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R Cherniak

Publications and source records attributed to R Cherniak.

71 records · Page 4Linked to original sources
PubMed1986

Ultrastructure of acapsular mutant Cryptococcus neoformans cap 67 and monosaccharide composition of cell extracts.

Acapsular mutant Cryptococcus neoformans cap 67 was grown in Pine's citrate broth medium for 3 days and the cells then transferred to a nitrogen-free medium for 6 days. The cells were subjected to a four stage extraction with buffered Triton-X100, cold dilute alkaline borohydride, hot dilute acetic acid, and a second alkaline extraction. Galactoxylomannan antigens were recovered from the culture supernates of both 3 day-old and 9 day-old yeast cells. The alkaline extracts contained water-soluble galactoxylomannan and a water-insoluble glucan. Dilute acid treatment released a minor amount of carbohydrate from the cells. The second alkaline extraction yielded increased amounts of glucan and galactoxylomannan from the 9 day-old cells. Soluble non-dialyzable cell extracts were antigenically identical in immunodiffusion with the culture supernate antigens. After the extraction sequence, all of the galactose, xylose, and mannose were removed from the cells. The walls retained their shape after extraction but their layers were loosened. Cells resuspended in nitrogen-free medium for six days developed thickened walls with alternating electron-dense and electron-lucent layers. The major constituent of the thickened 9 day-old cell walls was glucose, only 5% glucosamine was detected.

Antigens, Fungal↗
PubMed1985

Characterization of protein and mannan polysaccharide antigens of yeasts, moulds, and actinomycetes.

Antigens in coccidioidin were compared with purified subfractions via tandem immunoelectrophoresis (IEP) and by a combination of advancing line and crossed IEP. Rocket IEP was suitable for titrating the reactions and showing the relationship between column fractions. These techniques required multicomponent antisera produced by hyperimmunization over many months and by the use of known standard migration pairs. The IEP variations were used to chart the development of antisera against coccidioidin factors, to monitor antigen purifications, and to test the immunochemical homogeneity of an isolated antigen. Mannose-based heteroglycans of Cryptococcus neoformans were recovered from the culture filtrate. After precipitation of the major viscous glucuronoxylomannan (GXM) with ethanol or cetyltrimethylammonium bromide, the supernate is reserved because it contains a galactoxylomannan (GalXM). After removal of glucuronic acid from the GXM, the resulting xylomannan of serotype A was amenable to 13C-nuclear magnetic resonance (NMR) spectrometry; it revealed nonreducing xylose, alpha-1,3-mannose, and alpha-1,2/1,3 disubstituted mannose, thus confirming by an independent means what was previously known. The characterization sequence of GalXM included: (1) gas-liquid chromatography (GLC) of neutral sugars as peracetylated aldononitriles; (2) methylation-fragmentation GLC mass spectrometry to determine the glycosidic linkages; and (3) 13C-NMR showing similarities to mannan of Saccharomyces cerevisiae. Affinity chromatography of the GalXM on concanavalin A separated the galactoxylo component from an adsorbed mannoprotein. Selection of monoclonal antibodies (MAbs) relies on presumptive enzyme immunoassays (EIAs) or radioimmunoassays for rapid screening of clones and for determination of isotypes; however, higher resolution confirmatory tests are needed to obtain MAbs of desired specificity. MAbs against Candida tropicalis mannan were labeled with horseradish peroxidase to use for detecting mannan in serum. MAbs against the partially purified "m" factor of histoplasmin were characterized by the enzyme-linked immunoelectro-transfer blot technique (EITB), revealing unsuspected complexity in the antigen. Secreted proteins of Nocardia asteroides were isoelectrically focused; three proteins, identified by EITB as promising to be specific for that actinomycete, were cut out of gels and used to immunize mice for production of MAbs. The fimbriae of Actinomyces viscosus and A. naeslundii that mediate lactose-reversible coagglutination with Streptococcus sanguis have been used to evoke MAbs.(ABSTRACT TRUNCATED AT 400 WORDS)

Actinomycetales↗
PubMed1984

Enzyme immunoassay detection of IgM to galactoxylomannan of Cryptococcus neoformans.

