactobacillus plantarum, L. brevis and L. bulgaricus and the antibiotic in the intestines. These results can be used as a basis for further functional studies to prove the beneficial effect on limitation of the absorption of toxins and improvement of efflux of endogenous substances and xenobiotics when the combination of doxycycline and Lactobacillus spp. probiotics are administered to poultry. PMID:28224011

  2. Expression of cytokine mRNA in lentivirus-induced arthritis.

    PubMed Central

    Lechner, F.; Vogt, H. R.; Seow, H. F.; Bertoni, G.; Cheevers, W. P.; von Bodungen, U.; Zurbriggen, A.; Peterhans, E.

    1997-01-01

    Infection of goats with the lentivirus caprine arthritis encephalitis virus (CAEV) leads to persistent infection and development of chronic arthritis. We analyzed the expression of cytokines and viral RNA in the joints of goats at early time points after experimental infection with CAEV and in those of animals suffering from chronic arthritis as a result of natural infection. In situ hybridization experiments showed that the pattern of cytokine expression in caprine arthritis was similar to that found in rheumatoid arthritis (RA), with a few cells expressing the lymphocyte-derived cytokines interferon (IFN)-gamma and interleukin (IL)-2 and rather more cells expressing monocyte chemoattractant protein (MCP)-1, IL-6, and tumor necrosis factor (TNF)-alpha. IFN-gamma mRNA expression in experimentally infected joints peaked at day 12 and was mostly detected in areas containing viral RNA. At later time points, no IFN-gamma- or virus-expressing cells were found in inflamed joints but both were again detected in goats with severe arthritis. Interestingly, at the clinical stage of arthritis reflecting the chronic stage of infection, the inflammatory lesion was found to be immunologically compartmentalized. Humoral immune responses and cell-mediated immune responses appeared to concurrently occur in distinct areas of the synovial membrane. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9327739

  3. Myoepithelial mRNA expression profiling reveals a common tumor-suppressor phenotype.

    PubMed

    Barsky, Sanford H

    2003-04-01

    A series of myoepithelial cell lines and xenografts derived from benign human myoepithelial tumors of diverse sources (salivary gland, breast, and lung) exhibit common mRNA expression profiles indicative of a tumor-suppressor phenotype. Previously established myoepithelial cell lines and xenografts (HMS-#; HMS-#X) were compared to nonmyoepithelial breast carcinoma cells (MDA-MB-231 and MDA-MB-468, and inflammatory breast carcinoma samples, IBCr, and IBCw), a normal mammary epithelial cell line (HMEC) and individual cases of human breast cancer (zcBT#T), and matched normal human breast tissues (zcBT#N) (overall samples = 22). The global gene expression profile (22,000 genes) of these individual samples was examined using Affymetrix Microarray Gene Chips and subsequently analyzed with both Affymetrix and DChip algorithms. The myoepithelial cell lines/xenografts were distinct and very different from the nonmyoepithelial breast carcinoma cells and the normal breast and breast tumor biopsies. Two hundred and seven specifically selected genes represented a subset of genes that distinguished (P < 0.05) all the myoepithelial cell lines/xenografts from all the other samples and which themselves exhibited hierarchical clustering. Further analysis of these genes revealed increased expression in genes belonging to the classes of extracellular matrix proteins, angiogenic inhibitors, and proteinase inhibitors and decreased expression belonging to the classes of angiogenic factors and proteinases. Developmental genes were also differentially expressed (either over or underexpressed). These studies confirm our previous impression that human myoepithelial cells express a distinct tumor-suppressor phenotype.

  4. 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.

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

    PubMed Central

    Dzyubak, Ekaterina

    2016-01-01

    Members of the Erm methyltransferase family modify 23S rRNA of the bacterial ribosome and render cross-resistance to macrolides and multiple distantly related antibiotics. Previous studies have shown that the expression of erm is activated when a macrolide-bound ribosome stalls the translation of the leader peptide preceding the cotranscribed erm. Ribosome stalling is thought to destabilize the inhibitory stem-loop mRNA structure and exposes the erm Shine-Dalgarno (SD) sequence for translational initiation. Paradoxically, mutations that abolish ribosome stalling are routinely found in hyper-resistant clinical isolates; however, the significance of the stalling-dead leader sequence is largely unknown. Here, we show that nonsense mutations in the Staphylococcus aureus ErmB leader peptide (ErmBL) lead to high basal and induced expression of downstream ErmB in the absence or presence of macrolide concomitantly with elevated ribosome methylation and resistance. The overexpression of ErmB is associated with the reduced turnover of the ermBL-ermB transcript, and the macrolide appears to mitigate mRNA cleavage at a site immediately downstream of the ermBL SD sequence. The stabilizing effect of antibiotics on mRNA is not limited to ermBL-ermB; cationic antibiotics representing a ribosome-stalling inducer and a noninducer increase the half-life of specific transcripts. These data unveil a new layer of ermB regulation and imply that ErmBL translation or ribosome stalling serves as a “tuner” to suppress aberrant production of ErmB because methylated ribosome may impose a fitness cost on the bacterium as a result of misregulated translation. PMID:27645242

  6. mRNA Expression Signature of Gleason Grade Predicts Lethal Prostate Cancer

    PubMed Central

    Penney, Kathryn L.; Sinnott, Jennifer A.; Fall, Katja; Pawitan, Yudi; Hoshida, Yujin; Kraft, Peter; Stark, Jennifer R.; Fiorentino, Michelangelo; Perner, Sven; Finn, Stephen; Calza, Stefano; Flavin, Richard; Freedman, Matthew L.; Setlur, Sunita; Sesso, Howard D.; Andersson, Swen-Olof; Martin, Neil; Kantoff, Philip W.; Johansson, Jan-Erik; Adami, Hans-Olov; Rubin, Mark A.; Loda, Massimo; Golub, Todd R.; Andrén, Ove; Stampfer, Meir J.; Mucci, Lorelei A.

    2011-01-01

    Purpose Prostate-specific antigen screening has led to enormous overtreatment of prostate cancer because of the inability to distinguish potentially lethal disease at diagnosis. We reasoned that by identifying an mRNA signature of Gleason grade, the best predictor of prognosis, we could improve prediction of lethal disease among men with moderate Gleason 7 tumors, the most common grade, and the most indeterminate in terms of prognosis. Patients and Methods Using the complementary DNA–mediated annealing, selection, extension, and ligation assay, we measured the mRNA expression of 6,100 genes in prostate tumor tissue in the Swedish Watchful Waiting cohort (n = 358) and Physicians' Health Study (PHS; n = 109). We developed an mRNA signature of Gleason grade comparing individuals with Gleason ≤ 6 to those with Gleason ≥ 8 tumors and applied the model among patients with Gleason 7 to discriminate lethal cases. Results We built a 157-gene signature using the Swedish data that predicted Gleason with low misclassification (area under the curve [AUC] = 0.91); when this signature was tested in the PHS, the discriminatory ability remained high (AUC = 0.94). In men with Gleason 7 tumors, who were excluded from the model building, the signature significantly improved the prediction of lethal disease beyond knowing whether the Gleason score was 4 + 3 or 3 + 4 (P = .006). Conclusion Our expression signature and the genes identified may improve our understanding of the de-differentiation process of prostate tumors. Additionally, the signature may have clinical applications among men with Gleason 7, by further estimating their risk of lethal prostate cancer and thereby guiding therapy decisions to improve outcomes and reduce overtreatment. PMID:21537050

  7. 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

  8. Bacteria and Toll-like receptor and cytokine mRNA expression profiles associated with canine arthritis.

    PubMed

    Riggio, Marcello P; Lappin, David F; Bennett, David

    2014-08-15

    The major forms of inflammatory canine arthritis are immune-mediated arthritis (IMA) and septic arthritis (SA), although some cases of cruciate disease (CD) are associated with significant levels of synovitis. In this study, the bacteria associated with canine arthritis were identified and mRNA expression levels of Toll-like receptors (TLRs) and pro-inflammatory cytokines determined. Of the 40 synovial fluid samples analysed, bacteria were isolated from 12 samples by culture (2 CD, 10 SA) and detected in 4 samples (3 CD, 1 SA) using culture-independent methods. Statistically significant increases in TLR2, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-12 mRNA expression were seen in all disease groups compared to normal controls. All disease groups had decreased mRNA expression of other TLRs compared to normal controls, but this did not reach statistical significance. Synovial fluid cell counts revealed that the highest number and proportion of mononuclear cells and neutrophils were found in the IMA and SA samples, respectively. Age had an effect on the TLR and cytokine mRNA expression profiles: TNF-α (p=0.043) and IL-12 (p=0.025) mRNA expression was increased and TLR4 mRNA expression was reduced (p=0.033) in dogs up to 4 years of age compared to older animals. In the 10 SA samples from which bacteria were isolated, statistically significant increases in TLR2, TLR7, TNF-α and IL-6 mRNA expression were observed. It is concluded that canine arthritis is associated with increased mRNA levels of pro-inflammatory cytokines, which could in some cases be mediated by bacteria through activation of TLR2.

