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H Brade

Publications and source records attributed to H Brade.

At least 73 records · Page 4Linked to original sources
PubMed1997

Structural and serological characterisation of the O-specific polysaccharide from lipopolysaccharide of Acinetobacter calcoaceticus strain 7 (DNA group 1).

S-form lipopolysaccharide was isolated by phenol/water extraction from a strain of Acinetobacter calcoaceticus (DNA group 1 ). The structure of the O-antigenic polysaccharide was determined by compositional analysis and NMR spectroscopy of the de-O-acylated lipopolysaccharide. The isolated polysaccharide obtained after hydrolysis of lipopolysaccharide in 0.01 M trifluoroacetic acid has the following structure: [STRUCTURE IN TEXT] in which Pyr is pyruvate. The O-acetyl substitution of D-Gal was non-stoichiometric. The O-antigen was specifically recognised in western blots by polyclonal rabbit antisera.

Acinetobacter calcoaceticus↗
PubMed1997

Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide of the deep rough mutant I-69 Rd-/b+ of Haemophilus influenzae.

Monoclonal antibodies against the lipopolysaccharide (LPS) of the deep rough mutant I-69 Rd-/b+ of Haemophilus influenzae were obtained after immunization of mice with sheep erythrocytes which had been coated with de-O-acylated LPS. Characterization of antibodies was performed by enzyme immuno assay (EIA) using LPS or neoglycoconjugates containing partial structures of LPS as solid-phase antigens and by haemagglutination with sheep erythrocytes coated with de-O-acylated LPS. Binding data were confirmed by EIA inhibition experiments using deacylated LPS or synthetic partial structures thereof. Three antibodies were specific for 3-deoxy-D-manno-octulopyranosonic acid- (Kdo) 5-phosphate, one for Kdo-4-phosphate, and one required, in addition to a Kdo-phosphate, parts of the phosphorylated glucosamine backbone of lipid A. All antibodies also bound in (i) Western blots to bacterial whole-cell lysates or isolated LPS separated by SDS-PAGE, (ii) bacterial colony blots, and (iii) immunofluorescence with live bacteria. The latter result indicated that Kdo-4- and Kdo-5-phosphate are synthesized by the bacteria and are not the result of phosphate migration.

Animals↗
PubMed1997

Structural requirements of synthetic oligosaccharides to bind monoclonal antibodies against Chlamydia lipopolysaccharide.

Monoclonal antibodies were generated against a synthetic glycoconjugate containing the trisaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo (Kdo, 3-deoxy-D-manno-2-octulopyranosonic acid) which represents the genus-specific epitope of the lipopolysaccharide from the obligatory intracellular human pathogen Chlamydia. Antibodies of all immunoglobulin G isotypes were obtained and characterized by enzyme immunobinding and inhibition assays using the immunizing antigen as well as chemically synthesized derivatives of the Kdo trisaccharide. The latter contained (1) one of the three residues in beta- instead of alpha-linkage, (2) a Kdo residue the carboxyl group of which had been reduced to a CH2OH group (Kdo(C1-red)), or (3) changing the linkage of the terminal Kdo from 2-->8 to 2-->4. Only one compound, namely, alpha-Kdo-(2-->8)-alpha-Kdo(C1-red)-(2-->4)-alpha-Kdo exhibited binding to and inhibition of Kdo trisaccharide-specific antibodies, whereas all other compounds were not active. Structural and conformational investigations using NMR spectroscopy at high field on the allyl glycosides of the oligosaccharides 6-12 confirmed the conformational similarities between those structures 4, 5, and 10 which were able to bind to the antibodies investigated.

Animals↗
PubMed1997

Characterization of serum antibody response to chlamydiae in patients with sexually acquired reactive arthritis.

