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)

The endogenous expression in human embryonic kidney 293 (HEK293) cells of corticotropin-releasing factor (CRF) receptors was detected. High-affinity binding sites for human CRF (K(i)=3.6 nM), ovine CRF (K(i)=4.6 nM), rat urocortin (K(i)=2.2 nM), sauvagine (K(i)=2.4 nM) and astressin (K(i)=4.3 nM) with the pharmacological characteristics for CRF type 1 (CRF(1)) receptors and B(max) values of approximately 30 fmol/mg protein were determined. The four CRF receptor agonists nonselectively stimulated cAMP production in HEK293 cells at low agonist concentrations, whereas the antagonist astressin shifted the dose-response curve for ovine CRF significantly rightward. Transfection of the pcDNA3 vector into HEK293 cells strongly reduced the expression of the endogenous CRF receptor. Northern blot analysis revealed the expression of a CRF(1) transcript in human neuronal tissues, HEK293, human NTera-2 (NT2) carcinoma, Y-79 retinoblastoma and African green monkey kidney (COS-7) cells. Neither by Northern blot analysis nor by reverse transcriptase PCR (RT-PCR), the expression of CRF(2) could be detected. In cAMP stimulation experiments, functional CRF receptors were detected in these cell lines. These data show that HEK293 and other cell lines endogenously express CRF(1) receptors.
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PMID:Functional characterization of corticotropin-releasing factor type 1 receptor endogenously expressed in human embryonic kidney 293 cells. 1070 6

Calcium agonists induce membrane depolarization in endothelial cells through an unknown mechanism. Present studies tested the hypothesis that pulmonary artery endothelial cells express a cyclic nucleotide-gated (CNG) cation channel activated by store-operated calcium entry to produce membrane depolarization. In the whole-cell configuration, voltage-clamped cells revealed a large non-inactivating, outwardly rectifying cationic current in the absence of extra- or intracellular Ca(2+) that was reduced upon replenishment of Ca(2+). The inward current was non-selective for K(+), Na(+), Cs(+), and Rb(+) and was not inhibited by high tetraethylammonium concentrations. cAMP and cGMP stimulated the current and changed the cation permeability to favor Na(+). Moreover, 8-bromo-cAMP stimulated the current in voltage-clamped cells in the perforated patch mode. The cationic current was inhibited by the CNG channel blocker LY83,583, and reverse transcriptase-polymerase chain reaction cloning identified expression of a CNG channel resembling that seen in olfactory neurons. Activation of store-operated calcium entry using thapsigargin increased a current through the CNG channel. Stimulation of the current paralleled pulmonary artery endothelial cell membrane depolarization, and both the current and membrane depolarization were abolished using LY83,583. Taken together, these data demonstrate activation of store-operated calcium entry stimulates a CNG channel producing membrane depolarization. Such membrane depolarization may contribute to slow feedback inhibition of store-operated calcium entry.
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PMID:Cyclic nucleotide-gated channels mediate membrane depolarization following activation of store-operated calcium entry in endothelial cells. 1076 97

cDNAs encoding a novel phosphodiesterase, phosphodiesterase 11A (PDE11A), were isolated by a combination of reverse transcriptase-polymerase chain reaction using degenerate oligonucleotide primers and rapid amplification of cDNA ends. Their catalytic domain was identical to that of PDE11A1 (490 amino acids) reported during the course of this study. However, the cDNAs we isolated had N termini distinct from PDE11A1, indicating two novel N-terminal variants of PDE11A. PDE11A3 cDNA encoded a 684-amino acid protein including one complete and one incomplete GAF domain in the N-terminal region. PDE11A4 was composed of 934 amino acids including two complete GAF domains and shared 630 C-terminal amino acids with PDE11A3 but had a distinct N terminus containing the putative phosphorylation sites for cAMP- and cGMP-dependent protein kinases. PDE11A3 transcripts were specifically expressed in testis, whereas PDE11A4 transcripts were particularly abundant in prostate. Recombinant PDE11A4 expressed in COS-7 cells hydrolyzed cAMP and cGMP with K(m) values of 3.0 and 1.4 microm, respectively, and the V(max) value with cAMP was almost twice that with cGMP. Although PDE11A3 showed the same K(m) values as PDE11A4, the relative V(max) values of PDE11A3 were approximately one-sixth of those of PDE11A4. PDE11A4, but not PDE11A3, was phosphorylated by both cAMP- and cGMP-dependent protein kinases in vitro. Thus, the PDE11A gene undergoes tissue-specific alternative splicing that generates structurally and functionally distinct gene products.
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PMID:Isolation and characterization of two novel phosphodiesterase PDE11A variants showing unique structure and tissue-specific expression. 1090 26

