Language：English   Publishing type：Research paper (scientific journal)  

Stimulator of interferon genes (STING) contributes to anti-tumor immunity by activating antigen-presenting cells and inducing mobilization of tumor-specific T cells. A role for tumor-migrating neutrophils in the anti-tumor effect of STING-activating therapy has not been defined. We used mouse tumor transplantation models for assessing neutrophil migration into the tumor triggered by intratumoral treatment with STING agonist, 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Intratumoral STING activation with cGAMP enhanced neutrophil migration into the tumor in an NF-κB/CXCL1/2-dependent manner. Blocking the neutrophil migration by anti-CXCR2 monoclonal antibody impaired T cell activation in tumor-draining lymph nodes (dLNs) and efficacy of intratumoral cGAMP treatment. Moreover, the intratumoral cGAMP treatment did not show any anti-tumor effect in type I interferon (IFN) signal-impaired mice in spite of enhanced neutrophil accumulation in the tumor. These results suggest that both neutrophil migration and type I interferon (IFN) induction by intratumoral cGAMP treatment were critical for T-cell activation of dLNs and the anti-tumor effect. In addition, we also performed in vitro analysis showing enhanced cytotoxicity of neutrophils by IFN-β1. Extrinsic STING activation triggers anti-tumor immune responses by recruiting and activating neutrophils in the tumor via two signaling pathways, CXCL1/2 and type I IFNs.

DOI： 10.1007/s00262-021-02864-0

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In vitro fertilization (IVF) is an established technology that is widely used in reproductive engineering. However, in rats, successful application of IVF is difficult to achieve, and it has had poor reproducibility. In a previous study on the critical issues associated with successful IVF in Wistar rats, we investigated the influence of oocyte collection duration on fertilization rates by dividing the procedure into three steps (oviduct extraction from euthanized animals, oocyte collection from the ampullae of oviducts, and oocyte preincubation until insemination), and identified the appropriate times for each. Here we show that use of the same defined duration for oviduct extraction from superovulated Wistar rats and for oocyte collection from the oviducts also produced highly reproducible fertilization rates of more than 90% in other rat strains. Furthermore, the versatility of these criteria was demonstrated using another IVF protocol. Thus, this simple procedure has enabled the standardization of IVF in rats and will enhance further experimental studies.

DOI： 10.1016/j.theriogenology.2020.01.006

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Aim: Indoleamine 2,3-dioxygenase 1 (IDO1) metabolizes the essential amino acid tryptophan into kynurenine derivatives, which are involved in neural activity via the kynurenine pathway (KP). IDO1 is an initial rate-limiting enzyme in the KP and is activated by stress and/or inflammation. The perturbation of IDO1 activity, which causes KP imbalance, is associated with psychiatric and neurological disorders. It has been reported that wild-type (WT) mice under inflammatory conditions show increased anxiety-like behavior and decreased novel object recognition, whereas Ido1 knockout (KO) mice do not display these behaviors. However, the behavioral phenotypes of Ido1 KO mice have not yet been fully examined under non-inflammatory conditions.

Methods: We subjected Ido1 KO mice to a comprehensive behavioral test battery under normal conditions.

Results: Ido1 KO mice and WT mice showed similar locomotor activity, anxiety-like behavior, social behavior, depression-like behavior, and fear memory. In the T-maze test, Ido1 KO mice exhibited weak but nominally significant impairment in the working memory task of the T-maze, but this result failed to reach study-wide significance.

Conclusions: Ido1 KO mice did not show any clear behavioral abnormalities under normal conditions. Further studies may be necessary to investigate their behavioral phenotype under inflammatory conditions due to their known roles in inflammation.

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Aims: Tryptophan 2,3-dioxygenase (TDO2) is an initial rate-limiting enzyme of the kynurenine (Kyn) pathway in tryptophan (Trp) metabolism. The Trp-degrading enzymes, TDO2 and indoleamine 2,3-dioxygenase, are activated by stress and/or inflammation. Dysregulation of Trp metabolism, which causes shifts in the balance between Kyn and serotonin (5-HT) pathways, is associated with psychiatric and neurological disorders. In genetic studies, single-nucleotide polymorphisms in the TDO2 gene were shown to be involved in psychiatric disorders, such as schizophrenia and depression. It has been reported that targeted deletion of the Tdo2 gene in mice resulted in reduced anxiety-like behavior, enhanced exploratory activity and cognitive performance, and increased levels of Trp and 5-HT in the hippocampus and midbrain. However, the effect of Tdo2 gene deletion on behavioral phenotypes has not yet been investigated extensively.

Materials & methods: We conducted tests to further examine the behavioral effects of knockout (KO) of Tdo2 in mice.

Results: Deletion of Tdo2 resulted in seemingly lower anxiety-like behavior, higher locomotor activity, and abnormal gait pattern in mice, though none of them reached study-wide statistical significance. Tdo2 deficiency had no significant effects on other behaviors, such as prepulse inhibition, and depression-like and social behaviors.

Discussion and conclusion: He lack of clear phenotypes in Tdo2KO mice in this study might be due to the absence of stress and inflammatory conditions, which could induce expression of Tdo2 mRNA. Further studies are necessary to elucidate the roles of Tdo2 in behavioral phenotypes related to psychiatric disorders.

DOI： 10.1002/npr2.12006

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Authorship：Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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The catabolism of tryptophan to immunosuppressive and neuroactive kynurenines is a key metabolic pathway regulating immune responses and neurotoxicity. The rate-limiting step is controlled by indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO). IDO is expressed in antigen presenting cells during immune reactions, hepatic TDO regulates blood homeostasis of tryptophan and neuronal TDO influences neurogenesis. While the role of IDO has been described in multiple immunological settings, little is known about TDO's effects on the immune system. TDO-deficiency is neuroprotective in C. elegans and Drosophila by increasing tryptophan and specific kynurenines. Here we have determined the role of TDO in autoimmunity and neurodegeneration in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. We created reporter-TDO mice for in vivo imaging to show that hepatic but not CNS TDO expression is activated during EAE. TDO deficiency did not influence myelin-specific T cells, leukocyte infiltration into the CNS, demyelination and disease activity. TDO-deficiency protected from neuronal loss in the spinal cord but not in the optic nerves. While this protection did not translate to an improved overt clinical outcome, our data suggest that spatially distinct neuroprotection is conserved in mammals and support TDO as a potential target for treatment of diseases associated with neurodegeneration.

DOI： 10.1038/srep41271

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Adenosquamous carcinoma (ASC) is an uncommon variant of pancreatic neoplasm. We sought to trace the mode of tumor progression using specimens of ASC associated with intraductal papillary mucinous neoplasm (IPMN) of the pancreas. A resected specimen of the primary pancreatic ASC, developed in a 72-year-old man, was subjected to mutation profiling using amplicon-targeted sequencing and digital polymerase chain reaction. DNA was isolated from each histological compartment including noninvasive IPMN, squamous cell carcinoma (SCC), and adenocarcinoma (AC). Histologically, an IPMN with a large mural nodule was identified. The invasive tumor predominantly consisted of SCC, and a smaller AC was found around the lesion. Squamous metaplasias were sporadically distributed within benign IPMNs. Mutation alleles KRAS and GNAS were identified in all specimens of IPMN including the areas of squamous metaplasia. In addition, these mutations were found in SCC and AC. Clear transition from flat/low-papillary IPMN to SCC indicated a potent invasion front, and the SCC compartment was genetically unique, because the area has a higher frequency of mutation KRAS. The invasive tumors with distinct histological appearances shared the form of noninvasive IPMN as a common precursor, rather than de novo cancer, suggesting the significance of a genetic profiling scheme of tumors associated with IPMN.

DOI： 10.1097/MPA.0000000000000556

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Background: In mammals, the majority of the essential amino acid tryptophan is degraded via the kynurenine pathway (KP). Several KP metabolites play distinct physiological roles, often linked to immune system functions, and may also be causally involved in human diseases including neurodegenerative disorders, schizophrenia and cancer. Pharmacological manipulation of the KP has therefore become an active area of drug development. To target the pathway effectively, it is important to understand how specific KP enzymes control levels of the bioactive metabolites in vivo.

Methods: Here, we conducted a comprehensive biochemical characterization of mice with a targeted deletion of either tryptophan 2,3-dioxygenase (TDO) or indoleamine 2,3-dioxygenase (IDO), the two initial rate-limiting enzymes of the KP. These enzymes catalyze the same reaction, but differ in biochemical characteristics and expression patterns. We measured KP metabolite levels and enzyme activities and expression in several tissues in basal and immune-stimulated conditions.

Results and conclusions: Although our study revealed several unexpected downstream effects on KP metabolism in both knockout mice, the results were essentially consistent with TDO-mediated control of basal KP metabolism and a role of IDO in phenomena involving stimulation of the immune system.

DOI： 10.1016/j.bbagen.2016.07.002

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Tryptophan is an essential amino acid for hosts and pathogens. The liver enzyme tryptophan 2,3-dioxygenase (TDO) provokes, by its ability to degrade tryptophan to N-formylkynurenine, the precursor of the immune-relevant kynurenines, direct and indirect antimicrobial and immunoregulatory states. Up to now these TDO-mediated broad-spectrum effector functions have never been observed under hypoxia in vitro, although physiologic oxygen concentrations in liver tissue are low, especially in case of infection. Here we analysed recombinant expressed human TDO and ex vivo murine TDO functions under different oxygen conditions and show that TDO-induced restrictions of clinically relevant pathogens (bacteria, parasites) and of T cell proliferation are abrogated under hypoxic conditions. We pinpointed the loss of TDO efficiency to the reduction of TDO activity, since cell survival and TDO protein levels were unaffected. In conclusion, the potent antimicrobial as well as immunoregulatory effects of TDO were substantially impaired under hypoxic conditions that pathophysiologically occur in vivo. This might be detrimental for the appropriate host immune response towards relevant pathogens. © 2016 Frank Elbers et al.

DOI： 10.1155/2016/1638916

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Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of a polyglutamine (polyQ)-encoding tract within the androgen receptor (AR) gene. The pathologic features of SBMA are motor neuron loss in the spinal cord and brainstem and diffuse nuclear accumulation and nuclear inclusions of mutant AR in residual motor neurons and certain visceral organs. Hepatocyte growth factor (HGF) is a polypeptide growth factor which has neuroprotective properties. To investigate whether HGF overexpression can affect disease progression in a mouse model of SBMA, we crossed SBMA transgenic model mice expressing an AR gene with an expanded CAG repeat with mice overexpressing HGF. Here, we report that high expression of HGF induces Akt phosphorylation and modestly ameliorated motor symptoms in an SBMA transgenic mouse model treated with or without castration. These findings suggest that HGF overexpression can provide a potential therapeutic avenue as a combination therapy with disease-modifying therapies in SBMA. © 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2015.11.015

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Hepatocyte growth factor (HGF) is a pleiotropic cytokine that has been extensively studied over several decades, but was only recently recognized as a key player in mediating protection of many types of inflammatory and autoimmune diseases. HGF was reported to prevent and attenuate disease progression by influencing multiple pathophysiological processes involved in inflammatory and immune response, including cell migration, maturation, cytokine production, antigen presentation, and T cell effector function. In this review, we discuss the actions and mechanisms of HGF in inflammation and immunity and the therapeutic potential of this factor for the treatment of inflammatory and autoimmune diseases. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.autrev.2014.11.013

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Clonal populations originated from benign-looking 'founder cells' may spread widely within pancreas instead of being localized in situ before frank pancreatic ductal adenocarcinoma (PDA) can be detected. Metachronous PDA is not common event, and we here sought to define potent origin of multiple PDAs developed in a woman using advanced genetics technologies. Curative resection of pancreatic head tumour was performed; however, 'recurrent' lesions in the remnant pancreas were found 3.5 years later and total pancreatectomy was subsequently performed. The metachronous lesions were morphologically similar to the primary PDA. Using a next-generation sequencing and digital PCR, all three PDAs were shown to possess rare somatic mutations in KRAS (p.T58I & p.Q61H). Curiously, identical KRAS mutations were found in lowgrade 'intraepithelial' lesions, which localized in normal area of the pancreas and one of them possessed p53 mutation, which was also found in the PDAs. The footprint of the tumour evolution marked by mutational profiling supports a human correlate to the mouse models of 'dissemination' occurring at the earliest stages of pancreatic neoplasia.

DOI： 10.1002/cjp2.8

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DOI： 10.5702/massspectrometry.A0042

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Hepatocyte growth factor (HGF) is composed of an α-chain and a β-chain, and these chains contain four kringle domains and a serine protease-like structure, respectively. Activation of the HGF–Met pathway evokes dynamic biological responses that support morphogenesis (e.g., epithelial tubulogenesis), regeneration, and the survival of cells and tissues. Characterizations of conditional Met knockout mice have indicated that the HGF–Met pathway plays important roles in regeneration, protection, and homeostasis in various cells and tissues, which includes hepatocytes, renal tubular cells, and neurons. Preclinical studies designed to address the therapeutic significance of HGF have been performed on injury/disease models, including acute tissue injury, chronic fibrosis, and cardiovascular and neurodegenerative diseases. The promotion of cell growth, survival, migration, and morphogenesis that is associated with extracellular matrix proteolysis are the biological activities that underlie the therapeutic actions of HGF. Recombinant HGF protein and the expression vectors for HGF are biological drug candidates for the treatment of patients with diseases and injuries that are associated with impaired tissue function. The intravenous/systemic administration of recombinant HGF protein has been well tolerated in phase I/II clinical trials. The phase-I and phase-I/II clinical trials of the intrathecal administration of HGF protein for the treatment of patients with amyotrophic lateral sclerosis and spinal cord injury, respectively, are ongoing.