Sera from patients with sexually acquired reactive arthritis (SARA) with antibodies reacting with C. trachomatis and C. pneumoniae (group 1; n = 20) and also with C. psittaci (group 2; n = 19) were analyzed for antibody specificity. Sera from group 2 reacted significantly more often with C. trachomatis serotype E, H and K and had higher antibody titers to serotype E, as tested by microimmunofluorescence tests. Cross-reactivities occurring in microimmunofluorescence tests were related to the presence of antichlamydial lipopolysaccharide antibodies, adsorption of which by recombinant lipopolysaccharide removed microimmunofluorescence reactivity with C. psittaci antigen. In group 2, significantly more sera had antibodies to C. pneumoniae, remaining after lipopolysaccharide adsorption, as proved by adsorption with viable C. trachomatis and C. pneumoniae organisms. None of the sera had antibodies to Yersinia enterocolitica, Shigella flexneri, Sh. sonnei and Salmonella spp. It was observed that the frequency and titer of cross-reacting antibodies to chlamydial serotypes and species were related to the time period between the diagnosis of genital chlamydial infection and of SARA. Cross-reactivities were also related to the presence of lipopolysaccharide, but not heat shock protein 60- or neutralizing antibodies to chlamydiae. Antibody reactivity induced by antichlamydial lipopolysaccharide antibodies can be removed by lipopolysaccharide adsorption.

Adult↗
PubMed1996

Structural and serological characterisation of two O-specific polysaccharides of Acinetobacter.

Extraction of dry bacteria of Acinetobacter strain 34 (DNA group 2) or Acinetobacter strain 108 (DNA group 13) by phenol/water yielded a polymer that was identified by means of serological studies and fatty acid analysis as S-form lipopolysaccharide. Degradation of the lipopolysaccharides of strains 34 and 108 in 1% acetic acid and 5% acetic acid, respectively, and gel-permeation chromatography gave the respective O-antigenic polysaccharides, the structures of which were determined, by compositional analysis and NMR spectroscopy of the polysaccharide, as [Sequence: see text] for strain 108, where D-Fucp3NBuOH represents 3-[(R)-3-hydroxybutyramido] -3,6-dideoxy-D-galactose and D-GalpANAc represents 2-acetamido-2-deoxy-D-galacturonic acid. Both structures were specifically recognised in Western blots by polyclonal rabbit antisera and there was no cross-reaction between these two structures.

Acinetobacter↗
PubMed1995

The structure of the lipid A-core region of the lipopolysaccharides from Vibrio cholerae O1 smooth strain 569B (Inaba) and rough mutant strain 95R (Ogawa).

The lipopolysaccharides (LPS) from Vibrio cholerae 95R, a rough mutant strain of O1 V. cholerae 162 (Ogawa), and from smooth O1 V. cholerae 569B (Inaba) were de-O-acylated. In each case, one part of the products was treated with 48% aqueous HF which removed the phosphoryl and fructose residues, then reduced, de-N-acylated, and N-acetylated. Another part was de-N-acylated by treatment with hot KOH. The products of both degradation pathways were separated by high-performance anion-exchange chromatography. The major dephosphorylated and defructosylated product 1 was obtained in pure form, whereas the minor products 2 and 3 were eluted as a mixture, as were, from the second degradation, the phosphorylated oligosaccharides 4 (major product) and 5 (minor product). No phosphorylated component corresponding to oligosaccharide 3 could be identified by NMR spectroscopy in the latter mixture. The following structures of oligosaccharides 1-5 were established on the basis of monosaccharide and methylation analyses, Smith degradation, and 1H- and 13C-NMR investigations (correlated, total correlated, NOE and heteronuclear correlation spectroscopy; all sugars are present as alpha-D-pyranoses except where indicated otherwise; Hep, L-glycero-D-manno-heptose; Kdo, 3-deoxy-D-manno-2-octulosonic acid). [formula: see text] In the untreated lipopolysaccharide, the amino group of the non-reducing terminal glucosamine residue is not substituted.

