Abiotrophia defectiva adhere to saliva-coated hydroxyapatite beads via interactions between salivary proline-rich-proteins and bacterial glyceraldehyde-3-phosphate dehydrogenase.

Abiotrophia defectiva is a species of nutritionally variant streptococci that is found in human saliva and dental plaques and that has been associated with infective endocarditis. In our previous study, it was found that A. defectiva could bind specifically to saliva-coated hydroxyapatite beads (SHA). This study identified a cell surface component of A. defectiva that promotes adherence to SHA beads. The binding of A. defectiva to SHA was reduced in the presence of antibodies against human proline-rich protein (PRP); these results suggested that PRP may be a critical component mediating interactions between A. defectiva and the salivary pellicle. Two-dimensional gel electrophoresis of whole A. defectiva cells followed by Far-Western blotting was conducted by probing with synthetic peptides analogous to the binding region of PRP known as PRP-C. The results indicate that an A. defectiva protein of 37 kDa interacts with PRP-C. The results of amino-terminal sequencing of the adhesive A. defectiva protein revealed significant similarity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Recombinant GAPDH bound to immobilized PRP-C in a dose-dependent manner and binding of A. defectiva to SHA or to PRP was reduced in the presence of anti-GAPDH antiserum. Western blotting or electron immunomicroscopic observations with anti-GAPDH antiserum revealed that this protein was expressed in both cytosolic and cell wall fractions. These results suggest that A. defectiva could specifically bind to PRP via interactions with cell surface GAPDH; the findings suggest a mechanism underlying A. defectiva-mediated adherence to saliva-coated tooth surfaces.

Tyrosine tRNA synthetase as a novel extracellular immunomodulatory protein in Streptococcus anginosus.

Streptococcus anginosus is frequently detected in patients with infective endocarditis, abscesses or oral cancer. Although S. anginosus is considered the causative pathogen of these diseases, the pathogenic mechanisms of the bacterium have remained unclear. Previously, we suggested that an extracellular antigen from S. anginosus (SAA) serves as a pathogenic factor by inducing nitric oxide production in murine macrophages. In the present study, we identified SAA using LC-MS/MS and assessed the biological activities of His-tagged recombinant SAA in murine macrophages. SAA was identified as a tyrosine tRNA synthetase (SaTyrRS) that was isolated from the extracellular fraction of S. anginosus but not from other oral streptococci. In addition, inducible nitric oxide synthase and TNF-α mRNA expression was induced in recombinant SaTyrRS-stimulated murine macrophages. However, their mRNA expression was not induced in macrophages stimulated with truncated or heat-inactivated recombinant SaTyrRS, and the activation motif was identified as Arg264-Thr270. Consequently, these results indicated that SaTyrRS could be a novel and specific immunomodulatory protein in S. anginosus.

Distribution of dipeptidyl peptidase (DPP) 4, DPP5, DPP7 and DPP11 in human oral microbiota-potent biomarkers indicating presence of periodontopathic bacteria.

Dipeptidyl peptidase (DPP) 4, DPP5, DPP7 and DPP11, expressed in the periplasmic space, are crucial for energy production for Porphyromonas gingivalis, an asaccharolytic bacterium that causes periodontal disease. Bacterial DPP4 seems to be involved in regulation of blood glucose level via degradation of incretins. The present study aimed to identify four dpp orthologs in oral microbiota by database searches, and their enzymatic activities in periodontopathic and cariogenic bacteria, as well as oral specimens were determined. Search in the databases suggested that 43 species of 772 taxa possess dpp4 and other dpp genes. Most species are in the genera Bacteroides, Capnocytophaga, Porphyromonas, Prevotella and Tannerella, indicating a limited distribution of dpp orthologs in anaerobic periodontopathic rods. In accordance with those results, activities of all four DPPs were demonstrated in P. gingivalis, Porphyromonas endodontalis and Tannerella forsythia, while they were negligible in Treponema denticola, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans. Furthermore, DPP activities were also detected in subgingival dental plaque at different intensities among individual specimens, while DPP4 activity presumably derived from human entity was solely predominant in saliva samples. These findings demonstrated that DPP activities in dental plaque serve as potent biomarkers to indicate the presence of periodontopathic bacteria.

