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J C Richards

Publications and source records attributed to J C Richards.

At least 37 records · Page 2Linked to original sources
PubMed2000

Polymer-supported and chemoenzymatic synthesis of the Neisseria meningitidis pentasaccharide: a methodological comparison.

Neisseria meningitidis trisaccharide [GlcNAc[(1-->3)Galbeta(1-->4)Glc-R], tetrasaccharide [Galbeta(1-->4)GlcNAcbeta(1--> 3)Galbeta(1-->4)Glc-R], and a pentasaccharide [Neu5Acalpha(2-->3)Galbeta(1-->4)GlcNAcbeta(1-->3)G albeta(1-->4)Glc-SPh] were prepared via conventional chemical synthesis, polymer-supported synthesis, and chemoenzymatic methods, starting from D-lactose. The polymer polyethyleneglycol monomethylether (MPEG) and the linker dioxyxylene (DOX) were used with a lactose-bound acceptor to improve the purification process. Several enzymes (LgtA, GalE-LgtB fusion, and CMP-Neu5Ac synthetase/sialyltransferase fusion) were used for syntheses of these oligosaccharides. Excellent stereo- and regioselectivities as well as high yield (> 90% from Gal(1-->4)Glc-SPh) of the pentasaccharide were obtained. Both of the convenient processes are suitable for efficient preparation of target oligosaccharides.

Antigens, Bacterial↗
PubMed2000

Enzyme linked immunosorbent assay (ELISA) for the detection of serum antibodies to the inner core lipopolysaccharide of Neisseria meningitidis group B.

We have developed a solid-phase ELISA to study the human immune response to inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm) using structurally defined glycolipids from a genetically defined mutant (galE) of a serogroup B Nm strain. Previous studies had demonstrated that a galE (inner core) LPS epitope is conserved in approximately 70% Nm strains and was accessible to antibody in fully encapsulated wild-type Nm strains. A murine monoclonal antibody, MAb B5, raised to a galE mutant of serogroup B Nm strain, immunotype L3 (B.15.P1.7,16) was used to determine the specificity of the inner core LPS ELISA by inhibition studies using purified galE LPS and human sera. The intra-assay coefficient of variation (CV) was 5-6% and inter-assay CV was 19-22%. Using this ELISA, significant differences in the geometric mean titres (GMTs) of naturally occurring serum antibodies (specific to inner core LPS) between healthy adults (18-65 years, N=54) and healthy infants (3-4 months, N=144) of both IgG and IgM classes were found (P<0.0001). GMTs were expressed in galE arbitrary units (AU/ml) (95% confidence intervals): IgG antibodies in adults 5.7 (5. 0,6.9) and in infants 1.1 (1.0,1.3); IgM antibodies in adults 7.7 (5. 7,10.4), and in infants 0.85 (0.7,1.1). In age-matched children aged 26-113 months a difference (P=0.04) in specific IgG was found in healthy infants and infants in the acute phase of invasive Nm disease (GMT (95%CI) in AU/ml: in healthy infants 7.7 (5.3,11.0), in acute phase infants 4.2 (2.5,7.2). However, there was no difference in specific IgM (P=0.98) between these groups healthy infants 4.7 (3. 1,7.0), acute phase 4.6 (2.9, 7.4). In eleven children (5-181 months) there were differences in the GMTs of specific IgG and IgM (P=0.02, P=0.008 respectively) between paired acute and convalescent sera (GMT) (95%CI) in AU/ml: IgG acute 1.95 (0.98, 3.8), convalescent 5.2 (2.2,12.4); IgM acute 1.78 (1.05,3.0), convalescent 4.38 (2.6,7.3). We conclude that ELISA is a specific, sensitive and reproducible method for the detection of antibodies to inner core LPS of Nm and that an epitope defined by MAb B5 can be immunogenic in infants and adults. These findings are relevant to the potential candidacy of inner core LPS as a vaccine.

Acute Disease↗
PubMed2000

Serum lipids and their relationships with hostility and angry affect and behaviors in men.

Research into the relationship between the Type A behavior pattern and coronary heart disease suggests that the anger-hostility-aggression (AHA!) syndrome is directly related to total serum cholesterol and low-density lipoproteins. The present study involved an investigation of the specific components of the AHA! syndrome related to blood lipid levels in 98 healthy men. The disposition to experience and express anger when frustrated, criticized, or treated unfairly (angry reaction, a component of trait anger) was related to total serum cholesterol and to low-density lipoprotein levels. Age and diet also predicted levels of these lipids, but each was unrelated to angry reaction. These results suggest that in healthy men, the experience of strong angry affect in reaction to perceived rejection, criticism, or unfair treatment may be health-toxic because of its relationship to elevated unfavorable serum lipids.

