Lactobacillus helveticus SBT2171 Inhibits Lymphocyte Proliferation by Regulation of the JNK Signaling Pathway

Lactobacillus helveticus SBT2171 (LH2171) is a lactic acid bacterium with high protease activity and used in starter cultures in the manufacture of cheese. We recently reported that consumption of cheese manufactured using LH2171 alleviated symptoms of dextran sodium sulfate (DSS)-induced colitis in mice. In this study, we have examined whether LH2171 itself exerts an inhibitory effect on the excessive proliferation of lymphocytes. We found that LH2171 inhibited the proliferation of LPS-stimulated mouse T and B cells, and the human lymphoma cell lines, Jurkat and BJAB. Cell cycle analysis showed an accumulation of LH2171-treated BJAB cells in the G2/M phase. Further, phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun was reduced by LH2171 in BJAB cells. Subsequently, expression of cell division cycle 2 (CDC2), regulated by the JNK signaling pathway and essential for G2/M phase progression, was inhibited by LH2171. It was also demonstrated that intraperitoneal administration of LH2171 strongly alleviated symptoms of collagen-induced arthritis (CIA) in mice. These findings suggest that LH2171 inhibits the proliferation of lymphocytes through a suppression of the JNK signaling pathway and exerts an immunosuppressive effect in vivo.     

The performance of peat-filled subsurface flow filters treating landfill leachate and municipal wastewater

The main objective of this study was to determine the treatment capacity of well-mineralized peat in vertical and horizontal flow filters designed to reduce phosphorus, nitrogen and organic matter in municipal wastewater from the town of Tapa and landfill leachate in Väätsa, Estonia. Two identically designed onsite experiments were conducted using the following filter systems: (a) a vertical flow (VF) peat filter, (b) a vertical flow peat/ash sediment filter (both materials in equal volumes) followed by a horizontal flow (HF) peat filter. Sphagnum peat and hydrated oil-shale ash (ash sediment) was used. In our experiments, one treated municipal wastewater over 6 months and another treated landfill leachate over 12 months. In both cases, effluent from a conventional treatment (aerated activated sludge treatment) plant was used. The median value of total phosphorus (TP) concentration in Väätsa landfill leachate was 3.4 mg P L^?1 and in municipal wastewater from Tapa 4.9 mg P L^?1. The reduction of TP in VF peat filters during the first 6 months was 58% and 63%, and in peat/ash sediment filters 94% and 67% for the Tapa experiment and the Väätsa experiment, respectively. Both experiments demonstrated that the P-removal efficiency in VF peat filters begins to decrease after 6 months of operation. The purification efficiency in HF filters fluctuated, and no significant removal of TP was found. In the removal of organic matter (BOD, COD values) and nitrogen, the best results were obtained in VF peat filters.     

Infertility of CD9-deficient mouse eggs is reversed by mouse CD9, human CD9, or mouse CD81; polyadenylated mRNA injection developed for molecular analysis of sperm-egg fusion

CD9 is a membrane protein belonging to the tetraspanin family. Despite CD9's broad tissue distribution, the only abnormality observed in CD9-deficient mice was infertility of females, which was responsible for a defect in the sperm-egg fusion process. However, the function of CD9 in sperm-egg fusion is not clear at all because the technique to analyze the activity of molecules in sperm-egg fusion has not been established. We demonstrated that the exogenous mouse CD9, expressed by polyadenylated mRNA injection at the germinal-vesicle stage oocytes, was precisely localized to the egg plasma membrane, and the expression reversed the infertility of CD9(-/-) eggs. Then, two other tetraspanins, human CD9 and mouse CD81, overexpressed with this technique on CD9(-/-) eggs restored the fertilization rate up to approximately 90 and approximately 50% against that of wild type eggs, respectively. Moreover, in the presence of an anti-mouse CD9 mAb, which blocks sperm-egg fusion, expression of human CD9 or mouse CD81 on eggs also rescued the fusibility. These results suggested that human CD9 plays a crucial role in human fertilization, and mouse CD81 has the potential to compensate for CD9 function in sperm-egg fusion. In addition, the polyadenylated mRNA injection is effective for molecular analysis of sperm-egg fusion.     

