Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To determine the effect of hepatitis C virus (HCV) proteins on cell growth, Huh-7 cells were transfected with a full-length HCV cDNA (pMO9.6-T7 Rz) clone and HCV proteins were expressed using a replication-defective adenovirus that encodes the gene for the T7 RNA polymerase. Expression of HCV proteins from this full-length clone resulted in reduction in viability of transfected cells as measured by trypan blue viability assay. For identification and separation of cells expressing hepatitis C virus proteins by fluorescence microscopy and flow cytometry, GFP was cloned in the HCV full-length clone. Cells transfected with the HCV-GFP chimera clone produced high levels of accurately processed structural and nonstructural proteins similar to those of the HCV full-length clone, which could be detected by Western blot analysis. Cells expressing all HCV proteins lost membrane permeability and underwent apoptotic cell death, indicated by the appearance of a sub-G0 peak in cell cycle analysis, DNA fragmentation in a TUNEL assay, and microscopic detection of nuclear condensation. Using double-channel flow analysis we confirmed that high-level expression of HCV proteins affected membrane permeability and cell survival. These results suggest that expression of all structural and nonstructural proteins from HCV cDNA in hepatic cells induces apoptotic cell death, which might be an important event in chronic hepatitis infection in humans.
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PMID:Hepatitis C viral proteins affect cell viability and membrane permeability. 1173 45

Hepatitis C virus (HCV) is the most common cause of chronic viral hepatitis. The World Health Organization estimates that 170 million people world-wide are infected with HCV; 70% of them will develop chronic hepatitis and 20-30% cirrhosis in 10-30 years. Of those with cirrhosis, an estimated 25-30% will develop liver cancer. Since the identification and molecular characterization of HCV in 1989, a variety of diagnostic tests based on the detection of hepatitis virus antibodies or HCV RNA in the serum have been developed. The enzyme-linked immunosorbent assays (ELISA 3) and the recombinant immunoblot assays (RIBA 2nd and 3rd generation) exhibit improved sensitivity and specificity for HCV antibodies. Qualitative and quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) has allowed clinicians to track the natural history of HCV and to monitor the progress of therapy. This article reviews the state-of-the-art tests and assays developed for the diagnosis and management of HCV infection.
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PMID:[Diagnostic strategies in Hepatitis C virus infection]. 1209 56

The hepatitis C virus (HCV) is a small enveloped RNA virus belonging to the family flaviviridae and genus hepacivirus. The HCV RNA genome is 9,600 nucleotides in length and encodes a single polyprotein that is post-translationally cleaved into 10 polypeptides including t3 structural (C, E1, and E2) and multiple nonstructural proteins ([NS] NS2 to NS5). The NS proteins include enzymes necessary for protein processing (proteases) and viral replication (RNA polymerase). The virus replicates at a high rate in the liver and has marked sequence heterogeneity. There are 6 genotypes and more than 90 subtypes of HCV, the most common in the United States being 1a and 1b (approximately 75%), 2a and 2b (approximately 15%), and 3 (approximately 7%). Acute hepatitis C is marked by appearance of HCV RNA in serum within 1 to 2 weeks of exposure followed by serum alanine aminotransferase (ALT) elevations, and then symptoms and jaundice. Antibody to HCV (anti-HCV) tends to arise late. In acute resolving hepatitis, HCV RNA is cleared and serum ALT levels fall to normal. However, 55% to 85% of patients do not clear virus, but develop chronic hepatitis C. Chronic hepatitis C is often asymptomatic, but is usually associated with persistent or fluctuating elevations in ALT levels. The chronic sequelae of hepatitis C include progressive hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. Extra-hepatic manifestations include sicca syndrome, cryoglobulinemia, glomerulonephritis, and porphyria cutanea tarda. Knowledge of the course and outcome of hepatitis C is important in developing approaches to management and therapy.
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PMID:Course and outcome of hepatitis C. 1240 73

