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)

Zidovudine treatment of individuals infected with human immunodeficiency virus type 1 (HIV-1) results in HIV-1 isolates with a reduced zidovudine sensitivity in vitro. This reduction is due to mutations causing amino acid substitutions at five codons (41, 67, 70, 215, and 219) on the reverse transcriptase enzyme of HIV. HIV-1 isolates were obtained 8 to 69 weeks after therapy discontinuation from 10 patients at different stages of disease. Zidovudine sensitivity was determined by the HeLa CD4+ plaque assay. The presence of the resistance-conferring mutations was determined by using a selective polymerase chain reaction. Sensitivity could be determined for six isolate pairs: one showed a decline in the 50% inhibitory zidovudine concentration after therapy discontinuation; four pairs did not show a change. The majority of changes in the five codons in isolates from all 10 patients were the result of a relative increase in the wild-type sequence. Complete changes from mutant to the wild type were seen for only two codons in isolates from two patients. This study of isolates from a small group of individuals at different stages of disease, who had been taking zidovudine for 1 to 2 years, shows that a period of 1 year without zidovudine may be required to achieve a change from a mutant or mixed virus population to a wild-type virus population.
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PMID:Effects of discontinuation of zidovudine treatment on zidovudine sensitivity of human immunodeficiency virus type 1 isolates. 810 17

To investigate whether the oral cavity is a potential reservoir and possible sanctuary for Helicobacter pylori, supragingival and subgingival plaques were analyzed by a Helicobacter genus-specific reverse transcriptase-polymerase chain reaction based on the sequence data of H. pylori 16S rRNA. The amplified 500-bp DNA fragment was identified by ethidium bromide staining after agarose gel electrophoresis and by Southern hybridization. Twenty-five dyspeptic patients were studied. Histologic examination of gastric biopsy specimens revealed that 18 had H. pylori gastritis and 7 did not. For seven of the 18 (38.8%) patients with proven H. pylori gastritis, H. pylori was also identified in their dental plaque. None of the patients without H. pylori gastritis had H. pylori in their dental plaque. The detection of H. pylori in dental plaque suggests that this H. pylori colonization is not restricted to the gastric mucosa and that this ecological niche may serve as a possible sanctuary which may be responsible for reinoculation of the stomach after topical anti-H. pylori therapies such as bismuth.
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PMID:Detection of Helicobacter pylori in dental plaque by reverse transcription-polymerase chain reaction. 846 87

Ten mutations were generated in the env gene of Moloney murine leukaemia virus DNA. The mutations were made by site-directed mutagenesis to alter basic amino acids (lysine or arginine) in the surface glycoprotein gp70. Mutants were investigated following transfection into NIH/3T3 cells. All 10 mutants released virion particles into the medium, suggesting that none of the mutations affected overall viral gene expression or virion budding. Two mutants were positive in XC plaque assay, reverse transcriptase assay and re-infection experiments, showing that these mutations occurred in parts of the molecule not essential for infection. Three mutants were negative in both the XC plaque assay and re-infection experiments, suggesting that they make non-infectious virus particles. The results indicate a defect in the early phase of infection, perhaps in receptor binding or in the fusion of virion and host membranes. The other mutations resulted in reduced infectivity of released virion particles.
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PMID:Mutational analysis of Moloney murine leukaemia virus surface protein gp70. 846 56

This study developed a methodology to increase the sensitivity of enteric virus detection in tap water concentrates. Polymerase chain reaction (PCR) detection of virus in reduced volumes of virus-containing water concentrates was successful following removal of PCR inhibitory substances. Poliovirus 1 and coxsackievirus B3 were seeded into 378 l of tap water, concentrated with 1MDS filters, and reconcentrated by organic flocculation. The volume of concentrates was successfully reduced from 25 to 5 ml without loss of virus recovery. PCR detection of virus after treatment of a water concentrate (1.1 x 10(5)-fold concentration) with a Sephadex G-100 plus Chelex-100 column, or Sephadex G-50 plus Chelex-100 column, followed by heat treatment to release viral RNA, was compared with direct phenol-chloroform-isoamyl alcohol (PCI) extraction of viral RNA. The Sephadex G-50 plus Chelex-100 column did not remove inhibitory substances efficiently. The Sephadex G-100 plus Chelex-100 column could remove inhibitory substances, however, 99% of the viruses were also removed by the column. PCI extraction was found to be sufficient to remove inhibitory substances for reverse transcriptase (RT)-seminested PCR with a sensitivity of 0.2 plaque-forming units/10 microliters (0.2 PFU/l tap water).
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PMID:Increased sensitivity of poliovirus detection in tap water concentrates by reverse transcriptase-polymerase chain reaction. 860 95

