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Moyamoya disease (MMD) is a cerebrovascular disease characterized by progressive occlusion of the internal carotid arteries. Genetic studies originally identified RNF213 as an MMD susceptibility gene that encodes a large 591 kDa protein with a functional RING domain and dual AAA+ ATPase domains. As the functions of RNF213 and its relationship to MMD onset are unknown, we set out to characterize the ubiquitin ligase activity of RNF213, and the effects of MMD patient mutations on these activities and on other cellular processes. In vitro ubiquitination assays, using the RNF213 RING domain, identified Ubc13/Uev1A as a key ubiquitin conjugating enzyme that together generate K63-linked polyubiquitin chains. However, nearly all MMD patient mutations in the RING domain greatly reduced this activity. When full-length proteins were overexpressed in HEK293T cells, patient mutations that abolished the ubiquitin ligase activities conversely enhanced nuclear factor κB (NFκB) activation and induced apoptosis accompanied with Caspase-3 activation. These induced activities were dependent on the RNF213 AAA+ domain. Our results suggest that the NFκB- and apoptosis-inducing functions of RNF213 may be negatively regulated by its ubiquitin ligase activity and that disruption of this regulation could contribute towards MMD onset.

Neuromuscular junctions (NMJs) are cholinergic synapses characterized by ultrastructural specializations, including the presynaptic active zones, the acetylcholine (ACh) release sites of the motor nerve terminal, and the postsynaptic junctional folds of muscle membrane, where ACh receptors (AChRs) cluster for efficient neuromuscular transmission. The formation and maintenance of NMJs are governed by the muscle-specific receptor tyrosine kinase MuSK. We had previously demonstrated that the muscle cytoplasmic protein Dok-7 is an essential activator of MuSK, and its activation and NMJ formation are enhanced in the Dok-7 transgenic (Tg) mice, in which Dok-7 is specifically overexpressed in skeletal muscle. Although Dok-7 Tg mice develop abnormally large NMJs but show normal motor function, the forced expression of Dok-7 in the muscle improves impaired motor activity in mouse models of neuromuscular disorders with NMJ defects. However, the effect of Dok-7 overexpression in skeletal muscle on ultrastructure and neuromuscular transmission of NMJs is yet to be studied. Here, we investigated the structural and electrophysiological properties of NMJs in the diaphragm muscle of 8-week-old Dok-7 Tg mice. The areas of the presynaptic motor nerve terminals and postsynaptic muscle membrane of NMJs were 2.7 and 4.3 times greater in Dok-7 Tg mice than in WT mice, respectively. Electrophysiological analyses revealed that neuromuscular transmission via NMJs in Dok-7 Tg mice was significantly enhanced but not proportionally with the increased size of the synaptic contact. Consistent with this, the densities of active zones and synaptic vesicles (ACh carriers) in the presynaptic motor nerve terminals were reduced. In addition, the density and size of postsynaptic junctional folds in the muscle membrane were also reduced. Moreover, terminal Schwann cells exhibited significantly greater penetration of their processes into the synaptic clefts, which connect the pre- and post-synaptic specializations. Together, our findings demonstrate that transgenic overexpression of Dok-7 in the skeletal muscle enhances neuromuscular transmission with significant enlargement and ultrastructural alterations of NMJs, the latter of which might prevent toxic overactivation of AChRs at the abnormally enlarged NMJs.

Amyotrophic lateral sclerosis (ALS) is a progressive, multifactorial motor neurodegenerative disease with severe muscle atrophy. The glutamate release inhibitor riluzole is the only medication approved by the FDA, and prolongs patient life span by a few months, testifying to a strong need for new treatment strategies. In ALS, motor neuron degeneration first becomes evident at the motor nerve terminals in neuromuscular junctions (NMJs), the cholinergic synapse between motor neuron and skeletal muscle; degeneration then progresses proximally, implicating the NMJ as a therapeutic target. We previously demonstrated that activation of muscle‐specific kinase MuSK by the cytoplasmic protein Dok‐7 is essential for NMJ formation, and forced expression of Dok‐7 in muscle activates MuSK and enlarges NMJs. Here, we show that therapeutic administration of an adeno‐associated virus vector encoding the human DOK7 gene suppressed motor nerve terminal degeneration at NMJs together with muscle atrophy in the SOD1‐G93A ALS mouse model. Ultimately, we show that DOK7 gene therapy enhanced motor activity and life span in ALS model mice.

The motoneural control of skeletal muscle contraction requires the neuromuscular junction (NMJ), a midmuscle synapse between the motor nerve and myotube. The formation and maintenance of NMJs are orchestrated by the muscle-specific receptor tyrosine kinase (MuSK). Motor neuron-derived agrin activates MuSK via binding to MuSK's coreceptor Lrp4, and genetic defects in agrin underlie a congenital myasthenic syndrome (an NMJ disorder). However, MuSK-dependent postsynaptic differentiation of NMJs occurs in the absence of a motor neuron, indicating a need for nerve/agrin-independent MuSK activation. We previously identified the muscle protein Dok-7 as an essential activator of MuSK. Although NMJ formation requires agrin under physiological conditions, it is dispensable for NMJ formation experimentally in the absence of the neurotransmitter acetylcholine, which inhibits postsynaptic specialization. Thus, it was hypothesized that MuSK needs agrin together with Lrp4 and Dok-7 to achieve sufficient activation to surmount inhibition by acetylcholine. Here, we show that forced expression of Dok-7 in muscle enhanced MuSK activation in mice lacking agrin or Lrp4 and restored midmuscle NMJ formation in agrin-deficient mice, but not in Lrp4-deficient mice, probably due to the loss of Lrp4-dependent presynaptic differentiation. However, these NMJs in agrin-deficient mice rapidly disappeared after birth, and postsynaptic specializations emerged ectopically throughout myotubes whereas exogenous Dok-7-mediated MuSK activation was maintained. These findings demonstrate that the MuSK activator agrin plays another role essential for the postnatal maintenance, but not for embryonic formation, of NMJs and also for the postnatal, but not prenatal, midmuscle localization of postsynaptic specializations, providing physiological and pathophysiological insight into NMJ homeostasis.

