Hyperthermophilic archaea as a source for novel enzyme discovery

The Archaea are a group of microorganisms that are phylogenetically distinct to the Bacteria and Eucarya. Their size and shape resemble bacteria, but display stark differences in the structure of their membrane lipids and machinery that are responsible for DNA replication and transcription. In addition, Archaea seem to utilize metabolic pathways that differ to previously recognized, classical pathways in bacteria and eukaryotes. Based on the genome sequences of the Archaea, there are many cases in which a particular metabolic pathway seems to be absent or incomplete. The search for these “missing” pathways or enzymes has been an exciting field of research in the Archaea, and has led to the discovery of structurally novel enzymes or enzymes with novel activity. Until now, we have been focused on the metabolism of the hyperthermophilic archaeon Thermococcus kodakarensis. The organism is an obligate anaerobe and heterotroph, utilizing a wide range of organic compounds including peptides/amino acids, starch and maltooligosaccharides, and organic acids such as pyruvate. By searching for missing genes, we have identified a structurally novel fructose-1,6-bisphosphatase [1], a key enzyme in gluconeogenesis, and enzymes with novel activity, such as pantoate kinase and phosphopantothenate synthetase, both involved in coenzyme A biosynthesis in T. kodakarensis [2]. A number of genes predicted to encode kinases are present on the T. kodakarensis genome. Although more than half display similarity to characterized enzymes high enough to predict their substrates, there are still nearly 20 genes whose substrates are unknown. One of them turned out to be a myo-inositol 3-kinase [3]. Another was found to display an ADP-dependent ribose-1-phosphate kinase activity, which participates in a pentose bisphosphate pathway, a previously unidentified route to direct the ribose moieties of nucleosides to central carbon metabolism [4]. Another kinase was identified through studies on serine and cysteine metabolism in T. kodakarensis. The protein was initially annotated as a chromosome-partitioning protein ParB, but displayed ADP-dependent serine kinase activity. The enzyme was necessary for the conversion of Ser to Cys in vivo, and is most likely involved in Ser assimilation in this archaeon [5]. The structure of the enzyme explains the specificity of the enzyme towards Ser and ADP, and raises the possibility that structurally related proteins may also be present not only in archaea but also in bacteria [6]. These studies indicate the potential of archaea as a source for novel enzyme discovery demonstrate various means to identify gene function through genome sequence information. [1] Rashid N, Imanaka H, Kanai T, Fukui T, Atomi H, Imanaka T. A novel candidate for the true fructose-1,6-bisphosphatase in archaea. J. Biol. Chem., 277(34):30649-30655, 2002. [2] Yokooji Y, Tomita H, Atomi H, Imanaka T. Pantoate kinase and phosphopantothenate synthetase, two novel enzymes necessary for CoA biosynthesis in the Archaea. J. Biol. Chem., 284(41):28137-28145, 2009. [3] Sato T, Fujihashi M, Miyamoto Y, Kuwata K, Kusaka E, Fujita H, Miki K, Atomi H. An uncharacterized member of the ribokinase family in Thermococcus kodakarensis exhibits myo-inositol kinase activity. J. Biol. Chem., 288(29):20856-20867, 2013. [4] Aono R, Sato T, Imanaka T, Atomi H. A pentose bisphosphate pathway for nucleoside degradation in Archaea. Nat. Chem. Biol., 11(5):355-360, 2015. [5] Makino Y, Sato T, Kawamura H, Hachisuka SI, Takeno R, Imanaka T, Atomi H. An archaeal ADP-dependent serine kinase involved in cysteine biosynthesis and serine metabolism. Nat. Commun., 7:13446, 2016. [6] Nagata R, Fujihashi M, Kawamura H, Sato T, Fujita T, Atomi H, Miki K. Structural study on the reaction mechanism of a free serine kinase involved in cysteine biosynthesis. ACS Chem. Biol. Article ASAP, 2017. doi:10.1021/acschembio.7b00064

Low-temperature synthesis of oriented CoPtCu-MgO and CoFePt-Ag-SiO₂ nanocomposite thin films by rf-magnetron sputtering

