Dihydroxy and monohydroxy fatty acids in Legionella pneumophila

Five strains of Legionella pneumophila were examined for the presence of hydroxy fatty acid. The cellular distribution of the fatty acids was also determined, as was the variation of hydroxy acid production on five growth media. The strains tested all produced approximately 5 mol% of hydroxy fatty acid, most of which was found in the nonextractable, alkali-stable, acid-labile (wall-associated, amide-linked) fraction. Three major hydroxy acids were found, along with several minor components. The major hydroxy acids were analyzed by thin-layer chromatography, gas-liquid chromatography, mass spectrometry, and infrared spectrophotometry. These compounds were tentatively identified as 3-hydroxy-12-methyltridecanoate, 3-hydroxy-n-eicosanoate, and a novel dihydroxy acid, 2,3-dihydroxy-12-methyltridecanoate. The total amount of hydroxy acid produced, as well as the profile of the hydroxy acids, remained relatively unchanged with respect to strain and growth medium.     

Cellular distribution and linkage of D-(-)-3-hydroxy fatty acids in Bacteroides species.

Two strains of Bacteroides asaccharolyticus and two strains of Bacteroides fragilis were analyzed for total fatty acid, total lipid fatty acid, and total bound fatty acid profiles. Extracted lipids and defatted cell residues were subjected to sequential alkaline and acid methanolyses to distinguish ester- and amide-linked fatty acids in each fraction. In the lipid fractions, all the ester-linked fatty acids were nonhydroxylated, whereas all of the amide-linked fatty acids were hydroxylated. In the nonextractable fractions, both hydroxy and nonhydroxy fatty acids were found in both ester and amide linkage, although hydroxy acids predominated. The fatty acid profiles of the bound fractions differed widely from those of the lipid fractions. Bound fatty acid represented approximately 10% of the total cellular fatty acids.     

Structure determination of the polymorphism of acylgalactosylceramide in rat brain by gas chromatography/mass spectrometry and proton magnetic resonance

This paper describes the structure of acylcerebrosides isolated from rat brains. Three fractions (acylglycosylceramides I, II, III) were resolved according to their decreasing RF values on TLC. GLC analysis of acylglycosylceramides II and III indicates that their ester-linked fatty acids are short and rather unsaturated, while amide-linked fatty acids are longer and hydroxylated. Sugar GLC analysis indicates that acylglycosylceramides II and III contain only galactose. To determine the substitution position of the acyl group on the galactose moiety, the free hydroxyl groups of acylglycosylceramide were protected with dihydropyran, deacylated and subjected to permethylation. The methylated galactoside acetates obtained after hydrolysis and reduction were then analyzed by gas chromatography/mass spectrometry. Acylglycosylceramides II and III turned out to be complex mixtures of 2-O-acyl-, 3-O-acyl-, 4-O-acyl- and 6-O-acylgalactosylceramides. Moreover, the abundance of alpha-methylgalactoside reveals the existence of unsubstituted galactose, suggesting that some ester-linked fatty acids could be esterified to the hydroxyl group of hydroxy fatty acids linked to sphingosine. NMR spectrometry was used to confirm this ester linkage. The key spectral feature of the fatty acid-galactose linkage (4.45 ppm) did move to 4.15 ppm after saponification of acylglycosylceramide II; on the other hand, acylglycosylceramide III contained only the spectral feature 4.15 ppm, corresponding to a high percentage of unsubstituted galactose and consistent with the presence in the molecule of a fatty acid esterified by the omega-OH group of the hydroxy fatty acid (3.95 ppm).     

