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)

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

Sequences which control basal human T-cell lymphotropic virus type I (HTLV-I) transcription probably play an important role in initiation and maintenance of virus replication. We have identified and analyzed a 45-nucleotide sequence (downstream regulatory element 1 [DRE 1]) at the boundary of the R/U5 region of the long terminal repeat which is required for HTLV-I basal transcription. The basal promoter strength of constructs that contained deletions in the R/U5 region of the HTLV-I long terminal repeat were analyzed by chloramphenicol acetyltransferase assays following transfection of Jurkat T cells. We consistently observed a 10-fold decrease in basal promoter activity when sequences between +202 to +246 were deleted. By reverse transcriptase polymerase chain reaction RNA analysis, we confirmed that the drop in chloramphenicol acetyltransferase activity was paralleled by a decrease in the level of steady-state RNA. DRE 1 did not affect the level of Tax1 transactivation. Using a gel shift assay, we have purified a highly enriched fraction that could specifically bind DRE 1. This DNA affinity column fraction contained four detectable proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: p37, p50, p60, and p100. The affinity column fraction stimulated HTLV-I transcription approximately 12-fold in vitro. No effect was observed with the human immunodeficiency virus or adenovirus major late promoters. Following renaturation of the proteins isolated from an SDS-containing gel, p37, but not the other protein fractions, was able to specifically bind to DRE 1.
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PMID:Sequences downstream of the RNA initiation site regulate human T-cell lymphotropic virus type I basal gene expression. 847 78

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

Several monoclonal antibodies (mAbs) were produced against feline immunodeficiency virus (FIV) p24 capsid antigen. One of these, F2710, reacted strongly, not only with viral p24 and recombinant p24, but also with p50 Gag precursor protein in Western blot. Epitope mapping analysis revealed that mAb F2710 recognizes a heptapeptide, SFIDRLF, in the FIV p24 amino acid sequence. As this portion of FIV p24 is highly conserved among various FIV strains, the mAb seems to be a useful tool for detecting FIV p24 antigen in various samples. By means of this mAb and rabbit anti-p24 polyclonal antibody, an antigen capture ELISA was developed. The ELISA detected viral p24 antigen with good linearity. The lower detection limit of this assay is 40 pg/ml of recombinant p24 antigen, which is at least as sensitive as the reverse transcriptase assay in detecting FIV virion. Thus, this system is valuable for monitoring FIV replication in vitro.
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PMID:Characterization of one monoclonal antibody against feline immunodeficiency virus p24 and its application to antigen capture ELISA. 938 8

Fetal and neonatal lymphocytes are relatively resistant to activation and cytokine production when stimulated either via their T-cell antigen receptors or lectins. The molecular mechanism(s) responsible for this phenomenon have not been clearly elucidated. We have hypothesized that such defects in fetal/neonatal T-cell activation may be due to lack of expression of the transcriptional regulatory elements required for T-cell activation. We used reverse transcriptase-polymerase chain reaction to examine both fetal and term neonatal cord bloods for mRNA expression of three transcription factors implicated in T-cell activation: c-jun, c-fos, and NF kappa B (p50 subunit). We demonstrate that mRNAs for all three of these regulatory factors are expressed in fetal blood cells by the 27th week of gestation and in term cord bloods. Activation of term infant cord blood mononuclear cells with anti-CD3 monoclonal antibodies resulted in up-regulation of both c-jun and c-fos mRNAs within 15 min of stimulation. However, secretion of IL-2 by anti-CD3-stimulated cord blood mononuclear cells was still blunted compared with control cells from adults. We conclude that fetal nucleated blood cells constitutively express important genes for cytokine regulation and are able to increase intracellular accumulation of the mRNAs for these factors in response to anti-CD3 stimulation. Thus, qualitative differences in the capacity to regulate these factors could not be shown in fetal blood cells. Quantitative experiments comparing binding of these transcription factors to the IL-2 promoter are currently under investigation.
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PMID:Constitutive expression c-fos, c-jun, and NF kappa B mRNA is in nucleated fetal blood cells and up-regulation of c-fos and c-jun with anti-CD3 stimulation. 942 92

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

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

CD40-induced activation of cytokine gene expression in dendritic cells (DC) is an important process in the initiation of primary immune responses. We have determined the intracellular signaling events that lead to CD40 ligation-induced activation of interleukin-6 (IL-6) gene transcription in a murine DC line, FSDC, that is phenotypically representative of bone marrow-derived DC. IL-6 reverse transcriptase-PCR and promoter assays established the responsiveness of FSDC to anti-CD40 ligation. Further promoter assays showed that the transcription factors NF-kappaB and AP-1 are downstream transcriptional mediators of CD40-induced IL-6 gene expression. Anti-CD40 treatment of FSDC stimulated increased expression of specific NF-kappaB (p50:p65) and AP-1 (c-Jun, JunB, JunD, and c-Fos) DNA-protein complexes. Overexpression of an IkappaB-alpha super-repressor or a dominant negative JunD resulted in a strong inhibition of CD40-inducible IL-6 promoter activity supporting a role for both transcription factors. Upstream signal transduction events were studied by transfection of wild type and mutant human CD40 expression constructs into FSDC followed by stimulation with an anti-human CD40 antibody. These experiments revealed that anti-CD40 stimulation of NF-kappaB and IL-6 gene transcription requires specific amino acid residues in the cytoplasmic region of CD40 involved in the recruitment of TRAF2. Induction of IL-6 mRNA by anti-CD40 treatment was found to be a transient event (24 h) and was followed by a diminution of IL-6 transcript to levels below those found in unstimulated cells. This loss of IL-6 expression was associated with reduced p50:p65 NF-kappaB DNA binding and elevated binding of CBF1 to a site overlapping the NF-kappaB site. Overexpression of CBF1 resulted in a profound inhibition of basal and anti-CD40-induced IL-6 promoter activities indicating that prolonged induction of CBF1 may contribute to the transient nature of the IL-6 response. The physiological relevance of these molecular events to DC function is discussed.
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PMID:CD40 induces interleukin-6 gene transcription in dendritic cells: regulation by TRAF2, AP-1, NF-kappa B, AND CBF1. 1188 48

The cellular chaperone Hsp90 has been shown to associate with the reverse transcriptase (RT) of the duck hepatitis B virus and is required for RT functions. However, the molecular basis for the specific interaction between the RT and Hsp90 remains unknown. Comparison of protein compositional properties suggests that the RT is highly related to the protein kinase c-Raf, which interacts with Hsp90 via the cochaperone p50 (CDC37). We tested whether the RT, like c-Raf, is specifically recognized by p50. Immunoprecipitation and pull-down assays showed that p50 or p50deltaC, a p50 mutant defective in Hsp90 binding, could interact specifically with the RT both in vitro and in vivo, indicating that p50 can bind the RT independently of Hsp90. Furthermore, purified p50 and p50deltaC interacted directly with purified RT. The importance of p50-RT interaction for RT functions was underscored by 1) inhibition of protein-primed initiation of reverse transcription by p50deltaC in vitro and 2) stimulation of viral DNA replication and RNA packaging by p50 and their inhibition by p50deltaC in transfected cells. These results suggest that p50 can function as a cellular cofactor for the hepadnavirus RT by mediating the interaction between the RT and Hsp90.
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PMID:Role of p50/CDC37 in hepadnavirus assembly and replication. 1198 22


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