イデンセイ ナンチョウ ニ タイスル ユウコウ ナ ヤクザイ ノ スクリーニングホウ ノ カクリツ ト コウホ カゴウブツ ノ タンサク

1000人に1人の割合で生ずる先天性難聴は、遺伝的な原因が全体の約半数を占めており、難聴に関連する遺伝子はおよそ100種類が知られている。聴覚を司る内耳の蝸牛は非常に複雑な構造で、種々のタンパク質が役割分担をして聴覚受容を担っているが、これらの遺伝子に変異があることで難聴となる。遺伝性難聴の分類は、難聴以外の随伴症状の有無により症候群性難聴と非症候群性難聴に分けられる。両者に共通している原因遺伝子としてPendred症候群(PDS)の原因遺伝子SLC26A4が挙げられる。PDSでは難聴以外にその随伴症状として、甲状腺腫と内耳奇形が報告されており、一方の非症候群性難聴にもSLC26A4遺伝子の異常が共通してみられる。加えて、SLC26A4の遺伝子異常にともなう随伴症状は進行性である。このようなことから、SLC26A4の遺伝子変異を治療や創薬研究の目標とすることは、優先的であり、有効であると考えられる。SLC26A4からつくられるPendrinは、アミノ酸780個からなる膜貫通型タンパクで、主に内耳に発現し、塩化物イオン、重炭酸イオンなどの陰イオンとヨードの輸送を行っている。細胞内において変異型Pendrinは、正常なPendrinが小胞体で発現した後に細胞膜へと移行するのに対して小胞体に蓄積し、本来あるべき細胞膜へ移行することができない。このため、内耳コルチ器内のラセン隆起および外ラセン溝細胞においては陰イオンの輸送が障害され、水管拡大の症状をともない難聴を示す。そこでこれを改善する試みとして先行研究が行われ、古くから鎮痛作用として知られているサリチル酸が、変異型Pendrinに対しての分子シャペロン活性を示し、変異型Pendrinを小胞体から細胞膜へ移行させ再活性化することが明らかにされた。本研究では、サリチル酸による変異型Pendrinの細胞内局在変化に着目し、変異型Pendrinを恒常的に発現するStable細胞の確立、網羅的画像解析による迅速な薬剤スクリーニング法の開発を行い、サリチル酸とその類縁体から有効な細胞膜移行活性を有する化合物の探索を行った。　第二章では、変異型Pendrin(P123S)恒常発現(Stable)細胞を樹立した。先行研究によりSLC26A4遺伝子の変異のうち主要な10種類のミスセンスの変異型Pendrinの作製と、ミスフォールドからの薬剤によるレスキューが報告されている。10種類の変異型Pendrinを遺伝子導入したHEK293細胞に対するサリチル酸の分子シャペロン活性が調べられており、サリチル酸応答性を示す有用な4種類(P123S、M147V、S657N、H723R)の変異型Pendrin遺伝子が特定されている。ここで行われた一過性(Transient)の遺伝子導入と解析の場合、1)細胞播種、2)遺伝子導入、3)薬剤添加、4)免疫染色、5)細胞内局在変化　の過程を必要とし、実験ごとの遺伝子導入と遺伝子導入効率の安定性、化合物多数を用いた迅速なスクリーニングには課題があった。そこで本研究では、変異型Pendrin(P123S)を恒常的(Stable)に発現する細胞の樹立を検討した。具体的には、遺伝子導入後のHEK293細胞はネオマイシン耐性である性質を利用し、G-418 Sulfate存在下で10日間連続培養後、その生え抜きを選抜した。Pendrin発現とサリチル酸応答性を確認した後、これらを96well培養プレートで1細胞/1wellに限界希釈し、シングルコロニー由来の単クローンを得た。得られた単クローン細胞を再度、Pendrin発現・サリチル酸応答性の確認後、細胞のクローニング操作を合計2回行った。このようにして、野生型Pendrin発現細胞(Wt)と変異型Pendrin(P123S)発現細胞(PH1-1H1)を得たことで、遺伝子導入の段階を省くことが可能となり、第三章のMorphology解析に有用な細胞株であると判断した。第三章ではCellInsightTMを用いて細胞のMorphology解析のためのパラメーターを検討した。　　変異型Pendrin(P123S)は細胞質に集積し、野生型Pendrin(Wt)は細胞膜に局在する。一方、10 mMサリチル酸を加えることで改善し、Pendrinの細胞膜局在が上昇し細胞質局在は減少する。