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Biomedical subjects

J W Redmond

Publications and source records attributed to J W Redmond.

17 recordsLinked to original sources
PubMed1992

Characterisation of oligosaccharides from a glycoprotein variant of human serum albumin (albumin Casebrook) using high-performance anion-exchange chromatography and nuclear magnetic resonance spectroscopy.

The characterisation of oligosaccharides present on albumin Casebrook, a glycoprotein variant of human serum albumin, which contains an N-linked oligosaccharide at an attachment site formed by a point mutation of 494 Asp-->Asn, is described. The monosaccharide compositional analysis of purified glycopeptides suggested the presence of complex biantennary carbohydrate structures. The oligosaccharides which were released by N-glycosidase-F appeared to be a single molecular species according to their retention on high-performance anion-exchange chromatography. The structure of the oligosaccharide was suggested by sequential exoglycosidase digestions and confirmed by proton nuclear magnetic resonance spectroscopy. It was concluded that the oligosaccharides were essentially homogeneous and consisted of an alpha(2-6)-desialylated complex biantennary glycan.

Amidohydrolases↗
PubMed1991

Applications of automated amino acid analysis using 9-fluorenylmethyl chloroformate.

A rapid and sensitive fully automated method for the determination of primary and secondary amino acids in different matrices is described. Amino acids are derivatized with 9-fluorenylmethyl chloroformate using an automated precolumn derivatization technique. Data are presented to show that the technique is both reproducible and highly sensitive. Applications of the technique are presented, including the analysis of peptide and protein hydrolysates and the profiling of free amino acids in physiological fluids.

Adult↗
PubMed1991

Unusual structure of the exopolysaccharide of Rhizobium leguminosarum bv. viciae strain 248.

The exopolysaccharide from R. leguminosarum bv. viciae strain 248 differs from those of other Rhizobium strains with similar symbiotic behavior. 13C-N.m.r. spectroscopy of fragments generated by partial hydrolysis, together with methylation analysis and 13C-n.m.r. spectroscopy of the enzymically depolymerised exopolysaccharide, indicated the following nonasaccharide repeating-unit: [formula: see text] The locations of the acetyl and 3-hydroxybutanoyl substituents in the exopolysaccharide are assigned provisionally. R. leguminosarum bv. viciae strain 248, cured of its Sym plasmid pRL1JI, synthesised an exopolysaccharide in which the sites and degree of substitution were unchanged. A Tn5 mutant, derived from strain 248 and unable to induce nodules, synthesised small amounts of EPS that lacked galactose.

Carbohydrate Sequence↗
PubMed1991

Distribution of O-acetyl groups in the exopolysaccharide synthesized by Rhizobium leguminosarum strains is not determined by the Sym plasmid.

The patterns of O-acetylation of the exopolysaccharide (EPS) from the Sym plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii strain LPR5, R. leguminosarum bv. trifolii strain ANU843 and R. leguminosarum bv. viciae strain 248 were determined by 1H and 13C NMR spectroscopy. Beside a site indicative of the chromosomal background, these strains have one site of O-acetylation in common, namely residue b of the repeating unit. The O-acetyl esterification pattern of EPS of the Sym plasmid-cured derivatives of strains LPR5, ANU843, and 248 was not altered by the introduction of a R. leguminosarum bv. viciae Sym plasmid or a R. leguminosarum bv. trifolii Sym plasmid. The induction of nod gene expression by growth of the bacteria in the presence of Vicia sativa plants or by the presence of the flavonoid naringenin, produced no significant changes in either amount or sites of O-acetyl substitution. Furthermore, no such changes were found in the EPS from a Rhizobium strain in which the nod genes are constitutively expressed. The substitution pattern of the exopolysaccharide from R. leguminosarum is, therefore, determined by the bacterial genome and is not influenced by genes present on the Sym plasmid. This conclusion is inconsistent with the suggestion of Philip-Hollingsworth et al. (Philip-Hollingsworth, S., Hollingsworth, R. I., Dazzo, F. B., Djordjevic, M. A., and Rolfe, B. G. (1989) J. Biol. Chem. 264, 5710-5714) that nod genes of R. leguminosarum bv. trifolii, by influencing the acetylation pattern of EPS, determine the host specificity of nodulation.

