Immunochemical studies on levans from several strains of Actinomyces viscosus.

High molecular-weight levans elaborated by 8 separate strains of Actinomyces viscosus were purified: the inteactions of these levans with concanavalin A and anti-fructan myeloma immunoglobulins UPC-10 and J606 were examined by the quantitative precipitin method. Oligosaccharides released from the levans by partial acid hydrolysis were separated by partition chromatography on paper and characterized in situ by selective spray reagents. The liberated oligomers were compared with oligomers of known structure released from levans of Aerobacter levanicum and Leuconostoc mesenteriodes B512 as well as with plant inulin. The fragmentation analysis indicated a structure for Actinomyces levans comprising chains of beta (2----6)-linked fructofuranosyl units joined through multiple (1,2,6)-linked fructosyl branch units.

Plasma selenium-dependent glutathione peroxidase. Cell of origin and secretion.

Human plasma glutathione peroxidase (GSHPx) has been shown to be a glycosylated selenoprotein distinct enzymatically, structurally, and antigenically from known cellular glutathione peroxidases. The extracellular location of the enzyme and the fact that it is glycosylated suggested that it is a secreted protein. Utilizing mutually non-cross-reactive antibodies to human cellular and plasma GSHPx, we conducted a search to determine the tissue of origin for plasma GSHPx. The cells screened were endothelial cells because they are the main source of extracellular superoxide dismutase, HL-60 cells (myeloid cell line) because they are the main source of extracellular H2O2, and Hep G2 cells (hepatic cell line) because they are the source of many plasma proteins. Human umbilical vein endothelial cells were metabolically labeled with either [35S]methionine or [75Se]selenious acid, and HL-60 cells and Hep G2 cells were metabolically labeled with [75Se]selenious acid. Proteins were immunopurified from the labeled cells and their media with either anti-red blood cell (RBC) GSHPx IgG or with anti-plasma GSHPx IgG. Utilizing anti-RBC GSHPx IgG, only the cellular form of the enzyme was precipitated from all the cells tested but not from their media. When anti-plasma GSHPx IgG was applied to the cells and their media, a selenoprotein was precipitated only from the media of Hep G2 cells. When Hep G2 cells were incubated in the presence of the carboxylic ionophore monensin, an intracellular selenoprotein could be detected using anti-plasma GSHPx IgG. The precipitation of the cellular form from all three cell types was partially inhibited by preincubation of the anti-RBC GSHPx IgG with purified RBC GSHPx while the precipitation of the selenoprotein from the medium of Hep G2 cells by anti-plasma GSHPx IgG was prevented by preincubation of the antibody with purified plasma GSHPx. We suggest that plasma GSHPx is synthesized by and secreted from hepatic cells. This is, to the best of our knowledge, the only known selenoprotein with a defined function that has been shown to be synthesized for secretion by mammalian cells.

Antihuman plasma glutathione peroxidase antibodies: immunologic investigations to determine plasma glutathione peroxidase protein and selenium content in plasma.

Plasma glutathione peroxidase (GSHPx) (glutathione: H2O2 oxidoreductase) is a unique selenoglycoprotein. Treatment of this enzyme with glycopeptidase F partially deglycosylates it and establishes the presence of N-linked sugar moieties. Antibodies raised in a rabbit against the purified enzyme from plasma were found to be specific, noninhibitory, and capable of precipitating the enzymatic activity. The antibodies precipitated greater than 90% of the GSHPx activity of normal plasma, thus indicating that the selenoenzyme is the main if not the sole GSHPx activity of plasma. The antibodies did not precipitate RBC GSHPx. A slight cross-reactivity of the antibodies was found with rat plasma GSHPx. A GSHPx activity precipitation assay of normal plasma in the presence of selenium (Se)-deficient plasma indicates that no cross-reactive protein in the Se-deficient plasma interferes with the precipitation of the GSHPx activity from normal plasma. Thus, GSHPx protein as well as activity is deficient in plasma in the absence of Se. Antibodies against GSHPx either from RBCs or from plasma were used to specifically immunoprecipitate most of the GSHPx activity from RBCs or plasma, respectively, in healthy individuals to determine the amount of Se associated with the protein. GSHPx accounts for approximately 15% of the Se in RBCs and 12% of the Se in plasma. Thus, in normal individuals, these proteins account for only a fraction of plasma and RBC Se.