Nature and linkage type of fatty acids present in lipopolysaccharides of phase I and II Coxiella burnetii

The constituent fatty acids of lipopolysaccharides (LPS) of Coxiella burnetii (phase I and II) were qualitatively and quantitatively analysed by combined gas-liquid chromatography/mass spectrometry. The total fatty acid content (per mg LPS) was determined as 90.0 nmol (2.3 wt%) for LPS of phase I cells (LPS I) and 179.1 nmol (4.8 wt%) for LPS of phase II cells (LPS II). Of the 24 different acyl residues characterized (12 to 18 carbon atoms), nine were 3-hydroxy fatty acids (normal, iso- and anteiso-branched) which quantitatively predominated. All 3-hydroxylated fatty acids were found to possess the (R)-configuration, to be exclusively amide-linked and to be acylated at their 3-hydroxyl group. Ester-linked nonhydroxylated fatty acids (normal, iso- and anteiso-branched) were present but ester-bound 3-hydroxy- or 3-acyloxyacyl residues were lacking from C. burnetii LPS I and LPS II. As the major acyl group (R)-3-(12-methyl-tetradecanoyloxy)-12-methyl-tetradecanoic acid was identified. Our results show that the complex fatty acid spectrum of C. burnetii differs considerably from that of LPS of other Gram-negative bacteria. They further suggest an enormous heterogeneity of the lipid A component of C. burnetii LPS I and LPS II.     

A method for fractionation of cerebrosides into classes with different fatty acid compositions

A method is described for the separation of beef brain cerebrosides into three fractions containing different classes of fatty acids: nonhydroxy (I), unsaturated nonhydroxy (II), and hydroxy fatty acid cerebrosides (III). The procedure consists of benzoylation of either crude or purified cerebrosides, followed by column chromatographic separation of benzoylated derivatives containing nonhydroxy acids from those containing hydroxy fatty acids. The benzoyl groups are removed by sodium methoxide-catalyzed transesterification; from the reaction mixtures, fractions I and III precipitate. The fraction II present in mother liquor of I was shown to contain mainly short-chain and unsaturated nonhydroxy fatty acid cerebrosides. The fatty acid composition of each fraction was obtained by gas-liquid chromatography.     

Chemical and physical properties of lipopolysaccharide of Yersinia pestis

Lipopolysaccharide (LPS) prepared from Yersinia pestis 195/P contained d-glucose, d-glycero-d-mannoheptose, l-glycero-d-mannoheptose, glucosamine, 3-deoxyoctulosonic acid, lipid A, beta-hydroxymyristate, acetyl, phosphate, and protein. Traces of ethanolamine, mannose, and galactose were also detected. The lipid A moiety was composed of glucosamine substituted with phosphate, amide-linked beta-hydroxymyristate, and amide-bound acetate. The absence of significant amounts of additional fatty acids indicates a lipid A structure somewhat less complex than that of other gram-negative bacteria. The sugars identified are those generally found in the "core" region of LPS from the Enterobacteriaceae, with the exception of the d-glycero-d-mannoheptose. The molecular weight of the aggregated LPS was estimated to be 1.6 x 10(8).     

Chemical structure of the lipid A component of Plesiomonas shigelloides and its taxonomical significance

Abstract The chemical structure of the lipid A moiety of the lipopolysaccharide of the type strain of Plesiomonas shigelloides was elucidated. It consists of a β-(1 → 6)-linked glucosamine disaccharide carrying phosphate groups at C-1 of the reducing and at C-4' of the non-reducing glucosamine. It contains a total of 6 residues of fatty acids, 2 amide-linked and 4 ester-linked. The amino groups of the backbone disaccharide are N -acylated by substituted 3-hydroxyacyl residues: at the reducing glucosamine by 3-O-(14:0)14:0; and at the non-reducing glucosamine by 3-O-(12:0)14:0.
Two residues of 3-hydroxytetradecanoic acid are linked to C-3 and C-3' of the glucosamine residues; the hydroxy groups of these ester-linked 3-hydroxytetradecanoic acids are unsubstituted. In free lipid A, the hydroxyl groups at C-4 and C-6' are unsubstituted, indicating that the 2-keto-3-deoxyoctonic acid (KDO) is linked to C-6' of the non-reducing glucosamine, as was shown with enterobacterial lipid A. The taxonomical significance of these structural details is discussed.     