Mitogen-activated protein kinases-dependent induction of hepatocyte growth factor production in human dermal fibroblasts by the antibiotic polymyxin B

Hepatocyte growth factor (HGF) stimulates migration and proliferation of keratinocytes and has been suggested to be involved in wound healing. The cationic antibiotic polymyxin B (PMB) is commonly used as a topical antibiotic for wound care. If PMB possesses an HGF-inducing activity, the antibiotic is potentially beneficial for wound healing in addition to minimizing chances of infection. In this study, we found that PMB markedly induced HGF production from various types of cells including human dermal fibroblasts. Its effect was stronger than the effects of epidermal growth factor and cholera toxin and was comparable to the effect of 8-bromo-cAMP. Among the polymyxin family and polymyxin derivatives, colistin was also effective, whereas colistin methanesulfonate had only a marginal effect and PMB nonapeptide was ineffective. The stimulatory effect of PMB was accompanied by upregulation of HGF gene expression. Increase in phosphorylation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) was observed from 0.25h to 6h after the addition of PMB, while increase in phosphorylation of p38 mitogen-activated protein kinase (MAPK) was detected from 24h to 60h after PMB addition. The MAPK/ERK kinase inhibitor PD98059, the JNK inhibitor SP600125 and the p38 MAPK inhibitor SB203580 all potently inhibited PMB-induced HGF production. Lastly, proliferation of human dermal fibroblasts was significantly stimulated by PMB. These results indicate that PMB-induced HGF production and proliferation of human dermal fibroblasts and suggest that activation of MAPKs is involved in the induction of HGF production.     

Permanence of delayed population model with dispersal loss

Permanence of a dispersal single-species population model where environment is partitioned into several patches is considered. The species not only requires some time to disperse between the patches but also has some possibility to die during its dispersion. The model is described by delay differential equations. The existence of 'super' food-rich patch is proved to be sufficient to ensure partial permanence of the model. It is also shown that partial permanence implies permanence if each food-poor patch is chained to the super food-rich patch. Furthermore, it is proven that partial persistence is ensured if there exist food-rich patches and the dispersion of the species among the patches are small. When the dispersion is large, the partial persistence is realized under relatively small dispersion time.     

GroEL-substrate-GroES ternary complexes are an important transient intermediate of the chaperonin cycle

GroEL C138W is a mutant form of Escherichia coli GroEL, which forms an arrested ternary complex composed of GroEL, the co-chaperonin GroES and the refolding protein molecule rhodanese at 25 degrees C. This state of arrest could be reversed with a simple increase in temperature. In this study, we found that GroEL C138W formed both stable trans- and cis-ternary complexes with a number of refolding proteins in addition to bovine rhodanese. These complexes could be reactivated by a temperature shift to obtain active refolded protein. The simultaneous binding of GroES and substrate to the cis ring suggested that an efficient transfer of substrate protein into the GroEL central cavity was assured by the binding of GroES prior to complete substrate release from the apical domain. Stopped-flow fluorescence spectroscopy of the mutant chaperonin revealed a temperature-dependent conformational change in GroEL C138W that acts as a trigger for complete protein release. The behavior of GroEL C138W was reflected closely in its in vivo characteristics, demonstrating the importance of this conformational change to the overall activity of GroEL.     

