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 protein kinase C (PKC) family is composed of at least four conventional (alpha, beta I, beta II, and gamma) and several related novel (delta, epsilon, eta, and zeta) isoforms with different distribution and sensitivity to Ca2+ and phorbol esters. The enzyme is known to be present in cerebral endothelial cells. We have investigated the occurrence of seven isoforms (alpha, beta, gamma, delta, epsilon, eta, and zeta) by using reverse transcriptase-polymerase chain reaction in rat brain, in a freshly isolated brain microvessel fraction, in primary cultures of rat brain endothelial cells, in an immortalized rat brain endothelial cell line, and in aortic endothelial cell cultures. Brain tissue contained all seven investigated isoforms. A similar expression pattern was seen in freshly purified microvessels, but the PKC-gamma isoform could not be detected. Primary cultures of endothelial cells expressed PKC-alpha, -beta, -delta, -eta, and -epsilon isoenzymes, whereas the immortalized cell line expressed PKC-alpha, -delta, -epsilon, and -eta. The rat aortic endothelium contained only PKC-alpha and -delta isoforms. The variety of expression patterns of PKC family members in endothelial cells of different type may reflect differences in the functional responsiveness to environmental stimuli. Because PKC has been shown to be involved in the regulation of the blood-brain barrier permeability, the presence of different isoforms may confer a sophisticated intracellular regulatory mechanism to the brain endothelial cells.
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PMID:Expression of protein kinase C family members in the cerebral endothelial cells. 779 Aug 92

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

Integrins comprise a superfamily of alpha beta heterodimers that serve as cell signaling as well as adhesion molecules. We demonstrate that the alpha 3 beta 1 and alpha 6 beta 1 integrins are laminin/merosin receptors expressed in human thymocytes. By reverse transcriptase-PCR analysis, we determined that the alpha 3A beta 1, but not the alpha 3B beta 1, cytoplasmic structural variant of alpha 3 beta 1 is expressed in thymocytes. In contrast, both alpha 6A beta 1 and alpha 6B beta 1 cytoplasmic structural variants of alpha 6 beta 1 are expressed. A small percentage (10 to 15%) of human thymocytes bind to immobilized laminin, and even fewer (3 to 5%) bind to merosin, the laminin isoform normally present in the thymus. This binding, however, can be increased to 39 to 41% after activation of thymocytes with Mn2+ (or PMA). Binding to either laminin or merosin is completely inhibited by anti-beta 1 mAb or by a mixture of anti-alpha 3 and anti-alpha 6 mAbs, indicating that both alpha 3 beta 1 and alpha 6 beta 1 participate in thymocyte adhesion to the laminin family of extracellular matrix proteins. The protein kinase C inhibitors, calphostin C and staurosporine, inhibit Mn(2+)-enhanced thymocyte binding, suggesting that protein kinase C activity is crucial for the binding. Furthermore, the data indicate that at least two divalent cation binding sites serve to regulate integrin binding activity. Finally, we show that both immobilized laminin and merosin have costimulatory function for anti-CD3-induced thymocyte proliferation, and both anti-alpha 3 and anti-alpha 6 mAbs can block this proliferative response. The cooperative function of alpha 3 beta 1 and alpha 6 beta 1 evidenced in the laminin/merosin binding and proliferation assays suggests that thymocyte-merosin interactions may play an important role in thymic T cell development.
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PMID:Alpha 3 beta 1 and alpha 6 beta 1 integrins mediate laminin/merosin binding and function as costimulatory molecules for human thymocyte proliferation. 781 63

