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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Luzopeptin analogues, A, B and C (in decreasing degree of acetylation) were active, less active and inactive, respectively, against several animal tumour models. All bound to isolated DNA avidly. The present studies with cultured Novikoff hepatoma cells showed that luzopeptins A, B and C were inhibitory, less inhibitory and ineffective, respectively, on cell colony formation ability and whole-cell DNA and RNA synthesis. RNA synthesis was more sensitive than DNA synthesis to luzopeptins A and B. All had no direct effect on protein synthesis. All inhibited the DNA and RNA syntheses of isolated nuclei and luzopeptins B and C were slightly more active than luzopeptin A inhibition of RNA synthesis in isolated nuclei. All similarly inhibited DNA polymerase activity in vitro, but luzopeptin C was more active against RNA polymerase activity in vitro. It is concluded that luzopeptin cytotoxicity probably resulted from inhibition of DNA and RNA biosynthesis. Because all analogues bound avidly to isolated DNA and inhibited its functions, the differential anti-tumour activity may be attributed to the differential ability of these luzopeptins to traverse the cell membrane and, to some extent, other intracellular barriers. This probably is a result of difference in acetylation, resulting in differences in hydrophobicity.
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PMID:Effects of structural modifications of anti-tumour antibiotics luzopeptins on cell growth and macromolecule biosynthesis. 245 97

To investigate whether DNA replication in rat hepatoma cells is altered compared with that in normal rat liver, the main replicative enzyme, i.e. the DNA polymerase alpha complex, was partially purified from a slow-growing (TC5123) and a fast-growing (MH3924) Morris hepatoma cell strain as well as from normal rat liver. The purified DNA polymerase alpha complexes contained RNA primase. DNA polymerase alpha activities of these complexes were characterized with regard to both their molecular properties and their dNTP and DNA binding sites. The latter were probed with competitive inhibitors of dNTP binding, resulting in Ki values, and with DNA templates, yielding Km values. The sedimentation coefficients of native DNA polymerases alpha from Morris hepatoma cells were found to be lower than that of polymerase alpha from normal rat liver. Consequently, when following the procedure of Siegel and Monty for determination of molecular mass considerably smaller molecular masses were calculated for polymerases of hepatoma strains (TC5123, 127 kDa; MH3924, 138 kDa; rat liver, 168 kDa). Similar differences were found when the dNTP binding site was probed with inhibitors. Ki values obtained with butylphenyl-dGTP were higher for polymerases of the hepatoma strains than for that of normal rat liver. However, Ki values measured with aphidicolin and butylanilino-dATP were lower for DNA polymerase alpha from the fast-growing hepatoma cell strain than for that from normal rat liver, indicating a reduced affinity of the dNTP binding sites for dATP and dCTP. This reduced affinity could be responsible for lowered specificity of nucleotide selection in the base-pairing process which in turn may cause an enhanced error rate in DNA replication in malignant cells. Furthermore, when the DNA binding site was characterized by Michaelis-Menten constants using gapped DNA as a template, Km values were similar for all three DNA polymerases. In contrast, the Km value measured with single-stranded DNA as a template was found to be lower for DNA polymerase alpha from the fast-growing hepatoma MH3924 than for that from normal rat liver. Thus, the DNA-polymerizing complex from MH3924 combines both higher binding strength to single-stranded DNA templates and decreased nucleotide selection, properties which may enhance replication velocity and may lower fidelity.
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PMID:DNA polymerase alpha from normal rat liver is different than DNA polymerases alpha from Morris hepatoma strains. 250 1

DNA polymerase alpha (DNA-P alpha) in the nuclei of hepatocytes was visualized by the immunoperoxidase method to study the number of liver cells which were at the stage of G1, S, and G2 stage in the cell cycle. Seven liver specimens from patients with acute hepatitis (AH), 17 with chronic persistent hepatitis (CPH), 32 with chronic active hepatitis (CAH), 6 with liver cirrhosis (LC), 4 with hepatocellular carcinoma (HCC) and 4 with hospital controls were studied. The number of DNA-P alpha-positive hepatocytes in 1000 hepatocytes were as follows: 19.1 +/- 18.0 in AH, 8.8 +/- 6.1 in CPH, 27.3 +/- 23.8 in CAH, 21.8 +/- 14.3 in LC, 545.3 +/- 184.0 in HCC and 1.1 +/- 1.1 in hospital controls. The number of DNA-P alpha-positive hepatocytes in HCC were significantly increased compared with other liver diseases. Likewise, those in CAH and LC were higher than those in CPH and hospital controls. The liver cell necrosis was thought to be one of the secondary stimulators for cell division of hepatocytes.
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PMID:[A cell kinetic study of liver cells in various liver diseases by the detection of DNA polymerase alpha]. 268 24

