We recently identified the genes responsible for the serotype c-specific glucose side chain formation of rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans. These genes were located downstream from the rgpA through rgpF locus that is involved in the synthesis of RGP. In the present study, the corresponding chromosomal regions were isolated from serotype e and f strains and characterized. The rgpA through rgpF homologs were well conserved among the three serotypes. By contrast, the regions downstream from the rgpF homolog differed considerably among the three serotypes. Replacement of these regions in the different serotype strains converted their serotypic phenotypes, suggesting that these regions participated in serotypespecific glucose side chain formation in each serotype strain. Based on the differences among the DNA sequences of these regions, a PCR method was developed to determine serotypes. S. mutans was isolated from 198 of 432 preschool children (3 to 4 years old). The serotypes of all but one S. mutans isolate were identified by serotyping PCR. Serotype c predominated (84.8%), serotype e was the next most common (13.3%), and serotype f occured rarely (1.9%) in Japanese preschool children. Caries experience in the group with a mixed infection by multiple serotypes of S. mutans was significantly higher than that in the group with a monoinfection by a single serotype.Streptococcus mutans strains are classified into three serotypes (c, e, and f), and the serologic specificity is defined by rhamnose-glucose polysaccharide (RGP) on the cell wall (6). We have characterized the genes involved in RGP synthesis in S. mutans Xc (serotype c) in the course of our previous studies. Four rml genes (rmlA through rmlD) are directly related to the synthesis of dTDP-L-rhamnose (12, 13), and the gluA gene encodes the enzyme producing UDP-D-glucose (18). The rgpG gene is implicated in the initiation of RGP synthesis by transfer of N-acetylglucosamine-1-phosphate to a lipid carrier (16). Furthermore, six other genes (rgpA through rgpF) required for RGP synthesis were identified in the region downstream from rmlD, and these genes are likely to be involved in the transport and assembly of RGP (11,19).The RGPs are composed of ␣1,2-and ␣1,3-linked rhamnan backbones with glucose side chains linked to alternate rhamnoses. Each serotype-specific polysaccharide has unique linkages of its glucose side chains (serotype c, ␣1,2-linkage; serotype e, ␤1,2-linkage; and serotype f, ␣1,3-linkage) (5, 10). Recently, we identified and characterized the genes required for glucose side chain formation of the serotype c-specific RGP (9). However, the loci responsible for the determination of the other serotypes have not yet been elucidated.In this study, we identified the loci involved in the glucose side chain formation of RGP in serotypes e and f of S. mutans and confirmed that these regions determine serotype specificities. Furthermore, we designed three pairs of primers from specific DNA sequences within each serotype determinan...

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Tobacco smoking is an important risk factor for the development of several cancers, osteoporosis, and inflammatory diseases such as periodontitis. Nicotine is one of the major components of tobacco. In previous study, we showed that nicotine inhibits mineralized nodule formation by osteoblasts, and the culture medium from osteoblasts containing nicotine and lipopolysaccharide increases osteoclast differentiation. However, the direct effect of nicotine on the differentiation and function of osteoclasts is poorly understood. Thus, we examined the direct effects of nicotine on the expression of nicotine receptors and bone resorption-related enzymes, mineral resorption, actin organization, and bone resorption using RAW264.7 cells and bone marrow cells as osteoclast precursors. Cells were cultured with 10−5, 10−4, or 10−3 M nicotine and/or 50 µM α-bungarotoxin (btx), an 7 nicotine receptor antagonist, in differentiation medium containing the soluble RANKL for up 7 days. 1–5, 7, 9, and 10 nicotine receptors were expressed on RAW264.7 cells. The expression of 7 nicotine receptor was increased by the addition of nicotine. Nicotine suppressed the number of tartrate-resistant acid phosphatase positive multinuclear osteoclasts with large nuclei(≥10 nuclei), and decreased the planar area of each cell. Nicotine decreased expression of cathepsin K, MMP-9, and V-ATPase d2. Btx inhibited nicotine effects. Nicotine increased CA II expression although decreased the expression of V-ATPase d2 and the distribution of F-actin. Nicotine suppressed the planar area of resorption pit by osteoclasts, but did not affect mineral resorption. These results suggest that nicotine increased the number of osteoclasts with small nuclei, but suppressed the number of osteoclasts with large nuclei. Moreover, nicotine reduced the planar area of resorption pit by suppressing the number of osteoclasts with large nuclei, V-ATPase d2, cathepsin K and MMP-9 expression and actin organization.

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