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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The use of thiazide diuretics is associated with increased bone mineral density and, in some studies with reduced incidence of fractures, suggesting a potential role for these drugs in the treatment of osteoporosis. Our objective was to examine the effects of thiazides on osteoblast-like cells using the rat UMR-106 osteosarcoma cell line. Treatment of UMR-106 cells with chlorothiazide caused membrane depolarization and a rise of intracellular calcium but had no effect on adenosine 3,5'-cyclic monophosphate accumulation. The rise of intracellular calcium was partially inhibited by nifedipine and removal of extracellular calcium, indicating calcium uptake from the extracellular media, as well as by thapsigargin or dantrolene, indicating contributions from calcium release from intracellular stores. Reverse transcriptase-polymerase chain reaction was used to isolate a partial cDNA clone for the thiazide-sensitive sodium-chloride cotransporter from UMR-106 cells that hybridized to 5.0- and 11.0-kilobase mRNAs when Northern blot analysis was conducted. Antisense oligonucleotides to the sodium-chloride cotransporter specifically inhibited the chlorothiazide-induced depolarization and rise of intracellular calcium and reduced immunofluorescence staining for the sodium-chloride cotransporter protein in UMR-106 cells. We conclude that thiazide diuretics inhibit sodium-chloride cotransporter activity in UMR-106 cells, thereby altering intracellular calcium regulation. These results provide evidence for direct effects of thiazide diuretics on bone cells.
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PMID:Expression of the sodium-chloride cotransporter in osteoblast-like cells: effect of thiazide diuretics. 903 17

It has been long known that neoplastic transformation is accompanied by a lowered requirement for extracellular Ca2+ for growth. The studies presented here demonstrate that human fibroblastic cell lines produce the two commonly found 'housekeeping' isoforms of the plasma membrane Ca(2+)-ATPase (PMCA), PMCA1b and 4b, and at the expression of both is demonstrably lower in cell lines neoplastically transformed by SV40 than in the corresponding parental cell lines. Western blot analyses of lysates from control (GM00037) and SV40-transformed (GM00637) skin fibroblasts revealed a 138 kDa PMCA whose level was significantly lower in the SV40-transformed cells relative to either total cellular protein or alpha-tubulin. Similar analyses of plasma membrane preparations from control WI-38) and SV40-transformed (WI-38VA13) lung fibroblasts revealed 3-4-fold lower levels of PMCA in the SV40-transformed cells. Competitive ELISAs performed on detergent solubilized plasma membrane preparations indicated at least 3-4-fold lower levels of PMCA in the SV40-transformed cell lines compared to controls. Reverse transcriptase coupled-PCR analyses showed that PMCA1b and PMCA4b were the only isoforms expressed in all four cell lines. The PMCA4b mRNA level detected by Northern analysis also was substantially lower in SV40 transformed skin fibroblasts than in non-transformed fibroblasts. Quantitative RT-PCR analyses showed levels of PMCA1b and 4b mRNAs to be 5 and 10-fold lower, respectively, in GM00637 than in GM00037 when the levels of PCR products were normalized to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. These results demonstrate that the expression of these distinct PMCA genes is substantially lower in SV40 transformed human skin and lung fibroblasts and may be coordinately regulated in these cells.
Cell Calcium 1997 Jan
PMID:Analysis of plasma membrane Ca(2+)-ATPase expression in control and SV40-transformed human fibroblasts. 905 77

Acetylcholine (ACh) induces repetitive, propagating intracellular Ca2+ concentration ([Ca2+]i) oscillations in porcine tracheal smooth muscle (TSM) cells. Using real-time confocal microscopy, we examined the role of sarcoplasmic reticulum (SR) Ca2+ release through inositol 1,4,5-trisphosphate (IP3) receptor and ryanodine receptor (RyR) channels in ACh-induced [Ca2+]i oscillations. In beta-escin permeabilized TSM cells, exposure to ACh in the presence of GTP also resulted in [Ca2+]i oscillations. [Ca2+]i oscillations could not be initiated by IP3 alone; however, an elevation of [Ca2+]i was observed. During ongoing [Ca2+]i oscillations, exposure to heparin, an IP3 receptor antagonist, caused a slowing of oscillation frequency but not complete inhibition. In contrast, ruthenium red, a RyR antagonist, completely abolished ACh-induced [Ca2+]i oscillations. Reverse transcriptase-polymerase chain reaction of TSM mRNA demonstrated the expression of RyR-2 and RyR-3 isoforms of the RyR. These results indicate that SR Ca2+ release through RyR channels is critical for ACh-induced [Ca2+]i oscillations in porcine TSM cells.
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PMID:Role of ryanodine receptor channels in Ca2+ oscillations of porcine tracheal smooth muscle. 914 39

