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)

On the basis of the detection of the enteroviral RNA in the hearts of patients with healed myocarditis and dilated cardiomyopathy, we investigated cardiac viral persistence in experimental myocarditis. Weanling C3H/He mice were given myocarditis by inoculation with coxsackievirus B3 (Nancy strain), and their hearts were examined by genomic studies and viral isolation up to the 180th day after inoculation. The virus was isolated from the heart until the 9th day. By slot-blot hybridization, viral RNA was also only detected until the 9th day in the heart. Specific DNA amplification using the polymerase chain reaction (PCR) was performed after a reverse transcriptase reaction, then followed by Southern blot hybridization with a 32P-labelled oligomer probe. This technique achieved the type-specific detection of coxsackievirus B3 even at a level of less than one of the 50% tissue culture infective dose (TCID50). With this technique, viral RNA was detected up to the 28th day after inoculation. Thus, the viral RNA persisted in the hearts of these mice even when infectious virus could no longer be detected.
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PMID:Viral genomic detection in the hearts of C3H/He mice with experimental Coxsackievirus B3 myocarditis by gene amplification using the polymerase chain reaction. 131 49

In situ transcription (IST) was shown to be useful for the detection of human enteroviral RNA in cultured cells. A primer to detect a wide variety of enteroviral genomes and a coxsackievirus type B3 genome-specific primer were demonstrated to be efficient in IST assays. Transcription times greater than 10 to 30 min did not significantly improve the acquisition of a specific signal, whereas the signal-to-noise ratio decreased with time. Inclusion of actinomycin D to suppress DNA-dependent DNA polymerase activity in reverse transcriptase decreased the signal that was obtained without improving the signal-to-noise ratio. Use of RNase H-free murine leukemia virus reverse transcriptase in the IST reaction increased the signal versus that obtained by use of the avian myeloblastosis virus enzyme, which contains endogenous RNase H activity. Exogenous RNase H added to the transcription reaction ablated the signal. Background transcription because of poorly hybridized (mismatched) primers was reduced after primer hybridization and prior to the transcription reaction by rinsing fixed cells with 3 M tetramethylammonium chloride at temperatures which dissociate mismatched primer-template duplexes. The rapid detection time and the simplicity of application suggest that IST can be performed with a high specificity for the detection of enteroviral genomic sequences in cultured cells and may be more useful than in situ hybridization for the detection of enteroviral genomes.
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PMID:Detection of enteroviruses in cell cultures by using in situ transcription. 137 Aug 49

3'-Azido-2',3'-dideoxy-5-methylcytidine (CS-92, AzddMeC) is an antiviral nucleoside analogue structurally related to 3'-azido-3'-deoxythymidine (AZT). CS-92 is a potent and selective inhibitor of HIV-1 reverse transcriptase and HIV-1 replication in human lymphocytes and macrophages. The EC50 for CS-92 in HIV-1-infected human PBM cells was 0.09 microM. In HIV-1-infected human macrophages, the EC50 was 0.006 microM. This compound was also effective against human immunodeficiency virus type 2 in lymphocytes. The replication of Friend murine virus was only weakly inhibited, and no effect was observed against herpes simplex virus type 1 and type 2 and coxsackievirus B4. CS-92 was not toxic to PBM or Vero cells when tested up to 200 microM and was, furthermore, at least 40 times less toxic to granulocyte-macrophage and erythroid precursor cells in vitro than was AZT. The interaction of the 5'-triphosphate of CS-92 with HIV-1 reverse transcriptase indicated competitive inhibition (the inhibition constant, Kis, was 0.0093 microM) with a 30-fold greater affinity for CS-92-TP than for ddCTP. CS-92-TP inhibited HIV-1 reverse transcriptase by 50% at a concentration 6,000-fold lower than that which was required for a similar inhibition of DNA polymerase alpha. Pharmacokinetic studies showed that CS-92 was not deaminated to AZT in rats, but this compound was found to have a half-life of 2.7 hours. In rhesus monkeys, however, a compound with a retention time and ultraviolet spectra characteristics similar to AZT was detected. The mean half-life in rhesus monkeys for CS-92 was 1.52 and 1.74 h after intravenous and oral administration, respectively, and the oral bioavailability was about 21 percent. Additional preclinical studies with CS-92 will determine the ultimate utility of this antiviral agent for the treatment of HIV-1 infections.
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PMID:Antiretroviral activity, biochemistry, and pharmacokinetics of 3'-azido-2',3'-dideoxy-5-methylcytidine. 170 74