Immunochemical studies on pneumococcal type 37 capsular polysaccharide.

Pneumococcal type 37 capsular polysaccharide was obtained free of contaminants by affinity chromatography on Con-A, wheat germ agglutinin, Maclura pomifera lectin and HOPC-8 mouse myeloma protein affinity columns. The immunochemical reactivity of native and periodate oxidized borohydride reduced type 37 polysaccharide antigen with polyclonal rabbit and monoclonal mouse anti-Pn37 hybridoma antibodies was studied by quantitative precipitation. Quantitative hapten inhibition studies, employing the isomeric series of alpha- and beta-(1----2), (1----3), (1----4) and (1----6)-linked glucobioses as competitive inhibitors of antibody precipitation establish a specificity for anti-Pn37 antibody directed at least in part, against the Glc beta(1----2) Glc (sophorosyl) unit. A high mol. wt, D-glucose containing polysaccharide antigen, cross-reactive with rabbit anti-Pn37 is reported which was found to occur in the culture medium of 7 of 19 strains of Actinomyces examined.

Identification of the FTBL protein of Sumner and Dounce as a leucine aminopeptidase.

The crystalline beef liver protein of Sumner and Dounce (A. L. Dounce, P. Z. Allen, and G. A. Mourtzikos (1978) Arch. Biochem. Biophys. 188, 251-265) termed FTBL (football) protein because of the shape of its crystals, has been identified as a crystalline leucine aminopeptidase (LAP), on the basis of its high specific LAP activity and coincidence of its N terminal amino acid sequence (30 amino acids) with that of beef eye lens LAP. Amino acid analyses of the two proteins are also in reasonable agreement when based on the exact monomer molecular weight of beef eye lens protein obtained by the van Loon group ((1982) J. Biol. Chem. 257, 7077-7081). Our previously published monomer molecular weight of the FTBL protein was 25% too high, leading to the erroneous conclusion that the beef liver FTBL-LAP protein was a tetramer rather than a hexamer, as found by the van Loon group for beef lens LAP. The present report, taken together with our first paper on the FTBL protein establishes that the FTBL-LAP protein has been isolated from beef kidney and beef spleen as well as from beef liver. We now find that the properties of FTBL-LAP protein indicate that it is the same protein as beef eye lens LAP. The cellular and intracellular distributions of the FTBL-LAP protein have been considered in our first publication on the FTBL protein.

Interaction of Salmonella telaviv with Maclura pomifera lectin.

Salmonella telaviv, Salmonella tranoroa, and Salmonella illinois were examined for their ability to interact with 15 purified lectins of known sugar specificity. The only interaction observed was between the lectin of Maclura pomifera and S. telaviv. M. pomifera lectin specifically agglutinated suspensions of S. telaviv and precipitated with its purified lipopolysaccharide and isolated lipid A free O polysaccharide. Quantitative inhibition assays showing methyl-alpha-D-galactopyranoside and N-acetyl-D-galactosamine to be potent inhibitors of Maclura lectin precipitation by S. telaviv O polysaccharide suggest that the interaction is mediated by D-galactose or N-acetyl-D-galactosamine units of bacterial polysaccharide structure, or both.

Interaction of a streptococcal cell wall tetraheteroglycan and Salmonella telaviv lipopolysaccharide with polymeric McPC 870 and MOPC 384 mouse IgA myeloma proteins.

Interactions of polymeric McPC 870 and MOPC 384 mouse IgA myeloma proteins with a streptococcal cell wall tetraheteroglycan (T4-antigen), Salmonella tranoroa lipopolysaccharide (LPS), S. telaviv LPS and its lipid A free polysaccharide were examined by the quantitative precipitin method. Hapten inhibition of myeloma protein precipitation by T4-antigen or S. telaviv LPS indicates a complex anticarbohydrate specificity for 870 and 384 protein combining sites involving several sugars. While the combining site of 870 protein appears to bind alpha-D-mannopyranosyl, alpha-D-galactopyranosyl and alpha-D-glucopyranosyl residues, the combining site of 384 protein can accommodate both the alpha (1----2)-linked glucobiose (kojibiose) and alpha-D-galactopyranosyl but not the alpha-D-mannopyranosyl structure.