Analytical studies of lipopolysaccharide and its derivatives from Salmonella minnesota R595. III. Reappraisal of established methods

Established methods for analysis of components of lipopolysaccharides were assessed. Optimal release of glucosamine from lipopolysaccharide occurs after hydrolysis in 6 M hydrochloric acid at 100°C for 4 h and fatty acids are best released by treatment with boron trifluoride/methanol at 100°C for 6 h. The semicarbazide assay for 3-deoxy-d-manno-octulosonic acid was modified to give results comparable to those from the periodate/thiobarbituric acid method. It was concluded that each molecule of lipopolysaccharide from Salmonella minnesota R595 contains two octulosonic acid residues and only four fatty acids, on average. There are two amide-linked hydroxyacids, together with, on average, 0.5 residues of ester hydroxyacid and a total of 1.5 residues of ester-linked normal fatty acids. This conclusion differs from the accepted view of Salmonella lipid A, but is supported by NMR results.     

Biochemical studies on the cell wall lipopolysaccharides (O-antigens) of Vibrio cholerae 569 B (Inaba) and El-tor (Inaba).

Lipopolysaccharides were isolated from the cell walls of Vibrio cholerae 569 B (Inaba) and El-tor (Inaba). Chemical analysis revealed the presence of glucose, fructose, mannose, heptose, rhamnose, ethanolamine, fatty acids and glucosamine. The lipopolysaccharides do not contain 2-keto-3-deoxyoctonate, the typical linking sugar of polysaccharide and lipid moieties of enterobacterial lipopolysaccharides. Galactose, a typical core polysaccharide component of many gram-negative bacteria was also absent from lipopolysaccharides of these organisms. By hydrolysis in 1% acetic acid, the lipopolysaccharides have been separated into a polysaccharide part (degraded polysaccharide) and a lipid part (lipid A). Components of degraded polysaccharide and lipid A moiety were identified and determined. The lipid A fractions contained fatty acids, phosphorus and glucosamine. All the neutral sugars detected in lipopolysaccharides were shown to be the constituents of its polysaccharide moiety. The fatty acid analysis of lipopolysaccharide and lipid A showed the presence of both hydroxy and non hydroxy acids. They were different from those of lipids extracted from cell walls before the extraction of lipopolysaccharides. 3-Hydroxylauric and 3-hydroxymyristic acids predominated in lipopolysaccharide and lipid A of Vibrio cholerae and El-tor (Inaba).     

Chemical composition of a lipopolysaccharide from Legionella pneumophila

Lipopolysaccharide isolated from Legionella pneumophila (Phil. 1) was examined for chemical composition. The polysaccharide split off by mild acid hydrolysis contained rhamnose, mannose, glucose, quinovosamine, glucosamine and 2-keto-3-deoxyoctonate, in molar proportions 1.6:1.8:1.0:1.5:4.1:2.7. Heptoses were absent and glucose was probably mainly phosphorylated. The carbohydrate backbone of the lipid A part consisted of glucosamine, quinovosamine and glycerol, in the molar ratios 3.9:1.0:3.4, with glycerol as a phosphorylated moiety. A complex fatty acid substitution pattern comprising eight O-ester-linked, exclusively nonhydroxylated acids, and nineteen amide-linked, exclusively 3-hydroxylated acids was revealed. Both straight- and branched (iso and anteiso) carbon chains occurred. The major hydroxy fatty acid was 3-hydroxy-12-methyltridecanoic acid and six others were of a chain-length above 20 carbon atoms, with 3-hydroxy-20-methyldocosanoic acid as the longest. Two dihydroxy fatty acids, 2,3-dihydroxy-12-methyltridecanoic and 2,3-dihydroxytetradecanoic acids, were also detected. These results suggest that L. pneumophila contains a rather complex and unusual lipopolysaccharide structure of considerable biological and chemotaxonomic interest.Abbreviations LPS lipopolysaccharide - PS polysaccharide - KDO 2-keto-3-deoxy-octonate - GC gas chromatography - GC-MS gas chromatograph-mass spectrometer combined instrument - CI chemical ionization - EI electron impact - HF hydrofluoric acid - TFA trifluoroacetyl - TMS trimethylsilyl     

