To evaluate the role of 8/endobrevin in constitutive exocytosis, we have examined the exocytotic pathways of 8 and human growth hormone, both GFP-tagged, by total internal reflection fluorescence microscopy (TIRF-M). Human GH-GFP and 8-GFP were similarly expressed in small round vesicles and elongated tubular vesicles in HeLa cells, and were mostly exocytosed at the peripheral area of the cells. 8-GFP gave 2 types of exocytotic images: a burst type and a non-burst type. The burst type showed a sharp transient increase in the peak fluorescence intensity and a much slower decrease in the average intensity in the active windows, where exocytosis took place, as observed in the “full-fusion” type of exocytosis. The non-burst type showed a relatively long-lasting fusion to the plasma membrane with little transfer of 8-GFP to the plasma membrane, as observed in the so-called “kiss-and-run” type of exocytosis. Endogenous 8 and hGH-GFP were colocalized on the same vesicles at least in part. However, the constitutive exocytosis of hGH-GFP and CLuc, a secreted luciferase from Cypridina noctiluca, was normal, even when siRNAs for 8 and 3 robustly decreased their proteins. These results suggest that 8 is not essential for constitutive exocytosis, although it can be involved in the exocytosis.

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We examined the expression and intracellular localization of vesicle-associated membrane protein 2 (2) during the differentiation of skeletal muscle cells by immunofluorescence microscopy. In isolated single myofibers, 2 was expressed in quiescent satellite cells, downregulated in proliferating myoblastic cells, and re-expressed with differentiation. In the myoblastic cell line C2C12, 2 was expressed at a low level in the proliferating stage, and then increased after differentiation into myotubes. Based on these results, we propose that 2 can be used as a molecular marker for both quiescent satellite cells and myotubes, but not for proliferating myoblasts. We also found the partial colocalization of 2 with transferrin- or Rab11-labeled vesicles in myotubes, suggesting a role of 2 in the trafficking of recycling endosomes.

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Small extracellular vesicles (sEVs) are largely classified into two types, plasma-membrane derived sEVs and endomembrane-derived sEVs. The latter type (referred to as exosomes herein) is originated from late endosomes or multivesicular bodies (MVBs). In order to release exosomes extracellularly, MVBs must fuse with the plasma membrane, not with lysosomes. In contrast to the mechanism responsible for MVB–lysosome fusion, the mechanism underlying the MVB–plasma membrane fusion is poorly understood. Here, we systematically analyze soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family proteins and identify

5 as an MVB-localized SNARE protein required for exosome release. Depletion of 5 in HeLa cells impairs exosome release. Mechanistically, 5 mediates exosome release by interacting with SNAP47 and plasma membrane SNARE Syntaxin 1 (STX1) or STX4 to release exosomes. 5 is also found to mediate asymmetric exosome release from polarized Madin-Darby canine kidney (MDCK) epithelial cells through interaction with the distinct sets of Q-SNAREs, suggesting that 5 is a general exosome regulator in both polarized cells and non-polarized cells.

Key words: exosome, small extracellular vesicle (sEV), multivesicular body, SNARE,

5

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細胞内の膜交通システムにおいて、輸送小胞と標的オルガネラ膜の融合を制御するSNARE分子の中に、輸送小胞膜に局在するR-SNAREがある。R-SNAREは、アミノ末端にlongin domain と呼ばれる領域を持つlonginと、それ持たないbrevinに大別され、陸上植物のR-SNAREは全てlonginであることが知られている（SEC22、YKT6、7のグループが存在する）。シロイヌナズナゲノムには18個のlonginがコードされており、そのうち12個が7に分類される。我々は、7の機能分化が植物細胞における細胞内輸送経路の多様化に関与しているのではないかと考え、714というタンパク質に注目して解析を行った。シロイヌナズナ7は、さらに71と72の2種類に分類され、71の多くが液胞膜に局在するのに対し、714のみがゴルジ体に局在することが，シロイヌナズナ培養細胞での一過的発現解析により示されている。本大会では、シロイヌナズナ714と陸上植物の様々な系統から単離した714ホモログの詳細な細胞内局在解析の結果を報告するとともに、714がどのような細胞内輸送経路に関与しているのかについて、進化細胞生物学的な視点から議論したい。

