Hideaki Mayanagi

Synergistic inhibition by combination of fluoride and xylitol on glycolysis by mutans streptococci and its biochemical mechanism

The purpose of this study was to evaluate the combined inhibitory effect of fluoride and xylitol on acid production by mutans streptococci, Streptococcus mutans NCTC10449 and Streptococcus sobrinus 6715, from glucose under strictly anaerobic conditions at fixed pH 5.5 and 7.0. The bacteria were grown in a tryptone-yeast extract broth under strictly anaerobic conditions (N2: 80%; H2: 10%; CO2: 10%). Reaction mixtures for acid production from glucose contained bacterial cells with fluoride (0–6.4 mM) and/or xylitol (60 mM). Acidic end products of glucose fermentation and intracellular glycolytic intermediates were assayed. The combination of fluoride and xylitol inhibited acid production more effectively than fluoride or xylitol alone. In the presence of fluoride and xylitol, the proportion of lactic acid in the total amount of acidic end products decreased, while the proportion of formic and acetic acids increased. Analyses of intracellular glycolytic intermediates revealed that xylitol inhibited the upper part of the glycolytic pathway, while fluoride inhibited the lower part. This study indicates that fluoride and xylitol together have synergistic inhibitory effects on the acid production of mutans streptococci and suggests that xylitol has the potential to enhance inhibitory effects of low concentrations of fluoride.

Distinct spatiotemporal expression of EFA6D, a guanine nucleotide exchange factor for ARF6, among the EFA6 family in mouse brain.

The EFA6 family is a member of guanine nucleotide exchange factors (GEFs) that can activate ARF6 specifically in vitro. In this study, we determined the complete primary sequence of mouse EFA6D encoding a protein of 1004 amino acids with a calculated molecular weight of 111,207 Da. In ARF pull-down assay, EFA6D showed a preferential GEF activity toward ARF6. RT-PCR analysis revealed the widespread tissue distribution of EFA6D and the high expression of EFA6A, C and D in the brain. In situ hybridization analysis demonstrated a distinct spatiotemporal expression pattern of EFA6D from those of EFA6A and C in mouse brain. Furthermore, immunoblot analysis revealed that EFA6D was highly concentrated in the postsynaptic density fraction. These findings suggest differential spatiotemporal regulation of ARF6 by three members of the EFA6 family in the brain.

Xylitol Inhibition of Acid Production and Growth of Mutans Streptococci in the Presence of Various Dietary Sugars under Strictly Anaerobic Conditions

The aim of this study was to investigate the inhibitory effect of xylitol on the growth of and acid production by mutans streptococci in the presence of various dietary sugars, and the relationship between the inhibition and the accumulation of xylitol 5-phosphate (X5P) under strictly anaerobic conditions like those in the deep layers of dental plaque. Xylitol retarded the growth of mutans streptococci in the presence of glucose (G), galactose (Gal), maltose (M), lactose (L) or sucrose (S) as an energy source, though the inhibition of growth on fructose (Fr) was small. Xylitol inhibited acid production by washed cells of Streptococcus mutans from G, Gal, M, L or S (12–83% inhibition). S. mutans accumulated X5P intracellularly through activity of the phosphoenolpyruvate-xylitol phosphotransferase system (PEP-xylitol PTS) when they fermented these sugars in the presence of xylitol. However, in the presence of Fr, no inhibition of acid production was observed. In addition, the amounts of X5P during the fermentation of Fr were smaller than those of other sugars in spite of the presence of PEP-xylitol PTS activity. These results suggest that along with the intracellular accumulation of X5P, xylitol decreases the growth and acid production of mutans streptococci in the presence of various dietary sugars except Fr.

