Rat Inducible Nitric Oxide Synthase Research Articles

Construction and identification of recombinant adenovirus expressing short hairpin RNA of rat inducible nitric oxide synthase

Objective To construct an adenovirus vector expressing short hairpin RNA （shRNA） of rat inducible oxide synthase （iNOS） carrying enhanced green fluorescent protein （EGFP） and amplify the adenovirus vector in HEK-293 cells. Methods The shuttle plasmid pDC316-inos-shRNA-EGFP that was constructed at an earlier date, was cotransfected with the adenovirus skeleton plasmid pBHGlox-E1, 3Cre into 293 cells to obtain the produced replication defective recombinant adenovirus vector Ad5-inos- shRNA-EGFP. The recombinant adenovirus was propagated by repeat infection of 293 cells and purified by ion exchange method, then the virus particles were counted and the purity and titer were determined. Results Recombinant adenoviral vector Ad-ACE-shRNA was constructed successfully, which was confirmed by polymerase chain reaction （PCR） and GFP expression. After amplification and purification, the virus particle count, A260/A280 and titer of recombinant adenovirus were 3.6 × 10^11 VP/ml, 1.25 and 7.94 × 10^9 IU/ml, respectively. Conclusion Recombinant adenovirus vector Ad5-inos-shRNA-EGFP is successfully constructed, which laid a foundation for gene activation in erectile dysfunction treatment. Key words: RNA activation; Inducible nitric oxide synthase; Adenovirus; EGFP

Synthesis of N-benzyl- and N-phenyl-2-amino-4,5-dihydrothiazoles and thioureas and evaluation as modulators of the isoforms of nitric oxide synthase

Inhibition of the isoforms of nitric oxide synthase (NOS) has important applications in therapy of several diseases, including cancer. Using 1400W [ N-(3-aminomethylbenzyl)acetamidine], thiocitrulline and N δ-(4,5-dihydrothiazol-2-yl)ornithine as lead compounds, series of N-benzyl- and N-phenyl-2-amino-4,5-dihydrothiazoles and thioureas were designed as inhibitors of NOS. Ring-substituted benzyl and phenyl isothiocyanates were synthesised by condensation of the corresponding amines with thiophosgene and addition of ammonia gave the corresponding thioureas in high yields. The substituted 2-amino-4,5-dihydrothiazoles were approached by two routes. Treatment of simple benzylamines with 2-methylthio-4,5-dihydrothiazole at 180 °C afforded the corresponding 2-benzylamino-4,5-dihydrothiazoles. For less nucleophilic amines and those carrying more thermally labile substituents, the 4,5-dihydrothiazoles were approached by acid-catalysed cyclisation of N-(2-hydroxyethyl)thioureas. This cyclisation was shown to proceed by an S N2-like process. Modest inhibitory activity was shown by most of the thioureas and 4,5-dihydrothiazoles, with N-(3-aminomethylphenyl)thiourea (IC 50=13 μM vs rat neuronal NOS and IC 50=23 μM vs rat inducible NOS) and 2-(3-aminomethylphenylamino)-4,5-dihydrothiazole (IC 50=13 μM vs rat neuronal NOS and IC 50=19 μM vs human inducible NOS) being the most potent. Several thioureas and 4,5-dihydrothiazoles were found to stimulate the activity of human inducible NOS in a time-dependent manner.

Molecular cloning and characterization of the rat inducible nitric oxide synthase (iNOS) gene.

We have cloned and characterized the rat inducible nitric oxide synthase (iNOS) gene. It spans approx. 36kb and is divided into 27 exons and 26 introns. The distribution and length of exons are similar to those in the human iNOS gene. In the 5' flanking regulatory region of the rat iNOS gene, there are a number of putative transcription factor binding sites (>20), many of them probably indispensable for the gene's nuclear factor kappaB (NFkappaB)-dependent induction, but also many which may have a role in its NFkappaB-independent induction pathway. These include cyclic adenosine 3', 5'-monophosphate (cAMP) response elements (CRE), hypoxia responsive element (HRE) and GATA-core elements. Rat models are powerful tools in studies of neurological diseases. Because iNOS is most likely responsible for the harmful consequences of nitric oxide (NO) in general, the cloned rat iNOS gene will further reveal the mechanisms of iNOS inducibility in different cell types during development and disease, including brain diseases, and to promote studies of pharmacological intervention in cases where extensive NO production plays a critical role.

