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S C Holt

Publications and source records attributed to S C Holt.

At least 109 records · Page 6Linked to original sources
PubMed1989

The expression and function of a Bacteroides gingivalis hemagglutinin gene in Escherichia coli.

Eight Escherichia coli JM 109 transformants generated from a clone bank of Bacteroides gingivalis 381 genomic DNA, were found to express B. gingivalis antigens. Quantitation of antigen expression by ELISA indicated that isopropyl-beta-D-thiogalactopyranoside (IPTG) was not necessary for antigen expression for any of the clones but that expression in 2 of the clones, ST 2 and ST 3, was increased in cells grown in the presence of IPTG. Western blot analysis revealed that the expressed protein of clone ST 2 has a molecular weight of 125,000 Dal. and that clone ST 3 contains multiple bands of 30 to 50 kdal which react with the anti-B. gingivalis antiserum. Three of the transformants were found to agglutinate sheep erythrocytes. Polyclonal monospecific antiserum to one of the transformants, clone ST 2, was found to react to 2 major bands of MWs 43,000 and 38,000 and minor bands of 115,000, 105,000, 32,000, and 30,000 Dal. present in B. gingivalis cell lysate preparations. Adsorption of anti B. gingivalis antiserum with cells of clone ST 2 reduced the hemagglutination inhibition activity of the antiserum 4-fold whereas antiserum to the clone itself inhibited B. gingivalis hemagglutination at a titer of 8 times that of normal rabbit serum. Immunoelectronmicroscopic studies using the antiserum to clone ST 2 indicate that the product of the cloned gene (hemagglutinin) is located on the B. gingivalis cell surface. A restriction map generated of the cloned B. gingivalis DNA fragment confirms the insert to be 3.2 kbases and indicates the possibility of a repeated sequence in the fragment.

Bacteroides↗
PubMed1989

Chemical characterization and biologic properties of lipopolysaccharide from Bacteroides gingivalis strains W50, W83, and ATCC 33277.

The chemistry and selected biological activity of lipopolysaccharide (LPS) from Bacteroides gingivalis strains W50, W83, and ATCC 33277 were compared, as well as the role of this molecule as a mediator of selected inflammatory responses. Chemically, the LPSs consisted of 47-58% Lipid A, 5-10% carbohydrate, 0.05% 3-deoxy 2-octulosonic acid, 0.3% heptose, 3.8-5.2% hexosamine, and 2% phosphate. Rhamnose represented the dominant sugar (26-36%), with lesser amounts of glucose (18-34%), galactose (18-25%), mannose (9-12%), glucosamine (7-11%), and galactosamine (2-5%). The major fatty acids were: 13-methyl-tetradecanoate (42-45%), 3-OH-heptadecanoate (21-23%), hexadecanoate (16-19%), and 12-methyl-tetradecanoate (6-8%). SDS-PAGE and sodium deoxycholate-PAGE revealed the LPS to be a smooth chemotype. Differences in migration patterns between the virulent and avirulent strain LPSs also occurred. C3H/HeN macrophages (Mø) exposed to 1 microgram/ml of LPS released 3.2-4.2 ng of prostaglandin E (PGE)/ml of supernatant, representing 236-278% of control. Interleukin-1 (IL-1) activity in C3H/HeN and C3H/HeJ Mø exposed to 50 micrograms of LPS/ml was 382-724% and 270-300% of control, respectively; similar Mø exposed to 10 micrograms of LPS/ml released 1.6-2.0 ng and 0.3-0.5 ng of tumor necrosis factor (TNF)/ml of supernatant, respectively. Maximum TNF release in C3H/HeN Mø occurred in response to 50 micrograms of LPS/ml, and was sustained for up to 96 hours. These results suggest that LPS from the B. gingivalis strains stimulate cytokine production from Mø which, in turn, may play a role in orchestrating the inflammatory response for the development of periodontal diseases.

Bacterial Outer Membrane Proteins↗
PubMed1989

Characterization of selected gram-negative oral microorganisms by SDS-PAGE.