A sensitive method based on PCR followed by liquid-phase hybridization for detection of enterovirus and rhinovirus RNAs in clinical specimens and cell culture supernatants is described. RNA was extracted from stool samples, throat swabs, nasopharyngeal aspirates, cerebrospinal fluid, urine, and plasma with a commercial phenol-guanidinium-chloroform reagent and purified on a polysulfone membrane, on which the reverse transcriptase reaction was also done. Two sets of oligonucleotide primers from the 5' noncoding region of picornaviruses were selected for DNA amplification of 153-bp (enterovirus) and 120-bp (rhinovirus) regions. Double-stranded amplicons were digested into single strands with T7 gene 6 exonuclease and quantitated by an assay using a europium-labeled probe, streptavidin- and biotinylated probe-coated microtitration wells, and time-resolved fluorometry. The sensitivity of the assay was about one template molecule when purified coxsackievirus A9 RNA was used. All enterovirus prototype strains, except echoviruses 22 and 23, and clinical isolates grown in cell culture or suckling mice were strongly positive by the enterovirus PCR-hybridization, as were selected prototype strains and untyped isolates of rhinoviruses by the rhinovirus PCR-hybridization. In a series of 100 clinical specimens tested, the results for 92 agreed with virus culture results. The detection method described will be useful in etiopathogenic studies on enteroviruses and rhinoviruses.
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PMID:Detection of enteroviruses and rhinoviruses in clinical specimens by PCR and liquid-phase hybridization. 775 71

The exact cause of dilated cardiomyopathy (DCM) remains uncertain. However, a possibility of transition from coxsackievirus-infected myocarditis to DCM has been suspected. We investigated the role of enteroviral infection in the pathogenesis of DCM. The nested reverse transcriptase polymerase chain reaction (nRT-PCR) was used to detect enteroviral RNA in 45 endomyocardial biopsy tissues obtained from 35 patients with DCM and 10 patients (controls) with other non-infectious cardiac diseases. Enteroviral RNA was detected in 17 (49%) of the 35 patients with DCM. The progression to cardiac failure was rapid, usually within 12 months, and myocardial fibrosis and myocytic hypertrophy were marked in patients that were enteroviral RNA positive. Enteroviral RNA was not detected in any controls.
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PMID:Enteroviral RNA in dilated cardiomyopathy. 792 15

Mice infected with the Tucson strain of coxsackievirus B1 (CVB1T) develop chronic T cell-mediated polymyositis that is manifest as the acute infection resolves and is characterized by hindquarter weakness and muscle inflammation. This model system was used to study persistence of CVB1T RNA by using reverse transcriptase-polymerase chain reaction (RT-PCR). For the most part, RNA persistence reflected the myotropic and neurotropic nature of the virus. At 1 month after infection, infectious virus was not detected in muscle, but persistent viral RNA was found in both skeletal and cardiac muscle, brain, and spinal cord. The kidney was weakly positive for viral RNA, whereas the liver and spleen were negative. Hindquarter muscle was assayed for persistent viral RNA at 1, 3, 6, 9, and 12 months after infection. In a few cases, persistent viral RNA was detected as late as 12 months after infection. The incidence of persistent viral RNA was high at 1 month after infection and gradually declined until, at 6 months and beyond, it was maintained in 3% to 12% of the muscles tested. Long-term viral RNA persistence was not more common in severely weak animals. However, the degree of hindquarter weakness that developed by 1 month was static thereafter and did not change over the 12-month study period. In contrast, separate experiments revealed that typical mononuclear cell (MNC) infiltration of muscle followed a time course similar to that of viral RNA persistence, peaking at 1 month and gradually resolving by 6 months. Infiltrating polymorphonuclear leukocytes (PMNs) and mast cells were present at 3 to 12 months after infection, signifying that some inflammatory activity remained. Other signs of myopathy that persisted for 12 months included a lack of muscle regeneration, variations in fiber size, and myofiber atrophy with increased perimysial and endomysial connective tissue. These results demonstrate that coxsackievirus RNA can persist in muscle for extended periods of time and are compatible with the idea that persistent virus is involved in maintaining the chronic MNC inflammation observed in murine polymyositis.
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PMID:Duration of virus persistence and its relationship to inflammation in the chronic phase of coxsackievirus B1-induced murine polymyositis. 813 42

