Hiromasa Tsuda

Loop-Mediated Isothermal Amplification Method Targeting the lytA Gene for Detection of Streptococcus pneumoniae

ABSTRACT 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°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.

Characterization of NKT cells in human peripheral blood and decidual lymphocytes

PROBLEM: To examine whether natural killer (NKT) cells are present in human pregnancy decidua.
 METHOD OF STUDY: We calculated the percentage of CD3+CD161+Vα 24+‐NKT cells in peripheral blood and early pregnancy decidua, and analyzed intracellular cytokines, interleukin (IL)‐4 and interferon (IFN)γ in NKT cells using flow cytometry.
 RESULTS: A distinct subset of CD3+CD161+ lymphocytes expressing an invariant antigen receptor encoded by the Vα24 and Vβ11 segment was accumulated in the decidua. In pregnant subjects the percentages of NKT cells were significantly increased in the decidua compared with peripheral blood. Both NKT cells in the decidua and the peripheral blood had an ability to rapidly produce cytokine associated with Th1 (IFNγ) and Th2 (IL‐4). Interestingly, the percentages of IL‐4 and IFNγ producing NKT cells were significantly higher in the decidua compared with the peripheral blood.
 CONCLUSIONS: These findings suggest that NKT cells might control the Th1/Th2 balance by producing IL‐4 and IFNγ at the feto‐maternal interface.

Genes Involved in Bacitracin Resistance in Streptococcus mutans

ABSTRACT 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.

Role of serotype-specific polysaccharide in the resistance of Streptococcus mutans to phagocytosis by human polymorphonuclear leukocytes.

ABSTRACT To clarify the role of cell surface components ofStreptococcus mutans in resistance to phagocytosis by human polymorphonuclear leukocytes (PMNs), several isogenic mutants ofS. mutans defective in cell surface components were studied with a luminol-enhanced chemiluminescence (CL) assay, a killing assay, and a transmission electron microscope. The CL responses of human PMNs to mutant Xc11 defective in a major cell surface antigen, PAc, and mutant Xc16 defective in two surface glucosyltransferases (GTF-I and GTF-SI) were the same as the response to the wild-type strain, Xc. In contrast, mutant Xc24R, which was defective in serotype c-specific polysaccharide, induced a markedly higher CL response than the other strains. The killing assay showed that human PMNs killed more Xc24R than the parent strain and the other mutants. The transmission electron microscopic observation indicated that Xc24R cells were more internalized by human PMNs than the parental strain Xc. These results may be reflected by the fact that strain Xc24R was more phagocytosed than strain Xc. The CL response of human PMNs to a mutant defective in polysaccharide serotype e or f was similar to the response to Xc24R. Furthermore, mutants defective in serotype-specific polysaccharide were markedly more hydrophobic than the wild-type strains and the other mutants, suggesting that the hydrophilic nature of polysaccharides may protect the bacterium from phagocytosis. We conclude that the serotype-specific polysaccharide, but not the cell surface proteins on the cell surface of S. mutans, may play an important role in the resistance to phagocytosis.

Butyrate, a bacterial metabolite, induces apoptosis and autophagic cell death in gingival epithelial cells

BACKGROUND AND OBJECTIVE Butyrate is produced by some types of anaerobic periodontal bacteria. Millimolar concentrations of butyrate are found in mature dental plaque from periodontitis patients. Although butyrate reportedly has a variety of effects in many mammalian cells, its effect on gingival epithelial cells is not well known. In this study, we investigated the effect of butyrate on gingival epithelial Ca9-22 cell death. MATERIAL AND METHODS Death of Ca9-22 cells was assessed after treating the cells with or without butyrate. A SYTOX Green dye, which exhibits strong green fluorescence once it enters dead cells through ruptured cell membranes, was used for cell death detection. Phosphatidylserine redistribution was measured using fluorescein isothiocyanate-labeled annexin V. The activity of caspase-3 was measured as the amount of cleaved substrate peptide. Anti-apoptotic bcl-2 mRNA expression was measured using real-time RT-PCR. Western blotting and fluoromicroscopic analysis with anti-microtubule-associated protein 1 light chain 3 (LC3) antibodies were performed for detection of autophagy. RESULTS Stimulation with millimolar concentrations of butyrate for 48 h induced Ca9-22 cell death. The stimulation also caused increased caspase-3 activity, phosphatidylserine redistribution and bcl-2 down-regulation, suggesting butyrate-induced apoptosis. However, the pan-caspase inhibitor, Z-VAD-FMK, did not inhibit cell death completely. This implies the existence of other types of cell death. In addition, markers of autophagy, namely, the conversion of LC3-I to LC3-II and increased LC3 accumulation, were observed. Moreover, inhibition of autophagy by 3-methyladenine suppressed the butyrate-induced cell death, suggesting that butyrate could induce cell death through autophagy. CONCLUSION These data suggest that butyrate induces apoptosis and autophagic cell death.