Currently available therapies for the treatment of chronic hepatitis C are effective in half of patients, but are expensive, often poorly tolerated, and unsuitable for certain patient populations. The ideal therapy would be highly effective, orally bioavailable, have minimal side effects, be cost effective, and suitable for the majority of patients with hepatitis C. Recent advances in understanding the replication cycle of hepatitis C virus (HCV) and structural, crystallographic definitions of components of the viral polyprotein have improved the prospects for development of novel therapies. The lack of a small animal model of HCV infection continues to hamper progress in the preclinical evaluation of new antivirals and vaccines. Strategies to enhance response to current therapies include the development of novel interferons and delivery systems, nucleoside analogues that have reduced hemolysis compared with ribavirin, inosine 5' monophosphate dehydrogenase inhibitors, and other immunomodulators that are being evaluated as adjunctive therapy to interferon-based regimens. Compounds in preclinical or early phase human trials include small molecules that inhibit virus specific enzymes (such as the serine proteases, RNA polymerase and helicase), or those that prevent translation initiation (such as antisense molecules and ribozymes). Antifibrotic agents are also being developed in an attempt to prevent disease progression in patients in whom HCV RNA cannot be eradicated. While the advent of these newer compounds represent an exciting phase in the treatment of HCV, their safety and efficacy need to be established. Most of these newer therapies are unlikely to be available for routine clinical use in the next 3 to 5 years.
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PMID:Future therapy of hepatitis C. 1240

Patients with chronic hepatitis C frequently report tiredness, easy fatigability, and depression. The aim of this study is to determine whether hepatitis C virus (HCV) replication could be found in brain tissue in patients with hepatitis C and depression. We report two patients with recurrent hepatitis C after liver transplantation who also developed severe depression. One patient died of multiorgan failure and the other, septicemia caused by Staphylococcus aureussis. Both patients had evidence of severe hepatitis C recurrence with features of cholestatic fibrosing hepatitis. We were able to study samples of their central nervous system obtained at autopsy for evidence of HCV replication. The presence of HCV RNA-negative strand, which is the viral replicative form, was determined by strand-specific Tth-based reverse-transcriptase polymerase chain reaction. Viral sequences were compared by means of single-strand conformation polymorphism and direct sequencing. HCV RNA-negative strands were found in subcortical white matter from one patient and cerebral cortex from the other patient. HCV RNA-negative strands amplified from brain tissue differed by several nucleotide substitutions from serum consensus sequences in the 5' untranslated region. These findings support the concept of HCV neuroinvasion, and we speculate that it may provide a biological substrate to neuropsychiatric disorders observed in patients with chronic hepatitis C. The exact lineage of cells permissive for HCV replication and the possible interaction between viral replication and cerebral function that may lead to depression remain to be elucidated.
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PMID:Detection of hepatitis C virus sequences in brain tissue obtained in recurrent hepatitis C after liver transplantation. 1242 14

Antigen-presenting dendritic cells (DCs), which play a major role in the triggering of primary anti viral immune reactions, may also contribute, in some viral models, to the pathogenesis of persistent viral infection. In fact, impaired immune response to hepatitis B virus (HBV)-encoded antigens is seen in patients with chronic hepatitis B (CH-B). The aim of this study was to check the function of DCs in these patients and to investigate the underlying mechanism. DCs were enriched from peripheral blood mononuclear cells by culturing with interleukin (IL)-4 and granulocyte-macrophage colony stimulating factor for 7 days. The stimulatory capacity of DCs were checked in allogenic mixed leukocyte (MLR) reaction. The levels of IL-12 in the culture supernatants were measured by an enzyme-linked immunosorbent assay. HBV DNA and HBV RNA were localized in DCs by polymerase chain reaction (PCR) in situ hybridization and reverse-transcriptase (RT)-PCR in situ hybridization. The stimulatory capacity of DCs in allogenic MLR was significantly lower in patients with CH-B (36321+/-12523 cpm, n=18) compared to that of normal controls (65678+/-11174 cpm, n=18) (p<0.0001). Significantly lower levels of IL-12 were detected in cultures containing DCs from patients with CH-B than normal controls (46.7+/-25.6 versus 122.4+/- 37.1 pg/ml, p<0.0001). In situ hybridization revealed the localization of HBV DNA and HBV RNA in DCs from patients with CH-B. These results indicate that chronic infection by HBV is associated with functional defects of DCs. Localization of HBV DNA and HBV RNA indicates that DCs may constitute an extra hepatic reservoir and possibly of replication of HBV.
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PMID:Impaired function of antigen-presenting dendritic cells in patients with chronic hepatitis B: localization of HBV DNA and HBV RNA in blood DC by in situ hybridization. 1252 72