Adult↗
PubMed2000

The position of phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae affects binding and sensitivity to C-reactive protein-mediated killing.

The lic1 locus of Haemophilus influenzae controls the incorporation of environmental choline into lipopolysaccharide (LPS) as phosphorylcholine (ChoP) as well as the phase variation of this structure. ChoP is the target of an acute phase reactant in serum, C-reactive protein (CRP), which mediates killing through the activation of complement when bound to the organism. Structural analysis of the oligosaccharide region of the H. influenzae LPS showed that ChoP is linked to different hexose residues on different chain extensions in strains Rd and Eagan. Differences in the molecular environment of ChoP affect the epitope defined by monoclonal antibody 12D9 and were associated with polymorphisms within LicD, a putative diphosphonucleoside choline transferase. Exchanging the licD genes between the two strains with ChoP on different chain extensions was sufficient to switch its position. Allelic variants with ChoP on a hexose on heptose III rather than heptose I were sensitive to CRP-mediated serum bactericidal activity regardless of the genetic background. Differences in CRP-mediated killing correlated with differences in the binding of CRP from human serum to whole bacteria. This suggests that, in addition to the mechanism involving phase variation, the structural rearrangements within the oligosaccharide contribute to evasion of innate and acquired immunity.

Alleles↗
PubMed2000

Structural characterization of the outer core and the O-chain linkage region of lipopolysaccharide from Pseudomonas aeruginosa serotype O5.

The point of attachment of the O-chain in the outer core region of Pseudomonas aeruginosa serotype O5 lipopolysaccharide (LPS) was determined following a detailed analysis of the extended core oligosaccharide, containing one trisaccharide O-chain repeating unit, present in both the wild-type strain PAO1 and O-chain deficient mutant strains AK1401 and PAO-rfc. The structure of the extended core oligosaccharide was determined by various mass spectrometric methods as well as one-dimensional and two-dimensional NMR spectroscopy. Furthermore, the one-dimensional analogues of NOESY and TOCSY experiments were applied to confirm the structure of the outer core region in the O-chain polysaccharide. In both the extended core oligosaccharide and the core of the smooth LPS, a loss of one of the beta-glucosyl residues and the translocation of the alpha-rhamnosyl residue, followed by the attachment of the first O-chain repeating unit was observed. This process is complicated and could involve two distinct rhamnosyltransferases, one with alpha-1, 6-linkage specificity and another with alpha-1,3-linkage specificity. It is also plausible that an alpha-1,3 rhamnosyltransferase facilitates the addition of the 'new' alpha-rhamnosyl residue that will act as a receptor for the attachment of the single O-antigen repeating unit in the LPS of the semi-rough mutant. The 2-amino-2-deoxy-fucosyl residue of the first O-chain repeating unit directly attached to the core was found to have a beta-anomeric configuration instead of an alpha configuration, characteristic for this residue as a component of the O-chain polysaccharide. The results of this study provide the first example of the mechanistic implications of the structure of the outer core region in a fully assembled O-chain containing LPS, differing from the O-chain deficient rough LPS.

Bacterial Capsules↗
PubMed2000

Characterization of the phosphocholine-substituted oligosaccharide in lipopolysaccharides of type b Haemophilus influenzae.

