Bacillus subtilis var. natto increases the resistance of Caenorhabditis elegans to gram-positive bacteria.

AIMS: This study aimed to investigate the effect of Bacillus subtilis var. natto on the susceptibility of the model host, Caenorhabditis elegans, to bacterial infection. METHODS AND RESULTS: Caenorhabditis elegans worms were fed with a standard food consisting of Escherichia coli OP50 strain (control) or B. subtilis (natto) during their larval stage. The worms were then infected with pathogenic bacteria. We analyzed their survival time and RNA sequencing-based transcriptome. Upon infection with Staphylococcus aureus and Enterococcus faecalis, the survival time of B. subtilis (natto)-fed worms was longer than that of the control. Transcriptome analyses showed upregulation of genes associated with innate immunity and defense response to gram-positive bacteria in B. subtilis (natto)-fed worms. CONCLUSIONS: Bacillus subtilis (natto) conferred an increased resistance of C. elegans to gram-positive bacteria. Our findings provided insights into the molecular mechanisms underlying B. subtilis (natto)-regulated host immunity and emphasized its probiotic properties for preventing and alleviating infections caused by gram-positive bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first study to show that B. subtilis (natto) confers specific resistance against gram-positive bacteria.

Potential for acceleration of bone formation after implant surgery by using a dietary supplement: an animal study.

Dental implant treatment is an effective modality to restore lost aesthetic and masticatory functions. However, healing after implant surgery takes at least 3-6 months. This prolonged healing period poses several difficulties for individuals with a large edentulous area and decreases their quality of life. Consequently, shortening the healing period and accelerating final prosthesis placement after surgery is very clinically important. Peri-implant bone formation may be enhanced by systemic approaches, such as the use of osteoporosis supplements, to promote bone metabolism. To confirm whether intake of a supplement developed for osteoporosis, synthetic bone mineral (SBM), was effective in accelerating peri-implant bone formation as part of the healing process after implantation. Twenty-four 5-week-old female Wistar rats were randomly assigned to receive a standardised diet without (control group, n = 12) or with SBM (n = 12). The rats had implant surgery at 8 weeks of age under general anaesthesia. The main outcome measures were bone mineral density (BMD) and pull-out strength in the implant and femur, which were compared between the groups at 2 and 4 weeks after implantation using the Mann-Whitney U test. BMD was significantly greater in the SBM group at 2 and 4 weeks after implantation compared to the control group. Pull-out strength was significantly greater in the SBM groups at 2 and 4 weeks after implantation compared to the control group. This study demonstrated that SBM could be effective in accelerating peri-implant bone formation during the healing period after implantation.

The prevalence of rhinitis and its association with smoking and obesity in a nationwide survey of Japanese adults.

BACKGROUND: Rhinitis is a common disease, and its prevalence is increasing worldwide. Several studies have provided evidence of a strong association between asthma and rhinitis. Although smoking and obesity have been extensively analyzed as risk factors of asthma, associations with rhinitis are less clear. OBJECTIVE: The aims of our study were (i) to evaluate the prevalence of rhinitis using the European Community Respiratory Health Survey (ECRHS) questionnaire in Japanese adults and (ii) to evaluate the associations of smoking and body mass index (BMI) with rhinitis. METHODS: Following our study conducted in 2006-2007 to determine the prevalence of asthma using the ECRHS questionnaire, our present analysis evaluates the prevalence of rhinitis and its association with smoking and BMI in Japanese adults 20-79 years of age (N = 22819). We classified the subjects (20-44 or 45-79 years) into four groups as having (i) neither rhinitis nor asthma; (ii) rhinitis without asthma; (iii) asthma without rhinitis; or (iv) rhinitis with asthma. We then evaluated associations with smoking and BMI in each group. RESULTS: The overall age-adjusted prevalence of rhinitis was 35.1% in men and 39.3% in women. A higher prevalence was observed in the younger population than in the older population. Active smoking and obesity were positively associated with asthma without rhinitis. In contrast, particularly in the 20- to 44-year age-group, active smoking and obesity were negatively associated with rhinitis without asthma. CONCLUSION: The results of the present study suggest that smoking and obesity may have different effects on the development of rhinitis and asthma.

