Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Fragile X mental retardation protein (FMRP), a causative gene (FMR1) product of Fragile X syndrome (FXS), is an RNA-binding protein to regulate local protein synthesis in dendrites for postsynaptic functions. However, involvement of FMRP in local protein synthesis in axons for presynaptic functions remains unclear. Here we investigated role of FMRP in local translation of the active zone protein Munc18-1 during presynapse formation. We found that leucine-rich repeat transmembrane neuronal 2 (LRRTM2)-conjugated beads, which promotes synchronized presynapse formation, induced simultaneous accumulation of FMRP and Munc18-1 in presynapses of axons of mouse cortical neurons in neuronal cell aggregate culture. The LRRTM2-induced accumulation of Munc18-1 in presynapses was observed in axons protein-synthesis-dependently, even physically separated from cell bodies. The accumulation of Munc18-1 was enhanced in Fmr1-knockout (KO) axons as compared to wild type (WT), suggesting FMRP-regulated suppression for local translation of Munc18-1 in axons during presynapse formation. Using naturally formed synapses of dissociated culture, structured illumination microscope revealed that accumulation of Munc18-1 puncta in Fmr1-KO neurons increased significantly at 19 days in vitro, as compared to WT. Our findings lead the possibility that excessive accumulation of Munc18-1 in presynapses at early stage of synaptic development in Fmr1-KO neurons may have a critical role in impaired presynaptic functions in FXS.

DOI： 10.1016/j.neures.2018.09.013

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DOI： 10.3791/59893

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Numerous cell adhesion molecules, extracellular matrix proteins and axon guidance molecules participate in neuronal network formation through local effects at axo-dendritic, axo-axonic or dendro-dendritic contact sites. In contrast, neurotrophins and their receptors play crucial roles in neural wiring by sending retrograde signals to remote cell bodies. Semaphorin 3A (Sema3A), a prototype of secreted type 3 semaphorins, is implicated in axon repulsion, dendritic branching and synapse formation via binding protein neuropilin-1 (NRP1) and the signal transducing protein PlexinAs (PlexAs) complex. This review focuses on Sema3A retrograde signaling that regulates dendritic localization of AMPA-type glutamate receptor GluA2 and dendritic patterning. This signaling is elicited by activation of NRP1 in growth cones and is propagated to cell bodies by dynein-dependent retrograde axonal transport of PlexAs. It also requires interaction between PlexAs and a high-affinity receptor for nerve growth factor, toropomyosin receptor kinase A. We propose a control mechanism by which retrograde Sema3A signaling regulates the glutamate receptor localization through trafficking of cis-interacting PlexAs with GluA2 along dendrites; this remote signaling may be an alternative mechanism to local adhesive contacts for neural network formation.

DOI： 10.1080/19336918.2016.1210758

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

There is increasing evidence that localized mRNAs in axons and growth cones play an important role in axon extension and pathfinding via local translation. A few studies have revealed the presence of microRNAs (miRNAs) in axons, which may control local protein synthesis during axon development. However, so far, there has been no attempt to screen for axon-enriched miRNAs and to validate their possible localization to growth cones of developing axons from neurons of the central nervous system. In this study, the localization of miRNAs in axons and growth cones in cortical neurons was examined using a neuron ball culture method that is suitable to prepare axonal miRNAs with high yield and purity. Axonal miRNAs prepared from the neuron ball cultures of mouse cortical neurons were analyzed by quantitative real-time RT-PCR. Among 375 miRNAs that were analyzed, 105 miRNAs were detected in axons, and six miRNAs were significantly enriched in axonal fractions when compared with cell body fractions. Fluorescence in situ hybridization revealed that two axon-enriched miRNAs, miR-181a-1* and miR-532, localized as distinct granules in distal axons and growth cones. The association of these miRNAs with the RNA-induced silencing complex further supported their function to regulate mRNA levels or translation in the brain. These results suggest a mechanism to localize specific miRNAs to distal axons and growth cones, where they could be involved in local mRNA regulation. These findings provide new insight into the presence of axonal miRNAs and motivate further analysis of their function in local protein synthesis underlying axon guidance. (c) 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 397-406, 2014

DOI： 10.1002/dneu.22113

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The dendritic targeting of neurotransmitter receptors is vital for dendritic development and function. However, how such localization is established remains unclear. Here we show that semaphorin 3A (Sema3A) signalling at the axonal growth cone is propagated towards the cell body by retrograde axonal transport and drives AMPA receptor GluA2 to the distal dendrites, which regulates dendritic development. Sema3A enhances glutamate receptor interacting protein 1-dependent localization of GluA2 in dendrites, which is blocked by knockdown of cytoplasmic dynein heavy chain. PlexinA (PlexA), a receptor component for Sema3A, interacts with GluA2 at the immunoglobulin-like Plexin-transcription-factor domain (PlexA-IPT) in somatodendritic regions. Overexpression of PlexA-IPT suppresses dendritic localization of GluA2 and induces aproximal bifurcation phenotype in the apical dendrites of CA1 hippocampal neurons. Thus, we propose a control mechanism by which retrograde Sema3A signalling regulates the glutamate receptor localization through trafficking of cis-interacting PlexA with GluA2 along dendrites.

DOI： 10.1038/ncomms4424

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Nerve growth cones contain mRNA and its translational machinery and thereby synthesize protein locally. The regulatory mechanisms in the growth cone, however, remain largely unknown. We previously found that the calcium entry-induced increase of phosphorylation of eukaryotic elongation factor-2 (eEF2), a key component of mRNA translation, within growth cones showed growth arrest of neurites. Because dephosphorylated eEF2 and phosphorylated eEF2 are known to promote and inhibit mRNA translation, respectively, the data led to the hypothesis that eEF2-mediating mRNA translation may regulate neurite outgrowth. Here, we validated the hypothesis by using a chromophore-assisted light inactivation (CALI) technique to examine the roles of localized eEF2 and eEF2 kinase (EF2K), a specific calcium calmodulin-dependent enzyme for eEF2 phosphorylation, in advancing growth cones of cultured chick dorsal root ganglion (DRG) neurons. The phosphorylated eEF2 was weakly distributed in advancing growth cones, whereas eEF2 phosphorylation was increased by extracellular adenosine triphosphate (ATP)-evoked calcium transient through P2 purinoceptors in growth cones and resulted in growth arrest of neurites. The increase of eEF2 phosphorylation within growth cones by inhibition of protein phosphatase 2A known to dephosphorylate eEF2 also showed growth arrest of neurites. CALI of eEF2 within growth cones resulted in retardation of neurite outgrowth, whereas CALI of EF2K enhanced neurite outgrowth temporally. Moreover, CALI of EF2K abolished the ATP-induced retardation of neurite outgrowth. These findings suggest that an eEF2 phosphorylation state localized to the growth cone regulates neurite outgrowth. (c) 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013

DOI： 10.1002/dneu.22058

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Introduction: The semaphorins were initially described as axon guidance molecules that play important roles in the development of nervous system. Recent studies suggest that semaphorins and their receptors also exert such diverse functions as immune response, control of vascular endothelial cell motility and invasion of many types of cancer cells.
Areas covered: The available results concerning application of class 3 semaphorins and their inhibitors for the treatment in animal disease models.
Expert opinion: Because semaphorins are now recognized as key players in immune, cardiovascular, bone metabolism and neurological system, semaphorins and their receptors are most promising therapeutic targets for various disease states. As semaphorins exert their diverse or even opposing activities in vivo, more elaborate studies on pathophysiology and signal transduction mechanisms of semaphorins are required.

DOI： 10.1517/14728222.2012.710201

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

Semaphorin3A (Sema3A) exerts a wide variety of biological functions by regulating reorganization of actin and tubulin cytoskeletal proteins through signaling pathways including sequential phosphorylation of collapsin response mediator protein 1 (CRMP1) and CRMP2 by cyclin-dependent kinase-5 and glycogen synthase kinase-3 beta (GSK3 beta). To delineate how GSK3 beta mediates Sema3A signaling, we here determined the substrates of GSK3 beta involved. Introduction of either GSK3 beta mutants, GSK3 beta-R96A, L128A, or K85M into chick dorsal root ganglion (DRG) neurons suppressed Sema3A-induced growth cone collapse, thereby suggesting that unprimed as well as primed substrates are involved in Sema3A signaling. Axin-1, a key player in Wnt signaling, is an unprimed substrate of GSK3 beta. The phosphorylation of Axin-1 by GSK3 beta accelerates the association of Axin-1 with beta-catenin. Immunocytochemical studies revealed that Sema3A induced an increase in the intensity levels of beta-catenin in the DRG growth cones. Axin-1 siRNA knockdown suppressed Sema3A-induced growth cone collapse. The reintroduction of RNAi-resistant Axin-1 (rAxin-1)-wt rescued the responsiveness to Sema3A, while that of nonphosphorylated mutants, rAxin S322A/S326A/S330A and T485A/S490A/S497A, did not. Sema3A also enhanced the colocalization of GSK3 beta, Axin-1, and beta-catenin in the growth cones. The increase of beta-catenin in the growth cones was suppressed by the siRNA knockdown of Axin-1. Furthermore, either Axin-1 or beta-catenin RNAi knockdown suppressed the internalization of Sema3A. These results suggest that Sema3A induces the formation of GSK3 beta/Axin-1/beta-catenin complex, which regulates signaling cascade of Sema3A via an endocytotic mechanism. This finding should provide clue for understanding of mechanisms of a wide variety of biological functions of Sema3A.

