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)

Obelin mRNA obtained in vitro with the aid of SP6 RNA polymerase was translated in a wheat germ cell-free system. Only the polypeptide with a molecular mass of about 20 kDa was synthesized. The activation of apoobelin with a synthetic coelenterazine revealed a luminescence activity initiated by calcium. The specific activity was 3.6 +/- 0.4 x 10(15)photons per mg of the in vitro synthesized obelin (k=6.9s(-1)). The luminescence of the obelin was in a good correlation with the protein concentration calculated by the incorporation of [14C]Leu. The determination of the amount of de novo synthesized obelin based on measurement of its luminescence is one-thousand times more sensitive than the approach based on the incorporation of labeled amino acid. Thus, obelin mRNA has some advantages for evaluating the efficiency of cell-free translation when compared with standard methods.
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PMID:Obelin mRNA--a new tool for studies of translation in cell-free systems. 867 17

1. The biological activities of the proteinase-activated receptor number 2 (PAR-2)-derived peptides, SLIGRL (PP6) SLIGRL-NH2 (PP6-NH2) and SLIGR-NH2 (PP5-NH2) were measured in mouse and rat gastric longitudinal muscle (LM) tissue and in a rat aortic ring preparation and the actions of the PAR-2-derived peptides were compared with trypsin and with the actions of the thrombin receptor activating peptide, SFLLR-NH2 (TP5-NH2). 2. From a neonatal rat intestinal cDNA library, and from intestinal and kidney-derived cDNA, the coding region of the rat PAR-2 receptor was cloned and sequenced, thereby establishing its close sequence identity with the previously described mouse PAR-2 receptor; and this information, along with a reverse-transcriptase (RT) polymerase chain reaction (PCR) analysis of cDNA derived from gastric and aortic tissue was used to establish the concurrent presence of PAR-2 and thrombin receptor mRNA in both tissues. 3. In the mouse and rat gastric preparations, the PAR-2-derived polypeptides, PP6, PP6-HN2 and PP5-NH2 caused contractile responses that mimicked the contractile actions of low concentrations of trypsin (5 u/ml-1; 10 nM) and that were equivalent to contractions caused by TP5-NH2. 4. The cumulative exposure of the rat LM tissue to PP6-NH2 led to a desensitization of the contractile response to this polypeptide, but not to TP5-NH2 and vice versa, so as to indicate a lack of cross-desensitization between the receptors responsive to the PAR-2 and thrombin receptor-derived peptides. 5. In the rat gastric preparation, the potencies of the PAR-2-activating peptides were lower than the potency of TP5-NH2 (potency order: TP5-NH2 > > PP6-NH2 > or = PP6 > PP5-NH2); PP6 was a partial agonist in this preparation. 6. The contractile actions of PP6 and PP6-NH2 in the rat gastric preparation required the presence of extracellular calcium, were inhibited by nifedipine and were blocked by the cyclo-oxygenase inhibitor, indomethacin and by the tyrosine kinase inhibitor, genistein, but not by the kinase C inhibitor, GF109203X. The contractile responses were not blocked by atropine, chlorpheniramine, phenoxybenzamine, propranolol, ritanserin or tetrodotoxin. 7. In a precontracted rat aortic ring preparation, with an intact endothelium, all of the PAR-2-derived peptides caused a prompt relaxation response that was blocked by the nitric oxide synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME) but not by D-NAME; in an endothelium-free preparation, which possessed mRNA for both the PAR-2 and thrombin receptors, the PAR-2-activating peptides caused neither a relaxation nor a contraction, in contrast with the contractile action of TP5-NH2. The relaxation response to PP6-NH2 was not blocked by atropine, chlorpheniramine, genistein, indomethacin, propranolol or ritanserin. 8. In the rat aortic preparation, the potencies of PP6, PP6-NH2 and PP5-NH2 were greater than those of the thrombin receptor activating peptide, TP5-NH2 (potency order: PP6-NH2 > or = PP6 > PP5-NH2 > TP5-NH2). 9. In the rat aortic preparation, the relaxant actions of the PAR-2-derived peptides were mimicked by trypsin, at concentrations (0.5-1 u ml-1; 1-2 nM) lower than those that can activate the thrombin receptor. 10. The bioassay data obtained with the PAR-2 peptides and with trypsin, along with the molecular cloning/RT-PCR analysis, point to the presence of functional PAR-2 receptors that can activate distinct responses in the gastric and vascular smooth muscle preparations. These responses were comparable to those resulting from thrombin receptor activation in the same tissues, so as to suggest that the receptor for the PAR-2-activating peptides may play a physiological role as far reaching as the one proposed for the thrombin receptor.
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PMID:Rat proteinase-activated receptor-2 (PAR-2): cDNA sequence and activity of receptor-derived peptides in gastric and vascular tissue. 876 73

