From the cell envelope preparation of Sphingomonas paucimobilis two membrane fractions with diferent densities were separated by sucrose density gradient ultracentrifugation. The high-density fraction contained several major proteins, phospholipids, and glycosphingolipids, which are the only glycolipids of this lipopolysaccharide-lacking gram-negative bacterium. The low-density fraction showed many minor bands of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NADH oxidase activity was localized in this fraction. Combined with morphological data of vesicles formed by these membrane fractions, the high-density and low-density fractions were proposed to be an outer membrane and a cytoplasmic membrane, respectively. The localization of the glycosphingolipid was investigated also by means of immunoelectron microscopic analysis using a glycosphingolipid-specific antibody. The glycosphingolipid was shown to localize at the cell envelope, and the antigenic sugar portion was exposed to the bacterial cell surface. From these results the glycosphingolipid was assumed to have a function similar to that of the lipopolysaccharide of other gram-negative bacteria.Sphingomonas paucimobilis is an aerobic gram-negative rod formerly belonging to the genus Pseudomonas. The new genus Sphingomonas was recently proposed by Yabuuchi et al. (21) because many taxonomical data, including the base sequence of the 16S rRNA, revealed that S. paucimobilis is apart from the fluorescent group pseudomonads. The most remarkable difference of S. paucimobilis from other gram-negative bacteria is the presence of glycosphingolipid, which is usually a membrane component of eukaryotic cells (4), as the component of cellular lipids and the absence of lipopolysaccharide. Lipopolysaccharide is an amphiphilic molecule and a major component of the outer membrane of gram-negative bacteria, localizing at the outer leaflet of the membrane (17, 18). For this reason lipopolysaccharide was believed to be an essential component for all gram-negative bacteria. However, we reported in our previous paper (9) that S. paucimobilis harbored monosaccharide-type and tetrasaccharide-type glycosphingolipids and contained no lipopolysaccharide-like molecule. Those unexpected findings on the cellular lipids of S. paucimobilis prompted us to investigate the cell surface structure of this gram-negative bacterium and the physiological roles of the glycosphingolipids. In the present paper the separation of an outer and a cytoplasmic membrane and the biochemical and electron microscopic investigations for the localization of the glycosphingolipids are described.
MATERUILS AND METHODSBacterial strains and culture conditions. S. paucimobilis 1AM 12576 (originally NCTC 11030, type strain) was used in this study. Escherichia coli C600 (1) was used for the control experiment. Cells were grown in a medium and under conditions described previously (10).
284Isolation and separation of cell membranes. Membrane fractions were prepared by the method of Yamada et a...
This study examined the role of oxygen-derived free radicals in the pathogenesis of gastric mucosal lesions induced by HCl/ethanol. Superoxide dismutase, and catalase, and their combination reduced gastric lesion formation in mice. Gastric lesions were also reduced in mice treated with cyclophosphamide or anti-neutrophils, but not in mice treated with allopurinol or desulphated-carrageenan. Cobra venom factor did not reduce lesion formation. These results suggested that oxygen-free radicals may contribute to the formation of gastric mucosal lesions induced by HCl/ethanol, and that oxygen radicals were generated from neutrophils but not from xanthine oxidase. Anti-ulcer pectic polysaccharide, bupleuran 2IIc, which was recently isolated from the roots of Bupleurum falcatum L., showed potent inhibition of HCl/ethanol-induced gastric lesions in mice. Bupleuran 2IIc seemed to scavenge hydroxyl radical effectively. It was suggested that this anti-ulcer polysaccharide may provide protection to the gastric mucosa by scavenging oxygen-free radicals.
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