DOI： 10.1523/JNEUROSCI.6139-11.2012

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: The small non-coding microRNAs play an important role in development by regulating protein translation, but their involvement in axon guidance is unknown. Here, we investigated the role of microRNA-134 (miR-134) in chemotropic guidance of nerve growth cones.
Results: We found that miR-134 is highly expressed in the neural tube of Xenopus embryos. Fluorescent in situ hybridization also showed that miR-134 is enriched in the growth cones of Xenopus spinal neurons in culture. Importantly, overexpression of miR-134 mimics or antisense inhibitors blocked protein synthesis (PS)-dependent attractive responses of Xenopus growth cones to a gradient of brain-derived neurotrophic factor (BDNF). However, miR-134 mimics or inhibitors had no effect on PS-independent bidirectional responses of Xenopus growth cones to bone morphogenic protein 7 (BMP7). Our data further showed that Xenopus LIM kinase 1 (Xlimk1) mRNA is a potential target of miR-134 regulation.
Conclusions: These findings demonstrate a role for miR-134 in translation-dependent guidance of nerve growth cones. Different guidance cues may act through distinct signaling pathways to elicit PS-dependent and -independent mechanisms to steer growth cones in response to a wide array of spatiotemporal cues during development.

DOI： 10.1186/1756-6606-4-40

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Collapsin response mediator protein 5 (CRMP5) is one of the CRMP members that expresses abundantly in the developing brain. To examine the in vivo function of CRMP5, we generated crmp5-deficient (crmp5(-/-)) mice. Anti-calbindin immunofluorescence studies of crmp5(-/-) mice revealed aberrant dendrite morphology; specifically, a decrease in the size of soma and diameter of primary dendrite of the cerebellar Purkinje cells at postnatal day 21 (P21) and P28, but not at P14. Coincidentally, CRMP5 is detected in Purkinje cells at P21 and P28 from crmp5(+/-) mice. In cerebellar slices of crmp5(-/-) mice, the induction of long-term depression of excitatory synaptic transmission between parallel fibers and Purkinje cells was deficient. Given that brain-derived neurotrophic factor (BDNF) plays major roles in dendritic development, we tried to elucidate the possible roles of CRMP5 in BDNF signaling. The effect of BDNF to induce dendritic branching was markedly attenuated in cultured crmp5(-/-) neurons. Furthermore, CRMP5 was tyrosine phosphorylated when coexpressed with neurotrophic tyrosine kinase receptor type 2 (TrkB), a receptor for BDNF, in HEK293T cells. These findings suggest that CRMP5 is involved in the development, maintenance and synaptic plasticity of Purkinje cells.

DOI： 10.1523/JNEUROSCI.5337-10.2011

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The localization of specific mRNAs and their local translation in growth cones of developing axons has been shown to play an important mechanism to regulate growth cone turning responses to attractive or repulsive cues. However, the mechanism whereby local translation and growth cone turning may be controlled by specific mRNA-binding proteins is unknown. Here we demonstrate that brain-derived neurotrophic factor (BDNF) signals the Src-dependent phosphorylation of the beta-actin mRNA zipcode binding protein 1 (ZBP1), which is necessary for beta-actin synthesis and growth cone turning. We raised a phospho-specific ZBP1 antibody to Tyr396, which is a Src phosphorylation site, and immunofluorescence revealed BDNF-induced phosphorylation of ZBP1 within growth cones. The BDNF-induced increase in fluorescent signal of a green fluorescent protein translation reporter with the 3' untranslated region of beta-actin was attenuated with the Src family kinase-specific inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine]. Furthermore, a nonphosphorylatable mutant, ZBP1 Y396F, suppressed the BDNF-induced and protein synthesis-dependent increase in beta-actin localization in growth cones. Last, the ZBP1 Y396F mutant blocked BDNF-induced attractive growth cone turning. These results indicate that phosphorylation of ZBP1 at Tyr396 within growth cones has a critical role to regulate local protein synthesis and growth cone turning. Our findings provide new insight into how the regulated phosphorylation of mRNA-binding proteins influences local translation underlying growth cone motility and axon guidance.

DOI： 10.1523/JNEUROSCI.0499-10.2010

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Language：English   Publisher：JAPANESE PHARMACOLOGICAL SOC  

Li C, Bassell GJ, Sasaki Y, Frontiers in neural circuits, 2009, vol. 3, pp. 11, 2009

DOI： 10.3389/neuro.04.011.2009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr(32) residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr(32) of CRMP showed that Tyr(32)-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr(32) to Phe(32)) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr(32) is involved in Sema3A signaling.

DOI： 10.1074/jbc.M109.000240

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS RES FOUND  

Fragile X syndrome, the most frequent form of familial mental retardation, is caused by mutation of the Fmr1 gene. Fmr1 encodes the fragile X mental retardation protein (FMRP), an mRNA binding protein regulating local, postsynaptic mRNA translation along dendrites necessary for long-term synaptic plasticity. However, recent studies on FMRP localization in axons and growth cones suggest a possible function in the regulation of local protein synthesis needed for axon guidance. Here, we have demonstrated that FMRP is involved in axonal and growth cone responses induced by the axon guidance factor, Semaphorin-3A (Sema3A). In cultured hippocampal neurons from wild type mice, Sema3A-induced growth cone collapse was protein synthesis-dependent. In contrast, Sema3A-induced growth cone collapse was attenuated in Fmr1 knock-out (KO) neurons and insensitive to protein synthesis inhibitors, suggesting that FMRP is involved in protein synthesis-dependent growth cone collapse. Sema3A increased phosphorylation of eukaryotic initiation factor 4E (eIF4E), an indicator of local translation, in distal axons and growth cones of wild type, but not Fmr1 KO neurons. Furthermore, Sema3A rapidly induced a protein synthesis-dependent increase in levels of microtubule associated protein 1B (MAP1B) in distal axons of wild type neurons, but this response was attenuated in Fmr1 KO neurons. These results suggest a possible role of FMRP to regulate local translation and axonal protein localization in response to Sema3A. This study reveals a new link between FMRP and semaphorin signaling in vitro, and raises the possibility that FMRP may have a critical role in semaphorin signaling in axon guidance during brain development.

DOI： 10.3389/neuro.04.011.2009

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To visualize native or non-engineered RNA in live cells with single-molecule sensitivity, we developed multiply labeled tetravalent RNA imaging probes (MTRIPs). When delivered with streptolysin 0 into living human epithelial cancer cells and primary chicken fibroblasts, MTRIPs allowed the accurate imaging of native mRNAs and a non-engineered viral RNA, of RNA co-localization with known RNA-binding proteins, and of RNA dynamics and interactions with stress granules.