Acetylation↗
PubMed1991

Synthesis of a PAF immunogen and production of PAF-specific antibodies.

Platelet activating factor (PAF), a naturally occurring phospholipid with many potent physiological and pharmacological activities, is implicated as a mediator of many diseases. An immunoassay for PAF would greatly improve quantitation, and hence PAF-specific antibodies were required. Chemically-reactive analogs of PAF, containing an aldehyde group at the end of the 1-O-alkyl chain (hexyl or dodecyl), were synthesized from readily available materials. During the multi-step synthetic procedure, the aldehyde group was protected as an acetal, which was converted by mild acidic hydrolysis to the aldehyde immediately prior to protein coupling. These analogs were coupled to methylated bovine serum albumin and the resultant conjugates were injected into rabbits. Antibodies to PAF were detected using a solid phase radioimmunoassay based on Protein A-Sepharose. The dodecyl PAF conjugate proved to be the more immunogenic conjugate with more than half of the rabbits producing significant levels of antibodies (at least a 10-fold increase in radioactive uptake over pre-immune levels). Results from solid phase immunoassays employing nitrocellulose discs impregnated with PAF, lysoPAF, lecithin, lysolecithin and 2-O-methyl-lysoPAF indicated that the antibodies recognized only PAF. PAF-specific antibodies were isolated by affinity chromatography using a column of PAF-poly(lysine) conjugated to carboxy-activated polyacrylamide. The antibodies may be employed in a sensitive and specific immunoassay for PAF and for many other studies involving PAF.

Animals↗
PubMed1990

Rhizobium leguminosarum exoB mutants are deficient in the synthesis of UDP-glucose 4'-epimerase.

Rhizobium leguminosarum bv. viciae Exo- mutant strains RBL5523,exo7::Tn5,RBL5523,exo8::Tn5 and RBL5523,exo52::Tn5 are affected in nodulation and in the syntheses of lipopolysaccharide, capsular polysaccharide, and exocellular polysaccharide. These mutants were complemented for nodulation and for the syntheses of these polysaccharides by plasmid pMP2603. The gene in which these mutants are defective is functionally homologous to the exoB gene of Rhizobium meliloti. The repeating unit of the residual amounts of EPS still made by the exoB mutants of R. leguminosarum bv. viciae lacks galactose and the substituents attached to it. The R. leguminosarum bv. viciae and R. meliloti exoB mutants fail to synthesize active UDP-glucose 4'-epimerase.

Carbohydrate Sequence↗
PubMed1989

Production of antibodies to platelet activating factor.

Elucidation of the pathophysiological role of platelet activating factor (PAF) in health and disease is currently hampered by the lack of a sensitive, reproducible and easily applied assay for this potent phospholipid. This study describes the preparation of PAF in an immunogenic form, the production of antibodies to PAF and their use in the development of a preliminary RIA for PAF. Antibodies formed in response to a synthetic PAF analog coupled to a protein carrier were detected with two types of solid phases: PAF non-covalently adsorbed onto NC and the PAF analog covalently linked to PA. The latter was also used as a support for the isolation of anti-PAF antibodies by affinity chromatography. Quantitative hapten inhibition studies showed that the antibody combining sites were complementary to PAF and that cross-reactivity to lyso-PAF and some related phospholipids was negligible. Using these antibodies, [3H]PAF and Protein A-Sepharose as a means of separating bound and free tracer, the feasibility of developing a quantitative RIA for PAF was demonstrated.

Animals↗
PubMed1988

Identification of a nodD-dependent locus in the Rhizobium strain NGR234 activated by phenolic factors secreted by soybeans and other legumes.

