Comparison of Soybean Tissue Extracted with Different Solvents

The enantioselective hydrolysis of (R,S)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine (I) with enzymes was investigated. Optically active I and its hydrolyzate, 7,8-difluoro-2,3-dihydro-3-hydroxymethyl-4H-[1,4]benzoxazine (II), are the intermediates for preparing optically active ofloxacins, whose racemate is known to be an excellent antibacterial agent. Lipoprotein lipase from Pseudomonas fluorescens (LPL Amano 3) was found to predominantly hydrolyze (S)-I, giving (R)-I in 54% e.e. and (R)-II in 44% e.e. On the other hand, lipase from Candida cylindracea was found to predominantly hydrolyze (R)-I, giving (S)-I in 24% e.e. and (S)-II in 20% e.e. Since, the optical purities of I and II thus obtained were not particularly high, these optically active I and II were converted into 3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4-(3,5-dinitrobenzoyl)-4H-[1,4]benzoxazine (IV). After recrystallizing IV from ethyl acetate-hexane, (S)- and (R)-II were obtained with high enantiomeric excess by removing the crystallized racemic IV and subsequently hydrolyzing the resulting optically active IV with alkali. The reduction of II afforded 7,8-difluoro-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine (III), for which the optical purity was estimated to be >96%e.e. by HPLC analysis. (R)- and (S)-ofloxacin were prepared from (R)- and (S)-III with retention of their configuration.     

Comparative Study between Gas Chromatography and Ion-exchange Chromatography on Amino Acid Analysis of Foods

Lactobacillus brevis and Saccharomyces cerevisiae were completely sterilized by the supercritical (SC) CO2 micro-bubble method. Gaseous (G) and liquid (LQ) CO2 were used in a similar manner to compare the sterilizing effect. Among the three treatments, the microorganisms were only effectively sterilized by the SC CO2 treatment at 25 MPa and 35°C.     

Isolation of Escherichia coli B Mutant Deficient in Glutathione Biosynthesis

Certain edible large jellyfishes belonging to the order Rhizostomeae are consumed in large quantities in China and Japan. The exumbrella part of the edible jellyfish Stomolophus nomurai was cut and soaked in dilute hydrochloric acid solution (pH 3.0) for 12 h, and heated at 121 °C for 20 min. The immunostimulation effects of the jellyfish extract were examined. The jellyfish extract enhanced IgM production of human hybridoma HB4C5 cells 34-fold. IgM and IgG production of human peripheral blood lymphocytes (PBL) were also accelerated, 2.8- and 1.4-fold respectively. Moreover, production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α by human PBL was stimulated 100- and 17-fold respectively. Collagenase treatment inactivated the immunostimulation activity of the jellyfish extract. In addition, purified collagen from bovine Achilles’ tendon accelerated IgM production of hybridoma cells. These facts mean that collagen has an immunostimulation effect, and that the active substance in jellyfish extract is collagen.     

Growth Profile of Crown Gall Cells of Tobacco in Suspension Culture

In order to obtain a basic information of plant cell suspension culture as a step toward the development of large scale culture, culture conditions of crown gall cells (auxin non-requiring cells) were investigated. Addition of yeast extract to culture medium was significantly effective for the growth and cell dispersion.

In experiments on the ability of the cultured cells to utilize sugars as the carbon source, it was observed that galactose, added to the culture medium, markedly inhibited the cell growth.

Pasteurization of the medium containing fructose as carbon source made it brownish by Maillard reaction and the medium apparently restrained the cell growth. However, the fructose medium sterilized by filtration was excellent for the cell growth as well as sucrose or glucose medium. In a jar fermentor, even the glucose medium became brownish by heat sterilization and the brown colored medium restrained the cell growth. Under optimum conditions, the doubling time was 1.1 day in exponential phase and 2.0 g of cell (dry weight) per 100 ml culture was obtained as the maximum yield.     

HTLV-1 bZIP Factor Impairs Anti-viral Immunity by Inducing Co-inhibitory Molecule,T Cell Immunoglobulin and ITIM Domain (TIGIT)

Human T-cell leukemia virus type 1 (HTLV-1) infects CD4⁺ T cells and induces proliferation of infected cells in vivo, which leads to the onset of adult T-cell leukemia (ATL) in some infected individuals. The HTLV-1 bZIP factor (HBZ) gene, which is encoded in the minus strand of HTLV-1, plays critical roles in pathogenesis. In this study, RNA-seq and ChIP-seq analyses using HBZ transduced T cells revealed that HBZ upregulates the expression and promoter acetylation levels of a co-inhibitory molecule, T cell immunoglobulin and ITIM domain (TIGIT), in addition to those of regulatory T cells related genes, Foxp3 and Ccr4. TIGIT was expressed on CD4⁺ T cells from HBZ-transgenic (HBZ-Tg) mice, and on ATL cells and HTLV-1 infected CD4⁺ T cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in vivo. Expression of Blimp1 and IL-10 was upregulated in TIGIT⁺CD4⁺ cells of HBZ-Tg mice compared with TIGIT^-CD4⁺ T cells, suggesting the correlation between TIGIT expression and IL-10 production. When CD4⁺ T cells from HBZ-Tg mice were stimulated with TIGIT’s ligand, CD155, their production of the inhibitory cytokine IL-10 was enhanced. Furthermore, dendritic cells from HBZ-Tg mice produced high levels of IL-10 after stimulation. These data suggest that HBZ alters immune system to suppressive state via TIGIT and IL-10. Importantly, TIGIT suppressed T-cell responses to another HTLV-1 virus protein, Tax, in vitro. Blocking of TIGIT and PD-1 slightly increased anti-Tax T-cell activity in some HAM/TSP patients. These results suggest that HBZ-induced TIGIT on HTLV-1 infected cells impairs T-cell responses to viral antigens. This study shows that HBZ-induced TIGIT plays a pivotal role in attenuating host immune responses and shaping a microenvironment favorable to HTLV-1.     

Development of an optimized 5-stage protocol for the in vitro preparation of insulin-secreting cells from mouse ES cells

In order to produce insulin-secreting cells with a high value of glucose-stimulated insulin secretion (GSIS) from mouse embryonic stem cells, we have developed an optimized 5-stage protocol by referring to culture conditions so far reported elsewhere. This protocol is characterized by 4 points: (1) use of an activin-free medium in the first stage, (2) use of gelatin/fibronectin coated culture dishes in 1–4 stages throughout, (3) removal of undifferentiated cells by cell sorter at the end of 4th stage, and (4) sedimental culture in the 5th stage. GSIS value of the produced cells reached 2.4, that was at a higher rank of those so far reported. The produced cells were transplanted in diabetes model mice but no remedy effect was observed. Then transplantation was conducted in pre-diabetes model mice, in which GSIS was impaired without affecting insulin producing function. The transplantation of 5 × 10⁶ cells resulted in a marked improvement of glucose tolerance within 20 days. This effect decreased but was still observed at 120 days post-transplantation. This demonstrates the feasibility of the novel optimized protocol.