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.

Dependence of the V/III Ratio on Indium Incorporation in InGaN Films Grown by Metalorganic Vapour Phase Epitaxy.

InGaN epitaxial layers were grown on c-plane sapphire substrates using the metalorganic vapour phase epitaxy (MOVPE) system at 760 °C. By varying the total flow rate of group-III sources (TMI+TEG) with a fixed molar ratio of group-III sources [TMI/(TMI+TEG)], the influence of V/III ratio were investigated from 4500 to 20000. The grown N-polar InGaN layers were investigated by atomic force microscopy and it is found that the surface roughness decreases with increasing the V/III ratios. High resolution X-ray diffraction analyses show that the phase separation decreases with increasing the V/III ratios. Photoluminescence measurements reveal that the peak position of the band-edge emission shifted toward the shorter wavelength with increasing the V/III ratios. Reciprocal space mapping (RSM) analyses were carried out on InGaN films. At low V/III ratio, the phase separation can be detected in InGaN films.

Effect of sapphire nitridation and group-III source flow rate ratio on In-incorporation into InGaN grown by metalorganic vapor phase epitaxy.

In-composition of N-polar InGaN films on the sapphire substrate with the surface nitridation was investigated. By varying the ratio of the group-III source flow rate from 0.7 to 0.95, the In-composition and the surface morphologies of InGaN films were changed. The In-composition of N-polar InGaN films was affected by the strain relaxation and the surface morphologies.

A single-blood-sample method using inulin for estimating feline glomerular filtration rate.

BACKGROUND: Application of a multisample method using inulin to estimate glomerular filtration rate (GFR) in cats is cumbersome. OBJECTIVES: To establish a simplified procedure to estimate GFR in cats, a single-blood-sample method using inulin was compared with a conventional 3-sample method. ANIMALS: Nine cats including 6 clinically healthy cats and 3 cats with spontaneous chronic kidney disease. METHODS: Retrospective study. Inulin was administered as an intravenous bolus at 50 mg/kg to cats, and blood was collected at 60, 90, and 120 minutes later for the 3-sample method. Serum inulin concentrations were colorimetrically determined by an autoanalyzer method. The GFR in the single-blood-sample method was calculated from the dose injected, serum concentration, sampling time, and estimated volume of distribution on the basis of the data of the 3-sample method. RESULTS: An excellent correlation was observed (r = 0.99, P = .0001) between GFR values estimated by the single-blood-sample and 3-sample methods. CONCLUSIONS AND CLINICAL IMPORTANCE: The single-blood-sample method using inulin provides a practicable and ethical alternative for estimating glomerular filtration rate in cats.

Estimation of glomerular filtration rate in rabbits by a single-sample method using iodixanol.

To estimate the glomerular filtration rate (GFR) in conscious rabbits, a single-sample method using the non-ionic contrast medium iodixanol was compared with a three-sample method using the standard agent inulin. Iodixanol and inulin were co-administered intravenously to male New Zealand White rabbits at 60 mg I/kg and 40 mg/kg, respectively, and blood was collected 30, 60, 90 and 120 min later. Serum iodixanol and inulin concentrations were separately determined by high performance liquid chromatography and colorimetry, respectively. Serum urea nitrogen (UN) and creatinine concentrations were also determined. Based on the data from healthy and cisplatin-treated rabbits, the GFR estimated by iodixanol was well consistent with that by inulin. Further, when the GFR decreased to more than 60% of the reference value, serum creatinine concentrations became elevated. However, serum UN concentrations exhibited wide fluctuations, presumably due to a difference in renal handlings. The single-sample method using iodixanol was considered to be an expedient tool in both clinical and research settings, because the stress due to a multi-sample method was reduced.

Transforming growth factor-ß decreases the cancer-initiating cell population within diffuse-type gastric carcinoma cells.