DOI： 10.3390/biomedicines2040275

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Autoimmune neuroinflammation, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), a prototype for T cell-mediated autoimmunity, is believed to result from immune tolerance dysfunction leading to demyelination and substantial neurodegeneration. We previously showed that CNS-restricted expression of hepatocyte growth factor (HGF), a potent neuroprotective factor, reduced CNS inflammation and clinical deficits associated with EAE. In this study, we demonstrate that systemic HGF treatment ameliorates EAE through the development of tolerogenic dendritic cells (DCs) with high expression levels of glucocorticoid-induced leucine zipper (GILZ), a transcriptional repressor of gene expression and a key endogenous regulator of the inflammatory response. RNA interference-directed neutralization of GILZ expression by DCs suppressed the induction of tolerance caused by HGF. Finally, adoptive transfer of HGF-treated DCs from wild-type but not GILZ gene-deficient mice potently mediated functional recovery in recipient mice with established EAE through effective modulation of autoaggressive T cell responses. Altogether, these results show that by inducing GILZ in DCs, HGF reproduces the mechanism of immune regulation induced by potent immunomodulatory factors such as IL-10, TGF-β1, and glucocorticoids and therefore that HGF therapy may have potential in the treatment of autoimmune dysfunctions.

DOI： 10.4049/jimmunol.1302338

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Pneumococcal meningitis is a lethal form of bacterial infection in the central nervous system that often causes lifelong neurological sequelae, despite therapeutic advances. The contemporary view is that the inflammatory response to infection contributes to the functional disabilities among survivors of this disease. We previously have established a mouse model of neurobehavioural deficits, using an automated IntelliCage™ system, that revealed long-term behavioural and cognitive deficits in C57BL/6J female mice cured of meningitis by ceftriaxone treatment. We now have investigated the roles of two kynurenine pathway enzymes, indoleamine dioxygenase-1 (IDO1) and tryptophan dioxygenase-2 (TDO2), in the pathomechanisms of pneumococcal meningitis. Since tryptophan metabolism has long been implicated in behavioural and cognitive modulation through the production of neuroactive compounds, we hypothesised that preventing the actions of these enzymes through gene knockout would be beneficial in mice subjected to pneumococcal infection. We found no significant effect of IDO1 or TDO2 on mortality. Post-meningitic wild-type mice showed long-term diurnal hypoactivity and nocturnal hyperactivity when they were exposed to an Intellicage adaptation test throughout both the light and dark phases. These changes were not apparent in IDO1-/- survivors, but were present in the TDO2-/- survivors. Both IDO1-/- and TDO2-/- survivors were not protected against developing long-term cognitive deficits as measured in IntelliCage-based patrolling or reversal tasks. Collectively, these observations suggest (i) involvement of the kynurenine pathway in causing some behavioural sequelae of pneumococcal meningitis and (ii) that this pathway might operate synergistically with, or independently of, other pathways to cause other aspects of neurological sequelae.
Copyright © 2014. Published by Elsevier B.V.

DOI： 10.1016/j.bbr.2014.05.018

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness.

DOI： 10.1038/nature13323

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We investigated the contribution percentage of tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) to the conversion of D-trypto- phan to nicotinamide in TDO-knockout mice. The calculated percentage conversions indicated that TDO and IDO oxidized 70 and 30%, respectively, of the dietary L-tryptophan. These results indicate that both TDO and IDO biosynthesize nicotinamide from D-tryptophan and L-tryptophan in mice.

DOI： 10.1080/09168451.2014.905185

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Hepatocyte growth factor (HGF), which is a potent mitogen for mature hepatocytes and a mediator of the inflammatory responses to tissue injury, was recently highlighted as a potent neurotrophic factor in the central nervous system. We revealed that introducing exogenous HGF into the injured rat spinal cord using a herpes simplex virus-1 vector significantly reduces the area of damaged tissue and promotes functional recovery. However, this rat study did not examine the therapeutic effects of administering HGF after injury, which is the most critical issue for clinical application. To translate this strategy to human treatment, we induced a contusive cervical SCI in the common marmoset, a primate, and then administered recombinant human HGF (rhHGF) intrathecally. Motor function was assessed using an original open-field scoring system focusing on manual function, including reach-and-grasp performance and hand placement in walking. The intrathecal rhHGF preserved the corticospinal fibers and myelinated areas, thereby promoting functional recovery. This study demonstrates the therapeutic effects, safety, and clinical efficacy of intrathecal rhHGF treatment for SCI in adult nonhuman primates and the possibility that this novel therapy may be suitable for clinical application.

DOI： 10.1007/978-4-431-54502-6_14

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Authorship：Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

We hypothesized that the requirements of essential nutrients are dependent upon catabolic abilities. Mice lacking L-tryptophan 2,3-dioxygenase (TDO) are available. The body concentration of L-tryptophan (L-Trp) has been reported to be higher in TDO-deficient mice than in wild-type (WT) mice. We examined the requirement of an appropriate L-Trp level for TDO-deficient mice using several biomarkers. TDO-deficient mice were fed a 10% amino-acid mixture diet containing 0.06%, 0.08%, and 0.17% L-Trp. WT mice fed a 0.17% Trp diet (standard diet) were used as control mice. The concentrations of L-Trp and its metabolites via serotonin were higher in TDO-deficient mice fed the 0.17% L-Trp diet than in WT mice fed the standard diet, but the concentrations were almost identical between TDO-deficient mice fed the 0.06% L-Trp diet and WT mice fed the standard diet. Therefore, as hypothesized, requirements of essential nutrients are dependent on catabolic abilities.

DOI： 10.4137/IJTR.S12206

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In mammals, nicotinamide (Nam) is biosynthesized from L-tryptophan (L-Trp). The enzymes involved in the initial step of the L-Trp→Nam pathway are L-Trp-2,3-dioxygenase (TDO) and indoleamine-2,3-dioxygenase (IDO). We aimed to determine whether tdo-knockout (tdo-/-) mice fed a diet without preformed niacin can synthesize enough Nam to sustain optimum growth.Wild-type (WT) and tdo-/-mice were fed a chemically defined 20% casein dietwith orwithout preformed niacin (30mg nicotinic acid/kg) for 28 d. Body weight, food intake, and liver NAD concentrations did not differ among the groups. In the groups of mice fed the niacin-free diet, urinary concentrations of the upstream metabolites kynurenine (320% increase, P < 0.0001), kynurenic acid (270%increase, P < 0.0001), xanthurenic acid (770% increase, P < 0.0001), and 3-hydroxyanthranilic acid (3-HA; 450% increase, P < 0.0001) were higher in the tdo-/- mice than in the WT mice, while urinary concentrations of the downstream metabolite quinolinic acid (QA; 50% less, P = 0.0010) and the sum of Nam and its catabolites (10% less, P < 0.0001) were lower in the tdo-/- mice than in the WT mice. These findings show that the kynurenine formed in extrahepatic tissues by IDO and subsequent enzymes can be metabolized up to 3-HA, but not into QA. However, the tdo-/- mice sustained optimum growth even when fed the niacin-free diet for 1 mo, suggesting they can synthesize the minimum necessary amount of Nam from L-Trp, because the liver can import blood kynurenine formed in extrahepatic tissues and metabolize it into Nam via NAD and the resulting Nam is then distributed back into extrahepatic tissues. © 2013 American Society for Nutrition.

DOI： 10.3945/jn.113.176875

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant disorder associated with cerebellar neurodegeneration caused by expansion of a CAG repeat in the ataxin-7 gene. Hepatocyte growth factor (HGF), a pleiotrophic growth factor, displays highly potent neurotrophic activities on cerebellar neurons. A mutant c-met/HGF receptor knockin mouse model has revealed a role for HGF in the postnatal development of the cerebellum. The present study was designed to elucidate the effect of HGF on cerebellar neurodegeneration in a knockin mouse model of SCA7 (SCA7-KI mouse). SCA7-KI mice were crossed with transgenic mice overexpressing HGF (HGF-Tg mice) to produce SCA7-KI/HGF-Tg mice that were used to examine the phenotypic differences following HGF overexpression. The Purkinje cellular degeneration is thought to occur via cell-autonomous and non-cell autonomous mechanisms mediated by a reduction of glutamate transporter levels in Bergmann glia. The Purkinje cellular degeneration and reduced expression of glutamate transporters in the cerebellum of SCA7-KI mice were largely attenuated in the SCA7-KI/HGF-Tg mice. Moreover, phenotypic impairments exhibited by SCA7-KI mice during rotarod tests were alleviated in SCA7-KI/HGF-Tg mice. The bifunctional nature of HGF on both Purkinje cells and Bergmann glia highlight the potential therapeutic utility of this molecule for the treatment of SCA7 and related disorders. (C) 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2012.03.001

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Hepatocyte growth factor (HGF) and its receptor, c-Met, play pivotal roles in the nervous system during development and in disease states. However, the physiological roles of HGF in the adult brain are not well understood. In the present study, to assess its role in learning and memory function, we used transgenic mice that overexpress HGF in a neuron-specific manner (HGF-Tg) to deliver HGF into the brain without injury. HGF-Tg mice displayed increased alternation rates in the Y-maze test compared with age-matched wild-type (WT) controls. In the Morris water maze (MWM) test, HGF-Tg mice took less time to find the platform on the first day, whereas the latency to escape to the hidden platform was decreased over training days compared with WT mice. A transfer test revealed that the incidence of arrival at the exact location of the platform was higher for HGF-Tg mice compared with WT mice. These results demonstrate that overexpression of HGF leads to an enhancement of both short- and long-term memory. Western blot analyses revealed that the levels of N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B, but not NR1, were increased in the hippocampus of HGF-Tg mice compared with WT controls, suggesting that an upregulation of NR2A and NR2B could represent one mechanism by which HGF enhances learning and memory performance. These results demonstrate that modulation of learning and memory performance is an important physiological function of HGF that contributes to normal CNS plasticity, and we propose HGF as a novel regulator of higher brain functions.

DOI： 10.1002/jnr.23065

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Hepatocyte growth factor (HGF), which was originally identified and molecularly cloned as a potent mitogen for primary hepatocytes, exhibits multiple biological effects, such as mitogenic, motogenic, morphogenic, and anti- apoptotic activities, in the liver and other organs throughout the body by binding to the c-Met/HGF receptor tyrosine kinase (c-Met). In addition to hepatotrophic activities, HGF and c-Met are expressed in both developing and adult mature brains and nerves, and plays functional roles in the central as well as peripheral nervous systems. A large number of stud- ies have accumulated evidence showing that HGF is a multipotent growth factor that functions as a novel neurotrophic factor for a variety of neurons, including the hippocampal, cerebral cortical, midbrain dopaminergic, motor, sensory, sympathetic, parasympathetic and cerebellar granule neurons in vitro. In vivo, HGF exerts neuroprotective effects in the animal model of cerebrovascular diseases, spinal cord injury, neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), and neuroimmune diseases, preventing neuronal cell death and functioning on glial, vascular and immune cells. The multiple activities of HGF, in addition to highly potent neurotrophic activities, suggest that HGF is a potential therapeutic agent for the treatment of various diseases of the nervous system. Furthermore, the anxiolytic activity of HGF and an association of c-met with autism, as well as neurorecognition and schizophrenia, have been reported, suggesting a role for HGF in emotional and psychiatric status. This review describes the role of HGF in the nervous systems during development and focuses on the therapeutic potential of HGF for a variety of neurological, neuroimmunological and psychiatric diseases among adults.

DOI： 10.5692/clinicalneurol.52.942

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Many therapeutic interventions for spinal cord injury (SCI) using neurotrophic factors have focused on reducing the area damaged by secondary, post-injury degeneration, to promote functional recovery. Hepatocyte growth factor (HGF), which is a potent mitogen for mature hepatocytes and a mediator of the inflammatory responses to tissue injury, was recently highlighted as a potent neurotrophic factor in the central nervous system. We previously reported that introducing exogenous HGF into the injured rodent spinal cord using a herpes simplex virus-1 vector significantly reduces the area of damaged tissue and promotes functional recovery. However, that study did not examine the therapeutic effects of administering HGF after injury, which is the most critical issue for clinical application. To translate this strategy to human treatment, we induced a contusive cervical SCI in the common marmoset, a primate, and then administered recombinant human HGF (rhHGF) intrathecally. Motor function was assessed using an original open field scoring system focusing on manual function, including reach-and-grasp performance and hand placement in walking. The intrathecal rhHGF preserved the corticospinal fibers and myelinated areas, thereby promoting functional recovery. In vivo magnetic resonance imaging showed significant preservation of the intact spinal cord parenchyma. rhHGF-treatment did not give rise to an abnormal outgrowth of calcitonin gene related peptide positive fibers compared to the control group, indicating that this treatment did not induce or exacerbate allodynia. This is the first study to report the efficacy of rhHGF for treating SCI in non-human primates. In addition, this is the first presentation of a novel scale for assessing neurological motor performance in non-human primates after contusive cervical SCI.

DOI： 10.1371/journal.pone.0027706

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Hepatocyte growth factor (HGF) and glial cell line-derived neurotrophic factor (GDNF) are strong neurotrophic factors. However, their potentials in neurogenesis, angiogenesis, synaptogenesis, and antifibrosis have not been compared. Therefore, we investigated these effects of HGF and GDNF in cerebral ischemia in the rat. Wistar rats were subjected to 90 min of transient middle cerebral artery occlusion (tMCAO). Immediately after reperfusion, HGF or GDNF was given by topical application. BrdU was injected intraperitoneally twice daily 1, 2, and 3 days after tMCAO. On 14 day, we histologically evaluated infarct volume, antiapoptotic effect, neurogenesis, angiogenesis, synaptogenesis, and antifibrosis. Both HGF and GDNF significantly reduced infarct size and the number of TUNEL-positive cells, but only HGF significantly increased the number of BrdU-positive cells in the subventricular zone, and 5'-bromo-2'-deoxyuridine -positive cells differentiated into mature neurons on the ischemic side. Enhancement of angiogenesis and synaptogenesis at the ischemic boundary zone was also observed only in HGF-treated rats. HGF significantly decreased the glial scar formation and scar thickness of the brain pia mater after tMCAO, but GDNF did not. Our study shows that both HGF and GDNF had significant neurotrophic effects, but only HGF can promote the neurogenesis, angiogenesis, and synaptogenesis and inhibit fibrotic change in brains after tMCAO. (C) 2010 Wiley-Liss, Inc.

