Pseudomonas aeruginosa lectins interact with Escherichia coli strains O86B7 and O128B12, which possess B and H (O) blood group determinants, respectively. The interaction could be demonstrated by specific agglutination of the bacteria, by haemagglutination inhibition tests and by lectin-mediated peroxidase binding to the bacteria. The agglutination of E. coli O86B7 by the Pseudomonas galactose-binding lectin was inhibited by D-galactose and by the lipopolysaccharide extracted from E. coli O86B7. Similarly, the specific agglutination of E. coli O128B12 by the Pseudomonas mannose-binding lectin (which also binds L-fucose, L-galactose and D-fructose) was inhibited by D-mannose, L-fucose, L-galactose and D-fructose, as well as by athe lipopolysaccharide extracted from E. coli O128B12. The interaction between E. coli O128B12 and the Pseudomonas mannose-binding lectin was also demonstrated by lectin-mediated peroxidase binding to the bacterial surface. Peroxidase binding was also inhibited by the above-mentioned sugars and E. coli O128B12 lipopolysaccharide. Treatment of cells of the two E. coli strains with protein-denaturing agents did not reduce their agglutination by the Pseudomonas lectins. On the other hand, oxidation of the cell surface sugars by sodium metaperiodate or boiling the cells in the presence of 1% acetic acid for 1 h abolished their agglutination by the two lectins. It is, therefore, suggested that the Pseudomonas lectins interact with the B and H (O) blood group determinant sugars (D-galactose in E. coli O86B7 and L-fucose in E. coli O128B12) residing in the lipopolysaccharides of these E. coli strains.
A simple and rapid method for the quantitation of total cholesterol in lipid extracts using gas-liquid chromatography is presented here as a modification of an earlier saponification procedure (Ishikawa, T. T., J. MacGee, J. A. Morrison, and C. J. Glueck. 1974. Quantitative analysis of cholesterol in 5 to 20 microliters of plasma. J. Lipid Res. 15: 286-291). Using the original method, as well as a slightly modified version, we found a systematic loss of cholesterol measured as total cholesterol that was attributable to the formation of a byproduct during the procedure. Depending on the nature of the solvent mixture used for extraction after saponification, different byproducts were produced that had longer retention times than cholesterol. The byproducts were identified as cholesteryl butyrate (produced when methyl butyrate was included in the solvent mix) and cholesteryl propionate (with ethyl propionate in the solvent mix) by comparison to authentic standards using gas chromatography-mass spectroscopy. Using mixtures of cholesterol standards, we compared several solvents in lieu of the solvent mixture used in the original extraction procedure to identify those that eliminate the formation of the byproducts. Our optimized microsaponification procedure uses a single solvent, tetrachloroethylene, to extract lipids after the saponification reaction, and improves the accuracy of the cholesterol determination.
Plasma levels of high density lipoprotein (HDL) cholesterol and its major protein component apolipoprotein (apo) A-I are significantly reduced in both acute and chronic inflammatory conditions, but the basis for this phenomenon is not well understood. We hypothesized that secretory phospholipase A2(sPLA2), an acute phase protein that has been found in association with HDL, promotes HDL catabolism. A series of HDL metabolic studies were performed in transgenic mice that specifically overexpress human sPLA2 but have no evidence of local or systemic inflammation. We found that HDL isolated from these mice have a significantly lower phospholipid and cholesteryl ester and significantly greater triglyceride content. The fractional catabolic rate (FCR) of 125I-HDL was significantly faster in sPLA2 transgenic mice (4.08 ± 0.01 pools/day) compared with control wild-type littermates (2.16 ± 0.48 pools/day). 125I-HDL isolated from sPLA2transgenic mice was catabolized significantly faster than131I-HDL isolated from wild-type mice after injection in wild-type mice (p < 0.001). Injection of125I-tyramine-cellobiose-HDL demonstrated significantly greater degradation of HDL apolipoproteins in the kidneys of sPLA2 transgenic mice compared with control mice (p < 0.05). The fractional catabolic rate of [3H]cholesteryl ether HDL was significantly faster in sPLA2-overexpressing mice (6.48 ± 0.24 pools/day) compared with controls (4.80 ± 0.72 pools/day). Uptake of [3H] cholesteryl ether into the livers and adrenals of sPLA2 transgenic mice was significantly enhanced compared with control mice. In summary, these data demonstrate that overexpression of sPLA2 alone in the absence of inflammation causes profound alterations of HDL metabolism in vivo and are consistent with the hypothesis that sPLA2 may promote HDL catabolism in acute and chronic inflammatory conditions.
Differential scanning calorimetry and polarizing light microscopy have been used to investigate kinetic and thermodynamic properties of the phase behavior of cholesteryl ester contained in Fu5AH rat hepatoma cells and J774 murine macrophages. These cultured cells store cholesteryl esters as cytoplasmic inclusions of approximately 1-micron diameter and thus are models of the foam cells characteristic of atherosclerotic plaque. Simple binary mixtures of cholesteryl palmitate and cholesteryl oleate, the predominant cholesteryl esters in cellular inclusions in both cell types serve as models to explain important aspects of the phase behavior of these inclusions. Although inclusions should exist as stable crystals at 37 degrees C under conditions of thermodynamic equilibrium, microscopic examination of cells indicates that inclusions exist as metastable liquid crystals at 37 degrees C for extended periods of time. Using an analytical model based on nucleation theory, we predict that the cholesteryl ester inclusions should be liquid-crystalline in the cytoplasm of living cells. This may not be true either for lysosomal cholesteryl ester or for extracellular cholesteryl ester present in advanced atherosclerotic plaque where fusion of droplets can enhance the possibility of crystallization. The enhanced metastability of the relatively fluid liquid-crystalline state in cellular inclusions should result in increased activity of the neutral cholesteryl ester hydrolase in living cells.
