Biomedical subjects

Yoshio Nakano

Publications and source records attributed to Yoshio Nakano.

At least 37 records · Page 2Linked to original sources

PubMed2003

Outer membrane protein 100, a versatile virulence factor of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans (Aa) is one of the pathogenic bacteria involved in periodontal diseases. We have previously identified six major outer membrane proteins (Omps) of Aa Y4. Among them is an Omp with high molecular mass, designated Omp100, which has homology to a variety of virulence factors. Electron microscopic observation indicated that Omp100 is randomly localized on the cell surface of Aa. Aa Y4 has been shown to adhere and invade KB or normal human gingival keratinocytes. Anti-Omp100 antibody inhibited 50% of adhesion and 70% of invasion of Aa Y4 to KB cells. An Omp100 knock-out mutant had a decreased adhesion and invasion efficiency of 60%, compared with that of the wild type. Escherichia coli HB101 expressing Omp100 adhered twofold and invaded 10-fold more than the wild-type E. coli HB101. HB101 expressing Omp100 showed resistance to serum by trapping factor H, an inhibitor for C3b, with Omp100. Omp100 induced inflammatory cytokine responses of interleukin (IL)-8, IL-6 and tumour necrosis factor (TNF)alpha in epithelial cells, and induced IL-1beta and TNFalpha production in mouse macrophages. These results indicate that Omp100 is a versatile virulence factor that may demonstrate potential significance in the onset of periodontal diseases related to Aa.

Actinobacillus Infections↗

PubMed2003

Development of a 5' fluorogenic nuclease-based real-time PCR assay for quantitative detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis.

A 5' nuclease TaqMan PCR was developed for the quantitative detection of the periodontopathic bacteria Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. The relative numbers of bacteria were measured by the comparative threshold cycle method. This simplified method is a way of obtaining the relative quantities of these organisms from specimens and of monitoring the effect of therapy.

Aggregatibacter actinomycetemcomitans↗

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

Development of a 5' nuclease-based real-time PCR assay for quantitative detection of cariogenic dental pathogens Streptococcus mutans and Streptococcus sobrinus.

A 5' nuclease TaqMan PCR assay was developed for the quantitative detection of the major cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. The absolute and relative numbers of bacteria were measured by this method. This assay will be useful for quantifying these organisms in oral specimens and for analyzing biofilm formation.

Biofilms↗

PubMed2003

Removal of lead from aqueous solutions by condensed tannin gel adsorbent.

Lead has caused serious environmental pollution due to its toxicity, accumulation in food chains and persistence in nature. In this paper, lead removal from aqueous solutions was investigated using condensed tannin gel adsorbent synthesized from a natural tannin compound. It is found that the adsorption is strongly affected by pH values of aqueous solutions. Within pH range of 3.5-6, when initial lead concentration is 100 mg/L, removal efficiency is more than 90%. Adsorption equilibrium is reached within 150 minutes. The adsorption isotherm fits well with the Langmuir equation, by which the saturated adsorption uptake of 190 mg Pri2+ /g dry tannin gel adsorbent is obtained. By means of thermodynanamics analysis, it is revealed that the process is exothermic and the adsorption heat is up to 38.4 kJ/mol. With respect to high efficiency, moderate pH requirement and minimized second pollution, the tannin gel adsorbent exhibits a promising potential in the removal of lead from wastewater.

Adsorption↗

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

T cell-independent regulation of IgE antibody production induced by surface-linked liposomal antigen.

Control of IgE Ab production is important for the prevention of IgE-related diseases. However, in contrast to the existing information on the induction of IgE production, little is known about the regulation of the production of this isotype, with the exception of the well-documented mechanism involving T cell subsets and their cytokine products. In this study, we demonstrate an alternative approach to interfere with the production of IgE, independent of the activity of T cells, which was discovered during the course of an investigation intended to clarify the mechanism of IgE-selective unresponsiveness induced by surface-coupled liposomal Ags. Immunization of mice with OVA-liposome conjugates induced IgE-selective unresponsiveness without apparent Th1 polarization. Neither IL-12, IL-10, nor CD8(+) T cells participated in the regulation. Furthermore, CD4(+) T cells of mice immunized with OVA-liposome were capable of inducing Ag-specific IgE synthesis in athymic nude mice immunized with alum-adsorbed OVA. In contrast, immunization of the recipient mice with OVA-liposome did not induce anti-OVA IgE production, even when CD4(+) T cells of mice immunized with alum-adsorbed OVA were transferred. In the secondary immune response, OVA-liposome enhanced anti-OVA IgG Ab production, but it did not enhance ongoing IgE production, suggesting that the IgE-selective unresponsiveness induced by the liposomal Ag involved direct effects on IgE, but not IgG switching in vivo. These results suggest the existence of an alternative mechanism not involving T cells in the regulation of IgE synthesis.

Adjuvants, Immunologic↗

PubMed2002

Cloning and characterization of the L-cysteine desulfhydrase gene of Fusobacterium nucleatum.

