Immunochemical and structural analysis of the O polysaccharides of Salmonella zuerich [1,9,27,(46)].

Salmonella zuerich [1,9,27,(46)] has been shown to exhibit two levels of structural heterogeneity. The bacterium carries two distinct O-polysaccharide molecules with and without O:factor 1. Both sets of molecules (1+ and 1-) carry the two O:factors 27 and 46 on the same O chain, but they are expressed unevenly; in contrast to factor 27, O:factor 46 is always weakly expressed. Part of this weak expression was thought to be due to strong inhibition of factor 46 by factor 1. In this study, serological analysis gave more detailed information on the sizes of the different O:factor epitopes. Structural analysis of S. zuerich O polysaccharides showed that they are constructed of the expected chemical sequences characteristic of factors 1, 9, 27, and 46. No modification in the sugar sequence could account for the weak expression of O:factor 46. Factors 27 and 46 are present on oligosaccharides carrying either an alpha-Man (factor 27) or a beta-Man (factor 46) residue. In S. zuerich, the alpha-Man configuration is predominant, corroborating the expression of strong factor 27 and weak factor 46 on the bacteria. Questions raised by the existence of such heterogeneous O polysaccharides on the specificity of the O chain polymerase, as well as the place of S. zuerich in Salmonella evolution, are dicussed in this paper.

Structural analysis of a novel sialic-acid-containing trisaccharide from Rhodobacter capsulatus 37b4 lipopolysaccharide.

Sialic-acid-containing lipopolysaccharides from Rhodobacter capsulatus 37b4 (S-form lipopolysaccharide), KB-1 (R-type lipopolysaccharide) and Sp 18 (deep R-type lipopolysaccharide) were investigated for the linkage and substitution of sialic acids. Methylation analysis and behaviour towards acid and enzymic hydrolysis indicated a non-reducing terminal location of sialic acids in the R-type lipopolysaccharide of strain Sp 18, whereas an internal, chain-linked location of sialic acids was found in the lipopolysaccharides of strains 37b4 and KB-1. For these latter strains, methylation analysis revealed a substitution of sialic acids by other sugars at position 7 for strain 37b4 and positions 4 and 7 for strain KB-1. In accordance with the chain-linked position of sialic acids, mild hydrolysis of R. capsulatus 37b4 lipopolysaccharide with acetic acid released a trisaccharide with sialic acid at the reducing terminus. Structural investigation of this trisaccharide by methylation analysis, 1H- and 13C-NMR spectroscopy revealed the presence of the disaccharide Gal1-6Glc at the non-reducing end, probably with an alpha-anomeric configuration of the galactose residue, i.e. melibiose, beta-glycosidically linked to position 7 of sialic acid. Therefore the structure Gal alpha 1-6Glc beta 1-7Neu5Ac is proposed for this core oligosaccharide from R. capsulatus 37b4 lipopolysaccharide.

Structure of the capsular K3 antigen of Escherichia coli 04:K3:H4, a polysaccharide containing a 4-deoxy-2-hexulosonic acid.

The K3-antigenic capsular polysaccharide (K3 antigen) of Escherichia coli contains L-rhamnose, a 4-deoxy-2-hexulosonic acid, and an O-acetyl group in the molar ratio of 3:1:1. The backbone consists of a ----2)-O-alpha-L-rhamnopyranosyl-(1----3)-O-alpha-L-rhamnopyranosyl-(1----3)-O-alpha-L-rhamnopyranosyl-(1---- repeating unit. Either one of the 3-linked L-rhamnopyranosyl residues of each repeating unit may be substituted at O-2 with a 4-deoxy-2-hexulosonic acid, an isomer of the furanosyl form of KDO, about 90% of which is acetylated at 0-6. The 4-deoxy-2-hexulosonic acid residue is linked to the L-rhamnan backbone in a very labile linkage which is split by 1% acetic acid (30 min, 100 degrees). The K3 polysaccharide has a molecular weight of approximately 38,000, corresponding to approximately 60 repeating units.

Chemical characterization of enterobacterial common antigen isolated from Plesiomonas shigelloides ATCC 14029.

