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)

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

To investigate the purging effect of CD3AK/iNOS on primary leukemic cells from chronic myeloid leukemia patients in vitro, amphotropic packaging cell line PA317 transfected with the whole length of iNOS gene was cultivated, amplified and screened by G418. The viral titer was determined by the NIH3T3 cells. Human peripheral blood mononuclear cells were isolated and activated by anti-CD3 monoclonal antibody in vitro. CD3AK cells were incubated with viral supernatant and selected by G418. Resistant clones were assayed for iNOS gene expression by RT-RCR. The content of nitric oxide and the activity of iNOS in the culture supernatant of CD3AK/iNOS were evaluated by the method of Griess. After BMMNC or PBMNC from CML patients were co-cultured with CD3AK/iNOS, CD3AK/Neo and CD3AK/iNOS respectively, the expression of bcr/abl fusion gene was detected by serial dilution semi-quantitative net RT-PCR assay. The results showed that anti-G418 positive packaging cell line PA317 transfected with the whole length of iNOS gene clones could stably synthesize and excrete recombinant retroviral vectors. The titer of recombinant retroviral vectors was 1.0 x 10(5) CFU/ml. After being transfected by recombinant retroviral supernatant, the iNOS cDNA was expressed in CD3AK/iNOS. The content of NO and activity of iNOS that synthesized and excreted by CD3AK/iNOS were notably increased, compared with those of CD3AK. There were statistically significant differences in NO content and iNOS activity between two groups. After BMMNC or PBMNC from CML patients were co-cultured with CD3AK/iNOS, CD3AK/Neo and CD3AK/iNOS respectively, the expression of bcr/abl fusion gene in all of them was down-regulated by serial dilution semi-quantitative RT-PCR assay. It is concluded that construction of CD3AK/iNOS can markedly increase the content of NO and the activity of iNOS, which can be more efficient in in vitro purging leukemia cells for autologous hematopoietic stem cell transplantation.
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PMID:[Purging effects of CD3AK/iNOS in vitro on primary leukemic cells from chronic myeloid leukemia patients]. 1640 54