The presence of alpha, delta, epsilon, theta, and zeta protein kinase C isoforms in DS19 murine erythroleukemia cells has been established in this study. In addition, the mRNA levels of these isozymes have been measured by quantitative reverse transcriptase-polymerase chain reaction. Isoform delta has been found to be the most abundant isotype, whereas isoform zeta resulted to be present in only few copies. Furthermore, the expression levels of all five protein kinase C isozymes have been studied in three cell clones, derived from parental DS19 cells and characterized by different susceptibilities to differentiation. This comparative analysis indicated that the calcium-independent isozymes (delta, epsilon, zeta, and theta) display significantly higher expression levels in cells less prone to differentiation. On the other hand, the mRNA levels of the only calcium-dependent isoform present (alpha) fluctuate poorly from one cell clone to the other, but are the highest in the cell clone characterized by the fastest rate of differentiation. This study represents the first complete characterization of the basal levels of specific protein kinase C isotypes in different murine erythroleukemia cell clones and provides further evidence for the role of individual isozymes in the early events that trigger chemical induced murine erithroleukemia cell differentiation.
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PMID:Differential expression of protein kinase C isoform genes in three murine erythroleukemia cell variants: implication for chemical induced differentiation. 798 May 1

The protein kinase C (PKC) family of enzymes plays a key role in the regulation of cellular events, including cell proliferation and differentiation. Work from our laboratory has shown that the effects of dietary fat and fiber on colonic cell proliferation were positively correlated with membrane/cytosol PKC activity ratios (Chapkin et al., 1993, J. Nutr. 123, 649-655). The presence and subcellular distribution of specific PKC isoforms in rat and human colon were therefore determined in cytosolic and membrane extracts. Tissue extracts were probed with antibodies to individual PKC isoforms. PKC alpha, beta, delta, epsilon, and zeta were detected in both rat and human colonic mucosa, while PKC eta was detected in human colonic mucosa only. PKC alpha, beta, and zeta were predominantly localized in the cytosolic fraction, whereas the majority of PKC delta, epsilon, and eta were found in the membrane-associated fraction. Presence of mRNA for individual PKC isoforms was determined by reverse transcriptase PCR (RT-PCR). Using rat colonic mucosa, mRNA for PKC alpha, beta, delta, epsilon, eta, and zeta were detected by RT-PCR with identity confirmed by sequencing. The relative steady-state levels of PKC isoforms in human colon adenocarcinoma as compared with normal colonic mucosa were determined, with adenocarcinomas having higher amounts of cytosolic PKC beta, delta, epsilon, eta, and zeta. PKC isoforms were also detected in viable, exfoliated colonic cells isolated from human feces, demonstrating that this noninvasive method can be utilized to examine PKC expression in colonic cells. These results demonstrate that colonic mucosa expresses both calcium-dependent (classical) and calcium-independent (novel and atypical) PKC isoforms with distinct subcellular distributions for each. The dynamics of these PKC isoforms may have implications in the development of colon carcinogenesis.
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PMID:Protein kinase C isoforms in human and rat colonic mucosa. 803 70

The cDNA encoding mouse phosphatidylinositol transfer protein (PI-TP) was isolated by means of reverse transcriptase polymerase chain reaction. The nucleotide sequence of this cDNA has a high similarity (98%) with that of rat PI-TP; the predicted amino acid sequence is 99.6% identical to that of rat PI-TP. The cDNA encoding mouse PI-TP was cloned into the expression vector pET3d and the Escherichia coli strain BL21(DE3) was transformed with the resulting plasmid. After induction of the bacteria with isopropyl-beta-D-thiogalactopyranoside, PI-TP was efficiently expressed in the E. coli strain. It was estimated that 5% of the total soluble cell protein consisted of PI-TP. The recombinant mouse PI-TP was purified from the bacterial lysate in four steps: ammonium sulphate precipitation, anion-exchange chromatography, heparin-Sepharose affinity and gel filtration chromatography. Fractionation on the heparin-Sepharose affinity column yielded two forms: PI-TP Hepa1 and Hepa2. These two proteins have the same molecular mass of 35 kDa, both contain a phosphatidylglycerol molecule and both are recognized by anti-PI-TP antibody. Both recombinant proteins have an isoelectric point of 5.4 as compared to 5.5 for bovine brain PI-TP. Sequence analysis of the first 25 N-terminal amino acid residues showed that both forms are identical, except that PI-TP Hepa1 contains the initiator methionine which is lacking from PI-TP Hepa2. The two PI-TP forms have similar phospholipid-binding and transfer activity, comparable to that of bovine brain PI-TP. Both forms and bovine brain PI-TP are phosphorylated equally well in a Ca2+/phospholipid-dependent way by protein kinase C.
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PMID:Characterization of mouse phosphatidylinositol transfer protein expressed in Escherichia coli. 804 44