Phenytoin-Induced Bone Loss and Its Prevention with Alfacalcidol or Calcitriol in Growing Rats

Studies were carried out to determine the effects and mechanism of action of phenytoin on the bone metabolism in male rats. Administration of phenytoin, 20 mg/kg/ day for 5 weeks, did not affect the growth curve. Biochemical data indicated that the serum osteocalcin, a marker of bone formation, was decreased significantly but there were no significant differences in the levels of serum calcium, pyridinoline, 25-hydroxyvitamin D3 (25OHD) and parathyroid hormone (PTH) in the phenytoin-treated group compared with the vehicle-treated group. The values of bone mineral density (BMD) were decreased in all regions of bones tested (mandibular head, tibial metaphysis, tibial diaphysis, femoral metaphysis, and femoral diaphysis) in the phenytoin-treated group. In histomorphometric analysis, phenytoin decreased trabecular bone volume and trabecular thickness, and increased osteoclast numbers per area of bone surface in the secondary trabecular bone of the tibia. Additionally, there was no significant difference in osteoid thickness. Combined administration of either alfacalcidol or calcitriol with phenytoin for 5 weeks prevented the reduction of BMD induced by phenytoin. From these findings, it is unlikely that toxic effects on the growth curve caused the decreased BMD induced by phenytoin. It is also evident that repeated administration of phenytoin may cause osteopenia which may be due to bone loss by inhibiting bone formation and/or by accelerating bone resorption rather than osteoid accumulation. The bone loss is not rachitic because of the lack of increase in osteoid thickness. Moreover, combined administration of alfacalcidol or calcitriol with phenytoin showed a preventative effect against bone loss. The bone loss induced by phenytoin in this study may be a convenient model for further research into the problem of drug-induced osteopenia.

Expression of versican and ADAMTS during rat tooth eruption.

SummaryA disintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS) is a family of extracellular proteases and implicated in cleaving proteoglycans, such as aggrecan, versican and brevican. No information is available about expression or localization of these ADAMTSs in teeth. Versican is a large chondroitin sulfate proteoglycan that is present in a variety of connective tissue including dental pulp, dentin, cementum and periodontal ligaments. The present study was designed to investigate expression of ADAMTSs and versican during rat tooth eruption. Rat maxillary first molars in weeks 1, 2, 3, 4 and 6 were examined. The mRNA expression of ADAMTS1, ADAMTS4, ADAMTS5 and versican was localized using in situ hybridization. ADAMTS1, ADAMTS4, ADAMTS5 and versican were expressed in dental pulp cells, odontoblasts, cementoblasts, cementocytes, periodontal ligament cells, osteoblasts and osteocytes. The temporal and spatial expression pattern in these cellular phenotypes was comparable among ADAMTSs and versican. The present study suggests that dental pulp cells, odontoblasts, cementoblasts, cementocytes, periodontal ligament cells, osteoblasts and osteocytes may be involved in both production and degradation of versican with secreting ADAMTS1, ADAMTS4 and ADAMTS5.

Elevation of histidine decarboxylase activity in the mandible of mice by Prevotella intermedia lipopolysaccharide and its augmentation by an aminobisphosphonate

Lipopolysaccharide (LPS) produced by Gram-negative bacteria is an important cause of inflammation. Aminobisphosphonates are potent inhibitors of bone resorption but have inflammatory side-effects. Here, the effects of LPS from Prevotella intermedia (a prevalent Gram-negative bacterium both in periodontitis and endodontal infections) and alendronate (an aminobisphosphonate) on the activity of the histamine-forming enzyme, histidine decarboxylase (HDC), were examined in mouse mandible. Intravenous injection of P. intermedia LPS increased HDC activity in the mandible, maximal activity being induced within 3-6 h of the injection. The elevation of HDC activity was dependent on the dose of LPS, 10 microg/kg (0.25 microg/mouse) producing a significant elevation in enzyme activity. Intraperitoneal injection of alendronate (40 micromol/kg) also produced an increase in HDC activity. Moreover, the elevation of HDC activity induced by P. intermedia LPS was markedly augmented in mice given alendronate 3 days before the LPS injection. These results (i) suggest that P. intermedia LPS may stimulate the synthesis of histamine in the mandible and that the newly formed histamine may make at least some contribution to the development of inflammation (apical periodontitis and/or osteomyelitis); (ii) should encourage the clinical testing of antihistaminergic agents against inflammation; and (iii) confirm that care needs to be taken when administering aminobisphosphonates to patients.