The fibronectin-binding protein homologue Fbp62 of Streptococcus anginosus is a potent virulence factor.

Streptococcus anginosus appears to be able to adhere to cultured epithelial cells or fibronectin and this may be associated with bacterial pathogenicity. In the present study, the molecular characteristics and virulence of the fibronectin-binding protein (FBP), Fbp62, of S. anginosus were investigated in animal models to determine the role of the molecule in bacterial infection. fbp62 encodes a 549 amino acid residue with an apparent molecular mass of 62.8 kDa that lacks a membrane anchor motif and a leader peptide, suggesting that fbp62 codes for an atypical FBP. It has been observed that the S. anginosus Fbp62 is very similar to the FbpA of Streptococcus gordonii, PavA of Streptococcus pneumoniae, SmFnB of Streptococcus mutans and Fbp54 of Streptococcus pyogenes. Recombinant Fbp62 prepared from pGEX-4T-2 was found to bind to fibronectin in a dose-dependent manner and competitively inhibit the binding of S. anginosus to fibronectin. Furthermore, anti-Fbp62 antiserum abrogated the binding of S. anginosus to fibronectin. Adhesion of the isogenic mutant, Δfbp62, constructed from S. anginosus NCTC 10713 (wild-type, WT) by homologous recombination to HEp-2 cells and DOK cells was significantly weaker than that of S. anginosus WT. In addition, Δfbp62's lethality and ability to form abscesses were weaker in a mouse model of infection than in the WT strain. Taken together, these results suggest that Fbp62 is an important pathogenic factor of S. anginosus.

Aciduricity and acid tolerance mechanisms of Streptococcus anginosus.

Although Streptococcus anginosus constitutes a proportion of the normal flora of the gastrointestinal and genital tracts, and the oral cavity, it has been reported that S. anginosus infection could be closely associated with abscesses at various body sites, infective endocarditis, and upper gastrointestinal cancers. The colonization in an acidic environment due to the aciduricity of S. anginosus could be the etiology of the systemic infection of the bacteria. To elucidate the aciduricity and acid tolerance mechanisms of the microbe, we examined the viability and growth of S. anginosus under acidic conditions. The viabilities of S. anginosus NCTC 10713 and Streptococcus mutans ATCC 25175 at pH 4.0 showed as being markedly higher than those of Streptococcus sanguinis ATCC 10556, Streptococcus gordonii ATCC 10558, and Streptococcus mitis ATCC 49456; however, the viability was partially inhibited by dicyclohexylcarbodiimide, an H+-ATPase inhibitor, suggesting that H+-ATPase could play a role in the viability of S. anginosus under acidic conditions. In addition, S. anginosus NCTC 10713 could grow at pH 5.0 and showed a marked arginine deiminase (ADI) activity, unlike its ΔarcA mutant, deficient in the gene encoding ADI, and other streptococcal species, which indicated that ADI could also be associated with aciduricity. These results suggest that S. anginosus has significant aciduric properties, which can be attributed to these enzyme activities.

Degradation of Incretins and Modulation of Blood Glucose Levels by Periodontopathic Bacterial Dipeptidyl Peptidase 4.

Severe periodontitis is known to aggravate diabetes mellitus, though molecular events related to that link have not been fully elucidated. Porphyromonas gingivalis, a major pathogen of periodontitis, expresses dipeptidyl peptidase 4 (DPP4), which is involved in regulation of blood glucose levels by cleaving incretins in humans. We examined the enzymatic characteristics of DPP4 from P. gingivalis as well as two other periodontopathic bacteria, Tannerella forsythia and Prevotella intermedia, and determined whether it is capable of regulating blood glucose levels. Cell-associated DPP4 activity was found in those microorganisms, which was effectively suppressed by inhibitors of human DPP4, and molecules sized 73 kDa in P. gingivalis, and 71 kDa in T. forsythia and P. intermedia were immunologically detected. The kcat/Km values of recombinant DPP4s ranged from 721 ± 55 to 1,283 ± 23 µM-1s-1 toward Gly-Pro-4-methylcoumaryl-7-amide (MCA), while those were much lower for His-Ala-MCA. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis showed His/Tyr-Ala dipeptide release from the N termini of incretins, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide, respectively, with the action of microbial DPP4. Moreover, intravenous injection of DPP4 into mice decreased plasma active GLP-1 and insulin levels, accompanied by a substantial elevation in blood glucose over the control after oral glucose administration. These results are the first to show that periodontopathic bacterial DPP4 is capable of modulating blood glucose levels the same as mammalian DPP4; thus, the incidence of periodontopathic bacteremia may exacerbate diabetes mellitus via molecular events of bacterial DPP4 activities.

