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G D Shockman

Publications and source records attributed to G D Shockman.

At least 37 records · Page 2Linked to original sources
PubMed1984

Monoclonal antibodies to immunodeterminants of lipoteichoic acids.

Murine hybrid cell lines producing monoclonal antibodies directed against determinants present on lipoteichoic acids were generated. Hapten inhibition studies showed that one group of monoclonal antibodies was inhibited by deacylated cardiolipin, and the second group was inhibited by kojibiose. Thus, antibodies directed against the polyglycerophosphate chain, which is common to the lipoteichoic acids of many gram-positive species, and against the streptococcal group D antigen were obtained.

Animals↗
PubMed1984

Isolation and characterization of soluble peptidoglycan from several strains of Streptococcus faecium.

Two phenotypically autolysis-deficient strains of Streptococcus faecium ATCC 9790 were shown to produce high-molecular-weight, soluble, linear, uncross-linked peptidoglycan when incubated with benzylpenicillin in a wall medium which permits cell wall synthesis (wall thickening) but not balanced growth. This high-molecular-weight s-peptidoglycan was shown to have a molecular weight of 46,000 to 54,000, lack peptide cross-links, and be virtually devoid of accessory wall polymers. It was hydrolyzed by hen egg white lysozyme and the endogenous, autolytic N-acetylmuramidase of S. faecium, but was not attacked by proteinases. Chemical analyses of the polymer are consistent with the following structure, where n is the number of repeating disaccharide units: (formula; see text).

Amino Acid Sequence↗
PubMed1984

Hydrolysis of soluble, linear, un-cross-linked peptidoglycans by endogenous bacterial N-acetylmuramoylhydrolases.

Soluble, linear, uncross-linked peptidoglycans, prepared from two autolysis-defective mutants of Streptococcus faecium ATCC 9790 and from Micrococcus leuteus, were used as substrates for studies of hydrolysis by an N-acetylmuramoylhydrolase (muramidase). The kinetics of hydrolysis of these substrates and the ability of the muramidases isolated from S. faecium ATCC 9790 and from two autolysis-defective mutants, Lyt-14 and Aut-3, to carry out transglycosylation reactions were compared with the action of hen egg white lysozyme (EC 3.2.1.17). Hydrolysis of these substrates by the endogenous streptococcal muramidases resulted in the production of disaccharide-peptide monomers with the structure (formula; see text) as nearly the sole product. As estimated from increases in reducing groups, hydrolysis proceeded at a linear rate for extended intervals, with consumption of up to 75% of the substrate, even at substrate concentrations well below the Km value. Apparent Km and relative Vmax values for the three streptococcal enzymes were indistinguishable from each other or from those for hen egg white lysozyme. These results indicate that the autolysis-defective phenotype of these mutants cannot be attributed to differences in their muramidases. In contrast to the action of hen egg white lysozyme, the streptococcal muramidase failed to catalyze transglycosylations. The extended periods of hydrolysis at constant rates are consistent with the occurrence of multiple catalytic events after the formation of the enzyme-substrate complex.

Amino Acid Sequence↗
PubMed1984

Division of temperature-sensitive Streptococcus faecium mutants after return to the permissive temperature.

The regrowth of 27 temperature-sensitive division mutants of Streptococcus faecium ATCC 9790 was examined after various periods of incubation at the nonpermissive temperature. Several of the mutants blocked at various stages of septum formation or of daughter-cell separation divided in a partially or completely synchronous way after a short incubation at the nonpermissive temperature. All four lytic mutants blocked early in the cell division cycle divided at a normal rate after a brief lag.

Cell Division↗
PubMed1983

Purification and some properties of the endogenous, autolytic N-acetylmuramoylhydrolase of Streptococcus faecium, a bacterial glycoenzyme.

The latent form of the endogenous, autolytic N-acetylmuramoylhydrolase of Streptococcus faecium ATCC 9790 was purified to near homogeneity by affinity chromatography on concanavalin A-Sepharose 4B. The latent enzyme had Mr approximately 130,000 on sodium dodecyl sulfate-gel electrophoresis. Upon proteinase treatment (trypsin or endogenous proteinase), the latent form is converted to an active form Mr approximately 87,000. The enzyme was shown to be glycoprotein, containing monomeric and oligomeric glucose substituents. Some of the substrate specificity requirements of this enzyme are described.

Chromatography, Affinity↗
PubMed1983

Quantitative determination in human sera of vaccine-induced antibody to type-specific polysaccharides of group B streptococci using an enzyme-linked immunosorbent assay.

