Takashi Ooshima

Dentin matrix protein 1 is predominantly expressed in chicken and rat osteocytes but not in osteoblasts.

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.

Virulence Factors of Streptococcus mutans and Dental Caries Prevention

Streptococcus mutans possesses the abilities to adhere to pellicle-coated tooth surfaces and to form acids - two characteristics associated with the cariogenicity of this micro-organism. De novo synthesis of insoluble glucan by S. mutans glucosyltransferase from sucrose is essential in the adherence process. Therefore, agents which interfere with the adherence ability of S. mutans would be useful for controlling dental caries. In the present report, we have summarized our recent findings concerning virulence factors of S. mutans and means for prevention of S. mutans-induced dental caries.

Inhibitory effects of oolong tea extract on caries-inducing properties of mutans streptococci.

The inhibitory effects of oolong tea extract (OTE) on the caries–inducing properties of mutans streptococci were examined in vitro. OTE reduced the rate of acid production by mutans streptococci accompanied with the retardation of growth rate of mutans streptococci, while the action by chromatographically isolated oolong tea polyphenol (OTF6) was weak. On the other hand, both oolong tea products decreased cell surface hydrophobicity of almost all the oral streptococci examined in the present study, and also induced cellular aggregation of Streptococcus mutans, Streptococcus oralis, Streptococcus sanguis or Streptococcus gordonii. In these reactions, OTF6 showed a more pronounced activity than OTE. Furthermore, the oolong tea products inhibited the adherence of mutans streptococci to saliva–coated hydroxyapatite. These results suggest that OTF6 may inhibit bacterial adherence to the tooth surfaces by reducing the hydrophobicity of mutans streptococci, and OTE may inhibit caries–inducing activity of mutans streptococci by reducing the rate of acid production.

Detection of oral bacteria in cardiovascular specimens.

BACKGROUND/AIMS Oral bacteria, including cariogenic and periodontal pathogens, are thought to be etiological factors in the development of cardiovascular diseases. To define this relationship, we analyzed the distribution of oral bacterial species in cardiovascular specimens. METHOD Following acceptance into the study, 203 consecutive patients were analyzed, from whom 82 aortic valve specimens, 35 mitral valve specimens, and 86 aortic aneurysmal wall specimens, of which 16 contained aneurysmal thrombus tissues, were obtained. In addition, a total of 58 dental plaque specimens were collected from the same group of patients who underwent heart valve replacement or removal of aortic aneurysms. Bacterial DNA was extracted from both cardiovascular tissues and dental plaque in those cases and then species-specific polymerase chain reaction assays were used to analyze the occurrences of six oral streptococcal and six periodontal bacterial species. RESULTS Streptococcus mutans was the most frequently detected species in the cardiovascular specimens, followed by Aggregatibacter actinomycetemcomitans. As for dental plaque specimens from patients who underwent cardiovascular operations, most of the tested periodontitis-related species as well as oral streptococci were detected at high frequencies. Furthermore, the positive rate of S. mutans in cardiovascular specimens from patients whose dental plaque specimens were also positive for S. mutans was 78%, which was significantly higher than any other tested species when the same analysis was performed. CONCLUSION Our results suggest that specific oral bacterial species, such as S. mutans and A. actinomycetemcomitans, are related to bacteremia and may be etiologic factors for the development of cardiovascular diseases.

Caries inhibitory activity of cacao bean husk extract in in-vitro and animal experiments

Cacao bean husk extract (CBH) was examined for inhibitory effects on the caries-inducing properties of mutans streptococci in vitro and on caries development in specific pathogen-free Sprague-Dawley rats infected with mutans streptococci. CBH reduced the growth rate of almost all oral streptococci examined, which resulted in the reduction of acid production. Furthermore, insoluble glucan synthesis by the glucosyltransferases from Streptococcus mutans MT8148R and Streptococcus sobrinus 6715 was significantly inhibited by CBH. Hence, the sucrose-dependent cell adherence of mutans streptococci was also depressed by CBH. The administration of CBH in drinking water resulted in significant reductions of caries development and dental plaque accumulation in rats infected with either Strep. sobrinus 6715 or Strep. mutans MT8148R, and the minimum cariostatic concentration was 1.0 mg/ml. These results indicate that CBH possesses powerful anticariogenic potential.

