Although hyperhomocysteinemia has been recognized recently as a prevalent risk factor for myocardial infarction and stroke, the mechanisms by which it accelerates arteriosclerosis have not been elucidated, mostly because the biological effects of homocysteine can only be demonstrated at very high concentrations and can be mimicked by cysteine, which indicates a lack of specificity. We found that 10 -50 M of homocysteine (a range that overlaps levels observed clinically) but not cysteine inhibited DNA synthesis in vascular endothelial cells (VEC) and arrested their growth at the G 1 phase of the cell cycle. Homocysteine in this same range had no effect on the growth of vascular smooth muscle cells (VSMC) or fibroblasts. Homocysteine decreased carboxyl methylation of p21 ras (a G 1 regulator whose activity is regulated by prenylation and methylation in addition to GTP-GDP exchange) by 50% in VEC but not VSMC, a difference that may be explained by the ability of homocysteine to dramatically increase levels of S-adenosylhomocysteine, a potent inhibitor of methyltransferase, in VEC but not VSMC. Moreover, homocysteine-induced hypomethylation in VEC was associated with a 66% reduction in membrane-associated p21 ras and a 67% reduction in extracellular signal-regulated kinase 1/2, which is a member of the mitogen-activated protein (MAP) kinase family. Because the MAP kinases have been implicated in cell growth, the p21 ras

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Organophosphorus hydrolase (OPH) is a bacterial enzyme that hydrolyzes a variety of organophosphorus (OP) neurotoxins, including many widely used pesticides and chemical warfare agents containing P-0, P-F, P-CN and P-S bonds. It has extremely high efficiency in hydrolysis of many different phosphotriester and phosphothiolester pesticides (P-0 bond) such as paraoxon (k,,, > 3800s-') and coumaphos (kc,, =8OOs-') or phosphonate (P-F) neurotoxins such as DFP (kcat = 350 s-I) and the chemical warfare agent sarin (k,,, = 56 s-I).In contrast, the enzyme has much lower catalytic capabilities for phosphonothioate neurotoxins such as acephate (k,,, =2.8 s-') or the chemical warfare agent VX [0-ethyl S-(2-diisopropylaminoethyl) methylphosphonothioate] (k,,, = 0.3 s-'). This lower specificity for VX and its analogues are reflected by the specificity constants (kca,/Km values) for VX =0.75 x lo3 M-' s-' compared to 5.5 x 107MvI-'s-' for paraoxon. Different metal-associated forms of the enzyme demonstrated significantly varying hydrolytic capabilities for VX and its analogues, and the activity of OPH (Co+*) was consistently higher than that of OPH (Znf2) by five to twenty fold. Hydrolysis of the P-S bonds was determined by monitoring the formation of free -SH groups, and the specific cleavage of the P-S bond was verified by 31P NMR analysis.