DOI： 10.1038/NMETH.1316

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer US  

In the developing nervous system, post-mitotic neurons migrate and extend their neurites and form precise patterns of connections that emerge through the interaction between the growth cone and a myriad of environmental cues such as attractive or repulsive axon guidance molecules. Semaphorin3A (Sema3A) is the prototypical repulsive axon guidance molecule that potently induces growth cone collapse stalling neurite extension. Neuropilin-1 (NRP-1) and Plexin-As are ligand-binding and signal-transducing receptor components for Sema3A, respectively. Collapsin response mediator protein (CRMP) was identified as a signaling molecule of Sema3A. However, its molecular mechanisms have been ill-defined. CRMPs are now known to be composed of five homologous cytosolic proteins CRMP1-5
all of the family proteins are highly phosphorylated in developing brains. By screening pharmaceutical reagents and utilizing gene-deficient mice and through biochemical analysis, we found that Fyn and cyclin-dependent kinase 5 (Cdk5) mediate Sema3A-induced response in dorsal root ganglion (DRG) neurons. Cdk5 was associated with PlexA2 through the active state of Fyn. This raised the possibility that Sema3A induced growth cone collapse response through phosphorylation of CRMPs by Cdk5. The 2-D gel analysis of brain lysate from Cdk5-deficient mice revealed that CRMP2 was a substrate for Cdk5 in vivo. In vitro kinase assay revealed that Ser522 was the major site of CRMP1 and CRMP2 phosphorylation by Cdk5. Cdk5 primarily phosphorylated CRMP2 at Ser522, and GSK3β secondarily phosphorylates at Thr509. The dual-phosphorylated CRMP2 was recognized by the antibody 3F4, which is highly reactive with the neurofibrillary tangles of Alzheimer's disease. In DRG neurons, Sema3A stimulation enhanced the levels of the phosphorylated form of CRMP2 detected by 3F4. Overexpression of CRMP2 mutant substituting either Ser522 or Thr509 with Ala attenuated Sema3A-induced growth cone collapse. Knockdown of CRMP1 and CRMP2 inhibited Sema3A-induced growth cone collapse. The phosphorylation of CRMP1 and/or CRMP2 is therefore an essential step for Sema3A signaling. CRMP1 and CRMP2 were also good substrates for Fyn. The phosphorylation of CRMP1 by Cdk5 and Fyn also appears to be involved in Sema3A and Reelin signaling, contributing to spine maturation and the regulation of cell migration during the development of the cerebral cortex.

DOI： 10.1007/978-0-387-78887-6_2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

Subcellular regulation of protein synthesis requires the correct localization of messenger RNAs (mRNAs) within the cell. In this study, we investigate whether the axonal localization of neuronal mRNAs is regulated by extracellular stimuli. By profiling axonal levels of 50 mRNAs detected in regenerating adult sensory axons, we show that neurotrophins can increase and decrease levels of axonal mRNAs. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) regulate axonal mRNA levels and use distinct downstream signals to localize individual mRNAs. However, myelin-associated glycoprotein and semaphorin 3A regulate axonal levels of different mRNAs and elicit the opposite effect on axonal mRNA levels from those observed with neurotrophins. The axonal mRNAs accumulate at or are depleted from points of ligand stimulation along the axons. The translation product of a chimeric green fluorescent protein-p-actin mRNA showed similar accumulation or depletion adjacent to stimuli that increase or decrease axonal levels of endogenous p-actin mRNA. Thus, extracellular ligands can regulate protein generation within subcellular regions by specifically altering the localized levels of particular mRNAs.

DOI： 10.1083/jcb.200703209

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Axon pathfinding requires directional responses of growth cones to extracellular cues, which have been shown to involve local synthesis of protein. The identity and functions of the locally produced proteins remain, however, unclear. Here we report that Ca2+-dependent bidirectional turning of Xenopus laevis growth cones requires localized distribution and translation of beta-actin messenger RNA. Both beta-actin mRNA and its zipcode-binding protein, ZBP1, are localized at the growth cone and become asymmetrically distributed upon local exposure to brain-derived neurotrophic factor ( BDNF). Inhibition of protein synthesis or antisense interference with beta-actin mRNA-ZBP1 binding abolishes both Ca2+-mediated attraction and repulsion. In addition, attraction involves a local increase in beta-actin, whereas repulsion is accompanied by a local decrease in beta-actin; thus, both produce a synthesis- and ZBP1 binding-dependent beta-actin asymmetry but with opposite polarities. Together with a similar asymmetry in Src activity during bidirectional responses, our findings indicate that Ca2+-dependent spatial regulation of b-actin synthesis through Src contributes to the directional motility of growth cones during guidance.

DOI： 10.1038/nn1773

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

A member of semaphorin family, semaphorin3A (Sema3A), acts as a chemorepellent or chemoattractant on a wide variety of axons and dendrites in the development of the nervous systems. We here show that Sema3A induces clustering of both postsynaptic density-95 (PSD-95) and presynaptic synapsin I in cultured cortical neurons without changing the density of spines or filopodia. Neuropilin-1 (NRP-1), a receptor for Sema3A, is present on both axons and dendrites. When the cultured neurons are exposed to Sema3A, the cluster size of PSD-95 is markedly enhanced, and an extensive colocalization of PSD-95 and NRP-1 or actin-rich protrusion is seen. The effects of Sema3A on spine morphology are blocked by PP2, an Src type tyrosine kinase inhibitor, but not by the PP3, the inactive-related compound. In the cultured cortical neurons from fyn(-/-) mice, dendrites bear few spines, and Sema3A does not induce PSD-95 cluster formation on the dendrites. Sema3A and its receptor genes are highly expressed during the synaptogenic period of postnatal days 10 and 15. The cortical neurons in layer V, but not layer III, show a lowered density of synaptic bouton-like structure on dendrites in sema3A- and fyn-deficient mice. The neurons of the double-heterozygous mice show the lowered spine density, whereas those of single heterozygous mice show similar levels of the spine density as the wild type. These findings suggest that the Sema3A signaling pathway plays an important role in the regulation of dendritic spine maturation in the cerebral cortex neurons.

DOI： 10.1523/JNEUROSCI.5453-05.2006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Background. Ketamine has been reported to exert anti-inflammatory effects on macrophages stimulated with lipopolysaccharide (LPS) in vitro and in vivo. Several studies have reported conflicting results regarding the effects of propofol on cytokine production from immune cells. However, there have been no reports of the effects of these agents on inflammatory responses in glial cells. We investigated the effects of ketamine and propofol on LPS-induced production of nitric oxide, tumour necrosis factor-alpha (TNF-alpha) and prostaglandin E-2 (PGE(2)) from primary cultures of rat glial cells in vitro.
Methods. Glial cells were stimulated with LPS in the absence and presence of various concentrations of ketamine (30-1000 mu M) or propofol (30 and 300 mu M). Nitric oxide released into the culture media was determined by measuring nitrite using the Griess reaction, and concentrations of TNF-alpha and PGE(2) were measured by enzyme-linked immunosorbent assay (ELISA).
Results. Ketamine reduced LPS-induced TNF-alpha production without significant inhibition of nitrite release in mixed glial cells, astrocyte cultures and microglial cultures. Ketamine also inhibited LPS-induced production of PGE(2) in astrocyte cultures. In contrast, propofol had no effect on LPS-induced nitrite or TNF-alpha production in mixed glial cells.
Conclusions. The data demonstrate that ketamine inhibited some of the inflammatory responses of both astrocytes and microglial cells treated with LPS without causing major change in nitric oxide release. Propofol had no effect on the production of nitric oxide or TNF-alpha from LPS-stimulated glial cells.

DOI： 10.1093/bja/aei256

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

Collapsin response mediating protein-2 (CRMP2) has been identified as an intracellular protein mediating Semaphorin3A (Sema3A), a repulsive guidance molecule. In this study, we demonstrate that cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3beta (GSK3beta) plays a critical role in Sema3A signalling. In In vitro kinase assay, Cdk5 phosphorylated CRMP2 at Ser522, while GSK3beta did not induce any phosphorylation of CRMP2. Phosphorylation by GSK3beta was exclusively observed in Cdk5-phosphorylated CRMP2, but barely in CRMP2T509A. These results indicate that Cdk5 primarily phosphorylates CRMP2 at Ser522 and GSK3beta secondarily phosphorylates at Thr509. The dual-phosphorylated CRMP2, but not non-phosphorylated or single-phosphorylated CRMP2, is recognized with the antibody 3F4, which is highly reactive with the neurofibrillary tangles of Alzheimer's disease. 3F4 recognized the CRMP2 in the wild-type but not cdk5(-/-) mouse embryonic brain lysates. The phosphorylation of CRMP2 at Ser522 caused reduction of its affinity to tubulin. In dorsal root ganglion neurones, Sema3A stimulation enhanced the levels of the phosphorylated form of CRMP2 detected by 3F4. Over-expression of CRMP2 mutant substituting either Ser522 or Thr509 to Ala attenuates Sema3A-induced growth cone collapse response. These results suggest that the sequential phosphorylation of CRMP is an important process of Sema3A signalling and the same mechanism may have some relevance to the pathological aggregation of the microtubule-associated proteins.

