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本研究では、1948（昭和23）年に発行された「保育要領―幼稚園教育の手引き」から2017年（平成29）年に改訂となった現行の幼稚園教育要領に至るまでの領域「環境」に関する記述内容についての分析を行う。また、社会的な背景にも触れながら、領域「環境」の教育内容がどのような変遷の過程をたどってきたのかを探り、その特質についても明示していく。

本稿では、カナダ、ブリティッシュ・コロンビア州ビクトリア市のコープ式プリスクールを調査対象としている。調査の目的は、コープ式と呼ばれる親の参画が必須となる保育施設の活動の実践から、親参画の意義やニュージーランドのプレイセンター活動との相違を明らかにすることを通じて、日本における親参画型の保育や子育て支援活動への示唆を得ることである。カナダのコープ式プリスクールでは、有資格者となる保育教員が活動や運営を円滑にするための中心人物となっており、ニュージーランドのプレイセンターとは異なっていた。コープ式プリスクールでは、親の学習を義務づけ、保育者と共に運営されており、こうした特徴が組織を継続維していくための助力となっていた。以上から、親が主体的に活動する保育事業や子育て支援事業を実施するためには、運営の下支えに回る人材の確保と親に対する学習機会の提供が有効となることが明らかとなった。また、全ての親が一人一役の委員となり、保育者と家庭が相互補完しながら運営を担うことで、保育の質を維持する機能が働いていることが明らかとなった。

Adenocarcinoma is the most common type of lung cancer, and can be classified into various histologic subtypes. However, little is known about the subtype-dependent variations in lipid metabolism processes. We performed dual lipidomic analyses using liquid chromatography–mass spectrometry (LC-MS) and matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) to identify possible biomarkers to distinguish adenocarcinoma specimens from normal lung specimens, and to determine if there are any differences in lipid metabolism among the histologic subtypes (lepidic, acinar, papillary, micropapillary, solid, and mucinous). LC-MS was used to characterize the lipid profiles of lung adenocarcinoma and normal lung tissue, and MALDI-IMS analysis was performed to confirm the results with information on lipid localization within the lung. LC-MS analysis found significant differences in the relative abundances of phosphatidylcholine (PC)(16:0/16:0) (P = 0.0432) and sphingomyelin (SM)(42:2) (P P = 0.02221, P = 0.0004, and P = 0.0215, respectively). MALDI-IMS analysis confirmed that these ratios were significantly higher in adenocarcinoma regions of the lung. The ratio of PC(16:0–18:1)/PC(16:0–18:0) was significantly lower in solid subtypes than in other subtypes (P = 0.0028). The monounsaturated/saturated PC ratios may have applications in adenocarcinoma diagnoses and subtyping.

本研究は、ヨーロッパで開発された幼児教育カリキュラムに準じてデザインされたEuropean Educationall （sic） Group（以下、EEGと表記する）の玩具（以下、教育用マテリアルと呼ぶ）を保育の実践に持ち込み、保育を支える環境としての玩具のあり方を研究するものである。これまでの研究では、子どもたちの遊びの様子を検証した結果を考察し、各マテリアルに付属する「環境構成仕様書」及び「保育指導案」の開発を行った。本稿では、研究開発した「保育指導案」から３事例を取り上げ、遊びの検証と考察の結果と、開発後の留意点を考察する。検証の結果、「保育指導案」は保育者に好意的に受け止められ、保育を支える環境としての可能性が明らかとなった。保育者が求める指導案の具体的な傾向から、今後の教育用マテリアルの研究開発や販売展開の方向性が示唆された。

本稿は、ニュージーランドの保育所や幼稚園などの幼児教育施設を訪問し、記録や評価、保育計画のあり方について報告するものである。訪問した視察先ではどこの施設においてもラーニング・ストーリーを用いての保育記録を行っていた。改定後の保育所保育指針においては、乳児、１歳以上３歳未満児への保育についてのねらい及び内容が示されたとともに、保育者と家庭（保護者）との連携が強調されている。日本において、未満児の保育がますます一般化されていくなかで、保育者が担う役割として、保護者との関わりや子育て支援の重要性が増している。本報告では、ニュージーランドで実践されている記録・評価・カンファレンスの実際を紹介するとともに家庭との連携についても言及していく。

Worldwide prevalence of obesity is associated with the increase of lifestyle-related diseases. The accumulation of intermuscular adipose tissue (IMAT) is considered a major problem whereby obesity leads to sarcopenia and metabolic disorders and thus is a promising target for treating these pathological conditions. However, whereas obesity-associated IMAT is suggested to originate from PDGFRα+ mesenchymal progenitors, the processes underlying this adipogenesis remain largely unexplored. Here, we comprehensively investigated intra- and extracellular changes associated with these processes using single-cell RNA sequencing and mass spectrometry. Our single-cell RNA sequencing analysis identified a small PDGFRα+ cell population in obese mice directed strongly toward adipogenesis. Proteomic analysis showed that the appearance of this cell population is accompanied by an increase in galectin-3 in interstitial environments, which was found to activate adipogenic PPARγ signals in PDGFRα+ cells. Moreover, IMAT formation during muscle regeneration was significantly suppressed in galectin-3 knockout mice. Our findings, together with these multi-omics datasets, could unravel microenvironmental networks during muscle regeneration highlighting possible therapeutic targets against IMAT formation in obesity.

TRIF is an essential adaptor for Toll-like receptor 3/4 (TLR3/4) signaling to activate transcription factor interferon regulatory factor 3 (IRF-3). We examined the molecular mechanism of TLR3 signaling and found that TLR3 stimulation by double-stranded RNA (dsRNA) induces phosphorylation of TRIF at Ser210 and is required for IRF-3 recruitment. TANK-binding kinase 1 (TBK1) is known to be responsible for IRF-3 phosphorylation and activation. We found that TBK1 is also responsible for phosphorylation of Ser210 in TRIF. Unexpectedly, we discovered that IκB kinase β (IKKβ) plays an essential role in TLR3/4 signaling using a pharmacological inhibitor and gene deletion. Of note, IKKβ is essential in TLR3/4 but not in retinoic acid-inducible gene I (RIG-I) signaling. Mechanistically, IKKβ transiently associates with and induces the phosphorylation of TBK1 upon TLR3 stimulation. These results suggest a phosphorylation cascade of IKKβ and TBK1, where priming phosphorylation of TBK1 by IKKβ is required to surpass the threshold to induce signaling, thereby activating IRF-3.