Chemical studies on the lipopolysaccharide of Rhizobium meliloti 10406 and its lipid A region

The structure of the lipopolysaccharide from Rhizobium meliloti 10406, a derivative of the wild-type strain MVII-1, was examined. The compositional analysis of its polysaccharide moiety demonstrated lack of heptose(s), but high contents in glucose, galacturonic acid and 2-keto-3-deoxy-octonate (dOclA) as characteristic features. The lipid A moiety consisted of a -1,6 linked glucosamine disaccharide carrying ester (at C-4) and glycosidically (at C-1) linked phosphate residues, both present exclusively as monoester phosphates but not as phosphodiesters. Ester- and amidelinked 3-hydroxy fatty acids were mostly present as non-3-O-acylated residues. Laser desorption mass spectrometry (LD-MS) revealed heterogeneity in the fatty acid substitution, as was also indicated by the non-stoichiometric ratios obtained by quantitative fatty acid analysis. The predominating lipid A structure contained at the reducing glucosamine residue ester-linked 3-hydroxy-tetradecanoic acid (3-OH-14:0) and amide-linked 3-OH-18:0, or 3-OH-18:1, respectively. The distal (non-reducing) glucosamine carried ester-bound the recently discovered 27-hydroxyoctacosanoic acid and 3-OH-14:0 and, as amide-linked fatty acid, mostly 3-hydroxy-stearic acid (3-OH-18:0).The isolated lipopolysaccharide exhibited a high extent of lethal toxicity in galactosamine-treated mice, comparable to that of enterobacterial lipopolysaccharide. The structural relationship of LPS and lipid A of Rhizobium meliloti to other rhizobial lipopolysaccharides and lipid A's with respect to questions of taxonomy and of phylogenetic relationships will be discussed.Abbreviations LPS lipopolysaccharide - dOclA 3-deoxy-D-mannooctulosonic acid (KDO) - GalA galacturonic acid - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - LD-MS laser desorption-mass spectrometry     

Structural studies on the lipid A component of enterobacterial lipopolysaccharides by laser desorption mass spectrometry. Location of acyl groups at the lipid A backbone

In the present paper laser desorption mass spectrometry (LDMS) was applied to dephosphorylated free lipid A preparations obtained from lipopolysaccharides of Re mutants of Salmonella minnesota, Escherichia coli and Proteus mirabilis. The purpose of this study was to elucidate the location of (R)-3-hydroxytetradecanoic acid and 3-O-acylated (R)-3-hydroxytetradecanoic acid residues which are bound to amino and hydroxyl groups of the glucosamine disaccharide backbone of lipid A. Based on the previous finding from biochemical analyses that the amino group of the nonreducing glucosamine residue (GlcN II) of the backbone carries, in S. minnesota and E. coli, 3-dodecanoyloxytetradecanoic acid and, in P. mirabilis, 3-tetradecanoyloxytetradecanoic acid, a self-consistent interpretation of the LDMS was possible. It was found that: (a) in all three lipids A GlcN II is, besides the amide-linked 3-acyloxyacyl residue, substituted by ester-linked 3-tetradecanoyloxytetradecanoic acid; (b) the reducing glucosamine (GlcN I) is substituted by ester-linked 3-hydroxytetradecanoic acid; (c) the amino group of GlcN I carries a 3-hydroxytetradecanoic acid which is non-acylated in E. coli and which is partially acylated by hexadecanoic acid in S. minnesota and P. mirabilis. In lipids A which were obtained from the P. mirabilis Re mutant grown at low temperature (12 degrees C) LDMS analysis revealed that specifically the one fatty acid bound to the 3-hydroxyl group of amide-linked 3-hydroxytetra-decanoic acid at GlcN II is positionally replaced by delta 9-hexadecenoic acid (palmitoleic acid). It appears, therefore, that enterobacterial lipids A resemble each other in that the 3-hydroxyl groups of the two 3-hydroxytetradecanoic acid residues linked to GlcN II are fully acylated, while those of the two 3-hydroxytetradecanoic acid groups attached to GlcN I are free or only partially substituted.     

