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 aim was to explore whether biliary epithelial cells show muscarinic acetylcholine receptors and to investigate their role in ductular bile formation. In both, isolated rat biliary epithelial cells and Mz-Cha-1 cells, a biliary epithelial cell line, binding of [3H]N-methyl-scopolamine occurred with 0.718 +/- 0.08 and 0.482 +/- 0.05 fmol per 10(6) cells, respectively. To characterize the involved second messenger, intracellular Ca2+ levels were monitored by confocal microscopy. Stimulation of biliary epithelial cells with carbachol produced an increase in free cytosolic Ca2+ levels that declined to baseline values describing a sinusoidal oscillation curve. Increasing concentrations of the agonist decreased latency of the response and increased oscillation frequency. Similar results were obtained in Mz-Cha-1 cells. The intracellular Ca2+ originated from IP3 sensitive intracellular stores and from the extracellular medium. The Ca2+ response could partially be blocked by atropine and completely by pirenzepine, a specific muscarinic receptor-type M1 antagonist. The presence of M1 receptor messenger RNA (mRNA) in biliary epithelial cells was confirmed by reverse transcriptase polymerase chain reaction. In the isolated perfused guinea pig liver, a model with high ductular bile flow, carbachol induced a dose dependent decrease of bile flow by 79.6% +/- 9.8% at 50 mumol/L carbachol (P < .001), without affecting perfusion pressure or biliary electrolyte concentrations. It is concluded that biliary epithelial cells express muscarinic acetylcholine receptors. Stimulation of this receptor leads to cholestasis. This could be because of changes in peribiliary permeability and/or inhibition of biliary epithelial cell secretory function.
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PMID:Muscarinic acetylcholine receptor stimulation of biliary epithelial cells and its effect on bile secretion in the isolated perfused liver [corrected]. 909 80

Inspection of the intron splice junction of the mouse chemokine receptor fusin/CXC chemokine receptor R-4 (CXCR-4) revealed a potential in-frame alternative splice site in the region encoding the N-terminal ectodomain of the receptor. Both predicted splice products were detected by reverse transcriptase-PCR. Cell lines of T lymphocyte, B lymphocyte, and macrophage lineage, plus populations of elicited peritoneal exudate cells, thymocytes, and astrocytes coexpressed mRNA for both isoforms. The full length cDNA of the shorter alternate splice product, termed CXCR-4B, was cloned from peritoneal exudate cell RNA. Chinese hamster ovary cells transfected with either isoform, CXCR-4A or CXCR-4B, responded to stromal cell-derived factor-1alpha with a rise in intracellular calcium. Both alternate splice products are therefore functional stromal cell-derived factor-1 alpha receptors.
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PMID:Alternate splicing of mouse fusin/CXC chemokine receptor-4: stromal cell-derived factor-1alpha is a ligand for both CXC chemokine receptor-4 isoforms. 910 15

NIH-3T3 cells, a nontransformed murine fibroblast cell line previously found to be unresponsive to 5-hydroxytryptamine (5-HT) when cultured in 5-HT-free medium, became responsive to 5-HT, which induced an increase in intracellular calcium concentration. Pharmacological and ligand binding studies showed that NIH-3T3 cells endogenously express a 5-HT2A receptor that, when activated, mobilizes calcium from ionomycin-sensitive intracellular stores via coupling to a pertussis toxin-insensitive pathway. Using reverse transcriptase-PCR cloning and northern blot analysis, the presence of 5-HT2A receptor RNA with a similar nucleotide sequence (99% identity) and molecular size to that of murine brain was detected in NIH-3T3 cells. Responsiveness of the endogenous 5-HT2A receptor in nontransfected cells was completely desensitized after chronic treatment (half-time = 2 h) with 1 microM 5-HT and resensitized on removal of 5-HT. In contrast to NIH-3T3 cells transfected with 5-HT2A receptor cDNA under control of a viral promoter, the long-term agonist-induced functional desensitization in nontransfected NIH-3T3 cells was paralleled by a decrease in both 5-HT2A receptor density and RNA level. These results show that NIH-3T3 cells express an endogenous 5-HT2A receptor that is desensitized by agonist via down-regulation of both receptor number and mRNA. The NIH-3T3 cells provide a novel system for understanding 5-HT2A receptor regulation.
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PMID:Identification of an endogenous 5-hydroxytryptamine2A receptor in NIH-3T3 cells: agonist-induced down-regulation involves decreases in receptor RNA and number. 910 26