  9. Streamlining gene expression analysis: integration of co-culture and mRNA purification.

    PubMed

    Berry, Scott M; Singh, Chandresh; Lang, Jessica D; Strotman, Lindsay N; Alarid, Elaine T; Beebe, David J

    2014-02-01

    Co-culture of multiple cell types within a single device enables the study of paracrine signaling events. However, extracting gene expression endpoints from co-culture experiments is laborious, due in part to pre-PCR processing of the sample (i.e., post-culture cell sorting and nucleic acid purification). Also, a significant loss of nucleic acid may occur during these steps, especially with microfluidic cell culture where lysate volumes are small and difficult to access. Here, we describe an integrated platform for performing microfluidic cell culture and extraction of mRNA for gene expression analysis. This platform was able to recover 30-fold more mRNA than a similar, non-integrated system. Additionally, using a breast cancer/bone marrow stroma co-culture, we recapitulated stromal-dependent, estrogen-independent growth of the breast cancer cells, coincident with transcriptional changes. We anticipate that this platform will be used for streamlined analysis of paracrine signaling events as well as for screening potential drugs and/or patient samples.

  10. Evolution of Steroid-Inducible RP2 mRNA Expression in the Mouse Kidney

    PubMed Central

    Tseng-Crank, Julie; Berger, Franklin G.

    1987-01-01

    We have examined the structure and expression of mRNAs encoded by the androgen-inducible RP2 gene in the kidneys of nine mouse species within the genus Mus. There is considerable interspecies variation in the lengths of the major RP2 transcripts; some of this variation is due to the presence or absence of a B1 repetitive element in the 3'-untranslated region of the gene. In addition, the extent of RP2 mRNA induction by testosterone differs among the species. Two species show 10-20-fold induction, while others display a reduced response or none at all. Analysis of an interspecific hybrid indicates that the inducibility phenotype is inherited in an additive fashion. A correlation between RP2 inducibility and the time of formation of lineages within the Mus genus suggests that induction evolved in a stepwise fashion, with the acquisition of a modest hormonal response being followed by the appearance of a greater response. The interspecies variations in RP2 mRNA structure and regulation provide a useful model for the identification and study of genetic elements that elicit evolutionary alterations in steroid-modulated gene expression. PMID:3623081

  11. 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

  12. The vitamin D receptor localization and mRNA expression in ram testis and epididymis.

    PubMed

    Jin, Hui; Huang, Yang; Jin, Guang; Xue, Yanrong; Qin, Xiaowei; Yao, Xiaolei; Yue, Wenbing

    2015-02-01

    The objectives of present study were to investigate the presence of vitamin D receptor (VDR) in testis and epididymis of ram by polymerase chain reaction (PCR), to locate VDR in testis and epididymis by immunohistochemistry and to compare difference of VDR expression between testis and epididymis before and after sexual maturation by Real time-PCR and Western blot. The results showed that VDR exists in the testis and epididymis of ram while VDR protein in testis and epididymis was localized in Leydig cells, spermatogonial stem cells, spermatocytes, Sertoli cells and principal cells. For the adult ram, the amounts of VDR mRNA and VDR protein were less (p < 0.01) in testis than compared with caput, corpus and cauda epididymis. For prepubertal ram, the result showed the same trend (p < 0.01). However, the expression levels of VDR mRNA and VDR protein in caput, corpus, cauda epididymis and testis showed no significant difference (p > 0.05) between adult and prepubertal. In conclusion, VDR exists in testis and epididymis of ram, suggesting 1α,25-(OH)(2)VD(3) may play a role in ram reproduction.

  13. Molecular cloning and mRNA expression of duck invariant chain.

    PubMed

    Zhong, Dalian; Yu, Weiyi; Bao, Min; Xu, Zhiben; Li, Lin; Liu, Jing

    2006-04-15

    In the present study we identified a duck invariant chain (Ii) cDNA, named duck Ii-1, by RT-PCR and RACE. It was 1190 bp in length and contained a 669 bp open reading frame. An alternative transcript encoding a thyroglobulin (Tg)-containing form of Ii, named duck Ii-2, was also found in duck. The putative amino acid sequence of duck Ii-1 showed an 82% similarity to chicken Ii-1 and about 60% similarity to its mammalian homologues. The similarity of the Tg domain between duck and chicken Ii-2 was 96%, and about 70% between duck and mammalian Ii. The result of RT-PCR showed that Ii mRNA was extensively expressed in various tissues. High levels of both Ii-1 and Ii-2 mRNA were observed in the spleen and bursa of Fabricius. The predicted three-dimensional (3D) structures of duck Ii trimerization and Tg domain are similar to the corresponding regions of human Ii analyzed by comparative protein modeling. These findings indicate that the two isoforms of duck Ii, which strongly expressed in the major immune organs, share structural identity with human Ii.

  14. Effect of radiofrequency radiation on MRNA expression in cultured rodent cells

    SciTech Connect

    Parker, J.E.; Kiel, J.L.; Winters, W.D.

    1988-01-01

    Radiofrequency radiation (RFR) has been reported to induce adverse effects in biological systems, such as teratogenic and embryo lethal effects in mammals particularly during exposures producing significant hyperthermia. Other studies have implicated microwave exposure with causing changes in chromosome number and structure, formation of cataracts in humans rabbits and dogs; and promoting malignant tumor formation in rats, as well as increasing tumor production and leukemias. In addition, microwave exposures have been reported to change the structure of purified double-stranded plasmid DNA, causing it to become nicked and increasing the proportion of relaxed to super coiled molecules. In view of these reports of changes at different levels of cellular function and structure of mammalian systems to microwaves, the authors asked themselves if changes at the level of mRNA expression could be detected after microwave exposure of cultured rodent cells. They chose to look at the mRNA expression of certain oncogenes known to show elevated levels during cell replication, at the heat shock proteins known to respond to stresses other than heat, and at the long terminal repeat (LTR) region of mouse mammary tumor virus in four rodent cell lines.

  15. Cytokine mRNA expression in normal skin of various age populations before and after engraftment onto nude mice.

    PubMed

    Gilhar, A; Ullmann, Y; Shalagino, R; Weisinger, G

    1998-01-01

    Whether the impact of skin biological age on cytokine expression is a result of this tissue's proliferation potential or not is an important issue in dermatology. We investigated these questions by monitoring cytokine marker mRNA expression from human skin samples from healthy groups of individuals. The skin samples studied represented three age groups: fetal (17-21 weeks), young (18-35 years) and aged (76-88 years). Furthermore, upon skin transplantation of tissue from different age groups onto nude mice, we investigated whether cytokine marker RNA levels would change or normalize. Interestingly, both TNF-alpha and P53 mRNA showed a similar pattern of expression. Both were significantly higher in fetal skin (p < 0.0001 and p < 0.05, respectively), and no difference was noted between aged versus young skin. In contrast to this, IL1-alpha mRNA was expressed at its lowest and highest levels in fetal and young skin, respectively. Following skin transplantation, cytokines and P53 mRNA expression were normalized to similar levels in all age groups. This study implies that when cytokine expression was determined directly at the mRNA level, post-natal expression was not significantly different at either age group. Furthermore, it seems that the environmental conditions surrounding the grafted human skin found on nude mice encouraged normalization of donor cytokine expression.

  16. Circulating irisin levels and muscle FNDC5 mRNA expression are independent of IL-15 levels in mice.

    PubMed

    Quinn, LeBris S; Anderson, Barbara G; Conner, Jennifer D; Wolden-Hanson, Tami

    2015-11-01

    Interleukin-15 (IL-15) and irisin are exercise-induced myokines that exert favorable effects on energy expenditure and metabolism. IL-15 can induce PGC-1α expression, which in turn induces expression of irisin and its precursor, FNDC5. Therefore, the present study tested the hypothesis that increases in circulating irisin levels and muscle FNDC5 mRNA expression are dependent on IL-15. Circulating irisin levels and gastrocnemius muscle FNDC5 mRNA expression were examined following acute exercise in control and IL-15-deleted (IL-15 KO) mice, following injection of IL-15 into IL-15 KO mice, and in transgenic mice with elevated circulating IL-15 levels (IL-15 Tg mice). Circulating IL-15 levels and muscle PGC-1α and PPARδ mRNA expressions were determined as positive controls. No effect of IL-15 deletion on post-exercise serum irisin levels or muscle FNDC5 mRNA expression was detected. While serum IL-15 levels and muscle PGC-1α expression were elevated post-exercise in control mice, both serum irisin levels and muscle FNDC5 expression decreased shortly after exercise in both control and IL-15 KO mice. A single injection of recombinant IL-15 into IL-15 KO mice that significantly increased muscle PPARδ and PGC-1α mRNA expressions had no effect on circulating irisin release, but modestly induced muscle FNDC5 expression. Additionally, serum irisin and gastrocnemius muscle FNDC5 expression in IL-15 Tg mice were similar to those of control mice. Muscle FNDC5 mRNA expression and irisin release are not IL-15-dependent in mice.

