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)

Recent evidence indicates that membrane-bound costimulatory molecules of the B7 family are important for T-cell activation and are upregulated in IFN gamma-stimulated human microglia and in multiple sclerosis active lesions. In this study we have performed a detailed analysis of B7-1 and B7-2 expression and regulation in cultured mouse glial cells using immunocytochemical and semi-quantitative reverse transcriptase-polymerase chain reaction techniques. In an immortalized mouse microglial cell line (BV-2), expression of B7-1 and B7-2 was enhanced by interferon-gamma (IFN gamma). IFN gamma was a weak inducer of B7-2 mRNA and immunoreactivity in microglia primary cultures obtained from the neonatal mouse brain, whereas lipopolysaccharide, tumour necrosis factor-alpha, colony-stimulating factors and interleukin-1 beta did not affect microglial B7-2 expression. Combined IFN gamma and lipopolysaccharide treatment very effectively upregulated the B7-2 gene expression and immunoreactivity in microglia, but not in astrocytes. In both glial cell types, expression of B7-1 was not induced by any of the above agents. Among known microglia/macrophage deactivators, interleukin-10, prostaglandin E2 and cAMP-elevating agents, but not transforming growth factor-beta 1 and interleukin-4, inhibited B7-2 transcripts and immunoreactivity in IFN gamma/LPS-stimulated microglia, thus suggesting possible paracrine and autocrine mechanisms for regulating the expression of this important T-cell costimulatory signal in the brain.
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PMID:Analysis of B7-1 and B7-2 costimulatory ligands in cultured mouse microglia: upregulation by interferon-gamma and lipopolysaccharide and downregulation by interleukin-10, prostaglandin E2 and cyclic AMP-elevating agents. 900 48

The gene product affected in cystic fibrosis, the cystic fibrosis transmembrane conductance regulator (CFTR), is a chlorideselective ion channel that is regulated by cAMP-dependent protein kinase-mediated phosphorylation, ATP binding and ATP hydrolysis. Mutations in the CFTR gene may result in cystic fibrosis characterized by severe pathology (e.g. recurrent pulmonary infection, male infertility and pancreatic insufficiency) involving organs expressing the CFTR. Interestingly, in the kidney, where expression of the CFTR has been reported, impaired ion transport in patients suffering from cystic fibrosis could not be observed. To understand the role of the CFTR in chloride transport in the kidney, we attempted to identify an epithelial cell line that can serve as a model. We demonstrate that the CFTR is expressed constitutively in Madine-Darby canine kidney (MDCK) type I cells, which are thought to have originated from the distal tubule of the dog nephron. We show expression at the mRNA level, using reverse transcriptase-PCR, and at the protein level, using Western blot analysis with three different monoclonal antibodies. Iodide efflux measurements indicate that CFTR expression confers a plasma membrane anion conductance that is responsive to stimulation by cAMP. The cAMP-stimulated iodide release is sensitive to glybenclamide, diphenylamine carboxylic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, but not to 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid, an inhibitor profile characteristic of the CFTR chloride channel. Finally, the polarized localization of the CFTR to the apical plasma membrane was established by iodide efflux measurements and cell-surface biotinylation on MDCK I monolayers. Interestingly, MDCK type II cells, which are thought to have originated from the proximal tubule of the kidney, lack CFTR protein expression and cAMP-stimulated chloride conductance. In conclusion, we propose that MDCK type I and II cells can serve as convenient model systems to study the physiological role and differential expression of CFTR in the distal and proximal tubule respectively.
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PMID:Functional expression and apical localization of the cystic fibrosis transmembrane conductance regulator in MDCK I cells. 907 71