Lysine-specific demethylase 1 (LSD1) is a histone modifier for transcriptional repression involved in the regulation of hematopoiesis. We previously reported that a LSD1 inhibitor NCD38 induces transdifferentiation from erythroid lineage to granulomonocytic lineage and exerts anti-leukemia effect through de-repression of the specific super-enhancers of hematopoietic regulators including ERG in a human erythroleukemia cell line, HEL. However, the mechanistic basis for this specificity of NCD38 has remained unclear. Herein, we report major partners associated with LSD1 and clarify the mechanism in HEL cells. Proteome analysis identified 54 candidate proteins associated with LSD1, including several transcription factors such as GFI1B and RUNX1 as well as BRAF-histone deacetylase complex (BHC) components such as CoREST, HDAC1, and HDAC2. NCD38 selectively disrupted the interaction of LSD1 with GFI1B but not with RUNX1, CoREST, HDAC1 and HDAC2. Erg was downregulated in murine erythroid progenitors with prominent upregulation of Gfi1b. NCD38 induced ERG and attenuated an erythroid marker CD235a in HEL while this attenuation was mimicked by the lentiviral overexpression of ERG. The ERG super-enhancer contained the conserved binding motif of GFI1B and was actually occupied by GFI1B. NCD38 dissociated LSD1 and CoREST but not GFI1B from the ERG super-enhancer. Collectively, the selective separation of LSD1 from GFI1B by NCD38 restores the ERG super-enhancer activation and consequently upregulates ERG expression, inducing the transdifferentiation linked to the anti-leukemia effect.

ヨーロッパで開発された玩具（本稿では、教育マテリアルと記す）を日本の幼児教育および保育現場において活用できるのか、その可能性を探るため現場での実践を通じた検証をしている。ヨーロッパのいくつかの玩具メーカーが集まり組織化されたEuropean Educationall Group（以下、EEGと略す）においては、様々な国の就学前カリキュラムを踏襲し、独自のカリキュラムを考案している。そのカリキュラムをベースに子どもたちに学んでもらいたい6つ領域を示し、領域との結びつきを重要視しながら教育マテリアルの提供を行っている。本稿の目的は、EEGの6領域が日本の保育指針や幼稚園教育要領などが示している5つの領域にも分類が可能かどうかを調査し、日本での活用の可能性を探るための手がかりを示すことにある。検証の結果、日本国内の保育所等の教育・保育現場では、遊び手である子どもの姿からEEGのカリキュラムが提案する遊び方とは異なる遊びが展開されていることが明らかとなった。EEGでは、各玩具の制作者とは別に、幼児教育に特化した研究者や教育者が各マテリアルの基礎から応用まで遊び方・カリキュラムを開発している。そのため、子どもの学習面での教育的な効果に主眼が置かれ、子ども自身が遊びを発展させるという行為に積極的な傾向が見られた。逆に日本においては、子どもの主体的な遊びを保障することが重要視されていた。このことからも、日本においては、子どもの遊びを大切にし、環境構成をしていく際の一つの道標としてEEGの教育マテリアルを用い、個々の子どもの発達や学びの状況を確認するためのツールとして活用することが有効的であることが示される結果となった。

Objective.Anti-carbamylated protein (anti-CarP) antibodies are possible diagnostic biomarkers of anticitrullinated protein antibody (ACPA)-negative rheumatoid arthritis (RA). We aimed to elucidate the prevalence of anti-CarP antibodies in non-RA connective tissue diseases (CTD) because CTD are important in the differential diagnosis of ACPA-negative RA.Methods.The sera from 266 patients with RA and 616 patients with CTD and 80 healthy controls were examined using an in-house anti-CarP ELISA.Results.The prevalence and the level of anti-CarP antibodies in several CTD were comparable to those in ACPA-negative RA.Conclusion.Anti-CarP antibodies are not useful for differentiating ACPA-negative RA from CTD.

Objectives. Anti-carbamylated protein (anti-CarP) antibodies are detected in RA patients. Fetal calf serum is used as an antigen source in anti-CarP ELISA, and the precise target antigens have not been found. We aimed to identify the target antigens of anti-CarP antibodies. Methods. Western blotting of anti-CarP antibodies was conducted. Anti-carbamylated human albumin (CarALB) antibody was detected by in-house ELISA for 493 RA patients and 144 healthy controls (HCs). An inhibition ELISA of anti-CarP antibodies by CarALB and citrullinated albumin (citALB) was performed using eight RA patients’ sera. Serum CarALB was detected by liquid chromatography–tandem mass spectroscopy (LC/MS/MS), and the serum MPO concentration was measured by ELISA. Results. We focused on carbamylated albumin because it corresponded to the size of the thickest band detected by western blotting of anti-CarP antibodies. Anti-CarALB antibody was detected in 31.4% of RA patients, and the correlation of the titres between anti-CarALB and anti-CarP was much closer than that between anti-citALB and anti-CCP antibodies (ρ = 0.59 and ρ = 0.16, respectively). The inhibition ELISA showed that anti-CarP antibodies were inhibited by CarALB, but not by citALB. CarALB was detected in sera from RA patients by LC/MS/MS. The serum MPO concentration was correlated with disease activity and was higher in RA patients with anti-CarALB antibody than in those without. Conclusion. We found that carbamylated albumin is a novel target antigen of anti-CarP antibodies, and it is the first reported target antigen that has not been reported as the target of ACPA.

