Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA polymerase-beta was purified from Novikoff hepatoma and used as an antigen in an in vitro immunization system to produce monoclonal antibodies. These reagents surprisingly showed cross-reactivity to a number of proteins, including several DNA polymerases. Nearly all of these proteins possess nucleotide binding sites, which suggested the potential value of using the monoclonals to elucidate structure-function relationships within polymerase-beta. Furthermore, these antibodies were able to partially neutralize (40-50%) polymerase-beta activity, and this effect could be blocked by dNTP1 but not by dNMP or rNTP. The limited neutralization phenomenon is at least partially explained by the weak binding affinity of these antibodies. Scatchard analysis of immunoprecipitation data predicted a Kd of 1.8 x 10(-8) M. Epitope mapping studies showed that the region of polymerase-beta recognized by one of the monoclonal antibodies is within residues 235-335, and sequence homology studies indicated that the epitope is probably located in the region of amino acids 283-320. At least a portion of this area, namely residues 301-308 and 311-315, appears to be part of a nucleotide binding domain which has sequence homology with a portion of the highly conserved ATP binding site in adenylate kinase.
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PMID:Structure-function analysis of DNA polymerase-beta using monoclonal antibodies: identification of a putative nucleotide binding domain. 138 Aug 29

In retroviruses, the pol gene is expressed in the form of a gag-pol fusion protein by the mechanism of ribosomal frameshifting. In studies of the possible mechanism of hepadnaviral pol protein synthesis, recent results have ruled out core-pol fusion protein synthesis by ribosomal frameshifting. In this study, an in vitro transcription and translation coupling system was used to demonstrate that the HBV core and pol proteins could be synthesized independently using the pregenome RNA template. The result has led us to design experiments to distinguish between the involvement of a termination-reinitiation, internal initiation, or leaky scanning mechanism in the pol protein synthesis. In vitro experiments were then carried out to measure the amount of pol proteins being synthesized from (i) the preC mRNA, which contained an extra AUG and seven more nucleotides at the 5'-end in comparison with the pregenome RNA; (ii) the pregenome RNA in the presence of various amounts of antisense RNA annealing to the 5'-end of the pregenome RNA; and (iii) the pregenome RNA with an additional hairpin structure located upstream of the C gene. Results indicated that the synthesis of both core and pol proteins was concomitantly reduced in these three conditions, which suggested that leaky scanning is the most probable mechanism for pol protein synthesis in vitro. To further verify the mechanism in vivo, experiments were performed to assay the activity of DNA polymerase in virions, which were obtained from hepatoma cells transfected by plasmids containing either a wild-type sequence (5'-GGCATGG-3') or an optimal initiation context (5'-ACCATGG-3') of the C gene. Transfection results showed that the plasmid-containing mutations of the C gene significantly decreased the DNA polymerase activity in virions. This observation supports our hypothesis that the leaky scanning model is involved in the synthesis of pol protein.
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PMID:Evidence for involvement of a ribosomal leaky scanning mechanism in the translation of the hepatitis B virus pol gene from the viral pregenome RNA. 156 78

We have detected the in situ activities of DNA glycosylase, endonuclease, exonuclease, DNA polymerase, and DNA ligase using a novel polyacrylamide activity gel electrophoresis procedure. DNA metabolizing enzymes were resolved through either native or SDS-polyacrylamide gels containing defined 32P-labeled oligonucleotides annealed to M13 DNA. After electrophoresis, these enzymes catalyzed in situ reactions and their [32P]DNA products were resolved from the gel by a second dimension of electrophoresis through a denaturing DNA sequencing gel. Detection of modified (degraded or elongated) oligonucleotide chains was used to locate various enzyme activities. The catalytic and physical properties of Novikoff hepatoma DNA polymerase beta were found to be similar under both in vitro and in situ conditions. With 3'-terminally matched and mismatched [32P]DNA substrates in the same activity gel, DNA polymerase and/or 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I (large fragment), DNA polymerase III (holoenzyme), and exonuclease III were detected and characterized. In addition, use of matched and mismatched DNA primers permitted the uncoupling of mismatch excision and chain extension steps. Activities first detected in nondenaturing activity gels as either multifunctional or multimeric enzymes were also identified in denaturing activity gels, and assignment of activities to specific polypeptides suggested subunit composition. Furthermore, DNA substrates cast within polyacrylamide gels were successfully modified by the exogenous enzymes polynucleotide kinase and alkaline phosphatase before and after in situ detection of E. coli DNA ligase activity, respectively. Several restriction endonucleases and the tripeptide (Lys-Trp-Lys), which acts as an apurinic/apyrimidinic endonuclease, were able to diffuse into gels and modify DNA. This ability to create intermediate substrates within activity gels could prove extremely useful in delineating the steps of DNA replication and repair pathways.
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PMID:Characterization of DNA metabolizing enzymes in situ following polyacrylamide gel electrophoresis. 200 53

