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The recently chemically synthesized Escherichia coli lipid A and the natural free lipid A of E. coli were compared with respect to their endotoxic activities in the following test systems: lethal toxicity, pyrogenicity, local Shwartzman reactivity, Limulus amoebocyte lysate gelation capacity, tumour necrotizing activity, B cell mitogenicity, induction of prostaglandin synthesis in macrophages, and antigenic specificity. It was found that synthetic and natural free lipid A exhibit identical activities and are indistinguishable in all tests. [ABSTRACT FROM AUTHOR]

Lipopolysaccharides interact with complement only when they are present in a state of high aggregation with a high apparent molecular weight. Lipopolysaccharides in uniform salt forms prepared by electrodialysis and neutralization with different bases exhibited distinct differences in their anticomplementary activity which correlated with differences in their sedimentation co-efficients. Conversion of smooth (S) form lipopolysaccharides into the low-molecular-weight triethylamine form completely abolished their anti-complementary activity while conversion into the high-molecular-weight sodium form increased their activity. In contrast, a similar treatment of highly defective Re and Rd rough ® form lipopolysaccharides had no effect on their ability to interact with complement. Both the triethylamine and sodium forms were strongly anti-complementary despite large differences in their molecular weight. This was found to be due to the property of R lipopolysaccharides to reaggregate into a large-molecular-weight form through absorption of Mg2+ and Ca2+ present in the guinea pig serum used as complement source. Defective lipopolysaccharides derived from the Ra and Rb classes showed only negligible anti-complementary activity which did not increase by conversion into salt forms with high molecular weight. [ABSTRACT FROM AUTHOR]

Ions of low molecular weight like metal cations and basic amines are present in lipopolysaccharides regardless of the isolation procedure employed. They are present in salt form with the acidic groups of the molecule and, partly, bound by chelation. Electrodialysis which removed a large proportion of these basic materials led to acidic lipopolysaccharides often with reduced solubility. Electrodialyzed lipopolysaccharides could be rendered soluble by neutralizing with alkali or with a basic amine. Depending on the base employed for neutralization preparations were obtained which showed in water distinct differences in solubility, viscosity and opalescence. These differences were related to differences in the sedimentation coefficients of the various salt forms. Neutralization with triethylamine led in all cases to highly soluble preparations with low sedimentation coefficients, while, on the other hand, neutralization with Mg(OH)2 led in most cases to insoluble preparations. The acidic lipopolysaccharides obtained by electrodialysis deteriorate on storing in a freeze-dried form. On heating in distilled water autohydrolysis occurs and free lipid A is liberated. The lipid A which is so far known as a water-insoluble material showed increased solubility when prepared from electrodialyzed lipopolysaccharides. [ABSTRACT FROM AUTHOR]

A simple and quick procedure was used to analyse the carbohydrate composition of surface glycoproteins from chicken lymphocytes. The procedure included papain digestion of the cells, a two-step purification of the supernatant material and a sugar analysis by gaschromatography. The method made it possible to analyse samples of about 109 lymphocytes. The surface glycoproteins from different chicken lymphocyte preparations were found to differ significantly in their sugar composition. Lymphocytes from thymus, bursa, spleen or blood were characterized by typical relative amounts of glucose, galactosamine, fucose and sialic acid. The values for mannose, glucosamine and galactose, however, were approximately 1:1:1 for all four lymphocyte preparations. Bursectomy or thymectomy in combination with whole body irradiation altered the carbohydrate composition significantly. The results suggest the possibility that the carbohydrate composition can be used as a marker for different lymphocyte populations. The results are also discussed in respect to the hypothesis that carbohydrate determinants on the cell surface determine the migration of lymphocytes. [ABSTRACT FROM AUTHOR]

Reports on the in vitro deacylation of lipopolysaccharide of Salmonella minnesota by Acanthamoeba castellanii enzymes. Exhibition of amidase activity by the cell-free preparation; Binding to the oligosaccharide part of the lipopolysaccharide.