Correlation of the biologic responses of C3H/HEJ mice to endotoxin with the chemical and structural properties of the lipopolysaccharides from Pseudomonas aeruginosa and Escherichia coli

The basis of the biologic responses of C3H/HeJ mice to endotoxin administration in relation to the structural linkages in the lipid A portion of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa and Escherichia coli were investigated. P. aeruginosa LPS was found to be immunogenic, mitogenic, and toxic, but not lethal, in C3H/HeJ mice. The observed mitogenicity in spleen cells was directed toward immunoglobulin- (Ig) bearing cells, was present in response to isolated and solubilized lipid A, and was inhibitable by polymixin B. The P. aeruginosa LPS was chemically analyzed in order to define its composition and exclude the presence of contaminating proteins being responsible for the biologic responses of C3H/HeJ mice that were observed. Structural analysis of the linkages of the fatty acids to the glucosamine backbone in the lipid A of P. aeruginosa and E. coli revealed similarities in terms of the ratio of hydroxy fatty acids to straight chain fatty acids and the way in which these 2 types of fatty acids were linked to the backbone. Differences were seen in the carbon chain length of the fatty acid substituents, and the substituent on the hydroxy fatty acid that is directly ester linked to the glucosamine backbone. These data indicate that the refractivity of C3H/HeJ mice to the biologic effects after the administration of Gram-negative endotoxins may be limited to enterobacterial LPS. Those differences we found in the chain length and/or linkages of the fatty acid substituents in the lipid A portion of the LPS between P. aeruginosa and E. coli may be sufficient to render C3H/HeJ mice responsive to the biologic effects of nonenterobacterial endotoxins.     

Ammonium hydroxide hydrolysis: a valuable support in the MALDI-TOF mass spectrometry analysis of Lipid A fatty acid distribution

Lipid A is the lipophilic moiety of lipopolysaccharides (LPSs), the major components of the external membrane of almost all gram-negative bacteria. It is responsible for the toxicity of LPS and has a heterogeneous structure composed of a bis-phosphorylated glucosamine disaccharide backbone that is acylated at the positions 2, 3 of the GlcN I (proximal) and GlcN II (distal) residue with O- and N-linked 3-hydroxy fatty acids (primary substitution). These fatty acids are further acylated by means of their 3-hydroxy groups (secondary substitution). The toxicity of Lipid A is dependent on its primary structure; the number, the length, and the distribution of the fatty acids on the disaccharide backbone strongly influence the endotoxic activity. In this paper a general and easy methodology to obtain secondary fatty acid distribution, which is one of the most difficult issues in the structural determination of Lipid A, is proposed. The method combines ammonium hydroxide hydrolysis and matrix assisted laser desorption ionization (MALDI)-mass spectrometry analysis and has been successfully proven with five different Lipid A species. The procedure exploits the lower stability under mild alkaline conditions of acyl and acyloxyacyl esters with respect to that of the acyl and acyloxyacyl amides. The partially degraded Lipid A species obtained are analyzed by MALDI-MS. The generality of this approach was tested on five Lipid As, namely those arising from Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas reactans, and Burkholderia caryophylli.     

Nature and location of amide-bound (R)-3-acyloxyacyl groups in lipid A of lipopolysaccharides from various gram-negative bacteria

