Effects of antioxidants on homocysteine thiolactone-induced apoptosis in human umbilical vein endothelial cells

Background and objectives Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine thiolactone (HcyT), one of the homocysteine metabolites in vivo, is toxic both in vivo and in vitro. The aim of this study was to investigate the effect of HcyT on apoptotic damage in human umbilical vein endothelial cells (HUVECs) and the role of antioxidants in the reduction of HcyT-induced apoptosis. Methods HUVECs were cultured in DMEM supplemented with 20% heat inactivated fetal bovine serum cell cultures were maintained in a humidified 5% CO2 atmosphere at 37 ℃. Cytotoxicity was determined by MTT assay,which consists of hypodiploid cells with propidium iodide labeling and intracellular reactive oxygen species levels using 2',7'-dichlorofluorescein diacetate as the probe by flow cytometry. Results HcyT (250-2000μM) induced HUVECs apoptosis in a time- and concentration-dependent manner. Reactive oxygen species levels rose in response to increasing HcyT concentrations at 24-h incubation.The reduction of cell apoptosis by N-acetylcysteine, vitamin E, or pyrrolidine dithiocarbamate, occurred simultaneously with a significant decrease in intracellular reactive oxygen species levels. Conclusion HcyT exerts its cytotoxic effects on endothelial cells through an apoptotic mechanism involving cellular reactive oxygen species production. The capacity of N-acetylcysteine, vitamin E, and pyrrolidine dithiocarbamate to scavenge HcyT-induced cellular reactive oxygen species correlates well with their efficiency to protect against HcyT-promoted apoptotic damage. The protective effect of pyrrolidine dithiocarbamate on cell apoptosis indicates HcyT-generated hydrogen peroxide may provoke cell apoptosis via activating nuclear factor-kappa binding protein.

Facile Generation of Strained Peptidyl Thiolactones from Hydrazides and Its Application in Assembling MUC-1 VNTR OIigomers

An efficient method for the activation of C-terminal 4-mecaptoproline-or penicillamine-containing peptide hydrazides in ligation re-actions is reported herein.The corresp on ding peptide hydrazides can be readily prepared using solid-phase peptide synthesis,and subsequently activated by acetylacet one(acac)without exoge nous thiol additives.Strained peptidyl thiolactones could be the possible reactive in termediates that drastically accelerate the reacti on rates at the sterically demandi ng proline and valine sites.This developed protocol allows for sequential peptide ligations in a one-pot manner,and expedites the assembly of mucin 1(MUC-1)variable number tandem repeat(VNTR)trimers in various glycosylated forms.