Isomeric, anti-rhamnose antibodies having specificity for rhamnose-containing, streptococcal heteroglycans.

L-Rhamnose (6-deoxy-L-mannose) is a constituent carbohydrate unit of microbial, immunogenic heteroglycans and lipopolysaccharides, and often functions as the immunodeterminant group of such immunogens. Two types of anti-rhamnose antibody have now been isolated by affinity chromatography of immune sera obtained from rabbits immunized with vaccines of Streptococcus mutans, strain KI-R, and Streptococcus pneumoniae, type 32. The antibodies of one type were directed at a glycan of L-rhamnose, D-glucose, and D-galactose in the cell wall of S. mutans, and those of the other type, against a capsular glycan of L-rhamnose and D-glucose from S. pneumoniae. The two types of anti-rhamnose antibody were immunologically distinct, and showed no reciprocal cross-reactivity. Additional properties of the two types of antibody were determined; thus, both types of antibody were of the IgG class of immunoglobulins, both possessed molecular weights of 1.45 X 10(5), and both consisted of multiple or isomeric forms.

Antigenic specificity and heterogeneity of lipopolysaccharides from pyocin-sensitive and -resistant strains of Neisseria gonorrhoeae.

Homologous antisera were raised against lipopolysaccharides (LPSs) isolated from pyocin 103-sensitive JW31 strain Neisseria gonorrhoeae and its isogenic, pyocin-resistant variant, JW31R. Changes in immunochemical reactivity of LPS antigen associated with pyocin-resistance were examined by enzyme-linked immunosorbent assay, employing homologous and heterologous anti-LPS immune sera. The acquisition of pyocin 103 resistance is accompanied by a loss in LPS antigen reactivity with homologous anti-LPS. The variant LPS of pyocin 103-resistant mutants is immunogenic and displays a new, distinct antigenic specificity shared with other pyocin 103-resistant variant gonococcal strains. The acquisition of pyocin 103 resistance by JW31 strain gonococci is also accompanied by a striking loss of LPS cross-reactivity with antistreptococcal polysaccharide reagents having an antibody combining site specificity directed against the chemically defined lactose polymer from Streptococcus faecalis cell wall and pneumococcal type 14 capsular polysaccharide. When examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, JW31 and JW31R LPSs show banding patterns characteristic of microheterogeneous, rough-type LPS devoid of O-side chains. Immunoblot transfer analysis of gel-separated gonococcal LPS antigens shows a difference in the pattern of antibody binding by homologous versus cross-reactive anti-LPS, which suggests a heterogeneity in the distribution of cross-reactive determinants among LPS molecules.

Chemical and immunochemical studies on lipopolysaccharides from pyocin 103-sensitive and -resistant Neisseria gonorrhoeae.

The chemical and immunochemical properties of lipopolysaccharides (LPS) isolated from pyocin 103-sensitive and -resistant Neisseria gonorrheae were investigated. Marked differences were found in immunochemical behavior of LPS from pyocin-sensitive gonococcal strain JW31 and its isogenic pyocin-resistant variant JW31R. JW31 LPS readily precipitated wheat-germ agglutinin, soybean lectin, and rabbit anti-Streptococcus faecalis or horse anti-type 14 pneumococcal antibody. In contrast, JW31R LPS precipitated only soybean lectin. The combining-site specificity of anti-S. faecalis cross-precipitated by JW31 LPS, or type 14 pneumococcal capsular polysaccharide, was examined by hapten inhibition, and lactose found to be the most potent inhibitor. Horse anti-pneumococcal type 14 antibodies, cross-precipitated by JW31 LPS and streptococcal lactose polymer, exhibited heterogeneity with respect to combining site specificity. Gel filtration of LPS-derived core oligosaccharide showed both strain JW31 and JW31 R to possess R-type lipopolysaccharide with cores having a Mr approximately 1800. JW31R LPS contains more galactose but less hexosamine than JW31 LPS. Both JW31 and JW31R core oligosaccharides possess D-glucosamine and D-galactosamine, probably N-acetylated, as the only nonreducing end-groups, and (1 leads to 4)-linked D-glucose residues. Chemical data support immunochemical findings which indicate that lactose units occur as a structural feature of JW31 gonococcal LPS.