DOI： 10.1002/jnr.22524

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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New granule cells are continuously generated in the dentate gyrus of the adult hippocampus. During granule cell maturation, the mechanisms that differentiate new cells not only describe the degree of cell differentiation, but also crucially regulate the progression of cell differentiation. Here, we describe a gene, tryptophan 2,3-dioxygenase (TDO), whose expression distinguishes stem cells from more differentiated cells among the granule cells of the adult mouse dentate gyrus. The use of markers for proliferation, neural progenitors, and immature and mature granule cells indicated that TDO was expressed in mature cells and in some immature cells. In mice heterozygous for the alpha-isoform of calcium/calmodulin-dependent protein kinase II, in which dentate gyrus granule cells fail to mature normally, TDO immunoreactivity was substantially downregulated in the dentate gyrus granule cells. Moreover, a 5-bromo-2'-deoxyuridine labeling experiment revealed that new neurons began to express TDO between 2 and 4 wk after the neurons were generated, when the axons and dendrites of the granule cells developed and synaptogenesis occurred. These findings indicate that TDO might be required at a late-stage of granule cell development, such as during axonal and dendritic growth, synaptogenesis and its maturation.

DOI： 10.1186/1756-6606-3-26

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Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are strong neurotrophic factors, which function as antiapoptotic factors. However, the neuroprotective effect of GDNF and HGF in ameliorating ischemic brain injury via an antiautophagic effect has not been examined. Therefore, we investigated GDNF and HGF for changes of infarct size and antiapoptotic and antiautophagic effects after transient middle cerebral artery occlusion (tMCAO) in rats. For the estimation of ischemic brain injury, the infarct size was calculated at 24 hr after tMCAO by HE staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) was performed for evaluating the antiapoptotic effect. Western blot analysis of microtubule-associated protein 1 light chain 3 (LC3) and immunofluorescence analysis of LC3 and phosphorylated mTOR/Ser(2448) (p-mTOR) were performed for evaluating the antiautophagic effect. GDNF and HGF significantly reduced infarct size after cerebral ischemia. The amounts of LC3-I plus LC3-II (relative to beta-tubulin) were significantly increased after tMCAO, and GDNF and HGF significantly decreased them. GDNF and HGF significantly increased p-mTOR-positive cells. GDNF and HGF significantly decreased the numbers of TUNEL-, LC3-, and LC3/TUNEL double-positive cells. LC3/TUNEL double-positive cells accounted for about 34.3% of LC3 plus TUNEL-positive cells. This study suggests that the protective effects of GDNF and HGF were greatly associated with not only the antiapoptotic but also the antiautophagic effects; maybe two types of cell death can occur in the same cell at the same time, and GDNF and HGF are capable of ameliorating these two pathways.

DOI： 10.1002/jnr.22373

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Immune-mediated diseases of the CNS, such as multiple sclerosis and its animal model, experimental autoimmune encephalitis (EAE), are characterized by the activation of antigen-presenting cells and the infiltration of autoreactive lymphocytes within the CNS, leading to demyelination, axonal damage, and neurological deficits. Hepatocyte growth factor (HGF) is a pleiotropic factor known for both neuronal and oligodendrocytic protective properties. Here, we assess the effect of a selective overexpression of HGF by neurons in the CNS of C57BL/6 mice carrying an HGF transgene (HGF-Tg mice). EAE induced either by immunization with myelin oligodendrocyte glycoprotein peptide or by adoptive transfer of T cells was inhibited in HGF-Tg mice. Notably, the level of inflammatory cells infiltrating the CNS decreased, except for CD25(+)Foxp3(+) regulatory T (T(reg)) cells, which increased. A strong T-helper cell type 2 cytokine bias was observed: IFN-gamma and IL-12p70 decreased in the spinal cord of HGF-Tg mice, whereas IL-4 and IL-10 increased. Antigen-specific response assays showed that HGF is a potent immunomodulatory factor that inhibits dendritic cell (DC) function along with differentiation of IL-10-producing T(reg) cells, a decrease in IL-17-producing T cells, and down-regulation of surface markers of T-cell activation. These effects were reversed fully when DC were pretreated with anti-cMet (HGF receptor) antibodies. Our results suggest that, by combining both potentially neuroprotective and immunomodulatory effects, HGF is a promising candidate for the development of new treatments for immune-mediated demyelinating diseases associated with neurodegeneration such as multiple sclerosis.

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by progressive degeneration of motoneurons. We have demonstrated that hepatocyte growth factor (HGF) attenuates loss of both spinal and brainstem motoneurons of ALS model mice expressing mutated human SOD1(G93A) (G93A). This study was designed to assess disease-dependent regulatory mechanisms of c-Met/HGF receptor (c-Met) activation in the facial motoneurons of G93A mice. Using double transgenic mice expressing HGF and mutated SOD1(G93A)(G93A/HGF), we showed that phosphorylation of c-Met tyrosine residues at positions 1230, 1234 and 1235 (phospho-Tyr), and thereby its activation, was slightly evident in G93A and highly obvious in G93A/HGF mice (but absent in WT and HGF-Tg mice). Phosphorylation of the c-Met serine residue at position 985 (phospho-Ser), a residue involved in the negative regulation of its activation, was evident in WT and HGF-Tg mice. Protein phosphatase 2A (MA), which is capable of dephosphorylating c-Met phospho-serine, is upregulated in the facial motoneurons of G93A and G93A/HGF mice compared with WT and HGF-Tg mice. Thus, c-Met activation is reciprocally regulated by phosphorylation between c-Met serine and tyrosine residues through PP2A induction in the presence or absence of mutant SOD1 expression, and HGF functions more efficiently in ALS and ALS-related diseases. (C) 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2009.06.016

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The actin cytoskeleton inside extending axonal and dendritic processes must undergo continuous assembly and disassembly. Some extrinsic factors modulate actin turnover through controlling the activity of LIM kinase 1 (LIMK1), which phosphorylates and inactivates the actin depolymerizing factor cofilin. Here, we for the first time examine the function and regulation of LIMK1 in vivo in the vertebrate nervous system. Upon expression of wildtype or kinase-dead forms of the protein, dendrite growth by Xenopus retinal ganglion cells (RGCs) was unchanged. In contrast, maintaining a low, but significant level, of LIMK1 function in the RGC axon is critical for proper extension. Interestingly, bone morphogenetic protein receptor II (BMPRII) is a major regulator of LIMK1 in extending RGC axons, as expression of a BMPRII lacking the LIMK1 binding region caused a dramatic shortening of the axons. Previously, we found that BMPRIIs stimulate dendrite initiation in vivo. Thus, the fact that manipulation of LIMK1 activity failed to alter dendrite growth suggests that BMPs may activate distinct signalling pathways in axons and dendrites. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2009.03.027

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Tryptophan 2,3-dioxygenase (TDO), an initial and rate-limiting enzyme for the kynurenine pathway of tryptophan (Trp) metabolism, is thought to play an important role in systemic Trp metabolism as well as in emotional and psychiatric status. In contrast to its predominant expression in the liver, expression of TDO in the brain is poorly understood. Here, we show that tdo mRNA is expressed in various nervous tissues, including the hippocampus, cerebellum, striatum and brainstem. During development, tdo mRNA was differentially regulated in brain tissues. Further, we identified two novel variants of the tdo gene, termed tdo variant1 and variant2. Similar tetramer formation and enzymatic activity were obtained when these forms were expressed in wheat germ and COS-7 cells, respectively. Quantitative real-time RT-PCR revealed that tdo variants were expressed in various nervous tissues, with high expression in the cerebellum and hippocampus, followed by the midbrain. tdo variant2 was the only variant expressed in the cerebellum from postnatal day 4 (P4) to P7, suggesting a unique role for this variant during early postnatal development. Our findings indicate that tdo and its novel variants may play an important role in not only the liver but also in local areas in developing and adult brain.

DOI： 10.1016/j.neures.2009.02.004

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[Purpose] The present study examined satellite cell and hepatocyte growth factor (HGF) responses to exercise intensity in the rat soleus muscle. [Methods] HGF levels assessed during postnatal growth of the gastrocnemius muscle. Depression of HGF levels Occurred up to postnatal week 4, so 4-week-old rats were used the exercise training experiment. Rats walked or ran at speeds of 16 or 24 m/min, at -16% grade, 30 min/trial. Soleus muscles were removed after 72 h. Animals were injected with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) I h before sampling. The right soleus muscle was used for immunofluorescence, and the left soleus muscle was used to measure HGF protein levels. [Result] HGF levels were unchanged, although numbers of BrdU-positive nuclei increased 2.4-fold in rats with exercise training. [Conclusion] The relationship between activation of satellite cells and HGF production after exercise training remains unclear. However, this study indicates the exercise intensity necessary to activate satellite cells. In the future, this result may facilitate the creation of exercise training intensity as an index of satellite cell activity for muscle strength training.

DOI： 10.1589/jpts.21.141

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Although nutrients, including amino acids and their metabolites such as serotonin (5-HT), are strong modulators of anxiety-related behavior, the metabolic pathway(s) responsible for this physiological modulation is not fully understood. Regarding tryptophan (Trp), the initial rate-limiting enzymes for the kynurenine pathway of tryptophan metabolism are tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). Here, we generated mice deficient for tdo (Tdo(-/-)). Compared with wild-type littermates, Tdo(-/-)mice showed increased plasma levels of Trp and its metabolites 5-hydroxyindoleacetic acid (5-HIAA) and kynurenine, as well as increased levels of Trp, 5-HT and 5-HIAA in the hippocampus and midbrain. These mice also showed anxiolytic modulation in the elevated plus maze and open field tests, and increased adult neurogenesis, as evidenced by double staining of BrdU and neural progenitor/neuronal markers. These findings demonstrate a direct molecular link between Trp metabolism and neurogenesis and anxiety-related behavior under physiological conditions.

DOI： 10.1186/1756-6606-2-8

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HGF-like protein (HLP)/rnacrophage stimulating protein (MSP) is the only structural relative of hepatocyte growth factor (HGF), and is involved in the regulation of peripheral macrophage activation. However, the actions of HLP in microglia, a species of macrophage in the nervous system, which is closely involved in the neural degeneration and regeneration, is not yet understood. This study found that Ron, the receptor for HLP, is expressed in primary microglia using RT-PCR, immunocytochemical staining and Western blotting, and, thus, sought to elucidate the function of HLP on the primary microglia. HLP promoted microglial migration without affecting cell survival and proliferation. Furthermore, real-time quantitative RT-PCR analysis revealed that HLP greatly increased the mRNA of inflammatory cytokines, including IL-6 and GM-CSF, and NOS. These findings provide the first evidence that HLP has the potential to modulate inflammatory actions of microglia, which proposes novel aspects for the process of degeneration and/or regeneration of the brain.

DOI： 10.2220/biomedres.29.77

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To examine the effects of HGF on synaptic densities under excitotoxic conditions, we investigated changes in the number of puncta detected by double immunostaining with NMDA receptor subunits and presynaptic markers in cultured hippocampal neurons. Exposure of hippocampal neurons to excitotoxic NMDA (100 mu M) decreased the synaptic localization of NMDA receptor subunit NR213, whereas synaptic NR1 and NR2A clusters were not altered. Colocalization of PSD-95, a scaffolding protein of the receptor, with the presynaptic protein synapsin I was also decreased after excitotoxicity. Treatment with HGF attenuated these decreases in number. The decrease in the levels of surface NR2B subunits following the addition of the excitotoxic NMDA was also attenuated by the HGF treatment. The decrease in CREB phosphorylation in response to depolarization-evoked NMDA receptor activation was prevented by the HGF treatment. These results suggest that HGF not only prevented neuronal cell death but also attenuated the decrease in synaptic localization of NMDA receptor subunits and prevented intracellular signaling through the NMDA receptor. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.expneurol.2007.10.001

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of brainstem and spinal motoneurons. Although prevention of motoneuronal degeneration has been postulated as the primary target for a cure, accumulating evidence suggests that microglial accumulation contributes to disease progression. This study was designed to assess the ability of HGF to modulate microglial accumulation and motoneuronal degeneration in brainstem motor nuclei, using, double transgenic mice overexpressing mutated SODI(G93A) and HGF(G93A/HGF). Histological and immunohistochemical analyses of the tissues of G93A/HGF mice revealed a marked decrease in the number of microglia and reactive astrocytes and an attenuation of the loss of motoneurons in facial and hypoglossal nuclei compared with G93A mice. HGF overexpression attenuated monocyte chemoattractant protein-1 (MCP-1) induction, predominantly in astrocytes; suppressed activation of caspase-1, -3 and -9; and, increased X chromosome-linked inhibition of apoptosis protein (XIAP) in the motoneurons of G93A mice. The implication is that HGF reduces microglial accumulation by suppressing MCP-1 induction and prevents motoneuronal death through inhibition of pro-apoptotic protein activation. These findings suggest that, in addition to direct neurotrophic activity on motoneurons, HGF-suppression of gliosis may retard disease progression, making HGF a potential therapeutic agent for the treatment of ALS patients. (C) 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2007.08.017

