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Biomedical subjects

S C Holt

Publications and source records attributed to S C Holt.

At least 127 records · Page 7Linked to original sources
PubMed1985

Effects of peptidoglycans from periodontal pathogens on selected biological activities of CD-1 murine peritoneal macrophages.

Resident CD-1 murine peritoneal macrophages were exposed to various concentrations of purified peptidoglycan isolated from members of the genera Bacteroides, Eikenella, and Actinomyces. Macrophage viability, the release of lysozyme, acid phosphatase, and prostaglandins E1 and E2 were assayed as a function of peptidoglycan concentration and time. Macrophages responded as a function of peptidoglycan concentration with increased release of acid phosphatase and prostaglandins; all cells remained greater than 90% viable during the course of the experiments. However, concentrations of peptidoglycan greater than 50 micrograms/mL were toxic to the macrophages, while the peptidoglycan from B. capillus strain 925.08 and Actinomyces viscosus strain T14AV consumed complement by both the classical and the alternate pathways. Cellular lysozyme activity and phagocytosis of Saccharomyces cerevisiae were significantly reduced in the presence of peptidoglycan. When viewed by scanning electron microscopy, the activated macrophages were rounded, lacked distinct pseudopod extensions, and possessed an increased number of microvilli and plasma membrane associated vesicles. These morphological alterations occurred as early as 3 h. Transmission electron microscopy revealed the purified peptidoglycan to have been taken up into numerous phagosomes; however, even after 24 h incubation, it was only partially degraded.

Acid Phosphatase↗
PubMed1985

Characteristics of the outer membrane of selected oral Bacteroides species.

The outer membranes from selected oral Bacteroides species were isolated and characterized morphologically, chemically, and physically. Both sucrose density gradient centrifugation and sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) revealed outer membranes which varied slightly with the species, as well as showing complex polypeptide patterns after SDS--PAGE. The polypeptide distribution revealed a species-specific pattern; however, there was often a variation within a given species in many minor polypeptide bands, with heat-modifiable minor peptides occurring in nearly all species. Protein-associated carbohydrates occurred in several species. Outer membrane fragments (blebs) recovered by ultracentrifugation of the growth supernatants were either nearly identical to the outer membrane polypeptide patterns, or contained reduced amounts of specific polypeptides, again varying with the species. 125I-labelling of whole cells indicated possible surface exposure for several of the major polypeptides.

Autoradiography↗
PubMed1984

Isolation and characterization of the outer membrane and lipopolysaccharide from Eikenella corrodens.

The chemical composition of the outer membrane fractions (OMFs) of Eikenella corrodens strains 23834 and 470 as well as the strain 23834 lipopolysaccharide (LPS) was determined. The OMFs were obtained by Triton X-100 treatment of the heavier membrane fraction from sucrose density centrifugation of the total membrane fraction. The resulting OMFs of strains 23834 and 470, free of cytoplasmic membrane components, were found to contain 69.6 and 75.0% (wt/wt) protein, 4.8 and 9.2% lipid, 4.6 and 4.7% carbohydrate, and 2.0 and 4.6% muramic acid, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis both OMFs contained one major peptide determined to be 33,500 daltons for the strain 23834 OMF, and 37,500 daltons for the strain 470 OMF. Analysis of the OMF fatty acids revealed hexadecanoic, hexadecenoic, octadecenoic, and lesser amounts of octadecanoic acids. Transmission electron microscopic examination of the OMFs revealed typical large sheets of membrane. Structures (10 nm in diameter) resembling pores were also evident. The E. corrodens LPS was found to be composed of 34.5% (wt/wt) carbohydrate and 25.0% lipid A. Only minute amounts of 2-keto-3-deoxyoctonate and heptose could be detected. Fatty acid analysis revealed primarily octadecanoic and hexadecanoic acids, with lesser amounts of octadecenoic acid. No hydroxy fatty acids were detected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed the E. corrodens LPS to resemble other smooth-type LPSs. Transmission electron microscopic examination revealed a vesicle-like morphology. The E. corrodens LPS appears not to be a "classical," i.e., enteric, type of LPS.