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Tetanus neurotoxin (TeNT) produced by Clostridium tetani specifically cleaves /synaptobrevin () in central neurons, thereby causing inhibition of neurotransmitter release and ensuring spastic paralysis. Although polysialogangliosides act as components of the neurontoxin binding sites on neurons, evidence has accumulated indicating that a protein moiety is implicated as a receptor of TeNT. We have observed that treatment of cultured mouse neuronal cells with the phosphatidylinositol-specific phospholipase C (PIPLC) inhibited TeNT-induced cleavage of . Also, we have shown that the blocking effects of TeNT on neuroexocytosis can be prevented by incubation of Purkinje Cell preparation with PIPLC. In addition, treatment of cultured mouse neuronal cells with filipin, which disrupt clustering of GPI-anchored proteins in lipid refts, prevented intraneuronal cleavage by TeNT. Our results demonstrate that high sensitivity of neurons to TeNT requires rafts and one or more GPI-anchored protein(s) which act(s) as a pivotal receptor for neurotoxin. [Jpn J Physiol 54 Suppl:S142 (2004)]

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SNAREは膜交通経路において膜融合の実行を担う分子であり，その構造上の特徴から，Qa-，Qb-，Qc-，R-SNAREの4種に分類される．このうち陸上植物で著しい多様化が見られるR-SNARE，7ファミリーは，71と72という二つのサブグループで構成されている．72グループの中にはさらに，アミノ末端側のLonginドメインに約20アミノ酸残基からなる酸性配列が挿入された特徴的な分子種が存在する．当研究室ではこれまでに、シロイヌナズナの酸性挿入配列を持つ72分子，727が，エンドソームを中心としたポストゴルジ輸送経路で機能することや，挿入配列がエンドソームへの局在や液胞におけるSNARE複合体の形成に必須であることを明らかにしている．この挿入配列を持つ72分子は，これまで種子植物に特有のものと考えられていた．しかし，最近コケ植物苔類ゼニゴケにも類似の分子が存在していることが明らかとなり，その解析から，この挿入配列がオルタナティブスプライシングに由来することが新たに判明した．現在，このオルタナティブスプライシングが，陸上植物における72の進化において果たした役割を解明すべく研究を進めている．本講演では，これらゼニゴケ72スプライシングバリアントの細胞内局在やそれらが機能する輸送経路について解析した結果を報告する．

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Secretory granules (SGs) of salivary glands are considered to be generated as an immature granule and to mature by condensing their contents. Syntaxin 6 and vesicle associated membrane protein 4 (

4) are detected in the membrane fractions of immature but not mature SGs, suggesting that syntaxin 6 and 4 are transported from SGs to other organelles during the process of granule maturation. To study the role of syntaxin 6 in the biogenesis of granules, we transfected syntaxin 6 fused with enhanced green fluorescent protein(EGFP)into primary cultured parotid acinar cells. The EGFPsyntaxin 6 signal overlapped with that of an early endosome marker, early endosome antigen 1(EEA1), which was detected using immunofluorescence. The localization of EGFP-fused 4 was also similar to that of EEA1, suggesting that syntaxin 6 and 4 are transported from immature SGs to early endosomes. Transfection of a syntaxin 6 mutant that lacks a SNARE-binding domain caused a decrease in the number of SGs although the localization of the mutant was unchanged from that of the wild type syntaxin 6. These results suggest that syntaxin 6 has an important role in the maintenance of SGs in parotid acinar cells.

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SNAREは小胞輸送において膜融合を実行する因子であり，SNAREモチーフと呼ばれるcoiled-coil構造を形成するアミノ酸配列の特徴から，Qa，Qb，QcとRの4種に分類される．陸上植物種に保存された主要なR-SNAREである7ファミリーは，71と72という二つのサブグループで構成されている．さらに，種子植物の72グループには，longin domainに約20アミノ酸からなる酸性配列が挿入された特異な分子種が存在する．当研究室ではこれまでに，この酸性配列を有するシロイヌナズナ72分子，727がエンドソームを中心としたポストゴルジ輸送ネットワークで機能することを明らかにしてきた．現在はさらに，この種子植物特異的なR-SNAREの進化において挿入配列が果たした役割を解明すべく研究を進めている．本講演では，727からの酸性配列の欠失や，祖先型72分子への酸性配列の人為的挿入が，それぞれの分子機能に及ぼす影響について報告するとともに，種子植物の様々な系統から単離した72の機能解析の結果を報告する．

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This paper addresses pilot contamination in massive multiple-input multiple-output (MIMO) uplink. Pilot contamination is caused by reuse of identical pilot sequences in adjacent cells. To solve pilot contamination, the base station utilizes differences between the transmission frames of different users, which are detected via joint channel and data estimation. The joint estimation is regarded as a bilinear inference problem in compressed sensing. Expectation propagation (EP) is used to propose an iterative channel and data estimation algorithm. Initial channel estimates are attained via time-shifted pilots without exploiting information about large scale fading. The proposed EP modifies two points in conventional bilinear adaptive vector approximate message-passing (BAd-

). One is that EP utilizes data estimates after soft decision in the channel estimation while BAd- uses them before soft decision. The other point is that EP can utilize the prior distribution of the channel matrix while BAd- cannot in principle. Numerical simulations show that EP converges much faster than BAd- in spatially correlated MIMO, in which approximate message-passing fails to converge toward the same fixed-point as EP and BAd-.