Nanocomposite thin films composed of oriented Co₅₀Pt₄₄Cu₆ nanoparticles embedded in a MgO matrix has been synthesized by rf-magnetron co-sputtering onto NaCl(001) substrates kept at 620 K. As the sputtering power increases, (001) orientation is improved and atomic ordering is also promoted. However, superlattice reflections are quite weak and the ordered region is limited in local area of the nanoparticles, indicating low degree of order. Electron diffraction and elemental mapping revealed that additive Cu is alloyed with CoPt. We have also fabricated (Co₂₆Fe₂₀)Pt₄₄Ag₁₀SiO₂ nanocomposite thin films at 675K for comparison. Atomic ordering is promoted by Fe and Ag addition as well as higher substrate temperature; however, it was found that ternary element (Cu, Ag, Fe) addition into CoPt alloy is not so effective to promote atomic ordering. Origin of the slow ordering kinetics is discussed using thermodynamical parameters. © 2014 The Ceramic Society of Japan. All rights reserved.Kazuhisa SATO, Tamotsu KOSAKA, Toyohiko J. KONNO, Low-temperature synthesis of oriented CoPtCu–MgO and CoFePt–Ag–SiO₂ nanocomposite thin films by rf-magnetron sputtering, Journal of the Ceramic Society of Japan, 2014, 122, 1425, p. 317-321

成長発達過程におけるサイロトロピン放出ホルモンの生理的役割

金沢大医学部サイロトロピン放出ホルモン(TRH)とその代謝産物であるサイクロヒスチジンプロリン(CHP)が新生仔ラット体温調節機能の成熟に及ぼす影響を検討した。生后1週令の幼若ラットに【10^(-11)】〜【10^(-9)】モルのTRHを6日間髄注すると体温恒常性を獲得する3週令に有意の高体温が認められ、寒冷暴露からの体温回復が促進された。【10^(-10)】モルcHPは低体温を来し寒冷からの体温回復も遅延した。TRHの体温上昇作用はカテコールアミン(CA)合成阻害剤で消去されるので、中枢神経CAを介して発現すると考えられた。事実CA代謝回転率はTRHにより2〜4倍に促進され、TRH処置2週后の脳内ノルエピネフリン(NE)ドーパミン(DA)量は有意に低下した。この時期に一致してTRH処置ラットは低体温を示し、褐色脂肪ミトコンドリアのグアノシン2燐酸(GDP)結合能,α-グリセロ燐酸脱水素酵素,肝ミトコンドリアのチトクロムC還元酵素活性も減少した。以上の成績はTRHが中枢神経CA放出を介して熱産生を促進し、cHPはこれを抑制することを示している。一方TRHは甲状腺ホルモン分泌をも促進するので、次にCAと甲状腺ホルモンの熱産生に関する相互関係を検討した。ラットに甲状腺機能低下,亢進症を作成し、寒冷馴化過程における3CA代謝の変動をみると、機能低下ラットでは馴化につれて脳内DA,副腎CA,褐色脂肪NE含量は著明に増加した。さらに脳チロジン水酸化酵素活性,副腎DA-β-水酸化酵素活性,肝細胞膜βアドレナリン受容体特異結合能も増大した。逆に機能亢進ラットでは褐色脂肪NE量,ミトコンドリアGDP結合能,肝β受容体結合能は低下し、CAを介する熱産生の抑制が認められた。この結果から熱産生に関して甲状腺ホルモンとCAは相互補償的に作用するものと考えられる。中枢神経TRH-cHP系は両者の制禦因子として調節にあずかっている可能性が高いが、この点の検討は今後の課題として残された。The effects of thyrotropin-releasing hormone (TRH) and its metabolite, cyclo-histidine-proline (cHP), on the homeothermic development of neonatal rats were studied. The intrathecal administration of 10 - 10 moles of TRH during the second week of age produced a hyperthermia by 3 weeks of age; this was followed by a transient period of hypothermia. This effect was abolished by the simultaneous administration of 6-hydroxydopamine. In contrast, cHP decreased thermogenesis. TRH accelerated brain norepinephrine (NE) and dopamine (DA) release, resulting in a reduction of cerebral NE and DA at 4 weeks of age. In addition, mitochondrial [ ]-guanosine diphosphate(GDP) binding capacity and -glycerophosphate dehydrogenase activity in brown adipose tissue (BAT) were reduced. Liver cytochrome C reductase activity was also reduced. These results indicate that TRH stimulates central NE release, thereby enhancing thermogenesis while cHP decreases heat production.Since TRH stimulates both catecholamine (CA) and thyroid hormone release, the next experiment was investigated for the interrelation between CA and thyroid hormone during cold acclimation in rats. Hypothyroidism was induced by the administration of propylthiouracil and hyperthyroidism by the injection of thyroxine. Although hypothyroid rats maintained their body temperature within the normal range during cold exposure, the body weight gain was markedly impaired. They had increased cerebral DA, adrenal CA and BAT NE, enhanced cerebral tyrosine hydroxylase and adrenal DA- -hydroxylase activities and elevated [ ]-dihydroalprenolol (DHA) binding to liver plasma membranes. In contrast, BAT NE, [ ]-GDP binding and hepatic [ ]-DHA binding in hyperthyroid rats were reduced after 4 weeks of cold acclimation. These results indicate that thyroid hormone deficiency is associated with an accelerated CA synthesis and release, whereas the hyperthyroid state suppresses CA release, hepatic DHA binding and BAT heat production. Thus there is a close interrelationship between TRH, thyroid hormone and CA. TRH may play the role of a modulator in thermogenesis via CA and thyroid hormone regulation.研究課題/領域番号:59480228, 研究期間(年度):1984 – 1986出典：研究課題「成長発達過程におけるサイロトロピン放出ホルモンの生理的役割」課題番号59480228（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-59480228/594802281986kenkyu_seika_hokoku_gaiyo/）を加工して作