この局在変化について、従来の手法では、免疫染色の後共焦点レーザー顕微鏡にて得られた画像を画像解析ソフトFluoViewTMで取込し、細胞膜・細胞質の蛍光強度を無作為に100ポイント測定して評価していた。本研究では96well培養プレートを用いた迅速な薬剤スクリーニングを目的にこれを改良した。細胞イメージアナライザーCellInsightTMによる計測は、96well内を100フィールドに区分し、各フィールド内最大100個細胞を検出し、全細胞のタンパク質局在を網羅的に解析し蛍光強度を測定できる。そこで解析プログラムMorphology V4の解析パラメーターの検討を行った。複数の検討より、細胞の外周を解析の基準であるCh1とし、核をCh2とした。そしてCh1から内側へ5 pixel幅を細胞質Ch3(Cytoplasm:C)とし、Ch1から内側2 pixel幅を細胞膜(Plasma Membrane:M)Ch4とした。次にPH1-1H1細胞に対してサリチル酸による変異型Pendrin移行活性を評価した結果、Ch4(M)の上昇はみられなかったものの、サリチル酸濃度依存的Ch3(C)の減少を確認することができた。(Ch4は、細胞外周の微小な凹凸や輪郭の正確なトレースが困難で、また、背景の黒色領域も合わせて検出したため、正確な細胞膜の蛍光強度が検出できなかった。)　このことから、スクリーニングの基準を、Ch3(C)蛍光強度の減少として決定し、第四章のサリチル酸類縁体の移行活性スクリーニングに用いた。第四章では、サリチル酸類縁体による変異型Pendrin移行活性スクリーニングと候補化合物の探索を行った。サリチル酸とその類縁体は、96well培養プレート中PH1-1H1細胞に12時間添加し、免疫染色の後、第三章で確立したCellInsightTMによるMorphology V4でCh3を基準にスクリーニングした。その結果、化合物の濃度依存的にCh3(C)減少を示す6つの候補化合物を発見した。さらに、FluoViewTMによる詳細な蛍光強度の測定をし、細胞膜(Plasma Membrane:M) / 細胞質(Cytoplasm:C) = M/C比を調べた結果、サリチル酸は10 mMでM/C比が1.0であるのに比べ、M/C比が0.3 mMで1.5、0.1 mMで0.9を示す化合物(2-aminophenyl)methanolを見い出した(化合物番号8)。すなわち、化合物8はサリチル酸に対して活性がおよそ100倍高いことを示した。さらに、薬剤を培地から取り除いた後24時間までの細胞内薬剤持続性効果を検討した結果、サリチル酸は薬剤除去後6時間で効果が失われたのに対して、化合物8は12時間後まで効果が持続した。以上より、化合物8は変異型Pendrinに対して細胞膜移行活性を有する候補化合物であることが示唆された。本研究の結論1.限界希釈法による細胞のクローニングで、野生型Pendrin発現細胞(Wt)と変異型Pendrin(P123S)発現細胞(PH1-1H1)を得た。Wt細胞はG-418 Sulfate 存在下で恒常的に野生型Pendrinを発現し、細胞膜局在を示した。PH1-1H1細胞はG-418 Sulfate 存在下で恒常的に変異型Pendrinを発現し、細胞質に局在を示した。これに10 mMサリチル酸を12時間添加することで細胞膜局在を示した。得られたPH1-1H1細胞は、サリチル酸類縁体を用いた変異型Pendrin移行活性スクリーニングに有用な細胞である。2.CellInsightTMによるMorphology解析パラメーターの条件検討を行った。細胞外周を解析の基準であるCh1とし、Ch1より内側2 pixelから7 pixelの5 pixel幅を細胞質Ch3(Cytoplasm:C)として設定することで、サリチル酸濃度依存的なCh3(C)の減少が確認できた。すなわち、化合物濃度依存的なCh3(C)の減少に着目することで、96 well培養プレートを用いた迅速かつ網羅的な、変異型Pendrin移行活性スクリーニングが可能となった。3.変異型Pendrin移行活性スクリーニングより6つの有効な候補化合物を発見した。中でも化合物8 ((2-aminophenyl)methanol)はサリチル酸に対して細胞膜移行活性が100倍高く、加えて細胞内薬剤持続的効果も長く12時間を示した。すなわち、化合物8は変異型Pendrinの細胞膜移行活性を有する有用な候補化合物であることが示唆された。学習院大学Gakushuin Universit