Muscarinic receptor subtypes in the bovine corneal epithelial cells (BCE) were characterized on the basis of their: 1) ligand binding properties, 2) linkage to Ca2+ and cAMP cell signaling pathways, and 3) gene transcripts. Receptor subtypes, m1 and m2, are indicated by competition experiments using subtype-selective muscarinic receptor ligands. [3H]N-methylscopolamine ([3H]-MS) binding was displaced with IC50s of: 1) 1 microM for the m1 antagonist, pirenzipine; 2) 51 microM for the competitive m2 antagonist, AFDX-116; 3) 100 microM for the competitive m3 antagonist, 4-DAMP. In fural2 loaded BCE, carbachol (0.001 - 100 microM) increased intracellular Ca2+ concentration ([Ca2+]i), and these responses were significantly suppressed if they were preincubated with either atropine (1 microM) or 1 microM pirenzipine. In the absence of extracellular Ca2+, these carbachol-induced increases in [Ca2+]i were depressed. A considerable fall occurred with the presence of extracellular Ca2+ and 1 microM verapamil, an L-type Ca2+ channel blocker. These responses suggest that carbachol increases Ca2+ influx through an L-type Ca2+ channel in the plasma membrane, in addition to mobilizing Ca2+ from an intracellular store. BCE also possessed muscarinic receptors which were negatively linked to cAMP production insofar as: 1) preincubation with 10 microM carbachol significantly suppressed the increases in cAMP accumulation induced by isoproterenol (1 - 25 microM); 2) this blunting effect of carbachol on cAMP production was eliminated when the BCE were preincubated with either 1 microM AFDX-116, or 100 ng/ml pertussis toxin. The results of probing for muscarinic receptor gene expression are partially consistent with the ligand binding and functional assays. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of m2 but not m1, m3 or m4 gene transcripts. In summary, we obtained pharmacological and functional evidence for m1 and m2 receptors in BCE. However, only the m2 gene transcript could be detected.
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PMID:Characterization of the muscarinic receptor subtypes in the bovine corneal epithelial cells. 887 32

The overall goal of this study was to provide data on the function and physiologic significance of lens calpain I, a cysteine protease requiring low amounts of calcium for activation. Reverse-transcriptase polymerase chain reaction was used to detect mRNAs for calpains I and II in young rat lenses. An in vitro model of crystallin precipitation was used to assess the ability of calpain I to induce hydrolysis and precipitation of crystallins. We found that incubation of crystallins with purified calpain I was indeed a powerful inducer of crystallin precipitation. However, mRNA levels for calpain I in whole lens appeared to be lower compared to calpain-II mRNA. Participation of calpain I in crystallin precipitation during normal maturation of rodent lenses or cataract formation is thus theoretically possible, but unlikely, because of the low level of expression of calpain I.
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PMID:In vitro precipitation of rat lens crystallins by calpain I--a calpain requiring low amounts of calcium for activation. 888 97

The expression of muscarinic acetylcholine receptor (mAChR) subtypes in freshly isolated adult rat ventricular myocytes was investigated by reverse transcription of cellular mRNA followed by amplification of cDNA using the polymerase chain reaction (PCR). After reverse-transcriptase PCR, bands were obtained corresponding to the expected sizes for the m1 and m2 but not for the m3 to m5 mAChRs. The identity of the m1 and m2 bands was confirmed by single-cell PCR, restriction digest mapping, and Southern blot analysis. The presence of m1 and m2, but not m3, mAChR protein in these cells was shown by indirect immunofluorescence studies using subtype-specific antibodies. It was further investigated whether the identified m1 mAChR was responsible for the stimulatory effects on Ca2+ transients by high concentrations of carbachol ( > 10 mumol/L) known to occur in these cells. In pertussis toxin-treated ventricular myocytes electrically stimulated at 1 Hz, carbachol (300 mumol/L) increased the basal Ca2+ level from 96 +/- 7 to 118 +/- 8 nmol/L and the peak Ca2+ transient level from 519 +/- 32 to 640 +/- 36 nmol/L (mean +/- SEM P < .05 for both, n = 8). These effects of carbachol on Ca2+ transients were antagonized by 10 nmol/L pirenzepine, an m1 mAChR-selective antagonist. In contrast, the m2 mAChR-selective antagonist methoctramine (up to 100 nmol/L) did not inhibit the response. These results are the first to use single-cell PCR to probe cardiomyocyte-specific gene expression and indicate that m1 mAChRs are expressed on adult rat ventricular myocytes in addition to m2 mAChRs. The results further suggest that m1 mAChRs mediate the stimulatory responses on Ca2+ transients to high concentrations of cholinergic agonists seen in these cells.
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PMID:Molecular and functional identification of m1 muscarinic acetylcholine receptors in rat ventricular myocytes. 892 73

