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

Dynamin is an endocytic protein that functions in vesicle formation by scission of invaginated membranes. Dynamin maintains the structure of foot processes in glomerular podocytes by directly and indirectly interacting with actin filaments. However, molecular mechanisms underlying dynamin-mediated actin regulation are largely unknown. Here, biochemical and cell biological experiments were conducted to uncover how dynamin modulates interactions between membranes and actin in human podocytes. Actin-bundling, membrane tubulating, and GTPase activities of dynamin were examined in vitro using recombinant dynamin 2-wild-type (WT) or dynamin 2-K562E, which is a mutant found in Charcot-Marie-Tooth patients. Dynamin 2-WT and dynamin 2-K562E led to the formation of prominent actin bundles with constant diameters. Whereas liposomes incubated with dynamin 2-WT resulted in tubule formation, dynamin 2-K562E reduced tubulation. Actin filaments and liposomes stimulated dynamin 2-WT GTPase activity by 6- and 20-fold, respectively. Actin-filaments, but not liposomes, stimulated dynamin 2-K562E GTPase activity by 4-fold. Self-assembly-dependent GTPase activity of dynamin 2-K562E was reduced to one-third compared to that of dynamin 2-WT. Incubation of liposomes and actin with dynamin 2-WT led to the formation of thick actin bundles, which often bound to liposomes. The interaction between lipid membranes and actin bundles by dynamin 2-K562E was lower than that by dynamin 2-WT. Dynamin 2-WT partially colocalized with stress fibers and actin bundles based on double immunofluorescence of human podocytes. Dynamin 2-K562E expression resulted in decreased stress fiber density and the formation of aberrant actin clusters. Dynamin 2-K562E colocalized with α-actinin-4 in aberrant actin clusters. Reformation of stress fibers after cytochalasin D-induced actin depolymerization and washout was less effective in dynamin 2-K562E-expressing cells than that in dynamin 2-WT. Bis-T-23, a dynamin self-assembly enhancer, was unable to rescue the decreased focal adhesion numbers and reduced stress fiber density induced by dynamin 2-K562E expression. These results suggest that the low affinity of the K562E mutant for lipid membranes, and atypical self-assembling properties, lead to actin disorganization in HPCs. Moreover, lipid-binding and self-assembly of dynamin 2 along actin filaments are required for podocyte morphology and functions. Finally, dynamin 2-mediated interactions between actin and membranes are critical for actin bundle formation in HPCs.

DOI： 10.3389/fcell.2022.884509

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Elevation of soluble wild-type (WT) tau occurs in synaptic compartments in Alzheimer's disease. We addressed whether tau elevation affects synaptic transmission at the calyx of Held in slices from mice brainstem. Whole-cell loading of WT human tau (h-tau) in presynaptic terminals at 10-20 µM caused microtubule (MT) assembly and activity-dependent rundown of excitatory neurotransmission. Capacitance measurements revealed that the primary target of WT h-tau is vesicle endocytosis. Blocking MT assembly using nocodazole prevented tau-induced impairments of endocytosis and neurotransmission. Immunofluorescence imaging analyses revealed that MT assembly by WT h-tau loading was associated with an increased MT-bound fraction of the endocytic protein dynamin. A synthetic dodecapeptide corresponding to dynamin-1-pleckstrin-homology domain inhibited MT-dynamin interaction and rescued tau-induced impairments of endocytosis and neurotransmission. We conclude that elevation of presynaptic WT tau induces de novo assembly of MTs, thereby sequestering free dynamins. As a result, endocytosis and subsequent vesicle replenishment are impaired, causing activity-dependent rundown of neurotransmission.

DOI： 10.7554/elife.73542

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Other Link： https://cdn.elifesciences.org/articles/73542/elife-73542-v1.xml

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

DOI： 10.1002/humu.24307

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

Podosomes are actin-rich adhesion structures formed in a variety of cell types, such as monocytic cells or cancer cells, to facilitate attachment to and degradation of the extracellular matrix (ECM). Previous studies showed that dynamin 2, a large GTPase involved in membrane remodeling and actin organization, is required for podosome function. However, precise roles of dynamin 2 at the podosomes remain to be elucidated. In this study, we identified a BAR (Bin-Amphiphysin-Rvs167) domain protein pacsin 2 as a functional partner of dynamin 2 at podosomes. Dynamin 2 and pacsin 2 interact and co-localize to podosomes in Src-transformed NIH 3T3 (NIH-Src) cells. RNAi of either dynamin 2 or pacsin 2 in NIH-Src cells inhibited podosome formation and maturation, suggesting essential and related roles at podosomes. Consistently, RNAi of pacsin 2 prevented dynamin 2 localization to podosomes, and reciprocal RNAi of dynamin 2 prevented pacsin 2 localization to podosomes. Taking these results together, we conclude that dynamin 2 and pacsin 2 co-operatively regulate organization of podosomes in NIH-Src cells.

DOI： 10.1016/j.bbrc.2021.07.041

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>Elongated tubular endosomes play essential roles in diverse cellular functions. Multiple molecules have been implicated in tubulation of recycling endosomes, but the mechanism of endosomal tubule biogenesis has remained unclear. In this study, we found that JRAB/MICAL-L2 induces endosomal tubulation via activated Rab8A. In association with Rab8A, JRAB/MICAL-L2 adopts its closed form, which functions in the tubulation of recycling endosomes. Moreover, JRAB/MICAL-L2 induces liquid–liquid phase separation, initiating the formation of tubular recycling endosomes upon overexpression. Between its N-terminal and C-terminal globular domains, JRAB/MICAL-L2 contains an intrinsically disordered region, which contributes to the formation of JRAB/MICAL-L2 condensates. Based on our findings, we propose that JRAB/MICAL-L2 plays two sequential roles in the biogenesis of tubular recycling endosomes: first, JRAB/MICAL-L2 organizes phase separation, and then the closed form of JRAB/MICAL-L2 formed by interaction with Rab8A promotes endosomal tubulation.

DOI： 10.1038/s42003-021-02080-7

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Other Link： http://www.nature.com/articles/s42003-021-02080-7

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Membrane remodeling is required for dynamic cellular processes such as cell division, polarization and motility. BAR domain proteins and dynamins are key molecules in membrane remodeling that work together for membrane deformation and fission. In striated muscles, sarcolemmal invaginations termed T-tubules are required for excitation-contraction coupling. BIN1 and DNM2, which encode a BAR domain protein BIN1 and dynamin 2, respectively, have been reported to be causative genes of centronuclear myopathy (CNM), a hereditary degenerative disease of skeletal muscle, and deformation of T-tubules is often observed in the CNM patients. However, it remains unclear how BIN1 and dynamin 2 are implicated in T-tubule biogenesis, and how mutations in these molecules cause CNM to develop.Here, using an in cellulo reconstitution assay, we demonstrate that dynamin 2 is required for stabilization of membranous structures equivalent to T-tubules. GTPase activity of wild type dynamin 2 is suppressed through interaction with BIN1, whereas that of the disease-associated mutant dynamin 2 remains active due to lack of the BIN1-mediated regulation thus causing aberrant membrane remodeling. Finally, we show that in cellulo aberrant membrane remodeling by mutant dynamin 2 variants is correlated with their enhanced membrane fission activities, and the results can explain severity of the symptoms in patients. Thus, this study provides molecular insights into dysregulated membrane remodeling triggering the pathogenesis of DNM2-related centronuclear myopathy.

DOI： 10.1074/jbc.RA120.015184

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Toxoplasma gondii is an obligate intracellular protozoan parasite capable of infecting warm-blooded animals by ingestion. The organism enters host cells and resides in the cytoplasm in a membrane-bound parasitophorous vacuole (PV). Inducing an interferon response enables IFN-γ-inducible immunity-related GTPase (IRG protein) to accumulate on the PV and to restrict parasite growth. However, little is known about the mechanisms by which IRG proteins recognize and destroy T. gondii PV. We characterized the role of IRG protein Irgb6 in the cell-autonomous response against T. gondii, which involves vacuole ubiquitination and breakdown. We show that Irgb6 is capable of binding a specific phospholipid on the PV membrane. Furthermore, the absence of Irgb6 causes reduced targeting of other effector IRG proteins to the PV. This suggests that Irgb6 has a role as a pioneer in the process by which multiple IRG proteins access the PV. Irgb6-deficient mice are highly susceptible to infection by a strain of T. gondii avirulent in wild-type mice.

DOI： 10.26508/lsa.201900549

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Cell Biology  

DOI： 10.1247/csf.20020

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Vacuolar H+-ATPase-dependent (V-ATPase-dependent) functions are critical for neural proteostasis and are involved in neurodegeneration and brain tumorigenesis. We identified a patient with fulminant neurodegeneration of the developing brain carrying a de novo splice site variant in ATP6AP2 encoding an accessory protein of the V-ATPase. Functional studies of induced pluripotent stem cell-derived (iPSC-derived) neurons from this patient revealed reduced spontaneous activity and severe deficiency in lysosomal acidification and protein degradation leading to neuronal cell death. These deficiencies could be rescued by expression of full-length ATP6AP2. Conditional deletion of Atp6ap2 in developing mouse brain impaired V-ATPase-dependent functions, causing impaired neural stem cell self-renewal, premature neuronal differentiation, and apoptosis resulting in degeneration of nearly the entire cortex. In vitro studies revealed that ATP6AP2 deficiency decreases V-ATPase membrane assembly and increases endosomal-lysosomal fusion. We conclude that ATP6AP2 is a key mediator of V-ATPase-dependent signaling and protein degradation in the developing human central nervous system.

