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)

NF kappa B is a potent mediator of specific gene expression in human monocytes and has been shown to play a role in transcription of the HIV-1 genome in promonocytic leukemias. There is little information available on the response of NF kappa B to cytokines in normal human monocytes. We have used a 32P-labeled oligonucleotide derived from human immunodeficiency virus (HIV-1) long terminal repeat, which contains a tandem repeat of the NF kappa B binding sequence, as a probe in a gel retardation assay to study this transcription factor. Using this assay, we have detected NF kappa B in extracts of nuclei from normal human monocytes. Treatment of normal monocytes with 12-0-tetradecanoyl phorbol-13-acetate (TPA) for 4-24 h caused the complete disappearance of NF kappa B from nuclear extracts of monocytes. A similar result was obtained with the mature monocytic leukemia cell line THP-1. The constitutive transcription factor SP1 was unaffected by addition of TPA. The disappearance of NF kappa B from the nucleus was concentration dependent between 10 and 50 ng/ml of phorbol ester. In THP-1 cells, TPA also induced a new, faster-migrating NF kappa B species not induced in monocytes. Protein kinase C inhibitor staurosporine, but not cyclic nucleotide-dependent protein kinase inhibitor HA-1004, also dramatically reduced constitutive levels of nuclear NF kappa B. Finally, TPA addition to monocytes infected with HIV-1 inhibited HIV-1 replication, as determined by reverse transcriptase assays, in a concentration-dependent manner. These results are in striking contrast to the increase in nuclear NF kappa B and HIV-1 replication induced by phorbol esters in promonocytic leukemia cells U937 and HL-60, and emphasize the importance of studying cytokine regulation of HIV-1 in normal monocytes.
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PMID:Phorbol ester reduces constitutive nuclear NF kappa B and inhibits HIV-1 production in mature human monocytic cells. 146 36

The chronically infected promonocytic clone U1 expresses low-to-undetectable constitutive levels of human immunodeficiency virus (HIV). Virus replication in these cells can be increased up to 25-fold by phorbol esters (phorbol-12-myristate-13-acetate), recombinant cytokines such as tumor necrosis factor-alpha, and cytokine-enriched mononuclear cell supernatants. We have tested specific activators of protein kinases (PK) and PK inhibitors (isoquinolinesulfonamide derivatives), as well as calcium-mobilizing agents, for their effect on constitutive and induced virus expression in U1 cells. Virus expression was measured by reverse transcriptase, Western blot, and nuclear run-on analysis. Activation of PKC by 1-oleyl,2-acetylglycerol, a synthetic analog of the natural ligand 1,2-diacylglycerol, and bryostatin 1 (a recently described specific PKC activator) resulted in a two- to eightfold increase in virus production. In contrast, activators of cyclic-nucleotide-dependent PKs were not effective in inducing virus expression. PK inhibitors were tested for their effect on HIV upregulation by cytokines and other inducing agents. The isoquinolinesulfonamide derivative H7, a potent inhibitor of PKC activation, effectively blocked (70 to 90%) HIV induction by cytokines and phorbol-12-myristate-13-acetate. The derivative HA1004, which is more selective for cyclic-nucleotide-dependent kinases, did not suppress viral induction. In addition, increases in intracellular calcium levels dramatically enhanced HIV production induced by both specific PKC activators and cytokines. These results indicate that activation of PKC is a common pathway involved in the upregulation of HIV expression in chronically infected cells stimulated by cytokines and other inducing agents.
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PMID:Direct and cytokine-mediated activation of protein kinase C induces human immunodeficiency virus expression in chronically infected promonocytic cells. 220 Aug 85