Stem cells in normal tissues and cancer-initiating cells (CICs) are known to be enriched in side population (SP) cells. However, the factors responsible for the regulation of expression of ABCG2, involved in efflux of dyes, in SP cells have not been fully investigated. Here, we characterized the SP cells within diffuse-type gastric carcinoma, and examined the effects of transforming growth factor-ß (TGF-ß) on SP cells. Diffuse-type gastric carcinoma cells established from four independent patients universally contained SP cells between 1 and 4% of total cells, which displayed greater tumorigenicity than non-SP cells did. TGF-ß repressed the transcription of ABCG2 through direct binding of Smad2/3 to its promoter/enhancer, and the number of SP cells and the tumor-forming ability of cancer cells were decreased by TGF-ß, although ABCG2 is not directly involved in the tumor-forming ability of SP cells. Cancer cells from metastatic site expressed much higher levels of ABCG2 and included a greater percentage of SP cells than parental cancer cells did. SP cells are thus responsible for the progression of diffuse-type gastric carcinoma, and TGF-ß negatively contributes to maintain the CICs within the cancer.

Changes in the expression of myosins during postnatal development of masseter muscle in the microphthalmic mouse.

In the present study, to elucidate the influences of the deficiency of teeth on the masseter muscle, we analyzed changes in the expression of MyHC isoform mRNAs during postnatal development in mi/mi mice using real-time PCR. By 8 weeks of age, MyHC I had nearly disappeared in the +/+ mice, while it was still present in the mi/mi, and the level of MyHC I mRNA in the mi/mi was 5.1-fold higher than that in the +/+ (p<0.01). The levels of MyHC IIx mRNAs in the mi/mi mice were 41 ~ 55% lower than those in the +/+ at both 3 weeks and 4 weeks of age (p<0.05). No significant difference in the expression of MyHC IIa and IIb mRNAs in the masseter muscle was found between the mi/mi and +/+. From these results, we speculate that the deficiency of teeth affects the masseter muscles during the postnatal development.

[Investigation of the accurate measurement of the basic imaging properties for the digital radiographic system based on flat panel detector].

PURPOSE/AIM OF THE EXHIBIT: The purpose of this exhibit is: 1. To explain "resampling", an image data processing, performed by the digital radiographic system based on flat panel detector (FPD). 2. To show the influence of "resampling" on the basic imaging properties. 3. To present accurate measurement methods of the basic imaging properties of the FPD system. CONTENT ORGANIZATION: 1. The relationship between the matrix sizes of the output image and the image data acquired on FPD that automatically changes depending on a selected image size (FOV). 2. The explanation of the image data processing of "resampling". 3. The evaluation results of the basic imaging properties of the FPD system using two types of DICOM image to which "resampling" was performed: characteristic curves, presampled MTFs, noise power spectra, detective quantum efficiencies. CONCLUSION/SUMMARY: The major points of the exhibit are as follows: 1. The influence of "resampling" should not be disregarded in the evaluation of the basic imaging properties of the flat panel detector system. 2. It is necessary for the basic imaging properties to be measured by using DICOM image to which no "resampling" is performed.

Primary chondrosarcoma in the liver of a dog.

Primary chondrosarcoma was found in the quadrate lobe of the liver of a 6-year-old, intact, male Golden Retriever. At 6 months after partial hepatectomy, recurrence in the liver occurred. The dog died of its systemic metastases 10 months thereafter. Histologically, the hepatic mass revealed neoplastic chondrocytes with abundant chondroid matrix, and there were few myxoid areas where the cellularity and pleomorphism of the neoplastic cells were more prominent. The neoplastic cells were positive for periodic acid-Schiff and were immunohistochemically positive for vimentin and S-100 protein; the matrix was deeply stained for alcian blue and was metachromatic for toluidine blue stain. This tumor might be derived from pluripotent mesenchymal cells in the connective tissue of the liver. To the best of our knowledge, in all mammalians, including humans, this is the first report of extraskeletal chondrosarcoma primarily arising in the liver.

Repair of articular cartilage defects in rabbits using CDMP1 gene-transfected autologous mesenchymal cells derived from bone marrow.