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Hepatocyte growth factor (HGF) and its receptor are expressed in various regions of the brain and have protective effects against excitotoxic injuries. However, their effects on synapse formation remain to be elucidated. To determine whether HGF has the ability to alter synaptic function during development, we investigated changes in the number of synapse detected by double immunostaining for NMDA receptor subunits and a presynaptic marker in cultured young hippocampal neurons. Whereas application of HGF increased the number of cluster of synapsin, a presynaptic protein, the clusters of NMDA receptor subunits NR1 and NR2B were not altered. Interestingly, colocalization of PSD-95, a scaffolding protein of the receptor, with synapsin was increased by HGF treatment without a change in the total amount of it. In addition, we investigated the expression of surface NMDA receptor, neuroligin, and neurexin, which were assessed by use of a cell-surface biotinylation assay. The application of HGF did not change the surface expression of these proteins. Furthermore, we determined the release of glutamate in response to depolarization. Treatment with HGF promoted depolarization-evoked release of glutamate. These results suggest that HGF modulates the expression of the scaffolding protein of the NMDA receptor at the synapse and promotes maturation of excitatory synapses in young hippocampal neurons. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.expneurol.2007.06.007

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Hepatocyte growth factor (HGF) was initially cloned as a mitogen for hepatocytes and has been identified as a neurotrophic factor for a variety of neurons. However, few attempts have assessed the role of HGF in cells of oligodendrocyte lineage. The purpose of this study was to elucidate the role of HGF in such cells during development. Double immunostaining for either c-Met/HGF receptor or phospho-c-Met with either NG2 or RIP in rat striatum at postnatal day 3 (P3), P7, and P14 revealed that c-Met was phosphorylated on tyrosine residues and thereby activated in NG2(+) oligodendrocyte progenitor cells (OPCs) at P3-P14 and in RIP+ oligodendrocytes at P14. Intrastriatal injections of recombinant human HGF at both P7 and P10 revealed that the relative ratio of BrdU(+)/NG2(+) cells per total number of NG2(+) cells increased, while BrdU(+)/MBP+ oligodendrocyte numbers decreased. Western blot analysis showed a down-regulation of myelin basic protein (MBP) after HGF injection. Electron microscopy revealed that the numbers of myelinated nerve fibers decreased after HGF treatment. Furthermore, administration of anti-HGF IgG into the striatum increased the number of BrdU(+)/MBP+ oligodendrocytes. These findings demonstrated that HGF increases proliferation of OPCs and attenuates their differentiation into myelinating oligodendrocytes, presumably by favoring neurite outgrowth that may be inhibited by the myelin inhibitory molecules on oligodendrocytes. Down-regulation of HGF mRNA in the striatum from P7 to P14, as revealed by quantitative real-time RT-PCR, may be favorable for OPC differentiation into myelinating oligodendrocytes. Our findings suggest that c-Met signaling, together with HGF regulation, plays an important role in developmental oligodendrogenesis. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.brainres.2007.02.045

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Hepatocyte growth factor (HGF) is one of the most potent survival-promoting factors for motor neurons. We showed that introduction of the HGF gene into neurons of G93A transgenic mice attenuates motor neuron degeneration and increases the lifespan of these mice. Currently, treatment regimens using recombinant protein are closer to clinical application than gene therapy. To examine its protective effect on motor neurons and therapeutic potential we administered human recombinant HGF (hrHGF) by continuous intrathecal delivery to G93A transgenic rats at doses of 40 or 200 mu g and 200 mu g at 100 days of age (the age at which pathologic changes of the spinal cord appear, but animals show no clinical weakness) and at 115 days (onset of paralysis), respectively, for 4 weeks each. Intrathecal administration of hrHGF attenuates motor neuron degeneration and prolonged the duration of the disease by 63%, even with administration from the onset of paralysis. Our results indicated the therapeutic efficacy of continuous intrathecal administration of hrHGF in transgenic rats and should lead to the consideration for further clinical trials in amyotrophic lateral sclerosis using continuous intrathecal administration of hrHGF.

DOI： 10.1097/nen.0b013e318159886b

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Many therapeutic interventions using neurotrophic factors or pharmacological agents have focused on secondary degeneration after spinal cord injury (SCI) to reduce damaged areas and promote axonal regeneration and functional recovery. Hepatocyte growth factor (HGF), which was identified as a potent mitogen for mature hepatocytes and a mediator of inflammatory responses to tissue injury, has recently been highlighted as a potent neurotrophic and angiogenic factor in the central nervous system (CNS). In the present study, we revealed that the extent of endogenous HGF up-regulation was less than that of c-Met, an HGF receptor, during the acute phase of SCI and administered exogenous HGF into injured spinal cord using a replication-incompetent herpes simplex virous-1 (HSV-1) vector to determine whether HGF exerts beneficial effects and promotes functional recovery after SCI. This treatment resulted in the significant promotion of neuron and oligodendrocyte survival, angiogenesis, axonal regrowth, and functional recovery after SCI. These results suggest that HGF gene delivery to the injured spinal cord exerts multiple beneficial effects and enhances endogenous repair after SCI. This is the first study to demonstrate the efficacy of HGF for SCI. (C) 2007 Wiley-Liss, Inc.

DOI： 10.1002/jnr.21372

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Hepatocyte growth factor (HGF) is one of the prospective agents for therapy against a variety of neurologic and neurodegenerative disorders, although the precise mechanisms for the effect of HGF remain to be elucidated. We showed that treatment with HGF protected hippocampal cornu ammonis (CA) subregion 1 neurons from apoptotic cell death after transient forebrain ischemia. Accumulating evidence indicates that ischemia-induced neuronal damage occurs via caspase-independent pathways. In the present study, we focused on the localization of apoptosis-inducing factor (AIF), which is an important protein in the signal-transduction system through caspase-independent pathways, to investigate the possible mechanism for the protective effect of HGF after transient forebrain ischemia. Hepatocyte growth factor attenuated the increase in the expression of AIF protein in the nucleus after transient forebrain ischemia. We further explored the upstream components of AIF translocation. Primary DNA damage induced by Ca(2+) influx and subsequent NO formation are thought to be the initial events for AIF translocation, which results in the subsequent DNA damage by AIF. Hepatocyte growth factor prevented the primary oxidative DNA damage, as was estimated by using anti-8-OHdG (8-hydroxy-2 '-deoxyguanosine) antibody. Oxidative DNA damage after ischemia is known to lead to the activation of poly(ADP-ribose) polymerase (PARP) and p53, resulting in AIF translocation. Marked increases in the PAR polymer formation and the expression of p53 protein after ischemia were effectively prevented by HGF treatment. In the present study, we first showed that HGF was capable of preventing neuronal cell death by inhibiting the primary oxidative DNA damage and then preventing the activation of the PARP/p53/AIF pathway.

DOI： 10.1038/sj.jcbfm.9600287

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Early oxidative DNA damage is regarded to be an initiator of neuronal apoptotic cell death after cerebral ischemia. Although evidence suggests that HGF has the ability to protect cells from oxidative stress, it remains unclear as to how HGF suppresses oxidative DNA damage after cerebral ischemia. Apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) is a multifunctional protein in the DNA base repair pathway that is responsible for repairing apurinic/apyrimidinic sites in DNA after oxidation. We demonstrated that both the immunoreactivity and the number of APE/Ref-1-positive cells in the hippocampal CA1 region were decreased after transient forebrain ischemia and that treatment with HGF suppressed this reduction. The expression of Cu/ZnSOD and MnSOD in the hippocampal CA1 region did not change after ischemia, regardless of treatment with or not with HGF. The activity of NADPH oxidase was increased mainly in glia-like cells in the hippocampal CA1 region after ischemia, and this increase was attenuated by HGF treatment. These results suggest that the protective effects of HGF against cerebral ischemia-induced cell death in the hippocampal CA1 region are related to the improvement of neuronal APE/Ref-1 expression and the inhibition of NADPH oxidase activity in glia-like cells. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

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Hepatocyte growth factor (HGF) exerts its physiological activities as that of an organotropic factor for regeneration and can prevent ischemia-induced injuries: however, its effect and mechanism of action under in vivo pathophysiological conditions remains to be determined. Recently, we demonstrated that treatment with human recombinant HGF (hrHGF) attenuated the disruption of the blood-brain barrier (131313) observed after microsphere embolism-induced sustained cerebral ischemia. To see if tight junctional proteins were involved in this attenuation, in the present study, we investigated the effects of HGF on the levels of occludin and zonula occludens (ZO)-1 in cerebrovascular endothelial cells after microsphere embolism. Sustained cerebral ischemia was induced by the injection of 700 microspheres (48 mu m diameter) into the right internal carotid artery of rats. hrHGF was injected into the right ventricle of the brain by using an osmotic pump at a dose of 36 mu g/7 days per animal. The levels of tight junctional proteins in the endothelial cells were examined by immunohistochemical analysis. Treatment with hrHGF attenuated the decrease in the expression of occludin and ZO-1 proteins in the endothelial cells that occurred after sustained cerebral ischemia. Furthermore, treatment with hrHGF resulted in retention of these tight junctional proteins in fluorescein isothiocyanate (FITC)-albumin-perfused cerebral vessels, which did not leak FITC-albumin in the ipsilateral cortex. These results suggest that HGF-mediated maintenance of the tight junctional proteins in the endothelial cells may be a possible mechanism for the protective effect of HGF against the disruption of the BBB after cerebral ischemia. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2006.08.050

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Hepatocyte growth factor (HGF) exhibits strong neurotrophic activities on motoneurons both in vitro and in vivo. We examined survival-promoting effects of an adenoviral vector encoding human HGF (AxCAhHGF) on injured adult rat motoneurons after peripheral nerve avulsion. The production of HGF in COS1 cells infected with AxCAhHGF and its bioactivity were confirmed by ELISA, Western blot and Madin-Darby canine kidney (MDCK) cell scatter assay. The facial nerve or the seventh cervical segment (C7) ventral and dorsal roots of 3-month-old Fischer 344 male rats were then avulsed and removed from the stylomastoid or vertebral foramen, respectively, and AxCAhHGF, AxCALacZ (adenovirus encoding beta-galactosidase gene) or phosphate-buffered saline (PBS) was inoculated in the lesioned foramen. Treatment with AxCAhHGF after avulsion significantly prevented the loss of injured facial and C7 ventral motoneurons as compared to AxCALacZ or PBS treatment and ameliorated choline acetyltransferase immunoreactivity in these neurons. These results indicate that HGF may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.brainres.2006.06.104

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Accumulation of beta-amyloid protein (A beta) in the brain is a hallmark of Alzheimer's disease (AD), and A beta-mediated pathogenesis could result from increased production of A beta or insufficient A beta clearance by microglia, astrocytes, or the vascular system. Cell-surface receptors, such as scavenger receptors, might play a critical role in the binding and clearing of A beta; however, the responsible receptors have yet to be identified. We show that scavenger receptor with C-type lectin (SRCL), a member of the scavenger receptor family containing coiled-coil, collagen-like, and C-type lectin/carbohydrate recognition domains, is expressed in cultured astrocytes and microglia. In contrast to the low expression of SRCL in the wild-type mouse brain, in a double transgenic mouse model of AD (Tg-APP/PS1), immunohistochemistry showed that SRCL was markedly induced in A beta-positive astrocytes and A beta-positive vascular/perivascular cells, which are associated closely with cerebral amyloid angiopathy. In patients with AD, the distribution of SRCL was similar to that seen in the Tg-APP/PS1 temporal cortex. The presence of a large number of SRCL/A beta double-positive particles in the intracellular compartments of reactive astrocytes and vascular/perivascular cells in Tg-APP/PS1 mice and AD patients suggests a role for SRCL in A beta clearance. Moreover, CHO-K1 cells transfected with SRCL isoforms were found to bind fibrillar A beta(1-42). These findings suggest that SRCL could be the receptor involved in the binding or clearing of A beta by glial and vascular/perivascular cells in AD. (c) 2006 Wiley-Liss, Inc.

DOI： 10.1002/jnr.20992

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Occlusive cerebrovascular disease leads to brain ischemia that causes neurological deficits. Here we introduce a new strategy combining mesenchymal stromal cells (MSCs) and ex vivo hepatocyte growth factor (HGF) gene transferring with a multimutated herpes simplex virus type-1 vector in a rat transient middle cerebral artery occlusion (MCAO) model. Gene-transferred MSCs were intracerebrally transplanted into the rats' ischemic brains at 2 h (superacute) or 24 h (acute) after MCAO. Behavioral tests showed significant improvement of neurological deficits in the HGF-transferred MSCs (MSC-HGF)-treated group compared with the phosphate-buffered saline (PBS)-treated and MSCs-only-treated group. The significant difference of infarction areas on day 3 was detected only between the MSC-HGF group and the PBS group with the superacute treatment, but was detected among each group on day 14 with both transplantations. After the superacute transplantation, we detected abundant expression of HGF protein in the ischemic brain of the MSC-HGF group compared with others on day 1 after treatment, and it was maintained for at least 2 weeks. Furthermore, we determined that the increased expression of HGF was derived from the transferred HGF gene in gene-modified MSCs. The percentage of apoptosis-positive cells in the ischemic boundary zone (IBZ) was significantly decreased, while that of remaining neurons in the cortex of the IBZ was significantly increased in the MSC-HGF group compared with others. The present study shows that combined therapy is more therapeutically efficient than MSC cell therapy alone, and it may extend the therapeutic time window from superacute to acute phase.