The differential expression of Rho family of low molecular weight GTP-binding proteins and protein kinase C (PKC) isozymes were examined during differentiation of rat C6 glial cells to astrocytic phenotypes induced by dibutyryl cAMP (dbcAMP)/theophylline. The cells showed rapid and distinct morphological changes, resembling stellate astrocytes at 12 h after the treatment. The treated cells had a round cell body that extended several long processes each with a beaded appearance. In addition to morphological changes, Western blot analysis revealed that S-100 protein, known as a glial cell differentiation marker, increased and reached the maximal level (approximately 6-fold increase) at 24 h following the addition of dbcAMP. In the control experiments with cells cultured in the absence of serum but also without dbcAMP/theophylline, morphological changes were marginal and apparent increases of S-100 protein were not observed by Western blotting. In response to dbcAMP/theophylline treatment, RhoA showed increases in the mRNA level followed by the protein level, as inferred by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Rac1 and Cdc42 proteins were undetectable by Western blot analyses. In PKC isozymes, increases were observed in PKC beta 1, epsilon, and zeta by RT-PCR, and in beta 1 and epsilon by Western blotting. Among them, PKC epsilon showed the most distinct changes. Its mRNA level transiently increased from 3 to 6 h and then decreased even below the basal level at 18 h after the treatment. In contrast, Western blot analysis revealed that PKC epsilon gradually increased time-dependently to 24 h (approximately 6-fold increase), and remained elevated until 48 h. These results suggested that RhoA and PKC epsilon, and probably also PKC beta 1 and PKC zeta, were closely implicated in C6 cell differentiation.
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PMID:Differential expression of Rho family GTP-binding proteins and protein kinase C isozymes during C6 glial cell differentiation. 910 74

The immunohistochemical localization of interleukin-1 receptor antagonist protein (Il-1ra) was examined in brain tissues of neurologically normal controls as well as in cases of Alzheimer's disease (AD) and Pick's disease. In all control cases, immunoreactivity was observed in some normal-appearing neurons in the neocortex and hippocampus. In AD, there appeared to be increased numbers of positively staining neurons, and the staining of individual neurons was somewhat more intense. Il-1ra was additionally expressed in globular deposits in senile plaques and, weakly, in some extracellular neurofibrillary tangles. In Pick's disease, there was similar staining of normal-appearing neurons and intense staining in some degenerating neurons. Using reverse transcriptase-polymerase chain reaction techniques, the mRNA for IL-1ra was detected in cultured IMR-32 human neuroblastoma cells following differentiation with dibutyryl cAMP and bromodeoxyuridine. Taken together, these data suggest that IL-1ra is a product of normal neurons which may be upregulated in some pathological circumstances.
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PMID:Expression of interleukin-1 receptor antagonist protein in post-mortem human brain tissues of Alzheimer's disease and control cases. 911 7

Osteoblasts are established targets of estrogen action in bone. We screened 66 conditionally immortalized clonal human osteoblast cell lines for estrogen receptors (ERs) using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for ER alpha mRNA and transactivation of adenovirus-estrogen response element (ERE)-tk-luciferase by 17 beta-estradiol (17 beta-E2) for functional ER protein. One of these cell lines, termed HOB-03-CE6, was chosen for further characterization. The cells, which were conditionally immortalized with a temperature-sensitive SV40 large T antigen, proliferated at the permissive temperature (34 degrees C) but stopped dividing at the nonpermissive temperature (> or = 39 degrees C). Alkaline phosphatase activity and osteocalcin secretion were upregulated by 1 alpha, 25-dihydroxyvitamin D3 in a dose-dependent manner. The cells also expressed type I collagen and other bone matrix proteins, secreted a variety of growth factors and cytokines, formed mineralized nodules based on alizarin red-S and von Kossa histochemical staining, and responded to dexamethasone, all-trans retinoic acid, and transforming growth factor-beta 1. This cell line expressed 42-fold less ER message than MCF-7 human breast cancer cells, as determined by quantitative RT-PCR. However, adenovirus-ERE-tk-luciferase activity was upregulated three- to fivefold in these cells by 17 beta-E2 with an EC50 of 64 pM. Furthermore, this upregulation was suppressed by co-treatment with the anti-estrogen ICI-182, 780. Cytosolic extracts of these cells specifically bound [125I]-17 beta-E2 in a concentration-dependent manner with a Bmax of 2.7 fmoles/mg protein (approximately 1,200 ERs/cell) and a Kd of 0.2 nM. DNA gel-shift analysis using a [32P]-ERE demonstrated the presence of ERs in nuclear extracts of these cells. Moreover, binding of the extracts to this ERE was blocked by a monoclonal antibody to the human ER DNA-binding domain. We evaluated these cells for 14 of 20 reported endogenous responses to 17 beta-E2 in osteoblasts. Although most of these responses appeared to be unaffected by the steroid, 17 beta-E2 suppressed parathyroid hormone-induced cAMP production, as well as basal interleukin-6 mRNA expression; conversely, the steroid upregulated the steady-state expression of alkaline phosphatase message in these cells. In summary, we have identified a clonal, conditionally phenotypic, human osteoblast cell line that expresses functional ERs and exhibits endogenous responses to 17 beta-E2. This cell line will be a valuable in vitro model for exploring some of the molecular mechanisms of estrogen action in bone.
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PMID:Functional properties of a conditionally phenotypic, estrogen-responsive, human osteoblast cell line. 913 93

