Crystallization of Amorphous GdFe_2H_x Alloys Prepared by Hydrogen Absorption

Amorphous a-GdFe_2H_x alloys were prepared by two kinds of technique, i.e., the newly developed hydrogen-induced amorphization (HIA) of the Laves phase c-GdFe_2 and hyrogenation of the rapidly quenched amorphous a-Gd_Fe_ alloy. The formation of a GdFe_2H_x by hydrogen absorption was found to occur between 473 K and 573 K where the decomposition of c-GdFe_2 into the elemental hydride GdH_2 and α-Fe is suppressed. On the other hand, the rapidly quenched a-Gd_Fe_ alloy could absorb hydrogen in the amorphous state below 523 K. The crystallization behavior of the hydrogen-induced amorphous a-GdFe_2H_ alloy was similar to that of the hydrogenated amorphous a-Gd_Fe_H_ alloy. That is, the DSC curves of the a-GdFe_2H_x alloys showed a broad endothermic peak resulting partial desorption of hydrogen together with two exothermic peaks. The first exothermic peak was associated with the crystallization of the amorphous phase into GdH_2 and α-Fe, and the second one was attributed to the growth of them. On the other hand, a-Gd_Fe_ crystallized polymorphously to the Laves phase c-GdFe_2 showing an exothermic peak

Expression, Purification and Properties of Alanine Racemase from Thermus thermophillus HB8

An alanine racemase (EC 5.1.1.1) from an extreme thermophilic bacterium Thermus thermophilus HB8, was purified and characterized, and its gene was cloned. The cloned alanine racemase gene (alr) was expressed in Escherichia coli JM 109. The alr gene is composed of a 1080 bp and encoded a 360 amino acid, and was predicted to have a molecular weight of 38,596. The enzyme was purified by heat shock at 70°C for 10min and DEAE Toyopearl 650M column chromatography. The purified enzyme had an optimum pH9.0∼10.0 and an optimum temperature of 55°C∼60°C. Enzyme activity was retained 100% after incubation of the enzyme at 70°C for 10min. Alanine racemase from Thermus thermophilus HB8 is a monomeric enzyme with a molecular mass of 39 kDa.高度好熱性細菌 Thermus thermophilus HB8 由来アラニンラセマーゼ遺伝子を大腸菌中にクローニングし、発現させた後に、精製及び性質検討を行った。alr遺伝子は1080bpからなり360アミノ酸残基 HB8をコードしていたので、本酵素は38,596Daの分子量であると予想された。alr遺伝子の [ G + C ] 含量は、72%であり、Tm値は98.8℃であった。T.thermophilus HB8由来アラニンセマーゼを中等度好熱性細菌 Geobacillus stearothermophilus 及び赤痢菌 Shigella sonnei 由来アラニンラセマーゼと一次配列の比較をしたところ、G.stearothermophilus 由来のものと33%、赤痢菌由来の酵素を28%の相同性を示した。T.thermophilus HB8 由来アラニンラセマーゼを、70℃で10分間の熱処理後、DEAE-トーヨーパール陰イオン交換カラム等により精製した。精製酵素の最適温度は、d-アラニンからl-アラニンへの反応で55℃、l-アラニンからd-アラニンへの反応では60℃であり、最適pHは、9.0〜10.0であった。また、70℃で30分インキュベーションを行った後にも、活性の低下は見受けられず耐熱性を示した。更に、本酵素は分子量38,000モノマー酵素であると推定され、その反応機構に興味が持たれる

Successful Resolution of Fecal Impaction During Endoscopy Using a Looped Guidewire

Fecal impaction is the impaired excretion of a large fecal mass, and mild cases are treated by enema and osmotic laxatives. However, treatment-resistant cases need more invasive alternatives. A woman in her 60s presented with abdominal discomfort. Her abdomen was soft and without tenderness. Computed tomography revealed a large mass of feces in her sigmoid colon and no intestinal dilatation proximal to the mass. Endoscopy confirmed a fecal mass occupying the lumen. A glycerin enema, oral administration of polyethylene glycol, and enteral administration of amidotrizoic acid during colonoscopy were ineffective. We maneuvered a guidewire to form a loop at the tip of an endoscope, with which we subdivided the mass for successful removal. The patient’s abdominal discomfort disappeared immediately. Endoscopic disimpaction is far less invasive than surgery and should be considered when treating fecal impaction cases, without severe obstructive colitis, which are nonresponsive to conservative treatment

Purification and Characterization of Cystathionine γ-Synthase from Thermoacidophilic Archaea Sulfolobus tokodaii

The gene encoding a cystathionine γ-synthase from Sulfolobus tokodaii was cloned and expressed in Escherihia coli Rosetta-gami (DE3). Cystathionine γ-synthase ［EC 2. 5. 1. 48］ from Sulfolobus tokodaii (stCGS) was purified by heat treatment, DEAE- Toyopearl 650M and Sephacryl S-300 column chromatographies from E. coli transformants. stCGS shows optimum activity at pH 7.0, and is stable between pH5.0 and pH9.0. The optimum temperature of stCGS is above 100℃, and the enzyme showed the remaining activity of almost 100% up to 70℃. The K(m) and V(max) with O-phospho-L- homoserine as a substrate are 0.82 mM and 2.42 U/mg. To analyze the role of Phe 97 in the active site of stCGS, we constructed F97Y, R99C, and F97Y-R99C mutant enzymes. Although native stCGS has no activity toward l-methionine, F97Y mutant enzyme gained the elimination activity toward L-methionine.好熱好酸性アーキア Sulfolobus tokodaii 由来シスタチオニンγﾝシンターゼ（stCGS）遺伝子を pET-11a に組み込み pET-stCGS を構築した．このベクターでE. coli Rosettaﾝgami（DE3）を形質転換し，本遺伝子を発現させ，精製及び性質検討を行った．大腸菌で発現したシスタチオニンγﾝシンターゼの活性が無細胞抽出液で確認できた．S. tokodaii シスタチオニンγﾝシンターゼを70℃熱処理 DEAEﾝトヨパールイオン交換カラム等により単一精製した．精製酵素の最適温度は100℃以上であり，熱安定性は60分間処理で70℃までほぼ100ｵの残存活性を示した．また，最適pHについてはリン酸緩衝液やブリトンﾝロビンソン広域緩衝液の場合はpH7.0の時が最も活性が高く，トリス塩酸緩衝液の場合はpH9.0が最適であった．pH安定性についてはpH5.0～9.0において安定であった．O-ホスホ-l-ホモセリンに対するKm，Vmaxは，それぞれ0.82mM，2.42U/㎎であった．アポ酵素のホロ化実験により，本酵素活性がPLP に依存していることが明らかとなった．更に本酵素の脱離反応での基質特異性の検討を行った．変異酵素を用いた実験により，stCGSの基質特異性には，活性中心に存在するPhe97を含む領域が深く関わっていることが示唆された