DOI： 10.1038/sj.jcbfm.9600273

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Hepatocyte growth factor (HGF) has been implicated in protection against several types of cell injuries. We investigated the effects of human recombinant HGF (hrHGF) on the selective neuronal cell death in the hippocampal CA1 region after transient forebrain ischemia in rats and explored the nature of the intracellular signaling pathway for the protection against this neuronal injury. hrHGF was injected continuously into the hippocampal CA1 region directly using an osmotic pump from 10 min to 72 h after the start of reperfusion. The marked increase in the number of TUNEL-positive cells found in the CA1 region after ischernia was almost completely abolished by the hrHGF treatment. Akt phosphorylation as well as IKB phosphorylation, which has been implicated in events downstream of the Akt, was not affected by hrHGF treatment. Extracellular signal-regulated kinase (ERK) phosphorylation was decreased in the CA1 region with time after ischernia. hrHGF increased or recovered ERK phosphorylation without changing the total amount of ERK protein. Immunohistochemical analysis demonstrated that phosphorylated ERK was colocalized with a neuronal nucleus marker NeuN in the hippocampal CA1 region of ischemic rats with hrHGF treatment at the early period after reperfusion. These results suggest that the protective effects of hrHGF against neuronal death in the hippocampal CA1 after transient forebrain ischernia could be related to an ERK-dependent pathway. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ejphar.2006.01.037

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Hepatocyte growth factor (HGF) has been suggested as a mitogen for skeletal muscle satellite cells and participates in skeletal muscle hypertrophy. The present study assessed HGF levels in mouse soleus and plantaris muscles during 14 days of tail suspension and 3 days of reloading using the enzyme-linked immunosorbent assay. Immunohistochemical analyses were used to determine the locations of HGF, its receptor (c-Met) and proliferating cell nuclear antigen. In normal mice, HGF contents were 4.4 ± 0.5 ng/g tissue in the soleus muscle and 5.9 ± 1.2 ng/g tissue in the plantaris muscle, significantly higher than in the soleus muscle. HGF level in the soleus muscle was increased 314% from normal by reloading. HGF and c-Met were expressed in small cells contiguous to muscle fibers. Cells in similar positions displayed reactivity for PCNA, suggesting that these represent activated satellite cells. Thus, production of HGF protein appears to stimulated in satellite cells during recovery from disuse atrophy.

DOI： 10.1589/jpts.18.33

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The present study examined the localization of hepatocyte growth factor in rat skeletal muscle, and investigated whether levels of hepatocyte growth factor differ between skeletal muscles. Levels of hepatocyte growth factor in soleus and tibialis anterior muscles were measured using enzyme-linked immunosorbent assay. Localization of hepatocyte growth factor and proliferating cell nuclear antigen in the soleus muscle was visualized using immunofluorescence analysis. Level of hepatocyte growth factor was 3.2 ± 1.4 ng/g tissue in the soleus muscle and 3.4 ± 0.4 ng/g tissue in the tibialis anterior muscle. No significant differences were identified between muscles with differential contractile characteristics. Existence of hepatocyte growth factor was observed in cytoplasm of small cells conterminous to muscle fibers. Cells in a similar position displayed reactivity to proliferating cell nuclear antigen, suggesting that they represented activated skeletal muscle satellite cells. Hepatocyte growth factor is produced in normal rat skeletal muscle by activated skeletal muscle satellite cells.

DOI： 10.1589/jpts.17.109

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Although hepatocyte growth factor (HGF) and its receptor are expressed in various regions of the brain, their effects and mechanism of action under pathological conditions remain to be determined. Over-activation of the N-methyl-D-aspartate (NMDA) receptor, an ionotropic glutamate receptor, has been implicated in a variety of neurological and neurodegenerative disorders. We investigated the effects of HGF on the NMDA-induced cell death in cultured hippocampal neurons and sought to explore their mechanisms. NMDA-induced cell death and increase in the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells were prevented by HGF treatment. Although neither the total amounts nor the mitochondrial localization of Bax, Bcl-2 and Bcl-xL were affected, caspase 3 activity was increased after NMDA exposure. Treatment with HGF partially prevented this NMDA-induced activation of caspase 3. Although the amount of apoptosis-inducing factor (AIF) was not altered, translocation of AIF into the nucleus was detected after NMDA exposure. This NMDA-induced AIF translocation was reduced by treatment with HGF. In addition, increased poly(ADP-ribose) polymer formation after NMDA exposure was attenuated by treatment with HGF. These results suggest that the protective effects of HGF against NMDA-induced neurotoxicity are mediated via the partial prevention of caspase 3 activity and the inhibition of AIF translocation to the nucleus.

DOI： 10.1111/j.1471-4159.2005.03446.x

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The major pathological change in Fukuyama-type congenital muscular dystrophy brain is polymicrogyria. Pathological studies of Fukuyama-type congenital muscular dystrophy brain indicated that protrusion of neurons into the subarachnoid space through breaches in the glia limitans-basal lamina complex is a cardinal pathogenic process in this condition, It remains undetermined, however. whether the defect causing this abnormal migration resides in the migrating neurons or in the glia limitans-basal lamina complex, To elucidate the pathogenesis of brain abnormalities in Fukuyama-type congenital muscular dystrophy, we analyzed histologically and immunohistochemically the developing forebrain in fukutin-deficient chimeric mice and compared it with that in controls (n = 4 in each group). In chimeric embryos, ectopia became apparent as early as embryonic day 14, and laminar organization became progressively distorted. The basal lamina of the cortical surface in chimeras showed defects at E14, coinciding with the earliest time point at which ectopia were detected. Immunohistochemical analysis of glycosylated alpha-dystroglycan showed progressive defects coincidental with the disruption of the basal lamina. Neuronal migration was not affected in chimeras. as determined by detection of bromodeoxyuridine-labeled neurons. Extension of radial glial fibers was intact in chimeras. Taken together, disruption of the basal lamina, caused by the loss of interaction between hypoglycosylated alpha-dystroglycan and its ligands, plays a key role in the pathogenesis of cortical dysplasia in Fukuyama-type congenital muscular dystrophy. (c) 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.nmd.2005.03.009

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Background: Hepatocyte growth factor (HGF) has the capacity to selectively direct thalamocortical projections into an intermediate target, the pallidum, and eventually to their final cortical destination. HGF may have a role in the mediation of anxiety. Very little is known about other central behavioral effects of HGF. Objective: Our aim was to determine what effect HGF has on anxiety in rats. Methods: HGF was infused at a constant rate into cerebral lateral ventricles and its effect on anxiety in rats was monitored. Results: In the elevated plus maze test and the black and white box test, HGF administration caused all indicators of anxiety to increase. No significant effect on general locomotor activity was seen. Conclusion: HGF infusion into the brain produces an anxiolytic effect. Copyright (C) 2005 S. Karger AG, Basel.

DOI： 10.1159/000082853

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To clarify the trophic mechanism of residual anterior horn cells affected by sporadic amyotrophic lateral sclerosis (SALS) and familial ALS (FALS) with superoxide dismutase 1 (SOD1) mutations, we investigated the immunohistochemical expression of hepatocyte growth factor (HGF), a novel neurotrophic factor, and its receptor, c-Met. In normal subjects, immunoreactivity to both anti-HGF and anti-c-Met antibodies was observed in almost all anterior horn cells, whereas no significant immunoreactivity was observed in astrocytes and oligodendrocytes. Histologically, the number of spinal anterior horn cells in ALS patients decreased along with disease progression. Immunohistochemically, the number of neurons negative for HGF and c-Met increased with ALS disease progression. However, throughout the course of the disease, certain residual anterior horn cells co-expressed both HGF and c-Met with the same, or even stronger intensity in comparison with those of normal subjects, irrespective of the reduction in the number of immunopositive cells. Western blot analysis revealed that c-Met was induced in the spinal cord of a patient with SALS after a clinical course of 2.5 years, whereas the level decreased in a SALS patient after a clinical course of 11 years 5 months. These results suggest that the autocrine and/or paracrine trophic support of the HGF-c-Met system contributes to the attenuation of the degeneration of residual anterior horn cells in ALS, while disruption of the neuronal HGF-c-Met system at an advanced disease stage accelerates cellular degeneration and/or the process of cell death. In SOD1-mutated FALS patients, Lewy body-like hyaline inclusions (LBHIs) in some residual anterior horn cells exhibited co-aggregation of both HGF and c-Met, although the cytoplasmic staining intensity for HGF and c-Met in the LBHI-bearing neurons was either weak or negative. Such sequestration of HGF and c-Met in LBHIs may suggest partial disruption of the HGF-c-Met system, thereby contributing to the acceleration of neuronal degeneration in FALS patients.

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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motoneurons and degeneration of motor axons. We show that overexpression of hepatocyte growth factor (HGF) in the nervous system attenuates motoneuron death and axonal degeneration and prolongs the life span of transgenic mice overexpressing mutated Cu2+/Zn2+ superoxide dismutase 1. HGF prevented induction of caspase-1 and inducible nitric oxide synthase (iNOS) in motoneurons and retained the levels of the glial-specific glutamate transporter (excitatory amino acid transporter 2/glutamate transporter 1) in reactive astrocytes. We propose that HGF may be the first example of an endogenous growth factor that can alleviate the symptoms of ALS by direct neurotrophic activities on motoneurons and indirect activities on glial cells, presumably favoring a reduction in glutamatergic neurotoxicity.

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Development of the cerebral cortex is a series of precisely timed proliferative, migratory, and maturational processes. Hepatocyte growth factor (HGF) is a pleiotrophic cytokine, which plays important roles in the organogenesis and regeneration of various tissues, both during development and in the adult, due to its mitogenic, motogenic and morphogenic activities. In the present study, we examined expression and functional roles of HGF and c-Met during development of the rat cerebral cortex. Quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) revealed that expression levels of c-met and HGF mRNAs were increased in the cerebral cortex during late embryonic development and peaked at E18. Immunohistochemical analyses revealed that c-Met-immunoreactivity (IR) was localized to the preplate (PP), with weaker-IR in neuroepithelial layer (NE) at embryonic day 14 (E14). At E16, c-Met-IR was present in the cortical plate (CP) and the intermediate zone (IZ), with a weak presence in the ventricular zone (VZ). On the other hand, HGF-IR was present in NE and VZ at E14 and E16, respectively. HGF-IR appeared in cortical plate tissue from E16 onward. Double labeling immunofluorescent cytochemical studies revealed that c-Met-IR was localized both in TuJ-1-IR- and non-TuJ-1-IR-cells, purified from E18 cerebral cortex in vitro, suggesting the presence of c-Met-IR in postmitotic neurons as well as in neuroepithelial cells. c-Met-IR was strong in cell bodies and neurites shortly after in vitro culture, while at 7DIV c-Met-IR decreased in neurites and was evident in growth cones. HGF dose dependently supported neuronal survival in vitro under serum-deprived conditions. In a transwell culture chamber, HGF increased neuronal migration, and co-incubation with functional blocking antibody against HGF abrogated this motogenic effect of HGF. These lines of evidence suggest that HGF is involved in the development and maintenance of cortical neurons during differentiation, motogenesis, neuritogenesis and neuronal survival. (C) 2002 Elsevier Science B V All rights reserved.

DOI： 10.1016/S0169-328X(02)00168-7

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We isolated a cDNA encoding the Xenopus member of Sky/Axl/Mer receptor tyrosine kinase family (referred as Sky family), termed Xksy. The predicted Xksy protein has conserved structural characteristics of the Sky family: an unique extracellular domain of two immunoglobulin (Ig)-like repeats, two fibronectin type III (FNIII)-like repeats and an intracellular tyrosine kinase. Homology analysis of Xksy showed the highest identity to mammalian Sky protein. In contrast to the predominant expression of sky mRNA in the adult mammalian nervous system, Northern blot analysis showed ubiquitous expression of a single 5.2-kb Xksy mRNA in tissues of the adult Xenopus. RNase protection assays revealed that, during development, Xksy mRNA is expressed from mid neurulation stage. Levels increase through the tadpole stage and become restricted to the head region in embryos by stage 40. Whole-mount in situ hybridization analyses revealed that expression of Xksy is localized to the nervous system of the tadpole stage, including origins of sensory organs and branchial arches. When a chimeric receptor (EGFR-Xksy), composed of the extracellular region of epidermal growth factor (EGF) receptor and the transmembrane/ intracellular regions of Xksy, was expressed in a doxycycline repressive manner in HEK 293 cells, EGF-stimulus without doxycycline induced tyrosine phosphorylation of the chimeric receptor and evoke morphological changes. EGF treatment also induced growth modifications of EGFR-Xksy cells. And doxycycline pre-treatment eliminated these activities. These findings suggest that Xksy may play an important role in growth, differentiation and the accurate migration of cells during embryogenesis and early neural development. (C) 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-1119(02)00483-3

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Sky (also known as "Tyro3" and "Rse") is a member of the Axl/Sky/Mer receptor tyrosine kinase family and has two immunoglobulin-like repeats and two fibronectin type III-like repeats in the extracellular domain. Gash is a ligand for all members of the Axl family, each of which (Axl, Sky, and c-Mer) has different affinities to Gash. Physiological functions of Sky and Gash in the nervous system are not well understood, despite their importance, which is suggested by Sky structural features and its predominant expression in the brain. We found in the RNase protection assays that gash and sky mRNAs are expressed in the adult rat hippocampus and are similarly regulated during development. Expression levels were low during embryonic stages but gradually increased during development and reached the highest level in adulthood. Sky, but not Axl, immunoreactivity was observed in the adult hippocampus. Recombinant rat Gash attenuated hippocampal neuronal cell death that was caused by serum starvation in vitro, indicating that Gash is a novel neurotrophic factor for hippocampal neurons. Gash showed regulated expression in the sciatic nerve after nerve transection. Therefore, Sky and Gash have neurotrophic roles in the nervous system. (C) 2002 Wiley-Liss, Inc.

