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Sample records for hepatic mrna expression

  1. Altered mRNA expression of hepatic lipogenic enzyme and PPARalpha in rats fed dietary levan from Zymomonas mobilis.

    PubMed

    Kang, Soon Ah; Hong, Kyunghee; Jang, Ki-Hyo; Kim, Yun-Young; Choue, Ryowon; Lim, Yoongho

    2006-06-01

    Levan or high molecular beta-2,6-linked fructose polymer is produced extracellularly from sucrose-based substrates by bacterial levansucrase. In the present study, to investigate the effect of levan feeding on serum leptin, hepatic lipogenic enzyme and peroxisome proliferation-activated receptor (PPAR) alpha expression in high-fat diet-induced obese rats, 4-week-old Sprague-Dawley male rats were fed high-fat diet (beef tallow, 40% of calories as fat), and, 6 weeks later, the rats were fed 0%, 1%, 5% or 10% levan-supplemented diets for 4 weeks. Serum leptin and insulin level were dose dependently reduced in levan-supplemented diet-fed rats. The mRNA expressions of hepatic fatty acid synthase and acetyl CoA carboxylase, which are the key enzymes in fatty acid synthesis, were down-regulated by dietary levan. However, dietary levan did not affect the gene expression of hepatic malic enzyme, phosphatidate phosphohydrolase and HMG CoA reductase. Also, the lipogenic enzyme gene expression in the white adipose tissue (WAT) was not affected by the diet treatments. However, hepatic PPARalpha mRNA expression was dose dependently up-regulated by dietary levan, whereas PPARgamma in the WAT was not changed. The results suggest that the in vivo hypolipidemic effect of dietary levan, including anti-obesity and lipid-lowering, may result from the inhibition of lipogenesis and stimulation of lipolysis, accompanied with regulation of hepatic lipogenic enzyme and PPARalpha gene expression. PMID:16214330

  2. 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. PMID:25714618

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

    PubMed Central

    Yoon, Leena; Liu, Ya-Nan; Park, Hyunjin

    2015-01-01

    Abstract 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. PMID:25714618

  4. Expression of the mRNA encoding truncated PPAR alpha does not correlate with hepatic insensitivity to peroxisome proliferators.

    PubMed

    Hanselman, J C; Vartanian, M A; Koester, B P; Gray, S A; Essenburg, A D; Rea, T J; Bisgaier, C L; Pape, M E

    2001-01-01

    Two alternatively spliced forms of human PPAR alpha mRNA, PPAR alpha1 and PPAR alpha2, have been identified. PPAR alpha1 mRNA gives rise to an active PPAR alpha protein while PPAR alpha2 mRNA gives rise to a form of PPAR which lacks the ligand-binding domain. PPAR alpha2 is unable to activate a peroxisome proliferator response element (PPRE) reporter gene construct in transient transfection assays. Both PPAR alpha1 and PPAR alpha2 mRNA are present in human liver, kidney, testes, heart, small intestine, and smooth muscle. In human liver, PPAR alpha2 mRNA abundance is approximately half that of PPAR alpha1 mRNA; a correlation analysis of PPAR alpha1 and PPAR alpha2 mRNA mass revealed an r-value of 0.75 (n = 18). Additional studies with intact liver from various species, showed that the PPAR alpha2/PPAR alpha1 mRNA ratios in rat, rabbit, and mouse liver were less than 0.10; significantly lower than the 0.3 and 0.5 ratios observed in monkey and human livers, respectively. To determine if a high PPAR alpha2/PPAR alpha1 mRNA ratio was associated with insensitivity to peroxisome proliferators, we treated human, rat, and rabbit hepatocytes with WY14643, a potent PPAR alpha activator, and measured acyl CoA oxidase (ACO) mRNA levels. Rat ACO mRNA levels increased markedly in response to WY14643 while human and rabbit hepatocytes were unresponsive. Thus, although the PPAR alpha2/PPAR alpha1 mRNA ratio is low in rabbits, this species is not responsive to peroxisome proliferators. Further studies with male and female rats, which vary significantly in their response to peroxisome proliferators, showed little difference in the ratio of PPAR alpha2/PPAR alpha1 mRNA. These data suggest that selective PPAR alpha2 mRNA expression is not the basis for differential species or gender responses to peroxisome proliferators. PMID:11269670

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

    SciTech Connect

    Egloff, Caroline; Crump, Doug; Porter, Emily; Williams, Kim L.; Letcher, Robert J.; Gauthier, Lewis T.; Kennedy, Sean W.

    2014-09-15

    The organophosphate flame retardants tris(2-butoxyethyl) phosphate (TBOEP) and triethyl phosphate (TEP) are used in a wide range of applications to suppress or delay the ignition and spread of fire. Both compounds have been detected in the environment and TBOEP was recently measured in free-living avian species. In this study, TBOEP and TEP were injected into the air cell of chicken embryos at concentrations ranging from 0 to 45,400 ng/g and 0 to 241,500 ng/g egg, respectively. Pipping success, development, hepatic mRNA expression of 9 target genes, thyroid hormone levels, and circulating bile acid concentrations were determined. Exposure to the highest doses of TBOEP and TEP resulted in negligible detection of the parent compounds in embryonic contents at pipping indicating their complete metabolic degradation. TBOEP exposure had limited effects on chicken embryos, with the exception of hepatic CYP3A37 mRNA induction. TEP exposure decreased pipping success to 68%, altered growth, increased liver somatic index (LSI) and plasma bile acids, and modulated genes associated with xenobiotic and lipid metabolism and the thyroid hormone pathway. Plasma thyroxine levels were decreased at all TEP doses, including an environmentally-relevant concentration (8 ng/g), and gallbladder hypotrophy was evident at ≥ 43,200 ng/g. Tarsus length and circulating thyroxine concentration emerged as potential phenotypic anchors for the modulation of transthyretin mRNA. The increase in plasma bile acids and LSI, gallbladder hypotrophy, and discoloration of liver tissue represented potential phenotypic outcomes associated with modulation of hepatic genes involved with xenobiotic and lipid metabolism. - Highlights: • TBOEP is not embryolethal to chicken embryos. • TEP affected embryonic viability, morphometric endpoints, and thyroid hormone levels. • TEP altered mRNA levels of xenobiotic and lipid metabolism genes. • TEP increased plasma bile acids and caused gallbladder hypotrophy

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

    PubMed

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

    2010-12-01

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

  7. A competitive enzyme-linked immunosorbent assay specific for murine hepcidin-1: correlation with hepatic mRNA expression in established and novel models of dysregulated iron homeostasis

    PubMed Central

    Gutschow, Patrick; Schmidt, Paul J.; Han, Huiling; Ostland, Vaughn; Bartnikas, Thomas B.; Pettiglio, Michael A.; Herrera, Carolina; Butler, James S.; Nemeth, Elizabeta; Ganz, Tomas; Fleming, Mark D.; Westerman, Mark

    2015-01-01

    Mice have been essential for distinguishing the role of hepcidin in iron homeostasis. Currently, investigators monitor levels of murine hepatic hepcidin-1 mRNA as a surrogate marker for the bioactive hepcidin protein itself. Here, we describe and validate a competitive, enzyme-linked immunosorbent assay that quantifies hepcidin-1 in mouse serum and urine. The assay exhibits a biologically relevant lower limit of detection, high precision, and excellent linearity and recovery. We also demonstrate correlation between serum and urine hepcidin-1 values and validate the competitive enzyme-linked immunosorbent assay by analyzing plasma hepcidin response of mice to physiological challenges, including iron deficiency, iron overload, acute blood loss, and inflammation. Furthermore, we analyze multiple murine genetic models of iron dysregulation, including β-thalassemia intermedia (Hbbth3/+), hereditary hemochromatosis (Hfe−/−, Hjv−/−, and Tfr2Y245X/Y245X), hypotransferrinemia (Trfhpx/hpx), heterozygous transferrin receptor 1 deficiency (Tfrc+/−) and iron refractory iron deficiency anemia (Tmprss6−/− and Tmprss6hem8/hem8). Novel compound iron metabolism mutants were also phenotypically characterized here for the first time. We demonstrate that serum hepcidin concentrations correlate with liver hepcidin mRNA expression, transferrin saturation and non-heme liver iron. In some circumstances, serum hepcidin-1 more accurately predicts iron parameters than hepcidin mRNA, and distinguishes smaller, statistically significant differences between experimental groups. PMID:25425686

  8. The expression of IL-2, IL-4 and interferon-gamma (IFN-gamma) mRNA using liver biopsies at different phases of acute exacerbation of chronic hepatitis B.

    PubMed Central

    Fukuda, R; Ishimura, N; Nguyen, T X; Chowdhury, A; Ishihara, S; Kohge, N; Akagi, S; Watanabe, M; Fukumoto, S

    1995-01-01

    To investigate the hypothesis that Th1 phenotype cytokines are associated with the increasing activity of hepatitis and Th2 phenotype cytokines with decreasing activity in the liver of chronic viral hepatitis, expressions of the mRNA of the cytokines IL-2, IFN-gamma and IL-4 in the liver of 23 patients with chronic hepatitis B were investigated by reverse transcription polymerase chain reaction. Patients were divided into three groups according to the phase of acute exacerbation of hepatitis as increasing (n = 9), decreasing (n = 8), and stable phase (n = 6). Both IL-2 and IFN-gamma mRNA were preferentially expressed in increasing phase than in decreasing phase (P < 0.01, P < 0.05, respectively) and associated with the high serum alanine aminotransferase (ALT) level. On the other hand, IL-4 mRNA was detected in decreasing phase with significant frequency compared with increasing phase (P < 0.05). However, expression of IL-4 mRNA was not associated with serum ALT level. Our results suggest that Th1 phenotype cytokines up-regulate and Th2 phenotype cytokines down-regulate the liver inflammation of chronic viral hepatitis. PMID:7774054

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

    PubMed

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

    2015-12-01

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

  10. Thyroid hormone negatively regulates CDX2 and SOAT2 mRNA expression via induction of miRNA-181d in hepatic cells

    SciTech Connect

    Yap, Chui Sun; Sinha, Rohit Anthony; Ota, Sho; Katsuki, Masahito; Yen, Paul Michael

    2013-11-01

    Highlights: •Thyroid hormone induces miR-181d expression in human hepatic cells and mouse livers. •Thyroid hormone downregulates CDX2 and SOAT2 (or ACAT2) via miR-181d. •miR-181d reduces cholesterol output from human hepatic cells. -- Abstract: Thyroid hormones (THs) regulate transcription of many metabolic genes in the liver through its nuclear receptors (TRs). Although the molecular mechanisms for positive regulation of hepatic genes by TH are well understood, much less is known about TH-mediated negative regulation. Recently, several nuclear hormone receptors were shown to downregulate gene expression via miRNAs. To further examine the potential role of miRNAs in TH-mediated negative regulation, we used a miRNA microarray to identify miRNAs that were directly regulated by TH in a human hepatic cell line. In our screen, we discovered that miRNA-181d is a novel hepatic miRNA that was regulated by TH in hepatic cell culture and in vivo. Furthermore, we identified and characterized two novel TH-regulated target genes that were downstream of miR-181d signaling: caudal type homeobox 2 (CDX2) and sterol O-acyltransferase 2 (SOAT2 or ACAT2). CDX2, a known positive regulator of hepatocyte differentiation, was regulated by miR-181d and directly activated SOAT2 gene expression. Since SOAT2 is an enzyme that generates cholesteryl esters that are packaged into lipoproteins, our results suggest miR-181d plays a significant role in the negative regulation of key metabolic genes by TH in the liver.

  11. Seasonal changes in hepatic progesterone receptor mRNA, estrogen receptor mRNA, and vitellogenin mRNA in the painted turtle, Chrysemys picta.

    PubMed

    Custodia-Lora, Noemí; Callard, Ian P

    2002-10-01

    Previous studies using the fresh water turtle Chrysemys picta have demonstrated that progesterone (P) inhibits estradiol (E)-induced vitellogenin (vtg) secretion in this species. Further, there is evidence for the differential expression of the two P receptor isoforms (PRA and PRB) in the liver during the turtle seasonal cycle, correlating with hepatic vitellogenesis. In this study we report changes in the hepatic PR mPNA, ER mRNA, and vitellogenin (vtg) mRNA transcripts during the reproductive cycle of the turtle. Fragments of the turtle hepatic PR and ER cDNAs were cloned and sequenced and a previously cloned turtle vtg cDNA were used as probes in Northern blotting. No 3.7-kb PR mRNA, corresponding to the smaller PR transcript, PRA of other species was found, although, a smaller 1.8-kb transcript (putative PRC mRNA) was present. These observations suggest that the turtle as in the chicken and human, the 4.5-kb PR mRNA transcript encodes both PRA and PRB proteins. Only the larger PR mRNA transcript (4.5-kb), was found to vary significantly during the annual cycle, being highest when vitellogenesis was inhibited in winter and summer. Vtg mRNA could not be detected during the summer or winter, was highest during vitellogenesis in the spring, and reappeared during the fall period of vitellogenesis and ovarian recrudescence. ER mRNA followed a similar pattern, being highest during spring and early fall, when vtg synthesis is high. The data suggest that P/PR, as well as E/ER, may be involved in the seasonal regulation of hepatic vitellogenesis in this species. PMID:12392693

  12. Expression of Human Hemojuvelin (HJV) Is Tightly Regulated by Two Upstream Open Reading Frames in HJV mRNA That Respond to Iron Overload in Hepatic Cells

    PubMed Central

    Onofre, Cláudia; Tomé, Filipa; Barbosa, Cristina; Silva, Ana Luísa

    2015-01-01

    The gene encoding human hemojuvelin (HJV) is one of the genes that, when mutated, can cause juvenile hemochromatosis, an early-onset inherited disorder associated with iron overload. The 5′ untranslated region of the human HJV mRNA has two upstream open reading frames (uORFs), with 28 and 19 codons formed by two upstream AUGs (uAUGs) sharing the same in-frame stop codon. Here we show that these uORFs decrease the translational efficiency of the downstream main ORF in HeLa and HepG2 cells. Indeed, ribosomal access to the main AUG is conditioned by the strong uAUG context, which results in the first uORF being translated most frequently. The reach of the main ORF is then achieved by ribosomes that resume scanning after uORF translation. Furthermore, the amino acid sequences of the uORF-encoded peptides also reinforce the translational repression of the main ORF. Interestingly, when iron levels increase, translational repression is relieved specifically in hepatic cells. The upregulation of protein levels occurs along with phosphorylation of the eukaryotic initiation factor 2α. Nevertheless, our results support a model in which the increasing recognition of the main AUG is mediated by a tissue-specific factor that promotes uORF bypass. These results support a tight HJV translational regulation involved in iron homeostasis. PMID:25666510

  13. Zinc metallothionein (MT) induction by parenteral iron and endotoxin: A temporal analysis of hepatic MT mRNA changes

    SciTech Connect

    McCormick, C.C. )

    1991-03-15

    The present study was undertaken to compare the temporal characteristics of iron-induced hepatic MT mRNA accumulation to that effected by endotoxin. Young chicks were given (ip) either endotoxin, ferrous gluconate or an equivalent volume of saline. At various times following injections, liver was obtained from 5 chicks per treatment for total RNA extraction. Equal amounts of total hepatic RNA from each chick were pooled and 10 {mu}g separated by denaturing agarose gel electrophoresis. Hepatic MT mRNA and albumin mRNA were analyzed by Northern blot analysis using synthetic oligonucleotides. The results indicated little temporal difference in the accumulation of hepatic MT mRNA as affected by either endotoxin or iron. In both treatments, MT mRNA was minimally affected at 3 hours post-injection. Maximum accumulation was achieved during a 6 h period from 6 to 12 hours post-injection. At 24 hours, MT mRNA was considerably higher in liver of endotoxin-injected chicks when compared to that of iron-injection chicks. Albumin expression appeared not to be substantially affected by either treatment. The results suggest that the induction of hepatic MT by iron injection is not substantially different than that observed following endotoxin administration. It would be speculative to suggest that the processes by which MT is induced under these conditions are also similar.

  14. Chronic anemic hypoxemia increases plasma glucagon and hepatic PCK1 mRNA in late-gestation fetal sheep.

    PubMed

    Culpepper, Christine; Wesolowski, Stephanie R; Benjamin, Joshua; Bruce, Jennifer L; Brown, Laura D; Jonker, Sonnet S; Wilkening, Randall B; Hay, William W; Rozance, Paul J

    2016-07-01

    Hepatic glucose production (HGP) normally begins just prior to birth. Prolonged fetal hypoglycemia, intrauterine growth restriction, and acute hypoxemia produce an early activation of fetal HGP. To test the hypothesis that prolonged hypoxemia increases factors which regulate HGP, studies were performed in fetuses that were bled to anemic conditions (anemic: n = 11) for 8.9 ± 0.4 days and compared with control fetuses (n = 7). Fetal arterial hematocrit and oxygen content were 32% and 50% lower, respectively, in anemic vs. controls (P < 0.005). Arterial plasma glucose was 15% higher in the anemic group (P < 0.05). Hepatic mRNA expression of phosphonenolpyruvate carboxykinase (PCK1) was twofold higher in the anemic group (P < 0.05). Arterial plasma glucagon concentrations were 70% higher in anemic fetuses compared with controls (P < 0.05), and they were positively associated with hepatic PCK1 mRNA expression (P < 0.05). Arterial plasma cortisol concentrations increased 90% in the anemic fetuses (P < 0.05), but fetal cortisol concentrations were not correlated with hepatic PCK1 mRNA expression. Hepatic glycogen content was 30% lower in anemic vs. control fetuses (P < 0.05) and was inversely correlated with fetal arterial plasma glucagon concentrations. In isolated primary fetal sheep hepatocytes, incubation in low oxygen (3%) increased PCK1 mRNA threefold compared with incubation in normal oxygen (21%). Together, these results demonstrate that glucagon and PCK1 may potentiate fetal HGP during chronic fetal anemic hypoxemia. PMID:27170658

  15. Posttranscriptional regulation of collagen alpha1(I) mRNA in hepatic stellate cells.

    PubMed Central

    Stefanovic, B; Hellerbrand, C; Holcik, M; Briendl, M; Aliebhaber, S; Brenner, D A

    1997-01-01

    The hepatic stellate cell (HSC) is the primary cell responsible for the dramatic increase in the synthesis of type I collagen in the cirrhotic liver. Quiescent HSCs contain a low level of collagen alpha1(I) mRNA, while activated HSCs contain about 60- to 70-fold more of this mRNA. The transcription rate of the collagen alpha1(I) gene is only two fold higher in activated HSCs than in quiescent HSCs. In assays using actinomycin D or 5,6-dichlorobenzimidazole riboside collagen alpha1(I) mRNA has estimated half-lives of 1.5 h in quiescent HSCs and 24 h in activated HSCs. Thus, this 16-fold change in mRNA stability is primarily responsible for the increase in collagen alpha1(I) mRNA steady-state level in activated HSCs. We have identified a novel RNA-protein interaction targeted to the C-rich sequence in the collagen alpha1(I) mRNA 3' untranslated region (UTR). This sequence is localized 24 nucleotides 3' to the stop codon. In transient transfection experiments, mutation of this sequence diminished accumulation of an mRNA transcribed from a collagen alpha1(I) minigene and in stable transfections decreased the half-life of collagen alpha1(I) minigene mRNA. Binding to the collagen alpha1(I) 3' UTR is present in cytoplasmic extracts of activated but not quiescent HSCs. It contains as a subunit alphaCP, which is also found in the complex involved in stabilization of alpha-globin mRNA. The auxiliary factors necessary to promote binding of alphaCP to the collagen 3' UTR are distinct from the factors necessary for binding to the alpha-globin sequence. Since alphaCP is expressed in both quiescent and activated HSCs, these auxiliary factors are responsible for the differentially expressed RNA-protein interaction at the collagen alpha1(I) mRNA 3' UTR. PMID:9271398

  16. Mutual antagonism between hepatitis B viral mRNA and host microRNA let-7

    PubMed Central

    Takata, Akemi; Otsuka, Motoyuki; Ohno, Motoko; Kishikawa, Takahiro; Yoshikawa, Takeshi; Koike, Kazuhiko

    2016-01-01

    The interplay between viral and host factors plays a major role in viral pathogenesis. Hepatitis B virus (HBV) infection is a global health problem that leads to liver cirrhosis and hepatocellular carcinoma (HCC). Although HBV proteins have been studied extensively about their implication in hepatocarcinogenesis, the molecular mechanisms of oncogenesis are still largely unknown. A recent concept in gene regulation, in which competitive endogenous RNAs compete for common microRNAs (miRNAs), suggests that mRNA targets are key elements in the regulation of miRNA availability. Here, we show that HBV mRNA in the preS2 region can be targeted by host miRNA let-7 g. This leads to the sequestration of let-7 g and inhibition of let-7 g function. The expression of HBV transcripts, including the preS2 region, de-repressed let-7 g targets, which may contribute to long-term oncogenesis. HBV transcript-expressing transgenic mice, but not non-targeted transcript-expressing mice, were more prone to chemically induced hepatoocarcinogenesis. Let-7 target protein expression was upregulated in human HCC tissues derived from HBV-infected patients. On the other hand, let-7 g inhibited HBV preS2 protein expression and viral products. These results suggest that the interplay between viral intermediate transcripts during HBV replication and host miRNAs is crucial to the pathogenesis of chronic viral infection. PMID:26979389

  17. Mutual antagonism between hepatitis B viral mRNA and host microRNA let-7.

    PubMed

    Takata, Akemi; Otsuka, Motoyuki; Ohno, Motoko; Kishikawa, Takahiro; Yoshikawa, Takeshi; Koike, Kazuhiko

    2016-01-01

    The interplay between viral and host factors plays a major role in viral pathogenesis. Hepatitis B virus (HBV) infection is a global health problem that leads to liver cirrhosis and hepatocellular carcinoma (HCC). Although HBV proteins have been studied extensively about their implication in hepatocarcinogenesis, the molecular mechanisms of oncogenesis are still largely unknown. A recent concept in gene regulation, in which competitive endogenous RNAs compete for common microRNAs (miRNAs), suggests that mRNA targets are key elements in the regulation of miRNA availability. Here, we show that HBV mRNA in the preS2 region can be targeted by host miRNA let-7 g. This leads to the sequestration of let-7 g and inhibition of let-7 g function. The expression of HBV transcripts, including the preS2 region, de-repressed let-7 g targets, which may contribute to long-term oncogenesis. HBV transcript-expressing transgenic mice, but not non-targeted transcript-expressing mice, were more prone to chemically induced hepatoocarcinogenesis. Let-7 target protein expression was upregulated in human HCC tissues derived from HBV-infected patients. On the other hand, let-7 g inhibited HBV preS2 protein expression and viral products. These results suggest that the interplay between viral intermediate transcripts during HBV replication and host miRNAs is crucial to the pathogenesis of chronic viral infection. PMID:26979389

  18. Morphine Enhances Hepatitis C Virus (HCV) Replicon Expression

    PubMed Central

    Li, Yuan; Zhang, Ting; Douglas, Steven D.; Lai, Jian-Ping; Xiao, Wei-Dong; Pleasure, David E.; Ho, Wen-Zhe

    2003-01-01

    Little information is available regarding whether substance abuse enhances hepatitis C virus (HCV) replication and promotes HCV disease progression. We investigated whether morphine alters HCV mRNA expression in HCV replicon-containing liver cells. Morphine significantly increased HCV mRNA expression, an effect which could be abolished by either of the opioid receptor antagonists, naltrexone or β-funaltrexamine. Investigation of the mechanism responsible for this enhancement of HCV replicon expression demonstrated that morphine activated NF-κB promoter and that caffeic acid phenethyl ester, a specific inhibitor of the activation of NF-κB, blocked morphine-activated HCV RNA expression. In addition, morphine compromised the anti-HCV effect of interferon alpha (IFN-α). Our in vitro data indicate that morphine may play an important role as a positive regulator of HCV replication in human hepatic cells and may compromise IFN-α therapy. PMID:12937158

  19. Age-related changes in mRNA levels of hepatic transporters, cytochrome P450 and UDP-glucuronosyltransferase in female rats.

    PubMed

    Kawase, Atsushi; Ito, Ayami; Yamada, Ayano; Iwaki, Masahiro

    2015-06-01

    Hepatic transporters and metabolic enzymes affect drug pharmacokinetics. Limited information exists on the alteration in mRNA levels of hepatic transporters and metabolic enzymes with aging. We examined the effects of aging on the mRNA levels of representative hepatic drug transporters and metabolic enzymes by analyzing their levels in 10-, 30- and 50-week-old male and female rats. Levels of mRNA of drug transporters including multidrug resistance protein (Mdr)1a, multidrug resistance-associated protein (Mrp)2, breast cancer resistance protein (Bcrp) and organic anion-transporting polypeptide (Oatp)1a1, and the metabolic enzymes cytochrome P450 (CYP)3A1, CYP3A2 and UDP-glucuronosyltransferase (UGT)1A1 were analyzed using real-time reverse transcriptase polymerase chain reaction. The mRNA levels of transporters in male rats did not decrease with age, while the mRNA levels of Bcrp and Oatp1a1 in female rats decreased with age. The mRNA levels of CYP3A1 and CYP3A2 in male rats were higher than those in female rats. The mRNA levels of metabolic enzymes decreased with age in female but not male rats. In particular, the mRNA levels of UGT1A1 in 10-week-old female rats were higher than those in male rats. mRNA expression of hepatic transporters and metabolic enzymes are more susceptible to aging in female than male rats. The age-related decreases in the mRNA levels of Bcrp, Oatp1a1, CYP3A1 and CYP3A2 in female rats may affect the metabolism and transport of substrates. This study showed that aging affected the mRNA expression of hepatic transporters and metabolic enzymes in rats. PMID:24899460

  20. Endotoxin suppresses rat hepatic low-density lipoprotein receptor expression.

    PubMed Central

    Liao, W; Rudling, M; Angelin, B

    1996-01-01

    Endotoxin induces hyperlipidaemia in experimental animals. In the current study, we investigated whether endotoxin alters hepatic low-density lipoprotein (LDL) receptor expression in rats. Endotoxin treatment suppressed hepatic LDL receptor expression in a dose- and time-dependent manner. Eighteen hours after intraperitoneal injection of increasing amounts of endotoxin, LDL receptor and its mRNA levels were determined by ligand blot and solution hybridization respectively. LDL receptor expression was inhibited by about 70% at a dose of 500 micrograms/100 g body weight. However, LDL receptor mRNA levels were markedly increased in all endotoxin-treated groups at this time point (by 83-136%; P < 0.001). Time-course experiments showed that LDL receptor expression was already reduced by 48% 4 h after endotoxin injection and was maximally reduced (by 63-65%) between 8 and 18 h. Changes in hepatic LDL receptor mRNA showed a different pattern. By 4 h after endotoxin injection, LDL receptor mRNA had decreased by 78% (P < 0.001). However, by 8 h after endotoxin injection, LDL receptor mRNA had returned to levels similar to controls, and 18 and 24 h after endotoxin injection, they were increased by about 60% (P < 0.05). Separation of plasma lipoproteins by FPLC demonstrated that endotoxin-induced changes in plasma triacylglycerols and cholesterol were due to accumulation of plasma apolipoprotein B-containing lipoproteins among very-low-density lipoprotein, intermediate-density lipoprotein and LDL. It is concluded that endotoxin suppresses hepatic LDL receptor expression in vivo in rats. PMID:8611169

  1. Regulation of hepatic bile acid transporters Ntcp and Bsep expression.

    PubMed

    Cheng, Xingguo; Buckley, David; Klaassen, Curtis D

    2007-12-01

    Sodium-taurocholate cotransporting polypeptide (Ntcp) and bile salt export pump (Bsep) are two key transporters for hepatic bile acid uptake and excretion. Alterations in Ntcp and Bsep expression have been reported in pathophysiological conditions. In the present study, the effects of age, gender, and various chemicals on the regulation of these two transporters were characterized in mice. Ntcp and Bsep mRNA levels in mouse liver were low in the fetus, but increased to its highest expression at parturition. After birth, mouse Ntcp and Bsep mRNA decreased by more than 50%, and then gradually increased to adult levels by day 30. Expression of mouse Ntcp mRNA and protein exhibit higher levels in female than male livers. No gender difference exists in BSEP/Bsep expression in human and mouse livers. Hormone replacements conducted in gonadectomized, hypophysectomized, and lit/lit mice indicate that female-predominant Ntcp expression in mouse liver is due to the inhibitory effect of male-pattern GH secretion, but not sex hormones. Ntcp and Bsep expression are in general resistant to induction by a large battery of microsomal enzyme inducers. Administration of cholestyramine increased Ntcp, whereas chenodeoxycholic acid (CDCA) increased Bsep mRNA expression. In conclusion, mouse Ntcp and Bsep are regulated by age, gender, cholestyramine, and bile acid, but resistant to induction by most microsomal enzyme inducers. PMID:17897632

  2. mRNA modifications: Dynamic regulators of gene expression?

    PubMed Central

    Hoernes, Thomas Philipp; Hüttenhofer, Alexander; Erlacher, Matthias David

    2016-01-01

    ABSTRACT The expression of a gene is a tightly regulated process and is exerted by a myriad of different mechanisms. Recently, RNA modifications located in coding sequences of mRNAs, have been identified as potential regulators of gene expression. N6-methyladenosine (m6A), 5-methylcytosine (m5C), pseudouridine (Ψ) and N1-methyladenosine (m1A) have been found within open reading frames of mRNAs. The presence of these mRNA modifications has been implicated to modulate the fate of an mRNA, ranging from maturation to its translation and even degradation. However, many aspects concerning the biological functions of mRNA modifications remain elusive. Recently, systematic in vitro studies allowed a first glimpse of the direct interplay of mRNA modifications and the efficiency and fidelity of ribosomal translation. It thereby became evident that the effects of mRNA modifications were, astonishingly versatile, depending on the type, position or sequence context. The incorporation of a single modification could either prematurely terminate protein synthesis, reduce the peptide yield or alter the amino acid sequence identity. These results implicate that mRNA modifications are a powerful mechanism to post-transcriptionally regulate gene expression. PMID:27351916

  3. mRNA Composition and Control of Bacterial Gene Expression

    PubMed Central

    Liang, S.-T.; Xu, Y.-C.; Dennis, P.; Bremer, H.

    2000-01-01

    The expression of any given bacterial protein is predicted to depend on (i) the transcriptional regulation of the promoter and the translational regulation of its mRNA and (ii) the synthesis and translation of total (bulk) mRNA. This is because total mRNA acts as a competitor to the specific mRNA for the binding of initiation-ready free ribosomes. To characterize the effects of mRNA competition on gene expression, the specific activity of β-galactosidase expressed from three different promoter-lacZ fusions (Pspc-lacZ, PRNAI-lacZ, and PRNAII-lacZ) was measured (i) in a relA+ background during exponential growth at different rates and (ii) in relA+ and ΔrelA derivatives of Escherichia coli B/r after induction of a mild stringent or a relaxed response to raise or lower, respectively, the level of ppGpp. Expression from all three promoters was stimulated during slow exponential growth or at elevated levels of ppGpp and was reduced during fast exponential growth or at lower levels of ppGpp. From these observations and from other considerations, we propose (i) that the concentration of free, initiation-ready ribosomes is approximately constant and independent of the growth rate and (ii) that bulk mRNA made during slow growth and at elevated levels of ppGpp is less efficiently translated than bulk mRNA made during fast growth and at reduced levels of ppGpp. These features lead to an indirect enhancement in the expression of LacZ (or of any other protein) during growth in media of poor nutritional quality and at increased levels of ppGpp. PMID:10809680

  4. Regulation of hepatic bile acid transporters Ntcp and Bsep expression

    PubMed Central

    Cheng, Xingguo; Buckley, David; Klaassen, Curtis D.

    2009-01-01

    Sodium-taurocholate cotransporting polypeptide (Ntcp) and bile salt export pump (Bsep) are two key transporters for hepatic bile acid uptake and excretion. Alterations in Ntcp and Bsep expression have been reported in pathophysiological conditions. In the present study, the effects of age, gender, and various chemicals on the regulation of these two transporters were characterized in mice. Ntcp and Bsep mRNA levels in mouse liver were low in the fetus, but increased to its highest expression at parturition. After birth, mouse Ntcp and Bsep mRNA decreased by more than 50%, and then gradually increased to adult levels by day 30. Expression of mouse Ntcp mRNA and protein exhibit higher levels in female than male livers, which is consistent with the trend of human NTCP mRNA expression between men and women. No gender difference exists in BSEP/Bsep expression in human and mouse livers. Hormone replacements conducted in gonadectomized, hypophysectomized, and lit/lit mice indicate that female-predominant Ntcp expression in mouse liver is due to the inhibitory effect of male-pattern GH secretion, but not sex hormones. Ntcp and Bsep expression are in general resistant to induction by a large battery of microsomal enzyme inducers. Administration of cholestyramine increased Ntcp, whereas chenodeoxycholic acid increased Bsep mRNA expression. In silico analysis indicates that female-predominant mouse and human Ntcp/NTCP expression may be due to GH. In conclusion, mouse Ntcp and Bsep are regulated by age, gender, cholestyramine, and bile acid, but resistant to induction by most microsomal enzyme inducers. PMID:17897632

  5. Regulation of hepatic PPAR{gamma}2 and lipogenic gene expression by melanocortin

    SciTech Connect

    Poritsanos, Nicole J.; Wong, Davie; Vrontakis, Maria E.; Mizuno, Tooru M.

    2008-11-14

    The central melanocortin system regulates hepatic lipid metabolism. Hepatic lipogenic gene expression is regulated by transcription factors including sterol regulatory element-binding protein 1c (SREBP-1c), carbohydrate responsive element-binding protein (ChREBP), and peroxisome proliferator-activated receptor {gamma}2 (PPAR{gamma}2). However, it is unclear if central melanocortin signaling regulates hepatic lipogenic gene expression through the activation of these transcription factors. To delineate the molecular mechanisms by which the melanocortin system regulates hepatic lipid metabolism, we examined the effect of intracerebroventricular injection of SHU9119, a melanocortin receptor antagonist, on hepatic expression levels of genes involved in lipid metabolism in mice. SHU9119 treatment increased hepatic triglyceride content and mRNA levels of lipogenic genes, SREBP-1c, and PPAR{gamma}2, whereas it did not cause any changes in hepatic ChREBP mRNA levels. These findings suggest that reduced central melanocortin signaling increases hepatic lipid deposition by stimulating hepatic lipogenic gene expression at least partly through the activation of SREBP-1c and PPAR{gamma}2.

  6. Effects of starter feeding and early weaning on GHR mRNA expression in liver and rumen of lambs from birth to 84 days of age.

    PubMed

    Wang, Fangbin; Li, Chong; Li, Fadi; Wang, Weimin; Wang, Xiaojuan; Liu, Ting; Ma, Zhiyuan; Li, Baosheng

    2016-06-01

    The growth hormone receptor (GHR) is associated with animal growth and development. To investigate such effects on GHR gene expression, a total of 102 Hu lambs were randomly allocated to one of three groups (Group 1: starter diet from 7 d of age, weaning at 56 d of age; Group 2: starter diet from 42 d of age, weaning at 56 d of age; Group 3: starter diet from 7 d of age; weaning at 28 d of age). Six lambs from each group were sacrificed every 14 d to investigate the effects of starter feeding and weaning age on GHR mRNA expression in the liver and rumen. The results revealed that GHR mRNA expression was significantly higher in the liver and rumen (p < 0.05) than in other tissues. Early starter feeding up-regulated hepatic GHR mRNA expression on days 14, 28, 42 and 56 and ruminal GHR mRNA expression on days 28, 42, 70, and 84 (p < 0.05). Early weaning up-regulated hepatic GHR mRNA expression on days 56, 70 and 84 and ruminal GHR mRNA expression on days 42, 56, 70 and 84 (p < 0.05). Dietary and weaning regimes and age affected the hepatic and ruminal GHR mRNA expression. PMID:27032032

  7. Expression2Kinases: mRNA profiling linked to multiple upstream regulatory layers

    PubMed Central

    Gordonov, Simon; Lim, Maribel P.; Perkins, Matthew H.; Ma'ayan, Avi

    2012-01-01

    Motivation: Genome-wide mRNA profiling provides a snapshot of the global state of cells under different conditions. However, mRNA levels do not provide direct understanding of upstream regulatory mechanisms. Here, we present a new approach called Expression2Kinases (X2K) to identify upstream regulators likely responsible for observed patterns in genome-wide gene expression. By integrating chromatin immuno-precipitation (ChIP)-seq/chip and position weight matrices (PWMs) data, protein–protein interactions and kinase–substrate phosphorylation reactions, we can better identify regulatory mechanisms upstream of genome-wide differences in gene expression. We validated X2K by applying it to recover drug targets of food and drug administration (FDA)-approved drugs from drug perturbations followed by mRNA expression profiling; to map the regulatory landscape of 44 stem cells and their differentiating progeny; to profile upstream regulatory mechanisms of 327 breast cancer tumors; and to detect pathways from profiled hepatic stellate cells and hippocampal neurons. The X2K approach can advance our understanding of cell signaling and unravel drugs mechanisms of action. Availability: The software and source code are freely available at: http://www.maayanlab.net/X2K. Contact: avi.maayan@mssm.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22080467

  8. 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.

  9. Leptin mRNA expresses in the bull reproductive organ.

    PubMed

    Abavisani, A; Baghbanzadeh, A; Shayan, P; Tajik, P; Dehghani, H; Mirtorabi, M

    2009-12-01

    Leptin, a 167-amino acid hormone, is secreted mainly by fat tissue. It has some powerful effects on the regulation of metabolism and reproductive function through endocrine and probably paracrine mechanisms. The contribution rate of leptin function on the male reproductive system is not still clear. Characterization of leptin expression in reproductive organs will suggest that in addition to its endocrine action, leptin has also paracrine/autocrine effects on reproduction. The expression of functional leptin receptor mRNA has been already recognized in testis of rodents, human and cattle. Thus, the aim of the present study was to investigate the presence of leptin mRNA in the bovine testis, because it will be the first step for understanding of its paracrine/autocrine effects on the male reproductive organs in cattle. The present study was the first to showed leptin mRNA expression in the testis of Holstein cattle using reverse transcription and polymerase chain reaction (RT-PCR) analysis. RT-PCR products were amplified with nested PCR using inner leptin primer pairs to emphasis the first results. Besides, bovine beta actin gene was acted as an internal positive control as well as RNA purification marker. Our findings suggest that in addition to its endocrine actions at the hypothalamic-pituitary axis, leptin can has an autocrine and/or paracrine role in bull testicular function. PMID:19466574

  10. 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. PMID:24161824

  11. Effect of propionate on mRNA expression of key genes for gluconeogenesis in liver of dairy cattle.

    PubMed

    Zhang, Qian; Koser, Stephanie L; Bequette, Brian J; Donkin, Shawn S

    2015-12-01

    Elevated needs for glucose in lactating dairy cows are met through a combination of increased capacity for gluconeogenesis and increased supply of gluconeogenic precursors, primarily propionate. This study evaluated the effects of propionate on mRNA expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1), mitochondrial phosphoenolpyruvate carboxykinase (PCK2), pyruvate carboxylase (PC), and glucose-6-phosphatase (G6PC), key gluconeogenic enzymes, and capacity for glucose synthesis in liver of dairy cattle. In experiment 1, six multiparous mid-lactation Holstein cows were used in a replicated 3×3 Latin square design consisting of a 6-d acclimation or washout phase followed by 8h of postruminal infusion of either propionate (1.68mol), glucose (0.84mol), or an equal volume (10mL/min) of water. In experiment 2, twelve male Holstein calves [39±4 kg initial body weight (BW)] were blocked by birth date and assigned to receive, at 7d of age, either propionate [2mmol·h(-1)·(BW(0.75))(-1)], acetate [3.5mmol·h(-1)·(BW(.75))(-1)], or an equal volume (4mL/min) of saline. In both experiments, blood samples were collected at 0, 2, 4, 6, and 8h relative to the start of infusion and liver biopsy samples were collected at the end of the infusion for mRNA analysis. Liver explants from experiment 1 were used to measure tricarboxylic acid cycle flux and gluconeogenesis using (13)C mass isotopomer distribution analysis from (13)C3 propionate. Dry matter intake and milk yield were not altered by infusions in cows. Serum insulin concentration in cows receiving propionate was elevated than cows receiving water, but was not different from cows receiving glucose. Hepatic expression of PCK1 and G6PC mRNA and glucose production in cows receiving propionate were not different from cows receiving water, but tended to be higher compared with cows receiving glucose. Hepatic expression of PCK2 and PC mRNA was not altered by propionate infusion in cows. Blood glucose, insulin, and

  12. KSRP is critical in governing hepatic lipid metabolism through controlling Per2 expression

    PubMed Central

    Chou, Chu-Fang; Zhu, Xiaolin; Lin, Yi-Yu; Gamble, Karen L.; Garvey, W. Timothy; Chen, Ching-Yi

    2015-01-01

    Hepatic lipid metabolism is controlled by integrated metabolic pathways. Excess accumulation of hepatic TG is a hallmark of nonalcoholic fatty liver disease, which is associated with obesity and insulin resistance. Here, we show that KH-type splicing regulatory protein (KSRP) ablation reduces hepatic TG levels and diet-induced hepatosteatosis. Expression of period 2 (Per2) is increased during the dark period, and circadian oscillations of several core clock genes are altered with a delayed phase in Ksrp−/− livers. Diurnal expression of some lipid metabolism genes is also disturbed with reduced expression of genes involved in de novo lipogenesis. Using primary hepatocytes, we demonstrate that KSRP promotes decay of Per2 mRNA through an RNA-protein interaction and show that increased Per2 expression is responsible for the phase delay in cycling of several clock genes in the absence of KSRP. Similar to Ksrp−/− livers, both expression of lipogenic genes and intracellular TG levels are also reduced in Ksrp−/− hepatocytes due to increased Per2 expression. Using heterologous mRNA reporters, we show that the AU-rich element-containing 3′ untranslated region of Per2 is responsible for KSRP-dependent mRNA decay. These findings implicate that KSRP is an important regulator of circadian expression of lipid metabolism genes in the liver likely through controlling Per2 mRNA stability. PMID:25514904

  13. 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.

  14. Hepatic and renal Bcrp transporter expression in mice treated with perfluorooctanoic acid

    PubMed Central

    Eldasher, Lobna M.; Wen, Xia; Little, Michael S.; Bircsak, Kristin M.; Yacovino, Lindsay L.; Aleksunes, Lauren M.

    2013-01-01

    The breast cancer resistance protein (Bcrp) is an efflux transporter that participates in the biliary and renal excretion of drugs and environmental chemicals. Recent evidence suggests that pharmacological activation of the peroxisome proliferator activated receptor alpha (PPARα) can up-regulate the hepatic expression of Bcrp. The current study investigated the regulation of hepatic and renal Bcrp mRNA and protein in mice treated with the PPARα agonist perfluorooctanoic acid (PFOA) and the ability of PFOA to alter human BCRP function in vitro. Bcrp mRNA and protein expression were quantified in the livers and kidneys of male C57BL/6 mice treated with vehicle or PFOA (1 or 3 mg/kg/day oral gavage) for 7 days. PFOA treatment increased liver weights as well as the hepatic mRNA and protein expression of the PPARα target gene, cytochrome P450 4a14. Compared to vehicle-treated control mice, PFOA increased hepatic Bcrp mRNA and protein between 1.5- and 3-fold. Immunofluorescent staining confirmed enhanced canalicular Bcrp staining in liver sections from PFOA-treated mice. The kidney expression of cytochrome P450 4a14 mRNA, but not Bcrp, was increased in mice treated with PFOA. Micromolar concentrations of PFOA decreased human BCRP ATPase activity and inhibited BCRP-mediated transport in inverted membrane vesicles. Together, these studies demonstrate that PFOA induces hepatic Bcrp expression in mice and may inhibit human BCRP transporter function at concentrations that exceed levels observed in humans. PMID:23435180

  15. Ascorbic acid supplementation down-regulates the alcohol induced oxidative stress, hepatic stellate cell activation, cytotoxicity and mRNA levels of selected fibrotic genes in guinea pigs.

    PubMed

    Abhilash, P A; Harikrishnan, R; Indira, M

    2012-02-01

    Both oxidative stress and endotoxins mediated immunological reactions play a major role in the progression of alcoholic hepatic fibrosis. Ascorbic acid has been reported to reduce alcohol-induced toxicity and ascorbic acid levels are reduced in alcoholics. Hence, we investigated the hepatoprotective action of ascorbic acid in the reversal of alcohol-induced hepatic fibrosis in male guinea pigs (n = 36), and it was compared with the animals abstenting from alcohol treatment. In comparison with the alcohol abstention group, there was a reduction in the activities of toxicity markers and levels of lipid and protein peroxidation products, expression of α-SMA, caspase-3 activity and mRNA levels of CYP2E1, TGF-β(1), TNF-α and α(1)(I) collagen in liver of the ascorbic acid-supplemented group. The ascorbic acid content in liver was significantly reduced in the alcohol-treated guinea pigs. But it was reversed to normal level in the ascorbic acid-supplemented group. The anti-fibrotic action of ascorbic acid in the rapid regression of alcoholic liver fibrosis may be attributed to decrease in the oxidative stress, hepatic stellate cells activation, cytotoxicity and mRNA expression of fibrotic genes CYP2E1, TGF-β(1), TNF-α and α(1) (I) collagen in hepatic tissues. PMID:22149461

  16. Transforming growth factor (TGF)-beta stimulates hepatic jun-B and fos-B proto-oncogenes and decreases albumin mRNA.

    PubMed Central

    Beauchamp, R D; Sheng, H M; Ishizuka, J; Townsend, C M; Thompson, J C

    1992-01-01

    Transforming growth factor-beta (TGF-beta) modulates some components of the acute phase response in hepatic cells. The mechanisms for these actions of TGF-beta are largely unknown. The authors recently found that the decrease in albumin mRNA after TGF-beta 1 treatment required de novo RNA and protein synthesis, suggesting that TGF-beta acts through induction of another gene. The purpose of the current study was to determine whether TGF-beta 1 could regulate the expression of both the jun and fos genes that encode transcriptional regulatory proteins that constitute the AP-1 complex, and to determine whether expression of these genes may be coordinated with the decrease in albumin mRNA. Northern blot hybridization was used to determine levels of specific mRNAs. Transforming growth factor-beta 1 increased the levels of both jun-B and fos-B mRNA by 60 minutes after treatment of mouse hepatoma (BWTG3) cells. When TGF-beta 1 was removed from the media after 4 hours, there was a sustained effect of increased jun-B and decreased albumin mRNA (greater than 48 hours), and the subsequent decrease in jun-B levels coincided with the increase in albumin mRNA. The tumor-promoting phorbol ester (phorbol 12-myristate 13-acetate [PMA]), known to induce jun and fos gene expression, caused increases in jun-B and fos-B that preceded the decrease in albumin mRNA levels at 24 hours. These observations are consistent with our hypothesis that jun-B and fos-B induction may participate in downregulation of albumin synthesis as well as other hepatic responses to TGF-beta. Images FIG. 1. FIG. 2. FIG. 4. FIG. 5. FIG. 6. PMID:1417179

  17. Screening of mRNA Chemical Modification to Maximize Protein Expression with Reduced Immunogenicity.

    PubMed

    Uchida, Satoshi; Kataoka, Kazunori; Itaka, Keiji

    2015-01-01

    Chemical modification of nucleosides in mRNA is an important technology to regulate the immunogenicity of mRNA. In this study, various previously reported mRNA formulations were evaluated by analyzing in vitro protein expression and immunogenicity in multiple cell lines. For the macrophage-derived cell line, RAW 264.7, modified mRNA tended to have reduced immunogenicity and increased protein expression compared to the unmodified mRNA. In contrast, in some cell types, such as hepatocellular carcinoma cells (HuH-7) and mouse embryonic fibroblasts (MEFs), protein expression was decreased by mRNA modification. Further analyses revealed that mRNA modifications decreased translation efficiency but increased nuclease stability. Thus, mRNA modification is likely to exert both positive and negative effects on the efficiency of protein expression in transfected cells and optimal mRNA formulation should be determined based on target cell types and transfection purposes. PMID:26213960

  18. Screening of mRNA Chemical Modification to Maximize Protein Expression with Reduced Immunogenicity

    PubMed Central

    Uchida, Satoshi; Kataoka, Kazunori; Itaka, Keiji

    2015-01-01

    Chemical modification of nucleosides in mRNA is an important technology to regulate the immunogenicity of mRNA. In this study, various previously reported mRNA formulations were evaluated by analyzing in vitro protein expression and immunogenicity in multiple cell lines. For the macrophage-derived cell line, RAW 264.7, modified mRNA tended to have reduced immunogenicity and increased protein expression compared to the unmodified mRNA. In contrast, in some cell types, such as hepatocellular carcinoma cells (HuH-7) and mouse embryonic fibroblasts (MEFs), protein expression was decreased by mRNA modification. Further analyses revealed that mRNA modifications decreased translation efficiency but increased nuclease stability. Thus, mRNA modification is likely to exert both positive and negative effects on the efficiency of protein expression in transfected cells and optimal mRNA formulation should be determined based on target cell types and transfection purposes. PMID:26213960

  19. Protein kinase C-dependent regulation of human hepatic drug transporter expression.

    PubMed

    Mayati, Abdullah; Le Vee, Marc; Moreau, Amélie; Jouan, Elodie; Bucher, Simon; Stieger, Bruno; Denizot, Claire; Parmentier, Yannick; Fardel, Olivier

    2015-12-15

    Hepatic drug transporters are now recognized as major actors of hepatobiliary elimination of drugs. Characterization of their regulatory pathways is therefore an important issue. In this context, the present study was designed to analyze the potential regulation of human hepatic transporter expression by protein kinase C (PKC) activation. Treatment by the reference PKC activator phorbol 12-myristate 13-acetate (PMA) for 48h was shown to decrease mRNA expression of various sinusoidal transporters, including OATP1B1, OATP2B1, NTCP, OCT1 and MRP3, but to increase that of OATP1B3, whereas mRNA expression of canalicular transporters was transiently enhanced (MDR1), decreased (BSEP and MRP2) or unchanged (BCRP) in human hepatoma HepaRG cells. The profile of hepatic transporter mRNA expression changes in PMA-treated HepaRG cells was correlated to that found in PMA-exposed primary human hepatocytes and was similarly observed in response to the PKC-activating marketed drug ingenol mebutate. It was associated with concomitant repression of OATP1B1 and OATP2B1 protein expression and reduction of OATP, OCT1, NTCP and MRP2 activity. The use of chemical PKC inhibitors further suggested a contribution of novel PKCs isoforms to PMA-mediated regulations of transporter mRNA expression. PMA was finally shown to cause epithelial-mesenchymal transition (EMT) in HepaRG cells and exposure to various additional EMT inducers, i.e., hepatocyte growth factor, tumor growth factor-β1 or the HNF4α inhibitor BI6015, led to transporter expression alterations highly correlated to those triggered by PMA. Taken together, these data highlight PKC-dependent regulation of human hepatic drug transporter expression, which may be closely linked to EMT triggered by PKC activation. PMID:26462574

  20. Controlling mammalian gene expression by allosteric hepatitis delta virus ribozymes.

    PubMed

    Nomura, Yoko; Zhou, Linlin; Miu, Anh; Yokobayashi, Yohei

    2013-12-20

    We engineered small molecule responsive allosteric ribozymes based on the genomic hepatitis delta virus (HDV) ribozyme by replacing the P4-L4 stem-loop with an RNA aptamer through a connector stem. When embedded in the 3' untranslated region of a reporter gene mRNA, these RNA devices enabled regulation of cis-gene expression by theophylline and guanine by up to 29.5-fold in mammalian cell culture. Furthermore, a NOR logic gate device was constructed by placing two engineered ribozymes in tandem, demonstrating the modularity of the RNA devices. The significant improvement in the regulatory dynamic range (ON/OFF ratio) of the RNA devices based on the HDV ribozyme should provide new opportunities for practical applications. PMID:23697539

  1. The prognostic value of epidermal growth factor receptor mRNA expression in primary ovarian cancer.

    PubMed Central

    Bartlett, J. M.; Langdon, S. P.; Simpson, B. J.; Stewart, M.; Katsaros, D.; Sismondi, P.; Love, S.; Scott, W. N.; Williams, A. R.; Lessells, A. M.; Macleod, K. G.; Smyth, J. F.; Miller, W. R.

    1996-01-01

    The expression of mRNA for the epidermal growth factor (EGF) receptor, EGF and transforming growth factor alpha (TGF-alpha) was determined in 76 malignant, six borderline and 15 benign primary ovarian tumours using the reverse transcriptase-polymerase chain reaction and related to clinical and pathological parameters. Of the malignant tumours, 70% (53/76) expressed EGF receptor mRNA, 31% (23/75) expressed EGF mRNA and 35% (26/75) expressed TGF-alpha mRNA. For the borderline tumours, four of six (67%) expressed EGF receptor mRNA, 1/6 (17%) expressed TGF-alpha mRNA and none expressed EGF mRNA. Finally, 33% (5/15) of the benign tumours expressed EGF receptor mRNA, whereas 40% (6/15) expressed EGF mRNA and 7% (1/15) expressed TGF-alpha mRNA. The presence of the EGF receptor in malignant tumours was associated with that of TGF-alpha (P = 0.0015) but not with EGF (P = 1.00), whereas there was no relationship between the presence of EGF and TGF-alpha (P = 1.00). EGF receptor mRNA expression was significantly and positively associated with serous histology (P = 0.006) but not with stage or grade. Neither EGF nor TGF-alpha showed any link with histological subtype or stage. The survival of patients with malignant tumours possessing EGF receptor mRNA was significantly reduced compared with that of patients whose tumours were negative (P = 0.030 for all malignant tumours; P = 0.007 for malignant epithelial tumours only). In contrast, neither the expression of TGF-alpha nor EGF was related to survival. These data suggest that the presence of EGF receptor mRNA is associated with poor prognosis in primary ovarian cancer. Images Figure 1 PMID:8562334

  2. Enhanced glypican-3 expression differentiates the majority of hepatocellular carcinomas from benign hepatic disorders

    PubMed Central

    Zhu, Z; Friess, H; Wang, L; Abou-Shady, M; Zimmermann, A; Lander, A; Korc, M; Kleeff, J; Buchler, M

    2001-01-01

    BACKGROUND/AIMS—Hepatocellular carcinoma (HCC) is a common malignant tumour worldwide, and its differential diagnosis from benign lesions of the liver is often difficult yet of great clinical importance. In the present study, we analysed whether glypican-3 is useful in differentiating between benign and malignant liver diseases and whether it influences the growth behaviour of HCC.
METHODS—Northern blot analysis and in situ hybridisation.
RESULTS—Northern blot analysis indicated that expression of glypican-3 mRNA was either low or absent in normal liver, in focal nodular hyperplasia (FNH), and in liver cirrhosis. In contrast, expression of glypican-3 mRNA was markedly increased in 20 of 30 and moderately increased in five of 30 HCC samples. The average increase in glypican-3 mRNA expression in HCC was significant compared with expression in normal liver (21.7-fold increase, p<0.01). In comparison with FNH or liver cirrhosis, glypican-3 mRNA expression in HCC was increased 7.2- (p<0.05) and 10.8-fold (p<0.01), respectively. In addition, pushing HCCs exhibited significantly higher glypican-3 mRNA expression than invading tumours (p<0.05). In situ hybridisation analysis demonstrated weak expression of glypican-3 mRNA in normal hepatocytes and bile ductular cells, and weak to occasionally moderate signals in hepatocytes forming nodules of liver cirrhosis and in regenerated hepatic nodules of FNH. In contrast, glypican-3 in situ hybridisation signals were intense in hepatic cancer cells with even higher levels in pushing HCCs than in invading HCCs.
CONCLUSIONS—These findings suggest that glypican-3, in many cases, has the potential to differentiate between benign and malignant liver diseases.


Keywords: focal nodular hyperplasia; liver cirrhosis; hepatocellular cancer; glypican-3 PMID:11247902

  3. Chronic unpredictive mild stress leads to altered hepatic metabolic profile and gene expression

    PubMed Central

    Jia, Hong-mei; Li, Qi; Zhou, Chao; Yu, Meng; Yang, Yong; Zhang, Hong-wu; Ding, Gang; Shang, Hai; Zou, Zhong-mei

    2016-01-01

    Depression is a complex disease characterized by a series of pathological changes. Research on depression is mainly focused on the changes in brain, but not on liver. Therefore, we initially explored the metabolic profiles of hepatic extracts from rats treated with chronic unpredictive mild stress (CUMS) by UPLC-Q-TOF/MS. Using multivariate statistical analysis, a total of 26 altered metabolites distinguishing CUMS-induced depression from normal control were identified. Using two-stage receiver operating characteristic (ROC) analysis, 18 metabolites were recognized as potential biomarkers related to CUMS-induced depression via 12 metabolic pathways. Subsequently, we detected the mRNA expressions levels of apoptosis-associated genes such as Bax and Bcl-2 and four key enzymes including Pla2g15, Pnpla6, Baat and Gad1 involved in phospholipid and primary bile acid biosynthesis in liver tissues of CUMS rats by real-time qRT-PCR assay. The expression levels of Bax, Bcl-2, Pla2g15, Pnpla6 and Gad1 mRNA were 1.43,1.68, 1.74, 1.67 and 1.42-fold higher, and those of Baat, Bax/Bcl-2 ratio mRNA were 0.83, 0.85-fold lower in CUMS rats compared with normal control. Results of liver-targeted metabonomics and mRNA expression demonstrated that CUMS-induced depression leads to variations in hepatic metabolic profile and gene expression, and ultimately results in liver injury. PMID:27006086

  4. Cytochrome P450 mRNA Expression in the Rodent Brain: Species-, Sex-, and Region-Dependent Differences

    PubMed Central

    Stamou, Marianna; Wu, Xianai; Kania-Korwel, Izabela; Lehmler, Hans-Joachim

    2014-01-01

    Cytochrome P450 (P450) enzymes play a critical role in the activation and detoxication of many neurotoxic chemicals. Although research has largely focused on P450-mediated metabolism in the liver, emerging evidence suggests that brain P450s influence neurotoxicity by modulating local metabolite levels. As a first step toward better understanding the relative role of brain P450s in determining neurotoxic outcome, we characterized mRNA expression of specific P450 isoforms in the rodent brain. Adult mice (male and female) and rats (male) were treated with vehicle, phenobarbital, or dexamethasone. Transcripts for CYP2B, CYP3A, CYP1A2, and the orphan CYP4X1 and CYP2S1 were quantified in the liver, hippocampus, cortex, and cerebellum by quantitative (real-time) polymerase chain reaction. These P450s were all detected in the liver with the exception of CYP4X1, which was detected in rat but not mouse liver. P450 expression profiles in the brain varied regionally. With the exception of the hippocampus, there were no sex differences in regional brain P450 expression profiles in mice; however, there were marked species differences. In the liver, phenobarbital induced CYP2B expression in both species. Dexamethasone induced hepatic CYP2B and CYP3A in mice but not rats. In contrast, brain P450s did not respond to these classic hepatic P450 inducers. Our findings demonstrate that P450 mRNA expression in the brain varies by region, regional brain P450 profiles vary between species, and their induction varies from that of hepatic P450s. These novel data will be useful for designing mechanistic studies to examine the relative role of P450-mediated brain metabolism in neurotoxicity. PMID:24255117

  5. Cytochrome p450 mRNA expression in the rodent brain: species-, sex-, and region-dependent differences.

    PubMed

    Stamou, Marianna; Wu, Xianai; Kania-Korwel, Izabela; Lehmler, Hans-Joachim; Lein, Pamela J

    2014-02-01

    Cytochrome P450 (P450) enzymes play a critical role in the activation and detoxication of many neurotoxic chemicals. Although research has largely focused on P450-mediated metabolism in the liver, emerging evidence suggests that brain P450s influence neurotoxicity by modulating local metabolite levels. As a first step toward better understanding the relative role of brain P450s in determining neurotoxic outcome, we characterized mRNA expression of specific P450 isoforms in the rodent brain. Adult mice (male and female) and rats (male) were treated with vehicle, phenobarbital, or dexamethasone. Transcripts for CYP2B, CYP3A, CYP1A2, and the orphan CYP4X1 and CYP2S1 were quantified in the liver, hippocampus, cortex, and cerebellum by quantitative (real-time) polymerase chain reaction. These P450s were all detected in the liver with the exception of CYP4X1, which was detected in rat but not mouse liver. P450 expression profiles in the brain varied regionally. With the exception of the hippocampus, there were no sex differences in regional brain P450 expression profiles in mice; however, there were marked species differences. In the liver, phenobarbital induced CYP2B expression in both species. Dexamethasone induced hepatic CYP2B and CYP3A in mice but not rats. In contrast, brain P450s did not respond to these classic hepatic P450 inducers. Our findings demonstrate that P450 mRNA expression in the brain varies by region, regional brain P450 profiles vary between species, and their induction varies from that of hepatic P450s. These novel data will be useful for designing mechanistic studies to examine the relative role of P450-mediated brain metabolism in neurotoxicity. PMID:24255117

  6. Developmentally Regulated Expression of HDNF/NT-3 mRNA in Rat Spinal Cord Motoneurons and Expression of BDNF mRNA in Embryonic Dorsal Root Ganglion.

    PubMed

    Ernfors, Patrik; Persson, Håkan

    1991-01-01

    Northern blot analysis was used to demonstrate high levels of hippocampus-derived neurotrophic factor/neurotrophin-3 (HDNF/NT-3) mRNA in the embryonic day (E) 13 - 14 and 15 - 16 spinal cord. The level decreased at E18 - 19 and remained the same until postnatal day (P) 1, after which it decreased further to a level below the detection limit in the adult. In situ hybridization revealed that the NT-3 mRNA detected in the developing spinal cord was derived from motoneurons and the decrease seen at E18 - 19 was caused by a reduction in the number of motoneurons expressing NT-3 mRNA. The distribution of NT-3 mRNA-expressing cells in the E15 spinal cord was very similar to the distribution of cells expressing choline acetyltransferase or nerve growth factor receptor (NGFR) mRNA. Moreover, a striking similarity between the developmentally regulated expression of NT-3 and NGFR mRNA was noted in spinal cord motoneurons. A subpopulation of all neurons in the dorsal root ganglia expressed brain-derived neurotrophic factor (BDNF) mRNA from E13, the earliest time examined, to adulthood. These results are consistent with a trophic role of NT-3 for proprioceptive sensory neurons innervating the ventral horn, and imply a local action of BDNF for developing sensory neurons within the dorsal root ganglia. PMID:12106253

  7. Periconceptional undernutrition modifies endocrine profiles and hepatic gene expression in sheep.

    PubMed

    de Brun, V; Meikle, A; Casal, A; Sequeira, M; Contreras-Solís, I; Carriquiry, M; Forcada, F; Sosa, C; Abecia, J A

    2015-08-01

    This study investigated whether a 22-day period of undernutrition (half maintenance) could affect maternal endocrine responses and liver gene expression during early pregnancy (day 7). Thirty-five ewes were fed 1.5 (n = 15) or 0.5 (n = 20) their maintenance requirements and slaughtered on day 7 of the oestrus cycle or pregnancy (oestrus = day 0). Insulin, IGF, leptin and non-esterified fatty acids (NEFA) were determined on days -14, 0 and 7. Transcripts of the IGF family and adipokines receptors were determined in the liver by real-time RT-PCR. Underfed animals presented lower body weight and body condition, greater plasma concentration of NEFA, and lower plasma concentrations of leptin, insulin and IGF1 compared to adequately fed animals. Underfed ewes presented greater hepatic expression of IGFBP2 than well-fed ewes, but tended to have lesser expression of IGFBP5. While no effect of undernutrition on IGFBP4 and ADIPOR2 mRNA expressions was observed, they were increased by pregnancy in underfed animals. This study shows that undernutrition modifies endocrine profiles and hepatic gene expression of IGFBP2 and 5. The pregnancy status increased hepatic gene expression of IGFBP4 and ADIPOR2 mRNA in undernourished ewes. PMID:25319346

  8. Elevated TREM2 mRNA expression in leukocytes in schizophrenia but not major depressive disorder.

    PubMed

    Yoshino, Yuta; Kawabe, Kentaro; Yamazaki, Kiyohiro; Watanabe, Shinya; Numata, Shusuke; Mori, Yoko; Yoshida, Taku; Iga, Junichi; Ohmori, Tetsuro; Ueno, Shu-Ichi

    2016-06-01

    The pathological mechanisms of schizophrenia (SCZ) have not been clarified, but the microglia hypothesis has recently been discussed. We previously reported that the mRNA for a protein related to activation of microglia, triggering receptor expressed on myeloid cell 2 (TREM2), is expressed higher in peripheral leukocytes in SCZ than controls. In this study, we analyzed TREM2 mRNA expression in leukocytes from both SCZ and major depressive disorder (MDD) patients. We compared 50 SCZ patients and 42 MDD patients with age-matched controls. Levels of TREM2 mRNA in leukocytes were analyzed with quantitative real-time PCR method using TaqMan probe. TREM2 mRNA expression was significantly higher in leukocytes of SCZ subjects than controls, but the expression level was non-significantly different in MDD subjects. We observed a decrease in TREM2 mRNA expression in leukocytes from one SCZ patient after clozapine treatment. The expression did not change following ECT, but the expression level in this patient was still significantly higher than that in controls. We conclude that the high amount of TREM2 mRNA expression in leukocytes is specific to SCZ but not MDD and that changes in TREM2 mRNA expression may be a trait biomarker for SCZ. PMID:27130565

  9. Alteration of human hepatic drug transporter activity and expression by cigarette smoke condensate.

    PubMed

    Sayyed, Katia; Vee, Marc Le; Abdel-Razzak, Ziad; Jouan, Elodie; Stieger, Bruno; Denizot, Claire; Parmentier, Yannick; Fardel, Olivier

    2016-07-01

    Smoking is well-known to impair pharmacokinetics, through inducing expression of drug metabolizing enzymes. In the present study, we demonstrated that cigarette smoke condensate (CSC) also alters activity and expression of hepatic drug transporters, which are now recognized as major actors of hepatobiliary elimination of drugs. CSC thus directly inhibited activities of sinusoidal transporters such as OATP1B1, OATP1B3, OCT1 and NTCP as well as those of canalicular transporters like P-glycoprotein, MRP2, BCRP and MATE1, in hepatic transporters-overexpressing cells. CSC similarly counteracted constitutive OATP, NTCP and OCT1 activities in human highly-differentiated hepatic HepaRG cells. In parallel, CSC induced expression of BCRP at both mRNA and protein level in HepaRG cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B1, OATP2B1, OAT2, NTCP, OCT1 and BSEP, and enhanced that of MRP4. Such changes in transporter gene expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin, a reference activator of the aryl hydrocarbon receptor (AhR) pathway, and were counteracted, for some of them, by siRNA-mediated AhR silencing. This suggests that CSC alters hepatic drug transporter levels via activation of the AhR cascade. Importantly, drug transporter expression regulations as well as some transporter activity inhibitions occurred for a range of CSC concentrations similar to those required for inducing drug metabolizing enzymes and may therefore be hypothesized to be relevant for smokers. Taken together, these data established human hepatic transporters as targets of cigarette smoke, which could contribute to known alteration of pharmacokinetics and some liver adverse effects caused by smoking. PMID:27450509

  10. Transforming Growth Factor β1 (TGF-β1) Activates Hepcidin mRNA Expression in Hepatocytes.

    PubMed

    Chen, Simeng; Feng, Teng; Vujić Spasić, Maja; Altamura, Sandro; Breitkopf-Heinlein, Katja; Altenöder, Jutta; Weiss, Thomas S; Dooley, Steven; Muckenthaler, Martina U

    2016-06-17

    The hepatic hormone hepcidin is the master regulator of systemic iron homeostasis. Its expression level is adjusted to alterations in iron levels, inflammatory cues, and iron requirements for erythropoiesis. Bone morphogenetic protein 6 (BMP6) contributes to the iron-dependent control of hepcidin. In addition, TGF-β1 may stimulate hepcidin mRNA expression in murine hepatocytes and human leukocytes. However, receptors and downstream signaling proteins involved in TGF-β1-induced hepcidin expression are still unclear. Here we show that TGF-β1 treatment of mouse and human hepatocytes, as well as ectopic expression of TGF-β1 in mice, increases hepcidin mRNA levels. The hepcidin response to TGF-β1 depends on functional TGF-β1 type I receptor (ALK5) and TGF-β1 type II receptor (TβRII) and is mediated by a noncanonical mechanism that involves Smad1/5/8 phosphorylation. Interestingly, increasing availability of canonical Smad2/3 decreases TGF-β1-induced hepcidin regulation, whereas the BMP6-hepcidin signal was enhanced, indicating a signaling component stoichiometry-dependent cross-talk between the two pathways. Although ALK2/3-dependent hepcidin activation by BMP6 can be modulated by each of the three hemochromatosis-associated proteins: HJV (hemojuvelin), HFE (hemochromatosis protein), and TfR2 (transferrin receptor 2), these proteins do not control the ALK5-mediated hepcidin response to TGF-β1. TGF-β1 mRNA levels are increased in mouse models of iron overload, indicating that TGF-β1 may contribute to hepcidin synthesis under these conditions. In conclusion, these data demonstrate that a complex regulatory network involving TGF-β1 and BMP6 may control the sensing of systemic and/or hepatic iron levels. PMID:27129231

  11. Induction and characterization of hepatic metallothionein expression from cadmium-induced channel catfish (Ictalurus punctatus)

    SciTech Connect

    Zhang, Y.S.; Schlenk, D.

