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 cell line (BsT) established from neoplastic embryonal tissues of the platyfish (Xiphophorus maculatus) released spontaneously retrovirus-like particles. The particles have a buoyant density of 1.16 g/ml, a mean diameter of 100 nm and the morphology of immature retroviruses. The particle-associated proteins p70, p65, and p28 react with an antiserum directed against the major internal feline leukemia virus structural protein p27. The particles are associated with a reverse transcriptase. The purified enzyme has a molecular weight of about 70 kDa and prefers the template primers poly(rA):oligo(dT), poly(dC):oligo(dG), and poly(rC):oligo(dG) in the presence of Mn2+. The enzyme activity is inhibited by antibodies directed against the reverse transcriptase of feline leukemia virus and simian sarcoma virus. The particles contain a ribonucleic acid of about 70 S. In an endogenous reverse transcriptase reaction nucleic acids in the range of 0.2 to 0.4 kb were synthesized. In Northern blots with these nucleic acids as probe, three transcripts of about 8.5, 4.2, and 1.5 kb were detected in BsT cells. Southern blot analysis with the same probe demonstrates related sequences in the DNA of BsT cells and the platyfish and swordtail (Xiphophorus helleri). Hybridization experiments with the LTR-gag region of the feline leukemia virus show homologous sequences in the Xiphophorus genome.
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PMID:Isolation and characterization of a retrovirus from the fish genus Xiphophorus. 137 84

To investigate a non-RI test which is equivalent to the reverse transcriptase inhibiting antibody test, a reverse transcriptase inhibiting antibody was compared to the absolute number of CD-4 or CD-8 cells or CD-4/-8 ratio and also to photodensitometric analysis for western blotting. There is no correlation of the reverse transcriptase with any test for cell numbers and their ratio. In photodensitometry, relative units of anti-p65 and anti-p51 were compared with reverse transcriptase inhibiting antibody. The reverse transcriptase inhibiting antibody showed a higher correlation to the relative unit of p65 antibodies than that of p51 antibodies. The photodensitometric analysis of western blotting for a serum test may be a possible method to find a prognostic marker in HIV-1 infection.
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PMID:[Comparative evaluation of markers with reverse transcriptase inhibiting antibody in human immunodeficiency virus type 1 infection]. 247 51

Reactive oxygen species like hydrogen peroxide (H2O2) have been shown to serve as messengers in the induction of NF-kappa B and then in the activation and replication of HIV-1 in human cells. Because singlet oxygen (1O2) is another very important reactive oxygen species whose action in transcription factor activation is totally undetermined, we started to investigate its role in both NF-kappa B and HIV-1 activation. For provoking unbalanced redox conditions, 1O2 was generated by photosensitization using methylene blue as photosensitizer. Lymphocytes or monocytes (ACH-2 or U1 respectively) latently infected with HIV-1 were treated by photosensitization mediated by methylene blue and the production of reactive oxygen species was monitored through their cytotoxic effect in infected cells. The generation of 1O2 by methylene blue turns out to be very efficient in inducing NF-kappa B as a heterodimer composed of the p50 and p65 subunits. This induction appears specific since other transcription factors like AP-1 are only weakly activated by this treatment. In comparison with other inducing treatments such as phorbol esters or tumor necrosis factor alpha (TNF-alpha), the methylene-blue-mediated activation of NF-kappa B is slow, becoming optimal 180 min after treatment. These kinetic data were obtained by following, on the same samples, both the emergence of NF-kappa B in the nucleus and the disappearance of I kappa B-alpha in the cytoplasmic extracts. Conjugated with the induction of this transcription factor, HIV-1 reactivation from these latently infected cells was also observed by the measurement of reverse transcriptase activity in the cell supernatants. These data allow us to postulate that 1O2 is a biologically important reactive oxygen species which could play a role in the establishment of oxidative stress conditions leading to HIV-1 activation via the presence of NF-kappa B in the nucleus of infected cells.
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PMID:NF-kappa B transcription factor and human immunodeficiency virus type 1 (HIV-1) activation by methylene blue photosensitization. 770 61