It has previously been demonstrated [Eur. J. Biochem. 124, 191-198 (1982) and 137, 15-22 (1983)] that the lipid A component of Salmonella and Proteus lipopolysaccharides contains amide-linked (R)-3-acyloxyacyl residues. In the present study lipid A of other gram-negative bacteria was analysed for the presence of amide-bound 3-acyloxyacyl residues. It was found that such residues are constituents of all lipid A tested (Agrobacterium tumefaciens, Chromobacterium violaceum, Pseudomonas aeruginosa, Xanthomonas sinensis, Bacteroides fragilis, Vibrio cholerae, Fusobacterium nucleatum, Rhodospirillum tenue, Acinetobacter calcoaceticus, and Escherichia coli). Amide-linked (R)-3-acyloxyacyl groups, therefore, represent common and ubiquitous structural elements of bacterial lipid A. The composition of 3-acyloxyacyl groups differed considerably among different bacteria. As amide-bound (R)-3-hydroxy fatty acids straight chain and isobranched acyl groups with 10-17 carbon atoms were identified. The most frequently encountered fatty acids, substituting the 3-hydroxyl group of 3-hydroxy fatty acids, were nonhydroxylated straight chain and isobranched acyl residues with 10-17 carbon atoms as well as (S)-2-hydroxy fatty acids with 12 carbon atoms. In some cases, using laser desorption mass spectrometry, the distribution of 3-acyloxyacyl residues over the two available glucosamine amino groups of the lipid A backbone was investigated.     

Structural characterization of the lipid A component of Sinorhizobium sp. NGR234 rough and smooth form lipopolysaccharide. Demonstration that the distal amide-linked acyloxyacyl residue containing the long chain fatty acid is conserved in rhizobium and Sinorhizobium sp

A broad-host-range endosymbiont, Sinorhizobium sp. NGR234 is a component of several legume-symbiont model systems; however, there is little structural information on the cell surface glycoconjugates. NGR234 cells in free-living culture produce a major rough lipopolysaccharide (LPS, lacking O-chain) and a minor smooth LPS (containing O-chain), and the structure of the lipid A components was investigated by chemical analyses, mass spectrometry, and NMR spectroscopy of the underivatized lipids A. The lipid A from rough LPS is heterogeneous and consists of six major bisphosphorylated species that differ in acylation. Pentaacyl species (52%) are acylated at positions 2, 3, 2', and 3', and tetraacyl species (46%) lack an acyl group at C-3 of the proximal glucosamine. In contrast to Rhizobium etli and Rhizobium leguminosarum, the NGR234 lipid A contains a bisphosphorylated beta-(1' --> 6)-glucosamine disaccharide, typical of enterobacterial lipid A. However, NGR234 lipid A retains the unusual acylation pattern of R. etli lipid A, including the presence of a distal, amide-linked acyloxyacyl residue containing a long chain fatty acid (LCFA) (e.g. 29-hydroxytriacontanoate) attached as the secondary fatty acid. As in R. etli, a 4-carbon fatty acid, beta-hydroxybutyrate, is esterified to (omega - 1) of the LCFA forming an acyloxyacyl residue at that location. The NGR234 lipid A lacks all other ester-linked acyloxyacyl residues and shows extensive heterogeneity of the amide-linked fatty acids. The N-acyl heterogeneity, including unsaturation, is localized mainly to the proximal glucosamine. The lipid A from smooth LPS contains unique triacyl species (20%) that lack ester-linked fatty acids but retain bisphosphorylation and the LCFA-acyloxyacyl moiety. The unusual structural features shared with R. etli/R. leguminosarum lipid A may be essential for symbiosis.     

Characterization of lipid A and polysaccharide moieties of the lipopolysaccharides from Vibrio cholerae.

Lipid A and polysaccharide moieties obtained by mild acid hydrolysis of the lipopolysaccharides from Vibrio cholerae 569 B (Inaba) and Vibrio el-tor (Inaba) were characterized. Heterogeneity of lipid A fractions was indicated by t.l.c. and by gel filtration of the de-O-acylated products from mild alkaline methanolysis of the lipids. Presumably lipid A contains a glucosamine backbone, and the fatty acids are probably bound to the hydroxyl and amino groups of glucosamine residues. Approximately equal amounts of fatty acids C16:0, C18:1 and 3-hydroxylauric acid were involved in ester linkages, but 3-hydroxymyristic acid was the only amide-linked fatty acid. Sephadex chromatography of the polysaccharide moiety showed the presence of a high-molecular-weight heptose-free fraction and a low-molecular-weight heptose-containing fraction. Haemagglutination-inhibition assays of these fractions showed the heptose-free fraction to be an O-specific side-chain polysaccharide, whereas the heptose-containing fraction was the core polysaccharide region of the lipopolysaccharides. Identical results were obtained for both organisms.     