Haemophilus influenzae expresses heterogeneous populations of short-chain lipopolysaccharide (LPS) which exhibit extensive antigenic diversity among multiple oligosaccharide epitopes. These LPS oligosaccharide epitopes can carry phosphocholine (PCho) substituents, the expression of which is subject to high frequency phase variation mediated by genes in the lic1 genetic locus. The location and site of attachment of PCho substituents were determined by structural analysis of LPS from two type b H. influenzae strains, Eagan and RM7004. The lic2 locus is involved in phase variation of oligosaccharide expression. LPS obtained from the parent strains, from mutants generated by insertion of antibiotic resistance cassettes in the lic2 genetic locus, and from phase-variants showing high levels of PCho expression was characterized by electrospray ionization-mass spectrometry (ESI-MS) and 1H NMR spectroscopy of derived O-deacylated samples. ESI-MS of O-deacylated LPS from wild-type strains revealed mixtures of related glycoform structures differing in the number of hexose residues. Analysis of LPS from PCho-expressing phase-variants revealed similar mixtures of glycoforms, each containing a single PCho substituent. O-Deacylated LPS preparations from the lic2 mutants were much less complex than their respective parent strains, consisting only of Hex3 and/or Hex2 glycoforms, were examined in detail by high-field NMR techniques. It was found that the LPS samples contain the phosphoethanolamine (PEtn) substituted inner-core element, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1--> 3)-L-alpha-D-He pp-(1-->5)-alpha-Kdo in which the major glycoforms carry a beta-D-Glcp or beta-D-Glcp-(1-->4)-beta-D-Glcp at the O-4 position of the 3-substituted heptose (HepI) and a beta-D-Galp at the O-2 position of the terminal heptose (HepIII). LPS from the lic2 mutants of both type b strains were found to carry PCho groups at the O-6 position of the terminal beta-D-Galp residue attached to HepIII. In the parent strains, the central heptose (HepII) of the LPS inner-core element is also substituted by hexose containing oligosaccharides. The expression of the galabiose epitope in LPS of H. influenzae type b strains has previously been linked to genes comprising the lic2 locus. The present study provides definitive evidence for the role of lic2 genes in initiating chain extension from HepII. From the analysis of core oligosaccharide samples, LPS from the lic2 mutant strain of RM7004 was also found to carry O-acetyl substituents. Mono-, di-, and tri-O-acetylated LPS oligosaccharides were identified. The major O-acetylated glycoforms were found to be substituted at the O-3 position of HepIII. A di-O-acetylated species was characterized which was also substituted at the O-6 postion of the terminal beta-D-Glc in the Hex3 glycoform. This is the first report pointing to the occurrence of O-acetyl groups in the inner-core region of H. influenzae LPS. We have previously shown that in H. influenzae strain Rd, a capsule-deficient type d strain, PCho groups are expressed in a different molecular environment, being attached at the O-6 position of a beta-D-Glcp, which is in turn attached to HepI.

Acetylation↗
PubMed1999

The structure of the specific capsular polysaccharide of Rhodococcus equi serotype 4.

The specific capsular polysaccharide produced by Rhodococcus equi serotype 4 was found to be a high-molecular-weight acidic polymer composed of D-glucose, D-mannose, pyruvic acid and a previously unidentified 5-amino-3,5-dideoxynonulosonic (rhodaminic) acid in the proportions 2:1:1:1. Structural analysis, employing a combination of microanalytical methods, nuclear magnetic resonance spectroscopy, and mass spectrometric techniques, established that the polysaccharide consisted of linear repeating tetrasaccharide units having the sequence of residues shown below. In the native polysaccharide, the rhodaminic acid residues were present as their acetamido derivatives (RhoANAc) and carried 1-carboxyethylidene groups that bridged the O-7 and O-9 positions. Treatment of the capsular polysaccharide with dilute acetic acid and/or anhydrous hydrogen fluoride under hydrolytic/solvolytic conditions, resulted in the formation of four different oligosaccharide species. The 1H and 13C NMR resonances of these oligosaccharide fragments and of the native serotype 4 capsular polysaccharides were fully assigned by homo- and heteronuclear chemical shift correlation methods.

Animals↗
PubMed1999

Selective attention in obsessive-compulsive disorder.

Recent information-processing studies have suggested that a selective attention deficit may be involved in the symptomatology of obsessive-compulsive disorder (OCD). In this study, individuals diagnosed with OCD were distinguished from those with panic disorder and from control participants by their relatively poorer performance on a series of psychometric tasks of selective attention. These results are interpreted as supporting the hypothesis of a diminished ability of people with OCD to selectively ignore competing external (sensory) and internal (cognitive) stimuli, especially intrusive thoughts.

Adult↗
PubMed1999

Sialic acid in the lipopolysaccharide of Haemophilus influenzae: strain distribution, influence on serum resistance and structural characterization.

A survey of Haemophilus influenzae strains indicated that around one-third of capsular strains and over two-thirds of non-typeable strains included sialic acid in their lipopolysaccharides (LPS). Mutation of the CMP-Neu5Ac synthetase gene (siaB) resulted in a sialylation-deficient phenotype. Isogenic pairs, wild type and siaB mutant of two non-typeable strains were used to demonstrate that sialic acid influences resistance to the killing effect of normal human serum but has little effect on attachment to, or invasion of, cultured human epithelial cells or neutrophils. We determine for the first time the site of attachment of sialic acid in the LPS of a non-typeable strain and report that a small proportion of glycoforms include two sialic acid residues in a disaccharide unit.