Immunohistochemical expression of heat shock protein27 in the mouse dental pulp after immediate teeth separation

<p>Abstract</p> <p>Aim</p> <p>After immediate teeth separation, expression of HSP27 in the mouse dental pulp was examined. Immunohistochemistry was performed to examine the incidence of HSP27 expression.</p> <p>Materials and methods</p> <p>A total of 36 8-week-old ddY mice were used as experimental subjects and a wedge was inserted in between maxillary right molars. The wedge was removed 30 min or 3 h after insertion. Animals were immediately sacrificed after the removal of wedge or until 1 week later and serial sections from paraffin-embedded tissues were prepared. Immunohistochemistry was carried out to examine the expression of HSP27. The untreated side served as the control.</p> <p>Results</p> <p>In the control group, the endothelial cells and some pulp fibroblasts weakly expressed HSP27 suggesting that the expression is due to mechanical stress brought about by physiological masticatory force and pressure from the tongue. In both 30 min and 3 h experimental groups, HSP27 expression was highest at 24 h after wedge removal and the expression remained the same or started to decrease thereafter. The expression decreased at the same level as that of the control group 1 week after wedge removal.</p> <p>Conclusion</p> <p>HSP27 may serve as an indicator of stimulus strong enough to show its expression.</p

Immunohistochemical expression of hard tissue related factors in the mouse dental pulp after immediate teeth separation

We examined change of Runx2 and ALP expression in mouse tooth pulp which exposed to teeth separation experiment by immunohistochemistry as a model for conservative dentistry treatment. 8-week-old 36 male ddY mice were used and wedge was inserted between upper 1st and 2nd molars. The wedge was removed 30 minutes as well as 3 hours after the insertion and the samples were prepared extending up to 1 week of time period for regular histopathological and immunohistochemical examinations for ALP and Runx2 expression. The opposite sides without wedge insertion were taken as controls. In the control group pulp, weak expressions of Runx2 and ALP in the vessel endothelial cells as well as the pulp cells were revealed, suggesting the appearance of these genes upon mechanical stress induced by mastication and tongue pressure etc. On the other hand in the experiment group, Runx2 expression increased both in 30-minute and 3-hour teeth separation group. The expression became maximum at 24 hours. Then it gradually decreased and became similar level with the control group at 1-week after the wedge insertion. Similarly ALP expression increased after the wedge insertion and was maximum at 24 hours and then gradually decreased to the levels similar with the control group. These results suggest that when immunohistochemical expression of Runx2 as well as ALP was used as an index, no severe damage occur upon clinical application of wedge insertion

ASSESSORAMENTO DE PROJETOS COM NECESSIDADE DE COLETA DE DADOS AUTOMATIZADOS

A estatística é uma ferramenta importante para a tomada de decisão em diversas áreas porém, para se obter resultados confiáveis, é necessário um conjunto de dados consistente para as análises. Neste ponto, a coleta de dados pode ser um limitador para o andamento de muitos projetos. Desta forma, este trabalho teve por objetivo, apresentar os resultados obtidos por meio do assessoramento a projetos no processo de automatização da coleta de dados. Após a desenvolvimento do datalogger utilizando plataforma Arduíno, foi realizado reuniões com os responsáveis pelos projetos a serem atendido para definição do delineamento experimental, do cronograma e das ações que seriam realizada pelos membros. Com a diversidade de projetos atendidos, pode-se fortalecer os três pilares da Universidade Federal, Ensino – Pesquisa – Extensão, ou seja, foram atendidos projetos de TCC, Iniciação Científica, Extensão, Pesquisa, Mestrado, Industria e Disciplinas de Graduação. Os membro que trabalharam no projeto tiveram a oportunidade de conhecer o sistema de automatização no processo de coleta de dados utilizando plataforma open source, adquirir alguns soft skills, como comunicação efetiva e liderança e, publicar os resultado em artigo de periódicos, artigos em anais de eventos e capitulo de livros

Vα14 NK T cell–triggered IFN-γ production by Gr-1+CD11b+ cells mediates early graft loss of syngeneic transplanted islets