  17. 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

  18. [The extract of compound Radix Scutellariae on mRNA replication and IFN expression of influenza virus in mice].

    PubMed

    Chu, Ming; Chu, Zheng-yun; Wang, Dan-dan

    2007-01-01

    To study the extract of compound Radix Scutellariae on inhibiting the mRNA replication and inducing the INF expression of influenza virus in mice's vivo. Influenza virus infected mice pneumono-adaption stock A/FM/1/47 (H1N1) evoked mice pneumonia taken as animal model. RT-PCR was adopted to measure the content of influenza virus's mRNA and INF in mice to study the effect of the extract on mRNA replication and IFN expression. The extract of compound Radix Scutellariae could inhibit influenza virus' s mRNA replication (P < 0.01) and induce interferon expression in mice (P > 0.05). The extract of compound Radix Scutellariae can induce interferon and inhibit influenza virus replication in mice.

  19. Impact of nonsense-mediated mRNA decay on the global expression profile of budding yeast.

    PubMed

    Guan, Qiaoning; Zheng, Wei; Tang, Shijie; Liu, Xiaosong; Zinkel, Robert A; Tsui, Kam-Wah; Yandell, Brian S; Culbertson, Michael R

    2006-11-24

    Nonsense-mediated mRNA decay (NMD) is a eukaryotic mechanism of RNA surveillance that selectively eliminates aberrant transcripts coding for potentially deleterious proteins. NMD also functions in the normal repertoire of gene expression. In Saccharomyces cerevisiae, hundreds of endogenous RNA Polymerase II transcripts achieve steady-state levels that depend on NMD. For some, the decay rate is directly influenced by NMD (direct targets). For others, abundance is NMD-sensitive but without any effect on the decay rate (indirect targets). To distinguish between direct and indirect targets, total RNA from wild-type (Nmd(+)) and mutant (Nmd(-)) strains was probed with high-density arrays across a 1-h time window following transcription inhibition. Statistical models were developed to describe the kinetics of RNA decay. 45% +/- 5% of RNAs targeted by NMD were predicted to be direct targets with altered decay rates in Nmd(-) strains. Parallel experiments using conventional methods were conducted to empirically test predictions from the global experiment. The results show that the global assay reliably distinguished direct versus indirect targets. Different types of targets were investigated, including transcripts containing adjacent, disabled open reading frames, upstream open reading frames, and those prone to out-of-frame initiation of translation. Known targeting mechanisms fail to account for all of the direct targets of NMD, suggesting that additional targeting mechanisms remain to be elucidated. 30% of the protein-coding targets of NMD fell into two broadly defined functional themes: those affecting chromosome structure and behavior and those affecting cell surface dynamics. Overall, the results provide a preview for how expression profiles in multi-cellular eukaryotes might be impacted by NMD. Furthermore, the methods for analyzing decay rates on a global scale offer a blueprint for new ways to study mRNA decay pathways in any organism where cultured cell lines are

  20. Acute Myosin Heavy Chain Isoform mRNA Expression in Response to Two Resistance Exercise Intensities With Equal Volume Load in Resistance-Trained Men.

    PubMed

    Schwarz, Neil A; Spillane, Mike B; McKinley, Sarah K; Andre, Thomas L; Gann, Joshua J; Willoughby, Darryn S

    2015-08-01

    The purpose of this study was to determine if resistance exercise intensity, in the context of equal volume load, differentially affected myosin heavy chain (MHC) isoform messenger RNA (mRNA) expression in resistance-trained men. In a crossover, uniform-balanced design, 10 male participants (23.7 ± 2.8 years, 178.8 ± 5.9 cm, 85.9 ± 9.2 kg) completed 2 lower-body resistance exercise sessions of different intensities with equal volume load. For the higher-intensity exercise session, participants performed 5 sets of 6 repetitions at 80% of 1 repetition maximum (1RM). For the lower-intensity exercise session, participants performed 3 sets of 16 repetitions at 50% of 1RM. Muscle samples from the vastus lateralis were acquired before exercise (PRE), 45 minutes postexercise (45MINPE), 3 hours postexercise (3HRPE), 24 hours postexercise (24HRPE), and 48 hours postexercise (48HRPE). Statistical analyses of mRNA expression were performed using separate 2 × 5 two-way repeated-measures analyses of variance for each criterion variable (p ≤ 0.05). There were no statistically significant interactions between intensity and time. Likewise, there were no significant differences between exercise intensity in MHC expression. Expression of mRNA for all MHC isoforms decreased at all postexercise time points, except 3HRPE (p = 0.051), compared with PRE following both exercise bouts (p ≤ 0.05). The results of this study found no difference in mRNA expression of MHC isoforms as a function of resistance exercise intensity. In addition, in contrast to results found in previous studies of untrained men, MHC mRNA expression seems to decrease in response to acute resistance exercise in previously resistance-trained men.

  1. Differential expression of tyrosine hydroxylase mRNA in the developing rat mesencephalon.

    PubMed

    Solberg, Y; Pollack, Y; Silverman, W F

    1992-12-01

    1. With respect to the mesostriatal projection, the mesencephalon is composed of two dopaminergic (DA) cell populations, called dorsal tier and ventral tier. Strong evidence suggests differences in both the spatial and the temporal sequence of the innervation of the striatum between the two groups, with the ventral tier neurons innervating striatal patches prenatally and dorsal tier cells innervating striatal matrix postnatally. 2. Using in situ hybridization, we have examined the expression of the gene coding for tyrosine hydroxylase (TH) in mesencephalic DA neurons with respect to their postnatal development. Two ontogenic patterns of expression were observed: (a) dorsal tier neurons of the medial mesencephalon exhibited a sharp increase in expression beginning after birth, peaking on day 14, then decreasing and, finally, stabilizing; and (b) ventral tier neurons and dorsal tier cells from the lateral and the medial-dorsal mesencephalon showed only a slight increase in TH mRNA, reaching a plateau at P10. 3. The time course of the observed increase in TH gene expression in the first group, generally parallels the innervation of their target cells in the striatal matrix, suggesting that TH gene expression in these cells may be influenced by their postsynaptic cells or by the innervation process.

  2. Imipramine induces brain-derived neurotrophic factor mRNA expression in cultured astrocytes.

    PubMed

    Takano, Katsura; Yamasaki, Hiroshi; Kawabe, Kenji; Moriyama, Mitsuaki; Nakamura, Yoichi

    2012-01-01

    Depression is one of the most prevalent and livelihood-threatening forms of mental illnesses and the neural circuitry underlying depression remains incompletely understood. Recent studies suggest that the neuronal plasticity involved with brain-derived neurotrophic factor (BDNF) plays an important role in the recovery from depression. Some antidepressants are reported to induce BDNF expression in vivo; however, the mechanisms have been considered solely in neurons and not fully elucidated. In the present study, we evaluated the effects of imipramine, a classic tricyclic antidepressant drug, on BDNF expression in cultured rat brain astrocytes. Imipramine dose-dependently increased BDNF mRNA expression in astrocytes. The imipramine-induced BDNF increase was suppressed with inhibitors for protein kinase A (PKA) or MEK/ERK. Moreover, imipramine exposure activated transcription factor cAMP response element binding protein (CREB) in a dose-dependent manner. These results suggested that imipramine induced BDNF expression through CREB activation via PKA and/or ERK pathways. Imipramine treatment in depression might exert antidepressant action through BDNF production from astrocytes, and glial BDNF expression might be a target of developing novel antidepressants.

  3. Lesch-Nyhan syndrome: mRNA expression of HPRT in patients with enzyme proven deficiency of HPRT and normal HPRT coding region of the DNA.

    PubMed

    Nguyen, Khue Vu; Naviaux, Robert K; Paik, Kacie K; Nyhan, William L

    2012-08-01

    Inherited mutation of the purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch-Nyhan syndrome (LNS) or Lesch-Nyhan variants (LNV). We report a case of two LNS affected members of a family with deficiency of activity of HPRT in intact cultured fibroblasts in whom mutation could not be found in the HPRT coding sequence but there was markedly decreased HPRT expression of mRNA. Published by Elsevier Inc.

