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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]

Comparative chemical analyses have been performed on the lipopolysacharides of a Salmonella T2 form and of a T2-mutant derived from it and found to belong to the chemotype Ra. Methylation analysis showed that the T2 structure contains an N-acylglucosamine residue linked to position 4 of the subterminal glucose II unit of the core polysaccharide. The N-acyl group and a substituent which renders this glucosamine periodate resistant have not been identified. The structure of the T2-specifie region was further studied by the analysis of two fragments. One was obtained after Smith degradation and had the structure glucosaminyl-1,4-glucosyl-glyceraldehyde, the amino sugar carrying the N-acyl group and the unknown substituent. The other fragment obtained after hydrazino1ysis and acid hydrolysis was the trisaccharide GlcN-1,4-(GlcN-1,2)-Glc. The results show that the T2-specific structure is represented by a substituted N-acylglucosamine unit which is attached to the core at the same position as the T1- and the O-specific structures in the corresponding lipopolysaccharides. These results are in agreement with findings obtained with a T2,SR(4,12) form and the corresponding T2- mutant. [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]

In order to investigate the core structure of the lipopolysaccharide from an Escherichia coli 0100 strain, two derivatives were prepared from this strain by introducing genetic material from Salmonella abony. One, an SR hybrid, contained a lipopolysaccharide with short side chains consisting of simple repeating units of Salmonella group B specificity attached to the E. coli core. The other, an R form, contained only the E. coli core and no O side chains, due to a mutation in the Salmonella rfb chromosomal region, which determines the synthesis of the O repeating units. By partial acid hydrolysis of the SR and R lipopolysaccharides, oligosaccharide fragments were obtained which made it possible to reconstruct the hexose region of the core (see formula below). Methylation studies performed on the dephosphorylated polysaccharides confirmed and extended these results. It was found that only about 60% of the distal glucose units of the core chains were substituted by N-acetylglucosamine residues. Further, the heptose region was shown to form a branched trisaccharide. On mild acid hydrolysis of both lipopolysaccharide preparations a Gal-α1,7(8)-KDO disaccharide was formed, indicating that a galactose unit is linked to the 2-keto-3-deoxyoctonate (KDO) portion of the hpopolysaccharide. After Smith degradation of the SR lipopolysaccharide, the oligosaccharide Gal-β1,4-Glcglycerol was identified which derived from the linkage region between the Salmonella type repeating unit and the E. coli core. Only the core stubs substituted by N-acetylglucosamine residues carried an O-specific unit. On the basis of the these findings the partial structure of the E. coli 0100 core polysaccharide with a Salmonella group B-specific unit attached to it is proposed as given below. The relationships to core structures identified in lipopolysaccharides of other representatives of Enterobacteriaceae are discussed. [ABSTRACT FROM AUTHOR]

An investigation on the sugar compositions of lipopolysaccharides (O antigens) derived from 45 well—defined Citrobacter serotypes was carried out. As a result, a chemical classification into 20 chemotypes was achieved, of which 11 were identical with chemotypes occurring in Salmonella and Escherichia coli. All Citrobacter lipopolysaccharides contained glucosamine, glucose, heptose, 2-keto-3-deoxyoctonate and galactose, the latter, in some cases, in very small amounts. These sugars probably represent building blocks of the basal core polysaccharides, whose structures however may not be identical for all Citrobacter lipopolysaccharides. In addition to the basal sugars, the following monosaccharides were detected as constituents of the various Citrobacter lipopolysaccharides; D-xylose, D-mannose, fucose, rhamnose, 6-deoxylatose, 4-deoxy-D-idose, abequose, galactosamine, D-fucosamine, 3-amino-3,6-dideoxy-glucose and -galactose. The sugar compositions of serologically related Citrobacter and Salmonella O antigens were compared. While some cross-reacting pairs belonged to the same chemotype, other proved to be distinct with respect to their sugar constituents. [ABSTRACT FROM AUTHOR]

Lipopolysaccharides of Salmonella T1 forms contain ribose and galactose both present as furanosides. Mild periodate oxidation of the Ti lipopolysaccharide followed by treatment with alkali resulted m the formation of a main fragment which was identified as poly-ribose linked to the core polysaccharide. Poly-ribose was also identified as a degradation product of periodate oxidized T1 lipopolysaccharide which had been treated with mild acid. Another fragment formed under these conditions was identified as being derived from -6-Galf-1,3-Galf-1,3-Galf-1,6-Galf-1-. These results indicated that the T1-specific polysaccharide of T1 lipopolysaccharides contain poly-ribose and poly-galactose regions. Galactose-trisaccharide units may be present as repeating units of a poly-galactose chain. [ABSTRACT FROM AUTHOR]

