Demonstration and Partial Characterization of a Bacterial Growth Enhancer in sera

During our research into the pathogenesis of Vibrio parahaemolyticus, we noticed that the concentration of serum added to the tissue culture medium (Dulbecco’s modified Eagle’s medium: DMEM) greatly affected its growth. Using gel filtration column chromatography, we clearly demonstrated that serum contains not only a bacterial growth inhibitor (BGI) but also a bacterial growth enhancer (BGE) for Vibrio parahaemolyticus. Our data indicate that the BGI is transferrin, whereas the BGE seems to be an undescribed small molecule (molecular weight of 1,000–3,000 Da) and is associated with magnesium and molybdenum ions. BGE activity was not decreased by heat treatment (at 60 or 100°C for 30 min) and affected the growth rate of a wide range of Gram-negative and Gram-positive bacteria. The addition of EDTA into DMEM lowered the growth rate, whereas the addition of BGE restored the growth activity. This study suggests that sera contain a previously undescribed small BGE molecule.     

Ceiling culture-derived proliferative adipocytes retain high adipogenic potential suitable for use as a vehicle for gene transduction therapy

Adipose tissue is expected to provide a source of proliferative cells for regenerative medicine and cell-transplantation therapies using gene transfer manipulation. We have recently identified ceiling culture-derived proliferative adipocytes (ccdPAs) from the mature adipocyte fraction as cells suitable as a therapeutic gene vehicle because of their stable proliferative capacity. In this study, we examined the capability of adipogenic differentiation of the ccdPAs compared with stromal vascular fraction (SVF)-derived progenitor cells (adipose-derived stem cells, ASCs) with regard to their multipotential ability to be converted to another lineage and therefore their potential to be used for regenerative medicine research. After in vitro passaging, the surface antigen profile and the basal levels of adipogenic marker genes of the ccdPAs were not obviously different from those of the ASCs. However, the ccdPAs showed increased lipid-droplet accumulation accompanied with higher adipogenic marker gene expression after stimulation of differentiation compared with the ASCs. The higher adipogenic potential of the ccdPAs than the ASCs from the SVF was maintained for 42 days in culture. Furthermore, the difference in the adipogenic response was enhanced after partial stimulation without indomethacin. These results indicate that the ccdPAs retain a high adipogenic potential even after in vitro passaging, thus suggesting the commitment of ccdPAs to stable mature adipocytes after autotransplantation, indicating that they may have potential for use in regenerative and gene-manipulated medicine.     

Physiological stress exacerbates murine colitis by enhancing proinflammatory cytokine expression that is dependent on IL-18

Psychological stress is an environmental factor considered to be a precipitating factor of inflammatory bowel disease. Interleukin (IL)-18 plays a role in stress-induced aggravation in some diseases. The aim of this study was to establish a model of murine colitis exacerbated by psychological stress and to clarify the role of IL-18 in this model. Male C57Bl/6 mice and IL-18(-/-) mice were used for this study. The mice received dextran sulfate sodium (DSS) for induction of colitis. Some mice were exposed to psychological stress using a communication box. Body weight, colonic length, and histological inflammation were measured for assessment of colitis. Tumor necrosis factor (TNF)-α and IL-18 expression in the colon and IL-18 expression in the adrenal gland were analyzed using real-time PCR. The effect of anti-IL-18 antibody was also investigated. Effects of TNF-α and IL-18 on cytokine expressions were studied using the colonic epithelial cell line LS174T. Induction of psychological stress in DSS-treated wild-type mice significantly exacerbated colitis with enhanced expression of proinflammatory cytokines and IL-18. However, induction of psychological stress in DSS-treated IL-18(-/-) mice did not aggravate colitis compared with that in the IL-18(-/-) group given only DSS treatment. Stress-induced aggravation of colitis was ameliorated significantly by anti-IL-18 antibody treatment. IL-18 did not enhance TNF-α-induced expression of intercellular adhesion molecule-1 or IL-8 in LS174T. We established a model of colitis exacerbated by psychological stress. Psychological stress enhanced IL-18 expression and plays a proinflammatory role in stress-induced aggravation of colitis.     

