Structural elucidation of the Brucella melitensis M antigen by high-resolution NMR at 500 MHz

The Brucella M antigen from the species type strain Brucella melitensis 16M has been identified as a component of the cell wall lipopolysaccharide (LPS). O polysaccharide liberated from this LPS by mild acid hydrolysis exhibited M activity in serological tests and was shown to be a homopolymer of 4-formamido-4,6-dideoxy-alpha-D-mannopyranosyl residues arranged in an oligosaccharide repeating unit as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the native lipopolysaccharide. Structural analysis of the O polysaccharide by NMR methods was difficult due to apparent microheterogeneity of the repeating unit, which was in fact caused by the presence of rotational isomers of the N-formyl moiety. This problem was resolved by chemical modification of the polysaccharide to its amino and N-acetyl derivatives, the 500-MHz 1H and 125-MHz 13C NMR spectra of which could be analyzed in terms of a unique structure through application of pH-dependent beta-shifts and two-dimensional techniques that included COSY, relayed COSY, and NOESY experiments together with heteronuclear C/H shift correlation spectroscopy. On the basis of these experiments and supported by methylation and periodate oxidation data, the structure of the M polysaccharide was determined as a linear polymer of unbranched pentasaccharide repeating units consisting of four 1,2-linked and one 1,3-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl residues. The marked structural similarity of the M antigen and the A antigen, which is known to be a 1,2-linked homopolysaccharide of 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units, accounts for cross-serological reactions of the two and the long-standing confusion surrounding the nature of their antigenic determinants. Structural and serological considerations in conjuction with the sodium dodecyl sulfate banding pattern of Brucella A LPS suggest that its biosynthesis differs appreciably from that of the M antigen, which appears to be synthesized by regulated assembly of preformed oligosaccharide repeating units. Temperature, lysogenic phage may be responsible for such biosynthetic and structural variations.     

Preparation of polysaccharide-enzyme conjugates for competitive binding assays

A variety of bacterial O-polysaccharides were covalently linked to enzymes and it was demonstrated with three discrete monoclonal antibodies that enzyme-glycoconjugates function as convenient labelled antigens in direct enzyme immunoassays, particularly competitive assays that quantify bacterial O-antigens. Two strategies, each involving reductive amination, were used to couple O-polysaccharides to enzymes, while retaining high enzymic activity. Reduction of the Schiff base formed between, 1,3-diaminopropane and the terminal reducing ketodeoxyoctanoic acid (KDO) residue present in the majority of the lipopolysaccharide (LPS) core domains, following mild acid removal of Lipid A, offered the most direct route to mono-aminated polysaccharide. Alternatively, mild periodate oxidation of KDO and heptose residues generated multiple aldehyde targets for Schiff base formation, without affecting the O-antigenic determinant. Hetero- and homobifunctional coupling reagents, sulphosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and discuccinimidyl suberate, activated polysaccharide for coupling to enzymes at amino and sulphydryl sites and produced conjugates that retained at least 95% of the original enzymic activity. The most suitable enzyme conjugates, especially for competitive inhibition EIA were those bearing one polysaccharide chain, and these were easily prepared from horse-radish peroxidase. Although the extent of conjugation of activated polysaccharide to -galactosidase and alkaline phosphatase could be controlled by reaction stoichiometry, the use of these enzymes was a less effective utilization of valuable antigen and enzyme.Issued as NRCC No. 31634     

Rapid hydrophobic grid membrane filter-enzyme-labeled antibody procedure for identification and enumeration of Escherichia coli O157 in foods

An O-antigen-specific monoclonal antibody, labeled by horseradish peroxidase-protein A, was used in a hydrophobic grid membrane filter-enzyme-labeled antibody method for rapid detection of Escherichia coli O157 in foods. The method yielded presumptive identification within 24 h and recovered, on average, 95% of E. coli O157:H7 artificially inoculated into comminuted beef, veal, pork, chicken giblets, and chicken carcass washings. In food samples from two outbreaks involving E. coli O157:H7, the organism was isolated at levels of up to 10(3)/g. The lower limit of sensitivity was 10 E. coli O157 per g of meat. Specific typing for E. coli O157:H7 can be achieved through staining with labeled H7 antiserum or tube agglutination.     

DETERMINATION OF GUSTATORY SUCROSE THRESHOLD IN WATER BY USE OF A LINEAR SUCROSE GRADIENT

A novel experimental method was developed which allows the determination of the threshold concentration of sucrose by use of a linear sucrose gradient in water. With this method a continuous tasting of the test-liquid is possible. A panel of 15 persons experienced in taste-testing was used. Three gradients of different steepness were applied: 0 to 1.5% (w/w) sucrose in 2 min (I), 3 min (II) and 4 min (III). The results of the new method were compared with those of the standard method (DIN). With gradients I and II we found values which were significantly higher than those of the standard method (I: 0.49% (w/w); II: 0.46% (w/w); DIN: 0.31% (w/w)), whereas with gradient III the same threshold value was found as with the DIN-Method (III: 0.32% (w/w)).     

