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)

We developed microtiter well-based bioluminescence hybridization assays using the photoprotein aequorin as a reporter molecule. The target DNA was hybridized simultaneously with a capture probe and a detection probe. The capture probe was immobilized on the wells through digoxigenin/anti-digoxigenin interaction. The detection probe was biotinylated. The hybrids were determined by using aequorin covalently attached to streptavidin or complexes of biotinylated aequorin with streptavidin. The luminescence was then measured in the presence of excess Ca2+. The optimized protocols showed linearity in the range from 5 amol to 10 fmol of target DNA. In combination with reverse transcriptase polymerase chain reaction, the proposed assay was applied to the detection of the mRNA for prostate-specific antigen (PSA). PSA mRNA from a single cell, in the presence of one million cells that do not express PSA, was detected with a signal-to-background ratio of 2.5. Typical CVs obtained were 6%.
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PMID:Bioluminescence hybridization assays using recombinant aequorin. Application to the detection of prostate-specific antigen mRNA. 886 62

Previous studies have indicated the expression of multiple P2Y receptors by rat hepatocytes although they have not been identified. Here we show by reverse transcriptase-polymerase chain reaction (RT - PCR) that rat hepatocytes express mRNA encoding all of the four cloned rat P2Y receptors (P2Y(1), P2Y(2), P2Y(4) and P2Y(6)). The effects of UTP have been examined on single aequorin-injected rat hepatocytes. The [Ca(2+)](i) transients induced by UTP were indistinguishable from those induced by ATP in the same cell. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both UTP- and ATP-induced [Ca(2+)](i) transients. UDP, an agonist at the P2Y(6) receptor, failed to induce transients in hepatocytes, indicating that functional P2Y(6) receptors coupled to increased [Ca(2+)](i) are not expressed. The transients evoked by ADP were more sensitive to inhibition by suramin than those induced by either ATP or UTP. Within an individual cell, the transients induced by ATP and UTP were inhibited by the same concentration of suramin. This sensitivity of ATP and UTP responses to suramin suggests action through P2Y(2) rather than P2Y(4) receptors. Co-application of 30 microM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) caused a decrease in frequency and amplitude of transients induced by ADP. ATP- and UTP-induced transients also displayed a decrease in amplitude in response to addition of PPADS, but this was accompanied by an increase in frequency of transients. In conclusion the data presented here are consistent with the co-expression of P2Y(1) and P2Y(2) receptors by rat hepatocytes.
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PMID:Evidence that rat hepatocytes co-express functional P2Y1 and P2Y2 receptors. 1068 1

Five high affinity G-protein-coupled receptors for sphingosine 1-phosphate (S1P) have been characterised so far (S1P(1,2,3,4,5) formerly referred to as edg1,5,3,6,8). In this study, we show that S1P, dihydro-sphingosine 1-phosphate (dihydro-S1P) and dioleoylphosphatidic acid (doPA) are agonists for the orphan receptor GPR63. All three phospholipids mobilise intracellular calcium in CHO cells transiently transfected with GPR63. Calcium signals required cotransfection of a chimeric Galpha(q/i) protein in a fluorometric imaging plate reader (FLIPR) assay but did not require overexpressed G proteins in an aequorin assay, using a green fluorescent protein (GFP)-aequorin fusion protein as a bioluminescent Ca(2+) reporter. GPR63 expression in CHO cells confers proliferative responses to S1P in a pertussis toxin (PTX)-insensitive manner. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) indicated highest expression in brain, especially in the thalamus and the nucleus caudatus. In peripheral tissues, highest expression was observed in thymus, stomach and small intestine; lower abundance of transcripts was detected in kidney, spleen, pancreas and heart. The discovery that S1P, dihydro-S1P and dioleoylphosphatidic acid activate GPR63 will facilitate the identification of agonists and antagonists, and help to unravel the biological function of this receptor.
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PMID:Sphingosine 1-phosphate and dioleoylphosphatidic acid are low affinity agonists for the orphan receptor GPR63. 1261 18