Bacterial Outer Membrane Proteins↗
PubMed1984

Biological activities of Eikenella corrodens outer membrane and lipopolysaccharide.

Highly purified preparations of the outer membrane and lipopolysaccharide (LPS) of Eikenella corrodens strain ATCC 23834 and the outer membrane fraction (OMF) of strain 470 were tested in in vitro biological assays. The OMFs of both strains were found to be mitogenic for BDF and C3H/HeJ murine splenocytes. The E. corrodens LPS was mitogenic for BDF spleen cells; however, doses of LPS as high as 50 micrograms/ml failed to stimulate C3H/HeJ cells. When incubated with T-lymphocyte-depleted C3H/HeJ splenocytes, the strain 23834 OMF demonstrated significant mitogenic activity, indicating that the OMF is a B-cell mitogen by a mechanism other than that elicited by conventional LPS. The E. corrodens 23834 OMF and LPS were stimulators of bone resorption when tested in organ cultures of fetal rat long bones. In contrast, the strain 470 OMF was only weakly stimulatory. Both OMFs and LPSs demonstrated "endotoxic" activity, since as little as 0.062 micrograms of E. corrodens LPS and 0.015 micrograms of the OMFs induced gelation in the Limulus amebocyte clotting assay. Thus, despite having a "nonclassical" LPS biochemistry, the E. corrodens LPS elicits classical endotoxic activities. These results also indicate that the surface structures of E. corrodens have significant biological activities as measured in vitro. The expression of such activities in vivo may play an important role in the pathogenesis of periodontitis as well as other E. corrodens infections.

Animals↗
PubMed1983

Corncob formation between Fusobacterium nucleatum and Streptococcus sanguis.

Corncob formation in dental plaque was believed to be limited to strains of Bacterionema matruchotii and Streptococcus sanguis. We observed recently that strains of Fusobacterium nucleatum also interacted with S. sanguis to form corncobs. Since the fusobacteria are among the first anaerobic filaments to colonize subgingival plaque, these interactions could serve as a connecting link between the transformation of supra- to subgingival plaque. To further characterize these interactions, quantitative in vitro studies of the kinetics of corncob formation of the fusobacteria were undertaken. These studies indicated that fewer streptococci were needed to saturate F. nucleatum strain 364 compared to strain 10953. Corncob formation with both strains was enhanced with increasing pH up to pH 8, at which point autoaggregation of the streptococci occurred. Variation in ionic strength and divalent cations had little effect on the interaction, and EDTA suppressed aggregate formation only slightly. Detergents at concentrations above 0.05% also inhibited corncob formation. Electron micrographs suggested that attachment of the cocci to the fusiforms was mediated through localized tufts of fimbriae, as they are in the Bacterionema system. However, although both trypsin and heat treatment of the streptococci inhibited corncob formation with fusobacteria, the effects were not as complete as those seen in Bacterionema species. Unlike the Bacterionema model, trypsin and heat treatment of the fusobacteria resulted in inhibition of corncob formation. These results suggest that several different receptors may be involved in corncob formation.

Adhesiveness↗
PubMed1983

Acid and alkaline phosphatases of Capnocytophaga species. I. Production and cytological localization of the enzymes.

The two hydrolytic enzymes, acid (AcP; EC 3.1.3.2) and alkaline (AlP; EC 3.1.3.1) phosphatase, of the three types species of Capnocytophaga were examined. Both enzymes were produced constitutively, with their activity highest in C. ochracea strain 25. These two degradative enzymes (approximately 10% of the total activity) were released into the growth medium during the latter stages of growth, both as soluble and membrane-bound enzymes. When grown in the presence of high concentrations of organic phosphates, the synthesis and expression of AcP and AlP was unaltered. Cyto- and immuno-chemical localization situated the phosphatases in the periplasmic space, at the cell surface, and in membranous vesicles.