Prediction of periodontopathic bacteria in dental plaque of periodontal healthy subjects by measurement of volatile sulfur compounds in mouth air.

OBJECTIVES: The aim of this study was to determine whether measurements of volatile sulfur compounds (VSCs) are useful to predict colonization of periodontopathic bacteria. For this purpose, we assessed the relationships among distributions of 4 species of periodontopathic bacteria in tongue coating and dental plaque, oral conditions including VSC concentration in mouth air, and smoking habit of periodontal healthy young subjects. METHODS: The subjects were 108 young adults (mean age, 23.5±2.56 years) without clinical periodontal pockets. Information regarding smoking habit was obtained by interview. After VSC concentration in mouth, air was measured with a portable sulfide monitor (Halimeter(®)), non-stimulated saliva flow and dental caries status were assessed, and tongue coating and dental plaque samples were collected from the subjects. The tongue coating samples were weighed to determine the amount. The colonization of Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Treponema denticola in both tongue coating and plaque samples was investigated using species-specific polymerase chain reaction assays. RESULTS: Significant relationships were observed between the colonization of periodontopathic bacteria in tongue coating and plaque samples, especially that of P. gingivalis. VSC concentration showed the most significant association with colonization of P. gingivalis in both tongue coating and dental plaque. Logistic regression analysis demonstrated that the adjusted partial correlation coefficient [Exp(B)] values for VSC concentration with the colonization of P. gingivalis, P. intermedia, and T. denticola in dental plaque were 135, 35.4 and 10.4, respectively. In addition, smoking habit was also shown to be a significant variable in regression models [Exp(B)=6.19, 8.92 and 2.53, respectively]. Therefore, receiver operating characteristic analysis was performed to predict the colonization of periodontal bacteria in dental plaque in the subjects divided by smoking habit. Based on our results, we found cut-off values that indicated likelihood ratios (LR) within the efficient range for positive findings in both groups. CONCLUSION: The present results demonstrated that measurement of VSC concentration in mouth air is a useful method to predict the presence of colonization of some periodontopathic bacteria in dental plaque.

Reconsideration of the Iwasaki-Waggener iterative perturbation method for reconstructing high-energy X-ray spectra.

We have reviewed applicable ranges for attenuating media and off-axis distances regarding the high-energy X-ray spectra reconstructed via the Iwasaki-Waggener iterative perturbation method for 4-20 MV X-ray beams. Sets of in-air relative transmission data used for reconstruction of spectra were calculated for low- and high-Z attenuators (acrylic and lead, respectively) by use of a functional spectral formula. More accurate sets of spectra could be reconstructed by dividing the off-axis distances of R = 0-20 cm into two series of R = 0-10 cm and R = 10-20 cm, and by taking into account the radiation attenuation and scatter in the buildup cap of the dosimeter. We also incorporated in the reconstructed spectra an adjustment factor (f (adjust) ≈ 1) that is determined by the attenuating medium, the acceleration voltage, and the set of off-axis distances. This resulted in calculated in-air relative transmission data to within ±2 % deviation for the low-Z attenuators water, acrylic, and aluminum (Al) with 0-50 cm thicknesses and R = 0-20 cm; data to within ±3 % deviation were obtained for high-Z attenuators such as iron (Fe), copper (Cu), silver (Ag), tungsten (W), platinum (Pt), gold (Au), lead (Pb), thorium (Th), and uranium (U) having thicknesses of 0-10 cm and R = 0-20 cm. By taking into account the radiation attenuation and scatter in the buildup cap, we could analyze the in-air chamber response along a line perpendicular to the isocenter axis.

