Search PubMed⌕ Search

Biomedical subjects

H Brade

Publications and source records attributed to H Brade.

At least 181 records · Page 10Linked to original sources
PubMed1986

Chemical, biological, and immunochemical properties of the Chlamydia psittaci lipopolysaccharide.

The lipopolysaccharide (LPS) of Chlamydia psittaci was extracted from yolk sac-grown elementary bodies, purified, and characterized chemically, immunochemically, and biologically. The LPS contained D-galactosamine, D-glucosamine, phosphorus, long-chain fatty acids, and 3-deoxy-D-manno-2-octulosonic acid in the molar ratio of approximately 1:2:2:6:5. The antigenic properties of the isolated LPS were compared with those of the LPS from Chlamydia trachomatis and Salmonella minnesota Re by the passive hemolysis and passive hemolysis inhibition tests, absorption, hydrolysis kinetics, and Western blot analysis with rabbit polyclonal antisera against chlamydiae and with a mouse monoclonal antibody recognizing a genus-specific epitope of chlamydial LPS. Two antigenic determinants were identified, one of which was chlamydia specific and the other of which was cross-reactive with Re LPS. Both determinants were destroyed during acid hydrolysis, whereby a third antigen specificity was exposed which was indistinguishable from the lipid A antigenicity. In rabbit polyclonal antisera prepared against Formalin-killed elementary bodies or detergent-solubilized membranes, two antibody specificities were differentiated. One of these was chlamydia specific, and the other was cross-reactive with Re LPS. The LPS of C. psittaci was inactive within typical endotoxin parameters (lethal toxicity, pyrogenicity, local Shwartzman reactivity); it was, however, active in some in vitro assays, such as those testing for mouse B-cell mitogenicity and the induction of prostaglandin E2 in mouse peritoneal macrophages.

Abortion, Veterinary↗
PubMed1985

Identification of 2-keto-3-deoxy-1,7-dicarboxyheptonic acid as a constituent of the lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305.

A 3-deoxyaldulosonic acid, not previously described, was identified as a component of the lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305: 2-keto-3-deoxy-1,7-dicarboxyheptonic acid. This compound, whose steric configuration has not been determined, was released from lipopolysaccharide after acid hydrolysis as monosaccharide and in the form of a glucose-substituted disaccharide. It was identified by combined gas-liquid chromatography/mass spectrometry using various derivatization procedures. The presence of a 2-keto-3-deoxyaldulosonic acid was established from the typical fragmentation pattern of the C1-C6 moiety which was identical to that of similarly derivatized authentic 2-keto-3-deoxy-D-mannooctonic acid (dOclA). Location of the keto and deoxy function at C2 and C3, respectively, was deduced from the fragmentation pattern of the deuterolabelled derivatives after reduction of the keto and carboxy groups. The presence of two carboxy groups was ascertained after carboxy reduction with sodium boro-(2H)hydride of the carbonyl-reduced and permethylated derivative whereby the molecular masses were found to differ by 4 Da. Further proof for the presence of two carboxy groups was obtained by alkaline transesterification of the carbonyl-reduced and permethylated derivative with ethanolic sodium ethylate: it was shown that two methyl ester groups had been transesterified since the molecular mass was shifted higher by 28 Da. Substitution by a D-glucopyranosyl residue in position 4 was established by methylation analysis performed on the carbonyl- and carboxyl-reduced and permethylated disaccharide. The D-configuration of the glucosyl residue was determined by its reactivity with D-glucose oxidase. Thus, the structure of the disaccharide is 4-O-glucopyranosyl-2-keto-3-deoxy-1,7-dicarboxyheptonic acid.

Acetylation↗
PubMed1985

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 0.75 or 10mM sodium sulfate solutions as mobile phases. This method allowed the determination of KDO and sialic acids in amounts of 100 ng (0.5 nmol) and 200 pg (0.6 pmol), respectively.

Chromatography, High Pressure Liquid↗
PubMed1985

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.

