Determination of 3-deoxy-D-manno-octulosonic acid (KDO), N-acetylneuraminic acid, and their derivatives by ion-exchange liquid chromatography.

A liquid chromatography (1.6 MPa) system for the analysis of 3-deoxy-D-manno-2-octulosonic acid (KDO), N-acetylneuraminic acid (Neu5Ac), methyl alpha- and beta-glycosides of Neu5Ac and KDO, alpha-heptosyl-(1----5)-KDO, various sialyllactoses, alpha-KDO-(2----4)-KDO, alpha-KDO-(2----4)-KDO methyl alpha-glycoside, beta-KDO-(2----4)-KDO methyl beta-glycoside, D-glucuronic acid, D-glucurono-3,6-lactone, and D-galacturonic acid has been developed. Separation was achieved within 10 and 30 min by the use of a small column filled with a strongly basic, anion-exchange resin, Aminex A-29, and 0.

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.

Chemical characterization of Chlamydia trachomatis lipopolysaccharide.

The group-specific antigen of Chlamydia trachomatis serotype L2 was chemically analyzed. It is composed of typical lipopolysaccharide (LPS) components, i.e.

High state of order of isolated bacterial lipopolysaccharide and its possible contribution to the permeation barrier property of the outer membrane.

The conformational properties of the isolated S form of Salmonella sp. lipopolysaccharide (LPS), of Re mutant LPS, and of free lipid A were investigated by using X-ray diffraction and conformational energy calculations. The data obtained showed that LPS in a dried, in a hydrated, and probably also in an aqueous dispersion state is capable of forming bilayered lamellar arrangements similar to phospholipids.

Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities.

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.

Structural analysis of underivatized sialic acids by combined high-performance liquid chromatography-mass spectrometry.

Mass spectra of chemically ionized, positive ions of underivatized N,O-acylated sialic acids, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid and sialyl-alpha(2-3)-lactose were obtained by combined high-performance liquid chromatography--mass spectrometry, using a direct liquid inlet system. The mass spectra of the different compounds for which fragmentation schemes are proposed enable the differentiation between sialic acids, although the localization of O-substituents is not possible. However, since the various sialic acids separated well on high-performance liquid chromatography, combined high-performance liquid chromatography-mass spectrometry allowed their unequivocal characterization.

Newer aspects of the chemical structure and biological activity of bacterial endotoxins.

Gram-negative bacteria express at their surface various amphiphiles among which the lipopolysaccharides (endotoxins, O-antigens) have been studied most intensively. Lipopolysaccharides consist of a heteropolysaccharide portion (O-specific chain and core) which is responsible for the O- (and R-) antigenic properties and a covalently bound lipid component, termed lipid A, which contains the endotoxic principle of lipopolysaccharides. The detailed chemical structure of a large number of O-chains and the general architecture of the core oligosaccharide has been established.

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.

Alpha-2----4-interlinked 3-deoxy-D-manno-octulosonic acid disaccharide. A common constituent of enterobacterial lipopolysaccharides.

After mild acid hydrolysis, a disaccharide of 3-deoxy-D-manno-octulosonic acid (dOclA) was obtained from Re-mutant lipopolysaccharide of Salmonella minnesota, Salmonella godesberg, Proteus mirabilis, and Escherichia coli, and from the lipopolysaccharide of an S. minnesota Rb2 mutant. Combined gas-liquid chromatography/mass spectrometry of the reduced and permethylated derivatives indicated that the disaccharide is interlinked by a 2----4-glycosidic bond in all lipopolysaccharides tested.

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.

Structural alterations in the envelope of a gentamicin-resistant rough mutant of Pseudomonas aeruginosa.

Comparative studies of a gentamicin-sensitive strain (P28-0) of Pseudomonas aeruginosa and a gentamicin-resistant mutant (P23-800) have been carried out. No aminoglycoside-modifying enzymes were detected in extracts of the mutant. Electron microscopy of thin sections and the loss of O-antigenicity suggested that resistance of the mutant to gentamicin was related to an alteration in the outer membrane.

Concepts of the chemical structure of lipid A.

Lipid A represents the covalently bound lipid component of lipopolysaccharides (LPS), the endotoxins and O antigens of gram-negative bacteria. Lipid A's of endotoxically active LPS consist of a beta(1'----6)-linked D-glucosamine disaccharide that carries phosphoryl groups in positions 1 and 4'. Depending on the origin of lipid A, nonacylated, nitrogen-containing residues may be bound to these phosphoryl groups.

Lipopolysaccharides: structural principles and biologic activities.

Structural principles of the three regions of the lipopolysaccharide molecule are briefly discussed. One of the recently available, chemically synthesized lipid A analogues has been tested for biologic activities. The preparation was found to be toxic, pyrogenic, and mitogenic.

Biological activities of synthetic lipid A analogs: pyrogenicity, lethal toxicity, anticomplement activity, and induction of gelation of Limulus amoebocyte lysate.

Chemically synthesized lipid A analogs were investigated for several endotoxic activities, including pyrogenicity, lethal toxicity, anticomplement activity, and the capacity to gelate Limulus amoebocyte lysate in comparison to natural lipid A. The synthetic preparations contained D-glucosamine or D-glucosamine-beta-1,6-D-glucosamine disaccharide substituted by ester- and amide-bound hydroxylated or non-hydroxylated fatty acids and by phosphate groups in different combinations. Some preparations which were insoluble in water were succinylated and thus rendered more soluble.