Atelocollagen-mediated local and systemic applications of myostatin-targeting siRNA increase skeletal muscle mass.

RNA interference (RNAi) offers a novel therapeutic strategy based on the highly specific and efficient silencing of a target gene. Since it relies on small interfering RNAs (siRNAs), a major issue is the delivery of therapeutically active siRNAs into the target tissue/target cells in vivo. For safety reasons, strategies based on vector delivery may be of only limited clinical use. The more desirable approach is to directly apply active siRNAs in vivo. Here, we report the effectiveness of in vivo siRNA delivery into skeletal muscles of normal or diseased mice through nanoparticle formation of chemically unmodified siRNAs with atelocollagen (ATCOL). ATCOL-mediated local application of siRNA targeting myostatin, a negative regulator of skeletal muscle growth, in mouse skeletal muscles or intravenously, caused a marked increase in the muscle mass within a few weeks after application. These results imply that ATCOL-mediated application of siRNAs is a powerful tool for future therapeutic use for diseases including muscular atrophy.

Allergen-specific immunotherapy alters the expression of B and T lymphocyte attenuator, a co-inhibitory molecule, in allergic rhinitis.

BACKGROUND: B7/CD28 family co-signalling molecules play a key role in regulating T cell activation and tolerance. Allergen-specific immunotherapy (SIT) alters allergen-specific T cell responses. However, the effect of SIT on the expression of various co-signalling molecules has not been clarified. OBJECTIVE: We sought to determine whether SIT might affect the expression of three co-inhibitory molecules, programmed death (PD)-1, B7-H1 and B and T lymphocyte attenuator (BTLA), in Japanese cedar pollinosis (JCP). METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from JCP patients who had or had not received SIT. PBMC were cultured in the presence or absence of Cry j 1, after which the cell surface expression of PD-1, B7-H1 and BTLA, as well as IL-5 production, were determined. In addition, the effect of BTLA cross-linking on IL-5 production was examined. RESULTS: After Cry j 1 stimulation, no significant differences in PD-1 and B7-H1 expression were observed between SIT-treated and SIT-untreated patients. BTLA expression was down-regulated in untreated patients after Cry j 1 stimulation and up-regulated in SIT-treated patients. Up-regulation of BTLA in SIT-treated patients was particularly apparent in a CD4(+) T cell subset. IL-5 production was clearly reduced among SIT-treated patients, and the observed changes in BTLA expression correlated negatively with IL-5 production. Moreover, immobilization of BTLA suppressed IL-5 production in JCP patients. CONCLUSION: These results suggest that both IL-5 production and down-regulation of BTLA in response to allergen are inhibited in SIT-treated patients with JCP. BTLA-mediated co-inhibition of IL-5 production may contribute to the regulation of allergen-specific T cell responses in patients receiving immunotherapy.

Quantitative analysis of propionibacterial DNA in bronchoalveolar lavage cells from patients with sarcoidosis.

BACKGROUND AND AIM OF THE WORK: The causes of sarcoidosis are still unknown. Propionibacterial subspieces are thought to be one of the most likely sources of antigens. Here we attempted to measure the amount of propionibacterial DNA in bronchoalveolar lavage (BAL) cell samples from patients with sarcoidosis and other pulmonary diseases. METHODS: We examined BAL cells from 42 patients with sarcoidosis and 30 controls. Using quantitative polymerase chain reaction (PCR) for 16S rRNA of Propionibacterium acnes (P. acnes) and Propionibacterium granulosum (P. granulosum), we measured the amount of propionibacterial DNA in 500 ng of total DNA extracted from BAL cells from patients with sarcoidosis or other lung diseases. The correlation between clinical findings and the results of quantitative PCR were analyzed. RESULTS: The mean level of P. acnes DNA from patients with sarcoidosis was 59.9 genomes per 500 ng of total DNA, which was significantly higher than that in controls (20.7 genomes, p<0.000l). The mean level of P. granulosum DNA from patients with sarcoidosis was 1.2 genomes, which was similar to that in controls (1.0 +/-1.6 genomes, p=0.52). The number of genomes of P. acnes in BAL cells was correlated with the serum angiotensin-converting enzyme (ACE) level and the percentage of macrophages in BAL fluid from patients with sarcoidosis. CONCLUSIONS: The amount of P. acnes DNA in BAL cells from patients with sarcoidosis was significantly higher than that in BAL cells from patients with other pulmonary diseases. P. acnes may be involved in the pathogenesis of sarcoidosis.