A reverse transcriptase polymerase chain reaction (RT-PCR) assay was evaluated for the detection of eastern equine encephalomyelitis virus (EEEV). EEEV was detected by amplification of a 416-bp PCR product from within the E2 gene. Internal restriction endonuclease digestion and hybridizations to EEEV RNA demonstrated that the PCR product was amplified from EEEV. PCR amplifications from serial dilutions of an EEEV isolate identified by a neutralization test and titered by an infectious assay in cell culture indicated that this RT-PCR assay detected viral RNA at concentrations below 1 plaque forming unit(PFU) per reaction. The performance of the PCR assay in detection of EEEV was compared with an infectious assay detection procedure (IA/IFA) as part of the New Jersey 1993 vector surveillance program. During 1993, 7,007 field-collected Culiseta melanura (Coquillett) were assayed in 522 pools by both RT-PCR and IA/IFA. EEEV was detected in 95 pools by RT-PCR and 17 pools by IA/IFA; all IA/IFA positive pools were also positive by RT-PCR. During the 1993 field season, RT-PCR consistently detected virus at enzootic foci earlier that IA/IFA and in greater numbers of mosquito pools. The data indicated that viral RNA may be present earlier and in more mosquitoes than indicated by IA/IFA.
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PMID:Evaluation of reverse transcriptase polymerase chain reaction for the detection of eastern equine encephalomyelitis virus during vector surveillance. 866 94

Amino acid variation at reverse transcriptase (RT) codon 210 (generally Leu-210 to Trp [L210W], TTG-->TGG) is occasionally detected after the initiation of azidothymidine (AZT) therapy. The impact of this variation on AZT resistance and viral replication was addressed by four different approaches. The frequency and genetic background of the L210W mutation in vivo were assessed by analyzing sera of AZT-naive and AZT-experienced patients by RT-PCR and DNA sequencing. The degree of AZT resistance (50% infective concentration [IC50]) of recombinant viruses constructed by using the RT of 21 clinical isolates was stratified by the presence or absence of the 210 mutation. The AZT IC50S of a panel of mutant viruses (with or without W-210) constructed by site-directed mutagenesis in an HXB2 background were assayed by using a HeLa CD4 plaque reduction assay. Finally, the effect of the 210 mutation on viral replication was assessed by replication competition of an AZT-resistant virus, RTMN (L-41/Y-215), and RTMN with the W-210 mutation in the presence and in the absence of AZT. In AZT-naive patients, tryptophan at RT residue 210 was rare. After AZT exposure, W-210 appeared in a minority of those patients, most commonly in association with L-41 and Y-215. The presence of W-210 increased the AZTIC50 by two- to fourfold, as determined by both the recombinant virus assay and site-directed mutagenesis. A significant replication advantage in favor of the wild-type L-210 over W-210 was observed, although the selection against the 210 mutant was two- to threefold lower when the viruses were grown in the presence of 5 microM AZT. In summary, the L210W mutation appears to be of marginal significance, conferring approximately two- to fourfold-reduced sensitivity to AZT compared with similar AZT-resistant genomes with L-210. The selection pressure against W-210 may account for the modest proportion of patients in which W-210 appears in vivo.
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PMID:Significance of amino acid variation at human immunodeficiency virus type 1 reverse transcriptase residue 210 for zidovudine susceptibility. 870 14

Many bunyaviruses of the Bunyamwera and California serogroups are medically important human pathogens. The development of an effective technique to detect the viruses by using molecular biologic tools, such as PCR, improves not only clinical diagnosis but also virologic surveillance of mosquito vectors in the field. In this study, we evaluated eight pairs of primers for reactivity with 44 viruses of the genus Bunyavirus, using a reverse transcriptase PCR technique. With a pair of serogroup-specific primers we designed, all viruses of the serogroups tested could be detected. Further, virus-specific primer pairs were identified for California encephalitis virus, Jamestown Canyon virus, La Crosse virus, and snowshoe hare virus for use in North America. Using this technique, we could detect one La Crosse virus-infected mosquito in a pool of 100 mosquitoes with undetectable plaque titers.
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PMID:Detecting bunyaviruses of the Bunyamwera and California serogroups by a PCR technique. 872