Antibodies against the major capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), and a minor secreted polysaccharide, galactoxylomannan (GalXM), were surveyed by indirect enzyme immunoassay (EIA) in patients with cryptococcosis, with other mycoses, and in normal controls. Measurement of IgG levels against GalXM revealed cross reactions in candidiasis patients that were reduced by adsorption with Candida albicans cell walls. Measurement of IgM levels were subject to fewer cross reactions. The combination of adsorption with C albicans cell walls and measurement of IgM detected antibodies in 12 of 55 cryptococcosis patients. An end point equal to or greater than a titer of 1/16 excluded reactions in normals and limited cross reactivity in candidiasis patients to below 7%. This test has potential diagnostic significance in cryptococcosis patients who show no evidence of cryptococcal antigen circulating in the cerebrospinal fluid or serum. Reactions in this IgM assay were not spuriously due to rheumatoid factor. The major capsular GXM was much less serologically active and was subject to cross reactions with agents of bacterial sepsis. The specificity of the GalXM is directed mainly by the mannose and to a lesser extent by galactosyl residues.

Antibodies, Anti-Idiotypic↗
PubMed1983

Analysis of the common polysaccharide antigens from the cell envelope of Clostridium perfringens type A.

The major, common antigen of Clostridium perfringens type A, isolated and purified independently from three selected strains (Hobbs 5, Hobbs 9, and Hobbs 10), is composed of equimolar amounts of 2-acetamido-2-deoxy-D-mannose (Man-NAc) and 2-acetamido-2-deoxy-D-glucose (GlcNAc). The purified antigen gave a strong immunoprecipitin line by double immunodiffusion in gel. Smith degradation of the major, common antigen caused decomposition of all of the GlcNAc, without concomitant loss in ManNAc, or a perceptible change in serological activity. Therefore, the serological activity of the major, common antigen depended solely on the presence of ManNAc. Data obtained by the 13C-n.m.r.-spectral analysis of the Smith-degradation product revealed that it was a linear-backbone polysaccharide analogous to a Rhodotorula glutinis mannan, but composed of pairs of 2-acetamido-2-deoxymannopyranosyl residues alternately linked beta-(1 leads to 3) and beta-(1 leads to 4). The one-bond, carbon-hydrogen coupling-constant of 162 Hz for both anomeric centers was consistent with the proposed beta-linkages. A similar, 13C-n.m.r.-spectral analysis of the native, common antigen indicated that the GlcNAc residues were randomly connected to three of the four hydroxyl groups not already involved in linking the ManNAc backbone, the 4-hydroxyl group being the exception. A second, serologically inactive, polysaccharide composed of rhamnose, GalNAc, and galactose was identified, but not obtained in homogeneous state. The rhamnosyl residues were probably situated as nonreducing antennae, as they were quantitatively removed by Smith degradation without concomitant decomposition of the polymeric structure of the remaining residues.

Amino Acids↗
PubMed1981

Common polysaccharide antigens from the cell envelope of Clostridium perfringens type A.

Soluble antigens were obtained by extracting five serotype strains of Clostridium perfringens type A with water at 100 degrees C. The type-specific polysaccharides were precipitated with ethanol, and the common antigens were recovered from the ethanol supernatants by concentration, dialysis, and lyophilization. Refluxing the water-extracted cell residues with 1% acetic acid followed by concentration, dialysis, and lyophilization gave additional common antigen fractions. A comprehensive, side-by-side comparison of the antigen fractions, the ethanol precipitate, the ethanol supernatant, and the acetic acid supernatant, revealed that common antigens were recovered in all three fractions, and that three distinct entities were responsible for the formation of the observed common immunoprecipitin lines; whereas many fractions possessed all three immunoprecipitin lines, others contained only one or two. The serological homology observed between the various antigen fractions was apparently a consequence of N-acetylglucosamine- and N-acetylmannosamine-containing polymers. The common antigens were presumably associated with the cell envelope and may be the type of markers sought previously by others for the serological identification of C. perfringens.

