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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

MRC-5 human diploid cells infected with Simian Sarcoma Virus from woolly monkey (SSV-1) were not transformed but an efficient replication of non transforming SiLV was demonstrated. Increase of virus reverse transcriptase activity paralleled cell replication during successive passages. Preliminary results concerning the influence of viral infection on the life span and the karyotype of MRC-5 diploid cells will be reported and several implications of these findings discussed.
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PMID:Replication of primate oncornavirus SSV-1 on MRC-5 human diploid cells. 7 83

Continuous monitoring of enzymes, particularly those involved in nucleic acid synthesis could be a useful means of detecting infections and abnormalities in cells in culture. Model systems using mouse (3T3), human (MRC-5) and chick embryo cells infected with RNA tumour viruses were studied. Reverse transcriptase activities were determined by the incorporation of (3H) nucleotides into synthetic primer-templates or into complementary DNA of endogenous RNA and characterised by their specificity for primer-templates dT12-18.rAn, dG12-18.rCn, dT12-18.DAn and dG10.rCmn, their requirements for metal ions and inhibition by antisera. Measurement of reverse transcriptase is a more sensitive method than the COFAL test for the detection of RAV infection of chick cells. Iododeoxyuridine, bromodeoxyuridine and dexamethasone, which can induce latent C viruses, have no effect on MRC-5 cells; no increases in reverse transcriptase were detected and no C particles were seen by electron microscopy. Solid tumours developed in immunosuppressed mice injected s/c with 3T3 and MRC-5 cells chronically infected with MLV but none formed after injection of cells or virus suspension alone. Thymidine kinase activities of WI-38 and MRC-5 cells are greatly increased by infection with CMV or transformation with SV40. Mammalian tumours and tumour cell lines also show a high specific activity of cytoplasmic thymidine kinase.
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PMID:Reverse transcriptase and thymidine kinase as markers for tumorigenicity and viral contamination of cells. 7 84

Our studies have shown that the acyclic nucleotide analogues PMEA and HPMPC are able to penetrate into cells and are then activated to mono- and diphosphate derivatives. The latter correspond to triphosphate analogues and presumably serve an important role in the biological activity exerted by these antiviral agents. In support of this idea, the inhibitory effect of PMEApp on HIV reverse transcriptase has been demonstrated with both RNA and DNA template-primer systems. Further studies will be undertaken to determine the effect of HPMPCpp on viral DNA polymerases. Whereas the metabolism of PMEA in CEM cells gives rise to only PMEAp and PMEApp, additional metabolites were obtained in MRC-5 cells; the identity of these metabolites remains to be determined. In the case of HPMPC, a third metabolite was obtained in addition to HPMPCp and HPMPCpp, which has been tentatively assigned as a phosphate-choline adduct by analogy with activation of cytosine-based nucleoside derivatives. The metabolism of HPMPC was unchanged between uninfected and infected cells, indicating that viral enzymes are not necessary for the activation of HPMPC. The long intracellular half-lives of the HPMPC metabolites may have implications for the antiviral efficacy of this compound. The persistence of activated metabolites suggests that infrequent dosing may be possible due to a prolonged antiviral effect. Our results on the effectiveness of infrequent dosing schedules with HPMPC in the treatment of HSV 2 infections in mice support this hypothesis. It is also possible that HPMPCp-choline may serve as a reservoir for HPMPC and therefore for the presumed active metabolite HPMPCpp.
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PMID:Biochemical pharmacology of acyclic nucleotide analogues. 207 30

In transient gene expression assays we observed an increase in expression of the bacterial chloramphenicol acetyl-transferase (CAT) gene, under the transcriptional control of the HIV-1 LTR (pLTR-CAT), when this plasmid was cotransfected into Vero or MRC-5 cells with a plasmid containing either the HCMV immediate early 1 and 2 (E1, IE2) genes (pRL43a) or just the IE2 gene (pMP18). When the HCMV IE1 gene (pMP12) was cotransfected with pLTR-CAT into Vero cells the level of measurable CAT gene activity was below the level observed when pLTR-CAT was cotransfected with a nonspecific carrier plasmid (pGEM3). The negative influence of the HCMV IE1 gene product on the HIV-1 LTR in Vero cells was also observed when the HIV-1 tat gene (pLTR-TAT) was contransfected into Vero cells with pLTR-CAT and pMP12. However, when the HCMV IE1 gene was cotransfected into rhabdomyosarcoma (RD) cells with proviral HIV-1 DNA, an increase in viral production, as monitored by measurement of HIV-1 reverse transcriptase activity, was observed. In electrophoretic mobility shift assays, nuclear extracts obtained 15 hr post-HCMV infection (hpi) were found to contain a lower level of interaction with an oligonucleotide which corresponded to the HIV-1 LTR Sp-1 binding motif. Nuclear extracts obtained 40 hpi of MRC-5 cells had a greater level of interaction with, and changed the mobility of, the Sp-1 oligonucleotide relative to the uninfected nuclear extracts. HCMV-infected MRC-5 cell nuclear extracts also contain a factor(s) which interacted with the HIV-1 LTR between nucleotide positions -15 to -2 relative to the HIV-1 mRNA start site.
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PMID:Characterization of multiple molecular interactions between human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1). 215