DOI： 10.1172/JCI79990

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

Dynamin copolymerizes with cortactin to form a ring‑like complex that bundles and stabilizes actin filaments. Actin bundle formation is crucial for generation of filopodia and lamellipodia, which guide migration, invasion, and metastasis of cancer cells. However, it is unknown how the dynamin‑cortactin complex regulates actin bundle formation. The present study investigated phosphorylation of cortactin by cyclin‑dependent kinase 5 (CDK5) and its effect on actin bundle formation by the dynamin‑cortactin complex. CDK5 directly phosphorylated cortactin at T145/T219 in vitro. Phosphomimetic mutants in which one or both of these threonine residues was substituted by aspartate were used. The three phosphomimetic mutants (T145D, T219D and T145DT219D) had a decreased affinity for F‑actin. Furthermore, electron microscopy demonstrated that these phosphomimetic mutants could not form a ring‑like complex with dynamin 1. Consistently, the dynamin 1‑phosphomimetic cortactin complexes exhibited decreased actin‑bundling activity. Expression of the phosphomimetic mutants resulted in not only aberrant lamellipodia and short filopodia but also cell migration in NG108‑15 glioma‑derived cells. These results indicate that phosphorylation of cortactin by CDK5 regulates formation of lamellipodia and filopodia by modulating dynamin 1/cortactin‑dependent actin bundling. Taken together, these findings suggest that CDK5 is a potential molecular target for anticancer therapy.

DOI： 10.3892/ijo.2018.4663

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

Dynamin GTPase, an essential endocytotic protein, helically polymerizes at the neck of endocytic pits, and mechanically sever the membrane upon GTP hydrolysis. However, it is not known exactly how the dynamin disconnect the membrane. To clarify the mechanisms we analyzed structural changes of dynamin complexes during membrane fission using electron microscopy and high-speed atomic force microscopy (HS-AFM). Surprisingly, the dynamin ring complexes were clustered upon GTP hydrolysis and membrane constriction occurred at uncoated regions between the clusters, suggesting a novel mode of action of dynamin. In this commentary, we illustrate dynamin’s membrane fission models proposed thus far, and our novel “clusterase” model.

DOI： 10.2142/biophys.59.255

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Other Link： http://id.ndl.go.jp/bib/030043645

Language：English   Publishing type：Research paper (scientific journal)   Publisher：eLife Sciences Publications Ltd  

Dynamin is a mechanochemical GTPase essential for membrane fission during clathrin-mediated endocytosis. Dynamin forms helical complexes at the neck of clathrin-coated pits and their structural changes coupled with GTP hydrolysis drive membrane fission. Dynamin and its binding protein amphiphysin cooperatively regulate membrane remodeling during the fission, but its precise mechanism remains elusive. In this study, we analyzed structural changes of dynamin-amphiphysin complexes during the membrane fission using electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynamin-amphiphysin helices are rearranged to form clusters upon GTP hydrolysis and membrane constriction occurs at protein-uncoated regions flanking the clusters. We also show a novel function of amphiphysin in size control of the clusters to enhance biogenesis of endocytic vesicles. Our approaches using combination of EM and HS-AFM clearly demonstrate new mechanistic insights into the dynamics of dynamin-amphiphysin complexes during membrane fission.

DOI： 10.7554/eLife.30246

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

Cancer cell invasion is mediated by actin-based membrane protrusions termed invadopodia. Invadopodia consist of "core" F-actin bundles associated with adhesive and proteolytic machineries promoting cell invasion by degrading extracellular matrix (ECM). Formation of the F-actin core in invadopodia is regulated by various actin-binding proteins including Arp2/3 complex and cortactin. Dynamin GTPase localizes to the invadopodia and is implicated in cancer cell invasion, but its precise role at the invadopodia remained elusive.
In this study, we examined the roles of dynamin at the invadopodia of bladder cancer cells. Although all three dynamin isoforms (dynamin1, 2 and 3) are expressed in human bladder cancer cell line T24, only dynamin2 localizes to the invadopodia. Inhibition of dynamin2 function, using either RNA interference (RNAi) or the dynamin specific inhibitor Dynasore, caused defects in invadopodia formation and suppressed invasive activity of 124 bladder cancer cells. Structure-function analysis using dynamin2 deletion fragments identified the proline/arginine-rich domain (PRD) of dynamin2 as indispensable for invadopodia formation and invasiveness of 124 cells. Thus, dynamin2 contributes to bladder cancer invasion by controlling invadopodia formation in bladder cancer cells and may prove a valuable therapeutic target. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2016.10.063

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

The endocytic protein dynamin participates in the formation of actin-based membrane protrusions such as podosomes, pseudopodia, and invadopodia, which facilitate cancer cell migration, invasion, and metastasis. However, the role of dynamin in the formation of actin-based membrane protrusions at the leading edge of cancer cells is unclear. In this study, we demonstrate that the ubiquitously expressed dynamin 2 isoform facilitates cell migration by stabilizing F-actin bundles in filopodia of the lung cancer cell line H1299. Pharmacological inhibition of dynamin 2 decreased cell migration and filopodial formation. Furthermore, dynamin 2 and cortactin mostly colocalized along F-actin bundles in filopodia of serum-stimulated H1299 cells by immunofluorescent and immunoelectron microscopy. Knockdown of dynamin 2 or cortactin inhibited the formation of filopodia in serum-stimulated H1299 cells, concomitant with a loss of F-actin bundles. Expression of wild-type cortactin rescued the punctate-like localization of dynamin 2 and filopodial formation. The incubation of dynamin 2 and cortactin with F-actin induced the formation of long and thick actin bundles, with these proteins colocalizing at F-actin bundles. A depolymerization assay revealed that dynamin 2 and cortactin increased the stability of F-actin bundles. These results indicate that dynamin 2 and cortactin participate in cell migration by stabilizing F-actin bundles in filopodia. Taken together, these findings suggest that dynamin might be a possible molecular target for anticancer therapy.

DOI： 10.3892/ijo.2016.3592

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley-Blackwell Publishing Ltd  

It is unclear whether the improvement in diabetic nephropathy by sodium glucose cotransporter 2 (SGLT2) inhibitors is caused by a direct effect on SGLT2 or by the improvement in hyperglycemia. Here, we investigated the effect of dapagliflozin on early-stage diabetic nephropathy using a mouse model of type 1 diabetes and murine proximal tubular epithelial cells. Eight-week-old Akita mice were treated with dapagliflozin or insulin for 12 weeks. Body weight, urinary albumin excretion, blood pressure, as well as levels of blood glucose and hemoglobin A1c were measured. Expansion of the mesangial matrix, interstitial fibrosis, and macrophage infiltration in kidneys were evaluated by histology. Oxidative stress and apoptosis were evaluated in kidneys and cultured proximal tubular epithelial cells. Compared with nontreated mice, dapagliflozin and insulin decreased blood glucose and hemoglobin A1c levels equally. Urine volume and water intake increased significantly in the dapagliflozin-treated group compared with those in the insulin-treated group, but there were no differences in body weight or blood pressure between the two groups. Macrophage infiltration and fibrosis in renal interstitium improved significantly in the dapagliflozin group compared with the insulin group. Oxidative stress was attenuated by dapagliflozin, and suppression occurred in a dose-dependent manner. RNAi knockdown of SGLT2 resulted in reduced oxidative stress. Dapagliflozin ameliorates diabetic nephropathy by suppressing hyperglycemia-induced oxidative stress in a manner independent of hyperglycemia improvement in Akita mice. Our findings suggest that dapagliflozin may be a novel therapeutic approach for the treatment of diabetic nephropathy.

DOI： 10.1002/prp2.239

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

DOI： 10.1016/j.neulet.2016.06.030

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Glioblastoma multiforme (GBM) is the most common malignant brain tumor with a median survival time about one year. Invasion of GBM cells into normal brain is the major cause of poor prognosis and requires dynamic reorganization of the actin cytoskeleton, which includes lamellipodial protrusions, focal adhesions, and stress fibers at the leading edge of GBM. Therefore, we hypothesized that inhibitors of actin polymerization can suppress GBM migration and invasion. First, we adopted a drug repositioning system for screening with a pyrene-actin-based actin polymerization assay and identified fluvoxamine, a clinically used antidepressant. Fluvoxamine, selective serotonin reuptake inhibitor, was a potent inhibitor of actin polymerization and confirmed as drug penetration through the blood-brain barrier (BBB) and accumulation of whole brain including brain tumor with no drug toxicity. Fluvoxamine inhibited serum-induced ruffle formation, cell migration, and invasion of human GBM and glioma stem cells in vitro by suppressing both FAK and Akt/mammalian target of rapamycin signaling. Daily treatment of athymic mice bearing human glioma-initiating cells with fluvoxamine blocked tumor cell invasion and prolonged the survival with almost same dose of anti-depressant effect. In conclusion, fluvoxamine is a promising anti-invasive treatment against GBM with reliable approach.

DOI： 10.1038/srep23372

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

BACKGROUND INFORMATION: Cortactin contributes to growth cone morphogenesis by forming with dynamin, ring-shaped complexes that mechanically bundle and stabilise F-actin. However, the regulatory mechanism of cortactin action is poorly understood. RESULTS: Immunofluorescence microscopy revealed that protein kinase C (PKC) α colocalises with cortactin at growth cone filopodia in SH-SY5Y neuroblastoma cells. PKC activation by phorbol 12-myristate 13-acetate causes cortactin phosphorylation, filopodial retraction and F-actin-bundle loss. Moreover, PKCα directly phosphorylates cortactin in vitro at S135/T145/S172, mitigating both cortactin's actin-binding and actin-crosslinking activity, whereas cellular expression of a phosphorylation-mimetic cortactin mutant hinders filopodial formation with a significant decrease of actin bundles. CONCLUSIONS: Our results indicate that PKC-mediated cortactin phosphorylation might be implicated in the maintenance of growth cone.