A convolution/superposition method using primary and scatter dose kernels formed for energy bins of X-ray spectra reconstructed as a function of off-axis distance: comparison of calculated and measured 10-MV X-ray doses in thorax-like phantoms.

We performed experimental studies on the convolution/superposition method reported in the former companion paper (Iwasaki in Radiol Phys Technol 4, 2011) using 10-MV X-ray beams from open-jaw-collimated fields. The method uses primary and scatter dose kernels formed for energy bins of X-ray spectra reconstructed as a function of off-axis distance. We made a comparison of calculations and measurements in water phantoms and thorax-like phantoms with respect to percentage depth dose curves, tissue-phantom ratio curves, and dose profiles. We made the dose calculation by taking into account the beam-hardening effect with depth and the off-axis radiation-softening effect. We found that the method could be used, in general, for performing accurate dose calculations.

A convolution/superposition method using primary and scatter dose kernels formed for energy bins of X-ray spectra reconstructed as a function of off-axis distance: a theoretical study on 10-MV X-ray dose calculations in thorax-like phantoms.

A convolution/superposition method is proposed for use with primary and scatter dose kernels formed for energy bins of X-ray spectra reconstructed as a function of off-axis distance. It should be noted that the number of energy bins is usually about ten, and that the reconstructed X-ray spectra can reasonably be applied to media with a wide range of effective Z numbers, ranging from water to lead. The study was carried out for 10-MV X-ray doses in water and thorax-like phantoms with the use of open-jaw-collimated fields. The dose calculations were made separately for primary, scatter, and electron contamination dose components, for which we used two extended radiation sources: one was on the X-ray target and the other on the flattening filter. To calculate the in-air beam intensities at points on the isocenter plane for a given jaw-collimated field, we introduced an in-air output factor (OPF(in-air)) expressed as the product of the off-center jaw-collimator scatter factor (off-center S (c)), the source off-center ratio factor (OCR(source)), and the jaw-collimator radiation reflection factor (RRF(c)). For more accurate dose calculations, we introduce an electron spread fluctuation factor (F (fwd)) to take into account the angular and spatial spread fluctuation for electrons traveling through different media.

Proteome analyses of human macrophages exposed to low cytotoxic IC90 copper (2+) ions.

Dental noble alloys often contain copper (Cu). Eluted metal ions sometimes irritate oral tissues. The most eluted ions are Cu ions. The purpose of this study was to investigate the effects of low cytotoxic (IC90, 100 µmol/L) Cu ions on macrophages by proteome analyses consisting of two-dimensional (2D) electrophoresis and matrix-assisted laser desorption/ionization -time of flight (MALDI-TOF) mass spectrometry. The analyses revealed that stimulation with IC90 Cu ions for 1 day caused the macrophage to significantly increase five specific protein spots. Mascot peptide mass finger-print matching suggested that four of them were attributed to 70 kDa heat shock protein 1A/1B (HSP70). HSP70 expression was verified by expressions of corresponding HSPA1A and HSPA1B mRNAs of the macrophage in quantitative real-time PCR analyses. It was concluded that by producing abundant HSP70, the macrophage protected itself against intracellularly intruding cytotoxic Cu ions that might un-fold and crosslink cellular proteins.

Microscopic observations and inflammatory cytokine productions of human macrophage phagocytising submicron titanium particles.

This study was performed to microscopically observe and measure inflammatory cytokine production by human macrophages phagocytosing submicron titanium (Ti) particles. Observations with secondary electron microscopy (SEM), SEM/electron probe microanalysis (EPMA) and transmission electron microscopy (TEM) indicated that macrophages [phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells] at 24 h in culture actively phagocytosed and accumulated submicron Ti particles in intracellular phagosomes, in which refinement of Ti particles occurred. The macrophages were also cultured for 24 h in four media with and without submicron Ti particles and lipopolysaccharide (LPS; components of bacteria). Whilst neither stimulus reduced cell viability, submicron Ti particles and LPS activation independently and synergistically caused the macrophages to produce three inflammatory cytokines (TNF-alpha, IL-1beta and IL-6) at high levels in the culture supernatants. The inflammatory and osteolysis conditions caused by macrophages phagocytosing submicron Ti particles would be worsened by challenge with LPS in patients wearing Ti prostheses.