Crystal structure to 2.45 A resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen.

The atomic structure of an antibody antigen-binding fragment (Fab) at 2.45 A resolution shows that polysaccharide antigen conformation and Fab structure dictated by combinatorial diversity and domain association are responsible for the fine specificity of the Brucella-specific antibody, YsT9.1. It discriminates the Brucella abortus A antigen from the nearly identical Brucella melitensis M antigen by forming a groove-type binding site, lined with tyrosine residues, that accommodates the rodlike A antigen but excludes the kinked structure of the M antigen, as envisioned by a model of the antigen built into the combining site. The variable-heavy (VH) and variable-light (VL) domains are derived from genes closely related to two used in previously solved structures, M603 and R19.9, respectively. These genes combine in YsT9.1 to form an antibody of totally different specificity. Comparison of this X-ray structure with a previously built model of the YsT9.1 combining site based on these homologies highlights the importance of VL:VH association as a determinant of specificity and suggests that small changes at the VL:VH interface, unanticipated in modeling, may cause significant modulation of binding-site properties.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Delimitation of the maritime boundary in the gulf of Maine area

The compulsory dispute settlement regime included in the 1982 Law of the Sea Convention is recognized as one of the most comprehensive in a modern international convention. Yet, in the recent application of this regime, the question has arisen as to whether the procedural prerequisites associated with the LOS Convention's compulsory dispute settlement mechanism are so arduous as to avoid binding and compulsory jurisdiction in most instances. This article addresses that question by examining, in particular, the reasoning of the Southern Bluefin Tuna arbitration tribunal, which found Article 281 of Section 1 of the LOS Convention to bar jurisdiction to the compulsory dispute settlement mechanism prescribed by the Convention, and offers suggestions as to how states might distinguish or overcome the barriers imposed by the Southern Bluefin Tuna tribunal in future cases.     

Affinities of Shiga toxins 1 and 2 for univalent and oligovalent Pk-trisaccharide analogs measured by electrospray ionization mass spectrometry

The binding stoichiometry and affinities of the Shiga toxins, Stx1 and Stx2, for a series of uni- and oligovalent analogs of the Pk-trisaccharide were measured using the direct electrospray ionization mass spectrometry (ES-MS) assay. Importantly, it is shown that, for a given ligand, Stx1 and Stx2 exhibit similar affinities. The binding data suggest a high degree of similarity in the spatial arrangement and structural characteristics of the Pk binding sites in Stx1 and Stx2. The results confirm that both toxins recognize the alpha-D-Galp(1-->4)-beta-D-Galp(1-->4)-beta-D-Glcp carbohydrate motif of the cell surface glycolipid Gb3. This, taken together with the results of the chemical mapping study, suggests that the nature of the Pk binding interactions with Stx1 and Stx2 are similar. The affinities of Stx1-B(5) and Stx2 for the multivalent ligands reveals that site 2 of Stx2, which shares the same spatial arrangement as site 2 in Stx1, is the primary Pk binding site and that site 1 of Stx1 and of Stx2 can also participate in Pk binding.     

Molecular recognition of Candida albicans (1->2)-β-mannan oligosaccharides by a protective monoclonal antibody reveals the immunodominance of internal saccharide residues

A self-consistent model of β-mannan oligosaccharides bound to a monoclonal antibody, C3.1, that protects mice against Candida albicans has been developed through chemical mapping, NMR spectroscopic, and computational studies. This antibody optimally binds di- and trisaccharide epitopes, whereas larger oligomers bind with affinities that markedly decrease with increasing chain length. The (1→2)-β-linked di-, tri-, and tetramannosides bind in helical conformations similar to the solution global minimum. Antibody recognition of the di- and trisaccharide is primarily dependent on the mannose unit at the reducing end, with the hydrophobic face of this sugar being tightly bound. Recognition of a tetrasaccharide involves a frameshift in the ligand interaction, shown by strong binding of the sugar adjacent to the reducing end. We show that frameshifting may also be deliberately induced by chemical modifications. Molecular recognition patterns similar to that of mAb C3.1, determined by saturation transfer difference-NMR, were also observed in polyclonal sera from rabbits immunized with a trisaccharide glycoconjugate. The latter observation points to the importance of internal residues as immunodominant epitopes in (1→2)-β-mannans and to the viability of a glycoconjugate vaccine composed of a minimal length oligosaccharide hapten.