| Budget Amount *help |
¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1991: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1990: ¥800,000 (Direct Cost: ¥800,000)
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| Research Abstract |
New quantitative chemical methods for the detection of minute amounts of endotoxin has been developed using 3-hydroxytetradecanoic acid and L-glycero-D-manno-heptose as a chemical markers. After converting 3-hydroxytetradecanoic acid to methyl ester, it was coupled with a fluorescent probe, anthracene-9-carboxyl chloride, obtained by chlorization of 9-anthroic acid with oxalyl chloride. The resulting ester was isolated by HPLC on silica column. The purified product, methyl-3-0-(9-carboxy-anthracenyl)tetradecanoate(M/Z 462). was highly responsible to a fluorescence spectrophotometer, showing maximum emission with exitation wavelength at 252 nm and emission wavelength at 460 nm in dichloromethane, the limit of detection being as little as 10 f mol. Using this method it is possible to detect Salmonella abortus equi endotoxin in aqueous solution at a level of 40 pg. L-glycero-D-manno-heptose was also labeled by introducing aminopyridine. The limit of detection of the purified product, amin
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opyridylheptose, was 80 pg.
Chemically modified lipid A's of S., abortus equi were tested for mitogenicity on mouse spleen cells as well as antagonism of the mitogenicity of intact lipopolysaccharide(LPS). All the lipid A preparations deacylated by defferent alkaline treatments suffered a drastic loss of mitogenicity. The mitogenic activity of lipid A was also lost by introducing succinic residue on hydroxy group. Partially deacylated alkaline-treated preparations(but not completely deacylated)inhibited the activation of splenic B-cells by LPS. They were found to be toxic to spleen cells, however, and to suppress not only the sitogenicity of LPS but that of concanavalin A as well. This inhibitory action was not exhibited when all of the fatty acid was eliminated. Suceinylated lipid A, on the other hand, was not toxic to the cells and specifically inhibited the mitogenicity of lipopolysaccharide. Macrophages do not participate in this inhibition. Inhibition was observed when suceinylated lipid A was added to the culture 3 h before LPS. Inhibition was not affected by washing the cells before adding LPS. Inhibition increased as the ratio of suppressor to mitogen increased, suggesting that the suceinylated lipid A competes with intact lipopolysaccharide. Succinylated E coli type lipid A, which was synthesized chemically and has a complete structure of lipid A, was not a suppressor but exhibited rather stronger activity than the original. The chemical structure of the suppressor was suggested to be a monophosphorylated derivatives of natural Salmonella lipid A mixture. Less
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