土方 貴雄

Virus-encoded microRNAs (miRNAs) target viral and host mRNAs to repress protein production from viral and host genes, and regulate viral persistence, cell transformation, and evasion of the immune system. The present study demonstrated that simian virus 40 (SV40)-encoded miRNA miR-S1 targets a cellular miRNA miR-1266 to derepress their respective target proteins, namely, T antigens (Tags) and telomerase reverse transcriptase (TERT). An in silico search for cellular miRNAs to interact with viral miR-S1 yielded nine potential miRNAs, five of which, including miR-1266, were found to interact with miR-S1 in dual-luciferase tests employing reporter plasmids containing the miRNA sequences with miR-S1. Intracellular bindings of miR-1266 to miR-S1 were also verified by the pull-down assay. These miRNAs were recruited into the Ago2-associated RNA-induced silencing complex. Intracellular coexpression of miR-S1 with miR-1266 abrogated the downregulation of TERT and decrease in telomerase activity induced by miR-1266. These effects of miR-S1 were also observed in miR-1266-expressing A549 cells infected with SV40. Moreover, the infected cells contained more Tag, replicated more viral DNA, and released more viral particles than control A549 cells infected with SV40, indicating that miR-S1-induced Tag downregulation was antagonized by miR-1266. Collectively, the present results revealed an interplay of viral and cellular miRNAs to sequester each other from their respective targets. This is a novel mechanism for viruses to manipulate the expression of viral and cellular proteins, contributing to not only viral lytic and latent replication but also cell transformation observed in viral infectious diseases including oncogenesis.

Background and aims: Tsumura-Suzuki obese diabetic (TSOD) is a good model of metabolic syndrome showing typical lesions found in nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, and develops spontaneous hepatic tumors with a high frequency. Majority of the developing tumors overexpress glutamine synthetase (GS), which is used as a marker of hepatocellular carcinoma (HCC). The aim of this study is to assess the status of expression of metabolism-related genes and the level of bile acids in the TSOD mice-derived tumors and to determine the association with metabolic dysregulation between human HCC and TSOD mice-derived tumors. Methods: GS-positive hepatic tumors or adjacent normal tissues from 71-week-old male TSOD mice were subjected to immunohistochemical staining, quantitative RT-PCR (qRT-PCR), quantitation of cholic acid and taurocholic acid. Results: We found that downregulation of the rate-limiting enzyme for betaine synthesis (BADH), at both mRNA and protein levels in GS-positive TSOD mice-derived tumors. Furthermore, the bile acid receptor FXR and the bile acid excretion pump BSEP (Abcb11) were found to be downregulated, whereas BAAT and Akr1c14, involved in primary bile acid synthesis and bile acid conjugation, were found to be upregulated at mRNA level in GS-positive TSOD mice-derived tumors. BAAT and Akr1c14 were also overexpressed at protein levels. Total cholic acid was found to be increased in GS-positive TSOD mice-derived tumors. Conclusion: Our results strongly support the significance of TSOD mice as a model of spontaneously developing HCC.

miR-1, miR-133a, and miR-206 are muscle-specific microRNAs expressed in skeletal muscles and have been shown to contribute to muscle development. To gain insight into the pathophysiological roles of these three microRNAs in dystrophin-deficient muscular dystrophy, their expression in the tibialis anterior ( TA) muscles of mdx mice and CXMDJ dogs were evaluated by semiquantitative RT-PCR and in situ hybridization. Their temporal and spatial expression patterns were also analyzed in C2C12 cells during muscle differentiation and in cardiotoxin (CTX)-injured TA muscles to examine how muscle degeneration and regeneration affect their expression. In dystrophic TA muscles of mdx mice, miR-206 expression was significantly elevated as compared to that in control TA muscles of age-matched B10 mice, whereas there were no differences in miR-1 or miR-133a expression between B10 and mdx TA muscles. On in situ hybridization analysis, intense signals for miR-206 probes were localized in newly formed myotubes with centralized nuclei, or regenerating muscle fibers, but not in intact pre-degenerated fibers or numerous small mononucleated cells, possibly proliferating myoblasts and inflammatory infiltrates. Similar increased expression of miR-206 was also found in C2C12 differentiation and CTX- induced regeneration, in which differentiated myotubes or regenerating fibers showed abundant expression of miR-206. However, CXMDJ TA muscles contained smaller amounts of miR-206, miR-1, and miR-133a than controls. They exhibited more severe and more progressive degenerative alterations than mdx TA muscles. Taken together, these observations indicated that newly formed myotubes showed markedly increased expression of miR-206, which might reflect active regeneration and efficient maturation of skeletal muscle fibers.