    1995-08-01

    Two low-molecular-weight proteins of approximately 10,000 daltons (DA) were isolated from the hepatic tissue of catfish intraperitoneally injected with CdCl{sub 2} (5 mg/kg). Each protein was significantly induced following Cd treatment. Bound Cd, co-eluted from DEAE anion-exchange HPLC with rabbit hepatic metallothioneins I and II, did not possess UV absorption indicative of aromatic amino acids and corresponded to the expression of two mRNA transcripts of approximately 400 base pairs, which hybridized to a full-length cDNA probe to winter flounder metallothionein (MT). Because these data indicated that these two low molecular weight proteins were hepatic MT isoforms, induction of MT mRNA expression was studied in livers of channel catfish exposed to aqueous CdCl{sub 2} utilizing the ribonuclease protection assay. Following a 7-d exposure to 10, 50, and 100 {micro}g/L CdCl{sub 2}, expression of two MT mRNAs increased in a dose-dependent manner (MT-a, r{sup 2} = 0.95; MT-b, r{sup 2} = 0.98). Each transcript was maximally induced 48 h after exposure to 100 {micro}g/L CdCl{sub 2} and then gradually declined to approximately 20% above controls after 96 h and maintained this level of expression for 7 d. Although MT expression is rapidly induced in channel catfish by Cd, and MT mRNA measurement is a sensitive indicator of MT induction, the use of hepatic MT of channel catfish may not be a sensitive bioindicator of short-term, low-level aqueous exposures of Cd.

  12. Negative regulation of neuromedin U mRNA expression in the rat pars tuberalis by melatonin.

    PubMed

    Aizawa, Sayaka; Sakata, Ichiro; Nagasaka, Mai; Higaki, Yuriko; Sakai, Takafumi

    2013-01-01

    The pars tuberalis (PT) is part of the anterior pituitary gland surrounding the median eminence as a thin cell layer. The characteristics of PT differ from those of the pars distalis (PD), such as cell composition and gene expression, suggesting that the PT has a unique physiological function compared to the PD. Because the PT highly expresses melatonin receptor type 1, it is considered a mediator of seasonal and/or circadian signals of melatonin. Expression of neuromedin U (NMU) that is known to regulate energy balance has been previously reported in the rat PT; however, the regulatory mechanism of NMU mRNA expression and secretion in the PT are still obscure. In this study, we examined both the diurnal change of NMU mRNA expression in the rat PT and the effects of melatonin on NMU in vivo. In situ hybridization and quantitative PCR analysis of laser microdissected PT samples revealed that NMU mRNA expression in the PT has diurnal variation that is high during the light phase and low during the dark phase. Furthermore, melatonin administration significantly suppressed NMU mRNA expression in the PT in vivo. On the other hand, 48 h fasting did not have an effect on PT-NMU mRNA expression, and the diurnal change of NMU mRNA expression was maintained. We also found the highest expression of neuromedin U receptor type 2 (NMUR2) mRNA in the third ventricle ependymal cell layer, followed by the arcuate nucleus and the spinal cord. These results suggest that NMU mRNA expression in the PT is downregulated by melatonin during the dark phase and shows diurnal change. Considering that NMU mRNA in the PT showed the highest expression level in the brain, PT-NMU may act on NMUR2 in the brain, especially in the third ventricle ependymal cell layer, with a circadian rhythm. PMID:23843987

  13. Negative Regulation of Neuromedin U mRNA Expression in the Rat Pars Tuberalis by Melatonin

    PubMed Central

    Aizawa, Sayaka; Sakata, Ichiro; Nagasaka, Mai; Higaki, Yuriko; Sakai, Takafumi

    2013-01-01

    The pars tuberalis (PT) is part of the anterior pituitary gland surrounding the median eminence as a thin cell layer. The characteristics of PT differ from those of the pars distalis (PD), such as cell composition and gene expression, suggesting that the PT has a unique physiological function compared to the PD. Because the PT highly expresses melatonin receptor type 1, it is considered a mediator of seasonal and/or circadian signals of melatonin. Expression of neuromedin U (NMU) that is known to regulate energy balance has been previously reported in the rat PT; however, the regulatory mechanism of NMU mRNA expression and secretion in the PT are still obscure. In this study, we examined both the diurnal change of NMU mRNA expression in the rat PT and the effects of melatonin on NMU in vivo. In situ hybridization and quantitative PCR analysis of laser microdissected PT samples revealed that NMU mRNA expression in the PT has diurnal variation that is high during the light phase and low during the dark phase. Furthermore, melatonin administration significantly suppressed NMU mRNA expression in the PT in vivo. On the other hand, 48 h fasting did not have an effect on PT-NMU mRNA expression, and the diurnal change of NMU mRNA expression was maintained. We also found the highest expression of neuromedin U receptor type 2 (NMUR2) mRNA in the third ventricle ependymal cell layer, followed by the arcuate nucleus and the spinal cord. These results suggest that NMU mRNA expression in the PT is downregulated by melatonin during the dark phase and shows diurnal change. Considering that NMU mRNA in the PT showed the highest expression level in the brain, PT-NMU may act on NMUR2 in the brain, especially in the third ventricle ependymal cell layer, with a circadian rhythm. PMID:23843987

  14. Hepatitis B Virus X Protein Induces Hepatic Steatosis by Enhancing the Expression of Liver Fatty Acid Binding Protein

    PubMed Central

    Wu, Yun-li; Peng, Xian-e; Zhu, Yi-bing; Yan, Xiao-li; Chen, Wan-nan

    2015-01-01

    ABSTRACT Hepatitis B virus (HBV) has been implicated as a potential trigger of hepatic steatosis although molecular mechanisms involved in the pathogenesis of HBV-associated hepatic steatosis still remain elusive. Our prior work has revealed that the expression level of liver fatty acid binding protein 1 (FABP1), a key regulator of hepatic lipid metabolism, was elevated in HBV-producing hepatoma cells. In this study, the effects of HBV X protein (HBx) mediated FABP1 regulation on hepatic steatosis and the underlying mechanism were determined. mRNA and protein levels of FABP1 were measured by quantitative RT-PCR (qPCR) and Western blotting. HBx-mediated FABP1 regulation was evaluated by luciferase assay, coimmunoprecipitation, and chromatin immunoprecipitation. Hepatic lipid accumulation was measured by using Oil-Red-O staining and the triglyceride level. It was found that expression of FABP1 was increased in HBV-producing hepatoma cells, the sera of HBV-infected patients, and the sera and liver tissues of HBV-transgenic mice. Ectopic overexpression of HBx resulted in upregulation of FABP1 in HBx-expressing hepatoma cells, whereas HBx abolishment reduced FABP1 expression. Mechanistically, HBx activated the FABP1 promoter in an HNF3β-, C/EBPα-, and PPARα-dependent manner, in which HBx increased the gene expression of HNF3β and physically interacted with C/EBPα and PPARα. On the other hand, knockdown of FABP1 remarkably blocked lipid accumulation both in long-chain free fatty acids treated HBx-expressing HepG2 cells and in a high-fat diet-fed HBx-transgenic mice. Therefore, FABP1 is a key driver gene in HBx-induced hepatic lipid accumulation via regulation of HNF3β, C/EBPα, and PPARα. FABP1 may represent a novel target for treatment of HBV-associated hepatic steatosis. IMPORTANCE Accumulating evidence from epidemiological and experimental studies has indicated that chronic HBV infection is associated with hepatic steatosis. However, the molecular mechanism

  15. 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

  16. Effect of Cheonggukjang supplementation upon hepatic acyl-CoA synthase, carnitine palmitoyltransferase I, acyl-CoA oxidase and uncoupling protein 2 mRNA levels in C57BL/6J mice fed with high fat diet

    PubMed Central

    Soh, Ju-Ryoun; Shin, Dong-Hwa; Kwon, Dae Young

    2007-01-01

    This study investigated the effect of Cheonggukjang on mRNA levels of hepatic acyl-CoA synthase (ACS), carnitine palmitoyltransferase I (CPT-I), acyl-CoA oxidase (ACO) and uncoupling protein 2 (UCP2), and on serum lipid profiles in C57BL/6J mice. Thirty male C57BL/6J mice were divided into three groups; normal diet (ND), high fat diet (HD) and high fat diet with 40% Cheonggukjang (HDC). Energy intake was significantly higher in the HDC group than in the ND and HD groups. The HDC group normalized in weight gain, epididymal and back fat (g/100 g) accumulation which are increased by high fat diet. Serum concentrations of triglyceride and total cholesterol in the HDC were significantly lower than those in the HD group. These results were confirmed by hepatic mRNA expression of enzymes and protein (ACS, CPT-1, ACO, UCP2) which is related with lipid metabolism by RT-PCR. Hepatic CPT-I, ACO and UCP2 mRNA expression was increased by Cheonggukjang supplementation. We demonstrated that Cheonggukjang supplement leads to increased mRNA expressions of enzymes and protein involved in fatty acid oxidation in liver, reduced accumulation of body fat and improvement of serum lipids in high fat diet fed mice. PMID:18850232

  17. 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. PMID:7852311

  18. Oral MSG administration alters hepatic expression of genes for lipid and nitrogen metabolism in suckling piglets.

    PubMed

    Chen, Gang; Zhang, Jun; Zhang, Yuzhe; Liao, Peng; Li, Tiejun; Chen, Lixiang; Yin, Yulong; Wang, Jinquan; Wu, Guoyao

    2014-01-01

    This experiment was conducted to investigate the effects of oral administration of monosodium glutamate (MSG) on expression of genes for hepatic lipid and nitrogen metabolism in piglets. A total of 24 newborn pigs were assigned randomly into one of four treatments (n = 6/group). The doses of oral MSG administration, given at 8:00 and 18:00 to sow-reared piglets between 0 and 21 days of age, were 0 (control), 0.06 (low dose), 0.5 (intermediate dose), and 1 (high dose) g/kg body weight/day. At the end of the 3-week treatment, serum concentrations of total protein and high-density lipoprotein cholesterol in the intermediate dose group were elevated than those in the control group (P < 0.05). Hepatic mRNA levels for fatty acid synthase, acetyl-coA carboxylase, insulin-like growth factor-1, glutamate-oxaloacetate transaminase, and glutamate-pyruvate transaminase were higher in the middle-dose group (P < 0.05), compared with the control group. MSG administration did not affect hepatic mRNA levels for hormone-sensitive lipase or carnitine palmitoyl transferase-1. We conclude that oral MSG administration alters hepatic expression of certain genes for lipid and nitrogen metabolism in suckling piglets. PMID:24221354

  19. Intrahepatic mRNA Expression of FAS, FASL, and FOXP3 Genes Is Associated with the Pathophysiology of Chronic HCV Infection

    PubMed Central

    Amoras, Ednelza da Silva Graça; Gomes, Samara Tatielle Monteiro; Freitas, Felipe Bonfim; Santana, Bárbara Brasil; Ishak, Geraldo; Ferreira de Araújo, Marialva Tereza; Demachki, Sâmia; Conde, Simone Regina Souza da Silva; Ishak, Marluísa de Oliveira Guimarães; Ishak, Ricardo; Vallinoto, Antonio Carlos Rosário

    2016-01-01

    This study aimed to evaluate the relative mRNA expression of Fas receptor (FAS), Fas ligand (FASL), and forkhead box protein 3 (FOXP3) in liver biopsy specimens obtained from patients with viral and non-viral chronic hepatitis and correlate their expression with the fibrosis stage. A total of 51 liver biopsy specimens obtained from HBV (n = 6), HCV (n = 28), and non-viral hepatic disease (NVHD) (n = 9) patients and from individuals with normal liver histology (n = 8) (control—CT) were analyzed. Quantifications of the target genes were assessed using qPCR, and liver biopsies according to the METAVIR classification. The mRNA expression levels of FAS and FASL were lower in the CT group compared to the groups of patients. The increase in the mRNA expression of FAS and FASL was correlated with higher levels of inflammation and disease progression, followed by a decline in tissues with cirrhosis, and it was also associated with increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Higher mRNA expression of FOXP3 was observed in the HCV and NVHD groups, with the peak observed among patients with cirrhosis. The increased FOXP3 mRNA expression was positively correlated with increased FAS and FASL mRNA expression and the AST and ALT levels in all patients. Conclusions: These results suggest that regardless of the cause, the course of chronic liver disease may be modulated by the analyzed genes and correlated with an increase in regulatory T cells during the liver damage followed by hepatocyte destruction by Fas/FasL system and subsequent non specific lymphocytic infiltrate accumulation. PMID:27243827

  20. Glial cell line-derived neurotrophic factor protects against high-fat diet-induced hepatic steatosis by suppressing hepatic PPAR-γ expression.

    PubMed

    Mwangi, Simon Musyoka; Peng, Sophia; Nezami, Behtash Ghazi; Thorn, Natalie; Farris, Alton B; Jain, Sanjay; Laroui, Hamed; Merlin, Didier; Anania, Frank; Srinivasan, Shanthi

    2016-01-15

    Glial cell line-derived neurotrophic factor (GDNF) protects against high-fat diet (HFD)-induced hepatic steatosis in mice, however, the mechanisms involved are not known. In this study we investigated the effects of GDNF overexpression and nanoparticle delivery of GDNF in mice on hepatic steatosis and fibrosis and the expression of genes involved in the regulation of hepatic lipid uptake and de novo lipogenesis. Transgenic overexpression of GDNF in liver and other metabolically active tissues was protective against HFD-induced hepatic steatosis. Mice overexpressing GDNF had significantly reduced P62/sequestosome 1 protein levels suggestive of accelerated autophagic clearance. They also had significantly reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) and CD36 gene expression and protein levels, and lower expression of mRNA coding for enzymes involved in de novo lipogenesis. GDNF-loaded nanoparticles were protective against short-term HFD-induced hepatic steatosis and attenuated liver fibrosis in mice with long-standing HFD-induced hepatic steatosis. They also suppressed the liver expression of steatosis-associated genes. In vitro, GDNF suppressed triglyceride accumulation in Hep G2 cells through enhanced p38 mitogen-activated protein kinase-dependent signaling and inhibition of PPAR-γ gene promoter activity. These results show that GDNF acts directly in the liver to protect against HFD-induced cellular stress and that GDNF may have a role in the treatment of nonalcoholic fatty liver disease. PMID:26564715

  1. Virus-specific mRNA capping enzyme encoded by hepatitis E virus.

    PubMed

    Magden, J; Takeda, N; Li, T; Auvinen, P; Ahola, T; Miyamura, T; Merits, A; Kääriäinen, L

    2001-07-01

    Hepatitis E virus (HEV), a positive-strand RNA virus, is an important causative agent of waterborne hepatitis. Expression of cDNA (encoding amino acids 1 to 979 of HEV nonstructural open reading frame 1) in insect cells resulted in synthesis of a 110-kDa protein (P110), a fraction of which was proteolytically processed to an 80-kDa protein. P110 was tightly bound to cytoplasmic membranes, from which it could be released by detergents. Immunopurified P110 catalyzed transfer of a methyl group from S-adenosylmethionine (AdoMet) to GTP and GDP to yield m(7)GTP or m(7)GDP. GMP, GpppG, and GpppA were poor substrates for the P110 methyltransferase. There was no evidence for further methylation of m(7)GTP when it was used as a substrate for the methyltransferase. P110 was also a guanylyltransferase, which formed a covalent complex, P110-m(7)GMP, in the presence of AdoMet and GTP, because radioactivity from both [alpha-(32)P]GTP and [(3)H-methyl]AdoMet was found in the covalent guanylate complex. Since both methyltransferase and guanylyltransferase reactions are strictly virus specific, they should offer optimal targets for development of antiviral drugs. Cap analogs such as m(7)GTP, m(7)GDP, et(2)m(7)GMP, and m(2)et(7)GMP inhibited the methyltransferase reaction. HEV P110 capping enzyme has similar properties to the methyltransferase and guanylyltransferase of alphavirus nsP1, tobacco mosaic virus P126, brome mosaic virus replicase protein 1a, and bamboo mosaic virus (a potexvirus) nonstructural protein, indicating there is a common evolutionary origin of these distantly related plant and animal virus families. PMID:11413290

  2. 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

    2016-01-01

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

  3. Hepatic AQP9 expression in male rats is reduced in response to PPARα agonist treatment.

    PubMed

    Lebeck, Janne; Cheema, Muhammad Umar; Skowronski, Mariusz T; Nielsen, Søren; Praetorius, Jeppe

    2015-02-01

    The peroxisome proliferator receptor α (PPARα) is a key regulator of the hepatic response to fasting with effects on both lipid and carbohydrate metabolism. A role in hepatic glycerol metabolism has also been found; however, the results are somewhat contradictive. Aquaporin 9 (AQP9) is a pore-forming transmembrane protein that facilitates hepatic uptake of glycerol. Its expression is inversely regulated by insulin in male rodents, with increased expression during fasting. Previous results indicate that PPARα plays a crucial role in the induction of AQP9 mRNA during fasting. In the present study, we use PPARα agonists to explore the effect of PPARα activation on hepatic AQP9 expression and on the abundance of enzymes involved in glycerol metabolism using both in vivo and in vitro systems. In male rats with free access to food, treatment with the PPARα agonist WY 14643 (3 mg·kg(-1)·day(-1)) caused a 50% reduction in hepatic AQP9 abundance with the effect being restricted to AQP9 expressed in periportal hepatocytes. The pharmacological activation of PPARα had no effect on the abundance of GlyK, whereas it caused an increased expression of hepatic GPD1, GPAT1, and L-FABP protein. In WIF-B9 and HepG2 hepatocytes, both WY 14643 and another PPARα agonist GW 7647 reduced the abundance of AQP9 protein. In conclusion, pharmacological PPARα activation results in a marked reduction in the abundance of AQP9 in periportal hepatocytes. Together with the effect on the enzymatic apparatus for glycerol metabolism, our results suggest that PPARα activation in the fed state directs glycerol into glycerolipid synthesis rather than into de novo synthesis of glucose. PMID:25477377

  4. The Expression of Hepatic Carboxypeptidase E is Decreased in Patients with Cholesterol Gallstone

    PubMed Central

    Dai, Shu-Long; Zhou, Jin; Yang, Kun-Xing; Yang, Shi-Yong

    2015-01-01

    Background/Aims: Decreased carboxypeptidase E (CPE) expression is associated with numerous pathophysiological conditions. This study aimed to investigate the potential function of hepatic CPE in cholesterol gallstone formation. Patients and Methods: Patients with cholesterol gallstone (CGS group) and patients without cholesterol gallstones (non-CGS group) were enrolled. The serum total cholesterol, triglyceride, and biliary composition were analyzed. Eight liver samples from two patients without CGS and six patients with CGS were subjected to cDNA microarray analysis. Hepatic CPE expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemical analysis. Plasma CCK level was measured by ELISA. Results: cDNA microarray identified CPE as a gene downregulated in the CGS group. RT-PCR showed that CPE mRNA level was lower in CGS group than in control (P < 0.05, t-test). Moreover, Western blot and immunohistochemistry analysis showed that CPE protein level was significantly lower in CGS group than in the control group. In addition, plasma CCK level was lower in CGS group than in the control group. A positive correlation was found between serum CCK level and hepatic CPE mRNA level (r2 = 0.713, P = 0.003). Conclusions: Down-expression of liver CPE may reduce the secretion of serum CCK and contribute to the formation of cholesterol gallstone. PMID:26228366

  5. Ontogeny of Human Hepatic and Intestinal Transporter Gene Expression during Childhood: Age Matters

    PubMed Central

    Mooij, Miriam G.; Schwarz, Ute I.; de Koning, Barbara A. E.; Leeder, J. Steven; Gaedigk, Roger; Samsom, Janneke N.; Spaans, Edwin; van Goudoever, Johannes B.; Tibboel, Dick; Kim, Richard B.

    2014-01-01

    Many drugs prescribed to children are drug transporter substrates. Drug transporters are membrane-bound proteins that mediate the cellular uptake or efflux of drugs and are important to drug absorption and elimination. Very limited data are available on the effect of age on transporter expression. Our study assessed age-related gene expression of hepatic and intestinal drug transporters. Multidrug resistance protein 2 (MRP2), organic anion transporting polypeptide 1B1 (OATP1B1), and OATP1B3 expression was determined in postmortem liver samples (fetal n = 6, neonatal n = 19, infant n = 7, child n = 2, adult n = 11) and multidrug resistance 1 (MDR1) expression in 61 pediatric liver samples. Intestinal expression of MDR1, MRP2, and OATP2B1 was determined in surgical small bowel samples (neonates n = 15, infants n = 3, adults n = 14). Using real-time reverse-transcription polymerase chain reaction, we measured fetal and pediatric gene expression relative to 18S rRNA (liver) and villin (intestines), and we compared it with adults using the 2−∆∆Ct method. Hepatic expression of MRP2, OATP1B1, and OATP1B3 in all pediatric age groups was significantly lower than in adults. Hepatic MDR1 mRNA expression in fetuses, neonates, and infants was significantly lower than in adults. Neonatal intestinal expressions of MDR1 and MRP2 were comparable to those in adults. Intestinal OATP2B1 expression in neonates was significantly higher than in adults. We provide new data that show organ- and transporter-dependent differences in hepatic and intestinal drug transporter expression in an age-dependent fashion. This suggests that substrate drug absorption mediated by these transporters may be subject to age-related variation in a transporter dependent pattern. PMID:24829289

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

    PubMed

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

    2013-06-01

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

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

    PubMed Central

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

    2014-01-01

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

  8. Alternative splicing of parathyroid hormone-related protein mRNA: expression and stability

    PubMed Central

    Sellers, R S; Luchin, A I; Richard, V; Brena, R M; Lima, D; Rosol, T J

    2011-01-01

    Parathyroid hormone-related protein (PTHrP) is a multifunctional protein that is often dysregulated in cancer. The human PTHrP gene is alternatively spliced into three isoforms, each with a unique 3′-untranslated region (3′-UTR), encoding 139, 173 and 141 amino acid proteins. The regulation of PTHrP mRNA isoform expression has not been completely elucidated, but it may be affected by transforming growth factor-β1 (TGF-β1). In this study, we examined differences in the PTHrP mRNA isoform expression in two squamous carcinoma cell lines (SCC2/88 and HARA), an immortalized keratinocyte cell line (HaCaT), and spontaneous human lung cancer with adjacent normal tissue. In addition, the effect of TGF-β1 on PTHrP mRNA isoform expression and stability was examined. Cell-type specific expression of PTHrP mRNA isoforms occurred between the various cell lines, normal human lung, and immortalized human keratinocytes (HaCaT). PTHrP isoform expression pattern was significantly altered between normal lung tissue and the adjacent lung cancer. In vitro studies revealed that TGF-β1 differentially altered the mRNA steady-state levels and mRNA stability of the PTHrP isoforms. Protein–RNA binding studies identified different proteins binding to the 3′-UTR of the PTHrP isoforms (139) and (141), which may be important in the differential mRNA stability and response to cytokines between the PTHrP isoforms. The data demonstrate that there is cell-type specific expression of PTHrP mRNA isoforms, and disruption of the normal regulation during cancer progression may in part be associated with TGF-β1-induced changes in PTHrP mRNA isoform expression and stability. PMID:15291755

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

    PubMed Central

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

    2014-01-01

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

  10. 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.

  11. Microarray analysis of hepatic gene expression identifies new genes involved in steatotic liver

    PubMed Central

    Guillén, Natalia; Navarro, María A.; Arnal, Carmen; Noone, Enda; Arbonés-Mainar, José M.; Acín, Sergio; Surra, Joaquín C.; Muniesa, Pedro; Roche, Helen M.; Osada, Jesús

    2009-01-01

    Trans-10, cis-12-conjugated linoleic acid (CLA)-enriched diets promote fatty liver in mice, while cis-9, trans-11-CLA ameliorates this effect, suggesting regulation of multiple genes. To test this hypothesis, apoE-deficient mice were fed a Western-type diet enriched with linoleic acid isomers, and their hepatic gene expression was analyzed with DNA microarrays. To provide an initial screening of candidate genes, only 12 with remarkably modified expression between both CLA isomers were considered and confirmed by quantitative RT-PCR. Additionally mRNA expression of 15 genes involved in lipid metabolism was also studied. Ten genes (Fsp27, Aqp4, Cd36, Ly6d, Scd1, Hsd3b5, Syt1, Cyp7b1, and Tff3) showed significant associations among their expressions and the degree of hepatic steatosis. Their involvement was also analyzed in other models of steatosis. In hyperhomocysteinemic mice lacking Cbs gene, only Fsp27, Cd36, Scd1, Syt1, and Hsd3b5 hepatic expressions were associated with steatosis. In apoE-deficient mice consuming olive-enriched diet displaying reduction of the fatty liver, only Fsp27 and Syt1 expressions were found associated. Using this strategy, we have shown that expression of these genes is highly associated with hepatic steatosis in a genetic disease such as Cbs deficiency and in two common situations such as Western diets containing CLA isomers or a Mediterranean-type diet. Conclusion: The results highlight new processes involved in lipid handling in liver and will help to understand the complex human pathology providing new proteins and new strategies to cope with hepatic steatosis. PMID:19258494

  12. Expression of lipoprotein lipase mRNA and secretion in macrophages isolated from human atherosclerotic aorta.

    PubMed

    Mattsson, L; Johansson, H; Ottosson, M; Bondjers, G; Wiklund, O

    1993-10-01

    The expression of lipoprotein lipase (LPL) mRNA and the LPL activity were studied in macrophages (CD14 positive) from human atherosclerotic tissue. Macrophages were isolated after collagenase digestion by immunomagnetic isolation. About 90% of the cells were foam cells with oil red O positive lipid droplets. To analyze the mRNA expression, PCR with specific primers for LPL was used. Arterial macrophages were analyzed directly after isolation and the data showed low expression of LPL mRNA when compared with monocyte-derived macrophages. To induce the expression of LPL mRNA in macrophages, PMA was used. When incubating arterial macrophages with PMA for 24 h we could not detect any increase in LPL mRNA levels. Similarly, the cells secreted very small amounts of LPL even after PMA stimulation. In conclusion, these studies show a very low expression of LPL mRNA in the CD14-positive macrophage-derived foam cells isolated from human atherosclerotic tissue. These data suggest that the CD14-positive cells are a subpopulation of foam cells that express low levels of lipoprotein lipase, and the lipid content could be a major factor for downregulation of LPL. However, the cells were isolated from advanced atherosclerotic lesions, and these findings may not reflect the situation in early fatty streaks. PMID:8408628

  13. Expression of D2 dopamine receptor mRNA in the arterial chemoreceptor afferent pathway.

    PubMed

    Czyzyk-Krzeska, M F; Lawson, E E; Millhorn, D E

    1992-11-01

    Dopamine is a major neurotransmitter in the arterial chemoreceptor pathway. In the present study we wished to determine if messenger RNAs for dopamine D1 and D2 receptor are expressed in carotid body (type I cells), in sensory neurons of the petrosal ganglion which innervate the carotid body and in sympathetic neurons of the superior cervical ganglion. We failed to detect D1 receptor mRNA in any of these tissues. However, we found that D2 receptor mRNA was expressed by dopaminergic carotid body type I cells. D2 receptor mRNA was also found in petrosal ganglion neurons that innervated the carotid sinus and carotid body. In addition, a large number of sympathetic postganglionic neurons in the superior cervical ganglion expressed D2 receptor mRNA. PMID:1362730

  14. Differential Fmo3 Gene Expression in Various Liver Injury Models Involving Hepatic Oxidative Stress in Mice

    PubMed Central

    Rudraiah, Swetha; Moscovitz, Jamie E.; Donepudi, Ajay C.; Campion, Sarah N.; Slitt, Angela L.; Aleksunes, Lauren M.; Manautou, José E.

    2015-01-01

    Flavin-containing monooxygenase-3 (FMO3) catalyzes metabolic reactions similar to cytochrome P450 monooxygenase however, most metabolites of FMO3 are considered non-toxic. Recent findings in our laboratory demonstrated Fmo3gene induction following toxic acetaminophen (APAP) treatment in mice.The goal of this study was to evaluate Fmo3gene expression in diverseother mouse models of hepatic oxidative stress and injury. Fmo3 gene regulation by Nrf2 was also investigated using Nrf2 knockout (Nrf2 KO) mice. In our studies, male C57BL/6J mice were treated with toxic dosesof hepatotoxicants or underwent bile duct ligation (BDL, 10d). Hepatotoxicants included APAP (400 mg/kg, 24 to 72h), alpha-naphthylisothiocyanate (ANIT; 50 mg/kg, 2 to 48h), carbontetrachloride (CCl4;10 or 30 μL/kg, 24 and 48h) and allyl alcohol (AlOH; 30 or 60 mg/kg, 6 and 24h). Because oxidative stress activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2), additional studies investigated Fmo3 gene regulation by Nrf2 using Nrf2 knockout (Nrf2 KO) mice. At appropriate time-points, blood and liver samples were collected for assessment of plasma alanine aminotransferase (ALT) activity, plasma and hepatic bile acid levels, as well as liver Fmo3 mRNA and protein expression. Fmo3 mRNA expression increased significantly by 43-fold at 12h after ANIT treatment,and this increase translates to a 4-fold change in protein levels. BDL also increased Fmo3 mRNA expression by 1899-fold, but with no change in protein levels. Treatment of mice with CCl4decreased liver Fmo3gene expression, whileno change in expression was detected with AlOH treatment. Nrf2 KO mice are more susceptible to APAP (400 mg/kg, 72h) treatment compared to their wild-type (WT) counterparts, which is evidenced by greater plasma ALT activity. Fmo3 mRNA and protein expression increased in Nrf2 KO mice after APAP treatment. Collectively, not all hepatotoxicantsthat produce oxidative stress alter Fmo3gene expression. Along with APAP, toxic ANIT

  15. Changes in hepatic lipid parameters and hepatic messenger ribonucleic acid expression following estradiol administration in laying hens (Gallus domesticus).

    PubMed

    Lee, B K; Kim, J S; Ahn, H J; Hwang, J H; Kim, J M; Lee, H T; An, B K; Kang, C W

    2010-12-01

    Fatty liver hemorrhagic syndrome (FLHS) is characterized by increased hepatic triacylglycerol content associated with liver hemorrhages and results in a sudden decline in egg production. Genetic, environmental, nutritional, and hormonal factors have all been implicated in the etiology of FLHS, but the exact cause of FLHS is still unknown. Estrogens have been implicated in the development of excess fat content of the liver and in the etiology of FLHS. This study investigated estradiol (E(2)) administration in hens and its effect on lipid metabolism. Hy-Line Brown laying hens were intramuscularly injected with E(2) on a daily basis for 3 wk. The dosages were 0, 0.5, and 1.0 mg/kg of BW, with corn oil injections used as a control. Egg production and quality were measured among the groups, with no significant difference seen in egg production. Liver weights of hens treated with E(2) were greater than those of control hens, but the increase was not statistically significant. Serum glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase activities and E(2) plasma concentrations increased in a dose-dependent manner, with plasma concentration of E(2) increasing from 6,900 to 19,000 pg/mL. No significant differences in free cholesterol or phospholipids were observed, but there was a significant increase in hepatic triacylglycerol levels. Injection with E(2) showed an increased expression of mRNA for peroxisome proliferator-activated receptor γ (23-fold), but not for peroxisome proliferator-activated receptor α. A statistically significant increase was seen for fatty acid synthase, apolipoprotein B, and adenosine triphosphate citrate lyase, but not for acetyl coenzyme A carboxylase, apolipoprotein VLDL-II, microsomal triglyceride transport protein, or malic enzyme. For proteins involved in the oxidation of E(2), only cytochrome P450 3A37 showed a statistically significant increase. The present results suggest that E(2) upregulates the synthesis of fatty acids

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

    PubMed

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

    2016-03-01

    To evaluate the effects of dietary Molybdenum (Mo) or/and Cadmium (Cd) on trace elements and the mRNA expression levels of heat shock proteins (Hsps) and inflammatory cytokines in duck livers. 240 healthy 11-day-old ducks were randomly divided into six groups with 40 ducks in each group, which were treated with Mo or/and Cd at different doses on the basal diet for 120 days. On days 30, 60, 90 and 120, 10 birds in each group were randomly selected and euthanized and then the livers were collected to determine the contents of Mo, Cd, copper (Cu), iron (Fe), zine (Zn), Selenium (Se) and the mRNA expression levels of Hsps, inflammatory cytokines. In addition, liver tissues at 120 days were subjected to histopathological analysis with the optical microscope. The results showed that the mRNA expression of Hsp60, Hsp70, Hsp90, tumor necrosis factor-α (TNF-α), nuclear factor-kappa B (NF-κB), and cyclooxygenase-2 (COX-2) were significantly (P<0.01) upregulated in combination groups; Contents of Cu, Fe, Zn, and Se decreased in combined groups (P<0.05) in the later period of the test while contents of Mo and Cd significantly increased (P<0.01); Furthermore severe hepatocyte diffuse fatty, hepatic cords swelling, hepatic sinusoid disappeared, and inflammatory cells infiltrated around the hepatic central vein were observed in Mo combined with Cd groups. The results indicated that dietary Mo or/and Cd might lead to stress, inflammatory response, tissue damage and disturb homeostasis of trace elements in duck livers. Moreover the two elements showed a possible synergistic relationship. And the high mRNA expression of HSPs and inflammatory cytokines may play a role in the resistance of liver toxicity induced by Mo and Cd. PMID:26682514

  17. Ribozyme cleaves rex/tax mRNA and inhibits bovine leukemia virus expression.

    PubMed Central

    Cantor, G H; McElwain, T F; Birkebak, T A; Palmer, G H

    1993-01-01

    Bovine leukemia virus (BLV) encodes at least two regulatory proteins, Rex and Tax. Tax, the transactivating protein, stimulates the long terminal repeat to promote viral transcription and may be involved in tumorigenesis. Rex is involved in the transition from early expression of regulatory proteins to later expression of viral structural proteins. We have targeted ribozymes against the mRNA encoding Rex and Tax. The ribozymes consist of the hammer-head catalytic motif flanked by antisense sequences that hybridize with the complementary rex/tax mRNA. To evaluate cleavage in a cell-free system, we transcribed portions of rex/tax mRNA and incubated them with synthetic RNA ribozymes. A ribozyme was identified that cleaves > 80% of the target RNA. Synthetic DNA encoding this ribozyme was cloned into the expression vector pRc/RSV and transfected into BLV-infected bat lung cells. Intracellular cleavage of rex/tax mRNA was confirmed by reverse transcriptase PCR. In cells expressing the ribozyme, viral expression was markedly inhibited. Expression of the BLV core protein p24 was inhibited by 61%, and reverse transcriptase activity in supernatant was inhibited by 92%. Ribozyme inhibition of BLV expression suggests that cattle expressing these sequences may be able to control BLV replication. Images Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:7504287

  18. Unification of gene expression data applying standard mRNA quantification references for comparable analyses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High throughput quantitative measurements of gene expression data have problems of reproducibility and comparability due to a lack of standard mRNA quantification references. Efforts have been made to safeguard data fidelity, yet generating quality expression data of inherent value remains a challe...

  19. Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

    SciTech Connect

    Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI; Roskams, Tania; Oben, Jude A.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by

  20. Stability regulation of mRNA and the control of gene expression.

    PubMed

    Cheadle, Chris; Fan, Jinshui; Cho-Chung, Yoon S; Werner, Thomas; Ray, Jill; Do, Lana; Gorospe, Myriam; Becker, Kevin G

    2005-11-01

    Microarray technology has become highly valuable for identifying complex global changes in gene expression patterns. Standard techniques measure changes in total cellular poly(A) mRNA levels. The assumption that changes in gene expression as measured by these techniques are directly and well correlated with changes in rates of new gene synthesis form the basis of attempts to connect coordinated changes in gene expression with shared transcription regulatory elements. Yet systematic attempts at this approach remain difficult to demonstrate convincingly. One reason for this difficulty may result from the intricate convergence of both transcriptional and mRNA turnover events which, together, directly influence steady-state mRNA levels. Recent technical advances have led to the successful scale-up and application of nuclear run-on procedures directly to microarrays. This development has allowed a gene-by-gene comparison between new gene synthesis in the nucleus and measured changes in total cellular polyA mRNA. Results from these studies have begun to challenge the strict interpretation of changes in gene expression measured by conventional microarrays as being closely correlated with changes in mRNA transcription rate, but rather they tend to support the significant expansion of the role played by changes in mRNA stability regulation to standard analyses of gene expression. Gene expression profiles obtained from both polyA mRNA (whole-cell) and nuclear run-on (newly transcribed) RNA across a time course of one hour following the activation of human Jurkat T cells with PMA plus ionomycin revealed that regulation of mRNA stability may account for as much as 50% of all measurements of changes in total cellular polyA mRNA in this system. Stability regulation was inferred by the absence of corresponding regulation of nuclear gene transcription activity for groups of genes strongly regulated at the whole cell level and which were also resistant to inhibition by Actinomycin

  1. Gemin5 Binds to the Survival Motor Neuron mRNA to Regulate SMN Expression.

    PubMed

    Workman, Eileen; Kalda, Caitlin; Patel, Aalapi; Battle, Daniel J

    2015-06-19

    Reduced expression of SMN causes spinal muscular atrophy, a severe neurodegenerative disease. Despite the importance of maintaining SMN levels, relatively little is known about the mechanisms by which SMN levels are regulated. We show here that Gemin5, the snRNA-binding protein of the SMN complex, binds directly to the SMN mRNA and regulates SMN expression. Gemin5 binds with high specificity, both in vitro and in vivo, to sequence and structural elements in the SMN mRNA 3'-untranslated region that are reminiscent of the snRNP code to which Gemin5 binds on snRNAs. Reduction of Gemin5 redistributes the SMN mRNA from heavy polysomes to lighter polysomes and monosomes, suggesting that Gemin5 functions as an activator of SMN translation. SMN protein is not stoichiometrically present on the SMN mRNA with Gemin5, but the mRNA-binding activity of Gemin5 is dependent on SMN levels, providing a feedback mechanism for SMN to regulate its own expression via Gemin5. This work both reveals a new autoregulatory pathway governing SMN expression, and identifies a new mechanism through which SMN can modulate specific mRNA expression via Gemin5. PMID:25911097

  2. 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.

  3. 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. PMID:26681690

  4. 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

  5. Variations in cytokine mRNA expression during normal human pregnancy

    PubMed Central

    Kruse, N; Greif, M; Moriabadi, N F; Marx, L; Toyka, K V; Rieckmann, P

    2000-01-01

    Epidemiological data provide evidence that disease activity of T cell-mediated, organ-specific autoimmune diseases is reduced during pregnancy. Although there are several experimental animal studies on the effect of pregnancy on the immune system, the situation in humans is less clear. We therefore performed a prospective analysis of cytokine mRNA expression in whole blood by a new on-line reverse transcriptase-polymerase chain reaction technique and of serum hormone levels during pregnancy in healthy women. The control group included age-matched non-pregnant healthy women. Quantitativecytokine mRNA expression revealed significantly reduced IL-18, interferon-gamma (IFN-γ), and IL-2 mRNA levels in the first and second trimester in pregnancy compared with non-pregnant women. No difference between groups was detected for tumour necrosis factor-alpha (TNF-α) mRNA. IL-4 and IL-10 mRNA were detected at low levels in only 20% of pregnant women and were reduced to a statistically significant extent in the second and third trimester compared with the control group. Changes in IL-18 mRNA expression correlated inversely with serum values for human choriogonadotropin (HCG) and IL-10 serum levels correlated with increases in serum 17β-oestradiol levels. These data indicate immunomodulatory effects of pregnancy at the cytokine level which may be related to the variations in the clinical course of organ-specific, T cell-mediated autoimmune diseases during pregnancy. PMID:10632669

  6. Hepatic phosphoenolpyruvate carboxykinase expression after gastric bypass surgery in rats with type 2 diabetes mellitus.

    PubMed

    Wu, J; Hou, S S; Wang, W; Yin, M; Cheng, N; Ge, L L; Yin, J J; Xu, J

    2015-01-01

    The objective of this study was to investigate the mRNA expression of hepatic phosphoenolpyruvate carboxykinase (PEPCK) after gastric bypass surgery (GBS) in rats with type 2 diabetic mellitus (T2DM). Thirty-six male Goto-Kakizaki rats, aged 12 weeks, were randomly divided into the GBS, sham operation with diet restriction (SO), and sham operation alone (control) groups (N = 12 per group). Liver specimens from all rats were obtained during the operation and 8 weeks after operation. Blood lipid levels were measured before and 8 weeks after operation. Fasting blood glucose (FBG), food intake, and body weight were recorded at weekly time points after operation. The blood glucose area under the curve (AUC) was calculated, and insulin sensitivity indices (ISI) were assessed. The expression PEPCK mRNA and protein were measured by real-time polymerase chain reaction and western blot. Compared with those of the SO and control groups, the blood lipid levels and the FBG in the GBS group was significantly decreased (P < 0.05), as was the AUC (P < 0.05), whereas the ISI was significantly increased (P < 0.05). PEPCK mRNA and protein levels in the GBS group were lower than those in the control group, whereas those in the SO group were significantly higher than those in controls (P < 0.05). In conclusion, GBS can reduce blood glucose in T2DM rats while improving glucose tolerance and hyperglycemia, and the mechanism appears to be associated with a decrease of hepatic PEPCK mRNA and protein expression. PMID:26681041

  7. Insulin regulates enzyme activity, malonyl-CoA sensitivity and mRNA abundance of hepatic carnitine palmitoyltransferase-I.

    PubMed Central

    Park, E A; Mynatt, R L; Cook, G A; Kashfi, K

    1995-01-01

    The regulation of hepatic mitochondrial carnitine palmitoyltransferase-I (CPT-I) was studied in rats during starvation and insulin-dependent diabetes and in rat H4IIE cells. The Vmax. for CPT-I in hepatic mitochondrial outer membranes isolated from starved and diabetic rats increased 2- and 3-fold respectively over fed control values with no change in Km values for substrates. Regulation of malonyl-CoA sensitivity of CPT-I in isolated mitochondrial outer membranes was indicated by an 8-fold increase in Ki during starvation and by a 50-fold increase in Ki in the diabetic state. Peroxisomal and microsomal CPT also had decreased sensitivity to inhibition by malonyl-CoA during starvation. CPT-I mRNA abundance was 7.5 times greater in livers of 48-h-starved rats and 14.6 times greater in livers of insulin-dependent diabetic rats compared with livers of fed rats. In H4IIE cells, insulin increased CPT-I sensitivity to inhibition by malonyl-CoA in 4 h, and sensitivity continued to increase up to 24 h after insulin addition. CPT-I mRNA levels in H4IIE cells were decreased by insulin after 4 h and continued to decrease so that at 24 h there was a 10-fold difference. The half-life of CPT-I mRNA was 4 h in the presence of actinomycin D or with actinomycin D plus insulin. These results suggest that insulin regulates CPT-I by inhibiting transcription of the CPT-I gene. Images Figure 2 Figure 4 PMID:7575418

  8. 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

  9. 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

  10. Thyroid Hormone Regulates the mRNA Expression of Small Heterodimer Partner through Liver Receptor Homolog-1

    PubMed Central

    Ahn, Hwa Young; Kim, Hwan Hee; Kim, Ye An; Kim, Min; Ohn, Jung Hun; Chung, Sung Soo; Lee, Yoon-Kwang; Park, Do Joon; Park, Kyong Soo

    2015-01-01

    Background Expression of hepatic cholesterol 7α-hydroxylase (CYP7A1) is negatively regulated by orphan nuclear receptor small heterodimer partner (SHP). In this study, we aimed to find whether thyroid hormone regulates SHP expression by modulating the transcriptional activities of liver receptor homolog-1 (LRH-1). Methods We injected thyroid hormone (triiodothyronine, T3) to C57BL/6J wild type. RNA was isolated from mouse liver and used for microarray analysis and quantitative real-time polymerase chain reaction (PCR). Human hepatoma cell and primary hepatocytes from mouse liver were used to confirm the effect of T3 in vitro. Promoter assay and electrophoretic mobility-shift assay (EMSA) were also performed using human hepatoma cell line Results Initial microarray results indicated that SHP expression is markedly decreased in livers of T3 treated mice. We confirmed that T3 repressed SHP expression in the liver of mice as well as in mouse primary hepatocytes and human hepatoma cells by real-time PCR analysis. LRH-1 increased the promoter activity of SHP; however, this increased activity was markedly decreased after thyroid hormone receptor β/retinoid X receptor α/T3 administration. EMSA revealed that T3 inhibits specific LRH-1 DNA binding. Conclusion We found that thyroid hormone regulates the expression of SHP mRNA through interference with the transcription factor, LRH-1. PMID:26485468

  11. Achieving sustained virologic response after interferon-free hepatitis C virus treatment correlates with hepatic interferon gene expression changes independent of cirrhosis.

    PubMed

    Meissner, E G; Kohli, A; Virtaneva, K; Sturdevant, D; Martens, C; Porcella, S F; McHutchison, J G; Masur, H; Kottilil, S

    2016-07-01

    Chronic hepatitis C virus (HCV) infection can now be treated with oral directly acting antiviral agents, either with or without ribavirin (RBV). Virologic relapse after treatment can occur, and in some studies was more common in cirrhotic subjects. We previously observed changes in hepatic immunity during interferon (IFN)-free therapy that correlated with favourable outcome in subjects with early liver disease. Here, we compared changes in endogenous IFN pathways during IFN-free, RBV-free therapy between cirrhotic and noncirrhotic subjects. mRNA and microRNA (miRNA) expression analyses were performed on paired pre- and post-treatment liver biopsies from genotype-1 HCV subjects treated with sofosbuvir/ledipasvir (SOF/LDV) for 12 weeks (n = 4, 3 cirrhotics) or SOF/LDV combined with GS-9669 or GS-9451 for 6 weeks (n = 6, 0 cirrhotics). Nine of ten subjects achieved a sustained virologic response (SVR), while one noncirrhotic subject relapsed. Hepatic IFN-stimulated gene expression decreased with treatment in the liver of all subjects, with no observable impact of cirrhosis. Hepatic gene expression of type III IFNs (IFNL1, IFNL3, IFNL4-ΔG) similarly decreased with treatment, while IFNA2 expression, undetectable in all subjects pretreatment, was detected post-treatment in three subjects who achieved a SVR. Only the subject who relapsed had detectable IFNL4-ΔG expression in post-treatment liver. Other IFNs had no change in gene expression (IFNG, IFNB1, IFNA5) or could not be detected. Although expression of multiple hepatic miRNAs changed with treatment, many miRNAs previously implicated in HCV replication and IFN signalling had unchanged expression. In conclusion, favourable treatment outcome during IFN-free HCV therapy is associated with changes in the host IFN response regardless of cirrhosis. PMID:26840694

  12. MicroRNAs shape circadian hepatic gene expression on a transcriptome-wide scale

    PubMed Central

    Du, Ngoc-Hien; Arpat, Alaaddin Bulak; De Matos, Mara; Gatfield, David

    2014-01-01

    A considerable proportion of mammalian gene expression undergoes circadian oscillations. Post-transcriptional mechanisms likely make important contributions to mRNA abundance rhythms. We have investigated how microRNAs (miRNAs) contribute to core clock and clock-controlled gene expression using mice in which miRNA biogenesis can be inactivated in the liver. While the hepatic core clock was surprisingly resilient to miRNA loss, whole transcriptome sequencing uncovered widespread effects on clock output gene expression. Cyclic transcription paired with miRNA-mediated regulation was thus identified as a frequent phenomenon that affected up to 30% of the rhythmic transcriptome and served to post-transcriptionally adjust the phases and amplitudes of rhythmic mRNA accumulation. However, only few mRNA rhythms were actually generated by miRNAs. Overall, our study suggests that miRNAs function to adapt clock-driven gene expression to tissue-specific requirements. Finally, we pinpoint several miRNAs predicted to act as modulators of rhythmic transcripts, and identify rhythmic pathways particularly prone to miRNA regulation. DOI: http://dx.doi.org/10.7554/eLife.02510.001 PMID:24867642

  13. Glucose induces FGF21 mRNA expression through ChREBP activation in rat hepatocytes.

    PubMed

    Iizuka, Katsumi; Takeda, Jun; Horikawa, Yukio

    2009-09-01

    Fibroblast growth factor 21 (FGF21) has beneficial effects of improving the plasma glucose and lipid profiles in diabetic rodents. Here, we investigated carbohydrate response element binding protein (ChREBP) involvement in the regulation of FGF21 mRNA expression in liver. Glucose stimulation and adenoviral overexpression of dominant active ChREBP increased FGF21 mRNA. Consistently, adenoviral expression of dominant negative Mlx inhibited glucose induction of FGF21 mRNA. Furthermore, deletion studies of mouse FGF21 gene promoter (-2000 to +65 bp) revealed a glucose responsive region between -74 and -52 bp. These findings suggest that FGF21 expression is regulated by ChREBP. PMID:19660458

  14. 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.

  15. Effects of hepatitis C virus on suppressor of cytokine signaling mRNA levels: comparison between different genotypes and core protein sequence analysis.

    PubMed

    Pascarella, Stéphanie; Clément, Sophie; Guilloux, Kévin; Conzelmann, Stéphanie; Penin, François; Negro, Francesco

    2011-06-01

    Glucose metabolism disturbances, including insulin resistance and type 2 diabetes, are frequent and important cofactors of hepatitis C. Increasing epidemiological and experimental data suggest that all major genotypes of hepatitis C virus (HCV), albeit to a different extent, cause insulin resistance. The HCV core protein has been shown to be sufficient to impair insulin signaling in vitro through several post-receptorial mechanisms, mostly via the activation of suppressor of cytokine signaling (SOCS) family members and the consequent decrease of insulin receptor substrate-1 (IRS-1). The levels of IRS-1 and SOCS were investigated upon expression of the core protein of HCV genotypes 1-4. Furthermore, the core protein sequences were analyzed to identify the amino acid residues responsible for IRS-1 decrease, with particular regard to SOCS mRNA deregulation. The results suggest that the activation of SOCS family members is a general mechanism associated with the common HCV genotypes. A rare genotype 1b variant, however, failed to activate any of the SOCS tested: this allowed to analyze in detail the distinct amino acid sequences responsible for SOCS deregulation. By combining approaches using intergenotypic chimeras and site-directed mutagenesis, genetic evidence was provided in favor of a role of amino acids 49 and 131 of the HCV core-encoding sequence in mediating SOCS transactivation. PMID:21503913

  16. 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

    Background Berries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and

  17. Hepatic expression of tumour necrosis factor-alpha in chronic hepatitis B virus infection.

    PubMed Central

    Hussain, M J; Lau, J Y; Williams, R; Vergani, D

    1994-01-01

    AIM--To determine the hepatic expression of tumour necrosis factor-alpha (TNF alpha) in patients with chronic hepatitis B virus (HBV) infection. METHODS--Frozen liver biopsy sections from 19 patients with chronic HBV infection were studied, 12 of whom were HBeAg positive and 10 serum HBV DNA positive. Hepatic expression of TNF alpha was determined using immunohistochemistry. RESULTS--Only infiltrating mononuclear cells showed immunoreactive staining for TNF alpha (median 2, range 0-3; n = 19) which appeared as diffuse positive staining material in the cytoplasm. Patients with active liver disease, assessed histologically and biochemically, had a higher level of expression, both in the number of TNF alpha positive cells and the proportion of TNF alpha positive infiltrating mononuclear cells. There was no correlation between the expression of TNF alpha and serological parameters of viral infection (HBeAg and HBV DNA status and HBV DNA concentrations). CONCLUSION--Hepatic expression of TNF alpha is increased in chronic HBV infection and is related to the activity of liver disease and not to the level of HBV replication. Images PMID:7876386

  18. Liver X receptor agonist downregulates hepatic apoM expression in vivo and in vitro

    SciTech Connect

    Zhang Xiaoying; Zhu Zhaojin; Luo Guanghua; Zheng Lu; Nilsson-Ehle, Peter; Xu Ning

    2008-06-20

    It has been demonstrated that apolipoprotein M (apoM), a recently discovered HDL apolipoprotein, has antiatherosclerotic properties, which may be mediated by the enhancement of reversed cholesterol transportation and/or hepatic cholesterol catabolism. The detailed mechanisms are unknown yet. Liver X receptor (LXR) belongs to the nuclear receptor superfamily and is a ligand-activated transcription factor involved in the regulation of lipid metabolism and inflammation. Activation of LXR in the cell cultures results in an enhancement of cholesterol efflux to apoAI. In the present study, we investigated effects of the LXR agonist, T0901317 on hepatic apoM expression in vivo and in vitro. Serum apoM levels in mice given T0901317 at 10 mg or 100 mg/kg for 7 days were reduced by 12-17% (P < 0.05). In HepG2 cell cultures, apoM mRNA levels were significantly lower in presence of 25 {mu}M T0901317 (37.1%) than in control cells (P < 0.001). A similar reduction was found by the addition of 9-cis retinoic acid (RA). Twenty-five micromolar T0901317 together with 100 nM RA decreased apoM mRNA expression by 65% (P < 0.001). Thus, the LXR agonist T0901317 significantly downregulates apoM mRNA expression in vivo and in vitro, which indicates that apoM is another novel target gene regulated by the LXR. The combination of RA and T0901317 showed additive effects, which suggests that apoM expression can be modulated by LXR/RXR pathway.

  19. Peripheral blood mRNA expressions of stress biomarkers in manic episode and subsequent remission.

    PubMed

    Köse Çinar, Rugül; Sönmez, Mehmet Bülent; Görgülü, Yasemin

    2016-08-01

    Theoretical models of the neuroprogressive nature of bipolar disorder (BD) are based on the hypothesis that it is an accelerated aging disease, with the allostatic load playing a major role. Glucocorticoids, oxidative stress markers, inflammatory cytokines and neurotrophins play important roles in BD. The messenger ribonucleic acid (mRNA) expressions of brain-derived neurotrophic factor (BDNF), tissue plasminogen activator (tPA), glucocorticoid receptor (GR), heat shock protein 70 (HSP70), tumour necrosis factor-alpha (TNF-α) were examined in the peripheral blood of 20 adult male, drug-free BD patients during manic and remission periods and in 20 adult male, healthy controls. mRNA expression was measured using the quantitative real-time polymerase chain reaction (qRT-PCR). Compared to the controls, the expressions of BDNF and tPA mRNA were down-regulated in mania. In remission, BNDF and tPA mRNA levels increased, but they were still lower than those of the controls. Between mania and remission periods, only the change in mRNA levels of BDNF reached statistical significance. The results suggest that BDNF and tPA may be biomarkers of BD and that proteolytic conversion of BDNF may be important in the pathophysiology of BD. The change in BDNF levels between mania and remission could be adaptive and used to follow the progression of BD. PMID:27138695

  20. Regulation of Human Hepatic Drug Transporter Activity and Expression by Diesel Exhaust Particle Extract

    PubMed Central

    Le Vee, Marc; Jouan, Elodie; Stieger, Bruno; Lecureur, Valérie; Fardel, Olivier

    2015-01-01

    Diesel exhaust particles (DEPs) are common environmental air pollutants primarily affecting the lung. DEPs or chemicals adsorbed on DEPs also exert extra-pulmonary effects, including alteration of hepatic drug detoxifying enzyme expression. The present study was designed to determine whether organic DEP extract (DEPe) may target hepatic drug transporters that contribute in a major way to drug detoxification. Using primary human hepatocytes and transporter-overexpressing cells, DEPe was first shown to strongly inhibit activities of the sinusoidal solute carrier (SLC) uptake transporters organic anion-transporting polypeptides (OATP) 1B1, 1B3 and 2B1 and of the canalicular ATP-binding cassette (ABC) efflux pump multidrug resistance-associated protein 2, with IC50 values ranging from approximately 1 to 20 μg/mL and relevant to environmental exposure situations. By contrast, 25 μg/mL DEPe failed to alter activities of the SLC transporter organic cation transporter (OCT) 1 and of the ABC efflux pumps P-glycoprotein and bile salt export pump (BSEP), whereas it only moderately inhibited those of sodium taurocholate co-transporting polypeptide and of breast cancer resistance protein (BCRP). Treatment by 25 μg/mL DEPe was next demonstrated to induce expression of BCRP at both mRNA and protein level in cultured human hepatic cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B3, OATP2B1, OCT1 and BSEP. Such changes in transporter expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a reference activator of the aryl hydrocarbon receptor (AhR) pathway. This suggests that DEPe, which is enriched in known ligands of AhR like polycyclic aromatic hydrocarbons, alters drug transporter expression via activation of the AhR cascade. Taken together, these data established human hepatic transporters as targets of organic chemicals containing in DEPs, which may contribute to their

  1. Regulation of human hepatic drug transporter activity and expression by diesel exhaust particle extract.

    PubMed

    Le Vee, Marc; Jouan, Elodie; Stieger, Bruno; Lecureur, Valérie; Fardel, Olivier

    2015-01-01

    Diesel exhaust particles (DEPs) are common environmental air pollutants primarily affecting the lung. DEPs or chemicals adsorbed on DEPs also exert extra-pulmonary effects, including alteration of hepatic drug detoxifying enzyme expression. The present study was designed to determine whether organic DEP extract (DEPe) may target hepatic drug transporters that contribute in a major way to drug detoxification. Using primary human hepatocytes and transporter-overexpressing cells, DEPe was first shown to strongly inhibit activities of the sinusoidal solute carrier (SLC) uptake transporters organic anion-transporting polypeptides (OATP) 1B1, 1B3 and 2B1 and of the canalicular ATP-binding cassette (ABC) efflux pump multidrug resistance-associated protein 2, with IC50 values ranging from approximately 1 to 20 μg/mL and relevant to environmental exposure situations. By contrast, 25 μg/mL DEPe failed to alter activities of the SLC transporter organic cation transporter (OCT) 1 and of the ABC efflux pumps P-glycoprotein and bile salt export pump (BSEP), whereas it only moderately inhibited those of sodium taurocholate co-transporting polypeptide and of breast cancer resistance protein (BCRP). Treatment by 25 μg/mL DEPe was next demonstrated to induce expression of BCRP at both mRNA and protein level in cultured human hepatic cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B3, OATP2B1, OCT1 and BSEP. Such changes in transporter expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a reference activator of the aryl hydrocarbon receptor (AhR) pathway. This suggests that DEPe, which is enriched in known ligands of AhR like polycyclic aromatic hydrocarbons, alters drug transporter expression via activation of the AhR cascade. Taken together, these data established human hepatic transporters as targets of organic chemicals containing in DEPs, which may contribute to their

  2. mRNA expression pattern of gonadotropin receptors in bovine follicular cysts.

    PubMed

    Marelli, Belkis E; Diaz, Pablo U; Salvetti, Natalia R; Rey, Florencia; Ortega, Hugo H

    2014-12-01

    Follicular growth and steroidogenesis are dependent on gonadotropin binding to their receptors in granulosa and theca cells of ovarian follicles. The aim of the present study was to evaluate the expression patterns of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHCGR) in ovarian follicular structures from cows with cystic ovarian disease (COD) as compared with those of regularly cycling cows. Relative real-time RT-PCR analysis showed that the expression of FSHR mRNA in granulosa cells was highest in small antral follicles, then decreased significantly as follicles increased in size, and was lowest in cysts. FSHR mRNA was not detected in the theca cells of any follicular category, including cysts. LHCGR mRNA expression in granulosa cells was significantly higher in large antral follicles than in cysts, and not detected in granulosa cells of small and medium antral follicles. In theca cells, the expression level of LHCGR mRNA in medium antral follicles was higher than in small and large antral follicles, whereas that in follicular cysts it was similar to those in small and medium antral follicles, but higher than that in large antral follicles. Our findings provide evidence that there is an altered gonadotropin receptor expression in bovine cystic follicles, and suggest that in conditions characterized by altered ovulation, such as COD, changes in the signaling system of gonadotropins may play a fundamental role in their pathogenesis. PMID:25454493

  3. 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. PMID:26954186

  4. 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

  5. Steroid receptor mRNA expression in the ovarian follicles of cows with cystic ovarian disease.

    PubMed

    Alfaro, Natalia S; Salvetti, Natalia R; Velazquez, Melisa M; Stangaferro, Matías L; Rey, Florencia; Ortega, Hugo H

    2012-06-01

    Steroid receptors have been demonstrated to be important intra-ovarian regulators of follicular development and ovulatory processes. The aim of the present study was to determine the expression of steroid receptor mRNA in ovarian follicular structures from cows with cystic ovarian disease (COD) compared with ovarian structures from regularly cycling cows using reverse transcription polymerase chain reaction (RT-PCR). The cystic follicles showed a higher estrogen receptor α (ESR1) mRNA expression in the theca and granulosa and a lower estrogen receptor β (ESR2) expression. The cystic follicles also showed a strong expression of androgen receptor mRNA in the granulosa. No changes were observed in total progesterone receptor mRNA, but a very significant increase in the B isoform was found in the granulosa of the cystic follicles. The findings of the current study provide evidence that an altered steroid signaling system may be present in bovine follicular cysts, and we suggest that in conditions characterized by altered ovulation, such as COD, changes in the expression of ovarian steroid receptors could play a fundamental role in the pathogeny of this disease. PMID:21536311

  6. EWS represses cofilin 1 expression by inducing nuclear retention of cofilin 1 mRNA.

    PubMed

    Huang, L; Kuwahara, I; Matsumoto, K

    2014-06-01

    In Ewing's sarcoma family tumors (ESFTs), the proto-oncogene EWS that encodes an RNA-binding protein is fused by chromosomal translocation to the gene encoding one of the E-twenty six (ETS) family of transcription factors, most commonly friend leukemia virus integration 1 (FLI-1). Although EWS/FLI-1 chimeric proteins are necessary for carcinogenesis, additional events seem to be required for transformation to occur. We have previously reported that a protein product of an EWS mRNA target, whose expression is negatively regulated by EWS but not by EWS/FLI-1, contributes to ESFT development. However, the mechanism by which EWS represses protein expression remains to be elucidated. Here, we report that overexpression of full-length EWS repressed protein expression and induced nuclear retention of reporter mRNAs in a tethering assay. In contrast, when a mutant lacking the EWS C-terminal nuclear localization signal (classified as a PY-NLS) was expressed, reporter protein expression was upregulated, and the number of cells exporting reporter mRNA to the cytoplasm increased. EWS binds to the 3'-untranslated region in another mRNA target, cofilin 1 (CFL1), and negatively regulates the expression of CFL1. Overexpression of EWS induced nuclear retention of CFL1 mRNA. Furthermore, ESFT cell proliferation and metastatic potential were suppressed by small interfering RNA-mediated CFL1 knockdown. Together, our findings suggest that EWS induces nuclear retention of CFL1 mRNA, thereby suppressing expression of CFL1, and that CFL1 promotes development of ESFT. Targeting CFL1 might therefore provide another novel approach for treatment of this aggressive disease. PMID:23831569

  7. Connecting protein and mRNA burst distributions for stochastic models of gene expression

    NASA Astrophysics Data System (ADS)

    Elgart, Vlad; Jia, Tao; Fenley, Andrew T.; Kulkarni, Rahul

    2011-08-01

    The intrinsic stochasticity of gene expression can lead to large variability in protein levels for genetically identical cells. Such variability in protein levels can arise from infrequent synthesis of mRNAs which in turn give rise to bursts of protein expression. Protein expression occurring in bursts has indeed been observed experimentally and recent studies have also found evidence for transcriptional bursting, i.e. production of mRNAs in bursts. Given that there are distinct experimental techniques for quantifying the noise at different stages of gene expression, it is of interest to derive analytical results connecting experimental observations at different levels. In this work, we consider stochastic models of gene expression for which mRNA and protein production occurs in independent bursts. For such models, we derive analytical expressions connecting protein and mRNA burst distributions which show how the functional form of the mRNA burst distribution can be inferred from the protein burst distribution. Additionally, if gene expression is repressed such that observed protein bursts arise only from single mRNAs, we show how observations of protein burst distributions (repressed and unrepressed) can be used to completely determine the mRNA burst distribution. Assuming independent contributions from individual bursts, we derive analytical expressions connecting means and variances for burst and steady-state protein distributions. Finally, we validate our general analytical results by considering a specific reaction scheme involving regulation of protein bursts by small RNAs. For a range of parameters, we derive analytical expressions for regulated protein distributions that are validated using stochastic simulations. The analytical results obtained in this work can thus serve as useful inputs for a broad range of studies focusing on stochasticity in gene expression.