DOI： 10.1002/jnr.10211

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The cDNA clone encoding a mouse scavenger receptor with C-type lectin (SRCL), a novel member of the scavenger receptor fan-lily, has been isolated from a mouse embryonic cDNA library. The predicted cDNA sequence contains a 2226 bp open reading frame encoding a coiled-coil, collagen-like, C-type lectin/carbohydrate recognition domain with an overall sequence identity of 92% to human SRCL. In contrast to human, mouse SRCL mRNA was expressed ubiquitously in various adult tissues including the liver and spleen, in which human SRCL mRNA was under detection limits. Mouse SRCL mRNA was expressed in the macrophage cell line J774A.1 cells at a high level and in the embryo as early as E9. (C) 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0167-4781(01)00284-6

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HGF-like protein (HLP) is a member of the hepatocyte growth factor (HGF) family. Although HGF is shown to have neurotrophic activities on many of CNS and PNS neurons, the role of HLP in the nervous system is poorly understood despite the knowledge that Ron/HLP receptor is expressed in embryonic neurons. Here we show that HGF but not HLP promotes neurite extension and migration emanating from chick embryonic day 9 (E9) dorsal root ganglia (DRG) explants in the presence of low levels of NGF, however, HLP does promote neurite extension and cellular migration from E15 chick DRG explants with low levels of NGF. Ron-Fc, a chimeric molecule composed of the extracellular domain of Ron fused with immunoglobulin Fc, eliminated activities of HLP, such as cellular migration and long neurite extension emanating from E15 DRG explants in the presence of NGF, but did not eliminate short neurites, These results suggested that promotion of long neurite extension and migration depends on activities of HLP through its receptor/Ron, Taken together, we propose that HLP may play an important role in chick sensory ganglia at relatively late stages of development. This is the first evidence that HLP functions as a neurotrophic factor. (C) 2001 Academic Press.

DOI： 10.1006/bbrc.2001.4819

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Conventional RNA quantification methods such as Northern blots or RNase protection assays are often not sufficiently sensitive to measure mRNA levels in a small neuronal region. The reverse transcription-polymerase chain reaction (RT-PCR) is a sensitive alternative that can be used to determine the relative amount of mRNAs in tissues or cells. However, this method does not directly yield the absolute value of mRNA abundance because of the exponential nature of PCR. Using synthetic RNA competitors, we developed a competitive RT-PCR to evaluate the absolute amount of hepatocyte growth factor (HGF) and c-met/HGF receptor mRNAs in neural tissues. Here we describe the procedures we used to measure HGF and c-met mRNA levels in the punched ventral horn of the mouse spinal cord. This protocol provides a rapid, sensitive and accurate means of measuring mRNA levels and allows for comparison of the expression of related genes at one time and in a tiny piece of sample from a specific neuronal region. (C) 2000 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S1385-299X(00)00012-X

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Hepatocyte growth factor (HGF) is a pleiotrophic factor with mitogenic, motogenic, and morphogenic activities. Recent evidence has suggested that HGF plays an important role in the development and maintenance of. the nervous system. In this study, we examined spatial and temporal expression of HGF and its receptor, c-Met, during retinal development at RNA or protein levels. Competitive RT-PCR revealed that HGF and c-met mRNA expressions were up-regulated during the development and sustained at high levels in adulthood. By immunohistochemical analysis, we demonstrated that c-Met-immunoreactivity (IR) was present in the major classes of retinal neurons after their differentiation. In the adult, c-Met-IR was predominantly present in the photoreceptors. in contrast, HGF-IR was observed from P7 and thereafter in ganglion cells and the inner nuclear layer, but not in other layers. Differential or co-localization of HGF and c-Met indicates the autocrine or paracrine action of HGF depending on the cell types and developmental stages. Moreover, dynamic regulation of HGF and c-Met implicates their multiple roles in the development, maintenance and modification of retinal system. (C) 1999 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0006-8993(99)02097-1

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Sepsis and endotoxemia are involved in the development of fulminant hepatic failure, the prognosis of which is extremely poor and the mortality is high, with no available effective therapy Here, wt report that hepatocyte growth factor (HGF) exerts potent antiapoptotic effects in vivo and effectively prevents endotoxin-induced fulminant hepatic failure in mice. The animals were intraperitoneally injected three times with 120 mu g human recombinant HGF or saline 6 hours and 30 minutes before and 3 hours after an intraperitoneal injection of lipopolysaccharide (LPS) and D-galactosamine (GalN). Administration of LPS + GalN, without HGF, rapidly led to massive hepatocyte apoptosis and severe liver injury and all mice died of hepatic failure within 8 hours. In contrast, administration of human recombinant HGF strongly suppressed extensive progress of hepatocyte apoptosis and the liver injury induced by LPS + GalN, and 75% of the HGF-treated mice survived. Moreover, HGF strongly induced Bcl-xL expression and blocked apoptotic signal transduction upstream of CPP32 (caspase-3) in the liver, thereby leading to inhibition of massive hepatocyte apoptosis. We suggest that HGF may well have the potential to prevent fulminant hepatic failure, at least through its potent antiapoptotic action.

DOI： 10.1002/hep.510300102

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Peripheral nerve injury markedly regulates expression of neurotrophins and their receptors in the lesioned nerve. However, the role of endogenously produced neurotrophins in the process of nerve regeneration is unclear. Expression of a multifunctional neurotrophin, pan-neurotrophin-1 (PNT-1), was targeted to the peripheral nerves of transgenic mice by using a gene promoter that is specifically activated after nerve lesion but that is otherwise silent in all other tissues and during development. PNT-1 is a chimeric neurotrophin that combines the active sites of the neurotrophins nerve growth factor, brain derived neurotrophic factor, and neurotrophin-3 and binds and activates all known neurotrophin receptors, In adult transgenic mice, PNT-1 was highly expressed in transected but not in intact sciatic nerve. Morphometric analyses at the electron microscopy level showed increased and accelerated recovery of axon diameter of myelinated fibers in crushed peripheral nerves of transgenic mice compared with wild type. Examination of nerve bundles in target tissues indicated accelerated reinnervation of foot pad dermis and flexor plantaris muscle in transgenic mice. Moreover, transected sensory and motor axons of transgenic mice showed faster and increased return of neurophysiological responses, suggesting an accelerated rate of axonal elongation. Importantly, transgenic mice also showed a markedly ameliorated loss of skeletal muscle weight, indicating functional regeneration of motor axons. Together, these data provide evidence, at both the anatomical and functional levels, that neurotrophins endogenously produced by the lesioned nerve are capable of significantly accelerating the regeneration of both sensory and motor axons after peripheral nerve damage. In addition, our results indicate that exogenous PNT-1 administration may be an effective therapeutic treatment of peripheral nerve injuries.

DOI： 10.1073/pnas.95.9.5269

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Glial cell line-derived neurotrophic factor (GDNF), the most potent trophic factor yet described for both dopaminergic neurons of the substantia nigra and spinal motorneurons, has recently been shown to signal through a multireceptor complex composed of a novel glycosylphosphatidylinositol-anchored GDNF receptor-alpha (GDNFR-alpha) and the receptor tyrosine kinase product of the c-ret proto-oncogene (RET). Despite its importance, the individual expression patterns and the relationships between domains of expression of the different components of this trophic system are not understood. We show here by in situ hybridization that GDNF mRNA is expressed in the normal adult rat brain in several targets of substantia nigra neurons, including striatum, nucleus accumbens, thalamic nuclei, olfactory tubercle, hippocampus, cerebellum, and cingulate cortex as well as in the internal granular cell layer of the olfactory bulb. Within the basal ganglia we observe a pronounced segregation of regions expressing GDNF from those expressing GDNF receptors, suggesting that within these structures GDNF is functioning in its anticipated role as a target-derived trophic factor. In addition, the expression of GDNF and both GDNF receptors within the cerebellum, hippocampus, and olfactory bulb may indicate a paracrine mode of action. Importantly, we also see expression of RET mRNA in cellular populations within the cerebellum and the glomerular layer of the olfactory bulb, as well as in the subthalamic nucleus, which lack GDNFR-alpha expression, indicating that RET functions either independently of GDNFR-alpha or with GDNFR-alpha presented in trans. Conversely, GDNFR-alpha is widely expressed in many regions in which RET expression is absent, suggesting that GDNFR-alpha may associate with additional signaling receptors. Finally, RET and GDNFR-alpha show distinct patterns of regulated expression in the brain after kainic acid stimulation and in the sciatic nerve after nerve transection. Taken together these findings indicate that GDNF, RET, and GDNFR-alpha utilize multiple mechanisms to comprise physiologically relevant trophic circuits for different neuronal populations.Glial cell line-derived neurotrophic factor (GDNF), the most potent trophic factor yet described for both dopaminergic neurons of the substantia nigra and spinal motorneurons, has recently been shown to signal through a multireceptor complex composed of a novel glycosylphosphatidylinositol-anchored GDNF receptor-alpha (GDNFR-alpha) and the receptor tyrosine kinase product of the c-ret proto-oncogene (RET). Despite its importance, the individual expression patterns and the relationships between domains of expression of the different components of this trophic system are not understood. We show here by in situ hybridization that GDNF mRNA is expressed in the normal adult rat brain in several targets of substantia nigra neurons, including striatum, nucleus accumbens, thalamic nuclei, olfactory tubercle, hippocampus, cerebellum, and cingulate cortex as well as in the internal granular cell layer of the olfactory bulb. Within the basal ganglia we observe a pronounced segregation of regions expressing GDNF from those expressing GDNF receptors, suggesting that within these structures GDNF is functioning in its anticipated role as a target-derived trophic factor. In addition, the expression of GDNF and both GDNF receptors within the cerebellum, hippocampus, and olfactory bulb may indicate a paracrine mode of action. Importantly, we also see expression of RET mRNA in cellular populations within the cerebellum and the glomerular layer of the olfactory bulb, as well as in the subthalamic nucleus, which lack GDNFR-alpha expression, indicating that RET functions either independently of GDNFR-alpha or with GDNFR-alpha presented in trans. Conversely, GDNFR-alpha is widely expressed in many regions in which RET expression is absent, suggesting that GDNFR-alpha may associate with additional signaling receptors. Finally, RET and GDNFR-alpha show distinct patterns of regulated expression in the brain after kainic acid stimulation and in the sciatic nerve after nerve transection. Taken together these findings indicate that GDNF, RET, and GDNFR-alpha utilize multiple mechanisms to comprise physiologically relevant trophic circuits for different neuronal populations.

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Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic polypeptide, distantly related to transforming growth factor-beta (TGF-beta), originally isolated by virtue of its ability to induce dopamine uptake and cell survival in cultures of embryonic ventral midbrain dopaminergic neurons, and more recently shown to be a potent neurotrophic factor for motorneurons. The biological activities and distribution of this molecule outside the central nervous system are presently unknown. We report here on the mRNA expression, biological activities and initial receptor binding characterization of GDNF and a shorter spliced variant termed GDNF beta in different organs and peripheral neurons of the developing rat. Both GDNF mRNA forms were found to be most highly expressed in developing skin, whisker pad, kidney, stomach and testis. Lower expression was also detected in developing skeletal muscle, ovary, lung, and adrenal gland. Developing spinal cord, superior cervical ganglion (SCG) and dorsal root ganglion (DRG) also expressed low levels of GDNF mRNA. Two days after nerve transection, GDNF mRNA levels increased dramatically in the sciatic nerve. Overall, GDNF mRNA expression was significantly higher in peripheral organs than in neuronal tissues. Expression of either GDNF mRNA isoform in insect cells resulted in the production of indistinguishable mature GDNF polypeptides. Purified recombinant GDNF promoted neurite outgrowth and survival of embryonic chick sympathetic neurons. GDNF produced robust bundle-like, fasciculated outgrowth from chick sympathetic ganglion explants. Although GDNF displayed only low activity on survival of newborn rat SCG neurons, this protein was found to increase the expression of vasoactive intestinal peptide and preprotachykinin-A mRNAs in cultured SCG neurons. GDNF also promoted survival of about half of the neurons in embryonic chick nodose ganglion and a small subpopulation of embryonic sensory neurons in chick dorsal root and rat trigeminal ganglia. Embryonic chick sympathetic neurons expressed receptors for GDNF with K-d 1-5 X 10(-9) M, as measured by saturation and displacement binding assays. Our findings indicate GDNF is a new neurotrophic factor for developing peripheral neurons and suggest possible non-neuronal roles for GDNF in the developing reproductive system.

DOI： 10.1083/jcb.130.1.137

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The production of neurotrophin-4 (NT-4) in rat skeletal muscle was found to depend on muscle activity. The amounts of NT-4 messenger RNA present decreased after blockade of neuromuscular transmission with alpha-bungarotoxin and increased during postnatal development and after electrical stimulation in a dose-dependent manner. NT-4 immunoreactivity was detected in slow, type I muscle fibers. Intramuscular administration of NT-4 induced sprouting of intact adult motor nerves. Thus, muscle-derived NT-4 acted as an activity-dependent neurotrophic signal for growth and remodeling of about motor-neuron innervation. NT-4 may thus be partly responsible for the effects of exercise and electrical stimulation on neuromuscular performance.

DOI： 10.1126/science.7770776

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Acetylcholine, one of the main neurotransmitters in the nervous system, is synthesized by the enzyme choline acetyltransferase (ChAT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6), The molecular mechanisms controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo are largely unknown, A previous report showed; that a 3800-bp, but not a 1450-bp, 5' flanking segment from the rat ChAT gene promoter directed cell type-specific expression of a reporter gene in cholinergic cells in vitro. Now we have characterized a distal regulatory region of the ChAT gene that confers cholinergic specificity on a heterologous downstream promoter in a cholinergic cell line and in transgenic mice, A 2342-bp segment from the 5' flanking region of the ChAT gene behaved as an enhancer in cholinergic cells but as a repressor in noncholinergic cells in an orientation-independent manner. Combined with a heterologous basal promoter, this fragment targeted transgene expression to several cholinergic regions of the central nervous system of transgenic mice, including basal forebrain, cortex, pens, and spinal cord, In eight independent transgenic lines, the pattern of transgene expression paralleled qualitatively and quantitatively that displayed by endogenous ChAT mRNA in various regions of the rat central nervous system, In the lumbar enlargement of the spinal cord, 85-90% of the transgene expression was targeted to the ventral part of the cord, where cholinergic alpha-motor neurons are located. Transgene expression in the spinal cord was developmentally regulated and responded to nerve injury in a similar way as the endogenous ChAT gene, indicating that the 2342-bp regulatory sequence contains elements controlling the plasticity of the cholinergic phenotype in developing and injured neurons.