A rat hepatoma cell line (Q7) of Morris hepatoma origin was transfected with a construct containing the tandem dimer genome of human hepatitis B virus (HBV) and the neomycin-resistant selection marker. The culture medium of several neomycin-resistant single-cell clones was found to accumulate high levels of secreted HBV surface antigen and core-related e antigen. HBV-specific replication intermediates, including relaxed circular and single-stranded DNA with a minus-strand polarity, could be found in both the intracellular fraction and the extracellular culture medium by the Southern blot procedure. One of these clones, designated Q7 HBV-21, was characterized in further detail. DNA polymerase activity was present in the virus particles produced by Q7 HBV-21 cells. Characteristic transcripts of HBV, including the 3.5-, 2.5-, and 2.1-kilobase mRNA as well as a core-gene-related transcript of 2.2 kilobases could be detected. Electron microscopic examination of the conditioned medium from Q7 HBV-21 cells identified 42-nm Dane-like particles as well as 22-nm subviral particles with a spherical or filamentous shape. This Q7 HBV-21 cell line has been maintained in the absence of neomycin for 1 year without losing the properties of HBV DNA replication and Dane-like particle production. Our results strongly suggest that the species barrier of HBV infection is at an early step of viral absorption onto or penetration into the target hepatocytes. This nonhuman system for HBV production in culture could be used to complement the human HepG2 system.
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PMID:In vitro propagation of human hepatitis B virus in a rat hepatoma cell line. 276 28

The differentiated human hepatoma cell line Hep-G2 was transfected with cloned duck hepatitis B virus (DHBV) DNA. Introduction of closed circular DNA into the human liver cells resulted in the production of viral proteins: core antigen was detected in the cytoplasm, and e antigen, a related product, was secreted into the medium. Moreover, viral particles were released into the tissue culture medium which were indistinguishable from authentic DHBV by density, antigenicity, DNA polymerase activity, and morphology. Intravenous injection of tissue culture-derived DHBV particles into Pekin ducks established DHBV infection. In conclusion, transfection of human hepatoma cells with cloned DHBV DNA results in the production of infectious virus, as occurs with cloned human hepatitis B virus DNA. Human liver cells are therefore competent to support production of the avian and mammalian hepadnaviruses, indicating that liver-specific viral gene expression is controlled by evolutionarily conserved mechanisms. This new DHBV transfection system offers the opportunity to rapidly produce mutated DHBV which then can be further investigated in Pekin ducks.
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PMID:Production of infectious duck hepatitis B virus in a human hepatoma cell line. 283 23

Microtubule-associated protein-2 (MAP-2) isolated from porcine brains stimulated DNA synthesis catalyzed by the nuclear matrix isolated from Physarum polycephalum in the presence of activated DNA as exogenous templates. The degree of the stimulation depended on the amount of the nuclear matrix, but not on that of the template. MAP-2 also stimulated DNA polymerase alpha activity solubilized from nuclei, but not DNA polymerase beta activity. These results suggest that MAP-2 stimulates DNA synthesis by interacting with the putative DNA replication machinery including DNA polymerase alpha bound to the matrix. Similar stimulation occurred in the nuclear matrix isolated from HeLa and rat ascites hepatoma cells, which strongly suggests that MAP-2 is involved in the control of DNA replication in eukaryotic cells.
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PMID:Microtubule-associated protein-2 stimulates DNA synthesis catalyzed by the nuclear matrix. 293 May 45