  8. Omentin serum concentration and hepatic expression in chronic hepatitis C patients - together or apart?

    PubMed

    Kukla, Michał; Waluga, Marek; Adamek, Brygida; Zalewska-Ziob, Marzena; Kasperczyk, Janusz; Gabriel, Aandrzej; Bułdak, Rafał J; Sobala-Szczygieł, Barbara; Kępa, Lucjan; Ziora, Katarzyna; Żwirska-Korczala, Krystyna; Surma, Edward; Sawczyn, Tomasz; Hartleb, Marek

    2015-09-01

    Chronic hepatitis C (CHC) is accompanied by numerous metabolic disorders, partially associated with altered adipokine system regulation. Omentin (intelectin-1) is a novel adipokine known to play a pivotal role in metabolic regulation in CHC. In a group of 63 CHC patients (29 men/34 women) infected with genotype 1b, aged 6.6 ± 14.6 years, serum omentin levels and its gene expression in liver tissue were examined and their association with metabolic and histopathological features was assessed. Serum omentin levels were significantly higher in CHC patients compared to controls (p < 0.001), regardless of sex, body mass index (BMI), insulin sensitivity and lipid concentrations. There was no correlation between serum omentin and omentin hepatic expression. Neither parameter was associated with any histological features. Serum omentin in non-obese CHC patients seems not to be related to metabolic disorders or liver pathology. Omentin hepatic expression shows no relationship with either serum omentin levels or histopathological features. This suggests different mechanisms regulating circulating omentin concentration and omentin hepatic expression in CHC. PMID:26619101

  9. Effect of allyl alcohol on hepatic transporter expression: Zonal patterns of expression and role of Kupffer cell function

    PubMed Central

    Campion, Sarah N.; Tatis-Rios, Cristina; Augustine, Lisa M.; Goedken, Michael J.; van Rooijen, Nico; Cherrington, Nathan J.; Manautou, José E.

    2015-01-01

    During APAP toxicity, activation of Kupffer cells is critical for protection from hepatotoxicity and up-regulation of multidrug resistance-associated protein 4 (Mrp4) in centrilobular hepatocytes. The present study was performed to determine the expression profile of uptake and efflux transporters in mouse liver following treatment with allyl alcohol (AlOH), a periportal hepatotoxicant. This study also investigated the role of Kupffer cells in AlOH hepatotoxicity, and whether changes in transport protein expression by AlOH are dependent on the presence of Kupffer cells. C57BL/6J mice received 0.1 ml clodronate liposomes to deplete Kupffer cells or empty liposomes 48 h prior to dosing with 60 mg/kg AlOH, i.p. Hepatotoxicity was assessed by plasma ALT and histopathology. Hepatic transporter mRNA and protein expression were determined by branched DNA signal amplification assay and Western blotting, respectively. Depletion of Kupffer cells by liposomal clodronate treatment resulted in heightened susceptibility to AlOH toxicity. Exposure to AlOH increased mRNA levels of several Mrp genes, while decreasing organic anion transporting polypeptides (Oatps) mRNA expression. Protein analysis mirrored many of these mRNA changes. The presence of Kupffer cells was not required for the observed changes in uptake and efflux transporters induced by AlOH. Immunofluorescent analysis revealed enhanced Mrp4 staining exclusively in centrilobular hepatocytes of AlOH treated mice. These findings demonstrate that Kupffer cells are protective from AlOH toxicity and that induction of Mrp4 occurs in liver regions away from areas of AlOH damage independent of Kupffer cell function. These results suggest that Kupffer cell mediators do not play a role in mediating centrilobular Mrp4 induction in response to periportal damage by AlOH. PMID:19371622

  10. Effect of allyl alcohol on hepatic transporter expression: Zonal patterns of expression and role of Kupffer cell function

    SciTech Connect

    Campion, Sarah N.; Tatis-Rios, Cristina; Augustine, Lisa M.; Goedken, Michael J.; Rooijen, Nico van; Cherrington, Nathan J.; Manautou, Jose E.

    2009-04-01

    During APAP toxicity, activation of Kupffer cells is critical for protection from hepatotoxicity and up-regulation of multidrug resistance-associated protein 4 (Mrp4) in centrilobular hepatocytes. The present study was performed to determine the expression profile of uptake and efflux transporters in mouse liver following treatment with allyl alcohol (AlOH), a periportal hepatotoxicant. This study also investigated the role of Kupffer cells in AlOH hepatotoxicity, and whether changes in transport protein expression by AlOH are dependent on the presence of Kupffer cells. C57BL/6J mice received 0.1 ml clodronate liposomes to deplete Kupffer cells or empty liposomes 48 h prior to dosing with 60 mg/kg AlOH, i.p. Hepatotoxicity was assessed by plasma ALT and histopathology. Hepatic transporter mRNA and protein expression were determined by branched DNA signal amplification assay and Western blotting, respectively. Depletion of Kupffer cells by liposomal clodronate treatment resulted in heightened susceptibility to AlOH toxicity. Exposure to AlOH increased mRNA levels of several Mrp genes, while decreasing organic anion transporting polypeptides (Oatps) mRNA expression. Protein analysis mirrored many of these mRNA changes. The presence of Kupffer cells was not required for the observed changes in uptake and efflux transporters induced by AlOH. Immunofluorescent analysis revealed enhanced Mrp4 staining exclusively in centrilobular hepatocytes of AlOH treated mice. These findings demonstrate that Kupffer cells are protective from AlOH toxicity and that induction of Mrp4 occurs in liver regions away from areas of AlOH damage independent of Kupffer cell function. These results suggest that Kupffer cell mediators do not play a role in mediating centrilobular Mrp4 induction in response to periportal damage by AlOH.

  11. Replenishment of RANTES mRNA expression in activated eosinophils fromatopic asthmatics

    PubMed Central

    Velazquez, J R; Lacy, P; Moqbel, R

    2000-01-01

    Eosinophils have been shown to express the gene encoding regulated upon activation, normal T‐cell expressed and secreted (RANTES), a potent eosinophilotactic chemokine. RANTES protein expression in eosinophils has previously been shown to be up‐regulated by a number of agonists, including complement‐dependent factors (C3b/iC3b) and interferon‐γ (IFN‐γ). We hypothesized that gene expression of RANTES is regulated in these cells by eosinophil‐specific agonists. We analysed RANTES mRNA expression by reverse‐transcription polymerase chain reaction (RT‐PCR) in human peripheral blood eosinophils obtained from mild atopic asthmatics following stimulation over time. In resting eosinophils, a low level of RANTES mRNA was found to be constitutively expressed in all the atopic donors tested in this study (n = 6). Following stimulation with C3b/iC3b (serum‐coated surfaces), eosinophils released measurable levels of RANTES, while sustained transcript expression was detected for up to 24 hr of stimulation. In contrast, IFN‐γ (5 ng/ml) transiently and significantly (P < 0·05, n = 3) depleted relative amounts of RANTES PCR product (compared with β2‐microglobulin) after 1–4 hr of stimulation. RANTES transcript was again detectable after 24 hr of IFN‐γ incubation, suggesting that the pool of RANTES mRNA had been replenished. Other eosinophil‐active cytokines, interleukin‐3 (IL‐3), IL‐4, IL‐5 and granulocyte–macrophage colony‐stimulating factor, did not appear to modulate RANTES mRNA expression after 1 hr of incubation. The effect of IFN‐γ on RANTES mRNA was reversed by cycloheximide, suggesting that IFN‐γ may act by increasing the rate of translation of RANTES mRNA. These findings indicate that IFN‐γ may induce a rapid and transient effect on the translation and replenishment of RANTES mRNA in eosinophils. This novel observation supports the notion that eosinophils have the potential to replenish their stored and released

  12. RANKL, OPG and CTR mRNA expression in the temporomandibular joint in rheumatoid arthritis

    PubMed Central

    LIU, WEI-WEI; XU, ZHI-MIN; LI, ZHENG-QIANG; ZHANG, YAN; HAN, BING

    2015-01-01

    The calcitonin receptor (CTR) and receptor activator of nuclear factor κB ligand (RANKL) have been found to be involved in the differentiation of osteoclasts. The association between the RANKL:osteoprotegerin (OPG) expression ratio and the pathogenesis of bone-destructive rheumatoid arthritis (RA) has been described in several joints, but the available data for the temporomandibular joint (TMJ) are limited. The aim of the present study was to investigate the involvement of osteoclasts at sites of bone erosion by determining the CTR expression and the RANKL:OPG expression ratio in the TMJ in a collagen-induced arthritis (CIA) model. Forty-eight male Wistar rats were randomly divided into two groups: Control group, injected with saline solution for 6 weeks; and CIA group, injected with emulsion. The RANKL and OPG mRNA expression was significantly increased in immunized rats compared with that in non-immunized rats. The RANKL:OPG expression ratio on the trabecular bone surface was 9.0 and 6.4 in the CIA group at weeks 4 and 6, respectively, while the RANKL:OPG expression ratio in the controls was 1.0:2. CTR mRNA expression was significantly upregulated in immunized rats compared with that in non-immunized rats; the level of CTR mRNA in the CTR-positive osteoclasts on the trabecular bone surface was 10.9- and 7.8-fold higher in the CIA rats than that in the control rats at weeks 4 and 6, respectively. In conclusion, focal bone destruction in an experimental model of arthritis in the TMJ can be attributed to cells expressing CTR, a defining feature of osteoclasts. The expression of RANKL and OPG mRNA within the inflamed synovium provides an insight into the mechanism of osteoclast differentiation and function at the border of bone erosion in arthritis. PMID:26622411

  13. Serum KIBRA mRNA and Protein Expression and Cognitive Functions in Depression

    PubMed Central

    Talarowska, Monika; Szemraj, Janusz; Kowalczyk, Małgorzata; Gałecki, Piotr

    2016-01-01

    Background Genes participating in synaptic signalling or plasticity in brain regions such as the prefrontal cortex (PFC) and the hippocampus have been implicated in cognition. Recently, a new gene (KIBRA, WWC1) has been added to this group due to its impact on memory performance. Recurrent depressive disorder (rDD) is a multifactorial disease, that one of the typical features is cognitive impairment. The main objective of this study was to perform an analysis of the KIBRA gene on both mRNA and protein levels in patients suffering from rDD and to investigate the relationship between KIBRA expression and cognitive performance. Material/Methods The study comprised 236 subjects: patients with rDD (n=131) and healthy subjects (n=105, HS). Cognitive function assessment was based on: Trail Making Test, The Stroop Test, Verbal Fluency Test and Auditory Verbal Learning Test. Results Both mRNA and protein expression levels of KIBRA gene were significantly higher in healthy subjects when compared to rDD. The presented relationship is clear even after taking age, education and sex of the examined subjects into consideration. No statistically significant relationship was found in the experiments between any of the conducted tests and KIBRA gene expression on mRNA level for both the rDD and HS groups. The presented study has limitations related to the fact that patients were being treated with antidepressant. This is relevant due to the fact that some antidepressants may affect mRNA expression. Number of patients and healthy subjects may result in the lack of statistical significance in some cases. Conclusions 1. The results of our study show decreased expression of the KIBRA gene on both mRNA and protein levels in depression. 2. We did not find any significant relationship between KIBRA gene expression and cognitive functions in case of both the healthy subjects and the patients affected by rDD. PMID:26768155

  14. Expression of CCT6A mRNA in chicken granulosa cells is regulated by progesterone.

    PubMed

    Wei, Qingqing; Zhu, Guiyu; Cui, Xinxing; Kang, Li; Cao, Dingguo; Jiang, Yunliang

    2013-08-01

    CCT6A, the zeta subunit of the chaperonin containing TCP1 complex, is the only cytosolic chaperonin in eukaryotes and is estimated to assist in the folding of multiple proteins including actin, tubulin, cyclin E, myosin, transducin and the Von Hippel Lindau tumor suppressor. In this study, we examined the expression of CCT6A and progesterone receptor (PGR) mRNA in various tissues of chickens and the regulation of CCT6A and PGR mRNA in ovarian granulosa cells. Northern blot analysis revealed that CCT6A had one transcript and was highly expressed in the ovary tissues from chickens at both the sexually immature and mature stages. CCT6A mRNA expression was increased maximally from pre-hierarchy follicles to F5 follicles and subsequently declined in pre-ovulatory and post-ovulatory follicles. The expression of PGR mRNA exhibited the similar pattern to CCT6A. In granulosa cells isolated from pre-ovulatory follicles, follicle-stimulating hormone (FSH) inhibited the expression of CCT6A mRNA, whereas progesterone activated CCT6A and suppressed PGR expression in a time-dependent manner. We further investigated the regulation of CCT6A transcription by progesterone by constructing various progressive deletions and mutants and identified the core promoter element of CCT6A and the binding region of progesterone, which is located from -2056 to -2051. Taken together, our results indicate that CCT6A likely plays an important role in follicle growth, and in granulosa cells, progesterone activates CCT6A transcription via a progesterone response element (PRE) located in the distal promoter of CCT6A. PMID:23644154

  15. 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

  16. Differential expression of stress-inducible proteins in chronic hepatic iron overload

    SciTech Connect

    Brown, Kyle E. Broadhurst, Kimberly A.; Mathahs, M. Meleah; Weydert, Jamie

    2007-09-01

    Introduction:: Oxidative stress can trigger a cellular stress response characterized by induction of antioxidants, acute phase reactants (APRs) and heat shock proteins (HSPs), which are presumed to play a role in limiting tissue damage. In rodents, hepatic iron overload causes oxidative stress that results in upregulation of antioxidant defenses with minimal progressive liver injury. The aim of this study was to determine whether iron overload modulates expression of other stress-responsive proteins such as APRs and HSPs that may confer protection against iron-induced damage in rodent liver. Methods:: Male rats received repeated injections of iron dextran or dextran alone over a 6-month period. Hepatic transcript levels for a panel of APRs and HSPs were quantitated by real-time PCR and protein expression was evaluated by Western blot and immunohistochemistry. Results:: Hepatic iron concentrations were increased > 50-fold in the iron-loaded rats compared to controls. Iron loading resulted in striking increases in mRNAs for Hsp32 (heme oxygenase-1; 12-fold increase vs. controls) and metallothionein-1 and -2 (both increased {approx} 6-fold). Transcripts for {alpha}1-acid glycoprotein, the major rat APR, were increased {approx} 3-fold, while expression of other classical APRs was unaltered. Surprisingly, although mRNA levels for the HSPs were not altered by iron, the abundance of Hsp25, Hsp70 and Hsp90 proteins was uniformly reduced in the iron-loaded livers, as were levels of NAD(P)H:quinone oxidoreductase 1, an Hsp70 client protein. Conclusions:: Chronic iron administration elicits a unique pattern of stress protein expression. These alterations may modulate hepatic responses to iron overload, as well as other injury processes.

  17. Tumor necrosis factor alpha negatively regulates hepatitis B virus gene expression in transgenic mice.

    PubMed Central

    Gilles, P N; Fey, G; Chisari, F V

    1992-01-01

    It is well known that several inflammatory cytokines can modulate hepatocellular gene expression in a complex physiological process known as the hepatic acute-phase response. Since hepatitis B virus (HBV) characteristically induces a vigorous lymphomononuclear inflammatory response in the liver during acute and chronic hepatitis, it is possible that hepatocellular HBV gene expression may also be modulated by one or more of the cytokines produced by these cells. Using bacterial lipopolysaccharide (LPS) as a surrogate inducer of inflammatory cytokines in vivo, we have tested this hypothesis in a transgenic mouse model system. In experiments with two independent transgenic mouse lineages that express the HBV envelope region under the control of either HBV or cellular promoters, we observed a 50 to 80% reduction in the hepatic steady-state content of a 2.1-kb HBV mRNA following administration of a single intraperitoneal dose of LPS. The regulatory influence of several inflammatory cytokines known to be induced by LPS was also examined in this system. The negative regulatory effect of LPS was consistently reproduced by the administration of a single nontoxic dose of tumor necrosis factor alpha, and it was occasionally observed following the administration of high doses of alpha interferon and interleukin-6, while no effect was detectable in response to high-dose interleukin-1 alpha or to gamma interferon. These observations suggest that tumor necrosis factor alpha and perhaps other cytokines may activate a heretofore unsuspected intracellular pathway that negatively regulates HBV gene expression. The intracellular mechanism(s) responsible for this effect and its pathophysiologic relevance remain to be elucidated. Images PMID:1583737

  18. 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...

  19. Real time imaging of mRNA expression dynamics in live cells using protein complementation methods

    NASA Astrophysics Data System (ADS)

    Meller, Amit

    2009-03-01

    Traditional methods for mRNA quantification in cells, such as northern blots, quantitative PCR or microarrays assays, require cell lysis and therefore do not preserve its dynamics. These methods cannot be used to probe the spatio-temporal localization of mRNA in cells, which provide useful information for a wide range biomolecular process, including RNA metabolizim, expression kinetics and RNA interference. To probe mRNA dynamics in live prokaryotic and eukaryotic cells, we develop a method, which exploit the strong affinity of the eukaryotic initiation factor 4A (eIF4A) to specific RNA aptamers. Two parts of the eIF4A are fused to a split Green Fluorescence Protein (GFP), and are expressed in the cells at high abundance. However, only when the RNA apatmer is also present, the two protein parts complement and become fluorescent. Thus, the fluorescent background remains low, allowing us to directly image the expression of mRNA molecules in live e-coli cells from its early onset, over hours. We find that the expression kinetics can be classified in one out of at least three forms, which also display distinct spatial distributions. I will discuss the possible biological origin for these distributions and their time evolution.

  20. Hepatic miR-29ab1 expression modulates chronic hepatic injury

    PubMed Central

    Kogure, Takayuki; Costinean, Stefan; Yan, Irene; Braconi, Chiara; Croce, Carlo; Patel, Tushar

    2012-01-01

    MicroRNAs (miRNAs) are small, regulatory non-coding RNAs that have potent effects on gene expression. Several miRNA are deregulated in cellular processes involved in human liver diseases and regulation of cellular processes. Recent studies have identified the involvement of miR-29 in hepatic fibrosis and carcinogenesis. Although several targets of miR-29 have been identified, there is limited information regarding the cell-type specific roles of miR-29 in the liver, and we sought to evaluate the role of this miRNA in hepatic pathobiology. We report the generation of a tissue–specific knockout mouse to evaluate the role of miR-29 in hepatic fibrosis and carcinogenesis in response to injury. We hypothesized that miR-29 contributes to the hepatocyte driven response to chronic cellular injury that results in fibrosis. In support of this hypothesis, fibrosis and mortality were enhanced in miR29 knockout mice in response to carbon tetrachloride. Genome-wide gene expression analysis identified an over-representation of genes associated with fibrosis. The oncofetal RNA H19 was modulated in a miR-29 dependent manner following exposure to carbon tetrachloride in vivo. The impact of a hepatocyte specific miR-29 knockout on survival following chronic hepatic injury in vivo implicates this miRNA as a potential target for intervention. These results provide evidence of the involvement of miR-29 in chronic hepatic injury, and suggest a role for deregulated hepatocyte expression of miR-29 in the response to hepatic injury, fibrosis and carcinogenesis. PMID:22469499

  1. Hepatic miR-29ab1 expression modulates chronic hepatic injury.

    PubMed

    Kogure, Takayuki; Costinean, Stefan; Yan, Irene; Braconi, Chiara; Croce, Carlo; Patel, Tushar

    2012-11-01

    MicroRNAs (miRNAs) are small, regulatory non-coding RNAs that have potent effects on gene expression. Several miRNA are deregulated in cellular processes involved in human liver diseases and regulation of cellular processes. Recent studies have identified the involvement of miR-29 in hepatic fibrosis and carcinogenesis. Although several targets of miR-29 have been identified, there is limited information regarding the cell-type specific roles of miR-29 in the liver, and we sought to evaluate the role of this miRNA in hepatic pathobiology. We report the generation of a tissue-specific knockout mouse to evaluate the role of miR-29 in hepatic fibrosis and carcinogenesis in response to injury. We hypothesized that miR-29 contributes to the hepatocyte driven response to chronic cellular injury that results in fibrosis. In support of this hypothesis, fibrosis and mortality were enhanced in miR29 knockout mice in response to carbon tetrachloride. Genome-wide gene expression analysis identified an over-representation of genes associated with fibrosis. The oncofetal RNA H19 was modulated in a miR-29 dependent manner following exposure to carbon tetrachloride in vivo. The impact of a hepatocyte specific miR-29 knockout on survival following chronic hepatic injury in vivo implicates this miRNA as a potential target for intervention. These results provide evidence of the involvement of miR-29 in chronic hepatic injury, and suggest a role for deregulated hepatocyte expression of miR-29 in the response to hepatic injury, fibrosis and carcinogenesis. PMID:22469499

  2. hnRNP-U enhances the expression of specific genes by stabilizing mRNA.

    PubMed

    Yugami, Masato; Kabe, Yasuaki; Yamaguchi, Yuki; Wada, Tadashi; Handa, Hiroshi

    2007-01-01

    Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to be involved in pre-mRNA processing. hnRNP-U, also termed scaffold attachment factor A (SAF-A), binds to pre-mRNA and nuclear matrix/scaffold attachment region DNA elements. However, its role in the regulation of gene expression is as yet poorly understood. In the present study, we show that hnRNP-U specifically enhances the expression of tumor necrosis factor alpha mRNA by increasing its stability, possibly through binding to the 3' untranslated region. We also show that hnRNP-U enhances the expression of several other genes as well, including GADD45A, HEXIM1, HOXA2, IER3, NHLH2, and ZFY, by binding to and stabilizing these mRNAs. These results suggest that hnRNP-U enhances the expression of specific genes by regulating mRNA stability. PMID:17174306

  3. Impact of Cadmium Exposure during Pregnancy on Hepatic Glucocorticoid Receptor Methylation and Expression in Rat Fetus

    PubMed Central

    Castillo, Paula; Ibáñez, Freddy; Guajardo, Angélica; Llanos, Miguel N.; Ronco, Ana M.

    2012-01-01

    Adverse fetal environment due to maternal undernutrition or exposure to environmental chemicals alters glucocorticoid (GC) metabolism increasing the risk of metabolic disorders in adulthood. In this study, we investigated the effects of maternal exposure to cadmium (Cd, 50 ppm) during pregnancy in the methylation of fetal hepatic glucocorticoid receptor promoter (GR) and the correlation with its expression and that of the DNA methyltransferases (DNMT1a and 3a). We also studied the expression of liver phosphoenolpyruvate carboxykinase (PEPCK) and acyl-CoA oxidase (AOX), two enzymes involved in the metabolism of carbohydrates and lipids respectively. The methylation of the rat GR gene exon 110 (GR110) in nucleotides -2536 to -2361 was analyzed by pyrosequencing. Quantitative real time PCR was used to assess hepatic GR, PEPCK and AOX mRNA, and their protein levels using Western blotting analysis. Differential methylation was noted across groups at all CpG sites in the GR exon 110 in a sex-dependent manner. In males, CpG were more methylated than the controls (185±21%, p<0.001) but only CpG sites 1,6,7 and 9 showed a significantly different extent of methylation. In addition, a lower expression of GR (mRNA and protein) was found. On the contrary, in females, CpG were less methylated than the controls (62±11%, p<0.05) and overexpressed, affecting PEPCK and AOX expression, which did not change in males. The GR methylation profile correlates with DNMT3a expression which may explain epigenetic sex-dependent changes on GR110 promoter induced by Cd treatment. In conclusion, Cd exposure during pregnancy affects fetal liver DNMT3a resulting in sex-dependent changes in methylation and expression of GR110. Although these effects do not seem to be directly involved in the low birth weight and height, they may have relevant implications for long-term health. PMID:22957049

  4. 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.

  5. mRNA expression and protein localization of dentin matrix protein 1 during dental root formation.

    PubMed

    Toyosawa, S; Okabayashi, K; Komori, T; Ijuhin, N

    2004-01-01

    Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein. DMP1 was initially detected in dentin and later in other mineralized tissues including cementum and bone, but the DMP1 expression pattern in tooth is still controversial. To determine the precise localization of DMP1 messenger RNA (mRNA) and the protein in the tooth, we performed in situ hybridization and immunohistochemical analyses using rat molars and incisors during various stages of root formation. During root dentin formation of molars, DMP1 mRNA was detected in root odontoblasts in parallel with mineralization of the dentin. However, the level of DMP1 mRNA expression in root odontoblasts decreased near the coronal part and was absent in coronal odontoblasts. DMP1 protein was localized along dentinal tubules and their branches in mineralized root dentin, and the distribution of DMP1 shifted from the end of dentinal tubules to the base of the tubules as dentin formation progressed. During the formation of the acellular cementum, DMP1 mRNA was detected in cementoblasts lining the acellular cementum where its protein was localized. During the formation of the cellular cementum, DMP1 mRNA was detected in cementocytes embedded in the cellular cementum but not in cementoblasts, and its protein was localized in the pericellular cementum of cementocytes including their processes. During dentin formation of incisors, DMP1 mRNA was detected in odontoblasts on the cementum-related dentin, where its protein was localized along dentinal tubules near the mineralization front. The localization of DMP1 mRNA and protein in dentin and cementum was related to their mineralization, suggesting that one of the functions of DMP1 may be involved in the mineralization of dentin and cementum during root formation. PMID:14751569

  6. 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. PMID:12670714

  7. 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

  8. Decreased drebrin mRNA expression in Alzheimer disease: correlation with tau pathology.

    PubMed

    Julien, Carl; Tremblay, Cyntia; Bendjelloul, Farid; Phivilay, Alix; Coulombe, Marie-Andrée; Emond, Vincent; Calon, Frédéric

    2008-08-01

    To investigate the mRNA expression of the dendritic spine protein drebrin in Alzheimer's disease (AD), we performed post-mortem in situ hybridization studies in brain sections from 20 AD patients and 21 controls. AD diagnosis was confirmed by decreased drebrin protein and increased Abeta(40) (+464%; P < 0.05), Abeta(42) (+369%; P < 0.0001), Abeta(42/40) ratio (+226%; P < 0.01), total tau (+2,725%; P < 0.0001), and paired helical filament tau (PHFtau; +867%; P < 0.001) compared with controls. We found significant decreases in drebrin mRNA in the parietal cortex (-27%; P < 0.01), the temporal cortex (-22%; P < 0.05), and the hippocampus (-25%; P < 0.05) of AD patients compared with controls. Cortical levels of drebrin mRNA correlated positively with soluble total tau (r(2) = +0.244) but negatively with duration of symptoms (r(2) = -0.357) and PHFtau (r(2) = -0.248). Drebrin mRNA levels were correlated to a lesser degree with the drebrin protein content (r(2) = +0.136) and with sim2 (r(2) = +0.176), a potential modulator of drebrin transcription. Our results suggest that the down-regulation of drebrin mRNA expression plays an important role in AD and is closely related to the progression of the disease. PMID:18338803

  9. 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

  10. Beta-integrin of Anopheles gambiae: mRNA cloning and analysis of structure and expression.

    PubMed

    Mahairaki, V; Lycett, G; Blass, C; Louis, C

    2001-06-01

    We have isolated an mRNA encoding a beta integrin subunit of the malaria mosquito Anopheles gambiae. Our analysis predicts a protein that is very similar to betaPS, the fruitfly orthologue. The gene is expressed during all developmental stages and it is found in all body parts, including the midgut. Finally, the expression of the gene does not seem to be modulated during blood meals, except for a substantial increase 48 h posthaematophagy, when digestion is nearly complete. PMID:11437913

  11. Regulation of Hepatic Cholesteryl Ester Transfer Protein Expression and Reverse Cholesterol Transport by Inhibition of DNA Topoisomerase II*

    PubMed Central

    Liu, Mengyang; Chen, Yuanli; Zhang, Ling; Wang, Qixue; Ma, Xingzhe; Li, Xiaoju; Xiang, Rong; Zhu, Yan; Qin, Shucun; Yu, Yang; Jiang, Xian-cheng; Duan, Yajun; Han, Jihong

    2015-01-01

    Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from high density lipoprotein to triglyceride-rich lipoproteins. CETP expression can be transcriptionally activated by liver X receptor (LXR). Etoposide and teniposide are DNA topoisomerase II (Topo II) inhibitors. Etoposide has been reported to inhibit atherosclerosis in rabbits with un-fully elucidated mechanisms. In this study we determined if Topo II activity can influence cholesterol metabolism by regulating hepatic CETP expression. Inhibition of Topo II by etoposide, teniposide, or Topo II siRNA increased CETP expression in human hepatic cell line, HepG2 cells, which was associated with increased CETP secretion and mRNA expression. Meanwhile, inhibition of LXR expression by LXR siRNA attenuated induction of CETP expression by etoposide and teniposide. Etoposide and teniposide induced LXRα expression and LXRα/β nuclear translocation while inhibiting expression of receptor interacting protein 140 (RIP140), an LXR co-repressor. In vivo, administration of teniposide moderately reduced serum lipid profiles, induced CETP expression in the liver, and activated reverse cholesterol transport in CETP transgenic mice. Our study demonstrates a novel function of Topo II inhibitors in cholesterol metabolism by activating hepatic CETP expression and reverse cholesterol transport. PMID:25914138

  12. Use of mRNA expression to detect the induction of drug metabolising enzymes in rat and human hepatocytes

    SciTech Connect

    Richert, L. Tuschl, G.; Pekthong, D.; Mantion, G.; Weber, J.-C.; Mueller, S.O.

    2009-02-15

    It is important to investigate the induction of cytochrome P450 (CYP) enzymes by drugs. The most relevant end point is enzyme activity; however, this requires many cells and is low throughput. We have compared the CYP1A, CYP2B and CYP3A induction response to eight inducers in rat and human hepatocytes using enzyme activities (CYP1A2 (ethoxyresorufin), 2B (benzoxyresorufin for rat and bupropion for human) and CYP3A (testosterone)) and Taqman{sup TM} Low Density Array (TLDA) analysis. There was a good correlation between the induction of CYP1A2, CYP2B6 and CYP3A4 enzyme activities and mRNA expression in human hepatocytes. In contrast, BROD activities and mRNA expression in rat hepatocytes correlated poorly. However, bupropion hydroxylation correlated well with Cyp2b1 expression in rat hepatocytes. TLDA analysis of a panel of mRNAs encoding for CYPs, phase 2 enzymes, nuclear receptors and transporters revealed that the main genes induced by the 8 compounds tested were the CYPs. AhR ligands also induced UDP-glucuronosyltransferases and glutathione S-transferases in rat and human hepatocytes. The transporters, MDR1, MDR3 and OATPA were the only transporter genes significantly up-regulated in human hepatocytes. In rat hepatocytes Bsep, Mdr2, Mrp2, Mrp3 and Oatp2 were up-regulated. We could then show a good in vivo:in vitro correlation in the induction response of isolated rat hepatocytes and ex-vivo hepatic microsomes for the drug development candidate, EMD392949. In conclusion, application of TLDA methodology to investigate the potential of compounds to induce enzymes in rat and human hepatocytes increases the throughput and information gained from one assay, without reducing the predictive capacity.

  13. Expression of tilapia prepro-melanin-concentrating hormone mRNA in hypothalamic and neurohypophysial cells.

    PubMed

    Gröneveld, D; Eckhardt, E R; Coenen, A J; Martens, G J; Balm, P H; Wendelaar Bonga, S E

    1995-04-01

    Melanin-concentrating hormone (MCH) is a neuropeptide involved in background adaptation in teleost fish, and in multiple regulatory functions in mammals and fish. To study the expression of the MCH preprohormone (ppMCH) in teleosts, we first cloned a hypothalamic cDNA encoding the complete ppMCH of tilapia (Oreochromis mossambicus), and a cRNA probe derived from a 270 bp ppMCH cDNA fragment was used for the expression studies. The level of ppMCH mRNA expression in tilapia hypothalamus, measured by dot blot analysis, was significantly higher in fish adapted to a white background than in black-adapted animals, which is in accordance with the reported MCH plasma and tissue concentrations in fish. Northern blot analysis not only revealed a strong ppMCH mRNA signal in the hypothalamus, but also the presence of ppMCH mRNA in the neurointermediate lobe (NIL) of the pituitary. In situ hybridization and immunocytochemistry showed that ppMCH mRNA as well as MCH immunoreactivity are located in perikarya of two hypothalamic regions, namely in the nucleus lateralis tuberis (NLT) and the nucleus recessus lateralis (NRL). Quantitative analysis by dot blot hybridization revealed about eight times more ppMCH mRNA in the NLT than in the NRL and NIL of mature tilapias. ppMCH mRNA in the NIL could be localized to cell bodies of the neurohypophysis, which were also MCH immunoreactive. PMID:7619209

  14. 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

  15. Hepatic Aryl Hydrocarbon Receptor Attenuates Fibroblast Growth Factor 21 Expression.

    PubMed

    Girer, Nathaniel G; Murray, Iain A; Omiecinski, Curtis J; Perdew, Gary H

    2016-07-15

    The Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in many physiological processes. Several studies indicate that AHR is also involved in energy homeostasis. Fibroblast growth factor 21 (FGF21) is an important regulator of the fasting and feeding responses. When administered to various genetic and diet-induced mouse models of obesity, FGF21 can attenuate obesity-associated morbidities. Here, we explore the role of AHR in hepatic Fgf21 expression through the use of a conditional, hepatocyte-targeted AHR knock-out mouse model (Cre(Alb)Ahr(Fx/Fx)). Compared with the congenic parental strain (Ahr(Fx/Fx)), non-fasted Cre(Alb)Ahr(Fx/Fx) mice exhibit a 4-fold increase in hepatic Fgf21 expression, as well as elevated expression of the FGF21-target gene Igfbp1 Furthermore, in vivo agonist activation of AHR reduces hepatic Fgf21 expression during a fast. The Fgf21 promoter contains several putative dioxin response elements (DREs). Using EMSA, we demonstrate that the AHR-ARNT heterodimer binds to a specific DRE that overlaps binding sequences for peroxisome proliferator-activated receptor α (PPARα), carbohydrate response element-binding protein (ChREBP), and cAMP response element-binding protein, hepatocyte specific (CREBH). In addition, we reveal that agonist-activated AHR impairs PPARα-, ChREBP-, and CREBH-mediated promoter activity in Hepa-1 cells. Accordingly, agonist treatment in Hepa-1 cells ablates potent ER stress-driven Fgf21 expression, and pre-treatment with AHR antagonist blocks this effect. Finally, we show that pre-treatment of primary human hepatocytes with AHR agonist diminishes PPARα-, glucose-, and ER stress-driven induction of FGF21 expression, indicating the effect is not mouse-specific. Together, our data show that AHR contributes to hepatic energy homeostasis, partly through the regulation of FGF21 expression and signaling. PMID:27226639

  16. Chronic administration of caderofloxacin, a new fluoroquinolone, increases hepatic CYP2E1 expression and activity in rats

    PubMed Central

    Liu, Li; Miao, Ming-xing; Zhong, Ze-yu; Xu, Ping; Chen, Yang; Liu, Xiao-dong

    2016-01-01

    Aim: Caderofloxacin is a new fluoroquinolone that is under phase III clinical trials in China. Here we examined the effects of caderofloxacin on rat hepatic cytochrome P450 (CYP450) isoforms as well as the potential of caderofloxacin interacting with co-administered drugs. Methods: Male rats were treated with caderofloxacin (9 mg/kg, ig) once or twice daily for 14 consecutive days. The effects of caderofloxacin on CYP3A, 2D6, 2C19, 1A2, 2E1 and 2C9 were evaluated using a “cocktail” of 6 probes (midazolam, dextromethorphan, omeprazole, theophylline, chlorzoxazone and diclofenac) injected on d 0 (prior to caderofloxacin exposure) and d 15 (after caderofloxacin exposure). Hepatic microsomes from the caderofloxacin-treated rats were used to assess CYP2E1 activity and chlorzoxazone metabolism. The expression of CYP2E1 mRNA and protein in hepatic microsomes was analyzed with RT-PCR and Western blotting, respectively. Results: Fourteen-day administration of caderofloxacin significantly increased the activity of hepatic CYP2E1, leading to enhanced metabolism of chlorzoxazone. In vitro microsomal study confirmed that CYP2E1 was a major metabolic enzyme involved in chlorzoxazone metabolism, and the 14-d administration of caderofloxacin significantly increased the activity of CYP2E1 in hepatic microsomes, resulting in increased formation of 6-hydroxychlorzoxazone. Furthermore, the 14-d administration of caderofloxacin significantly increased the expression of CYP2E1 mRNA and protein in liver microsomes, which was consistent with the pharmacokinetic results. Conclusion: Fourteen-day administration of caderofloxacin can induce the expression and activity of hepatic CYP2E1 in rats. When caderofloxacin is administered, a potential drug-drug interaction mediated by CYP2E1 induction should be considered. PMID:26838075

  17. Alpha1-adrenoreceptor in human hippocampus: binding and receptor subtype mRNA expression.

    PubMed

    Szot, Patricia; White, Sylvia S; Greenup, J Lynne; Leverenz, James B; Peskind, Elaine R; Raskind, Murray A

    2005-10-01

    Alpha1-adrenoreceptors (AR), of which three subtypes exist (alpha1A-, alpha1B- and alpha1D-AR) are G-protein-coupled receptors that mediate the actions of norepinephrine and epinephrine both peripherally and centrally. In the CNS, alpha1-ARs are found in the hippocampus where animal studies have shown the ability of alpha1-AR agents to modulate long-term potentiation and memory; however, the precise distribution of alpha1-AR expression and its subtypes in the human brain is unknown making functional comparisons difficult. In the human hippocampus, 3H-prazosin (alpha1-AR antagonist) labels only the dentate gyrus (molecular, granule and polymorphic layers) and the stratum lucidum of the CA3 homogeneously. Human alpha1A-AR mRNA in the hippocampus is observed only in the dentate gyrus granule cell layer, while alpha1D-AR mRNA expression is observed only in the pyramidal cell layers of CA1, CA2 and CA3, regions where 3H-prazosin did not bind. alpha1B-AR mRNA is not expressed at detectable levels in the human hippocampus. These results confirm a difference in hippocampal alpha1-AR localization between rat and humans and further describe a difference in the localization of the alpha1A- and alpha1D-AR mRNA subtype between rats and humans. PMID:16039007

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

    PubMed

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

    2015-07-01

    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

  19. Effect of squalene synthase inhibition on the expression of hepatic cholesterol biosynthetic enzymes, LDL receptor, and cholesterol 7 alpha hydroxylase.

    PubMed

    Ness, G C; Zhao, Z; Keller, R K

    1994-06-01

    Squalene synthase catalyzes the committed step in the biosynthesis of sterols. Treating rats with zaragozic acid A, a potent inhibitor of squalene synthase, caused marked increases in hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, squalene synthase, and LDL receptor mRNA levels. The increase in HMG-CoA reductase mRNA fully accounted for the increases seen in enzyme protein and activity. Farnesyl pyrophosphate synthase mRNA and activity were only slightly increased by zaragozic acid A, while cholesterol 7 alpha hydroxylase mRNA levels were decreased substantially. When rats were pretreated with zaragozic acid A, there was no change in mRNA levels for the cholesterol biosynthetic enzymes or cholesterol 7 alpha hydroxylase upon subsequent treatment with mevalonolactone. Under these same conditions, the enzymatic activity of HMG-CoA reductase was also unaffected. Mevalonolactone treatment reduced the zaragozic acid A-mediated increase in hepatic LDL receptor mRNA levels. Feeding cholesterol eliminated the zaragozic acid A-induced increase in HMG-CoA reductase mRNA levels. These results suggest that inhibition of squalene synthase decreases the level of a squalene-derived regulatory product, resulting in altered amounts of several mRNAs and coordinate increases in HMG-CoA reductase mRNA, protein, and activity. The increase in HMG-CoA reductase gene expression was closely related to the degree of inhibition of cholesterol synthesis caused by zaragozic acid A. PMID:7911291

  20. Dithranol downregulates expression of Id1 mRNA in human keratinocytes in vitro.

    PubMed

    Ronpirin, C; Tencomnao, T

    2012-01-01

    The precise causes of psoriasis, a chronic skin disorder characterized by hyperproliferation of keratinocytes and incomplete keratinization, are unclear. It is known that expression of helix-loop-helix transcription factor Id1, which functions as an inhibitor of differentiation, is upregulated in psoriatic skin. We investigated the effect of the antipsoriatic drug dithranol on mRNA and protein expression levels of Id1 in the HaCaT keratinocyte cell line. Cultured HaCaT cells were treated with 0-0.5 μg/mL dithranol for 30 min. After 2 and 4 h, total cellular RNA and total proteins were isolated from HaCaT cells, and quantitative real-time reverse transcriptase (RT-PCR) and Western blot were used to determine the mRNA and protein levels of Id1, respectively. Changes in normalized Id1 mRNA levels were observed only after 4 h of dithranol treatment. There was reduced expression of Id1 mRNA transcripts in the HaCaT cells treated with 0.1 μg/mL dithranol, but the reduction was not significant. The expression of Id1 mRNA was significantly downregulated (almost 50%) when 0.25 or 0.5 μg/mL dithranol was applied to the HaCaT cells. However, the normalized Id1 protein levels were not significantly affected. The molecular mechanisms underlying this finding should be investigated further to help determine the therapeutic action of this drug. PMID:23079823

  1. c-kit mRNA expression in human and murine hematopoietic cell lines.

    PubMed

    André, C; d'Auriol, L; Lacombe, C; Gisselbrecht, S; Galibert, F

    1989-08-01

    The c-kit proto-oncogene belongs to the tyrosine kinase receptor family. Although its ligand is still unknown, there is increasing evidence to suggest its involvement in hematopoiesis. In order to detect lineage or differentiation related specificity, we have studied c-kit mRNA expression in both human and murine hematopoietic organs and cell lines. We show that c-kit mRNA expression is found at early stages of erythroid and myeloid differentiation. There is however, no evidence of c-kit expression in the lymphoid lineage. Our results suggest a possible role for c-kit as a receptor in the early stages of the erythroid/myeloid differentiation. PMID:2474787

  2. Leishmania amazonensis: Anionic currents expressed in oocytes upon microinjection of mRNA from the parasite.

    PubMed

    Lagos M, Luisa F; Moran, Oscar; Camacho, Marcela

    2007-06-01

    Transport mechanisms involved in pH homeostasis are relevant for the survival of Leishmania parasites. The presence of chloride conductive pathways in Leishmania has been anticipated since anion channel inhibitors limit the proton extrusion mediated by the H+ATPase, which is the major regulator of intracellular pH in amastigotes. In this study, we used Xenopus laevis oocytes as a heterologous expression system in which to study the expression of ion channels upon microinjection of polyA mRNA from Leishmania amazonensis. After injection of polyA mRNA into the oocytes, we measured three different types of currents. We discuss the possible origin of each, and propose that Type 3 currents could be the result of the heterologous expression of proteins from Leishmania since they show different pharmacological and biophysical properties as compared to endogenous oocyte currents. PMID:17328895

  3. 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.

  4. Gene-specific regulation of hepatic selenoprotein expression by interleukin-6.

    PubMed

    Martitz, J; Becker, N-P; Renko, K; Stoedter, M; Hybsier, S; Schomburg, L

    2015-11-01

    Sepsis is a severe inflammatory disease resulting in excessive production of pro-inflammatory cytokines including interleukin-6 (IL-6), causing oxidative stress, tissue damage and organ dysfunction. Health benefits have been observed upon selenium (Se) supplementation in severe sepsis. Selenium is incorporated into selenoproteins implicated in anti-oxidative defence, thyroid hormone metabolism and immunoregulation. Selenium metabolism is controlled by hepatocytes synthesizing and secreting the Se transporter selenoprotein P (SePP). The circulating SePP declines in sepsis causing low serum Se levels. Dysregulation of the hepatic selenoenzyme deiodinase type 1 (DIO1) potentially contributes to the low T3 (thyroid hormone) syndrome observed in severe diseases. We hypothesized that IL-6 affects hepatic selenoprotein biosynthesis directly. Testing human hepatocytes in culture, IL-6 reduced the concentrations of SePP mRNA and secreted SePP in a dose-dependent manner. In parallel, expression of DIO1 declined at the mRNA, protein and enzyme activity level. The effects of IL-6 on glutathione peroxidase (GPX) expression were isozyme-specific; GPX1 remained unaffected, while transcript concentrations of GPX2 increased and those of GPX4 decreased. This pattern of IL-6-dependent effects was mirrored in reporter gene experiments with SePP, DIO1, GPX1, and GPX2 promoter constructs pointing to direct transcriptional effects of IL-6. The redirection of hepatic selenoprotein biosynthesis by IL-6 may represent a central regulatory circuit responsible for the decline of serum Se and low T3 concentrations in sepsis. Accordingly, therapeutic IL-6 targeting may be effective for improving the Se and thyroid hormone status, adjuvant Se supplementation success and survival in sepsis. PMID:26399395

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

    PubMed

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

    2011-03-30

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

  6. Aromatase and estrogen receptor alpha mRNA expression as prognostic biomarkers in patients with astrocytomas.

    PubMed

    Dueñas Jiménez, J M; Candanedo Arellano, A; Santerre, A; Orozco Suárez, S; Sandoval Sánchez, H; Feria Romero, I; López-Elizalde, R; Alonso Venegas, M; Netel, B; de la Torre Valdovinos, B; Dueñas Jiménez, S H

    2014-09-01

    Estrogens are oncogenic hormones at a high level in breast, prostate, endometrial and lung cancer. Estrogens are synthesized by aromatase which has been used as a biomarker both in breast and lung cancer. Estrogen biological activities are executed by their classic receptors (ERα and ERβ). ERα has been described as a cancer promoter and ERβ, as a possible tumor suppressor. Both receptors are present at low levels in primary multiforme glioblastoma (GBM). The GBM frequency is 50 % higher in men than in women. The GBM patient survival period ranges from 7 to 18 months. The purpose of this pilot study was to evaluate aromatase and estrogen receptor expression, as well as 17ß-estradiol concentration in astrocytoma patients biopsies to obtain a prognosis biomarker for these patients. We analyzed 36 biopsies of astrocytoma patients with a different grade (I-IV) of malignity. Aromatase and estrogen receptor mRNA expression were analyzed by semiquantitative RT-PCR, and the E2 levels, by ELISA. E2 concentration was higher in GBM, compared to grade II or III astrocytomas. The number of cells immunoreactive to aromatase and estrogen receptors decreased as the grade of tumor malignity increased. Aromatase mRNA expression was present in all biopsies, regardless of malignity grade or patient age or gender. The highest expression of aromatase mRNA in GBM patients was associated to the worst survival prognostic (6.28 months). In contrast lowest expression of ERα mRNA in astrocytoma patients had a worst prognosis. In conclusion, aromatase and ERα expression could be used as prognosis biomarkers for astrocytoma patients. PMID:25005528

  7. Genetic organization and mRNA expression of enolase genes of Candida albicans.

    PubMed

    Postlethwait, P; Sundstrom, P

    1995-04-01

    In previous work, we cloned a Candida albicans cDNA for the glycolytic enzyme enolase and found a single, abundant enolase transcript on Northern (RNA) blots and a single protein on immunoblots, using antiserum raised against a recombinant enolase fusion protein. Because C. albicans enolase is abundantly produced during infection and elicits strong host immune responses, the mechanisms regulating enolase production are important for understanding the growth of C. albicans in vivo. To obtain more information on enolase gene expression by C. albicans, we used the enolase cDNA clone to investigate the genetic organization of enolase genes and the steady-state levels of enolase mRNA under several growth conditions. Gene disruption techniques in combination with Southern blot analyses of genomic DNA showed the presence of two enolase gene loci that could be distinguished by the locations of ClaI and Mn/I sites in their 3' flanking regions. Enolase steady-state mRNA levels were greatest during the middle phase of the logarithmic growth curve and were low during stationary phase. Minimal differences in enolase mRNA levels between yeast cells and hyphae were found. Propagation of C. albicans in glucose did not cause increased enolase mRNA levels compared with growth in a nonfermentable carbon source (pyruvate). It was concluded that two gene loci exist for C. albicans enolase and that enolase mRNA is constitutively produced at high levels during active metabolism. PMID:7896700

  8. Alpha-synuclein mRNA expression in oligodendrocytes in MSA

    PubMed Central

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

    2014-01-01

    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. PMID:24590631

  9. Physiology and mRNA expression in rainbow trout (Oncorhynchus mykiss) after long-term exposure to the new antifoulant medetomidine.

    PubMed

    Lennquist, Anna; Asker, Noomi; Kristiansson, Erik; Brenthel, Adam; Björnsson, Björn Thrandur; Kling, Peter; Hultman, Maria; Larsson, D G Joakim; Förlin, Lars

    2011-09-01

    Medetomidine is under evaluation for use as an antifouling agent, and its effects on non-target aquatic organisms are therefore of interest. In this study, rainbow trout was exposed to low (0.5 and 5.0nM) concentrations of medetomidine for up to 54 days. Recently we have reported on effects on paleness and melanophore aggregation of medetomidine in these fish. Here, specific growth rates were investigated together with a broad set of physiological parameters including plasma levels of growth hormone (GH), insulin-like growth factor-I (IGF-I) and leptin, glucose and haemoglobin (Hb), hematocrit (Ht), condition factor, liver and heart somatic indexes (LSI, HSI). Hepatic enzyme activities of CYP1A (EROD activity), glutathione S-transferases (GST) and glutathione reductase (GR) were also measured. Additionally, hepatic mRNA expression was analysed through microarray and quantitative PCR in fish sampled after 31 days of exposure. Medetomidine at both concentrations significantly lowered blood glucose levels and the higher concentration significantly reduced the LSI. The mRNA expression analysis revealed few differentially expressed genes in the liver and the false discovery rate was high. Taken together, the results suggest that medetomidine at investigated concentrations could interfere with carbohydrate metabolism of exposed fish but without any clear consequences for growth. PMID:21703361

  10. Regulation of cytochrome P450 mRNA expression in primary porcine hepatocytes by selected secondary plant metabolites from chicory (Cichorium intybus L.).

    PubMed

    Rasmussen, Martin Krøyer; Klausen, Christina Lindgaard; Ekstrand, Bo

    2014-03-01

    Chicory (Cichorium intybus) has been shown to induce enzymes of pharmacokinetic relevance (cytochrome P450; CYP). The aim of this study was to investigate the effects of selected secondary plant metabolites with a global extract of chicory root, on the expression of hepatic CYP mRNA (1A2, 2A19, 2C33, 2D25, 2E1 and 3A29), using primary porcine hepatocytes. Of the tested secondary plant metabolites, artemisinin, scoparone, lactucin and esculetin all induced increased expression of specific CYPs, while esculin showed no effect. In contrast, a global extract of chicory root decreased the expression of CYP1A2, 2C33, 2D25 and 3A29 at high concentrations. The results suggest that purified secondary metabolites from chicory affect CYP expression and thereby might affect detoxification in general, and that global extracts of plants can have effects different from individual components. PMID:24176340

  11. Regulation of luteinizing hormone receptor mRNA expression by a specific RNA binding protein in the ovary*

    PubMed Central

    Menon, K.M.J.; Nair, Anil K.; Wang, Lei; Peegel, Helle

    2009-01-01

    Summary The expression of LH receptor mRNA shows significant changes during different physiological states of the ovary. Previous studies from our laboratory have identified a post-transcriptional mechanism by which LH receptor mRNA is regulated following preovulatory LH surge or in response to hCG administration. A specific binding protein, identified as mevalonate kinase, binds to the open reading frame of LH receptor mRNA. The protein binding site is localized to nucleotides 203–220 of the LH receptor mRNA and exhibits a high degree of specificity. The expression levels of the protein show an inverse relationship to the LH receptor mRNA levels. The hCG-induced down-regulation of LH receptor mRNA can be mimicked by increasing the intracellular levels of cyclic AMP by a phosphodiesterase inhibitor. An in vitro mRNA decay assay showed that addition of the binding protein to the decay system caused accelerated LH receptor mRNA decay. Our results therefore show that LH receptor mRNA expression in the ovary is regulated post-transcriptionally by altering the rate of mRNA degradation by a specific mRNA binding protein. PMID:17055149

  12. Unmasking Upstream Gene Expression Regulators with miRNA-corrected mRNA Data

    PubMed Central

    Bollmann, Stephanie; Bu, Dengpan; Wang, Jiaqi; Bionaz, Massimo

    2015-01-01

    Expressed micro-RNA (miRNA) affects messenger RNA (mRNA) abundance, hindering the accuracy of upstream regulator analysis. Our objective was to provide an algorithm to correct such bias. Large mRNA and miRNA analyses were performed on RNA extracted from bovine liver and mammary tissue. Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%). Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%) and four levels of the magnitude of miRNA effect (ME) on mRNA expression (30%, 50%, 75%, and 83% mRNA reduction), we generated 17 different datasets (including the original dataset). For each dataset, we performed upstream regulator analysis using two bioinformatics tools. We detected an increased effect on the upstream regulator analysis with larger miRNA:mRNA pair bins and higher ME. The miRNA correction allowed identification of several upstream regulators not present in the analysis of the original dataset. Thus, the proposed algorithm improved the prediction of upstream regulators. PMID:27279737

  13. Elongation factor 1 gamma mRNA expression in oesophageal carcinoma.

    PubMed Central

    Mimori, K; Mori, M; Inoue, H; Ueo, H; Mafune, K; Akiyoshi, T; Sugimachi, K

    1996-01-01

    Elongation factor 1 gamma (EF1 gamma) is known to be a subunit of EF1, one of the G proteins that mediate the transport of aminoacyl tRNA to 80S ribosomes during translation. As little is known regarding the expression of EF1 gamma in human oesophageal carcinoma, this study looked at its expression using a northern blot analysis. Thirty six cases of oesophageal carcinoma and 15 oesophageal carcinoma cell lines were studied. The EF1 gamma mRNA overexpression at a level of twofold or more was seen in five (14%) of 36 carcinomatous tissues compared with the normal counterparts. All five overexpressed cases showed severe lymph node metastases compared with the non-overexpressed cases, and the difference was significant (p = 0.028). The stage of the disease of these five cases was far advanced compared with the nonoverexpressed cases (p = 0.012). All 15 oesophageal carcinoma cells expressed EF1 gamma mRNA relatively lower than the gastric or pancreatic carcinoma cell lines, in which EF1 gamma was originally isolated. As the expression of EF1 gamma mRNA could be detected even in the biopsy specimens, its overexpression in tumour tissue may provide preoperative useful information for predicting the aggressiveness of tumours. Images Figure 1 Figure 2 Figure 3 PMID:8566862

  14. 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

  15. Regional distribution of solute carrier mRNA expression along the human intestinal tract.

    PubMed

    Meier, Yvonne; Eloranta, Jyrki J; Darimont, Jutta; Ismair, Manfred G; Hiller, Christian; Fried, Michael; Kullak-Ublick, Gerd A; Vavricka, Stephan R

    2007-04-01

    Intestinal absorption of drugs, nutrients, and other compounds is mediated by uptake transporters expressed at the apical enterocyte membrane. These compounds are returned to the intestinal lumen or released into portal circulation by intestinal efflux transporters expressed at apical or basolateral membranes, respectively. One important transporter superfamily, multiple members of which are intestinally expressed, are the solute carriers (SLCs). SLC expression levels may determine the pharmacokinetics of drugs that are substrates of these transporters. In this study we characterize the distribution of 15 human SLC transporter mRNAs in histologically normal biopsies from five regions of the intestine of 10 patients. The mRNA expression levels of CNT1, CNT2, apical sodium-dependent bile acid transporter (ABST), serotonin transporter (SERT), PEPT1, and OCTN2 exhibit marked differences between different regions of the intestine: the first five are predominantly expressed in the small intestine, whereas OCTN2 exhibits strongest expression in the colon. Two transporter mRNAs studied (OCTN1, OATP2B1) are expressed at similar levels in all gut sections. In addition, ENT2 mRNA is present at low levels across the colon, but not in the small intestine. The other six SLC mRNAs studied are not expressed in the intestine. Quantitative knowledge of transporter expression levels in different regions of the human gastrointestinal tract could be useful for designing intestinal delivery strategies for orally administered drugs. Furthermore, changes in transporter expression that occur in pathological states, such as inflammatory bowel disease, can now be defined more precisely by comparison with the expression levels measured in healthy individuals. PMID:17220238

  16. Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone

    PubMed Central

    Brenmoehl, Julia; Walz, Christina; Ponsuksili, Siriluck; Schwerin, Manfred; Fuellen, Georg; Hoeflich, Andreas

    2016-01-01

    Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice. PMID:26799318

  17. Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone.

    PubMed

    Ohde, Daniela; Moeller, Mark; Brenmoehl, Julia; Walz, Christina; Ponsuksili, Siriluck; Schwerin, Manfred; Fuellen, Georg; Hoeflich, Andreas

    2016-01-01

    Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice. PMID:26799318

  18. Interferon-alpha inhibits murine macrophage transforming growth factor-beta mRNA expression.

    PubMed

    Dhanani, S; Huang, M; Wang, J; Dubinett, S M

    1994-06-01

    Transforming growth factor-beta (TGF-beta), a multifunctional polypeptide is produced by a wide variety of cells and regulates a broad array of physiological and pathological functions. TGF-beta appears to play a central role in pulmonary fibrosis and may contribute to tumor-associated immunosuppression. Alveolar macrophages are a rich source of TGF-beta and are intimately involved in lung inflammation. We therefore chose to study TGF-beta regulation in murine alveolar macrophages as well as an immortalized peritoneal macrophage cell line (IC-21). Murine macrophages were incubated with cytokines to evaluate their role in regulating TGF-beta mRNA expression. We conclude that IFN-alpha downregulates TGF-beta mRNA expression in murine macrophages. PMID:8088926

  19. Evaluation of Parkia pendula lectin mRNA differentially expressed in seedlings.

    PubMed

    Rêgo, M J B M; Santos, P B; Carvalho-Junior, L B; Stirling, J; Beltrão, E I C

    2014-05-01

    Parkia pendula (Willd.) Walp. (Fabaceae) is a neotropical species of the genus Parkia more abundantly distributed in Central to South America. From the seeds of P. pendula a glucose/mannose specific lectin (PpeL) was isolated that has been characterised and used as a biotechnological tool but until now this is the first manuscript to analyse P. pendula mRNA expression in seedlings. For this porpoise a Differential display reverse transcription polimerase chain reaction (DDRT-PCR) was used to evaluate the expression of P. pendula lectin mRNAs in non-rooted seedlings. No bands were observed in the agarose gel, indicating the absence of mRNA of PpeL seedlings. our findings confirm that lectins mRNAs are differently regulated among species even if they are grouped in the same class. PMID:25166336

  20. Requirement for nuclear autoantigenic sperm protein mRNA expression in bovine preimplantation development.

    PubMed

    Nagatomo, Hiroaki; Kohri, Nanami; Akizawa, Hiroki; Hoshino, Yumi; Yamauchi, Nobuhiko; Kono, Tomohiro; Takahashi, Masashi; Kawahara, Manabu

    2016-03-01

    Nuclear autoantigenic sperm protein (NASP) is associated with DNA replication, cell proliferation, and cell cycle progression through its specific binding to histones. The aim of this study was to examine the roles of NASP in bovine preimplantation embryonic development. Using NASP gene knockdown (KD), we confirmed the reduction of NASP messenger RNA (mRNA) expression during preimplantation development. NASP KD did not affect cleavage but significantly decreased development of embryos into the blastocyst stage. Furthermore, blastocyst hatching was significantly decreased in NASP KD embryos. Cell numbers in the inner cell mass of NASP KD blastocysts were also decreased compared to those of controls. These results suggest that NASP mRNA expression is required for preimplantation development into the blastocyst stage in cattle. PMID:26690724

  1. 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

  2. Expression profiling of Drosophila mitochondrial genes via deep mRNA sequencing

    PubMed Central

    Torres, Tatiana Teixeira; Dolezal, Marlies; Schlötterer, Christian; Ottenwälder, Birgit

    2009-01-01

    Mitochondria play an essential role in several cellular processes. Nevertheless, very little is known about patterns of gene expression of genes encoded by the mitochondrial DNA (mtDNA). In this study, we used next-generation sequencing (NGS) for transcription profiling of genes encoded in the mitochondrial genome of Drosophila melanogaster and D. pseudoobscura. The analysis of males and females in both species indicated that the expression pattern was conserved between the two species, but differed significantly between both sexes. Interestingly, mRNA levels were not only different among genes encoded by separate transcription units, but also showed significant differences among genes located in the same transcription unit. Hence, mRNA abundance of genes encoded by mtDNA seems to be heavily modulated by post-transcriptional regulation. Finally, we also identified several transcripts with a noncanonical structure, suggesting that processing of mitochondrial transcripts may be more complex than previously assumed. PMID:19843606

  3. Hyperinsulinemia Enhances Hepatic Expression of the Fatty Acid Transporter Cd36 and Provokes Hepatosteatosis and Hepatic Insulin Resistance*

    PubMed Central

    Steneberg, Pär; Sykaras, Alexandros G.; Backlund, Fredrik; Straseviciene, Jurate; Söderström, Ingegerd; Edlund, Helena

    2015-01-01

    Hepatosteatosis is associated with the development of both hepatic insulin resistance and Type 2 diabetes. Hepatic expression of Cd36, a fatty acid transporter, is enhanced in obese and diabetic murine models and human nonalcoholic fatty liver disease, and thus it correlates with hyperinsulinemia, steatosis, and insulin resistance. Here, we have explored the effect of hyperinsulinemia on hepatic Cd36 expression, development of hepatosteatosis, insulin resistance, and dysglycemia. A 3-week sucrose-enriched diet was sufficient to provoke hyperinsulinemia, hepatosteatosis, hepatic insulin resistance, and dysglycemia in CBA/J mice. The development of hepatic steatosis and insulin resistance in CBA/J mice on a sucrose-enriched diet was paralleled by increased hepatic expression of the transcription factor Pparγ and its target gene Cd36 whereas that of genes implicated in lipogenesis, fatty acid oxidation, and VLDL secretion was unaltered. Additionally, we demonstrate that insulin, in a Pparγ-dependent manner, is sufficient to directly increase Cd36 expression in perfused livers and isolated hepatocytes. Mouse strains that display low insulin levels, i.e. C57BL6/J, and/or lack hepatic Pparγ, i.e. C3H/HeN, do not develop hepatic steatosis, insulin resistance, or dysglycemia on a sucrose-enriched diet, suggesting that elevated insulin levels, via enhanced CD36 expression, provoke fatty liver development that in turn leads to hepatic insulin resistance and dysglycemia. Thus, our data provide evidence for a direct role for hyperinsulinemia in stimulating hepatic Cd36 expression and thus the development of hepatosteatosis, hepatic insulin resistance, and dysglycemia. PMID:26085100

  4. 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.

