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)

A simple approach for the determination of drug susceptibilities by using human immunodeficiency virus type 1 (HIV-1) RNA from the sera of patients is described. HIV-1 RNA was extracted from patient sera, and the 5' part of the reverse transcriptase (RT) gene was transcribed into DNA and amplified in a nested PCR. The amplified fragment covers the 3' part of the protease gene and amino acids 1 to 304 of the RT gene. This fragment can be introduced through homologous recombination, as described previously, into a novel HIV-1 reference strain (pHXB2 delta 2-261RT) from which amino acids 2 to 261 of RT have been deleted. The resulting recombinant virus expresses all properties of the HXB2 reference strain except for those encoded by the introduced part of the patient RT gene. Recombinant viruses were subsequently tested for drug susceptibility in a microtiter format killing assay [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay] as well as in the standard HeLa CD4+ plaque reduction assay. Similar susceptibility profiles were obtained by each assay with recombinant viruses derived from patients receiving alternating nevirapine and zidovudine treatment or lamivudine-zidovudine combination therapy. In conclusion, this approach enables high-through-put determination of the drug susceptibilities of serum RNA-derived RT genes, independent of the patient's viral background, and generates the possibility of relating changes in susceptibility to changes in viral genotypes.
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PMID:Human immunodeficiency virus type 1 drug susceptibility determination by using recombinant viruses generated from patient sera tested in a cell-killing assay. 889 Nov 52

Rauscher murine leukemia virus induces an erythroleukemia in susceptible strains of mice that is associated with splenomegaly and viremia. This animal model has been used for evaluating the in vivo efficacy of potential anti-HIV agents. The in vivo antiviral activity of therapeutic agents has usually been determined by measuring a reduction in the spleen weights of compound-treated mice or by quantitating viremia with the UV-XC plaque assay. The UV-XC assay, however, is time-consuming and labor-intensive. Virions of Rauscher murine leukemia virus, like other retroviruses, contain the enzyme reverse transcriptase. Quantitating the level of this enzyme in infected mouse sera provides a more rapid measure of viremia in the animal. We have examined the effects of several reagents, including detergent, KCl, EGTA, dGMP, spermine, as well as protease and RNase inhibitors, on the reverse transcriptase assay. The optimized assay method was effective in evaluating the antiviral activity of AZT in the Rauscher murine leukemia virus in vivo model. The assay is also amenable to automation if large numbers of assays are required.
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PMID:Optimization of the reverse transcriptase assay for the detection of viral burden in mice infected with Rauscher murine leukemia virus. 891 Jun 49

Cytokines are believed to play an important role in the pathogenesis of cutaneous T cell lymphoma. Data regarding the local cytokine pattern in mycosis fungoides (MF) are partly conflicting. Recent studies have suggested a shift from type 1 to type 2 cytokine pattern because IL-4 and IL-5 mRNA have been more frequently detected in lesions of advanced stages. Another study has described a type 1 cytokine pattern in MF lesions. None of the previous studies of cytokine mRNA expression in MF, however, used quantitative methods, and therefore only the presence of a cytokine, but not the level of expression, could be determined. To gain better insight into the development of cytokine pattern during tumor progression we used semiquantitative reverse transcriptase-polymerase chain reaction to analyze cytokine mRNA expression in MF skin lesions at different stages. Biopsies from patients with patch (n = 11), plaque (n = 6), and tumor (n = 3) stage MF were compared with biopsies from patients with pleomorphic T cell lymphoma (n = 5), psoriasis (n = 7), atopic dermatitis (n = 5), and nonlesional skin (n = 8). MF progression was associated with significantly higher IL-10 and lower interferon-gamma mRNA expression. Moreover, the stage-dependent increase in IL-10 mRNA expression was also found in paired samples from individual patients. Unlike in pleomorphic T cell lymphoma, however, typical T helper 2 cells did not seem to be the source of increasing IL-10 in advanced MF, because stage-independent IL-4 mRNA was rarely detected, suggesting contribution of nonlymphoid cells to local IL-10 production. The overexpression of IL-10 in MF may be of importance for tumor progression, because this immunosuppressive cytokine might be involved in downregulation of immunologic tumor surveillance.
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PMID:Progression of mycosis fungoides is associated with increasing cutaneous expression of interleukin-10 mRNA. 894 70