Free flow electrophoresis was shown to be a useful tool to enrich for mutants Conditionally defective in lipid A synthesis. The method was based on the observation that electrophoretic mobility of bacterial cells is dependent on the structure of lipopolysaccharides and is influenced by lesions in the synthesis of the O-specific chains as well as by lesion in the synthesis of the complete 3-deoxy-D-manno-octulosonic acid (dOclA) lipid A region. Using this procedure a new mutant conditionally defective in dOclA-8-P synthesis was isolated (mutant Ts5). Following a shift to nonpermissive conditions it accumulates a mixture of at least two equally represented lipid A precursor structures. One is made up of glucosamine, phosphate and 3-hydroxymyristic acid in a molar ratio 1.0:1.0:2.0 and lacks dOclA and the nonhydroxylated fatty acids lauric, myristic and palmitic acid (precursor I a). The other one (precursor I b) has the same basic composition with additional palmitic acid. The precursor preparation derived from mutant Ts5 titus differs from previously described lipid A intermediates by the relatively high substitution by palmitic acid. The implications of the above findings to the biosynthesis of lipid A are discussed. [ABSTRACT FROM AUTHOR]

It has previously been demonstrated [Eur. J. Biochem. 124, 191–198 (1982) and 137, 15–22 (1983)] that the lipid A component of Salmnonella 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, Bacteriodes 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. [ABSTRACT FROM AUTHOR]

Synthetic lipid A part structures corresponding structurally to a biosynthetic lipid A disaccharide precursor have been analyzed for endotoxic activity in several systems in vivo and in vitro. It was found that a synthetic β-1,6-linked D-glucosamine disaccharide, which carries four molar equivalents of (R)-3-hydroxyletradecanoyl residues in positions 2, 3, 2' and 3' and phosphoryl groups in positions 1 and 4' (preparation 406), exhibited lethal toxicity, B lymphocyte mitogenicity, the capacity to engender prostaglandin formation in macrophages and to induce endotoxic tolerance, as well as serological lipid A antigenicity. On a weight basis, preparation 406 was of comparable activity to lipid A precursor and bacterial free lipid A. Preparation 406, like lipid A precursor, lacked, however, the ability to induce the local Shwartzman phenomenon and both preparations were of moderate pyrogenicity. Two further synthetic analogues which contained only one phosphoryl group (preparation 404 at C-4', preparation 405 at C-1) showed comparable diminished activity depending on the test system employed, except in the capacity to inactivate complement where they exhibited, in contrast to preparation 406, significant activity. The results show that the endotoxic principle of lipopolysaccharides, as postulated previously is embedded in the lipid A component. Our results also suggest initial conclusions on the structured requirements for the expression of endotoxin activities. [ABSTRACT FROM AUTHOR]