Building connections by gene therapy Voluntary movement requires a molecular conversation between nerves and muscles. This conversation occurs at the neuromuscular junction, a structure where nerves and muscle physically connect. People with diseases characterized by muscle weakness have aberrantly small neuromuscular junctions. Arimura et al. used gene therapy to enlarge the neuromuscular junction, which made muscles stronger. Studying mouse models of two distinct neuromuscular disorders, they used an adenovirus vector to deliver DOK7 , a gene required for formation of the neuromuscular junction. The therapy improved the mice's motor activity and life span. Science , this issue p. 1505

Summary Highly topographic organization of neural circuits exists for the regulation of various brain functions in corticobasal ganglia circuits. Although neural circuit-specific refinement during synapse development is essential for the execution of particular neural functions, the molecular and cellular mechanisms for synapse refinement are largely unknown. Here, we show that protocadherin 17 (PCDH17), one of the nonclustered δ2-protocadherin family members, is enriched along corticobasal ganglia synapses in a zone-specific manner during synaptogenesis and regulates presynaptic assembly in these synapses. PCDH17 deficiency in mice causes facilitated presynaptic vesicle accumulation and enhanced synaptic transmission efficacy in corticobasal ganglia circuits. Furthermore, PCDH17 −/− mice exhibit antidepressant-like phenotypes that are known to be regulated by corticobasal ganglia circuits. Our findings demonstrate a critical role for PCDH17 in the synaptic development of specific corticobasal ganglia circuits and suggest the involvement of PCDH17 in such circuits in depressive behaviors.

The mammalian neuromuscular junction(NMJ), a cholinergic synapse between a motor neuron and a skeletal muscle fiber, is essential for neural control of muscle contraction. Impaired formation and/or maintenance of NMJs results in disorders of neuromuscular transmission such as myasthenia gravis(MG)and congenital myasthenic syndromes(CMSs). The formation and maintenance of NMJs are orchestrated by the muscle-specific receptor tyrosine kinase MuSK. Activation of MuSK involves its essential cytoplasmic activator Dok-7, the MuSK co-receptor Lrp4, and the motor neuron-derived MuSK activator agrin. Indeed, CMS-associated mutations are identified in the genes encoding these 4 proteins, and autoantibodies against MuSK, Lrp4, or agrin are found in MG patients, demonstrating the pathophysiological significance of the MuSK activation machinery. However, Lrp4 and agrin also play crucial roles separate from MuSK activation in NMJ formation and maintenance. Based on the finding that forced expression of Dok-7 in muscle activates MuSK and enlarges NMJs, we recently developed DOK7 gene therapy as a therapy aimed at enlarging the NMJ, which has potential for treating various neuromuscular disorders with defective NMJ structure.

As the synapse between a motor neuron and skeletal muscle, the neuromuscular junction (NMJ) is required for muscle contraction. The formation and maintenance of NMJs are controlled by the muscle-specific receptor kinase MuSK. Dok-7 is the essential cytoplasmic activator of MuSK, and indeed mice lacking Dok-7 form no NMJs. Moreover, DOK7 gene mutations underlie DOK7 myasthenia, an NMJ synaptopathy. Previously, we failed to detect MuSK activation in myotubes by Dok-7 mutated in the N-terminal pleckstrin homology (PH) or phosphotyrosine binding (PTB) domain or that lacked the C-terminal region (Dok-7-ΔC). Here, we found by quantitative analysis that Dok-7-ΔC marginally, but significantly, activated MuSK in myotubes, unlike the PH- or PTB-mutant. Purified, recombinant Dok-7-ΔC, but not other mutants, also showed marginal ability to activate MuSK's cytoplasmic portion, carrying the kinase domain. Consistently, forced expression of Dok-7-ΔC rescued Dok-7-deficient mice from neonatal lethality caused by the lack of NMJs, indicating restored MuSK activation and NMJ formation. However, these mice showed only marginal activation of MuSK and died by 3 weeks of age apparently due to an abnormally small number and size of NMJs. Thus, Dok-7's C-terminal region plays a key, but not fully essential, role in MuSK activation and NMJ formation.

Background: Breast-conserving surgery after primary systemic therapy (PST) has become a widely acceptable procedure for locally advanced breast cancers. Sometimes it is difficult to determine the appropriate resection-line at the time of surgical operation because of tumor size reduction or indistinct boundary of the tumor. Moreover we cannot detect tumors in cases with complete response. That was why we developed a new navigation technique using real-time image reconstruction. Materials and methods: Multi-detector row computed tomography (MD-CT) has been performed for all breast cancer patients in our institute. DICOM images of MD-CT were analyzed by image processing software ‘OsiriX'. OsiriX is an open source software which anyone can download from website (http://www. osirix-viewer.com). OsiriX has been specifically designed for navigation and visualization of multimodality and multidimensional images. We used 3D volume rendering images. At the time of surgery, pre-PST volume rendering images ware superimposed directly on the patients’ skin from projector which was connected to a personal computer in the operating room. Results: We could see ‘real-time’ rendering images (from skin to organ by layer to layer) on the patient's body. By using pre-PST imaging data, we could recognize precise tumor area and could perform breast conserving surgery safer and easier than before. Conclusion: Using 3D reconstruction MD-CT images which were obtained before PST, ‘real-time’ virtual navigation was helpful to determine the resection-line for clear surgical margin in breast-conserving surgery after PST. This new technique does not need some expensive equipments and easily performed by surgeon. Ordinary personal computer and projector make it possible to demonstrate a new environment in surgical operation. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-10-11.