DOI： 10.1073/pnas.92.9.4046

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Pan-neurotrophin 1 (PNT-1) is a synthetic trophic factor engineered by combining active domains of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) into an NT-3 backbone. This molecule was produced in transiently transfected COS cells or in baculovirus-infected insect cells and subsequently purified to homogeneity. Saturation binding in embryonic spinal sensory neurons demonstrated a greater number of high-affinity binding sites for PNT-1 than for its parental molecule NT-3. PNT-1 was shown to efficiently block the chemical crosslinking of NGF, BDNF, and NT-3 to their cognate Trk receptors and to the low-affinity NGF receptor expressed on neuronal and nonneuronal cells. PNT-1 stimulated survival and proliferation of MG87 fibroblasts expressing either TrkA, TrkB, or TrkC. PNT-1 also promoted survival of a greater number of embryonic dorsal root ganglion neurons than any of the other neurotrophins alone, and its effects were equivalent to a combination of NGF, BDNF, and NT-3. Analysis of receptor-specific neurotrophic activities demonstrated that PNT-1 efficiently rescued TrkA mRNA-containing sympathetic neurons and TrkB and TrkC mRNA-containing sensory neurons from the dorsal root and nodose ganglia. Finally, PNT-1 showed robust retrograde transport to DRG neurons in vivo after injection into the sciatic nerve. Radiolabeled PNT-1 accumulated in small-, medium-, and large-sized neurons. Coinjection with different unlabeled neurotrophins inhibited PNT-1 transport in distinct subpopulations of neurons of different sizes, suggesting that this molecule affects sensory neurons of different modalities. These results indicate that PNT-1 is a potent and multispecific neurotrophic factor that may be useful in the treatment of peripheral neuropathies and nerve damage.

DOI： 10.1073/pnas.92.2.607

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The structure of rat brain-derived neurotrophic factor (BDNF) gene is complex; four 5' exons are linked to separate promoters and one 3' exon is encoding the BDNF protein. To analyze the relative importance of the regulatory regions in vivo, we have generated transgenic mice with six different promoter constructs of the BDNF gene fused to the chloramphenicol acetyl transferase reporter gene. High level and neuronal expression of the reporter gene, that in many respects recapitulated BDNF gene expression, was achieved by using 9 kb of genomic sequences covering the promoter regions that lie adjacent to each other in the genome (promoters I and II and promoters III and IV, respectively) and by including sequences of BDNF intron-exon splice junctions and 3' untranslated region in the constructs. The genomic regions responsible for the in vivo upregulation of BDNF expression in the axotomized sciatic nerve and in the brain after kainic acid-induced seizures and KCl-induced spreading depression were mapped. These data show that regulation of the different aspects of BDNF expression is controlled by different regions in vivo, and they suggest that these promoter constructs may be useful for targeted expression of heterologous genes to specific regions of the central and peripheral nervous systems in an inducible manner.

DOI： 10.1083/jcb.128.1.185

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In this study we have shown, by in situ hybridization and RNase protection assay, a significant trkC mRNA increase confined to the dentate gyrus of hippocampus, both after seizures induced by intracerebroventricular injection of kainic acid and bicuculline. Moreover, after bicuculline treatment we observed an earlier increase of trkC mRNA level, which peaked after 3 h and returned back to normal levels by 12 h. In contrast, the kainic acid treatment produced a delayed increase of trkC mRNA, which initiated after 6 h, peaked at 12 h, and returned to normal levels at 24 h. This increase, which involves also trkC mRNA receptor with tyrosine kinase activity, was mediated by non-NMDA receptors and counteracted by GABA potentiating agent diazepam. Using embryonic neuronal cultures from cerebral hemispheres, including hippocampus, we found that glutamate receptor agonists, including glutamate, kainate, NMDA, and t-ACPD, increase trkC mRNA levels with the following rank order of efficacy: NMDA > t-ACPD > kainic acid > glutamate. In conclusion, our data show that trkC mRNA expression in granule cells of the hippocampus dentate gyrus is increased during seizure activity and that it is mediated by non-NMDA receptors.

DOI： 10.1007/BF02736755

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

Tyrosine protein kinases trkA, trkB and trkC are signal transduction receptors for a family of neurotrophic factors known as the neurotrophins. Here we report on changes in the expression of messenger RNAs for trkA, trkB and trkC in the brain following an injury caused by insertion of a 30-gauge needle into adult rat hippocampus or neocortex. Quantitative in situ hybridization revealed no change in the level of trkA messenger RNAs in any brain region following this insult. In contrast, increased levels of trkB messenger RNA compared to untreated animals were seen in the granule cell layer of the dentate gyrus ipsilateral to the injury already 30 min after the injury. The increase reached maximal levels (four-fold) between 2 and 4 h, but returned to control levels 8 h after the injury. No change was seen in the contralateral dentate gyrus. The levels of trkC messenger RNA increased in the same brain regions as trkB messenger RNA, though with a delayed response, reaching a maximal increase of 3.3-fold 4 h after the injury. As for trkB messenger RNA, the level of trkC messenger RNA then tapered off and reached control levels 8 h after the injury. However, 4h after the injury, a 1.7-fold increase of trkB and trkC messenger RNAs were seen in the ipsilateral piriform cortex. The increases of trkB and trkC messenger RNAs were confirmed using a nuclease protection assay. Increases of both trkB and trkC messenger RNAs were also seen in the piriform cortex, but not in the hippocampus, following needle insertion into the neocortex. Pretreatment of the animals with the non-competitive N-methyl-D-aspartate antagonist ketamine completely prevented the increases of trkB arid trkC messenger RNAs, suggesting that the brain injury caused a release of glutamate with subsequent activation of iv-methyl-D-aspartate receptors. In contrast, the anticonvulsive drug diazepam, the muscarinic antagonist atropine and the calcium-channel antagonist nimodipine had no effect on the increases of trkB and trkC messenger RNAs.

DOI： 10.1016/0306-4522(93)90036-F

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

The neurotrophin family includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Previous studies have demonstrated that expression of NGF and its low-affinity receptor is induced in non-neuronal cells of the distal segment of the transected sciatic nerve suggesting a role for NGF during axonal regeneration (Johnson, E. M., M. Taniuchi, and P. S. DeStefano. 1988. Trends Neurosci. 11:299-304). To assess the role of the other neurotrophins and the members of the family of Trk signaling neurotrophin receptors, we have here quantified the levels of mRNAs for BDNF, NT-3, and NT-4 as well as mRNAs for trkA, trkB, and trkC at different times after transection of the sciatic nerve in adult rats. A marked increase of BDNF and NT-4 mRNAs in the distal segment of the sciatic nerve was seen 2 wk after the lesion. The increase in BDNF mRNA was mediated by a selective activation of the BDNF exon IV promoter and adrenalectomy attenuated this increase by 50%. NT-3 mRNA, on the other hand, decreased shortly after the transection but returned to control levels 2 wk later. In Schwann cells ensheathing the sciatic nerve, only trkB mRNA encoding truncated TrkB receptors was detected with reduced levels in the distal part of the lesioned nerve. Similar results were seen using a probe that detects all forms of trkC mRNA. In the denervated gastrocnemius muscle, the level of BDNF mRNA increased, NT-3 mRNA did not change, while NT-4 mRNA decreased. In the spinal cord, only small changes were seen in the levels of neurotrophin and trk mRNAs. These results show that expression of mRNAs for neurotrophins and their Trk receptors is differentially regulated after a peripheral nerve injury. Based on these results a model is presented for how the different neurotrophins could cooperate to promote regeneration of injured peripheral nerves.

DOI： 10.1083/jcb.123.2.455

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：Bentham Science Publishers  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：Bentham Science Publishers  

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Authorship：Lead author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：Libertas Academica  

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Authorship：Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：Libertas Academica  

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Authorship：Corresponding author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：日本臨床社  

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Publisher：日本ビタミン学会  

DOI： 10.20632/vso.84.2_89

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：三輪書店  

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Language：Japanese   Publisher：日本アミノ酸学会  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：メジカルビュー社  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：中外医学社  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：三輪書店  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：医学書院  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：日本プランニングセンター  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：中外医学社  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：日本臨床社  

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Authorship：Lead author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：Elsevier  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：医学書院  

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：Kaeger  

DOI： 10.1159/000076815

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：星和書店  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：現代医療社  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：医学書院  

DOI： 10.11477/mf.1431901158

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：星和書店  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：日本臨床  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：医歯薬出版  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：医学書院  

DOI： 10.11477/mf.1431901008

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：中外医学社  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：羊土社  

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Application no：JP2000280081A  Date applied：2000.9

Announcement no：PCT/JP2001/001539  Date announced：2001.2

Patent/Registration no：CA2422774C  Date registered：2013.7 

Country of applicant：International (PCT) application  

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Applicant：中村 敏一

Application no：特願2000-280081 (P2000-280081)  Date applied：2000.9

Announcement no：特開2002-087983 (P2002-087983A)  Date announced：2002.3

Patent/Registration no：特許2000-280081  Date registered：2013.12 

Country of applicant：Domestic  

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Award type：International academic award (Japan or overseas)  Country：Japan

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Award type：International academic award (Japan or overseas)  Country：Sweden

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Award type：International academic award (Japan or overseas)  Country：France

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Country：Japan

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第17回教育研究推進センター講演会
「⾼齢者の健康寿命延伸をめざす基礎免疫⽼化研究」〜B細胞における加齢変化と免疫老化〜
丸山 光生先生（国立研究開発法人国立長寿医療研究センター研究所 副所長 老化機構研究部 部長）
主催：教育研究推進センター
⽇時：平成29年10⽉20⽇（⾦）17時00分より
会場：旭川医科⼤学教育研究推進センター
2階カンファレンスルーム（共⽤研究棟）

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北海道医学技術専門学校2年生が臨地実習の一環として、10月6日に旭川医科大学教育研究推進センター技術支援部門実験実習機器技術部門を見学（解説と簡単な実習）
日時：10月7日（金）13:00~16:00

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第16回教育研究推進センター講演会
「生殖細胞におけるDNAメチル化ダイナミクスの理解に向けて」～マウス受精卵へのエピゲノム編集技術の応用～
山﨑 大賀 先生（北里大学メディカルセンター研究部門上級研究員）
日時：平成29年9月27日（水） 17時00分より
会場：旭川医科大学教育研究推進センター
2階カンファレンスルーム（共用研究棟）

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東川・東神楽・美瑛地域の小中学校教員（26名）の方々に教育研究推進センター実験実習機器技術支援部門の機器を見学いただきました。
日時：2017年8月23日（水）14:30~16:00

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日時：平成２９年８月２日（水）〜８月４日（金）

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札幌西高等学校「高大病連携によるふるさと医療人育成の取組」の一貫として、旭川医科大学教育推進センター技術支援部実験実習機器技術支援部門を見学・体験実習しました。

日時：7月26日（水）12:55~15:15

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北見工業大学大学院医療工学専攻「医学総論Ⅰ」、「医学総論2」
旭川医科大学 教育研究推進、脳機能意向学研究センター＆外科学講座
日時：2017年4月7日〜8日

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「旭川医科大学の取り組んできた橋渡し研究の１０年」
船越 洋（旭川医科大学 教育研究推進センターセンター長）

日時：２０１７年２月１日（水）15:00~17:40
会場：ホテル札幌ガーデンパレス2階

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旭川西高等学校がスーパーサイエンスハイスクール（SSH：文部科学省による科学技術や理科・数学教育を重点的に行う高校を指定する制度）に採択されたプロジェクトの一貫として旭川医科大学看護学科での研修と旭川医科大学教育研究推進センター技術支援部門実験実習機器技術支援部門で研修しました。

日時：１月１１日（水）

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日時：平成２８年１０月１３日（木）16:00~18:00
場所：旭川医科大学 教育研究推進センター

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《サイエンス・リーダーズ・キャンプの概略》
受け入れ実施機関名「旭川医科大学」
プログラム名「先端生命科学研究の基盤技術を教育活動に活用できる教員育成講座」
実施責任者：旭川医科大学 学長 吉田晃敏 先生
全体統括責任者：旭川医科大学教育研究推進センター長 船越 洋
開催時期：平成２８年８月１日～平成２８年８月４日

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札幌西高等学校がスーパーサイエンスハイスクール（SSH：文部科学省による科学技術や理科・数学教育を重点的に行う高校を指定する制度）に採択されたプロジェクトの一貫として、旭川医科大学教育研究推進センター技術支援部実験実習機器技術支援部門を見学・実習を実施しました。

日時：7月13日（水）12：55～15：15

(1) 教育研究推進センター センター長挨拶
(2) 講演
(3) 教育研究推進センター技術支援部実験実習機器技術支援部訪問研修
(a) 遺伝子解析装置
(b) 共焦点顕微鏡装置
(c) 質量分析計

場所：教育研究推進センター技術支援部実験実習機器技術支援部門
1階 質量分析計
2階 共焦点レーザー顕微鏡室
2階 遺伝子解析装置室
3階 カンファレンスルーム

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《サイエンス・リーダーズ・キャンプの概略》
受け入れ実施機関名「旭川医科大学」
プログラム名「先端生命科学研究の基盤技術を教育活動に活用できる教員育成講座」
実施責任者：旭川医科大学 学長 吉田晃敏 先生
全体統括責任者：旭川医科大学教育研究推進センター長 船越 洋
開催時期：平成２７年８月３日～平成２７年８月６日