Carbohydrate Sequence↗
PubMed1995

The structures of oligosaccharide bisphosphates isolated from the lipopolysaccharide of a recombinant Escherichia coli strain expressing the gene gseA [3-deoxy-D-manno-octulopyranosonic acid (Kdo) transferase] of Chlamydia psittaci 6BC.

The lipopolysaccharide from the recombinant strain Escherichia coli F515-140 containing the cloned gene gseA [3-deoxy-D-manno-octulopyranosonic acid (Kdo) transferase] from Chlamydia psittaci 6BC was isolated and sequentially de-O-acylated and de-N-acylated. The products were separated by high-performance anion-exchange chromatography into three fractions, two of which contained a single compound. Their structures were elucidated by high-field NMR spectroscopy as alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN-(1-->6)-alpha-D-GlcN 1,4'-P2 (compound 1) (tetrasaccharide bisphosphate) [Holst, O., Broer, W., Thomas-Oates, J. E., Mamat, U. & Brade, H. (1993) Eur. J. Biochem. 214, 703-710] and alpha-Kdo-(2-->4)-[alpha-Kdo-(2-->8)-]-alpha-Kdo-(2-->4)-alpha-Kdo- (2-->6)-beta-D-GlcN-(1-->6)-alpha-D-GlcN 1,4'-P2 (compound 4) (hexasaccharide bisphosphate). The third fraction comprised two pentasaccharide bisphosphates, which could be separated by affinity chromatography using an immobilized monoclonal antibody specific for the trisaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo. The bound fraction was identified as alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D- GlcN-(1-->6)-alpha-D-GlcN 1,4'-P2 (compound 2) [Holst, O., Broer, W., Thomas-Oates, J. E., Mamat, U. & Brade, H. (1993) Eur. J. Biochem. 214, 703-710], whereas the unbound fraction was identified as alpha-Kdo-(2-->4)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN-(1-->6 )- alpha-D-GlcN 1,4'-P2 (compound 3). This novel Kdo tetrasaccharide extends our knowledge on multifunctional Kdo transferases.

Carbohydrate Sequence↗
PubMed1995

Diagnosis of ornithosis by cell culture and polymerase chain reaction in a patient with chronic pneumonia.

We report the case of a woman who had pneumonia due to Chlamydia psittaci. A Chlamydia species was determined to be the causative agent of the pneumonia because it was isolated from bronchoalveolar lavage fluid, because it could be detected in lung biopsy specimens by the direct immunofluorescence technique, and because Chlamydia-specific antibodies could be detected by ELISA and microimmunofluorescence. The infectious agent could not be identified at the species level with use of serological techniques, but the isolate was determined to be C. psittaci by PCR with use of species- and genus-specific sequences within the chlamydial lipopolysaccharide biosynthesis gene gseA. The case reported herein exemplifies the problems encountered in diagnosing ornithosis and shows that isolation of the etiologic agent followed by identification of the species by PCR is helpful in diagnosing this rare disease. In addition, the findings in our case show that laboratory personnel who are conducting tests for Chlamydia pneumoniae should be aware of the risk of accidentally isolating highly infectious C. psittaci organisms.

Aged↗
PubMed1995

Molecular cloning, sequence analysis, and functional characterization of the lipopolysaccharide biosynthetic gene kdtA encoding 3-deoxy-alpha-D-manno-octulosonic acid transferase of Chlamydia pneumoniae strain TW-183.

The gene kdtA of Chlamydia pneumoniae strain TW-183, encoding the enzyme 3-deoxy-alpha-D-manno-octulosonic acid (Kdo) transferase of lipopolysaccharide biosynthesis, was cloned and sequenced. A single open reading frame of 1314 bp was identified, the deduced amino acid sequence of which revealed 69% similarity and 43% identity with KdtA of Chlamydia trachomatis and Chlamydia psittaci. The gene was expressed in the Gram-positive host Corynebacterium glutamicum and the primary gene product was characterized as a multifunctional glycosyltransferase. Cell-free extracts generated in vitro the genus-specific epitope of Chlamydia composed of the trisaccharide alphaKdo(2-8)alphaKdo(2-4)alphaKdo. The results show that a single polypeptide affords three different glycosidic bonds, which is in contradiction to the dogma of glycobiology: 'one enzyme - one glycosidic bond'.

