Biomedical subjects

C A Hermerath

Publications and source records attributed to C A Hermerath.

4 recordsLinked to original sources

PubMed1988

Benzoquinone activation of Cryptococcus neoformans capsular polysaccharide for construction of an immunoaffinity column.

p-benzoquinone was used as a two-step coupling reagent for preparation of an immunoaffinity absorbent in which the capsular polysaccharide of Cryptococcus neoformans was linked to an agarose gel. Cryptococcal polysaccharide is a difficult subject for chemical modification because it contains immunogenic O-acetyl groups which are sensitive to alkaline hydrolysis. The polysaccharide was activated by treatment with benzoquinone. The 'activated' polysaccharide was reactive with amino groups on AH-Sepharose. A pH of 8-9 was optimal for activation of the polysaccharide. Once activated, the polysaccharide was reactive with a model substrate, L-alanine-4-nitroanilide, over a pH range of 6-10. Since the O-acetyl groups are hydrolyzed at pH above 8.0, an activation pH of 8.0 and a coupling pH of 7.5 were used to prepare the conjugated gel. The polysaccharide immunoaffinity column was used successfully for isolation of rabbit antibodies to cryptococcal polysaccharide.

Benzoquinones↗

PubMed1987

Cottonseed malate synthase : purification and immunochemical characterization.

Malate synthase (EC 4.1.3.2), an enzyme unique to the glyoxylate cycle, was purified to homogeneity from cotyledons of 72-hours, darkgrown cotton (Gossypium hirsutum L.) seedlings. Homogeneity of the enzyme was assessed by silver staining SDS-PAGE gels. Purification was accomplished by using a single buffer medium through six steps involving one ammonium sulfate fractionation and chromatography on three columns (Sephacryl S-300, DEAE Sephacel, Phenyl Sepharose). Large-scale preparation of glyoxysomes, a main step in all other published procedures, was not involved. The purified enzyme and that extracted from glyoxysomes appears to be a dodecamer with a native molecular weight of 750,000 (sedimentation coefficient of >20 Svedberg units [S] on sucrose gradients) composed of identical subunits (molecular weight approximately 63,000). The monomer (5S) occurs in the cytosol. Polyclonal antibodies raised in rabbits were judged to be monospecific for malate synthase by immunotitration, double immunodiffusion, and western blotting. Double immunodiffusion experiments revealed only partial immunological identity between the 5S (cytosolic) and 20S (glyoxysomal forms, although complete identity was observed between the 5S form in immature and germinated seeds, and the 20S form in immature and germinated seeds. Cross-reactivity of the cotton antimalate synthase serum was observed with extracts from five other oilseeds. Western blot analyses showed that malate synthase protein was not present in immature seeds prior to appearance of enzyme activity, but when present, subunit molecular weight was indistinguishable in immature, desiccated, and germinated seeds.

Journal Article↗

PubMed1984

Binding of cryptococcal polysaccharide to Cryptococcus neoformans.

Radioiodinated cryptococcal polysaccharide was used to study binding of the soluble polysaccharide to encapsulated and non-encapsulated cryptoccoci. Binding of polysaccharide to non-encapsulated cryptococci occurred rapidly over a 30-min period and was largely complete after 2 h. Bound, labeled polysaccharide was slowly eluted from Cryptococcus neoformans after the addition of unlabeled polysaccharide, indicating reversibility of binding. Non-encapsulated cryptococci bound polysaccharide in two ways. Specific binding to the yeast was saturable by ca. 82 ng of polysaccharide per 10(6) yeast cells. Nonspecific binding also occurred which was not saturable under the conditions used in our experiments. Phagocytosis of the non-encapsulated yeast strain was inhibited when the specific binding was ca. 50% saturated. Binding of polysaccharide to an encapsulated strain showed nonspecific, nonsaturable binding, but little specific binding occurred. Presumably the specific binding sites were saturated in the encapsulated strain. Polysaccharides obtained from a hypocapsular mutant (A61) and a normally encapsulated strain competed effectively with labeled serotype D polysaccharide for binding sites on non-encapsulated cryptococci and had identical phagocytosis-inhibiting properties. Similarly, polysaccharides from all four cryptococcal serotypes competed effectively with labeled serotype D polysaccharide for binding sites on the non-encapsulated strain, and all four polysaccharides inhibited phagocytosis of non-encapsulated Cryptococcus neoformans. Unmodified, de-O-acetylated, carboxyl-reduced, periodate-oxidized and reduced (polyalcohol), and Smith-degraded polysaccharides competed with labeled polysaccharide for binding sites on the cell. The unmodified, de-O-acetylated and carboxyl-reduced polysaccharides inhibited phagocytosis of non-encapsulated cells, but the polyalcohol and Smith product were unable to inhibit phagocytosis.

Animals↗

PubMed1981

Monoclonal antibody analysis of lipopolysaccharide from Neisseria gonorrhoeae and Neisseria meningitidis.

A hybridoma produced by the polyethylene glycol fusion of the NS-1 variant of the P3x63Ag8 BALB/c plasmacytoma to splenocytes harvested from a BALB/c mouse immunized with whole gonococci was found to be producing antibody to a common region on gonococcal lipopolysaccharide (LPS). Enzyme-linked immunosorbent assay inhibition systems were established by utilizing this antibody, designated 3F11, and 100% inhibition occurred with both LPS and the LPS-LPS and LPS-derived polysaccharides partially inhibited the enzyme-linked immunosorbent assay, whereas similar preparations isolated from Escherichia coli O:111, the J-5 mutant of this strain, and Salmonella minnesota Re595 failed to inhibit the assay. Studies utilizing whole gonococcal strains 4505 and the isogenic variant 4505r, which lacks both the LPS serotype and common determinants as inhibitors, demonstrated that the determinant recognized by the 3F11 antibody was present on the surface of 4505 and absent on 4505r. Inhibition studies were performed with beta-glucose, beta-galactose, D-glucosamine, D-galactosamine, heptose, 2-keto-3-deoxyoctanoate, N-acetylglucosamine, N-acetylgalactosamine, alpha-lactose, and beta-lactose. Complete inhibition of the enzyme-linked immunosorbent assay occurred with D-galactosamine, and partial inhibition was achieved with both alpha-lactose and beta-lactose. Based on these observations, the 3F11 antibody recognizes a site common to gonococcal LPS which is partially shared by meningococcal LPS. The chemical structure of the determinant appears to be a D-galactosamine-O-D-galactopyranosyl-(1-4)-D-glucopyranose. Additional specificity may be conferred by the steric relationship of the determinant on the intact LPS.

Antibodies, Bacterial↗