Contagious equine metritis: distribution of organisms in experimental infection of mares.

After contagious equine metritis bacteria were inoculated into the uterus of mares, genital tract tissues were examined for presence of the organism by bacteriologic cultural technique and an indirect immunofluorescent staining technique. Up to 14 days after mares were inoculated, the organism was frequently in the lumen of the uterus and in the cervix and, less frequently, in the vagina, vestibule, clitoral fossa, clitoral sinus, and uterine tubes. After 21 to 116 days, the organism was occasionally found on the ovarian surface, in the uterine tubes, uterus, cervix, and vagina and more frequently in the clitoral sinus and clitoral fossa. The distribution of organisms in the remainder of the genital tract was not different in mares that had been clitorectomized.

Identification of immunoglobulin heavy-chain isotypes of specific antibodies of horse 46 group B meningococcal antiserum.

Hyperimmune horse serum from a single animal (horse 46) immunized with group B (strain B-11) meningococcal vaccine provides a standardized, readily available diagnostic reagent used in primary isolation medium and for serogrouping of meningococci. Identification of the heavy-chain isotypes of specific anticapsular polysaccharide and anti-lipopolysaccharide isolated from horse 46 serum revealed a differential distribution in the occurrence of immunoglobulin classes. Meningococcal anticapsular antibodies of horse 46 serum were restricted predominately to the immunoglobulin M (IgM) class, with only trace amounts of IgGa present, whereas anti-lipopolysaccharide concomitantly produced showed a heterogeneity in its heavy-chain isotypes, consisting of IgM, IgGa, IgGb, moderate amounts of IgB, and a small amount of IgA.

Recovery of contagious equine metritis organisms and development of lesions in experimental infection of mares.

Twenty-three of 24 mares were infected experimentally with contagious equine metritis organisms by intrauterine inoculation, and killed 2-116 days later. From mares killed within 14 days after infection the organism could be recovered from many sites in the uterus, and most sites in the cervix, a few sites in the vagina and oviduct and from one clitoral sinus. At this time the endometrial folds were swollen and there were 10-20 ml of fluid in the uterus. In mares killed after 14 days, the organism was recovered from the ovarian surface (1 mare), oviduct (4 mares), uterus (2 mares) and the clitoral sinus (3 mares). Severe diffuse endometritis and cervicitis was initially acute, and became more severe subacute and predominantly plasmacytic by 14 days, then declined but persisted throughout the experiment.

Interaction with lectins and differential wheat germ agglutinin binding of pyocin 103-sensitive and -resistant Neisseria gonorrhoeae.

Strains of Neisseria gonorrhoeae were treated with pyocin 611 131 (pyocin 103) from Pseudomonas aeruginosa PA103, and isogenic resistant variants were isolated. The interaction of pyocin-sensitive and isogenic pyocin-resistant strains with wheat germ agglutinin (WGA) agglutinated all pyocin-sensitive, but not pyocin-resistant, strains. Binding of WGA to three pyocin-sensitive strains and their isogenic pyocin-resistant variants was examined quantitatively by using fluorescein-conjugated lectin. Pyocin-resistant strains maximally bound one-third to one-eighth the quantity of WGA bound by isogenic-sensitive strains. Linear Scatchard plots revealed homogeneous WGA-binding sites on three pyocin-sensitive and one pyocin-resistant strains. Biphasic Scatchard plots, obtained with two pyocin-resistant strains, show that WGA-binding sites in these strains are heterogeneous. The number of WGA-binding sites for pyocin-sensitive organisms ranged from 8 x 10(5) to 1 x 10(6) sites per coccus and from 1 x 10(5) to 3 x 10(5) sites per coccus for pyocin-resistant strains. The apparent association constant for WGA binding to pyocin-sensitive strains ranged from 3 x 10(6) to 6 x 10(6) liters/mol and from 6 x 10(6) to 1 x 10(7) liters/mol for pyocin-resistant strains. Gonococcal lipopolysaccharide was shown to serve as the pyocin 103 receptor by inhibition of pyocin activity. Lipopolysaccharide from a pyocin 103-resistant strain was not able to inhibit pyocin 103 activity. Pyocin 103 resistance was correlated with a structural alteration involving N-acetylglucosamine residues in gonococcal lipopolysaccharide. Based on interactions with wheat germ, soybean, and ricin lectins, a model of lipopolysaccharide structure in N. gonorrhoeae is presented.