An oligonucleotide probe to a conserved 3' region within the three identified ryanodine receptor-calcium release channel isoforms hybridized to a single clone from a rabbit kidney cDNA library. The kidney clone encoded the carboxyl-terminal 338 amino acids within the putative transmembrane domain of the type 2 ryanodine receptor sequence. Reverse transcriptase-polymerase chain reaction with isoform-specific oligonucleotide primers demonstrated the presence of the type 2 ryanodine receptor transcript in rabbit kidney, as well as in a non-excitable cell line, LLC-RK1, derived from rabbit kidney epithelial cells. Amplification by rapid amplification of 5' cDNA ends indicated the kidney type 2 ryanodine receptor transcript extended >7000 base pairs from the stop codon and is therefore not homologous to the short RyR-1 transcript of approximately 2500 base pairs previously observed in rabbit brain. [3H]Ryanodine binding and immunoblot analysis with a type 2 ryanodine receptor-specific antibody demonstrated that the native type 2 ryanodine receptor protein is expressed in the kidney. These observations suggest that the type 2 ryanodine receptor isoform may play a functional role in regulating intracellular calcium homeostasis in non-excitable cells.
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PMID:Ryanodine receptor expression in the kidney and a non-excitable kidney epithelial cell. 893 87

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel neuropeptide that produces its biological effects by interacting with G protein-coupled receptors. Molecular cloning of the PACAP receptor revealed the existence of five splice variant receptor forms differing in the third intracellular loop region, with four variants activating both adenylyl cyclase and phosphoinositide phospholipase C and one variant activating only adenylyl cyclase (Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H., and Journot, L. (1993) Nature 365, 170-175). Here, we report cloning of a novel PACAP receptor variant, designated PACAPR TM4 (transmembrane domain IV), that differs from the previously cloned short form of the PACAP receptor (PACAPR) primarily by discrete sequences located in transmembrane domains II and IV. Reverse transcriptase-polymerase chain reaction and primer extension analyses demonstrated tissue-specific differential expression of mRNAs encoding PACAPR TM4 and splice variant forms of the PACAP receptor. PACAPR TM4 and PACAPR possess identical intracellular domains, implicated as primary determinants of G protein recognition by rhodopsin-like receptors. However, unlike the PACAPR, PACAPR TM4 does not activate either adenylyl cyclase or phosphoinositide phospholipase C in response to PACAP in either transient or stable expression systems. However, PACAP stimulates increases in [Ca2+]i in cells expressing PACAPR TM4 by activating L-type Ca2+ channels, a response not elicited by stimulation with vasoactive intestinal polypeptide. The signaling phenotype of PACAPR TM4 is characteristic of the PACAP receptor involved in regulation of insulin secretion from pancreatic beta islets, a tissue expressing transcripts for PACAPR TM4 but not for PACAPR or its longer splice variant forms. These findings are consistent with a role of PACAPR TM4 in the physiological control of insulin release by PACAP in beta-islet cells. The finding that PACAPR TM4 has a unique signaling phenotype, although it possesses intracellular domains identical to those of the PACAPR, suggests that receptor-G protein recognition by rhodopsin-like receptors can be determined by sequences other than those located in intracellular receptor domains.
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PMID:Molecular cloning of a novel variant of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor that stimulates calcium influx by activation of L-type calcium channels. 894 80