We report here our effort of cloning and characterization of a novel human gene, which encodes a putative human endothelin receptor type B like protein (hET(B)R-LP), from a human hippocampus tissue cDNA library. hET(B)R-LP consists of 614 amino acids with seven putative transmembrane domains. The deduced amino acid sequence of hET(B)R-LP is 52% similar and 26.7% identical to human endothelin type B receptor. A 4.0 kb mRNA of hET(B)R-LP is abundantly expressed in the human brain. The results of in situ hybridization and reverse transcriptase in situ gene amplification reveal tissue distribution and cellular localization of signals of hET(B)R-LP mRNA in the neuronal cells, particularly concentrated in Purkinje cells of the cerebellum, and neuronal cells of the hippocampus of human brain, including pyramidal cells of Ammon's horn and granule cells of the dentate gyrus. A 4.0 kb mRNA of hET(B)R-LP is also less abundantly expressed in the liver and the placenta. Expression of recombinant protein, hET(B)R-LP/HA, in cells of COS7 and HEK293 transfected with plasmid DNA, hET(B)R-LP/HA/pcDNA1/Amp, was confirmed by Northern blot analysis and by immunofluorescence staining of cells with anti-HA antibody. Specific binding of radiolabeled ET-1 and ET-3 to membrane preparations and to intact cells expressing recombinant protein of hET(B)R-LP/HA did not show any significant difference of binding properties between cells transfected with plasmid DNA, hET(B)R-LP/HA/pcDNA1/Amp, and cells untransfected, including both COS7 cells and HEK293 cells. The results of assays of measuring Ca++ mobilization and cAMP production in HEK293 cells indicate that ET-1, ET-3, bombesin and neuropeptide Y are unable to produce any kind of significant difference of Ca++ mobilization and cAMP production between HEK293 cells expressing recombinant protein and HEK293 cells untransfected or HEK293 cells transfected with vector DNA only (pcDNA1/Amp) in functional assays performed. Therefore, its ligand and physiological significance of hET(B)R-LP remains to be discovered.
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PMID:A novel endothelin receptor type-B-like gene enriched in the brain. 914 77