Acid diffusion through extracellular polysaccharides produced by various mutants of Streptococcus mutans

Mutants of Streptococcus mutans V403, constructed by allelic exchange and altered in their capacity to produce enzymes involved in the production of extracellular polysaccharides from sucrose, were used to study the role of glucans and fructans in the diffusion of ions through cell concentrates. A 4.0mm diameter, 0.75 mm deep diffusion chamber with an ion-sensitive field-effect transistor electrode positioned at the base was used to monitor the diffusion of hydronium ions from a sodium lactate buffer using cell concentrates prepared from bacteria grown in various concentrations of sucrose and glucose. The wild-type strain V403 produced at least seven times as much water-insoluble glucan (ISG) from sucrose as mutants deficient in various combinations of glucosyltransferase B (GTF B), GTF C, GTF D and fructosyltransferase. The fastest diffusion of hydronium ions occurred with sucrose-grown cell concentrates of strain V403, and the time of diffusion to the bottom of the chamber was approximately 2.3 times longer when this strain was grown in glucose. The speed of diffusion with glucose-grown V403 was similar to that obtained with each of the mutants. When cells of strain V403 grown in sucrose and glucose were mixed, increases in diffusion speed were found to be directly related to the proportion of sucrose-grown cells. The mixing of ISG with several strains of S. mutans revealed that increases in diffusion speed were directly related to the quantity of ISG added.

The MMP activity in developing rat molar roots and incisors demonstrated by in situ zymography

Matrix metalloproteinases (MMPs) have been expressed during root development and periodontal tissue formation, whereas it is not known if these MMP molecules are enzymatically active to degrade the extracellular matrices (ECMs). The present study was designed to investigate the gelatinolytic and collagenolytic activity in rat molar root and incisor development. Three-week old rat mandibles were frozen and cut without fixation or decalcification and processed for in situ zymography using substrates gelatin and collagen. The enzymatic activity was assessed according to the intensity of fluorescence due to the lysis of the substrates. Odontoblasts, predentin, cementum, bone and the enamel matrix showed the high activity. The present study demonstrated MMP activity in calcified tissues using in situ zymography for the first time and the possible involvement of the MMP activity in molar root and incisor development and periodontal tissue formation.

Quantitative detection of Streptococcus mutans in the dental plaque of Japanese preschool children by real-time PCR

Aims:  To detect quantitatively the total bacteria and Streptococcus mutans in dental plaque by real‐time PCR with prbac, Sm and GTF‐B primers, and to compare their presence with the prevalence of dental caries in Japanese preschool children.

Inhibition of inflammatory and bone-resorption-inhibitory effects of alendronate by etidronate.

Among the bisphosphonates (BPs), the aminobisphosphonates (aminoBPs) have much stronger bone-resorption–inhibitory activities (BRIAs) than nonaminobisphosphonates (nonaminoBPs). However, aminoBPs have inflammatory effects. We previously reported that in mice: (i) all aminoBPs tested (10–40 μmol/kg) induced various inflammatory reactions (including induction of histidine decarboxylase), whereas clodronate (a non-aminoBP) (10–160 μmol/kg) inhibited these reactions; and (ii) a clear sclerotic line (tentatively called the BP line) was detectable in the tibia by radiography a few weeks after a single injection of either alendronate (a typical aminoBP) (1.6 μmol/kg) or clodronate (160 μmol/kg), and this BP-line formation (a marker for the BRIAs of BPs) was not reduced in mice given both alendronate and clodronate. In this study, using this murine model, we compared clodronate, etidronate (another typical non-aminoBP), alendronate, etidronate + alendronate, and clodronate + alendronate in terms of their inflammatory effects and/or BP-line formation. For BP-line formation, 480 μmol/kg etidronate was needed (single injection). At 160 μmol/kg, etidronate inhibited the histidine decarboxylase induction, but not the other inflammatory reactions induced by alendronate. However, etidronate (unlike clodronate) also inhibited alendronate-induced BP-line formation (even at 40 μmol/kg). Etidronate (160 μmol/kg) also inhibited the physicochemical changes in the tibia induced by six, weekly injections of alendronate. Therefore, depending on the dose, etidronate can inhibit alendronate’s inflammatory actions and its BRIA. These results, together with those reported previously, suggest that a strategy utilizing clodronate (but not etidronate) plus an aminoBP might prevent or reduce the inflammatory side effects induced by aminoBPs while preserving their powerful BRIAs. We discuss the mechanisms underlying the antagonism between aminoBPs and non-aminoBPs.