OBJECTIVE: Cartilage-derived morphogenetic protein 1 (CDMP1), which is a member of the transforming growth factor-beta superfamily, is an essential molecule for the aggregation of mesenchymal cells and acceleration of chondrocyte differentiation. In this study, we investigated whether CDMP1-transfected autologous bone marrow-derived mesenchymal cells (BMMCs) enhance in vivo cartilage repair in a rabbit model. METHODS: BMMCs, which had a fibroblastic morphology and pluripotency for differentiation, were isolated from bone marrow of the tibia of rabbits, grown in monolayer culture, and transfected with the CDMP1 gene or a control gene (GFP) by the lipofection method. The autologous cells were then implanted into full-thickness articular cartilage defects in the knee joints of each rabbit. RESULTS: During in vivo repair of full-thickness articular cartilage defects, cartilage regeneration was enhanced by the implantation of CDMP1-transfected autologous BMMCs. The defects were filled by hyaline cartilage and the deeper zone showed remodelling to subchondral bone over time. The repair and reconstitution of zones of hyaline articular cartilage was superior to simple BMMC implantation. The histological score of the CDMP1-transfected BMMC group was significantly better than those of the control BMMC group and the empty control group. CONCLUSION: Modulation of BMMCs by factors such as CDMP1 allows enhanced repair and remodelling compatible with hyaline articular cartilage.

The role of TNF-alpha in the pathogenesis of inflammation and joint destruction in rheumatoid arthritis (RA): a study using a human RA/SCID mouse chimera.

OBJECTIVE: In order to elucidate which cytokine preferentially stimulates the synovium in patients with rheumatoid arthritis (RA), we investigated the roles of tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) using SCID mice engrafted with human RA tissue (SCID-HuRAg). METHODS: The SCID-HuRAg mice were prepared according to our previously described method. First, SCID-HuRAg mice were treated with chimeric anti-TNF-alpha monoclonal antibody (mAb, 100 microg/mouse) and histological changes were examined 4 weeks after the initial treatment. Secondly, a total of 100 microg of recombinant TNF-alpha or IL-6 (0.6 microg/h) was administered daily to mice using an osmium pump. The histological changes and serum cytokine levels were examined 4 weeks after the initial administration. Human immunoglobulin G (IgG) was administered to mice as a control. RESULTS: Synovial inflammatory cells were significantly decreased after the anti-TNF-alpha mAb treatment; conversely, the degree of synovial inflammation was significantly exacerbated by TNF-alpha administration. The levels of both IL-6 and TNF-alpha in sera were significantly increased by recombinant TNF-alpha administration, while TNF-alpha levels were unchanged by IL-6 administration. This suggests that TNF-alpha controls IL-6 production. Despite the profound changes in inflammation, we found no effects on bone and no articular cartilage damage was produced by TNF-alpha. CONCLUSION: This study provides strong evidence that TNF-alpha is a key molecule in the control of the inflammatory changes that occur in the RA synovium. In addition, TNF-alpha regulates IL-6 production. However, other inflammatory pathways independent of TNF-alpha may contribute to the bone and cartilage damage seen in RA.

Quiet MRI with novel acoustic noise reduction.

Fast scan techniques, which are used to reduce scanning times, have raised scanning noise levels in magnetic resonance imaging (MRI) systems, resulting in greater patient discomfort and stress. It is well known that this noise is caused by vibration of the gradient coil due to the Lorentz forces generated by the current in the gradient coil, which is placed in a static magnetic field. We have confirmed that MRI noise can be substantially reduced by sealing the gradient coil in a vacuum chamber to block airborne vibration propagation, by supporting the gradient coil independently to block solid vibration propagation and by decreasing the eddy currents induced in RF coils, the RF shield and the static-field-magnet cryostat. Based on these findings, we have developed a silent MRI system in which scanning noise is markedly reduced under a wide range of scanning conditions.

Corticosteroid treatment induces chondrocyte apoptosis in an experimental arthritis model and in chondrocyte cultures.

OBJECT: In order to examine the mechanisms involved in steroid-induced arthropathy after intra-articular corticosteroid injection, a histological examination was performed in vivo using severe combined immunodeficiency (SCID) mice that were implanted with human articular cartilage into the back (SCID/hu model). In addition, the effect of corticosteroids on chondrocyte apoptosis was evaluated in vitro using cultured human chondrocytes. METHOD: Human articular cartilage was obtained during knee surgery and implanted subcutaneously into the backs of SCID mice. One month later, weekly injections of corticosteroid (hydrocortisone acatate: 1 mg/0.2 ml, triamcinolone acetonide: 0.2 mg/0.2 ml, dexamethasone acetate: 0.1 mg/0.2 ml) in the subcutaneous cavity around the grafted cartilage in SCID mice were initiated. After six weeks of treatment, the grafted cartilage pieces were removed from the SCID mice and examined histologically. Chondrocyte apoptosis after corticosteroid treatment was also investigated using cultured human chondrocytes. RESULT: In the corticosteroid treated, grafted articular cartilage, apoptotic chondrocytes were apparent in the superficial and middle layers of cartilage. But a reduced intensity of Safranin O staining was not remarkable. In the cultured chondrocytes, apoptotic changes were also observed after corticosteroid treatment. CONCLUSION: Corticosteroid treatment induces chondrocyte apoptosis and it may be important to understand the steroid-induced arthropathy.