DOI： 10.1111/boc.201500032

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Oxidative stress and inflammation play important roles in diabetic complications, including diabetic nephropathy. Metallothionein (MT) is induced in proximal tubular epithelial cells as an antioxidant in the diabetic kidney
however, the role of MT in renal function remains unclear. We therefore investigated whether MT deficiency accelerates diabetic nephropathy through oxidative stress and inflammation. Diabetes was induced by streptozotocin injection in MT-deficient (MT-/-) and MT+/+ mice. Urinary albumin excretion, histological changes, markers for reactive oxygen species (ROS), and kidney inflammation were measured. Murine proximal tubular epithelial (mProx24) cells were used to further elucidate the role of MT under high-glucose conditions. Parameters of diabetic nephropathy and markers of ROS and inflammation were accelerated in diabetic MT-/- mice compared with diabetic MT+/+ mice, despite equivalent levels of hyperglycemia. MT deficiency accelerated interstitial fibrosis and macrophage infiltration into the interstitium in the diabetic kidney. Electron microscopy revealed abnormal mitochondrial morphology in proximal tubular epithelial cells in diabetic MT-/- mice. In vitro studies demonstrated that knockdown of MT by small interfering RNA enhanced mitochondrial ROS generation and inflammation-related gene expression in mProx24 cells cultured under high-glucose conditions. The results of this study suggest that MT may play a key role in protecting the kidney against high glucose-induced ROS and subsequent inflammation in diabetic nephropathy. © 2014 the American Physiological Society.

DOI： 10.1152/ajprenal.00034.2013

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bbrc.2013.11.118

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Inhibition of sodium glucose cotransporter 2 (SGLT2) has been reported as a new therapeutic strategy for treating diabetes. However, the effect of SGLT2 inhibitors on the kidney is unknown. In addition, whether SGLT2 inhibitors have an anti-inflammatory or antioxidative stress effect is still unclear. In this study, to resolve these issues, we evaluated the effects of the SGLT2 inhibitor, dapagliflozin, using a mouse model of type 2 diabetes and cultured proximal tubular epithelial (mProx24) cells. Male db/db mice were administered 0.1 or 1.0 mg/kg of dapagliflozin for 12 weeks. Body weight, blood pressure, blood glucose, hemoglobin A1c, albuminuria and creatinine clearance were measured. Mesangial matrix accumulation and interstitial fibrosis in the kidney and pancreatic β-cell mass were evaluated by histological analysis. Furthermore, gene expression of inflammatory mediators, such as osteopontin, monocyte chemoattractant protein-1 and transforming growth factor-β, was evaluated by quantitative reverse transcriptase-PCR. In addition, oxidative stress was evaluated by dihydroethidium and NADPH oxidase 4 staining. Administration of 0.1 or 1.0 mg/kg of dapagliflozin ameliorated hyperglycemia, β-cell damage and albuminuria in db/db mice. Serum creatinine, creatinine clearance and blood pressure were not affected by administration of dapagliflozin, but glomerular mesangial expansion and interstitial fibrosis were suppressed in a dose-dependent manner. Dapagliflozin treatment markedly decreased macrophage infiltration and the gene expression of inflammation and oxidative stress in the kidney of db/db mice. Moreover, dapagliflozin suppressed the high-glucose-induced gene expression of inflammatory cytokines and oxidative stress in cultured mProx24 cells. These data suggest that dapagliflozin ameliorates diabetic nephropathy by improving hyperglycemia along with inhibiting inflammation and oxidative stress.

DOI： 10.1371/journal.pone.0100777

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Society for Neuroscience  

DOI： 10.1523/jneurosci.2762-12.2013

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Cancer stem cells (CSCs) are thought to be crucial for understanding the biological roots of cancer, and are of increasing importance as a target for new anticancer agents. According to an expression analysis of the cell surface antigens of various types of cancer, CD133 is considered to be a potential marker of cancer stemness. In this study, a human urinary bladder cancer cell line (J82) was used to analyze the cancer stem cell-like characteristics of CD133+ bladder cancer cells in vitro and in vivo. The CD133 expression in the J82 cells was examined and the cells were immunomagnetically categorized into positive and negative subsets. The CD133- and CD133+ subsets were phenotypically divergent with regard to the cell growth pattern, while CD133+ cells tended to colonize during their growth. In CD133+ cells, the pluripotent stem cell factors Oct-4 and Sox-2 were upregulated, and a statistically significant proliferation increase was observed when compared to CD133- cells. The CD133+ subpopulation was more tolerant to the chemotherapeutic agent cisplatin, and Bacillus Calmette-Guérin (BCG), an agent instilled intravesically to treat bladder cancer. In addition, CD133+ J82 cells were more resistant to radiation treatment when compared to CD133- cells. The in vivo tumorigenesis of the CD133- and CD133+ subsets of J82 cancer cells was also examined by subcutaneously injecting them into nude mice. The tumor growth was more aggressive in the CD133+ subpopulation, showing a significant difference in the tumorigenic potential in these subsets. In conclusion, J82 human bladder cancer cells include CD133- and CD133+ subpopulations, while the CD133 molecule is a potential marker of the potential malignancy of human bladder cancer. In the present study, the CD133+ subpopulation was herein demonstrated to have certain characteristics consistent with those of cancer stem cells.

DOI： 10.3892/mco.2012.29

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Bladder cancer is one of the most common urogenital malignancies. The intravesical instillation of anticancer agents is an attractive strategy to treat a superficial lesion or floating/disseminated cancer cells after transurethral operation. An adenovirus carrying REIC/Dkk-3, a tumor suppressor gene (Ad-REIC), exhibits cancer-specific apoptotic effects in various types of cancer cells. The aim of the present study was to examine the potential of Ad-REIC as a therapeutic agent for bladder cancer. KK47 and RT4 human bladder cancer cells were sensitive to the Ad-REIC treatment for apoptosis induction, but some human bladder cancer cell lines (T24, J82 and TccSup) were resistant. Significant cell growth inhibition was observed when these resistant cancer cell lines were treated with Ad-REIC in a condition of floating cells, which is clinically observed after transurethral operation and becomes a cause of intravesical cancer dissemination. The therapeutic potential of Ad-REIC for the treatment of multidrug-resistant bladder cancer was investigated. The adriamycin-resistant KK47 bladder cancer cells (KK47/ADM), which also present multidrug resistance, showed induction of significant apoptosis following Ad-REIC treatment. The Ad-REIC treatment induced downregulation of P-glycoprotein in KK47/ADM cells and restored the sensitivity to doxorubicin (adriamycin). Ad-REIC suppressed P-glycoprotein expression in a c-Jun-NH2-kinase (JNK)-dependent manner. Therefore, the current study indicated two therapeutic aspects of the Ad-REIC agent against human bladder cancer cells, as an apoptosis inducer/cell growth inhibitor and as a sensitizer of chemotherapeutic agents in multidrug-resistant cancer cells. The intravesical instillation of Ad-REIC could be an attractive therapeutic method in human bladder cancer where the treatment of superficial lesions and floating/disseminated or multidrug-resistant cancer cells is necessary.

DOI： 10.3892/ijo.2012.1503

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A novel transcriptional system was developed that can robustly enhance cancer-specific gene expression. In the system, hTERT promoter-driven gene expression was enhanced by an advanced two-step transcriptional amplification (TSTA). This construct was used to develop a novel system for detection of bladder cancer cells. The current study evaluated the advanced TSTA system by examining the cancer-specific gene transcription in various bladder cancer cell lines. The system significantly enhanced cancer-specific luciferase gene expression in the bladder cancer cell lines in comparison to the previous expression system of one-step or conventional TSTA. The fold gain of the enhancement was significantly correlated to the telomerase activity of the cell lines. A green fluorescent protein (GFP) gene encoding plasmid vector was constructed where hTERT promoter-driving transcription is enhanced by the advanced TSTA to utilize the system for the imaging and detection of viable bladder cancer cells. The advanced TSTA-hTERT-GFP plasmid successfully induced cancer-specific gene expression, showing robust GFP expression in human bladder cancer cell lines, but no visible GFP expression in normal bladder urothelial cells. The control GFP plasm id with a CMV promoter yielded GFP expression in both normal bladder cells and cancer cells. The advanced TSTA-hTERT-GFP plasmid allowed selective visualization of viable human bladder cancer cells in mixed cell culture containing 10- and 100-fold more normal bladder urothelial cells. These findings indicate that the advanced TSTA-hTERT expressional system is a valuable tool for detecting viable bladder cancer cells. The current system can be applied for in vitro detection of bladder cancer cells in urine and other types of cancer cells disseminated in vivo.

DOI： 10.3892/ijo.2012.1417

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The tumor suppressor REIC/Dkk-3 is a secretory protein which was originally identified to be downregulated in human immortalized cells In the present study, we investigated the expression pattern of REIC/Dkk-3 in various cell types to characterize its physiological functions We first examined the expression level of REIC/Dkk-3 in a broad range of cancer cell types and confirmed that it was significantly downregulated in all of the cell types We also examined the tissue distribution pattern in a variety of normal mouse organs Ubiquitous REIC/Dkk-3 protein expression was observed in the organs The expression was abundant in the liver, heart and brain tissue, but was absent in the spleen and peripheral blood mononuclear cells The immunohistochemical analyses revealed that the subcellular localization of REIC/Dkk-3 had a punctate pattern around the nucleus, indicating its association with secretory vesicles In cancer cells stably transfected with REIC/Dkk-3 the protein was predominantly localized to the endoplasmic reticulum (ER) under observation with confocal microscopy Because REIC/ Dkk-3 was found to be abundantly expressed in the acinar epithelial cells of the mouse prostate, we analyzed the effects of recombinant REIC/Dkk-3 protein on the acinar morphogenesis of RWPE-1 cells, which are derived from human normal prostate epithelium Statistically significant acinar growth was observed in the culture condition with 10 mu g/m1 REIC/Dkk-3 protein, implicating the soluble form m prostatic acinar development Current results suggest that REIC/Dkk-3 may play a role in regulating the morphological process of normal tissue architecture through an autocrine and/or paracrine manner

DOI： 10.3892/ijo_00000802

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Liposomes have been utilized for variety of membrane transport studies including clathrin-mediated endocytosis. Here we introduce clathrin-coated structures that are generated on large unilamellar liposomes by incubating with clathrin coat proteins. Large unilamellar liposomes are also used to reconstitute vesicle formation in a cell-free system, and the vesicle formation can be quantified by using dynamic light scattering (DLS). Furthermore, phagocytosis assay using liposome-conjugated styrene beads is demonstrated.