DOI： 10.1111/j.1365-2443.2005.00827.x

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An impressive body of evidence has been accumulated indicating that local protein synthesis is implicated in navigation of neurite extension induced by guidance cues, such as semaphorin3A (Sema3A). We found previously that a Src type tyrosine kinase Fyn and cyclin-dependent kinase 5 (Cdk5) mediate Sema3A-signaling. We also showed that Sema3A elicits axonal transport through neuropilin-1, a receptor for Sema3A, located at the growth cones. Here, we investigate the relationship between Sema3A-induced local signaling, protein synthesis, and axonal transport. Lavendustin A, a tyrosine kinase inhibitor, and olomoucine, a cyclin-dependent kinase inhibitor, suppressed Sema3A-induced facilitation of anterograde and retrograde axonal transport in dorsal root ganglion (DRG) neuron with and without the cell body. Sema3A-induced facilitation of axonal transport was attenuated in DRG neurons of fyn- (fyn(-/-)) and a Cdk5 activator, p35 (p35(-/-))-deficient mice when compared with those of wild-type or heterozygous mice. Inhibition of protein synthesis suppressed Sema3A-induced facilitation of axonal transport in the DRG neuron with and without the cell body. Sema3A enhanced the level of immunoreactivity of phosphorylated eukaryotic translation initiation factor 4E (eIF-4E) within 5 min in growth cones in a time course similar to that of the facilitated axonal transport. This enhanced signal for phospho-eIF4E was blocked by lavendustin A or olomoucine and was not detected in the fyn(-/-) and p35(-/-) neurons. These results provide evidence for a mutual regulatory mechanism between local protein synthesis and axonal transport.

DOI： 10.1523/JNEUROSCI.1476-04.2004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Ephrin-As are repulsive axonal guidance cues that regulate retinotectal projection. EphA tyrosine kinases, which are the receptors of ephrin-As, activate signaling cascades leading to cytosckeleton reorganization. Here, we address the role of cyclin-dependent kinase (Cdk) 5 in Eph receptor signaling induced by ephrin-A5. Ephrin-A5 induced a cell morphological response in PC-3M cells that endogenously express Cdk5 and EphA2, a receptor for ephrin-A5. This response was augmented by the transfection of p35, which is a neuronal regulator of Cdk5. While the morphological response of native PC-3M cells was not affected by olomoucine, an inhibitor of Cdk, the response was inhibited in the p35-transfected cells. In retinal ganglion cells, either olomoucine at 20 muM or Y-27632 at 10 muM, an inhibitor of Rho-kinase/ROKalpha/ROCKII, showed maximum inhibitory effect against ephrin-A5 (10 mug/ml)-induced growth cone collapse. Combined application of olomoucine and Y-27632 further suppressed the ephrin-A5 -induced response. Ephrin-A5 evoked phosphorylation of Cdk5 at Tyr15 and tau, a substrate of Cdk5 in retinal growth cones. Recombinant herpes simplex virus expressing Cdk5 mutant (kinase-negative or Tyr15 to Ala) showed a dominant-negative effect on the ephrin-A5-induced growth cone collapse. These findings demonstrate that both Cdk5 and the Rho kinase pathway independently contribute to the downstream of ephrin-A-induced signaling in retinal ganglion cells. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/S1044-7431(03)00220-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

Growth cone motility and morphology are based on actin-filament dynamics. Cofilin plays an essential role for the rapid turnover of actin filaments by severing and depolymerizing them. The activity of cofilin is repressed by phosphorylation at Ser3 by LIM kinase (LIMK, in which LIM is an acronym of the three gene products Lin-11, Isl-1, and Mec-3) and is reactivated by dephosphorylation by phosphatases, termed Slingshot (SSH). We investigated the roles of cofilin, LIMK, and SSH in the growth cone motility and morphology and neurite extension by expressing fluorescence protein-labeled cofilin, LIMK1, SSH1, or their mutants in chick dorsal root ganglion (DRG) neurons and then monitoring live images of growth cones by time-lapse video fluorescence microscopy. The expression of LIMK1 remarkably repressed growth cone motility and neurite extension, whereas the expression of SSH1 or a nonphosphorylatable S3A mutant of cofilin enhanced these events. The fan-like shape of growth cones was disorganized by the expression of any of these proteins. The repressive effects on growth cone behavior by LIMK1 expression were significantly rescued by the coexpression of S3A-cofilin or SSH1. These findings suggest that LIMK1 and SSH1 play critical roles in controlling growth cone motility and morphology and neurite extension by regulating the activity of cofilin and may be involved in signaling pathways that regulate stimulus-induced growth cone guidance. Using various mutants of cofilin, we also obtained evidence that the actin-filament-severing activity of cofilin is critical for growth cone motility and neurite extension.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Semaphorin-3A (Sema3A), a member of class 3 semaphorins, regulates axon and dendrite guidance in the nervous system. How Sema3A and its receptors plexin-As and neuropilins regulate neuronal guidance is unknown. We observed that in fyn- and cdk5-deficient mice, Sema3A-induced growth cone collapse responses were attenuated compared to their heterologous controls. Cdk5 is associated with plexin-A2 through the active state of Fyn. Sema3A promotes Cdk5 activity through phosphorylation of Tyr15, a phosphorylation site with Fyn. A Cdk5 mutant (Tyr15 to Ala) shows a dominant-negative effect on the Sema3A-induced collapse response. The sema3A gene shows strong interaction with fyn for apical dendrite guidance in the cerebral cortex. We propose a signal transduction pathway in which Fyn and Cdk5 mediate neuronal guidance regulated by Sema3A.

DOI： 10.1016/S0896-6273(02)00857-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

We have proposed the hypothesis that L-3,4-dihydroxyphenylalanine (DOPA) plays a role of neurotransmitter of the primary baroreceptor afferents terminating in the nucleus tractus solitarii (NTS). In the present study, we tried to clarify whether glutamate receptors and/or nitric oxide (NO), important modulators for central cardiovascular regulation, are involved in the DOPA-induced cardiovascular responses in the nucleus. Male Wistar rats were anesthetized with urethane and artificially ventilated. Compounds or antisense oligos (17-mer) for neuronal NO synthase were microinjected into depressor sites of the unilateral nucleus. DOPA 30-300 pmol microinjected into the nucleus dose-dependently induced depressor and bradycardic responses. Prior injection of kynurenic acid (600 pmol) suppressed DOPA (300 pmol)-induced responses by approximate to80%. Prior injection of N-G-monomethyl-L-arginine 100 nmol, a potent NO synthase inhibitor, reversibly attenuated by approximate to90% DOPA-induced responses, while the D-isomer 100 nmol produced no effect. Furthermore, prior injection of neuronal NO synthase antisense oligos (20 pmol) reversibly reduced by approximate to70% responses to DOPA. Sense or scrambled oligos produced no effect. A NO precursor L-arginine (30 nmol) induced depressor and bradycardic responses, but these responses were not affected by kynurenic acid. These results suggest important roles for glutamate receptors and NO in DOPA induced-depressor and bradycardic responses in the NTS. (C) 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/S0168-0102(02)00037-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CLINICAL INVESTIGATION INC  

Goshima Y, Ito T, Sasaki Y, Nakamura F, The Journal of clinical investigation, 2002, vol. 109, no. 8, pp. 993-998, 2002

DOI： 10.1172/JCI200215467

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：KLUWER ACADEMIC/PLENUM PUBL  

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Language：English   Publisher：The American Society for Clinical Investigation  

DOI： 10.1172/JCI0215467

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Language：Japanese   Publisher：(一社)日本神経化学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE AMERICA INC  

Semaphorin 3A is a chemorepulsive axonal guidance molecule that depolymerizes the actin cytoskeleton and collapses growth cones of dorsal root ganglia neurons. Here we investigate! the role of LIM-kinase 1, which phosphorylates an actin-depolymerizimg protein, cofilin, in semaphorin 3A-induced growth cone collapse. Semaphorin 3A induced phosphorylation and dephosphorylation of cofilin at growth cones sequentially. A synthetic cell-permeable peptide containing a cofilin phosphorylation site inhibited LIM-kinase in vitro and in vivo, and essentially suppressed semaphorin 3A-induced growth cone collapse. A dominant-negative LIM kinase, which could not be activated by PAK or ROCK, suppressed the collapsing activity of semaphorin 3A. Phosphorylation of cofilin by LIM-kinase may be a critical signaling event in growth cone collapse by semaphorin 3A.