The mammalian Dok protein family has seven members (Dok-1-Dok-7). The Dok proteins share structural similarities characterized by the NH2-terminal pleckstrin homology and phosphotyrosine-binding domains followed by SH2 target motifs in the COOH-terminal moiety, indicating an adapter function. Indeed, Dok-1 was originally identified as a 62 kDa protein that binds with p120 rasGAP, a potent inhibitor of Ras, upon tyrosine phosphorylation by a variety of protein tyrosine kinases. Among the Dok family, only Dok-1, Dok-2, and Dok-3 are preferentially expressed in hematopoietic/immune cells. Dok-1 and its closest relative Dok-2 act as negative regulators of the Ras-Erk pathway downstream of many immunoreceptor-mediated signaling systems, and it is believed that recruitment of p120 rasGAP by Dok-1 and Dok-2 is critical to their negative regulation. By contrast, Dok-3 does not bind with p120 rasGAP. However, accumulating evidence has demonstrated that Dok-3 is a negative regulator of the activation of JNK and mobilization of Ca2+ in B-cell receptor-mediated signaling, where the interaction of Dok-3 with SHIP-1 and Grb2 appears to be important. Here, we review the physiological roles and underlying mechanisms of Dok family proteins.

Major depressive and bipolar disorders are serious illnesses that affect millions of people. Growing evidence implicates glutamate signalling in depression, though the molecular mechanism by which glutamate signalling regulates depression-related behaviour remains unknown. In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to depression-related behaviour. The NR2A subunit of the NMDA receptor is tyrosine-phosphorylated, with Tyr 1325 as its one of the major phosphorylation site. We have generated mice expressing mutant NR2A with a Tyr-1325-Phe mutation to prevent the phosphorylation of this site in vivo. The homozygous knock-in mice show antidepressant-like behaviour in the tail suspension test and in the forced swim test. In the striatum of the knock-in mice, DARPP-32 phosphorylation at Thr 34, which is important for the regulation of depression-related behaviour, is increased. We also show that the Tyr 1325 phosphorylation site is required for Src-induced potentiation of the NMDA receptor channel in the striatum. These data argue that Tyr 1325 phosphorylation regulates NMDA receptor channel properties and the NMDA receptor-mediated downstream signalling to modulate depression-related behaviour.

We applied a new concept of “image overlay surgery” consisting of the integration of virtual reality (VR) and augmented reality (AR) technology, in which dynamic 3D images were superimposed on the patient’s actual body surface and evaluated as a reference for surgical navigation in gastrointestinal, hepatobiliary and pancreatic surgery. We carried out seven surgeries, including three cholecystectomies, two gastrectomies and two colectomies. A Macintosh and a DICOM workstation OsiriX were used in the operating room for image analysis. Raw data of the preoperative patient information obtained via MDCT were reconstructed to volume rendering and projected onto the patient's body surface during the surgeries. For accurate registration, OsiriX was first set to reproduce the patient body surface, and the positional coordinates of the umbilicus, left and right nipples, and the inguinal region were fixed as physiological markers on the body surface to reduce the positional error. The registration process was non-invasive and markerlesss, and was completed within 5 min. Image overlay navigation was helpful for 3D anatomical understanding of the surgical target in the gastrointestinal, hepatobiliary and pancreatic anatomies. The surgeon was able to minimize movement of the gaze and could utilize the image assistance without interfering with the forceps operation, reducing the gap from the VR. Unexpected organ injury could be avoided in all procedures. In biliary surgery, the projected virtual cholangiogram on the abdominal wall could advance safely with identification of the bile duct. For early gastric and colorectal cancer, the small tumors and blood vessels, which usually could not be found on the gastric serosa by laparoscopic view, were simultaneously detected on the body surface by carbon dioxide-enhanced MDCT. This provided accurate reconstructions of the tumor and involved lymph node, directly linked with optimization of the surgical procedures. Our non-invasive markerless registration using physiological markers on the body surface reduced logistical efforts. The image overlay technique is a useful tool when highlighting hidden structures, giving more information.

Transgastric access is a major route in natural orifice translumenal endoscopic surgery (NOTES); gastrotomy should be performed unless it would damage surrounding organs in the peritoneal cavity. This article describes a novel rendezvous gastrotomy technique over a direct percutaneous endoscopic gastrostomy (PEG). In six live porcines, the gastrotomy involved applying a direct PEG through the abdominal wall into the stomach and exchanging to a needle trocar. An endoscopic balloon catheter was passed through the trocar by rendezvous technique. Then the inflated balloon and endoscope were advanced to the peritoneal cavity through the gastrotomy. Transgastric cholecystectomy was performed with a hybrid needle grasper through the same percutaneous site and the gastrotomy was closed with endoscopic clips. The rendezvous gastrotomy technique could reduce guidewire exchange. The success rate was 100% (6/6). Mean times for transgastric peritoneoscopy and cholecystectomy were 25.5 and 83.5 min. Mortality and morbidity was 0%. The addition of the extra trocar was unnecessary in all procedures. The advantage of this introduction system includes the creation of controlled gastric perforation, which is easier to close. It provides reliable transgastric access and increases safety. It simplifies transgastric NOTES and provides less invasive hybrid NOTES procedure.

66歳男性，2型糖尿病（食事療法），脂肪肝にて近医通院中に，スクリーニング超音波にて肝腫瘍指摘され入院した．肝S5/6境界に辺縁ややirregularで径4cm大の腫瘍を認めた．HBs Ag，HBc Ab，HCV Ab陰性，腫瘍マーカー正常，入院時BMI 29.7kg/m2であった．肝腫瘍（胆管細胞癌疑い）と診断し肝S5/6切除術を行った．術後経過は良好で第8病日に退院した．病理では高～中分化型肝細胞癌であり，背景肝に中等度の脂肪変性と軽度の繊維化を認めた．飲酒歴を踏まえ非アルコール性脂肪肝炎（NASH）の非硬変肝に発生した肝細胞癌と診断した．術後13カ月現在無再発生存中である．近年，NASH硬変肝のHCC切除報告は散見されるが，NASH非硬変肝に発生したHCCに対する肝切除の報告は，われわれが医学中央雑誌（1983-2009）とPubMedにてNASH，肝細胞癌，肝切除をKey wordに検索したところ10例のみであり貴重な症例と思われたため報告する．

