Purification and pressure dependence of alanine racemase from the psychro- piezophilic bacterium shewanella violacea DSS 12

Shewanella violacea DSS12 (S. violacea) is a psychrophilic and piezophilic bacterium, isolated from mud of the Ryukyu Trench in Japan. The bacterium displays optimal growth at 8°C and 30 MPa. Alanine racemase is an enzyme which catalyses the interconversion of l-alanine and d-alanine, and is responsible for the synthesis of d-alanine contained in the peptidoglycan of bacterial cell wall. In this study, we purified alanine racemase from S. violacea and investigated the enzymological characteristics of alanine racemase. The bacterium was aerobically cultured using marine broth 2216 in a 5-liter medium bottle at 4°C for 3 days. The bacterial cells were lysed by applying of 100 MPa pressure using a French press, and the lysate was centrifuged. The supernatant obtained was ultracentrifuged at 141,000 g, and the supernatant obtained was applied to ammonium sulfate fractionation. The active fraction was dissolved and passed through a butyl-Toyopearl, phenyl-Sepharose, and shodex KW-200 columns to obtain a partially purified enzyme. Consequently, the enzyme was purified 540-fold and showed a specific activity of 2.68 μmol/min/mg. Alanine racemase exhibited high activity against l-Ala and l-Ser as substrates. The optimal pH and temperature of alanine racemase were 9.0 and 25°C, respectively. Please click Additional Files below to see the full abstract

Soybean-Derived Phosphatidylserine Improves Memory Function of the Elderly Japanese Subjects with Memory Complaints

Soybean-derived phosphatidylserine (Soy-PS) is a phosphatidylserine made from soybean lecithin by enzymatic reaction with L-serine. A double-blind, randomized controlled study was conducted to investigate the effects of Soy-PS on the cognitive functions of the elderly Japanese subjects with memory complaints. Seventy-eight elderly people with mild cognitive impairment (50–69 years old) were randomly allocated to take Soy-PS (100 mg, 300 mg/day) or placebo for 6 months. As a result, there was no difference in blood markers and vital signs during Soy-PS treatment and any side effect caused by Soy-PS treatment was not observed. Neuropsychological test scores were similarly increased in all groups including placebo group. However, in the subjects with relatively low score at baseline, the memory scores in PS treated groups were significantly increased against the baseline, while those of placebo group remained unchanged. And the memory improvements in Soy-PS-treated groups were mostly attributed to the increase in delayed verbal recall, a memory ability attenuated in the earliest stage of dementia. In conclusion, Soy-PS used in this study is considered as safety food ingredient and 6 months of Soy-PS supplementation could improve the memory functions of the elderly with memory complaints

HSP70 and HSP90 Differentially Regulate Translocation of Extracellular Antigen to the Cytosol for Cross-Presentation

Antigens (Ag) from cancer or virus-infected cells must be internalized by dendritic cells (DCs) to be presented to CD8+ T cells, which eventually differentiate into Ag-specific cytotoxic T lymphocytes (CTLs) that destroy cancer cells and infected cells. This pathway is termed cross-presentation and is also implicated as an essential step in triggering autoimmune diseases such as Type I diabetes. Internalized Ag locates within endosomes, followed by translocation through a putative pore structure spanning endosomal membranes into the cytosol, where it is degraded by the proteasome to generate antigen peptides. During translocation, Ag is believed to be unfolded since the pore size is too narrow to accept native Ag structure. Here, we show that paraformaldehyde-fixed, structurally inflexible Ag is less efficient in cross-presentation because of diminished translocation into the cytosol, supporting the “unfolded Ag” theory. We also show that HSP70 inhibitors block both endogenous and cross-presentation. ImageStream analysis revealed that the inhibition in cross-presentation is not due to blocking of Ag translocation because a HSP70 inhibitor rather facilitates the translocation, which is in marked contrast to the effect of an HSP90 inhibitor that blocks Ag translocation. Our results indicate that Ag translocation to the cytosol in cross-presentation is differentially regulated by HSP70 and HSP90

Bacterial adaptation to high pressure: a respiratory system in the deep-sea bacterium Shewanella violacea DSS12