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北見工業大学大学院医療工学専攻「医学総論Ⅰ」、「医学総論2」
旭川医科大学 教育研究推進センター＆脳機能医工学研究センター＆脳神経外科
日時：平成27年4月7日〜4月8日

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期間：平成２７年２月１３日〜１９日
組織委員として大会をサポートしました。

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日時：平成２７年２月１３日（金）18:00~20:00
場所：旭川市民文化会館大ホール

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《サイエンス・リーダーズ・キャンプの概略》
受け入れ実施機関名「旭川医科大学」
プログラム名「先端生命科学研究の基盤技術を教育活動に活用できる教員育成講座」
実施責任者：旭川医科大学 学長 吉田晃敏 先生
全体統括責任者：旭川医科大学教育研究推進センター長 船越 洋
開催時期：平成２６年８月４日～平成２６年８月７日

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アミノ酸とこころ 平成25年12月18日(水)13:30～15:30 旭川市旭川シニア大学

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詳細は（http://cent-scorpio.asahikawa-med.ac.jp/central/council/discussion/kyougikai.pdf）
参照してください。

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日時：10月4日（金）13：00～16：00

講義：13：05～13：45
船越 教授（教育研究推進センター センター長）

Aグループ 、Bグループ、Cグループ、Dグループで実施

見学：13：50～15：45
1階 電子顕微鏡 （担当：鈴木） 13：50～14：10 15：25～15：45 15：05～15：25 14：45～15：05
1階 質量分析計 （担当：阿久津） 14：10～14：30 13：50～14：10 15：25～15：45 15：05～15：25
3階 細胞自動解析分離装置 （担当：篠河） 14：45～15：05 14：10～14：30 13：50～14：10 15：25～15：45
3階 共焦点レーザー走査型顕微鏡 （担当：日下部） 15：05～15：25 14：45～15：05 14：10～14：30 13：50～14：10
3階 遺伝子解析装置 （担当：千葉） 15：25～15：45 15：05～15：25 14：45～15：05 14：10～14：30

場所：教育研究推進センター技術支援部実験実習機器技術支援部門
(1) 電子顕微鏡
(2) 質量分析計
(3) 細胞自動解析分離装置
(4) 共焦点レーザー走査型顕微鏡
(5) 遺伝子解析装置
(6) カンファレンスルーム

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日時：9月11日（水）13：30～16：45

13：35～14：05
講義（担当：教育研究推進センター センター長 船越 洋 教授）
14：10～16：35
Aグループ
3階 遺伝子解析装置 （担当：千葉） 14：10～14：30
3階 細胞自動解析分離装置 （担当：篠河） 14：30～14：50
3階 休憩（カンファレンスルーム） 14：50～15：05
1階 透過型電子顕微鏡 （担当：平） 15：05～15：50
1階 走査型電子顕微鏡 （担当：鈴木） 15：50～16：35
Bグループ
3階 細胞自動解析分離装置 （担当：篠河） 14：10～14：30
3階 遺伝子解析装置 （担当：千葉） 14：30～14：50
3階 休憩（カンファレンスルーム） 14：50～15：05
1階 走査型電子顕微鏡 （担当：鈴木） 15：05～15：50
1階 透過型電子顕微鏡 （担当：平） 15：50～16：35

場所：教育研究推進センター技術支援部実験実習機器技術支援部門
(1) 細胞自動解析分離装置
(2) 遺伝子解析装置
(3) 電子顕微鏡
(4) カンファレンスルーム

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《サイエンス・リーダーズ・キャンプの概略》
受け入れ実施機関名「旭川医科大学」
プログラム名「未来の生命科学のフロンティアにたつ教員育成」～先端実習を通じて～

受講対象者「基礎科学・医学進学者をめざす学生への教育者」
実施責任者「旭川医科大学 学長 吉田晃敏 先生」
提案申請者「旭川医科大学教育研究推進センター長 船越 洋」
会場：主会場 旭川医科大学教育研究推進センター
副会場 旭川医科大学遠隔医療センター・藤田観光ワシントンホテル旭川
全体統括責任者 「旭川医科大学教育研究推進センター長 船越 洋」

実施主担当者「旭川医科大学 顕微解剖学 平 義樹 先生」
実施副担当者「旭川医科大学 教育センター 教授 蒔田 芳男 先生」
開催時期：平成２５年８月５日～平成２５年８月８日」

(http://www.asahikawa-med.ac.jp/arec/SLC/)
(http://www.asahikawa-med.ac.jp/arec/local/info/2013/slc2013.html)

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札幌西高等学校がスーパーサイエンスハイスクール（SSH：文部科学省による科学技術や理科・数学教育を重点的に行う高校を指定する制度）に採択されたプロジェクトの一貫として『教育センター』と『教育研究推進センター』で受け入れて、旭川医科大学教育研究推進センター技術支援部実験実習機器技術支援部門を見学・実習を実施しました。

日時：8月7日（水）16：00～17：00

(1) 教育研究推進センター センター長挨拶
(2) 教育研究推進センター技術支援部実験実習機器技術支援部訪問研修
(a) 電子顕微鏡 （担当：鈴木）
(b) 質量分析計 （担当：阿久津）
(c) 細胞自動解析分離装置 （担当：篠河）
(d) 共焦点レーザー走査型顕微鏡 （担当：日下部）
(e) 遺伝子解析装置 （担当：千葉）

場所：教育研究推進センター技術支援部実験実習機器技術支援部門
1階 電子顕微鏡
1階 質量分析計
3階 細胞自動解析分離装置
3階 共焦点レーザー走査型顕微鏡
3階 遺伝子解析装置
3階 カンファレンスルーム

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8月10日（土）に独立行政法人科学技術振興機構の旭川西高等学校スーパーサイエンスハイスクール（SSH）事業 「Dohokuサイエンスフェスティバル」が旭川公会堂で開催した。

ーこの事業に協力するためー
旭川医科大学教育研究推進センターから「放射線を目で見てみよう！」「ジュースからDNAをとってみよう！」のテーマで参加した。

「Dohokuサイエンスフェスティバル」
日時 8月10日 10時00分～16時00分
会場 旭川市公会堂
ブース名 「放射線を目で見てみよう！」 演示者 阿久津 弘明 他
ブース名 「ジュースからDNAをとってみよう！」演示者 千葉 伸一 他

参加者 阿久津 弘明、千葉 伸一、篠河 栄利子、船越 洋

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２０１３年４月１０日
「北見工業大学大学院大学院医療工学専攻大学院講義および見学会」
ー教育研究推進センター＆センター技術支援部実験実習機器技術支援部門ー

１）開会の挨拶（３階会議室）１３：３０〜１３：０５
教育研究推進センター・センター長 船越 洋 教授

2）講演『再生医学と再生医療』１３：３５～１４：１０
教育研究推進センター・センター長 船越 洋 教授

3）実験実習機器技術支援部門見学順（各装置：１５分）
１４：１０～１４：３０ １階 電子顕微鏡 担当：鈴 木
１４：３０～１４：５０ １階 質量分析計 担当：阿久津
１４：５０～１５：００
休憩
１５：００～１５：２０ ３階 細胞自動解析分離装置 担当：篠 河
１５：２０～１５：４０ ３階 共焦点レーザ走査型顕微鏡 担当：日下部
１５：４０～１６：００ ３階 遺伝子解析装置 担当：千 葉

4）特別講演 １６：００〜１６：２０
脳機能医工学研究センター・センター長 高草木 薫 教授

5）閉会 (３階会議室) １６：２０～１６：２５
教育研究推進センター・センター長 船越 洋 教授

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旭川西高等学校がスーパーサイエンスハイスクール（SSH：文部科学省による科学技術や理科・数学教育を重点的に行う高校を指定する制度）に採択されたプロジェクトの一貫として臨床シミュレーションセンター と旭川医科大学教育研究推進センター技術支援部実験実習機器技術支援部門において講演会の実施と見学・実習を実施した。

日時：1月15日（火）10：00～14：00
（Aグループ、Bグループ、CグループとDグループの４グループで実施）

1階 電子顕微鏡 10：20～10：50
1階 質量分析計 10：50～11：20
3階 細胞自動解析分離装置 11：20～11：50
3階 共焦点レーザー走査型顕微鏡 13：00～13：30
3階 遺伝子解析装置 13：30～14：00

場所：教育研究推進センター技術支援部実験実習機器技術支援部門
(1) 電子顕微鏡
(2) 質量分析計
(3) 細胞自動解析分離装置
(4) 共焦点レーザー走査型顕微鏡
(5) 遺伝子解析装置
(6) カンファレンスルーム

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「アミノ酸とこころ」（旭川市）H25.1.25

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北海道医学技術専門学校２年生が臨地実習の一環として、9月28日に旭川医科大学教育研究推進センター技術支援部実験実習機器技術支援部門を見学（講演会を実施するとともに実際の先端機器を見て使用してみる実習を実施）

日時：9月28日（金）13：00～13：20 講演会
日時：9月28日（金）13：20～15：00 見学・実習
（Aグループ 、Bグループ、Cグループ、Dグループに分かれて実施）
1階 電子顕微鏡 13：20～13：35
1階 質量分析計 13：40～13：55
3階 細胞自動解析分離装置 14：00～14：15
3階 共焦点レーザー走査型顕微鏡 14：20～14：35
3階 遺伝子解析装置 14：40～14：55

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日時：9月11日（火）12：00～13：00
講義
日時：9月11日（火）13：00～15：00
Aグループ
3階 細胞自動解析分離装置 13：10～13：40
3階 共焦点レーザー走査型顕微鏡 13：40～14：10
3階 休憩（カンファレンスルーム） 14：10～14：25
3階 遺伝子解析装置 14：25～14：55
Bグループ
3階 遺伝子解析装置 13：10～13：40
3階 細胞自動解析分離装置 13：40～14：10
3階 休憩（カンファレンスルーム） 14：10～14：25
3階 共焦点レーザー走査型顕微鏡 14：25～14：55
日時：9月13日（木）11：00～14：00
Aグループ
1階 透過型電子顕微鏡 11：00～14：00
Bグループ
1階 走査型電子顕微鏡 11：00～14：00

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札幌日本大学学園 札幌日本大学高等学校がスーパーサイエンスハイスクール（SSH：文部科学省による科学技術や理科・数学教育を重点的に行う高校を指定する制度）に採択されたプロジェクトの一貫として8月4日に旭川医科大学教育研究推進センター技術支援部実験実習機器技術支援部門を見学（解説と簡単な実習）を実施した。

日時：8月4日（土）13：00～16：10
Aグループ
1階 電子顕微鏡 13：30～13：50
1階 質量分析計 13：55～14：15
3階 細胞自動解析分離装置 14：20～14：40
3階 休憩（カンファレンスルーム） 14：40～15：00
3階 共焦点レーザー走査型顕微鏡 15：05～15：25
3階 遺伝子解析装置 15：30～15：55
Bグループ
3階 細胞自動解析分離装置 13：30～13：50
3階 共焦点レーザー走査型顕微鏡 13：55～14：15
3階 遺伝子解析装置 14：20～14：40
3階 休憩（カンファレンスルーム） 14：40～15：00
1階 電子顕微鏡 15：05～15：25
1階 質量分析計 15：30～15：55

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旭川医科大学教育研究推進センターにおいて、スーパーサイエンスハイスクール（SSH：文部科学省による科学技術や理科・数学教育を重点的に行う高校を指定する制度）に採択された札幌日本大学学園 札幌日本大学高等学校に対して旭川医科大学教育研究推進センター主催で講演会とセンター技術支援部実験実習機器技術支援部門の先端機器の解説と実習を施行した。

対応職員は、船越センター長、日下部技術専門職員、阿久津技術専門職員、千葉技術専門職員、篠河技術補助員、宮川技術専門職員。

(1) 講演会 教育研究推進センター長・教授 船越 洋 12:50〜13:25
(2) 先端機器の解説と実習 13:30〜
Aグループ
1階 電子顕微鏡 （担当：宮川） 13：30～13：50
1階 質量分析計 （担当：阿久津） 13：55～14：15
3階 細胞自動解析分離装置 （担当：篠河） 14：20～14：40
3階 休憩（カンファレンスルーム） 14：40～15：00
3階 共焦点レーザー走査型顕微鏡 （担当：日下部） 15：05～15：25
3階 遺伝子解析装置 （担当：千葉） 15：30～15：55
Bグループ
3階 細胞自動解析分離装置 （担当：篠河） 13：30～13：50
3階 共焦点レーザー走査型顕微鏡 （担当：日下部） 13：55～14：15
3階 遺伝子解析装置 （担当：千葉） 14：20～14：40
3階 休憩（カンファレンスルーム） 14：40～15：00
1階 電子顕微鏡 （担当：宮川） 15：05～15：25
1階 質量分析計 （担当：阿久津） 15：30～15：55
(3) 質問コーナーと閉会式

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旭川医科大学教育研究推進センターおよび旭川医科大学生命科学教室に於いて、スーパーサイエンスハイスクール（SSH：文部科学省による科学技術や理科・数学教育を重点的に行う高校を指定する制度）に採択された旭川西高校を旭川医科大学生命科学（林要喜知教授） と旭川医科大学教育研究推進センター共同でセンター技術支援部実験実習機器技術支援部門を中心に先端機器の解説と実習を施行した。

日時：１１月３日 P.M.１：００～２：００（第１グループ）
１１月３日 P.M. ２：００～３：００（第２グループ）
１１月６日 P.M. １：００～４：００（質量分析計）

場所：教育研究推進センター技術支援部実験実習機器技術支援部門
(1) 電子顕微鏡
(2) DNAシークエンサー
(3) 超純水製造装置
(4) セルソーター
(5) 共焦点レーザー顕微鏡
(6) 質量分析計

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