Bacterial Adhesion↗
PubMed1999

Structural analysis of the lipopolysaccharide oligosaccharide epitopes expressed by a capsule-deficient strain of Haemophilus influenzae Rd.

Structural elucidation of the lipopolysaccharide (LPS) of Haemophilus influenzae, strain Rd, a capsule-deficient type d strain, has been achieved by using high-field NMR techniques and electrospray ionization-mass spectrometry (ESI-MS) on delipidated LPS and core oligosaccharide samples. It was found that this organism expresses heterogeneous populations of LPS of which the oligosaccharide (OS) epitopes are subject to phase variation. ESI-MS of O-deacylated LPS revealed a series of related structures differing in the number of hexose residues linked to a conserved inner-core element, L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp- (1-->4)-]- L-alpha-D-Hepp-(1-->5)-alpha-Kdo, and the degree of phosphorylation. The structures of the major LPS glycoforms containing three (two Glc and one Gal), four (two Glc and two Gal) and five (two Glc, two Gal and one GalNAc) hexoses were substituted by both phosphocholine (PCho) and phosphoethanolamine (PEtn) and were determined in detail. In the major glycoform, Hex3, a lactose unit, beta-D-Galp-(1-->4)-beta-D-Glcp, is attached at the O-2 position of the terminal heptose of the inner-core element. The Hex4 glycoform contains the PK epitope, alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp while in the Hex5 glycoform, this OS is elongated by the addition of a terminal beta-D-GalpNAc residue, giving the P antigen, beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-D-Glc p. The fully extended LPS glycoform (Hex5) has the following structure. [see text] The structural data provide the first definitive evidence demonstrating the expression of a globotetraose OS epitope, the P antigen, in LPS of H. influenzae. It is noteworthy that the molecular environment in which PCho units are found differs from that observed in an Rd- derived mutant strain (RM.118-28) [Risberg, A., Schweda, E. K. H. & Jansson, P-E. (1997) Eur. J. Biochem. 243, 701-707].

Carbohydrate Conformation↗
PubMed1999

Conservation and accessibility of an inner core lipopolysaccharide epitope of Neisseria meningitidis.

We investigated the conservation and antibody accessibility of inner core epitopes of Neisseria meningitidis lipopolysaccharide (LPS) because of their potential as vaccine candidates. An immunoglobulin G3 murine monoclonal antibody (MAb), designated MAb B5, was obtained by immunizing mice with a galE mutant of N. meningitidis H44/76 (B. 15.P1.7,16 immunotype L3). We have shown that MAb B5 can bind to the core LPS of wild-type encapsulated MC58 (B.15.P1.7,16 immunotype L3) organisms in vitro and ex vivo. An inner core structure recognized by MAb B5 is conserved and accessible in 26 of 34 (76%) of group B and 78 of 112 (70%) of groups A, C, W, X, Y, and Z strains. N. meningitidis strains which possess this epitope are immunotypes in which phosphoethanolamine (PEtn) is linked to the 3-position of the beta-chain heptose (HepII) of the inner core. In contrast, N. meningitidis strains lacking reactivity with MAb B5 have an alternative core structure in which PEtn is linked to an exocyclic position (i.e., position 6 or 7) of HepII (immunotypes L2, L4, and L6) or is absent (immunotype L5). We conclude that MAb B5 defines one or more of the major inner core glycoforms of N. meningitidis LPS. These findings support the possibility that immunogens capable of eliciting functional antibodies specific to inner core structures could be the basis of a vaccine against invasive infections caused by N. meningitidis.

Animals↗
PubMed1999

Examination of the AHA!-Illness relation in male and female university students from Australia, India, and the United States.

The search for the link between negative emotions and later health problems continues to be a dominant theme in behavioral medicine. Although researchers have identified significant relations between illness and anger, most studies focus selectively on one anger domain: either the affective, attitudinal, or behavioral component. In this study, we first related all three components of the AHA! (anger, hostility, and aggressive behavior) syndrome to somatic complaint reports in a male and female young adult population drawn from three countries. In subsequent analyses, we related multidimensional characteristics of anger to illness in those young adults who regularly use tobacco and alcohol. In both the total sample and the substance user analyses, we found that the anger-illness profile varied as a function of gender.

Journal Article↗
PubMed1998

Development of an on-line preconcentration method for the analysis of pathogenic lipopolysaccharides using capillary electrophoresis-electrospray mass spectrometry. Application to small colony isolates.

