Characterization of monoclonal antibodies specific for the V beta 3 family of the human T cell receptor generated using soluble TCR beta-chain.

A soluble, recombinant form of the human T cell receptor (TCR) beta-chain containing the V beta 3.1 sequence has been constructed, expressed in Chinese hamster ovary cells, amplified by dihydrofolate reductase selection, and purified in quantities appropriate for the generation of monoclonal antibodies (mAb). The V beta 3 sequence was chosen because of its reported elevated usage in the synovial T cells of rheumatoid arthritis patients but the approach described should be applicable to other known human V beta gene sequences. By this method, two mAb were prepared which reacted with up to 10% of normal, live peripheral blood T cells but with reactivity varying greatly among individual donors. Both mAb specifically bound to a murine T cell line transfected with a human TCR V beta 3.1 and immunoprecipitated a protein of the expected molecular weight for the TCR beta-chain. Both antibodies were mitogenic for T cells and analysis of peripheral blood lymphocyte cultures stimulated with the mAb suggested that both were specific for the V beta 3.1 subfamily and not D beta or J beta. Clones expressing V beta 3, which were derived from mAb-stimulated peripheral blood lymphocytes of a single individual, preferentially (8/13), but not exclusively, utilized the J beta 2.7 gene segment. The V beta 3.1 usage showed no preference for the CD8+ or CD4+ subpopulations of normal peripheral blood T cells.

Stimulation of lysyl oxidase (EC 1.4.3.13) activity by testosterone and characterization of androgen receptors in cultured calf aorta smooth-muscle cells.

Previous studies have indicated a greater incidence of atherosclerotic cardiovascular disease in men than in women of child-bearing age, suggesting that vascular interactions with sex steroids may effect pathogenesis in these cases. In the present study, it was found that the presence of 10-100 nM-testosterone in the growth medium of calf aortic smooth-muscle cells in culture stimulates lysyl oxidase activity approx. 2.5-fold in the medium and 5.5-fold in the fraction bound to the cell layer. Androgen receptors were identified in these cultured smooth-muscle cells, and their properties were very similar to those in the cytosolic fraction of whole bovine aortic tissue. These receptors appeared to be specific for androgen, of high affinity (Kd = 0.4 nM) and of low capacity (9000 sites/cell). The present results indicate that the aortic smooth-muscle cell is a cellular target for androgens, and thus raise the possibility that the development of fibrotic arterial lesions involving the deposition of excess collagen may in part be regulated by androgen-mediated stimulation of collagen cross-linkage formation as catalysed by lysyl oxidase.

Histone H1 is a substrate for lysyl oxidase and contains endogenous sodium borotritide-reducible residues.

Incubation of purified bovine aortic lysyl oxidase with rat liver or calf thymus H1 histone results in the catalytic formation of hydrogen peroxide, indicating the substrate potential of H1 for this connective tissue enzyme. Sodium borotritide-reducible residues consistent with aminoadipic semialdehyde and the lysinonorleucine crosslinkage were generated in H1 by incubation with lysyl oxidase. H1 histone also contains endogenous reducible functions including an unidentified prominent tritiated peak eluting near tyrosine as well as other lesser peaks, one of which is consistent with lysinonorleucine.

Vicinal diamines as pyrroloquinoline quinone-directed irreversible inhibitors of lysyl oxidase.

The observation that aliphatic diamines become poor substrates as the carbon chain length decreases and that ethylenediamine, the shortest diamine, is an irreversible inhibitor of lysyl oxidase led to the investigation of the mechanism of inhibition by ethylenediamine. The cis but not the trans isomer of 1,2-diaminocyclohexane was also a potent irreversible inhibitor of lysyl oxidase, consistent with the interaction of both amino groups of vicinal diamines with an enzyme moiety. Both cis-1,2-diaminocyclohexane and ethylenediamine but not trans-1,2-diaminocyclohexane markedly perturbed the spectrum of free pyrroloquinoline quinone (PQQ), a covalently linked form of which is the carbonyl cofactor of lysyl oxidase. cis-1,2-Diaminocyclohexane also induced similar changes in the spectrum of lysyl oxidase. The perturbations of the spectra of PQQ or of lysyl oxidase by cis-1,2-diaminocyclohexane or ethylenediamine as well as the development of irreversible inhibition of the enzyme by cis-1,2-diaminocyclohexane or ethylenediamine were all markedly reduced under anaerobic conditions. Moreover, approximately 1 mol of H2O2 was released per mol of PQQ or lysyl oxidase upon aerobic incubation with cis-1,2-diaminocyclohexane, while approximately 2 mol of 3H+ were released from cis-[1,2-3H] 1,2-diaminocyclohexane per mol of PQQ or lysyl oxidase under corresponding conditions. A proposal for the mechanism of inhibition of lysyl oxidase by vicinal diamines is presented which involves limited oxidation of the diamine linked to PQQ at the active site so that the PQQ-diamine complex is finally stabilized by a conjugated 6-membered ring.