Transfer of the strain NGR234nodD 1 gene into the narrow host range R. trifolii strain ANU843 on either a 6.7-kb HindIII or 17-kb XhoI fragment broadens the host range of this bacterium to include the tropical legumes Vigna unguiculata, Glycine ussuriensis, Leucaena leucocephala, and siratro (Macroptilium atropurpureum). Contrary to previous data (Bassam et al. 1986), mutagenesis of the 17-kb XhoI fragment with a mini-Mu lac transposon (Mu dII1734) showed that a functional nodD 1 gene was essential for extended host range. Gene expression studies using both Mu dII1734 fusions and a promoter-cloning vector indicated that several loci, including the nodD 1 gene, are constitutively expressed. No evidence was found for regulation of the strain NGR234 nodD 1 gene by its product. Another locus nod-81, was induced only in the presence of exudates from various plant species, including soybean (Glycine max). Whereas the expression of nod-81 was dependent on the presence of a functional nodD 1 gene product, a regulatory nod-box DNA sequence was not detected 5' to this gene by using available oligonucleotide hybridization probes. The nod-81 locus was induced by genistein, daidzein, naringenin, and coumestrol from both cotyledon and root tissue of freshly germinated soybean seedlings. A broad spectrum of commercially available phenolic compounds stimulated induction of the nod-81 locus, including some that antagonize nod gene induction in other Rhizobium species. The nodD 1 gene product from strain NGR234 was shown to determine the spectrum of compounds that induce nod-81 expression.

Base Sequence↗
PubMed1988

Induction of pathogenic-like responses in the legume Macroptilium atropurpureum by a transposon-induced mutant of the fast-growing, broad-host-range Rhizobium strain NGR234.

Mutant strain ANU2861, a transposon Tn5 mutant of the fast-growing, broad-host-range Rhizobium strain ANU280 (NGR234 Smr Rfr) overproduces polysaccharide, is an ade auxotroph, and induces poorly developed nodules on Leucaena leucocephala and Lablab purpureus (H.C. Chen, M. Batley, J.W. Redmond, and B.G. Rolfe, J. Plant Physiol. 120:331-349, 1985). Strain ANU2861 cannot form nodules on Macroptilium atropurpureum Urb. (siratro) or on Desmodium intortum and D. uncinatum and the nonlegume Parasponia. The parent strain, ANU280, effectively nodulates all these legume species except Parasponia, on which it forms ineffective nodules. Ultrastructural examination of infection sites on the legume siratro showed that mutant strain ANU2861 caused root hair curling (Hac+ phenotype), some cortical cell division (Noi+), but no infection threads (Inf-). Localized cellular responses, known to occur in phytopathological interactions, were observed in electron micrographs of the epidermal tissue at or near the infection zone after inoculation with strain ANU2861 but not the wild-type parental strain. These include (i) the rapid (within 20 h) accumulation of osmiophilic droplets attached to membranes at potential sites of strain ANU2861 penetration and (after 48 h) in the epidermal cells in the immediate region of the curled root hairs, and (ii) localized cell death of the epidermal cells. In addition, strain ANU2861 can initiate a systemic response in split-root siratro plants which prevents the successful nodulation of strain ANU280. A 6.3-kilobase fragment of wild-type genomic DNA, which includes the site of Tn5 insertion in strain ANU2861, was cloned and introduced to strain ANU2861. All the phenotypic defects of the mutant strain were corrected by the introduction of this DNA fragment. This indicates that the original Tn5 insertion is responsible for the phenotype.

Cloning, Molecular↗
PubMed1987

Nuclear magnetic resonance spectra of lipoteichoic acid.

Lipoteichoic acid acids with a range of chemical compositions have been studied using 1H; 13C- and 31P-nuclear magnetic resonance. Proton spectroscopy provided a rapid method for demonstrating whether alanine in a sample is covalently bound to the polyglycerophosphate chains and for monitoring hydrolysis of alanine. The nature of sugar substituents can be determined, with some limitations, from the 13C spectra, and the proportions of glycerol residues substituted by alanine and sugar can be measured. The 31P spectra of lipoteichoic acid provided information about both the degree of substitution and the distribution of the substituent along the polyglycerophosphate chain, except when the substituent was galactose. The polyglycerophosphate chains were shown to undergo rapid internal rotation and no evidence for tertiary structure was found either in the presence or absence of magnesium ions. Magnesium ions exchange rapidly between the bound and free state and the binding constant to lipoteichoic acid of 64 M-1 is typical for monophosphates in aqueous solution. There was no evidence that alanine substitution affects the binding constant for magnesium ions.