We have previously reported that carbon tetrachloride (CCI4) stimulates c-fos, c-jun, and Ca(2+)-activated neutral protease gene expression in rat hepatic tissue (Zawaski et al., Biochem. Biophys. Res. Comm. 197, 585-590, 1993). The proteins c-Fos and c-Jun constitute inducible transcription factors in signal transduction and regulate the transcriptional activation of a battery of genes involved in cell growth and division. The present study was initiated to characterize the role of cytochrome P450 expression and metabolic activation on the magnitude of immediate-early (i.e. c-fos and c-jun) gene expression. Animals were treated either with diallyl sulfide, N-acetylcysteine, pyridine, or phenobarbital before treatment with CCI4. Total and poly(A)+ RNA were isolated, and c-fos and c-jun mRNA levels were analyzed by Northern blot and reverse transcriptase-polymerase chain reaction analyses. Treatment of animals with CCI4 increased c-fos and c-jun mRNA levels from below the limit of detection in control tissue to intense bands within 30 min of treatment, with maximal expression monitored at 1 and 2 hr posttreatment. Treatment of animals with diallyl sulfide alone also elevated c-fos and c-jun mRNA expression to detectable levels. However, pretreatment of animals with diallyl sulfide before treatment with CCI4 produced a 76-92% decrease in c-fos and c-jun mRNA levels, relative to that monitored for CCI4-treated animals. Pretreatment with N-acetylcysteine did not affect c-fos or c-jun mRNA levels and diminished CCI4-stimulated c-fos and c-jun gene expression by 44 and 55%, respectively, relative to the immediate-early gene mRNA levels monitored in the hepatic tissue of CCI4-treated animals. Pretreatment of animals with the CYP2E1 inducer pyridine for 24 hr had only a marginal effect on c-fos mRNA levels, but increased CCI4-stimulated c-fos and c-jun mRNA levels by an additional approximately 2- to approximately 4-fold over those monitored in the uninduced hepatic tissue of CCI4-treated animals. Whereas phenobarbital treatment alone enhanced c-fos expression only marginally, CCI4 treatment of phenobarbital-pretreated animals increased c-fos expression by up to an additional approximately 8-fold and c-jun mRNA levels by up to an additional approximately 5-fold over the respective levels monitored in the hepatic tissue of CCI4-treated animals. Enhanced CYP2E1 or CYP2B1/2B2 levels after treatment with pyridine or phenobarbital elevated c-fos mRNA over untreated controls. This increase was marginal, however, and detectable only with reverse transcriptase-polymerase chain reaction. Examination of nuclear levels of the heterodimeric c-Fos and c-Jun AP-1 transcription factor complex revealed a time-dependent increase in AP-1 levels. AP-1 transcription factor binding was confirmed using competitor consensus sequences and antibody supershifts. Nuclear levels of NF-kappa B, a transcription factor complex implicated in hepatocyte proliferation and apoptotic or programmed cell death, were also examined. NF-kappa B, which consists of the p50 and p65/Rel A polypeptides, was increased in hepatic nuclear extracts at 2 and 24 hr after CCI4 administration, with a concomitant decrease in the p50 polypeptide. Thus, the magnitude of CCI4 stimulation of the immediate-early genes c-fos and c-jun is dependent on metabolic activation by the P450s, and the magnitude of the effect is dependent on the levels and isozyme composition of P450s in the tissue. Furthermore, nuclear transcription factor levels of AP-1 and NF-kappa B are elevated in response to this toxicant.
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PMID:Cytochrome P4502E1- and cytochrome P4502B1/2B2-catalyzed carbon tetrachloride metabolism: effects on signal transduction as demonstrated by altered immediate-early (c-Fos and c-Jun) gene expression and nuclear AP-1 and NF-kappa B transcription factor levels. 882 85

The inducible cyclooxygenase, COX-2, has been associated with vascular inflammation and cellular proliferation. We have discovered that hypoxia increases expression of the COX-2 gene in human vascular endothelial cells in culture independent of other stimuli. Western analysis of human umbilical vein endothelial cells (HUVEC) revealed a greater than 4-fold induction of protein by hypoxia (1% O2). The steady-state level of COX-2 mRNA was correspondingly elevated by both Northern blot and reverse transcriptase-polymerase chain reaction analysis. Using electrophoretic mobility shift assays with antibody supershifting, we also found that hypoxia causes increased binding of NF-kappaB p65 (Rel A) to the one out of the two NF-kappaB consensus elements in the COX-2 promoter which is closest to the transcription start site of the COX-2 gene. Transfection of an immortalized human microvascular endothelial cell line (HMEC-1) with mutation reporter gene constructs and HUVEC with both mutation and deletion reporter gene constructs suggested that transcription of the COX-2 gene was enhanced by hypoxia. In transcription factor decoy experiments, hypoxic HUVEC were exposed in culture to 20 microM of the same NF-kappaB element found to bind NF-kappaB protein. The wild type transcription factor decoy prevented hypoxic induction of COX-2, presumably by binding with cytoplasmic p65; however, mutated or scrambled oligonucleotides did not prevent the increase in COX-2 protein expression by hypoxia. Thus, the intracellular signaling mechanism that leads to induction of COX-2 by hypoxia includes binding of p65 to the relatively 3' NF-kappaB consensus element in the COX-2 upstream promoter region in human vascular endothelial cells.
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PMID:Hypoxia induces cyclooxygenase-2 via the NF-kappaB p65 transcription factor in human vascular endothelial cells. 899 3