DOI： 10.1007/978-1-60761-447-0_36

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry & Molecular Biology (ASBMB)  

DOI： 10.1074/jbc.m109.064204

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

DOI： 10.1016/j.bbrc.2009.10.105

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bbrc.2008.11.066

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

Testicular Sertoli cells highly express dynamin 2 and amphiphysin 1. Here we demonstrate that dynamin 2 is implicated in phosphatidylserine (PS)-dependent phagocytosis in Sertoli cells. Immunofluorescence and dual-live imaging revealed that dynamin 2 and amphiphysin 1 accumulate simultaneously at ruffles. These proteins are specifically bound in vitro. Over-expression of dominant negative dynamin 2 (K44A) inhibits liposome-uptake and leads to the mis-localization of amphiphysin 1. Thus, the cooperative function of dynamin 2 and amphiphysin 1 in PS-dependent phagocytosis is strongly suggested.

DOI： 10.18926/AMO/30954

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Cell Biology (ASCB)  

Amphiphysin 1 is involved in clathrin-mediated endocytosis. In this study, we demonstrate that amphiphysin 1 is essential for cellular phagocytosis and that it is critical for actin polymerization. Phagocytosis in Sertoli cells was induced by stimulating phosphatidylserine receptors. This stimulation led to the formation of actin-rich structures, including ruffles, phagocytic cups, and phagosomes, all of which showed an accumulation of amphiphysin 1. Knocking out amphiphysin 1 by RNA interference in the cells resulted in the reduction of ruffle formation, actin polymerization, and phagocytosis. Phagocytosis was also drastically decreased in amph 1 (−/−) Sertoli cells. In addition, phosphatidylinositol-4,5-bisphosphate–induced actin polymerization was decreased in the knockout testis cytosol. The addition of recombinant amphiphysin 1 to the cytosol restored the polymerization process. Ruffle formation in small interfering RNA-treated cells was recovered by the expression of constitutively active Rac1, suggesting that amphiphysin 1 functions upstream of the protein. These findings support that amphiphysin 1 is important in the regulation of actin dynamics and that it is required for phagocytosis.

DOI： 10.1091/mbc.e07-04-0296

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

Amphiphysin 1 (amph 1) is an endocytic protein enriched in the nerve terminals that functions in the clathrin-mediated endocytosis. It acts as membrane curvature sensor, a linker of clathrin coat proteins, and an enhancer of dynamin Guanosine Triphosphatase (GTPase) activity. Amph 1 undergoes phosphorylation by cyclin-dependent kinase 5 (Cdk5), at five phosphorylation sites, serine 262, 272, 276, 285, and threonine 310, as determined by mass spectrometry (MS). We show here that Cdk5-dependent phosphorylation of amph 1 is enhanced in the presence of lipid membranes. Analysis by tandem liquid chromatograph MS revealed that the phosphorylation occurs at two phosphorylation sites. The phosphorylation was markedly decreased by mutation either Ser276 or Ser285 of amph 1 to alanine (S276A and S285A). Furthermore, mutation of both sites (S276, 285A) completely eliminated the phosphorylation. Functional studies indicated that binding of amph 1 to lipid membrane was attenuated by Cdk5-dependent phosphorylation of wild type amph 1, but not of the S276, 285A form. Interestingly, endocytosis was increased in rat pheochromocytoma cells expressing amph 1 S276, 285A in comparison with wild type. These results suggest that Ser276 and Ser285 are regulatory Cdk5 phosphorylation sites of amph 1 in the lipid-bound state. Phosphorylation at these sites alters binding of amph 1 to lipid membranes, and may be an important regulatory aspect in the regulation of synaptic vesicle endocytosis.

DOI： 10.1111/j.1471-4159.2007.04507.x

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

Several species of ascidians, the so-called tunicates, accumulate extremely high levels of vanadium ions in their blood cells. We previously identified a family of vanadium-binding proteins, named Vanabins, from blood cells and blood plasma of a vanadium-rich ascidian, Ascidia sydneiensis samea. The 3-dimensional structure of Vanabin2, the predominant vanadium-binding protein in blood cells, has been revealed, and the vanadium-binding properties of Vanabin2 have been studied in detail. Here, we used Far Western blotting to identify a novel protein that interacts with Vanabin2 from a blood cell cDNA library. The protein, named Vanabin-interacting protein 1 (VIP1), was localized in the cytoplasm of signet ring cells and giant cells. Using a two-hybrid method, we revealed that VIP1 interacted with Vanabins 1, 2, 3, and 4 but not with Vanabin P. The N-terminal domain of VIP1 was shown to be important for the interaction. Further, Vanabin1 was found to interact with all of the other Vanabins. These results suggest that VIP1 and Vanabin1 act as metal chaperones or target proteins in vanadocytes. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbagen.2007.02.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC CELL BIOLOGY  

Tubulobulbar complexes (TBCs) are composed of several tubular invaginations formed at the plasma membrane of testicular Sertoli cells. TBCs are transiently formed at the contact region with spermatids at spermatogenic stage VII in rat and mouse, and such TBC formation is prerequisite for spermatid release. Since the characteristic structure of TBCs suggests that the molecules implicated in endocytosis could be involved in TBC formation, we here investigated the localization and physiological roles of endocytic proteins, amphiphysin 1 and dynamin 2, at TBCs. We demonstrated by immunofluorescence that the endocytic proteins were concentrated at TBCs, where they colocalized with cytoskeletal proteins, such as actin and vinculin. Immunoelectron microscopy disclosed that both amphiphysin 1 and dynamin 2 were localized on TBC membrane. Next, we histologically examined the testis from amphiphysin 1 deficient {Amph(-/-)} mice. Morphometric analysis revealed that the number of TBCs was significantly reduced in Amph(-/-). The ratio of stage VIII seminiferous tubules was increased, and the ratio of stage IX was conversely decreased in Amph(-/-). Moreover, unreleased spermatids in stage VIII seminiferous tubules were increased in Amph(-/-), indicating that spermatid release and the following transition from stage VIII to IX was prolonged in Amph(-/-) mice. These results suggest that amphiphysin 1 and dynamin 2 are involved in TBC formation and spermatid release at Sertoli cells.

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

Some species of the family Ascidiidae accumulate vanadium in concentrations in excess of 350 mM, which is about 10(7)-fold higher than the concentration of vanadium in seawater. In these species, signet ring cells with a single large vacuole in which vanadium ions are contained function as vanadium-accumulating cells. These have been termed vanadocytes. We recently isolated five vanadium-binding proteins, which we named Vanabin1, Vanabin2, Vanabin3, Vanabin4, and VanabinP, from vanadocytes of the vanadium-rich ascidian Ascidia sydneiensis samea. In this study, we analyzed localization of the Vanabins in the blood cells of A. sydneiensis samea using monoclonal antibodies and confocal microscopy. The Vanabin1 and Vanabin2 proteins were found in the cytoplasm and/or in some organelles of vanadocytes. Vanabin3 was also detected in the cytoplasm, while Vanabin4 was found exclusively in the cytoplasmic membrane.

DOI： 10.2108/zsj.23.909

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC CELL BIOLOGY  

Klotho mutant mouse (kl-/-), a mouse model for human aging, exhibits various phenotypes in a wide range of organs including arteriosclerosis, neural degeneration, skin and gonadal atrophy, pulmonary emphysema, calcification of soft tissues, and cognition impairment. Klotho mRNA, however, is expressed only in brain, kidney, reproductive organs, pituitary gland, and parathyroid gland. Therefore it remains to be elucidated how lack of Klotho protein in these limited organs leads to the variety of phenotypes. To shed light on mechanisms by which Klotho protein acts on distant targets, we examined localization of Klotho protein in brain, kidney, and reproductive organs, and analyzed brain and kidney in kl-/- mice searching for changes in target regions in these organs. In brain, Klotho proteins were localized at choroid plexus, where the proteins were dominantly localized at the apical plasma membrane of ependymal cells. In kl-/- brain, reduction of synapses was evident in hippocampus, suggesting a role of Klotho as a humoral factor in cerebrospinal fluid. Klotho proteins in kidney localized at distal renal tubules. Interestingly, in kl-/-mice, type IIa Na/phosphate (Pi) cotransporters, which function at the proximal renal tubules in reabsorption of phosphate ions, were translocated. This suggests that Klotho protein in kidney is implicated in calcium homeostasis which regulates localization of type IIa Na/Pi cotransporters via parathyroid hormone (PTH). Klotho proteins in reproductive organs were expressed only in mature germ cells, although in kl-/- mice germ cell maturation was arrested at earlier stages. Thus, Klotho proteins not only function as a humoral factor, but also are implicated in hormonal regulation, which may explain why mutation of klotho gene results in a variety of phenotypes.

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Language：Japanese   Publishing type：Part of collection (book)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Background-On exposure to oxidized low-density lipoprotein (oxLDL), vascular cells generally undergo apoptosis, which is one of the major pathogenic factors of atherosclerosis. In this study, we examined the role of dynamin ( a crucial GTPase protein in endocytosis) in oxLDL-induced apoptosis of vascular smooth muscle cells (VSMC).
Methods and Results-After oxLDL stimulation, dynamin-2 colocalized with LOX-1 around the cell surface, as well as oxLDL in the cytoplasm, suggesting that dynamin-2 was involved in scavenger receptor-mediated oxLDL endocytosis. Downregulation of dynamin-2 induced by dynamin-2 dominant negative plasmid (K44A) resulted in a decrease of oxLDL uptake and thereby in a reduction of apoptosis. These data demonstrated that dynamin-2 was involved in oxLDL-induced apoptosis via the oxLDL endocytotic pathway. On the other hand, dynamin-2 wild-type plasmid transfection promoted oxLDL-induced apoptosis without increasing oxLDL uptake. Interestingly, the p53 inhibitor pifithrin-alpha (PFT) significantly reduced apoptosis promoted by wild-type dynamin-2 (78% reduction compared with the PFT[-] condition). These results indicated that dynamin-2 enhanced oxLDL-induced apoptosis of VSMC by participating in the p53 pathway, probably as a signal transducer. Moreover, we demonstrated that, in advanced plaques of apolipoprotein E-/- mice, dynamin-2 expression was often enhanced in apoptotic VSMC, suggesting that dynamin-2 might participate in apoptosis of VSMC even in vivo.
Conclusions - Our data demonstrated that dynamin-2 at least partially regulated oxLDL-induced apoptosis of VSMC by participating in 2 independent pathways: the oxLDL endocytotic pathway and the p53 pathway. These findings suggest that dynamin-2 may serve as a new research or therapeutic target in vascular disease.

