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H Brade

Publications and source records attributed to H Brade.

At least 163 records · Page 9Linked to original sources
PubMed1987

Isolation and structural characterization of an 8-O-(4-amino-4-deoxy-beta-L-arabinopyranosyl)-3-deoxy-D-manno- octulosonic acid disaccharide in the lipopolysaccharide of a Proteus mirabilis deep rough mutant.

On short treatment of the lipopolysaccharide of a deep rough mutant of Proteus mirabilis (strain R45) with hydrochloric acid (0.01 M, 10 min, 100 degrees C) a compound was released which, on high-voltage paper electrophoresis, migrated slightly to the cathode (MGlcN = 0.19) and which stained with ninhydrin, alkaline silver nitrate and in the thiobarbituric acid assay. After purification and derivatization, its chemical structure was identified by combined gas-liquid chromatography/mass spectrometry and two-dimensional shift-correlated proton nuclear magnetic resonance spectroscopy as 8-O-(4-amino-4-deoxy-beta-L-arabinopyranosyl)-3-deoxy-D-manno-octu losonic acid. This disaccharide was also present in a wild-type strain of P. mirabilis.

Amino Sugars↗
PubMed1987

Investigations into the polymorphism of lipid A from lipopolysaccharides of Escherichia coli and Salmonella minnesota by Fourier-transform infrared spectroscopy.

The polymorphism of lipid A, the endotoxic principle of the lipopolysaccharides of gram-negative bacteria, has been investigated in the fully hydrated state at temperatures between 5 degrees and 58 degrees C via Fourier-transform infrared spectroscopy. These measurements were supplemented by X-ray diffraction, fluorescence intensity techniques and differential thermal analysis. Up to three distinct phase transitions could be detected, with the main transition temperatures lying at approximately 41 degrees, 46 degrees, 44 degrees and 47 degrees C for Escherichia coli lipid A, Salmonella minnesota lipid A, and the synthetic lipid A compounds 506 and 516, respectively. 4'-Monophosphoryl-lipid A samples exhibited their main transition temperatures at considerably higher temperatures (about 52 degrees C for E. coli lipid A). The analysis of greater than CH2 stretching absorption bands as well as the wide-angle scattering behaviour of the lipid A samples showed that the main transition apparently involved the completion of hydrocarbon chain melting of lipid A, as typically observed for phospholipids. However, the phase transition behaviour was found to be much more complex than that usually observed for model phospholipid systems. Even below the main transition temperature, considerable amounts of the methylene segments of the acyl chains of lipid A were found to assume gauche conformations. These conformational changes might be related to the occurrence of up to two further transitions located at about 22 degrees, 30 degrees, 27 degrees and 25.5 degrees C (first transition) and at about 34 degrees, 42 degrees, 38.5 degrees and 40.5 degrees C (second transition) for E. coli lipid A, S. minnesota lipid A and the synthetic lipid A compounds 506 and 516, respectively. Furthermore, by the analysis of some characteristic infrared absorption bands related to the hydrophilic backbone, it could be demonstrated that the temperature-induced conformational changes occurring within the hydrocarbon chains were constantly and simultaneously accompanied by detectable rearrangements within the interfacial region and the polar head group of lipid A. The following conclusions were drawn: Up to about 30 degrees C the lipid A assemblies were supposed to adopt virtually bilayered, true lamellar arrangements, as revealed by the analysis of greater than CH2 scissoring vibrations and X-ray diffraction pattern. However, as indicated by fluorometric techniques, no stable closed vesicles seemed to be formed even under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Calorimetry↗
PubMed1987

Studies on the chemical structure of the core-lipid A region of the lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305. Detection of a new 2-octulosonic acid interlinking the core oligosaccharide and lipid A component.

Lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305 was treated with acid (0.1 M HCl, 100 degrees C, 1 h). The product obtained (LPSdegr) was subjected to various modification and degradation procedures including reduction, hydrazinolysis and strong acid hydrolysis. Methylation analysis of purified part structures revealed the presence of a 4'-phosphorylated (beta 1'-6)-linked D-glucosamine disaccharide (lipid A backbone), which carried in position 6' a hitherto unknown 2-octulosonic acid (OclA) in highly acid-stable linkage. It was further shown that OclA is substituted in position 5 by a glucose tetramer, the reducing residue of which is phosphorylated. The hydrophilic region of the LPSdegr could thus be characterized as a phosphorylated heptasaccharide of the following structure: (Formula: see text).

Acinetobacter↗
PubMed1987

Chemical and serological investigations on the genus-specific lipopolysaccharide epitope of Chlamydia.