Protein kinase C (PKC), a widely-distributed enzyme implicated in the regulation of many physiological processes, consists of a family of at least twelve isoenzymes which differ in tissue distribution, subcellular localization, regulatory properties, etc. In addition to this heterogeneity at the protein level, we identify here for the first time a PKC zeta pseudogene (psi PKC zeta) transcript, specifically expressed in the brain, which is identical with PKC zeta except for sequence divergence within the first variable domain (V1). The authenticity of this unique V1 sequence (V1') in mRNA was confirmed by RNase protection and reverse transcriptase PCR (RT-PCR) analysis. When translated in-frame with PKC zeta, a stop codon is located 28 amino acids towards the N-terminus of the divergence point and the intervening sequence lacks an expected initiating methionine. psi PKC zeta is non-functional in terms of protein synthesis since Western blotting with an antibody directed against the C-terminus of PKC zeta failed to reveal a protein smaller than PKC zeta, and synthetic psi PKC zeta RNA failed to support protein synthesis in a translation system in vitro. PCR amplification of rat genomic DNA demonstrated lack of an intron at the junction between V1' and the first constant domain (the V1'-C1 border), and genomic DNA Southern blot analysis using PKC zeta and psi PKC zeta-specific probes indicated that they have different loci. psi PKC zeta, therefore, is not derived from the PKC zeta gene by alternative splicing, but rather is the product of a distinct gene. In Northern blot analysis, brain PKC zeta mRNA was identified as a low-abundance 3.1 kb transcript, while the abundant 2.5 and 4.7 kb mRNAs previously reported to encode PKC zeta are, in fact, psi PKC zeta transcripts. Analysis of rat brain, heart, lung, liver, kidney and skeletal muscle revealed psi PKC zeta mRNA only in brain. PKC zeta transcripts were most abundant in lung and kidney (2.7 and 4.7 kb mRNAs), correlating with the tissue profile of PKC zeta immunoreactivity in Western blots. Probes complementary to the common V5 and C1 domains detected both PKC zeta and psi PKC zeta transcripts. Interestingly, the C1 probe also detected an abundant novel 1.75 kb mRNA in brain and heart, suggesting the existence of an additional PKC zeta-related species. This work, therefore, also emphasizes the importance of careful choice of oligonucleotide and cDNA probes to study PKC zeta mRNA.
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PMID:Identification of a brain-specific protein kinase C zeta pseudogene (psi PKC zeta) transcript. 757 16

Upon withdrawal of interleukin-3 (IL-3) from human factor-dependent erythroleukemic cell line TF-1, bcl-2 mRNA and protein levels decrease within 8 to 24 hours. Accompanying this decrease is the onset of apoptosis as determined by flow cytometric analysis of DNA degradation. By 8 to 18 hours of deprivation approximately 70% to 80% of the cells have entered apoptosis. Downregulation of protein kinase (PK) by a 24-hour incubation in 100 nmol/L 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in the presence of IL-3 dramatically reduced bcl-2 mRNA levels, and induced apoptosis in the presence of IL-3. We have also found that even in the presence of IL-3, two inhibitors of PKC, light-activated calphostin and H-7, substantially reduced the levels of bcl-2 mRNA between 8 and 24 hours as measured by a semi-quantitative reverse transcriptase/polymerase chain reaction assay method; however, the cyclic nucleotide-dependent PK inhibitor HA 1004, that is a structural analog of H-7 but a poor inhibitor of PKC, did not reduce bcl-2 levels in the presence of IL-3. This decrease in bcl-2 mRNA was accompanied by a decline in bcl-2 protein levels by 8 to 24 hours after addition of light-activated calphostin. In addition to interfering with the maintenance of bcl-2 mRNA levels, inhibition of PKC with H-7 inhibited the induction of bcl-2 mRNA in factor-deprived TF-1 cells restimulated with IL-3. The cyclic nucleotide-dependent PK inhibitor HA 1004 did not inhibit IL-3-induced bcl-2 mRNA. Studies with actinomycin D showed that transcription plays a major role in maintaining bcl-2 levels in TF-1 cells, and it is therefore likely that IL-3 plays a role in maintaining bcl-2 transcription through activation of PKC in these cells.
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PMID:Human interleukin-3 receptor modulates bcl-2 mRNA and protein levels through protein kinase C in TF-1 cells. 779 58

Protein kinase C (PKC) appears to play a role in replication of human immunodeficiency virus type 1 (HIV-1). PKC is a family of at least 12 isozymes. In this study, we investigated a role of Ca(2+)-dependent PKC isozymes (alpha, beta, and gamma) in activation of latent HIV-1 in U1, a chronically infected promonocytic cell line, using polyclonal rabbit anti-PKC isozyme antibodies as specific inhibitors. Antibodies were introduced intracellularly by electroporation and then cells were stimulated with PMA. HIV-1 production was measured as p24 antigen using ELISA and reverse transcriptase activity. Anti-PKC beta antibody significantly inhibited PMA-induced HIV-1 production, whereas antibodies against PKC alpha and gamma had no significant effect. Furthermore, anti-PKC beta antibody inhibited PMA-induced activation of NF-kappa B and HIV-1 LTR. Preincubation of anti-PKC beta antibody with its antigenic peptide reversed the inhibitory effect of anti-PKC beta antibody. This study suggest that PKC beta plays a role in PMA-induced activation of latent HIV-1.
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PMID:Role of protein kinase C-beta isozyme in activation of latent human immunodeficiency virus type 1 in promonocytic U1 cells by phorbol-12-myristate acetate. 889 Nov 15

