Transformation of shikonin by a cell-free extract of Eubacterium sp. A-44, a human intestinal bacterium

For the purpose of investigating the metabolic processes of shikonin (1) by human intestinal bacteria, we prepared a sonicated bacterial cell suspension and a crude enzyme preparation from Eubacterium sp. A-44, one of the intestinal bacteria capable of transforming 1 to various metabolites. After anaerobic incubation with the suspension for 1 hr, most of 1 was transformed to prometaboshi-konin (2), and metaboshikonins I (3) and II (4). However, under aerobic conditions, the dimers, shikometabolins A (5) and B (6), were predominantly formed. In the presence of the crude enzyme preparation, formation of 2-4 was inhibited by oxygen, but markedly enhanced by the addition of NADH. On the other hand, formation of 5 and 6 was appreciably accelerated by the addition of NAD^+. In the absence of the crude enzyme preparation, NADH and/or NAD^+ showed no ability to transform 1 to the any metabolites, as in the case of a thermally inactivated preparation. Accordingly, the two different metabolic processes leading to compounds 2-4 and com-pounds 5 and 6 by Eubacterium sp. A-44 are concluded to be enzyme-dependent in the presence of NADH and NAD^+. ヒト腸内細菌による紫根成分shikoninの代謝過程を研究する目的でshikonin(1)を種々の代謝物に変換する腸内細菌Eubacterium sp.A-44の細胞破砕懸濁液及び粗酵素標品を調製した。この粗酵素標品とshikonin(1)を嫌気条件下で1時間インキュベーションすると,shikonin(1)のほとんどはprometaboshikonin(2)およびmetaboshikonin I(3),II(4)に変換された。しかしながら,好気条件下ではshikometabolin A(5),B(6)などの二量体が生成した。化合物2-4の生成は,酸素によって阻害され,NADHの添加により増強した。一方,化合物5,6の生成はNAD^+の添加により増加した。加熱処理した場合と同様,粗酵素標品を除去した場合は,NADH,NAD^+を添加してもshikoninの変換は起こらなかった。したがって,NADHやNAD^+の存在下での化合物2-4や化合物5,6への変換過程は酵素反応によって触媒されることを示している

Human intestinal Bacteroides spp. RHEIN-I and RHEIN-II capable of transforming rhein to rheinanthrone, induce rhein-dependent diarrhea in rats

Two rhein-metabolizing bacteria were isolated from human feces. The biochemical and morphological characteristics of both isolates were typical of Bacteroides spp. and named strains RHEIN-I and RHEIN-II, respectively. Rhein was effectively metabolized to rheinanthrone by both strains. In conventional male Wistar rats, diarrhea was not induced after oral administration of rhein at a dose of 100 mg/kg (fecal water content of 71 %), in spite of severe diarrhea with sennoside B at a dose of 40 mg/kg (increase of fecal water content to 89 %). Also in germ-free rats, rhein did not induce any diarrhea. Gnotobiote rats colonized with B. sp. strain RHEIN-I developed diarrhea (fecal water content increased to 85 %) 11 hr after the oral administration of rhein. These findings indicate that rhein-transforming bacteria are responsible for the laxative effect associated with the ingestion of rhein and rhein containing preparations. rhein, rheinanthrone, diarrhea, Bacteroides, gnotobiote. ヒト糞便から2種のrhein代謝細菌を単離した。これらは生化学的および形態学的特徴からBcteroidesに属する種と判断され,RHEIN-I,RHEIN-II株と仮称した。rheinは両菌株により完全にrheinanthroneに代謝された。通常の雄Wistarラットにおいてはrhein 100mg/kgの経口投与で下痢が生じなかったが(糞便の水分含量は71%),sennoside Bでは40mg/kgの用量で激しい下痢を生じた(水分含量89%)。又,無菌ラットではrheinは何ら下痢作用を示さなかった。ところが,Bacteroides sp. strain RHEIN-Iを消化管内に棲息させたラットではrhein経口投与の11時間後に下痢が生じた(糞便の水分含量は85%)。この結果は,これらrhein代謝菌がrheinやrheinを含む製剤を摂取した場合に生じる下痢作用に関与していることを示している

Three novel oligosaccharides synthesized using Thermoanaerobacter brockii kojibiose phosphorylase