幼児期の教育と小学校教育の円滑な接続を図っていくという観点から、「生活科」を中心とした合科的な指導のあり方を検討するために、幼児教育における領域「環境」と小学校教育「生活科」の学びの連続性に関する調査を行った。本研究では、EEG教育マテリアルを用い、幼児と小学校低学年の遊び方の特徴と傾向を分析している。その結果、幼児期においても遊び方の工夫が観察されるものの、児童期になるとより遊びを深めたいとする意欲や遊具の持つ可能性など知的な気付きが生まれていた。子どもの気付きは、次の自発的な遊びを誘発することから、子ども自身が幼児教育を通じて培ってきた経験や成果を踏まえながら、小学校教育における指導計画や授業実践を進めていくことが小学校始期の児童にとって極めて重要であることが本研究から明らかとなった。

幼児期の教育と小学校教育の円滑な接続を図っていくという観点から、「生活科」を中心とした合科的な指導のあり方を検討するために、幼児教育における領域「環境」と小学校「生活科」の学びの連続性に関する調査を行った。本研究では、保育領域「環境」と小学校「生活科」の内容に焦点を当て、保幼小連携の観点から教育用マテリアル（玩具）活用の可能性を検証することを目的とする。本稿では、基礎研究として、EEGの保育領域と日本の保育５領域の整合性を明らかにした上で、保育園における自由あそびの実践を行い、保育領域「環境」における教育用マテリアルの有用性を考察した。幼児期の子どもの教育用に考えられた教育用マテリアルは、日本の保育５領域と親和性があり、幼児教育の現場で活用可能性が高いことが確かめられた。

Pre-clinical animal tests are used to assess drug efficacy and safety, but are limited by factors such as their suitability as a model for humans, robustness, cost, and ethical issues. While an organ-on-a-chip using human cells is promising for recapitulating human physiological conditions, it is highly desirable in investigations of the side effects of drugs to integrate more than one type of tissue using a designed circulatory system. We have developed a microfluidic device—an Integrated Heart/Cancer on a Chip (iHCC)—using human healthy heart cells (hCMs) and liver cancer cells (HepG2) to recapitulate the side effects of an anti-cancer drug, doxorubicin (DXR), to achieve individual cultures of cells from different tissues on a single device with three sets of artificial blood circulation loops, microfabrication technology for micro valves and a pump provides accurate fluid operation. Using improved soft lithography adopting numerical optimization simulation, the microfluidic device was fabricated with on-chip integration of pneumatic valves and a peristaltic micropump establishing precision fluid flow. The iHCC developed allows modelling of the side effects of DXR on heart cells caused by the production of toxic metabolites (doxorubicinol; DXRol) by HepG2 cells and the delivery of DXRol to heart cells via the circulation loop. Our findings open the door towards the development of a “Body-on-a-Chip.”

本稿では、ニュージーランドのプレイセンターで始まった乳幼児向けの新たなプログラムとなるSPACEに着目し研究を進めている。調査の目的は、SPACEの活動を通じて1.参加する親子が地域の機関とつながり合いを持ち、2.仲間をつくりながら親としての自信を獲得しているのか、3.子どもの学ぶ権利を保障し、乳幼児期から幼児教育に参加する機会を提供しているのかを探求することにある。わが国では、2015年4月から子ども・子育て支援新制度が施行され、在宅親子や低年齢児がいる家庭に対する支援や地域を拠点とする子育て支援がますます強化される方向にある。本論文においては、SPACEの取り組みを報告しながら、日本国内にあるプレイセンター事業や新制度下の子ども・子育て支援事業への活用の可能性も踏まえて考察を加えていく。

Acute cardiac rupture and adverse left ventricular (LV) remodeling causing heart failure are serious complications of acute myocardial infarction (MI). While cardio-hepatic interactions have been recognized, their role in MI remains unknown. We treated cultured cardiomyocytes with conditioned media from various cell types and analyzed the media by mass spectrometry to identify α1-microglobulin (AM) as an Akt-activating hepatokine. In mouse MI model, AM protein transiently distributed in the infarct and border zones during the acute phase, reflecting infiltration of AM-bound macrophages. AM stimulation activated Akt, NFκB, and ERK signaling and enhanced inflammation as well as macrophage migration and polarization, while inhibited fibrogenesis-related mRNA expression in cultured macrophages and cardiac fibroblasts. Intramyocardial AM administration exacerbated macrophage infiltration, inflammation, and matrix metalloproteinase 9 mRNA expression in the infarct and border zones, whereas disturbed fibrotic repair, then provoked acute cardiac rupture in MI. Shotgun proteomics and lipid pull-down analysis found that AM partly binds to phosphatidic acid (PA) for its signaling and function. Furthermore, systemic delivery of a selective inhibitor of diacylglycerol kinase α-mediated PA synthesis notably reduced macrophage infiltration, inflammation, matrix metalloproteinase activity, and adverse LV remodeling in MI. Therefore, targeting AM signaling could be a novel pharmacological option to mitigate adverse LV remodeling in MI.

Collagen is frequently digested using pepsin in industries to produce a triple helical collagen without the N- and C-terminal telopeptides. However, kinetic analysis of this reaction is difficult because several Lys residues in the N- and in the C-terminal telopeptides form covalent bonds, leading to multiple substrates species, and pepsin cleaves collagen at various sites in the N-terminal and in the C-terminal telopeptides, yielding different products. Here we performed kinetic analysis of the digestion of bovine type I collagen with porcine pepsin. The reaction could be monitored by SDS-PAGE by measuring the intensity of the protein bands corresponding to the variant β11 chain. We obtained kinetic parameters relative to the decrease in the variant β11 chain upon digestion. At pH 4.0, the Km and kcat values increased with increasing temperature (30 to 65 °C), although the kcat /Km values were stable. Additional cleavage at the helical region was detected at 45 to 65 °C. At 37 °C, the Km and kcat values increased with decreasing pH, and the kcat /Km values at pH 2.1 to 4.5 were stable and higher than those at pH 5.0 and 5.5. No additional cleavage was detected at the examined pH. Thus, the optimal pH and temperatures for selective digestion of collagen telopeptides with pepsin are 2.1 to 4.5 and 30 to 40 °C, respectively. These results suggest that the method might be useful for the kinetic analysis of the digestion of collagen telopeptides with pepsin.