The protein kinase C (PKC) isoenzymes expressed by bovine tracheal smooth muscle (BTSM) were identified at the protein and mRNA levels. Western immunoblot analyses reliably identified PKCalpha, PKCbetaI and PKCbetaII. In some experiments immunoreactive bands corresponding to PKCdelta, PKCepsilon and PKCTheta were also labelled, whereas the gamma, eta and zeta isoforms of PKC were never detected. Reverse transcriptase PCR of RNA extracted from BTSM using oligonucleotide primer pairs designed to recognize unique sequences in the PKC genes for which protein was absent or not reproducibly identified by immunoblotting, amplified cDNA fragments that corresponded to the predicted sizes of PKCdelta, PKCepsilon and PKCzeta, which was confirmed by Southern blotting. Anion-exchange chromatography of the soluble fraction of BTSM following homogenization in Ca2+-free buffer resolved two major peaks of activity. Using epsilon-peptide as the substrate, the first peak of activity was dependent upon Ca2+ and 4beta-PDBu (PDBu=phorbol 12, 13-dibutyrate), and represented a mixture of PKCs alpha, betaI and betaII. In contrast, the second peak of activity, which eluted at much higher ionic strength, also appeared to comprise a combination of conventional PKCs that were arbitrarily denoted PKCalpha', PKCbetaI' and PKCbetaII'. However, these novel enzymes were cofactor-independent and did not bind [3H]PDBu, but were equally sensitive to the PKC inhibitor GF 109203X compared with bona fide conventional PKCs, and migrated on SDS/polyacrylamide gels as 81 kDa polypeptides. Taken together, these data suggest that PKCs alpha', betaI' and betaII' represent modified, but not proteolysed, forms of their respective native enzymes that retain antibody immunoreactivity and sensitivity to PKC inhibitors, but have lost their sensitivity to Ca2+ and PDBu when epsilon-peptide is used as the substrate.
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PMID:Protein kinase C isoenzymes in airway smooth muscle. 916 53

To explore the possible role of purinergic receptors in thymocyte development and in pathogenesis of adenosine deaminase SCID, we studied effects of extracellular adenosine triphosphate (ATP(ext)) and adenosine on TCR- and steroid hormone-triggered processes in mouse thymocytes. Reverse transcriptase-PCR analysis confirms the mRNA expression of several types of purinergic receptors, while the functioning of ATP receptors in thymocytes is reflected by ATP(ext)-induced intracellular calcium increases and by thymocyte subset-specific sensitivity to the effects of ATP(ext) and adenosine. Only ATP(ext), but not the ATP catabolites, adenosine, dexamethasone, or TCR cross-linking, was efficient in triggering rapid protein synthesis independent lysis of CD4+8- thymocytes and peripheral CD4+ T cells. In contrast, extracellular adenosine specifically induced the apoptosis of CD4+8+ thymocytes. ATP(ext) also induced a slower process of DNA fragmentation and protein synthesis-dependent apoptosis in all thymocyte subsets. ATP(ext) had an additive effect with TCR cross-linking in the induction of thymocyte death, but, unexpectedly, the effects of ATP(ext) at high concentration were antagonistic to steroid-induced apoptosis. Described here, the properties of ATP(ext) and adenosine are consistent with their involvement in the regulation of T cell development due to differential expression and signaling through purinergic receptors in different thymocyte subsets. The possible role of purinergic receptor signaling in T cell differentiation and adenosine deaminase SCID is discussed.
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PMID:Effects of extracellular ATP and adenosine on different thymocyte subsets: possible role of ATP-gated channels and G protein-coupled purinergic receptor. 916 24