DNA-dependent ATPase IV has been purified to near homogeneity from the Novikoff rat hepatoma. The enzyme is devoid of DNA polymerase, RNA polymerase, exonuclease, endonuclease, phosphomonoesterase, 3'- or 5'-phosphodiesterase, polynucleotide kinase, protein kinase, topoisomerase, helicase or DNA reannealing activities at a detection level of 10(-5) to 10(-7) relative to the ATPase activity. The enzyme is a monomer of Mr 110,000, has a sedimentation coefficient of 5.9 S, a Stokes radius of 40 A and a frictional coefficient of 1.32. In the presence of Mg2+ ion and a polynucleotide effector, ATPase IV hydrolyzes either ATP or dATP to the nucleoside diphosphate plus Pi. Other ribo- or deoxyribonucleoside triphosphates are not substrates. ATPase IV utilizes double-stranded DNA and single-stranded DNA as effector; however, it does not utilize poly(dT). The Km for dsDNA or ssDNA is 2.2 microM (nucleotide). A variety of ATP analogues were found to be competitive inhibitors of ATPase IV.
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PMID:Purification and enzymological characterization of DNA-dependent ATPase IV from the Novikoff hepatoma. 296 5

Sera from 31 HBsAg-positive Chinese patients with inoperable hepatocellular carcinoma (HCC) were tested for hepatitis B virus DNA (HBV DNA) by means of dot hybridisation and Southern blot technique. HBV DNA probes were prepared from human plasma. Eighteen of the patients were HBeAb-positive, 12 were HBeAg-positive and one case had neither marker. Serial specimens were obtained from 16 cases over 5-42 weeks, while the patients were treated with recombinant leukocyte A interferon (rIFN-A) or adriamycin. Seven patients (2 HBeAg-positive, 5 HBeAb-positive) were positive for HBV DNA. In two patients HBV DNA and HBV DNA polymerase (DNAp) appeared in serum weeks after rIFN-A or adriamycin treatment was started. In two other cases, HBV DNA that was initially present disappeared during rIFN-A treatment. In a fifth patient HBV DNA persisting after adriamycin treatment diminished after change of treatment to rIFN-A. With one possible exception the HBV DNA detectable by Southern blot technique was composed chiefly of sequences 2.2-3.2 kb size indicating the presence of unintegrated DNA forms. DNAp activities were raised in the presence of HBV DNA in 4 patients. These findings show that HBV replication can be activated or suppressed in advanced HCC. Treatment with rIFN-A may have been effective in suppressing HBV DNA synthesis, but the number of cases studied was too small to arrive at a definite conclusion on this point.
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PMID:Serum hepatitis B viral DNA in HBsAg-positive hepatocellular carcinoma treated with interferon or adriamycin. 301 83

Closed-circular HBV DNA was introduced into cells of the established human hepatoma culture HepG2. The culture medium of one of 40 single-cell clones contained HBV surface antigen (HBsAg), core-related antigens (HBc/eAg), and HBV DNA sequences. HBV DNA and DNA polymerase activity were detected in particles resembling both nucleocapsids and complete virions (Dane particles). Intracellular integrated and extrachromosomal HBV DNA sequences were detected. Relaxed-circular and single-stranded forms of viral DNA were identified as likely replicative intermediates of the HBV genome. In conclusion, in vitro production of Dane-like particles by transformed human hepatocytes has been achieved. This model should be valuable as a cell culture system for studying virus replication and virus-host cell interactions.
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PMID:Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA. 301 65

Transfection of human hepatoma cell lines with cloned HBV DNA resulted in the secretion of large amounts of hepatitis B surface antigen (HBsAg) and core-related antigens (HBc/HBeAg) if well-differentiated cell lines were employed. Synthesis of both viral antigens was the highest in cell line HuH-7 and continued for approximately 25 days. Particles resembling hepatitis B virions (Dane particles) by morphology, density and by the presence of the preS1 surface antigen were released from the transfected HuH-7 cells into the culture medium. These particles produced in vitro were also indistinguishable from the naturally occurring hepatitis B virions in containing the virus-associated DNA polymerase and mature HBV genomes. Restriction analysis of these DNA molecules was compatible with the nucleotide sequence of the transfecting HBV DNA sequence. Viral surface antigens and core proteins present in the culture medium were fractionated and characterized by immunoprecipitation and SDS--PAGE after labeling with [35S]methionine. Antisera specific for X-gene products identified in cell extracts two hitherto unknown HBV gene products. This system thus provides a new approach to open questions regarding HBV-related gene function and HBV replication.
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PMID:Production of hepatitis B virus in vitro by transient expression of cloned HBV DNA in a hepatoma cell line. 303 5


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