The humoral immune response of human volunteers vaccinated with highly purified type II-or type III-specific polysaccharide of group B streptococci was evaluated using an enzyme-linked immunosorbent assay standardized with quantitative precipitin analysis, a method which permits calculation of the micrograms of specific antibody protein per milliliter of serum, rather than expression of the data as titers. By inhibition studies, the assays were shown to be specific for antibody to the undegraded type II or III polysaccharide antigen. Purity of the antigens and the specificity of the immune response to them were evidenced by an increase in level of antibody only to the type-specific antigen used for immunization. The isotype of the antibody raised in the sera of immunized volunteers was primarily IgG, thus confirming the potential utility of vaccination against group B streptococci using polysaccharide vaccines to induce antibodies which will cross the human placenta.

Antibodies, Bacterial↗
PubMed1983

Effects of mecillinam and cefoxitin on growth, macromolecular synthesis, and penicillin-binding proteins in a variety of streptococci.

Although some strains of streptococci seem to be virtually inert to mecillinam, the growth of other strains, notably certain viridans streptococci (Streptococcus mutans and Streptococcus sanguis) was inhibited by relatively low concentrations of the drug. Inhibition of the synthesis of peptidoglycan, RNA, protein, and DNA in two tolerant strains, S. mutans FA-1 and GS-5, was studied over a wide range of concentrations of mecillinam, benzylpenicillin, and cefoxitin. The responses of both strains to all three beta-lactams were very similar; that is, synthesis of insoluble peptidoglycan was most susceptible. Inhibition of peptidoglycan synthesis was followed rapidly and sequentially by substantial but less severe inhibitions of RNA and protein synthesis. Significant inhibition of DNA synthesis was not observed. Binding studies with [14C]benzylpenicillin alone or after preexposure of membrane preparations to benzylpenicillin, mecillinam, or cefoxitin suggest that reasonably selective binding of a beta-lactam antibiotic to one or two of the major penicillin-binding proteins (PBP 1 or PBP 4) of S. mutans GS-5 and FA-1 may be the initial step in the series of events that results in the inhibition of growth and in the inhibition of insoluble peptidoglycan assembly and of RNA and protein synthesis.

Amdinocillin↗
PubMed1983

Quantitation of in vitro opsonic activity of human antibody induced by a vaccine consisting of the type III-specific polysaccharide of group B streptococcus.

Human antibody, induced by a vaccine consisting of undegraded and highly purified extracellular type III-specific polysaccharide of group B streptococcus, was shown to increase the rate of phagocyte-mediated killing of bacteria of the homologous type. The bactericidal effect was mediated by type III-specific antibody and was complement dependent. An assay which permitted quantitation of "opsonic activity" was developed. In this assay, loss of CFUs occurred at a constant rate, and the rate constant was used as a measure of opsonic activity of antisera. A linear relationship between type III-specific antibody concentration (40 to 500 ng/ml) and the rate constant of killing was observed. When sets of immune sera were tested, some sera reacted anomalously, mediating significantly higher or lower rates than expected on the basis of their antibody content. Since type III-specific antibody in immune sera was found almost exclusively in the immunoglobulin G class, we hypothesize that differences in immunoglobulin G subclass distribution of specific antibody may have been the source of this variation.

Antibodies, Bacterial↗
PubMed1983

Protoplast formation and localization of enzymes in Streptococcus mitis.

Cells of Streptococcus mitis ATCC 903 were converted to stable protoplasts by the cell wall-degrading M-1 enzyme of the mutanolysin complex isolated from Streptomyces globisporus. Over 90% of total glucokinase (EC 2.7.1.2), aminopeptidase (EC 3.4.11.1), and dextranglucosidase (EC 3.2.1.70) was recovered in the cytoplasmic fraction, whereas over 20% of total invertase (beta-fructofuranosidase: EC 3.2.1.26) was released during protoplast formation. ATPase (EC 3.6.1.3). chymotrypsin-like protease (EC 3.4.21.1), arginine aminopeptidase (EC 3.4.11.6), and lactate dehydrogenase (EC 1.1.1.27) were detected in Triton X-100 extracts of the cytoplasmic membrane fraction by crossed immunoelectrophoresis in combination with enzyme-staining procedures. By these methods, NADH dehydrogenase (EC 1.6.99.3), aminopeptidase, and lactate dehydrogenase were detected in the cytoplasmic fraction. Aminopeptidases in the cytoplasmic fraction differed from this activity in the membrane fractions in electrophoretic mobility and substrate specificity.

Cell Membrane↗
PubMed1983

Isolation, chemical composition, and molecular size of extracellular type II and type Ia polysaccharides of group B streptococci.