Endotoxic properties of chemically synthesized lipid A part structures. Comparison of synthetic lipid A precursor and synthetic analogues with biosynthetic lipid A precursor and free lipid A.

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 beta-1,6-linked D-glucosamine disaccharide, which carries four molar equivalents of (R)-3-hydroxytetradecanoyl 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.

Laser desorption mass spectrometry of synthetic lipid A-like compounds.

The applicability and the present limitations of the laser microprobe mass analyser LAMMA -500 as an instrument for the structural analysis of higher molecular weight, non-volatile, bio-organic compounds (less than or equal to 2000 u) were investigated. For this purpose mass spectra of various synthetic and natural compounds representing cell wall components of Gram-negative bacteria, e.g.

Zymosan-induced chemiluminescence of granulocytes in incubated human whole blood. The role of platelets.

Zymosan-induced chemiluminescence (ZI-CL) of whole blood which is mainly the response of granulocytes can be used as a rapid, simple and reliable diagnostic tool for hematological and allergic disorders. ZI-CL of whole blood remained unchanged when stored at 0 degree C for 4 hr, whereas it increased by incubation at 37 degrees C. This phenomenon points to platelet-granulocyte interaction during the incubation.

Chemical structure of the lipid A component of the lipopolysaccharide from a Proteus mirabilis Re-mutant.

The chemical structure of the lipid A component from the lipopolysaccharide of a Proteus mirabilis Re-mutant (strain R45) was analysed. It consists of a beta(1-6)-linked D-glucosamine disaccharide which carries two phosphate groups, one being ester-linked to position 4' of the nonreducing glucosaminyl residue and the other being bound to the glycosidic hydroxyl group of the reducing glucosaminyl residue. The ester-bound phosphate group is quantitatively substituted by a 4-amino-4-deoxy-L-arabinopyranosyl residue, the glycosidic phosphoryl group appears to be unsubstituted.

The primary structure of the glycan moiety of pseudomurein from Methanobacterium thermoautotrophicum.

After treatment of isolated cells walls of Methanobacterium thermoautotrophicum with sodium hydroxide or anhydrous hydrazine, water soluble glycan strands were obtained. These consisted of alternating (beta 1-3)-linked D-glucosamine and (alpha 1-3)-linked L-talosaminuronic acid residues and their length was about 25 disaccharides. Some of the L-talosaminuronic acid residues remained linked to either glutamic acid or the peptides N-gamma-glutamylalanine and N epsilon-(gamma-glutamylalanyl)lysine, indicating that the peptide moiety of pseudomurein is bound to the carboxyl group of talosaminuronic acid via the amino group of glutamic acid.

Fatty acid in lipopolysaccharides of Salmonella species grown at low temperature. Identification and position.

Salmonella minnesota R 595 (Re) and other Salmonella strains incorporate cis-delta 9-16:1 (palmitoleic acid) at the expense of mainly dodecanoic acid into the lipid-A portion of lipopolysaccharides, when the cells are grown at low temperature (12 degrees C). It has recently been shown, that in S. minnesota R 595 grown at 37 degrees C dodecanoic acid is linked to the 3-hydroxyl group of an amide-bound 3-hydroxytetradecanoic acid.

The role of prostaglandins in endotoxic activities.

Endotoxins elicit an extraordinary variety of biological effects in higher organisms. Mononuclear phagocytes are believed to be the cellular source of secondary mediators responsible for the host's reaction. Several findings indicate that among these endogenous mediators prostaglandins are of importance.

Lipid A, the lipid component of bacterial lipopolysaccharides: relation of chemical structure to biological activity.

Lipopolysaccharides are integral components of the outer membrane of Gram-negative bacteria and they participate in various membrane functions essential for bacterial growth and survival. Lipopolysaccharides also represent the endotoxins of Gram-negative bacteria and possibly play a role for the pathogenesis and manifestations of bacterial infections. These biological activities are mediated mainly by the lipid component of lipopolysaccharides, termed lipid A.

Involvement of prostaglandin E and adenosine 3', 5'-monophosphate in lipopolysaccharide-stimulated collagenase release by rat Kupffer cells.

Kupffer cells exposed to bacterial lipopolysaccharide in vitro synthesized collagenase and released the major portion of it into the extracellular space while the intracellular level of enzyme was not altered significantly. Cycloheximide prevented the appearance of collagenase in the medium indicating de novo synthesis. Indomethacin, an inhibitor of cyclooxygenase, also blocked collagenase synthesis.

The chemical structure of lipid A. Demonstration of amide-linked 3-acyloxyacyl residues in Salmonella minnesota Re lipopolysaccharide.

In Salmonella minnesota lipopolysaccharide the lipid A backbone, a substituted diphosphorylated beta 1,6-linked D-glucosamine disaccharide molecule, carries approximately seven residues of fatty acids: one each of dodecanoic, hexadecanoic, D-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 beta-elimination reaction, to the generation of amide-bound delta 2-tetradecanoic acid. This suggested that the 3-hydroxyl group of amide-bound hydroxy fatty acids carried a substituent.