Nitric oxide is known to participate in the immune and inflammatory processes. In this study, we investigated the production of nitric oxide (NO) in murine viral myocarditis induced by coxsackievirus B3. The expression of inducible NO synthase (iNOS) mRNA in the heart first appeared on day 4 after virus inoculation and it was detectable for one month by reverse transcriptase-polymerase chain reaction (RT-PCR) methods. iNOS activity which was determined by the conversion of L-[3H]arginine to L-[3H]citrulline was increased on day 4 and revealed its peak on day 8. Immunohistochemistry on day 7 showed increased iNOS staining mainly in infiltrating macrophages and polymorphonuclear leukocytes. Thus, NO is thought to be produced in the heart and play an important role in a murine model of coxsackievirus B3-induced viral myocarditis.
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PMID:Expression of nitric oxide synthase in a murine model of viral myocarditis induced by coxsackievirus B3. 860 80

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

Adolescent female mice were inoculated intraperitoneally with coxsackievirus B3 Nancy strain, sacrificed 3 and 5 days later and the livers harvested. A protocol for direct reverse transcriptase in situ PCR (RT-ISPCR) detection of enteroviral RNA in paraffin-embedded liver tissues was developed. The optimal conditions for the assay were determined. The best results were obtained when the tissue was fixed in formalin, prior to being embedded in paraffin, then cut in 5 micron thick sections, and mounted onto silanized slides. After deparaffination the slides were incubated in 1 microgram/m1 Proteinase K for 10 min and cDNA synthesis was carried out. For successful RT-ISPCR 40-50 cycles of amplification were necessary. The optimal concentrations of dNTP, primers and Taq Polymerase for RT-ISPCR were determined by serial dilution assays. Primers were selected from highly conserved sequences in the 5' non-coding region (5'NTR). To detect the viral RNA in the liver, digoxigenin-dUTP was incorporated during amplification, subsequently bound with an antidigoxigenin antibody conjugated to alkaline phosphatase (AP), followed by colorimetric detection with nitroblue tetrazolium salt (NBT) and 5-brom-4chloro-3indolyl-phosphate (BCIP). The result was a blue precipitate in the cytoplasm of hepatocytes from infected mice. Fibroblasts, endothelial cells, lymphocytes and the nuclei of hepatocytes were negative. Thus, RT-ISPCR is a specific method for the detection of enterovirus RNA in the hepatocytes of infected mice, and can be of use for the determination of EV liver disease in man.
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PMID:Direct in situ transcriptase polymerase chain reaction for the detection of Enterovirus genome in liver tissues. 912 62

Enteroviruses have been implicated in persistent infections of the nervous system and in certain paralytic motor neuron syndromes. Enteroviral persistence may depend on defective transcription, resulting in the abnormal production of equal amounts of genomic and template RNA strands rather than the normal ratio of 60-100:1. An in vitro model of a persistent coxsackie virus in human skeletal muscle cells was investigated using in situ hybridisation and a semiquantitative parallel, complementary, reverse transcriptase polymerase chain reaction. The ratio of genomic to template RNA was found to be approximately 60:1. We conclude that enteroviral persistence in this in vitro model is not dependent on altered transcription. In vivo, other viral and host factors should be considered.
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PMID:Genomic and template RNA transcription in a model of persistent enteroviral infection. 914 25


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