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Isolated cDNAs (isolated + cdna)
Selected AbstractsExpression of multiple P2Y receptors by MDCK-D1 cells: P2Y1 receptor cloning and signalingDRUG DEVELOPMENT RESEARCH, Issue 1 2003Richard J. Hughes The Madin Darby canine kidney (MDCK) cell line, a well-differentiated renal epithelial cell line, is a useful model to examine P2Y receptor signaling and response. Our studies with MDCK-D1, a clonal isolate, demonstrate that these cells release ATP in response to mechanical stimulation and activation of certain G-protein-coupled receptors. Reverse transcriptase-polymerase chain reaction (RT-PCR) studies document that MDCK cells express multiple P2Y receptors, including P2Y1, P2Y2, P2Y6, and P2Y11 receptors. We isolated cDNAs for several of the P2Y receptor genes and expressed these in cells, such as the 1321N1 astrocytoma cell line, that lack native P2Y receptor expression. We report here the molecular cloning of the MDCK P2Y1 receptor, heterologous expression in 1321N1 cells, and the ability of the heterologously expressed receptors to increase intracellular calcium and phosphoinositide hydrolysis. ADP, methylthioATP, and ADP,S are agonists with the greatest potency, while ATP and ATP,S show lower potency and efficacy, and benzoylbenzoylATP, UTP, and UDP lack efficacy at the cloned P2Y1 receptor. Several antagonists, including MRS2179, A3P5PS, suramin, and PPADS blocked response at the cloned P2Y1 receptors. With their ability to respond to ADP and ATP, P2Y1 receptors, along with other P2Y receptors expressed in MDCK cells, contribute to the response of these cells to ATP (or its breakdown product, ADP) released from the cells and to exogenously added nucleotides. Drug Dev. Res. 59:1,7, 2003. © 2003 Wiley-Liss, Inc. [source] Noxp20 and Noxp70, two new markers of early neuronal differentiation, detected in teratocarcinoma-derived neuroectodermic precursor cellsJOURNAL OF NEUROCHEMISTRY, Issue 2 2006M. Boucquey Abstract The murine 1C11 cell line, derived from F9 pluripotent teratocarcinoma cells, exhibits features of a bipotential neuronal precursor as it converts into serotonergic or catecholaminergic neurons under appropriate induction. In order to point out molecular markers expressed in this early neuroectodermic commitment, we used a cDNA subtractive hybridization method. The 105 different isolated cDNAs represented 75 known genes, expressed sequence tags (EST) or genomic fragments. A majority of known proteins encoded by these sequences are involved in cellular mobility or migration. We characterized two sequences showing identities with ESTs and we called them Noxp20 and Noxp70. The Noxp20 transcript encodes a putative protein with a predicted caspase recruitment domain and the Noxp70 transcript encodes a putative protein displaying a Zn-finger domain. Consistent with their roles in neuronal cell development, in situ hybridization showed that Noxp20 and Noxp70 are over-expressed in brain. At embryonic days 12 and 15, Noxp20 is strongly expressed in the ventricular and intermediate zones of the brain and of the spinal cord. At embryonic day 15, Noxp70 was found to be strongly expressed in the ventricular zone around the telencephalic ventricle, and to a lower extent in the thalamus and hypothalamus. At post-natal day 10, Noxp20 mRNA was detected in the dentate gyrus, the hippocampus, the cerebellum and the olfactory bulb. [source] Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesisTHE PLANT JOURNAL, Issue 2 2006Jörg Ziegler Summary Plants of the order Ranunculales, especially members of the species Papaver, accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the antitussive codeine. In this study, we investigated the molecular basis for this exceptional biosynthetic capability by comparison of alkaloid profiles with gene expression profiles between 16 different Papaver species. Out of 2000 expressed sequence tags obtained from P. somniferum, 69 show increased expression in morphinan alkaloid-containing species. One of these cDNAs, exhibiting an expression pattern very similar to previously isolated cDNAs coding for enzymes in benzylisoquinoline biosynthesis, showed the highest amino acid identity to reductases in menthol biosynthesis. After overexpression, the protein encoded by this cDNA reduced the keto group of salutaridine yielding salutaridinol, an intermediate in morphine biosynthesis. The stereoisomer 7- epi -salutaridinol was not formed. Based on its similarities to a previously purified protein from P. somniferum with respect to the high substrate specificity, molecular mass and kinetic data, the recombinant protein was identified as salutaridine reductase (SalR; EC 1.1.1.248). Unlike codeinone reductase, an enzyme acting later in the pathway that catalyses the reduction of a keto group and which belongs to the family of the aldo-keto reductases, the cDNA identified in this study as SalR belongs to the family of short chain dehydrogenases/reductases and is related to reductases in monoterpene metabolism. [source] Cloning and expression of the gene for an insect haemocyte anti-aggregation protein (VPr3), from the venom of the endoparasitic wasp, Pimpla hypochondriaca,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2009M. Paulina Dani Abstract A venom protein from the endoparasitic wasp, Pimpla hypochondriaca, was recently biochemically isolated. This protein possessed haemocyte anti-aggregation activity in vitro and shares the same N-terminal amino acid sequence as that deduced from a gene termed vpr3. The vpr3 gene was identified by sequence analysis of randomly isolated cDNAs from a P. hypochondriaca venom gland library. Presently, the gene for the full-length sequence of mature VPr3 protein was amplified from the P. hypochondriaca venom gland cDNA library by PCR. The amplicon was directionally cloned into a pET expression vector so that recombinant VPr3 (rVPr3) would have an N-terminal polyhistidine (His) tag. High levels of target protein expression were obtained following addition of IPTG (1,mM) and growth of the bacteria at 37°C for 5,h, or at 24°C for 20,h. Following lysis of bacteria grown at 37°C, the target protein partitioned into the insoluble fraction. However, at 24°C, a small amount of soluble protein was consistently detected. The amount of soluble rVPr3 was subsequently increased when the transformed bacteria were grown in Overnight Express Instant TB medium at 24°C. Soluble rVPr3 was purified utilizing the MagneHis Protein Purification System. Recombinant VPr3 was determined to have adverse effects on the cytoskeleton of Lacanobia oleracea haemocytes and to inhibit the ability of these cells to form aggregates in vitro. © 2009 Wiley Periodicals, Inc. [source] | ||||||||||