ラーニング・ストーリーとは、ニュージーランドの多くの教育や保育現場において用いられている観察記録の一つの方法である。本研究では、C保育園(後に対象児は、P保育園に転園。P保育園においても調査を継続)において、当時4歳児であったリリコの2年間を観察した。具体的には、K保育士にラーニング・ストーリーの手法に基づいた観察記録を蓄積してもらい、定期的に園全体で振り返る機会を設けた。そして、1 その記録をニュージーランドの幼児教育カリキュラムである「テ・ファリキ」と日本の「保育所保育指針」の双方と照合することによって保育士自身の保育観に影響や変化をもたらすのか、2 観察記録で得られた成果を次の日からの保育実践に活かすことにより、子どもの発達に効果が表れるのかを分析した。ラーニング・ストーリーに記されたリリコの活動を「テ・ファリキ」と照合した結果、保育者に対しては、「保育を客観的に観る力」と「子どもの内なる声を聴く力」を養成していたことが明確となった。また、対象児の発達面でも変化が見られ、とりわけ社会性と表現能力の両面が育まれていた。

Lysine-specific demethylase 1 (LSD1) is a histone modifier for transcriptional repression involved in the regulation of hematopoiesis. We previously reported that a LSD1 inhibitor NCD38 induces transdifferentiation from erythroid lineage to granulomonocytic lineage and exerts anti-leukemia effect through de-repression of the specific super-enhancers of hematopoietic regulators including ERG in a human erythroleukemia cell line, HEL. However, the mechanistic basis for this specificity of NCD38 has remained unclear. Herein, we report major partners associated with LSD1 and clarify the mechanism in HEL cells. Proteome analysis identified 54 candidate proteins associated with LSD1, including several transcription factors such as GFI1B and RUNX1 as well as BRAF-histone deacetylase complex (BHC) components such as CoREST, HDAC1, and HDAC2. NCD38 selectively disrupted the interaction of LSD1 with GFI1B but not with RUNX1, CoREST, HDAC1 and HDAC2. Erg was downregulated in murine erythroid progenitors with prominent upregulation of Gfi1b. NCD38 induced ERG and attenuated an erythroid marker CD235a in HEL while this attenuation was mimicked by the lentiviral overexpression of ERG. The ERG super-enhancer contained the conserved binding motif of GFI1B and was actually occupied by GFI1B. NCD38 dissociated LSD1 and CoREST but not GFI1B from the ERG super-enhancer. Collectively, the selective separation of LSD1 from GFI1B by NCD38 restores the ERG super-enhancer activation and consequently upregulates ERG expression, inducing the transdifferentiation linked to the anti-leukemia effect.

本稿では、ニュージーランド、ネルソン市のヴィクトリー・プレイセンターでJapanese Session(以下、日本語セッションと記す)を開催する「たんぽぽ」を調査対象としている。調査では、ニュージーランドにおいて日本人がプレイセンター活動をすることの意義や日本の活動者との相違を明らかにすることを通じて、日本におけるプレイセンター活動への示唆を得ることを目的としている。調査方法は、スーパーバイザーたちや参加する親たちへの聴き取り調査、グループインタビュー及び質問紙調査を行うこととし、日本語セッション設置に至るまでの経緯、対象者の属性と活動に対する意識、活動評価、課題や抱負などを確認した。その結果として、どちらの国の参加者も「親子が一緒に活動し、共に成長しあうこと」や「仲間づくりの拠点としてプレイセンターを活用すること」が活動を通じて成し遂げられていたことがわかった。今回調査を行った日本語セッション「たんぽぽ」では、セッションをリードするスーパーバイザーらが活動を円滑にするための中心人物となっていた。また、プレイセンターというシステムがすでにニュージーランド社会のなかで一定の地位を得ていることやプレイセンターの遊び環境が常設で確保されている点が「たんぽぽ」の活動発展への助力となっていた。以上のことから、親が主体的に活動できるように運営の下支えに回る人材を確保することやプレイセンター専用の施設を用意することが、日本国内のプレイセンター活動には求められることが明らかとなった。他方、日本語セッション「たんぽぽ」においては、親向けの学習がなかなか浸透しない傾向にあるため、「親学習」と「親による協働運営」をさらに徹底させていくことが、今後の「たんぽぽ」に課せられる課題であることが示される結果となった。

本調査は、親による協働保育活動であるニュージーランドのプレイセンターをモデルとして2002年より活動が始まった日本のプレイセンター活動に対する評価を、そこに通う参加者とセンター代表者を対象に質問紙によって実施した。参加する親たちは、プレイセンターの活動を通じて、子育ての仲間を増やし、学びあう経験を通じて、助け合いの精神や子どもに対する理解と知識を獲得していることがわかった。しかしながら、わが国では、プレイセンターが全国で10数か所と活動の場が少ないため、プレイセンターの活動が未だ一般には周知されておらず、参加を始めたばかりの親たちに活動の理念が理解されづらい現状があることが明らかとなった。今後はさらなるプレイセンターの普及活動や広報活動が求められるとともに、「親が成長する」ことを可能にするプレイセンターの価値を現在活動中の親を通じて世論に広げていく必要があることが示唆された。

Bovine type I collagen consists of two α1 and one α2 chains, containing the internal triple helical regions and the N- and C-terminal telopeptides. In industries, it is frequently digested with porcine pepsin to produce a triple helical collagen without the telopeptides. However, the digestion mechanism is not precisely understood. Here, we performed a mass spectrometric analysis of the pepsin digest of the N-terminal telopeptide pQLSYGYDEKSTGISVP (1–16) in the α1 chain. When purified collagen was digested, pQLSYGY (1–6) and pQLSYGYDEKSTG (1–12) were identified, while DEKSTG (7–12) was not. When the N-terminal telopeptide mimetic synthetic peptide pQLSK(MOCAc)GYDEKSTGISK(Dnp)P-NH2 was digested, pQLSK(MOCAc)GYDEKSTG (1–12) and ISK(Dnp)P-NH2 (13−16) were readily identified, pQLSK(MOCAc)GY (1−6) and DEKSTGISK(Dnp)P-NH2 (7−16) were weakly detected, and DEKSTG (7–12) was hardly identified. These results suggest that pepsin preferentially cleaves Tyr6–Asp7 and less preferentially Gly12–Ile13. They also suggest that the former cleavage requires native collagen structure, while the latter cleavage does not.