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病期I，病期II Aかつ巨大腫瘤を有しない小児の早期ホジキンリンパ腫（HL; Hodgkin lymphoma）に対する標準治療は，2–4コースの多剤併用化学療法と初発時に腫瘍が存在したリンパ節領域（IF; involved field）に対する低線量照射（IF照射）である．早期HLに対し，さまざまな化学療法が試みられている．また，晩期合併症の軽減を目的として，治療反応が良好だった場合，IF照射省略が試みられている．我々は2例の小児早期HLに対して，vinblastine，doxorubicin，methotrexate，prednisoloneによる

療法を実施した．療法2コース後に¹⁸F-fluorodeoxyglucose positron emission tomography/computed tomographyで完全寛解に到達したことを確認し，2コースの療法を追加してIF照射を省略した．肝酵素上昇と好中球減少症が治療中に認められた有害事象であった．2例は4年以上完全寛解を維持しており，晩期合併症を認めていない．療法は小児早期HLに対して安全な化学療法レジメンである可能性が示唆された．

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【目的】精巣上体通過中に様々な機能性分子を取り込んだ精子は，膜融合プロセスを介して尾部に残存する細胞滴を遊離する。以前我々は，マウス精子が精巣上体通過により細胞滴へPantophysin（Pphn）を獲得することを明らかにした。他細胞では，PphnはSNARE複合体の構成因子であるVesicle-associated membrane protein（

） 2と複合体を形成し，Ca²⁺依存性膜融合の制御に関与すると考えられている。今までの研究で，2および同じくSNARE複合体の構成因子である3は，先体反応機構に関与することが指摘されているが，細胞滴における存在は不明である。本研究では，マウス精子において，2および3の発現特性およびPphnとの関係性について調べ，さらに機能的役割を探った。【方法】実験①：ICRマウスの精巣上体尾部精子を免疫染色あるいは共免疫沈降に供し，2および3の局在あるいは両者とPphnとの関係性を調べた。実験②：精巣上体頭部，体部および尾部精子において，2の発現量および細胞滴遊離率を調べた。実験③：0あるいは10 μM EGTA/AM処理した精子において，既報に従いショ糖密度勾配遠心分離法で細胞滴を除去後，PI染色により原形質膜の正常性を調べることで，膜融合率を求めた。【結果】実験①：2および3は精子先体胞および尾部の細胞滴に局在した。共免疫沈降の結果，何れのタンパクもPphnと複合体を形成していることが分かった。実験②：精子における2発現量は精巣上体頭部から尾部にかけて低下した。一方，細胞滴遊離率は，精巣上体通過により増加した（P<0.05）。実験③：細胞滴除去後の膜融合率は細胞内Ca²⁺のキレートにより低下した（P<0.05）。以上の結果から， 2および3は細胞滴においてPphnと複合体を形成し，その遊離において重要な役割を果たす可能性が示唆された。

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Autophagy is a highly conserved intracellular degrading system and its dysfunction is considered related to the cause of neurodegenerative disorders. A previous study showed that the inhibition of endocytosis transport attenuates soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein transport to lysosomes and block autophagy. The other studies demonstrated oxidative stress, one of the inducers of neurodegenerative diseases inhibits endocytosis transport. Thus, we hypothesized that oxidative stress-induced endocytosis inhibition causes alteration of SNARE protein transport to lysosomes and impairs autophagy. Here, we demonstrated that oxidative stress inhibits endocytosis and decreased the lysosomal localization of

8, one of the autophagy-related SNARE proteins in a human neuroblastoma cell line. Moreover, this oxidative stress induction blocked the autophagosome-lysosome fusion step. Since we also observed decreased lysosomal localization of 8 and inhibition of autophagosome-lysosome fusion in endocytosis inhibitor-treated cells, oxidative stress may inhibit 8 trafficking by suppressing endocytosis and impair autophagy. Our findings suggest that oxidative stress-induced inhibition of 8 trafficking to lysosomes is associated with the development of neurodegenerative diseases due to the blocked autophagosome-lysosome fusion, and may provide a new therapeutic target for restoring the autophagic activity.