DOI： 10.1038/86011

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In rat striata, DOPA released is a causal factor for glutamate release and resultant delayed neuron death by four-vessel occlusion. Nanomolar DOPA cyclohexyl ester (CHE), a potent and relatively stable competitive DOPA antagonist, protects these events. We tried to clarify whether DOPA CHE protects these events in hippocampal CA1 pyramidal cell layers most vulnerable against ischemia. Five to 10 min ischemia caused slight to mild glutamate release in 10 min samples during microdialysis and mild to severe neuron death 96 h after reperfusion. DOPA and dopamine were under assay limit in this design, but were basally detected by 20 min sampling and released by 20 min ischemia. In 10 min samples, intrahippocampal perfusion of 100 nM DOPA CHE 10 min before ischemia for 70 min did not inhibit glutamate release by 10 min ischemia, while it abolished glutamate release and protected delayed neuron death by 5 min ischemia. DOPA CHE is neuroprotective under a mild ischemic condition in rat hippocampus CA1. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0304-3940(01)01520-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We previously proposed that L-3,4-dihydroxyphenylalanine (L-DOPA) is a neurotransmitter in the CNS. Receptor and transporter molecules for L DOPA, however, have not been determined. In the present study, in order to localize the uptake sites of L-DOPA in the CNS, we performed autoradiographic uptake studies using L-[C-14]DOPA and L-[H-3]DOPA in the uptake study on rat brain slice preparations, and further analyzed the properties of L-DOPA uptake. Image analysis of the L-[C-14]DOPA autoradiogram showed a unique heterogeneous distribution of uptake sites in the brain. The intensity was relatively high in the cerebral cortex, the hypothalamus. the cerebellum and the hippocampus, while the density was moderate or even low in the striatum and the substantia nigra, L-DOPA and phenylalanine. bur not dopamine (10 mM) were able to almost completely inhibit the uptake of L-[C-14]DOPA to basal levels. Microautoradiographic studies using L-[H]DOPA revealed accumulation of dense grains in the median eminence, the supraoptic nucleus of the hypothalamus, the cerebral cortex (layer I) and the hippocampus. In the cerebellum, grains formed in clusters surrounding the Purkinje cells. This grain accumulation was concluded to be in Bergmann glial cells, since the morphological pattern of grain accumulation was similar to that of the immunoreactivity of the glutamate aspartate transporter, a marker protein for Bergmann glial cells. In the hippocampus, the grain density significantly decreased under Nai-free conditions. In addition, grain density also decreased in the absence of Cl-. In contrast, grains in the choroid plexus and the ependymal cell layer, were not affected by the absence of Na+. These findings indicated that the uptake of L-DOPA occurs via various types of large neutral amino acid transport mechanisms.
It appears that neuronal and/or glial cells, which take up L-DOPA in a Na+-dependent manner. exist in the CNS. Our finding further supports the concept that L-DOPA itself may act as a neurotransmitter or neuromodulator. (C) 2001 IBRO. published by Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0306-4522(01)00008-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The vertebrate CRMP (collapsin-response-mediator protein) gene family comprises at least four members. These CRMPs exhibit about 60% amino acid identity with vertebrate dihydropyrimidinase (DHP), an amidohydrolase involved in the pyrimidine degradation pathway. CRMP is also referred to as DRP (DHP-related protein), TOAD-64 (turned on after division, 64 kDa) and Ulip (Unc-33-like phosphoprotein). These vertebrate CRMPs are expressed mainly in early neuronal differentiation, which suggests that they play a role in neuronal development. In this study we isolated two cDNA clones from nematode C. elegans based on their sequence homology to vertebrate CRMPs and DHP. These two molecules, termed CeCRMP/DHP-1 and -2, turned out to be Ulip-B and -A. respectively, which were previously identified in the C, elegans genomic database by Byk et al. (1998). These newly isolated molecules were believed to represent a common ancestral state before the gene duplication between CRMPs and DHP. CeCRMP/DHP-1 and -2 protein retained all putative zinc-binding residues thought to be essential for the amidohydrolase activity of DHP and exhibited a weak amidohydrolase activity when 5-bromo-dihydrouracil was used as a substrate. Whole-mount in situ hybridization and expression analysis using GFP fusions revealed that CeCRMP/DHP-1 was transiently expressed in the hypodermis of C. elegans during the early larva stage. CeCRMP/DHP-1 was also expressed in a single nerve cell between the pharynx and ring neuropil. On the other hand, expression of CeCRMP/DHP-2 was observed in the body wall muscle throughout the lifespan of C. elegans. These results indicate that a major site of CeCRMP/DHP-1 and -2 expression is non-neuronal. Targeted gene disruption of CeCRMP/DHP-2 caused no particular difference in appearance or movement phenotype. (C) 2000 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-1119(00)00494-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

This study characterized the in vitro pharmacological properties of a newly developed endothelin receptor antagonist, N-butane-sulfonyl-[N-(3,5-dimethylbenzoyl)-N-methyl-3-[4-(5-isoxazolyl)-phenyl]-(D)-alanyl]-(L)-valineamide sodium salt (IRL 3630A), and its in vivo effects on respiratory mechanics were determined. IRL 3630A showed highly balanced affinities to human endothelin ETA and ETB receptors, giving apparent K-i values of 1.5 and 1.2 nM, respectively. This compound also potently antagonized the endothelin-l-induced intracellular Ca2+ increases in both embryonic bovine tracheal (EBTr) cells expressing endothelin ETA receptors and human Girardi heart (BGH) cells expressing endothelin ETB receptors. In guinea pig isolated tracheas having both endothelin ETA and ETB receptors, IRL 3630A greatly inhibited endothelin-1-induced contraction (pA(2) = 7.1), which was partially or scarcely suppressed by the endothelin ETA receptor antagonist cyclo[-(D)-Trp-(o)-Asp-(L)-Pro-(D)-Val-(L)-Leu-] (BQ-123) or the endothelin ETB receptor antagonist N-(3,5-dimethylbenzoyl)-N-methyl-3-(4-phenyl)-(D)-(phenyl)-(D)-phenylalanyl-(L)-tryptophan (IRL 2500), respectively. Bolus i.v. injections of IRL 3630A administered into anaesthetized guinea pigs at 10 and 30 mu g/kg inhibited endothelin-1 (1.3 mu g/kg)-induced changes in respiratory resistance and compliance in a dose dependent manner, whereas both sodium 2-benzo[1,3]dioxol-5-yl-4-(4-methoxy-phemethoxy-benzyl)-but-2-enoate ate (an endothelin ETA receptor antagonist: PD 156707) and IRL 2500 at doses of up to 30 mu g/kg did not affect endothelin-l-induced changes in respiratory mechanics, reflecting the in vitro results. IRL 3630A is thus an effective bifunctional endothelin receptor antagonist, and will be useful in clarifying the role of endothelin in pulmonary diseases such as bronchial asthma. (C) 2000 Elsevier Science B.V. Ail rights reserved.

DOI： 10.1016/S0014-2999(00)00652-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Semaphorin III/collapsin-1 (Sema3A) guides a specific subset of neuronal growth cones as a repulsive molecule. In this study, we have investigated a possible role of non-neuronal Sema3A in lung morphogenesis. Expression of mRNAs of Sema3A and neuropilin-1 (NP-1), a Sema3A receptor, was detected in fetal and adult lungs. Sema3A-immunoreactive cells were found in airway and alveolar epithelial cells of the fetal and adult lungs. Immunoreactivity for NP-1 was seen in fetal and adult alveolar epithelial cells as well as endothelial cells. Immunoreactivity of collapsin response mediator protein CRMP (CRMP-2), an intracellular protein mediating Sema3A signaling, was localized in alveolar epithelial cells, nerve tissue and airway neuroendocrine cells. The expression of CRMP-2 increased during the fetal, neonate and adult periods, and this pattern paralleled that of NP-1. In a two-day culture of lung explants from fetal mouse lung (E11.5), with exogenous Sema3A at a dose comparable to that which induces growth cone collapse of dorsal root ganglia neurons, the number of terminal buds was reduced in a dose-dependent manner when compared with control or untreated lung explants. This decrease was not accompanied with any alteration of the bromodeoxyuridine-positive DNA-synthesizing fraction. A soluble NP-1 lacking the transmembrane and intracellular region, neutralized the inhibitory effect of Sema3A. The fetal lung explants from neuropilin-1 homozygous null mice grew normally in vitro regardless of Sema3A treatment, These results provide evidence that Sema3A inhibits branching morphogenesis in lung bud organ cultures via NP-I as a receptor or a component of a possible multimeric Sema3A receptor complex. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0925-4773(00)00401-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Although L-3,4-dihydroxyphenylalanine (L-DOPA) is claimed to be a neurotransmitter in the central nervous system (CNS), receptor or transporter molecules for L-DOPA have not been determined. In an attempt to identify a transporter for L-DOPA, we examined whether or not an active and high affinity L-DOPA transport system is expressed in Xenopus laevis oocytes injected with poly A(+) RNA prepared from several tissues. Among the poly A(+) RNAs tested, rabbit intestinal epithelium poly A(+) RNA gave the highest transport activity for L-[C-14]DOPA in the oocytes. The uptake was approximately five times higher than that of water-injected oocytes, and was partially Na+-dependent. L-Tyrosine, L-phenylalanine, L-leucine and L-lysine inhibited this transport activity, whereas D-DOPA, dopamine, glutamate and L-DOPA cyclohexylester, an L-DOPA antagonist did not affect this transport. Coinjection of an antisense cRNA, as well as oligonucleotide complementary to rabbit rBAT (NBAT) cDNA almost completely inhibited the uptake of L-[C-14]DOPA in the oocytes. On the other hand, an antisense cRNA of rabbit 4F2hc barely affected this L-[C-14]DOPA uptake activity. rBAT was thus responsible for the r-[C-14]DOPA uptake activity expressed in X. laevis oocytes injected with poly A(+) RNA from rabbit intestinal epithelium. As rBAT is localized at the target regions of L-DOPA in the CNS, rBAT might be one of the components involved in L-DOPAergic neurotransmission. (C) 2000 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0005-2736(00)00171-1