Gene expression analyses of human macrophage phagocytizing sub-micro titanium particles by allergy DNA chip (Genopal).

The purpose of this study was to examine gene expressions of macrophage phagocytizing sub-micro Ti particles by a DNA chip. Human monocytic cell line THP-1 was differentiated into macrophages by culturing for two days in medium supplemented with 200 nM phorbol ester (PMA). The macrophages were then cultured in four media: medium without PMA (control); medium with suspended sub-micro Ti particles (0.5 wt%); medium with 1.0 microg/ml lipopolysaccharide (LPS); and medium with LPS and Ti particles. After 6 hours' culture, total RNA were extracted and gene expressions were evaluated by DNA allergy chip with 205 allergy and inflammation related gene spots. We found that phagocytosis of sub-micro Ti particles and LPS independently and synergistically up-regulated 17 inflammation-related genes more than two-fold. The extensive expressions of four genes (CCL1, IL1B, IL6 and IL8) were further confirmed by real-time quantitative PCR. It turned out that dual stimulation of LPS and Ti particles most up-regulated three genes (IL1B, IL6 and IL8), followed by LPS while Ti particles moderately but least increased, suggesting that phagocytosis of sub-micro Ti particles induces moderate inflammation with its degree less than LPS, but phagocytosis of sub-micro Ti particles has the potential to worsen inflammation caused by LPS-stimulated macrophages.

Dose-dependent effects of Ni (II) ions on production of three inflammatory cytokines (TNF-alpha, IL-1beta and IL-6), superoxide dismutase (SOD) and free radical NO by murine macrophage-like RAW264 cells with or without LPS-stimulation.

The effect of Ni (II) ions on macrophages is not well understood. The purpose of this study was to examine the dose-dependent effects of Ni (II) ions up to 1,000 micromol/L on production of three inflammatory cytokines (TNF-alpha, IL-1beta and IL-6), superoxide dismutase (SOD) and nitric oxide (NO) by murine macrophage-like RAW264 cells with (+) or without (-) lipopolysaccharide (LPS) -stimulation. Ni (II) ions caused LPS (-) RAW264 cells to slightly increase production of TNF-alpha and IL-6, proportionally to the Ni (II) ion concentration while IL-1beta was not produced, and to slightly increase production of SOD and NO. It can be concluded that Ni (II) ions dose-dependently increased the inflammatory and oxidative stress conditions of LPS (-) RAW264 cells. LPS-stimulation caused RAW264 cells to produce in abundance the three inflammatory cytokines, SOD and NO. Ni (II) ions dose-dependently reduced the three cytokine quantities and NO amounts in LPS (+) RAW264 cells, while dose-independently increasing SOD amounts. It was noted that Ni (II) ions dose-dependently reduce the resistance power against bacteria of LPS (+) macrophages, because the production of volatile NO--bacteria killer is diminished proportionally to the Ni (II) ion concentration.

Accumulation of element Ti in macrophage-like RAW264 cells cultured in medium with 1 ppm Ti and effects on cell viability, SOD production and TNF-alpha secretion.

The adverse effect of Ti on body-defense macrophage is not well understood. The aims of this study were twofold: (1) to examine the intracellular accumulation of Ti element; and (2) to measure the cell viability, superoxide dismutase (SOD) production, and TNF-alpha secretion of macrophage-like RAW264 cells cultured for two days in medium with 1 ppm Ti prepared from acidic ICP Ti standard solution. PIXE analysis showed that element Ti was accumulated up to 7.3 ppm in RAW264 cells when cultured in the medium with 1 ppm Ti. Further, RAW264 cells cultured in the medium with 1 ppm Ti exhibited cell viability of about 60%, SOD production of about 180%, and TNF-alpha secretion of about 170% relative to those of control cells cultured in the medium without Ti. It was speculated that phagocytosis of minute Ti-containing complex (mostly TiO2) by macrophage caused oxidative stress and inflammatory reaction, leading to cell proliferation arrest and increased production of SOD and TNF-alpha.