<p>Abstract</p> <p>Background</p> <p>Recently synthesized novel oligosaccharides have been produced primarily by hydrolases and glycosyltransferases, while phosphorylases have also been subject of few studies. Indeed, phosphorylases are expected to give good results via their reversible reaction. The purpose of this study was to synthesis other novel oligosaccharides using kojibiose phosphorylase.</p> <p>Results</p> <p>Three novel oligosaccharides were synthesized by glucosyltransfer from β-D-glucose 1-phosphate (β-D-G1P) to xylosylfructoside [<it>O</it>-α-D-xylopyranosyl-(1→2)-β-D-fructofuranoside] using <it>Thermoanaerobacter brockii </it>kojibiose phosphorylase. These oligosaccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methyl derivatives, MALDI-TOF MS and NMR measurements were used for structural characterisation. The ¹H and ¹³C NMR signals of each saccharide were assigned using 2D-NMR including COSY (correlated spectroscopy), HSQC (herteronuclear single quantum coherence), CH₂-selected E-HSQC (CH₂-selected Editing-HSQC), HSQC-TOCSY (HSQC-total correlation spectroscopy) and HMBC (heteronuclear multiple bond correlation).</p> <p>Conclusion</p> <p>The structure of three synthesized saccharides were determined, and these oligosaccharides have been identified as <it>O</it>-α-D-glucopyranosyl-(1→2)-<it>O</it>-α-D-xylopyranosyl-(1→2)-β-D-fructofuranoside (saccharide <b>1</b>), <it>O</it>-α-D-glucopyranosyl-(1→2)-<it>O</it>-α-D-glucopyranosyl-(1→2)-<it>O</it>-α-D-xylopyranosyl-(1→2)-β-D-fructofuranoside (saccharide <b>2</b>) and <it>O</it>-α-D-glucopyranosyl-(1→[2-<it>O</it>-α-D-glucopyranosyl-1]₂→2)-<it>O</it>-α-D-xylopyranosyl-(1→2)-β-D-fructofuranoside (saccharide <b>3</b>).</p

慢性肺疾患の早産児におけるプロテインC経路 : 前向き研究

Background: Chronic lung disease (CLD) is a major neonatal pulmonary disorder associated with inflammation. Recent studies have shown that protein C anticoagulant pathways, such as those for protein C (PC), protein S (PS), and thrombomodulin (TM), could be useful indices for reflecting pulmonary injury. However, the involvement of these factors in preterm infants with very low birthweight (VLBW) who have developed CLD remains to be investigated. Here, we investigated whether PC pathway-related factors could predict the development of CLD in preterm infants with VLBW. Methods: We collected plasma samples from 26 preterm infants with VLBW (13 each from those with and without CLD) at the time of birth and measured TM, PC, and PS levels in their plasmas. We analyzed prospectively the relationship between these factors in infants with and without CLD. Results: There were significant differences in gestational age, birthweight, Apgar score (5 min), and duration of mechanical ventilation between the CLD and non-CLD groups. No significant differences in the PC and PS levels at birth were observed between the two groups, whereas the TM levels in the CLD group were significantly higher than those in the non-CLD group (P = 0.013). The TM levels correlated with gestational age and duration of mechanical ventilation. However, covariance analysis demonstrated that gestational age was significantly associated with TM levels, and consequently, development of CLD was not associated with TM level at birth. Conclusions: Thrombomodulin, PC, and PS levels at birth could not predict the development of CLD in preterm infants with VLBW.博士（医学）・甲第850号・令和4年9月28日© 2022 Japan Pediatric Society.This is the peer reviewed version of the following article: [https://onlinelibrary.wiley.com/doi/10.1111/ped.15221], which has been published in final form at [https://doi.org/10.1111/ped.15221]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.発行元が定める登録猶予期間終了の後、本文を登録予定（2023.01

Factors that contribute to long-term survival in patients with leukemia not in remission at allogeneic hematopoietic cell transplantation

<p>Abstract</p> <p>Background</p> <p>There has been insufficient examination of the factors affecting long-term survival of more than 5 years in patients with leukemia that is not in remission at transplantation.</p> <p>Method</p> <p>We retrospectively analyzed leukemia not in remission at allogeneic hematopoietic cell transplantation (allo-HCT) performed at our institution between January 1999 and July 2009. Forty-two patients with a median age of 39 years received intensified conditioning (n = 9), standard (n = 12) or reduced-intensity conditioning (n = 21) for allo-HCT. Fourteen patients received individual chemotherapy for cytoreduction during the three weeks prior to reduced-intensity conditioning. Diagnoses comprised acute leukemia (n = 29), chronic myeloid leukemia-accelerated phase (n = 2), myelodysplastic syndrome/acute myeloid leukemia (MDS/AML) (n = 10) and plasma cell leukemia (n = 1). In those with acute leukemia, cytogenetic abnormalities were intermediate (44%) or poor (56%). The median number of blast cells in bone marrow (BM) was 26.0% (range; 0.2-100) before the start of chemotherapy for allo-HCT. Six patients had leukemic involvement of the central nervous system. Stem cell sources were related BM (7%), related peripheral blood (31%), unrelated BM (48%) and unrelated cord blood (CB) (14%).</p> <p>Results</p> <p>Engraftment was achieved in 33 (79%) of 42 patients. Median time to engraftment was 17 days (range: 9-32). At five years, the cumulative probabilities of acute graft-versus-host disease (GVHD) and chronic GVHD were 63% and 37%, respectively. With a median follow-up of 85 months for surviving patients, the five-year Kaplan-Meier estimates of leukemia-free survival rate and overall survival (OS) were 17% and 19%, respectively. At five years, the cumulative probability of non-relapse mortality was 38%. In the univariable analyses of the influence of pre-transplant variables on OS, poor-risk cytogenetics, number of BM blasts (>26%), MDS overt AML and CB as stem cell source were significantly associated with worse prognosis (p = .03, p = .01, p = .02 and p < .001, respectively). In addition, based on a landmark analysis at 6 months post-transplant, the five-year Kaplan-Meier estimates of OS in patients with and without prior history of chronic GVHD were 64% and 17% (p = .022), respectively.</p> <p>Conclusion</p> <p>Graft-versus-leukemia effects possibly mediated by chronic GVHD may have played a crucial role in long-term survival in, or cure of active leukemia.</p