Acquired interferon γ responsiveness during Caco-2 cell differentiation: effects on iNOS gene expression

BACKGROUNDImpairment of intestinal barrier function occurs under a variety of inflammatory conditions and is mediated at least in part by interferon γ (IFN-γ) induced nitric oxide (NO) production. Previous in...

Molecular Cloning and Analysis of the Rat Inducible Nitric Oxide Synthase Gene Promoter in Aortic Smooth Muscle Cells

We have cloned five DNA fragments (−0.32, −0.48, −1.7, −3.2, and −5.1 kb) of the 5′-flanking region of the rat inducible nitric oxide synthase (iNOS) gene from rat genomic DNA. The functional importance of the 5′-flanking region was determined by transient expression of iNOS promoter-luciferase constructs in cultures of rat aortic smooth muscle cells. The −0.48 kb construct, containing one nuclear factor κB (NF-κB) binding site, expressed basal promoter activity but showed only a 1.5- and 1.7-fold increase in luciferase activity in response to lipopolysaccharide (LPS) or a cytokine mixture, respectively. However, the −3.2 kb construct (containing a second NF-κB binding site) showed full promoter activity with a 24-fold increase in response to LPS or cytokine mixture. The −5.1 kb construct showed no further increase in luciferase activity, suggesting that the 1.9 kb upstream of −3.2 kb may not be important in rat iNOS regulation. Rat iNOS promoter induction did not appear to be transcriptionally regulated by NO since NOS inhibitors did not affect induction. These data are in marked contrast to the mouse iNOS promoter in which a DNA sequence as short as a −85 bp, containing one NF-κB site, confers 10-fold inducibility by LPS. The present findings demonstrate that the rat iNOS gene is transcriptionally regulated by cytokines and LPS, but, unlike the mouse gene, the downstream NF-κB site does not appear to be a key region in responses to cytokines and LPS. These data suggest that the regulation of the rat gene may require the coexistence of at least two NF-κB sites or other elements upstream of −0.48 kb of the 5′-flanking region.

THE STRUCTURE OF THE PROMOTER REGION FOR RAT INDUCIBLE NITRIC OXIDE SYNTHASE GENE

The nucleotide sequences of the promoter region for rat inducible nitric oxide synthase (iNOS) were elucidated. The sequences were highly homologous with the mouse iNOS gene. The regulation of the iNOS gene may be distinguished between rodent and mammalian. © 1997 Elsevier Science Inc.

Molecular Cloning of the Rat Inducible Nitric Oxide Synthase Gene Promoter

The inducible nitric oxide synthase (iNOS) is not constitutively expressed but is induced in many types of mammalian cells by cytokines and bacterial endotoxins. Previously, we have reported the most interesting feature of rat iNOS gene that is up-regulated by cyclic AMP at transcriptional level as demonstrated by nuclear run-on assay. We now have isolated and sequenced the rat iNOS gene promoter and determined the transcription start site. Moreover, we have constructed a rat iNOS promoter-chloramphenicol acetyltransferase (CAT) fusion gene and analyzed its inducibility by interleukin 1β and cyclic AMP in transiently transfected Swiss 3T3 fibroblasts.

Implication of nitric oxide synthase in carcinogenesis: analysis of the human inducible nitric oxide synthase gene.

Nitric oxide (NO) is a newly identified, multifunctional biological mediator. However, it also has deleterious effects on biological materials. For instance, nucleic acids, proteins, and some prosthetic groups of enzymes can be modified by NO or its reaction products with other reactive oxygen species. Endogenous nitrosamine formation through the reaction of NO or its oxidized products with amines might be involved in carcinogenesis. These deleterious effects of NO are often associated with inflammatory processes both in experimental animals and human. We analyzed the molecular mechanism of control of expression of the inducible nitric oxide synthase (NOS) gene in mouse cells by cloning its putative promoter region. This promoter responded to various cytokines and endotoxin similarly to the endogenous NOS gene in mouse cells. No appreciable induction of NOS was observed in human peripheral blood cells, but induction was detected in a human glioblastoma cell line A-172. Therefore, the human inducible NOS cDNA was cloned from A-172 cells and its cDNA-deduced amino acid sequence found to have about 80% similarity to those of both mouse and rat inducible NOSs. The effects of various cytokines on the induction of the gene were somewhat different from those observed in mouse cells, but the mouse promoter responded to these cytokines similarly to the endogenous NOS gene in human cells, indicating functional similarity of cis-elements of the genes encoding both human and mouse inducible NOS. Structural analysis of the human inducible NOS gene by Southern blot analysis revealed putative genetic restriction fragment length polymorphism in intron 5.(ABSTRACT TRUNCATED AT 250 WORDS)