DOI： 10.1161/01.CIR.0000147828.86593.85

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Exposure of the skin to sunlight results in an increase of the content of epidermal trans-urocanic acid, a key metabolite of L-histidine, and also in occurrence of the isomerization of trans-urocanic acid to the cis isomer. S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (GS(CIE)), an adduct of urocanic acid and glutathione, is a presumed origin of a urinary compound S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteine (Cys(CIE)). The formation of GS(CIE) is stimulated by exposing the skin to sunlight irradiation. In this study we investigated an enzymatic formation of GS(CIE) from glutathione and cis-urocanic acid by incubation with rat liver extract that contained glutathione S-transferase (GST) at high activity. The formation of GS(CIE) was suppressed significantly when a liver extract depleted of GST activity was used. Enzymatic degradation of GS(CIE) with gamma -glutamyl transpeptidase resulted in the formation of N-[S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteinyl]glycine, a metabolic intermediate between the glutathione adduct and Cys(CIE). A hydrolyzed product of GS(CIE) by HCl was identical with the urinary Cys(CIE). Compounds were analyzed by high-voltage paper electrophoresis, capillary electrophoresis, and fast atom bombardment mass spectrometry. From these results, we suggest that GS(CIE) formed from cis-urocanic acid and glutathione is an origin of the urinary compound Cys(CIE) and that the formation reaction is catalyzed mostly by the action of GST.

DOI： 10.1002/elps.200305582

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

L-Glutamate is believed to function as an intercellular transmitter in the islets of Langerhans. However, critical issues, i.e. where, when and how L-glutamate appears, and what happens upon stimulation of glutamate receptors in the islets, remain unresolved. Vesicular glutamate transporter 2 (VGLUT2), an isoform of the vesicular glutamate transporter essential for neuronal storage of L-glutamate, is expressed in alpha cells (Hayashi, M., Otsuka, M., Morimoto, R., Hirota, S., Yatsushiro, S., Takeda, J., Yamamoto, A., and Moriyama, Y. (2001) J. Biol. Chem. 276, 43400-43406). Here we show that VGLUT2 is specifically localized in glucagon-containing secretory granules but not in synaptic-like microvesicles in alpha TC6 cells, clonal alpha cells, and islet alpha cells. VGLUT1, another VGLUT isoform, is also expressed and localized in secretory granules in alpha cells. Low glucose conditions triggered co-secretion of stoichiometric amounts of L-glutamate and glucagon from alpha TC6 cells and isolated islets, which is dependent on temperature and Ca(2+) and inhibited by phentolamine. Similar co-secretion of L-glutamate and glucagon from islets was observed upon stimulation of beta-adrenergic receptors with isoproterenol. Under low glucose conditions, stimulation of glutamate receptors facilitates secretion of gamma-aminobutyric acid from MIN6 m9, clonal beta cells, and isolated islets. These results indicate that co-secretion of L-glutamate and glucagon from alpha cells under low glucose conditions triggers GABA secretion from beta cells and defines the mode of action of L-glutamate as a regulatory molecule for the endocrine function. To our knowledge, this is the first example of secretory granule-mediated glutamatergic signal transmission.

DOI： 10.1074/jbc.M206758200

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

Dynamin 2 and dynamin 3 are highly expressed in testis. However, their functions in the tissue remain unclear. Considering that dynamin 1, neuron-specific isoform of dynamin, plays a pivotal role in endocytosis, functions of dynamin 2 and dynamin 3 in testis must be essential. Cellular expression and subcellular localization of dynamin 2 and dynamin 3 in testis were investigated. Dynamin 2 and dynamin 3 were highly expressed in germ cells and Sertoli cells, constituents of seminiferous tubules. By immunofluorescence it was revealed that dynamin 2 colocalizes with clathrin both at the plasmamembrane and at Golgi in a cell line of Sertoli cells. Immunoreactivity for dynamin 3, on the other hand, appeared as finer puncta, which did not colocalize with clathrin, suggesting that these two dynamins have distinct functions in Sertoli cells. In the klotho deficient mouse testis, which demonstrates disorder in spermatogenesis, expression of dynamin 2 and dynamin 3 was drastically reduced indicating possible association of these proteins with spermatogenesis.

DOI： 10.1016/S0006-291X(02)00470-9

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

5-hydroxytryptamine (5-HT) is a precursor and a putative modulator for melatonin synthesis in mammalian pinealocytes. 5-HT is present in organelles distinct from l-glutamate-containing synaptic-like microvesicles as well as in the cytoplasm of pinealocytes, and is secreted upon stimulation by norepinephrine (NE) to enhance serotonin N-acetyltransferase activity via the 5-HT2 receptor. However, the mechanism underlying the secretion of 5-HT from pinealocytes is unknown. In this study, we show that NE-evoked release of 5-HT is largely dependent on Ca2+ in rat pinealocytes in culture. Omission of Ca2+ from the medium and incubation of pineal cells with EGTA-tetraacetoxymethyl-ester inhibited by 59 and 97% the NE-evoked 5-HT release, respectively. Phenylephrine also triggered the Ca2+-dependent release of 5-HT, which was blocked by phentolamine, an alpha antagonist, but not by propranolol, a beta antagonist. Botulinum neurotoxin type E cleaved 25 kDa synaptosomal-associated protein and inhibited by 50% of the NE-evoked 5-HT release. Bafilomycin A1, an inhibitor of vacuolar H+-ATPase, and reserpine and tetrabenazine, inhibitors of vesicular monoamine transporter, all decreased the storage of vesicular 5-HT followed by inhibition of the NE-evoked 5-HT release. Agents that trigger L-glutamte exocytosis such as acetylcholine did not trigger any Ca2+-dependent 5-HT release. Vice versa neither NE nor phenylephrine caused synaptic-like microvesicle-mediated l-glutamate exocytosis. These results indicated that upon stimulation of a adrenoceptors pinealocytes secrete 5-HT through a Ca2+-dependent exocytotic mechanism, which is distinct from the exocytosis of synaptic-like microvesicles.

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

As a step toward the elucidation of mechanisms in vesicle budding, a cell-free assay that measures cytosol-induced vesicle generation from liposomes was established. This assay then was used to explore the role of phosphoinositides in vesicle formation. Liposomes incubated with brain cytosol in the presence of ATP and GTP massively generated small vesicles, as assessed both quantitatively and qualitatively by a dynamic light-scattering assay. Both ATP and GTP were required. Vesicle formation was inhibited greatly by the immunodepletion of dynamin 1 from the cytosol, indicating a major contribution of this GTPase in this reaction and suggesting that it mimics endocytic vesicle fission. Increasing the concentration of l-alpha-phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] but not of l-alpha-phosphatidylinositol 4-monophosphate or l-alpha-phosphatidylinositol in the lipid membranes enhanced vesicle formation. Lipid analysis revealed rapid degradation of PtdIns(4,5)P2 to l-alpha-phosphatidylinositol during the incubation with the reaction reaching a maximum within 5 sec, whereas vesicle formation proceeded with a longer time course. PtdIns(4,5)P2 degradation was independent of vesicle formation and occurred also in the presence of guanosine 5'-O-(thiotriphosphate), where few vesicle formations occurred. These results suggest that PtdIns(4,5)P2 plays a critical role in the early step of vesicle formation, possibly in the recruitment of coats and fission factors to membranes.

DOI： 10.1073/pnas.261715599

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

We have previously identified synaptojanin 1, a phosphoinositide phosphatase predominantly expressed in the nervous system, and synaptojanin 2, a broadly expressed isoform. Synaptojanin 1 is concentrated in nerve terminals, where it has been implicated in synaptic vesicle recycling and actin function. Synaptojanin 2A is targeted to mitochondria via a PDZ domain-mediated interaction. We have now characterized an alternatively spliced form of synaptojanin 2 that shares several properties with synaptojanin 1. This isoform, synaptojanin 2B, undergoes further alternative splicing to generate synaptojanin 2B1 and 2B2. Both amphiphysin and endophilin, two partners synaptojanin 1, bind synaptojanin 2B2, whereas only amphiphysin binds synaptojanin 2B1. Sequence similar to the endophilin-binding site in synaptojanin 1 is present only in synaptojanin 2B2, and this sequence was capable of affinity purifying endophilin from rat brain. The Sac1 domain of synaptojanin 2 exhibited phosphoinositide phosphatase activity very similar to that of the Sac1 domain of synaptojanin 1. Site-directed mutagenesis further illustrated its functional similarity to the catalytic domain of Sac1 proteins. Antibodies raised against the synaptojanin 2B-specific carboxyl-terminal region identified a 160-kDa protein in brain and testis. Immunofluorescence showed that synaptojanin 2B is localized at nerve terminals in brain and at the spermatid manchette in testis. Active Rac1 GTPase affects the intracellular localization of synaptojanin 2, but not of synaptojanin 1. These results suggest that synaptojanin 2B has a partially overlapping function with synaptojanin 1 in nerve terminals, with additional roles in neurons and other cells including spermatids.

DOI： 10.1074/jbc.M106404200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Exposure of the skin to sunlight results in an increase in the content of epidermal Urocanic acid, a key metabolite Of L-histidine, and some portions of the metabolite penetrate into the body fluid. S-[2-Carboxy-1 -(1H-imidazol-4-yl)ethyl]glutathione (GS(CIE)), an adduct of glutathione and urocanic acid, was proposed to be an origin of a urinary compound, S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteine (Cys(CIE)). Various catabolites of Cys(CIE) were also isolated from human urine previously. However, no direct evidence to show the existence of GS(CIE) as a biological material had been found. By using capillary electrophoresis, the glutathione adduct has now been found in the extracts of rat tissues from the kidney, liver, skin and blood when the rat was kept under conditions of sunlight irradiation after the fur on the dorsal skin had been clipped. On the other hand, no or a trace of GS(CIE) was determined in rat tissue extracts when the animal was kept indoor in usual manner. The glutathione adduct was isolated from the kidney extract of the sunlight-irradiated rat using ion-exchangers and high-voltage paper electrophoresis, and determined by fast-atom-bombardment mass spectrometry. These results indicate that GS(CIE) formation actually occurs in the body and that the formation is accelerated by exposing the rat to sunlight irradiation. From these findings, we propose an alternative pathway of histidine metabolism which is initiated by the adduction of urocanic acid to glutathione to form GS(CIE) and terminates with the formation of the urinary compounds via Cys(CIE).