We have used reverse transcriptase PCR, platelet mRNA and degenerate primers based on platelet peptide sequences, to amplify a fragment of platelet cGMP-inhibited phosphodiesterase (cGI-PDE; PDE3). Sequence analysis of this clone established that both the platelet and the cardiac forms of PDE3 were derived from the same gene (PDE3A). A RT-PCR product representing the C-terminal half of platelet PDE3 cDNA and corresponding to amino acid residues 560-1141 of the cardiac enzyme, was cloned and expressed in Escherichia coli cGI-PDEDelta1. Further deletion mutants were constructed by removing either an additional 100 amino acids from the N-terminus (cGI-PDEDelta2) or the 44-amino-acid insert characteristic of the PDE3 family, from the catalytic domain (cGI-PDEDelta1Deltai). In addition, site-directed mutagenesis was performed to explore the function of the 44-amino-acid insert. All mutants were evaluated for their ability to hydrolyse cAMP and cGMP, their ability to be photolabelled by [32P]cGMP and for the effects of PDE3 inhibitors. The Km values for hydrolysis of cAMP and cGMP by immunoprecipitates of cGI-PDEDelta1 (182+/-12 nM and 153+/-12 nM respectively) and cGI-PDEDelta2 (131+/-17 nM and 99+/-1 nM respectively) were significantly lower than those for immunoprecipitates of intact platelet PDE3 (398+/-50 nM and 252+/-16 nM respectively). Moreover, N-terminal truncations of platelet enzyme increased the ratio of Vmax for cGMP/Vmax for cAMP from 0.16+/-0.01 in intact platelet enzyme, to 0.37+/-0.05 in cGI-PDEDelta1 and to 0.49+/-0.04 in cGI-PDEDelta2. Thus deletion of the N-terminus enhanced hydrolysis of cGMP relative to cAMP, suggesting that N-terminal sequences may exert selective effects on enzyme activity. Removal of the 44-amino-acid insert generated a mutant with a catalytic domain closely resembling those of other PDE gene families but despite a limited ability to be photolabelled by [32P]cGMP, no cyclic nucleotide hydrolytic activities of the mutant were detectable. Mutation of amino acid residues in putative beta-turns at the beginning and end of the 44-amino-acid insert to alanine residues markedly reduced the ability of the enzyme to hydrolyse cyclic nucleotides. The PDE3 inhibitor, lixazinone, retained the ability to inhibit cAMP hydrolysis and [32P]cGMP binding by the N-terminal deletion mutants and the site-directed mutants, suggesting that PDE3 inhibitors may interact exclusively with the catalytic domain of the enzyme.
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PMID:Expression and mutagenesis of the catalytic domain of cGMP-inhibited phosphodiesterase (PDE3) cloned from human platelets. 917 84

Chronic catecholamine treatment induces beta-adrenergic receptor (beta AR) downregulation, i.e., a loss of total cell receptors. In the human respiratory tract, the mechanism(s) underlying beta AR downregulation remains poorly understood. The present study, therefore, examined the effects of 24 h of exposure to isoproterenol (Iso; 10 nM or 1 microM) on beta AR density and the rate of beta AR degradation, steady-state beta 2-adrenergic receptor (beta 2 AR) mRNA levels, and the content of Gs alpha and Gi alpha proteins in cultured human bronchial epithelial cells (i.e., the BEAS-2B cell line). beta AR density assessed by binding with [125I]iodopindolol decreased in a dose-dependent fashion with 24 h of Iso exposure. With Iso (1 microM), beta AR density decreased by approximately 82%. In contrast, forskolin (100 microM) and dibutyryl adenosine 3',5'-cyclic monophosphate (1 mM), agents that also increase adenosine 3',5'-cyclic monophosphate (cAMP) levels, had no significant effect on beta AR density. Iso exposure also elicited a concomitant decrease in Iso-stimulated cAMP but had no significant effect on the content of the G proteins G alpha i2 and Gs alpha assessed by immunoblotting and toxin-catalyzed ADP ribosylation. Of note, Iso exposure (1 microM) had no effect on steady-state levels of beta 2 AR mRNA measured both by Northern analysis and by reverse transcriptase-polymerase chain reaction. However, beta AR half-life assessed in the presence of the protein synthesis inhibitor cycloheximide was reduced by approximately 60% in Iso-treated cells (i.e., from 37 h in control to 16 h in 1 microM Iso). These results suggest that, in human airway epithelial cells, beta 2 AR downregulation 1) is not primarily driven by intracellular cAMP levels, 2) is not associated with significant decreases in steady-state levels of beta 2 AR mRNA, and 3) is largely posttranslationally regulated by increases in the rate of receptor protein degradation.
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PMID:Chronic effects of catecholamines on the beta 2-adrenoreceptor system in cultured human airway epithelial cells. 917 57