Serologically characterized samples of enterobacterial common antigen (ECA) from Plesiomonas shigelloides, Salmonella montevideo and Shigella sonnei were investigated by chemical methods including methylation and NMR techniques. All showed the same sugar composition and contained a lipid moiety with palmitic acid as main fatty acid and with a phosphodiester group. Additional enzymatic studies, reported in the preceding paper, provided evidence that the lipid moiety is an L-glycerophosphatidyl residue attached via a phosphodiester linkage to C-1 of GlcNAc as the reducing end of the ECA sugar chain. ECA of P. shigelloides showed the best-resolved 13C-NMR spectra, especially after the removal of non-stoichiometric O-acetyl groups at C-6 of GlcNAc of the ECA repeating unit and of the lipid moiety by mild acid hydrolysis (0.01 M HCl, 100 degrees C, 10 min). Subsequent 13C-NMR studies were therefore carried out with the mild-acid-treated ECA of P. shigelloides which allowed a tentative assignment of all resonances of the ECA repeating unit. 13C-NMR spectra of Salmonella and Shigella ECA were essentially the same as those obtained with Plesiomonas ECA. The same trisaccharide repeating unit was encountered as demonstrated previously in the cyclic form of ECA isolated from S. sonnei by Dell et al. [Carbohydr. Res. 133, 95-104 (1984)]. Methylation analysis, however, afforded small amounts of terminal GlcNAc thus proving, in combination with the demonstration of the attached lipid moiety, an acyclic nature of ECA from P. shigelloides and from the two enterobacterial species. The question of whether the cyclic form co-exists in S. sonnei phase I and possibly in other enterobacterial species or, whether it had been formed during extraction as an artifact, has not yet been answered. The way in which ECA was isolated in our studies would preclude the presence of a non-amphiphilic (cyclic) polysaccharide. The finding that the sugar chain of ECA is attached to an L-glycerophosphatidyl residue is in full corroboration with serological, enzymatic and gel electrophoretic studies shown in the preceding paper and with the character of ECA as a surface antigen being anchored by hydrophobic interactions in the outer membrane of Enterobacteriaceae and P. shigelloides.

An enzyme immunoassay for the screening of monoclonal antibodies to cyclosporin.

An enzyme-linked immunosorbent assay (ELISA) has been developed which allows hybridoma cell cultures to be tested for the presence of monoclonal antibodies specific for cyclosporin A. Immunization of mice with free cyclosporin was found to be preferable to immunization with cyclosporin-carrier conjugates. It is hoped that the availability of monoclonal antibodies to cyclosporin will clarify the contribution of cyclosporin metabolites to immunosuppression and nephrotoxicity.

Antibody-dependent cellular cytotoxicity and chemiluminescence as a tool for studying the mechanism of anti-glomerular basement membrane nephritis. The role of the cytotoxic potential of polymorphonuclear granulocytes and monocytes.

An in vitro system was elaborated to study the mechanisms inducing tissue injury in anti-glomerular basement membrane (GBM) nephritis. Collagenase-digested GBM (CGBM) was covalently attached to Fab' specific for chicken red blood cells (CRBC). The preparation of the CGBM-Fab' conjugate was effected by using iodoacetyl chloride coupling in analogy to a procedure described by Chiang & Koshland (1979). This conjugate was used for coating CRBC (CGBM-CRBC). In this system the antibody-dependent cellular cytotoxicity (ADCC) and the chemiluminescence mediated by purified bovine polymorphonuclear (PMN) and mononuclear cells (MNC) as well as rabbit MNC against CGBM-CRBC were compared in the presence of sheep anti-GBM IgG. All three cell populations were potent effectors in ADCC and chemiluminescence and evidence was obtained that the cytotoxic potential of MNC has to be attributed to monocytes. If compared at low effector target cell ratios in a 2 hr assay bovine PMN, however, were significantly more efficient than bovine MNC. The extent of both ADCC and chemiluminescence was directly related to the amount of anti-GBM IgG present in the system. Based on the inhibition experiments with oxygen intermediate scavengers, both ADCC and chemiluminescence by bovine PMN is dependent on generation of reactive oxygen species indicating that such radicals could play a role in vascular (endothelial) injury as documented in the loss of structural integrity of GBM.

Conformational changes of apigenin 7-O-(6-O-malonylglucoside), a vacuolar pigment from parsley, with solvent composition and proton concentration.

Malonylated apigenin 7-O-glucoside was prepared from malonyl-CoA and apigenin 7-O-glucoside using a malonyltransferase from parsley cell cultures. The enzyme product, as well as the flavonoid substrate, was analyzed by high-resolution nuclear magnetic resonance spectroscopy, confirming that malonic acid had been attached to the primary hydroxyl of the glucose moiety of apigenin 7-O-glucoside. During these studies in several solvents, it became apparent that, in particular, the chemical shift of H-6" A and the coupling constants J5",6"A and J5",6"B were dependent on solvent composition and proton concentration. Similar changes of sugar proton resonance frequencies were also observed with methyl 6-O-malonyl-beta-D-glucopyranoside, but not with apigenin 7-O-[6-O-(4-coumaroyl)glucoside]. The change of coupling constants is ascribed to a conformational modification of the sugar portion in the malonylglucosides. Malonylated flavonoid glycosides are exclusively located within the vacuoles of parsley cells. We propose that acylation of flavonoid glycosides which are synthesized in the cytoplasm facilitates transport of the substrates into the vacuole. The conformational modification which is induced by changes in proton concentration may provide a mechanism to trap flavonoids in situ.

Rapid effect of an elicitor on uptake and intracellular distribution of phosphate in cultured parsley cells.

Cell suspension cultures of parsley (Petroselinum hortense) grown in synthetic medium take up most of the inorganic phosphate supplied with the medium within the initial 5 days after transfer. Nuclear magnetic resonance spectra of intact parsley cells from this growth stage revealed that approximately half of the phosphate was located within the vacuoles, whereas after 7 days of growth phosphate content of the vacuoles was relatively low. At both times, addition of an elicitor preparation from Alternaria carthami, which is not toxic to the cells, led to a temporary increase of vacuolar phosphate at the expense of cytoplasmic phosphate, even when excess phosphate was added to the medium. The rapid decrease of cytoplasmic phosphate might play a role in the redirection of phenylpropanoid metabolism reported for elicitor-treated parsley cells.

