Immunohistopathological studies of blastomycosis: the use of labeled specific antigens in a hamster model of disease

We applied a modified immunofluorescence and immunoperoxidase method, utilizing labeled Blastomyces dermatitidis antigens, to look for specific antibody-bearing B/ plasma cells in the tissue infiltrates of blastomycosis lesions induced in hamsters. No specific anti-blastomyces antibodies were detectable by this method, although such antibodies were present in blood samples as demonstrated by routine immunodiffusion techniques. These studies suggest that humoral immune reactions do not play a major role in the pathogenesis of lesions of blastomycosis in hamsters.     

Isolation and properties of flagella of trypanosomatids.

A procedufe is described for the isolation of flagella of Crithidia fasciculata, Herpetomonas samuelpessoai and Leishmania tarentolae in a highly purified state and giving reasonably good yield. The 3 types of flagella give a similar electrophoretic pattern of proteins. It is shown that H. samuelpessoai and, to a lesser extent, C. fasciculata flagella confer protection against Trypanosoma cruzi infection.     

Prenylated flavonoids from seeds of Calopogonium mucunoides

The seeds of Calopogonium mucunoides furnished 7-O-γ,γ-dimethylallyl-8-methoxy-3′,4′-dioxymethylene-isoflavone, 7-O-γ,γ-dimethylallyl-3′-hydroxy-4′-methoxyisoflavone, 7-O-γ,γ-dimethylallyl-3′,4′-dimethoxyisoflavone and 2S-di[6′',6′'-dimethylpyrano (2′',3′':7,8;2′',3′':4′,3′)]-flavanone whose structures were established by spectroscopic means involving the use of 400 MHz ¹H NMR with double irradiation and INDOR techniques.     

Regulation, purification, and properties of xanthine dehydrogenase in Neurospora crassa.

Xanthine dehydrogenase (EC 1.2.1.37) is the first enzyme in the degradative pathway by which fungi convert purines to ammonia. In vivo, the activity is induced 6-fold by growth in uric acid. Hypoxanthine, xanthine, adenine, or guanine also induce enzyme activity but to a lesser degree. Immunoelectrophoresis using monospecific antibodies prepared against Neurospora crassa xanthine dehydrogenase shows that the induced increase in enzyme activity results from increased numbers of xanthine dehydrogenase molecules, presumably arising from de novo enzyme synthesis. Xanthine dehydrogenase has been purified to homogeneity by conventional methods followed by immunoabsorption to monospecific antibodies coupled to Sepharose 6B. Electrophoresis of purified xanthine dehydrogenase reveals a single protein band which also exhibits enzyme activity. The average specific activity of purified enzyme is 140 nmol of isoxanthopterine produced/min/mg. Xanthine dehydrogenase activity is substrate-inhibited by xanthine (0.14 mM), hypoxanthine (0.3 mM), and pterine (10 micron), is only slightly affected by metal binding agents such as KCN (6 mM), but is strongly inhibited by sulfhydryl reagents such as p-hydroxymercuribenzoate (2 micron). The molecular weight of xanthine dehydrogenase is 357,000 as calculated from a sedimentation coefficient of 11.8 S and a Stokes radius of 6.37 nm. Sodium dodecyl sulfate-gel electrophoresis of the enzyme reveals a single protein band having a molecular weight of 155,000. So the xanthine dehydrogenase protein appears to be a dimer. In contrast to xanthine dehydrogenases from animal sources which typically possess as prosthetic groups 2 FAD molecules, 2 molybdenum atoms, 8 atoms of iron, and 8 acid-labile sulfides, the Neurospora enzyme contains 2 FAD molecules, 1 molybdenum atom, 12 atoms of iron, and 14 eq of labile sulfide/molecule. The absorption spectrum of the enzyme shows maxima between 400 and 500 nm typical of a non-heme iron-containing flavoprotein.     

Dihydroisocoumarins and phthalide from wood samples infested by fungi

Wood samples, infested by fungi during storage, were shown to contain, besides the known 5-methyl-mellein, additional (3R)-8-hydroxy-3-methyl-3,4-dihydroisocoumarins substituted by 7-methyl, 5-formyl, 5-carboxy, 5-hydroxy, 5-methoxy, 6-methoxy-5-methyl and 6,7-dimethoxy-5-methyl groups, as well as 6-formyl-7-hydroxy-5-methoxy-4-methylphthalide. Several 2-methylchromanones were synthesized in order to show that this class of compounds can be distinguished from 3-methyl-3,4-dihydroisocoumarins by MS.     