Interaction of lectins with Neisseria gonorrhoeae.

Fifty-four strains of Neisseria gonorrhoeae were examined for their ability to be agglutinated by various lectins. Wheat germ agglutinin, ricin, soybean lectin, and peanut agglutinin agglutinated all strains tested. Dolichos biflorus, Sophora japonica, Maclura pomifera, Ulex Europaeus, Lens culinaris, Canavalia ensiformis, Phaseolus lunatus, and Bandeiraea simplicifolia BS I and BS II lectins either failed to agglutinate or agglutinated some strains but not others. Agglutination of gonococci by wheat germ agglutinin, ricin, and soybean lectin is sugar specific and most effectively inhibited by ligands known to interact with lectin combining sites. Lectin reactive groupings appear to be independent of antigenic determinants conferring Gc serogroup specificity. Interactions with wheat germ agglutinin and ricin suggest the occurrence of multiple beta-linked N-acetyl-D-glucosamine (D-GlcNAc) and beta-D-galactosyl (beta-D-Gal) units as common structural features of gonococcal cell envelope polysaccharide. Interactions with soybean agglutinin, peanut agglutinin, and Dolichos biflorus lectins suggest that alpha-N-acetyl-D-galactosamine (alpha-D-GalNAc) units and beta-D-Gal linked to GalNAc and (or) GlcNAc may also occur as structural features of the polysaccharide components of the cell envelope of some gonococci.

Evolutionary conservation of equine gc alleles and of Mammalian gc/albumin linkage.

Ancient origin of the equine vitamin D binding protein (Gc) polymorphism is suggested by the finding of two alleles, Gc(F) and Gc(S), in each of three equine subgenera, Equus, Asinus and Hippotigris. The equine Gc and albumin loci are closely linked (lod score = 6). Although no recombinants were observed, the data are not inconsistent with a map distance similar to the 2 centimorgans reported for the human albumin/Gc linkage relationship. Gametic association between the Gc(F) and Alb(F) alleles appears probable in the American Standardbred horse, perhaps as a result of population structure. Since Gc and albumin are both polymorphic in rodents and possibly other orders, this linkage group will be useful for studies of the evolution of mammalian linkage groups, as well as for a comparison of meiotic recombination frequencies and linkage disequilibria in different species.

Homologous and cross-reactive precipitins in anti-pneumococcal sera raised in mules.

Serial bleedings were obtained from two mules during prolonged immunization, one with type XXV the other with type VIII pneumococcal vaccine. IgGa, IgGb, IgGc, IgB, IgG(T) and IgM present among purified Pn anti-XXV and Pn anti-VIII immunoglobulin isolated from various bleedings were identified by use of rabbit anti-equine heavy chain specific reagents. Radioimmunodiffusion with 14C-labelled type XXV pneumococcal capsular polysaccharide and horse and donkey reagents with species specificity directed against donkey or horse IgGa respectively, demonstrated both parental horse and donkey IgGa heavy chain isotypes among the anti-PnXXV antibodies of the interspecies hybrid. Qualtitative and quantitative examination of the cross-precipitation of mule anti-PnXXV sera with the capsular polysaccharides of pneumococcal types IV, X and XA, with birch sap, ketha gum, and with polysaccharides of E. coli, Klebsiella and Rhizobium was carried out and compared with data obtained with anti-PnXXV raised in a horse. Analysis of supernatants from the cross-reactions showed that distinct subfractions had reacted. indicating a marked heterogeneity of the antibodies.