The alpha T3-1 cell line, an immortalized gonadotroph cell line, expresses high levels of the gonadotrophin-releasing hormone (GnRH) receptor. Sustained exposure of these cells to the GnRH receptor agonist des-Gly10-[D-Ala6]luteinizing hormone-releasing hormone ethylamide resulted in a substantial down-regulation of cellular levels of a combination of the alpha subunits of the phospholipase C-beta 1-linked G proteins Gq and G11, as assessed by immunoblotting with an antiserum able to identify these two proteins equally. This effect was dependent upon the concentration of agonist used (EC50 = 4 nM) and on the time of the treatment (t1/2 = 6 hr) when a maximally effective concentration of agonist (1 microM) was used. Comparison of agonist regulation of inositol phosphate generation and Gq alpha/G11 alpha down-regulation demonstrated that effects on inositol phosphate production were approximately 3-fold more potent. In contrast to Gq alpha/G11 alpha, membrane-associated levels of Gs alpha and G12 alpha, the G proteins that transduce stimulatory and inhibitory regulation, respectively, of adenylyl cyclase, were not altered by agonist treatment. Analysis of mRNA by reverse transcriptase/polymerase chain reaction indicated the coexpression by alpha T3-1 cells of mRNA corresponding to both Gq alpha and G11 alpha. Immunoblotting with antisera selective for either Gq alpha or G11 alpha confirmed their coexpression. Resolution of membranes from untreated and agonist-treated alpha T3-1 cells under sodium dodecyl sulfate-polyacrylamide gel electrophoresis conditions able to separate Gq alpha from G11 alpha indicated that G11 alpha was more prevalent than Gq alpha at steady state but that agonist treatment regulated cellular levels of both of these G proteins in a nonselective manner. Sustained activation of protein kinase C with phorbol myristate acetate was unable to mimic agonist regulation of cellular Gq alpha/G11 alpha levels, as was treatment of alpha T3-1 cells with the selective protein kinase C inhibitor chelerythrine. These data suggest that the GnRH receptor is able to interact functionally with both Gq alpha and G11 alpha in alpha T3-1 cells and that sustained exposure to a GnRH receptor agonist selectively regulates the cellular levels of the G proteins that interact with the receptor.
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PMID:The gonadotrophin-releasing hormone receptor of alpha T3-1 pituitary cells regulates cellular levels of both of the phosphoinositidase C-linked G proteins, Gq alpha and G11 alpha, equally. 805 44

Feline immunodeficiency virus (FIV) is a member of the genus Lentivirus of the family Retroviridae. FIV can infect T lymphocytes and monocytes/macrophages in vitro and in vivo, and causes an acquired immunodeficiency syndrome-like disease in cats. Several isolates of FIV from geographically distant countries have been molecularly cloned. There is considerable heterogeneity especially in Env gene among the FIV isolates and they can be divided into two or more subgroups. Like other lentiviruses, FIV has a complex genome structure. Gag gene encodes matrix, capsid and nucleocapsid proteins, and Pol gene encodes protease, reverse transcriptase, dUTPase and integrase. The dUTPase is not present in the primate lentiviruses but present in the non-primate lentiviruses. Env gene encodes surface and transmembrane envelope glycoproteins. In addition to the structural and enzymatic proteins, at least three more genes (Vif, ORF A, Rev) are present in FIV. Vif is related to the infectivity of the cell-free viruses. Rev functions in the stability and transport of incompletely spliced viral RNAs from the nucleus to cytoplasm and is indispensable for virus replication. Although the Tat protein of the primate lentiviruses is essential for virus replication, ORF A (putative Tat gene) of FIV is not essential for virus replication in established feline T lymphoblastoid cell lines. However, the ORF A gene product is related to the efficient replication of the virus in primary peripheral blood lymphocytes. In the long terminal repeat (LTR) of FIV, there are many putative binding sites for enhancer/promoter proteins. Among these binding sites, the putative AP-1 site is important for basal promoter activity of the LTR and responsible for the T cell activation signal through protein kinase C, however the site is not required for the virus replication in established feline T lymphoblastoid cell lines. Comparative study of the molecular biology of lentiviruses revealed that the genome structure, splicing pattern and functional enhancer protein-binding sites of FIV are more similar to those of the ruminant lentiviruses than those of the primate lentiviruses.
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PMID:The genome of feline immunodeficiency virus. 812 13