Antigens, Bacterial↗
PubMed1980

Concomitant but not causal association between surface charge and inhibition of phagocytosis by cryptococcal polysaccharide.

The mechanism by which capsular polysaccharides inhibit phagocytosis is not clearly understood. We investigated the association between a negative surface charge and inhibition of phagocytosis by the capsular polysaccharide of Cryptococcus neoformans. A two-polymer aqueous-phase system containing phosphate ions was used to assess surface charge. Opsonins such as normal bovine serum and normal human immunoglobulin G reduced the surface charge on non-encapsulated cryptococci and simultaneously enhanced phagocytosis. These same opsonins had no effect on phagocytosis or surface charge of encapsulated cryptococci. F (ab')(2) fragments of normal human immunoglobulin G neither enhanced phagocytosis nor altered the surface charge of non-encapsulated cryptococci. Addition of purified cryptococcal polysaccharide to non-encapsulated cells inhibited phagocytosis of the yeast and induced a strong negative charge at the yeast surface. Chemical modification to reduce the surface charge of either purified cryptococcal polysaccharide or intact encapsulated cryptococci produced a small loss of phagocytosis-inhibiting activity; however, all treated polysaccharide preparations retained a significant ability to inhibit phagocytosis of the yeast. These results indicated that the association between surface charge and inhibition of phagocytosis was largely circumstantial, and presence of a negative surface charge could not account for the powerful antiphagocytic action of cryptococcal polysaccharide.

Cell Membrane↗
PubMed1979

Biochemical and serological characteristics of soluble yeast phase antigens of Histoplasma capsulatum.

Soluble antigens of whole yeast-phase cells were extracted with a 0.1 M phosphate buffer containing 0.1 M sodium chloride and 0.02% iodacetate. After being separated by differential filtration into fractions less than or greater than 50,000 daltons these antigens were purified by molecular sieve and chromatographic separations on ionic exchange resins. Two high molecular weight fractions obtained from diethylaminoethyl-cellulose (DEAE) at pH 8.0 and 7.0 with tris (hydroxymethyl) aminomethane (Tris) buffer were M antigens; those obtained at pH 4.0 and 4.0 with salt were H antigens. The four fractions had protein to carbohydrate ratios of 7.3, 14.0, 8.4, and 6.5 respectively, and all had essentially the same amino acid composition with no methionine and tyrosine and little histodine, arginine, phenylalanine and lysine. They had high concentrations of glucose, less mannose and traces of galactose. The low molecular weight fractions had the new complex "Y antigen", M antigen with protein to carbohydrate ratios of 1.4, 1.4 and 0.3 respectively. The amino acid and sugar composition of Y antigen strongly resembled the composition of the low molecular weight H and M antigens. Unlike the high molecular weight antigens, these low molecular weight antigens had methionine in relatively high concentrations; they had the same sugars as their respective high molecular weight counterparts. The yeast phase antigens differed from their respective mycelial counterparts in the following ways: glucose was the major sugar in the yeast phase with less amounts of mannose and traces of galactose, whereas in the mycelial antigens, mannose was the major sugar, with lesser amounts of galactose, and hexosamine. The H and M antigens of the yeast phase had high concentrations of glycine and alanine, whereas in the mycelial phase, these antigens had high concentrations of threonine and proline; the H and M antigens of the yeast phase had 5 to 16 times the protein to carbohydrate ratio observed for the same antigens of histoplasmin.

Amino Acids↗
PubMed1979

Immunochemical evidence for multiple serotypes of Bacteroides fragilis.