Animals↗
PubMed1985

Structural investigations on the inner core region of lipopolysaccharides from Salmonella minnesota rough mutants.

The structure of the inner core region (L-glycero-D-mannoheptose/2-keto-3-deoxy-D-mannooctulosonic acid region) of lipopolysaccharides from Salmonella minnesota rough mutants was investigated. Using conventional methods (neutral sugar analysis, Smith degradation and methylation analysis) combined with gas chromatography/mass spectrometry (GC/MS) of higher oligosaccharides (up to tetrasaccharide), the linkages of the core sugars of lipopolysaccharides from S. minnesota rough mutants, strains R4 (Rd2P-), R7 (Rd1P-) and R5 (RcP-) were determined as: (formula see text) with R representing H in R4, L-glycero-D-mannoheptopyranosyl in R7, and D-glucopyranosyl-(1----3)-L-glycero-D-mannoheptopyranosyl in R5, respectively. In addition, it is shown that heterogeneity within the neutral sugar part of these lipopolysaccharides is low.

Carbohydrates↗
PubMed1985

Antigenic properties of Chlamydia trachomatis lipopolysaccharide.

The antigenic properties of the lipopolysaccharide (LPS) of Chlamydia trachomatis L2 were investigated. By means of passive hemolysis, passive hemolysis inhibition, and absorption experiments, it was shown that antiserum raised against chlamydial elementary bodies contained at least two different antibody specificities which reacted with different antigenic determinants of chlamydial LPS. One of these antibodies cross-reacted with enterobacterial Re LPS, recognizing a structure which is shared by both LPSs, whereas the reactivity of the second antibody was restricted to chlamydial LPS. The former antibody could be absorbed with Salmonella minnesota Re LPS, whereas the latter was not affected by this absorption. Therefore, chlamydial LPS possesses two distinct antigenic determinants, one of which is C. trachomatis specific, the other of which is responsible for the cross-reactivity with enterobacterial Re-type LPS. Both antigenic determinants were destroyed during mild acid-catalyzed hydrolysis. It was further shown that free chlamydial lipid A exhibits antigenicity that cross-reacts with free enterobacterial lipid A. This antigenicity, however, as in enterobacterial LPS, is present in a cryptic form, i.e., it is unmasked only after acid hydrolysis of LPS.

Chlamydia trachomatis↗
PubMed1985

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., D-glucosamine, long-chain 3-hydroxy fatty acids, 2-keto-3-deoxyoctonic acid, and phosphate in a molar ratio of approximately 2:5:3:2.6, respectively, resembling enterobacterial LPS of the Re chemotype. For the first time, 3-hydroxydocosanoic acid (3-OH C22:0) was found as an LPS constituent.

Chlamydia trachomatis↗
PubMed1985

Characterization of two different antibody specificities recognizing distinct antigenic determinants in free lipid A of Escherichia coli.

Antisera were raised in rabbits with acid-treated Re mutant bacteria from Salmonella minnesota and Escherichia coli and tested in a passive hemolysis assay with di- and monophosphorylated free lipid A of E. coli (LipA-Ac and LipA-HCl, respectively) coated onto sheep erythrocytes. Depending on the acid used to prepare the immunogen (acetic versus hydrochloric acid), different antibody specificities were obtained. Antiserum prepared against HCl-treated bacteria was found to react with both antigens to the same extent (i) in the passive hemolysis test, (ii) in the passive hemolysis inhibition test, and (iii) in absorption experiments, suggesting that antibodies in this antiserum recognize an antigenic determinant equally present in LipA-Ac and LipA-HCl. Antiserum raised against acetic acid-treated bacteria reacted with the homologous antigen (LipA-Ac) in the passive hemolysis and passive hemolysis inhibition test as well as in absorption experiments. However, the antiserum failed to react with the heterologous antigen (LipA-HCl) in the hemolysis test and during absorption, whereas in inhibition studies interaction of this antiserum with both antigens was observed. The inhibiting capacity of LipA-Ac was lower compared with that of LipA-HCl, indicating that the antigenic determinant of LipA-Ac is partly expressed by LipA-HCl in solution, but not when fixed on the surface of sheep erythrocytes. The role of glycosidically linked phosphate in lipid A is discussed with respect to antigenicity.

