Effects of 7S globulin 3 derived from the adzuki bean [Vigna angularis] on the CSP- and eDNA- dependent biofilm formation of Streptococcus mutans.

OBJECTIVE: Streptococcus mutans is a principal bacterium that forms pathogenic biofilm involved in the development of dental caries. S. mutans possesses a quorum sensing system (QS) stimulated by competence stimulating peptide (CSP), which is associated with bacteriocin production, genetic competency and biofilm formation. Inhibiting CSP-dependent QS is one of the aims leading to the inhibition of biofilm formation and is useful for establishing new prevention systems for dental caries. DESIGN: In this study, we selected adzuki bean [Vigna angularis] extract as a candidate component to inhibit CSP-dependent biofilm formation among various foods. To purify an inhibitory component from the adzuki extracts, we performed the salting-out method, two rounds of ion-exchange chromatography, and SDS and native PAGE. RESULTS: A primary protein band that inhibits CSP-dependent biofilm formation appeared at approximately 50 kDa and was identified as 7S globulin 3 (7S3), a major seed storage protein in adzuki bean. To determine the characteristics of 7S3 as an inhibitory component, aggregated proteins were extracted from the adzuki crude extracts at pH values lower than 6. The aggregated proteins inhibited CSP- and eDNA-dependent biofilm formation and showed 50 kDa band, which is identical with 7S3 in the purified sample. Moreover, 7S globulin 3 in the adzuki bean extract directly interacted with CSP at low pH conditions but not at neutral conditions, and inhibited CSP-dependent bacteriocin production. CONCLUSION: It was suggested that 7S3 might be a safe and useful material to prevent pathogenic activities in the biofilm formation of S. mutans.

Rapid Screening of Primary Aldosteronism by a Novel Chemiluminescent Immunoassay.

Measurement of plasma aldosterone and renin concentration, or activity, is useful for selecting antihypertensive agents and detecting hyperaldosteronism in hypertensive patients. However, it takes several days to get results when measured by radioimmunoassay and development of more rapid assays has been long expected. We have developed chemiluminescent enzyme immunoassays enabling the simultaneous measurement of both aldosterone and renin concentrations in 10 minutes by a fully automated assay using antibody-immobilized magnetic particles with quick aggregation and dispersion. We performed clinical validation of diagnostic ability of this newly developed assay-based screening of 125 patients with primary aldosteronism from 97 patients with essential hypertension. Results of this novel assay significantly correlated with the results of radioimmunoassay (aldosterone, active renin concentration, and renin activity) and liquid chromatography-tandem mass spectrometry (aldosterone). The analytic sensitivity of this particularly novel active renin assay was 0.1 pg/mL, which was better than that of radioimmunoassay (2.0 pg/mL). The ratio of aldosterone-to-renin concentrations of 6.0 (ng/dL per pg/mL) provided 92.0% sensitivity and 76.3% specificity as a cutoff for differentiating primary aldosteronism from essential hypertension. This novel measurement is expected to be a clinically reliable alternative for conventional radioimmunoassay and to provide better throughput and cost effectiveness in diagnosis of hyperaldosteronism from larger numbers of hypertensive patients in clinical settings.

NADH oxidase and alkyl hydroperoxide reductase subunit C (peroxiredoxin) from Amphibacillus xylanus form an oligomeric assembly.

The NADH oxidase-peroxiredoxin (Prx) system of Amphibacillus xylanus reduces hydroperoxides with the highest turnover rate among the known hydroperoxide-scavenging enzymes. The high electron transfer rate suggests that there exists close interaction between NADH oxidase and Prx. Variant enzyme experiments indicated that the electrons from ß-NADH passed through the secondary disulfide, Cys128-Cys131, of NADH oxidase to finally reduce Prx. We previously reported that ionic strength is essential for a system to reduce hydroperoxides. In this study, we analyzed the effects of ammonium sulfate (AS) on the interaction between NADH oxidase and Prx by surface plasmon resonance analysis. The interaction between NADH oxidase and Prx was observed in the presence of AS. Dynamic light scattering assays were conducted while altering the concentration of AS and the ratio of NADH oxidase to Prx in the solutions. The results revealed that the two proteins formed a large oligomeric assembly, the size of which depended on the ionic strength of AS. The molecular mass of the assembly converged at approximately 300 kDa above 240 mM AS. The observed reduction rate of hydrogen peroxide also converged at the same concentration of AS, indicating that a complex formation is required for activation of the enzyme system. That the complex generation is dependent on ionic strength was confirmed by ultracentrifugal analysis, which resulted in a signal peak derived from a complex of NADH oxidase and Prx (300 mM AS, NADH oxidase: Prx = 1:10). The complex formation under this condition was also confirmed structurally by small-angle X-ray scattering.

