Yoshiyuki Ogawa

Enhancement of Endotoxicity and Reactivity with Carbocyanine Dye by Sonication of Lipopolysaccharide

The specificity of endotoxin (lipopolysaccharide, LPS) in the carbocyanine dye reaction was investigated, and then a stoichiometric study of the dye‐LPS interaction was conducted with attention to the relationship of biological activities of LPS to the reactivity with the dye. Absorption maxima of some bacterial components in the dye reaction were as follows; LPS from both Escherichia coli and Pseudomonas aeruginosa and lipid A from E. coli LPS, 465 nm; Shigella flexneri LPS, 460 nm; Salmonella minnesota R595 glycolipid, 470 nm; polysaccharide from E. coli LPS, 650 nm; yeast RNA, 620 nm; streptococcal M protein and pyrogenic exotoxin, 610 nm; and free fatty acids, 445–450 nm. The absorbance at 465 nm was increased approximately threefold by sonicating LPS for 1–3 min, which roughly paralleled the decrease in turbidity of the LPS aqueous solution. The Limulus amoebocyte lysate (LAL) gelation activity of LPS increased 10‐fold when LPS was sonicated for 0.5–5 min, but it decreased to the control level after further treatment. This decrease, however, was overcome by sonication in the presence of 5 mmol of L‐ascorbic acid used as an antioxidant. The LAL gelation activity of LPS was inactivated in parallel with an increase in the ratio (w/w) of dye to LPS from 1.73 to 6.90 in the dye‐LPS mixture. Pyrogenicity of LPS was also clearly inactivated when the ratio was over 1.73. The ratios of the height of the β band at 465 nm (dye‐LPS complex) to that of the α band at 510 nm (free dye) were increased by sonicating LPS, indicating that the binding character, or stacking tendency, was increased by sonicating LPS.

Effect of streptococcal pyrogenic exotoxin on rabbit macrophage functions in vitro: mediation by splenic lymphocytes.

Streptococcal pyrogenic exotoxin (SPE) showed no direct effect on rabbit macrophage functions in vitro. However, when splenic lymphocytes were added to macrophage cultures, SPE caused marked augmentation of glucose consumption and superoxide anion production, and concomitant inhibition of phagocytosis without loss of cell viability. The SPE effects were demonstrated to be mediated by a soluble factor(s) released from the splenic lymphocytes in response to SPE stimulus.