Retinoic acid inhibits inducible nitric oxide synthase expression in 3T3-L1 adipocytes

The present study was undertaken to explore whether retinoids, which are known to have immunomodulatory actions, could attenuate tumor necrosis factor-alpha (TNF)-stimulated inducible nitric oxide synthase (iNOS) expression in 3T3-L1 adipocytes. Adipocytes incubated with TNF induced dose- and time-dependent accumulation of nitrite in the culture medium through the iNOS induction as confirmed by Western blotting. Treatment of cells with TNF in the presence of all-trans-retinoic acid (RA) significantly decreased their ability to produce nitrite and iNOS induction. Both 13-cis- and all- trans-RA-induced suppression was dose-dependent, and all-trans-RA was somewhat potent than 13-cis-RA. The inhibitory effect of RA on TNF-induced iNOS induction was reversible, completely recovered after 2 days, and was exerted through the inhibition of NF-kappaB activation. TNF also suppressed the lipoprotein lipase (LPL) activity of 3T3-L1 adipocytes. RA could not reverse the TNF- induced LPL suppression at RA levels causing near complete inhibition of the TNF-induced NO production. These results indicate that RAs attenuate iNOS expression reversibly in TNF-stimulated 3T3-L1 adipocytes, and that the TNF- induced LPL suppression is not the result of NO overproduction.

Expression and Purification of Human Tumor Necrosis FactorαFusing with Oligo- Histidine / 上海第二医科大学学报

ObjectiveExpression and purification of human tumor necrosis factorα(hTNFα)fusion protein with a stretch of six consecutive histidine residues(6×His) in E. coli.MethodshTNFα fusion proteins with 6×His at N and C terminus were expressed by using E. coli expression vectors pET- 28a(+) and pET-22b(+). The His6 tag allows the expression fusion protein purified in one step by immobilized metal Ni2+ chelation affinity chromatcgraphy in native state. Results The two construct expression vectors were expressed in E. coli respectively, the former with high level as insoluble protein, account for 45% of the total bacteria proteins and not purified; the later 8% as soluble protein, and characterized by SDS- PAGE, Westren-blot. By using affinity chromatography through Ni2+ - IDA Sepharose 6B, 100ml induction culture had 0.4mg hTNFα-6×His fusion proteins. Its purifity reached 90 %. ConclusionThe purification expression product can possess TNF activity and reach 5.42×104U/mg.