Different applications of the thiobarbituric acid assay were used to determine C4/5-unsubstituted, heptosyl-substituted and total 3-deoxy-D-mannooctulosonic acid (dOclA) in lipopolysaccharides of Salmonella minnesota rough mutants. C4/5-unsubstituted dOclA is determined without prior hydrolysis of the lipopolysaccharides by periodate oxidation near neutral pH at 4 °C using the methyl ketoside of dOclA as a standard. C4/5-unsubstituted dOclA, and dOclA which is substituted by other dOclA residues, is quantified after hydrolysis in 0.1 M acetate buffer pH 4.4. at 100deg;C/1 h. Total dOclA, including heptosyl-substituted dOclA, is measured after hydrolysis in 1 M HCl at 100°C for 1–4 h. For S. minnesota R595, 760–790 nmol/mg lipopolysaccharide are determined independently of the hydrolysis conditions employed. In R7, one half, and in R345, two thirds of the total dOclA content are detected after mild acid hydrolysis. Hydrolysis in 1 M HCl at 100 °C for 1 h (R7) and 4 h (R345) are required to liberate the heptosyl-substituted dOclA residues. For the three lipopolysaccharides tested, determination of C4/5-unsubstituted dOclA revealed that about one third of the total dOclA is reactive in this application of the thiobarbituric acid assay. By strong acid hydrolysis, a 3-deoxy-2-ketoaldonic acid is identified in the lipopolysaccharide of Vibrio cholerae. Upon mild and strong acid hydrolysis, dOclA undergoes degradation which is recognized by (a) decreased reactivity in the thiobarbituric acid assay, (b)changed ultraviolet spectra and (c) development of compounds which are reactive in the thiobarbituric acid assay after reduction with sodium borohydride. The rate of degradation depends on the strength of the acid but is the same for reference and lipopolysaccharide-liberated dOclA under a given hydrolysis condition. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide from a strain of Aeromonas salmonicida salmonicida was isolated from cells by the aqueous phenol method in 2.3% yield (based on dry weight of bacteria). Hydrolysis of the lipopolysaccharide in 1% acetic acid afforded O-polysaccharide (19% by weight), core-oligosaccharide (12.2%) and lipid A (44.6%). Analysis indicated that 3-deoxy-D-manno-2-octulosonic acid was absent from the lipopolysaccharide and that no low-molecular-weight compounds were released by the mild hydrolysis. The O-polysaccharide had the monosaccharide composition of rhamnose, glucose and N-acetylmannosamine in molar ratio of 1.0: 1.58:0.83. 75% of the N-acetylmannosamine residues were substituted at position 4 by O-acetyl groups. Hydrolysis of the methylated polysaccharide proved to be both difficult and dependent on the method of hydrolysis chosen, in all cases a partially methylated disaccharide of rhamnose and N-acetylmannosamine was identified in the hydrolysate. Methylation analysis, periodate oxidation and proton magnetic resonance analysis were used to confirm the structure of the repeating unit as: [ABSTRACT FROM AUTHOR]

Salmonella minnesota R 595 (Rc) and other Salmonella strains incorporate cis-Δ9-16:1 (paimitoleic acid) at the expense of mainly dodecanoic acid into the lipid-A portion of lipopolysaccharides, when the cells are grown at low temperature (12 °C). It has recently been shown, that in S. minnesota R 595 grown at 37 °C dodecanoic acid is linked to the 3-hydroxyl group of an amide-bound 3-hydroxytetradecanoic acid. The present data suggest, that cis-Δ9-16:1 occupies the same position in lipid A (12 °C). [ABSTRACT FROM AUTHOR]

Fractionation of the O-polysaccharide derived from Salmonella zuerich (1, 9, 27, 46) on a concanavalin A polymer permitted immunological and chemical analysis on the different fractions. The S. zuerich O-polysaccharide preparation is composed of two distinct populations of molecules: one, ZB1-, devoid of O-antigenic determinant 1, and the other, ZB1+, carrying the determinant 1. This determinant is linked to the presence of D-glucosyl residues on the side chain. O-polysaccharide molecules 1-, devoid of D-glucose, are shown to carry simultaneously both determinants 27 and 46. These determinants are not evenly distributed on the molecules. The expression of determinants 46 (-[Tyv]-βMan-, where Tyv = tyvelose) seems to he restricted to a distinct specific configuration, and it is altered by the presence of either determinant 27 (-[Tyv]-αMan-) or determinant 1 (Glc-Gal-) in the close neighbourhood. Molecules 1+ are partially or completely substituted by glucosyl residues and react with anti-1 antibodies. They are characterized by the same uneven distribution of determinants 27 and 46 as molecules ZB1-. In conclusion, the O-polysaccharide chains are heterogeneous. They contain simultaneously factors 27, 46, and often also 1. [ABSTRACT FROM AUTHOR]