  4. In situ hybridization analysis of leucomyosuppressin mRNA expression in the cockroach, Diploptera punctata.

    PubMed

    Fusé, M; Bendena, W G; Donly, B C; Tobe, S S; Orchard, I

    1998-06-08

    In the cockroach Diploptera punctata, sequencing of the cDNA for the insect myoinhibitory neuropeptide, leucomyosuppressin (LMS), has demonstrated that LMS is the only Phe-Met-Arg-Phe-amide (NH2) (FMRFamide)-related peptide to be encoded by this gene (Donly et al. [1996] Insect Biochem. Mol. Biol. 26:627-637). However, in the present study, high performance liquid chromatography analysis of brain extracts showed six discrete FMRFamide-like immunoreactive fractions, one of which co-eluted with LMS. This study compared the distribution of FMRFamide-related peptides visualized by immunohistochemistry with LMS mRNA expression demonstrated by in situ hybridization in D. punctata. Immunohistochemistry with a polyclonal antiserum generated against FMRFamide, but which recognizes extended RFamide peptides, demonstrated numerous RFamide-like immunoreactive cells and processes in both nervous and nonnervous tissues. RFamide-like immunoreactivity was found in cells and processes of the brain and optic lobes, the stomatogastric nervous system, including the frontal and ingluvial ganglia, and the suboesophageal ganglion. Immunoreactivity was also present in all ganglia of the ventral nerve cord and in the alimentary canal. Within the alimentary canal, positively stained processes were found in the crop, midgut, and hindgut, and immunoreactive endocrinelike cells were located in the midgut. In situ hybridization with a digoxigenin-labeled RNA probe spanning the entire LMS coding region showed cell bodies containing LMS mRNA in all ganglia studied, other than the ingluvial ganglion. Expression was most abundant in the brain and optic lobes and in the frontal and suboesophageal ganglia. LMS mRNA was also apparent, although less intensely, in all other ganglia of the ventral nerve cord. Within the alimentary canal, LMS mRNA-positive cells were only visible in the anterior portion of the midgut, in the endocrinelike cells. The appearance of LMS mRNA in the central nervous system

  5. Intronic SNP in ESR1 encoding human estrogen receptor alpha is associated with brain ESR1 mRNA isoform expression and behavioral traits.

    PubMed

    Pinsonneault, Julia K; Frater, John T; Kompa, Benjamin; Mascarenhas, Roshan; Wang, Danxin; Sadee, Wolfgang

    2017-01-01

    Genetic variants of ESR1 have been implicated in multiple diseases, including behavioral disorders, but causative variants remain uncertain. We have searched for regulatory variants affecting ESR1 expression in human brain, measuring allelic ESR1 mRNA expression in human brain tissues with marker SNPs in exon4 representing ESR1-008 (or ESRα-36), and in the 3'UTR of ESR1-203, two main ESR1 isoforms in brain. In prefrontal cortex from subjects with bipolar disorder, schizophrenia, and controls (n = 35 each; Stanley Foundation brain bank), allelic ESR1 mRNA ratios deviated from unity up to tenfold at the exon4 marker SNP, with large allelic ratios observed primarily in bipolar and schizophrenic subjects. SNP scanning and targeted sequencing identified rs2144025, associated with large allelic mRNA ratios (p = 1.6E10-6). Moreover, rs2144025 was significantly associated with ESR1 mRNA levels in the Brain eQTL Almanac and in brain regions in the Genotype-Tissue Expression project. In four GWAS cohorts, rs2104425 was significantly associated with behavioral traits, including: hypomanic episodes in female bipolar disorder subjects (GAIN bipolar disorder study; p = 0.0004), comorbid psychological symptoms in both males and females with attention deficit hyperactivity disorder (GAIN ADHD, p = 0.00002), psychological diagnoses in female children (eMERGE study of childhood health, subject age ≥9, p = 0.0009), and traits in schizophrenia (e.g., grandiose delusions, GAIN schizophrenia, p = 0.0004). The first common ESR1 variant (MAF 12-33% across races) linked to regulatory functions, rs2144025 appears conditionally to affect ESR1 mRNA expression in the brain and modulate traits in behavioral disorders.

  6. Extracellular inorganic phosphate regulates gibbon ape leukemia virus receptor-2/phosphate transporter mRNA expression in rat bone marrow stromal cells.

    PubMed

    Wada, Keinoshin; Mizuno, Morimichi; Komori, Takahide; Tamura, Masato

    2004-01-01

    In mammalian cells, several observations indicate not only that phosphate transport probably regulates local inorganic phosphate (Pi) concentration, but also that Pi affects normal cellular metabolism, which in turn regulates apoptosis and the process of mineralization. To elucidate how extracellular Pi regulates cellular functions of pre-osteoblastic cells, we investigated the expression of type III sodium (Na)-dependent Pi transporters in rat bone marrow stromal cells and ROB-C26 pre-osteoblastic cells. The mRNA expression level of gibbon ape leukemia virus receptor (Glvr)-2 was increased by the addition of Pi in rat bone marrow stromal cells, but not in ROB-C26 or normal rat kidney (NRK) cells. In contrast, the level of Glvr-1 mRNA was not altered by the addition of extracellular Pi in these cells. The induction of Glvr-2 mRNA by Pi was inhibited in the presence of cycloheximide (CHX). Moreover, mitogen-activated protein kinase (MEK) /extracellular-signal-regulated kinase (ERK) pathway inhibitors; U0126 (1.4-diamino-2, 3-dicyano-1, 4-bis [2-amino-phenylthio] butadiene) and PD98059 (2'-Amino-3'-methoxyflavone) inhibited inducible Glvr-2 mRNA expression, but p38 MEK inhibitor SB203580 [4-(4'-fluorophenyl)-2-(4'-methyl-sulfinylphenyl)-5-(4'pyridyl) imidazole] did not inhibit the induction of Glvr-2 mRNA expression, suggesting that extracellular Pi regulates de novo protein synthesis and MEK/ERK activity in rat bone marrow stromal cells, and through these, induction of Glvr-2 mRNA. Although Pi also induced osteopontin mRNA expression in rat bone marrow stromal cells but not in ROB-C26 and NRK cells, changes in cell viability with the addition of Pi were similar in both cell types. These data indicate that extracellular Pi regulates Glvr-2 mRNA expression, provide insights into possible mechanisms whereby Pi may regulate protein phosphorylation, and suggest a potential role for the Pi transporter in rat bone marrow stromal cells.

  7. Differential upregulation of aquaporin-4 mRNA expression in reactive astrocytes after brain injury: potential role in brain edema.

    PubMed

    Vizuete, M L; Venero, J L; Vargas, C; Ilundáin, A A; Echevarría, M; Machado, A; Cano, J

    1999-08-01

    Astrocytes and aquaporin-4 (AQP4) play a significant role in brain ion homeostasis. Consequently the regulation of AQP4 mRNA in the CNS after different neurological insults was of interest. A single intrastriatal injection of ringer or quinolinic acid strongly induced AQP4 mRNA in the striatum, specially at the core of the lesion. Colocalization studies demonstrated that AQP4 mRNA induction was restricted to hypertrophic astrocytes. The extent of striatal AQP4 mRNA induction did not correlate with neuronal degeneration, but it did with extravasation of Evans blue dye as a marker of BBB disruption. Distant lesions were additionally induced by either 6-OHDA or a knife cut in the medial forebrain bundle (MFB). The former, but not the latter, induced a high AQP4 mRNA expression in the lesioned substantia nigra. However, axotomy of the MFB induced a high AQP4 mRNA expression at the lesion site. We conclude that the induction of AQP4 mRNA expression is related to disruption of the blood-brain barrier and under brain edema conditions this water channel plays a key role in the reestablishment of the brain osmotic equilibrium. Copyright 1999 Academic Press.

  8. Iron chelation down-regulates dopamine transporter expression by decreasing mRNA stability and increasing endocytosis in N2a cells.

    PubMed

    Hegde, Narasimha V; Jensen, Gordon L; Unger, Erica L

    2011-02-15

    Cell surface expression of the dopamine transporter (DAT) is determined by the relative rates of its internalization and recycling. Changes in the cellular labile iron pool (LIP) affect many cellular mechanisms including those that regulate DAT trafficking. In this study, we analyzed DAT expression and posttranslational modifications in response to changes in cellular iron in transfected neuroblastoma cells (N2a). Iron chelation by desferrioxamine (DFO) altered DAT protein levels by decreasing the stability of DAT mRNA. Increased phosphorylation and ubiquitination of this transporter protein following DFO treatment were also observed. Cellular iron depletion elevated protein levels of the early endosomal marker Rab5. Moreover, confocal microscopy studies showed increased localization of DAT into the endosomal compartment in DFO-treated cells compared to control. Together, these findings suggest that cellular iron depletion regulates DAT expression through reducing mRNA stability as well as an increasing in endocytosis.