Cholinergic agonists stimulate isotonic fluid secretion in the parotid gland. This process is driven by the apical exit of Cl-, which enters the cells partly via Cl-/HCO-3 exchange across the basolateral membrane. Acidification of the cytosol by the extrusion of HCO-3 is prevented by the concomitant activation of the Na+/H+ exchanger (NHE), which is directly activated by cholinergic stimulation. Multiple isoforms of the NHE have been described in mammalian cells, but the particular isoform(s) present in salivary glands and their mechanism of activation have not been defined. Reverse transcriptase-polymerase chain reaction with isoform-specific primers was used to establish that NHE-1 and NHE-2, but not NHE-3 or NHE-4, are expressed in parotid glands. The presence of NHE-1 was confirmed by immunoblotting and immunofluorescence, which additionally demonstrated that this isoform is abundant in the basolateral membrane of acinar cells. The predominant role of NHE-1 in carbachol-induced Na+/H+ exchange was established pharmacologically using HOE694, an inhibitor with differential potency toward the individual isoforms. Because muscarinic agonists induce stimulation of protein kinases in acinar cells, we assessed the role of phosphorylation in the activation of the antiport. Immunoprecipitation experiments revealed that, although NHE-1 was phosphorylated in the resting state, no further phosphorylation occurred upon treatment with carbachol. Similar phosphopeptide patterns were observed in control and carbachol-treated samples. Together, these findings indicate that NHE-1, the predominant isoform of the antiporter in the basolateral membrane of acinar cells, is activated during muscarinic stimulation by a phosphorylation-independent event. Other processes, such as association of Ca2+-calmodulin complexes to the cytosolic domain of the antiporter, may be responsible for the activation of Na+/H+ exchange.
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PMID:Muscarinic agonists induce phosphorylation-independent activation of the NHE-1 isoform of the Na+/H+ antiporter in salivary acinar cells. 899 60

Chinese hamster ovary cells (CHO cells) do not exhibit any Ca2+/calmodulin-stimulated cAMP phosphodiesterase (PDE1) activity. Challenge of CHO cells with agonists for endogenous P2-purinoceptors, lysophosphatidic acid receptors and thrombin receptors caused a similar rapid transient induction of PDE1 activity in each instance. This was also evident on noradrenaline challenge of a cloned CHO cell line transfected so as to overexpress alpha 1B-adrenoceptors. This novel PDE1 activity appeared within about 15 min of exposure to ligands, rose to a maximum value within 30 min to 1 h and then rapidly decreased. In each case, the expression of novel PDE1 activity was blocked by the transcriptional inhibitor actinomycin D. Challenge with insulin of either native CHO cells or a CHO cell line transfected so as to overexpress the human insulin receptor failed to induce PDE1 activity. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1C isoform, did not amplify any fragment from RNA preparations of CHO cells expressing PDE1 activity, although they did so from the human thyroid carcinoma FTC133 cell line. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1A and PDE1B isoforms, successfully amplified a fragment of the predicted size from RNA preparations of both CHO cells expressing PDE1 activity and human Jurkat T-cells. Sequencing of the PCR products, generated using the PDE1A/B primers, yielded a novel sequence which, by analogy with sequences reported for bovine and murine PDE1B forms, suggests that the PDE1 species induced in CHO cells through protein kinase C activation and that expressed in Jurkat T-cells are PDE1B forms.
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PMID:Receptor-mediated stimulation of lipid signalling pathways in CHO cells elicits the rapid transient induction of the PDE1B isoform of Ca2+/calmodulin-stimulated cAMP phosphodiesterase. 900 15

Cells of two human follicular thyroid carcinoma cell lines (FTC133, FTC236) were stably transfected with a cDNA encoding the PDE4A cAMP-specific phosphodiesterase (PDE) splice variant RD1 (RNPDE4A1A) so as to generate the cloned cell lines, FTC133A and FTC236A. This allowed the expression of a novel rolipram-inhibited cAMP-specific PDE activity in these cells. Unlike the parent cell lines in which Ca2+/calmodulin caused a profound activation (approx. 3-4-fold) of homogenate PDE activity, no such stimulation was evident in the RD1-expressing cell lines, indicating loss of PDE1 activity. Reverse transcriptase-PCR analysis indicated that this was due to the down-regulation of the PDE1C isoform. The novel PDE4 activity in transfected cells was located exclusively in the membrane fraction, as was immunoreactive RD1. Low concentrations of the detergent Triton X-100, but not high NaCl concentrations, allowed RD1 to be solubilized. Laser scanning confocal immunofluorescence analyses identified RD1 immunoreactivity in a discrete perinuclear region of these RD1-expressing transfected cell lines. A similar pattern of labelling was observed using the antiserum Tex1, which specifically identified the Golgi apparatus. Treatment of FTC133A cells with the Golgi-perturbing agents monensin and brefeldin A led to a similar redistribution of immunoreactive species detected using both the Tex1 and anti-RD1 antisera. It is suggested that the PDE4A splice variant RD1 contains a membrane-association signal which allows the targeted expression of RD1 within the Golgi complex of these human follicular thyroid carcinoma cell lines.
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PMID:Intracellular localization of the PDE4A cAMP-specific phosphodiesterase splice variant RD1 (RNPDE4A1A) in stably transfected human thyroid carcinoma FTC cell lines. 900 17


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