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)

The genomes of both bacteria and eukaryotic organisms are known to encode selenoproteins, using the UGA codon for seleno-cysteine (SeC), and a complex cotranslational mechanism for SeC incorporation into polypeptide chains, involving RNA stem-loop structures. These common features and similar codon usage strongly suggest that this is an ancient evolutionary development. However, the possibility that some viruses might also encode selenoproteins remained unexplored until recently. Based on an analysis of the genomic structure of the human immunodeficiency virus HIV-1, we demonstrated that several regions overlapping known HIV genes have the potential to encode selenoproteins (Taylor et al. [31], J. Med. Chem. 37, 2637-2654 [1994]). This is provocative in the light of overwhelming evidence of a role for oxidative stress in AIDS pathogenesis, and the fact that a number of viral diseases have been linked to selenium (Se) deficiency, either in humans or by in vitro and animal studies. These include HIV-AIDS, hepatitis B linked to liver disease and cancer, Coxsackie virus B3, Keshan disease, and the mouse mammary tumor virus (MMTV), against which Se is a potent chemoprotective agent. There are also established biochemical mechanisms whereby extreme Se deficiency can induce a proclotting or hemorrhagic effect, suggesting that hemorrhagic fever viruses should also be examined for potential virally encoded selenoproteins. In addition to the RNA stem-loop structures required for SeC insertion at UGA codons, genomic structural features that may be required for selenoprotein synthesis can also include ribosomal frameshift sites and RNA pseudoknots if the potential selenoprotein module overlaps with another gene, which may prove to be the rule rather than the exception in viruses. One such pseudoknot that we predicted in HIV-1 has now been verified experimentally; a similar structure can be demonstrated in precisely the same location in the reverse transcriptase coding region of hepatitis B virus. Significant new findings reported here include the existence of highly distinctive glutathione peroxidase (GSH-Px)-related sequences in Coxsackie B viruses, new theoretical data related to a previously proposed potential selenoprotein gene overlapping the HIV protease coding region, and further evidence in support of a novel frameshift site in the HIV nef gene associated with a well-conserved UGA codon in the 1-reading frame.
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PMID:Genomic structures of viral agents in relation to the biosynthesis of selenoproteins. 915 12

A protocol for the in situ polymerase chain reaction (IS-PCR) detection of viral nucleic acid in the heart tissue of four-to-five-week-old CD1 mice infected with coxsackievirus B3 (CBV3) Nancy strain is described. To compare the effects of formalin concentration on the IS-PCR process, two different concentrations (10 and 37%) were employed. Using 37% formalin, 25 PCR cycles were sufficient and a permeabilization step could be omitted. However, postfixation of tissues with 4% paraformaldehyde and 100% ethanol after the deparaffinization, reverse transcriptase and amplification steps was required in order to minimize artefacts. When the tissues were fixed in 10% formalin, postfixation with 4% paraformaldehyde was not required, but a permeabilization step had to be employed and 40 cycles of PCR amplification were needed. To detect the PCR product in the 10% formalin-fixed samples, incubation with 0.3 U/ml of an anti-digoxigenin antibody conjugated to alkaline phosphatase was performed for 90 min. When 37% formalin-fixed samples were used, the concentration of the antibody conjugate had to be increased to 3 U/ml and the exposure time was decreased to 30 min. Enterovirus (EV) nucleic acid was detected in the cytoplasm of myocytes. Thus, IS-PCR was successful in localizing EV nucleic acid in the cytoplasm of myocytes in mice infected with a cardiotropic strain of CBV3. Using this technique, 10% formalin-fixed tissues gave better results than 37% formalin-fixed tissues.
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PMID:Comparison of procedures for the detection of enteroviruses in murine heart samples by in situ polymerase chain reaction. 949 12