  5. Metformin prevents hepatic steatosis by regulating the expression of adipose differentiation-related protein

    PubMed Central

    LIU, FANG; WANG, CHAO; ZHANG, LIJUN; XU, YUQIAO; JANG, LINA; GU, YU; CAO, XIANGMEI; ZHAO, XIN; YE, JING; LI, QING

    2014-01-01

    Non-alcoholic fatty liver disease (NAFLD) is a common liver disease, characterized by the excess accumulation of lipids in the liver. It has been demonstrated that the dysregulation of lipid droplet (LD)-associated proteins may be involved in the development of NAFLD. Adipose differentiation-related protein (ADRP), as one of the major LD-associated proteins, is expressed in normal and steatotic livers; however, the exact role of ADRP in the liver remains unknown. Previous studies have indicated that metformin, as an antidiabetic drug, effectively ameliorates NAFLD. However, its cellular and molecular mechanisms of action remain to be elucidated. Therefore, the aim of this study was to determine the role of ADRP in the metformin-mediated regulation of hepatic steatosis. We examined the effects of meformin in vivo and in vitro using ob/ob mice and primary hepatocytes, respectively. Lipid accumulation in the hepatocytes was induced by treatment with oleate. Our results revealed that metformin prevented hepatic steatosis in ob/ob mice and inhibited oleate-induced lipid accumulation in primary hepatocytes. Furthermore, using real-time PCR and western blot analysis, we examined the mRNA and protein expression of ADRP, respectively. We found that metformin significantly decreased the expression levels of ADRP. In addition, to further clarify the role of ADRP in lipid accumulation, we generated recombinant adenoviruses to induce the overexpression of ADRP and to knockdown ADRP. In the hepatocytes in which ADRP was overexpressed, the reducing effects of metformin on lipid accumulation were diminished. However, the knockdown of ADRP using siRNA targeting ADRP reduced the accumulation of triglycerides. Taken together, our data demonstrate that metformin prevents hepatic steatosis by regulating the expression of ADRP, which may be a key target in the treatment of NAFLD. PMID:24253218

  6. Comparison of IgE expression at the mRNA and protein levels in vitro.

    PubMed Central

    Turner, K J; Creany, J; Coelen, R J; Cameron, K J; Holt, B J; Beilharz, M W

    1991-01-01

    The regulating effects of IL-4 and pokeweed mitogen on IgE synthesis in vitro by human peripheral blood leucocytes has been compared with the corresponding effect of these regulators on the expression of IgE mRNA. The latter was measured by dot blot hybridization with an oligonucleotide coding for a unique six amino acid region of the CH epsilon 2 domain. Specificity of the oligonucleotide probe was established by its inability to hybridize with RNA extracted from HMY-2 (IgG) and XQ-15 (IgM) secreting cell lines whilst producing intense signals with RNA extracted from the IgE secreting cell line U266. Whilst IgE mRNA was detected in RNA extracted from PBL of both atopic and control subjects, spontaneous IgE synthesis was restricted to atopic PBL. IL-4 increased both IgE mRNA and IgE synthesis in all PBL samples but PWM, while significantly increasing IgE mRNA expression either failed to modify IgE synthesis or actively suppressed it. The assay system employed to quantitate IgE synthesis in vitro was shown to be inhibited by both IgE binding factors and IgG anti-IgE autoantibodies which are produced in PBL cultures. IgE mRNA levels might therefore more accurately monitor the regulatory effects of IL-4 and PWM on IgE synthesis than quantitation of the IgE by radioimmunoassay. Images Figure 1 PMID:1783428

  7. Association of differentially expressed genes with activation of mouse hepatic stellate cells by high-density cDNA mircoarray

    PubMed Central

    Liu, Xiao-Jing; Yang, Li; Luo, Feng-Ming; Wu, Hong-Bin; Qu-Qiang

    2004-01-01

    AIM: To characterize the gene expression profiles associated with activation of mouse hepatic stellate cell (HSC) and provide novel insights into the pathogenesis of hepatic fibrosis. METHODS: Mice HSCs were isolated from BALB/c mice by in situ perfusion of collagenase and pronase and single-step density Nycodenz gradient. Total RNA and mRNA of quiescent HSC and culture-activated HSC were extracted, quantified and reversely transcripted into cDNA. cDNAs from activated HSC were labeled with Cy5 and cDNAs from the quiescent HSC were labeled with Cy3, which were mixed with equal quantity, then hybridized with cDNA chips containing 4000 genes. Chips were washed, scanned and analyzed. Increased expression of 4 genes and decreased expression of one gene in activated HSC were confirmed by reverse transcription- polymerase chain reaction (RT-PCR). RESULTS: A total of 835 differentially expressed genes were identified by cDNA chip between activated and quiescent HSC, and 465 genes were highly expressed in activated HSC. The differentially expressed genes included those involved in protein synthesis, cell-cycle regulation, apoptosis, and DNA damage response. CONCLUSION: Many genes implicated in intrahepatic inflammation, fibrosis and proliferation were up-regulated in activated HSC. cDNA microarray is an effective technique in screening for differentially expressed genes between two different situations of the HSC. Further analysis of the obtained genes will help understand the molecular mechanism of activation of HSC and hepatic fibrosis. PMID:15162533

  8. Promoter Methylation and mRNA Expression of Response Gene to Complement 32 in Breast Carcinoma

    PubMed Central

    Eskandari-Nasab, Ebrahim; Hashemi, Mohammad; Rafighdoost, Firoozeh

    2016-01-01

    Background. Response gene to complement 32 (RGC32), induced by activation of complements, has been characterized as a cell cycle regulator; however, its role in carcinogenesis is still controversial. In the present study we compared RGC32 promoter methylation patterns and mRNA expression in breast cancerous tissues and adjacent normal tissues. Materials and Methods. Sixty-three breast cancer tissues and 63 adjacent nonneoplastic tissues were included in our study. Design. Nested methylation-specific polymerase chain reaction (Nested-MSP) and quantitative PCR (qPCR) were used to determine RGC32 promoter methylation status and its mRNA expression levels, respectively. Results. RGC32 methylation pattern was not different between breast cancerous tissue and adjacent nonneoplastic tissue (OR = 2.30, 95% CI = 0.95–5.54). However, qPCR analysis displayed higher levels of RGC32 mRNA in breast cancerous tissues than in noncancerous tissues (1.073 versus 0.959; P = 0.001), irrespective of the promoter methylation status. The expression levels and promoter methylation of RGC32 were not correlated with any of patients' clinical characteristics (P > 0.05). Conclusion. Our findings confirmed upregulation of RGC32 in breast cancerous tumors, but it was not associated with promoter methylation patterns. PMID:27118972

  9. Codon influence on protein expression in E. coli correlates with mRNA levels.

    PubMed

    Boël, Grégory; Letso, Reka; Neely, Helen; Price, W Nicholson; Wong, Kam-Ho; Su, Min; Luff, Jon D; Valecha, Mayank; Everett, John K; Acton, Thomas B; Xiao, Rong; Montelione, Gaetano T; Aalberts, Daniel P; Hunt, John F

    2016-01-21

    Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli. PMID:26760206

  10. 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

  11. 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.

  12. Cytokine mRNA expression in Peromyscus yucatanicus (Rodentia: Cricetidae) infected by Leishmania (Leishmania) mexicana.

    PubMed

    Loria-Cervera, Elsy Nalleli; Sosa-Bibiano, Erika Ivett; Van Wynsberghe, Nicole Raymonde; Saldarriaga, Omar Abdul; Melby, Peter C; Andrade-Narvaez, Fernando Jose

    2016-07-01

    Peromyscus yucatanicus, the main reservoir of Leishmania (Leishmania) mexicana in the Yucatan peninsula of Mexico, reproduces clinical and histological pictures of LCL in human as well as subclinical infection. Thus, we used this rodent as a novel experimental model. In this work, we analyzed cytokine mRNA expression in P. yucatanicus infected with L. (L.) mexicana. Animals were inoculated with either 2.5×10(6) or 1×10(2) promastigotes and cytokine expressions were analyzed by real-time RT-PCR in skin at 4 and 12weeks post-infection (wpi). Independently of the parasite inoculum none of the infected rodents had clinical signs of LCL at 4wpi and all expressed high IFN-γ mRNA. All P. yucatanicus inoculated with 2.5×10(6) promastigotes developed signs of LCL at 12wpi while the mice inoculated with 1×10(2) remained subclinical. At that time, both IFN-γ and IL-10 were expressed in P. yucatanicus with clinical and subclinical infections. Expressions of TNF-α and IL-4 were significantly higher in clinical animals (2.5×10(6)) compared with subclinical ones (1×10(2)). High TGF-β expression was observed in P. yucatanicus with clinical signs when compared with healthy animals. Results suggested that the clinical course of L. (L.) mexicana infection in P. yucatanicus was associated with a specific local pattern of cytokine production at 12wpi. PMID:27155064

  13. 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

  14. Quercetin induces hepatic γ-glutamyl hydrolase expression in rats by suppressing hepatic microRNA rno-miR-125b-3p.

    PubMed

    Wein, Silvia Anette; Laviano, Alessandro; Wolffram, Siegfried

    2015-12-01

    Exogenous factors such as food components including the flavonoid quercetin are suspected to influence micro RNA (miRNA) concentrations and thus possibly target enzymes involved in xenobiotic metabolism. This study therefore investigates the influence of orally administered quercetin on hepatic miRNA and the identification of enzyme target mRNAs relevant in drug metabolism. Male Wistar rats (n=16) were fed either a diet without (C) or with (Q) the addition of 100-ppm quercetin for 7 weeks and subsequently euthanized at the end of the dark phase. To avoid strong effects of food deprivation on hepatic metabolism, food was not removed until 5 h prior to the procedure. Liver was immediately dissected and snap-frozen in liquid nitrogen. Concentrations of 352 hepatic miRNA were measured in pool samples of each dietary group (n=8) using the RT(2) miRNA PCR Array System. Differential expression of miRNAs was assumed with fold changes ≥3. Target genes of differentially expressed miRNAs were identified using the database TargetScan. Because rno-miR-125b-3p showed the most prominent fold-change (-9) we further analyzed the expression of its top predicted target gene gamma-glutamyl hydrolase (GGH) by quantitative real-time PCR using hypoxanthine phosphoribosyltransferase 1 (hprt1) as endogenous control. Compared to controls, 23 miRNAs were differentially expressed in rats fed quercetin. A ninefold reduction in hepatic miRNA rno-miR-125b-3p was paralleled by significant induction of GGH mRNA in liver of quercetin fed rats. Because increased GGH expressions were repeatedly associated with resistance to methotrexate, concomitant intake with quercetin should be monitored carefully. PMID:26432773

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

    PubMed

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

    2001-03-30

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

  16. CART mRNA expression in rat monkey and human brain: relevance to cocaine abuse.

    PubMed

    Fagergren, Pernilla; Hurd, Yasmin

    2007-09-10

    The neuropeptide CART (cocaine and amphetamine regulated transcript) is suggested to be regulated by psychostimulant administration. We review here the localization of CART mRNA expression in the human brain and its possible relevance to human cocaine abuse. Except for strong hypothalamic expression, the CART transcript is predominately expressed in target regions of the mesocorticolimbic dopamine system, such as the nucleus accumbens shell, amygdala complex, extended amygdala and orbitofrontal, enthorhinal and piriform cortices. The discrete limbic localization strongly implies involvement in reward and reinforcement behaviors. We therefore examined CART mRNA expression in both Sprague Dawley rats and Rhesus monkeys that had self-administered cocaine. Cocaine self-administration in the rat (1.5 mg/kg/inj, on a fixed ratio 1 schedule of reinforcement for 1 week) and monkey (0.03 or 0.3 mg/kg/inj on a fixed 3 min interval schedule of reinforcement for 5 or 100 days) did not alter transcript levels in CART expressing nucleus accumbens (monkey not studied), amygdala nuclei or cortical areas. However, in the monkey sublenticular extended amygdala, low dose cocaine self-administration resulted in increased CART transcript levels after both 5 and 100 days of self-administration, whereas no difference was found after high dose self-administration. In conclusion, we found no substantial alterations CART mRNA expression during cocaine self-administration, but this neuropeptide has the anatomical and functional potential to modulate brain areas relevant for cocaine abuse. Further studies are needed to evaluate the involvement of CART in other components of the cocaine abuse cycle. PMID:17631364

  17. Hepatitis B Virus Expression and Replication in Ovum and the Influencing Factors

    PubMed Central

    Kong, Ying; Ye, Feng; Jin, Yan; Shi, Juanzi; Qiu, Hongtao; Lin, Shumei

    2016-01-01

    Background/Aim: The aim of this study was to investigate the factors that influence hepatitis B virus (HBV) expression and replication in the ovum. Materials and Methods: Immunohistochemistry and in situ hybridization techniques were used to assay the distributions of HBcAg, HBV DNA, and HBV mRNA in ovarian tissues and the ovum in 50 patients with chronic HBV infection. HBeAg and HBV DNA in the serum were also detected. Comparisons of categorical data were performed using McNemar test. Results: The positive rates of HBcAg, HBV DNA, and HBV mRNA in ovum and ovarian tissues of high replication group were significantly higher than low replication group (χ2 = 15.04, P < 0.05; χ2 = 12.96, P <0.05; χ2 = 19.36, P < 0.05; respectively). High positive rates of HBcAg, HBV DNA, and HBV mRNA in ovum and ovarian tissues were found in women with HBeAg-positive than HBeAg-negative (χ2 = 113.14,P < 0.05; χ2 = 11.13, P < 0.05; χ2 = 17.39, P < 0.05; respectively). Conclusion: HBV can infect and replicate in the ovary and ovum. Maternal HBeAg status and HBV DNA levels are important influencing factors. PMID:27184640

  18. 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

  19. Differential dynamics of the mammalian mRNA and protein expression response to misfolding stress.

    PubMed

    Cheng, Zhe; Teo, Guoshou; Krueger, Sabrina; Rock, Tara M; Koh, Hiromi W L; Choi, Hyungwon; Vogel, Christine

    2016-01-01

    The relative importance of regulation at the mRNA versus protein level is subject to ongoing debate. To address this question in a dynamic system, we mapped proteomic and transcriptomic changes in mammalian cells responding to stress induced by dithiothreitol over 30 h. Specifically, we estimated the kinetic parameters for the synthesis and degradation of RNA and proteins, and deconvoluted the response patterns into common and unique to each regulatory level using a new statistical tool. Overall, the two regulatory levels were equally important, but differed in their impact on molecule concentrations. Both mRNA and protein changes peaked between two and eight hours, but mRNA expression fold changes were much smaller than those of the proteins. mRNA concentrations shifted in a transient, pulse-like pattern and returned to values close to pre-treatment levels by the end of the experiment. In contrast, protein concentrations switched only once and established a new steady state, consistent with the dominant role of protein regulation during misfolding stress. Finally, we generated hypotheses on specific regulatory modes for some genes. PMID:26792871

  20. 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

  1. Analysis of hepatic deiodinase 2 mRNA levels in natural fish lake populations exposed to different levels of putative thyroid disrupters.

    PubMed

    Jarque, Sergio; Bosch, Carme; Casado, Marta; Grimalt, Joan O; Raldúa, Demetrio; Piña, Benjamin

    2014-04-01

    Hepatic mRNA levels of the dio2 gene (deiodinase 2), implicated in thyroid hormone homeostasis, were analyzed in trout from six remote lakes in the Pyrenees (Spain) and the Tatra Mountains (Slovakia). Highest levels corresponded to fish from the two coldest lakes in Pyrenees, whereas relatively low levels were found in the Tatra lakes. These values correlated with the presence of highly-brominated polybrominated diphenyl ethers (PBDE) congeners in the muscle of the same animals, reflecting the distribution of these compounds across European mountain ranges. In contrast, cyp1a expression levels, diagnostic for the presence of dioxin-like pollutants, mirrored the distribution of semi-volatile organochlorine compounds, indicating the specificity of the two types of biological responses. Exposure to PDBEs is known to increase transcription of dio2 and other thyroid-related genes in laboratory experiments; we propose that our data reflects the same phenomenon in natural populations, driven by anthropogenic pollutants at the environmental concentrations. PMID:24530182

  2. Urinary Hepcidin Levels in Iron-Deficient and Iron-Supplemented Piglets Correlate with Hepcidin Hepatic mRNA and Serum Levels and with Body Iron Status.

    PubMed

    Staroń, Robert; Van Swelm, Rachel P L; Lipiński, Paweł; Gajowiak, Anna; Lenartowicz, Małgorzata; Bednarz, Aleksandra; Gajewska, Małgorzata; Pieszka, Marek; Laarakkers, Coby M M; Swinkels, Dorine W; Starzyński, Rafał R

    2015-01-01

    Among livestock, domestic pig (Sus scrofa) is a species, in which iron metabolism has been most intensively examined during last decade. The obvious reason for studying the regulation of iron homeostasis especially in young pigs is neonatal iron deficiency anemia commonly occurring in these animals. Moreover, supplementation of essentially all commercially reared piglets with iron entails a need for monitoring the efficacy of this routine practice followed in the swine industry for several decades. Since the discovery of hepcidin many studies confirmed its role as key regulator of iron metabolism and pointed out the assessment of its concentrations in biological fluids as diagnostic tool for iron-related disorder. Here we demonstrate that urine hepcidin-25 levels measured by a combination of weak cation exchange chromatography and time-of-flight mass spectrometry (WCX-TOF MS) are highly correlated with mRNA hepcidin expression in the liver and plasma hepcidin-25 concentrations in anemic and iron-supplemented 28-day old piglets. We also found a high correlation between urine hepcidin level and hepatic non-heme iron content. Our results show that similarly to previously described transgenic mouse models of iron disorders, young pigs constitute a convenient animal model to explore accuracy and relationship between indicators for assessing systemic iron status. PMID:26323096

  3. 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. PMID:24080485

  4. mRNA expression profiles of calmodulin and liver receptor homolog-1 genes in chickens.

    PubMed

    Zhang, Z-C; Xiao, L-H; Wang, Y; Chen, S-Y; Yang, Z-Q; Zhao, X-L; Zhu, Q; Liu, Y-P

    2012-01-01

    Calmodulin (CALM), a calcium-binding protein, is expressed in the hypothalamic-pituitary-gonadal axis; it plays a pivotal role in the reproductive system by regulating gonadotropin-releasing hormone signaling. Downstream of hypothalamic-pituitary-gonadal signaling pathways, liver receptor homolog-1 (LRH-1) is involved in female gonadal hormone synthesis. In the chicken, although the two genes are known to be associated with reproductive traits, the interaction between gonadotropins and gonadal steroids remains unclear. We used quantitative real-time PCR to quantify the tissular (hypothalamus, pituitary, ovary, liver, kidney, oviduct, heart) and ontogenetic (12, 18, 32, and 45 weeks) mRNA expression profiles of CALM and LRH-1 in Erlang Mountainous chickens to determine their roles in the endocrine control of fertility, and compared these profiles with expression in Roman chickens. We found that the relative expressions of CALM and LRH-1 genes had the highest levels in the pituitary and ovary at 32 weeks. The expression level of CALM mRNA in the pituitary of Roman chickens was significantly higher than that in Erlang Mountainous chickens at 32 and 45 weeks, while the LRH-1 transcript level in the ovaries of Roman chickens was significantly lower than that of Erlang Mountainous chickens at 32 and 45 weeks. In summary, the transcript levels of CALM and LRH-1 genes are associated with chicken reproductive traits; in addition, we found that the CALM gene is the key regulator in the hypothalamic-pituitary-gonadal signaling network. PMID:23079841

  5. Variability in hepatic expression of organic anion transporter 7/SLC22A9, a novel pravastatin uptake transporter: impact of genetic and regulatory factors.

    PubMed

    Emami Riedmaier, A; Burk, O; van Eijck, B A C; Schaeffeler, E; Klein, K; Fehr, S; Biskup, S; Müller, S; Winter, S; Zanger, U M; Schwab, M; Nies, A T

    2016-08-01

    Human organic anion transporter 7 (OAT7, SLC22A9) is a hepatic transport protein poorly characterized so far. We therefore sought to identify novel OAT7 substrates and factors contributing to variable hepatic OAT7 expression. Using OAT7-expressing cells, pravastatin was identified as a substrate. Hepatic SLC22A9/OAT7 mRNA and protein expression varied 28-fold and 15-fold, respectively, in 126 Caucasian liver samples. Twenty-four variants in SLC22A9 were genotyped, including three rare missense variants (rs377211288, rs61742518, rs146027075), which occurred only heterozygously. No variant significantly affected hepatic SLC22A9/OAT7 expression. The three missense variants, however, showed functional consequences when expressed in vitro. Hepatic nuclear factor 4-alpha (HNF4α) emerged as a major transcriptional regulator of SLC22A9 by a series of in silico and in vitro analyses. In conclusion, pravastatin is the first identified OAT7 drug substrate. Substantial inter-individual variability in hepatic OAT7 expression, majorly driven by HNF4α, may contribute to pravastatin drug disposition and might affect response.The Pharmacogenomics Journal advance online publication, 4 August 2015; doi:10.1038/tpj.2015.55. PMID:26239079

  6. Complement gene expression in hepatic and extrahepatic tissues of NZB and NZB x W (F1) mouse strains.

    PubMed Central

    Passwell, J H; Schreiner, G F; Wetsel, R A; Colten, H R

    1990-01-01

    To study the role of local production of complement proteins during the evolution of a naturally occurring immune complex disease, C3, C4, C2 and Factor B mRNA expression was assessed in several tissues of the inbred mouse strains NZB and (NZB x W) F1 hybrid. In the NZB/W F1 hybrid strain, coincident with the development of glomerulonephritis a marked increase in kidney C3 and C4 mRNA was observed; Factor B mRNA, which is expressed as a doublet in kidney and intestine, showed an increase in expression of the smaller transcript. This alteration of kidney C3, C4 and Factor B mRNA is identical to that noted in association with lupus nephritis in the MRL lpr/lpr strain and following in vivo administration of endotoxin to the BALB/c strain. The development of systemic lupus erythematosis (SLE) in the NZB/W F1 was not associated with a marked change in hepatic complement gene expression. These findings support the hypothesis that local production of complement may play a role in the pathogenesis of glomerulonephritis and other tissue injury in SLE. Images Figure 1 Figure 2 PMID:2228028

  7. The Altered Renal and Hepatic Expression of Solute Carrier Transporters (SLCs) in Type 1 Diabetic Mice

    PubMed Central

    Xu, Chenghao; Zhu, Ling; Chan, Ting; Lu, Xiaoxi; Shen, Weiyong; Gillies, Mark C.; Zhou, Fanfan

    2015-01-01

    Diabetes mellitus is a chronic metabolic disorder that significantly affects human health and well-being. The Solute carrier transporters (SLCs), particularly the Organic anion/cation transporters (Oats/Octs/Octns), Organic anion transporting polypeptides (Oatps) and Oligopeptide transporters (Pepts) are essential membrane proteins responsible for cellular uptake of many endogenous and exogenous substances such as clinically important drugs. They are widely expressed in mammalian key organs especially the kidney and liver, in which they facilitate the influx of various drug molecules, thereby determining their distribution and elimination in body. The altered expression of SLCs in diabetes mellitus could have a profound and clinically significant influence on drug therapies. In this study, we extensively investigated the renal and hepatic expression of twenty essential SLCs in the type 1 diabetic Ins2Akita murine model that develops both hyperglycemia and diabetes-related complications using real-time PCR and immunoblotting analysis. We found that the renal expression of mOatp1a1, mOatp1a6, mOat1, mOat3, mOat5, mOct2 and mPept2 was decreased; while that of mPept1 was increased at the mRNA level in the diabetic mice compared with non-diabetic controls. We found up-regulated mRNA expression of mOatp1a4, mOatp1c1, mOctn2, mOct3 and mPept1 as well as down-regulation of mOatp1a1 in the livers of diabetic mice. We confirmed the altered protein expression of several SLCs in diabetic mice, especially the decreased renal and hepatic expression of mOatp1a1. We also found down-regulated protein expression of mOat3 and mOctn1 in the kidneys as well as increased protein expression of mOatp1a4 and mOct3 in the livers of diabetic mice. Our findings contribute to better understanding the modulation of SLC transporters in type 1 diabetes mellitus, which is likely to affect the pharmacokinetic performance of drugs that are transported by these transporters and therefore, forms the

  8. Reduced mRNA expression levels of MBD2 and MBD3 in gastric carcinogenesis.

    PubMed

    Pontes, Thaís Brilhante; Chen, Elizabeth Suchi; Gigek, Carolina Oliveira; Calcagno, Danielle Queiroz; Wisnieski, Fernanda; Leal, Mariana Ferreira; Demachki, Samia; Assumpção, Paulo Pimentel; Artigiani, Ricardo; Lourenço, Laércio Gomes; Burbano, Rommel Rodriguez; Arruda Cardoso Smith, Marília

    2014-04-01

    Aberrant methylation has been reported in several neoplasias, including gastric cancer. The methyl-CpG-binding domain (MBD) family proteins have been implicated in the chromatin remodeling process, leading to the modulation of gene expression. To evaluate the role of MBD2 and MBD3 in gastric carcinogenesis and the possible association with clinicopathological characteristics, we assessed the mRNA levels and promoter methylation patterns in gastric tissues. In this study, MBD2 and MBD3 mRNA levels were determined by RT-qPCR in 28 neoplastic and adjacent nonneoplastic and 27 gastritis and non-gastritis samples. The promoter methylation status was determined by bisulfite sequencing, and we found reduced MBD2 and MBD3 levels in the neoplastic samples compared with the other groups. Moreover, a strong correlation between the MBD2 and MBD3 expression levels was observed in each set of paired samples. Our data also showed that the neoplastic tissues exhibited higher MBD2 promoter methylation than the other groups. Interestingly, the non-gastritis group was the only one with positive methylation in the MBD3 promoter region. Furthermore, a weak correlation between gene expression and methylation was observed. Therefore, our data suggest that DNA methylation plays a minor role in the regulation of MBD2 and MBD3 expression, and the presence of methylation at CpGs that interact with transcription factor complexes might also be involved in the modulation of these genes. Moreover, reduced mRNA expression of MBD2 and MBD3 is implicated in gastric carcinogenesis, and thus, further investigations about these genes should be conducted for a better understanding of the role of abnormal methylation involved in this neoplasia. PMID:24338710

  9. Genomic Analysis and mRNA Expression of Equine Type I Interferon Genes

    PubMed Central

    Detournay, Olivier; Morrison, David A.; Wagner, Bettina; Zarnegar, Behdad

    2013-01-01

    This study aimed at identifying all of the type I interferon (IFN) genes of the horse and at monitoring their expression in equine cells on in vitro induction. We identified 32 putative type I IFN loci on horse chromosome 23 and an unplaced genomic scaffold. A phylogentic analysis characterized these into 8 different type I IFN classes, that is, putative functional genes for 6 IFN-α, 4 IFN-β, 8 IFN-ω (plus 4 pseudogenes), 3 IFN-δ (plus 1 pseudogene), 1 IFN-κ and 1 IFN-ɛ, plus 1 IFN-ν pseudogene, and 3 loci belonging to what has previously been called IFN-αω. Our analyses indicate that the IFN-αω genes are quite distinct from both IFN-α and IFN-ω, and we refer to this type I IFN as IFN-μ. Results from cell cultures showed that leukocytes readily expressed IFN-α, IFN-β, IFN-δ, IFN-μ, and IFN-ω mRNA on induction with, for example, live virus; while fibroblasts only expressed IFN-β mRNA on stimulation. IFN-κ or IFN-ɛ expression was not consistently induced in these cell cultures. Thus, the equine type I IFN family comprised 8 classes, 7 of which had putative functional genes, and mRNA expression of 5 was induced in vitro. Moreover, a relatively low number of IFN-α subtypes was found in the horse compared with other eutherian mammals. PMID:23772953

  10. Influence of neonatal hypothyroidism on hepatic gene expression and lipid metabolism in adulthood.

    PubMed

    Santana-Farré, Ruymán; Mirecki-Garrido, Mercedes; Bocos, Carlos; Henríquez-Hernández, Luis A; Kahlon, Nusrat; Herrera, Emilio; Norstedt, Gunnar; Parini, Paolo; Flores-Morales, Amilcar; Fernández-Pérez, Leandro

    2012-01-01

    Thyroid hormones are required for normal growth and development in mammals. Congenital-neonatal hypothyroidism (CH) has a profound impact on physiology, but its specific influence in liver is less understood. Here, we studied how CH influences the liver gene expression program in adulthood. Pregnant rats were given the antithyroid drug methimazole (MMI) from GD12 until PND30 to induce CH in male offspring. Growth defects due to CH were evident as reductions in body weight and tail length from the second week of life. Once the MMI treatment was discontinued, the feed efficiency increased in CH, and this was accompanied by significant catch-up growth. On PND80, significant reductions in body mass, tail length, and circulating IGF-I levels remained in CH rats. Conversely, the mRNA levels of known GH target genes were significantly upregulated. The serum levels of thyroid hormones, cholesterol, and triglycerides showed no significant differences. In contrast, CH rats showed significant changes in the expression of hepatic genes involved in lipid metabolism, including an increased transcription of PPARα and a reduced expression of genes involved in fatty acid and cholesterol uptake, cellular sterol efflux, triglyceride assembly, bile acid synthesis, and lipogenesis. These changes were associated with a decrease of intrahepatic lipids. Finally, CH rats responded to the onset of hypothyroidism in adulthood with a reduction of serum fatty acids and hepatic cholesteryl esters and to T3 replacement with an enhanced activation of malic enzyme. In summary, we provide in vivo evidence that neonatal hypothyroidism influences the hepatic transcriptional program and tissue sensitivity to hormone treatment in adulthood. This highlights the critical role that a euthyroid state during development plays on normal liver physiology in adulthood. PMID:22666351

  11. Influence of Neonatal Hypothyroidism on Hepatic Gene Expression and Lipid Metabolism in Adulthood

    PubMed Central

    Bocos, Carlos; Henríquez-Hernández, Luis A.; Kahlon, Nusrat; Herrera, Emilio; Norstedt, Gunnar; Parini, Paolo; Flores-Morales, Amilcar; Fernández-Pérez, Leandro

    2012-01-01

    Thyroid hormones are required for normal growth and development in mammals. Congenital-neonatal hypothyroidism (CH) has a profound impact on physiology, but its specific influence in liver is less understood. Here, we studied how CH influences the liver gene expression program in adulthood. Pregnant rats were given the antithyroid drug methimazole (MMI) from GD12 until PND30 to induce CH in male offspring. Growth defects due to CH were evident as reductions in body weight and tail length from the second week of life. Once the MMI treatment was discontinued, the feed efficiency increased in CH, and this was accompanied by significant catch-up growth. On PND80, significant reductions in body mass, tail length, and circulating IGF-I levels remained in CH rats. Conversely, the mRNA levels of known GH target genes were significantly upregulated. The serum levels of thyroid hormones, cholesterol, and triglycerides showed no significant differences. In contrast, CH rats showed significant changes in the expression of hepatic genes involved in lipid metabolism, including an increased transcription of PPARα and a reduced expression of genes involved in fatty acid and cholesterol uptake, cellular sterol efflux, triglyceride assembly, bile acid synthesis, and lipogenesis. These changes were associated with a decrease of intrahepatic lipids. Finally, CH rats responded to the onset of hypothyroidism in adulthood with a reduction of serum fatty acids and hepatic cholesteryl esters and to T3 replacement with an enhanced activation of malic enzyme. In summary, we provide in vivo evidence that neonatal hypothyroidism influences the hepatic transcriptional program and tissue sensitivity to hormone treatment in adulthood. This highlights the critical role that a euthyroid state during development plays on normal liver physiology in adulthood. PMID:22666351

  12. 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. PMID:25312047

  13. 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.

  14. Expression of SPARC by activated hepatic stellate cells and its correlation with the stages of fibrogenesis in human chronic hepatitis.

    PubMed

    Nakatani, Kazuki; Seki, Shuichi; Kawada, Norifumi; Kitada, Takuya; Yamada, Takao; Sakaguchi, Hiroki; Kadoya, Hirokazu; Ikeda, Kazuo; Kaneda, Kenji

    2002-11-01

    Secreted protein, acidic and rich in cysteine (SPARC), which functions in tissue remodeling, has been reported to be expressed by myofibroblasts in liver cirrhosis and hepatocellular carcinoma. This study aimed to reveal its expression in chronic hepatitis. Immuno-light and electron microscopy demonstrated that SPARC was expressed by nerve fibers and hepatic stellate cells (HSCs) in the liver parenchyma and myofibroblasts in the fibrous septa. Reaction products were localized in the rough endoplasmic reticulum and nuclear envelope. Serial section analysis demonstrated that SPARC, platelet-derived growth factor receptor-beta, and alpha-smooth muscle actin were co-expressed by HSCs. Quantitative analysis demonstrated that, while SPARC-positive HSCs were sparse in control livers, they significantly increased in number in the livers with chronic hepatitis. There were, however, no significant differences in number among the grades of activity, the stages of fibrosis, or etiology (virus-infected or autoimmune, hepatitis B virus or hepatitis C virus). In liver cirrhosis, however, they significantly decreased in number. The present results indicate that SPARC is expressed by activated HSCs in chronic hepatitis, suggesting the involvement of SPARC in hepatic fibrogenesis after chronic injuries. PMID:12447677

  15. Hepatitis

    MedlinePlus

    ... Got Homework? Here's Help White House Lunch Recipes Hepatitis KidsHealth > For Kids > Hepatitis Print A A A ... an important digestive liquid called bile . What Is Hepatitis? Hepatitis is an inflammation (say: in-fluh-MAY- ...

  16. 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. PMID:25967984

  17. Increased expression of C5a receptor (CD88) mRNA in canine mammary tumors.

    PubMed

    Hezmee, Mohd Noor Mohd; Kyaw-Tanner, Myat; Lee, Jia Yu Peppermint; Shiels, Ian A; Rolfe, Barbara; Woodruff, Trent; Mills, Paul C

    2011-01-01

    Mammary tumors are among the most common neoplastic conditions in dogs, and there is evidence that inflammation plays a role in the development of some tumor types in dogs. The complement system is a major participant in the inflammatory process and the complement activation component, C5a, is a potent inflammatory peptide. This study investigated the mRNA expression of the major receptor for C5a (C5aR; CD88) in histopathological samples of canine mammary tumors by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) using canine-specific primers for CD88. A total of seven canine mammary tumors (four malignant carcinomas, two benign mixed mammary tumors, and one myoepithelioma) and eight normal mammary glands were analysed. All the tumor samples expressed low levels of CD88 mRNA, while none of the normal mammary tissues showed any detectable expression. These preliminary results suggest that C5a-CD88 interaction may play a contributory role in the inflammatory response associated with mammary tumor development in dogs. Further studies investigating the mechanisms behind complement activation and C5a receptor expression in canine mammary tumors are warranted. PMID:20846729

  18. Effect of Huazhuojiedu medicated serum on the proliferation and activation of hepatic stellate cells and the expression of PI3K and p-Akt in rats

    PubMed Central

    Kang, Liang; Wang, Yangang; Zhang, Mingxi; Sun, Runxue; Lou, Yingying; Wang, Ying; Li, Diangui

    2014-01-01

    To observe the effect of Huazhuojiedu medicated serum on the proliferation and activation of hepatic stellate cells, as well as the expression of PI3K and p-Akt in rats, and to explore the underlying mechanism of Huazhuojiedu prescription against hepatic fibrosis. Hepatic stellate cells harvested from rats were resuscitated and subcultured, followed by the intervention of Huazhuojiedu equivalent dose, Huazhuojiedu double dose, and positive drug (Compound Biejiaruangan Troche) medicated serum of rats. After in vitro culture, hepatic stellate cells were stimulated with 5 ng/mL transforming growth factor-β1. At 24, 48, 72 hours, the proliferation of hepatic stellate cells was detected with MTT assay; at 48 hours, α-SMA mRNA and protein expression in hepatic stellate cells were determined with RT-PCR assay and western blot analysis, respectively, to evaluate the activation of hepatic stellate cells; in addition, PI3K and p-Akt protein expression levels were also assayed with western blot analysis at 48 hours. The results showed that, 24-hour transforming growth factor-β1 stimulation significantly promoted the proliferation of hepatic stellate cells (P < 0.01). Each medicated serum inhibited the proliferation of hepatic stellate cells (P < 0.01). Huazhuojiedu equivalent dose had the similar inhibition effect with positive drug (P > 0.05), and Huazhuojiedu double dose achieved more apparent inhibition effect (P < 0.01). After 48 and 72 hours of transforming growth factor-β1 stimulation, hepatic stellate cells still proliferated significantly (P < 0.01), which was inhibited by each medicated serum (P < 0.01). Huazhuojiedu equivalent dose showed a weaker inhibition effect than positive drug (P < 0.05), and Huazhuojiedu double dose exerted a strong inhibition effect (P < 0.05). After hepatic stellate cells were stimulated with transforming growth factor-β1 for 48 hours, the expression of α-SMA mRNA and protein in hepatic stellate cells was significantly increased (P

  19. Higher Hepatic miR-29 Expression in Undernourished Male Rats During the Postnatal Period Targets the Long-Term Repression of IGF-1.

    PubMed

    Sohi, Gurjeev; Revesz, Andrew; Ramkumar, Julie; Hardy, Daniel B

    2015-09-01

    A nutritional mismatch in postnatal life of low birth weight offspring increases the risk of developing the metabolic syndrome. Moreover, this is associated with decreased hepatic Igf1 expression, leading to impaired growth and metabolism. Previously, we have demonstrated that the timing of nutritional restoration in perinatal life can differentially program hepatic gene expression. Although microRNAs also play an important role in silencing gene expression, to date, the impact of a nutritional mismatch in neonatal life on their long-term expression has not been evaluated. Given the complementarity of miR-29 to the 3' untranslated region of Igf1, we examined how protein restoration in maternal protein restriction rat offspring influences hepatic miR-29 and Igf1 expression in adulthood. Pregnant Wistar rats were designated into 1 of 4 dietary regimes: 20% protein (control), 8% protein during lactation only (LP-Lact), 8% protein during gestation only (LP1) or both (LP2). The steady-state expression of hepatic miR-29 mRNA significantly increased in LP2 offspring at postnatal day 21 and 130, and this was inversely related to hepatic Igf1 mRNA and body weight. Interestingly, this reciprocal association was stronger in LP-Lact offspring at postnatal day 21. Functional relevance of this in vivo relationship was evaluated by transfection of miR-29 mimics in neonatal Clone 9 rat hepatoma cells. Transfection with miR-29 suppressed Igf1 expression by 12 hours. Collectively, these findings implicate that nutritional restoration after weaning (post liver differentiation) in maternal protein restriction rat offspring fails to prevent long-term impaired growth, in part, due to miR-29 suppression of hepatic Igf1 expression. PMID:26151354

  20. Hepatic Fgf21 Expression Is Repressed after Simvastatin Treatment in Mice.

    PubMed

    Ziros, Panos; Zagoriti, Zoi; Lagoumintzis, George; Kyriazopoulou, Venetsana; Iskrenova, Ralitsa P; Habeos, Evagelia I; Sykiotis, Gerasimos P; Chartoumpekis, Dionysios V; Habeos, Ioannis G

    2016-01-01

    Fibroblast growth factor 21 (Fgf21) is a hormone with emerging beneficial roles in glucose and lipid homeostasis. The interest in Fgf21 as a potential antidiabetic drug and the factors that regulate its production and secretion is growing. Statins are the most widely prescribed drug for the treatment of dyslipidemia. However, the function of statins is not limited to the lowering of cholesterol as they are associated with pleiotropic actions such as antioxidant, anti-inflammatory and cytoprotective effects. The recently described effect of statins on mitochondrial function and the induction of Fgf21 by mitochondrial stress prompted us to investigate the effect of statin treatment on Fgf21 expression in the liver. To this end, C57BL6J male mice and primary mouse hepatocytes were treated with simvastatin, and Fgf21 expression was subsequently assessed by immunoblotting and quantitative real-time PCR. Hepatic Fgf21 protein and mRNA and circulating levels of FGF21significantly decreased in mice that had received simvastatin in their food (0.1% w/w) for 1 week. This effect was also observed with simvastatin doses as low as 0.01% w/w for 1 week or following 2 intraperitoneal injections within a single day. The reduction in Fgf21 mRNA levels was further verified in primary mouse hepatocytes, indicating that the effect of simvastatin is cell autonomous. In conclusion, simvastatin treatment reduced the circulating and hepatic Fgf21 levels and this effect warrants further investigation with reference to its role in metabolism. PMID:27583452

  1. Expression of cytokine mRNA transcripts in renal cell carcinoma.

    PubMed

    Olive, C; Cheung, C; Nicol, D; Falk, M C

    1998-08-01

    Renal cell carcinoma (RCC) is a solid tumour of the kidney and is the most common renal neoplasm. Despite the presence of tumour infiltrating lymphocytes (TIL) in RCC, these tumours continue to progress in vivo suggesting a poor host immune response to the tumour, and the suppression of TIL effector function. Cytokines are key molecules that modulate the function of T cells. The possibility is investigated that the local production of cytokines in RCC contributes to immunosuppression of TIL. The expression of pro-inflammatory (IFN-gamma/IL-2) and immunosuppressive (IL-10/TGF-beta) cytokine mRNA transcripts was determined in RCC, normal kidney and peripheral blood of RCC patients using a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) with cytokine-specific primers. Following Southern blot hybridization of the PCR products with internal radiolabelled oligonucleotide probes, cytokine transcript levels were measured by densitometry and expressed relative to the glyceraldehyde-3-phosphate dehydrogenase densitometry score. With the exception of IL-10, there were no differences in expression of cytokine mRNA transcripts between the peripheral blood of patients and normal healthy individuals. It was found that TGF-beta transcripts were well represented in normal kidney and RCC. In contrast, the expression of IFN-gamma transcripts, while low in the majority of samples, was significantly increased in RCC when compared to normal kidney (P=0.05). The IL-2 and IL-10 transcripts showed a more variable expression in normal kidney and RCC, with no significant differences in expression between the sample groups. The data demonstrating pro-inflammatory and immunosuppressive cytokine expression in RCC do not support a prominent immunosuppressive cytokine profile in these tumours. PMID:9723777

  2. Decreased parvalbumin mRNA expression in dorsolateral prefrontal cortex in Parkinson’s disease

    PubMed Central

    Lanoue, Amélie C.; Blatt, Gene J.; Soghomonian, Jean-Jacques

    2013-01-01

    It has recently been shown that expression of the rate-limiting GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) is decreased in Brodmann area 9 (BA9) of the dorsolateral prefrontal cortex (DLPFC) in Parkinson’s disease (PD) compared to control brains (Lanoue, A.C., Dumitriu, A., Myers, R.H., Soghomonian, JJ., 2010. Exp Neurol. 206(1), 207–217). A subpopulation of cortical GABAergic interneurons expresses the calcium-binding protein parvalbumin and plays a critical role in the control of pyramidal neuron excitability and the generation of cortical gamma frequency oscillations. In view of its key role in the physiology of the cerebral cortex, we sought to determine whether the expression of parvalbumin and the number of parvalbumin-expressing neurons are altered in BA9 of PD brains. First, isotopic in situ hybridization histochemistry was used to examine mRNA expression of parvalbumin on post-mortem brain sections. Second, immunohistochemistry and design-based stereology were used to determine the density of parvalbumin-positive interneurons in BA9. Quantification of mRNA labeling at the single cell level showed a significant decrease in parvalbumin expression in PD cases. In contrast, neuronal density of parvalbumin-positive neurons was not significantly different between PD and controls. Results confirm that the GABAergic system is altered in the DLPFC in PD and identify the contribution of parvalbumin-expressing neurons in these alterations. We speculate that these effects could contribute to altered cortical excitability and oscillatory activity previously documented in PD. PMID:23891794

  3. 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. PMID:21969073

  4. Effects of glutamine supplementation on splenocyte cytokine mRNA expression in rats with septic peritonitis

    PubMed Central

    Yeh, Sung-Ling; Lai, Yu-Ni; Shang, Huey-Fang; Lin, Ming-Tsan; Chiu, Wan-Chun; Chen, Wei-Jao

    2005-01-01

    AIM: To investigate the effects of glutamine (GLN)-enriched diets before and GLN-containing total parenteral nutrition (TPN) after sepsis or both on the secretion of cytokines and their mRNA expression levels in splenocytes of rats with septic peritonitis. METHODS: Rats were assigned to a control group and 4 experimental groups. The control group and experimental groups 1 and 2 were fed a semipurified diet, while experimental groups 3 and 4 had part of the casein replaced by GLN which provided 25% of the total nitrogen. After rats were fed with these diets for 10 d, sepsis was induced by cecal ligation and puncture (CLP), whereas the control group underwent a sham operation, at the same time, an internal jugular vein was cannulated. All rats were maintained on TPN for 3 d. The control group and experimental groups 1 and 3 were infused with conventional TPN, while the TPN in experimental groups 2 and 4 was supplemented with GLN, providing 25% of the total nitrogen in the TPN solution. All rats were kiued 3 d after sham operation or CLP to examine their splenocyte subpopulation distribution and cytokine expression levels. RESULTS: Most cytokines could not be detected in plasma except for IL-10. No difference in plasma IL-10 was observed among the 5 groups. The IL-2, IL-4, IL-10, and TNF-α mRNA expression levels in splenocytes were significantly higher in experimental groups 2 and 4 than in the control group and group 1. The mRNA expression of IFN-γ was significantly higher in the GLN-supplemented groups than in the control group and experimental group 1. The proportion of CD45Ra+ was increased, while those of CD3+ and CD4+ were decreased in experimental group 1 after CLP was performed. There were no differences in spleen CD3+ lymphocyte distributions between the control and GLN-supplemented groups. CONCLUSION: GLN supplementation can maintain T-lymphocyte populations in the spleen and significantly enhance the mRNA expression levels of Th1 and Th2 cytokines and TNF

  5. Strain differences in hepatic cytochrome P450 1A and 3A expression between Sprague-Dawley and Wistar rats.

    PubMed

    Kishida, Tomoyuki; Muto, Shin-ichi; Hayashi, Morimichi; Tsutsui, Masaru; Tanaka, Satoru; Murakami, Makoto; Kuroda, Junji

    2008-10-01

    Expression of hepatic cytochrome P450 (CYP) isoforms was compared in Sprague-Dawley (SD) and Wistar (WI) rats, which are commonly used strains in preclinical studies. Basal CYP1A1, CYP1A2, and CYP3A2 mRNA levels were higher in WI rats than in SD rats (by 8-, 3- and 2-fold, respectively). Treatment with phenobarbital, a potent CYP inducer, increased the predominance of expression of these three mRNAs in WI rats (by 26-, 4-, and 2-fold, respectively) along with the predominance of increased microsomal total P450 contents and smooth-surface endoplasmic reticulum in the centrilobular hepatocytes. CYP1A enzymatic activity was also higher in WI rats than in SD rats. No strain differences were observed in phenobarbital induction of CYP2B1/2, CYP2C6, or CYP3A1. CYP3A2 mRNA was more strongly induced by dexamethasone, a typical inducer of CYP3A, together with CYP3A1 mRNA, in WI rats than in SD rats (by 2-fold), whereas the CYP1A1 and CYP1A2 mRNA expression induced by beta-naphtoflavone, a typical inducer of CYP1A, did not differ between the two strains. Furthermore, WI rats exhibited predominantly arylhydrocarbon receptor, pregnane X receptor, and constitutive androstane receptor mRNAs, responsible for CYP1A or CYP3A induction, with phenobarbital or dexamethasone induction. In conclusion, significant, predominant expression of hepatic CYP1A and CYP3A mRNAs in WI rats was observed, possibly related to nuclear receptor-mediated induction. Considering the pharmacokinetic and toxicological importance of CYP1A and CYP3A, different outcomes might arise depending on the rat strains used in preclinical studies of drugs metabolized typically or mainly by both isoforms. PMID:18827444

  6. Early Life Exposure to Fructose Alters Maternal, Fetal and Neonatal Hepatic Gene Expression and Leads to Sex-Dependent Changes in Lipid Metabolism in Rat Offspring

    PubMed Central

    Clayton, Zoe E.; Vickers, Mark H.; Bernal, Angelica; Yap, Cassandra; Sloboda, Deborah M.

    2015-01-01

    Aim Fructose consumption is associated with altered hepatic function and metabolic compromise and not surprisingly has become a focus for perinatal studies. We have previously shown that maternal fructose intake results in sex specific changes in fetal, placental and neonatal outcomes. In this follow-up study we investigated effects on maternal, fetal and neonatal hepatic fatty acid metabolism and immune modulation. Methods Pregnant rats were randomised to either control (CON) or high-fructose (FR) diets. Fructose was given in solution and comprised 20% of total caloric intake. Blood and liver samples were collected at embryonic day 21 (E21) and postnatal day (P)10. Maternal liver samples were also collected at E21 and P10. Liver triglyceride and glycogen content was measured with standard assays. Hepatic gene expression was measured with qPCR. Results Maternal fructose intake during pregnancy resulted in maternal hepatic ER stress, hepatocellular injury and increased levels of genes that favour lipogenesis. These changes were associated with a reduction in the NLRP3 inflammasome. Fetuses of mothers fed a high fructose diet displayed increased hepatic fructose transporter and reduced fructokinase mRNA levels and by 10 days of postnatal age, also have hepatic ER stress, and elevated IL1β mRNA levels. At P10, FR neonates demonstrated increased hepatic triglyceride content and particularly in males, associated changes in the expression of genes regulating beta oxidation and the NLRP3 inflammasome. Further, prenatal fructose results in sex-dependant changes in levels of key clock genes. Conclusions Maternal fructose intake results in age and sex-specific alterations in maternal fetal and neonatal free fatty acid metabolism, which may be associated in disruptions in core clock gene machinery. How these changes are associated with hepatic inflammatory processes is still unclear, although suppression of the hepatic inflammasome, as least in mothers and male neonates may

  7. Expression of hepatic mRNAs for insulin-like growth factors-I and -II during the development of hypothyroid rats.

    PubMed

    Gallo, G; de Marchis, M; Voci, A; Fugassa, E

    1991-12-01

    The effect of thyroid status on the expression of insulin-like growth factors-I and -II mRNAs in the liver of developing rats has been investigated. Northern blot analyses of the specific mRNA demonstrated the presence of four IGF-II mRNA species which were strongly expressed in fetal liver and progressively declined after birth, becoming undetectable after week 3. This decrease was markedly delayed in the liver of hypothyroid rats. In addition, expression of IGF-I mRNA, absent in fetal liver, began during week 1 after birth and progressively increased with age. This increase was markedly delayed in the liver of hypothyroid rats. The data suggest that thyroid hormones regulate rat development via the co-ordinate expression of hepatic IGF-II and IGF-I mRNAs. PMID:1783883

  8. 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

  9. Posttraumatic temporal TGF-β mRNA expression in lens epithelial cells of paediatric patients.

    PubMed

    Berezowski, P; Strzalka-Mrozik, B; Forminska-Kapuscik, M; Mazurek, U; Filipek, E; Nawrocka, L; Pieczara, E; Banasiak, P; Kimsa, M

    2012-01-01

    The aim of the study was to determine temporal TGFB1, TGFB2 and TGFB3 gene expression profiles in the anterior lens capsule of paediatric patients with posttraumatic cataract. The patient group comprised 22 children selected with a fragment of anterior lens capsule obtained during elective cataract surgery and sampled for molecular analysis. The levels of TGF-β isoforms in the anterior lens capsule were determined based on the number of mRNA copies per 1 μg total RNA by real-time qRTPCR. Three time-related result clusters were identified based on hierarchical cluster analysis: 2.2, 4.4 and 15.0 months (time span from injury to anterior capsule sampling during surgery) and compared with regard to temporal gene expression profile and quantitative relations of TGF-β1, 2 and 3 mRNAs. TGF-β1, TGF-β2, and TGF-β3 mRNAs were detected in all anterior lens capsule samples. A comparative analysis revealed: TGF-β1>TGF-β2>TGF-β3 during the entire observation period. The TGF-β mRNA levels continued to increase up to four months after injury, then returning close to the base levels after around 15 months. The expression patterns of TGF-β isoforms showed a similar tendency. Differences in the expression levels of TGF-β1 and TGF-β2 between the particular clusters were statistically significant. Posttraumatic transcriptional activities of TGF-β1 and TGF-β2 in the anterior lens capsule of paediatric patients depend on the time elapsing from injury. Our findings indicate that the transcriptional activities of TGFB family genes show a transient period of over-expression during the months after injury. TGF-β1 is a dominant isoform expressed in lens epithelial cells following injury. PMID:22464821

  10. Cloning, characterization and mRNA expression of interleukin-6 in blunt snout bream (Megalobrama amblycephala).

    PubMed

    Zhang, Chun-Nuan; Zhang, Ji-Liang; Liu, Wen-Bin; Wu, Qiu-Jue; Gao, Xiao-Chan; Ren, Hong-Tao

    2016-07-01

    In the present study, the interleukin-6 gene (IL-6) cDNA in blunt snout bream (Megalobrama amblycephala) was identified and its expression profiles under ammonia stress and bacterial challenge were investigated. The IL-6 sequence consisted of 1045 bp, including a 696 bp ORF which translated into a 232 amino acid (AA) protein. The protein contained a putative signal peptide of 24 AA in length. IL-6 expression analysis showed that the it is differentially expressed in various tissues under normal conditions and the highest IL-6 level was observed in the intestine tissue, followed by the liver, and then in the gills. Under ammonia stress, the IL-6 mRNA level both in spleens and intestine increased significantly (P < 0.05), with the maximum levels attained at 6 h, 12 h (72, 10-fold, respectively). Thereafter, they all significantly decreased (P < 0.01) and returned to the basal value within 48 h. Whereas, in livers it slightly decreased at 3 h firstly (0.5-fold), and then significantly (P < 0.05) increased with the maximum level attained 12 h (3-fold). Further expression analysis showed that the mRNA level of IL-6 in spleens, intestine and livers of blunt snout bream all increased significantly (P < 0.05), with maximum values attained at 6 h, 3 h, 6 h (10, 6, 18-fold, respectively) after Aeromonas hydrophila (A. hydrophila) injection, and then decreased to the basal value within 24 h which suggested that IL-6 was involved in the immune response to A. hydrophila. The cloning and expression analysis of the IL-6 provide theoretical basis to further study the mechanism of anti-adverseness and expression characteristics under stress conditions in blunt snout bream. PMID:26965748

  11. The Flavone Luteolin Suppresses SREBP-2 Expression and Post-Translational Activation in Hepatic Cells

    PubMed Central

    Wong, Tsz Yan; Lin, Shu-mei; Leung, Lai K.

    2015-01-01

    High blood cholesterol has been associated with cardiovascular diseases. The enzyme HMG CoA reductase (HMGCR) is responsible for cholesterol synthesis, and inhibitors of this enzyme (statins) have been used clinically to control blood cholesterol. Sterol regulatory element binding protein (SREBP) -2 is a key transcription factor in cholesterol metabolism, and HMGCR is a target gene of SREBP-2. Attenuating SREBP-2 activity could potentially minimize the expression of HMGCR. Luteolin is a flavone that is commonly detected in plant foods. In the present study, Luteolin suppressed the expression of SREBP-2 at concentrations as low as 1 μM in the hepatic cell lines WRL and HepG2. This flavone also prevented the nuclear translocation of SREBP-2. Post-translational processing of SREBP-2 protein was required for nuclear translocation. Luteolin partially blocked this activation route through increased AMP kinase (AMPK) activation. At the transcriptional level, the mRNA and protein expression of SREBP-2 were reduced through luteolin. A reporter gene assay also verified that the transcription of SREBF2 was weakened in response to this flavone. The reduced expression and protein processing of SREBP-2 resulted in decreased nuclear translocation. Thus, the transcription of HMGCR was also decreased after luteolin treatment. In summary, the results of the present study showed that luteolin modulates HMGCR transcription by decreasing the expression and nuclear translocation of SREBP-2. PMID:26302339

  12. Embedding mRNA Stability in Correlation Analysis of Time-Series Gene Expression Data

    PubMed Central

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

    2008-01-01

    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 R2 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 R2 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 R2 value among genes coding for a transient complex. PMID:18670596

  13. Lesion of the substantia nigra pars compacta downregulates striatal glutamate receptor subunit mRNA expression.

    PubMed

    Fan, X D; Li, X M; Ashe, P C; Juorio, A V

    1999-12-11

    This is a study of the effect of the unilateral administration of dopamine (DA) in the pars compacta of the substantia nigra (SN) of the rat on striatal glutamate receptor subunit (GluR1, GluR2 and NMDAR1) gene expression determined by in situ hybridization. The location of the nigral lesion was determined by tyrosine hydroxylase (TH) immunohistochemistry and its extent by the striatal DA and 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations. The DA-induced lesions produce significant bilateral reductions in the expression of GluR1 and NMDAR1 subunit mRNA in the medio-lateral striatum, whereas the expression of striatal GluR2 receptors was not changed. The reduction in GluR1 and NMDAR1 subunit mRNA may be the consequence of glutamatergic hyperactivity developed in the presence of a damaged nigro-striatal system and these may be associated with the genesis of some neurodegenerative diseases. PMID:10629751

  14. Down-regulated expression of transforming growth factor beta 1 mRNA in endometrial carcinoma.

    PubMed Central

    Perlino, E.; Loverro, G.; Maiorano, E.; Giannini, T.; Cazzolla, A.; Napoli, A.; Fiore, M. G.; Ricco, R.; Marra, E.; Selvaggi, L.

    1998-01-01

    Transforming growth factor beta1 (TGF-beta1) is a potent modulator of cell proliferation in vitro, and recent studies have demonstrated its overexpression in several different tumours; nevertheless, the molecular mechanisms of TGF-beta1 action on cell growth and differentiation have not been fully elucidated. To clarify the role of TGF-beta and its receptor in human endometrial proliferation and differentiation, TGF-beta1 expression at both the mRNA and protein levels has been evaluated by using Northern blotting and immunohistochemistry, in both normal (atrophic, proliferative and secretory) and neoplastic (adenocarcinoma) endometrial samples. This study demonstrates that TGF-beta1 mRNA expression is dramatically reduced in endometrial carcinomas with respect to non-neoplastic tissues, whereas the immunohistochemical expression of TGF-beta1 is enhanced in the epithelial component of endometrial carcinomas compared with non-neoplastic tissues. These data suggest that TGF-beta1 acts as a paracrine regulator of endometrial cell proliferation and that it may contribute to the carcinogenic mechanisms of endometrial carcinoma. Images Figure 1 Figure 5 Figure 6 Figure 8 PMID:9579831

  15. Down-regulated expression of transforming growth factor beta 1 mRNA in endometrial carcinoma.

    PubMed

    Perlino, E; Loverro, G; Maiorano, E; Giannini, T; Cazzolla, A; Napoli, A; Fiore, M G; Ricco, R; Marra, E; Selvaggi, L

    1998-04-01

    Transforming growth factor beta1 (TGF-beta1) is a potent modulator of cell proliferation in vitro, and recent studies have demonstrated its overexpression in several different tumours; nevertheless, the molecular mechanisms of TGF-beta1 action on cell growth and differentiation have not been fully elucidated. To clarify the role of TGF-beta and its receptor in human endometrial proliferation and differentiation, TGF-beta1 expression at both the mRNA and protein levels has been evaluated by using Northern blotting and immunohistochemistry, in both normal (atrophic, proliferative and secretory) and neoplastic (adenocarcinoma) endometrial samples. This study demonstrates that TGF-beta1 mRNA expression is dramatically reduced in endometrial carcinomas with respect to non-neoplastic tissues, whereas the immunohistochemical expression of TGF-beta1 is enhanced in the epithelial component of endometrial carcinomas compared with non-neoplastic tissues. These data suggest that TGF-beta1 acts as a paracrine regulator of endometrial cell proliferation and that it may contribute to the carcinogenic mechanisms of endometrial carcinoma. PMID:9579831

  16. 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

  17. Attenuated mRNA expression of inflammatory mediators in neonatal rat lung following lipopolysaccharide treatment

    PubMed Central

    Le Rouzic, Valerie; Wiedinger, Kari; Zhou, Heping

    2012-01-01

    Neonates are known to exhibit increased susceptibility to bacterial and viral infections and increasing evidence demonstrates that the increased susceptibility is related to their attenuated immune response to infections. The lung is equipped with an innate defense system involving both cellular and humoral mediators. The present study was performed to characterize the expression of inflammatory mediators in the lung of neonatal rats in comparison with older animals. Rats at postnatal day 1 (P1), P21, and P70 were treated with saline or 0.25 mg/kg lipopolysaccharide (LPS) via intraperitoneal injection. Two hours later, animals were sacrificed and the transcriptional response of key inflammatory mediators and enzyme activity of myeloperoxidase (MPO) in the lung of these animals were examined. LPS-induced messenger RNA (mRNA) expression of pro-inflammatory cytokines, namely interleukin (IL)-1β, IL-6, and tumor necrosis factor-α, antiinflammatory cytokines, namely IL-10 and IL-1 receptor antagonist (IL-1ra), and chemokines, namely macrophage inflammatory protein (MIP)-1β, MIP-2, and monocyte chemotactic protein-1, in P1 lung was much reduced compared to that in P21 and P70 animals at 2 hours postinjection. These data suggest that LPS-induced transcriptional response of cytokines and chemokines was much reduced in P1 lung even though the protein levels of these genes were not ascertained and mRNA levels of these genes may not reflect their final protein levels. MPO activity in LPS-treated P1 lung was also significantly attenuated compared to that in LPS-treated P70 lung, suggesting impaired neutrophil infiltration in P1 lung at 2 hours following LPS treatment. In parallel, the baseline mRNA expression of LPS-binding protein (LBP) in P1 lung was much lower than that in P21 and P70 lungs. While the protein level of LBP was not examined and the mRNA level of LBP may not reflect its final protein level, the reduced transcriptional response of cytokines and chemokines in

  18. Different Profile of mRNA Expression in Sinoatrial Node from Streptozotocin-Induced Diabetic Rat

    PubMed Central

    Ferdous, Zannatul; Qureshi, Muhammad Anwar; Jayaprakash, Petrilla; Parekh, Khatija; John, Annie; Oz, Murat; Raza, Haider; Dobrzynski, Halina; Adrian, Thomas Edward; Howarth, Frank Christopher

    2016-01-01

    Background Experiments in isolated perfused heart have shown that heart rate is lower and sinoatrial node (SAN) action potential duration is longer in streptozotocin (STZ)–induced diabetic rat compared to controls. In sino-atrial preparations the pacemaker cycle length and sino-atrial conduction time are prolonged in STZ heart. To further clarify the molecular basis of electrical disturbances in the diabetic heart the profile of mRNA encoding a wide variety of proteins associated with the generation and transmission of electrical activity has been evaluated in the SAN of STZ-induced diabetic rat heart. Methodology/Principal Findings Heart rate was measured in isolated perfused heart with an extracellular suction electrode. Expression of mRNA encoding a variety of intercellular proteins, intracellular Ca2+-transport and regulatory proteins, cell membrane transport proteins and calcium, sodium and potassium channel proteins were measured in SAN and right atrial (RA) biopsies using real-time reverse transcription polymerase chain reaction techniques. Heart rate was lower in STZ (203±7 bpm) compared to control (239±11 bpm) rat. Among many differences in the profile of mRNA there are some worthy of particular emphasis. Expression of genes encoding some proteins were significantly downregulated in STZ-SAN: calcium channel, Cacng4 (7-fold); potassium channel, Kcnd2 whilst genes encoding some other proteins were significantly upregulated in STZ-SAN: gap junction, Gjc1; cell membrane transport, Slc8a1, Trpc1, Trpc6 (4-fold); intracellular Ca2+-transport, Ryr3; calcium channel Cacna1g, Cacna1h, Cacnb3; potassium channels, Kcnj5, Kcnk3 and natriuretic peptides, Nppa (5-fold) and Nppb (7-fold). Conclusions/Significance Collectively, this study has demonstrated differences in the profile of mRNA encoding a variety of proteins that are associated with the generation, conduction and regulation of electrical signals in the SAN of STZ-induced diabetic rat heart. Data from this

  19. Electroacupuncture Enhances Preproenkephalin mRNA Expression in Rostral Ventrolateral Medulla of Rats

    PubMed Central

    Li, Min; Tjen-A-Looi, Stephanie C.; Longhurst, John C.

    2010-01-01

    Electroacupuncture (EA) causes prolonged suppression of reflex elevations in blood pressure for at least 60 minutes in anesthetized preparations. Thus, EA can modify sympathetic outflow and elevated blood pressure through actions in a number of hind brain regions, including the rostral ventrolateral medulla (rVLM). Since our previous data show that the opioid system plays a role in EA-related prolonged inhibition of presympathetic neuronal activity in the rVLM, we postulated that EA increases preproenkephalin (PPE) mRNA in this region, possibly for prolonged periods of time. Under α–chloralose anesthesia, rats received EA (1-2 mA, 2 Hz, 0.5 ms) at P5-P6 acupoints (overlying median nerves) or sham (needle placement without electrical stimulation) for 30 min. PPE mRNA in the rVLM also was evaluated in control rats that received surgery but no EA or sham treatment. 20 min, 1.5 h or 4 h following EA or sham treatment, PPE mRNA in the rVLM was analyzed by reverse transcription and quantitative real-time PCR. Relative ratios of PPE mRNA levels (normalized with 18s house keeping gene) were increased 1.5 h after EA stimulation (7.77 ± 1.39, n=6) relative to sham (2.84 ± 0.37, n=5) but were unchanged both 20 min and 4 h after EA, compared to the sham or surgery groups at the same time points. Thus, 30 min of EA transiently stimulates the production of enkephalin in a region of the brain that importantly regulates sympathetic outflow suggesting that even a single brief acupuncture treatment can increase the expression of this modulatory neuropeptide. PMID:20399834

  20. IPLA2 mRNA expression by human neutrophils in type 2 diabetes and chronic periodontitis.

    PubMed

    Ayilavarapu, Srinivas; Kantarci, Alpdogan; Hasturk, Hatice; Van Dyke, Thomas E

    2014-10-01

    Type 2 diabetes mellitus (T2D) is becoming increasingly prevalent worldwide and complications of T2D cause significant systemic and dental morbidity in the susceptible individual. Although T2D has been linked as a significant risk factor for chronic periodontitis (CP), molecular mechanisms explaining the pathogenesis and inflammatory impact of CP in T2D are lacking. iPLA2 is the calcium-independent form of phospholipase A2. In previous studies, we demonstrated that iPLA2 enzyme activity is altered in T2D. The purpose of this study was to elucidate the level of the iPLA2 abnormality in T2D by measuring messenger RNA levels in T2D-associated CP. A total of 53 healthy and T2D subjects with CP were recruited for this study. The clinical periodontal exam included probing pocket depth, clinical attachment levels and bleeding on probing. Peripheral venous blood was collected and neutrophils were isolated. Real time polymerase chain reaction was used to quantify iPLA2 mRNA in neutrophils from healthy controls and people with diabetes. Results revealed that the prevalence of moderate to severe CP was increased in people with T2D. The iPLA, mRNA levels in diabetics with different severity of CP were not significantly different compared to healthy controls; 1.07 vs 0.97 (mild CP), 1.07 vs 0.85 (moderate CP) and 1.07 vs 1.05 (severe CP). Collectively, the data suggest that levels of iPLA2 mRNA in T2D are not different than in health and are not directly influenced by periodontal disease status. The impact of inflammation on iPLA2 regulation is at the level of activation of the enzyme rather than expression at the mRNA level. PMID:25654966

  1. 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. PMID:19733250

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

    PubMed

    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

  3. 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

  4. Astaxanthin lowers plasma TAG concentrations and increases hepatic antioxidant gene expression in diet-induced obesity mice.

    PubMed

    Yang, Yue; Pham, Tho X; Wegner, Casey J; Kim, Bohkyung; Ku, Chai Siah; Park, Young-Ki; Lee, Ji-Young

    2014-12-14

    Non-alcoholic fatty liver disease (NAFLD) is significantly associated with hyperlipidaemia and oxidative stress. We have previously reported that astaxanthin (ASTX), a xanthophyll carotenoid, lowers plasma total cholesterol and TAG concentrations in apoE knockout mice. To investigate whether ASTX supplementation can prevent the development of NAFLD in obesity, male C57BL/6J mice (n 8 per group) were fed a high-fat diet (35%, w/w) supplemented with 0, 0.003, 0.01 or 0.03% of ASTX (w/w) for 12 weeks. The 0.03% ASTX-supplemented group, but not the other groups, exhibited a significant decrease in plasma TAG concentrations, suggesting that ASTX at a 0.03% supplementation dosage exerts a hypotriacylglycerolaemic effect. Although there was an increase in the mRNA expression of fatty acid synthase and diglyceride acyltransferase 2, the mRNA levels of acyl-CoA oxidase 1, a critical enzyme in peroxisomal fatty acid β-oxidation, exhibited an increase in the 0.03% ASTX-supplemented group. There was a decrease in plasma alanine transaminase (ALT) and aspartate transaminase (AST) concentrations in the 0.03% ASTX-supplemented group. There was a significant increase in the hepatic mRNA expression of nuclear factor erythroid 2-related factor 2 and its downstream genes, which are critical for endogenous antioxidant mechanism, in the 0.03% ASTX-supplemented group. Furthermore, there was a significant decrease in the mRNA abundance of IL-6 in the primary splenocytes isolated from the 0.03% ASTX-supplemented group upon lipopolysaccharide (LPS) stimulation when compared with that in the splenocytes isolated from the control group. In conclusion, ASTX supplementation lowered the plasma concentrations of TAG, ALT and AST, increased the hepatic expression of endogenous antioxidant genes, and rendered splenocytes less sensitive to LPS stimulation. Therefore, ASTX may prevent obesity-associated metabolic disturbances and inflammation. PMID:25328157

  5. 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.

