Phagocytosis of titanium particles and necrosis in TNF-alpha-resistant mouse sarcoma L929 cells.

In the oral cavity, titanium is an excellent biocompatible material. However, it is reported that high ratios of intracellular titanium particles can cause cell apoptosis or necrosis by as-yet unknown mechanisms. The purpose of this study was to investigate the response of tumor necrosis factor alpha (TNF-alpha)-resistant L929 fibroblasts to titanium particles. Cells were cultured in Eagle's medium supplemented with fetal bovine serum and L-glutamine. Titanium particle sizes were less than 9 micro. Cytotoxicity was assayed by a cell counting kit, trypan blue dye exclusion test and lactate dehydrogenase (LDH) leakage. The production of reactive oxygen species (ROS) was detected by a confocal laser scanning microscope (CLSM) using dichlorofluorescein diacetate as a fluorescent probe. Morphology was viewed by a CLSM and with an X-ray microanalyser (XMA). When titanium particles were added to cells, the viability decreased to around 50% at a particle concentration of 2.0%. The number of dead cells and LDH activity in the culture media increased significantly between 1 and 2 days. However, formation of active oxygen species did not occur, since no dichlorofluorescein fluorescence was observed. A scanning electron photomicrograph (SEM) revealed a large number of particles covering or adhering to cellular components in lysed cells compared with flattened control cells attached to the substrate. The XMA showed that the titanium accumulation was coincident with the deformed cell shape. The CLSM also confirmed that particles were within the cells. From these results it was concluded that titanium particles ingested in large quantities into the cell induced necrosis by a pathway other than by producing ROS.

Characterization of an outer membrane protein gene, pgmA, and its gene product from Porphyromonas gingivalis.

A gene upstream from fimA, the gene encoding fimbrilin, on the chromosome of Porphyromonas gingivalis was sequenced and shown to be the gene encoding an outer membrane protein in this organism based on homology and biochemical analyses. Therefore, the gene (formerly ORF5) was designated pgmA, the P. gingivalis outer membrane protein A gene. The gene product, PgmA, was sensitive to protease, and was detected as a 60-kDa protein from wild-type strains with trichloroacetic acid treatment, which was carried out to destroy intrinsic proteases, and from protease-deficient mutants without this treatment prior to electrophoresis. PgmA was indeed present in the membrane fraction. Its nature was determined to be that of outer membrane proteins in gram-negative bacteria based on attempts at differential extraction of inner membrane proteins with detergents. No evidence has been found thus far from functional analyses that this protein is related to fimbrial morphogenesis and functions or to serum resistance of this organism.

Serotype variation in Streptococcus anginosus, S. constellatus and S. intermedius.

Various serotypes reported in three serotyping systems for the 'Streptococcus milleri' group, i.e., Ottens I-IV, Osano I-IV and serotypes a-k, were compared with each other in a capillary precipitation test and by the double immunodiffusion test. Only two of the 19 serotypes, f and Ottens-III, were identical, and the other 17 serotypes were independent. Thus, 18 serotypes and another three candidates (a-, b- and g-cross-reactive) were found in the 'S. milleri' group. Of the 248 strains tested, 197 were serotypable, and 23 strains carried two type antigens. Generally, the Ottens I, II, III (serotype f), IV and Osano IV antigens, often together with the Lancefield group F antigen, were found in S. anginosus and S. constellatus. In addition, the serotype a, c, d, e and k antigens with the group A, C or G antigen were distributed in S. anginosus, the serotype b antigen in S. constellatus and the g, h, i, j, Osano I, II and III antigens in S. intermedius. Twenty-five of the untypable strains were Lancefield groupable (mostly F).

Molecular characterization of an enterobacterial metallo beta-lactamase found in a clinical isolate of Serratia marcescens that shows imipenem resistance.