The purpose of this study was to analyze hepatobiliary samples of patients with biliary atresia for rotavirus groups. A, B, and C, because group A rotavirus had been used to produce an animal model of the disease and group C rotavirus had been found in hepatobiliary samples from one group of patients. Biliary remnants and liver tissue from 10 biliary atresia and 14 control patients with other liver diseases were examined for rotavirus groups A, B, and C using nonisotopic, reverse transcriptase polymerase chain reaction enzyme immunoassay. Biliary atresia patients had a median age of 3 mo and were from a confined geographic area. Rotaviral stocks from groups A and C were used as polymerase chain reaction-positive controls. The limits of detection for rotaviral RNA from these two groups were respectively, 5 plaque-forming units and 50 tissue culture infectious doses (ID50). Tissue culture was 100-fold less sensitive for groups A and C than the polymerase chain reaction. The nested nonisotopic probes hybridized in solution only with their homologous target DNAs as determined by the enzyme immunoassay, or by Southern blot hybridization. Although it was possible to detect mRNA from a beta-actin housekeeping gene in all of the hepatobiliary samples, no evidence of rotaviral RNA was found in either the biliary atresia or the negative control group. In conclusion, rotavirus is not a common viral etiology of biliary atresia.
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PMID:Lack of evidence for rotavirus by polymerase chain reaction/enzyme immunoassay of hepatobiliary samples from children with biliary atresia. 902 44

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect Rift Valley fever (RVF) virus RNA in experimentally infected mosquitoes was developed. The specificity of the assay was evaluated with three other phleboviruses; sandfly fever Sicilian (Sabin), sandfly fever Naples (Sabin) and Punta Toro (MSP 3) viruses. The relative sensitivity of the assay, determined by using RVF virus RNA extracted from serial dilutions of virus culture, was approximately 50 plaque forming units. This sensitivity level was 100-fold higher when a nested PCR procedure was used. When the RT-PCR assay was used with coded samples of intrathoracically-infected and uninfected mosquito, the assay detected the virus in all infected mosquitoes. With this assay, it was possible to detect RVF virus RNA in a single infected mosquito in the background of 10, 25 or 50 uninfected mosquitoes.
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PMID:Detection of Rift Valley fever virus in mosquitoes by RT-PCR. 907 14

In this study we provide further evidence associating activated cells of the monocyte lineage with the lesions of multiple sclerosis (MS). Using a combination of immunohistochemistry and reverse transcriptase-dependent in situ polymerase chain reaction analysis, we have identified monocytes expressing inducible nitric oxide synthase (iNOS) to be prevalent in the plaque areas of post mortem brain tissue from patients with MS. In addition, we have obtained evidence of the nitration of tyrosine residues in brain areas local to accumulations of iNOS-positive cells. In parallel studies we have assessed the effects of inhibitors of iNOS induction, as well as scavengers of nitric oxide and peroxynitrite in the experimental allergic encephalomyelitis model. Significant therapeutic effects were seen with the inhibitor of iNOS induction, tricyclodecan-9-xyl-xanthogenate, a nitric oxide scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and a peroxynitrite scavenger, uric acid. In particular, treatment with high doses of uric acid virtually prevented clinical symptoms of the disease. Together with our demonstration of the presence of activated macrophages expressing high levels of iNOS and evidence of peroxynitrite formation in brain tissue from patients with MS, these findings are of importance in the development of approaches to treat this disease.
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PMID:Prevention of experimental allergic encephalomyelitis by targeting nitric oxide and peroxynitrite: implications for the treatment of multiple sclerosis. 912 29