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Language：English   Publisher：JAPANESE PHARMACOLOGICAL SOC  

The semaphorin family comprises secreted and transmembrane signaling proteins that function in the nervous, immune, respiratory and cardiovascular systems. Sema3A, a secreted type of semaphorin, is now recognized as the most potent repulsive molecule inhibiting or repelling neurite outgrowth. The biological actions of Sema3A are mediated via neuropilin (Npn)-1, a receptor or one of the components of a receptor complex for Sema3A. Although the molecular mechanisms of Sema3A-Npn-1 signaling are largely unknown, a pertussis toxin-sensitive trimeric G protein(s), Rac-1, collapsin response mediator protein (CRMP), cyclic nucleotides and tyrosine kinase(s) have been implicated as essential and/or modulatory components of these processes. As repulsive molecules could be impediments to axon outgrowth, determining how these repulsive molecules exert their actions has the potential of uncovering new therapeutic approaches to injury and/or degeneration of neuronal tissues.

DOI： 10.1254/jjp.82.273

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：ELSEVIER SCIENCE BV  

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：ELSEVIER SCIENCE BV  

We have proposed that L-3,4-dihydroxyphenylalanine (L-DOPA) is a neurotransmitter in the central nervous system. We examined L-DOPA uptake sites in rat brain slices using radiolabelled L-DOPA In the autoradiography with L-[C-14]-DOPA, the uptake activities were seen in cerebral cortex, hypothalamus and hippocampus, and these labeling were inhibited by cold L-DOPA or L-phenylalanine, but not by dopamine. In microautoradiography with L-[H-3]DOPA, we found that a high silver grain density in the hypothalamus, hippocampus, medulla oblongata and cerebellum, but low in the striatum. Under Nat-free condition, density of grains in hippocampus was significantly reduced. These data suggest that there exist some neural cells which uptake L-DOPA in a Na+-dependent manner in the CNS.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

Collapsin-1/Sema III, a member of the semaphorin family, has been implicated in axonal path-finding as a repulsive guidance cue. Cellular and molecular mechanisms by which collapsin-1 exerts its action are not fully understood. Collapsin-1 induces growth cone collapse via a pathway which may include neuropilin-1, a cell-surface collapsin-1 binding protein, as well as intracellular CRMP-62 and heterotrimeric G proteins. We previously identified a second action of collapsin-1, the facilitation of antero- and retrograde axoplasmic transport. This response occurs via a mechanism distinct from that causing growth cone collapse. To investigate the possible involvement of neuropilin-1 in the action of collapsin-1 on axoplasmic transport, we produced a soluble neuropilin-1 (sNP-1) lacking the transmembrane and intracellular region. sNP-1 progressively displaced the dose-response curve for collapsin-1 eo induce growth cone collapse to higher concentrations, sNP-1 also inhibited collapsin-1-induced augmentation of both antero- and retrograde axoplasmic transport. Furthermore, an anti-neuropilin-1 antibody blocked the collapsin-induced axoplasmic transport. These results together indicate that neuropilin-1 mediates collapsin-1 action on axoplasmic transport. To visualize collapsin-1 binding to endogenous neuropilin-1, we used a truncated collapsin-1-alkaline phosphatase fusion protein (CAP-4). CAP-4 stains the growth cone, neurite, and cell body. However, local application of collapsin-1 to growth cone but to neither neurite nor cell body promotes axoplasmic transport. Thus, growth cone NP-1 mediates the facilitatory action of collapsin-1 on antero- and retrograde axoplasmic transport, (C) 1999 John Wiley & Sons. Inc.

DOI： 10.1002/(SICI)1097-4695(19990615)39:4<579::AID-NEU11>3.0.CO;2-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：STOCKTON PRESS  

1 Signalling events responsible for endothelin(A) (ETA) and ETB receptor-induced contraction were examined in epithelium-denuded guinea-pig tracheal smooth muscle strips. Selective stimulation of each subtype was achieved by a combination of ET-1 (100 nM) and ETA and ETB receptor-selective antagonists, BQ-123 (10 mu M) and BQ-788 (3 mu M), respectively.
2 Both ETA and ETB receptors induced long-lasting contraction that was totally dependent on Ca2+ influx. Stimulation of ETA receptor induced both transient and sustained (Ca2+)(i) increases whereas that of ETB receptor induced only a sustained increase. Suppression of the transient (Ca2+)(i) increase by U73122 (3 mu M) did not affect the ETA-induced sustained (Ca2+)(i) increase and tension development. Stimulation of ETA receptor, but not ETB, induced phosphoinositide breakdown and protein kinase C (PKC). The activated PKC contributed to the contraction by increasing the Ca2+ sensitivity of the contractile apparatus.
3 Thus, ETA receptor is coupled both with phospholipase C/Ca2+/PKC signalling and Ca2+ influx pathways whereas ETB receptor was coupled only with the latter.
4 Stimulation. of ETB receptor, but not ETA, caused membrane depolarization measured with a fluorescent indicator, bis-(1,3 dibutylbarbituric acid)-trimethine oxonol. Both nifedipine (1 mu M) and verapamil (10 mu M) abolished ETB-induced Ca2+ influx and contraction, while they barely affected ETA-induced responses.
5 Therefore, the Ca2+ influx pathways activated by each subtype appeared to be completely different; ETA and ETB receptors opens voltage-independent Ca2+ channels and L-type voltage-dependent Ca2+ channels, respectively.

DOI： 10.1038/sj.bjp.0702365

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

We have previously obtained evidence that DOPA is probably involved in an upstream process of mechanisms for in vivo neuronal cell death in striatum. We attempted to clarify whether or not this is also the case in hippocampal region of conscious Wistar rats. Four vessels were occluded for 5 min during microdialysis of hippocampus. DOPA, dopamine and glutamate (Glu) in perfusates collected every l0 min were measured by HPLC-ECD and spectrophotometer. Delayed neuronal cell death in hippocampus was evaluated 96 hr after ischemia. Five-min transient brain ischemia induced Glu release, with the peak being 2.5-fold of a basal release at the fraction immediately after ischemia. The release of DOPA and dopamine was not consistently detectable, but an increase was sometimes observed during and after ischemia. Delayed neuronal cell death was slight to moderate with 5-min ischemia. Intrastriatal perfusion of DOPA cyclohexyl ester (DOPA CHE) at 100 nM, a novel stable potent competitive DOPA antagonist, almost completely inhibited the ischemia-induced glutamate release, and protected hippocampal neurons from delayed cell death. Endogenously released DOPA itself seems to act on its recognition site and to behave as a causal and/or deteriorating factor on glutamate release and resultant delayed neuronal cell death by transient ischemia in rats.

DOI： 10.1254/fpj.114.supplement_180

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PLENUM PUBL CORP  

1. We investigated the survival of neurons under serum-free conditions without any exogenous signal molecules, using primary cultures of rat cerebral cortex.
2. Survival activity, measured with Alamar Blue, showed a cell density dependency under serum-free conditions.
3. The addition of fetal bovine serum suppressed the apoptotic cell death accompanied by DNA-laddering and fragmentation specific in low-density cultures, resulting in the disappearance of the cell density dependency of survival.
4. These findings suggest that serum factors may substitute for endogenous survival factors from cortical neurons in high-density cultures.