The hepatic stellate cell (HSC), following a fibrogenic stimulus, is transformed from a quiescent to an activated cell. Cytokines induce NFkappaB activity in activated but not in quiescent HSCs with subsequent expression of NFkappaB-responsive genes, such as intercellular adhesion molecule (ICAM)-1 and interleukin (IL)-6. We investigated the effect of proteasome inhibitors and an IkappaB super-repressor on the cytokine mediated activation of NFkappaB, ICAM-1, and IL-6 in activated HSCs. Culture-activated HSCs were stimulated with IL-1beta or tumor necrosis factor alpha (TNFalpha) in the presence or absence of proteasome inhibitors, ALLN or MG-132, or after infection with an adenovirus expressing the IkappaB super-repressor (Ad5IkappaB) or beta-galactosidase (Ad5LacZ) as a control. NFkappaB activity was evaluated by immunofluorescence and by electrophoretic mobility shift assay. The steady state level of cytoplasmic IkappaB protein was measured by Western Blot. ICAM-1 and IL-6 expression was measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbant assay. Proteasome inhibitors, which block the degradation of IkappaB, and the Ad5IkappaB, which provides an exogenous nondegradable IkappaB, block the stimulation of NFkappaB activity by TNFalpha and IL-1beta in activated HSCs. These reagents block the subsequent nuclear translocation of p65 NFkappaB and induction of ICAM-1 and IL-6 by cytokines. The specificities of the proteasome inhibitors and the IkappaB super-repressor are demonstrated by their failure to block c-Jun N-terminal kinase induction by cytokines. Cytokine-induced stimulation of NFkappaB, ICAM-1, and IL-6 is blocked by proteasome inhibitors and Ad5IkappaB in activated HSCs. Inhibition of IkappaBalpha degradation is a potential target for anti-inflammatory therapy in the liver and might influence the activation process of HSCs following fibrotic stimuli.
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PMID:Inhibition of NFkappaB in activated rat hepatic stellate cells by proteasome inhibitors and an IkappaB super-repressor. 958 6

Nitric oxide (NO) plays an important role in inflammation and also in multiple stages of carcinogenesis. We investigated the effects of various tea polyphenols, including theaflavin, a mixture of theaflavin-3-gallate and theaflavin-3'-gallate, theaflavin-3,3'-digallate, thearubigin, and (-)-epigallocatechin-3-gallate on the induction of NO synthase in lipopolysaccharide-activated murine macrophages, RAW 264.7 cells. Theaflavin-3,3'-digallate was found to be stronger than (-)-epigallocatechin-3-gallate in inhibiting NO generation and inducible NO synthase protein in activated macrophages, while theaflavin, a mixture of theaflavin-3-gallate and theaflavin-3'-gallate and thearubigin were less effective. Inhibition of NO production was observed when cells were cotreated with theaflavin-3,3'-digallate and lipopolysaccharide. Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses demonstrated that significantly reduced 130-kDa protein and mRNA levels of inducible NO synthase were expressed in lipopolysacchride-activated macrophages with theaflavin-3,3'-digallate, compared to those without theaflavin-3,3'-digallate. Electrophoretic mobility shift assay (EMSA) indicated that theaflavin-3,3'-digallate blocked the activation of nuclear factor kappaB (NF-kappaB), a transcription factor necessary for inducible NO synthase induction. Theaflavin-3,3'-digallate also blocked phosphorylation of IkappaB from cytosolic fraction and reduced lipopolysacchride-induced nuclear accumulation of transcription factor NF-kappaB p65 and p50 subunits. These results suggest that theaflavin-3,3'-digallate decreases the protein levels of inducible NO synthase by reducing the expression of inducible NO synthase mRNA, and the reduction could be via preventing the activation of NF-kappaB, thereby inhibiting the induction of inducible NO synthase transcription. It was also demonstrated that the gallic acid moiety of theaflavin-3,3'-digallate is essential for their potent anti-inflammation activity.
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PMID:Theaflavin-3,3'-digallate from black tea blocks the nitric oxide synthase by down-regulating the activation of NF-kappaB in macrophages. 1007 14