Effects of Ni2+ ions on cell viability and NO production of murine peritoneal exudate cells (macrophages) with and without lipopolysaccharide stimulation.

The purpose of this study was to clarify the cytotoxicity of Ni2+ ions against murine peritoneal exudate cells (PEC) (macrophages). First, we examined the cell viability of PEC with and without lipopolysaccharide (LPS) stimulation in culture media containing Ni2+ ions up to 1000 micromol/L. Results showed that the cytotoxicity of Ni2+ ions against PEC was dose-dependent and accelerated by LPS stimulation, especially in media with Ni2+ ions exceeding 100 micromol/L. Second, we measured the production of nitric oxide (NO) from PEC and found that LPS caused the PEC to produce abundant NO. However, high dose of Ni2+ ions at concentration more than 200 micromol/L hindered and inhibited NO production. These results pointed out that the cytotoxicity of Ni2+ ions against macrophages depended on both the Ni2+ ion concentration and the presence of bacteria with LPS. Further, NO--a killer of bacteria--was lost when LPS-stimulated macrophages were exposed to high dose of Ni2+ ions.

Tolerance of the Rieske-type [2Fe-2S] cluster in recombinant ferredoxin BphA3 from Pseudomonas sp. KKS102 to histidine ligand mutations.

BphA3 from Pseudomonas sp. KKS102 is a Rieske-type [2Fe-2S] ferredoxin that transfers electrons from an NADH-dependent oxidoreductase, BphA4, to a biphenyl dioxygenase complex. A high-level expression and purification system for the recombinant BphA3 in Escherichia coli was constructed. Two histidine ligands of the Rieske-type cluster in BphA3, were each replaced with serine, cysteine, asparagine and tyrosine. The single mutants, in which either His44 or His65 was replaced with a cysteine residue (CH and HC mutants respectively), and the double mutant, in which both histidine residues were replaced with cysteine residue (CC mutant), accumulated to high levels in the E. coli cells, while the other single mutants did not. The purified WT (wild-type) protein showed characteristic near-UV and visible absorption and CD spectra of Rieske-type clusters. The X-ray absorption spectra were suggestive of the existence of [2Fe-2S] clusters, with one histidine and three cysteine ligands in the CH and HC mutants, and an [2Fe-2S] cluster with four cysteine ligands in the CC mutant. The BphA4-dependent cytochrome c reductase activities of the mutants were less than 0.3% of that of the WT protein. The redox potential of the WT protein determined by cyclic voltammetry was -180+/-5 mV compared with the standard hydrogen electrode, and that of the CH mutant was approx. 175 mV lower. The changes in the near-UV and visible absorption spectra of the mutants showed that the reduced iron-sulphur clusters in the mutants were unstable. His44 and His65 in BphA3 can be replaced with cysteine residues, but are required for the stabilization of the reduced form of the cluster.

Characterization and molecular cloning of a glutamyl endopeptidase from Staphylococcus epidermidis.

A novel extracellular endopeptidase, designated GluSE, was purified from Staphylococcus epidermidis ATCC 14990 cultured by the dialysis membrane technique, and the structural gene (gseA) was cloned. GluSE was a 27kDa, glutamic acid-specific protease, and the optimal pH was 8.0. The proteolytic activity was specifically inhibited with diisopropyl fluorophosphate, indicating that it is a serine protease. The gseA encoded a single polypeptide of 282 amino acids with a deduced molecular weight of 30,809, in which the first 19 N-terminal amino acids completely matched the deduced sequence starting at Val-67, suggesting that GluSE is synthesized with a propeptide. The amino acid sequence of GluSE exhibited 50.5% identity to Staphylococcus aureus V8-protease (GluV8). Although GluSE lacks a C-terminal 12 repeats of the PBN/PBZ tripeptide of GluV8, a catalytic triad of His-117, Asp-159 and Ser-235 was conserved in GluSE. Southern hybridization analysis revealed that gseA exists as a single copy on the chromosomal DNA. The finding that production of GluSE was obviously observed in the adherent culture conditions of the dialysis membrane technique, but not in the planktonic culture conditions, strongly suggests that GluSE could be involved in an important etiologic process in S.epidermidis infection leading to multiple tissue damages.