  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. Dietary proanthocyanidins boost hepatic NAD+ metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats

    PubMed Central

    Aragonès, Gerard; Suárez, Manuel; Ardid-Ruiz, Andrea; Vinaixa, Maria; Rodríguez, Miguel A.; Correig, Xavier; Arola, Lluís; Bladé, Cinta

    2016-01-01

    Proanthocyanidins (PACs) have been reported to modulate multiple targets by simultaneously controlling many pivotal metabolic pathways in the liver. However, the precise mechanism of PAC action on the regulation of the genes that control hepatic metabolism remains to be clarified. Accordingly, we used a metabolomic approach combining both nuclear magnetic resonance and mass spectrometry analysis to evaluate the changes induced by different doses of grape-seed PACs in the liver of healthy rats. Here, we report that PACs significantly increased the hepatic nicotinamide adenine dinucleotide (NAD+) content in a dose-dependent manner by specifically modulating the hepatic concentrations of the major NAD+ precursors as well as the mRNA levels of the genes that encode the enzymes involved in the cellular metabolism of NAD+. Notably, Sirtuin 1 (Sirt1) gene expression was also significantly up-regulated in a dose-response pattern. The increase in both the NAD+ availability and Sirt1 mRNA levels, in turn, resulted in the hepatic activation of SIRT1, which was significantly associated with improved protection against hepatic triglyceride accumulation. Our data clearly indicates that PAC consumption could be a valid tool to enhance hepatic SIRT1 activity through the modulation of NAD+ levels. PMID:27102823

  8. 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.

  9. 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

  10. mRNA Expression in Papillary and Anaplastic Thyroid Carcinoma: Molecular Anatomy of a Killing Switch

    PubMed Central

    Hébrant, Aline; Dom, Geneviève; Dewaele, Michael; Andry, Guy; Trésallet, Christophe; Leteurtre, Emmanuelle; Dumont, Jacques E.; Maenhaut, Carine

    2012-01-01

    Anaplastic thyroid carcinoma (ATC) is the most lethal form of thyroid neoplasia and represents the end stage of thyroid tumor progression. No effective treatment exists so far. ATC frequently derive from papillary thyroid carcinomas (PTC), which have a good prognosis. In this study, we analyzed the mRNA expression profiles of 59 thyroid tumors (11 ATC and 48 PTC) by microarrays. ATC and PTC showed largely overlapping mRNA expression profiles with most genes regulated in all ATC being also regulated in several PTC. 43% of the probes regulated in all the PTC are similarly regulated in all ATC. Many genes modulations observed in PTC are amplified in ATC. This illustrates the fact that ATC mostly derived from PTC. A molecular signature of aggressiveness composed of 9 genes clearly separates the two tumors. Moreover, this study demonstrates gene regulations corresponding to the ATC or PTC phenotypes like inflammatory reaction, epithelial to mesenchymal transition (EMT) and invasion, high proliferation rate, dedifferentiation, calcification and fibrosis processes, high glucose metabolism and glycolysis, lactate generation and chemoresistance. The main qualitative differences between the two tumor types bear on the much stronger EMT, dedifferentiation and glycolytic phenotypes showed by the ATC. PMID:23115614

  11. 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

  12. Hepatic expression of serum amyloid A1 is induced by traumatic brain injury and modulated by telmisartan.

    PubMed

    Villapol, Sonia; Kryndushkin, Dmitry; Balarezo, Maria G; Campbell, Ashley M; Saavedra, Juan M; Shewmaker, Frank P; Symes, Aviva J

    2015-10-01

    Traumatic brain injury affects the whole body in addition to the direct impact on the brain. The systemic response to trauma is associated with the hepatic acute-phase response. To further characterize this response, we performed controlled cortical impact injury on male mice and determined the expression of serum amyloid A1 (SAA1), an apolipoprotein, induced at the early stages of the acute-phase response in liver and plasma. After cortical impact injury, induction of SAA1 was detectable in plasma at 6 hours post-injury and in liver at 1 day post-injury, followed by gradual diminution over time. In the liver, cortical impact injury increased neutrophil and macrophage infiltration, apoptosis, and expression of mRNA encoding the chemokines CXCL1 and CXCL10. An increase in angiotensin II AT1 receptor mRNA at 3 days post-injury was also observed. Administration of the AT1 receptor antagonist telmisartan 1 hour post-injury significantly decreased liver SAA1 levels and CXCL10 mRNA expression, but did not affect CXCL1 expression or the number of apoptotic cells or infiltrating leukocytes. To our knowledge, this is the first study to demonstrate that SAA1 is induced in the liver after traumatic brain injury and that telmisartan prevents this response. Elucidating the molecular pathogenesis of the liver after brain injury will assist in understanding the efficacy of therapeutic approaches to brain injury. PMID:26435412

  13. IONOTROPIC GLUTAMATE RECEPTORS mRNA EXPRESSION IN THE HUMAN THALAMUS: ABSENCE OF CHANGE IN SCHIZOPHRENIA

    PubMed Central

    Dracheva, Stella; Byne, William; Chin, Benjamin; Haroutunian, Vahram

    2009-01-01

    Abnormalities in glutamate neurotransmission are thought to be among the major contributing factors to the pathophysiology of schizophrenia. Although schizophrenia has been regarded mostly as a disorder of higher cortical function, the cortex and thalamus work as a functional unit. Existing data regarding alterations of glutamate receptor subunit expression in the thalamus in schizophrenia remain equivocal. This postmortem study examined mRNA expression of ionotropic glutamate receptor (iGluR) subunits and PSD95 in 5 precisely defined and dissected thalamic subdivisions (medial and lateral sectors of the mediodorsal nucleus; and the ventrolateral posterior, ventral posterior, and centromedian nuclei) of persons with schizophrenia and matched controls using quantitative PCR with normalization to multiple endogenous controls. Among 15 genes examined (NR1 and NR2A-D subunits of NMDA receptor; GluR1-4 subunits of AMPA receptor; GluR5-7 and KA1-2 subunits of kainate receptor; PSD95), all but two (GluR4 and KA1) were expressed at quantifiable levels. Differences in iGluR gene expression were seen between different nuclei but not between diagnostic groups. The relative abundance of transcripts was: NR1≫NR2A>NR2B>NR2D>NR2C for NMDA, GluR2>GluR1>GluR3 for AMPA, and KA2>GluR5>GluR7>GluR6 for kainate receptors. The expression of PSD95 correlated with the expression of NR1, NR2A, NR2B, NR2D and GluR6 in all nuclei. These results provide detailed and quantitative information on iGluR subunit expression in multiple nuclei of the human thalamus but suggest that alterations in their expression are not a prominent feature of schizophrenia. PMID:18462708

  14. A distinct ERCC1 haplotype is associated with mRNA expression levels in prostate cancer patients.

    PubMed

    Woelfelschneider, Andreas; Popanda, Odilia; Lilla, Carmen; Linseisen, Jakob; Mayer, Claudia; Celebi, Oktay; Debus, Jürgen; Bartsch, Helmut; Chang-Claude, Jenny; Schmezer, Peter

    2008-09-01

    Both genetic variants and messenger RNA (mRNA) expression of DNA repair and tumor suppressor genes have been investigated as molecular markers for therapy outcome. However, the phenotypic impact of genetic variants often remained unclear, thus the rationale of their use in risk prediction may be limited. We therefore analyzed genetic variants together with anthropometric and lifestyle factors to see how these affect mRNA levels of ERCC1, MDM2 and TP53 in primary blood lymphocytes. mRNA expression was measured in 376 prostate cancer patients by quantitative real-time polymerase chain reaction after reverse transcription, and ERCC1 rs11615 T>C, ERCC1 rs3212986 C>A, MDM2 rs2279744 T>G and TP53 rs17878362 (p53PIN3) polymorphisms were determined. Considerable interindividual differences in mRNA expression were found (coefficients of variation: ERCC1, 45%; MDM2, 43% and TP53, 35%). ERCC1 expression was positively correlated with plasma levels of beta-carotene (P = 0.03) and negatively correlated with canthaxanthin (P = 0.02) and lutein (P = 0.02). Overall, the polymorphisms affected mRNA expression only weakly. Carriers of a distinct ERCC1 haplotype (CC) showed, however, significantly lower expression values than non-carriers (P = 0.001). Applying logistic regression, we found that CC haplotype carriers had a 1.69-fold increased odds ratio (95% confidence interval: 1.06-2.71) for reduced ERCC1 mRNA levels. This low ERCC1 expression might be associated with reduced DNA repair and better therapy response. In summary, the association we have found between ERCC1 genotype and mRNA expression supports recent clinical observations that genetic variation in ERCC1 can affect treatment outcome and prognosis. Our study further revealed a modulating effect by nutritional factors. PMID:18332046

  15. 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

  16. Increased neutrophil adherence and adhesion molecule mRNA expression in endothelial cells during selenium deficiency.

    PubMed

    Maddox, J F; Aherne, K M; Reddy, C C; Sordillo, L M

    1999-05-01

    Leukocyte aggregation and activation on endothelial cells (EC) are important preliminary events in leukocyte migration into tissue and subsequent inflammation. Thus, an increase in leukocyte adherence has the potential to affect inflammatory disease outcome. Selenium (Se) is an integral part of the antioxidant enzyme glutathione peroxidase (GSH-Px) and plays an important role in the maintenance of the redox state of a cell. Se supplementation in the bovine has been shown to improve the outcome of acute mastitis caused by coliform bacteria, in part by enhancing the speed of neutrophil migration into the affected mammary gland. However, the mechanisms by which Se modulates neutrophil migration have not been elucidated. Therefore, an in vitro model of Se deficiency in primary bovine mammary artery EC was used to examine the impact of Se status on the adhesive properties of EC. The effect of Se on functional activities was examined by measuring neutrophil adherence to Se-deficient and Se-supplemented EC. Se-deficient EC showed significantly enhanced neutrophil adherence when stimulated with tumor necrosis factor alpha (TNF-alpha) for 4 or 24 h, interleukin-1 for 12 h, or H2O2 for 20 min (P < 0.05). To determine the mechanisms underlying these changes in neutrophil adherence, the expression of EC adhesion molecules, ICAM-1, E-selectin, and P-selectin were examined at the molecular level by a competitive reverse transcription-polymerase chain reaction. Results revealed higher mRNA expression for E-selectin and ICAM-1 in Se-deficient EC stimulated with TNF-alpha for 3 and 6 h, and greater expression of P-selectin mRNA in Se-supplemented EC with 3-h TNF-alpha stimulation. These studies provide new information to establish the role of Se nutrition in the initiation of leukocyte adherence to endothelium. PMID:10331495

  17. 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

  18. Electroporation Enhances Immunogenicity of a DNA Vaccine Expressing Woodchuck Hepatitis Virus Surface Antigen in Woodchucks▿

    PubMed Central

    Liu, Katherine H.; Ascenzi, Mary A.; Bellezza, Christine A.; Bezuidenhout, Abraham J.; Cote, Paul J.; Gonzalez-Aseguinolaza, Gloria; Hannaman, Drew; Luxembourg, Alain; Evans, Claire F.; Tennant, Bud C.; Menne, Stephan

    2011-01-01

    The development of therapeutic vaccines for chronic hepatitis B virus (HBV) infection has been hampered by host immune tolerance and the generally low magnitude and inconsistent immune responses to conventional vaccines and proposed new delivery methods. Electroporation (EP) for plasmid DNA (pDNA) vaccine delivery has demonstrated the enhanced immunogenicity of HBV antigens in various animal models. In the present study, the efficiency of the EP-based delivery of pDNA expressing various reporter genes first was evaluated in normal woodchucks, and then the immunogenicity of an analog woodchuck hepatitis virus (WHV) surface antigen (WHsAg) pDNA vaccine was studied in this model. The expression of reporter genes was greatly increased when the cellular uptake of pDNA was facilitated by EP. The EP of WHsAg-pDNA resulted in enhanced, dose-dependent antibody and T-cell responses to WHsAg compared to those of the conventional hypodermic needle injection of WHsAg-pDNA. Although subunit WHsAg protein vaccine elicited higher antibody titers than the DNA vaccine delivered with EP, T-cell response rates were comparable. However, in WHsAg-stimulated mononuclear cell cultures, the mRNA expression of CD4 and CD8 leukocyte surface markers and Th1 cytokines was more frequent and was skewed following DNA vaccination compared to that of protein immunization. Thus, the EP-based vaccination of normal woodchucks with pDNA-WHsAg induced a skew in the Th1/Th2 balance toward Th1 immune responses, which may be considered more appropriate for approaches involving therapeutic vaccines to treat chronic HBV infection. PMID:21389124

  19. KIF14 mRNA expression is a predictor of grade and outcome in breast cancer.

    PubMed

    Corson, Timothy W; Gallie, Brenda L

    2006-09-01

    Gain of chromosome 1q is a hallmark of breast cancer, and likely reflects oncogene amplification. We previously identified mitotic kinesin KIF14 (kinesin family member 14) as an overexpressed candidate oncogene in the 1q31.3-1q32.1 minimal region of genomic gain in breast cancer cell lines. KIF14 also showed high expression in other cancers, notably an association with survival in lung tumors. We now report KIF14 expression in 99 primary breast tumors and 10 normal breast controls. Measured by real-time RT-PCR, KIF14 was overexpressed 10-fold on average in tumors relative to normals (t test p = 0.000054); expression increased with grade (ANOVA p = 0.000006). Infiltrating ductal carcinomas had higher KIF14 levels than lobular (p = 0.017), and estrogen receptor (ER) negative tumors had higher KIF14 levels than ER positive tumors (t test p = 0.030). KIF14 expression correlated positively with Ki-67 mRNA level (Spearman r = 0.692, p = 0.000001), fraction of positive nodes (r = 0.227, p = 0.024) and percent invasive cells (r = 0.360, p = 0.0002), and negatively with percent fatty stroma (r = -0.258, p = 0.010) and percent normal epithelium (r = -0.291, p = 0.003). KIF14 expression is thus tumor-specific and increased in more aggressive tumors. Indeed, KIF14 expression predicted overall survival (univariate Cox p = 0.010), with an odds ratio of 3.60 (1.37-9.48), in 50 tumors with available outcome data. KIF14 overexpression also predicted decreased disease-free survival (log-rank p = 0.049). These findings are the first evidence of association between expression of a mitotic kinesin and prognostic variables in breast cancer. PMID:16570270

  20. mRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma.

    PubMed

    Bache, Matthias; Rot, Swetlana; Keßler, Jacqueline; Güttler, Antje; Wichmann, Henri; Greither, Thomas; Wach, Sven; Taubert, Helge; Söling, Ariane; Bilkenroth, Udo; Kappler, Matthias; Vordermark, Dirk

    2015-06-01

    The roles of hypoxia-induced and stem cell-associated genes in the development of malignancy and tumour progression are well known. However, there are a limited number of studies analysing the impact of mRNA expression levels of hypoxia-induced and stem cell-associated genes in the tissues of brain tumours and glioblastoma patients. In this study, tumour tissues from patients with glioblastoma multiforme and tumour adjacent tissues were analysed. We investigated mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1) and osteopontin (OPN), and stem cell-associated genes survivin, epidermal growth factor receptor (EGFR), human telomerase reverse transcriptase (hTERT), Nanog and octamer binding transcription factor 4 (OCT4) using quantitative real-time polymerase chain reaction (qRT-PCR). Our data revealed higher mRNA expression levels of hypoxia-induced and stem cell-associated genes in tumour tissue than levels in the tumour adjacent tissues in patients with glioblastoma multiforme. A strong positive correlation between the mRNA expression levels of HIF-2α, CA9, VEGF, GLUT-1 and OPN suggests a specific hypoxia-associated profile of mRNA expression in glioblastoma multiforme. Additionally, the results indicate the role of stem-cell-related genes in tumour hypoxia. Kaplan-Maier analysis revealed that high mRNA expression levels of hypoxia-induced markers showed a trend towards shorter overall survival in glioblastoma patients (P=0.061). Our data suggest that mRNA expression levels of hypoxia-induced genes are important tumour markers in patients with glioblastoma multiforme. PMID:25963717

  1. Adverse early life experience and social stress during adulthood interact to increase serotonin transporter mRNA expression

    PubMed Central

    Gardner, Katherine L.; Hale, Matthew W.; Lightman, Stafford L.; Plotsky, Paul M.; Lowry, Christopher A.

    2009-01-01

    Anxiety disorders, depression and animal models of vulnerability to a depression-like syndrome have been associated with dysregulation of serotonergic systems in the brain. To evaluate the effects of early life experience, adverse experiences during adulthood, and potential interactions between these factors on serotonin transporter (slc6a4) mRNA expression, we investigated in rats the effects of maternal separation (180 min/day from days 2–14 of life; MS180), neonatal handing (15 min/day from days 2–14 of life; MS15), or normal animal facility rearing control conditions (AFR) with or without subsequent exposure to adult social defeat on slc6a4 mRNA expression in the dorsal raphe nucleus (DR) and caudal linear nucleus. At the level of specific subdivisions of the DR, there were no differences in slc6a4 mRNA expression between MS15 and AFR rats. Among rats exposed to a novel cage control condition, increased slc6a4 mRNA expression was observed in the dorsal part of the DR in MS180 rats, relative to AFR control rats. In contrast, MS180 rats exposed to social defeat as adults had increased slc6a4 mRNA expression throughout the DR compared to both MS15 and AFR controls. Social defeat increased slc6a4 mRNA expression, but only in MS180 rats and only in the “lateral wings” of the DR. Overall these data demonstrate that early life experience and stressful experience during adulthood interact to determine slc6a4 mRNA expression. These data support the hypothesis that early life experience and major stressful life events contribute to dysregulation of serotonergic systems in stress-related neuropsychiatric disorders. PMID:19781533

  2. Helicobacter pylori cag Pathogenicity Island Is Associated with Reduced Expression of Interleukin-4 (IL-4) mRNA and Modulation of the IL-4δ2 mRNA Isoform in Human Gastric Mucosa

    PubMed Central

    Orsini, Barbara; Ottanelli, Barbara; Amedei, Amedeo; Surrenti, Elisabetta; Capanni, Marco; Del Prete, Gianfranco; Amorosi, Andrea; Milani, Stefano; D'Elios, Mario Milco; Surrenti, Calogero

    2003-01-01

    Interleukin-4 (IL-4) and IL-4δ2 mRNA gastric expression was evaluated in healthy subjects and patients who did not have ulcers but were infected with Helicobacter pylori with or without the cag pathogenicity island (cag PAI). IL-4 mRNA was physiologically expressed by gastric epithelium and negatively influenced by H. pylori. Also, nonepithelial cells in the lamina propria of H. pylori-infected patients expressed IL-4 mRNA, whereas IL-4δ2 mRNA was found only in cag PAI-negative patients. Thus, gastric IL-4 takes part in the local immune response to H. pylori. PMID:14573693

  3. Altered expression of mRNA profiles in blood of early-onset schizophrenia

    PubMed Central

    Xu, Yong; Yao Shugart, Yin; Wang, Guoqiang; Cheng, Zaohuo; Jin, Chunhui; Zhang, Kai; Wang, Jun; Yu, Hao; Yue, Weihua; Zhang, Fuquan; Zhang, Dai

    2016-01-01

    To identify gene expression abnormalities in schizophrenia (SZ), we generated whole-genome gene expression profiles using microarrays on peripheral blood mononuclear cells (PBMCs) from 18 early-onset SZ cases and 12 controls. We detected 84 transcripts differentially expressed by diagnostic status, with 82 genes being upregulated and 2 downregulated. We identified two SZ associated gene coexpression modules (green and red), including 446 genes . The green module is positively correlated with SZ, encompassing predominantly up-regulated genes in SZ; while the red module was negatively correlated with disease status, involving mostly nominally down-regulated genes in SZ. The olfactory transduction pathway was the most enriched pathways for the genes within the two modules. The expression levels of several hub genes, including AKT1, BRCA1, CCDC134, UBD, and ZIC2 were validated using real-time quantitative PCR. Our findings indicate that mRNA coexpression abnormalities may serve as a promising mechanism underlying the development of SZ. PMID:26733343

  4. Amphetamine-induced c-fos mRNA expression is altered in rats with neonatal ventral hippocampal damage.

    PubMed

    Lillrank, S M; Lipska, B K; Bachus, S E; Wood, G K; Weinberger, D R

    1996-08-01

    To further characterize the mechanisms underlying enhanced dopamine-related behaviors expressed during adulthood in rats with neonatal excitotoxic ventral hippocampal (VH) damage, we studied the expression of c-fos mRNA in these rats after a single saline or amphetamine (AMPH) (10 mg/kg, i.p.) injection using in situ hybridization. The VH of rat pups was lesioned with ibotenic acid on postnatal day 7 (PD7). At the age of 90 days, rats were challenged with AMPH or saline, and the expression of c-fos mRNA using an oligonucleotide probe was assessed 30, 90, and 180 min later. AMPH significantly increased c-fos mRNA expression in medial prefrontal cortex, piriform cortex, cingulate cortex, septal region, and dorsolateral and ventromedial striatum in control and lesioned rats. However, this response to AMPH was attenuated 30 min after AMPH injection in all of these regions in the lesioned as compared to the sham-operated rats. No significant changes were seen at other time points. These results indicate that the neonatal VH lesion alters time-dependent intracellular signal transduction mechanisms measured by AMPH-induced c-fos mRNA expression in cortical and subcortical brain regions. Changes in c-fos mRNA expression in this putative animal model of schizophrenia may have implications for long-term alterations in cellular phenotype because of altered regulation of certain target genes. PMID:8855514

  5. Increase of hepatic fat accumulation by liver specific expression of Hepatitis B virus X protein in zebrafish.

    PubMed

    Shieh, Yun-Sheng; Chang, Yin-Shan; Hong, Jiann-Ruey; Chen, Li-Je; Jou, Luen-Kuang; Hsu, Chia-Chun; Her, Guor Mour

    2010-07-01

    The pathogenesis of fatty liver disease remains largely unknown. Here, we assessed the importance of hepatic fat accumulation on the progression of hepatitis in zebrafish by liver specific expression of Hepatitis B virus X protein (HBx). Transgenic zebrafish lines, GBXs, which selectively express the GBx transgene (GFP-fused HBx gene) in liver, were established. GBX Liver phenotypes were evaluated by histopathology and molecular analysis of fatty acid (FA) metabolism-related genes expression. Most GBXs (66-81%) displayed obvious emaciation starting at 4 months old. Over 99% of the emaciated GBXs developed hepatic steatosis or steatohepatitis, which in turn led to liver hypoplasia. The liver histology of GBXs displayed steatosis, lobular inflammation, and balloon degeneration, similar to non-alcoholic steatohepatitis (NASH). Oil red O stain detected the accumulation of fatty droplets in GBXs. RT-PCR and Q-rt-PCR analysis revealed that GBx induced hepatic steatosis had significant increases in the expression of lipogenic genes, C/EBP-alpha, SREBP1, ChREBP and PPAR-gamma, which then activate key enzymes of the de novo FA synthesis, ACC1, FAS, SCD1, AGAPT, PAP and DGAT2. In addition, the steatohepatitic GBX liver progressed to liver degeneration and exhibited significant differential gene expression in apoptosis and stress. The GBX models exhibited both the genetic and functional factors involved in lipid accumulation and steatosis-associated liver injury. In addition, GBXs with transmissible NASH-like phenotypes provide a promising model for studying liver disease. PMID:20416398

  6. Hepatitis C Virus Increases Occludin Expression via the Upregulation of Adipose Differentiation-Related Protein

    PubMed Central

    Branche, Emilie; Conzelmann, Stéphanie; Parisot, Clotilde; Bedert, Ludmila; Lévy, Pierre L.; Bartosch, Birke

    2016-01-01

    The hepatitis C virus (HCV) life cycle is closely associated with lipid metabolism. In particular, HCV assembly initiates at the surface of lipid droplets. To further understand the role of lipid droplets in HCV life cycle, we assessed the relationship between HCV and the adipose differentiation-related protein (ADRP), a lipid droplet-associated protein. Different steps of HCV life cycle were assessed in HCV-infected human Huh-7 hepatoma cells overexpressing ADRP upon transduction with a lentiviral vector. HCV infection increased ADRP mRNA and protein expression levels by 2- and 1.5-fold, respectively. The overexpression of ADRP led to an increase of (i) the surface of lipid droplets, (ii) the total cellular neutral lipid content (2.5- and 5-fold increase of triglycerides and cholesterol esters, respectively), (iii) the cellular free cholesterol level (5-fold) and (iv) the HCV particle production and infectivity (by 2- and 3.5-fold, respectively). The investigation of different steps of the HCV life cycle indicated that the ADRP overexpression, while not affecting the viral replication, promoted both virion egress and entry (~12-fold), the latter possibly via an increase of its receptor occludin. Moreover, HCV infection induces an increase of both ADRP and occludin expression. In HCV infected cells, the occludin upregulation was fully prevented by the ADRP silencing, suggesting a specific, ADRP-dependent mechanism. Finally, in HCV-infected human livers, occludin and ADRP mRNA expression levels correlated with each other. Alltogether, these findings show that HCV induces ADRP, which in turns appears to confer a favorable environment to viral spread. PMID:26731658

  7. Histidine-imbalanced diets stimulate hepatic histidase gene expression in rats.

    PubMed

    Torres, N; Beristain, L; Bourges, H; Tovar, A R

    1999-11-01

    A high protein concentration in the diet induces the gene expression of several amino acid degrading enzymes such as histidase (Hal) in rats. It is important to understand whether the amino acid pattern of the dietary protein affects the gene expression of these enzymes. The purpose of the present work was to study the effect of a histidine-imbalanced diet on the activity and mRNA concentration of rat hepatic histidase. Seven groups of six rats were fed one of the following diets: 1) 6% casein (basal), 2) 20% casein, 3) 35% casein, 4) an imbalance diet containing 6% casein plus a mixture of indispensable amino acids (IAA) equivalent to a 20% casein diet without histidine (I-20), 5) 6% casein plus a mixture of IAA equivalent to a 35% casein diet without histidine (I-35), 6) a corrected diet containing 6% casein plus IAA including histidine equivalent to a 20% casein diet, 7) a corrected diet containing 6% casein plus IAA including histidine equivalent to a 35% casein diet. Serum histidine concentration was inversely proportional to the protein content of the diet, and it was significantly higher in rats fed the corrected diets compared to their respective imbalanced diet groups. Hal activity increased as the protein content of the diet increased. Greater histidine imbalance resulted in lower food intake and higher Hal activity. Rats fed histidine-corrected diets had lower activity than their respective imbalanced groups. Differences in Hal activity were associated with differences in the concentration of Hal mRNA. These results indicate that rats fed a histidine-imbalanced diet exhibit reduced food intake and weight gain and increased Hal gene expression as a consequence of an increased amino acid catabolism. PMID:10539772

  8. Changes of hepatic lactoferrin gene expression in two mouse models of the acute phase reaction.

    PubMed

    Ahmad, Ghayyor; Sial, Gull Zareen Khan; Ramadori, Pierluigi; Dudas, Jozsef; Batusic, Danko S; Ramadori, Giuliano

    2011-12-01

    Lactoferrin (Ltf), an iron binding glycoprotein, is a pleiotropic molecule whose serum concentration increases under acute phase conditions. The physiological roles of this protein have been well elucidated, but the source and serum regulation of Ltf gene expression have not been investigated in detail as part of the acute phase reaction (APR). In the current work, the changes in hepatic Ltf-gene-expression during turpentine oil- (TO-) or LPS-induced APR were investigated. Ltf was upregulated at both the mRNA and protein levels in the liver of TO- and LPS-treated wild type (WT) mice. The pattern of induction however was different in both animal models indicating distinctive signalling patterns resulting in an acute phase reaction. Cytokines are the core regulators of APR. Among the major cytokines, IL-6 is an important signalling molecule, which also regulates iron homeostasis in response to an inflammatory situation. In this study, the administration of IL-6 induced Ltf gene expression in the liver of WT mice, in murine hepatocytes and in hepa 1-6 cells. Ltf-gene-expression was upregulated also in the liver of TO- and LPS-treated IL-6 knockout (KO) mice. The increase in serum Ltf after LPS injection was greater than after TO-injection both in WT and IL-6-KO mice. To evaluate the contribution of other acute phase cytokines in the regulation of Ltf-gene-expression in the liver, both in vitro and in vivo studies with IL-1β, TNF-α, or IFN-γ were performed. The results demonstrate that TNF-α and IFN-γ also upregulated Ltf-gene-expression, while IL-1β has no role in the regulation of Ltf-gene-expression. PMID:21963450

  9. Effect of maternal folic acid supplementation on hepatic one-carbon unit associated gene expressions in newborn piglets.

    PubMed

    Liu, Jing-Bo; Chen, Dai-Wen; Yu, Bing; Mao, Xiang-bing

    2011-08-01

    Intrauterine growth retardation (IUGR) induces alterations to hepatic gene expressions which might program poor postnatal growth and health status. Maternal folic acid supplementation was administered in gilt diets to test whether hepatic mRNA expressions of some important genes induced by IUGR could be rescued by folic acid supplementation. Thirty-two Yorkshire gilts were allotted to two treatment groups of control (C folic acid 1.3 mg/kg) or folic acid supplementation (FS folic acid 30 mg/kg) after mating, to study the effects of maternal folic acid supplementation on the mRNA expression of methionine adenosyltransferase (MAT), cystathionine-β-synthase (CBS), methylenetetrahydrofolate reductase (MTHFR), DNA methyltransferase1 (DNMT1), peroxisomal proliferator-activated receptor (PPARγ), glucocorticoid receptor (GR), obesity receptor (ob-R) and Acyl-CoA oxidase (AOX) in the liver of IUGR and NBW piglets. Blood and liver samples were collected for determinations of serum folic acid and gene expressions. The total number of born piglets, number of piglets born alive, average birth weight and 21 days average weight were not affected by dietary treatment (P>0.05), and serum folic acid concentration of piglets was greater in FS than C groups (P<0.05). Real-time PCR indicated that gene expression of MAT1A, MAT2A and DNMT1 were lower in IUGR piglets but could be elevated by maternal folic acid supplementation. Transcript expression levels of PPARγ, GR and AOX were higher in IUGR piglets, but were decreased to the level of normal piglets by maternal folic acid supplementation. Our results suggested that maternal folic acid supplementation be an effective way to rescue the gene expressions negatively induced by IUGR. PMID:21108044

  10. Changes in gene expression in liver tissue from patients with fulminant hepatitis E

    PubMed Central

    Naik, Anshu; Goel, Amit; Agrawal, Vinita; Sarangi, Aditya N; Chhavi, Nanda; Singh, Vineeta; Jameel, Shahid; Aggarwal, Rakesh

    2015-01-01

    AIM: To study host gene expression and number of immune cells in liver tissues from patients with fulminant hepatitis E (FH-E). METHODS: Microarray-based expression profiling was done using Illumina Human WG-6_v3_BeadChip arrays on post-mortem liver tissue from 5 patients with FH-E, and compared with similar tissue from 6 patients with fulminant hepatitis B (FH-B; disease controls) and normal liver tissue from 6 persons. Differential expression was defined as ≥ 2.0-fold change with Benjamini-Hochberg false discovery rate below 0.05 using t-test in liver tissue from FH-B and FH-E, than healthy liver tissue. For some genes that showed differential expression in FH-E, microarray data were validated using quantitative reverse transcription PCR. Differentially expressed gene lists were then subjected to “Gene Ontology” analysis for biological processes, and pathway analysis using BioCarta database on the DAVID server. In addition, tissue sections were stained for CD4+, CD8+ and CD56+ cells using indirect immunohistochemistry; cells staining positive for each of these markers were counted and compared between groups. RESULTS: Compared to normal livers, those from patients with FH-E and FH-B showed differential expression of 3377 entities (up-regulated 1703, downregulated 1674) and 2572 entities (up 1164, down 1408), respectively. This included 2142 (up 896, down 1246) entities that were common between the two sets; most of these belonged to metabolic, hemostatic and complement pathways, which are active in normal livers. Gene expression data from livers of patients with FH-E but not those of FH-B showed activation of several immune response pathways, particularly those involving cytotoxic T cells. The fold-change values of mRNA for selected genes in livers from FH-E than in normal liver tissue determined using quantitative reverse transcription PCR showed excellent concordance with microarray analysis. At immunohistochemistry, CD8+ T cells showed an increase in

  11. Expression of preproenkephalin-like mRNA and its peptide products in mammalian testis and ovary.

    PubMed Central

    Kilpatrick, D L; Howells, R D; Noe, M; Bailey, L C; Udenfriend, S

    1985-01-01

    The distribution of preproenkephalin mRNA and proenkephalin-derived peptides have been examined in gonadal tissues from rats, hamsters, and cattle. A preproenkephalin mRNA band was detected in the ovaries of all three species and in hamster testis that is identical in size to the 1450-nucleotide mRNA typically found in tissues that express proenkephalin. Rat testis, on the other hand, expresses at least one preproenkephalin-like mRNA that is substantially greater in size (1900 nucleotides). [Met]enkephalin-containing peptides were also detected in each of the gonadal tissues examined. Although the abundance of preproenkephalin-like mRNA in rat testis was comparable to that in rat brain, the testicular content of proenkephalin-derived [Met]enkephalin sequences was less than 4% of the rat brain content. Together these data suggest that preproenkephalin-like mRNA in rat testis is not efficiently translated, proenkephalin-derived peptides undergo rapid turnover in this tissue, or the mRNA in rat testis has a frameshift resulting in an altered coding sequence. Images PMID:3864164

  12. 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

  13. 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...

  14. Hepatic Gene Expression Profile in Mice Perorally Infected with Echinococcus multilocularis Eggs

    PubMed Central

    Gottstein, Bruno; Wittwer, Matthias; Schild, Marc; Merli, Michael; Leib, Stephen L.; Müller, Norbert; Müller, Joachim; Jaggi, Rolf

    2010-01-01

    Background Alveolar echinococcosis (AE) is a severe chronic hepatic parasitic disease currently emerging in central and eastern Europe. Untreated AE presents a high mortality (>90%) due to a severe hepatic destruction as a result of parasitic metacestode proliferation which behaves like a malignant tumor. Despite this severe course and outcome of disease, the genetic program that regulates the host response leading to organ damage as a consequence of hepatic alveolar echinococcosis is largely unknown. Methodology/Principal Findings We used a mouse model of AE to assess gene expression profiles in the liver after establishment of a chronic disease status as a result of a primary peroral infection with eggs of the fox tapeworm Echinococcus multilocularis. Among 38 genes differentially regulated (false discovery rate adjusted p≤0.05), 35 genes were assigned to the functional gene ontology group , while 3 associated with the functional group . Upregulated genes associated with could be clustered into functional subgroups including , , , and . Two downregulated genes related to and , respectively. The genes either associated with an or an pathway. From the overexpressed genes, 18 genes were subsequently processed with a Custom Array microfluidic card system in order to assess respective expression status at the mRNA level relative to 5 reference genes (Gapdh, Est1, Rlp3, Mdh-1, Rpl37) selected upon a constitutive and stable expression level. The results generated by the two independent tools used for the assessment of gene expression, i.e., microarray and microfluidic card system, exhibited a high level of congruency (Spearman correlation rho = 0.81, p = 7.87e-5) and thus validated the applied

  15. Vesnarinone suppresses TNFα mRNA expression by inhibiting valosin-containing protein.

    PubMed

    Hotta, Kentaro; Nashimoto, Akihiro; Yasumura, Eiji; Suzuki, Masafumi; Azuma, Motoki; Iizumi, Yosuke; Shima, Daisuke; Nabeshima, Ryusuke; Hiramoto, Masaki; Okada, Akira; Sakata-Sogawa, Kumiko; Tokunaga, Makio; Ito, Takumi; Ando, Hideki; Sakamoto, Satoshi; Kabe, Yasuaki; Aizawa, Shinichi; Imai, Takeshi; Yamaguchi, Yuki; Watanabe, Hajime; Handa, Hiroshi

    2013-05-01

    Vesnarinone is a synthetic quinolinone derivative used in the treatment of cardiac failure and cancer. It is also known to cause agranulocytosis as a side effect, which restricts its use, although the mechanism underlying agranulocytosis is not well understood. Here, we show that vesnarinone binds to valosin-containing protein (VCP), which interacts with polyubiquitinated proteins and is essential for the degradation of IκBα to activate nuclear factor (NF)κB. We show that vesnarinone impairs the degradation of IκBα, and that the impairment of the degradation of IκBα is the result of the inhibition of the interaction between VCP and the 26S proteasome by vesnarinone. These results suggest that vesnarinone suppresses NFκB activation by inhibiting the VCP-dependent degradation of polyubiquitinated IκBα, resulting in the suppression of tumor necrosis factor-α mRNA expression. PMID:23393163

  16. Direct evidence for cytotoxicity associated with expression of hepatitis delta virus antigen.

    PubMed

    Cole, S M; Gowans, E J; Macnaughton, T B; Hall, P D; Burrell, C J

    1991-05-01

    It has been postulated that hepatocyte injury resulting from infection with hepatitis D virus may be caused by a direct virus cytotoxicity in contrast to immune-mediated injury associated with hepatitis B virus. We have transfected HeLa and HepG2 continuous cell lines with a recombinant plasmid containing the hepatitis D antigen gene under the inducible control of the human metallothionein promoter. The addition of zinc to the cell culture medium then led to the expression of hepatitis D antigen associated with, in the short term, a significant reduction in the rate of RNA but not DNA synthesis and, in the longer term, cell death. The necrotic cells had pyknotic nuclei and shrunken eosinophilic cytoplasm; these necrotic cells resembled the apoptotic bodies seen in hepatitis D virus-related hepatitis. The level of hepatitis D antigen in individual cells that produced these changes was similar to the level of hepatitis D antigen in hepatocytes from a chimpanzee with acute hepatitis D virus infection. We conclude that the expression of hepatitis D antigen resulted in significant cytotoxic changes in these cells, providing strong support for the view that hepatitis D antigen may be specifically cytotoxic to infected hepatocytes in vivo. PMID:1709411

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

    PubMed Central

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

    2016-01-01

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

  18. Rev-erb beta regulates the Srebp-1c promoter and mRNA expression in skeletal muscle cells

    SciTech Connect

    Ramakrishnan, Sathiya N.; Lau, Patrick; Crowther, Lisa M.; Cleasby, Mark E.; Millard, Susan; Leong, Gary M.; Cooney, Gregory J.; Muscat, George E.O.

    2009-10-30

    The nuclear hormone receptor, Rev-erb beta operates as a transcriptional silencer. We previously demonstrated that exogenous expression of Rev-erb{beta}{Delta}E in skeletal muscle cells increased Srebp-1c mRNA expression. We validated these in vitro observations by injection of an expression vector driving Rev-erb{beta}{Delta}E expression into mouse tibialis muscle that resulted in increased Srebp-1c mRNA expression. Paradoxically, Rev-erb{beta} siRNA expression in skeletal muscle cells repressed Srebp-1c expression, and indicated that Rev-erb{beta} expression was necessary for Srebp-1c expression. ChIP analysis demonstrated that Rev-erb{beta} was recruited to the Srebp-1c promoter. Moreover, Rev-erb{beta} trans-activated the Srebp-1c promoter, in contrast, Rev-erb{beta} efficiently repressed the Rev-erb{alpha} promoter, a previously characterized target gene. Finally, treatment with the Rev-erb agonist (hemin) (i) increased the trans-activation of the Srebp-1c promoter by Rev-erb{beta}; and (ii) increased Rev-erb{beta} and Srebp-1c mRNA expression. These data suggest that Rev-erb{beta} has the potential to activate gene expression, and is a positive regulator of Srebp-1c, a regulator of lipogenesis.

  19. Translational control of germ cell-expressed mRNA imposed by alternative splicing: opioid peptide gene expression in rat testis.

    PubMed Central

    Garrett, J E; Collard, M W; Douglass, J O

    1989-01-01

    The three genes encoding the opioid peptide precursors (prodynorphin, proenkephalin, and proopiomelanocortin) are expressed in the rat testis. The sizes of the three opioid mRNAs in the testis differ from the sizes of the corresponding mRNAs in other rat tissues in which these genes are expressed. The smaller testicular proopiomelanocortin mRNA has previously been demonstrated to arise from alternative transcriptional initiation. In the present study, we found that the smaller testicular prodynorphin mRNA, expressed in Sertoli cells, results from alternative mRNA processing. Exon 2, which makes up 5' untranslated sequence, is removed from the mature transcript. Polysome analysis of brain and testis RNA indicates that the alteration of the prodynorphin leader sequence in the testis-specific transcript does not affect the efficiency of translation of this mRNA. The larger testicular proenkephalin transcript, expressed in developing germ cells, also results from alternative mRNA processing. Alternative acceptor site usage in the splicing of intron A results in a germ cell-specific proenkephalin transcript with a 491-nucleotide 5' untranslated leader sequence preceding the preproenkephalin-coding sequence. Polysome analysis indicates that this germ cell-specific proenkephalin mRNA is not efficiently translated. Mechanisms by which alternative mRNA splicing may serve to confer translational regulation upon the testicular proenkephalin transcript are discussed. Images PMID:2573832

  20. Influence of Cardiorespiratory Fitness on PPARG mRNA Expression Using Monozygotic Twin Case Control

    PubMed Central

    Queiroga, Marcos Roberto; Barbieri, Ricardo Augusto; Ferreira, Sandra Aires; Luchessi, André Ducati; Hirata, Rosario Dominguez Crespo; Hirata, Mario Hiroyuki; Kokubun, Eduardo

    2015-01-01

    The influence of cardiorespiratory fitness (VO2max) on anthropometric variables and PPARG mRNA expression was investigated. Monozygotic twin pairs aged 11–18 years were grouped into discordant (D) and concordant (C) high and low VO2max groups. VO2max was determined by progressive maximal exercise test on treadmill with gas exchange analysis. Body mass (BM), BMI, waist circumference (WC), triceps (TR), and subscapular (SB) skinfold thicknesses were measured. Twins from the discordant group had differences in VO2max values (D-high = 45.9 ± 10.0 versus D-low = 32.4 ± 10.6 mL·kg−1·min−1, P = 0.025), while no differences were found in the concordant group (C-high = 42.4 ± 9.2 versus C-low = 38.8 ± 9.8 mL·kg−1·min−1, P = 0.952). In discordant group, VO2max was negatively correlated with TR + SB (r = −0.540, P = 0.021) and positively correlated with PPARG expression in leukocytes (r = 0.952, P = 0.001). Moreover, PPARG expression was directly correlated with BM (r = 0.714, P = 0.047) and height (r = 0.762, P = 0.028). In concordant twins, VO2max was inversely correlated with BM (r = −0.290, P = 0.027), BMI (r = −0.472, P = 0.001), WC (r = −0.426, P = 0.001), and TR + SB (r = −0.739, P = 0.001). Twins D-high had 1.78-fold greater PPARG expression when compared with twins D-low (P = 0.048). In conclusion, the cardiorespiratory fitness may modulate PPARG expression in childhood and adolescence, independently of the genetic background. PMID:25879043

  1. Localized expression of mRNA for phagocyte-specific chemotactic cytokines in human periodontal infections.

    PubMed Central

    Tonetti, M S; Imboden, M A; Gerber, L; Lang, N P; Laissue, J; Mueller, C

    1994-01-01

    In bacterial infections, mononuclear and polymorphonuclear phagocytes are key components of host defenses. Recent investigations have indicated that chemokines are able to recruit and activate phagocytes. In particular, interleukin-8 (IL-8) attracts polymorphonuclear leukocytes (PMNs), while monocyte chemoattractant protein-1 (MCP-1) is selective for cells of the monocyte/macrophage lineage. In this investigation, we analyzed the in situ expression of IL-8 and MCP-1 mRNAs in human periodontal infections. Specific mRNA was detected by in situ hybridization using 35S-labeled riboprobes in frozen tissue sections. Phagocytes (PMNs and macrophages) were specifically detected as elastase-positive or CD68+ cells by a three-stage immunoperoxidase technique. Results indicated that expression of phagocyte-specific cytokines was confined to selected tissue locations and, in general, paralleled phagocyte infiltration. In particular, IL-8 expression was maximal in the junctional epithelium adjacent to the infecting microorganisms; PMN infiltration was more prominent in the same area. MCP-1 was expressed in the chronic inflammatory infiltrate and along the basal layer of the oral epithelium. Cells of the monocyte/macrophage lineage were demonstrated to be present in the same areas. The observed expression pattern may be the most economic way to establish a cell-type-selective chemotactic gradient within the tissue that is able to effectively direct polymorphonuclear phagocyte migration toward the infecting microorganisms and modulate mononuclear phagocyte infiltration in the surrounding tissues. This process may optimize host defenses and contribute to containing leukocyte infiltration to the infected and inflamed area, thus limiting tissue damage. Images PMID:8063420

  2. Isolation of an mRNA binding protein homologue that is expressed in nociceptors.

    PubMed

    Eilers, Helge; Trilk, Sharon L; Lee, Sook Young; Xue, Qing; Jong, Beverly E; Moff, Irene; Levine, Jon D; Schumacher, Mark A

    2004-11-01

    The peripheral detection of painful stimuli requires the activation of small-diameter primary afferent neurons known as nociceptors. We have exploited two features of nociceptor biology, expression of the high affinity receptor for nerve growth factor (TrkA) and sensitivity to capsaicin, to isolate novel proteins using a differential display cloning scheme. A resulting approximately 4.3-kb cDNA was isolated and sequence analysis inferred a approximately 157-kDa protein containing a signal/mitochondrial targeting peptide sequence. Due to its molecular weight and significant amino acid identity with 'human leucine-rich protein 130'[leucine-rich pentatricopeptide motif containing (LRPPRC)], we termed the cDNA candidate leucine-rich protein 157 (rLRP157). Western blot analysis of HEK293 cells over-expressing the candidate cDNA showed a single protein product of similar size to that found in rat dorsal root ganglion as well as in other neuronal tissues and cell lines. Although expressed in a wide variety of tissues, in situ hybridization and immunohistochemistry in dorsal root ganglion revealed that rLRP157 expression was restricted to the small-diameter neurons. Sequence identity with previously characterized mRNA binding proteins and its subcellular localization in sensory neurons suggest that rLRP157 is associated with mitochondrial function. Moreover, the genetic basis of French-Canadian Leigh syndrome, which confers a loss of mitochondrial cytochrome c oxidase and is characterized by neurodegeneration, was recently mapped to a mutation in the LRPPRC gene. Taken together with its expression in small-diameter sensory neurons, we hypothesize that rLRP157, the rat orthologue of the human LRPPRC, may play a role in the modulation of peripheral pain transduction and serve as a novel marker for nociceptor subtypes. PMID:15525270

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

    PubMed Central

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

    2013-01-01

    Without the age-regulating protein klotho, mouse lifespan is shortened and the rapid onset of age-related disorders occurs. Conversely, overexpression of klotho extends mouse lifespan. Klotho is most abundant in kidney and expressed in a limited number of other organs, including the brain, where klotho levels are highest in choroid plexus. Reports vary on where klotho is expressed within the brain parenchyma, and no data is available as to whether klotho levels change across postnatal development. We used in situ hybridization to map klotho mRNA expression in the developing and adult rat brain and report moderate, widespread expression across grey matter regions. mRNA expression levels in cortex, hippocampus, caudate putamen, and amygdala decreased during the second week of life and then gradually rose to adult levels by postnatal day 21. Immunohistochemistry revealed a protein expression pattern similar to the mRNA results, with klotho protein expressed widely throughout the brain. Klotho protein co-localized with both the neuronal marker NeuN, as well as, oligodendrocyte marker olig2. These results provide the first anatomical localization of klotho mRNA and protein in rat brain parenchyma and demonstrate that klotho levels vary during early postnatal development. PMID:23838326

  4. Transient and persistent expression of NT-3/HDNF mRNA in the rat brain during postnatal development.

    PubMed

    Friedman, W J; Ernfors, P; Persson, H

    1991-06-01

    Neurotrophin-3 (NT-3) is closely related to two known neurotrophic agents, NGF and brain-derived neurotrophic factor (BDNF), and acts upon overlapping, yet distinct, populations of peripheral ganglia. NT-3 mRNA expression in the adult rat brain is largely confined to the hippocampus. In this study, we have used in situ hybridization to examine expression of this novel neurotrophic factor during postnatal development. The striking observation was made that NT-3 mRNA was transiently expressed at high levels in the cingulate cortex during the first 2 weeks of age. In the hippocampus, the adult pattern of expression, in the CA2, medial CA1, and granule layer of the dentate gyrus, was detected at all ages examined. However, there were two major differences in NT-3 mRNA expression in the developing hippocampus: Labeled cells were detected in the hilar region of the dentate gyrus at postnatal day 1 (P1) and 1 week that were absent by 2 weeks of age. Further, the caudal hippocampus, which has a lower intensity of labeling than the rostral region in the adult, was devoid of NT-3-expressing cells in the P1 and 1-week-old rat brain. These data indicate a substantial plasticity in NT-3 mRNA expression and suggest that the spectrum of neurons supported by NT-3 during development is partially different from that in the mature rat brain. PMID:2045877

  5. 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. PMID:12417619

  6. Thy-1 Is Expressed in Hepatic Myofibroblasts and Not Oval Cells in Stem Cell-Mediated Liver Regeneration

    PubMed Central

    Dezső, Katalin; Jelnes, Peter; László, Viktória; Baghy, Kornélia; Bödör, Csaba; Paku, Sándor; Tygstrup, Niels; Bisgaard, Hanne Cathrine; Nagy, Peter

    2007-01-01

    Thy-1, a marker of hematopoietic stem cells, has been reported to be expressed by oval cells proliferating during stem cell-mediated regeneration in rat liver, suggesting a relationship between the two cell populations. Consequently, Thy-1 has become an accepted cell surface marker to sort hepatic oval cells. In the present study we used the well-characterized 2-acetylaminfluorene/partial hepatectomy model to induce transit-amplification of hepatic oval cells in the regenerating liver and characterized Thy-1 expression using Northern hybridization, quantitative reverse transcriptase-polymerase chain reaction analysis, immunofluorescence confocal microscopy, and immunoelectronmicroscopy. We found that Thy-1 expression was induced during transit-amplification of the oval cell population, but Thy-1 mRNA was not present in the α-fetoprotein-expressing oval cells. Thy-1 protein was consistently present outside the basement membrane surrounding the oval cells. It overlapped frequently with smooth muscle actin staining. A similar cellular localization of the Thy-1 protein was found on human liver specimens with ductular reactions obtained from patients with fulminant liver failure. Furthermore, Thy-1 was expressed by myofibroblasts in experimental liver fibrosis models without oval cell proliferation. We conclude that Thy-1 is not a marker of oval cells but is present on a subpopulation of myofibroblasts/stellate cells. PMID:17884967

  7. The angiotensin II receptor antagonist, losartan, enhances regulator of G protein signaling 2 mRNA expression in vascular smooth muscle cells of Wistar rats.

    PubMed

    Wu, Yaqiong; Nakagawa, Suguru; Takahashi, Hidenori; Kawabata, Yukari; Suzuki, Etsu; Uehara, Yoshio

    2016-05-01

    Angiotensin II (Ang II) reportedly enhances regulator of G-protein signaling 2 (RGS2), thus making a negative feedback loop for Ang II signal transduction. However, few studies have reported whether Ang II receptor (ATR) antagonists influence RGS2 mRNA expression. We investigated RGS2 mRNA expression when Ang II binding to ATR was blocked with Ang II subtype-1 receptor (AT1R) blockers using vascular smooth muscle cells from the thoracic aorta of male Wistar rats. RGS2 mRNA expression significantly increased with Ang II stimulation, and this increase was almost completely abolished by olmesartan, a potent AT1R-specific blocker. Ang II subtype-2 receptor (AT2R) was not involved in Ang II-mediated RGS expression. In contrast, the AT1R blocker, losartan, partially decreased Ang II-mediated RGS2 mRNA expression because this antagonist directly stimulated RGS2 mRNA expression in Ang II-free medium. EXP3174, which is an active metabolite of losartan, almost completely blunted Ang II-mediated RGS2 mRNA expression without direct stimulation of RGS2 mRNA expression. Moreover, pretreatment with olmesartan abolished Ang II-mediated RGS2 mRNA expression. Treatment with a protein kinase C inhibitor partially decreased losartan-mediated RGS2 mRNA expression. These results suggest that AT1R blockers inhibit RGS2 mRNA expression in response to Ang II via an AT1R-mediated mechanism. However, the AT1R blocker, losartan, behaves as a direct agonist for RGS2 mRNA expression via AT1R through protein kinase C-dependent and -independent pathways. In conclusion, losartan exhibits dual effects on RGS2 mRNA expression, and the direct upregulation of RGS2 mRNA expression may provide a new strategy for the treatment of hypertension. PMID:26763849

  8. IFNL3 mRNA structure is remodeled by a functional non-coding polymorphism associated with hepatitis C virus clearance

    PubMed Central

    Lu, Yi-Fan; Mauger, David M.; Goldstein, David B.; Urban, Thomas J.; Weeks, Kevin M.; Bradrick, Shelton S.

    2015-01-01

    Polymorphisms near the interferon lambda 3 (IFNL3) gene strongly predict clearance of hepatitis C virus (HCV) infection. We analyzed a variant (rs4803217 G/T) located within the IFNL3 mRNA 3′ untranslated region (UTR); the G allele (protective allele) is associated with elevated therapeutic HCV clearance. We show that the IFNL3 3′ UTR represses mRNA translation and the rs4803217 allele modulates the extent of translational regulation. We analyzed the structures of IFNL3 variant mRNAs at nucleotide resolution by SHAPE-MaP. The rs4803217 G allele mRNA forms well-defined 3′ UTR structure while the T allele mRNA is more dynamic. The observed differences between alleles are among the largest possible RNA structural alterations that can be induced by a single nucleotide change and transform the UTR from a single well-defined conformation to one with multiple dynamic interconverting structures. These data illustrate that non-coding genetic variants can have significant functional effects by impacting RNA structure. PMID:26531896

  9. Dysregulated hepatic expression of glucose transporters in chronic disease: contribution of semicarbazide-sensitive amine oxidase to hepatic glucose uptake

    PubMed Central

    Karim, Sumera; Liaskou, Evaggelia; Fear, Janine; Garg, Abhilok; Reynolds, Gary; Claridge, Lee; Adams, David H.; Newsome, Philip N.

    2014-01-01

    Insulin resistance is common in patients with chronic liver disease (CLD). Serum levels of soluble vascular adhesion protein-1 (VAP-1) are also increased in these patients. The amine oxidase activity of VAP-1 stimulates glucose uptake via translocation of transporters to the cell membrane in adipocytes and smooth muscle cells. We aimed to document human hepatocellular expression of glucose transporters (GLUTs) and to determine if VAP-1 activity influences receptor expression and hepatic glucose uptake. Quantitative PCR and immunocytochemistry were used to study human liver tissue and cultured cells. We also used tissue slices from humans and VAP-1-deficient mice to assay glucose uptake and measure hepatocellular responses to stimulation. We report upregulation of GLUT1, -3, -5, -6, -7, -8, -9, -10, -11, -12, and -13 in CLD. VAP-1 expression and enzyme activity increased in disease, and provision of substrate to hepatic VAP-1 drives hepatic glucose uptake. This effect was sensitive to inhibition of VAP-1 and could be recapitulated by H2O2. VAP-1 activity also altered expression and subcellular localization of GLUT2, -4, -9, -10, and -13. Therefore, we show, for the first time, alterations in hepatocellular expression of glucose and fructose transporters in CLD and provide evidence that the semicarbazide-sensitive amine oxidase activity of VAP-1 modifies hepatic glucose homeostasis and may contribute to patterns of GLUT expression in chronic disease. PMID:25342050

  10. Dysregulated hepatic expression of glucose transporters in chronic disease: contribution of semicarbazide-sensitive amine oxidase to hepatic glucose uptake.

    PubMed

    Karim, Sumera; Liaskou, Evaggelia; Fear, Janine; Garg, Abhilok; Reynolds, Gary; Claridge, Lee; Adams, David H; Newsome, Philip N; Lalor, Patricia F

    2014-12-15

    Insulin resistance is common in patients with chronic liver disease (CLD). Serum levels of soluble vascular adhesion protein-1 (VAP-1) are also increased in these patients. The amine oxidase activity of VAP-1 stimulates glucose uptake via translocation of transporters to the cell membrane in adipocytes and smooth muscle cells. We aimed to document human hepatocellular expression of glucose transporters (GLUTs) and to determine if VAP-1 activity influences receptor expression and hepatic glucose uptake. Quantitative PCR and immunocytochemistry were used to study human liver tissue and cultured cells. We also used tissue slices from humans and VAP-1-deficient mice to assay glucose uptake and measure hepatocellular responses to stimulation. We report upregulation of GLUT1, -3, -5, -6, -7, -8, -9, -10, -11, -12, and -13 in CLD. VAP-1 expression and enzyme activity increased in disease, and provision of substrate to hepatic VAP-1 drives hepatic glucose uptake. This effect was sensitive to inhibition of VAP-1 and could be recapitulated by H2O2. VAP-1 activity also altered expression and subcellular localization of GLUT2, -4, -9, -10, and -13. Therefore, we show, for the first time, alterations in hepatocellular expression of glucose and fructose transporters in CLD and provide evidence that the semicarbazide-sensitive amine oxidase activity of VAP-1 modifies hepatic glucose homeostasis and may contribute to patterns of GLUT expression in chronic disease. PMID:25342050

  11. Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury

    PubMed Central

    Wang, Shaogui; Ni, Hong-Min; Dorko, Kenneth; Kumer, Sean C.; Schmitt, Timothy M.; Nawabi, Atta; Komatsu, Masaaki; Huang, Heqing; Ding, Wen-Xing

    2016-01-01

    Chronic alcohol exposure increased hepatic receptor-interacting protein kinase (RIP) 3 expression and necroptosis in the liver but its mechanisms are unclear. In the present study, we demonstrated that chronic alcohol feeding plus binge (Gao-binge) increased RIP3 but not RIP1 protein levels in mouse livers. RIP3 knockout mice had decreased serum alanine amino transferase activity and hepatic steatosis but had no effect on hepatic neutrophil infiltration compared with wild type mice after Gao-binge alcohol treatment. The hepatic mRNA levels of RIP3 did not change between Gao-binge and control mice, suggesting that alcohol-induced hepatic RIP3 proteins are regulated at the posttranslational level. We found that Gao-binge treatment decreased the levels of proteasome subunit alpha type-2 (PSMA2) and proteasome 26S subunit, ATPase 1 (PSMC1) and impaired hepatic proteasome function. Pharmacological or genetic inhibition of proteasome resulted in the accumulation of RIP3 in mouse livers. More importantly, human alcoholics had decreased expression of PSMA2 and PSMC1 but increased protein levels of RIP3 compared with healthy human livers. Moreover, pharmacological inhibition of RIP1 decreased Gao-binge-induced hepatic inflammation, neutrophil infiltration and NF-κB subunit (p65) nuclear translocation but failed to protect against steatosis and liver injury induced by Gao-binge alcohol. In conclusion, results from this study suggest that impaired hepatic proteasome function by alcohol exposure may contribute to hepatic accumulation of RIP3 resulting in necroptosis and steatosis while RIP1 kinase activity is important for alcohol-induced inflammation. PMID:26769846

  12. FOXA2 mRNA expression is associated with relapse in patients with Triple-Negative/Basal-like breast carcinoma.

    PubMed

    Perez-Balaguer, Ariadna; Ortiz-Martínez, Fernando; García-Martínez, Araceli; Pomares-Navarro, Critina; Lerma, Enrique; Peiró, Gloria

    2015-09-01

    The FOXA family of transcription factors regulates chromatin structure and gene expression especially during embryonic development. In normal breast tissue FOXA1 acts throughout mammary development; whereas in breast carcinoma its expression promotes luminal phenotype and correlates with good prognosis. However, the role of FOXA2 has not been previously studied in breast cancer. Our purpose was to analyze the expression of FOXA2 in breast cancer cells, to explore its role in breast cancer stem cells, and to correlate its mRNA expression with clinicopathological features and outcome in a series of patients diagnosed with breast carcinoma. We analyzed FOXA2 mRNA expression in a retrospective cohort of 230 breast cancer patients and in cell lines. We also knocked down FOXA2 mRNA expression by siRNA to determine the impact on cell proliferation and mammospheres formation using a cancer stem cells culture assay. In vitro studies demonstrated higher FOXA2 mRNA expression in Triple-Negative/Basal-like cells. Further, when it was knocked down, cells decreased proliferation and its capability of forming mammospheres. Similarly, FOXA2 mRNA expression was detected in 10% (23/230) of the tumors, especially in Triple-Negative/Basal-like phenotype (p < 0.001, Fisher's test). Patients whose tumors expressed FOXA2 had increased relapses (59 vs. 79%, p = 0.024, log-rank test) that revealed an independent prognostic value (HR = 3.29, C.I.95% = 1.45-7.45, p = 0.004, Cox regression). Our results suggest that FOXA2 promotes cell proliferation, maintains cancer stem cells, favors the development of Triple-Negative/Basal-like tumors, and is associated with increase relapses. PMID:26298189

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

    PubMed Central

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

    2014-01-01

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

  14. Altered prefrontal cortical MARCKS and PPP1R9A mRNA expression in schizophrenia and bipolar disorder

    PubMed Central

    Konopaske, Glenn T.; Subburaju, Sivan; Coyle, Joseph T.; Benes, Francine M.

    2015-01-01

    Background We previously observed dendritic spine loss in the dorsolateral prefrontal cortex (DLPFC) from schizophrenia and bipolar disorder subjects. In the current study, we sought to determine if the mRNA expression of genes known to regulate the actin cytoskeleton and spines correlated with spine loss. Methods Five candidate genes were identified using previously obtained microarray data from the DLPFC from schizophrenia and control subjects. The relative mRNA expression of the genes linked to dendritic spine growth and function, i.e. IGF1R, MARCKS, PPP1R9A, PTPRF, and ARHGEF2, were assessed using quantitative real-time PCR (qRT-PCR) in the DLPFC from a second cohort including schizophrenia, bipolar disorder, and control subjects. Functional pathway analysis was conducted to determine which actin cytoskeleton-regulatory pathways the genes of interest interact with. Results MARCKS mRNA expression was increased in both schizophrenia and bipolar disorder subjects. PPP1R9A mRNA expression was increased in bipolar disorder subjects. For IGF1R, mRNA expression did not differ significantly among groups; however, it did show a significant, negative correlation with dendrite length. MARCKS and PPP1R9A mRNA expression did not correlate with spine loss, but interact with NMDA receptor signaling pathways that regulate the actin cytoskeleton and spines. Conclusions MARCKS and PPP1R9A might contribute to spine loss in schizophrenia and bipolar disorder through their interactions, possibly indirect ones, with NMDA signaling pathways that regulate spine structure and function. PMID:25757715

  15. Dynamics of c-fos and ICER mRNA expression in rat forebrain following lithium chloride injection.

    PubMed

    Spencer, C M; Houpt, T A

    2001-09-30

    Lithium is commonly used as a treatment for affective disorders in humans and as a toxin to produce conditioned taste aversions in rats. LiCl administration in rats has been correlated with activation of c-fos and cAMP-mediated gene transcription in many brain regions; however, little is known about the timing or duration of gene activation. We hypothesized that c-fos gene transcription is rapidly stimulated by LiCl, followed later by the expression of the inducible cAMP early repressor (ICER) transcription factor, a negative modulator of cAMP-mediated gene transcription. By in situ hybridization, we analyzed the timecourse of c-fos and ICER mRNA expression in the central nucleus of the amygdala (CeA), the paraventricular nucleus of the hypothalamus (PVN) and the supraoptic nucleus (SON) at seven time points (0, 0.3, 1, 3, 6, 9 and 12 h) after intraperitoneal LiCl injection (0.15 M, 12 ml/kg, 76 mg/kg). Expression of c-fos mRNA peaked between 20 min and 1 h and returned to baseline by 3 h in the CeA, PVN and SON. ICER mRNA was detected in these regions at 20 min, peaked at 1-3 h and returned to nearly baseline 9 h following LiCl injection. The time lag between c-fos mRNA expression and ICER mRNA expression within the same regions is consistent with ICER terminating c-fos gene transcription. However, no refractory period was detected for restimulation of c-fos transcription by a second injection of LiCl during the period of peak ICER mRNA expression, suggesting the involvement of other transcriptional modulators. PMID:11589989

  16. KIF14 and E2F3 mRNA expression in human retinoblastoma and its phenotype association

    PubMed Central

    Mitra, Moutushy; Mallikarjuna, Kandalam; Pranav, Oberoi; Srinivasan, Ramalingam; Nagpal, Amit; Venkatesan, Perumal; Kumaramanickavel, Govindasamy

    2009-01-01

    Purpose We quantified mRNA expression of candidate genes for proliferation (KIF14 and E2F3) in a large retinoblastoma tumor cohort and associated with disease phenotype. Methods KIF14 and E2F3 mRNA expression was quantified by real time PCR in 57 retinoblastoma (RB) tumors, 3 RB cell lines, and control samples that included 4 each fetal, age-matched, adult retinas. Immunohistochemistry was done to confirm KIF14 and E2F3 protein expression in tumor cells. The mRNA expression levels were correlated with disease phenotypes including the significance of chemotherapy on tumors. Results There was statistically significant overexpression of KIF14 and E2F3 mRNA in tumors compared with control retinas (p<0.0001). Further, E2F3 also showed a significant overexpression compared to RB cell lines (p=0.01). Immunohistochemistry confirmed KIF14 and E2F3 protein overexpression in tumor cells. KIF14 had significant mRNA overexpression with older age (p=0.01) in presenting patients and in unilateral RB patients (p=0.04). Chemotherapy-treated tumors showed a significant decrease in KIF14 and E2F3 expression compared to untreated tumors (p<0.01 and 0.001, respectively). Conclusions This report confirms significant mRNA overexpression of KIF14 and E2F3 together in a large cohort of RB tumors. The decreased expression in chemotherapy treated cases needs further validation in a large chemotherapy-treated cohort. PMID:19190782

  17. Integrated Expression Profiles of mRNA and miRNA in Polarized Primary Murine Microglia

    PubMed Central

    Freilich, Robert W.; Woodbury, Maya E.; Ikezu, Tsuneya

    2013-01-01

    Neuroinflammation contributes to many neurologic disorders including Alzheimer’s disease, multiple sclerosis, and stroke. Microglia is brain resident myeloid cells and have emerged as a key driver of the neuroinflammatory responses. MicroRNAs (miRNAs) provide a novel layer of gene regulation and play a critical role in regulating the inflammatory response of peripheral macrophages. However, little is known about the miRNA in inflammatory activation of microglia. To elucidate the role that miRNAs have on microglial phenotypes under classical (M1) or alternative (M2) activation under lipopolysaccharide (‘M1’-skewing) and interleukin-4 (‘M2a’-skewing) stimulation conditions, we performed microarray expression profiling and bioinformatics analysis of both mRNA and miRNA using primary cultured murine microglia. miR-689, miR-124, and miR-155 were the most strongly associated miRNAs predicted to mediate pro-inflammatory pathways and M1-like activation phenotype. miR-155, the most strongly up-regulated miRNA, regulates the signal transducer and activator of transcription 3 signaling pathway enabling the late phase response to M1-skewing stimulation. Reduced expression in miR-689 and miR-124 are associated with dis-inhibition of many canonical inflammatory pathways. miR-124, miR-711, miR-145 are the strongly associated miRNAs predicted to mediate anti-inflammatory pathways and M2-like activation phenotype. Reductions in miR-711 and miR-124 may regulate inflammatory signaling pathways and peroxisome proliferator-activated receptor-gamma pathway. miR-145 potentially regulate peripheral monocyte/macrophage differentiation and faciliate the M2-skewing phenotype. Overall, through combined miRNA and mRNA expression profiling and bioinformatics analysis we have identified six miRNAs and their putative roles in M1 and M2-skewing of microglial activation through different signaling pathways. PMID:24244499

  18. Effect of Danshao Huaxian capsule on Gremlin and bone morphogenetic protein-7 expression in hepatic fibrosis in rats

    PubMed Central

    Zhao, Xue-Ke; Cheng, Ming-Liang; Wu, Rong-Min; Yao, Yu-Mei; Mu, Mao; Zhu, Juan-Juan; Zhang, Bao-Fang; Zhou, Ming-Yu

    2014-01-01

    AIM: To observe the effect of Danshao Huaxian capsule (DHC) on the expression of Gremlin and bone morphogenetic protein-7 (BMP-7) in the liver of hepatic fibrosis rats. METHODS: A total of 75 male Wistar rats were randomly divided into a normal control group (A), a CCl4-induced hepatic fibrosis model group (B), a natural recovery group (C), a low-dose DHC-treated group (D), and a high-dose DHC-treated group (E), with 15 rats in each group. Liver fibrosis was induced by subcutaneous injections of carbon tetrachloride (CCl4) and a high-lipid/low-protein diet for 8 wk, except for the rats in group A. Then, the rats in the two DHC-treated groups were administered 0.5 and 1.0 g/kg DHC by gastrogavage once per day for 8 successive weeks, respectively. By the end of the experiment, the level of transforming growth factor β1 (TGF-β1) in the liver homogenate was determined by an enzyme-linked immunosorbent assay. The mRNA and protein expression of Gremlin and BMP-7 in the liver tissue was determined by reverse-transcription polymerase chain reaction, an immunohistochemical assay, and Western blot analysis. RESULTS: Compared with group A, the level of TGF-β1 and the mRNA and protein expression of Gremlin were significantly higher in group B (TGF-β1: 736.30 ± 24.40 μg/g vs 284.20 ± 18.32 μg/g, P < 0.01; mRNA of Gremlin: 80.40 ± 5.46 vs 49.83 ± 4.20, P < 0.01; positive protein expression rate of Gremlin: 38.46% ± 1.70% vs 3.83% ± 0.88%, P < 0.01; relative protein expression of Gremlin: 2.81 ± 0.24 vs 0.24 ± 0.06, P < 0.01), and the mRNA and protein expression of BMP-7 was significantly lower in group B (mRNA: 54.00 ± 4.34 vs 93.99 ± 7.03, P < 0.01; positive protein expression rate: 28.97% ± 3.14% vs 58.29% ± 6.02, P < 0.01; relative protein expression: 0.48 ± 0.31 vs 1.05 ± 0.12, P < 0.01). Compared with groups B and C, the degree of hepatic fibrosis was significantly improved, and the level of TGF-β1 and the mRNA and protein expression of Gremlin were

  19. Monitoring Cyp2b10 mRNA expression at cessation of 2-year carcinogenesis bioassay in mouse liver provides evidence for a carcinogenic mechanism devoid of human relevance: The dalcetrapib experience

    SciTech Connect

    Hoflack, J-C.; Mueller, L. Fowler, S.; Braendli-Baiocco, A.; Flint, N.; Kuhlmann, O.; Singer, T.; Roth, A.