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

D-Aspartate in mammalian neuronal and neuroendocrine cells is suggested to play a regulatory role(s) in the neuroendocrine function. Although D-aspartate is known to be released from neuroendocrine cells, the mechanism underlying the release is less understood. Rat pheochromocytoma PC12 cells contain an appreciable amount of D-aspartate (257 +/- 31 pmol/10(7) cells). Indirect immunofluorescence microscopy with specific antibodies against D-aspartate indicated that the amino acid is present within a particulate structure, which is co-localized with dopamine and chromogranin A, markers for secretory granules, but not with synaptophysin, a marker for synaptic-like microvesicles. After sucrose density gradient centrifugation of the postnuclear particulate fraction, about 80% of the D-aspartate was recovered in the secretory granule fraction. Upon the addition of KCI, an appreciable amount of D-aspartate (about 40 pmol/10(7) cells at 10 min) was released from cultured cells on incubation in the presence of Ca2+ in the medium. The addition of A23187 also triggered D-aspartate release. Botulinum neurotoxin type E inhibited about 40% of KCl- and Ca2+-dependent D-aspartate release followed by specific cleavage of 25-kDa synaptosomal-associated protein. alpha -latrotoxin increased the intracellular [Ca2+] and caused the Ca2+-dependent D-aspartate release. Bafilomycin Al dissipated the intracellular acidic regions and inhibited 40% of the Ca2+-dependent D-aspartate release. These properties are similar to those of the exocytosis of dopamine. Furthermore, digitonin-permeabilized cells took up radiolabeled D-aspartate depending on MgATP, which is sensitive to bafilomycin Al or 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile. Taken together, these results strongly suggest that D-aspartate is stored in secretory granules and then secreted through a Ca2+-dependent exocytotic mechanism. Exocytosis of D-aspartate further supports the role(s) of D-aspartate as a chemical transmitter in neuroendocrine cells.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Diabetes Association Inc.  

Pancreatic islet cells express receptors and transporters for L-glutamate and are thus believed to use L-glutamate as an intercellular signaling molecule. However, the mechanism by which L-glutamate appears in the islets is unknown. In the present study, we investigated whether L-glutamate is secreted through exocytosis by αTC6 cells (clonal mouse pancreatic α-cells). An appreciable amount of L-glutamate was released from cultured cells after the addition of KCl or A23187 in the presence of Ca2+ and 10 mmol/l glucose in the medium. The KCl-induced glutamate release was significantly reduced when assayed in the absence of Ca2+ or when the cells were pretreated with EGTA-AM. The KCl-induced Ca2+-dependent glutamate release was inhibited ∼40% by voltage-gated Ca2+ channel blockers, such as nifedipine at 20 μmol/l. The degree of KCl-induced Ca2+-dependent glutamate release was correlated with an increase in intracellular [Ca2+], as monitored by fura-2 fluorescence. Botulinum neurotoxin type E inhibited 55% of the KCl-induced Ca2+-dependent glutamate release, followed by specific cleavage of 25 kDa synaptosomal-associated protein. Furthermore, bafilomycin A1, a specific inhibitor of vacuolar H+-ATPase, inhibited 40% of the KCl-induced Ca2+-dependent glutamate release. Immunoelectronmicroscopy with antibodies against synaptophysin, a marker for neuronal synaptic vesicles and endocrine synaptic-like microvesicles, revealed a large number of synaptophysin-positive clear vesicles in cells. Digitonin-permeabilized cells took up L-glutamate only in the presence of MgATP, which is sensitive to bafilomycin A1 or 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (a proton conductor) but insensitive to either oligomycin or vanadate. From these results, it was concluded that αTC6 cells accumulate L-glutamate in the synaptophysin-containing vesicles in an ATP-dependent manner and secrete it through a Ca2+-dependent exocytic mechanism. The Ca2+-dependent glutamate release was also triggered when cells were transferred in the medium containing 1 mmol/l glucose, suggesting that low glucose treatment stimulates the release of glutamate. Our results are consistent with the idea that L-glutamate is secreted by α-cells through Ca2+-dependent regulated exocytosis.

DOI： 10.2337/diabetes.50.5.1012

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry and Molecular Biology Inc.  

Recent biochemical studies involving 2',7'-bis-(2-carboxyethyl)-5,6-carboxylfluorescein (BCECF)-labeled saponin-permeabilized and parasitized erythrocytes indicated that malaria parasite cells maintain the resting cytoplasmic pH at about 7.3, and treatment with vacuolar proton-pump inhibitors reduces the resting pH to 6.7, suggesting proton extrusion from the parasite cells via vacuolar H+-ATPase (Saliba, K. J., and Kirk, K. (1999) J. Biol. Chem. 274, 33213-33219). In the present study, we investigated the localization of vacuolar H+-ATPase in Plasmodium falciparum cells infecting erythrocytes. Antibodies against vacuolar H+-ATPase subunit A and B specifically immunostained the infecting parasite cells and recognized a single 67- and 55-kDa polypeptide, respectively. Immunoelectron microscopy indicated that the immunological counterpart of V-ATPase subunits A and B is localized at the plasma membrane, small clear vesicles, and food vacuoles, a lower extent being detected at the parasitophorus vacuolar membrane of the parasite cells. We measured the cytoplasmic pH of both infected erythrocytes and invading malaria parasite cells by microfluorimetry using BCECF fluorescence. It was found that a restricted area of the erythrocyte cytoplasm near a parasite cell is slightly acidic, being about pH 6.9. The pH increased to pH 7.3 upon the addition of either concanamycin B or bafilomycin A1, specific inhibitors of vacuolar H+-ATPase. Simultaneously, the cytoplasmic pH of the infecting parasite cell decreased from pH 7.3 to 7.1. Neither vanadate at 0.5 mM, an inhibitor of P-type H+-ATPase, nor ethylisopropylamiloride at 0.2 mM, an inhibitor of Na+/H+-exchanger, affected the cytoplasmic pH of erythrocytes or infecting parasite cells. These results constitute direct evidence that plasma membrane vacuolar H+-ATPase is responsible for active extrusion of protons from the parasite cells.

DOI： 10.1074/jbc.M003323200

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

Mammalian retinal photoreceptors and pinealocytes have common characteristic that they secrete melatonin and L-glutamate as chemical transmitters. Although pinealocytes express glutamate receptors and receive glutamate signals, whether or not photoreceptors express glutamate receptors is unknown. Here, we investigated the expression of the glutamate receptors in cultured Y79 clonal human retinoblastoma cells, as model systems of photoreceptors. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that GluR1, GluR5, GluR7, EAA2, NR1, NR2A and NR2D mRNAs were present in the cultured cells. Northern analysis confirmed the presence of GluR7, EAA2, NR1, NR2A and NR2D mRNAs, while other mRNAs were under the detection limit. Addition of (RS)-alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate increases intracellular (Ca2+) in Fura-2 loaded cells, which is blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. N-methyl-D-aspartate also increases intracellular (Ca2+). These results demonstrated the presence of functional ionotropic receptors in Y79 cells. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Rat pinealocytes receive noradrenergic innervation that stimulates melatonin synthesis. Besides melatonin, we showed previously that pinealocytes accumulate L-glutamate in microvesicles and secrete it through an exocytic mechanism. The secreted glutamate binds to the class II metabotropic glutamate receptor and inhibits norepinephrine-stimulated melatonin synthesis in neighboring pinealocytes through an inhibitory cyclic AMP cascade. In this study, it was found that, in addition to metabotropic receptors, pinealocytes express functional ionotropic receptors. RT-PCR and northern analyses indicated the expression of mRNA for GluR1, KA2, and NR2C in pineal gland. The presence of GluR1 protein was confirmed by immunological techniques, but neither KA2 nor NR2C was detected. Consistent with this observation, the presence of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate, non-N-methyl-D-aspartate receptor agonists, transiently stimulated increased the intracellular Ca2+ concentration of cultured pinealocytes, whereas N-methyl-D-aspartate did not. These responses were prevented by 6-cyano-7-nitroquinoxaline-2,3-dione, a selective antagonist for non-N-methyl-D-aspartate receptors, by L-type Ca2+ channel blockers such as nifedipine, or by omitting Ca2+ or Na+ in the medium. In the presence of Ca2+ and Na+, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxarolepropionic acid or kainate evoked glutamate secretion from the cultured cells, which was prevented by 6-cyano-7-nitroquinoxaline-2,3-dione, L-type Ca2+ channel blockers, type E or B botulinum neurotoxin, or incubation at &lt;20 degrees C. These results strongly suggest that GluR1 is functionally expressed in pinealocytes and triggers microvesicle-mediated exocytosis of L-glutamate via activation of L-type Ca2+ channels. It is possible that GluR1 participates in a signaling cascade that enhances and expands the L-glutamate signal throughout the pineal gland.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Microvesicles in endocrine cells are the morphological and functional equivalent of neuronal synaptic vesicles. Microvesicles accumulate various neurotransmitters through a transmitter-specific vesicular transporter energized by vacuolar H+-ATPase. We found that mammalian pinealocytes, endocrine cells that synthesize and secrete melatonin, accumulate L-glutamate in their microvesicles and secrete it through exocytosis. Pinealocytes use L-glutamate as either a paracrine- or autocrine-like chemical transmitter in a receptor-mediated manner, resulting in inhibition of melatonin synthesis. In this article, we briefly describe the overall features of the microvesicle-mediated signal-transduction mechanism in the pineal gland and discuss the important role of acidic organelles in a novel regulatory mechanism for hormonal synthesis and secretion.