はじめに：鼠径ヘルニア根治手術は若い外科医が基本手技を鍛錬する場であり，外科手術の入門編として位置していた．現在，初期研修医制度が実施され，外科系診療科志望でない研修医も外科をローテーションするカリキュラムが組まれている．今回，我々は鼠径ヘルニア根治術を初期研修医に執刀させる是非を検討する．方法：2005年4月から2007年12月に根治手術を施行した139例を対象とした．134例にmesh plug法が，5例にPROLENE hernia system法が行われた．初期研修医執刀例は72例（R群），外科医執刀例は67例（S群）だった．R群とS群において，患者背景，術中，術後因子を検討した．結果：患者背景においてR群とS群で有意差はなかった．術中因子として手術時間においてR群：S群に有意差を認めた（88.0分：64.2分，p＜0.001）．術後因子は，入院期間（3.8±2.1日：4.9±8.3日，p=0.14），合併症（9.8％：6.6％，p=0.64）に統計学的に有意差はなかったが，再発はR群7例（9.7％），S群1例（1.5％）で有意にR群において高かった（p=0.04）．考察：入院期間，合併症には有意差はなく，再発率は初期研修医術者が外科医と比較し有意に高かったが，助手として外科専門医が指導することで，再発率が抑えられる．よって，現在の研修医制度において外科系研修カリキュラムの手術執刀についての指導指診作成が望まれる．

There are several lines of evidence that the podocyte slit diaphragm (SD), which serves as a structural framework for the filtration barrier in kidney glomerulus, also plays an essential role as a signaling platform. Several SD components including nephrin and TRPC6 are known to be phosphorylated by a Src family tyrosine kinase (SFK), Fyn. Here we have characterized Neph1, another SD component, as a novel substrate of SFK. Fyn interacts with and phosphorylates the cytoplasmic domain of Neph1 in vitro and in intact cells. Peptide mass fingerprinting and site-directed mutagenesis identified several tyrosine phosphorylation sites. In pull-down assays using rat glomerular lysates, Neph1 but not nephrin specifically binds to adaptor protein Grb2 and tyrosine kinase Csk in a phosphorylation-dependent manner. Both tyrosine 637 and 638 of Neph1 are crucial for Neph1-Grb2 binding. Phosphorylation of tyrosine 637 is significantly up-regulated in in vivo models of podocyte injury. Furthermore, Neph1 attenuates ERK activation elicited by Fyn, and this inhibitory effect requires the intact binding motif for the Grb2 SH2 domain. Our results shown here demonstrate that Neph1 is a novel in vivo substrate of SFK and suggest that Neph1 modulates ERK signaling through phosphorylation-dependent interaction with Grb2. Thus, SFK orchestrates a wide spectrum of protein-protein interactions and intracellular signaling networks at SD through tyrosine phosphorylation.

結腸憩室炎に対する治療法は軽∼中等症の場合では絶食·抗生物質投与による治療が行われるが，穿孔ないし穿破をともなう場合，外科的治療が選択されることが多い．今回我々は穿破による膿瘍形成した憩室炎に対し，大腸内視鏡を用いて排膿術を施行，保存的治療にて軽快した2例を経験した．2症例は右側腹部痛，発熱を主訴に当院受診．CTにて上行結腸に広範な壁肥厚像，膿瘍形成を認め，穿破による憩室炎の診断となった．大腸内視鏡施行し上行結腸の広範な炎症を認め，表面に白苔をともなう排膿している憩室に対し，生理食塩水にて洗浄し，膿瘍ドレナージを施行した．術直後に腹痛，発熱が軽減し，保存的治療のみで退院した．結腸憩室炎に対する大腸内視鏡による膿瘍ドレナージは，外科的なサポート下において1つの治療オプションとして考慮できる手技と考えられた．

胆道再建20症例を対象にDIC併用造影MDCT（以下:Dual contrast DIC-CT）を行い肝内胆管から吻合部，挙上空腸および胆管壁とその周囲の形態的観察の可能性につき検討した．胆管から吻合部にかけての胆管短径および長径計測可能は右前枝15/20例（75％），右後枝15/20例（75％），左枝10/18例（55.5％），吻合部16/24吻合（66.6％）で，仮想的内腔面観察可能は順に15/20例（75％），11/20例（55％），10/18例（55.5％），11/20例（55％）であった．胆管径計測，内腔面観察不能の原因はpneumobiliaと造影剤による描出不良であった．Dual contrast DIC-CTによる胆管壁の観察から胆管炎を繰り返す症例で壁厚の測定が可能であった．また術後一過性の原因不明発熱を繰り返す症例で吻合部胆管に接する嚢胞性病変を認めその原因と推察された．Dual contrast DIC-CTを用いた検討により胆道再建後症例の病態解明に役立つ検査の一つになり得るものと思われた．

胆管閉塞を来した好酸球性胆管炎の2症例を経験した. 症例1は53歳の女性で, 検診にて肝異常陰影を指摘された. CT, ERCPで肝左葉の肝内胆管閉塞を認めた. 胆汁細胞診は良性であったが胆管癌も否定できず肝左葉切除術を施行した. 病理組織学的には胆管閉塞部位に多数の好酸球浸潤を認めた. 術後18か月再発の兆候はない. 症例2は87歳の女性で, 主訴は右上腹部痛. ERCPで下部胆管閉塞と総胆管結石を認め, 閉塞部位の組織診で好酸球主体の炎症細胞浸潤を認めた. 内視鏡的胆道拡張術, 切石術により症状は軽快した. 術後17か月再発の兆候はない. 文献上, 好酸球性胆管炎として報告されている症例は18例と少ない. 悪性疾患を念頭に高侵襲な治療が施行された報告もあるが, 近年臨床報告の増加から徐々に病態が解明されつつあり, 内視鏡的治療が主体となっていくと考えられる. 本疾患の知見と的確な診断により治療の低侵襲を維持できると考えられた.

The neuromuscular junction (NMJ) is a synapse between a motor neuron and skeletal muscle and is required for muscle contraction. The formation and maintenance of NMJs are governed by the muscle-specific receptor tyrosine kinase MuSK. We previously showed that the muscle cytoplasmic protein Dok-7 is an essential activator of MuSK. Indeed, mice lacking either Dok-7 or MuSK form no NMJs, and defects in the human DOK7 gene underlie a congenital myasthenic syndrome (an NMJ disorder). However, it remains unproven whether Dok-7 is required for the postnatal maintenance of NMJs. In this study, we generated recombinant adeno-associated virus (AAV) vectors encoding short hairpin RNAs targeting the mouse dok-7 gene (AAV-shD7). Systemic administration of AAV-shD7 into 2-week-old mice down-regulated dok-7 expression in muscle and induced myasthenic symptoms including reduction in body weight and motor function. Moreover, AAV-shD7 treatment suppressed MuSK-dependent gene expression of NMJ components and reduced the size of NMJs. These results demonstrate that correct, physiological levels of dok-7 expression are required for the postnatal maintenance of NMJs.