Persistent colonization of Candida albicans yeast on the tongue in NOD/SCID.e2f1-/- mice.

Candida albicans is a commensal fungus that commonly colonizes as opportunistic pathogens human mucosal surfaces. Our aim was to observe persistent infection of C. albicans on the tongue in NOD/SCID.e2f1(-/-) mice, which naturally was decreased saliva and undeveloped T and B cells. Using a cotton swab, a C. albicans suspension was applied to the tongue of wild type and mutant mice after disinfection using 0.2% Chlorhexidine (CHX). In our earlier report, it was found that many times inoculation per day and consecutive day inoculations without disinfection of indigenous microorganisms did not induce significant C. albicans infection for 48 h in the oral cavity. In this study, using inoculation of four sets {one inoculation after disinfection by CHX + interval (3 or 4 d)} induced longer term and higher numbers infection for 4 days on the tongue than results in a previous report in both NOD/SCID.e2f1(+/+) and NOD/SCID.e2f1(-/-) mice. Repeat of disinfection to indigenous microorganisms and inoculation with interval established and realized a new model for persistent infection of C. albicans yeast. However, decreased saliva and consecutive inoculations per day did not contribute to the persistent colonization on the tongue in the mice. It is suggested that the interaction between C. albicans and indigenous microorganisms is important for persistent colonization of C. albicans yeast on the tongue rather than decreased saliva in the oral cavity.

Actinomyces naeslundii GroEL-dependent initial attachment and biofilm formation in a flow cell system.

Actinomyces naeslundii is an early colonizer with important roles in the development of the oral biofilm. The effects of butyric acid, one of short chain fatty acids in A. naeslundii biofilm formation was observed using a flow cell system with Tryptic soy broth without dextrose and with 0.25% sucrose (TSB sucrose). Significant biofilms were established involving live and dead cells in TSB sucrose with 60mM butyric acid but not in concentrations of 6, 30, 40, and 50mM. Biofilm formation failed in 60mM sodium butyrate but biofilm level in 60mM sodium butyrate (pH4.7) adjusted with hydrochloric acid as 60mM butyric media (pH4.7) was similar to biofilm levels in 60mM butyric acid. Therefore, butyric acid and low pH are required for significant biofilm formation in the flow cell. To determine the mechanism of biofilm formation, we investigated initial A. naeslundii colonization in various conditions and effects of anti-GroEL antibody. The initial colonization was observed in the 60mM butyric acid condition and anti-GroEL antibody inhibited the initial colonization. In conclusion, we established a new biofilm formation model in which butyric acid induces GroEL-dependent initial colonization of A. naeslundii resulting in significant biofilm formation in a flow system.

On the origin of near-infrared extragalactic background light anisotropy.

Extragalactic background light (EBL) anisotropy traces variations in the total production of photons over cosmic history and may contain faint, extended components missed in galaxy point-source surveys. Infrared EBL fluctuations have been attributed to primordial galaxies and black holes at the epoch of reionization (EOR) or, alternately, intrahalo light (IHL) from stars tidally stripped from their parent galaxies at low redshift. We report new EBL anisotropy measurements from a specialized sounding rocket experiment at 1.1 and 1.6 micrometers. The observed fluctuations exceed the amplitude from known galaxy populations, are inconsistent with EOR galaxies and black holes, and are largely explained by IHL emission. The measured fluctuations are associated with an EBL intensity that is comparable to the background from known galaxies measured through number counts and therefore a substantial contribution to the energy contained in photons in the cosmos.

Adaptive response of Amphibacillus xylanus to normal aerobic and forced oxidative stress conditions.

Amphibacillus xylanus grows at the same rate and with the same cell yield under aerobic and anaerobic conditions. Under aerobic conditions, it exhibits vigorous oxygen consumption in spite of lacking a respiratory system and haem catalase. To understand the adaptive response of A. xylanus to oxidative stresses, a genomic analysis of A. xylanus was conducted. The analysis showed that A. xylanus has the genes of four metabolic systems: two pyruvate metabolic pathways, a glycolytic metabolic pathway and an NADH oxidase (Nox)-AhpC (Prx) system. A transcriptional study confirmed that A. xylanus has these metabolic systems. Moreover, genomic analysis revealed the presence of two genes for NADH oxidase (nox1 and nox2), both of which were identified in the transcriptional analysis. The nox1 gene in A. xylanus was highly expressed under normal aerobic conditions but that of nox2 was not. A purification study of NADH oxidases indicated that the gene product of nox1 is a primary metabolic enzyme responsible for metabolism of both oxygen and reactive oxygen species. A. xylanus was successfully grown under forced oxidative stress conditions such as 0.1 mM H2O2, 0.3 mM paraquat and 80 % oxygen. Proteomic analysis revealed that manganese SOD, Prx, pyruvate dehydrogenase complex E1 and E3 components, and riboflavin synthase ß-chain are induced under normal aerobic conditions, and the other proteins except the five aerobically induced proteins were not induced under forced oxidative stress conditions. Taken together, the present findings indicate that A. xylanus has a unique defence system against forced oxidative stress.