Inferring causal structures and comparing the causal effects among calving difficulty, gestation length and calf size in Japanese Black cattle.

The objectives of this study were to infer phenotypic causal networks involving gestation length (GL) and calving difficulty (CD) for the primiparity of 1850 Japanese Black heifers, and the birth weight (BWT), withers height (WH) and chest girth (CHG) of their full blood calves, and to compare the causal effects among them. The inductive causation (IC) algorithm was employed to search for causal links among these traits; it was applied to the posterior distribution of the residual (co)variance matrix of a multiple-trait sire-maternal grand sire (MGS) model. The IC algorithm implemented with 95% and 90% highest posterior density intervals detected only one structure with links between GL and BWT (WH or CHG) and between BWT (WH or CHG) and CD, although their directions were not resolved. Therefore, a possible causal structure based on the networks obtained from the IC algorithm [GL→BWT (WH or CHG)→CD] was fitted using a structural equation model to infer causal structure coefficients between the traits. The structural coefficients of GL on BWT and of BWT on GL on the observable scale showed that an extra day of GL led to a 270-g gain in BWT, and a 1-kg increase in BWT increased the risk for dystocia by 1.1%, in the causal structure. Similarly, an increase in GL by 1 day resulted in a 2.1 (2.0)-mm growth in WH (CHG), and a 1-cm increase in WH (CHG) increased the risk of dystocia by 1.2% (0.9%). The structural equation model was also fitted to alternative causal structures, which involved the addition of a directed link from GL to CD, or GL→CD to the structures described above. The inferred structural coefficients with the alternative structures were almost the same as the corresponding ones that had GL→BWT (WH or CHG)→CD. However, the direct causal effect of the extra link from GL on CD was similar to the indirect causal effect of GL through the mediating effect of BWT (WH or CHG) on CD and significant (P<0.05). This suggest that maternal genetic effects might not be removed completely from the residual variance components in the sire-MGS model, and the application of the IC algorithm to the variances from the model could detect an incorrect structure. Nonetheless, fitting the structural equation model to the causal structure provided useful information such as the magnitude of the causal effects between the traits.

Macromolecular crystallization in microgravity generated by a superconducting magnet.

About 30% of the protein crystals grown in space yield better X-ray diffraction data than the best crystals grown on the earth. The microgravity environments provided by the application of an upward magnetic force constitute excellent candidates for simulating the microgravity conditions in space. Here, we describe a method to control effective gravity and formation of protein crystals in various levels of effective gravity. Since 2002, the stable and long-time durable microgravity generated by a convenient type of superconducting magnet has been available for protein crystal growth. For the first time, protein crystals, orthorhombic lysozyme, were grown at microgravity on the earth, and it was proved that this microgravity improved the crystal quality effectively and reproducibly. The present method always accompanies a strong magnetic field, and the magnetic field itself seems to improve crystal quality. Microgravity is not always effective for improving crystal quality. When we applied this microgravity to the formation of cubic porcine insulin and tetragonal lysozyme crystals, we observed no dependence of effective gravity on crystal quality. Thus, this kind of test will be useful for selecting promising proteins prior to the space experiments. Finally, the microgravity generated by the magnet is compared with that in space, considering the cost, the quality of microgravity, experimental convenience, etc., and the future use of this microgravity for macromolecular crystal growth is discussed.

Movement and diffusion of paramagnetic ions in a magnetic field.

The magnetic movement and thermal diffusion have been studied for Cu(2+) ions in solution. The Cu(2+) ion solution was spotted on the silica gel support and exposed to the magnetic field of 410 kOe(2) cm(-1) intensity x gradient. The Cu(2+) ions were attracted toward the filed center. The moving distance and diffusing distance were observed at various time intervals. It is shown that the Cu(2+) ions move in a large group composed of Cu(2+) ions and H(2)O molecules. The size of the group is approximately estimated to be of 4.6 mum diameter by the analysis of the drift velocity of the group and the Cu(2+) ion concentration in the group.