Differences in host susceptibility to viral myocarditis caused by a given strain of coxsackievirus B3 (CVB3) are known to be largely related to host genetic factors. Little is known, however, about the key genes that encode determinants (mediators) of myocarditis development or the nature of injury. To identify these genes and further understand the molecular mechanisms of the disease process, we have used a murine model and the differential display technique to fingerprint mRNAs from CVB3-infected mouse hearts. Total RNA was extracted from hearts of 4- and 10-week-old A/J(H-2(a)) mice at day 4 after CVB3 infection, and mRNAs were detected by reverse transcriptase-polymerase chain reaction and subsequently analyzed on polyacrylamide DNA sequencing gels. The differentially displayed bands were confirmed by Northern hybridization using the bands as cDNA probes. Twenty-eight upregulated or downregulated bands were selected from the sequencing gels; among these, 2 upregulated and 3 downregulated cDNA fragments were confirmed by Northern hybridization. DNA sequence analysis and GenBank searching have determined that 4 of the 5 candidate genes are homologous to genes encoding Mus musculus inducible GTPase, mouse mitochondrial hydrophobic peptide (a subunit of NADH dehydrogenase), mouse beta-globin, and Homo sapiens cAMP-regulated response element binding protein (CREB) binding protein (CBP), respectively. The remaining candidate gene matches an unpublished cDNA clone, M musculus Nip21 mRNA (GenBank accession number, AF035207), which is homologous to human Nip2, a Bcl-2 binding protein. Our data suggest preliminarily that both structural and nonstructural genes are involved in myocarditis development. For the structural gene, beta-globin, we further confirmed its downregulation at the protein level by measuring the mean cell volume of red blood cells and found it was marginally reduced in the CVB3-infected group (P<0.06), with no change in hemoglobin concentration. Cardiac myoglobin concentration was also measured and found to be decreased (P<0.005), with a parallel decrease in total soluble protein in the CVB3-infected mouse myocardium (P<0.01). We also noted that the ratio of myoglobin to total protein was not significantly changed; this may be due to the downregulation of additional genes in the host heart, a number being observed on the differential display gels. The significant downregulation of beta-globin major gene expression in the heart may be relevant to impaired cardiac function in both the early and late postinfection period. The other identified nonstructural genes are known to be involved in regulation of gene expression, signal transduction pathways, and apoptotic cell death. The altered expression of structural and nonstructural genes may play important roles in the mediation of myocarditis development and perhaps other pathological processes in the heart.
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PMID:Viral myocarditis: identification of five differentially expressed genes in coxsackievirus B3-infected mouse heart. 1018 58

The Enterovirus may be the most common agent responsible for viral myocarditis and cardiomyopathy. Very little of the literature is available concerning the follow-up of patients who underwent transplantation with enteroviral positivity in native hearts. In the present study, 45 explanted hearts from patients who underwent orthotopic heart transplant at University of Padova were studied by reverse transcriptase (RT)-polymerase chain reaction (PCR): 27 patients had dilated cardiomyopathy (DC), 12 had ischemic cardiopathy (IC), 2 had valvular disease (VD), 2 had arrhythmogenic right ventricular cardiomyopathy (ARVC), 1 had giant cell myocarditis (GCM), and 1 had lymphocytic myocarditis (LM). Two sets of PCR primers from the highly conserved region of Enterovirus and Rhinovirus were used. Samples of both ventricles and septum were analyzed in every patients. The RT-PCR and nucleotide sequencing of amplicons were also performed on all post-transplantation follow-up biopsies in patients with Enterovirus positivity in the native heart. The viral genome was detectable in only 1 of 27 patients with DC (3%) and in 1 patient with LM. Nucleotide sequence analysis of the amplified product showed differences in nucleotide sequence of PCR samples compared with the sequence of the coxsackievirus B3 used in the current study. The patient with Enterovirus-positive DC showed a higher index of severe rejection (>3A) in the first 6 months, compared with the other patients tested. The patient with Enterovirus-positive LM died of disease recurrence 2 months after transplantation. The present study reveals a scarce presence of Enterovirus in the myocardium of patients with chronic myocardial disease. Because Enterovirus infection was predictive of a poor prognosis in these two patients, molecular studies are useful in excluding viral involvement in native hearts of transplanted patients.
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PMID:Enteroviral genome in native hearts may influence outcome of patients who undergo cardiac transplantation. 1040 92

