Substrate specificity of soluble methane monooxygenase. Mechanistic implications

Following the example set by studies of the mechanistic aspects of the substrate specificity of various cytochrome P-450 enzymes, we have undertaken a parallel investigation of the soluble methane monooxygenase from Methylococcus capsulatus (Bath). Soluble methane monooxygenase is a multicomponent enzyme with a broad substrate specificity. Using substrates previously tested with cytochrome P-450 enzymes and using purified enzyme preparations, this work indicates that soluble methane monooxygenase has a similar oxidative reaction mechanism to cytochrome P-450 enzymes. The evidence suggests that soluble methane monooxygenase oxidizes substrates via a nonconcerted reaction mechanism (hydrogen abstraction preceding hydroxylation) with radical or carbocation intermediates. Aromatic hydroxylation proceeds by epoxidation followed by an NIH shift.     

Transforming growth factor alpha: an aromatic side chain at position 38 is essential for biological activity.

Site-directed mutagenesis has been performed in the human transforming growth factor alpha gene. When tyrosine 38 is mutated into phenylalanine or tryptophane, biological activity is retained. In contrast, other alterations between cysteine 34 and cysteine 43 and disruption of disulfide bonds 8 to 21 and 34 to 43 resulted in loss of activities. The presence of an aromatic side chain at position 38 of transforming growth factor alpha seems to be essential for its activity.     

Inhibition of IFN-gamma-induced Ia antigen expression on synovial fibroblasts by IL-1

Naturally occurring substances capable of the negative regulation of class II molecules on synovial fibroblasts may play an important role in controlling the sustained immune processes ongoing in the rheumatoid joint. We report here that rIL-1 is capable of such a negative regulatory process. The simultaneous addition of rIL-1 and rIFN-gamma to rat synovial fibroblasts resulted in decreased Ia Ag and mRNA expression when compared with synovial fibroblasts treated with IFN-gamma alone. Both rIL-1 alpha and rIL-beta inhibited to a similar degree with the level of inhibition being dependent on both the concentration of IL-1 and IFN-gamma. Other cytokines, including IFN-alpha/beta, IL-2, and TNF, had no antagonistic effect on IFN-gamma-induced Ia expression. Time course experiments showed that IL-1 inhibited when present immediately before addition of IFN-gamma or when added during the first 24 h of IFN-gamma stimulation but not at later time points. Indomethacin failed to reverse the IL-1-mediated inhibition, despite the fact that exogenously added PGE2 also inhibited IFN-gamma-induced Ia expression. IL-1 treatment of synovial cells did not alter the ability of IFN-gamma to bind to the cells. These findings provide evidence for a negative regulatory role for IL-1 on synovial fibroblasts independent of PGE2 production and thus suggest that IL-1 is capable of both pro- and antiinflammatory actions within the rheumatoid joint.     

Structural homology of microtubule-associated proteins 1 and 2 demonstrated by peptide mapping and immunoreactivity

The major high molecular weight microtubule-associated polypeptides from hog brain (MAP-1 and MAP-2) were compared by one- and two-dimensional peptide mapping under varied conditions and by immunological techniques. Partial digestion of MAP-1 and MAP-2 with Staphylococcus aureus V8 protease and analysis in one dimension gave rise to very similar peptide maps independent of whether 125I-, 3H-, or 32P-labeled proteins were used. One-dimensional cleavage patterns of significant similarity were also obtained by partial digestion of MAP-1 and MAP-2 using trypsin or chymotrypsin. Furthermore, a pronounced similarity, although clear nonidentity, of MAP-1 and MAP-2 was also revealed after exhaustive digestion of 125I-labeled proteins with S. aureus V8 protease or trypsin followed by analysis of peptides in two dimensions. For immunological comparison, antisera were used that had been raised in rabbits using electrophoretically purified MAP-1 and MAP-2 components as immunogens. As determined by immunoprecipitation, the antiserum raised to MAP-1 was equally reactive with MAP-1 and MAP-2 components, whereas the antiserum to MAP-2 reacted primarily with MAP-2. Indicating the presence of common as well as unique antigenic determinants on MAP-1 and MAP-2, these results, therefore, were in agreement with the peptide mapping data. Implications of these results for biosynthetic mechanisms as well as differential distribution and functions of MAPs in cells are discussed.     

Toll-Like Receptor 4 Upregulation by Angiotensin II Contributes to Hypertension and Vascular Dysfunction through Reactive Oxygen Species Production

Hypertension is considered as a low-grade inflammatory disease, with adaptive immunity being an important mediator of this pathology. TLR4 may have a role in the development of several cardiovascular diseases; however, little is known about its participation in hypertension. We aimed to investigate whether TLR4 activation due to increased activity of the renin-angiotensin system (RAS) contributes to hypertension and its associated endothelial dysfunction. For this, we used aortic segments from Wistar rats treated with a non-specific IgG (1 µg/day) and SHRs treated with losartan (15 mg/kg·day), the non-specific IgG or the neutralizing antibody anti-TLR4 (1 µg/day), as well as cultured vascular smooth muscle cells (VSMC) from Wistar and SHRs. TLR4 mRNA levels were greater in the VSMC and aortas from SHRs compared with Wistar rats; losartan treatment reduced those levels in the SHRs. Treatment of the SHRs with the anti-TLR4 antibody: 1) reduced the increased blood pressure, heart rate and phenylephrine-induced contraction while it improved the impaired acetylcholine-induced relaxation; 2) increased the potentiation of phenylephrine contraction after endothelium removal; and 3) abolished the inhibitory effects of tiron, apocynin and catalase on the phenylephrine-induced response as well as its enhancing effect of acetylcholine-induced relaxation. In SHR VSMCs, angiotensin II increased TLR4 mRNA levels, and losartan reduced that increase. CLI-095, a TLR4 inhibitor, mitigated the increases in NAD(P)H oxidase activity, superoxide anion production, migration and proliferation that were induced by angiotensin II. In conclusion, TLR4 pathway activation due to increased RAS activity is involved in hypertension, and by inducing oxidative stress, this pathway contributes to the endothelial dysfunction associated with this pathology. These results suggest that TLR4 and innate immunity may play a role in hypertension and its associated end-organ damage.     

The effect of progesterone administration on sex hormone binding globulin binding capacity in women with severe premenstrual syndrome

Thirty-one women with severe premenstrual syndrome had low sex hormone binding globulin (SHBG) binding capacities 30.2 +/- 9.4 nmol DHT bound/l. The SHBG binding capacities rose when they were treated with three different doses of progesterone. On 400 mg (17 women) SHBG level was 45.11 +/- 11.80. On 800 mg (8 women) SHBG binding capacity rose to 64.75 +/- 14.30 and on the six women who took 1200 mg progesterone daily SHBG binding capacity was 78.5 +/- 23.10. These results are discussed.