Members of the bacterial genus Chlamydia are responsible for widespread disease among humans and animals, including endemic trachoma in developing countries, venereal disease in developed countries, and a variety of other diseases such as infantile pneumonia and lymphogranuloma venereum. Although there is little genetic relatedness between and large antigenic diversity between and among the two chlamydial species, one antigenic determinant has been preserved among all serovars: the genus-specific lipopolysaccharide epitope. In this report, the tools of molecular genetics, monoclonal antibodies, and analytical and synthetic chemistry have been combined to determine the structure of this epitope. This epitope is attributed to the presence of a trisaccharide of 3-deoxy-D-manno-octulosonic acid (KDO) of the sequence KDOp-(2----8)-KDOp-(2----4)-KDO. The structure includes a unique linkage of two KDO residues through a 2.8-linkage.

Animals↗
PubMed1987

The immunogenicity and antigenicity of lipid A are influenced by its physicochemical state and environment.

We investigated the immunogenicity and antigenicity of synthetic lipid A and partial structures thereof. Included in the study were compounds which varied in the position of phosphate (1-mono-, 4'-mono-, and 1,4'-bisphosphates) and in the acylation (type, number, and distribution of fatty acids) and, in the case of monosaccharide compounds, the nature of the backbone sugar (D-glucosamine, D-glucose, 3-amino-3-deoxy-D-glucose, and 2,3-diamino-2,3-dideoxy-D-glucose). With the aid of the passive-hemolysis and passive-hemolysis-inhibition assays and by absorption experiments, five distinct antibody specificities were detected in polyclonal rabbit antisera raised against sheep erythrocyte-coated lipid A and lipid A incorporated into the membrane of liposomes (liposome-incorporated immunogens). Three antibody specificities reacted with disaccharide antigens specific for a 1-mono-, 4'-mono-, and 1,4'-bisphosphorylated beta-1,6-linked D-glucosamine disaccharide. Two antibodies reacted with either 1- or 4-phosphates of acylated D-gluco-configured monosaccharides and exhibited no cross-reaction with each other. However, they cross-reacted with disaccharide antigens with phosphate groups in the appropriate positions. We found that the physicochemical state and the environment of lipid A modulated its immunoreactivity. The immunogenicity was best expressed by erythrocyte-coated and liposome-incorporated immunogens. The antigenicity of lipid A was also greatly influenced by its physical surroundings. The reaction pattern of the above antibodies was highly specific in the hemolysis assay and in absorption experiments (the antibody reacted with antigen embedded in a cell membrane), whereas some cross-reactivities were observed in inhibition studies (the antibody reacts with antigen in aqueous solution). By using liposome-incorporated antigens as inhibitors, nonspecific reactions were avoided and specific ones were enhanced. Thus the antibodies described above against lipid A recognize epitopes in the hydrophilic backbone, the exposure of which depends on the intrinsic physicochemical properties of lipid A on the one hand and the physical environment on the other.

Animals↗
PubMed1987

Use of synthetic antigens to determine the epitope specificities of monoclonal antibodies against the 3-deoxy-D-manno-octulosonate region of bacterial lipopolysaccharide.

Mouse monoclonal antibodies were raised against heat-killed bacteria of the Re mutant R595 of Salmonella minnesota and characterized by the passive hemolysis and passive hemolysis inhibition tests and by double immunodiffusion experiments using lipopolysaccharide (LPS) from different rough mutants of S. minnesota and synthetic antigens. The latter were copolymerization products of acrylamide with the alpha- and beta-allylglycosides of 3-deoxy-D-manno-octulosonic acid (KDO) and the alpha-2,4-linked KDO disaccharide [poly-alpha-KDO, poly-beta-KDO, and poly-(alpha-KDO)2, respectively], and sodium (3-deoxy-D-manno-octulopyranosyl)onate-(2----6)-(2-deoxy-2-[ (R)-3- hydroxytetradecanoylamino]- beta-D-glucopyranosyl)-(1----6)-(2-deoxy-2-[(R)-3-hydroxytetradecanoy lam ino]-D-glucose) [alpha-KDO-(GlcNhm)2], representing a part structure of Re LPS. One antibody (clone 20, immunoglobulin M) was found to recognize a terminal alpha-linked KDO residue, since it reacted in the passive hemolysis assay with alpha-KDO-(GlcNhm)2 and all LPS tested, it was inhibited by all synthetic antigens containing alpha-linked KDO residues, and it gave a reaction of identity with poly-alpha-KDO and poly-(alpha-KDO)2 in double immunodiffusion experiments. A second antibody (clone 25, immunoglobulin G3) was identified as specific for an alpha-2,4-linked KDO disaccharide, since it reacted in immunodiffusion exclusively with synthetic poly-(alpha-KDO)2 and not with the monosaccharide derivatives in either anomeric configuration, and it was inhibited only with poly-(alpha-KDO)2 and with LPS from S. minnesota R595 (Re) and R345 (Rb2). The reaction of this antibody with R345 LPS is attributed to the quantitative substitution with KDO disaccharide present as a side chain, which is not present in stoichiometric amounts in the other LPS.