Two lipopolysaccharide were analyzed comparatively: the lipopolysaccharide derived from a Salmonella typhimurium SR mutant, containing single repeating units linked to the core polysaccharide, and a lipopolysaccharide derived from a corresponding Ra mutant containing core polysaccharide only. Partial hydrolysis of these lipopolysaccharides led to the formation of a group of oligosaccharides occurring in both lipopolysaccharides. Their structural analysis provided information for the reconstruction of the hexose region of the core. From the SR lipopolysaccharide, four oligosaccharides were identified which were absent from the Ra lipopolysaccharide: (a) Man-α1,4-rhamnose (derived from the repeating units); (b) Gal-β1,4Glc; (c) Gal-β1,4-(GlcNAc-α1,2-)Glc; (d) Gal-β1,4Glc-α1,2-Gal. Oligosaccharides (b), (c), and (d) are derived from the linkage region between the repeating unit and the core. Methylation studies which were performed on the SR and Ra lipopolysaccharides led to the identification of the sugar linkages. On the basis of the present investigation, the partial structure of the SR lipopolysaccharide can be drawn as shown below, which confirms and extends previous results. The findings are discussed in regard to the biosynthesis and immunochemistry of SR and S form lipopolysaccharides. [ABSTRACT FROM AUTHOR]

Degradation studies performed on the original or dephosphorylated core oligosaccharides derived from lipopolysaccharides of Salmonella Ra, Rb, and RcP+ mutants revealed the presence of a branched heptose trisaccharide, -3(HepIII-1,7-)-HepII-1,3-HepI-. The branching heptose III is linked to position 7 of heptose II. Heptose II is also substituted (at either position 2, 4, or 6) by a phosphate group. Other previously identified substituents of the heptose units are a pyrophosphoryl-ethanolamine group linked to position 4 of heptose I and the hexose oligosaccharide of the core linked to position 3 of heptose II. The degree of substitution of the heptose main chain with heptose III varies in different lipopolysaccharides from 20 to 90%. A lipopolysaccharide of chemotype RcP- was analyzed and found not to contain the third heptose unit. [ABSTRACT FROM AUTHOR]

Salmonella typhimurium rfa-657 strains, including both mutants and transductants, were shown to contain in their lipopolysaccharides both the (usual) L and the D-isomer of glycero-D-manno-heptose. By the application, in sequence, of two different extraction procedures S and R form lipopolysaccharides could be isolated separately from a representative rfa-657 strain. The R lipopolysaccharide was predominantly of the Re chemotype, only a few 2-keto-3-deoxyoctonate (KDO) residues being substituted by heptose (Rd2 chemotype) or by longer core chains. The D-heptose was enriched in the R lipopolysaccharide, while the S lipopolysaccharide contained mainly the natural L-isomer. It was found that D-heptose can replace L-heptoses I to III of the wild-type core. However, an elongation to completeness of the core stubs containing D-heptose seldom occurs. The heterogeneity in structure of the lipopolysaccharide produced by this class of (leaky) mutants is assumed to be due to an altered 6-epimerase which, in the wild form, catalyses the conversion of NDP-D-glycero-o-manno-heptose into NDP-L-glycero-D-manno-heptose and which is determined by the gene rfa-657 situated between cysE and pyrE. The symbol rfaD is proposed for this gene. [ABSTRACT FROM AUTHOR]

Lipid A preparations from the lipopolysaccharides of four Salmonella minnesota R mutants and one Escherichia coli 0100 R mutant were assayed as soluble complexes with bovine serum albumin for lethality in mice, pyrogenicity in rabbis and complement inactivation. Although generally less active than endotoxic lipopolysaceharides, these lipid · albumin complexes nevertheless exhibited strong biological activity. These results indicate that lipid A contains the endotoxic principle of bacterial lipopolysaccharides. [ABSTRACT FROM AUTHOR]

Lauric, myristic, palmitic, and 3-D-(—)-myristic acid in a ratio of about 1:1:1:3 are present in the lipid A part of the glycolipid derived from the Re mutant Salmonella, minnesota R595. An additional fatty acid, Δ2-tetradecenoic acid found in larger amounts after alkaline treatment was recognized as an artifact formed by a β-elimination reaction from ester-linked 3-myristoxymyristic acid. Various methods for the stepwise liberation of fatty acids were used to identify their distribution on the backbone of lipid A which consists β-1,6-linked glucosamine disaccharide units substituted at position 3' with 2-keto-3-deoxyoctonate acid at positions 1 and 4' with phosphate. It was found that the amino groups of the glucosamine residues are substituted by 3-D.(—)-hydroxymyristic acid whose hydroxyl group is free. The three available hydroxyl groups at positions 3, 4, and 6' of the glucosamine disaccharide are substituted by equal amounts of lauric, palmitic, and 3-D-myristoxymyristic acid and, possibly, by smaller amounts of myristic and unsubstituted 3-D-hydroxymyristic acid. A similar structure of lipid A seems to exist in lipopolysaccharides of other Salmonella species. [ABSTRACT FROM AUTHOR]

