Enhancing the methyl-donor activity of methylcobalamin by covalent attachment of DNA.

The preparation of a covalent DNA conjugate of vitamin B12 by means of heterogeneous solid-phase synthesis is reported. The cyano-corrinoid made available, dipotassium Co(beta)-cyanocobalamin-(3''-->2'),(3''-->5')-bis-2''-deoxythymidyl-3''-ate (K(2)-4), was cleanly methylated at the Co center by electrosynthetic means. Aqueous solutions of the resulting organometallic DNA-B12 conjugate K(2)-5 exhibited spectroscopic properties indicative of significant weakening of the axial (Co-N) bond, together with a 25-times higher basicity relative to Co(beta)-methylcobalamin (2).

Identification of atherosclerosis-associated conformational heat shock protein 60 epitopes by phage display and structural alignment.

Autoimmune reactions to HSP60 are believed to play a key role during development of early atherosclerosis. Due to the high degree of phylogenetic conservation between microbial and human HSP60, bacterial infections might be responsible for inducing cross-reactivity to self HSP60, which is expressed on the surface of arterial endothelial cells stressed by classical atherosclerosis risk factors. Conformational epitopes recognized by polyclonal anti-mycobacterial HSP60 antibodies from subjects with atherosclerosis were identified using a phage displayed random library of cyclic constrained 7mer peptides.

Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy.

The highly conserved and ubiquitous heat shock proteins (HSP) are essential for the cellular homeostasis and efficiently trigger cellular responses to stress conditions. Both microbial and human HSP act as dominant antigens in numerous infectious and autoimmune diseases such as atherosclerosis, inducing a strong immune-inflammatory response. In the present study, the surface localization of HSP60 on stressed and unstressed human umbilical venous endothelial cells (HUVECs) was investigated using sensitive high resolution microscopy methods and flow cytometry.

Cross-reactive B-cell epitopes of microbial and human heat shock protein 60/65 in atherosclerosis.

Objective: Growing evidence suggests that immune reactions to heat shock protein 60 (HSP60) are involved in atherogenesis. Because of the high phylogenetic conservation between microbial and human HSP60, bacterial infections might be responsible for breaking the tolerance to self-HSP60, which is expressed on the surface of stressed arterial endothelial cells.

Methods And Results: We purified serum antibodies to Escherichia coli HSP60 (GroEL), the 60-kD chlamydial HSP, and HSP65 of Mycobacterium tuberculosis by affinity chromatography from clinically healthy subjects with sonographically proven carotid atherosclerosis.