Methotrexate inhibits rheumatoid synovitis by inducing apoptosis.

OBJECTIVE: To clarify the pharmacological action of methotrexate (MTX) on the synovium of patients with rheumatoid arthritis (RA) using severe combined immunodeficient (SCID) mice in which human RA synovial tissue had been grafted (SCID-HuRAg). METHODS: One month after engraftment of human RA tissue into SCID mice, MTX (0.3 mg/kg) was administered orally, then the appearance of apoptosis in the grafted tissue was examined by TdT mediated dUTP nick end labeling (TUNEL) staining and electron microscopy at various time points after MTX administration. In cultured synovial cells, synovial apoptotic changes after MTX treatment were studied by agarose gel electrophoresis and flow cytometric analysis. To compare the histological changes induced by MTX with those induced by other disease modifying antirheumatic drugs (DMARD) and a nonsteroidal antiinflammatory drug, histological examination of the grafted synovial tissues from SCID-HuRAg mice was conducted after 4 weeks of oral administration of MTX (0.3 mg/kg/week), salazosulfapyridine (30 mg/kg/day), auranofin (0.2 mg/kg/day), bucillamine (10 mg/kg/day), or indomethacin (2 mg/kg/day). RESULTS: A significant decrease in the number of inflammatory cells was observed in the grafted synovial tissue of MTX treated SCID-HuRAg. A similar antiinflammatory effect was not observed with the other DMARD. Induction of apoptosis was noted with MTX treatment but not with the others. The pro-apoptotic effect of MTX was also observed in synovial cell cultures. CONCLUSION: MTX induces apoptosis in RA synovium that, in turn, may contribute to its antiinflammatory effect on RA synovitis.

Reconstituting telomerase activity using the telomerase catalytic subunit prevents the telomere shorting and replicative senescence in human osteoblasts.

The rate of bone formation is largely determined by the number of osteoblasts, which in turn is determined by the rate of replication of progenitors and the life span of mature cells, reflecting the timing of death by apoptosis. However, the exact age-dependent changes of the cellular activity, replicative potential, and life span of osteoblasts have not been investigated to date. Here, we present evidence that the cellular activity, telomere lengths, and replicative life span of osteoblastic cells obtained from juxta-articular bone marrow gradually decrease with the advance of donor age. Recently, telomerase reverse transcriptase (hTERT) has been identified as a human telomerase catalytic subunit. We transfected the gene encoding hTERT into telomerase-negative human osteoblastic cells from donors and osteoblastic cell strain NHOst 54881 cells and showed that expression of hTERT induces telomerase activity in these osteoblastic cells. In contrast to telomerase-negative control cells, which exhibited telomere shortening and senescence after 10-15 population doublings, telomerase-expressing osteoblastic cells had elongated telomere lengths and showed continued alkaline phosphatase activity and procollagen I C-terminal propeptide (PICP) secretion for more than 30 population doublings. These results indicate that osteoblasts with forced expression of hTERT may be used in cell-based therapies such as ex vivo gene therapy, tissue engineering, and transplantation of osteoblasts to correct bone loss or osteopenia in age-related osteoporotic diseases.

Decreased cellular activity and replicative capacity of osteoblastic cells isolated from the periarticular bone of rheumatoid arthritis patients compared with osteoarthritis patients.