Amino Acid Sequence↗
PubMed1995

Gonococcal rfaF mutants express Rd2 chemotype LPS and do not enter epithelial host cells.

We have investigated the function of the Isi-1 gene of Neisseria gonorrhoeae previously implicated in lipopolysaccharide (LPS)-inner-core biosynthesis (Petricoin et al., 1991). Disruption of the gene in gonococcal strain MS11 resulted in the production of LPS that migrated faster than that from an isogenic galE mutant, typical for a mutation that influences the inner-core region. Complementation of a panel of Salmonella typhimurium mutants with defined defects in rfa loci demonstrated conclusively that the Isi-1 gene of MS11 is functionally homologous to the rfaF gene, which encodes heptosyltransferase II in both E. coli and S. typhimurium. Comparison of deduced amino acid sequences of the gonococcal and the Salmonella RfaF demonstrated 70% similarity, including 47% identical amino acid residues. Immunochemical analysis of the LPS using monoclonal antibodies directed against chemically defined inner-core glycoconjugates revealed that the gonococcal and Salmonella Rd2-chemotypes were antigenically similar, further extending the genetic and functional homology. Infection experiments in vitro demonstrated that the Isi-1 mutant could not invade human Chang epithelial cells despite expression of a genetically defined invasion-promoting gonococcal opacity protein. These data imply that the LPS phenotype is a critical factor for gonococcal invasiveness.

Amino Acid Sequence↗
PubMed1995

Binding of lipopolysaccharide (LPS) to an 80-kilodalton membrane protein of human cells is mediated by soluble CD14 and LPS-binding protein.

Activation of cells by bacterial lipopolysaccharide (LPS) plays a key role in the pathogenesis of gram-negative septic shock. The 55-kDa glycoprotein CD14 is known to bind LPS and initiate cell activation. However, there must be additional LPS receptors because CD14 is linked by a glycosylphosphatidyl inositol anchor to the cell membrane and therefore unable to perform transmembrane signalling. Searching for potential LPS receptors, we investigated the binding of LPS to membrane proteins of the human monocytic cell line Mono-Mac-6. Membrane proteins were electrophoretically separated under reducing conditions, transferred to nitrocellulose, and exposed to LPS, which was visualized with anti-LPS antibody. Smooth- and rough-type LPS, as well as free lipid A, bound to a variety of proteins in the absence of serum. However, in the presence of serum, additional or preferential binding to a protein of approximately 80-kDa was observed. Experiments with differently acylated lipid A structures showed that the synthetic tetraacyl compound 406 was still able to bind, whereas no binding was detected with the bisacyl compound 606. The 80-kDa membrane protein was also detected on human peripheral blood monocytes and endothelial cells. The serum factors mediating the binding of lipid A to the 80-kDa membrane protein were identified as soluble CD14 and LPS-binding protein. From these results, we conclude that this 80-kDa protein is a candidate for the hypothetical molecule for LPS and/or LPS-CD14 recognition and signal transduction.

Acute-Phase Proteins↗
PubMed1994

Isolation and structural analysis of oligosaccharide phosphates containing the complete carbohydrate chain of the lipopolysaccharide from Vibrio cholerae strain H11 (non-O1).