SDS-PAGE analysis of bacterial cell proteins was used to examine a diverse group of oral microorganisms. Most of the species examined, including physiologically similar microorganisms, produced distinct protein patterns which are characterized by the distribution of their major protein bands. The SDS-PAGE system described provides a rapid, simple and standardized identification technique to be used in consort with routine taxonomic procedures.

Bacterial Proteins↗
PubMed1989

Characterization of coaggregation between Bacteroides gingivalis T22 and Fusobacterium nucleatum T18.

Bacterial adherence is a key factor in the colonization of the oral ecosystem, yet little is known about the mechanisms by which the pathogen Bacteroides gingivalis adheres in the periodontal environment. We examined the ability of strains of B. gingivalis to coaggregate with selected microorganisms isolated from the subgingival microbiota of the cynomolgus monkey. A strong interaction was demonstrated between strains of B. gingivalis and Fusobacterium nucleatum, whereas less pronounced or no interaction was observed with other oral isolates. Electron microscopic examination of coaggregates revealed large masses of bacteria, in which the fusiform F. nucleatum T18 and coccobacillary B. gingivalis T22 cells formed a woven pattern. To investigate this interaction and the nature of the bacterial cell surface molecules involved, we used a microcoaggregation assay. Galactose and galactose-related sugars blocked coaggregation, in contrast with the lack of effect of glucose or glucose-related sugars. The ability of F. nucleatum T18 cells to coaggregate was diminished by pretreatment with pronase. Pretreatment of B. gingivalis T22 cells with pronase resulted in an inhibition of coaggregation, whereas pretreatment with sodium metaperiodate completely abolished coaggregation. These data suggest that the coaggregation between B. gingivalis T22 and F. nucleatum T18 represents a carbohydrate-lectin interaction, mediated by a galactose-containing carbohydrate on B. gingivalis T22 and a protein on F. nucleatum T18.

Amylases↗
PubMed1988

Implantation of Bacteroides gingivalis in nonhuman primates initiates progression of periodontitis.

Although periodontitis is a bacterial disease, its multidimensional nature and its bacterial complexity have made it difficult to definitively prove that specific microorganisms initiate the disease process. The successful implantation of a rifampin-resistant strain of the putative periodontal pathogen Bacteroides gingivalis into the periodontal microbiota of monkeys (Macaca fascicularis) resulted in an increase in the systemic levels of antibody to the microorganism and rapid and significant bone loss.

Animals↗
PubMed1988

Inhibition of superoxide production in human polymorphonuclear leukocytes by oral treponemal factors.

The inhibition of superoxide (O2-) production by human peripheral blood polymorphonuclear leukocytes (PMNs) in the presence of oral treponemes, their cellular components, and their culture supernatants was investigated. Superoxide production was inhibited 56% by a 25-microgram/ml phenol extract of a human clinical isolate. Inhibition by culture supernatants of both the clinical isolate and a reference strain was related to the bacterial phase of growth and viability, though inhibition also persisted in the decline phase. Inhibition of superoxide production was not evident when either opsonized or nonopsonized whole spirochetes were reacted with PMNs. The suppressive activity depended, therefore, on the treponemes either being disrupted or growing and releasing the inhibitory factor into the culture medium. These results suggest that oral treponemes possess factors which interfere with the activity of PMNs and thereby alter the inflammatory process in the diseased periodontal pocket.

Cell Survival↗
PubMed1988

Chemical and biological characterization of the lipopolysaccharide of the oral pathogen Wolinella recta ATCC 33238.