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In this paper, in order to reduce power dissipation at MOSFET gate port of class E amplifier, we propose gate signal peaking using third harmonic component. With PSPICE simulation, it is shown that higher power efficiency and higher output power of power stage can be obtained using third harmonic component than pure sinusoidal gate signal with same level of gate power dissipation.

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Acinar cells of salivary parotid gland produce saliva by two types of secretion; water fluid and exocytosis of proteins like amylase. Exocytosis of amylase is mainly regulated by intracellular cAMP, and 2, one of the SNARE proteins, is essential for cAMP-dependent amylase secretion. However, it is difficult to investigate the molecular mechanism of parotid acinar cells since there is no stable cell line that has the ability to release amylase upon stimulation. To examine the functions of parotid acinar cells, we tried to establish a transfection system of exogenous genes to primary culture. Acinar cells were dispersed from rat parotid glands by enzyme digestion, and cultured in Waymouth's medium that contained rat serum. Cells that cultured in dishes coated with various extracellular matrix kept amylase activity, and have the ability to release amylase in response to isoproterenol at least for 48 h. The cells spread on the dish bases and formed filopodia although they have granules that contain amylase, suggesting they are derived from acinar cells. 2 gene fused with enhanced green fluorescence protein (EGFP-2) was transfected to dispersed acinar cells and cultured for 48 h. EGFP-2 protein was observed to be correctly localized at granules that contained amylase. Therefore, the cells cultured by this method can be used to examine the effects of exogenous genes on functions of parotid acinar cells like regulated exocytosis and maturation of secretory granules. [Jpn J Physiol 54 Suppl:S78 (2004)]

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本研究では，上体を有する受動走行に内在する安定化メカニズムの解明を目的とする．シミュレーションの結果，(1) 上体を有する受動走行の安定解が存在する，(2) 重心の位置が受動走行の安定性に大きな影響を与える，(3) 上体の慣性モーメントが受動走行の安定性に影響を与える，ことが確認された．これらの結果は，上体を活用することで受動走行の安定性が向上することを示唆する結果である．

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我々は，小胞輸送，特にエンドサイトーシスが植物の高次現象に果たす役割を明らかにするべく，エンドソームに局在するR-SNAREである727に注目し研究を行っている．727はシロイヌナズナで唯一エンドソームにほぼ特異的に局在することから （Ueda et al., 2004; Uemura et al., 2004），エンドサイトーシスおいて重要な機能を担うと考えられるが， 変異体に目立った表現型は見られない．そこで，PVCと液胞に局在するQa-SNARE，VAM3との遺伝学的相互作用を調べたところ， が胚致死となる一方， の過剰発現がvam3 の表現型を抑圧することが明らかとなった．さらに，細胞内局在解析の結果，これら2つのSNAREが液胞とPVCが接する場所で共局在することも見いだした．VAM3はVTI11，SYP51 （いずれもQ-SNARE） と複合体を作ることが報告されているが (Sanderfoot et al., 2001; Yano et al., 2003)，727はこれら全てと共免疫沈降する．これらの結果から，727がVAM3，VTI11，SYP51と複合体を形成して機能するとともに，この複合体が液胞とPVCの融合を制御することにより，胚発生に不可欠な役割を果たしていることが明らかになった．

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Double immunohistochemistry of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins [synaptosomal-associated protein of 25kDa (SNAP-25), syntaxin and vesicle-associated protein-2 (-2)], and specific cell markers of taste buds cells [α-gustducin and phospholipase Cβ2 (PLCβ2) for type II cells; neural cell adhesion molecule (NCAM) for type III cells] was applied to gustatory epithelia of the rat circumvallate papillae. All three SNARE proteins were present in some elongated taste buds cells as well as intra-, peri- and subgemmal nerve fibers. Double immunohisotochemistry revealed that nearly all α-gustducin and PLCβ2 immunoreactive cells expressed SNAP-25, syntaxin, and -2. A majority of NCAM immunoreactive cells showed immunoreactivity for these SNARE proteins. These results indicate that these synapse-associated proteins (SNAP-25, syntaxin and -2) are present in both type II cells and type III cells. Moreover, more than 50% of intragemmal cells containing SNARE proteins showed immunoreactivities for α-gustducin, PLCβ2, and NCAM, suggesting the possible presence of transitional cells having histochemical proterties of both type II and type III cells.

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