Immunochemical studies on blood groups: The internal structure and immunological properties of water-soluble human blood group A substance studied by Smith degradation,liberation, and fractionation of oligosaccharides and reaction with lectins

Carbohydrate structures in the interior of a blood group A active substance (MSS) were exposed by one and by two Smith degradations. Reactivities of the original glycoprotein and its Smith degraded products with 13 different lectins and with anti-I Ma were studied by quantitative precipitin assay. MSS and its first Smith degraded product completely precipitated Ricinus communis hemagglutinin with five times less of the first Smith degraded glycoprotein being required for 50% precipitation. The second Smith degraded material precipitated only 90% of the lectin. MSS did not precipitate peanut lectin, whereas its first and second Smith degraded products completely precipitated the lectin. The first Smith degraded glycoprotein also reacted well with Wistaria floribunda, Maclura pomifera, Bauhinia purpurea alba, and Geodia lectins indicating that its carbohydrate moiety could contain dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 4dGlcNAc, dGalβ1 → 3dGlcNAcβ1 → 3dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGalNAc determinants at nonreducing ends. The second Smith degraded material precipitated well with Ricinus communis hemagglutinin, Arachis hypogaea, Geodia cydonium, Maclura pomifera, and Helix pomatia lectins showing that dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 4dGlcNAc residues at terminal nonreducing ends could be involved. Monoclonal anti-I Ma (group 1) serum reacted strongly with the first Smith degraded product indicating large numbers of anti-I Ma determinants, dGalβ1 → 4dGlcNAcβ1 → d 6dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGalNAc at nonreducing ends. The comparable activities of the native and Smith degraded products with wheat germ lectin indicate capacity to react with DGlcNAc residues at nonreducing ends and/or at positions in the interior of the chain. The totality of lectin reactivities indicates heterogeneity of the carbohydrate side chains. Oligosaccharides with ³H at their reducing ends released from the protein core of the first and second Smith degraded products were obtained by treatment with 0.05 m NaOH and 1 M NaB³H₄ at 50 °C for 16 h (Carlson degradation). The liberated reduced oligosaccharides were fractionated by dialysis, followed by retardion, Bio-Gel P-2, P-4, and P-6 columns. They were further purified on charcoal-celite columns, and by preparative paper chromatography and high-pressure liquid chromatography. Their distribution by size was estimated by the yields on dialysis, Bio-Gel P-2, and Bio-Gel P-6 chromatography, and from the radioactivity of the reduced sugars. Of the oligosaccharide fractions from the first Smith degraded product, about 77% of the carbohydrate side chain residues contained from 1 to 6 sugars, 13% from 7 to perhaps 12 sugars, and 10% was nondialyzable (polysaccharides and glycopeptide fragments). Of the second Smith degraded product, approximately 82% of carbohydrate residues had from 1 to 6 sugars, 14% from 7 to perhaps 20 sugars and 4% was nondialyzable. The biological activity profile of the two Smith degraded products together with the size distributions of the oligosaccharides indicated that their carbohydrate side chains, comprised a heterogeneous population ranging in size from 1 to about 12 sugars. When most of these chains that are shorter than hexasaccharides are fully characterized it may be possible to reconstruct the overall structure of the carbohydrate moiety of the blood group substances and account for their biological activities.     

Membrane lipid composition modulates the binding specificity of a monoclonal antibody against liposomes

A hybridoma secreting a monoclonal IgM 'anti-liposome' antibody was produced after injecting a mouse with liposomes containing dipalmitoylphosphatidylcholine, cholesterol, dicetyl phosphate, and lipid A. The antibody was selected by assaying for complement-dependent damage to liposomes lacking lipid A. The monoclonal antibody reacted best with liposomes containing the original immunizing mixture of lipids. Deletion of individual lipid constituents from liposomes diminished the ability of the liposomes to bind (adsorb) the antibody. Binding of the antibody was enhanced by including lipid A or galactosylceramide in the lipid bilayer, or by substituting egg phosphatidylcholine for dimyristoyl- (or dipalmitoyl-) phosphatidylcholine. Sphingomyelin could be substituted for dimyristoylphosphatidylcholine without altering the adsorption of antibody. Although the monoclonal anti-liposome antibody was completely inhibited by phosphocholine, it was probably not a conventional anti-phosphocholine antibody. The antibody apparently had a partial specificity for phosphate, and was inhibited by glycerophosphocholine, glycerophosphate, sodium phosphate, sodium sulfate, and inositol hexaphosphate, but not by choline or inositol.     

Detection of a Cytosolic Glutamine Synthetase in Leaves of Nicotiana tabacum L. by Immunocytochemical Methods

Two glutamine synthetase (GS) polypeptides (44 and 39 kD) were immunodetected on western blots of leaf extracts from tobacco (Nicotiana tabacum L.), a plant that has been reported to contain only chloroplast GS in the leaves. By immunocytochemical methods, we confirmed the localization of GS in the cytosol of cells in the vascular tissue and in the chloroplasts of mesophyll cells.