To investigate the potential pathogenic mechanisms of the oral periodontopathogen Wolinella recta ATCC 33238, we have isolated its lipopolysaccharide (LPS) and determined the chemical composition and selected in vitro biological activities of the molecule. Sodium desoxycholate-polyacrylamide gel electrophoresis revealed the W. recta LPS to be an atypical smooth LPS with short O-antigenic side chains. Chemically the LPS consisted of 47.2% lipid A, 19.6% polysaccharide, 9.0% heptose, 8.5% hexosamine, 3.2% phosphate, and 0.6% 2-keto-3-deoxyoctanoate. The major fatty acids were hexadecanoic acid (25.0%), 3-OH tetradecanoic acid (23.8%), tetradecanoic acid (15.4%), 3-OH hexadecanoic acid (11.6%), and octadecenoic acid (10.9%). Rhamnose constituted 87.8% of the carbohydrates generally associated with the O antigen, with smaller amounts of glucose (5.5%), mannose (4.9%), and an unidentified sugar (1.9%). CD-1 and C3H/HeN macrophages (M phi) exposed to 1 microgram of W. recta LPS per ml released 6.0 and 10.5 ng of prostaglandin E per ml of supernatant, representing 625% and 1,306% of prostaglandin E release by the control (without LPS). Maximum prostaglandin E release occurred in CD-1 M phi exposed to 100 micrograms of LPS per ml and was equivalent to 1,542% of release by the control. Interleukin-1 (IL-1) activities in CD-1 and C3H/HeN M phi exposed to 1 micrograms of LPS per ml were 257% and 1,941% of activities in the control, respectively. Maximum IL-1 release in CD-1 M phi occurred in response to 50 micrograms of LPS per ml and represented a 927% increase over release in the control, while 100 micrograms LPS per ml stimulated maximum IL-1 release in C3H/HeN M phi that was greater than 5,000% of release by the control.

Acid Phosphatase↗
PubMed1988

Lipopolysaccharide-stimulated PGE2 release from human monocytes. Comparison of lipopolysaccharides prepared from suspected periodontal pathogens.

Lipopolysaccharides (LPS) prepared from the suspected periodontal pathogens Actinobacillus actinomycetemcomitans (A. a.), Bacteroides gingivalis, B. intermedius and Wolinella recta were compared to Salmonella typhimurium LPS for their capacity to stimulate prostaglandin E2 (PGE2) release from human monocytes. Counterflow isolated monocytes were cultured with control medium or media containing 10 micrograms/ml LPS. Media were then exchanged every 24 hours for a total of 72 hours. Salmonella and Wolinella LPS preparations demonstrated seven-fold greater PGE2 release than B. gingivalis and two-fold greater than A. a. and B. intermedius. PGE2 release was found to decrease over time with all LPS preparations except Wolinella. The potency of the LPS preparations is tentatively ranked as follows: Wolinella greater than or equal to Salmonella greater than A. a. greater than B. intermedius greater than or equal to B. gingivalis. These findings demonstrate that LPS preparations from suspected periodontal pathogens are capable of stimulating PGE2 release from human monocytes. The high potency and prolonged stimulation of PGE2 release with Wolinella LPS suggests unusual toxic properties that may exert a greater influence in the pathogenesis of destructive periodontal diseases.

Actinobacillus↗
PubMed1987

Immunoelectron microscopic analysis of elongation of type 1 fimbriae in Escherichia coli.

Using 10- and 20-nm-diameter gold particles conjugated to an antifimbrial monoclonal antibody, we analyzed the location of assembly of newly formed subunits on growing type 1 fimbriae of Escherichia coli. Fimbriae were removed from an E. coli K-12-derived strain, CSH50, by blending. Blended cells were allowed to regenerate their fimbriae in growth medium for approximately 25 min, after which they were labeled with a 20-nm-gold-monoclonal antibody probe. Continued outgrowth of these labeled fimbriae was allowed for additional time intervals, after which they were labeled with a 10-nm-gold-monoclonal antibody probe. The resulting fimbriae, double labeled with 10- and 20-nm-diameter gold particles, were examined in an electron microscope. The pattern of labeling on individual fimbrial organelles indicated morphologically that newly synthesized subunits are added to a growing organelle at its base.

Antibodies, Monoclonal↗
PubMed1987

Identification of a 32-kilodalton protein of Mycoplasma pneumoniae associated with hemadsorption.