Several observations suggest that bacteria induce autoimmunity in primary biliary cirrhosis (PBC). Since no PBC-specific bacterial species could be identified, it can be speculated that the triggers are non-species-specific bacterial proteins. This hypothesis would imply that several or even all bacterial species can trigger PBC. Therefore, we investigated whether PBC exhibits immune reactions to non-species-specific bacterial antigens. Yersinia enterocolitica O3 was screened for the presence of proteins that were labeled by immunoblotting using PBC sera. We focused our investigations on a 160-kDa protein, which was further enriched and characterized by partial N-terminal amino acid sequencing. The prevalence of antibodies to this protein was determined by immunoblotting in a variety of diseases. The 160-kDa protein was identified as the beta-subunit of bacterial RNA-polymerase, a highly conserved bacterial protein with a very high degree of sequence identity among all bacterial species. Antibodies to the beta-subunit of bacterial RNA polymerase were specific for this protein. Until now no mammalian protein could be found that cross-reacts with these antibodies. The prevalence of antibodies to the beta-subunit of bacterial RNA polymerase (ARPA) using the protein from Yersinia enterocolitica O3 (serum dilution 1:1000) was: healthy controls (HC, N = 101) 7.9%, primary biliary cirrhosis (PBC, N = 61) 32.8%, autoimmune hepatitis type 1 (AIH, N = 46) 26.1%, alcoholic liver cirrhosis (ALC, N = 44) 9.1%, Crohn's disease (CD, N = 38) 7.9%, ulcerative colitis (UC, N = 24) 8.3%, primary sclerosing cholangitis + UC (PSC/UC, N = 11) 0%, acute yersiniosis (Yers, N = 36) 19.4%, acute infection with Campylobacter jejuni (Camp, N = 10) 0%, acute Q-fever (QF, N = 16) 6.25%, chronic hepatitis C (HCV, N = 39) 7.7%, c-ANCA-positive vasculitis (Vasc, N = 40) 15%, systemic lupus erythematosus (SLE, N = 28) 10.7%, and malaria tropica (MT, N = 24) 16.7%. There was no significant difference between PBC and AIH. The group of autoimmune liver diseases (PBC + AIH, N = 107, 29.9%) differed highly significantly from HC, chronic inflammatory bowel diseases (CD + UC + PSC/UC, N = 73, 6.8%), ALC, and HCV and also differed significantly (P = 0.01) from the group with bacterial and parasitic diseases (Yers + Camp + QF + MT, N = 86,13.95%) and from the group with Vasc + SLE (N = 68,13.2%). Testing of ARPA using the protein from E. coli yielded nearly identical results. In conclusion, an increased prevalence of antibodies to the beta-subunit of bacterial RNA polymerase, a highly conserved non-species-specific bacterial protein, can be found in primary biliary cirrhosis, but also in autoimmune hepatitis type I. These findings do not add an argument for a bacterial trigger of PBC. Rather, they suggest that ARPA belong to the pool of natural antibodies that are up-regulated in autoimmune liver diseases.
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PMID:Identification of beta-subunit of bacterial RNA-polymerase--a non-species-specific bacterial protein--as target of antibodies in primary biliary cirrhosis. 1275 71