An apparently healthy 7-year-old boy attempted to demonstrate his ability to dive into a whirlpool but was retrieved from the water in a state of unconsciousness after several minutes. Resuscitation was unsuccessful. No characteristic signs of drowning were found at the autopsy but examination of the lymph nodes and the cardiac muscle indicated a pre-existent infection. The histological examination revealed a slight degree of predominantly lymphocytic infiltration of the cardiac muscle. IgM antibodies against Coxsackie virus were detected in the serum sample by means of ELISA. The reverse transcriptase polymerase chain reaction (RT-PCR) performed on an extract of formalin-fixed, paraffin-embedded cardiac muscle tissue revealed a DNA sequence specific for Coxsackie B3 virus. Therefore, cardiac failure was due to a myocardial virus infection and the additional strain caused by diving. This case report emphasizes the importance of modern molecular biological methods in cases of sudden death including death by hydrocution.
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PMID:Hydrocution in a case of Coxsackie virus infection. 1055 May 96

The instability of RNA in solutions during storage and travel is an impediment to its utilization in routine diagnostics. A robust and simple approach to the problem of RNA protection and processing is offered by storage of RNA desiccated with processing procedures that do not solublize the RNA until the beginning of reverse transcription. The feasibility of this general approach was tested with coxsackievirus B4 (CVB-4) from blood or culture fluid held on a storage and transport medium (FTA(R)) and analysed by reverse transcriptase PCR (RT-PCR) without removing the RNA from the FTA(R) until reverse transcription. Phase-trapping techniques based on water-miscible solvents such as ethanol or phenol were compared with simple buffers and concentrated lithium chloride solutions. RT-PCR detection of viral RNA reached a sensitivity of approximately 0.1 fg, which is comparable with other non-nested PCR techniques. Whole blood as a virus vehicle significantly interfered with CVB-4 detection, but to an acceptable degree. Desiccation-storage of the RNA of CVB-4 appears to be unaffected by weeks on the storage medium under ambient conditions. These characteristics indicate that this approach forms a credible developmental base for RNA-based pathogen diagnostics with particular application to the problem of transporting potentially infectious body fluids to a centralized laboratory for analysis.
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PMID:Reverse transcription of an RNA genome from databasing paper (FTA(R)). 1081 92

The activation of the interferon (IFN)-alpha system and its relationship with coxsackievirus B (CVB) infection has been analyzed in 56 patients with insulin-dependent diabetes mellitus (IDDM; 25 children and 31 adults). Elevated levels of IFN-alpha were found in plasma of 70% of patients (39/56), and a positive detection of IFN-alpha mRNA in blood cells by reverse transcriptase-polymerase chain reaction (RT-PCR) was observed in 75% of patients (42/56). Enterovirus (EV) RNA assayed by seminested RT-PCR was detected in the blood of 50% of IFN-alpha-positive patients but not in any IFN-alpha-negative patients. The results of genotype analysis of amplified EV RNA sequences (5 CVB2, 8 CVB3, and 8 CVB4) were concordant with the results of CVB-neutralization tests. The comparison between IFN-alpha, EV RNA, and serology suggested that the proportion of CVB infection associated with IFN-alpha positivity might be higher than is predicted from the investigation of EV RNA. Together, the results suggest that, in a majority of cases, a CVB infection is associated with clinical IDDM.
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PMID:Increased level of interferon-alpha in blood of patients with insulin-dependent diabetes mellitus: relationship with coxsackievirus B infection. 1083 72