プレクチンと様々なタンパク質の相互作用とその生理機能意義について解説したもの。

ジストロフィンの欠損により起こる筋ジストロフィーの治療ではジストロフィンを強制発現させることが必要であるが。そのための遺伝子治療の戦略とデリバリーシステムについて解説した。（湯浅の文章に基づき、全体を添削し足りない部分を補った）

プレクチン遺伝子、タンパク質とそのアイソフォーム、さらにそれらの機能を説明し、遺伝子変異によりプレクチンタンパク質機能が損なわれると様々な分子病(単純型先天性表皮水疱症、筋ジストロフィーなど)の発症につながることを解説したもの。

新規にクローニングされたデスマスリン分子の特色や細胞内での局在を解説した。

プレクチンと筋形質膜との関係を筋形質膜裏打ちタンパク質であるジストロフィンやピンキュリンとの関係を中心に解析した.プレクチンはZ線レベルと縦ストランドからなる格子状の領域の筋形質膜下にジストロフィンとビンキュリンと共に共存し,ジストロフィンとビンキュリンはM線領域に相当する筋形質膜下にも局在したが,プレクチンは存在しなかった.ジストロフィン,ピンキュリンは共にプレクチン・ストランドが結合しているdense plaques内に存在していた.プレクチンは主としてメタピンキュリンと連関していることを示唆していた

Plectin is a versatile cytoskeletal linker protein and preferentially localized at interfaces between intermediate filaments and the plasma membrane in muscle, epithelial cells and other tissues. Its deficiency leads to disorganization of myofibrils and disruption of sarcolemmal membrane integrity in skeletal muscle. To get a better understanding of plectin's functional roles at the sarcolemma of skeletal muscles and to gain some insights into the underlying mechanism of plectin-deficient muscular dystrophy, we investigated both the ultrastructural localization and the molecular relationships of plectin to subsarcolemmal cytoskeletal components, such as desmin, dystrophin and vinculin, in rat skeletal muscles. Immunoelectron microscopy revealed that plectin fine threads tethered desmin intermediate filaments onto subsarcolemmal dense plaques overlying Z-lines. These subsarcolemmal dense plaques were found to contain dystrophin and vinculin, and thus may be the structural basis of costameres. Furthermore, the costameric dense plaques were apparently in register with -dystroglycan-specific gold labels on the outer surface of sarcolemma. Immunoprecipitation experiments showed in vivo association of plectin with desmin, vinculin, meta-vinculin, dystrophin and actin. Treatment of plectin immunoprecipitates with gelsolin reduced actin, dystrophin, vinculin, and meta-vinculin but not desmin, implicating that subsarcolemmal actin could at least partly mediate the interaction between plectin and dystrophin or (meta-) vinculin. Altogether, our data suggest that plectin, along with desmin intermediate filaments, might serve a vital structural role in the stabilization of the subsarcolemmal cytoskeleton, contributing to the maintenance of sarcolemmal integrity against shear stresses imposed during muscle contraction. © 2002, The Kitakanto Medical Society. All rights reserved.

骨格筋の中間径フィラメントネットワークはZ線間やZ線と筋形質膜とをつないでいるが、このネットワークの分子構築について自己のデータに基づき解説した。さらにこのネットワーク構造が力の効率的産生に必要不可欠であることに言及し、ネットワーク構造の欠落が筋疾患の原因となることも述べた。

中間径フィラメント結合タンパク質を紹介し、そのノックアウトマウスの特徴を解説した。

筋細胞表面膜標品についての生化学的及び免疫電子顕微鏡分析を行い,ジストロフィンを含む裏打ち領域に一致して見られる膜表面の微細な突起構造がα-DGであり,ここにラミニンが再構成されることを示し,ジストロフィン-DG複合体-ラミニンの一連のタンパク質連鎖からなる分子軸を超微形態学的に証明した.更に,デスミンからなる中間径フィラメントがプレクチン分子によってZ板に連結されることを免疫電子顕微鏡的に示し,中間径フィラメントは更にプレクチンを介して裏打ち構造に結合し,そこにジストロフィンの局在も示すことができ,裏打ちの少なくとも一部はジストロフィンを含むことを証明した

プレクチンが中間径フィラメントとZ板を連結していると推論される.筋形質膜が特殊化した筋腱連結部や神経筋接合部でもプレクチンの存在が認められた

食虫目スンクスの神経成長因子の組織間での比活性の検討や顎下腺や前立腺内での組織内分布を詳細に述べた総説。（免疫組織化学や電子顕微鏡による解析で貢献した。）

骨格筋線維間にみられる様々な連結様式や連結構造を紹介し、その連結様式や構造をもつ筋の力学的特性を考察した。さらに、運動単位や運動制御と筋線維構築との関係ならびに問題点についても言及した総説。

スンクス鼻粘膜下の終神経を光学顕微鏡・電子顕微鏡で精査し、２種類の異なる神経節が存在することを明らかにした。（電子顕微鏡の解析に関して貢献した。）

原猿類ガラゴにおいて、ヒト大腿二頭筋の短頭に相当するM.gluteocruralisの存在有無を支配神経の観点から精査し、独立した筋腹は存在しないが長頭相当部分に融合した形で存在することを示唆した。