The interaction between a chronically human immunodeficiency virus type 1 (HIV-1)-infected promonocytic line (U1) and a normal human embryonic lung fibroblast line (MRC-5) on HIV-1 expression was investigated. Coculture of U1 cells with MRC-5 cells induced HIV-1 reverse transcriptase (RT) activities 40- to 50-fold higher than those of parallel control cultures of U1 cells. Culture of U1 cells in the presence of media conditioned by MRC-5 cell culture supernatants resulted in a 30- to 40-fold greater HIV-1 RT activity over a 6-day period. HIV-1 RT activity, however, was not increased in the chronically infected T lymphocyte cell line (ACH-2) by either coculture with MRC-5 cells or when cultured in the MRC-5 cell culture supernatant-conditioned media. A polyclonal antibody against interleukin-6 (IL-6) blocked HIV-1 induction in the U1 cells by MRC-5 culture supernatants, indicating that IL-6 plays an important role in the HIV-1 induction. The magnitude of HIV-1 induction by the MRC-5 cell culture supernatant-conditioned media was proportional to the concentration of IL-6. In addition, the supernatants from three other normal human lung fibroblast (HLF) cell lines induced HIV-1 RT expression in U1 cells. Thus, normal unstimulated HLFs stimulate HIV-1 expression in chronically infected promonocytic cells by secreting IL-6, suggesting that the interaction of HLFs and macrophages may play an important role in the development of HIV-1 infection in the lungs.
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PMID:Induction of HIV-1 expression in chronically infected promonocytic cells cocultured with human lung fibroblasts. 876 62

All-trans retinoic acid (ATRA) is an essential component of the treatment of acute promyelocytic leukemia (APL), but the optimal timing and duration remain to be determined. Molecular characterization of this disease can refine the diagnosis and could be potentially useful in monitoring response to treatment. Patients defined morphologically to have APL were randomized to receive a 5-day course of ATRA before commencing chemotherapy or to receive daily ATRA commencing with chemotherapy and continuing until complete remission (CR). The chemotherapy was that used in current MRC Leukaemia Trials. Outcome comparisons were by intention to treat with additional analysis for relevant risk factors. Patients were characterized by molecular techniques for the fusion products of the t(15;17) and monitored by reverse transcriptase-polymerase chain reaction (RT-PCR) during and after treatment. Two hundred thirty-nine patients were randomized. Treatment with extended ATRA resulted in a superior remission rate (87% v 70%, P <.001), due to fewer early and induction deaths (12% v 23%, P =.02), and less resistant disease (2% v 7%, P =.03), which was associated with a significantly more rapid recovery of neutrophils and platelets. Extended ATRA reduced relapse risk (20% v 36% at 4 years, P =.04) and resulted in superior survival (71% v 52% at 4 years, P =.005). Presenting white blood cell count (WBC) was a key determinant of outcome. The 70% of patients who presented with a WBC less than 10 x 10(9)/L had a better CR (85% v 62%, P =.0001) and reduced relapse risk (22% v 42%, P =.002) and superior survival (69% v 43%, P <. 0001). Within the low count group, extended ATRA resulted in a better CR (94% v 76%, P =.001), reduced relapse risk (13% v 35%, P =. 04), and improved survival (80% v 57%, P =.0009). There was no evidence of benefit in patients presenting with a higher WBC (>10 x 10(9)/L). Molecular monitoring after the third chemotherapy course had a correlation with risk of relapse. The relapse risk was 57% if the RT-PCR was positive versus 27% if the RT-PCR was negative (P =. 006). APL patients who present with a low WBC derive substantial benefit from combining ATRA with induction chemotherapy until remission is achieved, whereas patients with a higher WBC did not benefit. Molecular characterization of disease can improve diagnostic precision and a positive RT-PCR after consolidation identifies patients at a higher risk of relapse.
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PMID:Presenting white blood cell count and kinetics of molecular remission predict prognosis in acute promyelocytic leukemia treated with all-trans retinoic acid: result of the Randomized MRC Trial. 1036 Nov 10