In the central nervous system (CNS), myelination of axons occurs when oligodendrocyte progenitor cells undergo terminal differentiation, and initiate process formation and axonal ensheathment. Although Fyn, a member of the Src-family kinases (SFKs), plays an important role in this differentiation process, the substrates of Fyn in oligodendrocytes are largely unknown. Using mass spectrometric analysis, we identified focal adhesion kinase (FAK) as a tyrosine-phosphorylated protein in the rat-derived CG4 oligodendrocyte cell line. Tyrosine phosphorylation of FAK was enhanced during differentiation of CG4 cells in a Fyn-dependent manner. In addition, phosphorylation of FAK was stimulated by laminin, one of the ligands for integrin. Knockdown of FAK expression in CG4 cells suppressed process outgrowth on laminin. Rac1 and Cdc42 activities, which are required for oligodendrocyte process formation, were down-regulated in FAK-knockdown cells. Expression of wild-type (WT) FAK in FAK-knockdown CG4 cells restored outgrowth of processes, but the Y397F mutant lacking the autophosphorylation site did not. These results suggest that FAK/Fyn-mediated activation of Rac1 and Cdc42 is critical for laminin-induced outgrowth of oligodendrocyte processes.

Although protein-tyrosine phosphorylation is important for hippocampus-dependent learning, its role in cerebellum-dependent learning remains unclear. We previously found that PTPMEG, a cytoplasmic protein-tyrosine phosphatase expressed in Purkinje cells (PCs), bound to the carboxyl-terminus of the glutamate receptor delta2 via the postsynaptic density-95/discs-large/ZO-1 domain of PTPMEG. In the present study, we generated PTPMEG-knockout (KO) mice, and addressed whether PTPMEG is involved in cerebellar plasticity and cerebellum-dependent learning. The structure of the cerebellum in PTPMEG-KO mice appeared grossly normal. However, we found that PTPMEG-KO mice showed severe impairment in the accelerated rotarod test. These mice also exhibited impairment in rapid acquisition of the cerebellum-dependent delay eyeblink conditioning, in which conditioned stimulus (450-ms tone) and unconditioned stimulus (100-ms periorbital electrical shock) were co-terminated. Moreover, long-term depression at parallel fiber-PC synapses was significantly attenuated in these mice. Developmental elimination of surplus climbing fibers and the physiological properties of excitatory synaptic inputs to PCs appeared normal in PTPMEG-KO mice. These results suggest that tyrosine dephosphorylation events regulated by PTPMEG are important for both motor learning and cerebellar synaptic plasticity.

Brek/Lmtk2 (brain-enriched kinase/lemur tyrosine kinase 2) is a member of the Aatyk family of kinases that comprises Aatyk1, Brek/Lmtk2/Aatyk2, and Aatyk3. Although several potential roles have been proposed for Brek and other Aatyk family members, the physiological functions of these kinases remain unclear. Here, we report that Brek −/− male mice are infertile, with azoospermia. Detailed histological analysis revealed that Brek −/− germ cells differentiated normally until the round-spermatid stage, but failed to undergo the normal change in morphology to become elongated spermatids. Testicular somatic cells appeared normal in these mice. Expression of Brek in testis was restricted to the germ cells, suggesting that the maturations of germ cells in Brek −/− mice are affected in a cell-autonomous manner. On the basis of these findings, we concluded that Brek is essential for a late stage of spermatogenesis. Further clarification of the mechanism by which Brek regulates spermatogenesis may help identify new targets for reproductive contraceptives and treatments against infertility.

Nogo-A is a neurite outgrowth inhibitor protein associated with myelin in the central nervous system. Unexpectedly, targeted disruption of Nogo-A in mice results in little or no improvement of axonal regeneration, suggesting that Nogo-A has other functions and/or receives complex regulations to exert its inhibitory functions. Here, we have found that Nogo-A becomes phosphorylated at Tyr-694 in the N-terminal region. The phosphorylation is mediated co-operatively by Src-family tyrosine kinases, which play many important roles in the nervous system. Levels of tyrosine phosphorylation of Nogo-A seem to be irrelevant to developmental stages of oligodendrocytes, and might be regulated by specific extracellular stimuli. Identification of tyrosine phosphorylation of Nogo-A will introduce an additional level of complexity into Nogo-A functions.

Fyn, a member of the Src family of tyrosine kinases, is implicated in both brain development and adult brain function. In the present study, we identified a Rho GTPase-activating protein (GAP), TCGAP (Tc10/Cdc42 GTPase-activating protein), as a novel Fyn substrate. TCGAP interacted with Fyn and was phosphorylated by Fyn, with Tyr-406 in the GAP domain as a major Fyn-mediated phosphorylation site. Fyn suppressed the GAP activity of wild-type TCGAP but not the Y406F mutant of TCGAP in a phosphorylation-dependent manner, suggesting that Fyn-mediated Tyr-406 phosphorylation negatively regulated the TCGAP activity. In situ hybridization analyses showed that TCGAP mRNA was expressed prominently in both immature and adult mouse brain, with high levels in cortex, corpus striatum, hippocampus, and olfactory bulb. Overexpression of wild-type TCGAP in PC12 cells suppressed nerve growth factor-induced neurite outgrowth, whereas a GAP-defective mutant of TCGAP enhanced the neurite outgrowth. Nerve growth factor enhanced tyrosine phosphorylation of TCGAP through activation of Src family kinases. These results suggest that TCGAP is involved in Fyn-mediated regulation of axon and dendrite outgrowth.