Animals↗
PubMed1985

A 28,000-dalton protein of normal mouse serum binds specifically to the inner core region of bacterial lipopolysaccharide.

Normal mouse serum was found to contain a protein, referred to here as factor, which binds to the inner core region of lipopolysaccharides (LPSs) of various bacterial families. Since factor-LPS interactions resulted in activation of guinea pig complement, factor activity could be assayed by a passive hemolysis test with sheep erythrocytes coated with LPS or lipid A from Acinetobacter calcoaceticus (which was found earlier to bind particularly well to factor). Factor was purified by G-50 and hydroxyapatite chromatography whereby the specific hemolytic activity was enriched 1,675-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed the presence of a 28,000-dalton protein as the main band. The identity of this band was determined by absorption experiments with LPS-coated sheep erythrocytes or latex beads, whereby the 28,000-dalton band disappeared after specific absorption and could be recovered from the absorbent. The binding specificity of factor was determined in a passive hemolysis inhibition assay with defined oligosaccharides representative for the inner core region of LPS. Thus, the di- and trisaccharides alpha-D-mannoheptopyranosyl-(1----5)-2-keto-3-deoxy-D-mannoocto nic acid and alpha-D-mannoheptopyranosyl-(1----3)-alpha-D-mannoheptopyranosy l-(1----5)-2- keto-3-deoxy-D-mannooctonic acid, respectively, were able to inhibit binding of factor to LPS. The results are in accordance with our earlier observation that the heptose-2-keto-3-deoxy-D-mannooctonic acid region represents a common antigen of bacterial LPS. Rabbit hyperimmune serum directed against this common antigen and purified factor was found to exhibit the same specificity for LPS. Factor activity was followed in mice in vivo after injection of LPS; it disappeared completely 15 min after the injection of LPS and reappeared within 1 h.

Animals↗
PubMed1985

Occurrence of 2-keto-deoxyoctonic acid 5-phosphate in lipopolysaccharides of Vibrio cholerae Ogawa and Inaba.

A phosphorylated 2-keto-3-deoxyoctonic acid (KDO) was released from the lipopolysaccharides of Vibrio cholerae Ogawa and Inaba after strong acid hydrolysis. The phosphorylated KDO was identified by gas-liquid chromatography and mass spectrometry after reduction and permethylation as KDO-5-phosphate and an isomer of it being phosphorylated at position 7 or 8. After treatment with alkaline phosphatase, KDO was detected by gas-liquid chromatography and mass spectrometry. It was indistinguishable from authentic 2-keto-3-deoxy-D-manno-octonic acid.