Genomic DNA sequencing in the vicinity of the pstA-1 gene from Mycobacterium tuberculosis allowed us to clone, sequence and identify a gene encoding a 70-kDa protein. The size of the protein was confirmed by in vitro coupled transcription/translation. Its N-terminal domain shows extensive sequence similarity with the catalytic domain of eukaryotic serine/threonine protein kinases, and the protein was therefore called Mbk (mycobacterial protein kinase). The deduced amino acid sequence contains two transmembrane segments, which flank a highly repetitive region, suggesting a receptor-like anchoring. The mbk gene was overexpressed in Escherichia coli and the gene product (Mbk) was purified as a fusion protein with gluthatione S-transferase. Recombinant Mbk was found to be autophosphorylated on threonine residues and capable of phosphorylating myelin basic proteins from bovine brain and histones from calf thymus on serine residues, both in a manganese-dependent manner. The phosphorylation of myelin basic proteins by Mbk was inhibited by calcium and by staurosporine, a widely used inhibitor of eukaryotic protein serine/threonine kinases. A similar gene was found in Mycobacterium bovis BCG DNA by Southern blot analysis. Its expression was detected in cultures of M. bovis BCG by reverse transcriptase/PCR. Although its biological role is unknown, it is the first serine/threonine protein kinase characterized in Mycobacteria.
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PMID:A serine/threonine protein kinase from Mycobacterium tuberculosis. 911 30

1. We have investigated the mechanism by which L-arginine stimulates membrane depolarization, an increase of intracellular calcium ([Ca2+]i) and insulin secretion in pancreatic beta-cells. 2. L-Arginine failed to affect beta-cell metabolism, as monitored by NAD(P)H autofluorescence. 3. L-Arginine produced a dose-dependent increase in [Ca2+]i, which was dependent on membrane depolarization and extracellular calcium. 4. The cationic amino acids L-ornithine, L-lysine, L-homoarginine (which is not metabolized) and NG-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor) produced [Ca2+]i responses similar to that produced by L-arginine. The neutral nitric oxide synthase inhibitors NG-nitro-L-arginine (L-NNA) and N omega-monomethyl-L-arginine (L-NAME) also increased [Ca2+]i. D-Arginine was ineffective. 5. L-Arginine did not affect whole-cell Ca2+ currents or ATP-sensitive K+ currents, but produced an inward current that was carried by the amino acid. 6. The reverse transcriptase-polymerase chain reaction demonstrated the presence of messenger RNA for the murine cationic amino acid transporters mCAT2A and mCAT2B within the beta-cell. 7. L-Arginine did not affect beta-cell exocytosis as assayed by changes in cell capacitance. 8. Our data suggest that L-arginine elevates [Ca2+]i and stimulates insulin secretion as a consequence of its electrogenic transport into the beta-cell. This uptake is mediated by the mCAT2A transporter.
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PMID:Electrogenic arginine transport mediates stimulus-secretion coupling in mouse pancreatic beta-cells. 913 Jan 59

We recently identified a developmental decline in protein kinase C (PKC) isoform expression, at the level of the protein, in rat ventricular myocardium. To investigate mechanisms regulating PKC isoform expression in cardiac tissue, this study uses Northern blot analysis to compare the abundance of PKC isoform mRNAs in neonatal and adult rat ventricular myocardium. PKC-epsilon protein and mRNA were detected in both neonatal and adult rat ventricular myocardial preparations. In contrast, coordinate postnatal declines in the abundance of PKC-alpha and PKC-delta proteins and transcripts were identified. An antiserum raised against the COOH-terminal sequence of PKC-zeta detected abundant immunoreactivity in neonatal, but not adult, ventricular myocytes. However, PKC-zeta transcripts were not detectable in the heart either by Northern blot analysis or a reverse transcriptase-polymerase chain reaction approach, indicating that neither the myocytes nor the contaminating cellular elements in the heart express PKC-zeta. Rather, PKC-lambda, another atypical PKC isoform that is structurally highly homologous to PKC-zeta, was detected at the protein and mRNA level in neonatal, but not adult, ventricular myocardium. Taken together, these results establish that developmental declines in calcium-sensitive, novel, and atypical PKC isoforms are paralleled by changes in the levels of the mRNAs encoding these proteins, suggesting transcriptional regulation of PKC during normal cardiac development. The results of this study further identify PKC-lambda as the atypical PKC isoform expressed by the immature ventricle.
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PMID:PKC-lambda is the atypical protein kinase C isoform expressed by immature ventricle. 913 45

Expression of calcium/calmodulin-activated adenylyl cyclase type I (ACI) mRNA has been determined in the cochlea and in an organ-of-Corti subdissected tissue fraction by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Amplification products of predicted size were obtained from the mouse cochlea and rat organ of Corti with nucleotide sequences corresponding to respective ACI brain transcripts. In addition, ACI template was detected in a rat inner hair cell cDNA library by PCR. Immunoreactivity to ACI has been localized within the organ of Corti to the inner hair cell, with diaminobenzidine staining found in both the cell body and in the stereocilia. Evidence, thus, has been obtained that both ACI transcript and protein are expressed in the inner hair cell, the primary mechanosensory receptor cell of the cochlea. We hypothesize that ACI is activated by calcium influx through a calcium/calmodulin interaction and that this adenytyl cyclase isoform may have a role in modulation of receptoneural afferent transmission and/or mechanosensory transduction in the cochlea.
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PMID:Expression of adenylyl cyclase type I in cochlear inner hair cells. 914 8