  9. 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

  10. 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

  11. Effect of sevoflurane anesthesia on the comprehensive mRNA expression profile of the mouse hippocampus.

    PubMed

    Hayase, Tomo; Tachibana, Shunsuke; Yamakage, Michiaki

    2016-01-01

    Postoperative nausea and vomiting (PONV) is a common complication after general anesthesia. Recent studies suggested that the hippocampus is involved in PONV. Hypothesising that hippocampal dopaminergic neurons are related to PONV, we examined the comprehensive mRNA profile of the hippocampus, using a sevoflurane-treated mouse model to confirm this. This study was conducted after approval from our institutional animal ethics committee, the Animal Research Center of Sapporo Medical University School of Medicine (project number: 12-033). Eight mice were assigned to two groups: a naïve group and a sevoflurane group (Sev group). In the Sev group, four mice were anesthetised with 3.5% sevoflurane for 1 hour. Subsequently, mRNA was isolated from their hippocampal cells and RNA sequencing was performed on an Illumina HiSeq 2500 platform. Mapping of the quality-controlled, filtered paired-end reads to mouse genomes and quantification of the expression levels of each gene were performed using R software. The Rtn4rl2 gene that encodes the Nogo receptor was the most up-regulated gene in the present study. The expression levels of dopamine receptor genes and the tachykinin gene were increased by sevoflurane exposure, while the genes related to serotonin receptors were not altered by sevoflurane exposure. The expression levels of LIM-homeodomain-related genes were highly down-regulated by sevoflurane. These findings suggest that sevoflurane exposure induces dopaminergic stimulation of hippocampal neurons and triggers PONV, while neuronal inflammation caused by LIM-homeodomain-related genes is down-regulated by sevoflurane.

  12. Effect of sevoflurane anesthesia on the comprehensive mRNA expression profile of the mouse hippocampus

    PubMed Central

    Hayase, Tomo; Tachibana, Shunsuke; Yamakage, Michiaki

    2016-01-01

    Postoperative nausea and vomiting (PONV) is a common complication after general anesthesia. Recent studies suggested that the hippocampus is involved in PONV. Hypothesising that hippocampal dopaminergic neurons are related to PONV, we examined the comprehensive mRNA profile of the hippocampus, using a sevoflurane-treated mouse model to confirm this. This study was conducted after approval from our institutional animal ethics committee, the Animal Research Center of Sapporo Medical University School of Medicine (project number: 12-033). Eight mice were assigned to two groups: a naïve group and a sevoflurane group (Sev group). In the Sev group, four mice were anesthetised with 3.5% sevoflurane for 1 hour. Subsequently, mRNA was isolated from their hippocampal cells and RNA sequencing was performed on an Illumina HiSeq 2500 platform. Mapping of the quality-controlled, filtered paired-end reads to mouse genomes and quantification of the expression levels of each gene were performed using R software. The Rtn4rl2 gene that encodes the Nogo receptor was the most up-regulated gene in the present study. The expression levels of dopamine receptor genes and the tachykinin gene were increased by sevoflurane exposure, while the genes related to serotonin receptors were not altered by sevoflurane exposure. The expression levels of LIM-homeodomain-related genes were highly down-regulated by sevoflurane. These findings suggest that sevoflurane exposure induces dopaminergic stimulation of hippocampal neurons and triggers PONV, while neuronal inflammation caused by LIM-homeodomain-related genes is down-regulated by sevoflurane. PMID:27867470

  13. Acute heat stress and dietary methionine effects on IGF-I, GHR, and UCP mRNA expression in liver and muscle of quails.

    PubMed

    Del Vesco, A P; Gasparino, E; Zancanela, V; Grieser, D O; Guimarães, S E F; Nascimento, C S; Voltolini, D M; Constantin, J; Gasparin, F S

    2014-02-13

    This study evaluated the expression of insulin-like growth factor I (IGF-I), growth hormone receptor (GHR), and uncoupling protein (UCP) mRNA in muscle and liver of quails that were in thermal comfort or exposed to heat stress and that were fed diets with or without methionine supplementation. Meat quails were fed a diet that either met the nutritional demands for methionine (MS) or did not meet this demand (methionine-deficient diet, MD). The animals were either kept at a thermal comfort temperature (25°C) or exposed to heat stress (38°C for 24 h starting on the 6th day). RNA was extracted from liver and breast muscle, and cDNA was synthesized and amplified using quantitative reverse transcription-polymerase chain reaction. Animals that were fed the MS diet and remained at the thermal comfort temperature exhibited increased IGF-I mRNA expression in the liver (0.56 AU). The GHR mRNA expression in the liver and muscle was influenced by both the study variables. Animals receiving the MS diet showed higher GHR expression, while increased expression was observed in animals at the thermal comfort temperature. The UCP mRNA expression in the muscle was influenced by both methionine supplementation and heat stress. Higher expression was observed in animals that received the MD diet (2.29 vs 3.77 AU) and in animals kept in thermal comfort. Our results suggest that heat stress negatively affects the expression of growth-related genes and that methionine supplementation is necessary to appropriately maintain the levels of IGF-I, GHR, and UCP transcripts for animal metabolism.

  14. 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

  15. 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.

  16. Successful TB treatment induces B-cells expressing FASL and IL5RA mRNA.

    PubMed

    van Rensburg, Ilana C; Wagman, Chandre; Stanley, Kim; Beltran, Caroline; Ronacher, Katharina; Walzl, Gerhard; Loxton, Andre G

    2017-01-10

    Activated B-cells increase T-cell behaviour during autoimmune disease and other infections by means of cytokine production and antigen-presentation. Functional studies in experimental autoimmune encephalomyelitis (EAE) indicate that B-cell deficiencies, and a lack of IL10 and IL35 leads to a poor prognosis. We hypothesised that B-cells play a role during tuberculosis. We evaluated B-cell mRNA expression using real-time PCR from healthy community controls, individuals with other lung diseases and newly diagnosed untreated pulmonary TB patients at three different time points (diagnosis, month 2 and 6 of treatment).We show that FASLG, IL5RA, CD38 and IL4 expression was lower in B-cells from TB cases compared to healthy controls. The changes in expression levels of CD38 may be due to a reduced activation of B-cells from TB cases at diagnosis. By month 2 of treatment, there was a significant increase in the expression of APRIL and IL5RA in TB cases. Furthermore, after 6 months of treatment, APRIL, FASLG, IL5RA and CD19 were upregulated in B-cells from TB cases. The increase in the expression of APRIL and CD19 suggests that there may be restored activation of B-cells following anti-TB treatment. The upregulation of FASLG and IL5RA indicates that B-cells expressing regulatory genes may play an important role in the protective immunity against M.tb infection. Our results show that increased activation of B-cells is present following successful TB treatment, and that the expression of FASLG and IL5RA could potentially be utilised as a signature to monitor treatment response.

  17. BORIS/CTCFL mRNA isoform expression and epigenetic regulation in epithelial ovarian cancer.

    PubMed

    Link, Petra A; Zhang, Wa; Odunsi, Kunle; Karpf, Adam R

    2013-01-01

    Cancer germline (CG) genes are normally expressed in germ cells and aberrantly expressed in a variety of cancers; their immunogenicity has led to the widespread development of cancer vaccines targeting these antigens. BORIS/CTCFL is an autosomal CG antigen and promising cancer vaccine target. BORIS is the only known paralog of CTCF, a gene intimately involved in genomic imprinting, chromatin insulation, and nuclear regulation. We have previously shown that BORIS is expressed in epithelial ovarian cancer (EOC) and that its expression coincides with promoter and global DNA hypomethylation. Recently, 23 different BORIS mRNA variants have been described, and have been functionally grouped into six BORIS isoform families (sf1-sf6). In the present study, we have characterized the expression of BORIS isoform families in normal ovary (NO) and EOC, the latter of which were selected to include two groups with widely varying global DNA methylation status. We find selective expression of BORIS isoform families in NO, which becomes altered in EOC, primarily by the activation of BORIS sf1 in EOC. When comparing EOC samples based on methylation status, we find that BORIS sf1 and sf2 isoform families are selectively activated in globally hypomethylated tumors. In contrast, CTCF is downregulated in EOC, and the ratio of BORIS sf1, sf2, and sf6 isoform families as a function of CTCF is elevated in hypomethylated tumors. Finally, the expression of all BORIS isoform families was induced to varying extents by epigenetic modulatory drugs in EOC cell lines, particularly when DNMT and HDAC inhibitors were used in combination.