Hydrogen sulfide and methyl mercaptan are the two major compounds associated with oral malodor. These compounds are highly toxic, and are thought to play an important role in periodontal disease. Fusobacterium nucleatum, an oral bacterium, produces large amounts of hydrogen sulfide from L-cysteine by the enzymatic action of L-cysteine desulfhydrase. We cloned and sequenced the cdl gene encoding L-cysteine desulfhydrase from F. nucleatum ATCC 10953, and revealed that the structural cdl gene consists of 921 bp and encodes a 33.4-kDa protein. The cloned gene was inserted into an expression vector, pDEST17, and expressed in Escherichia coli as a fused protein. The purified enzyme was tested for substrate specificity using various SH-containing compounds. Only L-cysteine served as a substrate for L-cysteine desulfhydrase to produce hydrogen sulfide.

Amino Acid Sequence↗

PubMed2002

3-Chloro-DL-alanine resistance by L-methionine-alpha-deamino-gamma-mercaptomethane-lyase activity.

The antibacterial agent 3-chloro-DL-alanine (3CA) is an inhibitor of peptidoglycan synthesis. Fusobacterium nucleatum and Porphyromonas gingivalis, the bacteria responsible for oral malodor, are shown to be resistant to 1 mM 3CA, whereas Streptococcus mutans and Escherichia coli are sensitive to this antibacterial agent at the same concentration. We isolated the 3CA resistance gene from F. nucleatum and showed that the gene encodes an L-methionine-alpha-deamino-gamma-mercaptomethane-lyase that catalyzes the alpha,gamma-elimination of L-methionine to produce methyl mercaptan. The enzyme also exhibits 3CA chloride-lyase (deaminating) activity. This antibacterial agent is expected to be useful for specific selection of malodorous oral bacteria producing high amounts of methyl mercaptan.

Anti-Bacterial Agents↗

PubMed2002

Immunization against dental caries.

Dental caries is one of the most common infectious diseases. Of the oral bacteria, mutans streptococci, such as Streptococcus mutans and S. sobrinus, are considered to be causative agents of dental caries in humans. There have been numerous studies of the immunology of mutans streptococci. To control dental caries, dental caries vaccines have been produced using various cell-surface antigens of these organisms. Progress in recombinant DNA technology and peptide synthesis has been applied to the development of recombinant and synthetic peptide vaccines to control dental caries. Significant protective effects against dental caries have been shown in experimental animals, such as mice, rats and monkeys, which have been subcutaneously, orally, or intranasally immunized with these antigens. Only a few studies, however, have examined the efficacy of dental caries vaccines in humans. Recently, local passive immunization using murine monoclonal antibodies, transgenic plant antibodies, egg-yolk antibodies, and bovine milk antibodies to antigens of mutans streptococci have been used to control the colonization of the organisms and the induction of dental caries in human. Such immunization procedures may be a safer approach for controlling human dental caries than active immunization.

Animals↗

PubMed2002

L-Methionine-gamma-lyase, as a target to inhibit malodorous bacterial growth by trifluoromethionine.

Methyl mercaptan is a major component responsible for oral malodor. This compound arises from the bacterial metabolism of methionine. Here we show that the growth of Porphyromonas gingivalis, a periodontal microorganism that produces large amounts of methyl mercaptan, was strongly inhibited by l-trifluoromethionine (TFM), a fluorinated derivative of methionine. In contrast, TFM had no effect on the growth of bacteria which do not produce methyl mercaptan. In addition, the survival rate of P. gingivalis-infected mice was remarkably increased by the co-injection of TFM. These results suggest that TFM is a promising antibacterial agent specific to the malodorous oral bacteria.

Animals↗

PubMed2002

Removal of phosphate by layered double hydroxides containing iron.

Iron-based layered double hydroxides (M2+(a)Fe3+(b) (OH)2(a+b) CO3(2-) b/2mH2O) were synthesized. Removal of phosphate by the compounds was studied from the viewpoint of buffering pH effect of the compounds and buffering capacity of solution. The compounds released metal cations (Mg2+, Ca2 , Fe3+) and/or their hydroxides responding to various water environments due to their buffering pH function. The released cations and/or hydroxides worked effectively as coagulants for the phosphate removal. The removal of phosphate depended on the buffering capacity of the solution that is the function of the solution pH and the concentration of phosphate. The removal of phosphate from the solution with small buffering capacity followed a Langmuir-type isotherm. The removal of phosphate from the solution with larger buffering capacity was largely increased. The removal of phosphate by the compounds was analyzed based on the model describing the buffering pH effect of the compounds from which the amount of released cations (CB) can be determined. The removal was well correlated with the amount of dissolution of the compounds, CB. The mechanism of phosphate removal was examined based on the removal efficiency (mol of removed phosphate/mol of released alkali). The efficiencies showed below one in the solution with large buffering capacity and above one in the solution with small buffering capacity. The efficiency below one showed the removal of phosphate through coagulation by the released metal cations and hydroxides. The successful removal of dilute phosphate (0.2mg P/l) from the drain water was also demonstrated.