Animals↗
PubMed1987

Antigenic and immunogenic properties of recombinants from Salmonella typhimurium and Salmonella minnesota rough mutants expressing in their lipopolysaccharide a genus-specific chlamydial epitope.

Rough mutants from Salmonella typhimurium and Salmonella minnesota were transformed with a plasmid containing a 6.5-kilobase insert of DNA from Chlamydia trachomatis assumed to encode a glycosyltransferase. Transformation resulted in the expression of a genus-specific chlamydial epitope on the lipopolysaccharide (LPS) of the recombinant strains. Proteinase K-digested whole-cell lysates of the recombinants and of controls were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining or Western blot analysis. Two LPS populations were detected in the recombinants, the parent LPS and a faster-migrating component. The latter stained with monoclonal antibody against the genus-specific chlamydial epitope and was not seen in the controls. LPS was extracted and purified from recombinants of S. minnesota R595 and R4 and characterized by the passive hemolysis and passive hemolysis inhibition assays and by hydrolysis kinetics. Different antigenic determinants could be distinguished from each other by the passive hemolysis inhibition test with monospecific antigen-antibody reactions. Rabbits were immunized with heat-killed recombinant bacteria to study the immunogenic properties of the recombinants. In all animals, antibodies were raised against the parent core specificity and against the chlamydia-specific epitope. The data show that the recombinant bacteria are useful as immunogens to prepare polyclonal antisera against chlamydiae and that LPS isolated from them exhibits the same antigenic determinants as chlamydial LPS and may thus be used as a substitute for chlamydial LPS in serological assays.

Animals↗
PubMed1986

Methylation analysis of the heptose/3-deoxy-D-manno-2-octulosonic acid region (inner core) of the lipopolysaccharide from Salmonella minnesota rough mutants.

A modified methylation analysis is described which allows the elucidation of the structure of the inner core region [heptose/3-deoxy-D-manno-2-octulosonic acid (KDO)] of enterobacterial lipopolysaccharides (LPS) of Salmonella minnesota rough mutants (Re, strain R595; and Rd2P-, strain R4). Methylation, carboxyl-reduction, remethylation, hydrolysis, carbonyl-reduction, and acetylation of the Re-mutant LPS yielded the 2,6-di-O-acetyl and 2,4,6-tri-O-acetyl derivatives of partially methylated 3-deoxyoctitol in equimolar amounts, indicating the presence of a terminal and a 4-linked pyranosidic KDO residue. For Rd2P- LPS, the hydrolysis step involved 0.1M trifluoroacetic acid at 100 degrees for 1 h which cleaved ketosidic linkages, and the final products included the foregoing acetyl derivatives in the molar ratio of 1:02 and a partially methylated and acetylated 3-deoxyoctitol derivative which was substituted at O-5 by a methylated heptopyranosyl residue. Trideuteriomethylation of the latter product followed by methanolysis and acetylation gave 5-O-acetyl-3-deoxy-1,7,8-tri-O-methyl-2,4,6-tri-O-trideuteriomethyl++ +-D- glycero-D-talo/galacto-octitol and 1,5-di-O-acetyl-2,3,4,6,7-penta-O-methyl-L-glycero-D-manno-heptitol++ +. These results prove the presence of a (2----4)-linked KDO disaccharide in Re LPS and show that the core region of Rd2P- LPS contains a terminal alpha-L-glycero-D-manno-heptopyranosyl group and a non-substituted, a 4-O-, and a 4,5-di-O-substituted pyranosidic KDO residue in the molar ratios 1:1:0.2:1.

Colorimetry↗
PubMed1986

The effect of removal of D-fructose on the antigenicity of the lipopolysaccharide from a rough mutant of Vibrio cholerae Ogawa.

The lipopolysaccharide (LPS) of a rough mutant (95R) of Vibrio cholerae Ogawa has been investigated chemically and serologically. D-Fructose was released from LPS under conditions (10mM trifluoroacetic acid, 60 degrees, 1 h) that liberated no other sugar constituent of the LPS (2-amino-2-deoxy-D-glucose, D-glucose, L-glycero-D-manno-heptose). Upon periodate oxidation, D-fructose and D-glucose were oxidised quantitatively, whereas approximately 50% of heptose was periodate-resistant. The data indicate that D-fructose does not link the polysaccharide and lipid A portion as proposed earlier, and suggest that D-fructose is present as a branch. By passive hemolysis inhibition, it was shown that the release of D-fructose paralleled the exposure of an antigenic determinant cryptic in LPS.