Chlorella vulgaris aldehyde reductase is capable of functioning as ferric reductase and of driving the fenton reaction in the presence of free flavin.

The free flavin-dependent Fenton reaction was detected in cell-free extracts of Chlorella. The corresponding enzyme was purified to homogeneity, and its N-terminal sequence was highly homologous to those of aldo-keto reductase family enzymes. The purified enzyme displayed aldehyde reductase activity in the presence of NADPH. Additionally, it showed ferric reductase activity and drove the Fenton reaction in the presence of free FAD and NADH.

Taxonomical and physiological comparisons of the three species of the genus Amphibacillus.

Amphibacillus is a genus for Gram-positive, spore-forming, rod-shaped, facultatively anaerobic bacteria with low-G+C content of DNA, established by Niimura et al. in 1990. Amphibacillus xylanus, the type species of the genus, grows well under both strictly anaerobic and aerobic conditions in spite of lacking any isoprenoid quinones, cytochromes, and catalase. Amphibacillus fermentum and Amphibacillus tropicus were later proposed by Zhilina et al. in 2001 for the isolates from a soda lake. In this paper, we revealed the latter two species also lacked isoprenoid quinones, cytochrome and catalase, and that they grew well under strictly anaerobic and aerobic conditions. The consistent growth of A. xylanus under both conditions is due to the presence of anaerobic and aerobic pathways for glucose metabolism in the organism. Although A. fermentum and A. tropicus are supposed to have a side enzymatic pyruvate pathway to produce lactate under both conditions, the two species have two major pyruvate metabolic pathways as observed in A. xylanus. Analysis data indicated that NADH formed both by the aerobic pyruvate pathway and by the glycolytic pathway was re-oxidized by the NADH oxidase in A. fermentum and A. tropicus as well as A. xylanus, and furthermore that the NADH oxidase-Prx (AhpC) system, i.e., NADH oxidase scavenging hydrogen peroxide with Prx, also functions in A. tropicus as observed with A. xylanus. Not only the taxonomical character of the genus Amphibacillus but also the growth characterization based on the two metabolic pathways and unique oxygen metabolism are distinctive in those traits from other facultative anaerobes.

IL-1ß stimulates IL-8 production, including prostaglandin E2 receptor EP4-triggered pathways, in synoviocyte MH7A cells.

Prostaglandin E2 (PGE2) is an important modulator of cytokine-driven inflammation. Using GeneChip analysis, we found that interleukin (IL)-1ß induces the gene expression of PTGER4, which encodes the PGE2 receptor subtype EP4 (PGE2EP4). This subtype is one of four PGE2 receptors occurring in synoviocyte MH7A cells. Immunofluorescence microscopy revealed a corresponding upregulation in the production of PGE2EP4 protein in IL-1ß-pretreated MH7A cells. PGE2 alone has no effect on IL-8 production, but in cells pretreated with IL-1ß it markedly enhances IL-8 production. Moreover, a stimulatory effect of PGE2 on IL-8 production in the synoviocyte MH7A cells was observed. These results indicate that, in the synovial tissues of patients with rheumatoid arthritis, PGE2 stimulates the release of IL-8 from the fibroblastic cells classified as present, thereby exacerbating inflammation.

An O2-inducible rubrerythrin-like protein, rubperoxin, is functional as a H2O2 reductase in an obligatory anaerobe Clostridium acetobutylicum.

Clostridium acetobutylicum, an obligatory anaerobe, is able to grow microoxically with the accumulation of two functionally unknown O2-induced proteins identified by two-dimensional electrophoresis. One was determined to be a novel type rubrerythrin-like protein, named rubperoxin (Rpr) in this study, that conserves one rubredoxin-type Fe(SCys)(4) site per polypeptide in the N-terminus. Recombinant rubperoxin expressed in E. coli purified in its oxidized form is a dimer with optical absorption maxima at 492, 377, and 277nm. Reduced rubperoxin is rapidly and fully oxidized by a half molar ratio of H2O2 per mole protein, and slowly oxidized by t-butyl hydroperoxide and O2. Cell-free extracts from microoxically grown cells efficiently reduce rubperoxin when NAD(P)H is used as the electron donor (preferentially reduced by NADH). These results strongly suggest that rubperoxin is involved in NAD(P)H-dependent H2O2 detoxification in vivo.