Acid Phosphatase↗
PubMed1983

Acid and alkaline phosphatases of Capnocytophaga species. II. Isolation, purification, and characterization of the enzymes from Capnocytophaga ochracea.

Capnocytophaga ochracea acid (AcP; EC 3.1.3.2) and alkaline (AlP; EC 3.1.3.1) phosphatase was isolated by Ribi cell disruption and purified by sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE.) Both phosphatases eluted from Sephadex G-150 consistent with molecular weights (migration) of 140 000 and 110 000. SDS-PAGE demonstrated a 72 000 and 55 000 subunit molecular migration for AcP and AlP, respectively. The kinetics of activity of purified AcP and AlP on p-nitrophenol phosphate and phosphoseryl residues of the phosphoproteins are presented.

Acid Phosphatase↗
PubMed1983

Acid and alkaline phosphatases of Capnocytophaga species. III. The relationship of the enzymes to the cell wall.

Acid (AcP) and alkaline phosphatase (AlP) were localized by physicobiochemical techniques. Greater than 53% of the phosphatases were detected, following sonication, in a low speed centrifugation pellet while osmotically shocked and spheroplasted Capnocytophaga species released only 9-28% and 11-43% of the cellular phosphatases, respectively. French pressure cell disruption was more effective in releasing the phosphatases. Cell fragments were separated into cell wall, cytoplasmic membrane, and soluble fractions as determined by marker enzyme, chemical composition, transmission electron microscopy, sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) protein analysis, and gel diffusion. AcP and AlP was partitioned between the isolated cell wall (31-46%) and soluble material (33-61%), with greater than 60% of the phosphatases remaining with the cell wall following Triton X-100 treatment. The amount of phosphatase at the surface of intact C. ochracea was quantitated by specific 125I-protein labelling.

Acid Phosphatase↗
PubMed1983

The in vitro effect of Actinobacillus actinomycetemcomitans strain Y4 lipopolysaccharide on murine peritoneal macrophages.

Actinobacillus actinomycetemcomitans strain Y4 lipopolysaccharide (LPS) was examined for its in vitro effect on murine peritoneal macrophage morphology, viability, and lysosomal enzyme activity. Fifty micrograms of Y4 LPS per 10(6) macrophages resulted in macrophage activation, eliciting the release of acid phosphates (AcP), as well as the accumulation of intracellular AcP, without a loss in viability. There was also an increase in the number of organelles and cell-surface ruffles. One hundred or 250 microgram of Y4 LPS caused flattening, rounding, and blebbing of the cell surface, as well as the release of large quantities of AcP and lactate dehydrogenase (LDH), within 5 min of exposure to the LPS. Alteration of cell ultrastructure occurred within 30 min, and extensive autophagocytosis by 24 h, indicative of cell injury. Short-term (less than 24 h) experiments appear necessary to distinguish between the effects of cell activation and cell death caused by A. actinomycetemcomitans Y4 LPS.

Acid Phosphatase↗
PubMed1982

Effects of lysozyme and inorganic anions on the morphology of Streptococcus mutans BHT: electron microscopic examination.

