The fatty-acylation-deficient bovine endothelial NO synthase (eNOS) mutant (Gly-2 to Ala-2, G2AeNOS) was purified from a baculovirus overexpression system. The purified protein was soluble and highly active (0.2-0.7 micromol of l-citrulline. mg-1.min-1), contained 0. 77+/-0.01 equivalent of haem per subunit, showed a Soret maximum at 396 nm, and exhibited only minor uncoupling of NADPH oxidation in the absence of l-arginine or tetrahydrobiopterin. Radioligand binding studies revealed KD values of 147+/-24.1 nM and 52+/-9.2 nM for specific binding of tetrahydrobiopterin in the absence and presence of 0.1 mM l-arginine respectively. The positive co-operative effect of l-arginine was due to a pronounced decrease in the rate of tetrahydrobiopterin dissociation (from 1.6+/-0.5 to 0. 3+/-0.1 min-1). Low-temperature SDS gel electrophoresis showed that approx. 80% of the protein migrated as haem-containing dimer after preincubation with l-arginine and tetrahydrobiopterin. Gel-filtration chromatography yielded one peak with a Stokes radius of 6.8+/-0.04 nm, corresponding to a hydrodynamic volume of 1. 32x10(-24) m3, whereas haem-deficient preparations (approx. 0.3 equivalent per subunit) contained an additional protein species with a hydrodynamic radius of 5.1+/-0.2 nm and a corresponding volume of 0.55x10(-24) m3, suggesting that haem availability regulates eNOS dimerization.

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Balneotherapy employing sulphurous thermal water is still applied to patients suffering from diseases of musculoskeletal system like osteoarthritis (OA) but evidence for its clinical effectiveness is scarce. Since the gasotransmitter hydrogen sulphide (H2S) seems to affect cells involved in degenerative joint diseases, it was the objective of this study to investigate the effects of exogenous H2S on fibroblast-like synoviocytes (FLS), which are key players in OA pathogenesis being capable of producing pro-inflammatory cytokines and matrix degrading enzymes. To address this issue primary FLS derived from OA patients were stimulated with IL-1β and treated with the H2S donor NaHS. Cellular responses were analysed by ELISA, quantitative real-time PCR, phospho-MAPkinase array and Western blotting. Treatment-induced effects on cellular structure and synovial architecture were investigated in three-dimensional extracellular matrix micromasses. NaHS treatment reduced both spontaneous and IL-1β-induced secretion of IL-6, IL-8 and RANTES in different experimental settings. In addition, NaHS treatment reduced the expression of matrix metallo-proteinases MMP-2 and MMP-14. IL-1β induced the phosphorylation of several MAPkinases. NaHS treatment partially reduced IL-1β-induced activation of several MAPK whereas it increased phosphorylation of pro-survival factor Akt1/2. When cultured in spherical micromasses, FLS intentionally established a synovial lining layer-like structure; stimulation with IL-1β altered the architecture of micromasses leading to hyperplasia of the lining layer which was completely inhibited by concomitant exposure to NaHS. These data suggest that H2S partially antagonizes IL-1β stimulation via selective manipulation of the MAPkinase and the PI3K/Akt pathways which may encourage development of novel drugs for treatment of OA.

Bioactive recombinant human bone morphogenetic protein-2 (rhBMP-2) was obtained using Escherichia coli pET-25b expression system: 55 mg purified rhBMP-2 were achieved per g cell dry wt, with up to 95% purity. In murine C2C12 cell line, rhBMP-2 induced an increase in the transcription of Smads and of osteogenic markers Runx2/Cbfa1 and Osterix, measured by semi-quantitative RT-PCR. Bioassays performed in human fat-derived stem cells showed an increased activity of the early osteogenic marker, alkaline phosphatase, and the absence of cytotoxicity.

The ERG1 gene of Saccharomyces cerevisiae encodes squalene epoxidase, a key enzyme in the ergosterol pathway. ERG1 is an essential gene. Disruption of the gene with URA3 results in a lethal phenotype when cells are grown under aerobic conditions, even in the presence of ergosterol. However, cells are viable in the presence of ergosterol under anaerobic growth conditions during which ergosterol is taken up by cells. Physical and genetic mapping data reveal that ERG1 is located on the right arm of chromosome VII proximal to QCR9 at a distance of 14·6 cM from ADE3.

Rat bone morphogenetic protein-4 (rBMP-4) cDNA was cloned from rat osteoblasts by RT-PCR and expressed in E. coli. Monomeric, dimeric and polymeric forms of recombinant rat BMP-4 (rrBMP-4) were obtained from inclusion bodies after solubilization with urea. The dimer was separated from the remaining polymer and host cell contaminants using size exclusion chromatography. Furthermore, purified rrBMP-4 was stabilized at low urea concentration (40 mM) and at pH 8.5 through the addition of bovine serum albumin. Both, rrBMP-4 dimer and polymer were biologically active as tested by the induction of alkaline phosphatase activity in MC3T3-E1 cells.

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