Hepatitis B virus (HBV) variants with a nonsense mutation in the distal pre-C region have been detected in patients positive for anti-HBe, and the complete nucleotide sequence of one cloned pre-C variant has been determined. Transfection of this HBV variant clone into the human hepatoma cell line HepG2 resulted in the appearance of major HBV transcripts, replicative forms of viral DNA evidenced by both molecular hybridization and endogenous DNA polymerase assay, as well as the expression and secretion of HBsAg and HBcAg particles. Western blotting revealed only the 21-kDa HBcAg but not the 17-kDa HBeAg. These results demonstrate the replication capacity of the HBV variant with a nonfunctional pre-C region despite its inability to express HBeAg.
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PMID:In vitro replication competence of a cloned hepatitis B virus variant with a nonsense mutation in the distal pre-C region. 201 46

A 5 bp insertion was introduced into the BstEII site at nucleotide 2815 in DNA of hepatitis B virus (HBV) and a mutant HBV genome was produced, which coded for envelope and core proteins, but not for DNA polymerase, due to a frameshift. Cultured hepatoma cells (HepG2) were simultaneously transfected with a plasmid harbouring a tandem dimer of the mutant HBV DNA and another plasmid harbouring a tandem dimer of DNA of woodchuck hepatitis virus or duck hepatitis B virus. The replication of mutant HBV DNA, incapable of encoding DNA polymerase, was accomplished by cotransfecting woodchuck hepatitis virus DNA, but not by duck hepatitis B virus DNA. These results indicated a trans-complementation of the C and P genes in mammalian hepadnaviruses beyond a species barrier.
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PMID:Trans-complementation of the C gene of human and the P gene of woodchuck hepadnaviruses. 215 4

A viricide capable of eliminating hepatitis B virus (HBV) from chronic carriers should, theoretically, decrease the risk of primary hepatocellular carcinoma. Extracts of Phyllanthus amarus have been shown to inhibit the DNA polymerase of HBV and woodchuck hepatitis virus (WHV) in vitro. Three of four recently infected WHV carriers treated i.p. with P. amarus extract lost WHV, animals infected for greater than or equal to 3 months showed a decrease in virus levels. Preliminary results in human carriers treated orally with P. amarus for 1 month indicated that approximately 60% of the carriers lost HBV during the observation period.
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PMID:Hepatitis B virus and primary hepatocellular carcinoma: treatment of HBV carriers with Phyllanthus amarus. 215 92

An in vitro system for production of hepatitis B virus (HBV) was established by infection of human hepatoma (HepG2) cells. HBV particles obtained from the serum of a chronic hepatitis B surface antigen (subtype ad) carrier were used to inoculate HepG2 cells. HBV envelope and core proteins were synthesized de novo by the infected cells and secreted into the medium 3 to 6 days postinfection. Viral covalently closed circular DNA, the putative template for viral RNA transcription, accumulated in the cells with increasing time postinfection. The HBV-infected HepG2 cells were maintained for several months (HepG2-BV cell line) and continued producing viral antigens. Both HBV DNA replicative intermediates and major HBV transcripts were identified in HepG2-BV cells. Complete HBV particles, which contain HBV DNA and DNA polymerase activity and express the three antigenic specificities of the envelope (hepatitis B surface antigen, pre-S2, and pre-S1), were released into the culture supernatant. Thus, successful in vitro infection of transformed human hepatocytes raising stable HBV-producing cells was achieved for the first time. This strongly suggests that HepG2 cells have a receptor(s) for virus attachment and penetration. Such a system represents a significant advance for the study of HBV-target cell interactions as the early events of HBV infection.
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PMID:In vitro infection of human hepatoma (HepG2) cells with hepatitis B virus. 215 60