The effect of regucalcin, a Ca2+-binding protein isolated from rat liver cytosol, on ribonucleic acid (RNA) synthesis in the nuclei of normal rat liver and of regenerating rat liver was investigated. The liver weight at 1 day after partial hepatectomy was increased about 50% of that of sham-operated (control) rats. Calcium chloride (1.0-20 microM Ca2+ as final concentration) was added into the reaction mixture of nuclear RNA synthesis. RNA synthesis was established by incorporation of [3H]-uridine 5'-triphosphate (UTP) into the nuclear RNA. Addition of Ca2+ (5 and 10 microM) caused a significant increase of RNA synthesis in the nuclei from control rat liver. Such effect of Ca2+ was potentiated in the nuclei of regenerating liver; nuclear RNA synthesis was increased about 2 fold by the 1.0 and 2.5 microM Ca2+ addition. The stimulatory effect of Ca2+ was significantly inhibited by the presence of alpha-amanitin (10(-8) M), an inhibitor of RNA polymerase II. The presence of regucalcin (0.25 and 0.5 microM) significantly inhibited RNA synthesis in the nuclei from control rat liver and from regenerating rat liver. The inhibitory effect of regucalcin was remarkable in the presence of EGTA (0.5 mM), and it was weakened by the addition of Ca2+ (5 microM). Such regucalcin effect was not seen in the presence of alpha-amanitin. The presence of anti-regucalcin IgG in the reaction mixture significantly increased RNA synthesis in the nuclei from control rat liver, indicating that the endogenous regucalcin may be involved in nuclear RNA synthesis. The present results demonstrate that regucalcin can inhibit nuclear RNA synthesis in rat liver. Regucalcin may have an inhibitory role in the regulation of liver nuclear RNA synthesis.
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PMID:Inhibitory effect of calcium-binding protein regucalcin on ribonucleic acid synthesis in isolated rat liver nuclei. 927 68

Porcine articular chondrocytes have the capacity to release superoxide in response to the addition of the calcium ionophore ionomycin in a concentration-dependent manner. This activity was not stimulated by the addition of fMetLeuPhe or the kinase activator phorbol myristate acetate (PMA). However, this release of superoxide was inhibited by iodonium diphenyl (IDP), suggesting the involvement of NADPH oxidase. Reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotides designed against the known sequences for the human phagocyte NADPH oxidase showed the expression of p22-phox, p40-phox, and p47-phox mRNA, while Western blot analysis of chondrocyte extracts using polyclonal antisera raised against the human phagocyte NADPH oxidase suggested the presence of the p67-phox polypeptide. These results suggest that porcine articular chondrocytes can release reactive oxygen species using a NADPH oxidase-like complex.
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PMID:Detection of superoxide and NADPH oxidase in porcine articular chondrocytes. 929 50

Aggregation of high affinity IgE receptors (Fc epsilonRI) expressed on mast cells and basophils is a potent stimulus for the release of inflammatory mediators from cytoplasmic granules. Fc epsilonRI-dependent exocytosis requires activation of protein kinase C and mobilization of calcium from intra- and extracellular stores. However, how these events ultimately regulate the membrane fusion step between cytoplasmic granules and the plasma membrane still remains unclear. In this study, we investigated the role of the small GTPases of the rab3 subfamily in the regulated exocytosis following stimulation of rat basophilic leukemia cells (RBL-2H3). Analysis using reverse-transcriptase-based PCR showed that RBL-2H3 cells expressed rab3a and rab3d isoforms, with a predominance of rab3d at the mRNA level. Investigation of the subcellular distribution using isoform-specific Abs demonstrated that the majority of rab3a was expressed in the cytosol, whereas rab3d was found predominantly in the membrane fraction. To determine whether these proteins play a role in Fc epsilonRI-triggered exocytosis, we established RBL-2H3 transfectants that overexpressed wild-type and expressed GTP-binding mutant forms (N135I) of rab3a and rab3d. Whereas expression of rab3a proteins did not significantly affect degranulation as tested by beta-hexosaminidase release, those of both wild-type and mutant rab3d proteins inhibited degranulation. Calculations of the initial fast and of the second slow release rates showed that they are both inhibited about twofold, suggesting that rab3d interferes with a rate-limiting step in Fc epsilonRI-stimulated exocytosis.
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PMID:Involvement of the ras-like GTPase rab3d in RBL-2H3 mast cell exocytosis following stimulation via high affinity IgE receptors (Fc epsilonRI). 930 Jul 4