DOI： 10.1023/A:1026375225275

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

Endothelin (ET) is known to be a potent mitogen in astrocytes, However, the contribution and signalling pathway of ETA and/or ETB receptor to the proliferation of astrocytes remain unclear. We investigated ET-induced DNA synthesis in astrocytes using ETB receptor-deficient mutant rats (aganglionosis rats: sl/sl). Western blotting with anti-ET receptor subtype-specific antibodies and Scatchard analysis of binding revealed that ETB receptor expression in astrocytes depended on gene dosage (+/+: sl/+: sl/sl = 2:1:0), whereas ETA receptor expression was unchanged among the three genotypes. ET-1 (10 nM) stimulated [H-3]thymidine incorporation and mitogen-activated protein kinase (MAP kinase) activity not only in +/+ via both ETA and ETB receptors, but also in sl/sl astrocytes via ETA receptor with about half the extent of those observed in +/+ astrocytes. Treatment with pertussis toxin (PTX) suppressed the ET-1-induced increases in the incorporation and MAP kinase activity in +/+, but not sl/sl astrocytes, indicating that the ETB receptor-, but not the ETA receptor-, mediated pathway to DNA synthesis involves PTX-sensitive G proteins, e.g. G(i) and/or G(o) (G(i/o)). in +/+ astrocytes, ET-1 (1 nM) stimulated cAMP accumulation, and the ETB receptor-selective agonist IRL 1620 (1 nM) suppressed 10 mu M forskolin-induced cAMP accumulation, suggesting G(s) coupling to the ETA receptor and Oil, coupling to the ETB receptor. On the other hand, ET-1 did not increase cAMP accumulation in sl/sl astrocytes, although ET-1 (1 nM) suppressed the forskolin-induced response, suggesting G(i/o) coupling to the ETA receptor, Our results suggest the possibility that the selectivity of G protein for ETA receptor is changed from G(s) to G(i/o) in ETB receptor-deficient astrocytes.

DOI： 10.1111/j.1460-9568.1998.00305.x

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Nitric oxide (NO), initially identified as an endothelium-derived relaxing factor, is a molecular mediator that has been implicated in many physiological and pathological processes. In primary cultured rat glial cells, a combination of inflammatory cytokines (tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta)) and bacterial lipopolysaccharide (LPS) stimulates production of nitrite via expression of the inducible form of nitric oxide synthase (iNOS). In these cells, simultaneous addition of endothelin (ET) markedly inhibited TNF-alpha/IL-1 beta-induced and LPS-induced nitrite production and iNOS expression, although ET by itself had no effect. The inhibitory effect of ETs appears to be mediated by ETB receptors. Forskolin also inhibited the iNOS expression. By contrast, pretreatment with ET for 24 hours enhanced LPS-induced nitrite production and iNOS expression. This stimulatory effect of ETs was suppressed by calphostin C, a protein kinase C inhibitor, and pretreatment with phorbol ester enhanced LPS-induced iNOS expression. Our findings present the possibility that ET has dual effects on iNOS expression in glial cells.

DOI： 10.1016/S0024-3205(98)00095-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Lipopolysaccharide is known to stimulate production of nitrite via expression of inducible nitric oxide (NO) synthase in not only macrophages but also glial cells. We found that in glial cell cultures lipopolysaccharide-stimulated inducible NO synthase expression and nitrite accumulation were synergistically enhanced by pretreatment with endothelin, whereas endothelin itself did not induce these responses. Pretreatment with endothelin-1, endothelin-3, and the selective endothelin type B (ETB) receptor agonist IRL 1620 caused the same effect with similar potencies, suggesting that the synergism was mediated via the endothelin ETB receptor. A protein kinase C inhibitor, calphostin C, suppressed endothelin-3-enhanced inducible NO synthase expression. Pretreatment with either endothelin-3 or phorbol ester enhanced lipopolysaccharide-induced production of tumor necrosis factor-alpha (TNF-alpha). Simultaneous addition of TNF-alpha increased lipopolysaccharide-stimulated inducible NO synthase expression. These results suggest that the increase in inducible NO synthase expression by endothelin was due to the elevated TNF-alpha production via protein kinase C. Our findings present the possibility that endothelin is implicated in neurotoxicity via enhancement of inducible NO synthase expression. (C) 1997 Elsevier Science B.V.

DOI： 10.1016/S0014-2999(97)01369-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT-RAVEN PUBL  

Excessive release of glutamate, from glial cells as well as neurons, is thought to be a major cause of neuronal death in ischemia. To investigate glutamate release from glial cells, we measured glutamate efflux from cultures of rat astrocytes preloaded with L-[H-3]-glutamate. Glutamate efflux was induced by either 60 mM KCl or Na+-free medium, suggesting that the efflux is due to the reversed operation of a Na+- and K+-coupled glutamate uptake machinery. While investigating various neuropeptides and neurotransmitters, we found that endothelin (ET) specifically induced efflux of glutamate. Northern blot analysis and binding study showed that the ET type B receptor (ETB-R) subtype was expressed two to three times more densely than the ET type A receptor (ETA-R) in astrocytes. The ETA-R antagonist IRL 2500 partially inhibited efflux of glutamate induced by 1 nM ET-1 in a concentration-dependent manner, causing a maximal inhibition of 60% at 1 mu M. However, the ETA-R antagonist BQ-123 did not cause significant inhibition even at 10 mu M. Combination of both antagonists completely inhibited the ET-1-induced efflux. These results indicate that both receptor subtypes are involved in efflux of glutamate with a major contribution from the ETA-R. Our findings suggest that ET, which is known to be released in ischemia, may exacerbate neurodegeneration by stimulating efflux of glutamate.

DOI： 10.1046/j.1471-4159.1997.68052194.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ENDOCRINE SOC  

It is known that serum promotes squamous differentiation of airway epithelial cell culture in vitro. We investigated which types of epithelial cells produce endothelins (ETs) and ET-evoked cellular responses in cultured human bronchial epithelial cells (HBECs). Squamous cells, not basal cells (nondifferentiated), secrete ET-1 actively. Specific binding of [I-125]ET-1, not [I-125]ET-3, was observed in squamous as well as basal cells, demonstrating the existence of only the ET(A) receptor. ET-1 together with epidermal growth factor stimulated synergistic DNA synthesis in basal cells, whereas ET-1 alone did not. Serum failed to induce DNA synthesis in HBECs, indicating terminal differentiation into squamous cells. ET-1 (1-100 nM) dose-dependently stimulated PGE(2)(0.6-2.8 ng/10(5) cells) and thromboxane B-2 release (4-30 pg/10(5) cells) from squamous HBECs, but not from basal cells. Western blot analysis showed that both squamous and basal HBECs expressed inducible and endothelial nitric oxide synthase, whereas ET-1 failed to stimulate nitric oxide synthase expression. Our findings suggest that secreted ET-1 from squamous cells evokes the release of prostanoids in an autocrine manner, and stimulates DNA synthesis in basal cells as a comitogen in a paracrine manner. Thus, it is likely that ET-1 secreted from HBECs plays an important role as a local, autocrine, and paracrine modulator in airway responses.

DOI： 10.1210/en.137.11.4542

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

We investigated the contribution of endothelin type A (ET(A)) and ET(B) receptors on ET-induced DNA synthesis in CCD-18Lu cells, a human lung cell line possessing both ET(A) and ET(B) (ET(A)/ET(B) ratio: 9:1). ET-1 (0.05-2 nM) potently induced [H-3]thymidine incorporation by 2- to 14-fold over the basal level. An ET(A)-selective antagonist, FR139317, inhibited 0.2 nM ET-1-induced DNA synthesis dose dependently, showing complete inhibition at 1 mu M. ET-3 was inactive up to 2 nM. In contrast, ET(B)-selective antagonists, 100 nM of BQ-788 or IRL 2500, partially (30-60%) inhibited 0.2 nM ET-1-induced DNA synthesis. Stimulation of either ET(A) or ET(B) evoked the increases in intracellular Ca2+ concentration ([Ca2+](i)). ET(B)-mediated but not ET-1-induced [Ca2+](i) increase was pertussis toxin (PTX) sensitive. Adenosine 3',5'-cyclic monophosphate (cAMP) formation via ET(A) was observed in PTX-treated cells, whereas the inhibition of isoproterenol-stimulated cAMP formation via ET(B) was observed in PTX-untreated cells. Like the ET(B)-selective antagonists, PTX treatment or dibutyryl cAMP partially (50-70%) inhibited ET-1-induced DNA synthesis. These data suggest that 1) ET-1 induces DNA synthesis predominantly through ET(A), via PTX-insensitive G protein; 2) ET(A)-mediated cAMP formation inhibits DNA synthesis; and 3) stimulation of ET(B) coupling to G(i) protein modulates ET(A)-mediated DNA synthesis by inhibiting cAMP formation.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

We have developed the coexpression system of both delta-opiold receptor (DOR1) and M(2)-muscarinic receptor (M(2)) which mediate agonist-evoked currents due to common post-receptor mechanisms including G(i1) and phospholipase C (PLC) activation in Xenopus oocytes reconstituted with G(i1)alpha. The DOR1-currents by 100 nM D-Ser(2)-leu-enkephalin-Thr(6) (DSLET) were selectively desensitized by 10 nM phorbol 12-myristate 13-acetate (PMA). The PMA-desensitization of DSLET-currents was abolished in the presence of calphostin C, a protein kinase C inhibitor, or reversed by an intracellular injection of calcineurin, a protein phosphatase 2B. When a higher concentration (3 mu M) of DSLET was used, DSLET-currents were rapidly desensitized by repeated challenges of DSLET itself. However, repeated challenges of 10 mu M ACh caused no influence on such DSLET- or M(2)-currents. The desensitization of DSLET-currents was selectively reversed by protein kinase C inhibitors. Similar results were also obtained with various delta-opioid agonists. These resutis suggest that protein kinase C is involved in the homologous desensitization of delta-opioid receptors.