Pestiviruses are potential contaminants of biological products produced in bovine or porcine cells or manufactured via processes using animal-derived raw materials such as bovine serum. In order to investigate possible contamination of products including those manufactured and/or licensed in the US, 38 lots of viral vaccines and five lots of interferon alpha (IFNalpha) were tested by reverse transcriptase polymerase chain reaction (RT-PCR) for the presence of bovine viral diarrhoea virus (BVDV). All vaccines and interferons were negative for contaminating BVDV RNA when tested by RT-PCR, with the exception of an experimental live viral vaccine that had been produced in BVDV contaminated rabbit kidney cells. Cell lines commonly used to produce biological products and vaccines were experimentally infected with the NADL strain of BVDV to determine if they were permissive for virus replication. MRC-5 and WI-38 cells were not infected. In contrast, Vero, CHO and CEF cells showed evidence of pestivirus infection. Taken together these data suggested that currently licensed viral vaccines were unlikely to be contaminated with pestiviruses. However, cell banks derived from non-human primate, hamster or rabbit kidney cell lines, or cultures of primary chick embryo fibroblasts, may be infected with BVDV if exposed to pestivirus contaminated raw materials during manufacture.
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PMID:Evaluation of vaccines, interferons and cell substrates for pestivirus contamination. 1079 55

This study compares the performance of a line probe assay (LiPA) for the detection of the major mutations associated with reduced sensitivity to nucleoside analogues with a well characterised point mutation assay (PMA). Plasma samples obtained from patients in a trial of four reverse transcriptase inhibitors (MRC Quattro Trial) were tested by both LiPA and PMA at baseline, 32nd and 64th weeks for the presence of drug resistance associated mutations in the reverse transcriptase (RT) gene. HIV-1 RNA was extracted from plasma by the Boom method and amplified by RT-PCR prior to being tested by LiPA or PMA. Assay discrepancies were further investigated by sequencing of the RT gene. Of 275 samples available from 98 trial subjects, 246 samples were successfully amplified by PCR and analysed by LiPA and PMA for six mutations. Of the 1476 individual codons analysed, LiPA successfully assayed 1444 (97.8%) and PMA gave a result with 1418 (96.1%). LiPA failed to give a result for 32 codons from 22 samples and PMA failed with 58 codons from 38 samples. Gross differences between the two assays, in which one scored a codon as wild-type only and the other as mutant only or vice versa, occurred at 28 codons analysed (1.9%) representing 26 samples from 20 subjects. Sequencing of 22 of the 26 samples confirmed the LiPA result in nine cases, the PMA result in 11 and detected a novel variant at codon 215 in four cases. The PMA and LiPA approach to the detection of the major mutations that are genotypically associated with reduced sensitivity to nucleoside analogues can correctly detect mutations in 97% of the cases.
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PMID:Comparison of a point mutation assay with a line probe assay for the detection of the major mutations in the HIV-1 reverse transcriptase gene associated with reduced susceptibility to nucleoside analogues. 1096 Jun 99

To assess the clinical significance of AML1/ETO gene detected by nested reverse transcriptase polymerase chain reaction, the outcome of 7 patients with acute myeloblastic leukemia between 3 and 14 years of age were presented. All patients had complete remission (CR) at the end of induction (AML-MRC 10 protocol) and 4 underwent unpurged autologous, 2 allogeneic (from matched siblings) non-T-cell-depleted bone marrow transplantations (BMT) in first CR. One patient died due to allogeneic BMT-related complications, and 4 patients relapsed at 13, 17, 18, and 26 months. Only one patient achieved second CR. All relapsed patients died between 18 and 36 months with resistant disease (n = 3) or infection during salvage chemotherapy (n = 1). Two patients who had autologous BMT are alive and disease free at 44 and 50 months. Although statistical significance could not be shown, event-free survival and overall survival rates of AML1/ETO-positive patients (28.57 and 28.57%, respectively) at 3.5 years were even lower than those of AML1/ETO-negative patients. The results confirm some previous reports that AML1/ETO gene in children and adolescents is not a favorable prognostic factor.
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PMID:Is AML1/ETO gene expression a good prognostic factor in pediatric acute myeloblastic leukemia? 1103 33

An outbreak of aseptic meningitis occurred in the northern area of Jiangsu Province in China from January to July in 2003. A total of 1,681 cases were involved in this outbreak, and 99% of patients were <15 years of age. To identify the etiologic agent, 66 cerebrospinal fluid specimens were tested by cell culture. Eighteen showed an enteroviruslike cytopathic effect on MRC-5 human fetal diploid lung cells. An enterovirus primer-mediated reverse transcriptase-polymerase chain reaction, a standard neutralization assay, and sequencing of the complete capsid-encoding (VP1) gene identified the 18 isolates (FDJS03) as echovirus 30. At least a 10% difference was seen in nucleotide sequences of VP1 between FDJS03 isolates and other global strains of echovirus 30. Phylogenetic analysis based on complete sequences of VP1 was performed to further characterize the FDJS03 isolates. This report is the first to identify a distinct lineage of echovirus 30 as a probable cause of this outbreak.
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PMID:Echovirus 30, Jiangsu Province, China. 1582 94


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