Biomedical subjects

Yoshihisa Yamashita

Publications and source records attributed to Yoshihisa Yamashita.

At least 19 recordsLinked to original sources

PubMed2006

Adiponectin inhibits osteoclast formation stimulated by lipopolysaccharide from Actinobacillus actinomycetemcomitans.

Previous epidemiologic studies have suggested that periodontal disease is closely related to obesity and glucose tolerance. As the level of adiponectin, an adipocyte-derived cytokine, in plasma had been reported to decrease in obese and type 2 diabetes patients, we explored the role of adiponectin in the etiology of periodontitis using the D clone of RAW264, a clone that exhibits highly efficient osteoclast formation, to determine whether adiponectin acts as a regulatory molecule in osteoclast formation stimulated by lipopolysaccharide of periodontopathic bacteria. We observed that adiponectin acted as a potent inhibitor of osteoclast formation stimulated by Toll-like receptor 4 (TLR4) ligand and receptor activator of NF-kappaB ligand (RANKL). Because NF-kappaB is an important transcription factor in osteoclast formation, we examined the effect of adiponectin on its transcriptional activity. A luciferase assay showed that adiponectin was able to inhibit the TLR4-mediated NF-kappaB activity in RAW264 cells. In addition, we observed that the cytokine was actually able to inhibit TLR4-mediated expression of the gene for inducible nitric oxide synthase and production of nitric oxide in the cells. These observations strongly suggest that adiponectin may function as a negative regulator of lipopolysaccharide/RANKL-mediated osteoclast formation in periodontal disease.

Actinobacillus Infections↗

PubMed2006

TRFMA: a web-based tool for terminal restriction fragment length polymorphism analysis based on molecular weight.

UNLABELLED: TRFMA provides a Web environment for analyzing T-RFLP results based on molecular weights of the fragments, rather than the numbers of nucleotides, to increase accuracy. The 16S rRNA data are saved as an XML file containing around 650 sequences (light version) and a MySQL database containing around 50 000 sequences (full version), which are connected to Web server via PHP5 and manipulated on an Internet browser. AVAILABILITY: TRFMA is freely available at http://myamagu.dent.kyushu-u.ac.jp/bioinformatics/trfma/index.html and can be downloaded from the same site.

Algorithms↗

PubMed2006

The influence of current and former smoking on gingival bleeding: the Hisayama study.

BACKGROUND: Previous studies have shown that smoking is a risk factor for periodontitis and that it has a suppressive effect on gingival bleeding. This study examined the relationship between smoking, including past smoking, and periodontal conditions, mainly gingival bleeding, in a community-based health investigation. METHODS: Smoking status was examined in 958 subjects, along with the quantity of tobacco currently or previously smoked (never, former light, former heavy, current light, and current heavy). We analyzed the influence of smoking on probing depth (PD), clinical attachment loss (CAL), and gingival bleeding on probing (BOP). RESULTS: In multivariate logistic regression analyses, current heavy smokers were at a significantly greater risk for having a higher proportion of teeth with PD > or =4 mm and a higher proportion of teeth with CAL > or =5 mm; however, they had a lower risk for having a high BOP than did those who had never smoked. Moreover, both former light and former heavy smokers had significantly lower risks for high BOP. When the analysis was limited to subjects with PD > or =4 mm, former heavy and current heavy smokers showed a significant suppression of high BOP compared to never smokers. CONCLUSION: This study suggests that smoking is significantly associated with PD and CAL and that current and past smoking has a suppressive effect on BOP.

Adult↗

PubMed2005

Adiponectin inhibits Toll-like receptor family-induced signaling.

Recent studies have shown that adiponectin, an adipocyte-derived cytokine, acts as a potent inhibitor of inflammatory responses. It has been also demonstrated that bacterial and viral signalings in host cells are triggered via Toll-like receptor (TLR) molecules. Therefore, in the present study, we investigated whether globular adiponectin (gAd) would be able to inhibit TLR-mediated nuclear factor-kappaB (NF-kappaB) signaling in mouse macrophages (RAW264). gAd predominantly bound to the AdipoR1 receptor and suppressed TLR-mediated NF-kappaB signaling. gAd-mediated inhibition of TLR-mediated IkappaB phosphorylation and NF-kappaB activation was eliminated by the pretreatment of cycloheximide. Also their inhibitions of gAd were blocked by preincubation of the cells with an antibody against AdipoR1, but not with an antibody against AdipoR2. Taken together, these findings indicate that adiponectin negatively regulates macrophage-like cell response to TLR ligands via an unknown endogenous product(s).