For the first time, an oligosaccharide has been prepared comprising the lipid A backbone, the core oligosaccharide and one repeating unit of the O-specific polysaccharide (O-chain) of a lipopolysaccharide. Lipopolysaccharide from Vibrio cholerae strain H11 (non-O1) was deacylated and the products were separated by high-performance anion-exchange chromatography. Major fractions were a hexadecasaccharide trisphosphate 1, representing the core-lipid A oligosaccharide substituted by one modified repeating unit of the O-antigenic polysaccharide, a dodecasaccharide trisphosphate 2 and an undecasaccharide trisphosphate 3, representing the core-lipid A region. Oligosaccharide 1 originated from beta-elimination upon alkaline hydrolysis of alpha-galacturonic acid of the O-chain; oligosaccharides 2 and 3 were most likely obtained from naturally occurring lipopolysaccharide species carrying no O-chain. The structures of these compounds were elucidated on the basis of monosaccharide composition, and NMR investigations comprising correlation spectroscopy, total correlation spectroscopy and nuclear Overhauser enhancement spectroscopy experiments, as well as heteronuclear 13C, 1H correlation spectroscopy. The structures are as follows: [formula: see text] where R is beta-L-threo-hex-4-enuronopyranosyl-(1-4)-alpha-Neu-(2-3)-beta-Gal A-(1-3)- beta-QuiN-(1-4)-beta-Sedf-(2- in 1, beta-Sedf-(2- in 2, and H in 3. Where not stated otherwise, sugars are pyranoses of the D-series. Hep is L-glycero-D-manno-heptose, QuiN is 2-amino-2,6-dideoxy-glucose, Kdo is 3-deoxy-D-manno-2-octulosonic acid, Sed is D-altro-heptulose and GalA is galacturonic acid.

Carbohydrate Sequence↗
PubMed1994

Crystal and molecular structure of allyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->8)-O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosidonate)-monohydrate.

The alpha-(2-->8)-linked Kdo disaccharide derivative allyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->8)-O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosidonate)-monohydrate C19H28O15Na2.H2O, M(r) = 542.32, is orthorhombic, P2(1)2(1)2(1) with a = 9.229(1), b = 12.036(1), c = 21.671(1) A, and Z = 4. The structure was solved by direct methods and refined to R = 0.040 for 2677 observed reflections. The torsion angles about the (2-->8)-glycosidic bond are stabilized by an intramolecular hydrogen bond between the carboxylate group at the anomeric carbon atom of the terminal Kdo residue and the hydroxyl group O-17 of the second Kdo moiety.

Carbohydrate Conformation↗
PubMed1994

Endotoxin and lipid A stimulate proliferation of human T cells in the presence of autologous monocytes.

In this paper we describe a new activity of LPS and partial structures: the induction of DNA synthesis and lymphokine production of human T lymphocytes. The LPS-induced T cell proliferation is dose dependent and requires 100 to 10,000 ng/ml of LPS or synthetic lipid A (compound 506) for optimal stimulation. In contrast, the synthetic lipid A precursor Ia (compound 406) is not active but rather antagonizes LPS-induced proliferation. The proliferation is accompanied by the expression of mRNA for the Th1 cell-derived lymphokines IFN-gamma and IL-2, but not for the Th2 lymphokines IL-4, IL-5, or IL-10. Highly enriched T lymphocyte preparations with less than 0.1% monocytes are not stimulated by LPS, showing that monocytes are required for T cell proliferation. Reconstitution experiments show that only monocytes, but not B lymphocytes, are able to support induction of DNA synthesis. Separating LPS-stimulated monocytes from T lymphocytes by a membrane, permeable for cytokines but not for cells, abolishes T cell proliferation. Fixation of monocytes with paraformaldehyde also abrogates their accessory function for T cells. If the monocytes are preincubated for 2 h at 37 degrees C with LPS and then washed, they still are able to induce T cell proliferation in the absence of additional LPS. Our results indicate that human T cells respond in a monocyte-supported manner to LPS exposure by proliferation and lymphokine production. We hypothesize that this reactivity of T lymphocytes to LPS may be of clinical relevance.

Adult↗