Production of the placental hormone, chorionic gonadotropin (CG), increases dramatically as cytotrophoblasts fuse to form syncytiotrophoblasts. The CG alpha- and beta-promoters are both responsive to cAMP, although the kinetics of cAMP stimulation are different. In an effort to understand the mechanisms of coordinate induction of these genes, AP-2 binding sites were identified in the promoter regions of the alpha and CGbeta genes. AP-2 bound to the upstream regulatory element (-186 to -156 base pairs (bp)) in the alpha-promoter and to several different regions of the CGbeta promoter, including footprints 2 and 4B (FP2, -311 to -279 bp; FP4B, 221 to -200 bp). AP-2 antibodies induced supershifts of these complexes, confirming the identity of the protein-DNA complex. In JEG-3 cells, which contain abundant AP-2, mutations in these CGbeta AP-2 sites reduced basal activity and decreased cAMP stimulation. In AP-2-deficient Hep-G2 cells, co-transfection of AP-2 stimulated expression of the CGbeta promoter 10-20-fold, and the alpha-promoter was induced by 3-6-fold. Mutations that eliminate AP-2 binding to CGbeta FP4B reduced AP-2 stimulation by more than 80%, whereas mutations in FP2 reduced AP-2 stimulation by less than 50%. Analyses of AP-2 mutants revealed a requirement for the DNA binding/dimerization domain and the amino-terminal proline-rich and acid-rich transactivation domains for stimulation of the CGbeta promoter. Primary cultures of placental cytotrophoblasts were differentiated into syncytiotrophoblasts in vitro to examine AP-2 expression by reverse transcriptase-polymerase chain reaction. AP-2 mRNA levels increased by day 2 and continued to rise in parallel with a marked increase in alpha and CGbeta gene expression. We conclude that both the alpha and CGbeta promoters contain binding sites for AP-2 and suggest that this transcription factor provides a mechanism for coordinating the induction of these genes during placental cell differentiation.
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PMID:Regulation of the human chorionic gonadotropin alpha- and beta-subunit promoters by AP-2. 918 71

A ClC-2G(2 alpha) Cl- channel was identified to be present in human lung and stomach, and a partial cDNA for this Cl- channel was cloned from a human fetal lung library. A full-length expressible human ClC-2G(2 alpha) cDNA was constructed by ligation of mutagenized expressible rabbit ClC-2G(2 alpha) cDNA with the human lung ClC-2G(2 alpha) cDNA, expressed in oocytes, and characterized at the single-channel level. Adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) treatment increased the probability of opening of the channel (Po). After PKA activation, the channel exhibited a linear (r = 0.99) current-voltage curve with a slope conductance of 22.1 +/- 0.8 pS in symmetric 800 mM tetraethylammonium chloride (TEACl; pH 7.4). Under fivefold gradient conditions of TEACl, a reversal potential of +21.5 +/- 2.8 mV was measured demonstrating anion-to-cation discrimination. As previously demonstrated for the rabbit ClC-2G(2 alpha) Cl- channel, the human analog, hClC-2G(2 alpha), was active at pH 7.4 as well as when the pH of the extracellular face of the channel (trans side of the bilayer; pHtrans) was asymmetrically reduced to pH 3.0. The extent of PKA activation was dependent on pHtrans. With PKA treatment, Po increased fourfold with a pHtrans of 7.4 and eightfold with a pHtrans of 3.0. Effects of sequential PKA addition followed by pHtrans reduction on the same channel suggested that the PKA- and pH-dependent increases in channel Po were separable and cumulative. Northern analysis showed ClC-2G(2 alpha) mRNA to be present in human adult and fetal lung and adult stomach, and quantitative reverse transcriptase-polymerase chain reaction showed this channel to be present in the adult human lung and stomach at about one-half the level found in fetal lung. The findings of the present study suggest that the ClC-2G(2 alpha) Cl- channel may play an important role in Cl- transport in the fetal and adult human lung.
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PMID:Characterization of the human pH- and PKA-activated ClC-2G(2 alpha) Cl- channel. 927 36


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