Protein kinase C (PKC) plays an integral role in control of many type II cell functions, including regulation of surfactant phospholipid secretion. To determine which isozymes of PKC may regulate type II cell functions, we identified those PKC isozymes activated in type II cells in association with surfactant phospholipid secretion after phorbol ester treatment. Transcripts encoding PKC-alpha, -beta, -delta, -epsilon, -eta, and -zeta were detected in type II cells by reverse transcriptase-polymerase chain reaction, whereas PKC-alpha, -beta, -delta, -eta, and -zeta were detected in type II cells by immunoblotting. PKC-alpha and -beta were only present in the cytosol in unstimulated type II cells, whereas PKC isozymes delta, eta, and zeta were found in cytosol and membrane fractions in unstimulated type II cells. 12-O-tetradecanoylphorbol-13-acetate stimulated surfactant secretion and activated PKC-alpha, -beta, -delta, and -eta isozymes in a dose-dependent manner. The inactive analogue 4alpha-phorbol 12,13-didecanoate neither activated PKC isozymes nor stimulated surfactant phospholipid secretion. PKC-zeta was not activated by any of the phorbol esters. PKC isozymes alpha, beta, delta, and eta are present in purified type II epithelial cells and are activated in a dose-dependent manner in alveolar type II cells in association with surfactant phospholipid secretion after phorbol ester treatment.
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PMID:Surfactant phospholipid secretion from rat alveolar type II cells: possible role of PKC isozymes. 912 66

Airway goblet cells secrete mucin in response to ATP and uridine 5'-triphosphate (UTP), but the underlying signal transduction pathways are poorly understood. Cultures of SPOC1 cells (L. H. Abdullah, S. W. Davis, L. Burch, M. Yamauchi, S. H. Randell, P. Nettesheim, and C. W. Davis. Biochem. J. 316: 943-951, 1996) secreted mucin on exposure to phorbol 12-myristate 13-acetate (PMA) [apparent affinity (K0.5) approximately 100 nM] and ionomycin (K0.5 approximately 5 microM) almost fivefold over baseline. Thapsigargin also elicited secretion (K0.5 approximately 20 nM). Ionomycin and PMA together elicited approximately twice the secretion of either agent alone. Overnight exposure to half-maximal PMA abolished the response to maximal doses of UTP and PMA, whereas ionomycin was fully effective. Protein kinase C (PKC) activity in the membrane fraction was increased by maximal doses of PMA and UTP, whereas ionomycin had no effect. PKC inhibitors were relatively ineffective against PMA- and UTP-induced mucin secretion. Human and canine goblet cells in epithelial explants, by video microscopy, underwent exocytosis with ionomycin (1 microM) and PMA (0.1 or 1 microM). SPOC1 cell mucin secretion was not stimulated by forskolin, 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate, or 8-bromoguanosine 3',5'-cyclic monophosphate. Cystic fibrosis transmembrane conductance regulator was not detected in SPOC1 cells by Western blotting, and its mRNA was detected by reverse transcriptase polymerase chain reaction (PCR) only as a very weak band and after 55 PCR cycles. Multidrug resistance (MDR1), however, was readily detected by Western blotting, and its mRNA was detected as a major band after 35 PCR cycles. Thus airway goblet cell mucin secretion, distal to receptor activation, may be regulated independently by Ca(2+)- and PKC-dependent pathways. Cystic fibrosis transmembrane conductance regulator and cyclic nucleotides, however, may not play a major role in this secretion.
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PMID:Protein kinase C and Ca2+ activation of mucin secretion in airway goblet cells. 925 57

One component of antigen receptor diversity shared by all gnathostomes characterized to date is mediated by a unique DNA polymerase, terminal deoxynucleotidyl transferase (TdT), which generates significant functional diversity during immunoglobulin and T-cell receptor rearrangement. To gain further insight into the evolutionary origin(s) of this unique enzyme and the immune system, a thymic cDNA clone encoding TdT was isolated from rainbow trout. The 2.3 kilobase (kb) full-length clone contained an open reading frame of 1 506 base pairs with a deduced protein product of Mr 57 000. Sequence comparisons demonstrate that TdT has been highly conserved in both sequence (>70% aa similarity, >50 aa identity) and overall structure during the course of vertebrate evolution. An amino acid alignment of all known TdT sequences (chicken, Xenopus, mouse, human, cattle, and trout) reveals that some, but not all, structural motifs believed to be critical for TdT activity have been conserved. The TdT alignment, in conjuction with the recently solved crystal structure for rat beta-polymerase, supports the hypothesis that both may have evolved from a common ancestral repair gene. In addition, four PKC phosphorylation sites are conserved, and hence may be involved in TdT regulation. Because TdT contributes to the generation of junctional diversity in antigen receptors of immature lymphocytes, its expression serves as a developmental marker for the sites of teleost lymphopoiesis. Northern blot (2.3 kb message) analysis shows that TdT mRNA is highly expressed within the thymus and to a lesser extent in the pronephros. In addition, reverse transcriptase-polymerase chain reaction analysis detected transcipts of both RAG1 and TdT in the thymus, pronephros, mesonephros, spleen, and intestine, but not within muscle, liver, or brain. Finally, TdT cDNA was amplified from embryos at 20 days post-fertilization (pf), which correlates with the appearence of the thymus and pronephros anlage during trout ontogeny.
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PMID:Characterization of rainbow trout terminal deoxynucleotidyl transferase structure and expression. TdT and RAG1 co-expression define the trout primary lymphoid tissues. 927 26