Phosphorylation of neural proteins in response to a diverse array of external stimuli is one of the main mechanisms underlying dynamic changes in neural circuitry. The NR2B subunit of the NMDA receptor is tyrosine-phosphorylated in the brain, with Tyr-1472 its major phosphorylation site. Here, we generate mice with a knockin mutation of the Tyr-1472 site to phenylalanine (Y1472F) and show that Tyr-1472 phosphorylation is essential for fear learning and amygdaloid synaptic plasticity. The knockin mice show impaired fear-related learning and reduced amygdaloid long-term potentiation. NMDA receptor-mediated CaMKII signaling is impaired in YF/YF mice. Electron microscopic analyses reveal that the Y1472F mutant of the NR2B subunit shows improper localization at synapses in the amygdala. We thus identify Tyr-1472 phosphorylation as a key mediator of fear learning and amygdaloid synaptic plasticity.

The aggregation of high affinity IgE receptors (Fcepsilon receptor I [FcepsilonRI]) on mast cells is potent stimulus for the release of inflammatory and allergic mediators from cytoplasmic granules. However, the molecular mechanism of degranulation has not yet been established. It is still unclear how FcepsilonRI-mediated signal transduction ultimately regulates the reorganization of the cytoskeleton and how these events lead to degranulation. Here, we show that FcepsilonRI stimulation triggers the formation of microtubules in a manner independent of calcium. Drugs affecting microtubule dynamics effectively suppressed the FcepsilonRI-mediated translocation of granules to the plasma membrane and degranulation. Furthermore, the translocation of granules to the plasma membrane occurred in a calcium-independent manner, but the release of mediators and granule-plasma membrane fusion were completely dependent on calcium. Thus, the degranulation process can be dissected into two events: the calcium-independent microtubule-dependent translocation of granules to the plasma membrane and calcium-dependent membrane fusion and exocytosis. Finally, we show that the Fyn/Gab2/RhoA (but not Lyn/SLP-76) signaling pathway plays a critical role in the calcium-independent microtubule-dependent pathway.

1. Fyn, a member of Src-family tyrosine kinases, is implicated in both brain development and adult brain function. Recent studies have identified some Fyn substrates, however, little is known about the transcriptional targets for Fyn mediated signaling pathways. In the present study, we sought to identify targets downstream of Fyn in vivo. 2. We compared genes expressed in adult hippocampi of wild-type and fyn-deficient mice using gene chips containing more than 12,000 genes. 3. The results showed that 559 transcripts were expressed differentially between these mice. Expression of 20 genes including a substantial number of myelin-associated genes was strongly repressed in fyn-deficient mice. 4. Reduced expression of these myelin-associated genes, such as MBP and MOG, in fyn-deficient mice was also confirmed by real-time PCR and northern blotting, arguing that Fyn is important for function and development of oligodendrocytes. 5. Further analysis of the genes that are differently expressed in fyn-deficient mice may shed light on the molecular mechanism by which Fyn regulates adult neural function.

Post-synaptic density-95 (PSD-95) is a neuronal scaffolding protein that associates with N -methyl-D-aspartate (NMDA) receptors and links them to intracellular signalling molecules. In neurons, neuronal nitric oxide synthase (nNOS) binds selectively to the second PDZ domain (PDZ2) of PSD-95, thereby exhibiting physiological activation triggered via NMDA receptors. We have demonstrated previously that Ca(2+)/calmodulin-dependent protein kinase IIalpha (CaM-K IIalpha) directly phosphorylates nNOS at residue Ser(847), and can attenuate the catalytic activity of the enzyme in neuronal cells [Komeima, Hayashi, Naito and Watanabe (2000) J. Biol. Chem. 275, 28139-28143]. In the present study, we examined how CaM-K II participates in the phosphorylation by analysing the functional interaction between nNOS and PSD-95 in cells. The results showed that PSD-95 directly promotes the nNOS phosphorylation at Ser(847) induced by endogenous CaM-K II. In transfected cells, this effect of PSD-95 required its dual palmitoylation and the PDZ2 domain, but did not rely on its guanylate kinase domain. CaM-K Ialpha and CaM-K IV failed to phosphorylate nNOS at Ser(847) in transfected cells. Thus PSD-95 mediates cellular trafficking of nNOS, and may be required for the efficient phosphorylation of nNOS at Ser(847) by CaM-K II in neuronal cells.

The neuromuscular junction (NMJ) is the synapse between a motor neuron and skeletal muscle. Defects in NMJ transmission cause muscle weakness, termed myasthenia. The muscle protein Dok-7 is essential for activation of the receptor kinase MuSK, which governs NMJ formation, and DOK7 mutations underlie familial limb-girdle myasthenia (DOK7 myasthenia), a neuromuscular disease characterized by small NMJs. Here, we show in a mouse model of DOK7 myasthenia that therapeutic administration of an adeno-associated virus (AAV) vector encoding the human DOK7 gene resulted in an enlargement of NMJs and substantial increases in muscle strength and life span. When applied to model mice of another neuromuscular disorder, autosomal dominant Emery-Dreifuss muscular dystrophy, DOK7 gene therapy likewise resulted in enlargement of NMJs as well as positive effects on motor activity and life span. These results suggest that therapies aimed at enlarging the NMJ may be useful for a range of neuromuscular disorders.

Accumulating evidence indicates that the Cbl protein plays a negative role in immune receptor signaling; however, the mode of Cbl action in B cell receptor (BCR) signaling still remains unclear. DT40 B cells deficient in Cbl showed enhanced BCR-mediated phospholipase C (PLC)-gamma2 activation, thereby leading to increased apoptosis. A possible explanation for the involvement of Cbl in PLC-gamma2 activation was provided by findings that Cbl interacts via its Src homology 2 (SH2) domain with B cell linker protein (BLNK) after BCR ligation. BLNK is a critical adaptor molecule for PLC-gamma2 tyrosine phosphorylation through its binding to the PLC-gamma2 SH2 domains. As a consequence of the interaction between Cbl and BLNK, the BCR-induced recruitment of PLC-gamma2 to BLNK and the subsequent PLC-gamma2 tyrosine phosphorylation were inhibited. Thus, our data suggest that Cbl negatively regulates the PLC-gamma2 pathway by inhibiting the association of PLC-gamma2 with BLNK.