Cytidine 5'-triphosphate (CTP):phosphocholine cytidylyltransferase (EC 2.7.7.15) catalyses the synthesis of the active metabolic intermediate cytidine diphosphocholine (which is mainly used in the synthesis of choline-containing phospholipids). It is a rate-limiting reaction in choline phospholipid biosynthesis in many cells. In this study, a microassay is reported for the detection of this enzyme in small numbers of cells. This enzyme was present in mouse oocytes and at all stages during preimplantation development. Enzyme activity was destroyed by boiling but increased with time and number of embryos in the reaction. Activity in two-cell embryos was dependent on Mg2+ but independent of Ca2+ and was enhanced by the addition of 1 microgram lysophosphatidylethanolamine ml-1 to the reaction mixture. Activity was apparently dependent upon the phosphorylation status of the enzyme since the absence of the phosphatase inhibitor NaF caused a significant inhibition of activity. The enzyme in oocytes had a specific activity of 2.8 +/- 0.3 fmol cytidine diphosphocholine (CDP-choline) per oocyte min-1 (mean +/- SEM). The specific activity in two-cell and eight-cell embryos and blastocysts was not different from that of oocytes. Fertilized one-cell embryos had significantly less activity (1.4 +/- 0.05 fmol CDP-choline produced per embryo min-1) than other stages studied. Furthermore, the enzyme present in one-cell embryos was not capable of being further activated by the addition of exogenous lysophosphatidylethanolamine to the reaction. The increase in activity from the one-cell to the two-cell stage was not inhibited by alpha-amanitin (an inhibitor of RNA polymerase II), cycloheximide (a protein synthesis inhibitor) [1-(5-isoquinolinesulfonyl)-2-methylpiperazine, HCl]dihydrochloride (H-7; a protein kinase inhibitor) and was independent of cell-cycle progression; these results suggest that enzyme activity is independent of transcription, protein synthesis and the action of some kinases, including cell-cycle-dependent kinases. This study provides the first description of cytidylyltransferase in the early mammalian embryo.
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PMID:CTP:phosphocholine cytidylyltransferase activity in the preimplantation mouse embryo. 930 73

A human monocyte-activating CC chemokine has been identified based on sequences in an expressed sequence tag (EST) cDNA database. The protein shows highest sequence identity to the macrophage inflammatory protein (MIP) group of chemokines, particularly MIP-3 (76.7%) and MIP-1alpha (75.4%), and has been named MIP-5. Model building confirms that the protein has a similar three dimensional structure to other chemokines, but has an additional third disulphide bond. Northern blot analysis and reverse-transcriptase PCR show that the mRNA for MIP-5 is expressed at a high levels in liver, intestine and in lung leukocytes. MIP-5 induces chemotaxis of human monocytes, T-lymphocytes and, to a lesser degree, eosinophils at nanomolar concentrations; it has no effect on neutrophil migration. In receptor-binding assays, MIP-5 shows IC50 values of 12 nM for competition with 125I-MIP-1alpha for binding to CC-chemokine receptor (CCR)1, and 2.5 nM for competition with 125I-MCP-3 for binding to CCR3. It shows no ability to compete with ligand for binding to the two interleukin (IL)-8 receptors (CXC-chemokine receptors 1 and 2) or to CCR2, CCR4 or CCR5. Consistent with this binding data, MIP-5 was only able to induce calcium fluxes in CHO cells stably transfected with CCR1 or CCR3.
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PMID:Characterisation of macrophage inflammatory protein-5/human CC cytokine-2, a member of the macrophage-inflammatory-protein family of chemokines. 934 9


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