The purpose of this paper was investigate the effect of parents' Playcentre participation in Japan, and to compare the impact of different Japan and New Zealand Playcentre participants. The focus was on social networks, and theformation of trust and norm of reciprocity generating social capitals which operates to empower parents themselves, their families and communities. Japanese parents with preschool aged children in the Picasso Playcentre (Tokyo area) and Eniwa Playcentre (Hokkaido, the northern island of Japan) were interviewed. The findings are interpreted as follows: Japanese parents being involved in adult education and Playcentre activities was significantly associated with positive outcomes with regards to child-rearing attitudes and diverse other contemporary Japanese social problems. These were compared with Satoh's own research in New Zealand and findings from the NZPF commissioned research Powell et al. (2005), The Effect of Adult Playcentre Participation on the Creation of Social capital in Local Communities.1)

わが国では、社会構造の変化とともに、子育てをめぐる環境も大きく変容した。かつて、子育ては、地域共同体のなかでの成員同士による相互扶助や拡大家族における祖父母のサポートにより成立していた。現代では、都市部を中心に核家族世帯が多くを占め、在宅親子の地域における「孤立化」が社会問題となっている。近年になると、改正児童福祉法や新保育所保育指針において、保育所が地域全体の中核となる保育資源であると明記されるなど、保育所における子育て支援の役割期待が大きく広がっている。すなわち、このことは、保育士に対する業務の多様化を意味しており、保育士の役割に保育所の入所児やその親だけでなく、地域の親子に対するサポート業務が含まれるようになったことを示している。そこで、本稿では、在宅親子に向けた保育所の子育て支援事業に着目し、ある首都圏の短期大学を卒業した保育士を対象とするアンケート調査をもとに、その実態を明らかにする作業を試みた。結果として、保育所における支援体制の限界が確認され、今後の子育て支援活動において、保育士だけに帰着しない、親自身・地域成員そして保育者の連携による相互扶助活動の実施がますます重要となることが示された。

わが国の社会構造は少子高齢社会となり、その進行は世界でも例がないスピードである。厚生労働省や関連審議会等は90年代に社会福祉基礎構造改革を提唱し、従来の措置制度から利用契約制度への転換、競争原理の導入や規制緩和による企業参入を図ってきた。そのことによって福祉・介護・保育等のサービスの質と量が保障され、専門職の人材養成や確保に関してもそれに見合う状況が政策的にも展開されるものとの期待があった。しかし、果して期待された保育や介護労働の実態はどのようなものであろうかについてここで検証するものである。

Collagen is frequently digested using pepsin in industries to produce a triple helical collagen without the N- and C-terminal telopeptides. However, kinetic analysis of this reaction is difficult because several Lys residues in the N- and in the C-terminal telopeptides form covalent bonds, leading to multiple substrates species, and pepsin cleaves collagen at various sites in the N-terminal and in the C-terminal telopeptides, yielding different products. Here we performed kinetic analysis of the digestion of bovine type I collagen with porcine pepsin. The reaction could be monitored by SDS-PAGE by measuring the intensity of the protein bands corresponding to the variant β11 chain. We obtained kinetic parameters relative to the decrease in the variant β11 chain upon digestion. At pH 4.0, the Km and kcat values increased with increasing temperature (30 to 65 °C), although the kcat /Km values were stable. Additional cleavage at the helical region was detected at 45 to 65 °C. At 37 °C, the Km and kcat values increased with decreasing pH, and the kcat /Km values at pH 2.1 to 4.5 were stable and higher than those at pH 5.0 and 5.5. No additional cleavage was detected at the examined pH. Thus, the optimal pH and temperatures for selective digestion of collagen telopeptides with pepsin are 2.1 to 4.5 and 30 to 40 °C, respectively. These results suggest that the method might be useful for the kinetic analysis of the digestion of collagen telopeptides with pepsin.

Patients who develop YMDD mutant during lamivudine therapy for hepatitis B virus (HBV) infection exhibit various clinical courses. Some patients show normal ALT levels, whereas others develop severe hepatitis exacerbations (SHEs) due to YMDD mutants. We studied 136 patients with YMDD mutant among 362 Japanese adult patients on lamivudine therapy. Clinical and virological features of patients without elevated HBV DNA after emergence of YMDD mutant (non-elevated group) were investigated. Moreover, virological analysis was also performed in patients with SHE due to YMDD mutants. Patients in the non-elevated group were characterized by HBeAg-negative pretreatment, HBeAg loss during therapy, a longer duration from commencement of therapy until emergence of YMDD mutant, and no mixed-type YMDD mutants. Patients with SHE had more substitutions in the reverse transcriptase (rt) region within the polymerase gene at the time of exacerbation than those without SHE, although no specific substitutions were noted. Sequence analysis of full-length HBV genome showed more substitutions in X, rt, and surface proteins in patients with SHE than in those without elevated HBV DNA level. In conclusion, negativity for HBeAg at commencement of therapy or before emergence of YMDD mutant was an important factor among non-elevated group. More substitutions in the rt region and the other proteins may be related to the emergence of severe hepatitis caused by lamivudine-resistant virus.