Polysaccharides carrying the type II- and type Ia-specific determinants of Lancefield group B streptococci were isolated and purified by anion-exchange chromatography and gel filtration from the supernatant culture medium after growth of strain 18RS21/67/1 (type II) and strain DS/1204/78 (type Ia), respectively. The average molecular weights of these polysaccharides were 97,000 (type II) and 94,000 (type Ia), as determined by reducing end group analyses. These molecular weights were in reasonably good agreement with molecular weights determined by gel filtration at high ionic strength on calibrated columns. The polysaccharides did not cross-react with antisera specific for the other type-specific determinants or with group B-specific antisera. Their content of galactose, glucose, glucosamine, and neuraminic acid (the last two calculated as N-acetyl derivatives) accounted for over 96% of their dry weight. The two polysaccharides differed from each other (and from type III polysaccharide) in their relative content of these monosaccharides. The molar ratios of galactose, glucose, and neuraminic acid to glucosamine were 3.3:2.3:1.35:1.0 for the type II polysaccharide and 2.0:0.8:1.4:1.0 for the type Ia polysaccharides. The results obtained indicate that these extracellular type II and Ia polysaccharides contain larger amounts of neuraminic acid than can be accounted for by previously proposed structures of their repeating units.

Antigens, Bacterial↗
PubMed1983

Increased carbohydrate substitution of lipoteichoic acid during inhibition of protein synthesis.

Decreases in electrophoretic mobilities of intracellular lipoteichoic acid, intracellular deacylated lipoteichoic acid, and extracellular deacylated lipoteichoic acid were observed during inhibition of protein synthesis in Streptococcus faecium after exposure to chloramphenicol or valine deprivation. Increased carbohydrate content, and thus an increased mass-to-charge ratio, rather than changes in ester alanine content or novel fatty acid substitutions, appeared to account for the decreased electrophoretic mobilities. The increase in carbohydrate content, as judged from mobility measurements, was progressive over time and appeared to occur on biosynthetically new lipoteichoic acid as well as on lipoteichoic acid made before inhibition of protein synthesis.

Chloramphenicol↗
PubMed1983

Division blocks in temperature-sensitive mutants of Streptococcus faecium (S. faecalis ATCC 9790).

Two hundred nine temperature-sensitive growth or division (or both) mutants of Streptococcus faecium ATCC 9790 were isolated. These strains were examined for timing of the division block in the cell division cycle. About 42% of the isolates were blocked at terminal stages of cell division. A second large group appeared to be blocked at various stages of septation. Only five of the temperature-sensitive isolates were blocked at a stage before the completion of chromosome replication. Thirty temperature-sensitive isolates lysed after one or more doublings at the nonpermissive temperature.

Cell Division↗
PubMed1982

Group B, type III streptococcal cell wall: composition and structural aspects revealed through endo-N-acetylmuramidase-catalyzed hydrolysis.

Cell walls from a group B, type III streptococcus strain were prepared, purified by extraction with sodium dodecyl sulfate, and solubilized by the M-1 fraction of mutanolysin, an endo-N-acetylmuramidase obtained from Streptomyces globisporus. The lysate was resolved into three fractions by ion-exchange chromatography: a fraction containing peptidoglycan (PG) fragments, free of neutral and acidic sugars and of phosphate; a complex of PG fragments and group B-specific polysaccharide; and a complex of PG fragments and group B-specific polysaccharide and type III-specific polysaccharide. The PG-polysaccharide complexes were large and heterogeneous in molecular size. When subjected to base-catalyzed beta-elimination, both complexes were disintegrated, and polysaccharides and low-molecular-weight PG fragments could then be separated by gel filtration. The low-molecular-weight PG fragment-containing fraction contained muramic acid, glucosamine, alanine, lysine, glutamic acid, and serine in molar ratios (to lysine) of 0.92:0.98:3.01:1.00:1.00:0.05. Wall-derived, purified group polysaccharide contained rhamnose, galactose, glucosamine, and phosphorus in molar ratios (to galactose) of 5.03:1.00:1.00:1.05. It also contained an unidentified sugar. Wall-derived, purified type III polysaccharide contained galactose, glucosamine, glucose, and N-acetylneuraminic acid in molar ratios (to glucose) of 1.94:0.85:1.00:1.39. On a dry-weight basis, the whole wall lysate contained 19.8 and 20.6% of group and type polysaccharide, respectively. Neither glycerol nor ribitol was found, and all of the cell wall phosphorus was accounted for as polysaccharide, indicating the absence of a wall teichoic acid.

Amino Acids↗
PubMed1982

Type-specific protection of neonatal rats from lethal group B streptococcal infection by immune sera obtained from human volunteers vaccinated with type III-specific polysaccharide.