Cations↗

PubMed2002

Acute toxicity of chlorophenols to earthworms using a simple paper contact method and comparison with toxicities to fresh water organisms.

An acute toxicity test of chlorophenols on earthworms (Eisenia fetida) was performed using a simple paper contact method proposed by OECD testing guideline no. 207, that were applied as an earthworm toxicity test. The median lethal concentration, EC50, had significant correlation with logP(ow) (1-octanol/water partition coefficient) of the chemicals. The toxicity of chlorophenols on E. fetida was compared with toxicities for other species: an algae (Selenostrum capricornutum), a crustacean (Daphnia magna), and a fish (Oryzias latipes). It was found that the toxicity of chlorophenols was almost same for E. fetida and for fresh water organisms. These results suggest the possibility of drawing correlations between the effects of pollutants on living things in different environments, fresh water and soil.

Animals↗

PubMed2002

Correlation between oral malodor and periodontal bacteria.

Volatile sulfur compounds (VSCs), including hydrogen sulfide, methyl mercaptan, and dimethyl sulfide, are primarily responsible for oral malodor. Recently, the mgl gene encoding L-methionine-alpha-deamino-gamma-mercaptomethane-lyase, which produces methyl mercaptan, was cloned from Porphyromonas gingivalis. This article discusses the mechanism and pathogenic role of the formation of VSCs by oral bacteria.

Adolescent↗

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

lcd from Streptococcus anginosus encodes a C-S lyase with alpha,beta-elimination activity that degrades L-cysteine.

Hydrogen sulfide is highly toxic to mammalian cells. It has also been postulated that hydrogen sulfide modifies haemoglobin resulting in haemolysis. The enzyme that produces hydrogen sulfide from L-cysteine was purified from Streptococcus anginosus. Using the N-terminal amino acid sequence of the purified enzyme, the lcd gene encoding L-cysteine desulfhydrase was cloned; the recombinant protein was then purified to examine its enzymic and biological characteristics. This L-cysteine desulfhydrase had the Michaelis-Menten kinetics K(m)=0.62 mM and V(max)=163 micro mol min(-1) mg(-1). DL-Cystathionine, L-cystine, S-(2-aminoethyl)-L-cysteine, 3-chloro-DL-alanine and S-methyl-L-cysteine were substrates for the enzyme, whereas D-cysteine, DL-homocysteine, L-methionine, DL-serine, DL-alanine, L-cysteine methyl ester, L-tryptophan, L-tyrosine and L-phenylalanine were not. These findings suggest that this L-cysteine desulfhydrase is a C-S lyase that catalyses the alpha,beta-elimination (alphaC-N and betaC-S) reaction. In addition, it is demonstrated that the hydrogen sulfide produced by this enzyme caused the modification and release of haemoglobin in sheep erythrocytes.

Amino Acid Sequence↗

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↗

PubMed2002

Expression and characterization of streptococcal rgp genes required for rhamnan synthesis in Escherichia coli.

Six genes (rgpA through rgpF) that were involved in assembling the rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans were previously identified (Y. Yamashita, Y. Tsukioka, K. Tomihisa, Y. Nakano, and T. Koga, J. Bacteriol. 180:5803-5807, 1998). The group-specific antigens of Lancefield group A, C, and E streptococci and the polysaccharide antigen of Streptococcus sobrinus have the same rhamnan backbone as the RGP of S. mutans. Escherichia coli harboring plasmid pRGP1 containing all six rgp genes did not synthesize complete RGP. However, E. coli carrying a plasmid with all of the rgp genes except for rgpE synthesized the rhamnan backbone of RGP without glucose side chains, suggesting that in addition to rgpE, another gene is required for glucose side-chain formation. Synthesis of the rhamnan backbone in E. coli required the initiation of transfer of N-acetylglucosamine to a lipid carrier and the expression of the rgpC and rgpD genes encoding the putative ABC transporter specific for RGP. The similarities in RGP synthesis between E. coli and S. mutans suggest common pathways for rhamnan synthesis. Therefore, we evaluated the rhamnosyl polymerization process in E. coli by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the lipooligosaccharide (LOS). An E. coli transformant harboring rgpA produced the LOS modified by the addition of a single rhamnose residue. Furthermore, the rgpA, rgpB, and rgpF genes of pRGP1 were independently mutated by an internal deletion, and the LOS chemotypes of their transformants were examined. The transformant with an rgpA deletion showed the same LOS profile as E. coli without a plasmid. The transformant with an rgpB deletion showed the same LOS profile as E. coli harboring rgpA alone. The transformant with an rgpF deletion showed the LOS band with the most retarded migration. On the basis of these results, we speculated that RgpA, RgpB, and RgpF, in that order, function in rhamnan polymerization.

Acetylglucosamine↗