Alteration of cholesterol biosynthetic pathways in the skin of mice administered polycyclic aromatic hydrocarbons.

When polycyclic aromatic hydrocarbons were applied solely or together with a tumor promoter (12-O-tetradecanoylphorbol-13-acetate) to the skin of mice, a marked decrease in the level of lathosterol was observed, reflecting a significant change in the metabolism of sterols. Yet the total amount of cholesterol was not changed. When diazacholesterol (a metabolic inhibitor) was administered to mice, both desmosterol and 5 alpha-cholesta-7,24-dien-3 beta-ol accumulated in the skin, whereas the level of lathosterol decreased. These results seem to suggest that a significant portion of lathosterol is formed via 5 alpha-cholesta-7,24-dien-3 beta-ol in addition to the pathway through methostenol. When polycyclic aromatic hydrocarbon was applied to the skin of the mouse treated with diazacholesterol, a significant increase of desmosterol and a marked drop of the level of 5 alpha-cholesta-7,24-dien-3 beta-ol were observed. These results strongly suggest that polycyclic aromatic hydrocarbons perturb the metabolism of sterol in the skin of mice while keeping the total amount of cholesterol unchanged. A similar metabolism also seems to be operating in tumor tissue itself.

Occlusal force and condylar motion in patients with anterior open bite.

Patients with open bite often show a weak occlusal force and temporomandibular disorders (TMDs). If these are the main cause of open bite, it may be hypothesized that both pre-pubertal and adult open-bite patients would show a weak occlusal force and abnormal condylar motion. The purpose of this study was to test this hypothesis. Test group subjects consisted of 13 consecutive pre-pubertal and 13 adult patients with anterior open bite. They were compared with age-matched normal subjects. The adult open-bite group showed a weaker occlusal force and a shorter range of condylar motion compared with the control subjects. In the pre-pubertal subjects, however, there were no significant differences in the occlusal force and range of condylar motion between the open-bite and control groups. Therefore, these results suggest that a weak occlusal force or TMDs may not be the main cause of open bite.

Sodium benzylideneascorbate induces apoptosis in HIV-replicating U1 cells.

U1 cells, a subclone of U937 cells chronically infected with human immunodeficiency virus type 1 (HIV-1), produced HIV-1 only in the presence of inducers such as 12-O-tetradecanoxylphorbol 13-acetate (TPA) or tumor necrosis factor (TNF)-alpha. The expression of HIV-antigen on U1 cells induced by TPA or TNF-alpha was found to be prevented by sodium 5,6-benzylidene-L-ascorbate (SBA) in a concentration-dependent manner. Treatment of U1 cells with SBA in the presence of inducers resulted in cell death with cell shrinkage, chromatin condensation and DNA fragmentation into nucleosomal oligomers, characteristics of apoptosis. In contrast, SBA had scarcely any apoptotic effect on U1 cells in the absence of inducers. SBA did not also induce apoptosis in parental U937 cells in the presence or absence of inducers. These results suggest that HIV-replicating U1 cells selectively undergo apoptosis on treatment with SBA.

New flow cytometric method for surface phenotyping basophils from peripheral blood.

To clarify the role of basophils in the pathogenesis of allergic disease, we developed a new method for performing surface phenotyping of these cells in centrifugation-enriched mononuclear cell fraction. This method identified basophils on the basic of a negative reactivity with mixed FITC-conjugated monoclonal anti-bodies (mAbs) (anti-CD2, -CD14, -CD16, and -CD19) with analysis performed by flow cytometry. The validity of this approach was confirmed by sorting experiments. Various PE-conjugated mAbs were also used to examine binding to FITC-negative basophils. Basophils from asthmatic patients (n = 14) as well as from normal subjects (n = 6) were shown to express CDw32 (Fc gamma RII), CD25 (IL-2R), but not CD64 (Fc gamma RI). We also detected binding of IgG1 and IgG4 to basophils. This method of phenotyping was very rapid and simple. It thus appears to be useful in the study of allergic disease, as well as of the biology of the basophil.