Pancreatic islet transplantation is a highly promising approach for the treatment of insulin-dependent diabetes mellitus. However, the procedure remains experimental for several reasons, including its low efficiency caused by the early graft loss of transplanted islets. We demonstrate that Gr-1+CD11b+ cells generated by transplantation and their IFN-γ production triggered by Vα14 NKT cells are an essential component and a major cause of early graft loss of pancreatic islet transplants. Gr-1+CD11b+ cells from Vα14 NKT cell–deficient (Jα281−/−) mice failed to produce IFN-γ, resulting in efficient islet graft acceptance. Early graft loss was successfully prevented through the repeated administration of α-galactosylceramide, a specific ligand for Vα14 NKT cells, resulting in dramatically reduced IFN-γ production by Gr-1+CD11b+ cells, as well as Vα14 NKT cells. Our study elucidates, for the first time, the crucial role of Gr-1+CD11b+ cells and the IFN-γ they produce in islet graft rejection and suggests a novel approach to improving transplantation efficiency through the modulation of Vα14 NKT cell function

Validation of leaf cover analysis software (LCAS) to monitor lettuce cultivated with organic fertiliser

Composting and vermicomposting are techniques used to stabilise organic waste and produce nutrient-rich fertilisers. Experiments that analyse cultures and different fertilisers applied to soil require a heavy workload and time to monitor plant development. Thus, alternative methods to facilitate this monitoring need to be created. The aim of this paper was to assess the development of lettuce cultivated with eight types of fertilisers derived from composting and vermicomposting, and to compare the traditional data collection method with leaf cover analysis software (“LCAS”). The assessed items were growth, diameter, number of leaves, fresh mass, dry mass, and leaf cover obtained using the software. Overall, the lettuce cultivars that received organic fertiliser developed better than the witness, especially in relation to treatments containing ash, such as the C4 compost (Mud, Ash and Pruning litter) and V2 vermicompost (Mud, Ash and Coffee husk). LCAS proved to be an effective tool to monitor the growth of lettuce cultivars in comparison with the traditional method since statistically the pattern of behaviour of the treatments was similar for plant cover, diameter, length, and fresh and dry masses

Metastasis to the gluteus maximus muscle from renal cell carcinoma with special emphasis on MRI features

<p>Abstract</p> <p>Background</p> <p>The skeletal muscle is an unusual site for metastasis from renal cell carcinoma (RCC). Metastatic RCC must be differentiated from benign primary soft-tissue tumors because aggressive surgical resection is necessary.</p> <p>Case presentation</p> <p>We present the case of a 65-year-old man with metastatic RCC in the gluteus maximus muscle (3.8 cm in diameter) found on enhanced computed tomography (CT) 6 years after nephrectomy. Retrospectively, the small mass (1 cm in diameter) was overlooked 5 years earlier on enhanced CT. Because the growth of the lesion was slow, benign tumor was a differential diagnosis. However, magnetic resonance imaging (MRI) showed that the mass had high-signal intensity on T1- and T2-weighted images (WIs) compared to that of skeletal muscle, with mild enhancement by Gadolinium. The MRI features were unusual for most soft-tissue tumors having low-signal intensity on T1-WI and high-signal intensity on T2-WI. Therefore, under a diagnosis of metastatic RCC, the lesion was resected together with the surrounding skeletal muscle. The histology was confirmed to be metastatic RCC.</p> <p>Conclusion</p> <p>MRI features of metastatic RCC may be beneficial in differentiating it from primary soft-tissue tumor.</p

Molecular cloning and characterization of the germline-restricted chromosome sequence in the zebra finch

The zebra finch (Taeniopygia guttata) germline-restricted chromosome (GRC) is the largest chromosome and has a unique system of transmission in germ cells. In the male, the GRC exists as a single heterochromatic chromosome in the germline and is eliminated from nuclei in late spermatogenesis. In the female, the GRC is bivalent and euchromatic and experiences recombination. These characteristics suggest a female-specific or female-beneficial function of the GRC. To shed light on the function of GRC, we cloned a portion of the GRC using random amplified polymorphic DNA–polymerase chain reaction and analyzed it using molecular genetic and cytogenetic methods. The GRC clone hybridized strongly to testis but not blood DNA in genomic Southern blots. In fluorescent in situ hybridization analysis on meiotic chromosomes from synaptonemal complex spreads, the probe showed hybridization across a large area of the GRC, suggesting that it contains repetitive sequences. We isolated a sequence homologous to the GRC from zebra finch chromosome 3 and a region of chicken chromosome 1 that is homologous to zebra finch chromosome 3; the phylogenetic analysis of these three sequences suggested that the GRC sequence and the zebra finch chromosome 3 sequence are most closely related. Thus, the GRC sequences likely originated from autosomal DNA and have evolved after the galliform–passeriform split. The present study provides a foundation for further study of the intriguing GRC