Increased tumor necrosis factor-a activity has been reported in patients with alcoholic hepatitis and is implicated in its pathogenesis. The aim of this study was to investigate potential mechanisms of increased tumor necrosis factor-a activity in alcoholic hepatitis. Monocyte nuclear factor-kB activity was assessed by electrophoretic mobility shift assay, monocyte tumor necrosis factor-a mRNA was semi-quantitatively assessed by reverse transcriptase polymerase chain reaction, and tumor necrosis factor-a in monocyte culture supernatants was measured. There was significantly greater spontaneous nuclear factor-kB activity in the monocytes of 6 patients with alcoholic hepatitis as compared with that in the monocytes of control subjects. There was spontaneous tumor necrosis factor-a mRNA and tumor necrosis factor-a release from the monocytes of patients with alcoholic hepatitis but not from the monocytes of normal subjects. Endotoxin increased nuclear factor-kB activity and induced tumor necrosis factor-a mRNA and tumor necrosis factor-a release from normal subjects' monocytes. Endotoxin further increased nuclear factor-kB activity, tumor necrosis factor-a mRNA, and tumor necrosis factor-a release from the monocytes of patients with alcoholic hepatitis. Supershift assays indicate that the monocyte nuclear factor-kB activation involves the p50 and p65 subunits. Dysregulated tumor necrosis factor-a metabolism in alcoholic hepatitis monocytes is associated with increased nuclear factor-kB activity and tumor necrosis factor-a mRNA expression.
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PMID:Increased monocyte nuclear factor-kappaB activation and tumor necrosis factor production in alcoholic hepatitis. 1081 Oct 50

Neoplastically transformed mouse and human keratinocytes elevate transactivation of both activator protein 1 (AP-1) and nuclear factor kappaB (NFkappaB) transcription factors. The present study addresses the question of whether elevated NFkappaB in addition to elevated AP-1-dependent gene expression is necessary for maintaining the tumor cell phenotype. When a tetracycline-regulatable dominant-negative c-jun (TAM67, having a truncated transactivation domain) was expressed in tumorigenic human keratinocytes, AP-1- and NFkappaB- but not p53-dependent reporter activity was inhibited by 40-60%. Tumor phenotype, as measured by anchorage-independent growth, was inhibited by 90%. Neither AP-1/NFkappaB activation nor expression of tumor phenotype was inhibited in TAM67-harboring keratinocytes under noninducing conditions. Electrophoretic mobility shift analysis showed that induction of TAM67 expression slightly increased AP-1- but reduced NFkappaB DNA-binding activity. Immunoprecipitation showed that TAM67 interacted in keratinocyte nuclei with NFkappaB p65, suggesting that inhibition of NFkappaB by TAM67 is mediated by direct protein-protein interactions, possibly producing decreased binding to DNA or inactivating p65. To analyze the putative effector genes that may be targeted by TAM67, expression of genes responsive to AP-1 or NFkappaB was measured by reverse transcriptase-polymerase chain reaction in TAM67 transfectants with or without TAM67 induction. Induction of TAM67 inhibited or reduced the expression of collagenase I, stromelysin I (AP-1 responsive), and interleukins 1 and 6 (NFkappaB responsive). These results indicate that genes controlled by NFkappaB and by AP-1 may be transformation-relevant targets of TAM67 and that TAM67 may inhibit NFkappaB activation through direct interaction with NFkappaB p65. Moreover, the findings provide proof for the principle of using inducible TAM67 as a gene therapy to suppress tumor phenotype in human carcinoma cells.
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PMID:Induced expression of dominant-negative c-jun downregulates NFkappaB and AP-1 target genes and suppresses tumor phenotype in human keratinocytes. 1110 61

Tumor necrosis factor receptor type 1 (TNFR1) and c-Myc are important in signal transduction in tumor necrosis factor-alpha (TNF-alpha)-induced cytotoxicity, whereas activation of nuclear factor-kappa B (NF-kappa B) protects against TNF-alpha-induced apoptosis. This study investigated the expression of NF-kappa B, TNFR1, and c-Myc in human astrocytoma tissues by reverse transcriptase-polymerase chain reaction (PCR) and immunohistochemical analysis. TNFR1 messenger ribonucleic acid (mRNA) and c-Myc mRNA were frequently expressed in malignant astrocytomas, especially in glioblastomas, compared with low-grade astrocytomas by PCR analysis. TNFR1 and c-Myc mRNAs were barely detectable in normal brain tissues. NF-kappa B p50 and p65 subunit mRNAs were detected in various grades of astrocytomas, with frequent expression in malignant astrocytomas. The presence of activated NF-kappa B was confirmed by nuclear localization in neoplastic astrocytes as determined by immunohistochemistry. Both p50 and p65 subunits were inhomogeneously expressed in neoplastic astrocytes of glioblastoma, but only in a few scattered tumor cells in low-grade astrocytoma, and almost undetectable in normal brain tissues. These results indicate that TNFR1 and c-Myc are overexpressed in malignant astrocytomas, and this may increase the cellular sensitivity to the cytotoxic action of TNF-alpha. NF-kappa B p50 and p65 were simultaneously induced and activated in malignant astrocytomas. Our results suggest that the constitutive activation of NF-kappa B subunits in malignant astrocytoma, especially in glioblastoma, could be associated with the resistance to TNF-alpha immunotherapy, and indicates new therapeutic strategies for malignant astrocytomas.
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PMID:Expression of nuclear factor-kappa B, tumor necrosis factor receptor type 1, and c-Myc in human astrocytomas. 1138 77


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