Monoclonal antibodies directed against a 32-kilodalton (kDa) protein of Mycoplasma pneumoniae have been used to characterize a hemadsorption-negative (HA-) mutant class whose protein profile was previously indistinguishable from the wild-type, hemadsorbing (HA+) strain. Electron microscopy and colloidal gold labeling techniques were applied for ultrastructural analysis of the 32-kDa protein. Results indicate that this protein clusters in the tip structure of M. pneumoniae (HA+) wild-type organisms. Additionally, the protein is precipitated by infected hamster sera.

Animals↗
PubMed1986

Capillary hemangioblastoma. An immunohistochemical study.

Previous studies using immunohistochemical methods to determine the presence of glial fibrillary acidic protein (GFAP) and Factor VIII-related antigen (FVIIIR:Ag) in stromal cells of capillary hemangioblastomas have yielded conflicting results with respect to the possible astrocytic and endothelial origins of these cells. This study included Ulex europaeus I lectin (UEAI), a more sensitive marker of endothelial cells. Antibodies were also used as markers of pericytes and a variety of markers were employed to identify different populations of histiocytes. Results of this investigation indicate that FVIIIR:Ag and UEAI are limited only to endothelial cells, and that GFAP is present in entrapped astrocytes only. Positivity of stromal cells was found with some of the histiocytic markers, but the authors were unable to conclude that these cells have a histiocytic origin. It was concluded that currently there is no evidence that stromal cells are derived from endothelial, pericytic, or astrocytic cells-their origin remains uncertain.

Antibodies, Monoclonal↗
PubMed1986

Penicillin resistance in the subgingival microbiota associated with adult periodontitis.

In this investigation, the penicillin-resistant and beta-lactamase-producing subgingival microbiota associated with adult periodontitis was identified, and the impact of a recent exposure to penicillin on the recovery of resistant organisms from this microbiota was assessed. Subjects with adult periodontitis were examined clinically and microbiologically. Twenty-one subjects had a documented history of penicillin therapy within the previous 6 months whereas an additional 21 subjects had no history of antibiotic use within 1 year. Subgingival plaque samples were cultured anaerobically on nonselective and penicillin-containing elective media. MICs and beta-lactamase production were determined for the isolates from the elective medium. The penicillin-resistant microbiota consisted primarily of gram-negative organisms, including Bacteroides, Veillonella, Haemophilus, Eikenella, and Capnocytophaga species. The prevalence (P less than 0.05) and proportions (P less than 0.005) of both penicillin-resistant pigmented Bacteroides and Veillonella species were significantly greater in subjects with recent penicillin exposure. Of the penicillin-resistant genera identified, beta-lactamase production was detected in species of pigmented Bacteroides, Capnocytophaga, and Streptococcus. The prevalence of beta-lactamase-producing Bacteroides species was significantly greater in subjects with recent penicillin exposure (P less than 0.05). Of the antibiotics examined, no single agent was uniformly effective against all of the penicillin-resistant strains, but metronidazole and clindamycin were active against all of the penicillin-resistant pigmented Bacteroides strains.

Adult↗
PubMed1985

Isolation and characterization of the peptidoglycans from selected gram-positive and gram-negative periodontal pathogens.

The peptidoglycans from several Gram-negative and Gram-positive periodontal pathogens were isolated, purified, and characterized both morphologically and chemically. In addition, the effects of the mureolytic enzymes, lysozyme, M-1 N-acetyl-muramidase, and the AM-3 endopeptidase, on the peptidoglycans were examined. These enzymes were found to be highly effective in the degradation of the purified peptidoglycans; however, a Bacteroides capillus peptidoglycan-protein complex exhibited a greater resistance to these enzymes. Morphologically, the peptidoglycans consisted of large saccular sheets which, when viewed by scanning electron microscopy, contained numerous holes and tears. Chemically, the peptidoglycans consisted of muramic acid, glucosamine, alanine, glutamic acid, and meso-diaminopimelic acid (DAP). One Bacteroides species, Bacteroides gingivalis strain W, contained glycine and LL-DAP, suggestive of an indirectly cross-linked A3 gamma peptidoglycan.

Actinomyces↗