3T3-L1 Cells↗

PubMed2005

High production of methyl mercaptan by L-methionine-alpha-deamino-gamma-mercaptomethane lyase from Treponema denticola.

Methyl mercaptan is derived from l-methionine by the action of l-methionine-alpha-deamino-gamma-mercaptomethane lyase (METase) and is a major component of oral malodor. This compound is highly toxic and is thought to play an important role in periodontal disease. We found that Treponema denticola, a member of the subgingival biofilm at periodontal disease sites, produced a large amount of methyl mercaptan even at low concentration of l-methionine. METase activity in a cell-free extract from T. denticola was detected by two-dimensional electrophoresis under non-denaturing conditions, and the protein spot that exhibited high METase activity was identified using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The identified gene produced a METase with a K(m) value for l-methionine (0.55mM) that is much lower than those of METases previously identified in the other organisms. This result suggests that T. denticola is an important producer of methyl mercaptan in the subgingival biofilm.

Bacterial Proteins↗

PubMed2005

Decreasing caries prevalence in Japanese preschool children is accompanied with a reduction in mutans streptococci infection.

OBJECTIVE: To evaluate the association between decreasing caries prevalence in Japanese preschool children and mutans streptococci (MS) infection. METHODS: The prevalence of caries and salivary MS was examined in 1- to 3-year-old preschool children in 1995 (n = 135) and 2000 (n = 118). RESULTS: The caries prevalence for 3-year-old subjects significantly declined from 65.1% to 37.5% (p < 0.01), reflecting the data seen in Japanese national oral health surveys in 1993 and 1999. Salivary MS detection for 3-year-old subjects significantly declined from 68.3% to 37.5% (p < 0.05). On the other hand, the decrease in the prevalence of caries and salivary MS was not significant in 1- and 2-year-olds. The correlation between caries experience (dft) and the salivary MS score for 3-year-old subjects with low caries prevalence in the later examination (r(s) = 0.479, p < 0.001) was comparable with that in 1995 (r(s) = 0.462, p = 0.001). CONCLUSIONS: The recent decrease in decay in Japan is accompanied with a reduction in MS infection in the deciduous dentition. MS levels are closely related to caries prevalence in preschool children regardless of the extent of caries.

Child, Preschool↗

PubMed2005

Identification and characterization of an autolysin-encoding gene of Streptococcus mutans.

We identified a gene (atlA) encoding autolytic activity from Streptococcus mutans Xc. The AtlA protein predicted to be encoded by atlA is composed of 979 amino acids with a molecular weight of 107,279 and has a conserved beta-1,4-N-acetylmuramidase (lysozyme) domain in the C-terminal portion. Sodium dodecyl sulfate extracts of strain Xc showed two major bacteriolytic bands with molecular masses of 107 and 79 kDa, both of which were absent from a mutant with inactivated atlA. Western blot analysis revealed that the 79-kDa band was derived from the 107-kDa peptide by cleavage of its N-terminal portion. The inactivation of atlA resulted in a marked decrease in autolysis and the formation of very long chains of cells compared to the case for the parent strain. Although both the parent and mutant strains formed biofilms in the presence of sucrose, the biofilms formed by the mutant had a sponge-like architecture with large gaps and contained 30% less biomass than those formed by the parent strain. Furthermore, strain Xc formed glucose-dependent, loose biofilms in the absence of sucrose, but the mutant lost this ability. These results suggest that AtlA may play an important role in biofilm formation by S. mutans. The antibody produced against the C-terminal peptide containing the beta-1,4-N-acetylmuramidase domain drastically inhibited the autolytic activity of strain Xc. This inhibition was specific among the oral streptococci to S. mutans. These results indicate that the catalytic domain of AtlA is located at the C terminus, suggesting that further characterization of this domain may provide a means to control cariogenic dental plaque formation.

Base Sequence↗

PubMed2005

Loop-mediated isothermal amplification method targeting the lytA gene for detection of Streptococcus pneumoniae.