  18. Effect of Radix Isatidis on the expression of moesin mRNA induced by LPS in the tissues of mice.

    PubMed

    Li, Jing; Liu, Yunhai; Fang, Jianguo; Chen, Xin; Xie, Wei

    2007-04-01

    To investigate the effect of the anti-endotoxic part of Radix Isatidis on the expression of moesin mRNA in murine tissues induced by lipopolysaccharide (LPS), the sample solution of F(022) part from Radix Isatidis was intraperitoneally administered to experimental mice, and the lipopoly-saccharide (LPS) were injected into the tail vein, and then the tissues of liver, kidney and spleen were colleted and cut into slices. The mRNA was detected by moesin mRNA hybridization in situ. The staining results were observed under microscope. It was found that moesin mRNA expression was increased in the tissues of liver, kidndy and spleen in mice treated with LPS, while in the mice pre-treated with F(022) part from Radix Isatidis, the LPS-induced moesin mRNA expressions in these tissues were inhibited in a dose-dependant manner. Our study showed that F(022) part from Radix Isatidis can inhibit the LPS-induced expression of moesin mRNA in the tissues of liver, kidney and spleen in mice.

  19. Chronic social subordination stress modulates glutamic acid decarboxylase (GAD) 67 mRNA expression in central stress circuits

    PubMed Central

    Makinson, Ryan; Lundgren, Kerstin H.; Seroogy, Kim B.; Herman, James P.

    2015-01-01

    Chronic social subordination is a well-known precipitant of numerous psychiatric and physiological health concerns. In this study, we examine the effects of chronic social stress in the visible burrow system (VBS) on the expression of glutamic acid decarboxylase (GAD) 67 and brain-derived neurotropic factor (BDNF) mRNA in forebrain stress circuitry. Male rats in the VBS system form a dominance hierarchy, whereby subordinate males exhibit neuroendocrine and physiological profiles characteristic of chronic exposure to stress. We found that social subordination decreases GAD67 mRNA in the peri-paraventricular nucleus region of the hypothalamus and the interfascicular nucleus of the bed nucleus of the stria terminalis (BNST), and increases in GAD67 mRNA in the hippocampus, medial prefrontal cortex, and dorsal medial hypothalamus. Expression of BDNF mRNA increased in the dorsal region of the BNST, but remained unchanged in all other regions examined. Results from this study indicate that social subordination is associated with several region-specific alterations in GAD67 mRNA expression in central stress circuits, whereas changes in the expression of BDNF mRNA are limited to the BNST. PMID:26066725

  20. Identify signature regulatory network for glioblastoma prognosis by integrative mRNA and miRNA co-expression analysis.

    PubMed

    Bing, Zhi-Tong; Yang, Guang-Hui; Xiong, Jie; Guo, Ling; Yang, Lei

    2016-12-01

    Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor in adults. Patients with this disease have a poor prognosis. The objective of this study is to identify survival-related individual genes (or miRNAs) and miRNA -mRNA pairs in GBM using a multi-step approach. First, the weighted gene co-expression network analysis and survival analysis are applied to identify survival-related modules from mRNA and miRNA expression profiles, respectively. Subsequently, the role of individual genes (or miRNAs) within these modules in GBM prognosis are highlighted using survival analysis. Finally, the integration analysis of miRNA and mRNA expression as well as miRNA target prediction is used to identify survival-related miRNA -mRNA regulatory network. In this study, five genes and two miRNA modules that significantly correlated to patient's survival. In addition, many individual genes (or miRNAs) assigned to these modules were found to be closely linked with survival. For instance, increased expression of neuropilin-1 gene (a member of module turquoise) indicated poor prognosis for patients and a group of miRNA -mRNA regulatory networks that comprised 38 survival-related miRNA -mRNA pairs. These findings provide a new insight into the underlying molecular regulatory mechanisms of GBM.

  1. 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.

  2. mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii.

    PubMed

    Nakamoto, Margaret A; Lovejoy, Alexander F; Cygan, Alicja M; Boothroyd, John C

    2017-08-29

    RNA contains over 100 modified nucleotides that are created post-transcriptionally, among which pseudouridine (Ψ) is one of the most abundant. Although it was one of the first modifications discovered, the biological role of this modification is still not fully understood. Recently, we reported that a pseudouridine synthase (TgPUS1) is necessary for differentiation of the single-celled eukaryotic parasite Toxoplasma gondii from active to chronic infection. To better understand the biological role of pseudouridylation we report here gel-based and deep-sequencing methods to identify TgPUS1-dependent Ψs in Toxoplasma RNA, and the use of TgPUS1 mutants to examine the effect of this modification on mRNAs. In addition to identifying conserved sites of pseudouridylation in Toxoplasma rRNA, tRNA, and snRNA, we also report extensive pseudouridylation of Toxoplasma mRNAs, with the Ψs being relatively depleted in the 3'-UTR but enriched at position 1 of codons. We show that many of the Ψs in tRNA and mRNA are dependent on the action of TgPUS1 and that TgPUS1-dependent mRNA Ψs are enriched in developmentally regulated transcripts. RNA-Seq data obtained from wild-type and TgPUS1-mutant parasites shows that genes containing a TgPUS1-dependent Ψ are relatively more abundant in mutant parasites while pulse/chase labeling of RNA with 4-thiouracil shows that mRNAs containing TgPUS1-dependent Ψ have a modest but statistically significant increase in half-life in the mutant parasites. These data are some of the first evidence suggesting that mRNA Ψs play an important biological role. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  3. Effects of Aflatoxin B1 on T-Cell Subsets and mRNA Expression of Cytokines in the Intestine of Broilers

    PubMed Central

    Jiang, Min; Peng, Xi; Fang, Jing; Cui, Hengmin; Yu, Zhengqiang; Chen, Zhengli

    2015-01-01

    This study was conducted to investigate the effects of aflatoxin B1 (AFB1) on T-cell subsets and mRNA expression of cytokines in the small intestine of broilers. One hundred and fifty-six one-day-old healthy Cobb broilers were randomly divided into control group (0 mg/kg AFB1) and AFB1 group (0.6 mg/kg AFB1) with three replicates per group and 26 birds per replicate for 21 days, respectively. At 7, 14, and 21 days of age, the duodenum, jejunum and ileum were sampled for analyzing T cell subsets (CD3+, CD3+CD4+ and CD3+CD8+) by flow cytometry as well as IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ and TNF-α mRNA expression by qRT-PCR. The percentages of T-cells in the intra-epithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) of duodenum, jejunum and ileum in the AFB1 group showed a decreased tendency in comparison to the control group. The mRNA expression of cytokines in the three intestinal segments in the AFB1 group presented a general decline compared with the control groups. Our data demonstrated that 0.6 mg/kg AFB1 in the broilers diet could reduce the percentages of T-cell subsets and the expression level of cytokine mRNA in the small intestine, implying that the immune function of the intestinal mucosa might be affected. The reduction of cytokines mRNA expression may be closely associated with the decreased proportions of T cells subsets induced by AFB1. PMID:25826527

  4. PAI-1 mRNA expression and plasma level in rheumatoid arthritis: relationship with 4G/5G PAI-1 polymorphism.

    PubMed

    Muñoz-Valle, José Francisco; Ruiz-Quezada, Sandra Luz; Oregón-Romero, Edith; Navarro-Hernández, Rosa Elena; Castañeda-Saucedo, Eduardo; De la Cruz-Mosso, Ulises; Illades-Aguiar, Berenice; Leyva-Vázquez, Marco Antonio; Castro-Alarcón, Natividad; Parra-Rojas, Isela

    2012-12-01

    Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting the synovial membrane, cartilage and bone. PAI-1 is a key regulator of the fibrinolytic system through which plasminogen is converted to plasmin. The plasmin activates the matrix metalloproteinase system, which is closely related with the joint damage and bone destruction in RA. The aim of this study was to investigate the relationship between 4G/5G PAI-1 polymorphism with mRNA expression and PAI-1 plasma protein levels in RA patients. 113 RA patients and 123 healthy subjects (HS) were included in the study. The 4G/5G PAI-1 polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism method; the PAI-1 mRNA expression was determined by real-time PCR; and the soluble PAI-1 (sPAI-1) levels were quantified using an ELISA kit. No significant differences in the genotype and allele frequencies of 4G/5G PAI-1 polymorphism were found between RA patients and HS. However, the 5G/5G genotype was the most frequent in both studied groups: RA (42%) and HS (44%). PAI-1 mRNA expression was slightly increased (0.67 fold) in RA patients with respect to HS (P = 0.0001). In addition, in RA patients, the 4G/4G genotype carriers showed increased PAI-1 mRNA expression (3.82 fold) versus 4G/5G and 5G/5G genotypes (P = 0.0001), whereas the sPAI-1 plasma levels did not show significant differences. Our results indicate that the 4G/5G PAI-1 polymorphism is not a marker of susceptibility in the Western Mexico. However, the 4G/4G genotype is associated with high PAI-1 mRNA expression but not with the sPAI-1 levels in RA patients.