Enteric infection of mice with respiratory enteric orphan virus (reovirus) type 1, strain Lang elicits both humoral and cellular immune responses. To investigate the role of CD8+, alpha/beta T-cell receptor (TCR)+ T cells in mucosal immunity to an enteric pathogen, we examined immune responses and viral clearance following enteric reovirus infection in C57BL/6, B6129F2, and beta2-microglobulin-deficient (beta2m-/-) mice. Analysis of Peyer's patch and lamina propria culture supernatants revealed a two- to threefold increase in levels of reovirus-specific immunoglobulin A in beta2m-/- mice compared to normal controls. These data corresponded to a similar increase in the frequency of virus-specific immunoglobulin A-producing cells in Peyer's patches and lamina propria and an increase in immunoglobulin G-producing cells in spleens from beta2m-/- mice compared to controls. These increased humoral immune responses were not due to a difference in B-cell populations because cell counts and flow cytometric analyses showed that beta2m-/- and control mice had similar numbers and percentages of B cells in mucosal and systemic tissues. Analysis of cytokine message by reverse transcriptase-PCR 5 and 10 days after infection revealed no difference in message level for transforming growth factor beta, gamma interferon, interleukin-4, interleukin-5, or interleukin-6 for all mouse strains. Virus tissue titers determined by plaque assay at 5 and 10 days after infection demonstrated that beta2m-/- mice cleared reovirus from the small intestines with the same efficiency as control mice. Collectively, these data suggest that CD8+, alpha/beta TCR+ T cells may regulate mucosal and systemic humoral immune responses to oral infection with reovirus.
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PMID:Enhanced mucosal and systemic immune responses to intestinal reovirus infection in beta2-microglobulin-deficient mice. 922 66

Lipophilic esters of 3'-azido-3'-deoxy-5'-O-(carboxyphosphinyl)thymidine (PFA-AZT) were synthesized and tested for antiretroviral activity in CD4+ HT4-6C cells infected with either wild-type HIV-1LAI, a PFA-resistant strain encoding a single-point mutation in reverse transcriptase (E89K), or an AZT-resistant clinical isolate (A018-post). Arbuzov condensation of 1-octadecyl, 1-eicosanyl, and 1-docosanyl chloroformate with trimethyl phosphite yielded the corresponding dimethyl long-chain alkyl triesters of PFA. Selective removal of one methyl group from the triesters with sodium iodide yielded monosodium salts, whereas treatment with bromotrimethylsilane cleaved both methyl groups while leaving the long-chain alkyl group intact. Neutralization of the resulting [(alkyloxy)carbonyl]phosphonic acids with 2 equiv of sodium methoxide afforded disodium salts of the phosphonic acid moiety. Similar chemistry was used to obtain the mono- and disodium salts of the cholesterol ester of PFA. Reaction of the triesters with phosphorous pentachloride, followed by coupling with AZT and O-demethylation with sodium iodide, afforded 3'-azido-3'-deoxy-5'-O-[[(1-octadecyloxy)carbonyl]phosphinyl ]thymidine (9a), 3'-azido-3'-deoxy-5'-O-[[(1-eicosanyloxy)carbonyl]phosphinyl ]thymidine (9b), 3'-azido-3'-deoxy-5'-O-[[(1-docosanyloxy)carbonyl]phosphinyl ]thymidine (9c), and 3'-azido-3'-deoxy-5'-O-[[(3 beta-cholest-5-enyloxy)carbonyl]phosphinyl]thymidine (9d). Concentrations of 9a-d found to inhibit replication of wild-type HIV-1LAI by 50% (EC50 values) as measured in a plaque reduction assay were in the 0.1-0.3 microM range as compared with 0.013 microM for AZT and 133 microM for PFA. The concentration at which toxicity was observed in 50% of the host cells (TC50 values) as measured by a visual grading scale of cellular morphology was 10 microM for 9a and 9d, 32 microM for 9b, and 320 microM for 9c. Thus, the TC50/EC50 ratio or selectivity index (SI) was 100 for 9a, 230 for 9b, and 1000 for 9c but only 33 for 9d, suggesting that the straight-chained fatty alcohol esters were more therapeutically selective. Similar TC50 and SI values were obtained for rapidly dividing CEM lymphoblasts as for HT4-6C cells. In assays against E89K, 9a-c had mean EC50 values of 0.13, 0.009, and 0.17 microM, whereas the EC50 of PFA was > 1000 microM and that of AZT was 0.009 microM; thus, E89K was highly resistant to PFA but not cross-resistant to either AZT or the lipophilic PFA-AZT conjugates. In viral replication assays against the A018C-post isolate, the mean EC50 values of 9a-c were 0.30, 0.53, and 0.77 microM as compared with 2.9 microM for AZT and 65 microM for PFA; thus, the virus recovered from a patient pretreated with AZT was not cross-resistant to either PFA or 9a-c. A notable feature of these results was that, in addition to being > 1000-fold more potent than PFA against the PFA-resistant mutant, the lipophilic PFA-AZT conjugates were more potent than PFA, as well as AZT, against AZT-resistant HIV-1.
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PMID:Synthesis and in vitro activity of long-chain 5'-O-[(alkoxycarbonyl)phosphinyl]-3'-azido-3'-deoxythymidines against wild-type and AZT- and foscarnet-resistant strains of HIV-1. 925 55