Thermoanaerobacter brockii Kojibiose Phosphorylaseによって合成された新規三および四糖の分離および同定

Novel tri- and tetra-saccharides were synthesized by glucosyltransfer from β-D-glucose 1-phosphate (β-D-G1P) to palatinose using Thermoanaerobacter brockii kojibiose phosphorylase. There saccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methyl derivatives, MALDI-TOF MS and NMR measurements were used for structural confirmation of the saccharides. The ^1H and ^13C NMR signals of the saccharides were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY and HMBC. These oligosaccharides were identified as 2^G-α-D-glucopyranosyl-palatinose; O-α-D-glucopyranosyl-(1→2)-O-α-D-glucopyranosyl-(1→6)-D-fructofuranose and 2^G(2-α-D-glucopyranosyl)_2-palatinose; O-α-D-glucopyranosyl-(1→2)-O-α-D-glucopyranosyl-(1→2)-O-α-D-glucopyranosyl-(1→6)-D-fructofuranose. パラチノースは抗う蝕性のようなさまざまな機能をもつことが知られている．しかしながら，パラチノースはゆっくりではあるが小腸で加水分解をうけるため，糖尿病のような疾患をもつ患者への使用は薦められない．さらに，この糖のような低分子のオリゴ糖は比較的高浸透圧になりやすいため生体にとってよくない影響を及ぼすことがある．本研究では，二糖であるパラチノースを用い，Thermoanaerobacter brockii kojibiose phosphorylaseのグルコシル転移作用を利用し，グルコース1リン酸とパラチノースから新規オリゴ糖を合成した．反応は糖1および糖2が効率よく生成した48時間で止めた (Fig. 1)．また，転移生成物である糖1は反応10時間で最大となった (Fig. 2)．活性炭-セライトカラムおよび調製用HPLCを用いて糖1および糖2を単離し，MALDI-TOF-MS分析およびメチル誘導体のガスクロマトグラフィー分析を行い構造の推定を行った (Fig. 3)．さらにCOSY，HSQC，HSQC-TOCSYおよびHMBC (Fig. 4) の各手法を用いた2次元NMR解析により糖1を2^G-α-D-glucopyranosyl-palatinose; O-α-D-glucopyranosyl-(1→2)-O-α-D-glucopyranosyl- (1→6)-D-fructofuranose，糖2を2^G(2-α-D-glucopyranosyl)_2-palatinose; O-α-D-glucopyranosyl-(1→2)-O-α-D-glucopyranosyl- (1→2)-O-α-D-glucopyranosyl-(1→6)-D-fructofuranoseと同定した (Table 1)．今後，これらの糖の栄養機能について明らかにする必要があ

Pb/Cd Ratios in Fresh Snow Derived from Cold Air Mass from Northern North Eastern China and Russian Maritime Province along the Coast of the Japan Sea in Honshu Island and a Long-range Transport Mechanism during Winter

粗大粒子の影響を避けるために近隣の地方工業都市から15 km以上離れた遠隔地において降雪・雨の試料を採取した．2014年及び2015年に日本の本州日本海沿いの遠隔地で採取した新雪中のPbとCdの濃度を2010，2014及び2015年に四国の遠隔地で採取された新雪と降雨試料中の濃度と共に報告した．降雪または降雨中の大気塊の起源は後方流跡線上の24時間前の位置から推定した．PbとCdの相関関係から北東中国の黒竜江省-極東ロシア（沿海地方）からの寒気塊に原因した降雪中では[Pb]/[Cd]＝27.4±2.8（R2＝0.979）が得られた．日本起源の場合の降水では[Pb]/[Cd]＝2.8±0.4（R2＝0.732）であった．PbとCdの発生地域を推定できる方法を提案した．中国華北，北東中国南部，朝鮮半島，北東中国北部-沿海地方，日本を起源にもつ大気に対するPb-Cd二元系相関図に本州での雪試料中の（[Pb], [Cd]）をプロットすることで，最も近くの回帰直線を示す地域を発生域に帰属した．無機小球形粒子（φ 1～3 μm）と硫酸塩としてのPbとCdを含むエアロゾル（φ 0.5±0.4 μm）の長距離輸送機構の相違が原因して，両者をトレーサーに用いて帰属した大気塊の起源が相互に異なるケースがあった．その相違は，降雪時間帯の後方流跡線の変動により説明できた