Cytomegalovirus (CMV) infection constitutes a serious threat to patients with acquired immune deficiency syndrome. Recently we reported that human immunodeficiency virus (HIV) infection of CD4+ cells was associated with sustained elevation of cellular levels of cAMP. Moreover, cyclic nucleotide modulators enhanced HIV replication by increasing intracellular levels of cAMP. In this study, the effect of CMV on HIV replication in CMV/HIV mixed infection and its relationship to cAMP were examined. MT-4 cells, CMV strain AD169, and HIV strain IIIB were used. Optimal enhancement (4.4-fold increase) of HIV replication was observed when MT-4 cells were infected with CMV at Day 0 followed by HIV on Day 4 after infection, as determined by reverse transcriptase activity on Day 11 after infection. cAMP (measured by radioimmunoassay) levels in cells infected with CMV alone, HIV alone, or CMV/HIV together were 2-, 3-, and 5-fold above untreated cells, respectively. CMV also enhanced the replication of UV-irradiated HIV 4-fold and this was associated with a 2-fold increase in cAMP as well. Moreover, UV-irradiated CMV enhanced HIV replication 8.8-fold. The same dose of viable and UV-irradiated CMV used in the above experiments increased protein kinase C activity in these cells 3.0- and 8.0-fold, respectively. These findings might suggest that cAMP and protein kinase C are involved in CMV enhancement of HIV replication. These findings may have relevance to the identification of novel target sites for development of antiviral therapeutics.
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PMID:Involvement of cAMP and protein kinase C in cytomegalovirus enhancement of human immunodeficiency virus replication. 841 79

The MCF-7 doxorubicin-resistant cell line MCF-7/Dox has been used extensively for studies of the multidrug resistance phenomenon. Using fluorescence-activated cell sorting (FACS), these cells were separated into two populations on the basis of rhodamine 123 (R123) accumulation. We designated these as low P-glycoprotein (LP-gp) and high P-gp (HP-gp) cells on the basis of their P-gp content. Using the reverse transcriptase polymerase chain reaction technique controlled by homologous internal standards, we analysed levels of MDR1 and MDR2 mRNA in each cell type. LP-gp and HP-gp cells had MDR1 mRNA levels of 2.17 +/- 0.17 and 6.65 +/- 2.29 amol ng-1 total RNA respectively, compared with 0.00088 +/- 0.00005 amol ng-1 in wild-type MCF-7 cells (MCF-7/WT). MCF-7/WT cells additionally contained 0.023 +/- 0.016 amol ng-1 of MDR2 mRNA, which was unchanged in LP-gp cells, but lower than in HP-gp cells, which contained 0.42 +/- 0.08 amol ng-1. Both LP-gp and HP-gp cells contained increased copies of the MDR1 gene. However, the degree of gene amplification did not correlate with the changes in MDR1 mRNA levels, indicating further regulatory levels of gene expression. The level of P-gp detected by MRK 16 correlated with R123 accumulation. HP-gp cells expressed a 10-fold higher level of P-gp1 than LP-gp cells. However, there was only a 3-fold increase in MDR1 mRNA level in HP-gp cells compared with LP-gp cells. These data suggest that some regulation of P-gp1 expression also occurred at the post-translational level. Phosphorylation of P-gp by protein kinase C (PKC)-alpha is necessary for its activity. Our analysis of PKC-alpha, 0 and epsilon isozyme levels, and subcellular distribution, shows a co-regulation of expression with P-gp, suggesting a necessary role for PKC in P-gp regulation.
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PMID:Regulation of P-glycoprotein 1 and 2 gene expression and protein activity in two MCF-7/Dox cell line subclones. 856 35


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