Sox family proteins are characterized by a unique DNA-binding domain, a HMG box which shows at least 50% sequence similarity with mouse Sry, the sex-determining factor. At present almost 30 Sox genes have been identified. Members of this family have been shown to be conserved during evolution and to play key roles during animal development. Some are involved in human diseases, including sex reversal. Here we report the isolation of a novel member of the Sox gene family, Sox30, which may constitute a distinct subgroup of this family. Using a bacterially expressed DNA-binding domain of Sox30, we show that it is able to specifically recognize the ACAAT motif. Furthermore, Sox30 is capable of activating transcription from a synthetic promoter containing the ACAAT motif. The specific expression of Sox30 in normal testes, but not in maturing germ cell-deficient testes, suggests the involvement of Sox30 in differentiation of male germ cells. Mapping analyses revealed that the Sox30 gene is located on human chromosome 5 (5q33) and on mouse chromosome 11.

直腸腺扁平上皮癌, S状結腸粘膜内癌, 横行結腸低分化腺癌の3重複癌を発症した11歳, 男児例を経験した. 20歳未満の若年者多発大腸癌および直腸腺扁平上皮癌はともにまれであるので報告する.症例は11歳の男児で, 主訴は粘血便であった. 平成5年8月, 直腸腺扁平上皮癌 (第1癌) に対しHartmann手術を施行した. 標本上S状結腸に高分化腺癌 (第2癌) を認めた. 平成6年1月, 人工肛門周囲膿瘍に対しドレナージ術を施行, 膿瘍壁より低分化腺癌を認めた. 平成6年3月, 第3回手術を行い術中に横行結腸低分化腺癌 (第3癌) を認めたため左半結腸切除術, 人工肛門再造設術を施行した. 術後3年経過した現在も再発なく健在である.今回は3重複癌を認め, いずれも異なった組織所見を呈し同時性多発癌と思われた. また直腸の腺扁平上皮癌は腺癌の扁平上皮化生によると思われ, 本症例では多彩な発癌様式が関与しているものと思われた.

Low-density lipoprotein receptor-related protein 4 (Lrp4) is essential for pre- and post-synaptic specialization at the neuromuscular junction (NMJ), an indispensable synapse between a motor nerve and skeletal muscle. Muscle-specific receptor tyrosine kinase MuSK must form a complex with Lrp4 to organize postsynaptic specialization at NMJs. Here, we show that the chaperon Mesdc2 binds to the intracellular form of Lrp4 and promotes its glycosylation and cell-surface expression. Furthermore, knockdown of Mesdc2 suppresses cell-surface expression of Lrp4, activation of MuSK, and postsynaptic specialization in muscle cells. These results suggest that Mesdc2 plays an essential role in NMJ formation by promoting Lrp4 maturation.

To identify novel signal transducers involved in signaling mediated by the Src-family protein tyrosine kinases (PTKs), we used a yeast two-hybrid system with a probe corresponding to the regulatory region of p56lyn, a member of Src-family PTKs. One of the isolated clones contained the COOH-terminal 470 amino acid residues of p120c-cbl, the product of the cellular homologue of the v-cbl retroviral oncogene. p120c-cbl is a cytoplasmic protein with nuclear protein-like motifs. Here we show in vivo association of p120c-cbl with p53/56lyn. After stimulation of the B cell antigen receptor (BCR), p120c-cbl was rapidly tyrosine phosphorylated. Studies with lyn- or syk-negative chicken B cells demonstrated that p53/56lyn, but not p72syk, was crucial for tyrosine phosphorylation of p120c-cbl upon stimulation of the BCR. We also show the importance of p59fyn in tyrosine phosphorylation of p120c-cbl in the T-cell receptor-mediated signaling using fyn-overexpressing T cell hybridomas and splenic T cells from fyn-deficient mice. These results suggest that p120c-cbl is an important substrate of Src-family PTKs in the intracellular signaling mediated by the antigen receptors

Overexpression of the ErbB2/HER2 receptor tyrosine kinase contributes to tumorigenesis. However, mechanisms regulating ErbB2 protein levels remain largely unclear. Here, we identified novel mechanisms of ErbB2 downregulation. ErbB2 constitutively binds to an adaptor protein FRS2β. We found that FRS2β bound to CD2AP and CIN85, which induces endosomal trafficking that targets lysosomes. FRS2β colocalized with CIN85 in the cytoplasm. Expression of wild type FRS2β but not its CIN85 non-binding mutant, downregulated the ErbB2 protein and inhibited anchorage-independent cell growth. Moreover, the E3 ubiquitin-protein ligase Cbl was contained within a complex of FRS2β and CIN85. Knockdown of both CIN85 and CD2AP or of Cbl, or treatment with lysosomal degradation inhibitors diminished FRS2β downregulation of ErbB2. In addition, knockdown of endogenous FRS2β caused upregulation of ErbB2 in primary neural cells. Finally, immunohistochemical analysis showed that human breast cancer tissues that overexpress ErbB2 expressed low levels of FRS2β. Thus, an FRS2β-CIN85/CD2AP-Cbl axis for downregulation of ErbB2 may regulate ErbB2 protein levels in physiological and pathological settings. Molecular targeting drugs that can increase or stabilize the ErbB2-FRS2β-CIN85/CD2AP-Cbl axis may have promise for the control of ErbB2-overexpressing tumors.

We evaluated the efficacy and safety of neoadjuvant chemotherapy using modified OPTIMOX1 plus bevacizumab for advanced rectal cancer.Nine cases with highly advanced rectal cancer for which curative surgery was potentially difficult were enrolled(clinical T4 in 7 cases, lateral node metastasis in 3 cases, M1 in 2 cases).The number of courses of modified OPTIMOX1(mFOLFOX6 and sLV5FU2, alternating administration)plus bevacizumab ranged from 1 to 21(median: 10). Surgical procedures consisted of internal sphincter resection(ISR)in 4 patients, ultra-low anterior resection(ULAR)in 2 patients, pelvic exenteration(TPE)in 2 patients, and Hartmann's procedure in 1 patient. Liver resection was conducted in 2 patients. RM1 was confirmed in 2 patients, but curative surgery was performed in the other patients. Histological efficacy of grade x/1a/1b/2were seen in the above 1/4/2/2 cases, respectively. Neurotoxicity associated with oxaliplatin was mild; no grade 3 neurotoxicity was noted. Recurrence has been confirmed in 5 patients at the median follow-up period of 650 days.It was suggested that modified OPTIMOX1 plus bevacizumab is effective and safe to administer as a neoadjuvant chemotherapy for curative resection or anus-preserving surgery in patients with highly advanced rectal cancer.