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Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese  

J-GLOBAL

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

Rat pinealocytes use L-glutamate as a modulator for melatonin synthesis. Upon binding of L-glutamate to the class II metabotropic glutamate receptor, norepinephrine (NE)-dependent formation of cAMP was inhibited, resulting in decreased serotonin-N-acetyltransferase (NAT) activity and melatonin output. Although L-glutamate at 1 mM caused 90% inhibition of melatonin synthesis, about 30% of the NAT activity remained, suggesting the presence of another target for L-glutamate. In this study, we found that L-glutamate also inhibits hydroxyindole-O-methyltransferase (HIOMT). The inhibition is reversible and dose-dependent: the maximal inhibition was obtained with more than 0.4 mM L-glutamate. Contrary to L-glutamate-evoked inhibition of NAT, agonists for class Il metabotropic receptors such as (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) had no effect on HIOMT. Neither (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), an specific antagonist for class II mGluRs, nor dibutyryl cAMP restored the L-glutamate-evoked inhibition of HIOMT. Northern blot analyses revealed that L-glutamate significantly inhibits the expression of mRNA of NAT, but not that of HIOMT. These results indicated that HIOMT is an another target for L-glutamate due to its inhibition of melatonin synthesis, and the signaling pathway toward the inhibition is distinct from that of NAT. (C) 1999 Elsevier Science B.V. All rights reserved.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

MAMMALIAN pinealocytes, endocrine cells for melatonin, express class II metabotropic glutamate receptors (mGluRs), which are involved in negative regulation of melatonin synthesis through an inhibitory cAMP cascade. We investigated whether mGluRs other than class II receptors are expressed in rat pinealocytes. RT-PCR analysis and Northern blotting indicated that the mRNA of mGluR5, a class I receptor, was present in pineal glands. Quisqualate and 1-aminocyclopentane- 1,3-dicarboxylate (1S,3R-ACPD), class I receptor agonists, increased the intracellular [Ca2+] of fura-2 loaded cultured pinealocytes in the absence of extracellular Ca2+ which is blocked by methylcarboxyphenylglycine, a class I receptor antagonist. These results suggest that mGluR5 is functionally expressed in pinealocytes and triggers Ca2+ efflux from intracellular stores. NeuroReport 10:1599-1603 (C) 1999 Lippincott Williams & Wilkins.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC / PLENUM PUBL  

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

Rat pinealocytes, melatonin-secreting endocrine cells, contain peripheral glutaminergic systems. L-Glutamate is a negative regulator of melatonin synthesis through a metabotropic receptor-mediated inhibitory cAMP cascade. Previously, we reported that depolarization of pinealocytes by externally added KCl and activation of L-type Ca2+ channels resulted in secretion of L-glutamate by microvesicle exocytosis. What is unknown is how and what kinds of stimuli trigger glutamate exocytosis under physiological conditions. Here, we report that the nicotinic acetylcholine receptor can trigger glutamate exocytosis from cultured rate pinealocytes. Moreover, acetylcholine or nicotine inhibited norepinphrine-dependent serotonin N-acetyltransferase activity, which results in decreased melatonin synthesis. These activities were blocked by (2S,3S,4S)-2-methyl-2- (carboxycyclopropyl)glycine, an antagonist of the metabotropic glutamate receptor. These results suggest that cholinergic stimulation initiates the glutaminergic signaling cascade in pineal glands and that parasympathetic neurons innervating the gland exert negative control over melatonin synthesis by way of the glutaminergic systems.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Microvesicles are endocrine counterparts of neuronal synaptic vesicles, and accumulate and secrete classic neurotransmitters. In mammalian pinealocytes, microvesicles accumulate L-glutamate through a vesicular glutamate transporter and secrete it through exocytosis, To characterize the molecular organization of microvesicles in more detail, we investigated in this study the expression and localization of synaptic vesicle protein 2 (SV2) in rat pinealocytes. RT-PCR analysis indicated that transcripts specific for two isoforms, SV2A, a ubiquitous form present in neuronal and endocrine cells, and SV2B, a neuron-specific form, are amplified in pineal RNAs, Northern blotting with specific transcripts indicated that the mRNA for SV2B is predominantly expressed, whereas that for SV2A is below the detection limit. Site-specific antibodies against SV2B recognized a single 72-kDa polypeptide in the pineal membrane fraction, whereas anti-SV2A antibodies did not recognize any polypeptides, Immunohistochemical analysis of cultured cells indicated that SV2B is expressed in pinealocytes but not in other types of cells. SV2B is present in somata and is especially rich in processes, which are filled with microvesicles. SV2B is colocalized with synaptophysin and synaptotagmin, markers for microvesicles, Immunoelectron microscopy indicated that SV2B is associated with microvesicles. These results indicated that SV2B, but not SV2A, is expressed in rat pinealocytes and associated with microvesicles, As SV2B is also expressed in cultured alpha TC6 clonal pancreatic alpha cells, SV2B is not a protein specific for neurons.

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

It has been known that pinealocytes contain the highest level of D-aspartate among various neuroendocrine cells in the rat. Here, we report that exogenous D-aspartate strongly inhibited norepinephrine-dependent melatonin synthesis in the rat pineal gland, the concentration required for 50% inhibition being 75 mu M. This inhibition was due at least partly to decreased norepinephrine-dependent serotonin N-acetyltransferase activity. Upon incubation, D-aspartate was gradually released from pinealocytes and accumulated in the incubation medium as determined by high-performance liquid chromatography on a Pirkle-type chiral column, These results suggest that D-aspartate acts as a negative regulator for melatonin synthesis in the pineal gland. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

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

D-Aspartate is now known to be present in mammalian neuronal and endocrine cells in vivo, and may play some role(s) in neurocrine and endocrine functions. However, origin of D-aspartate is unknown. Here, we report that free D-aspartate (108 pmoles/3 x 10(7) cells) is present in the cultured PC12 cells, a rat pheochromocytoma cell line, as determined with immunohistochemical techniques as well as high performance liquid chromatography (HPLC) on a Pirkle-type chiral column. The amount of D-aspartate does not change with the passage. The culture medium does not contain D-aspartate. These results strongly suggest the presence of a de novo biosynthetic pathway for D-aspartate in the endocrine cells. (C) 1998 Elsevier Science Ireland Ltd.

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

Rat pinealocytes receive noradrenergic innervation that stimulates melatonin synthesis in a cAMP-mediated manner. In addition to melatonin, we showed previously that pinealocytes secrete L-glutamate through an exocytic mechanism. The released glutamate inhibits norepinephrine (NE)-dependent melatonin synthesis. Consistent with this observation, specific agonists of class II metabotropic glutamate receptors (mGluRs), including 1-(1S,3R)-aminocyclopentane-1,3-dicarboxylic acid (tACPD), inhibited NE-dependent melatonin synthesis, whereas agonists for other types of glutamate receptors did not. Furthermore, reverse transcription-PCR, Northern blotting, and immunohistochemistry analyses indicated expression of class II mGluR3 in pinealocytes. Inhibitory guanine nucleotide-binding protein (G(i)) was also detected in pinealocytes. L-Glutamate or agonists of class II receptors decreased NE- or forskolin-dependent increase of cAMP and serotonin-N-acetyltransferase activities to similar extents. These effects were blocked by pertussis toxin or dibutyryl cAMP, These results indicate that the inhibitory cAMP cascade is involved in the glutamate-evoked inhibition of melatonin synthesis, We propose that the glutaminergic system negatively regulates NE-dependent melatonin synthesis in rat pinealocytes.

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Language：Japanese  

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

Pinealocytes, the neuroendocrine cells that produce melatonin, accumulate glutamate in microvesicles through a specific vesicular transporter energetically coupled with vacuolar-type proton ATPase. The glutamate is secreted into the extracellular space through microvesicle-mediated exocytosis and then stimulates neighboring pinealocytes, resulting in inhibition of norepinephrine-dependent melatonin synthesis. In this study, we identified and characterized the plasma membrane-type glutamate transporter in rat pinealocytes. The [H-3]-glutamate uptake by cultured pinealocytes was driven by extracellular Na+, saturated with the [H-3] glutamate concentration used, and significantly inhibited by L-glutamate, L-aspartate, beta-threo-hydroxyaspartale, pyrrolidine dicarboxylate, and L-cysteine sulfinate, substrates or inhibitors of the plasma membrane glutamate transporter. Consistently, the clearance of extracellular glutamate, as measured by HPLC, was also dependent on Nai and inhibited by beta-threo-hydroxyaspartate and L-cysteine sulfinate. Immunological studies with site-specific antibodies against three isoforms of the Na+-dependent glutamate transporter (GLT-1, GLAST, and EAAC1) revealed the expression of only the GLT-1 type transporter in pineal glands. Expression of the GLT-1 type transporter in pineal glands was further demonstrated by means of reverse transcription-polymerase chain reaction with specific DNA probes. Immunohistochemical analysis indicated that the immunological counterpart(s) of the GLT-1 is localized in pinealocytes. These results suggested that the GLT-1-type Na+-dependent transporter is expressed and functions as a reuptake system for glutamate in rat pinealocytes. The physiological role of the transporter in the termination of the glutamate signal in the pineal gland is discussed.

