Hiromitsu Yokohama

E5531, a synthetic non-toxic lipid A derivative blocks the immunobiological activities of lipopolysaccharide

The major pathological responses to Gram‐negative bacterial sepsis are triggered by endotoxin or lipopolysaccharide. As endotoxin is shed from the bacterial outer membrane, it induces immunological responses that lead to release of a variety of cytokines and other cellular mediators. As part of a program aimed at developing a therapeutic agent for septic shock, we have developed E5531, a novel synthetic lipopolysaccharide antagonist. As measured by release by tumour necrosis factor‐α, human monocytes or whole blood can be activated by lipopolysaccharide, lipid A, and lipoteichoic acid (from Gram‐positive bacteria). E5531 potently antagonizes activation by all these agents while itself being devoid of agonistic activity. The inhibitory activity of E5531 was dependent on time of addition. When 10 nM E5531 was added simultaneously with lipopolysaccharide or 1–3 h before addition of lipopolysaccharide, production of tumour necrosis factor‐α was inhibited by more than 98%. The addition of E5531 1 h after lipopolysaccharide reduced the efficacy of E5531 by 47%. Antagonistic activity of E5531 was specific for lipopolysaccharide as it was ineffective at inhibiting interferon‐γ mediated NO release of RAW 264.7 cells, phorbor 12‐myristate 13‐acetate stimulated superoxide anion production in human neutrophils, concanavalin A stimulated mitogenic activity in murine thymocytes and tumor necrosis factor‐α induced E‐selectin expression in human umbilical vein endothelial cells. E5531 as well as MY4, an anti‐CD14 antibody, inhibited radiolabelled lipopolysaccharide binding in human monocytes. These results support our contention that E5531 is a potent antagonist of lipopolysaccharide‐induced release of tumour necrosis factor‐α and other cellular mediators and may be an effective therapeutic agent for human septic shock due to Gram‐negative bacteria.

High-performance liquid chromatographic method for the determination of dolichols in tissues and plasma

High-performance liquid chromatographic methods for the determination of dolichols in tissues and plasma have been developed. The tissue concentration of dolichols was measured by high-performance liquid chromatography with uv detection and plasma levels of dolichols were determined fluorometrically after derivatization with anthracene-9-carboxylic acid. In both methods, 2,2-didecaprenylethanol was used as an internal standard. The method with fluorescence detection was sufficiently sensitive to measure the concentration of dolichols in human plasma.

A high-performance liquid chromatographic method for the assay of cholesterol 7α-hydroxylase activity

Abstract A high-performance liquid chromatographic method has been developed for the measurement of cholesterol 7α-hydroxylase activity in liver microsomes. 7α-Hydroxycholesterol generated from endogenous choleserol was derivatized with anthroyl 1-carbonitrile, chromatographed on a reversephase column, and detected fluorometrically. The detection limit of 7α-hydroxycholesterol was 1 ng/tube. The cholesterol 7α-hydroxylase activity in rat liver microsomes was assayed by this method, and the effects of some detergents and of the addition of exogenous cholesterol together with detergents on the enzyme activity were investigated. The endogenous 7α- and 7β-hydroxy-cholesterol could be also measured by this method.