The rat brain alpha1A calcium channel clone has been expressed in COS-7 cells together with the neuronal accessory subunits beta1b and alpha2-delta. From reverse transcriptase polymerase chain reaction (RT-PCR), immunocytochemistry and electrophysiology experiments, we have obtained no evidence that these cells contain any endogenous calcium channels. Transfected cells were identified by co-expression of a cDNA for the reporter Green Fluorescent Protein. From immunocytochemical evidence, a high degree of co-expression was obtained between Green Fluorescent Protein and individual calcium channel subunits. When all three calcium channel subunits (alpha1, alpha2-delta and beta1b) were co-expressed, evidence was obtained that all subunits were present at the cell membrane. Voltage-dependent calcium currents were observed between 24 and 72 h after transfection with the three calcium channel subunits. The current density for the combination alpha1A/alpha2-delta/beta1b was 4.19 +/- 0.69 pA.pF(-1) and the current produced was slowly inactivating. The time constant of inactivation of the maximum I(Ba) was 332 +/- 46 ms (n = 5). The voltage-dependence of activation and steady-state inactivation had voltages of half activation and inactivation of 9.5 +/- 2.5 mV and -30.4 +/- 1.5 mV respectively, and there was little overlap between the two curves. The alpha1A current was completely blocked by 100 microM Cd2+ and was also blocked by omega-conotoxin MVIIC (500 nM). Dose-inhibition curves and analysis of k(on) and k(off) for omega-agatoxin IVA both revealed apparent K(D) values of approximately 11 nM for alpha1A currents, with a k(on) of 7.8 x 10(4) M(-1).s(-1). The results suggest that alpha1A expressed in these cells has some resemblance to the P type component of calcium current observed in native neurons, although it shows a somewhat greater degree of inactivation than native P current, more similar to the Q type current component. It also has an affinity for omega-agatoxin IVA 2-5 fold lower than reported for P current, but approximately 9-fold higher than reported for Q current.
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PMID:Properties of cloned rat alpha1A calcium channels transiently expressed in the COS-7 cell line. 915 80

Vascular smooth muscle cells (VSMC) contribute to the pathophysiology of hypertension through cell growth and contraction, and phospholipase C (PLC) is a critical effector enzyme in growth factor and vasoconstrictor signaling. There is indirect evidence that angiotensin II (ANG II) receptors are linked to the PLC-beta isoform signaling pathways. However, recent studies suggest that PLC-beta isoforms may not be expressed in VSMC. Our data demonstrate that in human aortic VSMC, PLC-beta 1 and PLC-gamma 1 proteins were detected by immunoblot analysis, and PLC-beta 1 mRNA was identified by reverse transcriptase-polymerase chain reaction in rat aortic VSMC. Incubation of permeabilized VSMC with anti-PLC-beta 1 or anti-Gq alpha antibodies inhibited ANG II-dependent inositol polyphosphate (IP) formation, while anti-PLC-gamma 1 antibodies did not inhibit ANG II-regulated IP formation. Conversely, anti-PLC-gamma 1 antibodies completely abolished platelet-derived growth factor (PDGF)-dependent IP generation, whereas anti-PLC-beta 1 antibodies had no effect on PDGF-induced PLC activation. Inhibition of tyrosine phosphorylation with genistein or herbimycin A did not diminish ANG II-stimulated IP formation or cytosolic free Ca2+ concentration transients, thereby confirming that ANG II signals via a PLC-gamma 1-independent mechanism. In summary, PLC-beta 1 and PLC-gamma 1 are expressed in human aortic VSMC, and PLC-beta 1 is the isoform that is critical for ANG II-regulated PLC signaling in these cells.
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PMID:Angiotensin II activates the beta 1 isoform of phospholipase C in vascular smooth muscle cells. 917 47

Large conductance, calcium-activated (BK) potassium channels play a central role in the excitability of cochlear hair cells. In mammalian brains, one class of these channels, termed Slo, is encoded by homologues of the Drosophila 'slowpoke' gene. By homology screening with mouse Sla cDNA, we have isolated a full-length clone (cSlo1) from a chick's cochlear cDNA library, rSlol had greater than 90% identity with mouse Slo at the amino acid level, and was even better matched to a human brain Slo at the amino and carboxy termini. cSlol had none of the additional exons found in splice variants from mammalian brain. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to show expression of cSlal in the microdissected hair cell epithelium basilar papilla. Transient transfection of HIEK 293 cells demonstrated that cSlol encoded a potassium channel whose conductance averaged 224 pS at +60 mV in symmetrical 140 mM K. Macroscopic currents through cSlol channels were blocked by scorpion toxin or tetraethyl ammonium, and were voltage and calcium dependent. cSlol is likely to encode BK-type calcium-activated potassium channels in cochlear hair cells.
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PMID:CSlo encodes calcium-activated potassium channels in the chick's cochlea. 917 44


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