Galectin-9 Enhances Cytokine Secretion,but Suppresses Survival and Degranulation,in Human Mast Cell Line

Galectin-9 (Gal-9), a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells.     

Insights into the Classification of Myasthenia Gravis

Background and Purpose

Myasthenia gravis (MG) is often categorized into thymoma-associated MG, early-onset MG with onset age <50 years, and late-onset MG with onset age ≥50 years. However, the boundary age of 50 years old between early- and late-onset MG remains controversial, and each category contains further subtypes. We attempted to classify MG from a statistical perspective.

Methods

We analyzed 640 consecutive MG patients using two-step cluster analysis with clinical variables and discrimination analysis, using onset age as a variable.

Results

Two-step cluster analyses categorized MG patients into the following five subtypes: ocular MG; MG with thymic hyperplasia (THMG); generalized anti-acetylcholine receptor antibody (AChR-Ab)-negative MG; thymoma-associated MG; and generalized AChR-Ab-positive (SP) MG without thymic abnormalities. Among these 5 subtypes, THMG showed a distribution of onset age skewed toward a younger age (p<0.01), whereas ocular MG and SPMG without thymic abnormalities showed onset age skewed toward an older age (p<0.001 and p<0.0001, respectively). The other 2 subtypes showed normal distributions. THMG appeared as the main component of early-onset MG, and ocular MG and SPMG without thymic abnormalities as the main components of late-onset MG. Discrimination analyses between THMG and ocular MG and/or SPMG without thymic abnormalities demonstrated a boundary age of 45 years old.

Conclusions

From a statistical perspective, the boundary age between early- and late-onset MG is about 45 years old.     

Isolation and characterization of phytase isozymes produced by Aspergillus oryzae

A mutant strain (KL-38) of Aspergillus oryzae was obtained by UV irradiation. Phytase activity of KL-38 in molded rice (koji rice) was about 2.7-fold of that obtained from the parent strain (BP-1). Phytase activity of KL-38 in the submerged culture was similar to that of BP-1. Two types of phytase were produced from koji culture: phytase I (Phy I) was produced during incubation of both koji and submerged cultures, and phytase II (Phy II) was obtained only from koji culture. Phy II production was increased in KL-38 compared with BP-1, whereas the production of Phy I was similar for both KL-38 and BP-1. This finding indicates that A. oryzae has at least two types of phytase isozyme.     

Structure of Novel Enzyme in Mannan Biodegradation Process 4-O-β-d-Mannosyl-d-Glucose Phosphorylase MGP

The crystal structure of a novel component of the mannan biodegradation system, 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP), was determined to a 1.68-Å resolution. The structure of the enzyme revealed a unique homohexameric structure, which was formed by using two helices attached to the N-terminus and C-terminus as a tab for sticking between subunits. The structures of MGP complexes with genuine substrates, 4-O-β-d-mannosyl-d-glucose and phosphate, and the product d-mannose-1-phosphate were also determined. The complex structures revealed that the invariant residue Asp131, which is supposed to be the general acid/base, did not exist close to the glycosidic Glc-O4 atom, which should be protonated in the catalytic reaction. Also, no solvent molecule that might mediate a proton transfer from Asp131 was observed in the substrate complex structure, suggesting that the catalytic mechanism of MGP is different from those of known disaccharide phosphorylases.     

Predictive indicators for the therapeutic effect of OK-432 in patients with chronic active type B hepatitis

Twenty-two patients with chronic type B hepatitis were treated with OK-432. Immunological parameters were serially measured to find predictive indicators for the seroconversion from hepatitis B envelope antigen(HBe Ag) to anti-HBe. In patients who achieved the disappearance of HBe Ag associated with or without the appearance of anti-HBe, the numbers of CD8⁺DR⁺ and CD4⁺DR⁺T cells in peripheral blood increased gradually during OK-432 therapy and then reduced subsequently to the seroconversion from HBe Ag positive to anti-HBe positive. Increases of DR-positive T cells in numbers were significantly correlated with increased amounts of IFN- produced in response toin vitro OK-432 stimulation.In vitro OK-432-stimulated IFN- production and the increase of CD8⁺DR⁺T cells in number in peripheral blood could be proposed as predictive indicators for the disappearance of HBe Ag.