It is widely accepted that receptor protein-tyrosine kinases (RTKs) are activated upon dimerization by binding to their extracellular ligands. However, EGF receptor (EGFR) dimerization per se does not require ligand binding. Instead, its cytoplasmic kinase domains have to form characteristic head-to-tail asymmetric dimers to become active, where one 'activator' domain activates the other 'receiver' domain. The non-catalytic, cytoplasmic regions of RTKs, namely the juxtamembrane and carboxy terminal portions, also regulate kinase activity. For instance, the juxtamembrane region of the RTK MuSK inhibits the kinase domain probably together with a cellular factor(s). These findings suggest that RTKs could be activated by cytoplasmic proteins. Indeed, Dok-7 and cytohesin have recently been identified as such activators of MuSK and EGFR, respectively. Given that failure of Dok-7 signaling causes myasthenia, and inhibition of cytohesin signaling reduces the proliferation of EGFR-dependent cancer cells, cytoplasmic activators of RTKs may provide new therapeutic targets.

Previous studies found that the NMDA receptor-mediated signaling regulates thermal nociception, though the underlying molecular mechanism remains unclear. The GluN2B subunit of the NMDA receptor is tyrosine-phosphorylated, Tyr-1472 being the major phosphorylation site. In this study, we have found that homozygous knock-in mice that express a Tyr-1472-Phe mutant of GluN2B display defects in the nociceptive response in the hot plate test. Expression of the neurotensin receptor subtype 2 (NTSR2), which is relevant to the regulation of thermal nociception, is decreased in the amygdala of GluN2B Tyr-1472-Phe knock-in mice. In addition, NTSR2-mediated c-fos induction is impaired in the amygdala of these mice. These data suggest that Tyr-1472 phosphorylation on GluN2B is involved in thermal nociception through regulating the NTSR2 mRNA expression in the amygdala.

The phosphoinositide 3-kinase (PI3K) pathway has been extensively studied in neuronal function and morphogenesis. However, the precise molecular mechanisms of PI3K activation and its downstream signalling in neurons remain elusive. Here, we report the identification of the Neuronal tYrosine-phosphorylated Adaptor for the PI 3-kinase (NYAP) family of phosphoproteins, which is composed of NYAP1, NYAP2, and Myosin16/NYAP3. The NYAPs are expressed predominantly in developing neurons. Upon stimulation with Contactin5, the NYAPs are tyrosine phosphorylated by Fyn. Phosphorylated NYAPs interact with PI3K p85 and activate PI3K, Akt, and Rac1. Moreover, the NYAPs interact with the WAVE1 complex which mediates remodelling of the actin cytoskeleton after activation by PI3K-produced PIP(3) and Rac1. By simultaneously interacting with PI3K and the WAVE1 complex, the NYAPs bridge a PI3K-WAVE1 association. Disruption of the NYAP genes in mice affects brain size and neurite elongation. In conclusion, the NYAPs activate PI3K and concomitantly recruit the downstream effector WAVE complex to the close vicinity of PI3K and regulate neuronal morphogenesis.

The objective of this study was to clarify the surgical outcome of patients with palliative surgery for malignant bowel obstruction.This study investigated the clinical features, operative procedures and postoperative course of 35 patients who underwent a palliative surgery for malignant bowel obstruction. And then the patients were divided into two groups; Patients in A group were consisted of 4 patients with hospital death and 7 patients with postoperative complications. Patients in B group were consisted of 24 patients without hospital death or postoperative complications.Eighteen patients who had been inserted nasogastric tube or ileus tube in the preoperative state could be removed. Thirty-three of 35 patients(94.3%)could become an oral ingestion. Four of 35 patients(11.4%)could not be discharged; 3 patients died of cancer and 1 patient died of acute myocardial infarction. Postoperative complications were seen in 7 patients except 4 patients with hospital death. The median postoperative stay was 18 days(3-58). Twenty six of 35 patients(74.3%) underwent chemotherapy. The median survival time was 137 days(3-1,614). The patients in A group showed a lower level of albumin(p=0.0071)and hemoglobin,(p=0.0006)and poorer performance status(p=0.0178)than the patients in B group. The median hospital stay of the patients in A group and B group were 28 days and 16 days, respectively(p=0.0823). The median survival time of the patients in A group and B group were 42 days and 119 days, respectively(p=0.0035).We concluded that the palliative surgery made an oral ingestion possible and improved a quality of life of the patients with malignant bowel obstruction. However, the surgical indication should be carefully decided for the patients with low albumin, hemoglobin and poor performance status.

Background Anxiety disorders are a highly prevalent and disabling class of psychiatric disorders. There is growing evidence implicating the glutamate system in the pathophysiology and treatment of anxiety disorders, though the molecular mechanism by which the glutamate system regulates anxiety-like behavior remains unclear. Results In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to anxiety-like behavior. The GluN2B subunit of the NMDA receptor is tyrosine-phosphorylated: Tyr-1472 is the major phosphorylation site. Homozygous knock-in mice that express a Tyr-1472-Phe mutant of GluN2B, which prevents phosphorylation of this site, show enhanced anxiety-like behavior in the elevated plus-maze test. Expression of corticotropin-releasing factor (CRF), which is important for the regulation of anxiety-like behavior, is increased in the amygdala of the knock-in mice. Furthermore, injection of CRF receptor antagonist attenuated the enhanced anxiety-like behavior of the knock-in mice. We also show that elevated plus-maze exposure simultaneously induced de-phosphorylation of Tyr-1472 and increased CRF expression. Conclusions These data suggest that Tyr-1472 phosphorylation on GluN2B is important for anxiety-like behavior by negative regulation of CRF expression in the amygdala.