We have studied the population dynamics in response to selective drug pressure of mixtures of wild-type and mutant HIV viruses exposed to either an inhibitor of the viral protease or a nonnucleoside allosteric inhibitor of the viral reverse transcriptase. In order to quantitate mutant virus present in a mixed population, we developed a selective plaque assay, which appears to be generally applicable to population dynamics studies where the viruses in question differ in the sensitivity to a given drug by at least 10-fold. In this assay system, the titer of virus in a mixture is measured in the absence and presence of a concentration of a specific inhibitor known to suppress virus replication by 99%. Virus detected in the presence of inhibitor corresponds to mutant virus, whereas detection in the absence of drug results in quantitation of the total virion population. Wild-type virus is then estimated by difference. Utilizing this system we studied the fate of mixtures of wild-type and the protease-resistant mutant variant I84V in the presence and absence of the cyclic urea HIV protease inhibitor, DMP 450. We also examined the dynamics of mixtures of wild-type and the resistant mutant variant, L100I, in the presence and absence of the drug DMP 266. In both systems we demonstrated that in the absence of drug, mutant virus is at a selective disadvantage for growth compared to wild-type, whereas in the presence of a specific inhibitor, mutant virus exhibits the selective growth advantage over wild-type virus. Better understanding of HIV population dynamics may allow the development of superior inhibitors and the careful application of combination therapy in the clinical setting.
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PMID:Population dynamics studies of wild-type and drug-resistant mutant HIV in mixed infections. 929 20

Genomic segment A of the variant infectious bursal disease virus (IBDV) strain MD was amplified by reverse transcriptase/polymerase chain reaction and further characterized by baculovirus expression. Three different baculovirus clones were constructed containing the genes encoding VP2, VP2/VP4, and the complete polyprotein cloned into the baculovirus transfer vector pVL1392. Baculovirus recombinants were identified by dot blot hybridization and were plaque purified three times. Baculovirus expression of the recombinants produced IBDV-specific proteins that were comparable in molecular size to native MD IBDV viral proteins VPX (48 kD), VP2 (45 kD), VP3 (32 kD), and VP4 (28 kD) as determined by sodium dodecyl sulfare-polyacrylamide gel electrophoresis and western immunoblot analysis. All three recombinants produced a 48-kD protein that possibly represents VPX, the precursor product of VP2. In addition to the 48-kD protein, the VP2/VP4 recombinant produced an IBDV-specific protein corresponding to the 28-kD VP4. The baculovirus-expressed polyprotein gene produced, in addition to the 48-kD protein, a 32-kD (VP3) IBDV-specific protein and a 29-kD protein that migrated slightly slower than MD VP4. The baculovirus-expressed proteins were used as antigens in an indirect enzyme-linked immunosorbent assay (ELISA). The ELISAs detected antibodies against the variant IBDV strains MD, GLS, and IN and the classic IBDV strains SAL and STC but did not detect antibodies against the variant Del-A and classic IBDV strain BVM or the serotype 2 IBDV strain OH.
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PMID:Expression of MD infectious bursal disease viral proteins in baculovirus. 935 8


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