DOI： 10.1046/j.1471-4159.1997.69041491.x

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

Rat pinealocytes accumulate glutamate in microvesicles and secrete it through exocytosis so as to transmit signals intercellularly. Glutamate is involved in the negative regulation of norepinephrine-stimulated melatonin production. In this study, we found that aspartate is also released from cultured rat pinealocytes during the exocytosis of glutamate. The release of aspartate was triggered by addition of KCl or A23187 (a Ca2+ ionophore) in the presence of Ca2+ and was proportional to the amount of L-glutamate released. Furthermore, the release of aspartate was inhibited by both botulinum neurotoxin type E and L- or N-type voltage-gated Ca2+ channel blockers. Bay K 8644, an agonist for the L-type Ca2+ channel, stimulated the release of aspartate 2.1-fold. Immunohistochemical analyses with antibodies against aspartate and synaptophysin revealed that aspartate is colocalized with synaptophysin in a cultured pinealocyte. HPLC with fluorometric detection indicated that the released aspartate is of the L form, although pinealocytes also contain the D form in their cytoplasm, corresponding to similar to 30% of the total free aspartate, Radiolabeled L-aspartate was taken up by the microsomal fraction from bovine pineal glands in a Na+-dependent manner. The Na+-dependent uptake of L-aspartate was strongly inhibited by L-cysteine sulfinate, beta-hydroxyaspartate, and L-serine-O-sulfate, inhibitors for the Na+-dependent glutamate/aspartate transporter on the plasma membrane, Na+-dependent sequestration of L-aspartate was also observed in cultured rat pinealocytes, which was inhibited similarly by these transporter inhibitors. These results strongly suggest that L-aspartate is released through microvesicle-mediated exocytosis from pinealocytes and is taken up again through the Na+-dependent transporter at the plasma membrane. The possible role of L-aspartate as an intercellular chemical transmitter in the pineal gland is discussed.

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

Our previous studies in rat indicated that pinealocytes secrete L-glutamate through microvesicle-mediated exocytosis to regulate negatively melatonin production. Recently, we further found that pinealocytes secrete L-aspartate through microvesicle-mediated exocytosis. In the present study, we investigated the role of L-aspartate in the melatonin production in isolated rat pineal glands. It was found that L-aspartate inhibits norepinephrine-stimulated melatonin production as well as serotonin N-acetyltransferase activity reversibly and dose dependently, the concentrations required for 50% inhibition being 150 and 175 mu M, respectively. L-Asparagine and oxaloacetate, metabolites of L-aspartate, had no effect on the melatonin production. These results suggest that pinealocytes use L-aspartate, as well as L-glutamate, as a negative regulator for melatonin production. (C) 1997 Elsevier Science Ireland Ltd.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

Escherichia coli H+-ATPase subunit a is a hydrophobic F0 subunit. To investigate the topology of the subunit in the membrane, we prepared site-specific polyclonal antibodies against amino-terminal (Ser-3 to Leu-16), middle loop (Lys-167 to Gln-181), and carboxyl-terminal (Thr-259 to His-271) peptide segments. Enzyme-linked immunosorbent assay revealed that these antibodies specifically reacted with subunit a of inside-out membrane vesicles, but not with that of right-side-out spheroplasts. Full reactivity appeared when spheroplasts were disrupted with Triton X-100 (0.5%) or by sonication. These results suggest that at least parts of the three peptide segments of subunit a face the cytoplasm. Based on these observations, we propose a novel transmembrane topology of subunit a.

DOI： 10.1016/0014-5793(96)00621-7

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

The microvesicles (MVs) in bovine posterior pituitaries contain the reserpine-sensitive vesicular monoamine transporter (Moriyama et al. (1995) J. Biol. Chem. 270, 11424-11429). An antibody against the N-terminal region of the monoamine transporter from bovine chromaffin granules recognized a polypeptide in the MVs with a similar molecular mass to the chromaffin granule counterpart. 1-Methyl-4-phenylpyridinium inhibited the norepinephrine uptake by the MVs and chromaffin vesicles, the concentrations required for 50% inhibition being 8 and 150 mu M, respectively. Histamine also showed similar effect. These results indicated that the monoamine transporter in MVs is immunologically similar to, but distinguishable pharmacologically, from the chromaffin granule counterpart, and suggested dir polymorphism of the transporter in bovine tissues. (C) 1996 Academic Press, Inc.

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Microvesicles (MVs) in endocrine cells are morphologically similar to neuronal synaptic vesicles. MVs were shown to contain proteins involved in neurotransmitter storage such as vacuolar H(+)-ATPase and neurotransmitter transporters, and ones in vesicular trafficking such as synaptobrevins and N-ethylmaleimide-sensitive fusion protein. Isolated MVs accumulate cell-specific neurotransmitters in an energy-dependent manner. Upon stimulation, the MVs may fuse with the plasma membrane and secrete the internal neurotransmitters. Thus, endocrine cells possess an MV-mediated secretion system as an intercellular signal transducing system.

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

Pinealocytes, parenchymal cells of the pineal gland, secrete glutamate through microvesicle-mediated exocytosis upon depolarization by KCl in the presence of Ca2+, which is involved in a novel paracrine-like intercellular signal transduction mechanism in neuroendocrine organs. In the present study, we investigated whether or not the L-type Ca2+ channel is involved in the microvesicle-mediated glutamate secretion from cultured rat pinealocytes. Nifedipine, a specific antagonist of the L-type Ca2+ channel, inhibited the Ca2+-dependent glutamate exocytosis by 48% at 20 μM. Other L-type Ca2+ channel antagonists, such as nitrendipine, showed similar effects. 1,4-Dihydro-2,6-dimethyl-5-nitro-4[2-(trifluoromethyl)-phenyl]-3- pyridinecarboxylic acid methyl ester (BAY K8644), an agonist of the L-type Ca2+ channel, at 1 μM, on the other hand, stimulated the glutamate exocytosis about 1.6-fold. Consistently, these Ca2+ channel antagonists inhibited about 50% of the Ca2+ uptake, whereas BAY K8644 increased the uptake 5.3-fold. An antibody against the carboxyl-terminal region of the rabbit L-type Ca2+ channel recognized polypeptides of pinealocytes with apparent molecular masses of 250 and 270 kDa, respectively, and immunostained the plasma membrane region of the pinealocytes. These results strongly suggested that the entry of Ca2+ through L-type Ca2+ channel(s), at least in part, triggers microvesicle-mediated glutamate exocytosis in pinealocytes. © Munksgaard.

DOI： 10.1111/j.1600-079X.1996.tb00284.x

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

Mammalian pinealocytes are neuroendocrine cells that synthesize and secrete melatonin, these processes being positively controlled by norepinephrine derived from innervating sympathetic neurons. Previously, we showed that pinealocytes contain a large number of microvesicles (MVs) that specifically accumulate L-glutamate through a vesicular glutamate transporter and contain proteins for exocytosis such as synaptobrevin 2 (VAMP2). These findings suggested that the MVs are counterparts of synaptic vesicles and are involved in paracrine-like chemical transduction in the pineal gland. Here, we show that pinealocytes actually secrete glutamate upon stimulation by KCl in the presence of Ca2+ at 37°C. The ability of glutamate secretion disappeared when the cells were incubated at below 20°C. Loss of the activity was also observed on successive stimulation, but it was recovered after 12 hr incubation. A low concentration of cadmium chloride or ω-conotoxin GVIA inhibited the secretion. Botulinum neurotoxin E cleaved synaptic vesicle-associated protein 25 (SNAP-25) and thus inhibited the secretion. The released L-glutamate stimulated pinealocytes themselves via glutamate receptor(s) and inhibited norepinephrine-stimulated melatonin secretion. These results strongly suggest that pinealocytes are glutaminergic paraneurons, and that the glutaminergic system regulates negatively the synthesis and secretion of melatonin. The MV-mediated paracrine-like chemical transduction seems to be a novel mechanism that regulates hormonal secretion by neuroendocrine cells. © Munksgaard.

DOI： 10.1111/j.1600-079X.1996.tb00285.x

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

Histochemical study indicated that the posterior pituitary possesses numerous microvesicles (MVs) containing synaptophysin, a marker protein specific for brain synaptic vesicles (Navone, F., Di Gioia, G., Jahn, R., Browning, M., Greengard, P. and De Camilli, P. (1989) J. Cell Biol. 109, 3425-2433). By monitoring cross-reactivity with anti-synaptophysin antibody, the MVs were highly purified from bovine posterior pituitaries by a combination of differential and sucrose density gradient centrifugations. The purified MVs had an average diameter of about 60 nm and were associated with synaptophysin as revealed by immunoelectron microscopy. The vesicles contained ATPase activity partially sensitive to bafilomycin A1 and to vanadate. The membrane fraction immunoisolated with anti-synaptophysin antibody also exhibited similar ATPase activity. The two ATPases could be purified separately
the vanadate-sensitive enzyme was identified as a 115- kDa polypeptide immunochemically similar to chromaffin granule P-ATPase (forming phosphoenzyme intermediate), and the bafilomycin A1-sensitive ATPase showed essentially the same properties as those of vacuolar type H+- ATPases. Upon addition of ATP, the MVs formed an electrochemical gradient of protons and took up norepinephrine in a reserpine-sensitive manner, indicating the presence of secondary monoamine transporter coupled with vacuolar type H+-ATPase. No uptake of L-glutamate, γ-aminobutyrate, glycine, or acetylcholine was observed. The identification of MVs as organelles responsible for storage of monoamines is important for understanding the physiological function of the posterior pituitary.

DOI： 10.1074/jbc.270.19.11424

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2-(4-Phenylpiperidino)cyclohexanol (AH-5183) and 2-bromo-α-ergocryptine, known inhibitors of the transport of acetylcholine and l-glutamate, respectively, into synaptic vesicles, inhibited the ATP-dependent uptake of dopamine in parallel with the dissipation of the electrochemical gradient of protons in chromaffin granule membrane vesicles. These compounds induced the release of accumulated dopamine from the vesicles. They also inhibited the ATP-dependent formation of the electrochemical gradient of protons in liposomes reconstituted with chromaffin H+-ATPase without affecting the activities for ATP hydrolysis, and ATP-dependent uptakes of dopamine, γ-aminobutyrate, and glutamate into synaptic vesicles. These results indicated that 2-(4-phenylpiperidino)cyclohexanol and 2-bromo-α-ergocryptine acted as uncouplers in the secretory vesicles. © 1991.

DOI： 10.1016/0003-9861(91)90614-O

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Authorship：Lead author   Language：Japanese  

J-GLOBAL

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Language：English   Publisher：日本神経化学会  

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Language：English   Publisher：(公社)日本生化学会  

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Language：English   Publisher：(一社)日本細胞生物学会  

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Language：English   Publisher：(一社)日本細胞生物学会  

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