It is difficult to separate Streptococcus pneumoniae from the genotypically similar species Streptococcus mitis and Streptococcus oralis, which are commensals of the human oral cavity. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63 degrees C) with high specificity, efficiency, and rapidity, was examined regarding its applicability for detecting S. pneumoniae. An S. pneumoniae-specific LAMP primer targeting the lytA gene was designed. The primer specificity was validated using 10 Streptococcus and 7 non-Streptococcus species. Within 60 min, the assay could detect 10 or more copies of purified S. pneumoniae DNA with a sensitivity 1,000 times that of conventional PCR. Clinical isolates of 21 other strains (3 S. oralis, 17 S. mitis, and 1 Streptococcus species) that harbor virulence-factor-encoding genes (lytA or ply) were tried to differentiate S. pneumoniae. The detection of S. pneumoniae in clinical isolates was more selective using the LAMP method than using conventional PCR. Therefore, LAMP appears to be a sensitive and reliable means of diagnosing S. pneumoniae infection.

Bacterial Typing Techniques↗

PubMed2005

Discrimination of Streptococcus pneumoniae from viridans group streptococci by genomic subtractive hybridization.

Two oligonucleotide primer sets for the discrimination of Streptococcus pneumoniae from "pneumococcus-like" oral streptococcal isolates by PCR were developed. Genomic subtractive hybridization was performed to search for differences between Streptococcus pneumoniae strain WU2 and the most closely related oral streptococcus, Streptococcus mitis strain 903. We identified 19 clones that contained S. pneumoniae-specific nucleotide fragments that were absent from the chromosomal DNA of typical laboratory strains of S. mitis and other oral bacteria. Subsequently, oligonucleotide PCR primers for the detection of S. pneumoniae were designed from the sequences of the subtracted DNA fragments, and the specificities of the 19 primer sets were evaluated by PCR using chromosomal DNAs extracted from four S. pneumoniae clinical isolates and from 20 atypical organisms classified as S. mitis or S. oralis, which harbored genes encoding the pneumococcal virulence factors autolysin (lytA) or pneumolysin (ply), as templates. Of the 19 primer sets, two (Spn9802 and Spn9828) did not amplify PCR products from any of the pneumococcus-like streptococcal strains that we examined. The genes containing the Spn9802 and Spn9828 sequences encoded proteins of unknown function that did not correspond to any previously described proteins in other bacteria. These new oligonucleotide primers may be very useful for early and correct diagnosis of S. pneumoniae infections.

Bacterial Proteins↗

PubMed2005

Relationship between drinking and periodontitis: the Hisayama Study.

BACKGROUND: Although recent studies suggest a relationship between alcohol consumption and periodontal disease, the dose-response relationship between drinking and the severity of periodontitis is unclear. METHODS: Alcohol consumption was evaluated using the frequency of drinking and the daily alcohol intake for 961 individuals aged 40 to 79 years. Periodontal status was evaluated using probing depth (PD) and clinical attachment loss (CAL). RESULTS: Alcohol consumption was linearly associated with the extent of PD and CAL in univariate analyses (P <0.001). In multivariate logistic regression analyses, the subjects drinking 15 to 29.9 g alcohol per day (odds ratio [OR] = 2.7; 95% confidence interval [CI] = 1.1 to 6.6) or more than 30 g per day (OR = 2.5; 95% CI = 1.1 to 5.7) had a significantly higher risk of having more than 35% of their teeth with PD > or = 4 mm than non-drinkers, independent of other confounding variables. No significant relationship between drinking and CAL was observed in the multivariate analysis. CONCLUSION: These results suggest that the effect of drinking on periodontal condition is limited to subjects with deep periodontal pockets associated with more than one-third of their teeth.

Adult↗

PubMed2004

Real-time TaqMan PCR for quantifying oral bacteria during biofilm formation.

A TaqMan PCR was developed for quantifying early colonizer microorganisms in dental biofilms. To design species-specific primers and TaqMan probes, genomic subtractive hybridization was used. This quantitative assay in combination with subtractive hybridization may be of value in the study of microbial ecosystems consisting of related species that are involved in the formation and etiology of biofilms.

Actinomyces↗

PubMed2003

Selection of hemoperfusion therapy for patients with septic shock on the basis of the primary disease.