Permanent murine fibroblasts (LTK-) were transfected with a dimer of hepatitis B virus (HBV) DNA and a neomycin resistance gene which were both linked to the simian virus 40 (SV40) early promoter/enhancer. One of the stably transfected clones, LTK4/36, which secreted HBsAg, HBeAg, and HBV DNA was further analyzed. It contained eight to nine copies of integrated HBV DNA per haploid genome and low amounts of episomal HBV DNA. The secreted viral DNA was covalently linked to protein and was associated with particles which had the characteristic density of natural virions from serum of human viremic carriers. The particles contained an endogenous DNA polymerase, small and middle surface proteins, but in contrast to natural virions very little core protein and large surface protein. Instead of core protein, they contained incompletely processed HBe protein which is colinear to core protein. The fibroblast-derived virions were less stable than virions from human carriers or from transfected hepatoma cells. After several days of storage, their DNA was only partially protected against DNase. Obviously, nonhepatic cells can express HBV-like particles, even if liver-dependent gene products like large surface protein and core protein are missing.
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PMID:Replication of hepatitis B virus in transfected nonhepatic cells. 221 25

We have studied antibodies (anti-pol antibody) against the polymerase gene product of hepatitis B virus by solid-phase enzyme immunoassay using synthetic peptides coded for by this gene. Sera from six patients with acute hepatitis B, 112 chronic hepatitis B virus carriers and six healthy individuals with naturally acquired immunity to hepatitis B virus were tested for anti-pol antibody. In acute hepatitis B virus infection, anti-pol antibody was detected in three of six patients. In chronic hepatitis B virus infection, anti-pol antibody was detected in 17 of 29 (59%), in 23 of 33 (70%) of cirrhotic patients and in 18 of 24 (75%) patients with cirrhosis complicated by hepatocellular carcinoma, compared with 4 of 19 (21%) asymptomatic carriers and 2 of 7 (29%) patients with chronic persistent hepatitis. Titers of anti-pol antibody were higher in cirrhotic patients with and without hepatocellular carcinoma than in patients with chronic active hepatitis. The presence of anti-pol antibody, however, had no relationship with hepatitis B virus-associated DNA polymerase activities and other viral replicative markers. As for sera from six healthy individuals with naturally acquired immunity to hepatitis B virus, two (33%) were positive for anti-pol antibody. These results indicate that the immune response toward the polymerase gene product is induced during acute and chronic hepatitis B virus infection. In chronic hepatitis B virus infection, anti-pol antibody may serve as a new marker indicative of a long period of hepatitis B virus-induced hepatitis.
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PMID:Detection of antibodies against the polymerase gene product in hepatitis B virus infection. 239 Oct 62

The effects of suramin, an antiparasitic agent, upon in vitro hepatitis B surface antigen production by the human hepatoma cell line PLC/PRF/5 and hepatitis B virus associated DNA polymerase activity in the serum of a chronically infected patient were examined. Treatment with suramin resulted in decreases in hepatitis B surface antigen production and hepatitis B-virus associated DNA polymerase activity. The decrease in hepatitis B surface antigen production was paralleled by a general decrease in hepatoma cell viability and cellular protein synthesis. Although the inhibitory effects of suramin for hepatitis B virus appear to be nonspecific as demonstrated in these two in vitro systems, the recently announced trial of suramin for the treatment of the acquired immunodeficiency syndrome should afford an unusual opportunity to evaluate the effectiveness of suramin in the treatment of chronic hepatitis B virus infection.
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PMID:Effects of suramin on in vitro HBsAg production by PLC/PRF/5 cells and hepatitis B virus DNA polymerase activity. 242 68


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