N-ethylmaleimide-sensitive factor: a redox sensor in exocytosis.

Abstract Vascular injury triggers endothelial exocytosis of granules, releasing pro-inflammatory and pro-thrombotic mediators into the blood. Nitric oxide (NO) and reactive oxygen species (ROS) limit vascular inflammation and thrombosis by inhibiting endothelial exocytosis. NO decreases exocytosis by regulating the activity of the N-ethylmaleimide-sensitive factor (NSF), a central component of the exocytic machinery. NO nitrosylates specific cysteine residues of NSF, thereby inhibiting NSF disassembly of the soluble NSF attachment protein receptor (SNARE). NO also modulates exocytosis of other cells; for example, NO regulates platelet activation by inhibiting α-granule secretion from platelets. Other radicals besides NO can regulate exocytosis as well. For example, H2O2 inhibits exocytosis by oxidizing NSF. Using site-directed mutagenesis, we have defined the critical cysteine residues of NSF, and found that one particular cysteine residue, C264, renders NSF sensitive to oxidative stress. Since radicals such as NO and H2O2 inhibit NSF and decrease exocytosis, NSF may act as a redox sensor, modulating exocytosis in response to changes in oxidative stress.

A novel mechanism for glucose side‐chain formation in rhamnose‐glucose polysaccharide synthesis1

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.

Bacitracin upregulates mbrAB transcription via mbrCD to confer bacitracin resistance in Streptococcus mutans.

Abstract. Streptococcus mutans is a bacterial cause of dental caries that is resistant to bacitracin. The aim of this study was to elucidate the mbrABCD-related bacitracin resistance mechanism of S. mutans. Transcriptome data demonstrated that the expression levels of 33 genes were induced more than twofold by bacitracin. Fourteen genes were selected from the upregulated genes, and defective mutants of these genes were constructed for measurement of their sensitivity to bacitracin. Among the mutants, only the mbrA- or mbrB-deficient mutants exhibited 100- to 121-fold greater sensitivity to bacitracin when compared with the wild-type strain. Moreover, knockout of the mbrC and mbrD genes abolished the bacitracin-induced mbrAB upregulation. These results suggest that both mbrC and mbrD are required for mbrAB upregulation that confers the bacitracin-resistant phenotype on S. mutans. [Supplementary Table: available only at http://dx.doi.org/10.1254/jphs.11052SC]

Chaetocin inhibits RANKL-induced osteoclast differentiation through reduction of Blimp1 in Raw264.7 cells

AIMS Periodontitis is one of the most common bone-destructive diseases. Osteoclast is differentiated from hematopoietic macrophage-like cells through receptor activator of NFκB ligand (RANKL)-RANK signaling system, and the reduction in osteoclast formation may result in prevention of bone-resorptive diseases. Chaetocin is a compound isolated from fungal cultures and has been reported as a potent and selective inhibitor of suppressor of variegation 3-9 homolog 1 (Suv39h1), which catalyzes histone methylation on histone H3 lysine 9 (H3K9) residues. However, the effect of chaetocin on osteoclast differentiation is uncertain. In this study, we examine the effect of chaetocin on RANKL-induced osteoclast differentiation and cell growth. MAIN METHODS Mouse macrophage-like Raw264.7 cells were treated with RANKL in the presence or absence of chaetocin, and tartrate-resistant acid phosphatase (TRAP) staining was performed. Cell growth was measured as the amount of DNA stained with SYTOX Green dye. Expression and production of osteoclast differentiation markers, anti-osteoclastogenic genes, B lymphocyte-induced maturation protein-1 (Blimp1), and cell growth suppressors were examined by qRT-PCR or/and Western blot analysis. KEY FINDINGS Here we show that chaetocin dose-dependently reduced RANKL-induced osteoclast differentiation and cell growth via Blimp1 downregulation which results in the upregulation of osteoclast differentiation inhibitors and cell growth suppressors. These effects were not derived from the chaetocins inhibitory effect of Suv39h1. SIGNIFICANCE These results suggest that chaetocin suppresses RANKL-induced osteoclastogenesis and cell growth through blimp1 downregulation, followed by induction of anti-osteoclastogenic genes and cell growth suppressors, without inhibition of Suv39h1. Thus, chaetocin might be a drug candidate for the prevention of bone resorption in bone-destructive diseases.

Magnetic Resonance Imaging Finding in Divergence Paralysis

We report magnetic resonance imaging (MRI) findings in two patients with divergence paralysis that was secondary to multiple sclerosis and pontine infarction, respectively. In both patients, cranial MRI showed lesions in the unilateral tegmentum of the caudal pons. Therefore, these MRI findings suggested that involvement of the nucleus reticularis tegmenti pontis might be responsible for the divergence paralysis in our cases.