  5. Zip1, Zip2, and Zip8 mRNA expressions were associated with growth hormone level during the growth hormone provocation test in children with short stature.

    PubMed

    Sun, Ping; Wang, Shifu; Jiang, Yali; Tao, Yanting; Tian, Yuanyuan; Zhu, Kai; Wan, Haiyan; Zhang, Lehai; Zhang, Lianying

    2013-10-01

    Short stature of children is affected by multiple factors. One of them is growth hormone (GH) deficiency. Growth hormone therapy can increase the final height of children with growth hormone deficiency. Zinc is found to induce dimerization and to enhance the bioactivity of human GH. Two gene families have been identified involved in zinc homeostasis. Previous studies in our laboratory have shown that Zip1, Zip2, Zip6, and ZnT1 mRNA were associated with zinc level in established human breast cancer in nude mice model; Zip8 was significantly lower in zinc-deficient Wistar rats in kidney. In this study, five zinc transporters: Zip1, Zip2, Zip6, Zip8, and ZnT1 were chosen. We aimed to investigate the mRNA expression of zinc transporters and to explore the relationship between zinc transporters and growth hormone in short stature children. Growth hormone provocation test is used to confirm the diagnosis of growth hormone deficiency. Six short children for the test were enrolled. At the same time, 15 sex- and age-matched normal children were enrolled as control. The expression levels of zinc transporters in peripheral blood mononuclear cells were determined by quantitative real-time PCR. Zip1 and Zip2 mRNA expression positively correlated with growth hormone level (r = 0.5133, P = 0.0371; r = 0.6719, P = 0.0032); Zip8 mRNA expression negatively correlated with growth hormone level (r = -0.5264, P = 0.0285) during the test in short stature children. The average expression level of Zip2 was significantly higher and Zip6, Zip8 mRNA levels were significantly lower in short stature children than in health controls at 0 min (P < 0.05, P < 0.05).

  6. KLK11 mRNA expression predicts poor disease-free and overall survival in colorectal adenocarcinoma patients.

    PubMed

    Alexopoulou, Dimitra K; Kontos, Christos K; Christodoulou, Spyridon; Papadopoulos, Iordanis N; Scorilas, Andreas

    2014-01-01

    Dysregulated expression of several KLK family members has been observed in colorectal adenocarcinoma. In the present study, the prognostic value of KLK11 mRNA expression as a molecular tissue biomarker in colorectal adenocarcinoma was examined. Using quantitative real-time PCR, KLK11 mRNA expression was studied in 120 cancerous and 41 paired noncancerous colorectal specimens obtained from 120 patients with primary colorectal adenocarcinoma. A significant upregulation of KLK11 transcripts in colorectal tumors was observed. KLK11 mRNA expression was associated with the depth of tumor invasion and the histological grade. Furthermore, KLK11 mRNA expression predicted poor disease-free and overall survival, independently of patient gender, age, tumor size, location, histological subtype, grade, venous invasion, lymphatic invasion, TNM stage, radiotherapy and chemotherapy treatment. KLK11 mRNA expression could be considered as a new molecular prognostic biomarker in colorectal adenocarcinoma, with additional prognostic value in patients with highly invasive tumors and/or positive lymph nodes.

  7. Increased ceramide synthase 2 and 6 mRNA levels in breast cancer tissues and correlation with sphingosine kinase expression.

    PubMed

    Erez-Roman, Racheli; Pienik, Reut; Futerman, Anthony H

    2010-01-01

    Intervention in the ceramide metabolic pathway is emerging as a novel means to regulate cancer and to modify the activity of chemotherapeutic drugs. We now study mRNA expression levels of the six ceramide synthase (CerS) genes in breast cancer tissue. CerS2 and CerS6 mRNA was significantly elevated in breast cancer tissue compared to paired normal tissue, with approximately half of the individuals showing elevated CerS2 and CerS6 mRNA. A significant correlation was found between CerS2 and CerS6 expression, and between CerS4 and CerS2/CerS6 expression. Moreover, patients that expressed higher CerS2 or 4 mRNA levels tended to show no changes in sphingosine kinase 1 levels, and likewise patients that expressed no change in CerS2 or CerS4 mRNA levels tended to express higher levels of sphingosine kinase 1. Together these results suggest an important role for the CerS genes in breast cancer etiology or diagnosis.

  8. 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.

  9. The mRNA expression of SATB1 and SATB2 in human breast cancer

    PubMed Central

    Patani, Neill; Jiang, Wen; Mansel, Robert; Newbold, Robert; Mokbel, Kefah

    2009-01-01

    Background SATB1 is a nuclear protein that has been recently reported to be a 'genome organizer' which delineates specific epigenetic modifications at target gene loci, directly up-regulating metastasis-associated genes while down-regulating tumor-suppressor genes. In this study, the level of mRNA expression of SATB1 and SATB2 were assessed in normal and malignant breast tissue in a cohort of women with breast cancer and correlated to conventional clinico-pathological parameters. Materials and methods Breast cancer tissues (n = 115) and normal background tissues (n = 31) were collected immediately after excision during surgery. Following RNA extraction, reverse transcription was carried out and transcript levels were determined using real-time quantitative PCR and normalized against β-actin expression. Transcript levels within the breast cancer specimens were compared to the normal background tissues and analyzed against TNM stage, nodal involvement, tumour grade and clinical outcome over a 10 year follow-up period. Results The levels of SATB1 were higher in malignant compared with normal breast tissue (p = 0.0167). SATB1 expression increased with increasing TNM stage (TNM1 vs. TNM2 p = 0.0264), increasing tumour grade (grade1 vs. grade 3 p = 0.017; grade 2 vs. grade 3 p = 0.0437; grade 1 vs. grade 2&3 p = 0.021) and Nottingham Prognostic Index (NPI) (NPI-1 vs. NPI-3 p = 0.0614; NPI-2 vs. NPI-3 p = 0.0495). Transcript levels were associated with oestrogen receptor (ER) positivity (ER(-) vs. ER(+) p = 0.046). SABT1 expression was also significantly correlated with downstream regulated genes IL-4 and MAF-1 (Pearson's correlation coefficient r = 0.21 and r = 0.162) and SATB2 (r = 0.506). After a median follow up of 10 years, there was a trend for higher SATB1 expression to be associated with shorter overall survival (OS). Higher levels of SATB2 were also found in malignant compared to background tissue (p = 0.049). SATB2 expression increased with increasing tumour

  10. Microassay for glucose-induced preproinsulin mRNA expression to assess islet functional potency for islet transplantation

    PubMed Central

    Omori, Keiko; Mitsuhashi, Masato; Todorov, Ivan; Rawson, Jeffrey; Shiang, Keh-Dong; Kandeel, Fouad; Mullen, Yoko

    2009-01-01

    Background The capacity for insulin synthesis in islets is important for islet transplantation to succeed. We developed a micro assay that evaluates the potency of human islets by measuring changes in glucose induced insulin gene (INS) expression using a single islet in octuplicate samples. Methods Poly (A)+ mRNA was purified from a set of single handpicked human islets. Glucose induced mature (post-spliced) and premature (pre-spliced) insulin mRNA were quantified by RT-PCR using several insulin mRNA primers designed at different locations including, intron, exon, and an exon-intron junction. Results The synthesis of premature INS mRNA was significantly increased in islets exposed to high glucose for 16 hours (vs. 4-hour, p<0.01), whereas mature INS mRNA showed no difference. Glucose-induced premature INS mRNA synthesis was attenuated in heat-damaged islets. Stimulation index (SI) calculated by normalizing premature by mature INS mRNA (SI_INS mRNA) positively correlated with SI of insulin release (SI_16h-insulin) from the same set of islets during 16-hour incubation in high or low glucose media, as well as SI of glucose-mediated insulin release obtained from the same islet lot in a perifusion system (N=12). Furthermore, linear multiple regression analysis using SI_INS mRNA and SI_16h-insulin predicted islet transplantation outcome in NODscid mice (N=8). Conclusion The measurement of glucose induced premature INS mRNA normalized by mature INS mRNA can be used to assess the functional quality of human islets and may predict islet function after transplantation in type 1 diabetic patients. PMID:20098276

  11. Quantitative assessment of hTERT mRNA expression in dysplastic nodules of HBV-related hepatocarcinogenesis.

    PubMed

    Oh, Bong-Kyeong; Kim, Young-Joo; Park, Young Nyun; Choi, Jinsub; Kim, Kyung Sik; Park, Chanil