IA-2, a member of the protein tyrosine phosphatase family, represents a major target autoantigen in type 1 diabetes. To study the regulation of IA-2 gene expression, we used INS-1 insulinoma cells to analyze beta-cell signal transduction pathways as well as the effect of metabolic and hormonal factors involved in the regulation of the insulin secretory pathway. Quantitative competitive reverse transcriptase-polymerase chain reaction revealed that an increase of cellular cAMP mediated by forskolin (10 micromol/l, 24 h) or 3-isobutyl-1-methylxanthine (100 micromol/l, 24 h) induced maximal stimulation of IA-2 mRNA levels (451 +/- 85 and 338 +/- 86% compared with basal conditions; P < 0.001). In contrast, activation of protein kinase C (PKC) by short-term treatment with phorbol 12-myristate 13-acetate (PMA) (1 micromol/l, 6 h) did not alter IA-2 expression, whereas depletion of PKC by prolonged culturing (24 h) exerted a significant inhibition (57 +/- 24%; P < 0.05). cAMP-dependent upregulation was confirmed by the findings that glucagon (10 micromol/l, 24-48 h) increased levels of IA-2 mRNA (190 +/- 35%; P < 0.05), whereas short-term incubation with high glucose concentration showed no effect. However, prolonged incubation in high glucose (21 mmol/l) induced a time- and dose-dependent increase of IA-2 mRNA expression, reaching maximal values after 144 h (285 +/- 68%; P < 0.05). These studies demonstrate that stimuli of insulin secretion that operate by activation of adenylate cyclase generating cAMP significantly increase IA-2 gene expression. In contrast, activation of PKC by high glucose concentration or PMA exerted no effect, suggesting that IA-2 gene expression is not simply coupled to insulin secretion, but may be involved in the fine regulation of beta-cell function. These findings may be important to clarify the function of IA-2 in beta-cells and elucidate mechanisms involved in the induction of autoimmunity to IA-2.
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PMID:Regulation of the diabetes-associated autoantigen IA-2 in INS-1 pancreatic beta-cells. 1090 70

Macrophage colony-stimulating factor (M-CSF) is a multifunctional cytokine attributed with key biological functions beyond the first discovered role in promoting proliferation of myeloid cell lineage. The human pancreatic cancer cell line MIA PaCa-2, from which the M-CSF gene was originally cloned, was used to study regulation of M-CSF expression. Expression of M-CSF was inducible by interleukin-1alpha (IL-1alpha), lipopolysaccharide (LPS) and PMA as demonstrated by a biological activity assay, Northern-blot analysis and reverse transcriptase (RT) PCR. Treatment of the cells with forskolin or dibutyryl-cAMP attenuated the expression of M-CSF induced by IL-1alpha or LPS, but not by PMA. Electromobility shift assays showed that IL-1alpha predominantly activated nuclear factor kappaB (NF-kappaB), while PMA preferentially activated activator protein-1 (AP-1). The activation of NF-kappaB, but not AP-1, could be attenuated by cAMP elevation. Relative RT-PCR demonstrated that the expression of a 1.6-kb M-CSF mRNA transcript was more effectively induced by IL-1alpha than a 4.0-kb transcript. By and large the induced expression of both mRNA transcripts could be attenuated by cAMP. M-CSF promoter-driven luciferase reporter-gene assays revealed that cAMP elevation attenuated the IL-1-induced transcription activation of the M-CSF promoter, but it had no effect on PMA-induced transcription. Our findings suggest that cAMP regulates M-CSF gene expression at the transcriptional level and that its inhibitory effect involves NF-kappaB signalling pathway.
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PMID:cAMP attenuates interleukin-1-stimulated macrophage colony-stimulating factor (M-CSF) expression. 1092 34