We have generated a line of mutant mouse that lacks betaKlotho, a protein that structurally resembles Klotho. The synthesis and excretion of bile acids were found to be dramatically elevated in these mutants, and the expression of 2 key bile acid synthase genes, cholesterol 7alpha-hydroxylase (Cyp7a1) and sterol 12alpha-hydroxylase (Cyp8b1), was strongly upregulated. Nuclear receptor pathways and the enterohepatic circulation, which regulates bile acid synthesis, seemed to be largely intact; however, bile acid-dependent induction of the small heterodimer partner (SHP) NR0B2, a common negative regulator of Cyp7a1 and Cyp8b1, was significantly attenuated. The expression of Cyp7a1 and Cyp8b1 is known to be repressed by dietary bile acids via both SHP-dependent and -independent regulations. Interestingly, the suppression of Cyp7a1 expression by dietary bile acids was impaired, whereas that of Cyp8b1 expression was not substantially altered in betaklotho mice. Therefore, betaKlotho may stand as a novel contributor to Cyp7a1-selective regulation. Additionally, betaKlotho-knockout mice exhibit resistance to gallstone formation, which suggests the potential future clinical relevance of the betaKlotho system.

In Japan, there are few studies of long-term (more than 1 month) interferon (IFN) therapy for chronic hepatitis B (CHB). In this retrospective study, we investigated the efficacy and predictors of response to 6-month IFN therapy.We analyzed 66 Japanese patients with CHB who were treated with IFN for 6 months. They comprised patients who were hepatitis B e antigen (HBeAg)-positive (n=45) and -negative (n=21). One (2%), 8 (12%), and 51 (77%) patients were infected with hepatitis B virus (HBV) genotypes A, B, and C, respectively. Responders in patients positive for HBeAg were defined as those who showed normalization of serum alanine aminotransferase (ALT) level, HBeAg loss, and HBV DNA negativity at 6 months after completion of IFN therapy. In patients negative for HBeAg, responders were defined as those patients who showed normalization of ALT level and HBV DNA negativity at the same 6-month time point.Of the 45 patients with HBeAg at the commencement of IFN therapy, 9 (20%) were responders. Young patients, especially those with a high serum ALT level, were significantly more likely to respond to IFN therapy. Of the 21 patients negative for HBeAg, 13 (62%) were responders. There were no significant differences in clinical characteristics between responders and nonresponders among patients negative for HBeAg. Multivariate analyses identified HBeAg negativity and young age as independent factors associated with a positive response to 6-month IFN therapy. However, long-term follow-up of the treated patients showed a fall in the response rate.The response rate to 6-month IFN therapy among HBeAg-positive patients was low. However, young patients may require long-term IFN therapy.

Lamivudine is used for the treatment of chronic hepatitis B (CH-B), and exhibits excellent antiviral activity. However, longterm administration increases the likelihood of the emergence of resistant viruses, with an accompanying relapse of hepatitis. However, recent studies have reported lamivudine-resistant viruses in patients with CH-B before such treatment. The aim of this study was to investigate whether YMDD mutants occur in nature. The existence of lamivudine-resistant viruses was examined in 20 asymptomatic carriers of hepatitis B virus (ASC), 10 patients who lost hepatitis B surface antigen (HBsAg) during follow-up and in 20 lamivudine-treated patients with and without breakthrough hepatitis. Both polymerase chain reaction (PCR) restriction fragment length polymorphism and SMITEST hepatitis B virus (HBV)-YMDD mutation detection methods were used to detect resistant viruses. No YMDD mutants were detected in the sera of the 20 ASC at the initial and final medical examinations, nor were YMDD mutants detected in sera collected at the initial medical examination, about 6 months before, or immediately after the loss of HBsAg in the 10 patients. In the 20 patients treated with lamivudine, YMDD mutants were not detected in any of them before treatment, whereas mutants were detected in the sera of 10 patients during treatment. Our results suggest that lamivudine-resistant YMDD mutant viruses were present in a few patients with HBV infection who before they have been treated with lamivudine.

Among patients with chronic hepatitis C virus (HCV) infection, serum alanine aminotransferase (ALT) rarely increases above 500 IU/L. We examined the clinical and virological features of untreated patients with serum ALT ≥ 500 IU/L. One thousand seven hundred and sixty adult patients with chronic HCV infection were followed-up. Among these patients, 22 developed ALT flare-up (M:F = 13:9, median age, 50.5 years). We evaluated liver function tests, genotype, and viral titer in these patients and 44 randomly selected age- and sex-matched control without ALT flare-up. In four patients with ALT flare-up, we examined changes in viral loads and sequential changes in amino acid sequences of the core region, hypervariable region 1 (HVR1), and interferon sensitivity determining region (ISDR) before and after ALT flare-up. Multivariate analysis identified genotype 2 as the only significant determinant of ALT flare-up. ALT flare-up occurred in three of four patients without increase in viral load. Several alterations in amino acids were noted in HVR1 before and within 6 months of ALT flare-up. One or two alterations in the core region and many alterations in HVR1 were noted after ALT flare-up in some patients. Genotype 2 is an important factor for ALT flare-up. However, we could not directly relate ALT flare-up to these alterations in amino acids of the core region, HVR1, and ISDR. J. Med. Virol. 75:240–248, 2005. © 2004 Wiley-Liss, Inc.

HBe antigen (HBeAg) loss or seroconversion can occur during lamivudine therapy. The purpose of this study was to analyze nucleotide sequences in precore and core promoter regions, and examine the influence of mutations in these regions on the disappearance of HBeAg during lamivudine therapy. Serial serum samples were obtained from 51 patients (HBeAg loss in 26 patients) at commencement of therapy (baseline) and after 1 year of lamivudine therapy. Serum samples were amplified with PCR and nucleotide sequences of HBV were analyzed. At baseline, a precore stop codon mutation (A1896) was identified in 8 of 26 HBeAg loss patients and in 8 of 25 HBeAg non-loss patients. At 1 year, precore mutation was observed in 4 of 14 patients analyzed who showed HBeAg loss. At 1 year, however, a precore mutation was observed also in 3 of 9 analyzed patients who showed no HBeAg loss. Core promoter mutations were noted in 21 of 26 HBeAg loss patients and in 20 of 25 HBeAg non-loss patients. At 1 year, core promoter mutations were noted in 11 of 14 HBeAg loss patients and in 8 of 9 HBeAg non-loss patients. Our data suggested that during lamivudine therapy, core promoter and precore mutations do not influence HBeAg loss or seroconversion but may reduce the viral level upon HBeAg loss or seroconversion.