    2006-04-01

    Telomerase reverse transcriptase (hTERT) is the rate-limiting determinant of telomerase, which is critical for carcinogenesis. Dysplastic nodules (DNs) appear to be preneoplastic lesions of hepatocellular carcinomas (HCCs). In this study, in order to characterize DNs, hTERT mRNA, hTERT gene dosage, and mRNA for c-myc, a transcriptional activator of hTERT were studied in human multi-step hepatocarcinogenesis. Fifty four hepatic nodules including 5 large regenerative nodules, 14 low-grade DNs, 7 high-grade DNs, 11 DNs with HCC foci and 17 HCCs, 23 livers with chronic hepatitis/cirrhosis, and 6 normal livers were examined. Transcript levels were measured by real-time quantitative RT-PCR and gene dosages by real-time PCR and Southern blotting. The hTERT mRNA levels increased with the progression of hepatocarcinogenesis, and a significant induction in the transition between low- and high-grade DNs was seen. Most high-grade DNs strongly expressed hTERT mRNA at levels similar to those of HCCs. Twenty-one percent of low-grade DNs had high levels of hTERT mRNA, up to those of high-grade DNs and there was no difference in the pathological features between low-grade DNs with and without increased hTERT mRNA levels. No correlation was found between hTERT mRNA levels, hTERT gene dosage, and c-myc mRNA levels. These results suggest that the induction of hTERT mRNA is an important early event and that its measurement by real-time quantitative RT-PCR is a useful tool to detect premalignant/malignant tendencies in hepatic nodules. However, hTERT gene dosage and c-myc expression are not the main mechanisms regulating hTERT expression in hepatocarcinogenesis.

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

    SciTech Connect

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

    2015-12-15

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

  13. 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

  14. 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.

  15. Reelin mRNA expression during embryonic brain development in the turtle Emys orbicularis.

    PubMed

    Bernier, B; Bar, I; Pieau, C; Lambert De Rouvroit, C; Goffinet, A M

    1999-10-25

    The expression of reelin messenger ribonucleic acid (mRNA) was studied during embryonic brain development in the turtle Emys orbicularis, by using radioactive in situ hybridization. A high expression was consistently found in the olfactory bulb and in a few neurons in the marginal zone and, to a lesser extent, in the subplate of the dorsal and medial cortical sectors. In the diencephalon, the ventral division of lateral geniculate nuclei and the prospective reticular thalamic nuclei were strongly positive. High reelin signal was also associated with some layers of the tectum and with the external granule cell layer of the cerebellum. A more moderate signal was detected in the septal nuclei, striatum, dorsal ventricular ridge, retina, habenular nuclei, and hypothalamus, and in some reticular nuclei of the midbrain and hindbrain and in ventral spinal cord. The cortical plate, basal forebrain, amygdala, and tegmentum were weakly labeled. When they are compared to reelin expression during mammalian brain development, our data reveal an evolutionarily conserved canvas of reelin expression and significant differences, particularly in developing cortical fields. Most significantly, the developing turtle cortex does not display the heavy reelin expression in subpial Cajal-Retzius cells that is so typical of its mammalian counterpart. Given the key role of reelin in laminar cortical development, our data suggest that the increase in the number of reelin-producing cells and/or the amplification of reelin expression in the cortical marginal zone might have been a driving factor during the evolution of the laminated cerebral cortex from stem reptiles to mammals, as indicated in previous comparative analyses. Copyright 1999 Wiley-Liss, Inc.

  16. Gene expression profile of IGF1 and MSTN mRNA and their correlation with carcass traits in different breeds of geese at 70 d of age.

    PubMed

    Tang, Q; Song, C; Zhang, S; Hu, Y; Zhao, D; Zou, J

    2014-02-01

    1. The expression of insulin-like growth factor-1 (IGF1) and myostatin (MSTN) mRNA in breast and leg muscle was quantified in 70-d-old Taihu and Wanxi geese by using a Multiplex Competitive Fluorescent-PCR method and the correlations between mRNA levels and carcass traits were analysed. 2. IGF1 mRNA expression in breast muscle in Taihu geese was significantly higher than that in Wanxi geese and the MSTN mRNA level in leg muscle in Taihu geese was significantly higher than that in Wanxi geese. 3. There was no significant difference in breast muscle MSTN or leg muscle IGF1 mRNA expression between the two breeds. 4. Within the same breed, the IGF1 mRNA expression in leg muscle of male geese was significantly higher than that in female geese, and MSTN mRNA expression in leg muscle was significantly higher than that in breast muscle. 5. There was no difference in the IGF1 mRNA expression between tissues. 6. There was a positive correlation between IGF1 mRNA and MSTN mRNA and a negative correlation between IGF1 mRNA expression of breast muscle and leg muscle ratio. 7. In Wanxi geese, MSTN mRNA expression in leg muscle was negatively associated with body weight and leg muscle weight.

  17. Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

    PubMed

    Xie, Fang-Fei; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Hong; Wu, Jian; Guo, Yu-Fan; Zeng, Ke-Qin; Wang, Ming-Jun; Zhu, Xiao-Wei; Xia, Wei; Wang, Lan; He, Pei; Bing, Peng-Fei; Lu, Xin; Zhang, Yong-Hong; Lei, Shu-Feng

    2017-07-22

    DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P < 0.05 and more than 5.96% genes presented very strong correlation (R T4 > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

  18. 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...

  19. Brain and heart sodium channel subtype mRNA expression in rat cerebral cortex.

    PubMed Central

    Yarowsky, P J; Krueger, B K; Olson, C E; Clevinger, E C; Koos, R D

    1991-01-01

    The expression of mRNAs coding for the alpha subunit of rat brain and rat heart sodium channels has been studied in adult and neonatal rat cerebral cortex using the reverse transcription-polymerase chain reaction (RT-PCR). Rat brain sodium channel subtype I, II, IIA, and III sequences were simultaneously amplified in the same PCR using a single oligonucleotide primer pair matched to all four subtype sequences. Identification of each subtype-specific product was inferred from the appearance of unique fragments when the product was digested with specific restriction enzymes. By using this RT-PCR method, products arising from mRNAs for all four brain sodium channel subtypes were identified in RNA extracted from adult rat cerebral cortex. The predominant component was type IIA with lesser levels of types I, II, and III. In contrast, the type II and IIA sequences were the predominant RT-PCR products in neonatal rat cortex, with slightly lower levels of type III and undetectable levels of type I. Thus, from neonate to adult, type II mRNA levels decrease relative to type IIA levels. Using a similar approach, we detected mRNA coding for the rat heart sodium channel in neonatal and adult rat cerebral cortex and in adult rat heart. These results reveal that mRNAs coding for the heart sodium channel and all four previously sequenced rat brain sodium channel subtypes are expressed in cerebral cortex and that type II and IIA channels may be differentially regulated during development. Images PMID:1658783

  20. Differential expression of arc mRNA and other plasticity-related genes induced by nicotine in adolescent rat forebrain.

    PubMed

    Schochet, T L; Kelley, A E; Landry, C F

    2005-01-01

    Relatively little attention has been focused on mechanisms related to neural plasticity and drug abuse in adolescence, compared with abundant research using adult animal models. As smoking is typically initiated in adolescence, an important question to address is whether the adolescent brain responds differently to nicotine compared with the adult. To investigate this question, we examined the expression of a number of early response genes (arc, c-fos and NGFI-B) that have been implicated in synaptic plasticity and addiction, following acute nicotine in adolescent and adult rats. Baseline expression of arc and c-fos was higher in adolescent brains compared with adults. Following acute nicotine treatment (0.1, 0.4mg/kg), we found a marked induction of arc mRNA in the prefrontal cortex of nicotine-treated adolescents compared with a less pronounced increase of arc in the adult. c-fos and NGFI-B were also upregulated by nicotine, but not in an age-related manner. In contrast, nicotine induced less arc, c-fos, and NGFI-B expression in the somatosensory cortex of adolescents compared with adults. A fourth gene, quinoid dihydropteridine reductase was expressed at lower levels in white matter of the adolescent forebrain compared with the adult, but was not affected by nicotine. These results suggest that in adolescence, the activity of specific early response genes is higher in brain regions critical for emotional regulation and decision-making. Further, nicotine affects key plasticity molecules in these areas in a manner different from the adult. Thus, adolescence may represent a neurobiologically vulnerable period with regard to nicotine exposure.

  1. 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

  2. The -844 G/A PAI-1 polymorphism is associated with mRNA expression in rheumatoid arthritis.

    PubMed

    Torres-Carrillo, Nora Magdalena; Torres-Carrillo, Norma; Vázquez-Del Mercado, Mónica; Delgado-Rizo, Vidal; Oregón-Romero, Edith; Parra-Rojas, Isela; Muñoz-Valle, José Francis