    2012-03-15

    Introduction: Dalcetrapib is a cholesteryl ester transfer protein (CETP) modulator in clinical assessment for cardiovascular outcome benefits. In compliance with regulatory requirements, dalcetrapib was evaluated in rodent 2-year carcinogenesis bioassays. In the mouse bioassay, male mice demonstrated increased liver weight and statistically increased incidences of hepatocellular adenoma/carcinoma. Hepatic cytochrome p450 (Cyp) 2b10 mRNA induction and increased Cyp2b10 enzyme activity signify activation of hepatic nuclear receptor constitutive androstane receptor (CAR), a widely established promoter of rodent-specific hepatic tumors. We therefore monitored hepatic Cyp2b10 mRNA and its enzyme activity in a subset of dalcetrapib-treated male mice from the bioassay. Methods: Liver samples were obtained from ∼ 1/3 of male mice from each dose group including vehicle-controls (mean and earliest study day of death 678 and 459 respectively). Quantitative real time PCR (qRT-PCR) was performed to determine Cyp2b10 mRNA expression and Cyp1a-, Cyp2b10- and Cyp3a-selective activities were monitored. Results: Cyp2b10 mRNA was strongly induced by dalcetrapib with an expected wide inter-individual variation (5–1421-fold). Group average fold-induction versus vehicle-controls showed a dose-related increase from 48-fold (250 mg/kg/day) to 160-fold (750 mg/kg/day), which declined slightly at 2000 mg/kg/day (97-fold). Cyp enzyme activities showed approximate doubling of total Cyp P450 content per milligram protein and a 9-fold increase in Cyp2b10-selective pentoxyresorufin O-dealkylase activity (750 mg/kg/day). Discussion: These data from hepatic Cyp2b10 monitoring are strongly suggestive of CAR activation by dalcetrapib, a mechanism devoid of relevance towards hepatocarcinogenesis in humans; results show feasibility of Cyp2b10 as a surrogate marker for this mechanism at cessation of a carcinogenesis bioassay. -- Highlights: ► Liver tumors were induced in male mice by dalcetrapib

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

    PubMed Central

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

    2014-01-01

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

  1. mRNA expression of DOK1-6 in human breast cancer

    PubMed Central

    Ghanem, Tamara; Bracken, James; Kasem, Abdul; Jiang, Wen G; Mokbel, Kefah

    2014-01-01

    AIM: To examine the expression of downstream of tyrosine kinase (DOK)1-6 genes in normal and breast cancer tissue and correlated this with several clinico-pathological and prognostic factors. METHODS: DOK1-6 mRNA extraction and reverse transcription were performed on fresh frozen breast cancer tissue samples (n = 112) and normal background breast tissue (n = 31). Tissues were collected between 1991 and 1996 at two centres and all patients underwent mastectomy and ipsilateral axillary node dissection. All tissues were randomly numbered and the details were only made known after all analyses were completed. Transcript levels of expression were determined using real-time polymerase chain reaction and analyzed against TNM stage, tumour grade and clinical outcome over a 10-year follow-up period. RESULTS: DOK-2 and DOK-6 expression decreased with increasing TNM stage. DOK-6 expression decreased with increasing Nottingham Prognostic Index (NPI) [NPI-1 vs NPI-3 (mean copy number 15.4 vs 0.22, 95%CI: 2.7-27.6, P = 0.018) and NPI-2 vs NPI-3 (mean copy number 7.6 vs 0.22, 95%CI: 0.1-14.6, P = 0.048)]. After a median follow up period of 10 years, higher levels of DOK-2 expression were found among patients who remained disease-free compared to those who developed local or distant recurrence (mean copy number 3.94 vs 0.0000096, 95%CI: 1.0-6.85, P = 0.0091), and distant recurrence (mean copy number 3.94 vs 0.0025, 95%CI: 1.0-6.84, P = 0.0092). Patients who remained disease-free had higher levels of DOK-6 expression compared to those who died from breast cancer. CONCLUSION: Decreasing expression levels of DOK-2 and DOK-6 with increased breast tumour progression supports the notion that DOK-2 and DOK-6 behave as tumour suppressors in human breast cancer. PMID:24829863

  2. Selenium Deficiency Influences the mRNA Expression of Selenoproteins and Cytokines in Chicken Erythrocytes.

    PubMed

    Luan, Yilin; Zhao, Jinxin; Yao, Haidong; Zhao, Xia; Fan, Ruifeng; Zhao, Wenchao; Zhang, Ziwei; Xu, Shiwen

    2016-06-01

    Selenium (Se) deficiency induces hemolysis in chickens, but the molecular mechanism for this effect remains unclear. Se primarily elicits its function through the activity of selenoproteins, which contain the unique amino acid selenocysteine (Sec). In this study, we aimed to investigate the effect of Se deficiency on the expression of 24 selenoproteins and 10 cytokines. One hundred eighty chickens were randomly divided into 2 groups (90 chickens per group). During the entire experimental period, chickens were allowed ad libitum consumption of feed and water. The chickens were fed either a Se-deficient diet (0.008 mg Se/kg; produced in the Se-deficient area of Heilongjiang, China) or a Se-supplemented diet (as sodium selenite) at 0.2 mg/kg for 35 days. At the 35th day, the messenger RNA (mRNA) levels of 24 selenoproteins and 10 cytokines were examined in erythrocytes of 5 chickens per group, and the correlation was analyzed. The results showed that the expression of 24 selenoproteins and 7 cytokines (IL-2, IL-4, IL-8, IL-10, IL-12β, TGF-β4, and IFN-γ) decreased (P < 0.05), and the expression of 3 cytokines (IL-1γ, IL-6 and IL-7) was higher in the Se-deficient group. In both groups, glutathione peroxidase (GPX), thioredoxin 1 (Txnrd1), selenoprotein P1 (SELP), and selenoprotein synthetase (SPS2) were highly expressed compared to the other selenoproteins in chicken erythrocytes (P < 0.05). These data suggest that GPXs, Txnrd1, SELP, and SPS2 possibly play a more important role than the other selenoproteins. The increase of pro-inflammatory cytokines (IL-1γ, IL-6, and IL-7) suggested that the immune system of chickens was damaged by the Se deficiency. Correlation analysis suggested that although the expression of 24 selenoproteins and 7 cytokines decreased and that of 3 cytokines increased, there was a close correlation between their expression levels and a Se diet. These results suggested that Se deficiency influenced the expressions of 24 selenoproteins

  3. Prognostic values of four Notch receptor mRNA expression in gastric cancer

    PubMed Central

    Wu, Xiaoyu; Liu, Wentao; Tang, Ding; Xiao, Haijuan; Wu, Zhenfeng; Chen, Che; Yao, Xuequan; Liu, Fukun; Li, Gang

    2016-01-01

    Notch ligands and receptors are frequently deregulated in several human malignancies including gastric cancer. The activation of Notch signaling has been reported to contribute to gastric carcinogenesis and progression. However, the prognostic roles of individual Notch receptors in gastric cancer patients remain elusive. In the current study, we accessed the prognostic roles of four Notch receptors, Notch 1–4, in gastric cancer patients through “The Kaplan-Meier plotter” (KM plotter) database, in which updated gene expression data and survival information include a total of 876 gastric cancer patients. All four Notch receptors’ high mRNA expression was found to be correlated to worsen overall survival (OS) for all gastric cancer patients followed for 20 years. We further accessed the prognostic roles of individual Notch receptors in different clinicopathological features using Lauren classification, pathological grades, clinical grades, HER2 status and different choices of treatments of gastric cancer patients. These results indicate that there are critical prognostic values of the four Notch receptors in gastric cancer. This information will be useful for better understanding of the heterogeneity and complexity in the molecular biology of gastric cancer and to develop tools to more accurately predict their prognosis. PMID:27363496

  4. In vivo and in vitro CYP1B mRNA expression in channel catfish.

    PubMed

    Willett, Kristine L; Ganesan, Shobana; Patel, Monali; Metzger, Christine; Quiniou, Sylvie; Waldbieser, Geoff; Scheffler, Brian

    2006-07-01

    Our goal was to study the induction of CYP1B mRNA expression in channel catfish (Ictalurus punctatus). CYP1B belongs to the cytochrome P450 superfamily of genes, is involved in the oxidation of endogenous and exogenous compounds, and could potentially be a useful biomarker in fish for exposure to AhR ligands. The full-length catfish CYP1B cDNA is 2417 nt to the polyA tail and encodes a putative protein of 536 amino acids. It has 67% amino acid similarity to carp and zebrafish CYP1B and 68% similarity to carp CYP1B2. Male channel catfish were collected from three Mississippi Delta sites: Lake Roebuck, Itta Bena; Bee Lake, Thornton; and Sunflower River, Indianola. Total RNA was isolated from wild-caught catfish gill, blood, gonad and liver tissues. Quantitative real-time reverse transcriptase PCR was used to determine relative induction of CYP1B in wild catfish compared to laboratory control and BaP-exposed catfish (20mg/kg i.p. after 4 days). BaP exposure significantly induced CYP1B message in blood, gonad, and liver of laboratory catfish. In these same tissues of wild catfish from sites with relatively low sediment contaminants, CYP1B message was not statistically increased relative to laboratory control catfish. CYP1B transcript abundance was higher in gills compared to other tissues in both laboratory and wild catfish. When primary cultured gill cells were treated with increasing concentrations of BaP, TCDD, and PCBs 77, 126 and 169, CYP1B mRNA was induced more than 10-fold while PCB153 and 4,4'DDT did not cause significant CYP1B induction. Our results suggest that catfish CYP1B is induced by the classic AhR ligands. PMID:16697458

  5. Hepatic Expression of Detoxification Enzymes Is Decreased in Human Obstructive Cholestasis Due to Gallstone Biliary Obstruction

    PubMed Central

    Chai, Jin; Feng, Xinchan; Zhang, Liangjun; Chen, Sheng; Cheng, Ying; He, Xiaochong; Yang, Yingxue; He, Yu; Wang, Huaizhi; Wang, Rongquan; Chen, Wensheng

    2015-01-01

    Background & Aims Levels of bile acid metabolic enzymes and membrane transporters have been reported to change in cholestasis. These alterations (e.g. CYP7A1 repression and MRP4 induction) are thought to be adaptive responses that attenuate cholestatic liver injury. However, the molecular mechanisms of these adaptive responses in human obstructive cholestasis due to gallstone biliary obstruction remain unclear. Methods We collected liver samples from cholestatic patients with biliary obstruction due to gallstones and from control patients without liver disease (n = 22 per group). The expression levels of bile acid synthetic and detoxification enzymes, membrane transporters, and the related nuclear receptors and transcriptional factors were measured. Results The levels of bile acid synthetic enzymes, CYP7B1 and CYP8B1, and the detoxification enzyme CYP2B6 were increased in cholestatic livers by 2.4-fold, 2.8-fold, and 1.9-fold, respectively (p<0.05). Conversely, the expression levels of liver detoxification enzymes, UGT2B4/7, SULT2A1, GSTA1-4, and GSTM1-4, were reduced by approximately 50% (p<0.05) in human obstructive cholestasis. The levels of membrane transporters, OSTβ and OCT1, were increased 10.4-fold and 1.8-fold, respectively, (p<0.05), whereas those of OSTα, ABCG2 and ABCG8 were all decreased by approximately 40%, (p<0.05) in human cholestatic livers. Hepatic nuclear receptors, VDR, HNF4α, RXRα and RARα, were induced (approximately 2.0-fold, (p<0.05) whereas FXR levels were markedly reduced to 44% of control, (p<0.05) in human obstructive cholestasis. There was a significantly positive correlation between the reduction in FXR mRNA and UGT2B4/7, SULT2A1, GSTA1, ABCG2/8 mRNA levels in livers of obstructive cholestatic patients (p<0.05). Conclusions The levels of hepatic detoxification enzymes were significantly decreased in human obstructive cholestasis, and these decreases were positively associated with a marked reduction of FXR levels. These findings

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

    SciTech Connect

    Sun, Lixin; Zhang, Shuang; Jiang, Zhenzhou; Huang, Xin; Wang, Tao; Huang, Xiao; Li, Han; Zhang, Luyong

    2011-12-09

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

  7. Assessment of potential biomarkers, metallothionein and vitellogenin mRNA expressions in various chemically exposed benthic Chironomus riparius larvae

    NASA Astrophysics Data System (ADS)

    Park, Kiyun; Kwak, Inn-Sil

    2012-12-01

    The objective of this study was conducted to identify the possibility of using Chironomus metallothionein (MT) and vitellogenin (VTG) as biomarkers of stress caused by endocrinedisrupting chemicals (EDCs), heavy metals, herbicides and veterinary antibiotics. We characterized the MT and VTG cDNA in Chironomus riparius and evaluated their mRNA expression profiles following exposure to different environmental pollutants. The gene expression analysis showed that the MT mRNA levels increased significantly after long-term exposure to cadmium (Cd), copper (Cu), Lead (Pb), di(2-ethylhexyl) phthalate (DEHP), and 2,4-dichlorophenoxyacetic acid (2,4-D). Moreover, the VTG mRNA expression increased significantly in C. riparius larvae exposed to BPA, NP, DEHP, Cd, 2,4-D and fenbendazole. Evaluation of the long-term effects of environmental pollutants revealed up regulation of Chironomus MT mRNA in response to DEHP exposure among EDCs, and the level of the VTG mRNA was increased significantly following treatment with Cd and herbicide 2,4-D at all concentrations in a dose-dependent manner. These results indicate that VTG could be used as a potential biomarker of herbicide and Cd as well as EDCs, while MT was a potential biomarker of heavy metals such as Cd, Cu, and Pb in aquatic environments.

  8. Senegenin Attenuates Hepatic Ischemia-Reperfusion Induced Cognitive Dysfunction by Increasing Hippocampal NR2B Expression in Rats

    PubMed Central

    Gu, Xiaoping; Zheng, Yaguo; Sun, Yu-e; Liang, Ying; Bo, Jinhua; Ma, Zhengliang

    2012-01-01

    Background The root of Polygala tenuifolia, a traditional Chinese medicine, has been used to improve memory and intelligence, while the underlying mechanisms remain largely unknown. In this study, we investigated the protective effects of senegenin, an component of Polygala tenuifolia root extracts, on cognitive dysfunction induced by hepatic ischemia-reperfusion. Methodology/Principal Findings Initially, we constructed a rat model of hepatic ischemia-reperfusion (HIR) and found that the memory retention ability of rats in the step-down and Y maze test was impaired after HIR, paralleled by a decrease of N-methyl-D-aspartate (NMDA) receptor NR2B subunit mRNA and protein expressions in hippocampus. Furthermore, we found that administration of senegenin by gavage attenuated HIR-induced cognitive impairment in a dose and time dependent manner, and its mechanisms might partly due to the increasing expression of NR2B in rat hippocampus. Conclusions/Significance Cognitive dysfunction induced by HIR is associated with reduction of NR2B expression. Senegenin plays a neuroprotective role in HIR via increasing NR2B expression in rat hippocampus. These findings suggest that senegenin might be a potential agent for prevention and treatment of postoperative cognitive dysfunction (POCD) or other neurodegenerative diseases. PMID:23029109

  9. BayMiR: inferring evidence for endogenous miRNA-induced gene repression from mRNA expression profiles

    PubMed Central

    2013-01-01

    Background Popular miRNA target prediction techniques use sequence features to determine the functional miRNA target sites. These techniques commonly ignore the cellular conditions in which miRNAs interact with their targets in vivo. Gene expression data are rich resources that can complement sequence features to take into account the context dependency of miRNAs. Results We introduce BayMiR, a new computational method, that predicts the functionality of potential miRNA target sites using the activity level of the miRNAs inferred from genome-wide mRNA expression profiles. We also found that mRNA expression variation can be used as another predictor of functional miRNA targets. We benchmarked BayMiR, the expression variation, Cometa, and the TargetScan “context scores” on two tasks: predicting independently validated miRNA targets and predicting the decrease in mRNA abundance in miRNA overexpression assays. BayMiR performed better than all other methods in both benchmarks and, surprisingly, the variation index performed better than Cometa and some individual determinants of the TargetScan context scores. Furthermore, BayMiR predicted miRNA target sets are more consistently annotated with GO and KEGG terms than similar sized random subsets of genes with conserved miRNA seed regions. BayMiR gives higher scores to target sites residing near the poly(A) tail which strongly favors mRNA degradation using poly(A) shortening. Our work also suggests that modeling multiplicative interactions among miRNAs is important to predict endogenous mRNA targets. Conclusions We develop a new computational method for predicting the target mRNAs of miRNAs. BayMiR applies a large number of mRNA expression profiles and successfully identifies the mRNA targets and miRNA activities without using miRNA expression data. The BayMiR package is publicly available and can be readily applied to any mRNA expression data sets. PMID:24001276

  10. Uncoupling protein-2 mRNA expression in mice subjected to intermittent hypoxia*

    PubMed Central

    Vieira, Luciana Rodrigues; Martinez, Denis; Forgiarini, Luiz Felipe; da Rosa, Darlan Pase; de Muñoz, Gustavo Alfredo Ochs; Fagundes, Micheli; Martins, Emerson Ferreira; Montanari, Carolina Caruccio; Fiori, Cintia Zappe

    2015-01-01

    Objective: To investigate the effect of intermittent hypoxia-a model of obstructive sleep apnea (OSA)-on pancreatic expression of uncoupling protein-2 (UCP2), as well as on glycemic and lipid profiles, in C57BL mice. Methods: For 8 h/day over a 35-day period, male C57BL mice were exposed to intermittent hypoxia (hypoxia group) or to a sham procedure (normoxia group). The intermittent hypoxia condition involved exposing mice to an atmosphere of 92% N and 8% CO2 for 30 s, progressively reducing the fraction of inspired oxygen to 8 ± 1%, after which they were exposed to room air for 30 s and the cycle was repeated (480 cycles over the 8-h experimental period). Pancreases were dissected to isolate the islets. Real-time PCR was performed with TaqMan assays. Results: Expression of UCP2 mRNA in pancreatic islets was 20% higher in the normoxia group than in the hypoxia group (p = 0.11). Fasting serum insulin was higher in the hypoxia group than in the normoxia group (p = 0.01). The homeostasis model assessment of insulin resistance indicated that, in comparison with the control mice, the mice exposed to intermittent hypoxia showed 15% lower insulin resistance (p = 0.09) and 21% higher pancreatic β-cell function (p = 0.01). Immunohistochemical staining of the islets showed no significant differences between the two groups in terms of the area or intensity of α- and β-cell staining for insulin and glucagon. Conclusions: To our knowledge, this is the first report of the effect of intermittent hypoxia on UCP2 expression. Our findings suggest that UCP2 regulates insulin production in OSA. Further study of the role that UCP2 plays in the glycemic control of OSA patients is warranted. PMID:25909153

  11. 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

  12. Mechanical stretch of human uterine smooth muscle cells increases IL-8 mRNA expression and peptide synthesis.

    PubMed

    Loudon, J A Z; Sooranna, S R; Bennett, P R; Johnson, M R

    2004-12-01

    Labour is associated with increased synthesis of interleukin-8 (IL-8) by the fetal membranes and myometrium, which leads to an inflammatory infiltrate. Stretch has been shown to increase the expression of contraction-associated proteins in animal models of labour and in human myocytes in vitro. In this study, we tested the hypothesis that mechanical stretch of human myometrial cells increases IL-8 messenger ribonucleic acid (mRNA) expression. We isolated myocytes from non-pregnant women undergoing hysterectomy and pregnant women undergoing Caesarean section before and after the onset of labour. Myocytes in culture were subjected to stretch of varying intensity (6-16%) and duration (1 or 6 h) using the Flexercell system. IL-8 mRNA expression was lowest in myocytes from pregnant women not in labour, intermediate in those from non-pregnant women and greatest in those from pregnant women in labour. Stretch increased IL-8 mRNA expression independent of reproductive state. The stretch-induced increase in IL-8 mRNA expression was associated with higher IL-8 levels in the culture supernatant and enhanced promoter activity. These data suggest that stretch contributes to the increase in myometrial IL-8 synthesis associated with the onset of labour in humans. PMID:15489245

  13. Expressions and clinical significances of CD133 protein and CD133 mRNA in primary lesion of gastric adenocacinoma

    PubMed Central

    2010-01-01

    Background To study on expressions and clinical significances of CD133 protein and CD133 mRNA in primary lesion of gastric adenocarcinoma (GC). Methods Expressions of CD133 protein by immunostaining (99 cases) and CD133 mRNA by semi-quantitative RT-PCR (31 cases) were detected in primary lesion and in noncancerous gastric mucosa tissue (NCGT). Correlations of CD133 protein expression with clinicopathological parameters and post-operative survival were analyzed. Relations of CD133 mRNA level with Ki-67 labeling index (LI), and lymphatic metastasis were assessed too. Results Brown particles indicating CD133 protein positivity occurred in some parts of tumor cells and epithelium. Expressive percentage of CD133 protein positivity was significantly higher in subgroups with >5 cm diameter (P = 0.041), later TNM stage (P = 0.044), severer lymph node metastasis (P = 0.017), occurrences of lymphatic invasion (P = 0.000) and vascular invasion (P = 0.000) respectively. Severer invasion depth (P = 0.011), lymph node metastasis occurrence (P = 0.043) and later TNM stage (P = 0.049) were the independent risk factors for CD133 protein expression. Average brightness scale value (BSV) of CD133 mRNA was significantly higher in subgroups with >5 cm diameter (P = 0.041), lymph node metastasis occurrence (P = 0.004) and in lower Ki-67 LI (P = 0.02). Relative analysis revealed that BSV of CD133 mRNA related positively to metastatic lymphatic nodes ratio (P = 0.008) and metastatic lymph node number (P = 0.009), but negatively to Ki-67 LI (P = 0.009). Survival of positive subgroup of CD 133 protein was significantly poorer (P = 0.047). Lymph node metastasis occurrence (P = 0.042), later TNM stage (P = 0.046) and CD 133 protein positive expression (P = 0.046) were respectively the independent risk factors to survival. Conclusion Higher expressive level of CD133 mRNA is associated to lower Ki-67 LI and severer lymphatic metastasis. Therefore, the expressive level of CD133 mRNA can play an

  14. 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

  15. 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. PMID:27407180

  16. Hepatitis C virus genotype and HIV coinfection affect cytokine mRNA levels in unstimulated PBMC but do not shift the T1/T2 balance.

    PubMed

    Lee, Silvia; Watson, Mark W; Clark, Ben; Flexman, James P; Cheng, Wendy; French, Martyn A H; Price, Patricia

    2006-08-01

    Rapid progression of hepatitis C virus (HCV) disease in patients with HIV/HCV may reflect different cytokine responses and be influenced by HCV genotype. This is addressed by a study of patients with HIV/HCV coinfection and infection with HCV genotype 2 or 3 (2/3). They are compared with coinfected patients infected with genotype 1 and HCV monoinfected patients matched for HCV genotype. IFN-gamma, IL-10, IL-4 and IL-4delta2 mRNA were quantified by real-time PCR in unstimulated PBMC and after in vitro stimulation with HCV core or nonstructural 3/4A antigen. In unstimulated PBMC, levels of IFN-gamma and IL-4 mRNA were lowest in HIV/HCV genotype 1 patients, intermediate in HIV/HCV genotype 2/3 patients and highest in HCV genotype 2/3 patients. Neither HCV genotype nor HIV affected levels of IL-10 mRNA in unstimulated PBMC or IFN-gamma, IL-4 and IL-10 mRNA in PBMC stimulated with HCV antigens. Levels of IL-4 and IL-4delta2 mRNA correlated in mitogen-stimulated PBMC from all patient groups but both were low in HIV/HCV genotype 1 patients. Serum soluble CD30 levels (a putative marker of a T2 cytokine environment) did not differ between patient groups. The data do not suggest a shift in the T1/T2 balance driven by HIV coinfection or HCV genotype but either may affect IL-4 bioavailability. PMID:16834574

  17. Associations of MMP-2, BAX, and Bcl-2 mRNA and Protein Expressions with Development of Atrial Fibrillation.

    PubMed

    Diao, Shu-Ling; Xu, Hui-Pu; Zhang, Bei; Ma, Bao-Xin; Liu, Xian-Liang

    2016-01-01

    BACKGROUND To examine changes of mRNA and protein expressions of MMP-2, Bcl-2, and BAX in atrial fibrillation (AF) patients, and investigate the correlations among these 3 biomarkers. MATERIAL AND METHODS Rheumatic heart disease patients (n=158) undergoing cardiac surgical procedures for mitral valve repair or replacement were included as the AF group (n=123), containing paroxysmal AF (n=42), persistent AF (n=36), and permanent AF (n=45). Rheumatic heart disease patients with sinus rhythm (SR) (n=35) were enrolled as the SR group (control group). Immunohistochemistry, Western blot, and real-time polymerase chain reaction (PCR) were applied to detect the protein and mRNA expression levels of MMP-2, Bcl-2, and BAX. Apoptosis was observed with light and electron microscopes and detected by TdT-mediated dUTP nick-end labeling (TUNEL). RESULTS Compared with the SR group, the left atrial diameters (LADs), protein and mRNA expression levels of MMP-2 and BAX, apoptotic index (AI), and Bcl-2/BAX ratio were evidently increased in the 3 AF groups, but protein and mRNA expression levels of Bcl-2 decreased in the AF groups (all P<0.05). Correlation analysis found that MMP-2 protein expression levels was positively correlated with BAX expression, but negatively correlated with Bcl-2 expression levels. CONCLUSIONS Our study results suggest that elevated MMP-2 expression and disturbance balance of Bcl-2/BAX expressions may be associated with the development and maintenance of AF. MMP-2 may be involved in the development of AF through promoting BAX expressions and inhibiting Bcl-2. PMID:27141955

  18. Associations of MMP-2, BAX, and Bcl-2 mRNA and Protein Expressions with Development of Atrial Fibrillation

    PubMed Central

    Diao, Shu-Ling; Xu, Hui-Pu; Zhang, Bei; Ma, Bao-Xin; Liu, Xian-Liang

    2016-01-01

    Background To examine changes of mRNA and protein expressions of MMP-2, Bcl-2, and BAX in atrial fibrillation (AF) patients, and investigate the correlations among these 3 biomarkers. Material/Methods Rheumatic heart disease patients (n=158) undergoing cardiac surgical procedures for mitral valve repair or replacement were included as the AF group (n=123), containing paroxysmal AF (n=42), persistent AF (n=36), and permanent AF (n=45). Rheumatic heart disease patients with sinus rhythm (SR) (n=35) were enrolled as the SR group (control group). Immunohistochemistry, Western blot, and real-time polymerase chain reaction (PCR) were applied to detect the protein and mRNA expression levels of MMP-2, Bcl-2, and BAX. Apoptosis was observed with light and electron microscopes and detected by TdT-mediated dUTP nick-end labeling (TUNEL). Results Compared with the SR group, the left atrial diameters (LADs), protein and mRNA expression levels of MMP-2 and BAX, apoptotic index (AI), and Bcl-2/BAX ratio were evidently increased in the 3 AF groups, but protein and mRNA expression levels of Bcl-2 decreased in the AF groups (all P<0.05). Correlation analysis found that MMP-2 protein expression levels was positively correlated with BAX expression, but negatively correlated with Bcl-2 expression levels. Conclusions Our study results suggest that elevated MMP-2 expression and disturbance balance of Bcl-2/BAX expressions may be associated with the development and maintenance of AF. MMP-2 may be involved in the development of AF through promoting BAX expressions and inhibiting Bcl-2. PMID:27141955

  19. Clinical usefulness of WT1 mRNA expression in bone marrow detected by a new WT1 mRNA assay kit for monitoring acute myeloid leukemia: a comparison with expression of WT1 mRNA in peripheral blood.

    PubMed

    Kitamura, Kunio; Nishiyama, Takahiro; Ishiyama, Ken; Miyawaki, Shuichi; Miyazaki, Kanji; Suzuki, Kenshi; Masaie, Hiroaki; Okada, Masaya; Ogawa, Hiroyasu; Imai, Kiyotoshi; Kiyoi, Hitoshi; Naoe, Tomoki; Yokoyama, Yasuhisa; Chiba, Shigeru; Hata, Tomoko; Miyazaki, Yasushi; Hatta, Yoshihiro; Takeuchi, Jin; Nannya, Yasuhito; Kurokawa, Mineo; Ueda, Yasunori; Koga, Daisuke; Sugiyama, Haruo; Takaku, Fumimaro

    2016-01-01

    We have previously shown the clinical usefulness of Wilms' tumor 1 gene (WT1) mRNA expression in peripheral blood (PB) as a minimal residual disease (MRD) monitoring marker in 191 acute myeloid leukemia (AML) patients using the WT1 mRNA assay kit "Otsuka" (Otsuka Pharmaceutical Co., Ltd.; "former kit"). In contrast, the usefulness of WT1 mRNA expression in bone marrow (BM) has been investigated in only a limited number of subjects using former kit. Following that previous study, a next-generation kit, WT1 mRNA assay kit II "Otsuka" (Otsuka Pharmaceutical Co., Ltd.; "new kit") has been newly developed. In the present study, we aimed to evaluate the performance of the new kit and to investigate the clinical usefulness of WT1 mRNA expression in BM. The PB and BM were collected on the same day from 164 blood disease patients, including 118 AML patients. WT1 mRNA expression was determined using the new and former kits and the values obtained were compared. The performance of new kit was shown to be equivalent to that of former kit. As reported in PB, WT1 mRNA expression in BM was found to be a useful marker for monitoring disease status as well as for a diagnosis of early stage relapse in AML patients. PMID:26520650

  20. Hepatitis

    MedlinePlus

    ... has been associated with drinking contaminated water. Hepatitis Viruses Type Transmission Prognosis A Fecal-oral (stool to ... risk for severe disease. Others A variety of viruses can affect the liver Signs and Symptoms Hepatitis ...

  1. Expression of Toll-Like Receptors 2 and 4 and Related Cytokines in Patients with Hepatic Cystic and Alveolar Echinococcosis

    PubMed Central

    Tuxun, Tuerhongjiang; Ma, Hai-Zhang; Apaer, Shadike; Zhang, Heng; Aierken, Amina; Li, Yu-Peng; Lin, Ren-Yong; Zhao, Jin-Ming; Zhang, Jin-Hui; Wen, Hao

    2015-01-01

    Several studies have demonstrated the important role of Toll-like receptors in various parasitic infections. This study aims to explore expression of Toll-like receptors (TLRs) and related cytokines in patients with human cystic echinococcosis (CE) and alveolar echinococcosis (AE). 78 subjects including AE group (N = 28), CE group (N = 22), and healthy controls (HC, N = 28) were enrolled in this study. The mRNA expression levels of TLR2 and TLR4 in blood and hepatic tissue and plasma levels related cytokines were detected by using ELISA. Median levels of TLR2 mRNA in AE and CE groups were significantly elevated as compared with that in healthy control group. Median levels of TLR4 expression were increased in AE and CE. Plasma concentration levels of IL-5, IL-6, and IL-10 were slightly increased in AE and CE groups compared with those in HC group with no statistical differences (p > 0.05). The IL-23 concentration levels were significantly higher in AE and CE groups than that in HC subjects with statistical significance. The increased expression of TLR2 and IL-23 might play a potential role in modulating tissue infiltrative growth of the parasite and its persistence in the human host. PMID:26635448

  2. Peripheral Blood Mononuclear Cell Gene Expression Remains Broadly Altered Years after Successful Interferon-Based Hepatitis C Virus Treatment

    PubMed Central

    Waldron, Paul Ravi; Holodniy, Mark

    2015-01-01

    Background. Inflammatory gene expression in peripheral blood mononuclear cells (PBMCs) is altered in chronic Hepatitis C Virus (HCV) infection. Duration of changes after pegylated interferon- (peg-IFN-) based HCV treatment is unclear. Methods. PBMC mRNA expression of 184 inflammatory response genes was analyzed (nCounter GX Human Inflammation Kit, Nanostring) from peg-IFN treatment nonresponders (NR, n = 18), sustained virologic responders (SVR, n = 22), and spontaneous clearers (SC, n = 15). Logistic regression was used for comparison. Results. Median time from last treatment was 2 and 2.7 years in SVR and NR, respectively (p = NS). Mean mRNA counts were significantly different for 42 and 29 genes comparing SVR to SC patients and NR to SC, respectively, and no genes comparing SVR to NR. Differential expression of 24 genes was significantly different in both SVR and NR groups compared to SC. Among these 24 acute and chronic inflammatory cascade genes, significant upregulation was noted for proinflammatory transcription regulators Fos, CEBPB, and MyD88 in SVR and NR compared to SC. HDAC4 was significantly downregulated in SVR and NR compared to the SC group. Conclusions. PBMC inflammatory gene expression patterns in SVR resemble NR more than SC patients. A generalized inflammatory response persists in PBMCs long after successful peg-IFN treatment for HCV infection. PMID:26568966

  3. Piperine Induces Hepatic Low-Density Lipoprotein Receptor Expression through Proteolytic Activation of Sterol Regulatory Element-Binding Proteins

    PubMed Central

    Ochiai, Ayasa; Miyata, Shingo; Shimizu, Makoto; Inoue, Jun; Sato, Ryuichiro

    2015-01-01

    Elevated plasma low-density lipoprotein (LDL) cholesterol is considered as a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) uptakes plasma lipoproteins and lowers plasma LDL cholesterol, the activation of LDLR is a promising drug target for atherosclerosis. In the present study, we identified the naturally occurring alkaloid piperine, as an inducer of LDLR gene expression by screening the effectors of human LDLR promoter. The treatment of HepG2 cells with piperine increased LDLR expression at mRNA and protein levels and stimulated LDL uptake. Subsequent luciferase reporter gene assays revealed that the mutation of sterol regulatory element-binding protein (SREBP)-binding element abolished the piperine-mediated induction of LDLR promoter activity. Further, piperine treatments increased mRNA levels of several SREBP targets and mature forms of SREBPs. However, the piperine-mediated induction of the mature forms of SREBPs was not observed in SRD–15 cells, which lack insulin-induced gene–1 (Insig–1) and Insig–2. Finally, the knockdown of SREBPs completely abolished the piperine-meditated induction of LDLR gene expression in HepG2 cells, indicating that piperine stimulates the proteolytic activation of SREBP and subsequent induction of LDLR expression and activity. PMID:26431033

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

    SciTech Connect

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

    1988-02-01

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

  5. The fatty liver dystrophy (fld) mutation: Developmentally related alterations in hepatic triglyceride metabolism and protein expression

    SciTech Connect

    Reue, K.; Rehnmark, S.; Cohen, R.D.; Leete, T.H.; Doolittle, M.H. |; Giometti, C.S.; Mishler, K.; Slavin, B.G.

    1997-07-01

    Fatty liver dystrophy (fld) is an autosomal recessive mutation in mice characterized by hypertriglyceridemia and development of a fatty liver in the early neonatal period. Also associated with the fld phenotype is a tissue-specific deficiency in the expression of lipoprotein lipase and hepatic lipase, as well as elevations in hepatic apolipoprotein A-IV and apolipoprotein C-II mRNA levels. Although these lipid abnormalities resolve at the age of weaning, adult mutant mice exhibit a peripheral neuropathy associated with abnormal myelin formation. The fatty liver in fld/fld neonates is characterized by the accumulation of large triglyceride droplets within the parenchymal cells, and these droplets persist within isolated hepatocytes maintained in culture for several days. To identify the metabolic defect that leads to lipid accumulation, the authors investigated several aspects of cellular triglyceride metabolism. The mutant mice exhibited normal activity of acid triacylglycerol lipase, an enzyme thought to be responsible for hydrolysis of dietary triglycerides in the liver. Metabolic labeling studies performed with oleic acid revealed that free fatty acids accumulate in the liver of 3 day old fld/fld mice, but not in adults. This accumulation in liver was mirrored by elevated free fatty acid levels in plasma of fld/fld neonates, with levels highest in very young mice and returning to normal by the age of one month. Quantitation of fatty acid oxidation in cells isolated from fld/fld neonates revealed that oxidation rate is reduced 60% in hepatocytes and 40% in fibroblasts; hepatocytes from adult fld/fld mice exhibited an oxidation rate similar to those from wild-type mice.

  6. Diurnal variation in hepatic expression of the rat S14 gene is synchronized by the photoperiod

    SciTech Connect

    Kinlaw, W.B.; Fish, L.H.; Schwartz, H.L.; Oppenheimer, J.H.

    1987-04-01

    We have analyzed the factors responsible for the circadian variation in rat hepatic mRNA-S14. Regulation of this sequence, which is found in lipogenic tissues and encodes a protein (S14) believed to be associated with fatty acid synthesis, is an excellent model of the interaction of thyroid hormone and dietary factors at the hepatocellular level. The mRNA exhibits a 3-fold diurnal variation (peak, approximately 2000 h; nadir, 0800 h) in ad libitum feeding rats on a 12-h light, 12-h dark photoschedule. We studied the effects of the photoschedule, periodic food intake, hypophysectomy, and induction by thyroid hormone (T3) on the mRNA-S14 rhythm. Adaptation to feeding restricted to either light or dark periods for 15 days did not greatly affect the diurnal rhythm. Photoreversal resulted in a 180 degrees phase shift, whereas the rhythm persisted in the presence of constant light. Oscillation continued around a higher baseline after a receptor-saturating dose of T3 in both normal and hypophysectomized rats. Our results indicate primary entrainment of the mRNA-S14 diurnal rhythm to the photoperiod, rather than to periodic food intake. Moreover, the circadian regulatory signal, which probably originates in the central nervous system, appears capable of antagonizing a maximal T3-inductive stimulus and does not originate in the pituitary gland. Persistence of the oscillation in constant light rules out circulating melatonin as the mediator. Synchronization of the rhythm by the photoschedule suggests that neuroendocrine factors are important determinants of rhythmic changes in hepatic gene expression.

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

    PubMed

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

    2009-06-30

    Nicotine, the psychoactive ingredient in tobacco, can be neuroprotective but the mechanism is unknown. In the adult hippocampus, chronic nicotine can increase expression of growth factors which could contribute to nicotine's neuroprotective effects. During development, nicotine could also increase expression of neurotrophic factors. Therefore, we determined whether chronic neonatal nicotine (CNN) exposure increased mRNA expression levels of brain-derived neurotrophic factor (BDNF), nerve-growth factor (NGF), neurotrophin-3 (NT-3), fibroblast growth factor-2 (FGF-2), and insulin-like growth factor-1 (IGF-1). Nicotine (6 mg/kg/day in milk formula) or milk formula (controls) were delivered in three daily doses via oral gastric intubation to rat pups from postnatal day (P)1 to P8, and then sacrificed. Brains were processed for in situ hybridization using specific (35)S-labeled cRNA probes. At P8, CNN had a significant stimulant treatment effect on the expression of BDNF, FGF-2, NT-3 and IGF-1 [p<0.01], but not NGF. Specifically, BDNF mRNA expression, detected in CA1, CA3 stratum (s.) pyramidal and granule cell layer of the dentate gyrus (DG), was increased by 27.4%, 23.26% and 27.3%, respectively. FGF-2 mRNA expression, detected in neurons and astrocytes in CA1 s. radiatum, CA2 and CA3 s. pyramidale, and molecular layer of the DG, was increased by 34.0%, 8.9%, 31.0% and 23.1%, respectively. NT-3 mRNA expression in CA2 s. pyramidale was increased by 80.0%, and CNN increased the number of IGF-1-expressing cells in CA1 (18.0%), CA3 (20.9%) and DG (17.7%). Thus, nicotine exposure during early postnatal development differentially up-regulated expression of neurotrophic factor mRNAs in the hippocampus, which could increase neurotrophic tone and alter developmental processes. PMID:19410565

  8. Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor.

    PubMed

    Caruso, Hillary G; Torikai, Hiroki; Zhang, Ling; Maiti, Sourindra; Dai, Jianliang; Do, Kim-Anh; Singh, Harjeet; Huls, Helen; Lee, Dean A; Champlin, Richard E; Heimberger, Amy B; Cooper, Laurence J N

    2016-06-01

    Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue. PMID:27163741

  9. Changes in mRNA expression of arcuate nucleus appetite-regulating peptides during lactation in rats

    PubMed Central

    Suzuki, Yoshihiro; Nakahara, Keiko; Maruyama, Keisuke; Okame, Rieko; Ensho, Takuya; Inoue, Yoshiyuki; Murakami, Noboru

    2014-01-01

    The contribution of hypothalamic appetite-regulating peptides to further hyperphagia accompanying the course of lactation in rats was investigated by using PCR array and real-time PCR. Furthermore, changes in the mRNA expression for appetite-regulating peptides in the hypothalamic arcuate nucleus (ARC) were analyzed at all stages of pregnancy and lactation, and also after weaning. Food intake was significantly higher during pregnancy, lactation, and after weaning than during non-lactation periods. During lactation, ARC expression of mRNAs for agouti-related protein (AgRP) and peptide YY was increased, whereas that of mRNAs for proopiomelanocortin (POMC) and cholecystokinin (CCK) was decreased, in comparison with non-lactation periods. The increase in AgRP mRNA expression during lactation was especially marked. The plasma level of leptin was significantly decreased during the course of lactation, whereas that of acyl-ghrelin was unchanged. In addition, food intake was negatively correlated with the plasma leptin level during lactation. This study has clarified synchronous changes in the expression of many appetite-regulating peptides in ARC of rats during lactation. Our results suggest that hyperphagia during lactation in rats is caused by decreases in POMC and CCK expression and increases in AgRP expression in ARC, the latter being most notable. Together with the decrease in the blood leptin level, such changes in mRNA expression may explain the further hyperphagia accompanying the course of lactation. PMID:24299740

  10. Sorting Live Stem Cells Based on Sox2 mRNA Expression

    PubMed Central

    Larsson, Hans M.; Lee, Seung Tae; Roccio, Marta; Velluto, Diana; Lutolf, Matthias P.; Frey, Peter; Hubbell, Jeffrey A.

    2012-01-01

    While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB+SSEA1+ cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB+ cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB− cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner. PMID:23209609

  11. Expression of fluorescent proteins in Branchiostoma lanceolatum by mRNA injection into unfertilized oocytes.

    PubMed

    Hirsinger, Estelle; Carvalho, João Emanuel; Chevalier, Christine; Lutfalla, Georges; Nicolas, Jean-François; Peyriéras, Nadine; Schubert, Michael

    2015-01-01

    We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution. PMID:25650764

  12. Molecular cloning and mRNA expression analysis of sheep MYL3 and MYL4 genes.

    PubMed

    Zhang, Chunlan; Wang, Jianmin; Wang, Guizhi; Ji, Zhibin; Hou, Lei; Liu, Zhaohua; Chao, Tianle

    2016-02-15

    Using longissimus dorsi muscles of Dorper sheep as the experimental materials, the complete cDNAs of ovine MYL3 (Myosin light chain 3) and MYL4 (Myosin light chain 4) genes were cloned using RT-PCR, 5' RACE and 3' RACE. We obtained 925-bp and 869-bp full-length cDNAs and submitted their sequences to GenBank as accession numbers of KJ710703 and KJ768855, respectively. The cDNAs contained 600-bp and 582-bp open reading frames (ORFs) and encoded proteins comprising 199 and 193 amino acid residues, respectively. Neither protein was predicted to have a signal peptide, but both were predicted to have several N-glycosylation, O-glycosylation, and phosphorylation sites. The secondary structures of MYL3 and MYL4 were predicted to be 40.70% and 48.70% α- helical, respectively. Sequence alignment showed that the MYL3 and MYL4 proteins of Ovis aries both shared more than 91% amino acid sequence similarity with those of Mus musculus, Homo sapiens, Rattus norvegicus, Bos taurus, and Sus scrofa. The levels of MYL3 and MYL4 mRNA in various sheep tissues were determined using qRT-PCR. The results showed that both mRNAs were highly expressed in the heart. This study has established a foundation for further investigation of the ovine MYL3 and MYL4 genes. PMID:26656596

  13. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  15. RELATIONSHIP BETWEEN BRAIN AND OVARY AROMATASE ACTIVITY AND ISOFORM-SPECIFIC AROMATASE MRNA EXPRESSION IN THE FATHEAD MINNOW (PIMEPHALES PROMELAS)

    EPA Science Inventory

    There is growing evidence that some chemicals present in the environment have the capacity to inhibit, or potentially induce, aromatase activity. This study compared aromatase activities and isoform-specific mRNA expression in brain and ovary tissue from non-exposed fathead min...

  16. Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation

    SciTech Connect

    Trempe, J.P.; Carter, B.J.

    1988-01-01

    The authors studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p/sub 40/ promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p/sub 40/. When the rep gene was present in cis or in trans, cat expression from p/sub 40/ was decreased 3- to 10-fold, but there was a 2- to 10-fold increase in the level of p/sub 40/ mRNA. Conversely, cat expression increased and the p/sub 40/ mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p/sub 40/ mRNA levels. They also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p/sub 5/ promoter in a fashion independent of rep.

  17. Effect of atmospheric fine particles on epidermal growth factor receptor mRNA expression in mouse skin tissue.

    PubMed

    Han, X; Liang, W L; Zhang, Y; Sun, L D; Liang, W Y

    2016-01-01

    We investigated the effect of atmospheric fine particles on epidermal growth factor receptor (Egfr) mRNA expression in mouse skin tissue and explored the effect of atmospheric fine particles on skin aging. Forty female BALB/c mice were randomly divided into four groups (each comprising 10 mice) as follows: a saline control group and low-, medium-, and high-dose atmospheric fine particle groups (1.6, 8.0, and 40.0 mg/kg, respectively) (fine particles were defined as those with a diameter of £2.5 mm, i.e., PM2.5). Each dose group was exposed to intratracheal instillation for 3 days. Twenty-four hours after the last exposure, real-time quantitative polymerase chain reaction was used to detect the expression of Egfr mRNA in the skin tissue of each mouse. The expression levels of Egfr mRNA in the medium- and high-dose PM2.5 groups were significantly higher (P < 0.05) than that in the control group, and were positively correlated with the dose. Medium and high concentrations of PM2.5 can induce the expression of Egfr mRNA and promote skin aging. PMID:27050971

  18. Serum leptin concentrations, leptin mRNA expression, and food intake during the estrous cycle in rats.

    PubMed

    Fungfuang, Wirasak; Nakada, Tomoaki; Nakao, Nobuhiro; Terada, Misao; Yokosuka, Makoto; Gizurarson, Sveinbjorn; Hau, Jann; Moon, Changjong; Saito, Toru R

    2013-03-01

    The aim of this study was to investigate food intake, serum leptin levels, and leptin mRNA expression during the sexual cycle in rats. Female Wistar-Imamichi rats aged 8-10 weeks were used in this experiment. Food intake was measured during the light and dark phases (light on at 07:00 and off at 19:00) of the 4-day estrous cycle in female rats. Serum leptin levels were measured by ELISA, and leptin mRNA expression levels were analyzed using real-time PCR on diestrous- and proestrous-stage rats. Our results revealed that during the sexual cycle, food intake was significantly higher in the dark phase compared with the light phase. Food intake in proestrous females was significantly lower in the light and dark phases compared with the other groups. Serum leptin concentrations were significantly higher in both phases in proestrous rats compared with diestrous rats. There was a significant increase in leptin mRNA expression in adipose tissue during the proestrous period compared with the diestrous period. These findings suggest that increased leptin mRNA expression and serum leptin levels, which are induced by estrogen during the proestrous stage, may play a role in regulating appetitive behavior. PMID:23573101

  19. Expression levels of estrogen receptor α mRNA in peripheral blood cells are an independent biomarker for postmenopausal osteoporosis☆

    PubMed Central

    Chou, Chi-Wen; Chiang, Tsay-I; Chang, I-Chang; Huang, Chung-Hung; Cheng, Ya-Wen

    2016-01-01

    Background The up- and down-regulation of the osteoclastogenesis response depends on the estrogen/estrogen receptor (ER) signaling pathway. Previous reports have shown that the promoter hypermethylation and gene polymorphism of ERα are risks for menopausal osteoporosis. No previous study has evaluated the expression levels of ERα mRNA in menopausal osteoporosis using human subjects. We hypothesized that ERα mRNA expression may show less resistance to postmenopausal osteoporosis. Methods In this study, we enrolled 107 women older than 45 years without menstruation and classified them into control, osteopenia, and osteoporosis groups depending on their T-scores. The ERα mRNA levels in peripheral blood cells (PBCs) were analyzed via quantitative real-time reverse-transcription polymerase chain reaction (QRT-PCR), and estrogen in the serum was detected via ELISA. Results ERα mRNA levels in PBCs had a negative correlation with age and a positive correlation with estrogen and BAP in the osteopenia and osteoporosis groups, but not in the control group. Additionally, multivariate analysis showed that older age (> 55 years), and low ERα mRNA levels in PBLs (≦ 250.39 copies/μg DNA) were associated with an approximately 9.188-, and 31.25-fold risk of osteoporosis. Conclusion We conclude that ERα mRNA levels in PBLs could be used as an independent risk factor for postmenopausal osteoporosis. General significance Our findings suggested that ERα mRNA levels in PBLs may be more important than age and serum estrogen levels. PMID:27051599

  20. Effects of jump training on procollagen alpha(1)(i) mRNA expression and its relationship with muscle collagen concentration.

    PubMed

    Ducomps, Christophe; Larrouy, Dominique; Mairal, Aline; Doutreloux, Jean-Paul; Lebas, Francois; Mauriege, Pascale

    2004-04-01

    The aim of this study was to examine the effects of a prolonged high-intensity exercise, jumping, on procollagen alpha(1)(I) mRNA level and collagen concentration in different muscles of trained (T) and control (C) rabbits. Procollagen alpha(1)(I) mRNA expression was much higher (2.8 to 23.5 times) in semimembranosus proprius (SMP), a slow-twitch oxidative muscle, than in extensor digitorum longus (EDL), rectus femoris (RF), and psoas major (Psoas) muscles, both fast-twitch mixed and glycolytic, whatever group was considered (p < 0.001). Procollagen alpha(1)(I) mRNA level also decreased significantly between 50 and 140 days in all muscles (0.001< p < 0.01). However, mRNA levels were 16 to 97% greater at 140 days in all muscles of T animals compared to C ones (0.01< p <0.05). Collagen concentrations of EDL and RF muscles were also higher (14 to 19%) in T than in C rabbits at 90 and 140 days (0.001 < p < 0.05). In the whole sample, collagen concentration was negatively associated with the procollagen alpha(1)(I) mRNA level in EDL and RF muscles (- 0.49 < r < (- 0.44, p < 0.05), while being positively related to mRNA expression in SMP and Psoas muscles (0.65 < r < 0.85, p < 0.01). It is concluded that jump training clearly restricts the decrease of procollagen (I) mRNA level and probably affects collagen synthesis level. In trained rabbit muscles, the maintenance of a better synthesis level could partly explain the higher collagen concentrations found in EDL and RF at 140 days. Nevertheless, the collagen degradation process seems to play the main role in the increase of total collagen concentration with age in EDL and RF muscles. PMID:15064425

  1. Brain-derived peptides increase blood-brain barrier GLUT1 glucose transporter gene expression via mRNA stabilization.

    PubMed

    Boado, R J

    1998-10-23

    The present investigation studied the effect of the brain-derived peptide preparation Cerebrolysin (CI, EBEWE, Austria) on the turnover rate and gene expression of the blood-brain barrier (BBB) GLUT1 glucose transporter mRNA. Studies were performed in brain endothelial cultured cells transfected with the human (h) GLUT1 transcript. In control cells, the full length 2.8 Kb hGLUT1 mRNA was rapidly degraded following transfection, and the abundance of this transcript at 4 and 6 h was comparable to background mRNA levels seen in cells transfected without hGLUT1 mRNA. On the contrary, the decay of the hGLUT1 mRNA was stabilized in CI-treated cells resulting in a marked reduction in the fractional turnover rate (72.4 and 4.0%/h, control and CI, respectively). In parallel experiments, CI induced a significant increase in the levels of immunoreactive GLUT1 protein measured by enzyme-linked immunosorbent assay (ELISA). In conclusion, data presented here demonstrate that factors in CI increase BBB-GLUT1 transcript stability, and that this is associated with an induction of BBB-GLUT1 gene expression in brain endothelial cultured cells. PMID:9832194

  2. [Induction of rat hepatic CYP2E1 expression by arecoline in vivo].

    PubMed

    Huang, Xiang-tao; Xiao, Run-mei; Wang, Ming-feng; Wang, Jun-jun; Chen, Yong

    2016-01-01

    The regulation mechanism of arecoline on rat hepatic CYP2E1 was studied in vivo. After oral administration of arecoline hydrobromide (AH; 4, 20 and 100 mg x kg(-1) x d(-1)) to rats for one week, the hepatic CYP2E1 mRNA level remained unchanged, but the hepatic CYP2E1 protein content was dose-dependently increased. Additionally, although the hepatic CYP2E1 activity was induced by AH treatment, the induction was attenuated with the increase in dosage. The results indicate that the effect of arecoline on rat hepaticdoes not involve transcriptional activation of the gene, but largely involves the stabilization of CYP2E1 protein against degradation or increased efficiency of CYP2E1 mRNA translation, and additionally involve the post- ranslational modification of CYP2E1 protein. Furthermore, the CYP2E1 response is fairly equal among the different species, the induction of rat hepatic CYP2E1 by arecoline suggests that there is a risk of metabolic interaction among the substrate drugs of CYP2E1 in betel-quid use human. PMID:27405178

  3. Effect of erythropoietin on hepatic cytochrome P450 expression and function in an adenine-fed rat model of chronic kidney disease

    PubMed Central

    Feere, D A; Velenosi, T J; Urquhart, B L

    2015-01-01

    BACKGROUND AND PURPOSE Erythropoietin (EPO) is used to treat anaemia associated with chronic kidney disease (CKD). Hypoxia is associated with anaemia and is known to cause a decrease in cytochrome P450 (P450) expression. As EPO production is regulated by hypoxia, we investigated the role of EPO on P450 expression and function. EXPERIMENTAL APPROACH Male Wistar rats were subjected to a 0.7% adenine diet for 4 weeks to induce CKD. The diet continued for an additional 2 weeks while rats received EPO by i.p. injection every other day. Following euthanasia, hepatic P450 mRNA and protein expression were determined. Hepatic enzyme activity of selected P450s was determined and chromatin immunoprecipitation was used to characterize binding of nuclear receptors involved in the transcriptional regulation of CYP2C and CYP3A. KEY RESULTS EPO administration decreased hepatic mRNA and protein expression of CYP3A2 (P < 0.05), but not CYP2C11. Similarly, EPO administration decreased CYP3A2 protein expression by 81% (P < 0.001). A 32% decrease (P < 0.05) in hepatic CYP3A enzymatic activity (Vmax) was observed for the formation of 6βOH-testosterone in the EPO-treated group. Decreases in RNA pol II recruitment (P < 0.01), hepatocyte nuclear factor 4α binding (P < 0.05) and pregnane X receptor binding (P < 0.01) to the promoter region of CYP3A were also observed in EPO-treated rats. CONCLUSIONS AND IMPLICATIONS Our data show that EPO decreases the expression and function of CYP3A, but not CYP2C in rat liver. PMID:25219905

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

    PubMed Central

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

    2015-01-01

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

  5. The effects of high fat diet and estradiol on hypothalamic prepro-QRFP mRNA expression in female rats.

    PubMed

    Schreiber, Allyson L; Arceneaux, Kenneth P; Malbrue, Raphael A; Mouton, Alan J; Chen, Christina S; Bench, Elias M; Braymer, H Douglas; Primeaux, Stefany D

    2016-08-01

    Estradiol (E2) is a potent regulator of feeding behavior, body weight and adiposity in females. The hypothalamic neuropeptide, QRFP, is an orexigenic peptide that increases the consumption of high fat diet (HFD) in intact female rats. Therefore, the goal of the current series of studies was to elucidate the effects of E2 on the expression of hypothalamic QRFP and its receptors, QRFP-r1 and QRFP-r2, in female rats fed a HFD. Alterations in prepro-QRFP, QRFP-r1, and QRFP-r2 expression across the estrous cycle, following ovariectomy (OVX) and following estradiol benzoate (EB) treatment were assessed in the ventral medial nucleus of the hypothalamus/arcuate nucleus (VMH/ARC) and the lateral hypothalamus. In intact females, consumption of HFD increased prepro-QRFP and QRFP-r1 mRNA levels in the VMH/ARC during diestrus, a phase associated with increased food intake and low levels of E2. To assess the effects of diminished endogenous E2, rats were ovariectomized. HFD consumption and OVX increased prepro-QRFP mRNA in the VMH/ARC. Ovariectomized rats consuming HFD expressed the highest levels of QRFP. In the third experiment, all rats received EB replacement every 4days following OVX to examine the effects of E2 on QRFP expression. Prepro-QRFP, QRFP-r1 and QRFP-r2 mRNA were assessed prior to and following EB administration. EB replacement significantly reduced prepro-QRFP mRNA expression in the VMH/ARC. Overall these studies support a role for E2 in the regulation of prepro-QRFP mRNA in the VMH/ARC and suggest that E2's effects on food intake may be via a direct effect on the orexigenic peptide, QRFP. PMID:26823127

  6. Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading

    NASA Technical Reports Server (NTRS)

    Carlson, C. J.; Booth, F. W.; Gordon, S. E.

    1999-01-01

    Transgenic mice lacking a functional myostatin (MSTN) gene demonstrate greater skeletal muscle mass resulting from muscle fiber hypertrophy and hyperplasia (McPherron, A. C., A. M. Lawler, and S. -J. Lee. Nature 387: 83-90, 1997). Therefore, we hypothesized that, in normal mice, MSTN may act as a negative regulator of muscle mass. Specifically, we hypothesized that the predominately slow (type I) soleus muscle, which demonstrates greater atrophy than the fast (type II) gastrocnemius-plantaris complex (Gast/PLT), would show more elevation in MSTN mRNA abundance during hindlimb unloading (HU). Surprisingly, MSTN mRNA was not detectable in weight-bearing or HU soleus muscle, which atrophied 42% by the 7th day of HU in female ICR mice. In contrast, MSTN mRNA was present in weight-bearing Gast/PLT muscle and was significantly elevated (67%) at 1 day but not at 3 or 7 days of HU. However, the Gast/PLT muscle had only atrophied 17% by the 7th day of HU. Because the soleus is composed only of type I and IIa fibers, whereas the Gast/PLT expresses type IId/x and IIb in addition to type I and IIa, it was necessary to perform a more careful analysis of the relationship between MSTN mRNA levels and myosin heavy-chain (MHC) isoform expression (as a marker of fiber type). A significant correlation (r = 0.725, P < 0. 0005) was noted between the percentage of MHC isoform IIb expression and MSTN mRNA abundance in several muscles of the mouse hindlimb. These results indicate that MSTN expression is not strongly associated with muscle atrophy induced by HU; however, it is strongly associated with MHC isoform IIb expression in normal muscle.

  7. LPS-induced imbalanced expression of hepatic vascular stress genes in cirrhosis: possible mechanism of increased susceptibility to endotoxemia.

    PubMed

    Baveja, Rajiv; Keller, Steve; Yokoyama, Yukihiro; Sonin, Natalie; Clemens, Mark G; Zhang, Jian X

    2002-04-01

    Cirrhosis predisposes the liver to secondary stresses such as endotoxemia possibly via dysregulation of the hepatic portal circulation secondary to imbalanced upregulation of vascular stress genes. In this study we determined the effect of cirrhosis on hepatic vasoregulatory gene expression in response to endotoxin (LPS, i.p., 1 mg/kg). Cirrhosis was induced by bile duct ligation (BDL) for 21 days in male Sprague-Dawley rats. Plasma and liver samples were taken 6 h following an injection of LPS for alanine aminotransferase (ALT) assays and RT-PCR analysis of mRNA levels for genes of interest: endothelin (ET-1), its receptors ET(A) and ET(B), endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and heme oxygenase-1 (HO-1). ALT release increased by 5.5-fold in the BDL animals and 9.9-fold in BDL + LPS compared to sham. ET-1 mRNA was increased by either LPS or BDL treatment alone and increased significantly more in BDL + LPS compared to sham + LPS. mRNA levels for ET(B) receptors showed no change, whereas ETA transcripts decreased in BDL animals compared to sham, with no significant difference between the saline and LPS treatment groups. The resultant increased ratio of ET(B) over ET(A) in BDL animals was reflected functionally in the portal pressure responses to ET(A) and ET(B) agonists ET-1 and IRL-1620 (a specific ETB receptor agonist). The pressor response to ET-1 was attenuated, while the response to IRL-1620 was similar in BDL and sham. eNOS mRNA levels did not increase in response to either BDL or LPS or a combination of both compared to sham. The increase in iNOS mRNA was attenuated in BDL + LPS compared to sham + LPS. HO-1 expression increased significantly in sham + LPS, but failed to increase in BDL + LPS. Taken collectively, significantly greater induction of the constrictor ET-1 over the dilation forces (i.e., eNOS, iNOS, and HO-1) was observed in BDL + LPS. This suggests a compromised ability of the cirrhotic liver to upregulate sufficient

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

    PubMed

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

    2006-10-01

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

  9. Enhancement of mRNA expression of survivin and human beta-defensin-3 in lesions of psoriasis vulgaris.

    PubMed

    Wang, Fang; Zhang, Xia; Xia, Ping; Zhang, Li; Zhang, Zhenghua

    2016-02-01

    Suppression of apoptosis is one of the pathogenetic mechanisms for psoriasis vulgaris (PV). Survivin has the function of regulating cell division and inhibiting apoptosis. Patients with PV have an increased resistance to cutaneous infections. Human β-defensin-3 (hBD-3) is a kind of antimicrobial peptide with antimicrobial activities. To assess and compare the transcript levels of survivin and hBD-3 in pairwise skin from PV. A total of 20 patients, 10 with mild PV and 10 with severe PV, and 10 healthy control donors were recruited in the study. Real-time PCR was conducted to determine survivin and hBD-3 mRNA expression in skin lesions and normal-appearing skin of PV patients, and normal skin of healthy controls. Compared with normal control skin, the survivin mRNA expression of normal-appearing skin in the mild PV group, lesions of the mild PV group and the severe PV group were significantly elevated (P<0.05). hBD-3 mRNA expression was statistically increased in both normal-appearing skin and in lesions in mild and severe PV groups, in contrast to normal skin (P<0.001). Significant differences of hBD-3 mRNA were also found between lesions and non-lesional skin in the mild PV group and severe PV group (P<0.05). Survivin mRNA levels were mildly correlated with hBD-3 mRNA levels (rs = 0.398; P<0.05) in skin lesions from 20 PV patients. Survivin and hBD-3 may be involved in the pathogenesis of psoriasis. PMID:26771657

  10. [Regulatory effect of thymosin α1 on expression of tlr9/ido mRNA in bone marrow mesenchymal stem cells from children with aplastic anemia].

