Although osteocytes are the most abundant cells in bone, little is known about their function, and no specific marker protein for osteocytes has been described. Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein expressed in tooth organ and bone. Our previous work showed that in the chicken, which is not capable of forming tooth, DMP1 messenger RNA (mRNA) is highly expressed in bone by Northern blot analysis. To clarify the significance of DMP1 expression in bone, the expression of DMP1 mRNA and its protein was examined in the chicken and rat. In the chicken, DMP1 mRNA was detected only in bone tissues and was localized in osteocytes and preosteocytes but not in osteoblasts. Similarly, in the rat, DMP1 mRNA was predominantly expressed in osteocytes and preosteocytes in bone matrix but not in osteoblasts located at the bone surface. Antiserum was raised against the peptide from rat DMP1, and the localization of DMP1 was examined by immunohistochemistry. In the development of bone, DMP1 was first detected in newly formed bone matrix after osteoblastic cells had been embedded within it. After the appearance of typical osteocytes, DMP1 was localized in the pericellular bone matrix of osteocytes, including their processes. These data show that DMP1 is a bone matrix protein specifically expressed in osteocytes and preosteocytes and suggest that DMP1 plays a role in bone homeostasis because of its high calcium ion-binding capacity.

The aim of this study was to evaluate molecular and phenotypic methods for the identification of nonhemolytic streptococci. A collection of 148 strains consisting of 115 clinical isolates from cases of infective endocarditis, septicemia, and meningitis and 33 reference strains, including type strains of all relevant Streptococcus species, were examined. Identification was performed by phylogenetic analysis of nucleotide sequences of four housekeeping genes, ddl, gdh, rpoB, and sodA; by PCR analysis of the glucosyltransferase (gtf) gene; and by conventional phenotypic characterization and identification using two commercial kits, Rapid ID 32 STREP and STREPTOGRAM and the associated databases. A phylogenetic tree based on concatenated sequences of the four housekeeping genes allowed unequivocal differentiation of recognized species and was used as the reference. Analysis of single gene sequences revealed deviation clustering in eight strains (5.4%) due to homologous recombination with other species. This was particularly evident in S. sanguinis and in members of the anginosus group of streptococci. The rate of correct identification of the strains by both commercial identification kits was below 50% but varied significantly between species. The most significant problems were observed with S. mitis and S. oralis and 11 Streptococcus species described since 1991. Our data indicate that identification based on multilocus sequence analysis is optimal. As a more practical alternative we recommend identification based on sodA sequences with reference to a comprehensive set of sequences that is available for downloading from our server. An analysis of the species distribution of 107 nonhemolytic streptococci from bacteremic patients showed a predominance of S. oralis and S. anginosus with various underlying infections.The genus Streptococcus currently consists of more than 50 species, most of which belong to one of six phylogenetic clusters that are revealed by comparative analysis of 16S rRNA gene sequences. In addition to the pyogenic group, which includes the traditional pathogenic species (i.e., hemolytic streptococci), these clusters are the anginosus group, the mitis group, the salivarius group, the bovis group, and the mutans group (30,34). Many of the species of these five clusters are major constituents of the commensal microbiota of the human oral cavity and upper respiratory tract and are occasionally implicated in various pathologies. The anginosus group, formerly called "Streptococcus milleri" in some parts of the world (16), includes three recognized species (Streptococcus anginosus, Streptococcus intermedius, and Streptococcus constellatus) that are primarily associated with suppurative infections of tissues of the mouth and various body sites, including the meninges (9,37,44,54,56). The mitis group currently includes

The prevalence of caries and the number and species distribution of salivary mutans streptococci in 356 children (aged 0-2 yr old) in Japan were examined twice at an interval of 1 yr. No mutans streptococci were isolated from the predentate children. The detection rate of mutans streptococci and the prevalence of caries increased with age. Mutans streptococci were isolated from 39.9% of the subjects. The majority of isolates were identified as Streptococcus mutans. The concentration of mutans streptococci correlated with the number of erupted teeth (r = 0.339). In addition, the concentration of mutans streptococci was more closely correlated to the caries prevalence of the next year (r = 0.465) than that of the year when the salivary concentration of mutans streptococci was first evaluated (r = 0.368). The children who had no caries but harbored mutans streptococci in the first year showed significantly higher caries prevalence in the next year than did the children with neither caries nor mutans streptococci. These results indicate that the establishment of mutans streptococci is associated with caries initiation in early childhood.