Immunochemical localization of the C-terminal hexapeptide of histone H3 at the surface of chromatin subunits.

The C-terminal hexapeptide of histone H3 of chicken erythrocytes (residues 130-135) corresponding to the sequence Ile-Arg-Gly-Glu-Arg-Ala ( IRGERA ) was prepared by solid-phase peptide synthesis and, after coupling to bovine serum albumin, was used to elicit antibodies in rabbits. The antigenic activity of the synthetic peptide IRGERA was found to be very similar to that of the natural CN3 fragment (residues 121-135), and it inhibited the H3-anti H3 reaction in complement fixation, solid-phase radioimmunoassay, and enzyme-linked immunosorbent assay. Antibodies induced by IRGERA were found to bind equally well to IRGERA coupled to hemocyanin, to the intact H3 molecule, and to chromatin subunits (nucleosomes and core particles). The results demonstrate that the C-terminal hexapeptide of histone H3 is located at the surface of chromatin subunits and agree with current models proposed for the spatial organization of the chromatin core particle.

Immunochemical properties of oligosaccharide-protein conjugates with Klebsiella-K2-specificity. II. Protective capacity of the conjugates and anti-conjugate antibodies against infection with Klebsiella pneumoniae 01:K2 in mice.

Oligosaccharide subunits from Klebsiella pneumoniae B5055 capsular polysaccharide, covalently linked to protein carriers, the native polysaccharide, and killed whole bacteria, were compared with respect to their immunogenicity in mice and their capacity to protect mice against bacterial infection. The protection was studied (a) by active immunization and (b) by passive administration of antisera raised in rabbits using the different immunogens. The results were as follows: in the active protection experiments the octa- and dodecasaccharide conjugates were as effective as the polysaccharide in increasing the LD50 by a factor of 10(5) as compared to nonimmunized animals. Thus, these antigens were only slightly less effective than killed bacteria. In contrast the tetrasaccharide conjugate was at least 1000-fold less effective than all other antigens used. In the passive protection experiments these results were paralleled in that the antiserum against the tetrasaccharide conjugate also showed the lowest degree of protection, though, for methodical reasons, the differences in the LD50s were less pronounced. The drastically lower efficiency of the tetrasaccharide in comparision to its higher oligomers is in agreement with serological findings previously published (see first communication), which showed that two repeating units are the minimum requirement for a substantial representation of polysaccharide serological specificity.

MOPC 104E IgM/anti-idiotype solid-phase inhibition assay as a model for screening myeloma proteins for ligand binding specificity.

The inhibition of binding of 125I-labeled MOPC 104E IgM anti-idiotype to the controlled pore glass-bound IgM by various oligo- and polysaccharides was investigated as a model for the screening of myeloma proteins with unknown hapten binding specificity. The inhibitory efficiencies of the different haptens in this system were found to correlate well with their known abilities to bind to MOPC 104E IgM. Thus B1355S dextran, nigerosyl-alpha (1-3)-nigerose, and nigerosyl-alpha (1-3)-glucose, all known for specific binding to MOPC 104E IgM, were efficient inhibitors in the assay, in contrast to a series of more or less unrelated carbohydrate compounds, which showed only weak inhibitory capacity. According to these results there seems to be no obstacle to applying the anti-idiotype assay system to screening myeloma proteins whose binding specificity has not yet been determined.

Separation of anti-dinitrophenyl antibodies according to affinity by fractionated elution from immunoadsorbent using thiocyanate.

The possibility of fractionating anti-DNP-antibodies according to their affinity by successive elution from a DNP-immunoadsorbent with increasing concentrations of thiocyanate (KSCN) was investigated. Using KSCN at molarities of 0.5 M, 2.0 M and 3.0 M in a stepwise elution procedure, the mean affinity of the eluted antibody fractions was found to increase with increasing molarity of the eluent. The method described is a simple, reliable means of separating antibodies on an affinity basis which is of special value in systems where haptens are not available.

Binding site of a dextran-specific homogeneous IgM: thermodynamic and spectroscopic mapping by dansylated oligosaccharides.

The hapten binding properties of the homogeneous mouse IgM secreted by MOPC-104E were investigated. Hapten-association constants were determined either by equilibrium and displacement equilibrium dialysis or by fluorometric titrations of the protein with the fluorescent derivatives of the haptens. For the latter type of measurements, several oligosaccharides were derivatized to the corresponding dansylhydrazones. The synthesis, generally applicable to oligosaccharides with free reducing ends, is described. Analysis of the thermodynamic parameters for the binding of 18 haptens forms the basis for proposing a model of the binding site of MOPC-104E. This model is supported and refined by results of the measurements of linear and circular polarization of the fluorescence of the dansylated haptens. The binding site is proposed to consist of a cavity with about 12-A depth, complementary to a terminal nonreducing nigerosyl group. At the entrance to this cavity, a further subsite is identified forming interactions of lower specificity with an additional glucose unit.