Demonstration of Streptococcus mutans with a Cell Wall Polysaccharide Specific to a New Serotype, k, in the Human Oral Cavity

ABSTRACT Streptococcus mutans organisms are occasionally isolated from the blood of patients with infective endocarditis, though the mechanisms of invasion and survival remain to be elucidated. Two of four blood isolates from patients with bacteremia or infective endocarditis (strains TW295 and TW871) were serologically untypeable by immunodiffusion testing, which was due to a lack of the glucose side chain of the serotype-specific polysaccharide antigen of S. mutans. Immunodiffusion analyses using antisera against these strains demonstrated that 2 of 100 isolates from 100 subjects showed a positive reaction, while further analysis of 2,500 isolates from 50 subjects revealed that all 50 isolates from a single subject were not reactive with anti-c, -e, and -f antisera, though they were reactive with anti-TW295 and -TW871 antisera. The oral isolates showed biological properties similar to those of the reference S. mutans strain MT8148, including high levels of sucrose-dependent adhesion and cellular hydrophobicity, along with expression of glucosyltransferases and a protein antigen, PA. We designated these organisms serotype k. A glucose side chain-defective mutant strain was then constructed by insertional inactivation of the gluA gene of strain MT8148, which showed biological properties similar to those of serotype k of S. mutans. Serotype k oral isolates were less susceptible to phagocytosis, as were the gluA-inactivated mutant of strain MT8148 and blood isolates. These results indicate that S. mutans serotype k strains are present in the oral cavity in humans and may be able to survive longer in blood owing to their low susceptibility to phagocytosis.

Contributions of Three Glucosyltransferases to Sucrose-dependent Adherence of Streptococcus mutans

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:l 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.

Oolong Tea Polyphenols Inhibit Experimental Dental Caries in SPF Rats Infected with Mutans Streptococci

An extract of oolong tea (semifermented tea leaves of Camellia sinensis) and its chromatographically isolated polyphenolic compound was examined for in vitro inhibitory effects on glucosyltransferases (GTases) of mutans streptococci and on caries development in Sprague-Dawley rats infected with mutans streptococci. The samples showed no detectable effect on the growth of mutans streptococci. However, insoluble glucan synthesis from sucrose by the GTases of Streptococcus mutans MT8148R and Streptococcus sobrinus 6715 was markedly inhibited, as was sucrose-dependent cell adherence of these mutans streptococci. The administration of the oolong tea extract and the isolated polyphenol compound into diet 2000 and drinking water resulted in significant reductions in caries development and plaque accumulation in the rats infected with mutans streptococci. The active components in the oolong tea extract were presumptively identified as polymeric polyphenols which were specific for oolong tea leaves. These results indicate that the oolong tea polyphenolic compounds could be useful for controlling dental caries.

Virulence of Porphyromonas gingivalis is altered by substitution of fimbria gene with different genotype.

Porphyromonas gingivalis is a periodontal pathogen whose fimbriae are classified into six genotypes based on the diversity of the fimA genes encoding each fimbria subunit. It was suggested that P. gingivalis strains with type II fimbriae were more virulent than type I strains. For the present study, we generated the mutants in which fimA was substituted with different genotypes to study virulence of type II fimbriae. Using plasmid vectors, fimA of ATCC33277 (type I strain) was substituted with type II fimA, and that of OMZ314 (type II strain) with type I fimA. The substitution of type I fimA with type II enhanced bacterial adhesion/invasion to epithelial cells, whereas substitution with type I fimA resulted in diminished efficiency. Following bacterial invasion, type II clones swiftly degraded cellular paxillin and focal adhesion kinase, and inhibited cellular migration, whereas type I clones and ΔfimA mutants did not. BIAcore analysis demonstrated that type II fimbriae possess greater adhesive abilities for their receptor α5β1‐integrin than those of type I. In a mouse abscess model, the type II clones significantly induced serum IL‐1β and IL‐6, as well as other infectious symptoms. These results suggest that type II fimbriae are a critical determinant of P. gingivalis virulence.

Production and properties of bacteriocins (Mutacins) from Streptococcus Mutans

Studies using the stab culture technique determined that almost all of the reference Streptococcus mutans strains of serotypes c and e produced bacteriocins (mutacins) against more than one of the 10 indicator organisms which included strains of Strep. mutans, Strep. salivarius, Strep. sanguis and group A Strep. pyogenes. However, reference strains of Strep. mutans types a and d never elaborated mutacins against more than one indicator strain. Among 113 clinical isolates of Strep. mutans obtained from Japanese children, 84 strains (74 per cent) inhibited at least one of the indicator strains. Type c strains accounted for 85 of the 113 clinical isolates, and these strains produced mutacins more frequently than strains of other serotypes. Very few strains produced a cell-free form of mutacin in Trypticase Soy broth enriched with 2 per cent yeast extract. Mutacins were generally heat-stable, but some were destroyed by treatment with proteolytic and/or lipolytic enzymes. Part of the mutacin activity diffused through cellophane paper. These results suggest that mutacins contain at least 2 kinds of inhibitory substances ranging from low molecular weight materials to higher molecular weight, protein-lipid complexes. Mutacinogeny was not due to the accumulation of hydrogen peroxide. Production of mutacin was influenced markedly by the culture media employed.