Isolation of two human fibroblastic cell populations with multiple but distinct potential of mesenchymal differentiation by ceiling culture of mature fat cells from subcutaneous adipose tissue

Adipose tissue is a source of adult multipotent stem cells that can differentiate along mesenchymal lineage. When mature fat cells obtained from human subcutaneous adipose tissue were maintained with attachment to the ceiling surface of culture flasks filled with medium, two fibroblastic cell populations appeared at the ceiling and the bottom surface. Both populations were positive to CD13, CD90, and CD105, moderately positive to CD9, CD166, and CD54, negative to CD31. CD34, CD66b, CD106, and CD117, exhibited potential of unlimited proliferation, and differentiated along mesenchymal lineage to produce adipocytes, osteoblasts, and chondrocytes. The population that appeared at the ceiling surface showed higher potential of adipogenic differentiation. These observations showed that the cells tightly attached to mature fat cells can generate two fibroblastic cell populations with multiple but distinct potential of differentiation. Since enough number of both populations for clinical transplantation can be easily obtained by maintaining fat cells from a small amount of subcutaneous adipose tissue, this method has an advantage in preparing autologous cells for patients needing repair of damaged tissues by reconstructive therapy.     

Histopathological and electron microscopy studies on sleepy disease of koi Cyprinus carpio koi in Japan

Koi sleepy disease (KSD) usually occurs when koi Cyprinus carpio koi are taken from nursing earthen ponds and placed in cement-lined ponds containing fresh water in spring and autumn in Japan. We transfered koi from an earthen pond to tanks containing fresh water and 0.5 % salt water in an attempt to replicate KSD and prevent the onset of KSD, respectively, in the laboratory. KSD broke out after 4 to 5 d, followed by mass mortality (76%: 95/125 fish) within 17 d, in the fresh water. Diseased fish died within a few days. Examination revealed enlarged cells in the respiratory epithelia of gill lamellae; hyperplasia of interlammellar epithelia resulted in clubbing of gill filaments, which caused hypoxia when severe. Electron microscopy showed that enlarged cells contained immature particles (416-450 nm diameter) or mature virions (333-400 x 400-413 nm) of a pox-like virus in the cytoplasm. Mature virions were transported to the periphery of the cells. Hepatocytes, renal tubular epithelial cells, muscle cells of the lateral musculature and cardiac muscle cells were cloudy with mitochondrial degeneration. PCR assay using primer sets for a pox-like virus causing 'carp edema' determined that KSD-virus is the same as the carp edema virus. None of the koi held in 0.5% salt water showed sleepy disease during a 25 d experimental period; PCR assay revealed no KSD-virus in gills of koi in the same treatment.     

Stearoyl-CoA desaturase 1 deficiency increases insulin signaling and glycogen accumulation in brown adipose tissue

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of oleate (C18:1) and palmitoleate (C16:1), which are the main monounsaturated fatty acids of membrane phospholipids, triglycerides, wax esters, and cholesterol esters. Previously, we showed that SCD1 deficiency elevates insulin-signaling components and downregulates protein-tyrosine phosphatase-1B (PTP-1B) in muscle, a major insulin-sensitive tissue. Here we found that, in brown adipose tissue (BAT), another insulin-sensitive tissue, the basal tyrosine phosphorylations of insulin receptor (IR) and IR substrates (IRS-1 and IRS-2) were upregulated in SCD1(-/-) mice compared with wild-type mice. The association of IRS-1 and IRS-2 with the alpha-p85 subunit of phosphatidylinositol 3-kinase as well as Akt-Ser(473) and Akt-Thr(308) phosphorylation is also elevated in the SCD1(-/-) mice. The mRNA expression, protein levels, and activity of PTP-1B implicated in the attenuation of the insulin signal are reduced in the SCD1(-/-) mice. The content of GLUT4 in the plasma membrane increased 2.5-fold, and this was accompanied by a 6-fold increase in glucose uptake in BAT of SCD1(-/-) mice. The increased glucose uptake was associated with higher glycogen synthase activity and glycogen accumulation. In the presence of insulin, [U-(14)C]glucose incorporation into glycogen was increased in BAT of SCD1(-/-) mice. Taken together, these studies illustrate increased insulin signaling and increased glycogen metabolism in BAT of SCD1(-/-) mice.