Carbohydrates↗
PubMed1986

Molecular requirement for interleukin 1 induction by lipopolysaccharide-stimulated human monocytes: involvement of the heptosyl-2-keto-3-deoxyoctulosonate region.

Experiments were undertaken to localize in the lipopolysaccharide (LPS) the minimal structural determinants sufficient to initiate the signal leading to interleukin 1 (IL 1) secretion by human monocytes. Our results clearly demonstrated that this signal is triggered by structures present in the so-called inner-core region which chemically consists of 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) and heptose in many LPS of gram-negative bacteria. Thus, the isolated polysaccharide region of Bordetella pertussis endotoxin as well as fragments derived therefrom containing the reducing KDO unit were able to induce similar levels of IL1 induction as the native LPS. Similarly, the trisaccharide alpha-D-manno-heptopyranosyl-(1-3)-alpha-D-manno-heptopyranosyl -(1-5)-3 -deoxy-D-manno-octulosonic acid (hep-hep-KDO), representative for the inner-core region of a large number of enterobacterial LPS, was a very potent IL 1 inducer. Neither KDO monosaccharide, nor the alpha-(2-4)-linked 3-deoxy-D-manno-octulosonic acid disaccharide isolated from Salmonella rough-form LPS promoted the signal indicating that the minimal structure of endotoxin able to induce IL 1 secretion resides in the hep (1-5)-KDO disaccharide.

Carbohydrate Sequence↗
PubMed1986

Induction of human interleukin 1 by bacterial and synthetic lipid A.

Lipopolysaccharides (LPS) are well known inducers of interleukin 1 (IL 1). Here we show that synthetic heptaacyl Salmonella minnesota (compound 516) and synthetic E. coli type (compound 506) lipid A, as well as monodephospho part structures thereof, are able to induce IL 1 production in human mononuclear cells. The 4'-monodephospho part structure (compound 505) was found to be the most active IL 1 inducer as compared to compound 506 and the 1-monodephospho part structure (compound 504). Synthetic lipid A precursor Ia, lacking nonhydroxylated fatty acids (compound 406), and its 1- or 4'-monodephospho part structures (compounds 404 and 405) did not induce IL 1 production. IL 2 is not produced during stimulation with the synthetic compounds. IL 1 activity was partially heat labile and could be inhibited by a rabbit antiserum against human leukocytic pyrogen. These results show that the lipid A component of LPS is active in inducing IL 1 and that the presence of 3-acyloxyacyl residues is necessary for IL 1 induction.

Blood Physiological Phenomena↗
PubMed1986

Immunogenicity and antigenicity of synthetic Escherichia coli lipid A.

The immunogenicity and antigenicity of synthetic Escherichia coli lipid A (compound 506) and its 1- and 4'-monophosphorylated derivatives (compounds 505 and 504, respectively) and nonphosphorylated derivative (compound 503) were compared with those of bis- and 4'-monophosphorylated natural free lipid A from E. coli. The synthetic compounds under study were either coated onto sheep erythrocytes (except for the water-insoluble preparation 503) or incorporated into liposomes and used for the immunization of rabbits. Both types of immunogens (the latter representing fully synthetic immunogens) resulted in high-titered polyclonal antisera which were characterized before or after absorption in a passive hemolysis assay as well as in a passive hemolysis inhibition assay with the synthetic compounds as test antigens. All antisera were found to react with their corresponding homologous antigens coated onto sheep erythrocytes, with titers of up to 2,048, and were comparable to those antisera obtained after immunization with natural lipid A exposed on the bacterial surface after acid hydrolysis. Antisera against bisphosphorylated compound 506 were highly specific for the homologous antigens, showing no interaction with compounds 504 and 505 in the passive hemolysis test. The same held true for the absorption experiments in which glutaraldehyde-fixed sheep erythrocytes were sensitized with the respective antigens. Antisera against monophosphorylated compounds 504 and 505 exhibited, besides their expected homologous reactivity, complete cross-reactivity with compound 506, but they did not cross-react with each other. Thus, anti-504 and anti-505 antibodies recognized distinct antigenic determinants, being related to the ester linked 4'-phosphate or the glycosidically linked 1-phosphate, respectively. Both antigenic determinants were also expressed by bisphosphorylated compound 506 used as an antigen; however, upon immunization, only antibodies against compound 506 were elicited.

Epitopes↗