Sera obtained from human volunteers at 6 weeks after vaccination with highly purified type III polysaccharide antigen prepared from a group B Streptococcus, strain M732, were found to protect neonatal rats from otherwise lethal infection by the homologous strain. The specific antibody content of the sera, expressed in micrograms of antibody protein per milliliter, was determined by an enzyme-linked immunosorbent assay in conjunction with quantitative precipitin analysis. For two sera studied in detail, the protective dose of antibody for 50% of the animals was 0.4 micrograms. Immune serum obtained from a volunteer who received type II polysaccharide vaccine was not protective against type III infection. Absorption of anti-type III serum by quantitative precipitation of antibodies with type III polysaccharide completely removed the passive protective activity of the serum. The results show that antibodies induced in humans by purified type II polysaccharide give serotype-specific protection in an animal model of neonatal infection.

Adult↗
PubMed1982

Effects of penicillin on synthesis and excretion of lipid and lipoteichoic acid from Streptococcus mutans BHT.

Cultures of Streptococcus mutans BHT grown for at least eight generations in a chemically defined medium containing [1(3)-14C]glycerol, when treated with growth-inhibitory concentrations (0.2 micrograms/ml) of benzylpenicillin (Pen G), produced and excreted increased amounts of lipid and lipoteichoic acid per unit of cells. Cellular lysis was not observed. Compared with untreated controls, lipid excretion increased 15-fold, and lipoteichoic acid excretion increased 6-fold, 4 h after the addition of Pen G. All lipid species showed increased synthesis and excretion after exposure to Pen G. Although the same lipid types were found in both the Pen G-treated and the untreated cultures, the percent composition was altered after treatment with Pen G. The most dramatic example of this was the percentage of intracellular diphosphatidylglycerol found in the Pen G-treated cultures, 22.6%, in contrast to 5.3% found in the untreated cultures.

Glycerol↗
PubMed1981

Peptidoglycan loss during hen egg white lysozyme-inorganic salt lysis of Streptococcus mutans.

Streptococcus mutans BHT was grown in Todd-Hewitt dialysate medium containing N-acetyl[(14)C]glucosamine for 6 to 11 generations. After treatment with cold and hot trichloroacetic acid and trypsin, 52 to 65% of the radioactivity remained present in insoluble peptidoglycan-containing residues. Hen egg white lysozyme or mutanolysin treatment of the peptidoglycan residues resulted in the release of 80 and 97%, respectively, of the (14)C label to the supernatant fraction. Hydrochloric acid hydrolysates of such supernatants showed that essentially all of the radioactivity present in insoluble peptidoglycan fractions was present in compounds that comigrated on paper chromatography with glucosamine ( approximately 60%) or muramic acid ( approximately 30%). Treatment of whole cells with low and high concentrations of lysozyme alone resulted in losses of 45 and 70% of the insoluble peptidoglycan, respectively, yet release of deoxyribonucleic acid from cells was not detected. Sequential addition of appropriate concentrations of selected inorganic salts after lysozyme treatment did result in the liberation of deoxyribonucleic acid. Deoxyribonucleic acid release was correlated with a further release of peptidoglycan from the insoluble fraction. However, the total amount of peptidoglycan lost effected by the low concentration of lysozyme and NaSCN (lysis) was significantly less than the amount of peptidoglycan hydrolyzed by high concentrations of lysozyme alone (no lysis), suggesting that the overall amount of peptidoglycan lost did not correlate well with cellular lysis. The total amount of insoluble peptidoglycan lost at the highest salt concentrations tested was found to be greater than could be accounted for by lysozyme-sensitive linkages of the peptidoglycan, possibly implicating autolysins. The results obtained suggested that hydrolysis of peptidoglycan bonds in topologically localized, but strategically important, sites was a more significant factor in the sequence that results in loss of cellular integrity (lysis).

Anions↗
PubMed1980

Evidence that Streptococcus mutans constructs its membrane with excess fluidity for survival at suboptimal temperatures.

When cells from cultures of Streptococcus mutans strain FA-1 grown at 37 degrees C were exposed to incubation temperatures of 26 degrees C or less for 5 min or more, an extensive aggregation of particles was observed on the convex fracture faces of their freeze-cleaved membranes. Aggregation of particles was accompanied by a parallel increase in the activation energy for growth. By shifting the growth temperature from 37 to 24 degrees C for one doubling of culture mass, the transition temperature for membrane particle aggregation could be lowered from about 26 to 0 degrees C. Although membrane lipids became enriched with unsaturated fatty acids during this period of growth at 24 degrees C, this enrichment was not accompanied by an increased growth rate of the culture. However, the period of growth at 24 degrees C did result in bacteria that could grow more rapidly at 10 degrees C than could bacteria directly transferred from cultures grown at 37 degrees C. These observations suggest that the increase in membrane fluidity that occurs when bacteria are grown at 24 degrees C doses not allow bacteria to grow faster at 24 degrees C, but rather allows them to adapt more readily to further decreases in growth temperature.

Cell Membrane↗