A method is described for quantitation of enterovirus RNA in experimentally infected murine tissues. Viral RNA was extracted from tissue samples and amplified by reverse transcriptase PCR in the presence of an internal standard RNA. The ratio of PCR product derived from viral RNA and internal standard RNA was then determined using specific probes in a post-PCR electrochemiluminescent hybridization assay. This provided an estimate of the viral RNA copy number in the original sample, and detection of PCR product derived from internal standard RNA validated sample processing and amplification procedures. RNA copy number correlated with viral infectivity of cell culture-derived virus, and one tissue culture infective dose was found to contain approximately 10(3) genome equivalents. The ratio of RNA copy number to infectivity in myocardial tissue taken from mice during the acute phase of coxsackievirus B3 myocarditis was more variable ranging from 10(4)-10(7), and was dependent on the stage of infection, reflecting differential rates of clearance for viral RNA and viral infectivity. The assay is rapid, and could facilitate investigations which currently rely upon enterovirus quantitation by titration in cell culture. This would be useful for experimental studies of viral pathogenesis, prophylaxis and antiviral therapy.
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PMID:Development and evaluation of quantitative-competitive PCR for quantitation of coxsackievirus B3 RNA in experimentally infected murine tissues. 1089 31

A hospital-acquired outbreak with febrile illness and/or rash occurred in our neonatal special care nursery (SCN) from September 1995 to September 1996. A total of 23 infants developed symptoms. We could not detect the etiological agents by routine virus isolation. In a retrospective study, however, enterovirus RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) from four out of six cerebrospinal fluid (CSF) samples and from two of 12 sera. Thus six out of 16 patients from whom samples were obtained were diagnosed retrospectively as having enterovirus infection. Furthermore, we detected the enterovirus genome from four of 20 serum samples obtained from patients who had other clinical symptoms, and from infants hospitalized without noticeable clinical illness during the same periods. This outbreak was caused by two different enteroviruses, which we assumed were echovirus type 7 (Echo 7) and coxsackievirus B3 (Cox B3), because of the sequence results. We demonstrated the clinical advantage of the analysis of nucleotide sequencing as supportive evidence of transmission.
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PMID:Diagnosis of horizontal enterovirus infections in neonates by nested PCR and direct sequence analysis. 1097 45

Nitric oxide (NO) is an important biologically active molecule that plays a key part in host defence against bacteria, protozoa, and tumour cells. NO has antiviral effects against several DNA viruses, such as murine poxvirus, herpes simplex virus, and Epstein-Barr virus, and some RNA viruses, such as coxsackievirus. In several studies, in vitro and in vivo, overproduction of NO has been noted in the presence of HIV-1 infection. Furthermore, increased NO production may contribute directly to the pathogenesis of HIV-1-associated dementia. The mechanisms of virus infection mediated by NO may be related to: direct antiviral effects of NO; impairment of antiviral defence mediated by T-helper-1 immune response by suppressing T-helper-1 functions; NO-induced cytotoxic effects by oxidative injury with cellular and organ dysfunctions; and NO-induced oxidative stress leading to rapid viral evolution with productions of drug-resistant and immunologically tolerant mutants. By contrast, there is some evidence of NO activity--directly, indirectly, or both--decreasing or blocking HIV-1 replication, through inhibition of viral enzymes, such as reverse transcriptase, protease, or cellular nuclear transcription factor (NF-kappa B) and long-terminal repeat-driven transcription. Therefore, although NO surely plays an important part in HIV-1 infection, that role is sometimes helpful and other times damaging to the host. Future challenges are to learn more about the beneficial and harmful effects of NO in HIV-1 infection, and how to selectively inhibit excessive NO production or to use NO-releasing drugs to decrease viral replication. This review discusses the role of NO in the pathogenesis of HIV-1 infection, inasmuch as its role against HIV-1 is unequivocal in inhibiting or increasing viral replication.
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PMID:Role of nitric oxide in HIV-1 infection: friend or foe? 1261 27


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