The carcinoembryonic antigen (CEA) family member CEACAM1 (previously called biliary glycoprotein or CD66a) was previously shown to function as a receptor that can mediate the binding of Opa protein-expressing Neisseria meningitidis to both neutrophils and epithelial cells. Since neutrophils and polarized epithelia have both been shown to coexpress multiple CEACAM receptors, we have now extended this work to characterize the binding specificity of meningococcal Opa proteins with other CEA family members. To do so, we used recombinant Escherichia coli expressing nine different Opa variants from three meningococcal strains and stably transfected cell lines expressing single members of the CEACAM family. These infection studies demonstrated that seven of the nine Opa variants bound to at least one CEACAM receptor and that binding to each of these receptors is sufficient to trigger the Opa-dependent bacterial uptake by these cell lines. The other two Opa variants do not appear to bind to either CEACAM receptors or heparan sulfate proteoglycan receptors, which are bound by some gonococcal Opa variants, thus implying a novel class of Opa proteins. We have also extended previous studies by demonstrating induction of CEACAM1 expression after stimulation of human umbilical vein endothelial cells with the proinflammatory cytokine tumor necrosis factor alpha, which is present in high concentrations during meningococcal disease. This induced expression of CEACAM1 leads to an increased Opa-dependent bacterial binding and invasion into the primary endothelia, implying that these interactions may play an important role in the pathogenesis of invasive meningococcal disease.

Streptococcus mutans produces 3 types of glucosyltransferase (GTF), whose cooperative action is considered to be essential for its cellular adherence to the tooth surface. However, the precise mechanisms for synthesizing adhesive glucans and the specific roles of each GTF in cellular adherence to smooth surfaces have not been elucidated. In the present study, seven types of isogenic mutants of S. mutans MT8148 lacking GTFB, GTFC, and/or GTFD activities were constructed by inactivation of the genes encoding GTFB, GTFC, and/or GTFD. Furthermore, recombinant GTFB, GTFC, and GTFD were prepared from Escherichia coli cells harboring recombinant plasmids containing each of the gtf genes. Using these GTF-deficient mutants and rGTFs, we reconstituted sucrose-dependent adherence of S. mutans resting cells and examined the role of each GTF in vitro. The highest level of sucrose-dependent adherence was found at the ratio of 20 rGTFB:1 rGTFC:4 rGTFD in both the resting cells of GTF-deficient mutants and insoluble glucan synthesized by rGTFs. Moreover, when rGTFC and rGTFD were both present at concentrations of 1.5 mU and 6 mU, respectively, the insoluble glucan synthesized from sucrose by the rGTFs showed a high level of adhesiveness to smooth surfaces, even without rGTFB. These results suggest that the presence of all three GTFs at the optimum ratio is necessary for sucrose-dependent adherence of S. mutans, and that GTFC and GTFD may play significant roles in the synthesis of adhesive and insoluble glucan from sucrose.

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Lipopolysaccharides (LPSs) were isolated from Bacteroides gingivalis and Escherichia coli by the phenolwater and butanol-water procedures. The phenol-water-extracted LPS from B. gingivalis 381 was composed of 46% carbohydrate, 23% hexosamine, 18% fatty acid, and 5% protein. The major component sugars of this preparation were glucose, glucosamine, rhamnose, galactose, galactosamine, and mannose, and their molecular ratio was 1:0.9:0.7:0.6:0.6:0.4, respectively. Neither heptose nor 2-keto-3-deoxyoctonate was detected. the butanol-water-extracted LPS from this strain was composed of 76% glucose, 7% fatty acid, and 13% protein, and it was associated with a number of polypeptides (13 to 56 kilodaltons). The main fatty acid of both LPS preparations was palmitic acid. It was found that biological activities of LPS from B. gingivalis were comparable to those of LPS from E. coli in terms of activation of the clotting enzyme of Limulus amebocyte lysate, mitogenicity, polyclonal B cell activation, and stimulation of interleukin 1 production in BALB/c mice. Furthermore, LPS-nonresponsive C3H/HeJ spleen cells were found to yield good mitogenic responses to both phenol-water-extracted LPS and butanol-water-extracted LPS from B. gingivalis or butanol-water-extracted LPS from E. coli. On the other hand, spleen cells from LPS-responsive C3H/HeN mice responded well to all these LPS preparations. * Corresponding author. MATERIALS AND METHODS Bacterial strains and growth conditions. B. gingivalis 381 (originally isolated by S. Sockransky, Forsyth Dental Center, Boston, Mass.) and E. coli strain K235 (19) were obtained from Y.

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