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Language：English   Publisher：ELSEVIER SCIENCE BV  

In the Xenopus oocytes expressing mu- or kappa-opioid receptors, agonist-induced currents were observed only when the oocyte was coinjected with Gi1 alpha RNA and pretreated with K-252a, a potent inhibitor of protein kinases. The evoked currents were abolished by intracellular injection of EGTA or inositol 1,4,5-trisphosphate and the current-voltage relationship revealed that they are mediated through typical calcium-dependent chloride channels. These findings suggest that the mu(-) and kappa-receptors mediate phospholipase C activation through Gi1 alpha, and that these receptor mechanisms including downstream signalings might be inhibited by phosphorylation in vivo in the Xenopus oocyte.

DOI： 10.1016/0169-328X(95)00077-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

2-Lysophosphatidylcholine and cis-unsaturated fatty acids such as linoleic and linolenic acids, which are the products of the hydrolysis of phosphatidylcholine catalyzed by phospholipase A2 (EC 3.1.1.4), significantly potentiate the differentiation of HL-60 cells to macrophages that is induced by either a membrane-permeant diacylglycerol or a phorbol ester. The cell differentiation was assayed by measuring the expression of CD11b, one of the cell surface markers of macrophages, and also by the appearance of phagocytic activity. Snake venom phospholipase A2 added directly to the cells is also active for this potentiation. Neither lysophosphatidylcholine, fatty acid, nor phospholipase A2 is active unless a membrane-permeant diacylglycerol or a phorbol ester is present. The results presented provide further evidence that activation of phospholipase A2 may be intimately related to the signal transduction pathway through protein kinase C.

DOI： 10.1073/pnas.90.11.4917

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Lysophospholipid, particularly 2-lysophosphatidylcholine (lysoPtdCho), significantly potentiates the diacylglycerol (DAG)-induced activation of protein kinase C (PKC) in vitro. LysoPtdCho shows no effect, unless DAG and phosphatidylserine (PtdSer) are present. This lysoPtdCho action also depends on its own as well as on Ca2+ concentration. At physiological Ca2+ concentrations, the activation of the alpha-, beta-, and gamma-subspecies (cPKC) is enhanced by lysoPtdCho in the 10(-6) M range, but inversely inhibited in the 10(-5) M range. The delta- and epsilon-subspecies (nPKC), which are enzymatically insensitive to Ca2+, are mostly inhibited by lysoPtdCho at its low concentrations. The enhancement of cPKC activation by lysoPtdCho is due to the increase in an apparent affinity of the enzyme for PtdSer but not for DAG. The results may account, at least in part, for the previous observations made with intact cell systems that lysoPtdCho significantly potentiates the DAG-induced cellular responses such as T-lymphocyte activation and HL-60 cell differentiation [(1992) Trends Biochem. Sci. 17, 414-417].

DOI： 10.1016/0014-5793(93)81655-J

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Both 2-lysophosphatidylcholine and cis-unsaturated fatty acids were previously shown to intensify agonist-induced cellular responses by enhancing the diacylglycerol-dependent activation of protein kinase C. Consistent with these observations, extracellular, secretory group II phospholipase A2, when added directly to human resting T lymphocytes, greatly potentiates their activation that was induced by a membrane-permeant diacylglycerol and ionomycin, as determined by the expression of the a subunit of the interleukin 2 receptor and thymidine incorporation into DNA. Diacylglycerol and ionomycin were essential for this cellular response, and phospholipase A2 alone showed no effect. The amount of 2-lysophosphatidylcholine produced in these cells by the exogenous phospholipase A2 was greatly increased in the presence of diacylglycerol and ionomycin, suggesting that the membrane phospholipids became susceptible to the phospholipase A2 when protein kinase C was activated. The results suggest that phospholipase A2 secreted into inflammatory sites plays a role in the propagation of cellular responses. Protein kinase C may function in the hydrolysis of membrane phospholipids by the exogenous phospholipase A2, and the products of this phospholipid hydrolysis enhance agonist-induced protein kinase C activation, thereby intensifying cellular responses.

DOI： 10.1073/pnas.90.2.716

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：RAVEN PRESS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD PRESS  

2-Lysophosphatidylcholine (lysoPtdCho), a product of the hydrolysis of phosphatidylcholine catalyzed by phospholipase A2, greatly potentiates the activation of human resting T lymphocytes that is induced by a membrane-permeant diacylglycerol plus a calcium ionophore, as determined by the expression of the alpha-subunit of the interleukin 2 receptor and thymidine incorporation into DNA. LysoPtdCho per se is inactive unless both diacylglycerol and a calcium ionophore are present. This effect of lysoPtdCho is also observed when diacylglycerol is replaced by a tumor-promoting phorbol ester. Other lysophosphatides including lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidic acid are inert except for lysophosphatidylethanolamine, which is far less effective than lysoPtdCho. Tracer experiments with radioactive choline indicate that, when T lymphocytes are stimulated with an antigenic signal, lysoPtdCho is indeed produced in a time-dependent fashion, although the concentration of this lysophospholipid accumulated remains to be quantitated. It suggests that phospholipase A2 is directly involved in the signal transduction pathway through protein kinase C to induce long-term cellular responses.

DOI： 10.1073/pnas.89.14.6447

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Four derivatives of hen lysozyme, each lacking one native disulfide bond of the four in authentic lysozyme, were produced in Escherichia coli by expressing synthetic mutant genes. In thc reoxidation reaction of the reduced derivatives purified from inclusion bodies, the addition of glycerol significantly enhanced the efficiency of folding and 'correct' disulfide bond formation. This enabled simple chromatographical purification of refolded materials. Purified 3SS-derivatives all showed lytic activities and secondary structures comparable to authentic lysozyme, which directly showed that none of the four native disulfide bonds is a prerequisite for 'correct' in vitro folding.

DOI： 10.1016/0014-5793(92)80466-T

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Authorship：Lead author,　Corresponding author   Language：Japanese  

DOI： 10.15105/j01677.2012267058

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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DOI： 10.1016/j.neures.2011.07.507

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DOI： 10.1016/j.neures.2011.07.978

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DOI： 10.1016/j.neures.2010.07.1570

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An impressive body of evidence has been accumulated indicating that local protein synthesis is implicated in navigation of neurite extension induced by guidance cues, such as semaphorin3A (Sema3A). We found previously that a Src type tyrosine kinase Fyn and cyclin-dependent kinase 5 (Cdk5) mediate Sema3A-signaling. We also showed that Sema3A elicits axonal transport through neuropilin-1, a receptor for Sema3A, located at the growth cones. Here, we investigate the relationship between Sema3A-induced local signaling, protein synthesis, and axonal transport. Lavendustin A, a tyrosine kinase inhibitor, and olomoucine, a cyclin-dependent kinase inhibitor, suppressed Sema3A-induced facilitation of anterograde and retrograde axonal transport in dorsal root ganglion (DRG) neuron with and without the cell body. Sema3A-induced facilitation of axonal transport was attenuated in DRG neurons of fyn- (fyn(-/-)) and a Cdk5 activator, p35 (p35(-/-))-deficient mice when compared with those of wild-type or heterozygous mice. Inhibition of protein synthesis suppressed Sema3A-induced facilitation of axonal transport in the DRG neuron with and without the cell body. Sema3A enhanced the level of immunoreactivity of phosphorylated eukaryotic translation initiation factor 4E (eIF-4E) within 5 min in growth cones in a time course similar to that of the facilitated axonal transport. This enhanced signal for phospho-eIF4E was blocked by lavendustin A or olomoucine and was not detected in the fyn(-/-) and p35(-/-) neurons. These results provide evidence for a mutual regulatory mechanism between local protein synthesis and axonal transport.

DOI： 10.1523/JNEUROSCI.1476-04.2004

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