A clinical isolate of Serratia marcescens (TN9106) produced a metallo beta-lactamase (IMP-1) which conferred resistance to imipenem and broad-spectrum beta-lactams. The blaIMP gene providing imipenem resistance was cloned and expressed in Escherichia coli HB101. The IMP-1 was purified from E. coli HB101 that harbors pSMBNU24 carrying blaIMP, and its apparent molecular mass was calculated to be about 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic studies of IMP-1 against various beta-lactams revealed that this enzyme hydrolyzes not only various broad-spectrum beta-lactams but also carbapenems. However, aztreonam was relatively stable against IMP-1. Although clavulanate or cloxacillin failed to inhibit IMP-1, Hg2+, Fe2+, or Cu2+ blocked the enzyme's activity. Moreover, the presence of EDTA in the reaction buffer resulted in a decrease in the enzyme's activity. Carbapenem resistance was not transferred from S. marcescens TN9106 to E. coli CSH2 by conjugation. A hybridization study confirmed that blaIMP was encoded on the chromosome of S. marcescens TN9106. By nucleotide sequencing analysis, blaIMP was found to encode a protein of 246 amino acid residues and was shown to have considerable homology to the metallo beta-lactamase genes of Bacillus cereus, Bacteroides fragilis, and Aeromonas hydrophila. The G+C content of blaIMP was 39.4%. Four consensus amino acid residues, His-95, His-97, Cys-176, and His-215, which form putative zinc ligands, were conserved in the deduced amino acid sequence of IMP-1. By determination of the amino acid sequence at the N terminus of purified mature IMP-1, 18 amino acid residues were found to be processed from the N terminus of the premature enzyme as a signal peptide. These results clearly show that IMP-1 is an enterobacterial metallo beta-lactamase, of which the primary structure has been completely determined, that confers resistance to carbapenems and other broad-spectrum beta-lactams.

Genetic and physiological characteristics of oral Streptococcus intermedius.

The taxonomic relationships among strains of oral Streptococcus intermedius and the related streptococci were determined by G + C mol%, DNA-DNA hydridization, phenotypical and serological tests. As to the results, group 1 strains of S. intermedius including the type strain ATCC 27335 and five isolates behaved distinctively with respect to genetic, phenotypic and serological characteristics compared with group 2 strains of S. intermedius including ATCC 31412 and two isolates. Therefore, nine strains of oral S. intermedius could be separated at least into two homology groups.

Serogrouping of oral Streptococcus intermedius.

Employing twenty fresh oral isolates of Streptococcus intermedius, studies were carried out to characterize serological relations among the isolates and also between the isolates and the strains of bacterial species closely related to S. intermedius. The Rantz-Randall extracts from the cells were used as antigens. The anti-rabbit serum raised against S. intermedius ATCC 27335T reacted with the cell extracts from only three strains of the isolates, which were designated serogroup I strains. The other isolates were classified into four serogroups, I, III, IV, and V, which specifically reacted with the cell extracts from the homologous serogroup strains. However, the serogroup II antiserum formed in immunodiffusion a common precipitin line between the extracts from the cells of serogroups II and I. The serogroups I, III, IV, and V antisera reacted with none of the extracts from the bacterial cells closely related to S. intermedius, which included Streptococcus anginosus ATCC 33397T, Streptococcus constellatus ATCC 27823T, three NCTC strains of "Streptococcus milleri," and three ATCC strains of Streptococcus MG. The precipitin line formed by the homologous reaction of the serogroup II antiserum was found to be a reaction of identity with that formed by the extract from "S. milleri" NCTC 10708. Conversely, the antiserum against NCTC 10708 strain did not react with the cell extracts of serogroup II.

[Bacteriological examination of saliva of institutionalized severely mentally retarded persons with chronic rumination].

The purpose of this study was to investigate dental caries prevalence, the pH and the presence of bacteria in the saliva of institutionalized severely mentally retarded persons with chronic rumination. The results obtained were as follows: The prevalence of dental caries among the group with chronic rumination was higher than among the group not suffering from rumination. The viable cell number of mutans streptococci, lactobacilli and yeasts in the group with chronic rumination was significantly higher than that of group without rumination. These results suggested that the saliva of the group with chronic rumination creates a favourable environment for aciduric bacterial growth.

Chondroitinase C activity of Streptococcus intermedius.

Chondroitin 6-sulfate depolymerizing activity was examined in the culture supernatant of Streptococcus intermedius ATCC 27335. 2-Acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose was split from the substrate. The enzyme(s) was not active upon chondroitin 4-sulfate or dermatan sulfate, which indicated that the enzyme responsible for the depolymerization is chondroitinase C.

Chondroitin sulfate-depolymerizing activity in Streptococcus intermedius and other streptococci.

The type strain (ATCC 27335) and 18 human oral isolates of Streptococcus intermedius and some other related streptococcal species were tested for chondroitin sulfate C-depolymerizing activity employing a modified screening plate method of Smith and Willett. As the results, S. intermedius strains except for ATCC 31412 strain were found to possess this activity. Propionibacterium acnes ATCC 11828 used as a positive control strain demonstrated strong activity, whereas S. intermedius strains showed only slightly detectable activity. This finding might be interesting in view of the classification of this species as well as its pathogenicity.