Chronic hepatitis C virus (HCV) infection is the cause of an emerging global epidemic of chronic liver disease. Current combination therapies are at best 80% efficacious and are often poorly tolerated. Strategies to improve the therapeutic response include the development of novel interferons, nucleoside analogues with reduced haemolysis compared with ribavirin and inosine 5'-monophosphate dehydrogenase inhibitors. Compounds in preclinical or early clinical trials include small molecules that inhibit virus-specific enzymes (such as the serine proteases, RNA polymerase and helicase) or interfere with translation (including anti-sense molecules, iRNA and ribozymes). Advances in understanding HCV replication, obtaining a sub-genomic replicon and contriving potential small animal models, in addition to solving crystallographic structures for the replication enzymes, have improved prospects for developing novel therapies. This review summarizes current and evolving treatments for chronic hepatitis C infection. In addition, progress in HCV targets and drug discovery tools valuable in the search for novel anti-HCV agents is detailed.
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PMID:Hepatitis C virus therapies: current treatments, targets and future perspectives. 1279 May 12

The bidirectional activity of the precore/core promoter of hepatitis B virus (HBV) has been demonstrated in cultured cell lines. However, HBV antisense transcripts (asRNAs) have not been demonstrated in vivo. In the present study using liver tissue from patients with chronic hepatitis, an anchored 5'RACE mapping the 5' ends at position 1680/1681, 1655 or 1609/1602 was carried out. In limited cases, RLM-3'RACE detected asRNAs to terminate at four or five consecutive dT residues in the 0.7 kb downstream region. PCR of oligo(dT)-primed cDNA did not amplify a typical polyadenylated asRNA. RT-PCR using various primers did not detect any spliced forms. Competitive RT-PCR estimated the copy numbers of the asRNAs to be 0.05-0.4 % of total sense RNAs. All sequenced asRNAs had ORF6 but, in one patient, the asRNA initiating at position 1680/1681 had additional initiation and termination codons in front of ORF6. Therefore, asRNAs are transcribed by RNA polymerase III at a low level, encompass a dispensable ORF6 gene and might be retained in the nucleus. The endogenous asRNAs complementary to the common ends of all sense RNAs suggest antisense-mediated self-regulation of hepadnavirus.
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PMID:Antisense RNAs transcribed from the upstream region of the precore/core promoter of hepatitis B virus. 1281 Aug 86

The "D drug" HIV reverse-transcriptase inhibitors zalcitabine, didanosine, and stavudine are relatively strong inhibitors of polymerase-gamma compared with the "non-D drugs" zidovudine, lamivudine, and abacavir. D drugs deplete mitochondrial DNA (mtDNA) in cultured hepatocytes. This mtDNA depletion is associated with an increased in vitro production of lactate. To investigate the origin of hyperlactatemia in HIV-infected patients and the effects of antiretroviral therapy on liver mtDNA, we biopsied liver tissue from 94 individuals with chronic hepatitis C virus (HCV) infection. Eighty subjects were coinfected with HIV. Serum lactate was measured at the time of biopsy. Hepatic mtDNA and liver histology were centrally assessed. Liver mtDNA content of HIV-infected patients receiving D drugs at the time of biopsy (n = 34) was decreased by 47% (P<.0001) compared with those without D drugs (n = 35). Aside from a possible association between HCV genotype I status and mtDNA depletion in multivariate analysis, there were no other virologic, immunologic, histologic, demographic or treatment-related variables that could explain the mtDNA depletion. Lactate was above the upper limit of normal in only three patients, all of whom were treated with D drugs. The mtDNA in each of them was lower than in any non-D drug patient and significantly (P =.017) depleted compared with D drug patients with normal lactate. In conclusion, D drug treatment is associated with decreased hepatic mtDNA in HIV-infected patients with chronic HCV infection. Moderate mtDNA depletion in liver does not necessarily lead to hyperlactatemia, but more pronounced decreases in hepatic mtDNA may be an important contributor to lactate elevation.
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PMID:Depletion of mitochondrial DNA in liver under antiretroviral therapy with didanosine, stavudine, or zalcitabine. 1476 83


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