Abstract Shewanella violacea DSS12 is a psychrophilic facultative piezophile isolated from the deep sea. In a previous study, we have shown that the bacterium adapted its respiratory components to alteration in growth pressure. This appears to be one of the bacterial adaptation mechanisms to high pressures. In this study, we measured the respiratory activities of S. violacea grown under various pressures. There was no significant difference between the cells grown under atmospheric pressure and a high pressure of 50 MPa relative to oxygen consumption of the cell-free extracts and inhibition patterns in the presence of KCN and antimycin A. Antimycin A did not inhibit the activity completely regardless of growth pressure, suggesting that there were complex III-containing and -eliminating pathways operating in parallel. On the other hand, there was a difference in the terminal oxidase activities. Our results showed that an inhibitor-and pressure-resistant terminal oxidase was expressed in the cells grown under high pressure. This property should contribute to the high-pressure adaptation mechanisms of S. violacea

深海に生息する好圧性微生物における酵素の高水圧環境下への適応戦略と環境問題への応用

深海という環境は、恒常的な高水圧・低温といった極限環境にさらされた世界である。また、全海洋底に拡がっているプレート境界域には、熱水活動が繰り広げられており、水圧の影響により400℃を超える熱水の噴出が起こっている。そうした極限世界にも、現場環境に適応した実に多様な生物の世界が拡がっている。 　こうした深海世界には、高水圧下に適応した「好圧性微生物」が存在すると信じられてきており、その存在は、1979年、Yayanosらにより分離・培養され、証明された1)。その後、1990年に我が国の海洋科学技術センター(現、海洋研究開発機構)において、深海環境プログラム(DEEPSTAR計画)が組織されると、その知見は爆発的に拡がっていった2)。しかしながら、多様な「好圧性微生物」の世界が明らかになるに従い、いくつかの疑問が生じた。「なぜ、好圧菌は高圧下でよく生育できるのか？その分子生物学的なメカニズムは陸上微生物と比較してどこが違うのか？」、更には「好圧菌って役に立つの？」といった質問が投げかけられるようになった。 　本報告では、こうした疑問点に答えるために私たちの研究の現状を紹介し、未開拓の微生物資源でもある、深海微生物の隠された能力について解説する。発表要旨, 日本農芸化学会2013年度第1回関東支部例会（2013年7月6日, 東京農業大学世田谷キャンパス

Novel psychropiezophilic Oceanospirillales species Profundimonas piezophila gen. nov., sp. nov., isolated from the deep-sea environment of the Puerto Rico trench

The diversity of deep-sea high-pressure-adapted (piezophilic) microbes in isolated monoculture remains low. In this study, a novel obligately psychropiezophilic bacterium was isolated from seawater collected from the Puerto Rico Trench at a depth of ~ 6,000 m. This isolate, designated YC-1, grew best in a nutrient rich marine medium with an optimal growth hydrostatic pressure of 50 MPa (range 20-70 MPa) at 8 °C. Under these conditions the maximum growth rate was extremely slow, 0.017 h-1, and the maximum yield was 3.51 × 107 cells ml-1. Cell size changed with pressure, shifting from 4.0-5.0 μm in length and 0.5-0.8 μm in width at 60 MPa, to 0.8-1.0 μm diameter coccoid cells under 20 MPa, the minimal pressure required for growth. YC-1 is a Gram-negative, non-flagellum forming, facultative anaerobic heterotroph. Its predominant cellular fatty acids are the monounsaturated fatty acids (MUFAs) C16:1 and C18:1. Unlike many other psychropiezophiles YC-1 does not synthesize any polyunsaturated fatty acids (PUFAs). Phylogenetic analysis placed YC-1 within the family of Oceanospirillaceae, closely related to the uncultured symbiont of the deep-sea whale bone-eating worms of the genus Osedax. In common with some other members of the Oceanospirillales, including those enriched during the Deepwater Horizon oil spill, YC-1 is capable of hydrocarbon utilization. Based on its characteristics, YC-1 appears to represent both a new genus and a new species which we name “Profundimonas piezophila” gen. nov., sp. nov