The effects of hen egg white lysozyme and the inorganic salt sodium thiocyanate on the integrity of Streptococcus mutans BHT were studied by transmission electron microscopy. Both control cells and cells exposed to NaSCN possessed thick outer cell walls and densely staining inner cell walls juxtaposed to the plasma membranes. In the presence of NaSCN, however, the S. mutans BHT nucleoid was coagulated into thick electron-dense filaments. Exposure of S. mutans BHT to 150 mug of hen egg white lysozyme per ml resulted in the progressive destruction of both the cell walls and the plasma membranes. The enzyme appeared to affect the region of the cell wall septum, and exposure to 150 mug of hen egg white lysozyme per ml for as short a time as 10 min resulted in visible morphological cell wall alterations. At 30 min, ultrastructural observations revealed that the majority of the cells were in the process of expelling a portion of their cytoplasmic contents from the septal and other regions of the cells at the time of fixation. After 3 h of incubation in the presence of this high lysozyme concentration, gelled protoplasmic masses, which were free from the cells, were evident. In addition, extensive damage to the outer and inner cell walls and to the plasma membranes was apparent, although the cells maintained their shape. On some areas of the cell surface, the outer cell wall and plasma membrane were completely absent, whereas at other locations the outer cell wall was either split away from the inner cell wall and plasma membrane or distended from an area free of inner cell wall and plasma membrane. Upon addition of NaSCN to the hen egg white lysozyme-treated cells, both the gelled protoplasmic masses and the damaged cells exhibited an exploded appearance and existed as membrane ghosts, cell wall fragments, or dense aggregates of cytoplasmic components. The effects of a low lysozyme concentration (22.5 mug/ml) on S. mutans morphology were less pronounced at short incubation times (i.e., 10 and 30 min) than those that were observed with a high enzyme concentration; however, breaks in the cell walls and dissolution of the plasma membranes with resulting cell lysis were visible after a prolonged (3-h) incubation and after subsequent addition of NaSCN.

Cell Membrane↗
PubMed1980

Quantitative in vitro assay for "corncob" formation.

The interaction of Bacterionema matruchotii with strains of Streptococcus sanguis produces a structure which morphologically resembles a corncob. To determine the specific bacterial surface receptors involved in the interaction, we developed a quantitative assay. The assay consisted of mixing saline suspensions of [CH(3)-(3)H]thymidine-labeled streptococci and B. matruchotii, incubating at 37 degrees C for 2 h, and filtering the mixture through a 5-mum polycarbonate membrane filter. The free cocci and filaments passed through the filter, but the corncobs were retained. Estimates of the number of corncobs formed were obtained by quantitating the radioactivity retained on the membranes relative to that of controls of streptococci alone. Although saturation of the Bacterionema occurred at a ratio of streptococci to Bacterionema of 10:1 (Klett units), a 2:1 ratio was chosen because of the increased sensitivity of the assay at this ratio. The percentage of streptococci binding at this ratio was 18.6 +/- 8.1 (standard deviation). All five Bacterionema strains tested formed corncobs; in contrast, only three strains of S. sanguis were positive. These were serotype 1 strains which had localized surface "fuzz." Although scanning electron microscopic observations revealed an almost random distribution of cocci along the filament surface, transmission electron microscopy revealed that the streptococci were attached to the Bacterionema by the surface fuzz. No differences in corncob formation were observed in sodium phosphate buffer, pH 6 to 8, at phosphate concentrations ranging from 0.005 to 0.05 M. Concentrations of NaCl or KCl up to 0.25 M did not affect corncob formation, and low concentrations of CaCl(2) increased corncob formation slightly, whereas MgCl(2), ethylenediaminetetraacetic acid, and citrate buffers reduced the number of streptococci binding to the filaments. These results suggest that divalent cations may play a role in this process.

Actinomycetaceae↗
PubMed1980

Morphology and ultrastructure of oral strains of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

Selected human oral and nonoral strains of the genera Actinobacillus and Haemophilus were examined by transmission and scanning electron microscopy. The strains examined were morphologically identical to recognized Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus. By transmission electron microscopy, the cells were typically gram negative in morphology, with several strains possessing some extracellular ruthenium red-staining polymeric material. Numerous vesicular structures, morphologically identical to lipopolysaccharide vesicles, were seen to originate from and be continuous with the surface of the outer membrane. Large numbers of these vesicles were also found in the external environment. Scanning electron microscopic observations revealed that both actinobacilli and haemophili possessed surface projections and an amorphous surface material which connected and covered adjacent cells.

Actinobacillus↗