To keep an eye on severe nosocomial infection and to evaluate the clinical difference of blood-stream infection between community-acquired and hospital-acquired infection, a survey of blood culture was performed in National Tokyo Medical Center from the period between November 2000 and October 2001. There were 252 episodes detected in 219 patients (80 community-acquired episodes in 80 patients and 172 hospital-acquired episodes in 139 patients). The three most common foci of infection/pathogens were as follows: in the community-acquired cases; urinary tract, pneumonia, infective endocarditis/Escherichia coli, viridant group of streptococci, Streptococcus pneumoniae, and in the hospital-acquired cases; intra-venous catheter, urinary tract, neutropenia-related bacteremia/Staphylococcus aureus, coagulase negative Staphylococcus, Enterococcus. Fifteen patients with community-acquired bacteremia and 37 patients with hospital-acquired bacteremia had been died within a month of the episode; the mortality was not significantly different between the both. The average of peak serum concentrations of C-reactive protein during the episodes of community-acquired bacteremia was higher than that of hospital-acquired bacteremia. These findings probably show that life threatening bloodstream infections seemed to be more common in the community. The rate of nosocomial bacteremia was approximately 1%, and no outbreak was observed during the period. Targeted bacteremia surveillance is maybe useful and efficient method to detect severe hospital-acquired infections.

Maximizing the amount of protein translated per unit mRNA is an important goal in establishing expression systems. The 5′ untranslated region (5′-UTR) of mRNA is known to play an important role in determining the rate of translation. The full length 5′-UTR from theArabidopsis thaliana heat shock protein (HSP) gene,HSPI8.2 gene, was inserted into the cloning site between the cauliflower mosaic virus 35S RNA promoter and β-glucuronidase (GUS) gene in the pBI221 plasmid. When this construct was transfected intoNicotiana tabacum (tobacco) BY-2 protoplasts, the level of protein was about 10-fold higher than that of unmodified pB1221. The accumulation of each transcript was the same level. We also demonstrated that the 5′-UTR of theHSP18.2 gene enhances the rate of translation in stable transgenic BY-2 clones andArabidopsis T87 protoplasts. The 5′-UTRs of the otherArabidopsis HSP genes —HSP17.4, HSP81-1,HSP81-2, andHSP81-3 — also conferred efficient translation. These 5′-UTRs ofHSP genes may be of use in increasing the expression of foreign proteins. In combination with a strong promoter, it can be used in the development of efficient protein production systems.

Rapid advances in the field of molecular genetics have led to the need for a analytical assay. A number of methods have devised for this purpose, a material used the most widely is used the leukocytes DNA. In this study, we have reported the cases of three types. First, we found the cases who were heterozygotes for adenine phosphoribosyltransferase (APRT) gene mutations with APRT deficiency. Second, we evaluated the usefulness of a real-time reversed transcriptase polymerase chain reaction (RTPCR) system to detect minimal residual diseases. This method was applied a case who had t (8; 21) chimeric transcript in peripheral blood. Third, we evaluated a method to determine human cytomegalovirus (CMV) DNA levels in blood cells in a case transplaned liver. Thus, these methods were indicated possible applications for analysis using blood leukocytes.

The 5'-untranslated region (5'-UTR) functions as a translational enhancer in monocotyledonous plant cells is necessary to express a foreign gene efficiently. Here, we show that the 5'-UTR of the rice alcohol dehydrogenase gene contributes to efficient translation in not only dicotyledonous plant cells, but also in monocotyledonous rice cells.

A non-radioisotopic PCR-SSCP method using fluorescently labeled primers was developed for use in a clinical setting. In order to establish PCR-SSCP method for the clinical use, we developed a non-radioisotopic PCR-SSCP method using fluorescence-labeled primers. Electrophoresis in buffer containing with MgCl2 provided good separation of the bands and use of an image analyser gave more sensitive detection than conventional method. The mutations of K-ras gene and androgen receptor gene were confirmed by DNA sequencing analysis of the known mutations of K-ras gene and the unknown mutations of androgen receptor gene. The fluorescence-mediated PCR-SSCP system made it possible to detect mutations more rapidly and with more sensitivity than conventional PCR. Thus, the improved PCR-SSCP method will be useful for the clinical screening of unknown mutatios.

Dynamical aspects of three chloroplast promoters responding to change in light condition were examined in mature chloroplasts of wheat (Triticum aestivum) by in vitro transcription. The wheat psbD/C operon has four distinct promoters, two of which named as D/C-3 and D/C-4 promoters dominantly function in mature chloroplasts to produce the mRNAs encoding D2/CP43 and CP43 alone, respectively. Activity of the D/C-3 promoter in mature chloroplasts was reduced to less than 30% by 24 h dark adaptation and recovered by re-illumination to the original level within 30 to 60 min. The activation of the D/C-3 promoter which requires de novo cytoplasmic protein synthesis was induced by low fluence of light (e.g. 16 microE m(-2) s(-1)), but the extent of activation increased with increasing light fluence. The accumulation of mRNAs from the D/C-3 promoter saturated at 2- to 3-fold higher level within 2 h when the dark-adapted seedlings were transferred to the light at 72 microE m(-2) s(-1), concomitant with the increase in rate of D2 synthesis, suggesting that synthesis of D2 in mature chloroplasts is controlled via the D/C-3 promoter activity in a light-dependent way. Activity of the D/C-4 promoter slightly increased in the dark and decreased in the light. Effect of light on the psbA promoter activity was not observed at all in mature chloroplasts. In vitro transcriptional analysis of the D/C-3 promoter with 5' deletion mutations revealed that at least two cis elements which are located within the sequences of -78 to -47 and -46 to -29 of the transcription initiation site, respectively, act as enhancing elements in the D/C-3 promoter. The light-switching element of the transcription, however, was suggested to be located in the core promoter sequence downstream of the -35 element.