Gene expression, protein expression, and function of amiloride-sensitive sodium channels were examined in human lymphocytes from normal individuals and individuals with Liddle's disease. Using reverse transcriptase polymerase chain reactions, expression of all three cloned epithelial sodium channel (ENaC) subunits was detected in lymphocytes. Polyclonal antibodies to bovine alpha-ENaC bound to the plasma membrane of normal and Liddle's lymphocytes. A quantitative analysis of fluorescence-tagged ENaC antibodies indicated a 2.5-fold greater surface binding of the antibodies to Liddle's lymphocytes compared with normal lymphocytes. The relative binding intensity increased significantly (25%; p < 0.001) for both normal and Liddle's cells after treatment with 40 microM 8-CPT-cAMP. Amiloride-sensitive whole cell currents were recorded under basal and cAMP-treated conditions for both cell types. Liddle's cells had a 4.5-fold larger inward sodium conductance compared with normal cells. A specific 25% increase in the inward sodium current was observed in normal cells in response to cAMP treatment. Outside-out patches from both cell types under both treatment conditions revealed no obvious differences in the single channel conductance. The P(open) was 4.2 +/- 3.9% for patches from non-Liddle's cells, and 27.7 +/- 5.4% in patches from Liddle's lymphocytes. Biochemical purification of a protein complex, using the same antibodies used for the immunohistochemistry, yielded a functional sodium channel complex that was inhibited by amiloride when reconstituted into lipid vesicles and incorporated into planar lipid bilayers. These four independent methodologies yielded findings consistent with the hypotheses that human lymphocytes express functional, regulatable ENaC and that the mutation responsible for Liddle's disease induces excessive channel expression.
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PMID:Expression and regulation of normal and polymorphic epithelial sodium channel by human lymphocytes. 1111 30

Type II secreted phospholipase A(2) (sPLA(2)) releases precursors of important inflammatory lipid mediators from phospholipids. Some observations have indicated that the sPLA(2), which has been implicated in chronic inflammatory conditions such as arthritis, contributes to atherosclerosis in the arterial wall. sPLA(2) was not detected in control vascular smooth muscle cells (VSMC). Treatment of VSMC with agents that increase intracellular cAMP (eg, forskolin, dibutyryl [db]-cAMP) resulted in a time- and concentration-dependent increase in sPLA(2) gene expression. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) showed a marked dose-dependent inhibition of forskolin-induced mRNA by protein kinase A inhibitor. Electrophoretic mobility shift analysis of nuclear proteins from forskolin-treated and db-cAMP-treated VSMC with C/EBP consensus oligonucleotides and C/EBP oligonucleotides from the rat promoter revealed greater binding than in control VSMC. Incubation of VSMC with H89, a specific protein kinase inhibitor, also blocked the binding of nuclear C/EBP to the C/EBP site of the rat promoter induced by db-cAMP and forskolin. Binding was unchanged with the use of CRE consensus oligonucleotides. Antibodies revealed the specific formation of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP-ss and -delta antibodies. Functional activation of C/EBP was confirmed by a luciferase reporter gene assay. A construct comprising 4 tandem repeat copies of the C/EBP element from the rat sPLA(2) promoter linked to luciferase was transcriptionally activated in VSMC by cotransfection with expression vector for the protein kinase A catalytic subunit. It was also significantly activated in transfected VSMC treated by forskolin or db-cAMP. H89 inhibited this activations. We therefore conclude that the increases in sPLA(2) mRNA and enzyme activity produced by cAMP-elevating agents is controlled by a mechanism involving nuclear C/EBP-ss and -delta acting through a protein kinase A signaling pathway.
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PMID:Protein kinase A-dependent stimulation of rat type II secreted phospholipase A(2) gene transcription involves C/EBP-beta and -delta in vascular smooth muscle cells. 1111 53

Renin secretion can be stimulated by ATP via purinergic P2Y receptors. ATP is a cotransmitter with norepinephrine and is released from the cytosol during cell damage. Such release could account for the de novo renin expression seen in the proximal tubule in renal disease and in myocardial infarct borders. Whereas most P2Y purinoceptor subtypes utilize phosphoinositide signal-transduction pathways, the effector mechanisms of the subtype P2Y(11) also involve increases in cAMP, a well-known renin secretagogue and stimulus to renin production. The present study tested the effect of ATP on human renin gene (REN) promoter activity and the role of P2Y(11). By means of reverse transcriptase-polymerase chain reaction, we found that renin-expressing Calu-6 cells express P2Y(11) mRNA. Expression was also detected in the brain, kidney, testis, muscle, liver, and spleen. We made a novel cell line (Calu-6/P2Y11) in which P2Y(11) cDNA, under the control of a strong promoter, was stably integrated into genomic DNA. These cells produced P2Y(11) mRNA during culture. Treatment of Calu-6/P2Y11 cells with 1 mmol/L ATP caused a 3-fold increase in renin mRNA and protein over 36 hours. Transient transfection of Calu-6/P2Y11 cells with constructs containing 896 bp of human REN 5'-flanking DNA linked to the luciferase reporter gene led to a 5.8+/-0.6-fold increase (mean+/-SEM) in reporter activity in response to ATP (P=0.0015). In contrast, UTP produced only a 1.4+/-0.1-fold increase (P=0.016). For ADP, it was 1.7+/-0.1-fold (P=0.011). The response profile was ATP>ADP>AMP=adenosine=0, consistent with a P2Y(11) effect. Mutation of the cAMP response element (CRE) located at -222 in the REN promoter DNA abolished the effect of ATP. Furthermore, ATP induced a rapid, time-dependent increase in the phosphorylation of CRE binding protein (CREB) and activating transcription factor-1. These data implicate a cAMP pathway in mediation of the P2Y(11) effect. In conclusion, we have made a novel cell line that overexpresses the P2Y(11) purinoceptor. Stimulation of these cells by ATP activates a cAMP signal-transduction pathway that phosphorylates CREB and stimulates renin promoter activity via the CRE at -222. The data raise the possibility of a contribution of ATP/P2Y(11) effects to sympathetic stimulation of renin, as well as to responses in renin seen after tissue damage, such as in kidney disease and myocardial infarction.
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PMID:Capacity for purinergic control of renin promoter via P2Y(11) receptor and cAMP pathways. 1111 31