Purification and some properties of bovine liver cytosol thioltransferase.

A cytosol thioltransferase was purified 37,000-fold from bovine liver by essentially the same procedure as reported for rat liver enzyme by Axelsson et al. [1978) Biochemistry 17, 2978-2984). The purified enzyme appears to be homogeneous on sodium dodecyl sulfate (SDS)-gel electrophoresis and has a molecular weight (Mr) of 11,000, an isoelectric point (pI) of 8.1, and an optimum pH with S-sulfocysteine and GSH as substrates of 8.5. It is specific for disulfides including L-cystine, S-sulfocysteine, ribonuclease A, trypsin, soybean kunitz trypsin inhibitor, soybean Bowman Birk trypsin inhibitor and insulin, and converts Bowman Birk trypsin inhibitor to an inactive form. The enzyme does not act as a protein : disulfide isomerase, as measured by reactivation of "scramble" ribonuclease and Kunitz soybean trypsin inhibitor. Thioltransferase activity was found in cytosol of various bovine tissues.

Peptidyl aldehyde inhibitors of proteasome induce apoptosis rapidly in mouse lymphoma RVC cells.

Proteases play an important role in regulation of apoptosis. To elucidate the role of proteasome in apoptosis, we examined the effects of the proteasome inhibitors, carbobenzoxy-L-isoleucyl-gamma-t-butyl-L-glutamyl-L-alanyl-L-leucinal and carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal on RVC lymphoma cells. Cells exposed to the proteasome inhibitors arrested at G2/M phase followed by internucleosomal DNA cleavage, chromatin condensation, and formation of apoptotic bodies dose- and time-dependently. Ubiquitinated histone H2A levels decreased in the exposed cells, suggesting a relationship between deubiquitination of histone H2A and the chromatin disarray seen in apoptosis. Northern blots showed an increase in expression of polyubiquitin genes early in the incubation. These findings suggest that the ubiquitin-mediated proteasome-proteolytic system is involved in regulating the cell cycle and apoptosis in RVC cells.

Permeability of dormant spores of Bacillus subtilis to gramicidin S.

Gramicidin S, dissolved in ethanol, penetrated into the inside of the dormant spores of Bacillus subtilis, had a partial inhibitory effect on L-alanine-initiated germination and completely inhibited their outgrowth and vegetative growth. The activity of particulate NADH oxidase of the antibiotic-treated dormant spores was also influenced significantly. Abnormal morphological changes were observed in germinated spores from gramicidin S-treated dormant spores. An immunoelectron microscopy method with colloidal gold-IgG complex showed that the penetration site of gramicidin S inside dormant spores was mainly the core region. These facts suggest that gramicidin S induces the damage of not only the outer membrane-spore coat complex but also the inner membrane surrounding the spore protoplast, and is able to penetrate into the core region of B. subtilis dormant spores.

Left ventricular motion after bypass operation for coronary artery disease with collaterals.

Left ventricular ejection changes obtained from left ventricle roentgenograms were analyzed before and after coronary artery bypass grafting in 22 consecutive patients with chronic obstructive left anterior descending coronary artery disease receiving collaterals before surgical revascularization. The collateral vessels all disappeared after surgical revascularization. After operation, ejection changes of anterobasal, anterolateral and apical walls supplied by the left anterior descending coronary artery improved from 43.6% +/- 9.7% to 48.5% +/- 8.6% (p < 0.05), from 35.2% +/- 10.9% to 39.4% +/- 9.5% (p < 0.05), and from 46.0% +/- 10.6% to 50.0% +/- 8.7% (p < 0.05), respectively. The improvement in left ventricular wall motion did not appear to be related to the extent of preoperative collateralization. Thus, left ventricular wall motion was impaired in the area supplied by collaterals and was improved by myocardial revascularization. These results suggest that coronary blood flow, even through well-developed collaterals, may not be sufficient, which may produce chronic active ischemia and impaired left ventricular wall motion.