Gas Chromatography-Mass Spectrometry↗
PubMed1985

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. Recent analyses of the enterobacterial inner core region indicate the presence of a linear trisaccharide of alpha 2.4-linked 3-deoxy-D-manno-2-octulosonic acid (KDO) residues of which only the reducing group is believed to be located in the main core chain. This KDO residue which provides the link between the polysaccharide and the lipid A component appears to be involved in a recently detected ubiquitous immunodeterminant expressed by lipopolysaccharides of various origin. The chemical structure of enterobacterial lipid A's is now known in some detail. Lipid A of Salmonella, Escherichia coli and Proteus consists of a beta 1.6-linked D-glucosamine disaccharide which carries four (R)-3-hydroxytetradecanoyl groups in positions 2, 3, 2' and 3' and two phosphoryl residues in positions 1 and 4'. Up to three of the hydroxy fatty acids (positions 2, 2' and 3') are, at their 3-hydroxyl groups, acylated by non-hydroxylated acyl residues, and to phosphoryl groups non-acylated, nitrogen-containing residues such as 4-amino-4-deoxy-L-arabinopyranose and phosphorylethanolamine may be bound. The hydroxyl group in position 4 of the glucosamine disaccharide is free and that in position 6' serves as the attachment site for KDO (i.e. the polysaccharide component) in lipopolysaccharide. Based on this structure lipid A analogues have been chemically synthesized and analysed for endotoxic activity in vivo and in vitro. In two test systems (pyrogenicity, local Shwartzman reaction) the synthetic part structures exhibited weak or no endotoxic activity, as did a precursor of lipid A biosynthesis which is structurally identical to one of the analogues. In many other systems, however, including lethal toxicity, B-lymphocyte mitogenicity, macrophage activation, induction of cross tolerance, expression of lipid A antigenicity, the synthetic materials were of comparable activity as bacterial free lipid A. These findings support the structural proposals made for lipid A and they prove the previous hypothesis that the endotoxic principle is embedded in lipid A.(ABSTRACT TRUNCATED AT 400 WORDS)

Endotoxins↗
PubMed1984

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. In addition, it was shown by gas-liquid chromatography of appropriate synthetic standards and a previously characterized alpha 2----4-linked dOclA disaccharide (derived from lipopolysaccharide of S. godesberg) that the non-reducing dOclA residue possesses the alpha configuration. In the case of lipopolysaccharide of S. minnesota Rb2 mutant, this result, together with earlier findings, suggests that it contains a linear dOclA trisaccharide of the sequence dOclA(alpha 2-4)dOclA. The results show that a dOclA(alpha 2-4)dOclA disaccharide represents a common architectural principle in enterobacterial lipopolysaccharides.

Chromatography, Gas↗
PubMed1984

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. 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 or 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 structural requirements for the expression of endotoxin activities.

Animals↗
PubMed1983

A method to detect 2-keto-3-deoxyoctanat and related compounds on pherograms and chromatograms.

A variation of the thiobarbituric acid spray reagent to detect 2-keto-3-deoxyoctanat (KDO), KDO derivatives, 2-deoxysugars, and N-acetylneuraminic acid on pherograms and chromatograms is described. Using all reagents in organic solvents and developing the color at room temperature, accurate locations of the spots without spreading and a low background stain are achieved.

Chromatography, Thin Layer↗
PubMed1983

Isolation of a 3-deoxy-D-mannooctulosonic acid disaccharide from Salmonella minnesota rough-form lipopolysaccharides.

Lipopolysaccharides of Salmonella minnesota rough mutants were treated with 20 mM acetate buffer pH 4.4 at 70 degrees C/3 h. After 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 compound 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.

Disaccharides↗
PubMed1983

A new lipopolysaccharide antigen identified in Acinetobacter calcoaceticus: occurrence of widespread natural antibody.

Serological investigation of the lipopolysaccharide (LPS) of Acinetobacter calcoaceticus revealed a new antigen to which antibody in high titre is present in the serum of many mammalian species. The passive haemolysis test showed that antibody, in titres ranging from 32-4096, was invariably present in the serum of mice, rats, guinea-pigs, and horses. Rabbits and human beings had lower and more variable titres (less than 2-512). The antigen persisted after prolonged hydrolysis of the LPS in 1% acetic acid at 100 degrees C. Acinetobacter lipid A, which resembled antigenically the lipid A of many gram-negative bacteria, could be distinguished from the new antigen by inhibition and absorption experiments. Antibody to the new antigen could be completely absorbed with acinetobacter lipid A but not with enterobacterial lipid A; moreover, the latter failed to react with the antibody in the passive haemolysis test. Immunisation of rabbits with lipid A-immunogenic acinetobacter cells gave rise to antibodies against the new antigen and to lipid-A antibodies. Absorption of the immune serum with acinetobacter lipid A removed antibody to both antigens, but absorption with enterobacterial lipid A removed only the lipid -A antibodies.

Acinetobacter↗