Alanine↗
PubMed1987

Nitrogen fixation ability of exopolysaccharide synthesis mutants of Rhizobium sp. strain NGR234 and Rhizobium trifolii is restored by the addition of homologous exopolysaccharides.

Several transposon Tn5-induced mutants of the broad-host-range Rhizobium sp. strain NGR234 produce little or no detectable acidic exopolysaccharide (EPS) and are unable to induce nitrogen-fixing nodules on Leucaena leucocephala var. Peru or siratro plants. The ability of these Exo- mutants to induce functioning nodules on Leucaena plants was restored by coinoculation with a Sym plasmid-cured (Nod- Exo+) derivative of parent strain NGR234, purified EPS from the parent strain, or the oligosaccharide from the EPS. Coinoculation with EPS or related oligosaccharide also resulted in formation of nitrogen-fixing nodules on siratro plants. In addition, an Exo- mutant (ANU437) of Rhizobium trifolii ANU794 was able to form nitrogen-fixing nodules on white clover in the presence of added EPS or related oligosaccharide from R. trifolii ANU843. These results demonstrate that the absence of Rhizobium EPSs can result in failure of effective symbiosis with both temperate and subtropical legumes.

Carbohydrate Conformation↗
PubMed1986

Synthesis of methyl pyranosides and furanosides of 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) by acid-catalysed solvolysis of the acetylated derivatives.

Treatment of methyl 2,4,5,7,8-penta-O-acetyl-3-deoxy-alpha-D-manno-oct- 2-ulopyranosonic acid, or its methyl ester, with refluxing methanolic 0.1 M hydrogen chloride for 16 h gave 95% of methyl (methyl 3-deoxy-alpha-D-manno-oct-2-ulopyranosid)onate. Acetylation of the methyl ester of 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) gave mainly methyl 2,4,6,7,8-penta-O-acetyl-3-deoxy-alpha,beta-D-manno-oct-2-ulofuranoso nate. Treatment of this mixture with methanolic 0.02 M hydrogen chloride at room temperature gave methyl (methyl 3-deoxy-alpha, beta-D-manno-oct-2-ulofuranosid)onate and the corresponding 4-acetates which were isolated by reverse-phase column chromatography of their 7,8-O-isopropylidene derivatives. Confirmation of the position of the isopropylidene group was obtained by acetylation to give methyl (methyl 4,6-di-O-acetyl-3-deoxy-7,8-O-isopropylidene-alpha,beta-D-manno-oct-2-ul ofuranosid)onate. The furanose anomers were differentiated primarily by J3,4 values (alpha approximately 6.1 Hz, beta approximately 2.2 Hz). The anomeric configuration in the furanose series has been assigned on the basis of optical rotation.

Bordetella pertussis↗
PubMed1979

The structure of the O-antigenic side chain of the lipopolysaccharide of Vibrio cholerae 569B (Inaba).

Mineral acid hydrolysis of the lipopolysaccharide from Vibrio cholerae 569B (Inaba) gives an oligosaccharide fraction which was shown, by use of 13C NMR and chemical methods, to be a regular alpha-(1 leads to 2) linked chain of D-perosamine (4-amino-4,6-dideoxy-D-mannose) units. This chain represents the O-antigen of the lipopolysaccharide, in which the amino functions are acylated with 3-hydroxypropionyl groups. The chromatographic properties of some hydroxamic acids are described and used to characterize these acyl groups.

Carbohydrates↗
PubMed1978

The 4-amino sugars present in the lipopolysaccharides of vibro cholerae and related vibrios.

Brief treatment, with 10 M hydrochloric acid, of the lipopolysaccharides of Vibrio cholerae and related organisms led to the release of 4-amino-4, 6-dideoxy-D-mannose (D-perosamine) and 4-amino-4-deoxyl-L-arabinose. These sugar are highly unstable and undergo complex changes in neutral and basic media. Of the strains examined, the lipopolysaccharides from representatives of the Inaba group contain only D-perosamine, while those of the Ogawa biotype have 4-amino-4-deoxyl-L-arabinose as an additional component. The other Vibrios produce lipopolysaccharides which lack these sugars.

Amino Sugars↗