The objective of this study was to analyze retrospectively the efficacy of polymyxin-B immobilized fiber (PMX-F) alone and in combination with continuous venovenous hemofiltration (CHF) on the prognosis of critically ill patients with sepsis using a retrospective chart review in a university hospital in Japan. A cohort of 246 patients meeting the criteria of sepsis, septic shock, or both, according to the American College of Chest Physicians/Society of Critical Care Medicine (ACCP/ACCM) Consensus Conference, were examined in this study. From these patients, 48 were selected who were found to have definitive causative bacteria and whose primary diseases were clearly identified. According to the charts, two major primary diseases were identified: one related to cardiovascular disease and the other to gastrointestinal disease. Other diseases were excluded from this study because of the small numbers of patients in categories such as malignant, hematological, genitourinary, and other diseases. Furthermore, patients who had levels of serum creatinine above 2.0 mg/dl were excluded. The prevalence of diabetes mellitus (up to 63%) was very high in both groups. There were no significant differences between the two groups in age or the Apache II scores at the start of hemoperfusion treatment; however, the gender ratio varied: 72% of the cardiovascular group were male, compared to 46% of the gastrointestinal group. The causative bacteria were markedly different between the two groups. For half of the gastrointestinal group the causative bacterium was Escherichia coli, while for half of the cardiovascular group the causative bacterium was Pseudomonas aeruginosa. The survival rate differed significantly between the two groups. The patients in the cardiovascular group survived longer than those in the gastrointestinal group. Moreover, for the patients with cardiovascular disease, there was no significant difference in the survival rate between treatment with PMX-F alone and with PMX-F and CHF in combination. In contrast, for the patients with gastrointestinal disease, there was a significant difference between treatment with PMX-F alone and with PMX-F and CHF in combination. When a patient with sepsis or septic shock is treated with hemoperfusion, the decision as to whether PMX-F should be given alone or in combination with CHF might be determined on the basis of the primary disease of the patient.

APACHE↗

PubMed2003

Insertional inactivation of pac and rmlB genes reduces the release of tumor necrosis factor alpha, interleukin-6, and interleukin-8 induced by Streptococcus mutans in monocytic, dental pulp, and periodontal ligament cells.

Streptococcus mutans possesses different cell wall molecules, such as protein of the I/II family, the serotype f polysaccharide rhamnose glucose polymer (RGP), and lipoteichoic acid (LTA), which act as adhesins and modulins, allowing S. mutans to colonize teeth and cause dental caries and pulpitis. We tested several isogenic mutants of S. mutans defective in protein I/II and/or RGP, as well as purified modulins such as protein I/II, RGP, and LTA, for their binding and activation abilities on monocytic, dental pulp (DP), and periodontal ligament (PDL) cells. Our results demonstrate that both protein I/II and RGP play important roles in streptococcal adherence to human monocytic and fibroblastic cells, whereas LTA is only a minor adhesin. In the activation process, the cytokine response elicited is polarized toward a Th1 response which seems principally due to protein I/II and RGP. Even if protein I/II seems to be more efficient in its purified form in triggering cells to release interleukin-8 (IL-8), RGP is the most efficient cytokine-stimulating component in intact bacteria, while LTA plays only a minor role. In cell activation, we showed, by using either cytochalasin D or coated ligands, that internalization of either S. mutans, S. mutans isogenic mutants, or purified ligands is not necessary to trigger cells to release IL-8. We also showed that, besides the implication of monocytes in pulpal inflammation, fibroblast-like cells such as DP and PDL cells are also actively implicated in local inflammation and in the generation of a Th1 response after stimulation with S. mutans cells or antigens.

Adhesins, Bacterial↗

PubMed2003

Analysis of loci required for determination of serotype antigenicity in Streptococcus mutans and its clinical utilization.

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 serotype-specific 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.

Child, Preschool↗

PubMed2003

Does a clinical evaluation of oral cleanliness correlate with caries incidence in preschool children? Findings from a cohort study.

The aim of this study was to assess whether a clinical evaluation of oral cleanliness reflects subsequent caries incidence. Oral examination of 180 children (1- to 4-year-olds) was carried out twice in a six-month period. Caries prevalence at baseline (dfs) in 1- to 2-year-olds (group A) and 3- to 4-year-olds (group B) correlated significantly with oral cleanliness as well as salivary mutans streptococci count (MS). Caries increment (delta(dfs)) correlated significantly with oral cleanliness in group A but not in group B, while delta(dfs) significantly correlated with MS in group B and slightly correlated with that in group A. ANCOVA revealed that dfs was significantly higher at the second examination than at baseline in group B, even after adjusting for oral cleanliness. This finding was confirmed by Wilcoxon test when group B was divided in three categories (low, middle and high) based on oral cleanliness. This suggests that the relationship between delta(dfs) and oral cleanliness decreased with age and that the significant positive correlation found in group B by our point-prevalence survey is derived from the remainder of the positive correlation that occurred at a younger age.