[Background]Periplakin (PPL) is a rod-shaped cytolinker protein thought to connect cellular adhesion junctional complexes to cytoskeletal filaments. PPL serves as a structural component of the cornified envelope in the skin and interacts with various types of proteins in cultured cells; its level decreases dramatically during tumorigenic progression in human epithelial tissues. Despite these intriguing observations, the physiological roles of PPL, especially in non-cutaneous tissues, are still largely unknown. Because we observed a marked fluctuation of PPL expression in mouse liver in association with the bile acid receptor farnesoid X receptor (FXR) and cholestasis, we sought to characterize the role of PPL in the liver and determine its contributions to the etiology and pathogenesis of cholestasis. [Methods]Time- and context-dependent expression of PPL in various mouse models of hepatic and renal disorders were examined by immunohistochemistry, western blotting, and quantitative real-time polymerase chain reactions. [Results]The hepatic expression of PPL was significantly decreased in Fxr−/− mice. In contrast, the expression was dramatically increased during cholestasis, with massive PPL accumulation observed at the boundaries of hepatocytes in wild-type mice. Interestingly, the hepatic accumulation of PPL resulting from cholestasis was reversible. In addition, similar accumulation of PPL at cellular boundaries was found in epithelial cells around renal tubules upon ureteral obstruction. [Conclusions]PPL may be involved in the temporal accommodation to fluid stasis in different tissues. Further examination of the roles for PPL may lead to the discovery of a novel mechanism for cellular protection by cytolinkers that is applicable to many tissues and in many contexts.

We previously reported that HER2 is transferred from HER2 overexpressing breast cancer cells to CD14+ monocytes via trastuzumab dependent trogocytosis (Suzuki et al. BMC cancer 2015) and EGFR of MDA-MB-231 (triple negative human breast cancer cell line) is also transferred to immune cells via trogocytosis and those immune cells express EGFR on the cell surface without therapeutic antibody administration (AACR 2015 #2349). There are several reports that suggest that trogocytosis might act to stimulate immunological tolerance or immune effector cell activation, however its immunological roles still remain unclear. Therefore, in order to clarify the role of trogocytosis, we study the profile of proteins that potentially are transferred from cancer cell to immune cell via trogocytosis. Human triple negative breast cancer cell line, MDA-MB-231 that was cultured with the medium containing arginine labeled with 13C instead of normal 12C (SILAC method, ThermoFisher Scientific) was co-cultured with human peripheral blood mononuclear cell (PBMC) that was cultured with normal medium. The cell mixture was labeled with CD45+ magnetic beads and CD45+ cell (PBMC) and CD45- cell (MDA-MB-231) were separated by MACS cell separation kit (Miltenyi Biotec). Proteins of PBMC that were passed the trogocytosis were resolved on SDS-PAGE and LC-MS/MS was carried out to identify the cancer specific trogocytosed proteins in PBMC by SILAC method. We found that proteins with higher H/L ratio were included in many of cytoskeletal proteins and interestingly mitochondrial metabolism related proteins of cancer cells were also detected in PBMC. Those up-regulated proteins in PBMC were reduced in MDA-MB-231. The findings suggested that the role of trogocytosis of PBMC on breast cancer cell might be involved in metabolisms of both cancer cell and immune cell. Thus, we are exploring the effect of trogocytosis on cancer cell metabolisms especially on mitochondrial related metabolism that could be reduced by immune cell trogocytosis and also on metabolism of immune cells themselves that capture the cancer cell proteins via trogocytosis. Citation Format: Eiji Suzuki, Masashi Inoue, Shinji Ito, Junko Satoh, Kosuke Kawaguchi, Ayane Yamaguchi, Moe Tsuda, Masakazu Toi. Proteomics analysis of breast cancer cell-specific proteins that are transferred to immune cells via trogocytosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4142.

Major anthocyanins of root peels of atsumi-kabu (red turnip, Brassica campestris L.) were isolated and identified as cyanidin-3-diglucoside-3-monoglucoside (I, rubrobrassicin), cyanidin-3,5-diglucoside (II), and cyanidin-3-monoglucoside (III). The effects of these anthocyanins on serum cholesterol levels were examined in rats fed for 3 weeks on either a control diet based on lard-cholesterol or experimental diets containing 0.03% I or 0.045% of a mixture of II and III (I for experiment 1, and mixture of II and III for experiment 2). Significant increases in serum high density lipoprotein (HDL)-cholesterol levels and significant decreases in the atherogenic index ((total cholesterol – HDL-cholesterol)/HDL-cholesterol) were observed in rats fed on a diet containing I (experiment 1). In experiment 1, total cholesterol – HDL-cholesterol (VLDL- + LDL-choIesterol) somewhat decreased in rats fed on a diet containing I. A significant decrease in atherogenic index was observed in rats fed on a diet containing the ...

Cytochrome P450 1A1 (CYP1A1) is one of the most important detoxification enzymes due to its broad substrate specificity and wide distribution throughout the body. On the other hand, CYP1A1 can also produce highly carcinogenic intermediate metabolites through oxidation of polycyclic aromatic hydrocarbons. We describe what we believe to be a novel regulatory system for whole-body CYP1A1 expression by a factor originating in the gut. A mutant mouse was generated in which the arylhydrocarbon receptor nuclear translocator (Arnt) gene is disrupted predominantly in the gut epithelium. Surprisingly, CYP1A1 mRNA expression and enzymatic activities were markedly elevated in almost all non-gut tissues in this mouse line. The induction was even observed in early-stage embryos in pregnant mutant females. Interestingly, the upregulation was CYP1A1 selective and lost upon administration of a synthetic purified diet. Moreover, the increase was recovered by addition of the natural phytochemical indole-3-carbinol to the purified diet. These results suggest that an Arnt-dependent pathway in gut has an important role in regulation of the metabolism of dietary CYP1A1 inducers and whole-body CYP1A1 expression. This machinery might be involved in naturally occurring carcinogenic processes and/or other numerous biological responses mediated by CYP1A1 activity.