Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphodiesterases (PDEs) play a critical role in the regulation of intracellular cyclic nucleotide concentration and, consequently, regulate the state of cellular differentiation. We have reported that the Src-selective tyrosine kinase inhibitor, herbimycin A, potentiates luteinizing hormone (LH)-stimulated cAMP accumulation in culture media by ovarian thecal-interstitial cells (TIC; see Taylor, C and Terranova, P.F. (1995) Lipopolysaccharide inhibits rat ovarian thecal-interstitial cell steroid secretion in vitro. Endocrinology 136, 5527-5532). The present study was conducted to investigate the effects of herbimycin, and changes in Src tyrosine kinase activity, on PDE activity in rat TIC an in the mouse TM3 Leydig cell line. Treatment of TIC with herbimycin (1 microM) for 24 h inhibited basal and LH-stimulated PDE activity (approximately 50 and 70%, respectively) and was associated with an increase in cAMP and progesterone accumulation in culture media. Treatment of TM3 cells with herbimycin inhibited PDE activity and increased cAMP accumulation in a dose- and time-dependent manner. TM3 cell cultures challenged with herbimycin had lower Src tyrosine kinase activity than controls (approximately 50%); however, protein kinase A activity was unaffected. TM3 cells stably transfected with a dominant negative Src tyrosine kinase (TM3Srck-) had lower PDE activity than cells transfected with a G418 resistance gene alone (TM3pSV2neo) which served as control cells. Conversely, TM3 cells expressing a temperature-sensitive Src kinase had significantly greater PDE activity at the Src active temperature (35 degrees C; the temperature at which the enzyme is active) than TM3pSV2neo control cells grown at the same temperature. TM3 cell lysates hydrolyzed minimal amounts of cGMP, indicating a cAMP-specific PDE. Phosphodiesterase activity in both TM3 and rat TIC was sensitive to the PDE4-selective inhibitor RO20-1724, indicating the predominant active enzyme is probably a member of the cAMP-specific PDE4 family. From the present data, we conclude that a tyrosine kinase of the Src family may play an important role in regulating phosphodiesterase activity in thecal and Leydig cells, and thus regulate intracellular cAMP and the state of cellular differentiation.
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PMID:Src tyrosine kinase activity in rat thecal-interstitial cells and mouse TM3 Leydig cells is positively associated with cAMP-specific phosphodiesterase activity. 902 67

The role of an inducible nitric oxide synthase (iNOS)expression in the mechanisms of opioid tolerance and dependence was investigated. A recombinant retroviral expression vector containing a cDNA fragment of iNOS was transfected into the neuroblastomaxglioma NG108-15 cells by lipofectamine gene transferring technique. G418-resistant clones were selected and were named NG-LNCXiNOS cells. Using Southern blot, PCR amplification for Neo gene, RT-PCR and Western blot analysis, NG-LNCXiNOS cells were confirmed to have an integral exogenous iNOS gene which was being transcribed and translated into protein. NADPH-diaphorase (NADPH-d) histochemical staining and immunohistochemical staining with iNOS-specific antibody demonstrated that high-level expression of iNOS protein was present in the cytoplasm of NG-LNCXiNOS cells. The catalytic activity and NO( )(2) content in supernatant medium were obviously enhanced in iNOS gene-transfected cells. The results show that the biochemical and pharmacological properties of the recombinant enzyme were similar to those of native enzyme. The recombinant enzyme activity was completely dependent on NADPH and failed to be stimulated by the addition of calcium and calmodulin. Chelating agents failed to decrease its activity. NOS inhibitors could markedly reduce NO( )(2) production at a concentration-dependent manner. The expression of iNOS gene was involved in the up-regulation of NO-cGMP signal transduction cascade. Therefore, an iNOS gene-modified neuronal cell line was successfully established, offering an excellent model system for seeking and screening new drugs to treat opioid tolerance and dependence.
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PMID:Expression of Inducible Nitric Oxide Synthase Gene in Neuronal Cells Mediated by Retrovirus Vector. 1213 69

The neuroblastoma x glioma NG108-15 cells were transfected with recombinant eukarytic expression plasmid pCMViNOS containing the full-length cDNA encoding inducible nitric oxide synthase (iNOS). A lot of G418-resistant clones were screened at 600 micrograms/ml of geneticin. In the 2# clone expressing iNOS gene, iNOS catalytic activity in the cytosol fraction displayed to have an increasing trend, accompanying with the accumulation of NO2- content in the supernantant of cultured cells and the intracellular cGMP concentration, which suggested that NO-cGMP signal pathway was mediated by the expression of iNOS gene and blocked by NG-nitro-L-arginine (L-NNA) and methylene blue (MB). Activity of iNOS was concentration-dependently inhibited by NOS inhibitors such as L-NNA and aminoguanidine. The result of measurement of NADPH diaphorase activity and immunocytochemical staining showed that localization of the function expression of iNOS protein mainly existed in the cytoplasm of NG108-15 cells transfected with pCMViNOS. Furthermore, the chromosomal integration, transcript and protein translation of foreign iNOS gene were identified by Southern hybridization, RT-PCR and Western blot, respectively. The results indicated that iNOS gene-transfected cells had mRNA transcription and specific protein expression at high level. Given the above results, the engineering cell line with stable expression of iNOS gene was successfully established. The new neuronal cell line may serve as a source of iNOS and provide a useful cell model for studying iNOS biological function and developing novel iNOS-selective inhibitors.
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PMID:[Stable expression of recombinant inducible nitric oxide synthase in NG108-15 cells and its biological characterization]. 1254 60