The present investigation describes the use of on-line chromatographic preconcentration coupled to capillary zone electrophoresis-electrospray mass spectrometry (cPC-CZE-ES-MS) for trace level analysis of negatively charged lipopolysaccharides (LPS) obtained from pathogenic strains of Haemophilus influenzae. The analytical performance of two different types of adsorption media [i.e., C18 irregular particles and poly(styrene-divinylbenzene) membrane] for anionic analytes was first evaluated using a mixture of peptide standards to determine the overall sensitivity of this approach. These chromatographic preconcentrators provided an enhancement of sample loadings of up to 5 microliters with good linear response and low nM concentration detection limits for most peptides investigated. The application of cPC-CZE-ES-MS is further demonstrated for extracts of O-deacylated LPS obtained from H. influenzae strain Eagan. In combination with novel enzymatic releasing methods using proteinase K, this technique provides unparalleled sensitivity and enabled the identification of LPS surface antigens from as little as five bacterial colonies.

Amino Acid Sequence↗
PubMed1998

Structural elucidation of the lipopolysaccharide core regions of the wild-type strain PAO1 and O-chain-deficient mutant strains AK1401 and AK1012 from Pseudomonas aeruginosa serotype O5.

Lipopolysaccharide (LPS) of the Pseudomonas aeruginosa serotype O5 wild-type strain PAO1 and derived rough-type mutant strains AK1401 and AK1012 was isolated by a modified phenol/chloroform/petroleum-ether extraction method. Deoxycholate/PAGE of the LPS from the rough mutant AK1401 indicated two bands near the dye front with mobilities similar to those of the parent strain, indicating that both LPS contain a complete core and a species comprising a core and one repeating unit. Composition analysis of the LPS from strains PAO1 and AK1401 indicated that the complete core oligosaccharide was composed of D-glucose (four units), L-rhamnose (one unit), 2-amino-2-deoxy-D-galactose (one unit), L-glycero-D-manno-heptose (Hep; two units), 3-deoxy-D-manno-octulosonic acid (Kdo; two units), L-alanine (one unit) and phosphate (three units). The glycan structure of the LPS was determined by one-dimensional and two-dimensional (2D) NMR techniques in combination with MS-based methods on oligosaccharide samples obtained from the LPS by delipidation procedures. The locations of three phosphomonoester groups on the first heptose residue were established by a two-dimensional 31P (omega1)-half-filtered COSY experiment on the reduced core oligosaccharide sample of the LPS from the wild-type strain. The presence of a 7-O-carbamoyl substituent was observed on the second heptose. The structure of the core region of the O-chain-deficient LPS from P. aeruginosa serotype 05 is as follows: [structure: see text] where R1 is beta-D-Glcp-(1-->2)-alpha-L-Rhap-(1-->6)-alpha-D-Glcp-(1--> and R2 is alpha-D-Glcp-(1-->6)-beta-D-Glcp-(1->. A structural model is presented that is also representative of that for P. aeruginosa serotype O6 LPS. A revised structure for the serotype O6 mutant strain A28 is presented.

Lipopolysaccharides↗
PubMed1998

Structure of an alpha-2,6-sialylated lipooligosaccharide from Neisseria meningitidis immunotype L1.

The recent cloning of the lipooligosaccharide (LOS) a-2,3-sialyltransferase from Neisseria meningitidis immunotype L3 permitted us to examine other immunotypes for this structural gene. We identified the gene and measured the enzyme activity in the L1 immunotype strain which had previously been reported to lack sialic acid in its LOS because it contains a terminal alpha-linked galactose which was thought not to be an acceptor for the sialyltransferase. This finding prompted us to re-examine the structure of the LOS from the L1 immunotype, which revealed the presence of sialic acid on the terminal alpha-linked galactose. Oligosaccharides derived from the LOS were shown to be sialylated by composition and methylation analysis, mass spectrometry and nuclear magnetic resonance. The detailed structural analysis showed the sialic acid to occur only at 06 of the terminal a-D-galactopyranose residue of the alpha-D-Gal-1,4-beta-D-Gal-1,4-beta-D-glc trisaccharide (Pk epitope) chain of the LOS, in the alpha-D configuration. These data are the first report of a alpha-2,6-linked sialic acid in a bacterial LOS or lipopolysaccharide, and also the first report of a sialylated Pk epitope.

Carbohydrate Conformation↗