Extractable and lipopolysaccharide fatty acid and hydroxy acid profiles from Desulfovibrio species

An analysis of the phospholipid ester-linked and the lipopolysaccharide (LPS) fatty acids and hydroxy fatty acids of six lactate-utilizing Desulfovibrio-type sulfate-reducing bacteria (SRB) has been performed using capillary gas-liquid chromatography-mass spectrometry (GLC-MS). The concentrations of normal fatty acids were essentially similar, with the possible exception of a high content of normal fatty acids in the LPS of Desulfovibrio gigas. Determination of monounsaturated acid double bond configuration was performed by GLC-MS analysis of the derivatized fatty acids. A total of nine branched chain and eight straight chain monounsaturated fatty acids was detected in the Desulfovibrio species analyzed. The major component detected in five Desulfovibrio was the 17-carbon iso-branched monoenoic acid which showed cis unsaturation [i17:1(n-7)c] seven carbons from the terminal methyl group of the fatty acid chain. D. gigas, in contrast, contained almost no unsaturated fatty acids and was greatly enriched in iso-branched 15:0. Major differences between strains were found in the phospholipid and LPS hydroxy fatty acids. These components, in addition to the i17:1(n-7)c and other characteristic branched chain unsaturated acids, can possibly be utilized as signatures of the lactate-utilizing SRB.     

Characteristic Constituents of Glycolipids from Frog Brain and Sciatic Nerve

Cerebroside, sulfatide, monoglycosyl glyceride, and ester cerebroside were isolated from frog brain and sciatic nerve, and their distribution and chemical constituents were determined. The long-chain base compositions of cerebroside, sulfatide, and ester cerebroside were unique in the presence of branched-base components (5-15% of the total bases) and in the abundance of saturated dihydroxy base components (15-45% of the total). The amount of branched long-chain bases was greater in sciatic nerve than in brain. The hexose composition of the glycolipids consisted entirely of galactose except for brain cerebroside, in which a small amount of glucose was detected. Monogalactosyl glyceride consisted of the diacyl and alkylacyl forms, in a molar ratio of 81:19 for brain and 62:38 for sciatic nerve. The fatty acid composition of glycosphingolipids was characterized by the predominance of hydroxy and nonhydroxy 24:1 acids, and the concentration of 24:0 was extremely low. The proportion of unsaturated fatty acids accounted for 80% of the total. Major fatty acids of monogalactosyl glyceride were palmitic, oleic, stearic, and palmitoleic acids; the highest concentration was that of palmitic acid. Ester cerebroside was separated into three subfractions mainly on the basis of the proportion of hydroxy and nonhydroxy components in the amide-linked fatty acids.     

Fatty acid composition and Shwartzman activity of lipopolysaccharides from oral bacteria

The composition and the nature of the linkage of fatty acids and the Shwartzman activity of lipopolysaccharide (LPS) preparations derived from oral gram-negative bacteria including Bacteroides gingivalis, Bacteroides loesheii, Eikenella corrodens, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans were examined. 3-Hydroxylated and nonhydroxy fatty acids of various chain lengths were found in all of the LPS preparations. All nonhydroxy fatty acids were found to be ester-bound, and part of the 3-hydroxy fatty acids in the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans were shown to be involved in ester linkage. It was also suggested that the hydroxy group of the ester-bound 3-hydroxy fatty acid of the LPS of F. nucleatum and A. actinomycetemcomitans is at least partly substituted by another fatty acid, but in the LPS of B. gingivalis and E. corrodens it is not. The main amide-linked fatty acid of the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans was 3-hydroxyheptadecanoic, 3-hydroxydodecanoic, 3-hydroxyhexadecanoic, and 3-hydroxytetradecanoic acid, respectively. The results of the Shwartzman assay showed that the E. corrodens LPS was the most active among the preparations tested, and that the Shwartzman toxicity of Bacteroides LPS is extremely low.