Age Factors↗

PubMed2002

A novel mechanism for glucose side-chain formation in rhamnose-glucose polysaccharide synthesis.

We have cloned two genes (rgpH and rgpI) that encode proteins for the formation of the glucose side-chains of the Streptococcus mutans rhamnose-glucose polysaccharide (RGP), which consists of a rhamnan backbone with glucose side-chains. The roles of rgpH and rgpI were evaluated in a rhamnan-synthesizing Escherichia coli. An E. coli strain that harbored rgpH reacted with antiserum directed against complete RGP, whereas the E. coli strain that carried rgpI did not react with this antiserum. Although E. coli:rgpH reacted strongly with rhamnan-specific antiserum, co-transformation of this strain with rgpI increased the number of glucose side-chains and decreased immunoreactivity with the rhamnan-specific antiserum significantly. These results suggest that two genes are involved in side-chain formation during S. mutans RGP synthesis in E. coli: one gene encodes a glucosyltransferase, and the other gene probably controls the frequency of branching. This is the first report to identify a gene that is involved in regulation of branching frequency in polysaccharide synthesis.

Bacterial Proteins↗

PubMed2002

Guanosine diphosphate-4-keto-6-deoxy-d-mannose reductase in the pathway for the synthesis of GDP-6-deoxy-d-talose in Actinobacillus actinomycetemcomitans.

The serotype a-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans is an unusual sugar, 6-deoxy-d-talose. Guanosine diphosphate (GDP)-6-deoxy-d-talose is the activated sugar nucleotide form of 6-deoxy-d-talose, which has been identified as a constituent of only a few microbial polysaccharides. In this paper, we identify two genes encoding GDP-6-deoxy-d-talose synthetic enzymes, GDP-alpha-d-mannose 4,6-dehydratase and GDP-4-keto-6-deoxy-d-mannose reductase, in the gene cluster required for the biosynthesis of serotype a-specific polysaccharide antigen from A. actinomycetemcomitans SUNYaB 75. Both gene products were produced and purified from Escherichia coli transformed with plasmids containing these genes. Their enzymatic reactants were analysed by reversed-phase HPLC (RP-HPLC). The sugar nucleotide produced from GDP-alpha-d-mannose by these enzymes was purified by RP-HPLC and identified by electrospray ionization-MS, 1H nuclear magnetic resonance, and GC/MS. The results indicated that GDP-6-deoxy-d-talose is produced from GDP-alpha-d-mannose. This paper is the first report on the GDP-6-deoxy-d-talose biosynthetic pathway and the role of GDP-4-keto-6-deoxy-d-mannose reductase in the synthesis of GDP-6-deoxy-d-talose.

Actinobacillus↗

PubMed2002

Genes involved in bacitracin resistance in Streptococcus mutans.

Streptococcus mutans is resistant to bacitracin, which is a peptide antibiotic produced by certain species of Bacillus. The purpose of this study was to clarify the bacitracin resistance mechanism of S. mutans. We cloned and sequenced two S. mutans loci that are involved in bacitracin resistance. The rgp locus, which is located downstream from rmlD, contains six rgp genes (rgpA to rgpF) that are involved in rhamnose-glucose polysaccharide (RGP) synthesis in S. mutans. The inactivation of RGP synthesis in S. mutans resulted in an approximately fivefold-higher sensitivity to bacitracin relative to that observed for the wild-type strain Xc. The second bacitracin resistance locus comprised four mbr genes (mbrA, mbrB, mbrC, and mbrD) and was located immediately downstream from gtfC, which encodes the water-insoluble glucan-synthesizing enzyme. Although the bacitracin sensitivities of mutants that had defects in flanking genes were similar to that of the parental strain Xc, mutants that were defective in mbrA, mbrB, mbrC, or mbrD were about 100 to 120 times more sensitive to bacitracin than strain Xc. In addition, a mutant that was defective in all of the mbrABCD genes and rgpA was more sensitive to bacitracin than either the RGP or Mbr mutants. We conclude that RGP synthesis is related to bacitracin resistance in S. mutans and that the mbr genes modulate resistance to bacitracin via an unknown mechanism that is independent of RGP synthesis.

Anti-Bacterial Agents↗