Recent studies have shown that the homeobox gene Hex plays an important role in inducing differentiation of vascular endothelial cells. In this study, we examined the expression of Hex in vascular smooth muscle cells (VSMCs) in vitro and in vivo. Immunohistochemistry showed a marked induction of Hex protein in neointimal VSMCs after balloon injury in rat aorta. Western and reverse transcriptase-polymerase chain reaction analyses demonstrated that Hex was abundantly expressed in cultured VSMCs, whereas it was undetectable in other cell types or in normal aorta. The expression pattern of Hex was similar to that of SMemb/NMHC-B, a nonmuscle isoform of myosin heavy chain that we have previously reported to be a molecular marker of dedifferentiated VSMCs. We next examined the role of Hex in SMemb gene transcription. Promoter analysis demonstrated that the sequence identical to consensus cAMP-responsive element (CRE) located at -481 of the SMemb promoter was critical for Hex responsiveness. Mutant Hex expression vector, which lacks the homeodomain, failed to stimulate SMemb gene transcription, suggesting the requirement of the homeodomain for its transactivation. Elecrophoretic mobility shift assay showed that Hex binds to a consensus binding sequence for homeobox proteins, but not to CRE. Cotransfection of protein kinase A expression vector increased the ability of Hex to stimulate SMemb promoter activity in a CRE-dependent manner. Overexpression of CRE binding protein (CREB), but not Mut-CREB which contains mutation at Ser133, strongly activated Hex-induced SMemb promoter activity. These results suggest that Hex mediates transcriptional induction of the SMemb/NMHC-B gene via its homeodomain, and Hex can function as a transcriptional modulator of CRE-dependent transcription in VSMCs.
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PMID:Homeobox protein Hex induces SMemb/nonmuscle myosin heavy chain-B gene expression through the cAMP-responsive element. 1113 66

In the present study, we examined whether prostaglandin (PG) E2 and PGI2 regulated intercellular adhesion molecule-1 (ICAM-1) expression in human oral gingival epithelial cells stimulated with tumor necrosis factor alpha (TNF alpha). TNF alpha potently induced ICAM-1 expression in a dose- and time-dependent fashion. PGE2 and carbacyclin (a stable analogue of PGI2) significantly decreased ICAM-1 expression in TNF alpha-challenged oral gingival epithelial cells. Next, of the four subtypes of PGE2 receptors (EP1, EP2, EP3 and EP4), we examined which subtype(s) mediated inhibition of TNF alpha-induced ICAM-1 expression by PGE2. 11-deoxy-PGE2, an EP2/EP4 agonist, significantly suppressed TNF alpha-induced ICAM-1 expression, whereas butaprost, an EP2 agonist, sulprostone, an EP1/EP3 agonist, and ONO-AP-324, an EP3 agonist, caused no effect on it. By reverse transcriptase-polymerase chain reaction, expression of EP4 mRNA was detected in oral gingival epithelial cells. Dibutyryl cAMP, a cAMP analogue, and forskolin, a direct activator of adenylate cyclase, significantly inhibited TNF alpha-induced ICAM-1 expression in oral gingival epithelial cells. From these results, we suggest that PGE2 and PGI2 inhibit TNF alpha-elicited ICAM-1 expression by cAMP-dependent pathways via EP4 receptors and IP receptors, respectively.
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PMID:Prostaglandins E2 and I2 downregulate tumor necrosis factor alpha-induced intercellular adhesion molecule-1 expression in human oral gingival epithelial cells. 1115 20


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