    PubMed

    Hou, Fang; Huang, Jian-Ming; Li, Ge

    2010-12-01

    The purpose of this study was to explore the regulatory effect of thymosin α1 (Tα1) on expression of TOLL-like receptor 9 (TLR9)/indoleamine2, 3-dioxygenase (ido) mRNA in bone marrow mesenchymal stem cells (MSC) from children with aplastic anemia (AA). Culture system of bone marrow MSC from AA children and normal children in vitro was established, and the effects of Tα1 on expressions of tlr9 mRNA and ido mRNA of MSC from AA children and normal children were determined by RT-PCR. The results showed that the bone marrow MSC from normal children did not express tlr9 and ido mRNA. Bone marrow MSC from children with AA obviously expressed tlr9 mRNA , but did not express ido mRNA; AA children's MSC treated with Tα1 for 18 hours markedly down-regulated tlr9 mRNA expression, but up-regulated ido mRNA expression in the concentration- and time-dependent ways. It is concluded that Tα1 can up-regulate the expression of ido mRNA in bone marrow MSC from children with AA. PMID:21176371

  11. Hyperoxia increases hepatic arginase expression and ornithine production in mice

    SciTech Connect

    Malleske, Daniel T.; Rogers, Lynette K.; Velluci, Sean M.; Young, Tamara L.; Park, Min S.; Long, Donald W.; Welty, Stephen E.; Smith, Charles V.; Nelin, Leif D. . E-mail: NelinL@pediatrics.ohio-state.edu

    2006-08-15

    Hyperoxic exposure affects the levels and activities of some hepatic proteins. We tested the hypothesis that hyperoxic exposure would result in greater hepatic .NO concentrations. C3H/HeN mice were exposed to >95% O{sub 2} for 72 or 96 h and compared to room air-breathing controls. In contrast to our working hypothesis, exposure to >95% O{sub 2} for 96 h decreased hepatic nitrite/nitrate NO {sub X} concentrations (10.9 {+-} 2.2 nmol/g liver versus 19.3 {+-} 2.4 nmol/g liver in room air, P < 0.05). The hepatic levels of endothelial NO synthase (eNOS) and inducible NOS (iNOS) proteins were not different among the groups. The arginases, which convert L-arginine to urea and L-ornithine, may affect hepatic NOS activities by decreasing L-arginine bioavailability. Hepatic ornithine concentrations were greater in hyperoxic animals than in controls (318 {+-} 18 nmol/g liver in room air, and 539 {+-} 64, and 475 {+-} 40 at 72 and 96 h of hyperoxia, respectively, P < 0.01). Hepatic arginase I protein levels were greater in hyperoxic animals than in controls. Hepatic carbamoyl phosphate synthetase (CPS) protein levels and activities were not different among groups. These results indicate that increases in hepatic levels of arginase I in mice exposed to hyperoxia may diminish .NO production, as reflected by lower liver levels of NO {sub X}. The resultant greater hepatic ornithine concentrations may represent a mechanism to facilitate tissue repair, by favoring the production of polyamines and/or proline.

  12. Circulating and Hepatic Fas Expression in HCV-Induced Chronic Liver Disease and Hepatocellular Carcinoma

    PubMed Central

    El Bassiouny, Azza E. I.; El-Bassiouni, Nora E. I.; Nosseir, Mona M. F.; Zoheiry, Mona M.K.; El-Ahwany, Eman G.; Salah, Faten; Omran, Zeinab S.O.; Ibrahim, Raafat A.

    2008-01-01

    Apoptosis is central for control and elimination of viral infections. In chronic hepatitis C virus (HCV) infection, enhanced hepatocyte apoptosis and upregulation of the death-inducing ligands CD95/Fas occur. This study aimed to study the role of serum soluble Fas and hepatic Fas expression as early predictors of advancement of chronic hepatitis C disease. The current study included 50 cases of chronic hepatitis C (CHC) (and negative hepatitis B virus infection), 30 cases of liver cirrhosis (LC) and HCV, and 20 cases of hepatocellular carcinoma (HCC) and HCV admitted to Theodor Bilharz Research Institute, Giza, Egypt. Fifteen wedge liver biopsies, taken during laparoscopic cholecystectomy, were included in the study as normal controls. Assessment of serum soluble Fas level (sFas) and other laboratory investigations, including liver function tests, serologic markers for viral hepatitis, and serum alpha-fetoprotein level (alpha-FP), were determined for all cases. Histopathologic study and immunohistochemistry using monoclonal antibody for CD95 were also done. The sFas was significantly increased in CHC, LC, and HCC cases compared with normal controls (P < .01). The increase of sFas in HCC was also significantly higher than that of CHC (P < .01). However, positive hepatic expression of Fas antigen was higher in CHC than LC with no significant difference; meanwhile, it was significantly lower in HCC (P < .01) compared with CHC. In conclusion, circulating and hepatic Fas expression in chronic hepatitis C infection illustrate the mechanism of liver injury caused by death receptors throughout the multistep process of fibrosis/carcinogenesis. Not only the higher degree of hepatic fibrosis, but also the lower expression of Fas protein, are correlated with the increased incidence of HCC. PMID:18679533

  13. Expression and identification of hepatitis C virus polyprotein cleavage products.

    PubMed Central

    Grakoui, A; Wychowski, C; Lin, C; Feinstone, S M; Rice, C M

    1993-01-01

    Hepatitis C virus (HCV) is the major cause of transfusion-acquired non-A, non-B hepatitis. HCV is an enveloped positive-sense RNA virus which has been classified as a new genus in the flavivirus family. Like the other two genera in this family, the flaviviruses and the pestiviruses, HCV polypeptides appear to be produced by translation of a long open reading frame and subsequent proteolytic processing of this polyprotein. In this study, a cDNA clone encompassing the long open reading frame of the HCV H strain (3,011 amino acid residues) has been assembled and sequenced. This clone and various truncated derivatives were used in vaccinia virus transient-expression assays to map HCV-encoded polypeptides and to study HCV polyprotein processing. HCV polyproteins and cleavage products were identified by using convalescent human sera and a panel of region-specific polyclonal rabbit antisera. Similar results were obtained for several mammalian cell lines examined, including the human HepG2 hepatoma line. The data indicate that at least nine polypeptides are produced by cleavage of the HCV H strain polyprotein. Putative structural proteins, located in the N-terminal one-fourth of the polyprotein, include the capsid protein C (21 kDa) followed by two possible virion envelope proteins, E1 (31 kDa) and E2 (70 kDa), which are heavily modified by N-linked glycosylation. The remainder of the polyprotein probably encodes nonstructural proteins including NS2 (23 kDa), NS3 (70 kDa), NS4A (8 kDa), NS4B (27 kDa), NS5A (58 kDa), and NS5B (68 kDa). An 82- to 88-kDa glycoprotein which reacted with both E2 and NS2-specific HCV antisera was also identified (called E2-NS2). Preliminary results suggest that a fraction of E1 is associated with E2 and E2-NS2 via disulfide linkages. Images PMID:7679746

  14. Expression of CD134 and CXCR4 mRNA in term placentas from FIV-infected and control cats.

    PubMed

    Scott, Veronica L; Burgess, Shane C; Shack, Leslie A; Lockett, Nikki N; Coats, Karen S

    2008-05-15

    Feline immunodeficiency virus (FIV) causes a natural infection of domestic cats that resembles HIV-1 in pathogenesis and disease progression. Feline AIDS is characterized by depression of the CD4+ T cell population and fatal opportunistic infections. Maternal-fetal transmission of FIV readily occurs under experimental conditions, resulting in infected viable kittens and resorbed or arrested fetal tissues. Although both FIV and HIV use the chemokine receptor CXCR4 as a co-receptor, FIV does not utilize CD4 as the primary receptor. Rather, CD134 (OX40), a T cell activation antigen and co-stimulatory molecule, is the primary receptor for FIV. We hypothesized that placental expression of CD134 and CXCR4 may render the placenta vulnerable to FIV infection, possibly facilitating efficient vertical transmission of FIV, and impact pregnancy outcome. The purpose of this project was to quantify the relative expression of CD134 and CXCR4 mRNA from the term placentas of three groups of cats: uninfected queens producing viable offspring, experimentally-infected queens producing only viable offspring, and experimentally-infected queens producing viable offspring among mostly non-viable fetuses. Total RNA was extracted from term placental tissues from all groups of cats. Real-time one-step reverse transcriptase-PCR was used to measure gene expression. The FIV receptors CD134 and CXCR4 were expressed in all late term feline placental tissues. Placentas from FIV-infected queens producing litters of only viable offspring expressed more CD134 and CXCR4 mRNA than those from uninfected queens, suggesting that infection may cause upregulation of the receptors. On the other hand, placentas from FIV-infected cats with non-successful pregnancies expressed similar levels of CD134 mRNA and slightly less CXCR4 mRNA than those from uninfected queens. Thus, it appears that cells expressing these receptors may play a role in pregnancy maintenance. PMID:18295905

  15. Hepatitis B virus induces hypoxia-inducible factor-2α expression through hepatitis B virus X protein.

    PubMed

    Hu, Jian-Li; Liu, Li-Ping; Yang, Sheng-Li; Fang, Xiefan; Wen, Lu; Ren, Quan-Guang; Yu, Chao

    2016-03-01

    A growing number of studies suggest that the hepatitis B virus X protein (HBx) enhances the protein stability of the hypoxia-inducible factor-1α (HIF-1α). However, the relationship between hepatitis B virus (HBV), HBx and hypoxia-inducible factor-2α (HIF-2α) has not yet been fully elucidated. Immunohistochemical analysis was employed to detect the expression of HIF-2α in normal liver, HBV-related chronic hepatitis, and HBV-related and non-HBV-related hepatocellular carcinoma (HCC) tissues. Quantitative real‑time PCR (qPCR) and western blotting were used to investigate the impact of HBV and HBx on the expression of HIF‑2α. Immunoprecipitation and immunofluorescence were applied to explore the underlying mechanisms. The HIF‑2α expression was found to be higher in HBV‑related chronic hepatitis tissues than in normal liver tissues. Furthermore, it was higher in HBV‑related HCC tissues and HBV‑integrated HepG2 cells than in the corresponding non‑HBV‑related HCC tissues and HepG2 cells. Both HBV and HBx enhanced the protein stability of HIF‑2α. HBx‑mediated upregulation of HIF‑2α resulted mainly from an inhibition of the degradation of HIF‑2α due to the binding of HBx to the von Hippel‑Lindau protein (pVHL). In addition, HBx upregulated the expression of HIF‑2α by activating the NF‑κB signaling pathway. Thus, the present study identified that HBV induces the HIF‑2α expression through its encoded protein HBx. This upregulates the HIF-2α expression by binding to the pVHL activating the NF-κB signaling pathway. PMID:26647960

  16. Distribution of Androgen Receptor mRNA Expression in Vocal, Auditory, and Neuroendocrine Circuits in a Teleost Fish

    PubMed Central

    Forlano, Paul M.; Marchaterre, Margaret; Deitcher, David L.; Bass, Andrew H.

    2010-01-01

    Across all major vertebrate groups, androgen receptors (ARs) have been identified in neural circuits that shape reproductive-related behaviors, including vocalization. The vocal control network of teleost fishes presents an archetypal example of how a vertebrate nervous system produces social, context-dependent sounds. We cloned a partial cDNA of AR that was used to generate specific probes to localize AR expression throughout the central nervous system of the vocal plainfin midshipman fish (Porichthys notatus). In the forebrain, AR mRNA is abundant in proposed homologs of the mammalian striatum and amygdala, and in anterior and posterior parvocellular and magnocellular nuclei of the preoptic area, nucleus preglomerulosus, and posterior, ventral and anterior tuberal nuclei of the hypothalamus. Many of these nuclei are part of the known vocal and auditory circuitry in midshipman. The midbrain periaqueductal gray, an essential link between forebrain and hindbrain vocal circuitry, and the lateral line recipient nucleus medialis in the rostral hindbrain also express abundant AR mRNA. In the caudal hindbrain-spinal vocal circuit, high AR mRNA is found in the vocal prepacemaker nucleus and along the dorsal periphery of the vocal motor nucleus congruent with the known pattern of expression of aromatase-containing glial cells. Additionally, abundant AR mRNA expression is shown for the first time in the inner ear of a vertebrate. The distribution of AR mRNA strongly supports the role of androgens as modulators of behaviorally defined vocal, auditory, and neuroendocrine circuits in teleost fish and vertebrates in general. PMID:20020540

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

    PubMed Central

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

    1995-01-01

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

  18. The metastatic potential of canine mammary tumours can be assessed by mRNA expression analysis of connective tissue modulators.

    PubMed

    Lamp, O; Honscha, K U; Schweizer, S; Heckmann, A; Blaschzik, S; Einspanier, A

    2013-03-01

    Metastases are the crucial factor for the prognosis of canine mammary tumours (CMTs). In women, the peptide hormone relaxin is linked with metastatic breast cancer. Therefore, the impact of relaxin and its receptors on matrix metalloproteinase (MMP) expression, metastatic disease and survival was analysed using qRT-PCR and immunohistochemistry of CMT samples from 59 bitches. The expression of relaxin and its receptor RXFP1 (relaxin family peptide receptor 1) was discovered on gene and protein levels. Intratumoural relaxin mRNA expression and relaxin plasma levels had no prognostic value. High mRNA levels RXFP1 were an independent marker of metastatic potential, with a more than 15-fold risk increase, and a predictor for shorter survival. Also, MMP-2 expression was associated with early death because of CMT. The mRNA expressions of relaxin, RXFP1 and MMP-2 were positively correlated indicating a common pathogenetic linkage. Thus, RXFP1 is proposed as a new early marker of metastatic potential in CMT and a possible therapeutic target. PMID:22235833

  19. Regulation of NMDA receptor subunit mRNA expression in the guinea pig vestibular nuclei following unilateral labyrinthectomy.

    PubMed

    Sans, N; Sans, A; Raymond, J

    1997-10-01

    The localization of neurons expressing mRNAs for the NR1 and NR2A-D subunits of the glutamatergic NMDA receptor was examined by non-radioactive in situ hybridization throughout the guinea pig vestibular nuclei. After deafferentation of the vestibular nuclei by unilateral labyrinthectomy, modifications of the mRNA distributions were followed for 30 days. A quantitative analysis was performed in the medial vestibular nucleus by comparison of the labelled neurons in the ipsi- and contra-lateral nuclei. In vestibular nuclei, the NR1 subunit mRNA was found in various populations of neurons. The NR2A and NR2C subunit mRNAs were less widely distributed, whereas little NR2D mRNA was detected and only rare cells contained NR2B mRNA. NR1 and NR2A-D mRNAs were colocalized in some but not other neuronal types. Twenty hours after the lesion, there was a transient ipsilateral increase of NR1 mRNA level in the medial vestibular nucleus, followed by a decrease 48 h after the lesion and, at 3 days, by recovery to the control level. An ipsilateral increase in the mRNA level of NR2C subunit was detected 20 h after lesion and maintained at 48 h. No significant changes were apparent in NR2A, NR2B and NR2D mRNA levels. The distributions and the differential signal intensities of NR2A-D mRNAs suggest various subunit organizations of the NMDA receptors in different neurons of the vestibular nuclei. Neuronal plasticity reorganizations in the vestibular nuclei following unilateral labyrinthectomy appear to include only changes in NR1 and NR2C mRNA levels modifying the functional diversity of the NMDA receptor in the ipsilateral medial vestibular nucleus neurons. The transient changes in NR1 and the NR2C subunit mRNA expressions in response to sensory deprivation are consistent with an active role for NMDA receptors in the appearance and development of the vestibular compensatory process. PMID:9421163

  20. Effects of arginine supplementation on splenocyte cytokine mRNA expression in rats with gut-derived sepsis

    PubMed Central

    Shang, Huey-Fang; Hsu, Chun-Sen; Yeh, Chiu-Li; Pai, Man-Hui; Yeh, Sung-Ling

    2005-01-01

    AIM: To investigate the effects of arginine (Arg)-enriched diets before sepsis and/or Arg-containing total parenteral nutrition (TPN) after sepsis or both on cytokine mRNA expression levels in splenocytes of rats with gut-derived sepsis. METHODS: Rats were assigned to four experimental groups. Groups 1 and 2 were fed with a semipurified diet, while groups 3 and 4 had part of the casein replaced by Arg which provided 2% of the total calories. After the rats were fed with these diets for 10 d, sepsis was induced by cecal ligation and puncture (CLP), at the same time an internal jugular vein was cannulated. All rats were maintained on TPN for 3 d. Groups 1 and 3 were infused with conventional TPN, while groups 2 and 4 were supplemented with Arg which provided 2% of the total calories in the TPN solution. All rats were killed 3 d after CLP to examine their splenocyte subpopulation distribution and cytokine expression levels. RESULTS: Plasma interleukin (IL)-2, IL-4, tumor necrosis factor-α (TNF-α) and interferon (IFN-γ) were not detectable 3 d after CLP. There were no differences in the distributions of CD45Ra+, CD3+, CD4+, and CD8+ cells in whole blood and splenocytes among the four groups. The splenocyte IL-2 mRNA expression in the Arg-supplemented groups was significantly higher than that in group 1. IL-4 mRNA expression in groups 3 and 4 was significantly higher than that in groups 1 and 2. The mRNA expression of IL-10 and IFN-γ was significantly higher in group 4 than in the other three groups. There was no difference in TNF-α mRNA expression among the four groups. CONCLUSION: The influence of Arg on the whole blood and splenic lymphocyte subpopulation distribution is not obvious. However, Arg administration, especially before and after CLP, significantly enhances the mRNA expression levels of Th1 and Th2 cytokines in the spleen of rats with gut-derived sepsis. PMID:16437653

  1. Evaluating hypoxia-inducible factor-1α mRNA expression in a pelagic fish, Pacific herring Clupea pallasii, as a biomarker for hypoxia exposure.

    PubMed

    Froehlich, Halley E; Roberts, Steven B; Essington, Timothy E

    2015-11-01

    Hypoxia [dissolved oxygen (DO)<2 mg L(-1)] is a major environmental perturbation for many aquatic ecosystems, particularly highly productive estuaries. Most research attention and understanding about the impacts of hypoxia on estuarine species has focused on the benthos, where hypoxia is most common. Although the pelagic zone is also susceptible to the effects of hypoxia, the biological interactions and consequences are not as well understood in marine environments because documenting exposure or avoidance of hypoxia is often difficult. Physiological biomarkers may provide a way to gain more detailed spatiotemporal information regarding species' exposure to hypoxia. Here, we identified and tested a hypoxia-specific responsive gene, hypoxia-inducible factor-1α (hif-1α), to evaluate its potential as a biomarker for hypoxia exposure in Pacific herring (Clupea pallasii). We conducted controlled laboratory experiments to establish the level of hepatic hif-1α elevated gene expression (>1 sd normoxic mean), exposure amplification (≥2 hours), reduction rate (ca. 24 hours), and some evidence of a lethal hypoxic limit (ca. 2 mg L(-1), ≥4 hours). We then used these findings to evaluate the spatiotemporal patterns of hif-1α for Pacific herring in a seasonally hypoxia estuary, Hood Canal, Washington, USA. Although expression did not parallel the local hypoxic conditions in the estuary, herring from the more severe hypoxic year (2013) had a higher probability of having elevated mRNA levels. These patterns indicate that hepatic hif-1α levels may not be directly indicative of local DO levels for pelagic marine fish, but rather provide insight into hypoxia exposure over broader scales. PMID:26241022

  2. FATHEAD MINNOW VITELLOGENIN: CDNA SEQUENCE AND MRNA AND PROTEIN EXPRESSION AFTER 17 BETA-ESTRADIOL TREATMENT

    EPA Science Inventory

    In the present study, a sensitive ribonuclease protection assay (RPA) for VTG mRNA was developed for the fathead minnow (Pimephales promelas), a species proposed for routine endocrine-disrupting chemical (EDC) screening.

  3. Hepatic transcriptome analysis of hepatitis C virus infection in chimpanzees defines unique gene expression patterns associated with viral clearance.

    PubMed

    Nanda, Santosh; Havert, Michael B; Calderón, Gloria M; Thomson, Michael; Jacobson, Christian; Kastner, Daniel; Liang, T Jake

    2008-01-01

    Hepatitis C virus infection leads to a high rate of chronicity. Mechanisms of viral clearance and persistence are still poorly understood. In this study, hepatic gene expression analysis was performed to identify any molecular signature associated with the outcome of hepatitis C virus (HCV) infection in chimpanzees. Acutely HCV-infected chimpanzees with self-limited infection or progression to chronicity were studied. Interferon stimulated genes were induced irrespective of the outcome of infection. Early induction of a set of genes associated with cell proliferation and immune activation was associated with subsequent viral clearance. Specifically, two of the genes: interleukin binding factor 3 (ILF3) and cytotoxic granule-associated RNA binding protein (TIA1), associated with robust T-cell response, were highly induced early in chimpanzees with self-limited infection. Up-regulation of genes associated with CD8+ T cell response was evident only during the clearance phase of the acute self-limited infection. The induction of these genes may represent an initial response of cellular injury and proliferation that successfully translates to a "danger signal" leading to induction of adaptive immunity to control viral infection. This primary difference in hepatic gene expression between self-limited and chronic infections supports the concept that successful activation of HCV-specific T-cell response is critical in clearance of acute HCV infection. PMID:18927617

  4. Gene expression profiling in hepatic tissue of newly weaned pigs fed pharmacological zinc and phytase supplemented diets

    PubMed Central

    Martínez-Montemayor, Michelle M; Hill, Gretchen M; Raney, Nancy E; Rilington, Valencia D; Tempelman, Robert J; Link, Jane E; Wilkinson, Christopher P; Ramos, Antonio M; Ernst, Catherine W

    2008-01-01

    Background Zinc (Zn) is an essential trace element. However, Zn bioavailability from commonly consumed plants may be reduced due to phytic acid. Zn supplementation has been used to treat diarrheal disease in children, and in the U.S. swine industry at pharmacological levels to promote growth and fecal consistency, but underlying mechanisms explaining these beneficial effects remain unknown. Moreover, adding supplemental phytase improves Zn bioavailability. Thus, we hypothesized that benefits of pharmacological Zn supplementation result from changes in gene expression that could be further affected by supplemental phytase. The goal of this study was to investigate the effects of feeding newly weaned pigs dietary Zn (150, 1,000, or 2,000 mg Zn/kg) as Zn oxide with or without phytase [500 phytase units (FTU)/kg] for 14 d on hepatic gene expression. Liver RNA from pigs fed 150, 1,000, or 2,000 mg Zn/kg, or 1,000 mg Zn/kg with phytase (n = 4 per treatment) was reverse transcribed and examined using the differential display reverse transcription polymerase chain reaction technique. Liver RNA from pigs fed 150 or 2,000 mg Zn/kg (n = 4 per treatment) was also evaluated using a 70-mer oligonucleotide microarray. Results Expressed sequence tags for 61 putatively differentially expressed transcripts were cloned and sequenced. In addition, interrogation of a 13,297 element oligonucleotide microarray revealed 650 annotated transcripts (FDR ≤ 0.05) affected by pharmacological Zn supplementation. Seven transcripts exhibiting differential expression in pigs fed pharmacological Zn with sequence similarities to genes encoding GLO1, PRDX4, ACY1, ORM1, CPB2, GSTM4, and HSP70.2 were selected for confirmation. Relative hepatic GLO1 (P < 0.0007), PRDX4 (P < 0.009) and ACY1 (P < 0.01) mRNA abundances were confirmed to be greater in pigs fed 1,000 (n = 8) and 2,000 (n = 8) mg Zn/kg than in pigs fed 150 (n = 7) mg Zn/kg. Relative hepatic HSP70.2 (P < 0.002) mRNA abundance was confirmed to

  5. The Prognostic Value of BRCA1 mRNA Expression Levels Following Neoadjuvant Chemotherapy in Breast Cancer

    PubMed Central

    Margeli, Mireia; Cirauqui, Beatriz; Castella, Eva; Tapia, Gustavo; Costa, Carlota; Gimenez-Capitan, Ana; Barnadas, Agusti; Ronco, Maria Sanchez; Benlloch, Susana; Taron, Miquel; Rosell, Rafael

    2010-01-01

    Background A fraction of sporadic breast cancers has low BRCA1 expression. BRCA1 mutation carriers are more likely to achieve a pathological complete response with DNA-damage-based chemotherapy compared to non-mutation carriers. Furthermore, sporadic ovarian cancer patients with low levels of BRCA1 mRNA have longer survival following platinum-based chemotherapy than patients with high levels of BRCA1 mRNA. Methodology/Principal Findings Tumor biopsies were obtained from 86 breast cancer patients who were candidates for neoadjuvant chemotherapy, treated with four cycles of neoadjuvant fluorouracil, epirubicin and cyclophosphamide. Estrogen receptor (ER), progesterone receptor (PR), HER2, cytokeratin 5/6 and vimentin were examined by tissue microarray. HER2 were also assessed by chromogenic in situ hybridization, and BRCA1 mRNA was analyzed in a subset of 41 patients for whom sufficient tumor tissue was available by real-time quantitative PCR. Median time to progression was 42 months and overall survival was 55 months. In the multivariate analysis for time to progression and overall survival for 41 patients in whom BRCA1 could be assessed, low levels of BRCA1 mRNA, positive PR and negative lymph node involvement predicted a significantly lower risk of relapse, low levels of BRCA1 mRNA and positive PR were the only variables associated with significantly longer survival. Conclusions/Significance We provide evidence for a major role for BRCA1 mRNA expression as a marker of time to progression and overall survival in sporadic breast cancers treated with anthracycline-based chemotherapy. These findings can be useful for customizing chemotherapy. PMID:20209131

  6. Chronic morphine exposure during puberty induces long-lasting changes in opioid-related mRNA expression in the mediobasal hypothalamus.

    PubMed

    Byrnes, Elizabeth M

    2008-01-23

    Substance abuse in developing females may have significant long-term effects on reproductive competency. Chronic morphine exposure during puberty has been shown to reduce prolactin secretion in lactating rats. Opioid activity within the mediobasal hypothalamus (MBH) regulates suckling-induced prolactin secretion. Thus, the current study was conducted to determine whether chronic pubertal morphine exposure alters the expression of mu- and/or kappa-opioid receptor mRNA or pro-opioimelanocortin (POMC) mRNA within the MBH. Using an increasing dose regimen, female Sprague-Dawley rats were injected twice daily for a total of 20 days with morphine sulfate or saline beginning at 30 days of age. Several weeks later, quantitative RT-PCR was used to determine mRNA expression within the MBH in diestrus, never pregnant (nulliparous) controls, postpartum day 5 (PPD5), PPD10, PPD18, and diestrus, reproductively experienced (primiparous) females. Pubertal morphine exposed females had increased mu- and kappa-receptor mRNA expression as well as decreased POMC mRNA expression on diestrus. During lactation, mu- and kappa-receptor mRNA expression in the MBH decreased while POMC mRNA expression increased in similarly treated females. No changes in mRNA expression were observed during lactation in pubertal saline-treated females; however, increased mu- and kappa-receptor mRNA expression as well as decreased POMC mRNA expression was observed in primiparous, pubertal saline-treated females when compared to nulliparous controls. Thus, chronic morphine exposure during puberty results in long-term alterations in mu- and kappa-receptor as well as POMC mRNA expression in the MBH which are similar to the changes observed following reproductive experience. These changes do not correlate with the decreased prolactin secretion observed during early lactation; however, they do demonstrate the enduring nature of the effects of chronic opiate exposure during puberty on hypothalamic opioid systems in

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

    NASA Technical Reports Server (NTRS)

    Fitzgerald, J.; Hughes-Fulford, M.

    1996-01-01

    Serum-deprived mouse osteoblastic cells (MC3T3-E1a) were centrifuged under a regime designed to simulate a space shuttle launch (maximum of 3g). Messenger RNA levels for eight genes involved in bone growth and maintenance were determined using RT-PCR. Following 30 min of centrifugation, mRNA level for early response gene c-fos was significantly increased 89% (P < 0.05). The c-fos induction was transient and returned to control levels after 3 h. The mRNA level for the mineralization marker gene osteocalcin was significantly decreased to 44% of control level (P < 0.005) 3 h after centrifugation. No changes in mRNA levels were detected for c-myc, TGFbeta1, TGFbeta2, cyclophilin A, or actin. No basal mRNA level for TGFbeta3 was detected. In addition, no change in the steady-state synthesis of prostaglandin E2 was detected, possibly due to lack of lipid substrates in serum-deprived cells, suggesting that the increase in c-fos mRNA in response to gravitational loading is a result of mechanical stimulation. These results indicate that a small magnitude mechanical loading, such as that experienced during a shuttle launch, can alter mRNA levels in quiescent osteoblastic cells.

  8. Mangiferin treatment inhibits hepatic expression of acyl-coenzyme A:diacylglycerol acyltransferase-2 in fructose-fed spontaneously hypertensive rats: a link to amelioration of fatty liver

    SciTech Connect

    Xing, Xiaomang; Li, Danyang; Chen, Dilong; Zhou, Liang; Chonan, Ritsu; Yamahara, Johji; Wang, Jianwei; Li, Yuhao

    2014-10-15

    Mangiferin, a xanthone glucoside, and its associated traditional herbs have been demonstrated to improve abnormalities of lipid metabolism. However, its underlying mechanisms remain largely unclear. This study investigated the anti-steatotic effect of mangiferin in fructose-fed spontaneously hypertensive rat (SHR)s that have a mutation in sterol regulatory element binding protein (SREBP)-1. The results showed that co-administration of mangiferin (15 mg/kg, once daily, by oral gavage) over 7 weeks dramatically diminished fructose-induced increases in hepatic triglyceride content and Oil Red O-stained area in SHRs. However, blood pressure, fructose and chow intakes, white adipose tissue weight and metabolic parameters (plasma concentrations of glucose, insulin, triglyceride, total cholesterol and non-esterified fatty acids) were unaffected by mangiferin treatment. Mechanistically, mangiferin treatment suppressed acyl-coenzyme A:diacylglycerol acyltransferase (DGAT)-2 expression at the mRNA and protein levels in the liver. In contrast, mangiferin treatment was without effect on hepatic mRNA and/or protein expression of SREBP-1/1c, carbohydrate response element binding protein, liver pyruvate kinase, fatty acid synthase, acetyl-CoA carboxylase-1, stearoyl-CoA desaturase-1, DGAT-1, monoacyglycerol acyltransferase-2, microsomal triglyceride transfer protein, peroxisome proliferator-activated receptor-alpha, carnitine palmitoyltransferase-1 and acyl-CoA oxidase. Collectively, our results suggest that mangiferin treatment ameliorates fatty liver in fructose-fed SHRs by inhibiting hepatic DGAT-2 that catalyzes the final step in triglyceride biosynthesis. The anti-steatotic effect of mangiferin may occur independently of the hepatic signals associated with de novo fatty acid synthesis and oxidation. - Highlights: • We investigated the anti-steatotic effect of mangiferin (MA) in fructose-fed SHR. • MA (15 mg/kg/day for 7 weeks) ameliorated fructose-induced fatty liver in

  9. Hepatic expression of the woodchuck hepatitis virus X-antigen during acute and chronic infection and detection of a woodchuck hepatitis virus X-antigen antibody response.

    PubMed

    Jacob, J R; Ascenzi, M A; Roneker, C A; Toshkov, I A; Cote, P J; Gerin, J L; Tennant, B C

    1997-12-01

    The expression and localization of the woodchuck hepatitis virus X-antigen (WHxAg) was examined and compared with other markers of a woodchuck hepatitis virus (WHV) infection using rabbit antisera generated against recombinant WHxAg produced in bacteria. Cellular fractionation studies showed that WHxAg was localized to the soluble and cytoskeletal fractions of the cell when assayed by immunoprecipitation of [35S]-met-cys labeled extracts derived from primary cultures of acute WHV-infected hepatocytes. Immunohistochemical examination of liver from chronic WHV-infected animals showed WHV core antigen (WHcAg) and WHxAg expression in non-neoplastic tissue. The WHxAg was found localized to the cytoplasm of infected cells, similar to WHcAg. WHxAg expression was diminished in the foci of altered hepatocytes and in hepatocellular adenomas but was found in only 1 of 11 hepatocellular carcinomas (HCC). Hepatic biopsies from woodchucks experimentally inoculated with WHV were examined during the acute phase of infection and during convalescence for WHcAg and WHxAg expression by immunohistochemistry. Concurrent expression of WHcAg and WHxAg was observed during the viremic phase of infection. The two antigens exhibited similar localization to the cell cytoplasm, similar distribution within the liver lobule, and similar patterns of clearance during convalescence. An immune response to WHxAg was documented in some woodchucks following acute WHV infection. These studies further define the woodchuck model of HBV infection and should allow for the investigation of the role of hepadnaviral X-antigen expression in the pathogenesis of chronic hepatitis and HCC. PMID:9398005

  10. Infusion of ACTH stimulates expression of adrenal ACTH receptor and steroidogenic acute regulatory protein mRNA in fetal sheep.

    PubMed

    Carey, Luke C; Su, Yixin; Valego, Nancy K; Rose, James C

    2006-08-01

    The late-gestation plasma cortisol surge in the sheep fetus is critical for stimulating organ development and parturition. Increased adrenal responsiveness is one of the key reasons for the surge; however, the underlying mechanisms are not fully understood. Our recent studies suggest that ACTH-mediated increased expression of ACTH receptor (ACTH-R) and steroid acute regulatory protein (StAR) may play a role in enhancing responsiveness. Hence, we examined effects of ACTH infusion in fetal sheep on mRNA expression of these two mediators of adrenal responsiveness and assessed the functional consequences of this treatment in vitro. Fetuses of approximately 118 and 138 days of gestational age (dGA) were infused with ACTH-(1-24) for 24 h. Controls received saline infusion. Arterial blood was sampled throughout the infusion. Adrenals were isolated and analyzed for ACTH-R and StAR mRNA, or cells were cultured for 48 h. Cells were stimulated with ACTH, and medium was collected for cortisol measurement. Fetal plasma ACTH and cortisol concentrations increased over the infusion period in both groups. ACTH-R mRNA levels were significantly higher in ACTH-infused fetuses in both the 118 and 138 dGA groups. StAR mRNA increased significantly in both the 118 and 138 dGA groups. Adrenal cells from ACTH-infused fetuses were significantly more responsive to ACTH stimulation in terms of cortisol secretion than those from saline-infused controls. These findings demonstrate that increases in circulating ACTH levels promote increased expression of ACTH-R and StAR mRNA and are coupled to heightened adrenal responsiveness. PMID:16478774

  11. mRNA expression is a relevant tool to identify developmental neurotoxicants using an in vitro approach.

    PubMed

    Hogberg, Helena T; Kinsner-Ovaskainen, Agnieszka; Coecke, Sandra; Hartung, Thomas; Bal-Price, Anna K

    2010-01-01

    So far, only a few industrial chemicals have been identified as developmental neurotoxicants. Because the current developmental neurotoxicity (DNT) guideline (Organisation for Economic Co-operation and Development TG 426) is based entirely on in vivo studies that are both time consuming and costly, there is a need to develop alternative in vitro methods for initial screening to prioritize chemicals for further DNT testing. In this study, gene expression at the mRNA level was evaluated to determine whether this could be a suitable endpoint to detect potential developmental neurotoxicants. Primary cultures of rat cerebellar granule cells (CGCs) were exposed to well known (developmental) neurotoxicants (methyl mercury chloride, lead chloride, valproic acid, and tri-methyl tin chloride) for different time periods. A significant downregulation of the mRNA level for the neuronal markers (NF-68, NF-200, N-methyl D-aspartate glutamate receptor, and gamma-amino butyric acid receptor) was observed after exposure to methyl mercury chloride, valproic acid, and tri-methyl tin chloride. Moreover, a significant increase of the neural precursor marker nestin mRNA was also observed. The mRNA expression of the astrocytic markers (glial fibrillary acidic protein [GFAP] and S100beta) was unchanged. In contrast, exposure to lead chloride significantly decreased the mRNA level of the astrocytic marker GFAP, whereas the neuronal markers were less affected. These results suggest that gene expression could be used as a sensitive tool for the initial identification of DNT effects induced by different mechanisms of toxicity in both cell types (neuronal and glial) and at various stages of cell development and maturation. PMID:19651682

  12. Mutation-dependent effects on mRNA and protein expressions in cultured keratinocytes of Hailey-Hailey disease.

    PubMed

    Matsuda, Mitsuhiro; Hamada, Takahiro; Numata, Sanae; Teye, Kwesi; Okazawa, Hiromi; Imafuku, Shinichi; Ohata, Chika; Furumura, Minao; Hashimoto, Takashi

    2014-07-01

    Hailey-Hailey disease (HHD) is a dominantly inherited skin disease caused by mutations in ATP2C1 gene, which encodes secretory pathway Ca(2+) /Mn(2+) -ATPase protein 1. The precise mechanism remains unclear. In this study, to understand molecular basis of HHD, we examined expression of mRNA and protein in cultured keratinocytes derived from three HHD patients with different mutations. We showed that reduced expression of mRNA and protein in patient with p.Gln504X, but not in patients with p.Pro307His and c.1308+1G>A. RT-PCR analysis for patient with c.1308+1G>A revealed in-frame exon skipping. Reduction of mRNA and protein in p.Gln504X was considered to be caused by nonsense-mediated mRNA decay. p.Pro307His located adjacent to Ca(2+) -binding residue may induced conformational change, which leads to defective Ca(2+) transport. In-frame shorter transcript caused by c.1308+1G>A may have slightly reduced activity, which accounted for mild phenotype of the patient. These results clarified the pathogenic effects of different causative mutations in development of skin lesions. PMID:24698124

  13. The somatotropic axis during the physiological estrus cycle in dairy heifers--Effect on hepatic expression of GHR and SOCS2.

    PubMed

    Mense, K; Meyerholz, M; Gil Araujo, M; Lietzau, M; Knaack, H; Wrenzycki, C; Hoedemaker, M; Piechotta, M

    2015-04-01

    Pituitary growth hormone (GH) release and hepatic insulin-like growth factor-I (IGF-I) production increase after an injection of 17β-estradiol (E2) in ovariectomized dairy cattle. However, whether endogenous sexual steroid hormones also influence the hepatic GH receptor (GHR) signaling pathway during a physiological estrus cycle remains unclear. The aim of this study was to analyze the hepatic GHR signaling pathway during the luteal phase and after a period of increased E2 concentrations (after ovulation) as well as in 7 heifers before ovulation. Ovarian ultrasounds were performed daily during repeated physiological cycles (n = 56) of 30 Holstein Friesian heifers to determine ovulation [before ovulation (n = 7, bOv) and after ovulation 24-60 h after the appearance of estrus signs (n = 49, aOv)] and luteal phase (CLP; d 12 ± 1 after ovulation). Blood samples and liver biopsies were obtained, and blood concentrations of E2, P4, insulin-like growth factor (IGF)-I, IGF-II, and GH were measured. In the liver biopsies, we determined mRNA expression of the estrogen receptor α (ERα), GHR, Janus kinase 2 (JAK2), signal transducer and activator of transcription 5B (STAT5B), suppressor of cytokine signaling (SOCS)2 and 3, IGF-I, and IGF-II by quantitative reverse transcription-PCR. The concentration of E2 was higher bOv than aOv and CLP, as expected. The concentrations of IGF-I and GH were higher bOv and aOv compared with CLP. In contrast, concentrations of IGF-II were lower aOv compared with bOv and CLP. The mRNA expression of GHR was higher in liver biopsies obtained bOv compared with aOv and CLP. Notably, the expression of SOCS2 was higher bOv than aOv and in the CLP. Increased hepatic expression of SOCS2 during estrus was detectable when IGF-I concentrations were high; this result might indicate that SOCS2 expression attenuates the GHR signal transduction pathway during the phase of increased pituitary GH release. In conclusion, hepatic GHR and SOCS2 mRNA expression

  14. Expression of Interferon Lambda 4 Is Associated with Reduced Proliferation and Increased Cell Death in Human Hepatic Cells

    PubMed Central

    Onabajo, Olusegun O.; Porter-Gill, Patricia; Paquin, Ashley; Rao, Nina; Liu, Luyang; Tang, Wei; Brand, Nathan

    2015-01-01

    Interferon lambda 4 (IFN-λ4) is a novel type-III interferon that can be generated only in individuals carrying a ΔG frame-shift allele of an exonic genetic variant (rs368234815-ΔG/TT). The rs368234815-ΔG allele is strongly associated with decreased clearance of hepatitis C virus (HCV) infection. Here, we further explored the biological function of IFN-λ4 expressed in human hepatic cells—a hepatoma cell line HepG2 and fresh primary human hepatocytes (PHHs). We performed live confocal imaging, cell death and proliferation assays, mRNA expression profiling, protein detection, and antibody blocking assays using transient and inducible stable in vitro systems. Not only did we observe significant intracellular retention of IFN-λ4 but also detected secreted IFN-λ4 in the culture media of expressing cells. Secreted IFN-λ4 induced strong activation of the interferon-stimulated genes (ISGs) in IFN-λ4-expressing and surrounding cells in transwell assays. Specifically, in PHHs, secreted IFN-λ4 induced expression of the CXCL10 transcript and a corresponding pro-inflammatory chemokine, IP-10. In IFN-λ4-expressing HepG2 cells, we also observed decreased proliferation and increased cell death. All IFN-λ4-induced phenotypes—activation of ISGs, decreased proliferation, and increased cell death—could be inhibited by an anti-IFN-λ4-specific antibody. Our study offers new insights into biology of IFN-λ4 and its possible role in HCV clearance. PMID:26134097

  15. Expression of BDNF and TH mRNA in the brain following inhaled administration of α-pinene.

    PubMed

    Kasuya, Hikaru; Okada, Narumi; Kubohara, Mika; Satou, Tadaaki; Masuo, Yoshinori; Koike, Kazuo

    2015-01-01

    Essential oils are mainly administered by inhalation. Administration by inhalation is considered to occur through two pathways, neurological transfer and pharmacological transfer. However, the relationship between the two routes is not clear. To clarify this relationship, we administered α-pinene, which has an anxiolytic-like effect, to mice. Emotional behavior and accumulation and expression of relevant mRNAs in the brain (brain-derived neurotrophic factor (BDNF); tyrosine hydroxylase (TH)) were examined following inhaled administration of α-pinene (10 μL/L air for 60 or 90min). To evaluate the anxiolytic-like effect, the elevated plus maze (EPM) test was used. Inhalation of α-pinene for 60 min produced a significant increase in the total distance traveled in the EPM test compared with control (water). The concentration of α-pinene in the brain after 60 min of inhalation was significantly increased compared with that after 90 min of inhalation. The expression of BDNF mRNA in the olfactory bulb and in the hippocampus was almost the same after 60 min of inhalation compared to that after 90 min of inhalation. The expression of TH mRNA in the midbrain after 60 min of inhalation was significantly increased compared with that of the control. Thus, an increase in α-pinene in the brain induces an increase in TH mRNA expression and increases locomotor activity. The anxiolytic-like effect may be related to both neurological transfer and pharmacological transfer. PMID:25230317

  16. Oligodendrocyte Morphometry and Expression of Myelin – Related mRNA in Ventral Prefrontal White Matter in Major Depressive Disorder

    PubMed Central

    Rajkowska, Grazyna; Mahajan, Gouri; Maciag, Dorota; Sathyanesan, Monica; Iyo, Abiye H.; Moulana, Mohadetheh; Kyle, Patrick B.; Woolverton, William L.; Miguel-Hidalgo, Jose Javier; Stockmeier, Craig A.; Newton, Samuel S.

    2015-01-01

    White matter disturbance in the ventral prefrontal cortex (vPFC) in major depressive disorder (MDD) has been noted with diffusion tensor imaging (DTI). However, the cellular and molecular pathology of prefrontal white matter in MDD and potential influence of antidepressant medications is not fully understood. Oligodendrocyte morphometry and myelin-related mRNA and protein expression was examined in the white matter of the vPFC in MDD. Sections of deep and gyral white matter from the vPFC were collected from 20 subjects with MDD and 16 control subjects. Density and size of CNPase-immunoreactive (−IR) oligodendrocytes were estimated using 3-dimensional cell counting. While neither density nor soma size of oligodendrocytes was significantly affected in deep white matter, soma size was significantly decreased in the gyral white matter in MDD. In rhesus monkeys treated chronically with fluoxetine there was no significant effect on oligodendrocyte morphometry. Using quantitative RTPCR to measure oligodendrocyte-related mRNA for CNPase, PLP1, MBP, MOG, MOBP, Olig1 and Olig2, in MDD there was a significantly reduced expression of PLP1 mRNA (which positively correlated with smaller sizes) and increased expression of mRNA for CNPase, OLIG1 and MOG. The expression of CNPase protein was significantly decreased in MDD. Altered expression of four myelin genes and CNPase protein suggests a mechanism for the degeneration of cortical axons and dysfunctional maturation of oligodendrocytes in MDD. The change in oligodendrocyte morphology in gyral white matter may parallel altered axonal integrity as revealed by DTI. PMID:25930075

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

    PubMed Central

    Heijtz, Rochellys Diaz; Alexeyenko, Andrey; Castellanos, F Xavier

    2007-01-01

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

  18. Changes in apoptotic microRNA and mRNA expression profiling in Caenorhabditis elegans during the Shenzhou-8 mission.

    PubMed

    Gao, Ying; Li, Shuai; Xu, Dan; Wang, Junjun; Sun, Yeqing

    2015-11-01

    Radiation and microgravity exposure have been proven to induce abnormal apoptosis in microRNA (miRNA) and mRNA expression, but whether space conditions, including radiation and microgravity, activate miRNAs to regulate the apoptosis is undetermined. For that purpose, we investigated miRNome and mRNA expression in the ced-1 Caenorhabditis elegans mutant vs the wild-type, both of which underwent spaceflight, spaceflight 1g-centrifuge control and ground control conditions during the Shenzhou-8 mission. Results showed that no morphological changes in the worms were detected, but differential miRNA expression increased from 43 (ground control condition) to 57 and 91 in spaceflight and spaceflight control conditions, respectively. Microgravity altered miRNA expression profiling by decreasing the number and significance of differentially expressed miRNA compared with 1 g incubation during spaceflight. Alterations in the miRNAs were involved in alterations in apoptosis, neurogenesis larval development, ATP metabolism and GTPase-mediated signal transduction. Among these, 17 altered miRNAs potentially involved in apoptosis were screened and showed obviously different expression signatures between space conditions. By integrated analysis of miRNA and mRNA, miR-797 and miR-81 may be involved in apoptosis by targeting the genes ced-10 and both drp-1 and hsp-1, respectively. Compared with ground condition, space conditions regulated apoptosis though a different manner on transcription, by altering expression of seven core apoptotic genes in spaceflight condition, and eight in spaceflight control condition. Results indicate that, miRNA of Caenorhabditis elegans probably regulates apoptotic gene expression in response to space environmental stress, and shows different behavior under microgravity condition compared with 1 g condition in the presence of space radiation. PMID:26286471

  19. Changes in apoptotic microRNA and mRNA expression profiling in Caenorhabditis elegans during the Shenzhou-8 mission

    PubMed Central

    Gao, Ying; Li, Shuai; Xu, Dan; Wang, Junjun; Sun, Yeqing

    2015-01-01

    Radiation and microgravity exposure have been proven to induce abnormal apoptosis in microRNA (miRNA) and mRNA expression, but whether space conditions, including radiation and microgravity, activate miRNAs to regulate the apoptosis is undetermined. For that purpose, we investigated miRNome and mRNA expression in the ced-1 Caenorhabditis elegans mutant vs the wild-type, both of which underwent spaceflight, spaceflight 1g-centrifuge control and ground control conditions during the Shenzhou-8 mission. Results showed that no morphological changes in the worms were detected, but differential miRNA expression increased from 43 (ground control condition) to 57 and 91 in spaceflight and spaceflight control conditions, respectively. Microgravity altered miRNA expression profiling by decreasing the number and significance of differentially expressed miRNA compared with 1 g incubation during spaceflight. Alterations in the miRNAs were involved in alterations in apoptosis, neurogenesis larval development, ATP metabolism and GTPase-mediated signal transduction. Among these, 17 altered miRNAs potentially involved in apoptosis were screened and showed obviously different expression signatures between space conditions. By integrated analysis of miRNA and mRNA, miR-797 and miR-81 may be involved in apoptosis by targeting the genes ced-10 and both drp-1 and hsp-1, respectively. Compared with ground condition, space conditions regulated apoptosis though a different manner on transcription, by altering expression of seven core apoptotic genes in spaceflight condition, and eight in spaceflight control condition. Results indicate that, miRNA of Caenorhabditis elegans probably regulates apoptotic gene expression in response to space environmental stress, and shows different behavior under microgravity condition compared with 1 g condition in the presence of space radiation. PMID:26286471

  20. Differential effects of binge methamphetamine injections on the mRNA expression of histone deacetylases (HDACs) in the rat striatum

    PubMed Central

    Omonijo, Oluwaseyi; Wongprayoon, Pawaris; Ladenheim, Bruce; McCoy, Michael T.; Govitrapong, Piyarat; Jayanthi, Subramaniam; Cadet, Jean Lud

    2014-01-01

    Methamphetamine use disorder is characterized by recurrent binge episodes. Humans addicted to methamphetamine experience various degrees of cognitive deficits and show evidence of neurodegenerative processes in the brain. Binge injections of METH to rodents also cause significant toxic changes in the brain. In addition, this pattern of METH injections can alter gene expression in the dorsal striatum. Gene expression is regulated, in part, by histone deacetylation. We thus tested the possibility that METH toxic doses might cause changes in the mRNA levels of histone deacetylases (HDACs). We found that METH did produce significant decreases in the mRNA expression of HDAC8, which is a class I HDAC. METH also decreased expression of HDAC6, HDAC9, and HDAC10 that are class II HDACs. The expression of the class IV HDAC, HDAC11, was also suppressed by METH. The expression of Sirt2, Sirt5, and Sirt6 that are members of class III HDACs was also downregulated by METH injections. Our findings implicate changes in HDAC expression may be an early indicator of impending METH-induced neurotoxicity in the striatum. This idea is consistent with the accumulated evidence that some HDACs are involved in neurodegenerative processes in the brain. PMID:25452209

  1. The expression of histamine H4 receptor mRNA in the skin and other tissues of normal dogs.

    PubMed

    Eisenschenk, Melissa N C; Torres, Sheila M F; Oliveira, Simone; Been, Clint S

    2011-10-01

    The histamine 4 (H(4)) receptor was first cloned and characterized in 2000 using the human H(3) receptor DNA sequence. The H(4) receptor has been shown to participate in various aspects of inflammation, such as chemotaxis, upregulation of adhesion molecule expression and modulation of cytokine secretion. The primary goal of this study was to determine whether H(4) receptor mRNA is expressed in normal canine skin by performing an RT-PCR. An additional goal was to determine the expression of this receptor in the colon, liver, spleen and kidney. Tissues were collected from five healthy, young-adult pit bull dogs. Samples were immediately placed in RNAlater(®) solution and stored at -20°C until processed. The amplified products in all skin samples in addition to the colon, liver, spleen and kidney (variable expression) had the expected size of 400-500 bp. The sequenced amplicons matched the National Center for Biotechnology Information published sequence for the canine H(4) receptor. The study results showed that canine normal skin expresses the H(4) receptor mRNA. Further studies using immunohistochemistry should be conducted to demonstrate the expression of the H(4) receptor at the protein level and to localize the expression of this receptor in the skin. PMID:21392139

  2. Hepatitis

    MedlinePlus

    ... be serious. Some can lead to scarring, called cirrhosis, or to liver cancer. Sometimes hepatitis goes away by itself. If it does not, it can be treated with drugs. Sometimes hepatitis lasts a lifetime. Vaccines can help prevent some viral forms.

  3. Ephrin/Ephrin receptor expression in ammonia-treated rat astrocytes and in human cerebral cortex in hepatic encephalopathy.

    PubMed

    Sobczyk, Karmela; Jördens, Markus S; Karababa, Ayse; Görg, Boris; Häussinger, Dieter

    2015-02-01

    Hepatic encephalopathy (HE) represents a neuropsychiatric syndrome, which evolves as a consequence of a low grade cerebral edema and a concomitant oxidative/nitrosative stress response. Ephrin receptors (EphR) and their ligands (ephrins) regulate astrocytic glutamate uptake and gliotransmitter release thereby governing neurotransmission, but their role in HE and ammonia toxicity is unclear. We therefore tested effects of ammonia on expression levels of EphR/ephrin isoforms in cultured rat astrocytes and analysed underlying mechanisms. NH4Cl induced mRNA expression changes of several EphR/ephrin isoforms in a methionine sulfoximine-, NADPH oxidase- and NO synthase-dependent manner in cultured astrocytes. A prominent upregulation was noted for EphR A4 mRNA and protein in NH4Cl-treated astrocytes. NH4Cl-treatment decreased EphR A4 molecular mass to similar extent as found in astrocytes treated with the N-glycosylation inhibitor tunicamycin. Knockdown of EphR A4 by siRNA, or treating astrocytes with NH4Cl or tunicamycin abolished fibroblast growth factor-induced and EphR A4-dependent astrocyte proliferation. NH4Cl-treatment also decreased GLAST mRNA levels in cultured astrocytes. This effect was sensitive to inhibitors of NAPDH oxidase or glutamine synthetase, but was insensitive to siRNA-mediated EphR A4 knockdown. Eph/ephrin gene expression changes were also found in post mortem brain samples of cirrhotic patients without or with HE compared to controls suggesting a potential in vivo relevance of the present findings. The present study suggests that ammonia modulates EphR/ephrin signaling in astrocytes and in the brain of cirrhotic patients with HE with potential implications for deranged neurotransmission in HE. PMID:25064044

  4. Inhibition of hepatitis B virus gene expression & replication by crude destruxins from Metarhizium anisopliae var. dcjhyium

    PubMed Central

    Dong, Cong; Yu, Jiuru; Zhu, Ying; Dong, Changjin

    2013-01-01

    Background & objectives: Destruxin A, destruxin B and destruxin E isolated from entomopathogenic fungus Metarhizium anisopliae showed a strong suppressive effect on the replication of hepatitis B virus (HBV) in human hepatoma cells. In this study, the anti-HBV effects of the crude destruxins extracted from M. anisopliae var. dcjhyium were detected both in vitro and in vivo. Methods: HepG2.2.15 cells were cultured to observe the inhibitory effects of the crude destruxins on the gene expression and replication of HBV by radioimmunoassay detection and real-time quantitative PCR. In vivo, duck HBV (DHBV)-infected ducks were treated with the crude destruxins at 2.0, 4.0, 6.0 μg/kg once a day for 15 days, DHBV DNA was examined by real-time quantitative PCR. Results: The crude destruxins suppressed the replication of HBV-DNA and the production of HBsAg and HBeAg with IC50 of about 1.2 and 1.4 μg/ml. Transcript of viral mRNA was significantly suppressed by the crude destruxins in HepG2.2.15 cells. In vivo, the duck serum DHBV-DNA levels were markedly reduced in the group of the crude destruxins. Interpretation & conclusions: The crude destruxins inhibited the gene expression and replication of HBV both in vitro and in vivo, and their anti-HBV effect was stronger than that with destruxin B. Our results indicate that the crude destruxins from M.anisopliae var. dcjhyium may be potential antivirus agents. Further studies need to be done to confirm these findings. PMID:24521644

  5. Hepatitis B viral replication influences the expression of natural killer cell ligands

    PubMed Central

    Koumbi, Lemonica; Pollicino, Teresa; Raimondo, Giovanni; Kumar, Naveenta; Karayiannis, Peter; Khakoo, Salim I.

    2016-01-01

    Background Hepatitis B virus (HBV) is accounting for over one million deaths annually due to immune-mediated chronic liver damage. Natural killer (NK) cells are abundant in the liver and contribute in HBV persistence. NK cytotoxic effects are controlled by signals from activating and inhibitory receptors. HBV may circumvent host antiviral immunity via the regulation of NK receptors and their ligands. We investigated the effect of viral replication and HBeAg mutations on NK mediators expression in the livers of chronic HBV (CHB) patients and in cell cultures. Methods HBV monomers bearing hotspot mutations in the basal core promoter and precore region were transfected into HepG2 cells using a plasmid-free assay. Serum viremia and liver HBV RNA were measured in 19 CHB patients. The expression of HBV RNA and of NKG2D ligands, B7H6, DNAX accessory molecule-1, lectin-like transcript 1 (LLT1), LFA-1 and TRAIL was measured in the livers of CHB patients and transfected cells. Results In general, high HBV replication in CHB patients and cell lines upregulated the mRNA of all NK cell ligands and particularly the inhibitory NK cell ligand, LLT1. The exception was the NKG2D ligand, MICA, that was significantly decreased in patients with high serum viremia and intrahepatic HBV RNA levels. Conclusions HBV replication has differential effects on NK cell ligands suggesting a potential escape mechanisms through up-regulation of LLT1 and down-regulation of MICA. A general trend towards upregulating NK cell ligands can be counteracted by decreasing MICA and hence weakening NK surveillance. PMID:27366037

  6. Co-expression analysis of differentially expressed genes in hepatitis C virus-induced hepatocellular carcinoma.

    PubMed

    Song, Qingfeng; Zhao, Chang; Ou, Shengqiu; Meng, Zhibin; Kang, Ping; Fan, Liwei; Qi, Feng; Ma, Yilong

    2015-01-01

    The aim of the current study was to investigate the molecular mechanisms underlying hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) using the expression profiles of HCV-infected Huh7 cells at different time points. The differentially expressed genes (DEGs) were identified with the Samr package in R software once the data were normalized. Functional and pathway enrichment analysis of the identified DEGs was also performed. Subsequently, MCODE in Cytoscape software was applied to conduct module analysis of the constructed co-expression networks. A total of 1,100 DEGs were identified between the HCV-infected and control samples at 12, 18, 24 and 48 h post-infection. DEGs at 24 and 48 h were involved in the same signaling pathways and biological processes, including sterol biosynthetic processes and tRNA amino-acylation. There were 22 time series genes which were clustered into 3 expression patterns, and the demarcation point of the 2 expression patterns that 401 overlapping DEGs at 24 and 48 h clustered into was 24 h post-infection. tRNA synthesis-related biological processes emerged at 24 and 48 h. Replication and assembly of HCV in HCV-infected Huh7 cells occurred mainly at 24 h post-infection. In view of this, the screened time series genes have the potential to become candidate target molecules for monitoring, diagnosing and treating HCV-induced HCC. PMID:25339452

  7. Co-expression analysis of differentially expressed genes in hepatitis C virus-induced hepatocellular carcinoma

    PubMed Central

    SONG, QINGFENG; ZHAO, CHANG; OU, SHENGQIU; MENG, ZHIBIN; KANG, PING; FAN, LIWEI; QI, FENG; MA, YILONG

    2015-01-01

    The aim of the current study was to investigate the molecular mechanisms underlying hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) using the expression profiles of HCV-infected Huh7 cells at different time points. The differentially expressed genes (DEGs) were identified with the Samr package in R software once the data were normalized. Functional and pathway enrichment analysis of the identified DEGs was also performed. Subsequently, MCODE in Cytoscape software was applied to conduct module analysis of the constructed co-expression networks. A total of 1,100 DEGs were identified between the HCV-infected and control samples at 12, 18, 24 and 48 h post-infection. DEGs at 24 and 48 h were involved in the same signaling pathways and biological processes, including sterol biosynthetic processes and tRNA amino-acylation. There were 22 time series genes which were clustered into 3 expression patterns, and the demarcation point of the 2 expression patterns that 401 overlapping DEGs at 24 and 48 h clustered into was 24 h post-infection. tRNA synthesis-related biological processes emerged at 24 and 48 h. Replication and assembly of HCV in HCV-infected Huh7 cells occurred mainly at 24 h post-infection. In view of this, the screened time series genes have the potential to become candidate target molecules for monitoring, diagnosing and treating HCV-induced HCC. PMID:25339452

  8. Lifelong Parental Voluntary Wheel Running Increases Offspring Hippocampal Pgc-1α mRNA Expression But Not Mitochondrial Content or Bdnf Expression

    PubMed Central

    Venezia, Andrew C.; Guth, Lisa M.; Spangenburg, Espen E.; Roth, Stephen M.

    2015-01-01

    When exercise is initiated during pregnancy, offspring of physically active mothers have higher hippocampal expression of brain derived neurotrophic factor (Bdnf) and other plasticity and mitochondrial-associated genes, resulting in hippocampal structural and functional adaptations. In the present study, we examined the effects of lifelong parental voluntary wheel running (before, during, and after pregnancy) on offspring hippocampal mRNA expression of genes implicated in the exercise-induced improvement of cognitive function. C57BL/6 mice were individually housed at 8 weeks of age with (EX; n=20) or without (SED; n=20) access to a computer-monitored voluntary running wheel (VRW) for 12 weeks prior to breeding. EX breeders maintained access to the VRW throughout breeding, pregnancy, and lactation. Male offspring were housed in sedentary cages, regardless of parental group, and were sacrificed at 8 (n=18) or 28 weeks (n=19). PCR was used to assess mRNA expression of several genes and mitochondrial content (ratio of mitochondrial to nuclear DNA) in hippocampal homogenates. We found significantly higher peroxisome proliferator-activated receptor γ coactivator 1 alpha (Pgc-1α) mRNA expression in EX offspring compared to SED offspring at 8 wks (p=0.04), though the effect was no longer present at 28 wks. There was no difference in mitochondrial content or expression of Bdnf or any other mRNA targets between offspring at 8 or 28 wks. In contrast to exercise initiated during pregnancy, parental voluntary physical activity initiated early in life and maintained throughout pregnancy has little effect on offspring mRNA expression of genes implicated in exercise-induced hippocampal plasticity. PMID:25919993

  9. Electroacupuncture-regulated neurotrophic factor mRNA expression in the substantia nigra of Parkinson's disease rats.

    PubMed

    Wang, Shuju; Fang, Jianqiao; Ma, Jun; Wang, Yanchun; Liang, Shaorong; Zhou, Dan; Sun, Guojie

    2013-02-25

    Acupuncture for the treatment of Parkinson's disease has a precise clinical outcome. This study investigated the effect of electroacupuncture at Fengfu (GV16) and Taichong (LR3) acupoints in rat models of Parkinson's disease induced by subcutaneous injection of rotenone into rat neck and back. Reverse transcription-PCR demonstrated that brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression was significantly increased in the substantia nigra of rat models of Parkinson's disease, and that abnormal behavior of rats was significantly improved following electroacupuncture treatment. These results indicated that electroacupuncture treatment upregulated brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression in the substantia nigra of rat models of Parkinson's disease. Thus, electroacupuncture may be useful in the treatment of Parkinson's disease. PMID:25206697

  10. merA gene expression in aquatic environments measured by mRNA production and Hg(II) volatilization.

    PubMed Central

    Nazaret, S; Jeffrey, W H; Saouter, E; Von Haven, R; Barkay, T

    1994-01-01

    The relationship of merA gene expression (specifying the enzyme mercuric reductase) to mercury volatilization in aquatic microbial communities was investigated with samples collected at a mercury-contaminated freshwater pond, Reality Lake, in Oak Ridge, Tenn. Levels of merA mRNA transcripts and the rate of inorganic mercury [Hg(II)] volatilization were related to the concentration of mercury in the water and to heterotrophic activity in field samples and laboratory incubations of pond water in which microbial heterotrophic activity and Hg(II) concentration were manipulated. Levels of merA-specific mRNA and Hg(II) volatilization were influenced more by microbial metabolic activity than by the concentration of mercury. merA-specific transcripts were detected in some samples which did not reduce Hg(II), suggesting that rates of mercury volatilization in environmental samples may not always be proportional to merA expression. PMID:7527625

  11. Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing

    SciTech Connect

    Kaye, F.; Battey, J.; Nau, M.; Brooks, B.; Seifter, E.; De Greve, J.; Birrer, M.; Sausville, E.; Minna, J.

    1988-01-01

    The authors' analyzed in detail the structure of the L-myc gene isolated from human placental DNA and characterized its expression in several small-cell lung cancer cell lines. The gene is composed of three exons and two introns spanning 6.6 kilobases in human DNA. Several distinct mRNA species are produced in all small-cell lung cancer cell lines that express L-myc. These transcripts are generated from a single gene by alternative splicing of introns 1 and 2 and by use of alternative polyadenylation signals. In some mRNAs that is a long open reading frame with a predicted translated protein of 364 residues. Amino acid sequence comparison with c-myc and N-myc demonstrated multiple discrete regions with extensive homology. In contrast, other mRNA transcripts, generated by alternative processing, could encode a truncated protein with a novel carboxy-terminal end.

  12. Higher decidual EBI3 and HLA-G mRNA expression in preeclampsia: Cause or consequence of preeclampsia.

    PubMed

    Prins, J R; van der Hoorn, M L P; Keijser, R; Ris-Stalpers, C; van Beelen, E; Afink, G B; Claas, F H J; van der Post, J A M; Scherjon, S A

    2016-01-01

    The maternal immune system must adapt to tolerate the invasion of the allogeneic feto-placental unit. It is generally accepted that improper adaptation causes pregnancy complications like preeclampsia. The Epstein-Barr virus-induced gene 3 (EBI3) protein is a subunit of immune-modulatory cytokines interleukin 27 (IL-27) and IL-35. EBI3 has been reported to associate with HLA-G. In this small pilot study we find higher decidual EBI3 (p<0.05) and HLA-G (p<0.01) mRNA expression in preeclampsia (n=7) compared to normotensive (n=8) pregnancies. Whether the higher EBI3 and HLA-G mRNA expression is a consequence or cause of preeclampsia remains to be answered. Further research to determine the effects on IL-27 and IL-35 is needed. PMID:26472010

  13. Expression of insulin-like growth factors at mRNA levels during the metamorphic development of turbot (Scophthalmus maximus).