Protein kinase C (PKC) inhibitors decrease alpha1(I) collagen mRNA in stellate cells exposed to 200 micromol/liter of acetaldehyde. The purpose of these studies was to determine whether PKC activation plays a role in transcriptional activation of the alpha2(I) collagen gene. Cultured stellate cells were exposed to 200 micromol/liter of acetaldehyde. PKC, inositol triphosphate, diacylglycerol (DAG), and intracellular free calcium (Ca2+i) were measured. Alpha1(I) and alpha2(I) collagen messages were determined by reverse transcriptase-polymerase chain reaction. Activation of the alpha2(I) collagen promoter was determined in transiently transfected stellate cells. Acetaldehyde exposure enhanced PKC activity translocation to the particulate fraction at 20 min. Acetaldehyde did not increase Ca2+i, or inositol triphosphate but increased DAG levels at 20 min and 3 hr. Acetaldehyde increased both the alpha1(I) and alpha2(I) collagen messages in stellate cells. Calphostin C, a specific PKC inhibitor, which blocks DAG binding, eliminated both activation of the alpha2(I) collagen promoter by acetaldehyde and mRNA production by reverse transcriptase-polymerase chain reaction analysis. Similarly, D609, an inhibitor of DAG production, also inhibited alpha2(I) collagen gene expression. This study shows that collagen production by acetaldehyde is mediated by a calcium-independent PKC mechanism.
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PMID:Acetaldehyde enhances murine alpha2(I) collagen promoter activity by Ca2+-independent protein kinase C activation in cultured rat hepatic stellate cells. 1006 57

Parathyroid hormone (PTH)-related protein (PTHrP) acts as a local regulator of osteoblast function via mechanisms that involve PTH/PTHrP receptors linked to protein kinase A (PKA) and C (PKC). However, the regulation of PTHrP production and mRNA expression in human osteoblasts is poorly understood. Here we have characterized alternative PTHrP mRNA 3' splicing variants, encoding PTHrP isoforms of 139, 141, and 173 amino acids, and studied the regulation of PTHrP and its mRNAs by activated PKA and PKC in two human osteoblast-like cell lines (KPDXM and TPXM). Using exon-specific Northern analysis and reverse transcriptase-coupled polymerase chain reaction, we identified mRNAs encoding PTHrP(1-139) and PTHrP(1-141) in both cell lines. PTHrP(1-139) mRNAs predominated in TPXM cells and PTHrP(1-173) mRNAs were only detected in TPXM cells. Activation of PKA or PKC resulted in different effects on PTHrP and its mRNAs in the two cell lines. In TPXM cells, peptide-specific immunoassays detected high basal levels of PTHrP, increasing by 2-fold in cell extracts and 4-fold in culture media at 7 h and 24 h after exposure to forskolin, respectively, paralleling changes in PTHrP mRNA expression. Phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), a PKC activator, had no effect. In KPDXM cells, PTHrP was not detected in culture media under basal experimental conditions, and barely detectable amounts were present in cell extracts of TPA-treated cells, although the mRNA levels increased substantially in response to TPA. In the responsive cell lines, the effects on mRNA levels were dose dependent, and increased by 6.9- to 10.5-fold and 2.0- to 4.1-fold at 4 h in TPXM and KPDXM cells after exposure to 10 microM forskolin and 150 nM TPA, respectively. PTHrP mRNA levels then declined but were sustained above controls also at 12 h in both cell lines, albeit at considerably higher levels in TPXM cells. The different responsiveness to agents activating PKA- and PKC-dependent pathways may depend on the cellular state of differentiation, or alternatively, cancer cell line-specific defects. Our data demonstrating distinct differences in mRNA species and the amounts of PTHrP produced by the two cell lines as compared with roughly equivalent overall mRNA levels may suggest that post-transcriptional mechanisms play an important role in limiting the production of intracellular and secreted PTHrPs in human osteoblastic cells.
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PMID:Two human osteoblast-like osteosarcoma cell lines show distinct expression and differential regulation of parathyroid hormone-related protein. 1035 98


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