低エネルギー宇宙線加速源の解明に向けた楯座腕超新星残骸の系統的な研究

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 大橋 正建, 東京大学教授 寺澤 敏夫, 東京大学教授 山本 智, 東京大学教授 手嶋 政廣, 東京大学准教授 吉越 貴紀University of Tokyo(東京大学

Flow Cytometric Assessment of Neutrophil Oxidative Metabolism in Chronic Granulomatous Disease on Small Quantities of Whole Blood: Heterogeneity in Female Patients

A rapid and sensitive flow cytometric assay is presented for the quantitative estimation of the oxidative metabolic activity of individual polymorphonuclear leukocytes (PMN) on less than 100 td of whole blood. This procedure is a simplified version using whole blood of the method of Bass et al (J. Immunol. 130:1910, 1983) that estimated the metabolic burst activity of phorbol myristate acetate (PMA)-stimulated individual PMN as the intracellular generation of a fluorescence product by a flow cytometric assay. With this method, almost all the PMN from normal subjects responded to PMA as a single cell population generating bright intracellular fluorescence. PMN from a boy with chronic granulomatous disease (CGD), could not respond to PMA with any increase of their fluorescence intensity. His mother had two distinct PMN populations one functionally normal and the other defective, indicating a random lyonization in the carrier mother and the X-linked recessive mode of inheritance. In two female patients with CGD from unrelated families, their PMN responded to PMA, as a whole, with a minimal increase in the fluorescence intensity, but the metabolic defects in their PMN were not so complete as seen in a classical X-linked CGD boy. But, PMN from two female sibling patients from the other family responded to PMA as a single uniform cell population with a weak but definite fluorescence intensity. However, the genetic background of these female patients with CGD remains unclear, since PMN dysfunction could not be identified in their mothers with this method.This work was supported in part by a grant (No. 58440046) from the Ministry of Education of Japan

Chaotic Itinerancy in Regional Business Cycle Synchronization

Chaotic itinerancy is complex behavior in high-dimensional dynamical systems　characterized by itinerant motion among many different ordered states through chaotic transition. In this study, we robustly observe this behavior in a model of regional business cycles, in which all regions are homogeneous and connected through producers' adaptive behavior based on global information. Although producers adjust their output quite slowly toward the average level announced by the government, regional business cycles begin to synchronize due to the entrainment effect. Moreover, the economy is more likely to exhibit chaotic itinerancy when the producers emphasize the expected-profit-maximization and when they adjust their expectations more slowly toward the average. It is also clarified that behind the dynamics of chaotic itinerancy exist cycles among periodic orbits with different unstable directions, which is called　unstable dimension variability