OBJECTIVE: Periarticular osteopenia is frequently observed in rheumatoid arthritis (RA). Bone loss has been considered to be at least partly due to inadequate bone formation, which in turn, is largely dependent on the number of osteoblasts and the osteoblastic activity. Normal human somatic cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that the telomere, the terminal sequence of chromosomes, is the mitotic clock that triggers senescence. In the present study, we sought to clarify the relationship between periarticular osteopenia and osteoblast replicative senescence in RA. METHODS: We examined age-related changes in cellular activity (alkaline phosphatase activity, osteocalcin and C-terminal type I procollagen secretion, and cAMP response to parathyroid hormone), replicative capacity, and senescent cell expression in osteoblasts from periarticular bone samples obtained from 15 patients with RA and 15 age-matched patients with osteoarthritis (OA). Cellular replicative capacity was analyzed by the mean telomere length and in vitro remaining replicative lifespan of the cells. RESULTS: In both OA and RA groups, the cell proliferation rate, the levels of osteoblastic markers, mean telomere length, and replicative lifespan in osteoblastic cells gradually decreased with the increasing age of the donor. The percentage of senescent osteoblastic cells in the periarticular bone increased with age in both groups, and the rate of expression of senescent cells was higher in RA patients than in age-matched OA patients. The osteoblastic activities and replicative capacity of osteoblastic cells from RA patients were lower than those from OA patients at any donor age. The age-related decreases in the osteoblastic activity and replicative capacity of osteoblastic cells from periarticular bone were greater in RA patients than in OA patients. CONCLUSION: Our results suggest that osteoblast replicative senescence in periarticular bones occurs more rapidly with aging in RA than in OA patients and contributes to periarticular osteopenia in RA.

Dual-wavelength optical head with a wavelength-selective filter for 0.6- and 1.2-mm-thick-substrate optical disks.

A dual-wavelength optical head, believed to be new in design, for 0.6- and 1.2-mm-thick-substrate optical disks was developed by use of 650- and 780-nm-wavelength light and a wavelength-selective filter, which reduces the spherical aberration that is due to the difference in substrate thicknesses and restricts the numerical aperture for 780-nm-wavelength light. According to this configuration, both high light power efficiency and wide image field characteristics are obtained, which are suitable not only for read-only but also for recordable or rewritable optical disk systems. A reading operation for a digital video disc and for a compact disc and a recording operation for a phase change optical disk were successfully demonstrated.

[Clinical evaluation of enzyme immuno assay systems for HCV antibody--synthetic peptide antigen and recombinant antigen].

To evaluate the frequency of the false positive results, we examined 168 patients for the presence of HCV using two HCV antibody assay systems, Synpep HCV-EIA (Synpep) and Abbott EIA II (Abbott). The results obtained by the two methods were significantly different in 22 patients. Cases in 17 of these patients, the results were positive with Abbott but negative with Synpep, and there were no clinical signs or delectable virus RNA. However, in 2 cases, the results were markedly positive with Abbott and weakly positive with Synpep. The presence of virus RNA and the increase of transaminase were observed in one case but both were noted in the other case. The serum of these two patients reacted with the C33C and C22-3 regions in RIBA II. We observed another 2 cases in which the elevation of the cut-off index with Synpep preceded that with Abbott at the early stage of acute hepatitis C. We also compared the cut-off index with the histology activity index (HAI) score determined by liver biopsy. The average cut-off index with Synpep was proportional to the HAI score in the range between 0 and 13. Based on the cut-off index/HAI score relationship, we suggest that patients with inactive chronic hepatitis show a Synpep cut-off index less than 11.

[Current alteration of Helicobacter pylori in gastro-duodenal diseases].

The current alteration of the detection rate of Helicobacter pylori (H. pylori) on mucous membranes of the stomach has been surveyed and reviewed for 49 cases of gastro-duodenal diseases. The survey has been conducted for 3 years and an endoscopic examinations have been performed 2-8 times. On the first visit, the diagnosis can be made endoscopically, and H2 blockers and another agents were used as therapeutic drugs. The detection rate was 67.3% (33 cases) for positive cases and positive reaction cases, while the rate was 32.6% (16 cases) in negative cases and negative reaction cases. Among them, the most commonly observed disease was atrophic gastritis, followed by duodenal ulcer, and acute gastric mucous lesions (AGML). Peptic ulcer was not seen. After administration of H2 blockers and anti-ulcer agents, the H. pylori-detection rate was found to fluctuate according to the diseases and alterations in the morbidity period. Some cases indicated the disappearance of H. pylori in accordance with the improvement of the lesion site.