    PubMed

    Meng, Zhen; Hu, Peng; Lei, Jilin; Jia, Yudong

    2016-09-01

    Insulin-like growth factors I and II (IGF-I and IGF-II) are important regulators of vertebrate growth and development. This study characterized the mRNA expressions of igf-i and igf-ii during turbot (Scophthalmus maximus) metamorphosis to elucidate the possible regulatory role of the IGF system in flatfish metamorphosis. Results showed that the mRNA levels of igf-i significantly increased at the early-metamorphosis stage and then gradually decreased until metamorphosis was completed. By contrast, mRNA levels of igf-ii significantly increased at the pre-metamorphosis stage and then substantially decreased during metamorphosis. Meanwhile, the whole-body thyroxine (T4) levels varied during larval metamorphosis, and the highest value was observed in the climax-metamorphosis. The mRNA levels of igf-i significantly increased and decreased by T4 and thiourea (TU, inhibitor of endogenous thyroid hormone) during metamorphosis, respectively. Conversely, the mRNA levels of igf-ii remained unchanged. Furthermore, TU significantly inhibited the T4-induced mRNA up-regulation of igf-i during metamorphosis. The whole-body thyroxine (T4) levels were significantly increased and decreased by T4 and TU during metamorphosis, respectively. These results suggested that igf-i and igf-ii may play different functional roles in larval development stages, and igf-i may have a crucial function in regulating the early metamorphic development of turbot. These findings may enhance our understanding of the potential roles of the IGF system to control flatfish metamorphosis and contribute to the improvement of broodstock management for larvae. PMID:27255364

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

    PubMed

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

    2010-04-01

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

  15. Regulation of VEGF and bFGF mRNA expression and other proliferative compounds in skeletal muscle cells.

    PubMed

    Jensen, L; Schjerling, P; Hellsten, Y

    2004-01-01

    The role of muscle contraction, prostanoids, nitric oxide and adenosine in the regulation of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and endothelial cell proliferative compounds in skeletal muscle cell cultures was examined. VEGF and bFGF mRNA, protein release as well as the proliferative effect of extracellular medium was determined in non-stimulated and electro-stimulated rat and human skeletal muscle cells. In rat skeletal muscle cells these aspects were also determined after treatment with inhibitors and/or donors of nitric oxide (NO), prostanoids and adenosine. Electro-stimulation caused an elevation in the VEGF and bFGF mRNA levels of rat muscle cells by 33% and 43% (P < 0.05), respectively, and in human muscle cells VEGF mRNA was elevated by 24%. Medium from electro-stimulated human, but not rat muscle cells induced a 126% higher (P < 0.05) endothelial cell proliferation than medium from non-stimulated cells. Cyclooxygenase inhibition of rat muscle cells induced a 172% increase (P < 0.05) in VEGF mRNA and a 104% increase in the basal VEGF release. Treatment with the NO donor SNAP (0.5 microM) decreased (P < 0.05) VEGF and bFGF mRNA by 42 and 38%, respectively. Medium from SNAP treated muscle cells induced a 45% lower (P < 0.05) proliferation of endothelial cells than control medium. Adenosine enhanced the basal VEGF release from muscle cells by 75% compared to control. The present data demonstrate that contractile activity, NO, adenosine and products of cyclooxygenase regulate the expression of VEGF and bFGF mRNA in skeletal muscle cells and that contractile activity and NO regulate endothelial cell proliferative compounds in muscle extracellular fluid. PMID:15609080

  16. SREBP2 Activation Induces Hepatic Long-chain Acyl-CoA Synthetase 1 (ACSL1) Expression in Vivo and in Vitro through a Sterol Regulatory Element (SRE) Motif of the ACSL1 C-promoter.

    PubMed

    Singh, Amar Bahadur; Kan, Chin Fung Kelvin; Dong, Bin; Liu, Jingwen

    2016-03-01

    Long-chain acyl-CoA synthetase 1 (ACSL1) plays a key role in fatty acid metabolism. To identify novel transcriptional modulators of ACSL1, we examined ACSL1 expression in liver tissues of hamsters fed a normal diet, a high fat diet, or a high cholesterol and high fat diet (HCHFD). Feeding hamsters HCHFD markedly reduced hepatic Acsl1 mRNA and protein levels as well as acyl-CoA synthetase activity. Decreases in Acsl1 expression strongly correlated with reductions in hepatic Srebp2 mRNA level and mature Srebp2 protein abundance. Conversely, administration of rosuvastatin (RSV) to hamsters increased hepatic Acsl1 expression. These new findings were reproduced in mice treated with RSV or fed the HCHFD. Furthermore, the RSV induction of acyl-CoA activity in mouse liver resulted in increases in plasma and hepatic cholesterol ester concentrations and reductions in free cholesterol amounts. Investigations on different ACSL1 transcript variants in HepG2 cells revealed that the mRNA expression of C-ACSL1 was specifically regulated by the sterol regulatory element (SRE)-binding protein (SREBP) pathway, and RSV treatment increased the C-ACSL1 abundance from a minor mRNA species to an abundant transcript. We analyzed 5'-flanking sequence of exon 1C of the human ACSL1 gene and identified one putative SRE site. By performing a promoter activity assay and DNA binding assays, we firmly demonstrated the key role of this SRE motif in SREBP2-mediated activation of C-ACSL1 gene transcription. Finally, we demonstrated that knockdown of endogenous SREBP2 in HepG2 cells lowered ACSL1 mRNA and protein levels. Altogether, this work discovered an unprecedented link between ACSL1 and SREBP2 via the specific regulation of the C-ACSL1 transcript. PMID:26728456

  17. Gene expression profiles of hepatic cell-type specific marker genes in progression of liver fibrosis

    PubMed Central

    Takahara, Yoshiyuki; Takahashi, Mitsuo; Wagatsuma, Hiroki; Yokoya, Fumihiko; Zhang, Qing-Wei; Yamaguchi, Mutsuyo; Aburatani, Hiroyuki; Kawada, Norifumi

    2006-01-01

    AIM: To determine the gene expression profile data for the whole liver during development of dimethylni-trosamine (DMN)-induced hepatic fibrosis. METHODS: Marker genes were identified for different types of hepatic cells, including hepatic stellate cells (HSCs), Kupffer cells (including other inflammatory cells), and hepatocytes, using independent temporal DNA microarray data obtained from isolated hepatic cells. RESULTS: The cell-type analysis of gene expression gave several key results and led to formation of three hypotheses: (1) changes in the expression of HSC-specific marker genes during fibrosis were similar to gene expression data in in vitro cultured HSCs, suggesting a major role of the self-activating characteristics of HSCs in formation of fibrosis; (2) expression of mast cell-specific marker genes reached a peak during liver fibrosis, suggesting a possible role of mast cells in formation of fibrosis; and (3) abnormal expression of hepatocyte-specific marker genes was found across several metabolic pathways during fibrosis, including sulfur-containing amino acid metabolism, fatty acid metabolism, and drug metabolism, suggesting a mechanistic relationship between these abnormalities and symptoms of liver fibrosis. CONCLUSION: Analysis of marker genes for specific hepatic cell types can identify the key aspects of fibrogenesis. Sequential activation of inflammatory cells and the self-supporting properties of HSCs play an important role in development of fibrosis. PMID:17072980

  18. Amylase mRNA transcripts in normal tissues and neoplasms: the implication of different expressions of amylase isogenes.

    PubMed

    Seyama, K; Nukiwa, T; Takahashi, K; Takahashi, H; Kira, S

    1994-01-01

    To understand the cellular origin and mechanism of gene expression in amylase-producing cancers, the phenotyping of amylase isogenes by the polymerase chain reaction and restriction-fragment-length polymorphism using restriction endonucleases TaqI, DdeI, HinfI, and AfaI were performed for 3 amylase-producing lung adenocarcinomas, 16 lung cancers without hyperamylasemia, other human malignant neoplasms, cultured cell lines, and normal tissues. In addition, amylase mRNA transcripts were semi-quantified by the limited polymerase chain reaction. Amylase mRNA transcripts were detected in all of the tissues examined. The AMY1 gene (salivary type) was exclusively and highly expressed in the salivary glands and the amylase-producing lung adenocarcinomas. Coexpression of the AMY1 gene and AMY2 gene (pancreatic type) was observed in most of the lung cancers without hyperamylasemia, lung tissue, and cells scraped from the tracheal epithelium, thyroid, and female genital tract (ovary, fallopian tube, and uterus cervix), while minimal levels of mRNA transcripts of the AMY2 gene were detected in other malignant neoplasms, various normal tissues, and the cultured cell lines. All mRNA transcripts identified as being those of the AMY2 gene were further identified as being from the AMY2B gene except for the transcripts from the pancreas, in which the AMY2A gene and AMY2B gene were coexpressed. On the basis of these results, the clinical occurrence of amylase-producing cancer likely relates to the tissues expressing the AMY1 gene, while the AMY2B gene, which evolutionarily is the oldest gene among human amylase isogenes, is constitutively expressed in various tissues. PMID:7507116

  19. Lung squamous cell carcinoma mRNA expression subtypes are reproducible, clinically important and correspond to different normal cell types

    PubMed Central

    Wilkerson, Matthew D.; Yin, Xiaoying; Hoadley, Katherine A.; Liu, Yufeng; Hayward, Michele C.; Cabanski, Christopher R.; Muldrew, Kenneth; Miller, C. Ryan; Randell, Scott H.; Socinski, Mark A.; Parsons, Alden M.; Funkhouser, William K.; Lee, Carrie B.; Roberts, Patrick J.; Thorne, Leigh; Bernard, Philip S.; Perou, Charles M.; Hayes, D. Neil

    2010-01-01

    Purpose Lung squamous cell carcinoma (SCC) is clinically and genetically heterogeneous and current diagnostic practices do not adequately substratify this heterogeneity. A robust, biologically-based SCC subclassification may describe this variability and lead to more precise patient prognosis and management. We sought to determine if SCC mRNA expression subtypes exist, are reproducible across multiple patient cohorts, and are clinically relevant. Experimental Design Subtypes were detected by unsupervised consensus clustering in five published discovery cohorts of mRNA microarrays, totaling 382 SCC patients. An independent validation cohort of 56 SCC patients was collected and assayed by microarrays. A nearest-centroid subtype predictor was built using discovery cohorts. Validation cohort subtypes were predicted and evaluated for confirmation. Subtype survival outcome, clinical covariates, and biological processes were compared by statistical and bioinformatic methods. Results Four lung SCC mRNA expression subtypes, named primitive, classical, secretory, and basal, were detected and independently validated (P < 0.001). The primitive subtype had the worst survival outcome (P < 0.05) and is an independent predictor of survival (P < 0.05). Tumor differentiation and patient sex were associated with subtype. The subtypes’ expression profiles contained distinct biological processes (primitive – proliferation, classical – xeniobiotics metabolism, secretory – immune response, basal – cell adhesion) and suggested distinct pharmacologic interventions. Comparison to lung model systems revealed distinct subtype to cell type correspondence. Conclusions Lung SCC consists of four mRNA expression subtypes that have different survival outcomes, patient populations, and biological processes. The subtypes stratify patients for more precise prognosis and targeted research. PMID:20643781

  20. Profile of expression of certain markers of apoptosis in chronic hepatitis C and hepatitis B patients in an Egyptian population.

    PubMed

    El-Bendary, Mahmoud; Hawas, Samia; El-Hammady, Dina; Al-Hadidy, Al-Hadidy Mohammed; Eldegla, Heba

    2016-09-01

    Increased peripheral blood mononuclear cell (PBMC) apoptosis during viral hepatitis has been suggested to cause impaired regulation of the immune response and maintenance of the infection. The purpose of this work was to study the expression of some apoptotic markers in chronic hepatitis B (CHB) and C (CHC) infections in order to understand the underlying mechanisms of immune failure and viral persistence. This study aims to evaluate the level of PBMC apoptosis and the expression of the apoptosis-related proteins Fas and Bcl-2 in CHB and CHC patients. This case control study was carried out on 38 cases (group I: 20 chronic HCV patients; group II: 18 chronic HBV patients) attending the Tropical Medicine Clinic, Mansoura University Hospital, in addition to 10 healthy controls. Morphological assessment of apoptosis of cultured PBMCs was done. The level of Fas and Bcl-2 expression by PBMCs was detected using flow cytometry. An increased level of apoptosis correlated with increased Fas expression, but no increase in Bcl-2 expression was found on the surface of PBMCs in CHC and CHB patients compared to controls. No significant difference in the level of apoptosis, Fas, or Bcl2 expression between CHC and CHB patients was detected. Modulation of apoptosis, particularly by manipulation of Fas receptor activation, may be of therapeutic benefit in chronic CHB and CHC. PMID:27262945

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  2. Effects of ketamine exposure on dopamine concentrations and dopamine type 2 receptor mRNA expression in rat brain tissue

    PubMed Central

    Li, Bing; Liu, Mei-Li; Wu, Xiu-Ping; Jia, Juan; Cao, Jie; Wei, Zhi-Wen; Wang, Yu-Jin

    2015-01-01

    Objective: To explore the effects of ketamine abuse on the concentration of dopamine (DA), a monoamine neurotransmitter, and the mRNA expression of dopamine type 2 (D2) receptors in brain tissue, we used male Wistar rats to model ketamine abuse through chronic intraperitoneal infusion of ketamine across different doses. Methods: The rats were sacrificed 45 minutes and 1, 2, and 3 weeks after initiating the administration of ketamine or normal saline, as well as 3 days following discontinuation. Brain tissue was harvested to examine the concentration of 2,5-dihydroxyphenylacetic acid and homovanillic acid, the primary metabolites of DA, as well as the expression of D2 receptor mRNA. In addition, behavioral changes were observed within 30 minutes of administration, and withdrawal symptoms were also documented. A factorial experimental design was used to investigate variations and correlations in the primary outcome measures across the four doses and five time points. Brain DA concentrations were significantly higher in the ketamine-treated groups compared with the saline-treated group, with 30 mg/kg > 10 mg/kg > 60 mg/kg > saline (P < 0.05). The D2 receptor mRNA expression exhibited an inverse downregulation pattern, with 30 mg/kg < 10 mg/kg < 60 mg/kg < saline (P < 0.05). In the 10 mg/kg and 30 mg/kg ketamine-treated groups, the DA concentration and D2 receptor mRNA level in the brain tissue correlated with the dose of ketamine (r = 0.752, r = -0.806), but no significant correlation was found in the 60 mg/kg group. Result: These findings indicated that chronic dosing with ketamine increased the concentration of DA in rat brain tissue by increasing DA release or interrupting DA degradation. D2 receptor mRNA expression likely decreased because of stimulation with excessive DA. Conclusion: High-dose (60 mg/kg) ketamine had potent paralyzing effects on the central nervous system of rats and weakened the excitatory effects of the limbic system. Brain DA and D2 receptor mRNA

  3. mRNA and microRNA expression profiles of the NCI-60 integrated with drug activities

    PubMed Central

    Liu, Hongfang; D’Andrade, Petula; Fulmer-Smentek, Stephanie; Lorenzi, Philip; Kohn, Kurt W.; Weinstein, John N.; Pommier, Yves; Reinhold, William C.

    2010-01-01

    As part of the Spotlight on Molecular Profiling series, we present here new profiling studies of mRNA and microRNA expression for the 60 cell lines of the NCI DTP drug screen (NCI-60) using the 41,000-probe Agilent Whole Human Genome Oligo Microarray and the 15,000-feature Agilent Human microRNA Microarray V2. The expression levels of ~21,000 genes and 723 human microRNAs were measured. These profiling studies include quadruplicate technical replicates for six and eight cell lines for mRNA and microRNA, respectively, and duplicates for the remaining cell lines. The resulting data sets are freely available and searchable online in our CellMiner database. The result indicates high reproducibility for both platforms and an essential biological similarity across the various cell types. The mRNA and microRNA expression levels were integrated with our previously published 1,429-compound database of anticancer activity obtained from the NCI DTP drug screen. Large blocks of both mRNAs and microRNAs were identified with predominately unidirectional correlations to ~1,300 drugs including 121 drugs with known mechanisms of action. The data sets presented here will facilitate the identification of groups of mRNAs, microRNAs and drugs that potentially affect and interact with one another. PMID:20442302

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

    PubMed Central

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

    2012-01-01

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

  5. Chronic stress alters glucocorticoid receptor and mineralocorticoid receptor mRNA expression in the European starling (Sturnus vulgaris) brain.

    PubMed

    Dickens, M; Romero, L M; Cyr, N E; Dunn, I C; Meddle, S L

    2009-10-01

    Although the glucocorticoid response to acute short-term stress is an adaptive physiological mechanism that aids in the response to and survival of noxious stimuli, chronic stress is associated with a negative impact on health. In wild-caught European starlings (Sturnus vulgaris), chronic stress alters the responsiveness of hypothalamic-pituitary-adrenal (HPA) axis as measured by the acute corticosterone response. In the present study, we investigated potential underlying neuroendocrine mechanisms by comparing glucocorticoid receptor and mineralocorticoid receptor mRNA expression in the brains of chronically and nonchronically-stressed starlings. Hypothalamic paraventricular nucleus, but not hippocampal, glucocorticoid receptor mRNA expression in chronically-stressed birds was significantly lower compared to controls, suggesting changes in the efficacy of corticosterone negative feedback. In addition, chronically-stressed birds showed a significant decrease in hippocampal MR mRNA expression. Together, these results suggest that chronic stress changes the brain physiology of wild birds and provides important information for the understanding of the underlying mechanisms that result in dysregulation of the HPA axis in wild animals by chronic stress. PMID:19686439

  6. An mRNA expression signature for prognostication in de novo acute myeloid leukemia patients with normal karyotype

    PubMed Central

    Chou, Wen-Chien; Hou, Hsin-An; Tseng, Mei-Hsuan; Kuo, Yi-Yi; Chen, Yidong; Chuang, Eric Y.; Tien, Hwei-Fang

    2015-01-01

    Although clinical features, cytogenetics, and mutations are widely used to predict prognosis in patients with acute myeloid leukemia (AML), further refinement of risk stratification is necessary for optimal treatment, especially in cytogenetically normal (CN) patients. We sought to generate a simple gene expression signature as a predictor of clinical outcome through analyzing the mRNA arrays of 158 de novo CN AML patients. We compared the gene expression profiles of patients with poor response to induction chemotherapy with those who responded well. Forty-six genes expressed differentially between the two groups. Among them, expression of 11 genes was significantly associated with overall survival (OS) in univariate Cox regression analysis in 104 patients who received standard intensive chemotherapy. We integrated the z-transformed expression levels of these 11 genes to generate a risk scoring system. Higher risk scores were significantly associated with shorter OS (median 17.0 months vs. not reached, P < 0.001) in ours and another 3 validation cohorts. In addition, it was an independent unfavorable prognostic factor by multivariate analysis (HR 1.116, 95% CI 1.035~1.204, P = 0.004). In conclusion, we developed a simple mRNA expression signature for prognostication in CN-AML patients. This prognostic biomarker will help refine the treatment strategies for this group of patients. PMID:26517675

  7. Lack of correlation between mRNA expression and enzymatic activity of the aspartate aminotransferase isoenzymes in various tissues of the rat.

    PubMed

    Abruzzese, F; Greco, M; Perlino, E; Doonan, S; Marra, E

    1995-06-12

    Little is known about control of expression of basal levels of the aspartate aminotransferases which are ubiquitous 'house keeping' enzymes in vertebrates. We have measured both mRNA and activity levels for both isoenzymes in various rat tissues as a function of age. Patterns of mRNA expression for the two isoenzymes were similar in a particular tissue about differed widely between tissues. Surprisingly, there was no simple correlation between mRNA levels and specific activities of the enzyme products. We conclude that translation for mRNA for these two isoenzymes is subject to tissue-specific, and in some cases age-related, regulation. PMID:7789537

  8. Distribution of PTPN22 polymorphisms in SLE from western Mexico: correlation with mRNA expression and disease activity.

    PubMed

    Machado-Contreras, Jesús René; Muñoz-Valle, José Francisco; Cruz, Alvaro; Salazar-Camarena, Diana Celeste; Marín-Rosales, Miguel; Palafox-Sánchez, Claudia Azucena

    2016-08-01

    Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune inflammatory disease characterized by loss of self-tolerance with hyperactivation of autoreactive T and B cells. Protein tyrosine phosphatase non-receptor type 22 (PTPN22) encodes for lymphoid-specific phosphatase (Lyp), which is a key negative regulator of T lymphocyte activation. The aim of this study was to evaluate the genetic contribution of PTPN22 -1123G>C and +1858C>T polymorphisms and their haplotypes in SLE patients, as well as mRNA expression according to -1123G>C promoter polymorphism and disease activity. One hundred and fifty SLE patients and 150 unrelated healthy controls (HC), both Mexican mestizos, were genotyped by PCR-RFLP technique for the PTPN22 -1123G>C and +1858C>T polymorphisms. PTPN22 mRNA expression levels were determined by real-time PCR from PBMCs of thirty patients with SLE and fifteen HC carrying different genotypes. Distributions of genotype and allelic frequencies were similar between SLE and HC. The most frequent alleles were -1123 G and +1858 C in both groups (69 vs. 66 % and 97 vs. 98 %, in SLE and HC, respectively). However, the recessive model of inheritance analysis showed a lower frequency of -1123 CC genotype in SLE patients (7 vs. 15 %), suggesting a protection effect to develop SLE (OR 0.41, CI 1.10-5.28, p = 0.02). Haplotype analysis showed strong linkage disequilibrium D' = 0.98 for PTPN22 -1123G>C and +1858C>T polymorphisms, but haplotypes were not associated with SLE. The PTPN22 mRNA expression did not show differences among -1123G>C genotypes; nevertheless, a significant negative correlation with disease activity was found (r = -0.64, p < 0.01). SLE inactive patients showed similar PTPN22 mRNA expression levels to healthy controls, whereas in patients with severe flare, the expression was nearly depleted. In conclusion, we found a lack of association of PTPN22 -1123G>C and +1858C>T polymorphisms with the risk of developing SLE in a Mexican

  9. Characteristics of mRNA dynamic expression related to spinal cord ischemia/reperfusion injury: a transcriptomics study.

    PubMed

    Qi, Zhi-Ping; Xia, Peng; Hou, Ting-Ting; Li, Ding-Yang; Zheng, Chang-Jun; Yang, Xiao-Yu

    2016-03-01

    Following spinal cord ischemia/reperfusion injury, an endogenous damage system is immediately activated and participates in a cascade reaction. It is difficult to interpret dynamic changes in these pathways, but the examination of the transcriptome may provide some information. The transcriptome reflects highly dynamic genomic and genetic information and can be seen as a precursor for the proteome. We used DNA microarrays to measure the expression levels of dynamic evolution-related mRNA after spinal cord ischemia/reperfusion injury in rats. The abdominal aorta was blocked with a vascular clamp for 90 minutes and underwent reperfusion for 24 and 48 hours. The simple ischemia group and sham group served as controls. After rats had regained consciousness, hindlimbs showed varying degrees of functional impairment, and gradually improved with prolonged reperfusion in spinal cord ischemia/reperfusion injury groups. Hematoxylin-eosin staining demonstrated that neuronal injury and tissue edema were most severe in the 24-hour reperfusion group, and mitigated in the 48-hour reperfusion group. There were 8,242 differentially expressed mRNAs obtained by Multi-Class Dif in the simple ischemia group, 24-hour and 48-hour reperfusion groups. Sixteen mRNA dynamic expression patterns were obtained by Serial Test Cluster. Of them, five patterns were significant. In the No. 28 pattern, all differential genes were detected in the 24-hour reperfusion group, and their expressions showed a trend in up-regulation. No. 11 pattern showed a decreasing trend in mRNA whereas No. 40 pattern showed an increasing trend in mRNA from ischemia to 48 hours of reperfusion, and peaked at 48 hours. In the No. 25 and No. 27 patterns, differential expression appeared only in the 24-hour and 48-hour reperfusion groups. Among the five mRNA dynamic expression patterns, No. 11 and No. 40 patterns could distinguish normal spinal cord from pathological tissue. No. 25 and No. 27 patterns could distinguish simple

  10. Characteristics of mRNA dynamic expression related to spinal cord ischemia/reperfusion injury: a transcriptomics study

    PubMed Central

    Qi, Zhi-ping; Xia, Peng; Hou, Ting-ting; Li, Ding-yang; Zheng, Chang-jun; Yang, Xiao-yu

    2016-01-01

    Following spinal cord ischemia/reperfusion injury, an endogenous damage system is immediately activated and participates in a cascade reaction. It is difficult to interpret dynamic changes in these pathways, but the examination of the transcriptome may provide some information. The transcriptome reflects highly dynamic genomic and genetic information and can be seen as a precursor for the proteome. We used DNA microarrays to measure the expression levels of dynamic evolution-related mRNA after spinal cord ischemia/reperfusion injury in rats. The abdominal aorta was blocked with a vascular clamp for 90 minutes and underwent reperfusion for 24 and 48 hours. The simple ischemia group and sham group served as controls. After rats had regained consciousness, hindlimbs showed varying degrees of functional impairment, and gradually improved with prolonged reperfusion in spinal cord ischemia/reperfusion injury groups. Hematoxylin-eosin staining demonstrated that neuronal injury and tissue edema were most severe in the 24-hour reperfusion group, and mitigated in the 48-hour reperfusion group. There were 8,242 differentially expressed mRNAs obtained by Multi-Class Dif in the simple ischemia group, 24-hour and 48-hour reperfusion groups. Sixteen mRNA dynamic expression patterns were obtained by Serial Test Cluster. Of them, five patterns were significant. In the No. 28 pattern, all differential genes were detected in the 24-hour reperfusion group, and their expressions showed a trend in up-regulation. No. 11 pattern showed a decreasing trend in mRNA whereas No. 40 pattern showed an increasing trend in mRNA from ischemia to 48 hours of reperfusion, and peaked at 48 hours. In the No. 25 and No. 27 patterns, differential expression appeared only in the 24-hour and 48-hour reperfusion groups. Among the five mRNA dynamic expression patterns, No. 11 and No. 40 patterns could distinguish normal spinal cord from pathological tissue. No. 25 and No. 27 patterns could distinguish simple

  11. Effects of the PPARα agonist WY-14,643 on plasma lipids, enzymatic activities and mRNA expression of lipid metabolism genes in a marine flatfish, Scophthalmus maximus.

    PubMed

    Urbatzka, R; Galante-Oliveira, S; Rocha, E; Lobo-da-Cunha, A; Castro, L F C; Cunha, I

    2015-07-01

    Fibrates and other lipid regulator drugs are widespread in the aquatic environment including estuaries and coastal zones, but little is known on their chronic effects on non-target organisms as marine fish. In the present study, turbot juveniles were exposed to the PPARα model agonist WY-14,643 for 21 days by repeated injections at the concentrations of 5mg/kg (lo-WY) and 50mg/kg (hi-WY), and samples taken after 7 and 21 days. Enzyme activity and mRNA expression of palmitoyl-CoA oxidase and catalase in the liver were analyzed as first response, which validated the experiment by demonstrating interactions with the peroxisomal fatty acid oxidation and oxidative stress pathways in the hi-WY treatment. In order to get mechanistic insights, alterations of plasma lipids (free cholesterol, FC; HDL associated cholesterol, C-HDL; triglycerides, TG; non-esterified fatty acids, NEFA) and hepatic mRNA expression of 17 genes involved in fatty acid and lipid metabolism were studied. The exposure to hi-WY reduced the quantity of plasma FC, C-HDL, and NEFA. Microsomal triglyceride transfer protein and apolipoprotein E mRNA expression were higher in hi-WY, and indicated an increased formation of VLDL particles and energy mobilization from liver. It is speculated that energy depletion by PPARα agonists may contribute to a higher susceptibility to environmental stressors. PMID:25974001

  12. Hepatitis C virus (HCV) proteins induce NADPH oxidase 4 expression in a transforming growth factor beta-dependent manner: a new contributor to HCV-induced oxidative stress.

    PubMed

    Boudreau, Howard E; Emerson, Suzanne U; Korzeniowska, Agnieszka; Jendrysik, Meghan A; Leto, Thomas L

    2009-12-01

    Viral hepatitis-induced oxidative stress accompanied by increased levels of transforming growth factor beta (TGF-beta) and hepatic fibrosis are hallmarks of hepatitis C virus (HCV) infection. The mechanisms of redox regulation in the pathogenesis of HCV-induced liver disease are not clearly understood. The results of our current studies suggest that reactive oxygen species (ROS) derived from Nox4, a member of the NADPH oxidase (Nox) family, could play a role in HCV-induced liver disease. We found that the expression of HCV (genotype 1a) cDNA constructs (full-length and subgenomic), core protein alone, viral RNA, or replicating HCV (JFH-AM2) induced Nox4 mRNA expression and ROS generation in human hepatocyte cell lines (Huh-7, Huh-7.5, HepG2, and CHL). Conversely, hepatocytes expressing Nox4 short hairpin RNA (shRNA) or an inactive dominant negative form of Nox4 showed decreased ROS production when cells were transfected with HCV. The promoters of both human and murine Nox4 were used to demonstrate transcriptional regulation of Nox4 mRNA by HCV, and a luciferase reporter tied to an approximately 2-kb promoter region of Nox4 identified HCV-responsive regulatory regions modulating the expression of Nox4. Furthermore, the human Nox4 promoter was responsive to TGF-beta1, and the HCV core-dependent induction of Nox4 was blocked by antibody against TGF-beta or the expression of dominant negative TGF-beta receptor type II. These findings identified HCV as a regulator of Nox4 gene expression and subsequent ROS production through an autocrine TGF-beta-dependent mechanism. Collectively, these data provide evidence that HCV-induced Nox4 contributes to ROS production and may be related to HCV-induced liver disease. PMID:19812163

  13. Analysis of early hepatic stage schistosomula gene expression by subtractive expressed sequence tags library.

    PubMed

    Wang, Xinzhi; Gobert, Geoffrey N; Feng, XinGang; Fu, Zhiqiang; Jin, Yamei; Peng, Jinbiao; Lin, Jiaojiao

    2009-07-01

    Schistosome parasites require a complex lifecycle requiring two hosts and aquatic phases of development. The schistosomula is a key phase of parasite development within the mammalian host, however relatively little is understood about the molecular processes underlying this stage. In this study 5723 subtractive expressed sequence tags (ESTs) were randomly selected from a 7 day hepatic schistosomula enriched library constructed using suppression subtractive hybridization method. Sequence analysis of these ESTs identified 1762 unique genes (contigs). Among them, 989 contigs were annotated with known genes, 311 contigs were homologous to established genes, 101 contigs were similar to established genes, 72 contigs were weakly similar to established genes and 289 sequences did not match any published sequences. Genes identified related to metabolism, cellular development, immune evasion and host-parasite interactions were identified as enriched in the hepatic schistosomula stage. The future identification of poorly annotated but stage-specific genes may potentially represent new drugs or vaccine targets, applicable for the future controlling of schistosomiasis. PMID:19428674

  14. The Promoter Methylation Status and mRNA Expression Levels of CTCF and SIRT6 in Sporadic Breast Cancer

    PubMed Central

    Wang, Da; Zhang, Xuemei

    2014-01-01

    Promoter hypermethylation causes gene silencing and is thought to be an early event in carcinogenesis. This study was to detect promoter methylation status and mRNA expression levels of CCCTC-binding factor (CTCF) and sirtuin 6 (SIRT6), and to explore the relationship between methylation and mRNA expression in breast cancer patient samples. Promoter methylation analysis and expression profile analysis of two genes were performed by methylation-specific PCR, bisulfite sequencing PCR, and quantitative real-time PCR in cancer lesions and matched normal tissues. The promoter region of CTCF has not been hypermethylated in all patient samples. In contrast, methylation of SIRT6 gene was present in invasive cancers (93.5%) and matched normal tissues (96.8%) from 62 patients. Promoter hypermethylation of SIRT6 was also observed in ductal carcinoma in situ (three of three) and matched normal tissues (two of three). mRNA expression of CTCF and SIRT6 in invasive tumors showed a lower level than that in paired normal tissues (p=0.008 and p=0.030, respectively). The fold change values of CTCF expression were significantly lower in invasive ductal cancer lesions with Ki-67-positive status (p=0.042). In conclusion, our data showed that the methylation status of CTCF and SIRT6 promoter regions was not statistically different in cancer lesions compared with matched normal tissues. No significant association between promoter methylation status and expression profiles of CTCF and SIRT6 was found in invasive breast cancers. PMID:24842653

  15. Molecular cloning, mRNA expression, and characterization of HSP90 gene from Chinese mitten crab Eriocheir japonica sinensis.

    PubMed

    Li, Peng; Zha, Jie; Zhang, Zhenhua; Huang, Hua; Sun, Hongying; Song, Daxiang; Zhou, Kaiya

    2009-07-01

    HSP90 is a highly conserved molecular chaperone important in the maturation of a broad spectrum of proteins. Using expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques, an HSP90 gene designated as EjsHSP90 was cloned and characterized from the Chinese mitten crab Eriocheir japonica sinensis. The full-length cDNA of EjsHSP90 is 2,517 bp and contains an open reading frame of 2157 bp which encodes a 718 amino acid polypeptide (82.8 kDa) bearing characteristics of the HSP90 family and an ATP binding domain. Sequence alignment shows that EjsHSP90 shared 79%-96% identity with HSP90 sequences reported in other animals, and it shares identical structural features. Fluorescent real-time quantitative RT-PCR approach was performed to examine the expression profiles of EjsHSP90 mRNA by testing its relative level in three types of tissues at three different developmental stages, respectively. We found that EjsHSP90 is expressed throughout the three developmental stages but expression levels varied among different body parts of crabs. EjsHSP90 mRNA expression in the abdomen of the first crab stage is consistently higher than that of the other two stages, suggesting that EjsHSP90 gene is involved in the crabs' early developmental process, especially in the crab brachyurization process. Results from quantitative RT-PCR excluded the possibility that the expression of EjsHSP90 mRNA is induced primarily by osmotic stress. Phylogenetic analyses reveal that HSP90 gene is informative and complementary for reconstruction of arthropod phylogenetic relationships. PMID:19166961

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

    PubMed Central

    NISHIYAMA, Yoshihiro; NAKAYAMA, Shouta M.M.; WATANABE, Kensuke P.; KAWAI, Yusuke K.; OHNO, Marumi; IKENAKA, Yoshinori; ISHIZUKA, Mayumi

    2016-01-01

    Rat cytochrome P450 (CYP) exhibits inter-strain differences, but their analysis has been scattered across studies under different conditions. To identify these strain differences in CYP more comprehensively, mRNA expression, protein expression and metabolic activity among Wistar (WI), Sprague Dawley (SD), Dark Agouti (DA) and Brown Norway (BN) rats were compared. The mRNA level and enzymatic activity of CYP1A1 were highest in SD rats. The rank order of Cyp3a2 mRNA expression mirrored its protein expression, i.e., DA>BN>SD>WI, and was similar to the CYP3A2-dependent warfarin metabolic activity, i.e., DA>SD>BN>WI. These results suggest that the strain differences in CYP3A2 enzymatic activity are caused by differences in mRNA expression. Cyp2b1 mRNA levels, which were higher in DA rats, did not correlate with its protein expression or enzymatic activity. This suggests that the strain differences in enzymatic activity are not related to Cyp2b1 mRNA expression. In conclusion, WI rats tended to have the lowest CYP1A1, 2B1 and 3A2 mRNA expression, protein expression and enzymatic activity among the strains. In addition, SD rats had the highest CYP1A1 mRNA expression and activity, while DA rats had higher CYP2B1 and CYP3A2 mRNA and protein expression. These inter-strain differences in CYP could influence pharmacokinetic considerations in preclinical toxicological studies. PMID:26806536

  17. Relation between mRNA expression and sequence information in Desulfovibrio vulgaris: Combinatorial contributions of upstream regulatory motifs and coding sequence features to variations in mRNA abundance

    SciTech Connect

    Wu, Gang; Nie, Lei; Zhang, Weiwen

    2006-05-26

    ABSTRACT-The context-dependent expression of genes is the core for biological activities, and significant attention has been given to identification of various factors contributing to gene expression at genomic scale. However, so far this type of analysis has been focused whether on relation between mRNA expression and non-coding sequence features such as upstream regulatory motifs or on correlation between mRN abundance and non-random features in coding sequences (e.g. codon usage and amino acid usage). In this study multiple regression analyses of the mRNA abundance and all sequence information in Desulfovibrio vulgaris were performed, with the goal to investigate how much coding and non-coding sequence features contribute to the variations in mRNA expression, and in what manner they act together...

  18. Expression of beta 3-adrenoceptor mRNA in rat brain.

    PubMed Central

    Summers, R. J.; Papaioannou, M.; Harris, S.; Evans, B. A.

    1995-01-01

    The reverse transcription/polymerase chain reaction was used to demonstrate beta 3-adrenoceptor mRNA in rat brain regions. Levels were highest in hippocampus, cerebral cortex and striatum and lower in hypothalamus, brainstem and cerebellum. Images Figure 1 PMID:8590968

  19. Genome-wide identification and analysis of mRNA expression in fibroblasts, ES cells, and iPS cells.

    PubMed

    Hirai, Hiroyuki; Kikyo, Nobuaki

    2016-03-01

    Genome-wide expression patterns of mRNA were compared between mouse embryonic fibroblasts (MEFs), embryonic stem cells (ESCs), and various types of induced pluripotent stem cells (iPSCs). iPSCs were established and maintained using modified Oct4 with or without exogenous leukemia inhibitory factor (LIF) and used to identify mRNAs that were potentially involved in the LIF-independence. The data have been deposited in the NCBI's Gene Expression Omnibus (GEO) database with the accession number GSE65563. PMID:26981399

  20. Genome-wide identification and analysis of mRNA expression in fibroblasts, ES cells, and iPS cells

    PubMed Central

    Hirai, Hiroyuki; Kikyo, Nobuaki

    2015-01-01

    Genome-wide expression patterns of mRNA were compared between mouse embryonic fibroblasts (MEFs), embryonic stem cells (ESCs), and various types of induced pluripotent stem cells (iPSCs). iPSCs were established and maintained using modified Oct4 with or without exogenous leukemia inhibitory factor (LIF) and used to identify mRNAs that were potentially involved in the LIF-independence. The data have been deposited in the NCBI's Gene Expression Omnibus (GEO) database with the accession number GSE65563. PMID:26981399

  1. Basal expression of nucleoside transporter mRNA differs among small intestinal epithelia of beef steers and is differentially altered by ruminal or abomasal infusion of starch hydrolysate.

    PubMed

    Liao, S F; Alman, M J; Vanzant, E S; Miles, E D; Harmon, D L; McLeod, K R; Boling, J A; Matthews, J C

    2008-04-01

    In ruminants, microbial-derived nucleic acids are a major source of N and are absorbed as nucleosides by small intestinal epithelia. Although the biochemical activities of 2 nucleoside transport systems have been described for cattle, little is known regarding the regulation of their gene expression. This study was conducted to test 2 hypotheses: (1) the small intestinal epithelia of beef cattle differentially express mRNA for 3 concentrative (CNT1, 2, 3) and 2 equilibrative (ENT1, 2) nucleoside transporters (NT), and (2) expression of these NT is responsive to small intestine luminal supply of rumen-derived microbes (hence, nucleosides), energy (cornstarch hydrolysate, SH), or both. Eighteen ruminally and abomasally catheterized Angus steers (260 +/- 17 kg of BW) were fed an alfalfa cube-based diet at 1.33x NE(m) requirement. Six steers in each of 3 periods were blocked by BW (heavy vs. light). Within each block, 3 steers were randomly assigned to 3 treatments (n = 6): ruminal and abomasal water infusion (control), ruminal SH infusion/abomasal water infusion, or ruminal water infusion/abomasal SH infusion. The dosage of SH infusion amounted to 20% of ME intake. After a 14-or 16-d infusion period, steers were slaughtered, and duodenal, jejunal, and ileal epithelia were harvested for total RNA extraction and the relative amounts of mRNA expressed were determined using real-time RT-PCR quantification methodologies. All 5 NT mRNA were found expressed by each epithelium, but their abundance differed among epithelia. Specifically, jejunal expression of all 5 NT mRNA was higher than that by the ileum, whereas jejunal expression of CNT1, CNT3, and ENT1 mRNA was higher, or tended to be higher, than duodenal expression. Duodenal expression of CNT2, CNT3, and ENT2 mRNA was higher than ileal expression. With regard to SH infusion treatments, ruminal infusion increased duodenal expression of CNT3 (67%), ENT1 (51%), and ENT2 (39%) mRNA and ileal expression of CNT3 (210%) and

  2. Thyroid Hormone Regulates Hepatic Expression of Fibroblast Growth Factor 21 in a PPARα-dependent Manner*

    PubMed Central

    Adams, Andrew C.; Astapova, Inna; Fisher, ffolliott M.; Badman, Michael K.; Kurgansky, Katherine E.; Flier, Jeffrey S.; Hollenberg, Anthony N.; Maratos-Flier, Eleftheria

    2010-01-01

    Thyroid hormone has profound and diverse effects on liver metabolism. Here we show that tri-iodothyronine (T3) treatment in mice acutely and specifically induces hepatic expression of the metabolic regulator fibroblast growth factor 21 (FGF21). Mice treated with T3 showed a dose-dependent increase in hepatic FGF21 expression with significant induction at doses as low as 100 μg/kg. Time course studies determined that induction is seen as early as 4 h after treatment with a further increase in expression at 6 h after injection. As FGF21 expression is downstream of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα), we treated PPARα knock-out mice with T3 and found no increase in expression, indicating that hepatic regulation of FGF21 by T3 in liver is via a PPARα-dependent mechanism. In contrast, in white adipose tissue, FGF21 expression was suppressed by T3 treatment, with other T3 targets unaffected. In cell culture studies with an FGF21 reporter construct, we determined that three transcription factors are required for induction of FGF21 expression: thyroid hormone receptor β (TRβ), retinoid X receptor (RXR), and PPARα. These findings indicate a novel regulatory pathway whereby T3 positively regulates hepatic FGF21 expression, presenting a novel therapeutic target for diseases such as non-alcoholic fatty liver disease. PMID:20236931

  3. Surface expression and cytolytic function of natural killer cell receptors is altered in chronic hepatitis C

    PubMed Central

    Nattermann, J; Feldmann, G; Ahlenstiel, G; Langhans, B; Sauerbruch, T; Spengler, U

    2006-01-01

    Introduction Impaired activity of natural killer (NK) cells has been proposed as a mechanism contributing to viral persistence in hepatitis C virus (HCV) infection. As the function of NK cells is primarily regulated by NK cell receptors (NKR), we analysed whether decreased NK cell function in hepatitis C may be related to dysregulated NKR expression. Patients and methods Expression of NK cell was analysed by flow cytometry on lymphocytes from HCV(+) subjects (n = 30), patients who became HCV(−) after antiviral therapy (n = 10), healthy individuals (n = 10), and hepatitis B virus (HBV) infected patients (n = 9). Cytolytic function of lymphocytes was studied in a redirected lysis assay and in a standard 51chromium release cytotoxicity assay, respectively. Results In patients with chronic hepatitis C, we found a significantly reduced proportion of NKp46 and NKp30 expressing NK cells compared with healthy and HBV infected subjects. Low expression of natural cytotoxicity receptor (NCR) was also confirmed in in vitro activated NK cell populations derived from HCV patients compared with uninfected donors. In contrast, patients who cleared HCV under antiviral therapy showed normal expression of NKp44, NKp30, and NKp46. Reduced NCR expression in chronic hepatitis C was associated with a parallel decrease in NCR mediated target cell killing. Furthermore, we found a significantly increased proportion of NKG2A expressing NK cells and CD8+ T cells in HCV positive patients, resulting in a reduced cytolytic activity against cells incubated with the HLA‐E stabilising peptide HCV core35–44. Conclusion The present study indicates that defective expression of NKR represents a novel mechanism contributing to impaired function of NK cells and CD8+ T cells in chronic hepatitis C. PMID:16322112

  4. Cysteine Sulfinic Acid Decarboxylase Regulation: A Role for FXR and SHP in Murine Hepatic Taurine Metabolism

    PubMed Central

    Kerr, Thomas A.; Matsumoto, Yuri; Matsumoto, Hitoshi; Xie, Yan; Hirschberger, Lawrence L.; Stipanuk, Martha H.; Anakk, Sayeepriyadarshini; Moore, David D.; Watanabe, Mitsuhiro; Kennedy, Susan

    2014-01-01

    Background Bile acid synthesis is regulated by nuclear receptors including farnesoid X receptor (FXR) and small heterodimer partner (SHP), and by fibroblast growth factor15/19 (FGF15/19). Because bile acid synthesis involves amino acid conjugation, we hypothesized that hepatic cysteine sulfinic acid decarboxylase (CSAD) (a key enzyme in taurine synthesis) is regulated by bile acids. Aims To investigate CSAD regulation by bile acids and CSAD regulatory mechanisms. Methods Mice were fed a control diet or a diet supplemented with either 0.5% cholate or 2% cholestyramine. To gain mechanistic insight into CSAD regulation, we utilized GW4064 (FXR agonist), FGF19, or T-0901317 (LXR agonist) and Shp−/− mice. Tissue mRNA expression was determined by qRT-PCR. Amino acids were measured by HPLC. Results Mice supplemented with dietary cholate exhibited reduced hepatic CSAD mRNA expression while those receiving cholestyramine exhibited increased hepatic CSAD mRNA expression. Activation of FXR suppressed CSAD mRNA expression whereas hepatic CSAD mRNA expression was increased in Shp−/− mice. Hepatic hypotaurine concentration (the product of CSAD) was higher in Shp−/− mice with a corresponding increase in serum (but not hepatic) taurine-conjugated bile acids. FGF19 administration suppressed hepatic CYP7A1 mRNA but did not change CSAD mRNA expression. LXR activation induced CYP7A1 mRNA yet failed to induce CSAD mRNA expression. Conclusion CSAD mRNA expression is physiologically regulated by bile acids in a feedback fashion via mechanisms involving SHP and FXR but not FGF15/19 or LXR. These novel findings implicate bile acids as regulators of CSAD mRNA via mechanisms shared in part with CYP7A1. PMID:24033844

  5. Changes in neurotransmitter levels and proinflammatory cytokine mRNA expressions in the mice olfactory bulb following nanoparticle exposure

    SciTech Connect

    Tin-Tin-Win-Shwe Mitsushima, Dai; Yamamoto, Shoji; Fukushima, Atsushi; Funabashi, Toshiya; Kobayashi, Takahiro; Fujimaki, Hidekazu

    2008-01-15

    Recently, there have been increasing reports that nano-sized component of particulate matter can reach the brain and may be associated with neurodegenerative diseases. Previously, our laboratory has studied the effect of intranasal instillation of nano-sized carbon black (CB) (14 nm and 95 nm) on brain cytokine and chemokine mRNA expressions and found that 14-nm CB increased IL-1{beta}, TNF-{alpha}, CCL2 and CCL3 mRNA expressions in the olfactory bulb, not in the hippocampus of mice. To investigate the effect of a single administration of nanoparticles on neurotransmitters and proinflammatory cytokines in a mouse olfactory bulb, we performed in vivo microdialysis and real-time PCR methods. Ten-week-old male BALB/c mice were implanted with guide cannula in the right olfactory bulb and, 1 week later, were instilled vehicle or CB (14 nm, 250 {mu}g) intranasally. Six hours after the nanoparticle instillation, the mice were intraperitoneally injected with normal saline or 50 {mu}g of bacteria cell wall component lipoteichoic acid (LTA), which may potentiate CB-induced neurologic effect. Extracellular glutamate and glycine levels were significantly increased in the olfactory bulb of CB-instilled mice when compared with vehicle-instilled control mice. Moreover, we found that LTA further increased glutamate and glycine levels. However, no alteration of taurine and GABA levels was observed in the olfactory bulb of the same mice. We also detected immunological changes in the olfactory bulb 11 h after vehicle or CB instillation and found that IL-1{beta} mRNA expression was significantly increased in CB- and LTA-treated mice when compared with control group. However, TNF-{alpha} mRNA expression was increased significantly in CB- and saline-treated mice when compared with control group. These findings suggest that nanoparticle CB may modulate the extracellular amino acid neurotransmitter levels and proinflammatory cytokine IL-1 {beta} mRNA expressions synergistically with LTA

  6. [Effects of PTK787 on cell proliferation and expression of fak mRNA in K562].

    PubMed

    Di, Xiao-Hua; Chen, Ri-Ling; Liu, Xiao-Li; Tian, Chuan; Guo, Ya-Nan

    2010-06-01

    The aim of this study was to investigate the effects of tyrosine kinase inhibitor PTK787 on cell proliferation, cell cycle and the expression of fak mRNA of human chronic myeloid leukemia (CML) cell line K562, and to explore the mechanism of PTK787 against acute myeloid leukemia. The MTT method was used to detect the effects of PTK787 in various concentrations and at different time points on proliferation of K562 cells; the flow cytometry was used to determine the effects of PTK787 in different concentrations on cell cycle of K562 cells; the RT-PCR was used to assay the expression of fak mRNA in K562 cells treated with PTK787 for 48 hours. The results showed that along with increasing of the concentration and prolonging of time, the inhibitory rate of PTK787 on K562 proliferation was gradually enhanced. The comparison between various concentration groups at same time or comparison between various time groups in same concentration showed significant differences (p < 0.05), in which the effect of 320 micromol/L PTK787 on cells was strongest, while the continuous increase of PTK787 concentration or prolong of action time did not enhance the inhibitory rate on K562 proliferation. With increasing of drug concentration, the cell proportion in G(1) phase gradually increased, the cell proportion in S phase gradually decreased, the comparison between various groups revealed significant differences (p < 0.05), however the continuous increase of drug concentration from 160 micromol/L did not obviously change the cell proportion in phases of cell cycle. With increasing of drug concentration, the expression of fak mRNA in K562 cells gradually reduced with significant differences between various groups (p < 0.05), but with continuous increase of drug concentration from 160 micromol/L, the effect of PTK787 on the expression of fak mRNA in K562 cells also did not obviously change. It is concluded that the PTK787 shows effect of anti-leukemia cells through inhibiting transformation

  7. Changes in neurotransmitter levels and proinflammatory cytokine mRNA expressions in the mice olfactory bulb following nanoparticle exposure.

    PubMed

    Tin-Tin-Win-Shwe; Mitsushima, Dai; Yamamoto, Shoji; Fukushima, Atsushi; Funabashi, Toshiya; Kobayashi, Takahiro; Fujimaki, Hidekazu

    2008-01-15

    Recently, there have been increasing reports that nano-sized component of particulate matter can reach the brain and may be associated with neurodegenerative diseases. Previously, our laboratory has studied the effect of intranasal instillation of nano-sized carbon black (CB) (14 nm and 95 nm) on brain cytokine and chemokine mRNA expressions and found that 14-nm CB increased IL-1 beta, TNF-alpha, CCL2 and CCL3 mRNA expressions in the olfactory bulb, not in the hippocampus of mice. To investigate the effect of a single administration of nanoparticles on neurotransmitters and proinflammatory cytokines in a mouse olfactory bulb, we performed in vivo microdialysis and real-time PCR methods. Ten-week-old male BALB/c mice were implanted with guide cannula in the right olfactory bulb and, 1 week later, were instilled vehicle or CB (14 nm, 250 microg) intranasally. Six hours after the nanoparticle instillation, the mice were intraperitoneally injected with normal saline or 50 mug of bacteria cell wall component lipoteichoic acid (LTA), which may potentiate CB-induced neurologic effect. Extracellular glutamate and glycine levels were significantly increased in the olfactory bulb of CB-instilled mice when compared with vehicle-instilled control mice. Moreover, we found that LTA further increased glutamate and glycine levels. However, no alteration of taurine and GABA levels was observed in the olfactory bulb of the same mice. We also detected immunological changes in the olfactory bulb 11 h after vehicle or CB instillation and found that IL-1 beta mRNA expression was significantly increased in CB- and LTA-treated mice when compared with control group. However, TNF-alpha mRNA expression was increased significantly in CB- and saline-treated mice when compared with control group. These findings suggest that nanoparticle CB may modulate the extracellular amino acid neurotransmitter levels and proinflammatory cytokine IL-1 beta mRNA expressions synergistically with LTA in the

  8. Versican gene expression in human articular cartilage and comparison of mRNA splicing variation with aggrecan.

    PubMed Central

    Grover, J; Roughley, P J

    1993-01-01

    The chondrocytes in human articular cartilage from subjects of all ages express mRNAs for both of the aggregating proteoglycans aggrecan and versican, although the level of expression of versican mRNA is much lower than that of aggrecan mRNA. Aggrecan shows alternative splicing of the epidermal growth factor (EGF)-like domain within its C-terminal globular region, but there is no evidence for a major difference in situ in the relative expression of this domain with age. At all ages studied from birth to the mature adult, a greater proportion of transcripts lacked the EGF domain. The relative proportions of the two transcripts did not change upon culture and passage of isolated chondrocytes. In contrast, the neighbouring complement regulatory protein (CRP)-like domain was predominantly expressed irrespective of age, but cell culture did result in variation of the splicing of this domain. Versican possesses two EGF-like domains and one CRP-like domain, but at all ages the three domains were predominantly present in all transcripts. This situation persisted upon culture and passage of the chondrocytes. Thus, unlike aggrecan, the versican expressed by human articular cartilage does not appear to undergo alternative splicing of its C-terminal globular region, either in cartilage in situ or in chondrocytes in culture. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8484718

  9. Expression of the hepatitis B virus genome in chronic hepatitis B carriers and patients with hepatocellular carcinoma

    SciTech Connect

    Bowyer, S.M.; Dusheiko, G.M.; Schoub, B.D.; Kew, M.C.

    1987-02-01

    The authors examined the methylation status of CCGG sites in hepatitis B virus (HBV) DNA to determine whether methylation could be responsible for the selective expression of the HBV surface gene in chronic hepatitis B infection and hepatocellular carcinoma. Infected liver tissue from patients with low levels of viral replication was analyzed for HBV DNA copy number per haploid cell genome. Total cellular DNA, with sufficient HBV DNA, was digested with the restriction endonucleases Msp I and Hpa II, to determine whether the HBV DNA was methylated, or HindIII, to determine whether the HBV DNA was integrated or episomal. The cleavage fragments were analyzed by Southern blotting and hybridization to /sup 32/P-labeled HBV DNA. In replicative chronic hepatitis B, hypomethylation of the HBV genome correlated with HBV expression in both virions and infected tissue. In carriers with nonreplicative infection, it was difficult to ascertain the role of methylation as copy number was low. HBV DNA copy number was also low in 17 out of 29 of the rumor tissues tested and as many as 14 out of 16 of the adjacent non-neoplastic tissues tested. Integrated sequences were hypermethylated in the PLC/PRF/5 cell line and in six of the tumor tissues suggesting that methylation plays a role in HBV gene repression. However, since DNA from five other tumors was hypomethylated, the belief that methylation per se is an absolute determinant of HBV core gene repression does not hold for human hepatocellular carcinoma tissue.

  10. Runx3 Expression Inhibits Proliferation and Distinctly Alters mRNA Expression of Bax in AGS and A549 Cancer Cells

    PubMed Central

    Torshabi, Maryam; Faramarzi, Mohammad Ali; Tabatabaei Yazdi, Mojtaba; Ostad, Seyyed Naser; Gharemani, Mohammad Hosein

    2011-01-01

    Runx3, a member of Runt-related transcription factor (Runx) proteins with tumor suppressor effect, is a tissue–restricted and cancer related transcription factor that regulate cell proliferation and growth, as well as differentiation. In the present study, exogenous Run3 was transiently expressed in AGS (human gastric adenocarcinoma), with undetectable Runx3 protein and in A549 (human lung carcinoma) with low levels of endogenous Runx3 protein. The GFP tagged Runx3 was transfected into AGS and A549 cells using fugene6 and PolyFect and Runx3 expression was confirmed by fluorescent microscopy and RT-PCR. The effect of Runx3 transfection on cell proliferation was determined by MTT assay and the results were confirmed by the trypan blue dye exclusion method. The effect of Runx3 expression on mRNA expression of BCL2-associated X protein (Bax) was evaluated using RT-PCR. In AGS and A549 cells, Runx3 expression inhibited cell proliferation (p < 0.01). The growth inhibition was less in A549 cells. We show that Runx3 expression increases Bax mRNA expression in AGS cells when compared with control (p < 0.05), but no significant differences in mRNA expression was observed in both examined cells. Runx3 expression has antiproliferative effect in AGS cell perhaps via increase in expression of Bax. The effect of Runx3 on A549 cells’ viability which has endogenous level of Runx3 is not related to Bax. These findings implicate a complex regulation by Runx3 in inhibition of cell proliferation utilizing Bax. PMID:24250365

  11. Striatal mRNA expression patterns underlying peak dose l-DOPA-induced dyskinesia in the 6-OHDA hemiparkinsonian rat.

    PubMed

    Smith, L M; Parr-Brownlie, L C; Duncan, E J; Black, M A; Gemmell, N J; Dearden, P K; Reynolds, J N J

    2016-06-01

    l-DOPA is the primary pharmacological treatment for relief of the motor symptoms of Parkinson's disease (PD). With prolonged treatment (⩾5years) the majority of patients will develop abnormal involuntary movements as a result of l-DOPA treatment, known as l-DOPA-induced dyskinesia. Understanding the underlying mechanisms of dyskinesia is a crucial step toward developing treatments for this debilitating side effect. We used the 6-hydroxydopamine (6-OHDA) rat model of PD treated with a three-week dosing regimen of l-DOPA plus the dopa decarboxylase inhibitor benserazide (4mg/kg and 7.5mg/kgs.c., respectively) to induce dyskinesia in 50% of individuals. We then used RNA-seq to investigate the differences in mRNA expression in the striatum of dyskinetic animals, non-dyskinetic animals, and untreated parkinsonian controls at the peak of dyskinesia expression, 60min after l-DOPA administration. Overall, 255 genes were differentially expressed; with significant differences in mRNA expression observed between all three groups. In dyskinetic animals 129 genes were more highly expressed and 14 less highly expressed when compared with non-dyskinetic and untreated parkinsonian controls. In l-DOPA treated animals 42 genes were more highly expressed and 95 less highly expressed when compared with untreated parkinsonian controls. Gene set cluster analysis revealed an increase in expression of genes associated with the cytoskeleton and phosphoproteins in dyskinetic animals compared with non-dyskinetic animals, which is consistent with recent studies documenting an increase in synapses in dyskinetic animals. These genes may be potential targets for drugs to ameliorate l-DOPA-induced dyskinesia or as an adjunct treatment to prevent their occurrence. PMID:26968766

  12. Effect of long real space flight on the whole genome mRNA expression properties in medaka Oryzias latipes

    NASA Astrophysics Data System (ADS)

    Kozlova, Olga; Gusev, Oleg; Levinskikh, Margarita; Sychev, Vladimir; Poddubko, Svetlana

    The current study is addressed to the complex analysis of whole genome mRNA expression profile and properties of splicing variants formation in different organs of medaka fish exposed to prolonged space flight in the frame of joint Russia-Japan research program “Aquarium-AQH”. The fish were kept in the AQH joint-aquariums system in October-December 2013, followed by fixation in RNA-preserving buffers and freezing during the space flight. The samples we returned to the Earth frozen in March 2013 and mRNAs from four fish were sequenced in organ-specific manner using HiSeq Illumina sequencing platform. The ground group fish treated in the same way was used as a control. The comparison between the groups revealed space group-specific specific mRNA expression pattern. More than 50 genes (including several types of myosins) were down-regulated in the space group. Moreover, we found an evidence for formation of space group-specific splicing variants of mRNA. Taking together, the data suggest that in spite of aquatic environment, space flight-associated factors have a strong effect on the activity of fish genome. This work was supported in part by subsidy of the Russian Government to support the Program of competitive growth of Kazan Federal University among world class academic centres and universities.

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

    PubMed

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

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation. PMID:27112822

  14. Upregulation of metabotropic glutamate receptor 8 mRNA expression in the rat forebrain after repeated amphetamine administration

    PubMed Central

    Parelkar, Nikhil K; Wang, John Q.

    2008-01-01

    Metabotropic glutamate receptors (mGluRs) are G-protein-coupled receptors and are densely expressed in the forebrain of adult rats. Accumulative evidence suggests a critical role of mGluRs in the regulation of normal physiological activity of neurons and pathogenesis of mental illnesses such as schizophrenia, depression, and substance addiction. In this study, we investigated alterations in mGluR8 subtype mRNA expression in the rat forebrain in response to repeated intraperitoneal administration of amphetamine (twice daily for 12 days, 5 mg/kg per injection) using quantitative in situ hybridization. We found that mGluR8 mRNA levels were profoundly increased in the dorsal (caudate putamen) and ventral (nucleus accumbens) striatum 1 day after the discontinuation of amphetamine treatments. Such increases were sustained up to 21 days of withdrawal. Increases in mGluR8 mRNAs were also found in the cerebral cortex, including the cingulate and sensory cortex but not the piriform cortex, at 1 and 21 days. These data demonstrate a positive response of mGluR8 in mRNA abundance in most forebrain regions to repeated stimulant exposure. PMID:18255232

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

    PubMed Central

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

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation. PMID:27112822

  16. Integration of mRNA expression profile, copy number alterations, and microRNA expression levels in breast cancer to improve grade definition.

    PubMed

    Cava, Claudia; Bertoli, Gloria; Ripamonti, Marilena; Mauri, Giancarlo; Zoppis, Italo; Della Rosa, Pasquale Anthony; Gilardi, Maria Carla; Castiglioni, Isabella

    2014-01-01

    Defining the aggressiveness and growth rate of a malignant cell population is a key step in the clinical approach to treating tumor disease. The correct grading of breast cancer (BC) is a fundamental part in determining the appropriate treatment. Biological variables can make it difficult to elucidate the mechanisms underlying BC development. To identify potential markers that can be used for BC classification, we analyzed mRNAs expression profiles, gene copy numbers, microRNAs expression and their association with tumor grade in BC microarray-derived datasets. From mRNA expression results, we found that grade 2 BC is most likely a mixture of grade 1 and grade 3 that have been misclassified, being described by the gene signature of either grade 1 or grade 3. We assessed the potential of the new approach of integrating mRNA expression profile, copy number alterations, and microRNA expression levels to select a limited number of genomic BC biomarkers. The combination of mRNA profile analysis and copy number data with microRNA expression levels led to the identification of two gene signatures of 42 and 4 altered genes (FOXM1, KPNA4, H2AFV and DDX19A) respectively, the latter obtained through a meta-analytical procedure. The 42-based gene signature identifies 4 classes of up- or down-regulated microRNAs (17 microRNAs) and of their 17 target mRNA, and the 4-based genes signature identified 4 microRNAs (Hsa-miR-320d, Hsa-miR-139-5p, Hsa-miR-567 and Hsa-let-7c). These results are discussed from a biological point of view with respect to pathological features of BC. Our identified mRNAs and microRNAs were validated as prognostic factors of BC disease progression, and could potentially facilitate the implementation of assays for laboratory validation, due to their reduced number. PMID:24866763

  17. Global RT-PCR and RT-qPCR Analysis of the mRNA Expression of the Human PTPome.

    PubMed

    Nunes-Xavier, Caroline E; Pulido, Rafael

    2016-01-01

    Comprehensive comparative gene expression analysis of the tyrosine phosphatase superfamily members (PTPome) under cell- or tissue-specific growth conditions may help to define their individual and specific role in physiology and disease. Semi-quantitative and quantitative PCR are commonly used methods to analyze and measure gene expression. Here, we describe technical aspects of PTPome mRNA expression analysis by semi-quantitative RT-PCR and quantitative RT-PCR (RT-qPCR). We provide a protocol for each method consisting in reverse transcription followed by PCR using a global platform of specific PTP primers. The chapter includes aspects from primer validation to the setup of the PTPome RT-qPCR platform. Examples are given of PTP-profiling gene expression analysis using a human breast cancer cell line upon long-term or short-term treatment with cell signaling-activation agents. PMID:27514798

  18. Human hepatocytes express absent in melanoma 2 and respond to hepatitis B virus with interleukin-18 expression.

    PubMed

    Pan, Xingfei; Xu, Haixia; Zheng, Changlong; Li, Mei; Zou, Xiaofang; Cao, Hong; Xu, Qihuan

    2016-08-01

    Absent in melanoma 2 (AIM2) is a recently recognized cytoplasmic receptor which could sense cytoplasmic double-stranded DNA (dsDNA). After AIM2 detects the presence of parasitic nucleic acids (dsDNA) derived from invasive bacteria or viral genomes (for example, vaccinia virus and cytomegalovirus) within infected cells, AIM2 inflammasome could be formed. The formed AIM2 inflammasome could induce innate immune response and increase expressions of IL-1β and IL-18. Hepatitis B virus (HBV) is a hepatotropic, non-cytopathic double-stranded DNA virus. The immune response to viral antigens or virus is thought to be responsible for both liver damage and viral clearance in patients with HBV infection. However, there are no reports about whether AIM2 inflammasome exists in hepatocytes. In the present study, we investigated the presence and activity of AIM2 inflammasome in human hepatocytes. We found that AIM2 was expressed in cytoplasm of hepatocytes, and IL-18 expression was increased after AIM2 sensed HBV in hepatocytes in vitro. These results showed that AIM2 inflammasome was active in hepatocytes. We also found that hepatic AIM2 expression of chronic hepatitis B (CHB) patients was higher than that of controls. Hepatic AIM2 expression levels were positively correlated to the severity of liver inflammation. IL-18 is already considered to be associated with hepatic injury during HBV infection. In conclusion, we, therefore, believe that AIM2 inflammasome in hepatocytes might play an important role in the development and maintenance of HBV-related hepatitis. PMID:27094165

  19. Amitriptyline induces brain-derived neurotrophic factor (BDNF) mRNA expression through ERK-dependent modulation of multiple BDNF mRNA variants in primary cultured rat cortical astrocytes and microglia.

    PubMed

    Hisaoka-Nakashima, Kazue; Kajitani, Naoto; Kaneko, Masahiro; Shigetou, Takahiro; Kasai, Miho; Matsumoto, Chie; Yokoe, Toshiki; Azuma, Honami; Takebayashi, Minoru; Morioka, Norimitsu; Nakata, Yoshihiro

    2016-03-01

    A significant role of brain-derived neurotrophic factor (BDNF) has been previously implicated in the therapeutic effect of antidepressants. To ascertain the contribution of specific cell types in the brain that produce BDNF following antidepressant treatment, the effects of the tricyclic antidepressant amitriptyline on rat primary neuronal, astrocytic and microglial cortical cultures were examined. Amitriptyline increased the expression of BDNF mRNA in astrocytic and microglial cultures but not neuronal cultures. Antidepressants with distinct mechanisms of action, such as clomipramine, duloxetine and fluvoxamine, also increased BDNF mRNA expression in astrocytic and microglial cultures. There are multiple BDNF mRNA variants (exon I, IIA, IV and VI) expressed in astrocytes and microglia and the variant induced by antidepressants has yet to be elaborated. Treatment with antidepressants increased the expression of exon I, IV and VI in astrocyte and microglia. Clomipramine alone significantly upregulated expression of exon IIA. The amitriptyline-induced expression of both total and individual BDNF mRNA variants (exon I, IV and VI) were blocked by MEK inhibitor U0126, indicating MEK/ERK signaling is required in the expression of BDNF. These findings indicate that non-neural cells are a significant target of antidepressants and further support the contention that glial production of BDNF is