In Salmonella minnesota lipopolysaccharide the lipid A backbone, a substituted diphosphorylated β, 6-linked D-glucosamine disaccharide molecule, carries approximately seven residues of fatty acids: one each of dodecanoic, hexadecanoic, n-3-hydroxytetradecanoic and D-3-O-(tetradecanoyl)-tetradecanoic acid in ester linkage and two of D-3-hydroxytetradecanoic acid in amide linkage. In the present study it is shown that treatment of the lipopolysaccharide with alkali at elevated temperature leads, through a β-elimination reaction, to the generation of amide- bound Δ2-tetradecenoic acid. This suggested that the 3-hydroxyl group of amide-bound hydroxy fatty acids carried a substituent. To elucidate the nature of the substituent, free Salmonella lipid A was methylated with methyl iodine in the presence of silver salts followed by mild acid hydrolysis, a procedure which is known to cleave amide (and not ester) bonds selectively. In the hydrolysate, by means of combined gas-liquid chromatography/mass spectrometry the methyl esters of 3-O-dodecanoyl)-tetradecanoic and 3-O-hexadecanoyl)-tetradecanoic acid were identified, This shows that in Lipid A amide-linked 3-hydroxytetradecanoic acid residues are 3-O-acylitted by dodecanoic and hexadecanoic acid, respectively. Quantitative analyses suggest that the Salmonella lipid A backbone is substituted by four D-3-hydroxytetra- decanoyl residues., two being present as esters and two as amicles. The nonhydroxylated fatty acids are not bound directly to the backbone. Rather, they are attached to hydroxyl groups of 3-hydroxytetradecanoyl residues: specifically, tetradecanoic acid substitutes ester-bound and dodecanoic and hexadecanoic acid amide-bound 3-hydroxytetradecanoic acid. [ABSTRACT FROM AUTHOR]

The chemical structure of the lipid A component of the lipopolysaccharide from Vibrio cholerae95R was studied. After sequential degradation a reduced D-glucosamine disaccharide was isolated from lipid A and, after permethylation, shown by combined gas-liquid chromatography/mass spectrometry to be β1,6-linked. The disaccharide is substituted with a phosphate group, ester-bound to the non-reducing glucosamine (GlcN) residue and a pyrophosphorylethanolamine group (PP-Etn) linked to C-1 of the reducing glucosamine residue. This backbone structure is shown in the following formula: P-GIcN(β1-6)GIcN-1-PP-Etn. The amino groups of the glucosamine disaccharide are substituted by D-3-hydroxytetradecanoic acid; tetradecanoic, hexadecanoic and a D-3-O-(D-3-hydroxydodecanoyl)-dodecanoic acid residue are linked to hydroxyl groups. A similar fatty acid composition was detected in lipopolysaccharides from Inaba, Ogawa and NAG strains of V. cholerae. [ABSTRACT FROM AUTHOR]

The article presents a study which examines the effect of endotoxin and lipoteichoic acid (LTA) on bone resorption. The goal of the study is mentioned, which was performed in order to ascertain the portion of the endotoxin and LTA molecules necessary to preserve their effect of encouraging bone resorption. Several endotoxins from Salmonella minnesota were compared, including endotoxins from the smooth form, the mutant form R-345, and the mutant form R-595. LTA was compared with a preparation lacking fatty acids, noting the goal of learning whether lipopolysaccharide-stimulated bone resorption is mediated by bone prostaglandin synthesis.

Lipopolysaccharides of Salmonella minnesota rough mutants were treated with 20 mM acetate buffer pH 4.4 at 70°C/3 h. Alter dialysis of the hydrolysates about one third of the total 3-deoxy-D-mannooctulosonic acid (dOclA) content but no neutral sugars were found in the dialysate. By high-voltage paper electrophoresis, a com- pound with the mobility of 1.2 relative to dOclA could be isolated from the dialysate. It was identified as a dOclA disaccharide by hydrolysis without or after reduction with sodium borohydride and by analysis with the thiobarbituric acid assay under different conditions. The ketosidic linkage in the disaccharide is assumed to be 2.4 or 2.5. [ABSTRACT FROM AUTHOR]