An immunochemical comparison of outer membrane antigens obtained from five select and biochemically defined strains indicated that there are several serotypes of Bacteroides fragilis. Each strain was serologically defined by individual or by combinations of determinant groups composed of carbohydrates in the form of polysaccharides or glycoproteins. The carbohydrate constituents were tentatively identified as glucose, galactose, fucose, rhamnose, glucosamine, galactosamine, and traces of mannose. Strains were observed to have minor qualitative and major quantitative variations in carbohydrate composition.

Antigens, Bacterial↗
PubMed1977

Capsular polysaccharide of Clostridium perfringens Hobbs 9.

Several closely related capsular polysaccharides were isolated from a strain of Clostridium perfringens Hobbs 9 type A by extraction of encapsulated cells with cold 0.85% NaCl. The soluble polymers were precipitated with alcohol and purified by (NH4)2SO4 fractionation, enzymatic digestion with papain and ribonuclease, and chromatography on diethylaminoethyl-Sephadex A25. The polysaccharides were composed mainly of glucose, galactose, and galactosamine. The major fraction contained these constituents (representing 77% of the dry weight) in a molar ratio of 1:1.6:1.1. All of the fractions contained phosphate and peptide material that was not removed during purification. The polysaccharides were closely related but not identical as indicated by double-diffusion-in-gel experiments. Immunoelectrophoresis in agarose demonstrated that the polysaccharides had identical mobilities and that no resolution into additional fractions occurred. The immunological activity of all the purified polysaccharides was destroyed by periodate oxidation but was unaffected by protease.

Antibody Specificity↗
PubMed1975

Composition of the capsular polysaccharides of Clostridium perfringens as a basis for their classification by chemotypes.

An analytical procedure, using gas-liquid chromatography, was developed for the identification of the per(trimethylsilyl) ethers of the constituent monosaccharides obtained from the capsular polysaccharides of Clostridium perfringens Hobbs 5, Hobbs 9, Hobbs 10, and NCTC 10578. Qualitative and quantitative differences between the major polysaccharide components enabled the differentiation of the four strains of C. perfringens investigated.

Acetylation↗
PubMed1974

Capsular polysaccharide of Clostridium perfringens Hobbs 10.

A capsular polysaccharide was isolated from a strain of Clostridium perfringens Hobbs 10 type A by cold-water extraction of whole, heavily encapsulated cells. The water-soluble polymer was isolated by alcohol precipitation and purified by treatment with chloroform-butanol, cetytrimethylammonium bromide, and column gel permeation chromatography by using Bio-Gel A-5m agarose. The formation of a single precipitin line, when the isolated polysaccharide was reacted with its homologous antisera by double diffusion in gel, was considered a criterion of immunochemical purity. The purified polymer appeared as a single peak when eluted from diethylaminoethyl-Sephadex with a linear gradient of NaCl. The polysaccharide was composed of glucose, galactose, galactosamine, and iduronic acid in a molar ratio of 4.1:5.1.7:1, respectively. These constituents accounted for 83% of the dry weight. The polysaccharide appeared to have a molecular weight of 40,000 and exhibited aggregation up to 120,000. A trace of peptide material could not be removed during purification.

Chemical Precipitation↗
PubMed1972

Immunochemistry of the capsular polysaccharides from Clostridium perfringens: selected Hobbs strains 1, 5, 9, and 10.

The properties of the immunochemically distinct substances isolated from four strains of Clostridium perfringens, mucoid variants of Hobbs 1, 5, 9, and 10, were shown to be compatible with the hypothesis that polysaccharides are generally responsible for the "type-specificity" of these organisms. The presence of "group-specific" substance was demonstrated by the cross-reaction of recent preparations of Hobbs 5 antisera with the isolated polysaccharides. The cross-reactions were not observed with specific antisera of the other strains studied and were independent of the "type-specific" phenomenon.

Animals↗