The French paradox is a dietary anomaly which has focused attention on the Mediterranean diet. Epidemiological studies revealed that this diet, replete in flavonoid-rich foods (Allium and Brassica vegetables, and red wine), correlated with the increased longevity and decreased incidence of cardiovascular disease seen in these populations. The most frequently studied flavonoid, quercetin, has been shown to have biological properties consistent with its sparing effect on the cardiovascular system. Quercetin and other flavonoids have been shown to modify eicosanoid biosynthesis (antiprostanoid and anti-inflammatory responses), protect low-density lipoprotein from oxidation (prevent atherosclerotic plaque formation), prevent platelet aggregation (antithrombic effects), and promote relaxation of cardiovascular smooth muscle (antihypertensive, antiarrhythmic effects). In addition, flavonoids have been shown to have antiviral and carcinostatic properties. However, flavonoids are poorly absorbed from the gut and are subject to degradation by intestinal micro-organisms. The amount of quercetin that remains biologically available may not be of sufficient concentration, theoretically, to explain the beneficial effects seen with the Mediterranean diet. The role of flavonoids may transcend their presence in food. The activity of flavonoids as inhibitors of reverse transcriptase suggests a place for these compounds in the control of retrovirus infections, such as acquired immunodeficiency syndrome (AIDS). In addition to specific effects, the broad-modulating effects of flavonoids as antioxidants, inhibitors of ubiquitous enzymes (ornithine carboxylase, protein kinase, calmodulin), and promoters of vasodilatation and platelet disaggregation can serve as starting material for drug development programmes.
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PMID:Review of the biology of Quercetin and related bioflavonoids. 884 3

We have selected and plaque purified a mutant of feline immunodeficiency virus (FIV) that is resistant to 2',3'-dideoxycytidine (ddC). This mutant was selected in cultured cells in the continuous presence of 25 microM ddC. The mutant, designated DCR-5c, was fourfold resistant to ddC, threefold resistant to 2',3'-dideoxyinosine, and more than fourfold resistant to phosphonoformic acid. DCR-5c displayed little or no resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine, 3'-azido-3'-deoxythymidine, or 9-(2-phosphonylmethoxyethyl) adenine. Reverse transcriptase purified from DCR-5c was less susceptible to inhibition by ddCTP, phosphonoformic acid, ddATP, or azido-dTTP than the wild-type FIV reverse transcriptase. Sequence analysis of DCR-5c revealed a single base change (G to C at nucleotide 2342) in the reverse transcriptase-encoding region of FIV. This mutation results in substitution of His for Asp at codon 3 of FIV reverse transcriptase. The role of this mutation in ddC resistance was confirmed by site-directed mutagenesis.
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PMID:Selection and characterization of a mutant of feline immunodeficiency virus resistant to 2',3'-dideoxycytidine. 884 58

We selected mutants of feline immunodeficiency virus (FIV) that are resistant to 2',3'-dideoxy-2',3'-didehydrothymidine (d4T). Two mutants were selected in cultured cells with a stepwise increase in d4T concentration, resulting in mutants able to replicate in 100 microM d4T. These mutants were three- to sixfold more resistant to d4T than wild-type FIV. They were also cross-resistant to 3'-azido-3'-deoxythymidine (AZT), 3'-fluoro-2',3'-dideoxythymidine, 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and 9-(2-phosphonylmethoxyethyl)adenine, and they were highly resistant to phosphonoformic acid (PFA). Plaque-purified mutants were isolated from each of the mutant populations. The mutant phenotype was stable, because both of the plaque-purified mutants remained d4T resistant even after three passages in the absence of d4T. One of the plaque-purified mutants, designated D4R-3c, was further characterized. Compared with wild-type reverse transcriptase (RT), RT purified from D4R-3c was 3-fold resistant to inhibition by the 5'-triphosphate of d4T, 10-fold resistant to inhibition by the 5'-triphosphate of AZT, and 6-fold resistant to PFA. D4R-3c had a single point mutation in the RT-encoding region of the pol gene at position 2474, resulting in a Val to Ile mutation at codon 47 of the FIV RT. The role of this mutation in d4T resistance was confirmed by site-directed mutagenesis.
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PMID:Mutants of feline immunodeficiency virus resistant to 2',3'-dideoxy-2',3'-didehydrothymidine. 887 67


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