The cell wall glycolipid (somatic antigen) of the Re mutant of Salmonella minnesota is comparable in its endotoxic activity to the complete wild type lipopolysaccharide. The glycolipid is composed mainly of lipid A and 2-keto-3-deoxyoctonate (KDO); smaller amounts of ethanolamine and 4-aminoarabinose, and traces of amino acids are also present. The serological specificity is determined by KDO residues. In order to determine if non-lipid A constituents contribute to endotoxicity, amino groups and KDO units in the glycolipid were chemically modified by the introduction of substituents. When free amino groups were dinitrophenylated or succinylated the serological specificity was unaffected. By contrast, the original serological specificity was completely lost when the free hydroxyl groups of the KDO region were substituted with succinyl residues or the carboxyl groups converted to the methyl esters. In biological tests, however, the modified preparations exhibited full endotoxic activity. It is concluded that the polar groups, which determine the serological specificity of the KDO region of the glycolipid, do not play a specific role in endotoxicity. [ABSTRACT FROM AUTHOR]

A procedure is described for the preparation of liposomes with either lecithin or sphingomyelin in the presence of various Salmonella minnesota S and R form lipopolysaccharides (antigens). These liposomes release trapped glucose marker when incubated with an appropriate rabbit antiserum (as source of antibodies) and native, is. unheated, guinea pig serum (as source of complement). The extent of marker loss is dependent on the amount of antigen added; treatment of R or S form lipopolysaccharide with alkali markedly reduces the amount necessary for half-maximal sensitization, I e. half-maximal glucose release. The alkali-treated lipopolysaccharides are also effective in "passive" sensitization of liposomes. In this procedure (in contrast to the above which has been designated "active" sensitization), the liposomes are first generated in the absence of any antigen and subsequently incubated with lipopolysaccharide, Both actively and passively sensitized liposomes lose a greater percentage of their trapped glucose when prepared with lecithin than with sphingomyelin. The properties of the lipopolysaccharide liposomes are compared with those containing various ceramide antigens as described previously. [ABSTRACT FROM AUTHOR]

The anomeric configuration of mannose is α in the O antigens of Salmonella groups B and D, and β in group E, i .e. directly opposite to what was anticipated earlier from data obtained with mannosidase from rat epididymis The present results were obtained with the aid of α-mannosidase from jack bean meal and of purified α- and β mannosidases from a marine gastropod, which were allowed to act upon the disaccharides mannosyl-rhamnose isolated from partial hydrolysates of the respective antigens. The new findings, however, do not alter the validity of previous conclusions. [ABSTRACT FROM AUTHOR]

A number of lipopolysaccharides derived from Salmonella and Escherichia coli S and R mutant strains were tested for toxicity and anticomplementary activity in the absence of added antiserum. Although all preparations were toxic, only a few exhibited high anticomplementary activity, while others proved to be of low or negligible activity. It was found that isolated lipid A from both active and reactive lipopolysaccharides was strongly anticomplementary as well as toxic, when made water-soluble with the aid of suitable carriers. Treatment of R form lipopolysaccharide with Mg2+ or Ca2+ led to complete precipitation of the lipopolysaccharide with consequent loss of toxicity and anticomplementary activity. This treatment had practically no effect on the anticomplementary activity and toxicity of S form lipopolysaccharides. When lipopolysaccharide, after reaction with complement, was reisolated and purified, the resulting preparation was found to be non-toxic and of negligible anticomplementary activity. No detectable alterations in either the sugar or the fatty acid composition could be detected. The only significant change was the loss of solubility in water. Treatment of the reisolated lipopolysaccharide with EDTA completely restored solubility m water, toxicity, and anti-complementary activity. [ABSTRACT FROM AUTHOR]

When the lipopolysaccharides of two Salmonella R mutants were hydrolyzed in 0.5 N HCl at 37° for 7 to 24 h, a previously undescribed sugar was liberated. This sugar has been identified as 4-amino-4-deoxy-L-arabinose by comparison of the free amino sugar, its two reduced derivatives, its two N-acetylated derivatives, and its methyl glycoside with the authentic amino sugar and its corresponding derivatives on thin layer, gas liquid, and column chromatography. A survey of about 50 enterobacterial lipopolysaccharides revealed that this amino sugar is present in a large percentage of the preparations. The preparation of 4-amino-4-deoxyarabinose by the reduction and limited periodate oxidation of 2-amino-2-deoxymannose is described. [ABSTRACT FROM AUTHOR]

4-Deoxy-D-arabino-hexose has been identified as a constituent of the four Citrobacter sero-types 4, 23, 27 and 36a. Its structure was evaluated by the aid of mass spectrometry and gas liquid chromatography in comparison with known deoxyhexoses. [ABSTRACT FROM AUTHOR]

Mild acid hydrolysis of the glycolipid derived @om a Salmonella Rd2 mutant led to the liberation of three major split products which, after separation by electrophoresis, were identified as free 2-keto-3-deoxyoctonate (KDO), KDO-7-phosphorylethanolamine, and L-glycero-α-D-mannoheptosyl-l,5-KDO. [ABSTRACT FROM AUTHOR]

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]

From the lipid A backbone of Salmonella lipopolysaceharide, β- 1,6-linked glucosamine disaccharides had been isolated previously, which were substituted at the non-reducing glucosamine unit with phosphate and 2-keto-3-deoxyoctonate residues. The results now obtained by periodate degradation show that the phosphate group is linked to the 4 position and the 2-keto-3-deoxyoctonate oligosaccharide probably to the 3 position of the non-reducing glucosamine unit. [ABSTRACT FROM AUTHOR]